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Porphyria cutanea tarda (pct) is the most common type of porphyria worldwide, affecting sun - exposed areas of the skin, manifested by cutaneous fragility, vesicobullae, atrophic scarring and formation of milia . This condition is a disorder of heme biosynthesis caused by a catalytic deficiency in uroporphyrinogen decarboxylase . There are inherited, acquired and toxic forms of the condition and diagnosis is confirmed based on skin symptoms and porphyrin analysis of urine, blood and stool . Without clinical suspicion for this important diagnosis the necessary investigations may not be performed, resulting in a delay of diagnosis and treatment . We present the cases of two patients who were initially misdiagnosed with epidermolysis bullosa acquisita (eba) predominantly based on histopathology but were subsequently diagnosed with pct on porphyrin studies due to clinical suspicion . A 36-year - old female presented with a 6-month history of blisters on the dorsal aspects of her hands bilaterally with associated fragility and photosensitivity . This was also associated with a 2-year history of increased hair growth on her cheeks . She had been previously treated with oral antibiotics and topical steroid with no obvious improvement . The patient had a medical background of asthma for which she was using twice daily fluticasone / salmeterol inhalations and salbutamol inhalations as required . Her only other medication was the oral contraceptive pill, which she had been taking long - term . The patient had multiple blisters and erosions distributed over the dorsa of her hands with a number of milia (fig . 1). A punch biopsy of the right hand index finger and a perilesional biopsy of the left hand were performed . Histopathology showed a partly regenerated subepidermal blister with a heavy inflammatory cell infiltrate between the old roof and the regenerating base (fig . The pattern was recognized as the u - serrated pattern, typical of antibodies to type vii collagen . Based on these findings, eba was diagnosed and salt split skin preparation was not performed . Although the initial attending doctor was dissuaded from a diagnosis of pct on the basis of histology findings, subsequent review favoured pct clinically and porphyrin studies later confirmed this (table 1). The patient had deranged liver function tests, but her liver screen did not demonstrate any abnormalities, including negative hepatitis b / c and hiv serologies and normal iron studies and alpha fetoprotein levels . Desmoglein 1 and 3 reactivity and subsequent collagen vii reactivity testing were all negative, excluding a concomitant diagnosis of pemphigus or eba . The patient was commenced on hydroxychloroquine 200 mg twice weekly and was given advice on mechanical and photoprotection . She was also commenced on regular venesections, aiming for a ferritin level of 50 g / l to improve pct symptoms . A 50-year - old female presented for a second opinion after a 3-year history of spontaneous blistering affecting the extensor surfaces of her hands and forearms with associated scarring . She had been diagnosed with eba by a previous clinician based on biopsy histopathology findings 3 months earlier . There was a cell - poor subepidermal vesicle containing only a few lymphocytes and with festooning of the dermal papillae and very little dermal inflammatory infiltrate seen (fig . Direct immunofluorescence studies revealed positive staining for igg in a linear pattern along the dermoepidermal junction . All other markers were negative and salt split skin studies could not be performed due to technical difficulties . The patient was initially treated with potent topical corticosteroids as well as low - dose oral corticosteroids and was still experiencing flares . She was then tried on 100 mg of oral doxycycline, which resulted in some improvement . She also reported a positive family history for blistering of the hands affecting her maternal grandmother but no known family history of liver disease . She had been taking the oral contraceptive pill for many years but had recently ceased taking this . On examination, the dorsal aspects of both hands were erythematous, with evidence of milia and post - inflammatory hyperpigmentation (fig . Based on this clinical appearance, further testing was performed, including full blood count, renal function, electrolytes and liver function, which were all normal . In addition no hfe mutations were detected and hepatitis b / c and hiv serologies were negative . Further urine and stool porphyrin studies showed elevated urine porphyrin, faecal porphyrin and porphyria / creatinine ratio, which confirmed the diagnosis of pct (table 1). Desmoglein 1 and 3 reactivity and collagen vii reactivity testing were negative, excluding a concomitant diagnosis of pemphigus or eba . She was also advised to use gloves and zinc oxide sunscreen to provide physical protection . A 36-year - old female presented with a 6-month history of blisters on the dorsal aspects of her hands bilaterally with associated fragility and photosensitivity . This was also associated with a 2-year history of increased hair growth on her cheeks . She had been previously treated with oral antibiotics and topical steroid with no obvious improvement . The patient had a medical background of asthma for which she was using twice daily fluticasone / salmeterol inhalations and salbutamol inhalations as required . Her only other medication was the oral contraceptive pill, which she had been taking long - term . The patient had multiple blisters and erosions distributed over the dorsa of her hands with a number of milia (fig . 1). A punch biopsy of the right hand index finger and a perilesional biopsy of the left hand were performed . Histopathology showed a partly regenerated subepidermal blister with a heavy inflammatory cell infiltrate between the old roof and the regenerating base (fig . The pattern was recognized as the u - serrated pattern, typical of antibodies to type vii collagen . Based on these findings, eba was diagnosed and salt split skin preparation was not performed . Although the initial attending doctor was dissuaded from a diagnosis of pct on the basis of histology findings, subsequent review favoured pct clinically and porphyrin studies later confirmed this (table 1). The patient had deranged liver function tests, but her liver screen did not demonstrate any abnormalities, including negative hepatitis b / c and hiv serologies and normal iron studies and alpha fetoprotein levels . Desmoglein 1 and 3 reactivity and subsequent collagen vii reactivity testing were all negative, excluding a concomitant diagnosis of pemphigus or eba . The patient was commenced on hydroxychloroquine 200 mg twice weekly and was given advice on mechanical and photoprotection . She was also commenced on regular venesections, aiming for a ferritin level of 50 g / l to improve pct symptoms . A 50-year - old female presented for a second opinion after a 3-year history of spontaneous blistering affecting the extensor surfaces of her hands and forearms with associated scarring . She had been diagnosed with eba by a previous clinician based on biopsy histopathology findings 3 months earlier . There was a cell - poor subepidermal vesicle containing only a few lymphocytes and with festooning of the dermal papillae and very little dermal inflammatory infiltrate seen (fig . Direct immunofluorescence studies revealed positive staining for igg in a linear pattern along the dermoepidermal junction . All other markers were negative and salt split skin studies could not be performed due to technical difficulties . The patient was initially treated with potent topical corticosteroids as well as low - dose oral corticosteroids and was still experiencing flares . She was then tried on 100 mg of oral doxycycline, which resulted in some improvement . She also reported a positive family history for blistering of the hands affecting her maternal grandmother but no known family history of liver disease . She had been taking the oral contraceptive pill for many years but had recently ceased taking this . On examination, the dorsal aspects of both hands were erythematous, with evidence of milia and post - inflammatory hyperpigmentation (fig . Based on this clinical appearance, further testing was performed, including full blood count, renal function, electrolytes and liver function, which were all normal . In addition no hfe mutations were detected and hepatitis b / c and hiv serologies were negative . Further urine and stool porphyrin studies showed elevated urine porphyrin, faecal porphyrin and porphyria / creatinine ratio, which confirmed the diagnosis of pct (table 1). Desmoglein 1 and 3 reactivity and collagen vii reactivity testing were negative, excluding a concomitant diagnosis of pemphigus or eba . She was also advised to use gloves and zinc oxide sunscreen to provide physical protection . Pct is a metabolic disorder which can result in the development of hepatocellular carcinoma, advanced liver fibrosis and also liver cirrhosis if cutaneous manifestations are not recognized and properly investigated . There are multiple genetic and environmental triggers of pct, most commonly including alcohol abuse, oestrogens and liver disease . Other triggering factors include polychlorinated hydrocarbons, iron, viral infections (such as hepatitis c and hiv), dialysis in patients with renal failure and the inheritance of specific mutations in the hfe gene which underlies classic haemochromatosis . Treatment of pct includes withdrawal of precipitating triggers, phlebotomy and oral anti - malarial agents . The most common symptomatic complaints of patients with pct are due to cutaneous manifestations which can occur after sun exposure or minor trauma . These include increased photosensitivity and skin fragility with vesicles / bullae and erosions / crusts on sun - exposed sites such as the dorsa of the hands . Crusting of lesions can then take weeks to heal, resulting in atrophic scarring with milia formation . Post - inflammatory hyperpigmentation, scarring alopecia, hypertrichosis and morpheaform / sclerodermoid changes can be also seen . Histological examination of involved skin is not required to confirm the diagnosis of any cutaneous porphyria, rather skin biopsies are obtained to exclude other entities in the differential diagnosis, including eba, drug - induced pseudoporphyria and phototoxic / bullous drug eruptions . As demonstrated by our cases, this can lead to potential misdiagnosis due to histological similarities between the vesiculobullous differentials . In pct, characteristic histopathological findings include subepidermal blisters, which lack inflammatory infiltrate, with preservation of dermal papillae in the lesion's floor (festooning). Under direct immunofluorescence microscopy, fibrinogen, complement and immunoglobulins, particularly igg, are often present surrounding blood vessels of the papillary dermis and at the dermal - epidermal junction . As shown in our cases, these findings can be easily confused with eba in particular, which typically shows similar cell - poor subepidermal blisters and direct immunofluorescence microscopy showing igg deposits at the basement membrane zone, however usually targeting type vii collagen . Thus, whilst histological examination is important in excluding other differential diagnoses, it is important for clinicians to recognize the potential for misdiagnosis of pct as a result of findings on histology . In our cases, both patients had skin biopsies reported as eba, and this could lead to inexperienced clinicians accepting this diagnosis and treating accordingly and inappropriately . Whilst case 1 was managed correctly with porphyrin studies performed despite eba histopathological findings, in case 2 the histopathological findings were initially accepted and led to misdiagnosis and delay in appropriate pct treatment . Compounding this diagnostic dilemma is the similar clinical appearances of bullae / milia in both eba and pct, which can falsely reassure the clinician not to perform the necessary porphyrin investigations . These typically include porphyrin analysis of urine, blood and stool which reveal increased urinary elimination of uroporphyrin, hepta - carboxylated porphyrins and coproporphyrin, and increased faecal excretion of isocoproporphyrin . In summary, our cases demonstrate the possible difficulty in differentiating pct from eba and the potentially significant consequences of this misdiagnosis . In patients with undifferentiated bullous / erosive skin conditions occurring in photo - distributed regions, pct should be considered in the differential diagnosis irrespective of histopathological findings on biopsies and further investigated appropriately . Without a healthy clinical suspicion for pct, which is generally more common than eba, diagnostic porphyrin screening of urine, blood and stool may not be performed, thus leaving the patient untreated and ultimately at increased risk of developing chronic liver disease and possible hepatocellular carcinoma.
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The prevalence of prostate cancer is rapidly increasing and prostate cancer is now the 7th most commonly diagnosed cancer in korea . Factors contributing to this increase include an aging population; westernization of the diet, such as higher intakes of dietary fat, meat, and excessive calories; and the availability of generalized diagnostic tools for prostate cancer . Metabolic syndrome (ms) is the combination of several metabolic abnormalities, including hypertension, dyslipidemia, central obesity, insulin resistance, and glucose intolerance . Ms is clearly related to cardiovascular disease or type ii diabetes; however, the relationship between ms and malignant diseases is unclear . There are sporadic reports showing that that the incidence and mortality rates of colon cancer, endometrial cancer, and breast cancer are related to the components of ms, such as obesity and insulin resistance . Previous studies on the relationship between prostate cancer and ms have reported contradictory results [6 - 8]. Most previous studies on the association of ms and prostate cancer were conducted in multi - racial groups or non - korean populations . In this study, we investigated whether there is a relationship between ms and prostate cancer in korean men who underwent a transrectal ultrasound - guided prostate biopsy . From october 2003 to may 2011, 432 korean men with a serum prostate - specific antigen (psa) level 4 ng / ml or an abnormal digital rectal examination finding underwent transrectal ultrasound - guided prostate biopsy . Patients who had been diagnosed as having prostate cancer before or who had undergone prostatic surgery and patients taking 5-alpha reductase inhibitors or whose data were insufficient for analysis were excluded . Prophylactic oral antibiotics were taken from 2 days before the prostate biopsy until 3 days after the biopsy . Transrectal ultrasound - guided prostate biopsy was performed by using a falcon ultrasound instrument (bk medical, peabody, ma, usa) and 12 cores were obtained . A 16-gauge biopsy needle (magnum 1000; bard, byran, co, usa) and spring - loaded biopsy gun (mg1522; bard) were used . For pain control, 25 to 50 mg meperidine (demerol) was injected intramuscularly before biopsy . Biopsy results were classified as benign prostatic hyperplasia, acute or chronic prostatitis, chronic inflammatory atrophy, prostatic intraepithelial neoplasia, atypical small acinar proliferation, or prostate cancer with gleason score . A biopsy gleason score 7 was regarded as high - grade prostate cancer and a gleason score <7 as low - grade . All patients received a physical examination including measurement of height, weight, and waist circumference before prostate biopsy . Body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared (kg / m). Fasting serum samples including high - density lipoprotein (hdl) cholesterol, triglyceride, blood glucose, total psa (tpsa), and free psa were analyzed . Ms was defined according to the national cholesterol education program adult treatment panel iii (ncep - atp iii) criteria as follows: 1) high fasting blood glucose level (110 mg / dl), 2) hypertriglyceridemia (150 mg / dl), 3) reduced hdl cholesterol (<40 mg / dl in men and <50 mg / dl in women), 4) high blood pressure (130/85 mmhg) or use of anti - hypertensive drugs, and 5) abdominal obesity (abdomen circumference> 90 cm in men or> 80 cm in women according to the 2000 world health organization criteria for abdominal obesity in the western pacific region). At least three of the five criteria needed to be met for a diagnosis of ms . Patients were divided into 2 groups according to presence of ms or diagnosis of prostate cancer . Patients with prostate cancer were subdivided according to gleason score: gleason score 7 as high - grade and <7 as low - grade prostate cancer groups . By use of multiple logistic regression with the enter method, the statistically significant variables as assessed in the univariate analysis were entered and investigated as predictors of prostate cancer presence versus absence and in a separate model comparing predictors of high - grade versus low - grade prostate cancer among men with cancer on biopsy . The logistic regression analysis was carried out by using data from patients for whom complete data were available . Spss ver . 18.0 (spss inc, chicago, il, usa) was used for the statistical analysis . The patients' mean age was 68.868.95 years, their mean tpsa was 13.9720.42 ng / ml, their mean prostate volume was 49.2324.89 ml, their mean waist circumference was 85.156.92 cm, their mean weight was 65.938.38 kg, and their mean bmi was 23.762.71 kg / m . Ninety patients (25.4%) were diagnosed with prostate cancer and 75 patients (21.2%) were diagnosed with ms according to the ncep - atp iii criteria . There was no significant difference in age, tpsa, or prostate volume between the groups with and without ms . Patients with ms had higher values of bmi, waist circumference, fasting blood sugar, and triglyceride, and presented with a lower hdl - cholesterol level, than did patients without ms . Patients diagnosed with prostate cancer had higher age, tpsa, and psa density (dpsa) than did patients without prostate cancer . Among the 90 patients with prostate cancer, 27 (30%) had ms . At the same time, among the patients without prostate cancer, 18% had ms . Twenty - seven patients had a gleason score 6 and 63 patients had a gleason score 7 . Prostate cancer patients diagnosed with ms had a mean gleason score of 6.631.92, which is significantly lower than that of prostate cancer patients without ms (7.541.71; p=0.029) (table 1). Both tpsa and dpsa were higher in patients with a high - grade gleason score than in those with a low - grade gleason score . In the logistic regression analysis, high tpsa and small prostate volume were significant predictors for prostate cancer diagnosis (odds ratio [or], 1.079 and 0.979, respectively). However, age, presence of ms, and bmi were not significantly related to increasing prostate cancer risk . In the logistic regression analysis, tpsa was significantly related to high - grade prostate cancer (or, 1.109; p=0.017) (table 2). Presence of ms was significantly associated with a decreased risk of high - grade prostate cancer (or, 0.101; p=0.004) (table 3). The prevalence of ms in korea, which ranges from 19.8 to 30.9% according to the ncep - atp iii, has increased up to 28.6% according to the national health nutrition survey in 2001 as compared with the previous survey in 1998 . Ms is related to cardiovascular disease or type ii diabetes; however, the relationship between ms and malignancy is unclear . It has been reported that the incidence and mortality rates of colon cancer, endometrial cancer, and breast cancer are associated with components of ms such as obesity and insulin resistance . Changes in neoplastic metabolism, dna oxidation damage or repair malfunction, local inflammation, and insulin - like growth factor 1 (igf-1) may contribute to the relationship between ms and malignant disease . Results of previous studies on the relationship between prostate cancer and ms are also controversial [13 - 16]. Data from an investigation in finland suggested a positive association between prostate cancer and ms; in that study with 507 prostate cancer cases, patients with at least three metabolic factors showed a 56% increased risk . Hammarsten and hogstedt reported that ms components including hypertension, obesity, dyslipidemia, and hyperinsulinemia are risk factors for the development of clinical prostate cancer and suggested that clinical prostate cancer could be a component of the ms . On the contrary, tande et al . Reported an inverse association; in their study in a large cohort of the atherosclerosis risk in communities study, men with ms showed a 23% reduction in risk of prostate cancer . They hypothesized that this finding reflects a decrease in bioavailable testosterone with the ms and a concomitant reduction in prostate cancer risk . The present study evaluated the relationship between ms and prostate cancer in korean patients who underwent transrectal ultrasound - guided prostate biopsy . Our results showed that the presence of ms did not significantly increase prostate cancer risk (or, 1.733; 95% confidence interval [ci], 0.851 to 3.530; p=0.13). De nunzio et al . Showed that ms was not associated with increased prostate cancer risk, but was associated with an increased risk (or, 3.8; 95% ci, 1.33 to 10.9) of high - grade prostate cancer (gleason score7) in patients with prostate cancer at biopsy . Hammarsten and hogstedt reported that patients with high - grade prostate cancer and psa<50 ng / ml were more obese, were more dyslipidemic, and showed a higher plasma insulin level than did those with low - grade prostate cancer and psa<50 ng / ml . In our results, however, the presence of ms was associated with a significantly decreased risk of high - grade prostate cancer (or, 0.101; 95% ci, 0.022 to 0.473; p=0.004) (table 3). Similar to our results, han et al . Reported that prostate cancer patients with ms had a tendency to show a lower gleason score, but the result was not statistically significant . Reported that patients with lower bmi were more likely to have high - grade cancer despite the lack of statistical significance . Moreira et al . Showed that diabetes mellitus was associated with a greater risk of high - grade prostate cancer . After stratification by obesity and race, the association was strongest among obese caucasian men, which suggests that the effect of diabetes on high - grade prostate cancer could be modified by race and obesity . They focused on the complex association among diabetes, serum hormonal levels, and obesity and suggested that compounded effects of lower free igf-1, lower testosterone in obese caucasian men, and lower insulin from diabetes perhaps created a very poor growth factor environment leading to the selection of aggressive tumors . Suggested that hormone - resistant prostate cancer can occur earlier in the case of obesity - induced hormonal changes including decreased levels of total testosterone, free testosterone, and sex hormone binding globulin and increased levels of estradiol . Insulin resistance, which is a fundamental component of ms, plays an important role in malignant formation regarding the intracellular insulin receptor or intrinsic hormone metabolism . Animal studies have also revealed that removal of igf-1 receptor or lowering of the igf-1 level decreases malignant formation . An epidemiologic investigation also linked increasing igf-1 level to prostate cancer and supported the suggestion that overactivity of male hormone levels induced by insulin resistance, increased igf-1 level, and decreased igf binding protein level would also be related to prostate cancer . It has been suggested that evaluating ms as a single condition may be an inappropriate approach to investigating prostate cancer risk . Specifically, combining all the multiple components of the syndrome into a single variable may confound or obscure the independent effects and interactions of these metabolic components on prostate cancer risk . Unfortunately, our study did not analyze the association between each component of ms and prostate cancer because of a lack of information, which is major limitation of retrospective review . On the basis of previous reports, however, the association of ms with a significantly decreased risk of high - grade prostate cancer in our study might result from a change in the sex steroid pathway or circulating levels of cytokines such as leptin and adiponectin . Although the exact function of androgen and estrogen in prostate cancer development is not definite, it has been suggested that low total and free testosterone are inversely associated with low - grade prostate cancer but positively associated with high - grade prostate cancer . A possible explanation for this relation is that high - grade prostate cancer is more aggressive and may be more androgen - independent than low - grade tumors, thereby continuing to progress despite the relative lack of testosterone . Circulating levels of cytokines such as leptin and adiponectin leptin stimulates the in vitro growth of androgen - insensitive prostate cancer cells, and increased serum leptin levels are associated with larger, high - grade, and more advanced tumors . Adiponectin showed antitumor activity via inhibition of angiogenesis, and lower adiponectin serum levels are associated with high - grade and more advanced prostate cancer . Chronic prostatic inflammation as observed in patients with ms is associated with a milieu rich in proinflammatory cytokines, inflammatory mediators, and growth factors, which may lead to an uncontrolled proliferative response with rapidly dividing cells that are more likely to undergo mutation, as observed in cancer . This investigation had limitations, including the retrospective review and lack of prostatectomy gleason scores, which limited the statistical power and generalizability of our results . In addition, in our study, information on each primary component of ms could not obtained, and thus an analysis of the relationship between each component of ms and prostate cancer was not performed . Despite these limitations, however, our results showing that the presence of ms was not significantly related to increasing prostate cancer risk but was associated with a significantly decreased risk of high - grade prostate cancer could play a role in disclosing the link between ms and prostate cancer . Further prospective studies in larger patient groups with long - term follow - up as well as basic research are needed to clarify the relationship between the components of ms and prostate cancer and to evaluate the possible implication of ms prevention on prostate cancer development . The results of our study suggest that high tpsa and small prostate volume are significant predictors for prostate cancer diagnosis . However, age, presence of ms, and bmi were not significantly related to increasing prostate cancer risk . Furthermore, the presence of ms was significantly associated with a decreased risk of high - grade prostate cancer . A larger, prospective, multicenter investigation
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The 2007 american medical association expert committee on prevention, assessment, and treatment of childhood obesity identified caregiver feeding behaviors as an important target for intervention, categorically stating that there is consistent evidence to support avoiding overly restrictive feeding behaviors . A growing body of the literature indicating that caregiver feeding behaviors, in particular overly restrictive or controlling behaviors, significantly reduce a child's ability to self - regulate their energy intake [24] and result in weight gain or a higher body mass index [46] shaped the expert committee's guidelines . One area related to caregiver feeding behaviors is feeding dynamics, which is centered on the roles, interactions, and balance of control between the caregiver and child in a feeding relationship . Food is offered, where food is eaten, and when food is eaten and allows the child to decide whether to eat as well as what and how much to eat of the food offered [7, 8]. Satter posits that children learn to trust and develop a healthy relationship with food when they and their caregivers each have an appropriate level of control and autonomy in the feeding relationship . The primary premise of feeding dynamics is based on nurturing optimal self - regulation of energy intake to support normal growth . Young children have an innate ability to self - regulate their energy intake in response to their metabolic needs [1012]. This natural aptitude weakens as children get older and become more responsive to external influences on eating [1315]. Children with the highest body fat and whose parents restrict their food choices and quantity of intake exhibit the weakest level of regulation . However, this relationship is complex, not always consistent, and probably bidirectional; that is, the child's characteristics, for example, age, gender, and weight, influence caregiver eating or feeding practices and vice versa [4, 1719]. For instance, mothers who eat regular meals and choose nutritious foods for themselves are more likely to extend these behaviors to their children . Mothers who eat intuitively (giving themselves unconditional permission to eat, eating for physical rather than emotional reasons, and relying on their internal hunger and satiety cues to guide eating) restrict their children's food intake less and allow their children to share feeding responsibilities . The child's perception of the caregiver's controlling behavior can contribute to the complexity of the relationship [21, 22]. Limiting the amount and types of snack foods available in the home, a covert controlling action, is associated with decreased intake of unhealthy snacks [22, 23], while feeding practices, like pressuring the child to eat, are associated with an aversion to healthy food choices and a higher risk of overweight [4, 24]. The challenge is to determine the right balance of control between the caregiver and the child . Despite the complexity of the relationship, a review of the literature supported the feeding dynamic approach as a viable paradigm for obesity prevention or treatment and is described in depth elsewhere . What remains uncertain is how best to teach mothers to adopt these feeding roles, whether mothers will be willing or able to implement the roles, and how these roles can be adapted in an obesogenic environment, as scholars have not yet developed or tested this approach as a comprehensive program . The goal of this paper is to describe the development of the feeding dynamics intervention (fdi), a parent - targeted program based on the feeding dynamics approach in addition to evidence - based lifestyle behaviors known to prevent or treat childhood obesity . In study 1, we tested the acceptability and implementation of the feeding dynamic components . Specifically, we initially piloted a 90-minute class to determine how best to teach the feeding dynamic roles and to obtain feedback from the mothers of 25-year - old children regarding their perception and experience implementing the feeding roles . We asked mothers of young children from two childcare centers if they would be willing to attend a 90-minute learning session with other mothers about how to feed their children . A total of 22 mothers indicated that they would like to attend a learning session . We invited mothers, as opposed to fathers, because mothers are typically the parent responsible for grocery shopping and meal preparation . Of these 22 mothers, 17 attended the class and fourteen mothers completed both the pre- and postsurveys . Mothers' mean age was 32.8 (sd = 4.2) years, and their children's mean age was 3.6 (sd = 1.2) years . They were identified as white (50%) and african american (50%). As their highest level of education, they had graduated from high school (47%) or from a 4-year college (50%); 3% did not graduate from high school . We assessed feeding behaviors using the child feeding questionnaire (cfq) and the caregiver feeding responsibility scale (cfrs). For the cfq restriction (8 items), monitoring (3 items), and pressure to eat (4 items) subscales, higher scores reflect greater levels of the measured feeding behavior . Restriction ranged from 1 (disagree) to 5 (agree), and monitoring and pressure to eat ranged from 1 (never) to 5 (always). Cronbach alphas were 0.73 for restriction, 0.73 for pressure to eat, and 0.82 for monitoring . We used the cfq parent perception of child's weight subscale to determine weight status . Responses are markedly underweight, underweight, normal weight, overweight, and markedly overweight . The cfrs measures the feeding dynamic roles for caregivers and children as proposed by satter . Specifically, items ask mothers about the extent to which they perform their responsibilities (e.g., feeding their children at regular times, serving meals with a variety of foods, and ensuring that the family eats together without distractions such as tv) and allow their children to perform their responsibilities (i.e., deciding what or how much to eat of what has been offered). With a response scale ranging from 1 (never) to 5 (always), higher scores indicate greater adherence to the feeding dynamic approach . The cfrs factor structure is unidimensional, demonstrates internal consistency reliability (= 0.70), test - retest reliability over a 5-week period (r = 0.80), and constructs validity among mothers of 2- to 5-year - old children . We also developed six independent questions on mothers' prior familiarity with, as well as understanding and acceptability of, the feeding dynamic approach (scale ranging from 1 (strongly disagree) to 5 (strongly agree)). Specifically, two of these six questions queried mothers on the extent to which they knew about and fed their children according to principles within the feeding dynamic approach before taking the class if they were more familiar with the feeding dynamic approach prior to the class, then they would have less to gain from the class . One question asked whether they understood the feeding dynamic approach after the class to determine whether it was delivered in a clear and informative manner . The remaining three questions asked about their acceptability of the feeding dynamic approach, that is, the extent mothers believed that this approach would be beneficial for children, started feeding their children according to this approach, and planned to continue feeding their children according to this approach . The class content, adapted from ellyn satter's work on caregiver / child division of feeding responsibility, focused on how to (a) implement each of the caregiver roles (i.e., what food is offered, where food is eaten, and when food is eaten) and (b) allow and support the child in carrying out his / her roles (whether to eat, what and how much to eat of the food offered) [7, 8]. She defined each role, emphasized the benefits of carrying out and supporting the roles, and provided step - by - step guidance on implementation . For example, in presenting the caregiver role of where food is eaten, the instructor recommended the caregiver designate an area in the home where all food and drink can be consumed except water . The instructor presented the benefit of undertaking such an action first from a caregiver perspective (e.g., you will have a cleaner home) and then from the child's perspective (e.g., if you maintain this rule in your home, your child will learn to associate eating with specific areas in the home at an early age and limit the likelihood he / she will eat in front of the tv or bedroom). She advised caregivers to have a family discussion about the action (e.g., limiting where food and drinks are consumed to a designated area) a couple of days before the change and present the change as an opportunity for the family to encourage each other for the shared benefit of a cleaner home . She recommended that the caregiver and family select a designated area in the home for food or drinks (preferably the kitchen). The instructor elicited input and problem - solved challenges and barriers raised by the participants, such as an uncooperative spouse or family, how to identify and prepare the physical space for the meal, or the absence of a kitchen or dining table in the home . She taught participants to share any potential exceptions to the rule ahead of implementation (e.g., we can eat in the living room but only on family fun nights). She advised them to try to implement each recommendation at least 80% of the time during the 46-week trial period . Participants provided informal feedback on each recommendation for a significant proportion of the class time . We used paired t - tests to assess change in feeding behavior, between responses on the pre- and postintervention surveys . We applied the bonferroni adjustment to avoid a type i error due to multiple comparisons . Because five items / scales on the maternal feeding behaviors from pre- to postintervention were skewed and/or kurtotic (defined as skewness / standard error> |2|), we transformed these variables using reflect and square root transformations for four variables that were moderately negatively skewed and square root transformations for one variable that was moderately positively skewed . These transformations reduced skewness and kurtosis to acceptable levels, so paired - sample t - tests could be performed . Effect size (hedges' g) for each feeding behavior determined the magnitude in change following the class . We used hedges' g in lieu of cohen's d to estimate effect size because hedges' g provides a better estimate for small sample sizes . However, the effect sizes are comparable in interpretation . Specifically, per cohen, we considered an effect size of 0.20 a small effect, 0.50 a medium effect, and 0.80 a large effect . Based on the independent questions developed by the research team (range 15 with higher scores indicating greater familiarity, acceptability, and experience), mothers did not feed their children according to the feeding dynamic approach (m = 2.00, sd = 0.7) and indicated that they were not familiar with the approach (m = 2.00, sd = 0.9) prior to the intervention . After intervention, mothers reported that they understood the feeding dynamic approach (m = 4.50, sd = 0.6), believed that children should be fed according to the feeding dynamic approach (m = 4.22, sd = 0.6), planned to make lasting changes in how they feed their children (m = 4.29, sd = 0.5), and were somewhat able to implement the changes (m = 3.45, sd = 1.0). We noted that an effect size (hedges' g) greater than 0.20 appeared in more than half (57%) of maternal feeding behaviors (table 1), with the largest effect sizes (hedges' g 0.8) occurring with behaviors that represent the mother adopting her responsibility for determining what food is served, not using food as a reward, and not controlling her child's intake . The mothers' lowest scores were related to allowing children to carry out their eating roles . Based on the cfq subscales, controlling behaviors such as restriction, pressure to eat, and monitoring decreased significantly from pre- to postintervention (table 1). At postintervention, mothers provided feedback on areas they thought would be challenging to implement or needed clarification . They recommended a guide on how much food to prepare or serve, without appearing to waste food . They anticipated that it would be difficult not to control or restrict their children's food intake, and they had a preference to plate their children's food at certain meals . Mothers reported that they needed information on nutrition label reading, were insistent that label reading would help them be more knowledgeable when purchasing food, and verbalized that label reading should be a key component of any nutrition intervention . They identified commonly anticipated barriers, which included how to implement the recommendations with older children and nonsupportive family members, how to share the approach with extended family members who may also care for the child, and how to talk with their children about hunger and satiety . Despite the small sample size, there was an improvement in behaviors aligning with the feeding dynamic approach following the class (table 1). Guided by our prior work in this area [9, 20, 30, 31] and results from study 1, a research team of two dietitians, a physician, a curriculum expert, and psychologist developed the 6-lesson fdi . We designed study 2 to evaluate the fdi program content in three evaluations over six months and finalize its content . An evaluation team of three dietitians, five health educators, and a public health specialist from the university extension program in eight rural and urban ohio counties conducted the initial program evaluation . Age of evaluators ranged from 33 to 56 years, and all were caucasian, had taught families for a minimum of five years, and currently worked with low - socioeconomic clientele of different ethnicities . In this initial evaluation, we assessed the evaluators' perceptions about the feeding dynamic approach, the likelihood of their clients adopting these roles, and how best to present the material and identify benefits and challenges . They evaluated each lesson for content, ease of teaching, applicability for families, and appropriateness of supporting materials . The evaluators spent one day with the research team reviewing the fdi framework and content . They completed written and verbal feedback on each lesson and provided feedback during discussions with the research team . Subsequently, a group of five reviewers, which included parents and two of the initial evaluators, conducted the second and third program evaluations . Their ages ranged from 26 to 35 years, two had children who were toddlers and preschoolers, and one parent had a child who was overweight . In these second and third evaluations, we refined the fdi content based on results from the literature reviews, the pilot study (study 1), feedback from the evaluators, and research on feeding dynamics [9, 20]. We structured each session to last 3 - 4 hours, not including time spent reviewing the material prior to the meeting . The evaluators reported all the caregiver and child roles in the feeding dynamic approach would be applicable for their clients . Two evaluators expressed concern that allowing children to determine how much to eat would be challenging for parents who have children with impaired satiation . However, they all reported they would feel comfortable teaching the fdi content after only a brief training session . The final fdi content included information that (a) caregivers are responsible for offering nutritious foods and exposing children to new foods (the what), providing structured snack and mealtimes to decrease indiscriminate snacking or grazing (the when), identifying designated areas for eating or drinking within the home (the where), and sitting and eating with children and keeping the eating atmosphere pleasant (family meals and role modeling) and (b) children are responsible for what to eat and how much (or even whether) to eat from the food provided (table 2). We also included evidence - based lifestyle messages, for example, reducing sweetened beverages, having family meals, serving balanced meals, decreasing fast food consumption and eating out, reducing tv viewing, and increasing physical activity in the fdi [1, 32]. In the fdi, we discouraged tactics such as drinking water before meals, waiting 20 minutes between servings, or putting down the fork between bites for the sole purpose of food restriction . We also discouraged offering effusive praise like clapping hands, making smiley faces, or offering rewards for eating, as these behaviors constitute pressure to eat and may promote food consumption in the absence of hunger . Instead, we incorporated techniques on how to use neutral phrases, such as you must have enjoyed your macaroni and cheese tonight, rather than wow, that's too much macaroni and cheese in the fdi curriculum . At each lesson these changes are essential as they may improve the mother's self - efficacy in carrying out the recommendations and strengthen her perception that the fdi will enable her to feed her child well and help her child learn to eat well . In the final fdi curriculum, we added a module to help mothers recognize their own hunger and satiety cues and be mindful of their eating behavior to each lesson in response to results from a prior study that found mothers who had positive eating behaviors (i.e., those who ate intuitively) were more likely to allow their children to carry out their own feeding roles . This addition provides an opportunity for role modeling, a social - cognitive theory (sct) construct used in the development of the fdi . We also incorporated feedback obtained from the 90-minute class (study 1). To address mothers' concern about how much food to prepare or serve without appearing to waste food and control or restrict their children's intake, we encouraged mothers to use at least 1.5 portions per child when cooking as a guide . After much debate, the team decided to allow mothers to plate some meals as family style meals may not be realistic for certain meals / settings and could have a varied effect on portion size and energy intake in some children . For plating, the fdi instructs mothers to provide approximately three - fourths of a portion size initially, allowing the child to have additional servings if requested . We used these guidelines to provide a consistent approach within the curriculum and facilitate mothers' adherence . Because mothers in study 1 were unanimous that nutrition label reading should be a key component of any nutrition intervention we presented nutrition label reading as a guide to help with balancing food choices through the day or week, rather than restricting intake or food choices . In response to their feedback about anticipated barriers and the diversity in feeding behaviors recommendations from the reviewers included incorporating more hands - on activities, use of video clips to demonstrate division of responsibility roles, strengthening the authoritative parenting component of the sessions, emphasizing issues around food security, and using more pictures in the handouts . There was significant debate about the use of video clips drawn from contemporary television shows to illustrate feeding and parenting styles, as some reviewers felt it would limit the use of the curriculum in resource - poor settings . The team decided to include these video clips as they demonstrated strong visual examples of the feeding roles . In addition, they felt that, with rapid technological advancement, this concern may be less relevant in the future even in resource - poor areas . With each review, we identified and incorporated learner objectives and processes in structuring the content (table 3). With these modifications, the fdi content deviated in significant ways from the trust model as proposed by satter [7, 8]. In the final study, we piloted the final curriculum of fdi to assess whether mothers who received the intervention adopted the caregiver roles and decreased excessive controlling feeding behavior (i.e., restriction and pressure to eat). We piloted the comprehensive 6-lesson fdi over 12 weeks with a convenience sample (n = 8) of mothers recruited through an email sent to a parent - teacher association for the local school district . We targeted this parent - teacher association because the mothers in this association had children in the targeted age range for the fdi, the school was situated in the same county as the childcare centers, and mothers lived near the facility where the fdi would take place to ensure that distance was not a deterrent to completing the fdi . The eighth mother withdrew prior to the start of the intervention due to family reasons . At each lesson, mothers set goals for home and provided feedback on the content in a pre- and posttest survey . We noted that sample characteristics in studies 1 and 3 are comparable in terms of child age, although mothers in study 3 were slightly younger and less educated than mothers in study 1 . The participants completed a survey prior to and following the classes that included items on demographics, maternal height and weight, the cfq, and the cfrs . The postsurvey included similar measures and the six questions on perception, acceptability, and experience with feeding dynamic approach . We used paired - sample t - tests to compare differences between responses on the pre- and postintervention surveys . Mothers reported that they were not familiar with the feeding dynamic approach before the intervention (m = 1.71, sd = 0.7 on a scale ranging from 1 = strongly disagree to 5 = strongly agree). After the intervention, they agreed that children should be fed according to the fdi (m = 4.00, sd = 1.0), strongly agreed that they understood the feeding dynamic approach (m = 4.57, sd = 0.5), and indicated a willingness (m = 4.14, sd = 1.46) and a plan (m = 4.43, sd = 0.8) to continue to feed their children according to the fdi . Mothers felt neutral with regard to whether it was difficult to feed their children according to the fdi (m = 2.86, sd = 1.2) and strongly agreed that the fdi should be used with all children, regardless of if they are overweight (m = 4.43, sd = 0.5), thin or underweight (m = 4.43, sd = 0.5), or average weight (m = 4.43, sd = 0.5). Therefore, these mothers' data show that they viewed the fdi as both acceptable and fairly feasible . Following the intervention, six of the seven mothers who completed the intervention reported less restrictive and pressuring practices on the cfq (table 4). Concern over their children's weight decreased following the intervention (preintervention: m = 3.2, sd = 1.4; postintervention m = 2.3, sd = 1.2). Six of the seven mothers reported that they were able to implement the following strategies: making one meal for all family members (instead of short order cooking), letting children choose what to eat from what is on the table, having children drink only water between meals and snacks, and having food off - limits between meals and snack times . Five of the seven mothers reported that they were able to let the children decide how much they want to eat from what was served and when they were done with eating . For the two participants that indicated otherwise, one had not yet tried and the other did not want to try it . Participant #3 disagreed with the roles in a feeding dynamic relationship, especially the child's roles . She was latino, normal weight, married, and college educated and had an 11-month - old child in addition to the 5-year - old child in the study . She expressed concerns about cultural appropriateness of some of the recommendations and shared it was rude for the child not to taste from what had been served . She strongly believed in the importance of parental control over all feeding decisions . In summary, all but one participant responded well to the 6-lesson fdi; mothers were able to implement the recommendations, and the level of controlling feeding behaviors improved . We used the results gleaned from these studies to further refine the program by including (a) a section on how feeding behaviors may be perceived by the child, (b) a discussion on the intersection between fdi recommendations and cultural beliefs, (c) a review of parental and child feeding roles in each lesson to reemphasize the core concepts of feeding dynamics (we used this review to obtain periodic feedback on participants' progress and to encourage mothers who have not yet tried the recommendations to do so), (d) a 3-dimensional child - friendly stomach model that illustrates three stomachs in different states (i.e., hungry, full, and stuffed) and a module to instruct mothers how to use the stomach model with their young children, (e) team exercises on meal planning using paper food models, and (f) physical activity resource handouts on relevant community physical activity resources and an indoor play activity sheet . The activity sheet consists of 4 - 5 aerobic activities such as running in place, which the child and family can perform in 3060-second intervals . The objectives of the fdi in delineating child and caregiver feeding responsibilities and incorporating what children are fed within the broader framework of how they are fed enable caregivers to achieve the overall goal of feeding their children well, an act that evokes strong emotions tied to a sense of accomplishment as a parent . First, we developed the fdi, an innovative program which incorporates evidence - based recommendations for childhood obesity and a core focus on feeding dynamics . From our studies, we modified and expanded on the feeding dynamic concepts by adapting them appropriately to be pragmatic, cognizant of the prevailing obesogenic environment, and responsive to the mother's needs and diversity in the child's eating behaviors and patterns . Second, we uncovered that mothers were receptive to the feeding dynamic approach concepts, found them understandable, and were willing and able to implement the intervention at home . The rigorous stepwise development of the studies allowed us to incorporate a wide range of data, prior research, and participant feedback which strengthened the final fdi; however, limitations exist . We used convenience samples to pilot test the intervention; thus, the mothers in our samples may have been more receptive to an intervention than the general population . We used the newly developed caregiver feeding responsibility scale to assess the feeding dynamic components, which needs further validation in different populations . Additional limitations include report bias because the study relied solely on maternal reports and some of the items and subscales had relatively large standard deviations, which could impact effect size levels . The fdi needs to be carried out in larger samples before definitive conclusions are made . Finally, based on the feedback from the mothers, the evolving research on feeding and eating behaviors in young children, and our research, the final fdi program contains significant deviations from the trust model [7, 8] these changes include the addition of a module on portion sizes to guide the amount of food to cook, lack of insistence on family style meals, and introducing label reading to guide selections of nutritious options . The addition of evidence - based lifestyle recommendations beyond feeding behaviors further strengthened the fdi, increasing its potential to be a promising and practical program for obesity prevention or treatment in young children . They include researchers (a) investigating the outcome of the fdi with a larger and diverse population, (b) studying the degree to which children can be taught to recognize hunger and satiety cues via parent - directed intervention versus a child - targeted intervention, (c) videotaping within the home to assess the feeding environment rather than relying on caregiver self - report, (d) using objective assessments of self - regulation such as the energy compensation and eating in the absence of hunger tests, (e) validating the acceptability and feasibility of the physical activity component, and (f) exploring short- and long - term effects of the fdi on the child's anthropometrics and eating behaviors.
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Large animal experiments with quadrupeds are frequently performed to investigate pathomechanisms of different cardiac pathologies (e.g., mitral regurgitation or heart failure) and/or to test novel treatment options (e.g., novel heart valve designs or mechanical support systems). The three - dimensional orientation of the heart with respect to the thorax is, however, fundamentally different in quadrupeds than in humans . As a consequence, surgical approaches to access individual structures of the heart in quadrupeds are not intuitive for a cardiac surgeon trained to operate on human patients . While there has been extensive research on the left heart and mitral valve in quadrupeds, including descriptions of surgical strategies, studies investigating the right heart and the tricuspid valve have been reported less frequently . One possible reason for the relatively small number of experimental studies focusing on mechanisms leading to tricuspid valve disease may be uncertainty about the optimal surgical access to approach the tricuspid valve in quadrupeds . We set out to develop and describe a surgical strategy that allows good visualization of all components of the tricuspid valve complex in sheep . Sheep cadavers were analyzed to determine relevant surgical structures (cardiac anatomy and peripheral vessels) from two different surgical access sites (right and left thoracotomy). Afterward, 6 female sheep (mean weight, 48 6 kg) were operated via right thoracotomy, and the strategy to surgically approach the tricuspid valve complex was subsequently refined (see results). By performing cardiopulmonary bypass and cardioplegic arrest, radiopaque markers (commercially available hollow silver balls; diameter, 2 mm; weight, 0.003 g) were attached to all components of the tricuspid valve complex . Sheep were premedicated with ketamine (10 mg / kg intramuscularly; zoetis, germany) and midazolame (0.1 mg / kg intramuscularly; rotexmedica, germany), intubated, and mechanically ventilated with inhalational isoflurane (1.0%2.5%; abbvie, germany). Analgesia was reached by intravenous application of 0.002 mg / kg fentanyl (rotexmedica) per hour . Sheep were relaxed with pancuronium (0.1 mg / kg intravenously; inresa, germany). 22 - 2684 - 04 - 02 - 032/11) by the thuringia animal welfare committee, thuringia, germany . Anatomical analyses revealed that a left thoracotomy provided optimal access to the aorta, but visualization of both caval veins and the tricuspid valve complex was significantly impaired . In contrast, a right thoracotomy provided good access to both caval veins and permitted straightforward visualization of the tricuspid valve complex . As a consequence, all in vivo studies were performed via a right thoracotomy with the sheep lying in a left lateral position (panel a in fig . 1). Panels b - d in fig . 1 show the surgical situs after cutting down the left femoral artery (panel b in fig . The surgical strategies used to approach the tricuspid valve, including potential surgical pitfalls, are described below in more detail . In order to surgically visualize the tricuspid valve complex, different positions from the surgeon with respect to the sheep were tested . Positioning the surgeon on the front / abdominal side of the sheep allowed straightforward opening of the thorax and placement of all cannulas, whereas a surgeon positioned on the back side of the sheep allowed best access to the tricuspid valve . 2 schematically show the surgeon standing either on the abdominal / front side (panel a in fig . The lower border of the scapula was a good anatomical landmark for the beginning of the skin incision . The fourth intercostal space provided the best access to the tricuspid valve complex . During the opening of that intercostal space after insertion of the rib retractor, the pericardium was opened and the heart suspended in a pericardial cradle (panel c in fig . 2). At that juncture, the right atrium and ventricle, as well as both caval veins, are clearly visible and easy to access . In this study, 1 demonstrates that the aorta cannot be seen via a right thoracotomy without additional exposure . In order to visualize the aorta, 2 shows the situs after manual retractions of the right atrial appendage, pulmonary atrium, and pulmonary artery . The ascending aorta has been dissected from the pulmonary artery and is encircled by vessel loops . As compared to humans, where the ascending aorta is in general about 45 cm long, the ascending aorta in sheep is significantly shorter (23 cm) due to early branching of the brachiocephalic trunk (similar to cattle, sheep have a common brachiocephalic trunk from which all supra - aortic vessels [right and left carotid and subclavian arteries] branch). It is therefore technically impossible to place the aortic and cardioplegic cannulae in the ascending aorta at the same time . Furthermore, the aortic arch and descending aorta were difficult to visualize from this surgical access . As a consequence, we preferred to use the peripheral vessels (carotid and femoral arteries) for placement of the aortic cannula (see anatomy and surgical access to the peripheral vessels). 2 illustrates the situs after cannulation of both caval veins and insertion of the cardioplegic cannula . After aortic cross clamping and cardioplegia, the surgeon switched to the back side of the sheep (panel b in fig . 2 shows the tricuspid valve annulus, leaflets, chords, and papillary muscles after implantation of the radiopaque markers . Cardioplegic arrest was induced by using warm, potassium - enriched blood cardioplegia in an antegrade fashion . At this time, it is of great importance to visualize the brachiocephalic trunk as it can either be misidentified as the descending aorta or may be overlooked (i.e., the cross clamp may either be placed on the trunk or the descending aorta erroneously). Both complications occurred in one of the six sheep in this study, resulting in insufficient cardiac arrest . Furthermore, unlike humans, sheep commonly have a vein that drains blood from the upper part of the body into the coronary sinus (equivalent to a left persistent caval vein in humans). This vein is, however, located on the left side of the heart and cannot be encircled or occluded from a right - sided surgical access . A retrograde delivery of cardioplegia, therefore, proved to be infeasible via a right thoracotomy . Preparation of peripheral vessels may be needed for invasive pressure monitoring and/or for peripheral placement of arterial or venous cannulae for the heart - lung machine . The locations of the left femoral and right carotid arteries are displayed in panels b and d in fig . The inguinal canal in sheep is not as easy to palpate as that in humans . Furthermore, it is of note that the positions of the femoral vessels change significantly depending on the degree of flexion / extension of the leg; moreover, the femoral vessels may be located underneath thick muscle bundles (panel b in fig . In four of the six sheep the carotid artery was used for arterial cannula placement and the femoral artery was punctured for arterial pressure monitoring . 1 shows the locations of the right jugular vein and carotid artery after surgical exposure . In sheep of this size (approximately 50 kg) the jugular vein is 2 to 3 cm in diameter, is easily palpable, and lies directly underneath the skin . The skin should be incised medially (towards the trachea) from the jugular vein to avoid damage to the vessel . The right common carotid artery lies deeper and is medial to the jugular vein (panel d in fig . 1). In one of the six sheep, implantation of venous cannulae through the femoral and carotid veins was tested; however, the femoral vein diameter was smaller than the cannula and the approach had to be abandoned . Table 1 shows a stepwise protocol that allows surgical access to the tricuspid valve through a right thoracotomy with total cardiopulmonary bypass and cardioplegic arrest . Table 2 summarizes potential pitfalls that should be considered when undertaking tricuspid valve surgery using cardioplegic arrest via a right thoracotomy in sheep . In order to surgically visualize the tricuspid valve complex, different positions from the surgeon with respect to the sheep were tested . Positioning the surgeon on the front / abdominal side of the sheep allowed straightforward opening of the thorax and placement of all cannulas, whereas a surgeon positioned on the back side of the sheep allowed best access to the tricuspid valve . Panels a and b in fig . 2 schematically show the surgeon standing either on the abdominal / front side (panel a in fig . The lower border of the scapula was a good anatomical landmark for the beginning of the skin incision . The fourth intercostal space provided the best access to the tricuspid valve complex . During the opening of that intercostal space after insertion of the rib retractor, the pericardium was opened and the heart suspended in a pericardial cradle (panel c in fig . 2). At that juncture, the right atrium and ventricle, as well as both caval veins, 1 demonstrates that the aorta cannot be seen via a right thoracotomy without additional exposure . In order to visualize the aorta, 2 shows the situs after manual retractions of the right atrial appendage, pulmonary atrium, and pulmonary artery . The ascending aorta has been dissected from the pulmonary artery and is encircled by vessel loops . As compared to humans, where the ascending aorta is in general about 45 cm long, the ascending aorta in sheep is significantly shorter (23 cm) due to early branching of the brachiocephalic trunk (similar to cattle, sheep have a common brachiocephalic trunk from which all supra - aortic vessels [right and left carotid and subclavian arteries] branch). It is therefore technically impossible to place the aortic and cardioplegic cannulae in the ascending aorta at the same time . Furthermore, the aortic arch and descending aorta were difficult to visualize from this surgical access . As a consequence, we preferred to use the peripheral vessels (carotid and femoral arteries) for placement of the aortic cannula (see anatomy and surgical access to the peripheral vessels). 2 illustrates the situs after cannulation of both caval veins and insertion of the cardioplegic cannula . After aortic cross clamping and cardioplegia, the surgeon switched to the back side of the sheep (panel b in fig . 2 shows the tricuspid valve annulus, leaflets, chords, and papillary muscles after implantation of the radiopaque markers . Cardioplegic arrest was induced by using warm, potassium - enriched blood cardioplegia in an antegrade fashion . At this time, it is of great importance to visualize the brachiocephalic trunk as it can either be misidentified as the descending aorta or may be overlooked (i.e., the cross clamp may either be placed on the trunk or the descending aorta erroneously). Both complications occurred in one of the six sheep in this study, resulting in insufficient cardiac arrest . Furthermore, unlike humans, sheep commonly have a vein that drains blood from the upper part of the body into the coronary sinus (equivalent to a left persistent caval vein in humans). This vein is, however, located on the left side of the heart and cannot be encircled or occluded from a right - sided surgical access . A retrograde delivery of cardioplegia, therefore, proved to be infeasible via a right thoracotomy . Preparation of peripheral vessels may be needed for invasive pressure monitoring and/or for peripheral placement of arterial or venous cannulae for the heart - lung machine . The locations of the left femoral and right carotid arteries are displayed in panels b and d in fig . The inguinal canal in sheep is not as easy to palpate as that in humans . Furthermore, it is of note that the positions of the femoral vessels change significantly depending on the degree of flexion / extension of the leg; moreover, the femoral vessels may be located underneath thick muscle bundles (panel b in fig . In four of the six sheep the carotid artery was used for arterial cannula placement and the femoral artery was punctured for arterial pressure monitoring . 1 shows the locations of the right jugular vein and carotid artery after surgical exposure . In sheep of this size (approximately 50 kg) the jugular vein is 2 to 3 cm in diameter, is easily palpable, and lies directly underneath the skin . The skin should be incised medially (towards the trachea) from the jugular vein to avoid damage to the vessel . The right common carotid artery lies deeper and is medial to the jugular vein (panel d in fig . 1). In one of the six sheep, implantation of venous cannulae through the femoral and carotid veins was tested; however, the femoral vein diameter was smaller than the cannula and the approach had to be abandoned . Table 1 shows a stepwise protocol that allows surgical access to the tricuspid valve through a right thoracotomy with total cardiopulmonary bypass and cardioplegic arrest . Table 2 summarizes potential pitfalls that should be considered when undertaking tricuspid valve surgery using cardioplegic arrest via a right thoracotomy in sheep . We describe a safe, reproducible strategy that allows good surgical exposure of the tricuspid valve complex in sheep by performing right thoracotomy with cardiopulmonary bypass and cardioplegic arrest . Tricuspid valve surgery in quadrupeds is associated with several pitfalls, which are mostly due to anatomic differences from human beings . The description of our applied surgical strategy and its potential pitfalls may help to simplify experimental tricuspid valve surgery in quadrupeds . Described experimental reconstruction of the tricuspid valve with autologous fascia lata, but no full cardiopulmonary bypass was used . Kinney et al . Described a trans - sternal bilateral thoracotomy in a canine model with acute, reversible tricuspid insufficiency . That access route appears to be relatively invasive and might impair the recovery of the animal in cases in which chronic studies are to be performed . Walter et al . Described the creation of a tricuspid regurgitation model in which a right thoracotomy was used for surgical access to the tricuspid annulus; however, the procedure was described only briefly and a cardiopulmonary bypass was not needed . In our study, since surgical placement of radiopaque markers required cardiopulmonary bypass and cardioplegic arrest, a strategy to induce and maintain cardioplegia was needed . We have described our surgical strategy in great detail and found it to be safe, straightforward, and reproducible with good visualization of all components of the tricuspid valve complex . With regard to the arterial cannulation site, we observed that, if a right thoracotomy is used, the ascending aorta lies behind the pulmonary artery and therefore is difficult to access . As a consequence, we recommend using peripheral vessels (carotid or femoral artery) for arterial cannulae placement . However, two things should be considered if arterial cannulae for a heart - lung machine are to be placed in peripheral vessels . First, the size of the vessels may be too small to allow placement of an arterial cannula of a sufficient size . In such cases, placement of a second arterial cannula in a different vessel may be an alternative . Second, if the size of the vessel is small, placement of an arterial cannula may result in temporary occlusion of the vessel with the risk of local clot formation and, eventually, thromboembolism after cannula removal . In order to avoid a cannulation - related thromboembolism, we recommend the placement of a vessel clamp distal from the cannulation site prior to the removal of the cannula . After the cannula has been taken out, the clot formation can be easily removed and the vessel reopened . The in vivo experiments reported herein were performed as acute investigations, i.e., the sheep were not recovered . Therefore, it cannot be determined whether the developed surgical strategy is feasible in a chronic experimental setting . Furthermore, anatomy in other quadrupeds may differ from those in sheep . In conclusion, tricuspid valve surgery via a right - sided thoracotomy and using a cardiopulmonary bypass allows good visualization of all components of the tricuspid valve complex in sheep . However, access to the ascending aorta is impaired when using that surgical approach . As a consequence the results of this study may stimulate further experimental research on the tricuspid valve in quadrupeds and, ultimately, help to identify more fully the pathomechanisms leading to tricuspid valve disease.
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Opipramol is an atypical anxiolytic and antidepressant drug which has been found to be effective in depressive disorder . Although it was developed by schindler and blattner in 1961, its use was limited and recently it has regained popularity among psychiatrists all over for the treatment of somatoform and depressive disorders . Structurally its nucleus is similar to that of tricyclic antidepressant imipramine and the attached side chain is identical to that of perphenazine . Although it is structurally similar to tricyclic antidepressants, it does not inhibit the neuronal uptake of norepinephrine and/or serotonin and is a sigma - receptor agonist, primarily at the sigma-1 receptor subtype, but also at the sigma-2 subtype with lower affinity . Sigma receptor agonist opipramol is not presumed to cause affective switch because of its relative sparing of monoamine receptors . Hereby, we describe a case of opipramol - induced mania in a patient with bipolar depression . A 39-yr - old male was brought to our opd with 4 weeks history of talking excessively even to unfamiliar people, being irritable to others, overspending, singing, dancing and reduced need for sleep . It was reported that he had a manic episode with psychotic symptoms 15 years back and was on lithium prophylaxis till 5 years ago . Four months ago, the patient consulted a psychiatrist with history of excessive sadness, inability to sleep, easy fatiquability, anhedonia, not being able to carry out his job and reduced libido, and was diagnosed to have moderate depressive episode and was restarted on lithium 900 mg / day . Two months ago, as patient did not improve on lithium monotherapy with serum lithium level of 0.7 meq / litre, opipramol 50 mg / day was added along with lithium . And after 1 month on opipramol treatment patient was brought to us with the current symptoms . Patient's investigations showed serum lithium level of 0.7 meq / litre and thyroid function was normal . On mental status examination patient was diagnosed as a case of bipolar affective disorder, current episode being treatment emergent manic switch due to opipramol as per the international society for bipolar disorders (isbd) criteria and patient was admitted . Opipramol was stopped and patient was continued on lithium 900 mg / day and risperidone titrated up to 4 mg / day . His manic symptoms started resolving by second week and remitted by fourth week and the patient was discharged . The total manic score on young's mania rating scale was 39 at the time of admission and it dropped to 28, 17 and 6 at the end of first, second and fourth week of treatment, respectively . Although being effective, almost all the antidepressants carry a risk of mania / hypomania in bipolar affective disorder . Treatment - emergent affective switch (teas) is not well defined and according to the international society for bipolar disorders (isbd) 2009 criteria, switch can be called as definite treatment emergent manic switch if: it occurs within a window period of 8 weeks from intervention, full syndromic hypomanic, manic, mixed symptoms emerge andif symptoms last for at least 2 consecutive days with daily occurrence of symptomatic periods lasting more than 50% of time each day . It occurs within a window period of 8 weeks from intervention, full syndromic hypomanic, manic, mixed symptoms emerge and if symptoms last for at least 2 consecutive days with daily occurrence of symptomatic periods lasting more than 50% of time each day . In the largest multicentre trial of antidepressants in bipolar depression step bd, when patients followed for up to 2 years, transition from depression directly to manic, hypomanic or mixed states was observed in 21% of individuals prospectively observed for a single episode . Tricyclic antidepressants (tcas) have consistently been associated with a high risk of teas compared to other antidepressants; incidence rates ranging from 9% to 69% . The question whether concomitant use of mood stabilizers would reduce the switch inducing property of antidepressants is unanswered and wide variation in rates of teas observed in studies is often attributed to concomitant administration of mood stabilizers . Studies show that when mood stabilizers are combined with tcas risk of switching to mania is significantly reduced . When used along with mood stabilizers very low switch rates ranging from 0% to 3.7%, manic symptoms emerged within 4 weeks of opipramol treatment even though he was on lithium and serum lithium level was optimal . The dose of opipramol in our case was 50 mg / day which indicate that even very low dose of opipramol has propensity to cause switch to mania . It does not have inhibitory action on reuptake of norepinephrine or serotonin and dopamine unlike other antidepressants . Opipramol also blocks histamine, serotonin, dopamine and alpha-1 adrenergic receptors, on the basis of which switch due to opipramol cannot be explained . Ssris, tcas and norepinephrine - dopamine reuptake inhibitor bupropion which are reported to cause switch also have sigma receptor agonism and only factor which is shared between these drugs and opipramol is the affinity for sigma receptors . In animal models, sigma-1 receptor mediates stimulant and appetitive properties of cocaine which is similar to hypomanic symptoms and sigma-1 receptor antagonists block the hyperlocomotion and appetitive effect of cocaine . Hence agonistic action on sigma receptor itself may be postulated to cause behavioral activation and manic switch in case of opipramol, though this correlation is yet to be understood in detail . In summarisation, the above - mentioned case of bipolar depression who developed treatment emergent manic switch due to opipramol while on concomitant treatment with lithium remitted by fourth week of opipramol discontinuation . Sigma-1 receptor agonism of opipramol may be hypothesized as the basis of manic switch due to opipramol . This is the first case of opipramol - induced manic switch published in the literature and further studies are essential to confirm our view.
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The proportion of overweight and obese adolescents has increased in the western world and the trend is predicted to continue . Finland is no exception, and depending on the definitions used, approximately 10 to 20% of finnish adolescents are considered overweight or obese . In a comparative study of 41 countries, the overweight and obesity prevalence in finnish 15-year - old adolescents was a bit higher than the average of 17% in boys and 10% in girls . Slightly higher percentages than average were also obtained in the proportion of adolescents regarding themselves as a bit or much too fat (males: 22% in finland versus 21% on average; females: 45% versus 41%). The proportions can be assumed to be even higher among the subgroup of overweight young people, who have been shown to be less content with their bodies than adolescents of normal weight [2, 4, 5]. Likely due to increased levels of obesity and the great value placed on a thin appearance, attempts to lose weight have become more widespread among adolescents . Paradoxically, repeated dieting to lose weight may lead to weight gain via long - term adoption of fasting followed by overeating or decreased breakfast consumption . Extreme dieting has been associated with eating disorders as well as other negative psychological outcomes, such as lower self - esteem in adolescents [69]. The prevalence of adolescents' weight control behaviour increases with higher body mass index [1012]. It has been stated that overweight adolescents may adopt extreme weight control practices because they are further from their ideal weight or have failed to lose weight by means of modest eating or changes in exercise . Obesity, body dissatisfaction, low self - esteem, a feeling of poor life management, and anxiety in young people are connected with weight - reduction behaviour [7, 14, 15]. Overweight and obese children and adolescents have again lower body satisfaction than their nonoverweight peers [16, 17]. However, the perception of overweight rather than the actual weight appears to be the potent force leading to weight - reduction behaviour . According to some studies, acceptance of body size and shape is common amongst overweight teenagers, factors that protect susceptible adolescents from adopting unhealthy weight - control practices include positive body image . It evolves and changes under biological, psychological, social, and cultural influences [20, 21]. The majority of body image disturbances begin during adolescence, although their occurrence has been reported at younger ages . Negative body image predicts weight control behaviour, which may manifest itself in unhealthy actions, for example, fasting, purging, smoking, extreme diets, or training [6, 23, 24]. Body image problems and weight concerns are related to eating disturbances [24, 25], low self - worth [2628], depressive moods [29, 30], and suicidal ideation . The increasing rate of overweight and obese adolescents has been a public health concern in finland, and several actions to tackle this problem have been taken . However, less attention has been paid to overweight adolescents' perceptions of their weight and their attempts and practices to control their weight . The purpose of this study was firstly, to monitor overweight 15-year olds' self - perceived weight from 1994 to 2010 and determine the prevalence of body dissatisfaction and secondly, to survey the prevalence of weight control behaviour (i.e., attempts to lose weight and use of specific weight control practices) in the same group during the 2000s in finland . The empirical data for the study were obtained from the finnish data of the health behaviour in school - aged children (hbsc) study . The hbsc study comprises cross - national research conducted by an international network of research teams in collaboration with the world health organization regional office for europe . The overall goal of the hbsc study is to gain new insights into and increasing the understanding of health behaviour, lifestyles, and their context in young people . The scope of the hbsc study covers the measurements of a comprehensive variety of behaviours, ranging from those that are a risk to health to those that promote health . As well as running the monitoring survey, the hbsc study also seeks to influence the development of programmes and policies in order to promote the health of young people at both national and international levels . The survey questions span a range of health indicators and health - related behaviour as well as the life circumstances of young people . The questions provide information on demographic factors; health behaviour including physical activity; eating and dieting; well - being indicators including body mass index and body image . The cross - sectional data were collected through school - based surveys; anonymous, standard questionnaires were issued to a nationally representative sample of 15-year - olds in finland between march and may in 1994, 1998, 2002, 2006, 2010 . The mean age of respondents has been 15 years and 10 months (sd = 4 months) throughout the sampling years of 1994 to 2010 . Standard cluster sampling was followed regionally and conducted in accordance with the structure of the national education system . The primary sampling unit was the school, and the participating class from the school was randomly selected . The number of the schools involved in the survey was 64 in 1994; 85 in 1998; 100 in 2002; 99 in 2006; and 128 in 2010 . The number of pupils in the research data as well as the response rate for each survey year is presented in table 1 . The data cleaning process and data management in detail are presented elsewhere [3, 34]. In the hbsc survey, heights and weights, as well as other measures, are based on self - reports . Body mass index was calculated by dividing weight in kilograms by the square of height in meters (kg / m) for each respondent . The adolescents' weight statuses were categorised by means of the iotf age- and gender - specific bmi cut - off points represented by cole et al . . In the present study, the group of overweight adolescents include obese participants if not otherwise noted . All bmi values under the thresholds for overweight were classified as nonoverweight (i.e., normal and underweight). Self - reported variables are subject to random error and to systematic reporting bias . However, strauss reported that over 90% of youth aged 1216 years were correctly classified as normal - weight or obese based on self - reported heights and weights . More modest results have been obtained in some other studies [35, 36], but the studies by himes et al . . Reported again high correlations between reported and measured bmi in adolescents . To evaluate the reliability of the present survey, the stability of the responses to the questions concerning weigh and height, as well as all measures presented below, was investigated by test - retest correlations . These correlations were based on two similar questionnaires completed within a fortnight by 13- and 15-year - old schoolchildren (n = 194) from the provinces of eastern and western finland in 2005 . Almost all the respondents indicated the same weight on both measurements (intraclass correlations icc = .99; 95% confidence interval ci = .99 .99), whereas the stability in self - reported height was found to be only acceptable (icc = .63; 95% ci = .54.70). To assess perceived weight much too thin; a bit too thin; about the right size; a bit too fat; or much too fat . For the clarity of presentation, we combined the replies of the first two options (much too thin and a bit too thin), as well as the last two options (a bit too fat or much too fat), and these joint replies are shown in the findings . The test - retest stability in self - perceived weight were found to be excellent (icc = .81; 95% ci = .76.85). To identify those adolescents who were trying to lose weight at the time of taking part in the survey, respondents were asked to indicate if they were at present on a diet or doing something else to lose weight . Possible responses were yes; no, but i should lose some weight; no, my weight is fine; and no, because i need to put on weight . The additional question, have you gone on a diet, changed your eating habits or done something else to control your weight during the last 12 months?, assessed the occurrence and duration of weight control practices . Yes, for a few days to yes, for 6 months or more . Those respondents whose answer was yes were then asked to indicate which of the listed practices they used to control their weight during the previous 12 months . Listed weight control practices were exercising; skipping meals; fasting (i.e., going without food for 24 or more hours); eating fewer sweets; eating less fat; drinking fewer soft drinks; eating less (smaller amounts); eating more fruits and/or vegetables; drinking more water; restricting diet to one or more food groups (i.e., eat only fruits and vegetables, liquids only, eat only bread, and water); vomiting; using diet pills or laxatives; smoking more; dieting under the supervision of a professional . The test - retest stability of the responses to weight control practices varied from excellent (skipping meals; fasting; restricting diet; vomiting; smoking more) to poor (eating fewer sweets; drinking fewer soft drinks). The stability of responses in attempts to control weight during the previous 12 months was found to be excellent (icc = .90; 95% ci=.87.92). The body image investment (bis), presented by orbach and mikulincer, is a measure of emotional investment in the body . The first subscale, which was included in the finnish survey in 2006 and 2010, refers to feelings and attitudes regarding body image and contains six items: (1) i am frustrated with my physical appearance; (2) i am satisfied with my appearance; (3) i hate my body; (4) i feel comfortable with my body; (5) i feel anger toward my body; (6) i like my appearance in spite of its imperfections . The respondents were instructed as follows: here are some statements about one's feelings of his / her body . Please evaluate how the statements relate to you by checking the degree to which you agree or disagree with each one of statement . Scale scores were obtained by summarising the items (items 1, 3, and 5 were scored in the reverse direction). The test - retest stability of the responses to the bis items varied from excellent (item 1: icc = .77; 95% ci = .70.82) to acceptable (item 6: icc = .68; 95% ci = .59.75). The prevalence estimates for data points 1994, 1998, 2002, 2006, and 2010 are presented separately for each gender . For analyses in this paper, pasw statistics software (version 18) however, more precise variance estimates can be calculated by specifying and using the exact sample design . Therefore, when calculating 95% confidence intervals to determine if the differences in the prevalences were significant, analyses were made by stata statistical software (version 12). The possible lack of precision in variance estimation due to cluster sampling of this study was thus taken into account by using stata procedures for complex survey designs . Within genders, none of the differences in the prevalence of overweight between survey years was statistically significant (table 2). However, the proportion of overweight boys increased linearly from 13% in 1994 to 20% in 2006 . Results from the 2010 survey revealed a turning point; linear increase in the proportions of overweight adolescents seemed subsided in both genders . Between genders, boys reported statistically significantly higher levels of overweight than girls throughout the study period . The prevalence of obesity increased from 1% in 1994 to 4% in 2006 in boys and from 1% in 1994 to 2% in 2010 in girls (table 2). Nevertheless, none of the differences between years or genders was statistically significant according to the prevalence estimates and their 95% confidence intervals . Due to low number of cases in the obesity group, we combined the preobese and obese cases and refer to them as overweight in the later text . There were no statistically significant differences in self - perception of weight among overweight adolescents between survey years of 19942010 (table 3). Two - thirds of overweight boys and some 90% of overweight girls reported perceiving their bodies as too fat . Corresponding prevalences for nonoverweight adolescents varied from 9 to 14% in boys and from 37 to 43% in girls (data not shown). Overweight boys reported that they believe their body is about the right size statistically more often than overweight girls throughout the study period (table 3). The mean values of emotional investment in the body (bis) scores were statistically significantly lower in overweight adolescents than in nonoverweight adolescents in both genders and in both 2006 and 2010 surveys (table 4). The bis mean values for girls were significantly lower than for boys in both weight status groups (i.e., in overweight and nonoverweight). Within genders or weight status groups, there were no statistically significant differences in the mean values of bis scores between years 2006 and 2010 . The proportions of overweight adolescents engaged in weight control behaviour at the time of filling in the survey surged from 3% in 1994 to 18% in 2006 in 15-year - old overweight boys and from 19% in 1994 to 39% in 2010 in 15-year - old overweight girls (table 5). The prevalences of current weight controlling for nonoverweight adolescents varied from 2 to 4% in boys and from 5 to 14% in girls during 19942010 (data not shown). Based to the prevalence estimates and their 95% confidence intervals presented in table 5, the difference in the prevalence rates of current weight controlling was statistically significant between years 1994 and 2006 in overweight boys . On the other hand, there was no statistically significant difference between years 2006 and 2010 . In contrast to boys, the highest prevalence (39%) in current attempts to lose weight during the study period was observed in 2010 in overweight girls . None of the differences in the prevalences between survey years was statistically significant in overweight girls . Between genders, the difference in the prevalence of current weight controlling was statistically significant in 1998, 2002, and 2010 . There were no statistically significant differences within genders in the proportions of overweight adolescents, who gave an affirmative answer to the question of have you gone on a diet, changed their eating habits, or done something else to control your weight during the last 12 months? Between years 2002, 2006, and 2010 (table 5). Based the prevalence estimates and their 95% confidence intervals, attempts to control weight during the previous 12 months were significantly more common among overweight girls (5671%) than boys (2227%) in 2002, 2006 and 2010 . The corresponding prevalences for nonoverweight adolescents varied from 5% in 2010 to 8% in 2002 in boys and from 38% in 2010 to 50% in 2002 in girls (data not shown). There was no statistically significant difference between years 2002, 2006, and 2010 in the prevalence rates of using specific weight control practices among overweight adolescents who had tried to control their weight (table 6). The most common indicated weight control practices were exercising (> 85%), eating fewer sweets (> 84%) and less fat (> 75%), and drinking fewer soft drinks (> 74%). Throughout the study, a higher proportion of overweight girls than boys, who have tried to control their weight, indicated to have used all the specific weight control practices with exception of skipping meals and eating fewer sweets in 2006 . However, statistically significant differences between genders were found only in eating smaller portions in 2002 and 2010, and smoking more in 2006 . The current study monitored the prevalence rates of self - perceived weight and weight control behaviour among overweight adolescents in finland . There were no significant differences in self - perception of weight between the survey years of 1994, 1998, 2002, 2006, and 2010 . It seems that the prevalence of self - perceived overweight has remained stable in the 2000s among the overweight finnish adolescents after the decrease during 19791999 reported in the adolescents health and lifestyle survey . It also appears that the increased prevalence of obesity in the surrounding society has not greatly influenced the criteria of an ideal body type among adolescents, that is, the reference for desired weight has not been made more relaxed . The vast majority of 15-year - old overweight adolescents perceived their bodies as too fat, their body image was lower than in nonoverweight adolescents, and attempts to lose weight were prevalent especially among overweight girls across the study period . The unhappiness with body weight and engagement in weight control behaviour showed a clear gender difference, as overweight girls reported more often their discontent than the overweight boys . The gender discrepancy in the self - perception of body weight was remarkable: some 90% of overweight 15-year - old females felt that they are either a bit too fat or much too fat, whereas the corresponding proportion for males remained under 70% throughout the study period of 19942010 . The above findings may be a positive indication of girls' better health awareness, or alternatively, the results can be interpreted as a negative indication of more intense appearance pressure to be thin . These indications may also be related to each other: adolescent girls are often focused on appearance and weight loss in describing the aspects of the healthy eating [15, 41]. The prevalence of weight - related teasing has remained stable among both overweight and nonoverweight adolescents in the usa during 19992004 . Some studies even claim that the stigmatisation of obesity by children and adolescents has risen [13, 42]. Bullying has been found to have a significant impact on the young people's desire to lose weight [17, 43]. Bullying because of body size has also been found to be associated with dieting and unhealthy weight loss practices, even regardless of adolescents' weight status . This might be one explanation why girls with and without overweight were discontent with their bodies and, more commonly than boys, tried to lose weight . Girls in particular are pointed out as overweight, and they will receive directives to lose weight from both girls and boys more often than boys do . Haines and her coauthors propose that the increase in obesity among youth may have resulted in relaxed body size and shape standards particularly for males, whereas the ideal female weight and shape has remained unchanged . The focus of body - related teasing among males may have moved to muscularity instead of body weight . Results from a study of muscle dissatisfaction in young adult men in finland also support this presumption . The prevalence of weight control behaviour at the time of filling in the survey was statistically significantly higher in 2006 compared to 1994 in 15-year - old overweight boys . However, the difference between genders was more notable . Even in 2006, when the highest prevalence (18%) of current dieting in boys was found, considerably higher proportion (26%) of overweight girls was engaged in weight control . In addition, compared to one in four overweight boys, every second of overweight girls answered that they have gone on a diet, changed their eating habits, or done something else to control their weight during the 12 months previous of the survey in 2006 . Even though the weight control behaviour was common among overweight adolescents, and especially among girls, the majority of the overweight adolescents' weight control practices could be considered safe: exercising and eating fewer sweets were the most commonly indicated weight control practices . Unhealthy weight control practices have been found to become more common among overweight adolescents in the usa during 19992004 . Our findings do not indicate the same development in finland, even when taking into account the difference in the manner of representation of the results (i.e., overall prevalence versus prevalence within those who have tried to control their weight). However, based on the prevalence of attempts to lose weight among overweight adolescents, and their responses regarding weight control practices in this study, weight control is difficult when you already have overweight even with the knowledge of practices and motivation induced by self - perceived fatness and body dissatisfaction . The current environment is obesogenic and carries conflicting demands for young people . Thinness is idolised and obesity stigmatised and concurrently, meals and snacks high in fat and sugar are encouraged to be eaten, and quick commercial solutions for weight loss are advertised . In finland, the prevention of childhood and adolescent obesity has received attention both nationally and locally in the 2000s, and the projects appear to be fruitful: there was a slight but not statistically significant decrease in the proportion of overweight in adolescents in 2010 compared to the rates in 2006 . However, it has been stated that obesity prevention projects may lead to stigmatisation or even to disordered eating and eating disorders, particularly among those with poor body image . There appears to be a false dichotomy between the concerns of adolescent obesity and the body discontent in young people, as these two concerns are tightly interwoven in overweight adolescents' attempts to lose weight . The findings of our study overweight adolescents self - perceived overweight, lower body satisfaction, and attempts to lose weight suggest that in the prevention of adolescent obesity, it is crucial to focus on methods that strengthen the adolescents' self - esteem and do not emphasize body shape and weight . Body satisfaction has been found to protect girls from weight gain even after stabilising the effects of body mass index and sociodemographic factors . It is essential to create favourable conditions for strengthening the factors that provide protection from poor body image such as good self - esteem, resilience to stress, and faith in self - efficacy in young people's health promotion . Protective factors reinforce each other, so that the more assets a young person has, the more likely he or she is to engage in health - promoting behaviour . The low prevalence of obesity (13%) together with limited sample sizes can be considered to be a limitation of this study; quantitative studies of only a few obese respondents were therefore not practical . Adolescents' self - reported and measured heights and weights have been found to be highly correlated, but generally a bias of underreporting body weight will contribute to an underestimation of the prevalence of overweight . Therefore, the prevalence of overweight in finnish adolescents may be higher than reported herein . The hbsc study relies on adolescents' self - report, which raises questions about the reliability of the answers . The stability of the responses was investigated by a test - retest study in 2005 in finland and was found to be acceptable . In addition, the self - assessment is in some cases the only way to obtain information about a phenomenon . However, adolescents may interpret the concepts of being on a diet and weight control differently than do adults or health professionals . Further research is also needed on how and on what grounds young people assess their body, and more versatile indicators of self - perceived weight and weight controlling might produce higher validity of the measurement . Kautiainen concluded that the implications of perceived weight for prevention of obesity and promotion of well - being are complex, because they may differ according to the severity of overweight, the adolescent's maturational, and psychological status as well as gender . It should be noted that while obesity is one of the most powerful predictors of body dissatisfaction, the level of discontent, and its consequences vary . Results of this study demonstrated not only the self - reported overweight in finnish 15-year olds, but also that the majority of overweight adolescents were aware of their weight status . Findings revealed apparent gender differences in self - perception of weight, body dissatisfaction, and in engagement in weight control behaviour among overweight adolescents . The results emphasise the need for further research into the impact of gender on the attitudes towards own weight and the importance of the size and shape of the body.
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This article reviews the magnetic resonance imaging (mri) techniques available for use in the diagnosis and management of patients with cerebral metastases . Particular attention is paid to advanced imaging methodologies from which quantitative parameters can be derived, the role of these imaging biomarkers in distinguishing metastases from primary central nervous system (cns) tumours and tumour mimics, and metrics that may be of value in predicting the origin of the primary tumour . Autopsy studies have reported an incidence of cerebral metastases of up to 25% in patients with systemic cancer . With advancements in the management of patients with systemic malignancy, there has been a decline in patients dying from uncontrolled systemic disease . Cancer patients are surviving longer and whilst their systemic disease is controlled with newer medications, an increase in cns dissemination has been reported . Cerebral metastases are a leading cause of mortality in patients with metastatic malignancy and the median survival for those patients receiving whole brain radiotherapy (wbrt) is 7 months . Surgical resection of solitary metastases has been shown to convey a survival advantage in patients with systemically controlled disease in a small number of studies, but this is diminished in the presence of small, additional, non - resectable metastases . In addition, stereotactic radiosurgery (srs) is increasingly being utilized in the treatment of multiple (up to 4) small (less than 3 cm maximum diameter) metastases, although the advantages of this technique over wbrt for the treatment of more than 2 lesions remains controversial . It is therefore imperative that imaging provides accurate diagnosis, identification, size information and localization of all intracranial lesions in patients with presumed cerebral metastatic disease in order to optimize their management . The differential diagnosis of the solitary enhancing cerebral mass, of which metastatic disease is one of the leading causes, can be a major diagnostic challenge in neuroradiology . Whilst multiplicity favours the diagnosis of metastatic disease, differentiation from multifocal glioblastoma multiforme (gbm) and tumour mimics such as infection or tumefactive demyelination can be difficult . Distinction between these entities and metastatic disease is important with regard to both immediate patient management, and the need for additional imaging in patients with no known malignancy to identify a primary site and treat appropriately . Classically, cerebral metastases are seen on either computed tomography (ct) scanning or mri as lesions occurring at the cortical interface at the grey and white matter junction, most commonly located within the cerebral hemispheres followed by the cerebellum . Lesions can vary from radiologically silent microscopic deposits to masses measuring several centimetres in diameter . Contrast enhancement is frequently seen and can be intense, punctate, nodular or ring enhancing . Haemorrhage can also be a feature, and occurs more frequently in certain underlying primary pathologies (see below). The degree of peri - tumoural oedema varies from virtually none to (more commonly) extensive surrounding oedema . Mri exhibits superior sensitivity to ct for small lesion identification, particularly in the posterior fossa, and double / triple dose contrast, delayed imaging and the use of magnetization transfer to suppress background signal from non - enhancing tissues can further improve the sensitivity of lesion detection . In addition, certain imaging characteristics such as high attenuation on non - contrast ct and low t2 signal intensity as seen in mucinous metastases (fig . 1) or high signal on non - contrast t1 imaging of melanoma metastases (fig . The utilization of alternative conventional sequences such as contrast - enhanced multi - shot echo planar fluid attenuated inversion recovery (flair) and pre- and post - contrast inversion recovery t1-weighted sequences instead of conventional t1 spin echo sequences have failed to demonstrate benefit in improving lesion conspicuity . Phenotypic descriptors such as non - enhancing cortical flair signal abnormality adjacent to the mass lesion have been described more frequently in glioma but this feature is also seen in some cases of cerebral metastases . Figure 1imaging appearances of a solitary mucinous metastases from a colonic carcinoma primary on (a) non - contrast ct (hyper - attenuating lesion); (b) t2-weighted imaging (markedly hypointense); (c) t1-weighted pre - contrast imaging (isointense); (d) post - gadolinium contrast t1-weighted imaging (rim enhancement). Figure 2imaging characteristics of melanoma metastases . (a) t2; (b) pre - contrast t1; (c) post - contrast t1 . Imaging appearances of a solitary mucinous metastases from a colonic carcinoma primary on (a) non - contrast ct (hyper - attenuating lesion); (b) t2-weighted imaging (markedly hypointense); (c) t1-weighted pre - contrast imaging (isointense); (d) post - gadolinium contrast t1-weighted imaging (rim enhancement). (a) t2; (b) pre - contrast t1; (c) post - contrast t1 . The relative volume of signal hyperintensity on flair or t2-weighted imaging to the enhancing lesion volume is frequently cited as a good discriminator of malignant glioma and cerebral metastases in radiology textbooks . A greater degree of peri - lesional signal change, commonly referred to as oedema although the underlying content in glioma is more complex, is reportedly indicative of metastatic disease rather than glioma . A small (n = 29) ct - based study from 1989 reported differences in the mean ratio of oedema volume to tumour volume between metastases (3.1) and primary lesions (1.4). A more recent mri study of 26 metastases and 22 high - grade gliomas (hgg) evaluated the peri - tumoural oedema to tumour area ratio, and found significant differences between hgg (0.69 0.41) and metastases (2.41 1.63), p <0.001 . More evidence actually supports the use of advanced imaging metrics in the peri - lesional oedema to discriminate these 2 entities as detailed below, reflecting the underlying pathological differences in the origin of this signal change . Dynamic contrast - enhanced (dce) imaging techniques have been developed that allow for a number of parameters to be estimated that are thought to reflect the microvascular environment . Detailed descriptions of these methodologies are beyond the scope of this review and interested readers are directed to a number of excellent review articles on the subject of perfusion and permeability imaging in neuro - oncology . In brief, a bolus of contrast agent is injected intravenously and tracked with a series of dynamic images . Baseline longitudinal relaxivity (t1) is measured, and changes in t1-weighted signal intensity are converted to changes in contrast agent concentration . Contrast agent concentration time courses are generated, and post - processing of the data with application of pharmacokinetic modelling techniques allows for the calculation of a number of parameters, the most widely used of which is k (contrast volume transfer coefficient, in effect the amount of contrast passing from the intravascular space to the extravascular, extracellular space) reflecting both local blood flow and capillary permeability . For dynamic susceptibility contrast techniques (dsc - mri), the contrast agent bolus is tracked using fast t2- or t2 * -weighted acquisitions . Models of differing complexity can be used but the fundamental goal is to derive estimates of cerebral blood volume (cbv) and cerebral blood flow (cbf). In distinguishing primary from metastatic cerebral tumours, the only parameter reported to have value in robustly separating the 2 entities is cbv . A number of studies have reported higher cbv values in the solid tumour component of hggs than in metastases . Furthermore, in the examination of the peri - tumoural so - called oedematous region (peri - lesional t2/flair signal hyperintensity seen adjacent to enhancing or clearly solid tumour mass), hggs are reported to demonstrate higher cbv values than metastases (fig . 3). This may reflect differences in the underlying mechanisms of the so - called peri - tumoural oedema, which in primary intrinsic tumour may contain infiltrating angiogenic tumour cells in addition to vasogenic oedema, and in metastatic disease is more likely to reflect pure vasogenic oedema . Figure 3perfusion imaging in (a) solitary cerebral metastasis and (b) gbm (axial imaging on the left and sagittal reformats on the right). Bottom row: cbv maps . Magnified images of cbv maps and corresponding t2-weighted images . (a) the peri - lesional non - enhancing tissue surrounding the metastasis exhibits a very low relative cbv (white arrowhead) and represents vasogenic oedema; (b) the peri - lesional non - enhancing tissue surrounding the gbm has a slightly higher relative cerebral blood volume (white arrow) and represents infiltrating glioma . Perfusion imaging in (a) solitary cerebral metastasis and (b) gbm (axial imaging on the left and sagittal reformats on the right). Top row: t2-weighted imaging . Bottom row: cbv maps . Magnified images of cbv maps and corresponding t2-weighted images . (a) the peri - lesional non - enhancing tissue surrounding the metastasis exhibits a very low relative cbv (white arrowhead) and represents vasogenic oedema; (b) the peri - lesional non - enhancing tissue surrounding the gbm has a slightly higher relative cerebral blood volume (white arrow) and represents infiltrating glioma . Diffusion - weighted imaging and tractography diffusion - weighted imaging (dwi) allows quantification of the movement of free water molecules occurring secondarily to random thermal motion . The apparent diffusion coefficient (adc) is a measure of the degree of random motion of water molecules due to thermal energy . The term apparent is used to convey that it is not truly the free diffusion of water that is being observed . On visual inspection of diffusion imaging and adc maps, there is considerable evidence suggesting that this is of value in distinguishing necrotic tumours (both primary hggs and metastases) from abscesses, with restricted diffusion seen in the core of abscesses . However, it should be noted that the distinction is not clear cut and restricted diffusion has been reported in cerebral metastases of certain histological types (both small and non - small - cell lung, breast, colon and testicular carcinoma) (fig . The literature regarding the usefulness of measurement of adc values and related metrics in distinguishing hgg from cerebral metastases is controversial . Some groups have reported higher values of adc and adc ratios in the solid tumour or peri - tumoural tissue in cerebral metastases than in hgg, while others have reported lower values in either tumour or peri - lesional tissue and some have reported no significant difference in adc values between hgg and metastatic disease . These conflicting results may be a reflection of the wide variation in different imaging acquisitions, post - processing analysis techniques and scanners used . In addition, all studies have had relatively small recruitment numbers, few containing more than 20 patients with cerebral metastases and none containing a single metastatic histological tissue type, thus making the data rather heterogeneous and limiting its interpretation and applicability . Figure 4restricted diffusion in cerebral metastases . (a) t2-weighted imaging; (b) post - contrast t1-weighted imaging; (c) dwi; (d) adc map . There is restricted diffusion within the medial aspect of the tumour which demonstrates restriction in the more solid, t2 hypointense, non - enhancing component . Restricted diffusion in cerebral metastases . (a) t2-weighted imaging; (b) post - contrast t1-weighted imaging; (c) dwi; (d) adc map . There is restricted diffusion within the medial aspect of the tumour which demonstrates restriction in the more solid, t2 hypointense, non - enhancing component . The use of diffusion tensor imaging (dti) allows assessment of different, more detailed, diffusion - based parameters such as mean diffusivity (md) and fractional anisotropy (fa). Md from dti takes into account the orientation of the diffusion ellipsoid in each voxel, while adc from dwi represents diffusion in the orthogonal measurement directions as applied to every voxel . Confusion can arise from the often interchangeable use of md and adc . Like studies evaluating the role of adc in cerebral metastases, the literature regarding md and fa measures is also controversial . In a study comparing 16 gbms with 12 metastases, measures of md in both tumoural and peri - tumoural tissue was found to be significantly different between metastases and gbm, with low md values in the solid tumour in metastatic disease (0.98 0.188) compared with gbm (1.22 0.284), and high md values in the peri - tumoural tissue of metastases (1.41 0.097) versus gbm (1.25 0.201). This study also reported differences in the fa values of the peri - tumoural region, but not the solid tumour between the 2 malignancy types with metastases having significantly lower fa values (0.159 0.02) than gbm (0.188 0.045). In comparison, a similar study that included 10 gbms and 6 metastases reported no significant difference between enhancing tumoural or peri - tumoural tissue md or fa values, although the md value of the cystic component was reported to be significantly lower in the metastatic group than the gbm group; however there was considerable overlap between the 2 entities . A larger study by wang et al . Of 63 patients with ring - enhancing lesions (38 gbms and 25 metastases) examined more detailed features of the diffusion tensor shape, namely linear and planar coefficients (cl and cp, respectively), in addition to measures of fa and adc in the tumour core, enhancing rim, immediate peri - tumoural region and distant peri - tumoural region . They reported significantly higher values of both fa and cp for all tissue regions and elevated cl in all but the distant peri - tumoural region in gbm tumours compared with cerebral metastases . No significant difference in adc measures was found between gbm or metastases but the model from this study that best predicted tumour type with a sensitivity of 92% and specificity of 100% included adc, fa and cp measures . Other scalar measures of directional diffusion that can be derived include p (pure isotropic diffusion), q (pure anisotropic diffusion) and l (the total magnitude of the diffusion tensor). In a comparative study of the value of these measures in distinguishing gbm from metastases, the only parameter found to have a significantly lower value in the cerebral metastases compared with gbm was the measure q in the peri - tumoural region . There is also evidence for the utility of dwi or dti in discriminating solitary, cystic metastases from cerebral abscesses . Since abscesses often exhibit significantly lower central adc, they can be confused with mucinous adenocarcinomas . Have recently suggested that a combination of central adc with rim fa helps to distinguish those neoplastic cysts with low central adc from abscesses . Proton magnetic resonance spectroscopy (mrs) allows tissue metabolites to be assessed non - invasively . The 2 main techniques use single voxel (e.g. Press, steam) or multivoxel chemical shift imaging (csi) techniques . A number of studies using csi have shown some potential in differentiating gbm from cerebral metastases, wherein examination of the peri - tumoural region has been reported to show a lower choline (cho)/creatine (cr) ratio in cerebral metastases than that seen in gbms . In a study of 53 hggs and 20 metastases, server et al . Reported 100% sensitivity, 88.9% specificity, a positive predictive value (ppv) of 80% and a negative predictive value (npv) of 100% using a cut - off value of 1.24 for the peri - tumoural cho / cr ratio to discriminate between hgg and metastases . Similarly, this group also reported the value of examining the peri - tumoural cho / naa ratio and, using a cut - off value of 1.11, found 100% sensitivity, 91.1% specificity, 83.3% ppv, and 100% npv in discriminating hgg from cerebral metastases . A recent report in 2 cases of mucinous adenocarcinoma has described the presence of a metabolite peak that mimics naa and may reduce the specificity of mrs in this tumour type . The use of short echo time proton spectroscopy allows lipid and macromolecule signals to be observed opstad et al . Studied the value of short echo time mrs in comparing lipid and macromolecule signals in cerebral metastases (n = 34) and gbm (n = 25) and reported significantly higher values in metastases than gbm, although there was considerable overlap between the 2 groups . In an attempt to address the multicenter applicability of single voxel spectroscopy for assisting differential diagnosis of brain tumours, garca - gmez et al . Assessed spectra from several sites in over 300 intracranial lesions with histological diagnosis . They reported around 90% accuracy in pairwise discriminant analysis for differentiation between meningiomas, low - grade gliomas, gbm and metastases . Unfortunately, the discrimination of gbm from metastasis was less accurate at 78%, and they recommend a combined approach in further work . The value of mrs in non - invasive discrimination of source tissue in cerebral metastases has been studied by 2 groups . Reported increased mobile lipid content and elevated lip / ncr in cerebral metastases arising from a colorectal primary when compared with metastases from histologically distinct primary tumours . In addition, huang et al . Compared non - small - cell lung carcinoma (nsclc) cerebral metastases (n = 40) with those from breast (n = 17) and melanoma (n = 9) using csi mrs, reporting the cho / cr ratio to be significantly lower in nsclc metastases than in either breast or melanoma metastases . Mrs can be performed ex vivo on biopsy samples for metabolic classification of tumours, and there is evidence for clinically useful differential characterization of cerebral metastases . While this is invasive, it provides evidence for the potential use of new metabolic and spectroscopic imaging techniques in vivo . As with many advanced mri techniques, where optimization of acquisition and analysis requires further work, there is often diagnostic overlap within a particular imaging parameter . Evidence suggests that the ability to distinguish primary hggs from solitary cerebral metastases is improved when a multi - parametric imaging approach is taken . Law et al . Reported significant differences in both rcbv and cho / cr in the peri - tumoural region of gbm and metastases and advocated that these imaging parameters could be used together to try and distinguish between the 2 tumour types . Similarly, bulakbasi et al . Found both adc values and mrs values were separately useful in differentiating cerebral tumours, but when combined there was added value in predicting tumour type . The combined use of perfusion - weighted imaging (pwi) and mrs has also shown value in differentiating cerebral metastases originating from primary lung carcinoma from lesions originating from either breast carcinoma or melanoma . Wang et al . Used a combination of dti and dsc to distinguish gbm, solitary metastases and primary cerebral lymphoma (pcl). They concluded that the best discriminative combination for distinguishing gbm from the other tumour types involved fa from the enhancing rim and rcbv from the peri - tumoural region . This finding is in keeping with the histopathological findings in gbm with pseudopallisading cellular rim, and angiogenesis occurring adjacent to microinvasive disease in the surrounding oedema . Again, the multiparametric approach has been used for the discrimination of infectious from cystic neoplastic lesions . In a relatively small study, the lower central adc was again found in infectious lesions, while the presence of amino acids on mrs was also indicative of infection . In addition, the blood volume in the rim of the neoplastic lesions was found to be higher . The evidence regarding the use of advanced imaging techniques in cerebral metastatic disease is largely controversial with a variety of conflicting results regarding the different advanced imaging modalities . The majority of studies have focused on discriminating hgg from solitary cerebral metastases, with fewer studies attempting to differentiate metastatic lesions according to histological subtype, and therefore inform further imaging . At best, the majority of evidence suggests that quantitative metrics derived from pwi, dwi, dti or mrs of the peri - tumoural tissue may provide a supplementary means of discriminating hgg from cerebral metastases, and the use of a multimodality approach combining parameters derived from each of the advanced imaging techniques is likely to improve both the diagnostic sensitivity and specificity . Unfortunately, most of the current studies are limited with regard to their small patient numbers (few have more than 30 patients for a given tumour type) and the heterogeneous collection of histological types of cerebral metastases that are frequently grouped together . A large multi - centre trial is required to evaluate the usefulness of these combined advanced mr parameters in discriminating both gbm from cerebral metastases, and metastases of different histological subtype . In order to achieve diagnostic currency in the setting of busy clinical scanners, a selection of optimized advanced mr techniques requiring limited post - processing is desirable . A combination of short echo time mrs, dti and dsc imaging is a likely combination, with attention paid to both the enhancing tumour itself and the peri - tumoural tissue . The limitations in the receiver operating characteristics of these approaches mean that the expert input of a suitably experienced radiologist with a complete clinical picture will still be crucial in each case.
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The affective component of pain includes feelings of annoyance, sadness, anxiety, and depression in response to a noxious stimulus . In particular, depression and pain share a high degree of comorbidity, and a large number of studies have examined the close relationship between pain and depression . The rate of depression has been reported to be between 21 and 50% in study populations with low (011.8%) levels of preoperative depression . Indeed, postoperative pain intensity is correlated with the degree of depressive symptoms [13]. High postoperative depression scores have also been associated with increased length of stay and poor functional outcomes after surgeries [3, 4]. Preoperative anxiety and catastrophization are two well - studied risk factors for the development of chronic postsurgical pain . Both of these factors are known to lead to worsening depressed mood in the postoperative period . Longitudinal studies have shown that depression is a risk factor for the onset of disabling or chronic pain [9, 10]. Conversely, in patients with chronic pain, the mean prevalence of major depression is reported to be between 18 and 85%, depending on the practice setting [11, 12]. In fact, pain is a major risk factor for the development of depression . In a longitudinal, cohort study with 12-year follow - up, pain at baseline as well as the severity and chronicity of pain was statistically significantly associated with the onset of depression . There is a significant correlation between the severity of pain and the degree of depression . In a study examining the long - term course of depression, greater severity of pain at baseline, greater number of pain locations, and longer duration of pain baseline pain severity prior to the initiation of antidepressant treatment has also been shown to be a strong negative predictor of treatment response . At the same time, depression also adversely affects prognosis in the treatment of chronic pain . Patients with chronic pain and depression report more pain complaints and increased severity and longer duration of pain symptoms . Some studies have reported that patients with comorbid pain and depression have poorer response to pain treatment than nondepressed patients . Comorbid pain and depression also lead to significant functional impairments . In a cross - sectional study, patients with major depressive disorder with chronic pain are found to be 2.14.6 times more likely to report interference in activities of daily living and family and social interactions than depressed patients without pain . They have also been found to be more likely to take sick leave because of pain . Thus, a wealth of clinical data suggests a high degree of comorbidity between pain and depression . Basic and translational studies utilizing imaging as well as animal models have begun to unravel the mechanistic basis for the relationship between pain and depression . In this review, we will examine the current understanding of the circuit and molecular plasticity that underlie this complex relationship and how such understanding can lead to successful therapies . In imaging studies of acute experimental pain in human subjects, areas most commonly activated include the primary somatosensory (s1) and secondary somatosensory cortex (s2), anterior cingulate cortex (acc), insular cortex (ic), prefrontal cortex (pfc), thalamus, nucleus accumbens (nac), and amygdala [1921]. S1 and s2 activations contribute to the sensory - discriminative dimension of pain . The acc, pfc, ic, nac, and amygdala, meanwhile, have been implicated in the affective component of pain (figure 1). For example, reductions in gray matter volume are observed in the ic, acc, and pfc, areas involved in the emotional and cognitive aspects of pain . Several studies have also directly examined brain changes in the transition from acute to chronic pain . Brain activity in patients with back pain for approximately two months showed activations in the ic, thalamus, acc, and pfc . In patients with back pain for more than ten years, meanwhile, abnormal activations in the perigenual acc, medial pfc (mpfc), and amygdala are observed in response to pain . Thus, the transition from acute to chronic pain may be accompanied by a shift from the sensory to affective - emotional circuitry for pain . Additionally, functional connectivity of the nac with the pfc has been found to be higher in patients with persistent back pain, indicating that the reward circuitry may also play a role in this switch from sensory to affective focus in chronic pain [23, 24]. Interestingly, the regions and circuits identified in acute and particularly chronic pain studies closely mirror those found in studies of depression . Thus, areas most commonly found to be dysregulated in depressed patients include the pfc, acc, nac, hippocampus, and amygdala [25, 26]. Gray matter volume loss and alterations in activity have been found in these areas, similar to changes that occur with chronic pain . The pfc and acc are both involved in the processing of negative mood and affect and thus are implicated in depression studies . The hippocampus and amygdala, meanwhile, play roles in the formation and retrieval of negative emotional memory that is associated with pain and depression . More recently, fmri studies of resting state networks, during task - free settings, have been done to examine changes in patients with chronic pain and depression . Compared to healthy controls, the default mode network (dmn) is altered in patients with chronic pain, with greater representation in the precuneus and posterior cingulate cortex and less representation in the pfc . Correlation of the pfc with the ic is increased, and connectivity of the pfc with posterior portions of the dmn is decreased in patients with chronic pain . The dmn consists of a set of brain regions active at rest and deactivated during a task . The precise function of the dmn is unclear, but it may be involved in the regulation of attention and arousal . Altered connectivity of the mpfc and ic may reflect changes in emotional circuits associated with chronic pain . For example, in a resting state fmri study of depressed patients, patterns of functional connectivity in the dmn and affective network based on multivariate pattern analysis are distinct between depressed patients and healthy controls . Thus, connections involving the pfc and acc are most likely to be altered in the comorbidity between chronic pain and depression . It is thought that the concept of suffering is central to the experience of pain as well as the experience of depression [2931]. Hence, depression has been interpreted as a state of being emotional as opposed to physical pain . The pattern of brain activity observed with emotional pain, such as intense social rejection which is known to cause depressed mood, is similar to activations in response to exposure to experimental pain, including somatosensory as well as affective areas [32, 33]. A recent study, however, suggests that although there is commonality at the gross anatomical level, distinct networks may actually underlie acute physical versus emotional pain . This study finds that experimental pain and intense social rejection activated similar brain regions in human subjects, including s2, ic, thalamus, and periaqueductal gray (pag). However, multivariate pattern analysis showed distinct physical and emotional representations in these regions . Nevertheless, some shared representations were found in areas not primarily involved with sensory pain processing, including the left parahippocampal and fusiform gyri, retrosplenial cortex, posterior cingulate cortex, and striatum . These shared representations may thus be associated with behavioral context, memory, and motivation, and they may form the network link between depression and pain . There is evidence that depressed mood or other negative emotional states can also modulate sensory neural responses . In an fmri study, subjects were exposed to nonpainful visceral stimulation during neutral and negative emotional contexts . Significantly greater activations were noted in the acc and anterior ic during increasingly greater negative emotional contexts and greater levels of reported anxiety that is commonly found in depressed patients . The interaction between chronic pain and depression is likely more complex than assessing acute, experimental conditions . As noted above, in the transition from acute to chronic back pain, a shift from nociceptive to emotional circuitry similar brain regions may be dysregulated in both chronic pain and depression, including areas involved in emotional and reward processing such as the acc, ic, pfc, and nac . However, in their study of patients with chronic pain, baliki et al . Investigated the effect of comorbid depression on spatial and connectivity properties of resting state networks and found only low correlations between depression and their network connectivity measurements . Further investigation is thus required to explore the relationship between chronic pain and depression at the circuit level . Depressive symptomatology has a wide spectrum, and different brain areas may regulate different symptom clusters . Similarly, chronic pain varies in etiology and disability, and different conditions may have distinct links with depressive symptomatology . Whereas imaging studies have established evidence of plasticity at the anatomic and circuit level that underlie the relationship between pain and depression, they do not reveal mechanistic insights into the cause and effect relationship between changes in neurocircuitry and the pathology of pain and depression . Rodent models, on the other hand, have enabled preclinical investigation into the mechanisms and symptoms of pain - induced depression . Two well - developed behavioral assays for depression in rodents are the sucrose preference test (spt) and the forced swim test (fst). Depressed rodents, however, display a decreased preference for sucrose and hence symptoms of anhedonia . The fst, meanwhile, assesses behavioral despair, another important feature of depression . On this test if the rodent is depressed, it will display a decreased motivation to swim during the second session . Other related tests that can be used to test depression - like behaviors include the open field test, elevated plus maze, and novelty - suppressed feeding test which are assays for anxiety - like behaviors that often accompany depression . Another behavioral assay, the conditioned place avoidance / preference test (cpa / cpp), allows additional insight into the aversive and motivational aspects of animals in pain or depressed states . In the cpa and cpp, an animal with no preexisting preference is trained to associate one chamber in a two - chamber set with either a rewarding or aversive stimulus, such as pain and or pain relief, or a pro- or antidepressant, and to associate the other chamber with the control condition . After a period of associative training, the animal this test has been used to test the efficacy of both antidepressants and analgesics [3841]. Models for depression and pain are both very well established [37, 4245]. On the one hand, widely accepted models for depression already rely on pain as a stressor to induce depression - like symptoms . In the social defeat model, for example, smaller animals are subjected to physical interactions with larger, more aggressive individuals, and pain resulting from such aggressive physical interactions plays an important role in inducing subsequent depression - like behaviors [37, 46, 47]. In a study combining the social defeat model with a neuropathic pain model in rats, five - day exposure to social stress was found to ameliorate pain sensitivity in chronic pain, but ten - day exposure worsened pain thresholds . Conversely, a study in which mice were housed in either an impoverished or enriched environment after developing chronic neuropathic pain symptoms demonstrated that the enriching environment improved chronic pain symptoms . At the same time, current rodent models for pain, including acute and persistent pain models, have been shown to induce depression - like behaviors . Incisions penetrating the skin, fascia, and muscle, such as the plantar incision model using the rodent hind paw, the gastrocnemius incision model using the posterior surface of the rodent hind limb, and the tail incision model, reliably induce allodynia, a pain behavior in response to a nonnoxious stimulus . Moreover, hyperalgesia, increased sensitivity to a noxious stimulus, can develop around the site of the incision . Acute incisional pain has been suggested to induce temporary depression - like symptoms as manifested by decreased sucrose preference, and these depressive symptoms resolve in a time frame similar to that of the recovery of sensory symptoms . An inflammatory model of pain, in which complete freund's adjuvant (cfa) is injected into the hind paw in rodents, reliably induces sensory symptoms of chronic inflammatory pain lasting at least two weeks . After cfa injection, rats with inflammatory pain have been found to show significantly diminished sucrose preference compared to control animals, and this depressive phenotype can last at least two to five weeks [50, 51]. In addition, cfa - treated rats can also develop behavioral despair, another feature of depression [52, 53]. Finally, inflammatory pain has been shown to induce place avoidance in rats [54, 55]. In this test, rats stayed away from the chamber associated with inflammatory pain, suggesting that pain serves as an aversive stimulus that the rats actively avoid . Similarly, acute or repeated stress has been shown as an aversive stimulus in the cpa test in a number of rodent depression studies [37, 56]. Thus, studies using the cpa have demonstrated that pain, similar to psychological stressors known to cause depression, can serve as powerful aversive signals to alter behavior . Chronic neuropathic pain models mimic human chronic pain - induced depression more closely than acute or inflammatory pain . The most commonly used models of neuropathic pain employ peripheral nerve injury, and these include the chronic constriction injury (cci), by which ligatures are wrapped around the sciatic nerve, the sciatic nerve ligation model (snl) of mononeuropathy, by which the l4 spinal nerve is ligated, and the spared nerve injury (sni) model, by which two of the three terminal branches of the sciatic nerve, the tibial and common peroneal nerves, are axotomized [42, 57]. Sni has been shown to induce anhedonia as well as behavioral despair in rats, as measured by the spt and fst [49, 58]. Certain types of pain treatment, meanwhile, have been shown to decrease these depressive symptoms . Snl, likewise, has been shown to cause depressive symptoms as well as anxiety in rodents [59, 60]. Thus, animal models for depression have already employed pain as an element of stress to induce depressive symptoms . More importantly, various rodent pain models have been validated to induce depression - like behaviors and hence serve as valuable models for the study of the comorbidity between pain and depression . The advancement of modern systems - level neuroscience tools has enabled the identification of brain regions involved in pain and depression processing and the investigation into their structure and function . Pain is a complex, multidimensional experience that involves the potential recruitment of a large, bilateral network of brain regions . These components may become activated dynamically depending on a number of factors: context, stimulus, cognition, and emotion [22, 61]. Recent efforts to identify a so - called pain signature have resulted in the identification of regions which are most essential to experiencing pain . These regions include the thalamus, the cortex, including the acc, ic, s1, s2, nac, and the pag [19, 21]. A much larger system involves the function of subsystems which include the descending pain modulatory system comprised of the pag and rostral ventromedial medulla (rvm), as well as the reward - motivation network, including the pfc, nac, and the ventral tegmental area (vta). Not surprisingly, this interconnected group of brain regions also forms the basis for understanding the pathophysiology of depression . Preclinical studies focusing on the affective consequences of neuropathic pain have begun to link the aforementioned circuit changes with behavior outcomes in pain states . These studies have provided insights into the potential function of regions involved in the anxio - depressive components of chronic pain most notably, the acc, ic, hippocampus, amygdala, the nac, and the vta (figure 1). The acc, which processes cognitive, emotional, and autonomic functions and interacts with the thalamus and amygdala, has been implicated in both pain processing and depression [63, 64]. Hypermetabolism and diminished volume of the acc have both been observed in depressed patients and acc changes, including altered membrane potential oscillations, have separately been observed in the cci neuropathic pain model . Recruitment of the acc may play multiple roles in the experience of pain and pain - induced depression, from anticipation to aversion . In the sni neuropathic pain model, for example, the volume of the acc was found to diminish only after a delay, at the same time as the onset of anxio - depressive symptoms, suggesting that structural changes in the acc potentially represent certain depressive changes in response to pain . The acc response to pain, moreover, has been shown to be potentiated by amputation . Lesion studies have moreover confirmed that the acc is necessary for the aversive component of neuropathic pain . The ic, meanwhile, has been strongly implicated in processing pain intensity and interacts with several other key pain regions, including the pfc, acc, s2, and amygdala (figure 1). Because of the complexity of the ic's connections with other relevant regions, its role in processing affective or depressive symptoms of pain remains a strong possibility . The hippocampus and amygdala are two additional structures that likely play a role in pain - induced depressive behaviors . The hippocampus, a region known for its role in learning and memory, has been well - studied in the regulation of depressive phenotype . Recent animal studies have shown that cognitive and affective processes are impaired in chronic neuropathic pain conditions and that this impairment corresponds with a decreased hippocampal volume . Neurometabolic changes that can result in decreased serotonin levels in the hippocampus have also been demonstrated to cause depressive symptoms in an inflammatory pain model in rodents . Moreover, studies have demonstrated that pain - induced changes in the hippocampus correlate with anxiety - like behaviors commonly associated with the depressed state . In contrast to decreased hippocampal volume, neuropathic pain has been observed to increase the volume and neuronal proliferation in both the central and basolateral nuclei of amygdala . Given the well documented role of the amygdala in fear and in mood disorders, the amygdala has been suggested as a strong candidate for mediating depression - like symptoms resulting from pain [7375]. Indeed, a number of studies have demonstrated that the amygdala is crucial in maintaining anxio - depressive and nocifensive behaviors in persistent inflammatory and neuropathic pain states [7680]. Recent studies of the mesolimbic pathway, including the pfc, vta, and nac, have indicated a prominent role for these regions in pain processing, particularly in the depression - like symptoms resulting from chronic pain . The pfc, vta, and nac play crucial roles in reward processing, and these regions are also activated by nociception [8183]. The pfc - vta - nac axis is critical for the maintenance of normal hedonic experience and motivation, and hence this reward circuitry also plays a part in the pathogenesis of depression . A recent study showed that chronic pain induces the synaptic incorporation of calcium permeable -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors in the nac and that transmission through these receptors has important antidepressant properties in the chronic pain state . Meanwhile, the pfc has been found to play a role in pain through its connection with the nac (figure 1). It has been shown recently that basal dendrites in the pfc of sni - treated rodents are longer and have more branches than those of control animals, and spine density is also selectively increased in basal dendrites of these neurons from sni rats . Thus, changes in the circuit that links the pfc, vta, and nac may play an important role in the relationship between pain and depression . The current glutamate hypothesis for depression posits that long lasting alterations in glutamate signaling contribute to the regulation of depressive phenotype . Ampa receptors are the primary mediators of excitatory synaptic transmission, and they are composed of four subunits, glua14 . Reduced glua1 levels in the amygdala, pfc, and hippocampus have been found in several rodent stress models of depression [8688]. In the nac, meanwhile, lower levels of glua2-containing ampa receptors have been described in depressive states [89, 90]. Second, antidepressants can increase glua1 and glua2 expression in the pfc and nac [92, 93]. Lastly, ampa potentiators which directly increase ampa receptor activities have been shown to have antidepressant properties . Glutamate signaling has been studied in animal pain models as well . In the classic pag - rvm - spinal descending pathway [9496], neurons from the pag form glutamatergic projections through ampa receptors on gabaergic cells in the rvm to inhibit dorsal horn neurons . Not surprisingly, the administration of glutamate into the pag is known to produce analgesia [98100]. In the rvm, meanwhile, ampa receptor upregulation mediates analgesia in inflammatory pain states [101, 102], whereas their downregulation in neuropathic pain causes hyperalgesia . Thus, transmission through ampa receptors is required for the intact pag - rvm descending pathway [104, 105]. The nac provides pain - induced analgesia, in part through its projection to the rvm . Intra - nac administration of ampa receptor antagonists, however, can disrupt this pain - induced analgesic mechanism . Furthermore, chronic pain has been shown to decrease vesicular glutamate transporter (vglut) 1 and 3 levels in the nac, suggesting a decrease in glutamate signaling in this region . The most direct evidence for the analgesic effects of glutamate signaling comes from a study that showed ampakines, positive allosteric modulators for ampa receptors, have antinociceptive properties in persistent inflammatory and neuropathic pain conditions . At the same time, however, chronic inflammatory pain increases trafficking of glua1 ampa receptor subunits but decreases glua2 delivery [109111], leading to the formation of glua2-lacking receptors . Similarly, ampa receptor signaling in the acc and amygdala has also been suggested to increase synaptic plasticity and confer hyperalgesia [113117]. Thus, in chronic pain conditions, ampa receptor signaling plays both pronociceptive and antinociceptive roles, depending on the target cns regions . An emerging number of studies have demonstrated that glutamate signaling also plays an important role in specifically mediating the depressive symptoms of pain . For example, a study on ampa receptor subunit trafficking showed that chronic neuropathic pain selectively increases glua1 levels at the synapse of the nac, leading to the formation of glua2-lacking, or calcium permeable ampa receptors (cpars). Glutamate transmission through these newly formed cpars, in turn, relieves the depressive symptoms of pain without altering pain sensitivities . In addition, pharmacologic studies using ketamine and ampakines, two classes of drugs that increase glutamate signaling through ampa receptors [92, 118, 119], have demonstrated that drugs that increase glutamate signaling in the brain can also treat the depressive symptoms of chronic pain [49, 53]. Thus, glutamate signaling, particularly signaling through ampa receptors, plays a key role in regulating pain, depression, and depression in the context of chronic pain . Norepinephrine (ne) neurons are found in the locus ceruleus (lc), and decreased ne signaling is known to be associated with depression [120124]. The lc releases ne into multiple regions in the brain, including the cerebral cortex, limbic system, and spinal cord . Originally thought to regulate attention and fight - or - flight responses, the ne system has been shown to affect a wide range of cognitive and affective functions . Activation of the descending modulatory pathway from the rvm and pag, meanwhile, can release ne into the spinal dorsal horn . The binding of ne to the spinal alpha2 receptors has been found to exert antinociceptive effects . Thus, noradrenergic system can have a profound influence on the pathogenesis of pain and depression . Dopamine dysfunction has also been associated with increased pain sensitivity in several chronic pain conditions, including headache, fibromyalgia, and osteoarthritis [131134]. Interestingly, parkinson's disease, which presents a classic hypodopaminergic state, is associated with increased incidence of both depression and chronic pain [135, 136]. While the molecular mechanism for the role of da signaling in pain - induced depression remains incompletely characterized, it is thought that dopaminergic signaling in the pfc and nac is critical for the maintenance of normal hedonic drive, as well as working memory, concentration, and locomotion . Thus, dysfunction of the da system results in symptoms of anhedonia, reduced concentration, sleep disturbance, and psychomotor retardation, all salient symptoms of pain - induced depression [127, 137]. The role of 5-ht in depression is well - described [120, 121, 138, 139]. 5-ht is projected widely throughout the brain to modulate synapses as well as the release and function of ne and da, and its activity in the pfc, acc, vta, and nac is thought to influence mood and affect . In addition, 5-ht has also been shown to be critical for descending modulation of pain [140144]. Activation of the descending projections from the rvm, which includes nucleus raphe magnus, produces 5-ht release in the spinal dorsal horns [97, 145148]. The rvm provides both descending inhibition and facilitation of pain, through the activation of on and off cells, respectively [148154]. Thus, 5-ht signaling can exert both antidepressant and antinociceptive properties by modulating synaptic connectivity and the other monoamine signaling in various regions in the brain . A recent study examined the role of brain indoleamine 2,3-dioxygenase (ido1) in the comorbidity of pain and depression . Ido1 is a rate - limiting enzyme in the metabolism of tryptophan, a precursor to serotonin, and ido1 activity has been linked to decreased 5-ht content . This study found that inhibition of hippocampal ido1 activity attenuated pain - induced depression, thus providing further evidence that 5-ht signaling can exert control over the comorbidity of pain and depression . Bdnf binds to two receptors on the surface of cells: trkb and the low - affinity nerve growth factor receptor . Trkb is a receptor tyrosine kinase, and the binding of bdnf to trkb promotes the formation of long - term potentiation at synapses as well as de novo neurogenesis . Decreased bdnf levels have been identified in patients who have suffered from major depressive disorder, whereas patients who underwent successful antidepressant treatment have shown increased bdnf levels [156158]. Elevated bdnf levels have been associated with fibromyalgia, whereas a decreased level has been found in patients with chronic migraine [159, 160]. Bdnf val66met polymorphism, in particular, has been identified in patients with chronic abdominal and pelvic pain [161, 162]. Bdnf has been shown to regulate both peripheral and spinal sensitivity to chronic pain in various animal models . Furthermore, in the hippocampus, decreased bdnf has been found in chronic inflammatory pain states, and this decrease has been shown to be responsible for depression - like behaviors . Thus, by modulating synaptic plasticity throughout the peripheral and central pain circuits, bdnf can alter pain sensitivity and, more importantly, the level of pain - induced depression . The endocannabinoid system is a signaling system comprising the g - protein coupled cannabinoid cb1 and cb2 receptors, their intrinsic lipid ligands, endocannabinoids such as the n - arachidonoyl ethanolamide (anandamide) and the 2-arachidonoyl glycerol, and associated proteins (transporters, biosynthetic, and degradative enzymes). The cb1 receptor couples to both gi / o proteins which function to inhibit adenylyl cyclase activity, activate potassium channels, and inhibit voltage - gated calcium channels, while the cb2 receptor is known to couple to gi proteins . The cb1 receptor is located predominantly on presynaptic axon terminals, and it regulates calcium influx and subsequent neurotransmitter release to influence synaptic plasticity . Cb1 knockout mice become anhedonic during chronic mild stress, and they also demonstrate decreased motivation, indicative of the depressive phenotype [166, 167]. These changes are likely caused by alterations in cb1 signaling that help to maintain normal synaptic efficiency in the pfc, amygdala, hippocampus, and nac [168171]. Both cb1 and cb2 receptors are abundantly expressed in motor and limbic regions and in areas that are involved in pain transmission and modulation, including the pag, rvm, spinal cord dorsal horn, and the peripheral nerve . In the rvm, cb1 signaling can alter the on and off cell balance to favor descending inhibition . In the spinal dorsal horn, it can decrease n - methyl - d - aspartic acid or n - methyl - d - aspartate (nmda) receptor activation and decrease noxious stimulus - evoked firing of wide dynamic range neurons . Endocannabinoid signaling can also inhibit neuropeptide release from primary afferent fibers in response to acute noxious stimulus . Interestingly, a recent study showed that a cb2-selective agonist gw405833 specifically relieves depressive symptoms of chronic neuropathic pain in rats, but it has no antidepressant effects in rats that do not experience pain . This finding not only indicates that pain can induce depression, but it provides evidence that pain - induced depression may have unique physiologic and molecular mechanisms . Transcriptional mechanisms have been well studied in animal models of depression, and genome wide arrays have begun to uncover genetic bases for depression in patients who suffer from major depressive disorders . Increased transcriptional activity in the hippocampus induced by creb has been demonstrated to have antidepressant effects . In contrast, creb signaling in the nac promotes stress susceptibility and induces anhedonic behaviors . Prominent targets for creb include potassium channels, glutamate receptors, dynorphin, and other neuropeptides [175177]. By acting on these receptors and peptides, creb can increase the intrinsic excitability of cortical, hippocampal, and accumbal neurons and promote glutamatergic plasticity, which will in turn serve as circuit level regulators for affect and behavior . A number of studies have shown that creb signaling in the hippocampus, cortex, and nac can alter pain sensitivity [178180]. Furthermore, a recent study demonstrated that creb signaling in the acc promotes the negative affective experience of pain, as manifested by conditioned place avoidance . Translational regulators, especially mtor, have been actively investigated in both pain and depression models in rodents . Mtor signaling in the pfc has been shown to give rise to dendritic growth and upregulation of the synaptic machinery, which underlie the antidepressant effect of ketamine [92, 182]. Recently, mtor signaling in the hippocampus has also been found to regulate pain - related synaptic plasticity . Thus, translational regulation provides another level of control for the comorbidity of pain and depression . In addition to transcriptional and translational control of synapse and neuronal excitability, proteins that posttranslationally modify histone, dna, and recruit large sets of coactivators or corepressors of genes have come under active scrutiny over the last two decades . Such epigenetic mechanisms have been identified to play significant roles in the pathogenesis and regulation of depression and pain . For example, local inhibition of certain histone deacetylases (hdacs) or dna methyltransferases (dnmts) and conversely local activation of histone methyltransferases (hmts) in the nac have been found to have antidepressant effects in various animal models of depression [184186]. Similarly, alterations of hdacs and dnmts in the cortex and hippocampus have also been implicated in depression - like behaviors [187189]. Epigenetic mechanisms have been demonstrated to alter pain sensitivity at both spinal and peripheral levels . In addition, studies have begun to reveal that changes in hdacs and dnmts in the hypothalamus, cortex, and brain stem can regulate both sensory and affective pain behaviors [191193]. Particularly, altered dna methylation of the corticotropin releasing - factor genes in the hypothalamic - pituitary - adrenal axis has been shown to result in stress - induced pain hypersensitivity, raising the possibility that central epigenetic mechanisms can play a role in the relationship between pain and depression . A line of clinical evidence that supports the close pathological relationship between chronic pain and depression comes from the drugs that can treat both conditions . Currently, two classes of antidepressants, serotonin, and norepinephrine reuptake inhibitors (snris) and tricyclic antidepressants (tcas) are federal drug administration (fda) approved for the treatment of depression as well as a variety of chronic pain conditions . Furthermore, experimental treatments, including ketamine and deep brain stimulation, have been demonstrated to have efficacy in treating both pain and depression . Serotonin reuptake inhibitors (ssris) and serotonin and norepinephrine reuptake inhibitors (snris) are mainstay treatment options for depression . By inhibiting the reuptake of serotonin while the exact targets for these drugs are not known, it is thought that they exert antidepressant effects by augmenting synaptic plasticity in the cortex, hippocampus, striatum, and a number of other brain regions . However, the efficacy of ssris in pain has been disappointing, with number needed to treat greater than 15 for most of the drugs in this class . Snris, in contrast, have been highly successful in treating a wide range of pain, particularly neuropathic pain, including diabetic neuropathy, postherpetic neuralgia, and fibromyalgia [196200]. Thus, likely snris modulate the ne signaling pathway in the cns to decrease pain and pain - induced depression . One possible mechanism is the activation of the descending modulatory pathway from the rvm and pag to the dorsal horn neurons . On the other hand, there is increasing evidence that the pathogenesis of fibromyalgia strongly involves abnormal processing in the brain, and the incidence for the comorbidity of fibromyalgia and depression is particularly high [201, 202]. Thus, snris likely can also alter ne signaling and synaptic transmission in the brain to decrease pain, depression, and other associated affective symptoms in fibromyalgia patients . Currently, duloxetine is the first - line treatment option for fibromyalgia . For neuropathic pain in general, it is interesting to note, however, that the dose of snris for chronic pain treatment is approximately half of what is used for the treatment of major depressive disorder . These drugs block the serotonin transporter and norepinephrine transporter to increase the synaptic concentration of these neurotransmitters . Tcas have been largely replaced by the ssris and snris as the first - line treatment for depression due to their more serious side effect profile and poorer tolerability at higher doses . Over the past two decades, however, tcas have been widely used in the treatment of a number of chronic pain conditions, most notably neuropathic pain conditions . They are used for essentially the same pain syndromes as snris, including diabetic neuropathy, postherpetic neuralgia, and fibromyalgia as well as chronic low back pain with an element of radiculopathy . The number needed to treat pain for the tcas is comparable to that of the snris . Similar to the snris, the dose of tcas for chronic pain treatment is less than half of what is used for depression . Ketamine is classified as a dissociative anesthetic, and it is fda approved as an anesthetic as well as an analgesic agent . More recently, ketamine has been shown to increase presynaptic glutamate release and to activate mtor to promote the translation of glua1 ampa receptor units at the postsynaptic site . Recently, ketamine has emerged as a fast onset, long - lasting, and potent antidepressant . A single subanesthetic dose of iv ketamine has rapidly and reproducibly decreased depressive symptoms in treatment - resistant depressed patients, with antidepressant responses detected within 1 - 2 hours after infusion, maintained in a majority of patients for at least 24 hours, and in some cases for up to 7 days [205207]. While the mechanism of antidepressant action for ketamine remains incompletely understood, it likely involves an increase in ampa receptor signaling, an increase in bdnf levels, and an overall increase in synaptic plasticity in the cortex and hippocampus [92, 208, 209]. In animal studies, a subanesthetic dose of ketamine has been shown to relieve pain - induced depression for up to five days after a single administration, and this time frame is comparable to studies of stress - induced depression models as well as human studies of depression . Recently, sub - anesthetic - dose ketamine infusion has been used for the treatment of refractory neuropathic pain syndromes with significant depressive comorbidities, most notably the complex regional pain syndrome [210213]. In these patients, repeated infusions of subanesthetic doses of ketamine have been shown to effectively treat pain as well as associated affective comorbidities, including depressed mood . In a number of these studies, the effect has lasted weeks to months . Despite of these drugs discussed above, pharmacologic options for the comorbidity of pain and depression remain fairly limited . Based on our current understanding of circuit and molecular mechanisms for the comorbidity of pain and depression, however, two future therapeutic approaches hold promise . The first approach is to use neuromodulation (such as deep brain stimulation (dbs) or transcranial magnetic stimulation or even biofeedback or cognitive behavioral therapy) to target the converging circuit pathways in the brain that are responsible for pain as well as depression . Our knowledge of the converging pain and depression circuitry suggests that certain brain regions, including the dorsolateral pfc, hippocampus, and nac, can be potential targets for neuromodulation . Dbs targeting some of these regions, in particular the pfc, can significantly alter long - term neural plasticity and has been under intense and active investigation for refractory depression [214216]. However, neuromodulation treatment for pain has been limited to spinal cord and peripheral afferent stimulations . In the future, dbs targeting the pfc and nac should be considered in preclinical and clinical studies for the comorbidity of pain and depression . A second hypothetical therapeutic approach that holds promise is targeting the excitatory neurotransmission that can treat both pain and depression . In this a recent study shows that ampakines, drugs that potentiate ampa receptor function, can relieve depressive symptoms of pain . Furthermore, ketamine, which increases ampa receptor signaling, has also been shown to relieve pain - induced depression in animal models . Thus, future clinical studies are needed to further evaluate the efficacy of drugs such as ampakines and ketamine in the treatment of comorbidity of pain and depression . Finally, an intriguing class of drugs are hdac inhibitors which, by modifying genomic expression at a large scale, can profoundly affect both the circuit and molecular basis of pain and depression . Hdac inhibitors have been shown to relieve pain in neuropathic and inflammatory models of pain [151, 218]. Hdac inhibitors have already been approved for the treatment of some cancers, and the hdac inhibitor valproic acid is used as a mood stabilizer . Future studies are needed to determine whether inhibition of a single or multiple hdac isoforms is most efficacious for the treatment of pain and depression . Pain and depression are important comorbidities . Both clinical and preclinical studies clearly indicate that pain can cause depression, and that depression can worsen pain behaviors . Circuit and molecular mechanisms that underlie this plasticity have begun to emerge . Some of the successful current therapeutic approaches have validated these mechanisms, and future therapies based on such mechanistic understanding can be developed to better serve clinical needs.
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Invasive procedures mainly chorionic villus sampling (cvs) and amniocentesis are very common for prenatal diagnosis . Nowadays, conventional prenatal chromosome analysis, fluorescence in situ hybridization (fish), and array comparative genomic hybridization (acgh) analyses depend on samples acquired from invasive procedures . Chromosomal abnormalities that are compatible with life but cause considerable morbidity occur in 0.65% of newborns, and apparently balanced structural chromosomal rearrangements that will eventually affect reproduction occur in 0.2% of newborns . The application of acgh or the single - nucleotide polymorphism (snp) chip has allowed the detection of submicroscopic abnormalities and genomic disorders; the most commonly seen recurrent genomic disorders occur in approximately 0.18% of newborns . In 2009, the american college of obstetricians and gynecologists (acog) recommended that conventional karyotyping should remain the principal cytogenetic tool in prenatal diagnosis, and that acgh should be an adjunct to prenatal care for women with abnormal ultrasound findings (aus) and a normal conventional karyotype . However, many factors such as the risk that the fetus will have a chromosomal abnormality from direct fetal ultrasound imaging and indirect maternal serum markers, the risk of procedure - related miscarriage, the consequences of having an affected child, anxiety, ethnic background, and religion can affect a pregnant woman s decision to accept or reject an invasive procedure [57]. We expect that future application of cell - free fetal dna (cff - dna) screening for cytogenetic disorders will dramatically improve the diagnostic yield and reduce unnecessary invasive procedures; therefore, it is time to summarize the clinical indications of invasive prenatal diagnosis . In addition, cff - dna screening has many limitations and many cytogenomic abnormalities, including chromosomal structural abnormalities and genomic abnormalities, cannot be screened with it . Therefore, clinical indications accurately predicting the risk of cytogenomic abnormalities play an important role in prenatal genetic counseling and diagnosis . Current prenatal clinical indications include aus, abnormal maternal serum screening (amss), advanced maternal age (ama), family history (fh) of chromosomal abnormalities, and other events that could affect fetal health . Prenatal diagnosis is expected to move forward with more effective non - invasive prenatal indications and genome - wide analysis of cytogenomic abnormalities . However, the efficacy of clinical indications in prenatal diagnosis could vary in different practice settings and different regions . This study aimed to analyze the efficacies of indications of cvs and amniocentesis from a regional obstetric unit . The results provide the diagnostic yields from single or combined clinical indications, and indicate which women need the invasive testing and future direction for better prenatal diagnosis of cytogenomic abnormalities . This information will be useful for the obstetricians, clinical geneticists, and laboratory staff to further improve the quality of prenatal diagnosis . Yale cytogenetics laboratory is a regional reference laboratory for prenatal diagnosis in new haven county and surrounding areas . From 2000 to 2012, the laboratory performed karyotype analysis on 3229 cvs and 4589 amniotic fluid (af) specimens (excluding 17 culture failure samples). Fish using probes for the hira gene at 22q11.21 had been performed on 55 patients with aus of cardiac defects or fh of digeorge syndrome . Since 2009, acgh has been validated and offered to high - risk pregnancies after pre - testing counseling of technical specifications and limitations . All test results and clinical indications of these 7818 cvs and amniocenteses were compiled from the laboratory s cytoaccess database . The clinical indications for prenatal diagnosis included: (1) ama defined by age equal or greater than 35 years for singlet, 33 years for twin, and 31 years for triplet pregnancies; (2) aus of increased nuchal translucency (3 mm), cystic hygroma and other kinds of suspected fetal anomalies; (3) amss from first or second trimester maternal serum quad screen, including human chorionic gonadotropin (hcg), -fetoprotein (afp), unconjugated estriol, and inhibin a; (4) fh of a chromosome abnormality in previous pregnancy, parents with abnormal karyotype, or history of abnormal offspring birth; (5) multiple pregnancy (mp), including twins and triplets; and (6) other indications such as intrauterine fetal death or demise, radiation or medication exposure during pregnancy, anxiety, and consanguineous marriage . Of the 7818 cases, 68%, 30%, and 2% were referred by single indication, 2 indications, and 3 indications, respectively . Single and combined indications occurring in more than 1% of the samples (except for mp) were used to evaluate diagnostic yields . Conventional chromosome analysis was performed on g - band metaphases prepared from cultured af and cvs cells . Fish tests using aneuvision probes for rapid screening of aneuploidies of chromosomes x, y, 21, 13, and 18 and targeted probes for genomic disorders were performed following manufacturer s protocols (vysis / abbott, abbott park, il). Genomic dna was extracted from cvs and cultured amniocytes using the gentra puregene kit (qiagen, valencia, ca). The oligonucleotide acgh analysis using the agilent human genome cgh microarray 44k kit (containing 44 913 60-mer oligonucleotides, using ncbi36/hg18 human genome assembly), and the 180k kit (177 873 60-mer oligonucleotides, using grch37/hg19 assembly) (agilent technologies, inc ., santa clara, ca) was performed as previously described . Diagnostic yield was defined as percentage of the abnormal cases from cases referred by single, combined, or all indications . Time series regression analyses were performed for variance to evaluate the significance of trends and chi - square tests to compare rates, using spss for windows (spss inc . From 2000 to 2012, the most commonly used indications were ama, amss, and aus . For cvs cases, ama referrals declined from 55.8% to 51.1% (p<0.05), amss referrals increased from 0.5% to 11.2% (p<0.05), and aus referrals remained at the 10.63.3% level (figure 1a). For af cases, ama referrals significantly declined from 55.9% to 35.2% (p<0.001), amss referrals remained at 26.82.4%, and aus referrals increased from 5% to 20% (p<0.05) (figure 1b). The indications of fh, mp, and others remained in less than 12.33.1% and showed a relatively stable trend except for a decline in fh referrals for cvs . The usage of multiple indications defined by the average number of indications per case showed an increasing trend during the 13 year period (p<0.01) and cvs referrals required more indications than af (p<0.01) (figure 1c). Although the annual caseload of cvs and af showed a significant 57.2% decrease from 853 cases in 2000 to 365 cases in 2012 (p<0.01), there was no significance change in the number of abnormal findings (figure 1d). The diagnostic yield for chromosomal abnormalities showed a significant increase from 7.2% in 2000 to 13.4% in 2012 (p<0.01) and the overall diagnostic yield was 9.6% . Clinical indications associated with detected chromosomal abnormalities were also counted and ordered as follows: ama 57.6%, aus 40.3%, amss 30.5%, fh 9.6%, mp 4.8%, and other indications 2.3% . The efficacy of single or combined indications for detecting chromosomal abnormalities is summarized in table 1 . The 3 highest diagnostic yields were noted in cases with ama / aus (51.4% for cvs, 24.2% for af), aus (34.7% for cvs, 14.5% for af), and ama / amss (17.8% for cvs, 9.9% for af). In this cohort, the most effective single indication was aus followed by amss or fh and then ama . Mp was referred as indication of prenatal diagnosis due to our regional practice . However, there were no chromosomal abnormalities detected from mp referrals . Fish tests were used to detect 22q11.2 deletion on prenatal cases with aus of cardiac defects and fh of digeorge syndrome . Previous validation of acgh analysis on 11 cases with prenatally detected structural abnormalities successfully defined the genomic imbalances and gene content . The acgh analysis performed on 237 prenatal cases with normal chromosome findings detected 6 pathogenic and likely pathogenic genomic abnormalities . Recurrent genomic disorders of 16p13.11 deletions inherited from a paternal carrier were detected in 2 unrelated families and a 22q11.2 duplication was seen in 1 case . The aus findings of iugr, cystic hygroma, echogenic bowel, and tetralogy of fallot (tof) were noted in cases with recurrent genomic disorders . The clinical indications and the genomic abnormalities from fish and acgh analyses are shown in table 2 . Variants of unknown significance (vous) were detected in 5 cases; 2 of them pursued parental study and were confirmed as familial variants of maternal origin (data not shown). The estimated diagnostic yield from the fish and acgh analyses for genomic abnormalities on tested prenatal cases was 4% (12/303). Our cytogenetics laboratory has provided prenatal chromosome, fish, and microarray analyses for yale affiliated hospitals with stable referrals from new haven county and surrounding areas . Consistent with previous studies, our data demonstrated a continuous trend of reduced invasive procedures . The caseload reduction and procedure shifting were largely attributed to better knowledge and more informative counseling from clinical indications . The revised 2007 acog screening and invasive testing guidelines recommended that genetic screening and invasive diagnostic testing for aneuploidy should be available to all pregnant women, regardless of maternal age . Ama, a known factor for increased risk of down syndrome, is still the leading single indication in many reports . In our practice, ama is the most common indication, followed by amss in second place and aus in third place . However, due to the development of sensitive ultrasonic technology and maternal serum markers, the indication of amss for cvs and aus for af increased from 2000 to 2012, with a significant decrease in ama . Analysis of diagnostic yields from single and combined clinical indications can provide useful information for prenatal genetic counseling and diagnosis . For single indications for cvs and af, ama, amss, aus, and fh accounted for 39%, 13%, 11%, and 4% of referrals, but 19%, 10%, 23%, and 4% of total abnormal findings, respectively . For combined indications, ama / aus, ama / amss, and ama / fh accounted for 3%, 13%, and 5% of referrals, but 13%, 17%, and 4% of total abnormal findings, respectively (table 1). It was obvious that ama / aus, aus, and ama / amss are the most effective indications and together they accounted for 26% of total referrals and 52% of total abnormal findings . However, the remaining 48% of chromosomal abnormalities from other single and combined indications are indispensable in prenatal practice . In the present cohort, only 40% of cases detected with chromosome abnormalities had aus findings, suggesting the absence of ultrasound visible fetal structural anomalies in about 60% of prenatal chromosomal abnormalities . Further advancement of prenatal ultrasonography with better imaging and the inclusion of soft markers will improve the diagnostic yield . However, some fetuses with chromosomal abnormalities could lack ultrasound - detectable fetal anomalies . With a population of 0.86 million in new haven county and 2.17 million in the surrounding counties and a 1.3% birth rate by the 2012 united states census, it is estimated that each year there are 11 180 newborns in new haven county and 27 430 newborns in the surrounding counties . In this prenatal cohort during a 13-year period, chromosome analysis detected 292 fetuses (average 22 cases per year) with trisomy 21 for the diagnosis of down syndrome; fish and acgh detected 7 fetuses with deletions and duplication in the digeorge syndrome region (0.5 case per year). In a general population, the occurrence of down syndrome (omim#190685) is about 1/650 to 1/1000 live births and occurrence of digeorge syndrome (omim#188400) is 1 in 4000, indicating a 6:1 to 4:1 ratio of down vs. digeorge . Using a 1/800 occurrence for down syndrome, prenatal diagnosis of 22 fetuses inferred an annual basis of 17 600 pregnant women, which could be translated to a population of 1.35 million, consisting of the 0.86 million in the new haven county and 23% of the 2.17 million from the surrounding counties . From the same population basis, postnatal cytogenomic analysis was performed on 1354 pediatric patients from 2006 to 2009 and detected 7 cases with trisomy 21 (1.75 cases per year), 13 cases with the 22q11.21 deletion, and 3 cases with the reciprocal duplication (4 cases per year). Combined the prenatal and postnatal data, the annual detection of 24 down syndrome and 4~5 digeorge region deletions and duplications matches the occurrence and down vs. digeorge ratio from the estimated 17 600 pregnancies . But only 10% (0.5/4~5) of digeorge deletions and duplications were detected prenatally in the current setting . The lower diagnostic yield of genomic disorders in prenatal practice is likely due to the lack of sensitive and specific clinical indications . Since 20082010 we found that the number of invasive procedures began to change significantly, perhaps because non - invasive prenatal testing became popular from that time . Recently, a non - invasive prenatal screening method using massive parallel sequencing of maternal plasma cff - dna has been validated and rapidly integrated into prenatal genetic evaluation . A rapid acmg statement for this newly introduced cff - dna - based screening has been issued . Currently, this newly integrated procedure has demonstrated high sensitivity and specificity in detecting common aneuploidies . This progress has also re - energized efforts to develop non - invasive prenatal diagnosis using maternal circulating fetal cells [8,1922]. Further improvement of cff - dna based screening for genomic imbalances is technically feasible and is expected to become a direct indication of genomic abnormalities in prenatal counseling for recommending further invasive procedures . From 2000 to 2012, with the development of sensitive ultrasonic technology and maternal serum markers, the indication of amss and aus were increasing while ama were significantly decreasing, although ama is the most common indication . Largely due to this change of13 years, prenatal evaluation using ama / aus was the most effective in detecting chromosomal abnormalities, but better indications for genomic abnormalities are needed . The occurrence of chromosomal morbidity, reproductive defects, and common genomic disorders in 0.65%, 0.2%, and 0.18%, respectively, of newborns indicated that the baseline of prenatal cytogenomic diagnosis should be set on at least 1% but most likely up to 3% of pregnant women if including other genomic abnormalities and gene mutations . Diagnostic prenatal cytogenomic tests should be performed on 176 to 528 fetuses from 17 600 pregnancies; the 2012 caseload of 365 in this laboratory is within the baseline level . The goal of invasive procedures for prenatal diagnosis is to achieve much higher diagnostic yield for cytogenomic abnormalities . Toward that end, prenatal genetic evaluation using multiple screening methods of ama, aus, amss, and cff - dna and combined diagnostic chromosome and acgh tests will become highly effective in detecting cytogenomic abnormalities.
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Increasing initial tension in the screw known as preload, without exceeding its yield strength stabilizes the screw joint . An integral part of this process in dental implant screws is tightening screws to an appropriate torque or target value . It is demonstrated that application of insufficient preload, affects the stability of screw joint at implant abutment connection . It is stated that connection - related complications (screw loosening or fracture) are among the most frequent technical complications that affect the survival rates of fixed implant supported prosthesis [59]. Hand, electronic and mechanical torque drivers are available for tightening screws and applying torque to the screw joint . It is demonstrated that hand held drivers are dependent on the experience level of the operator and are inconsistent, unpredictable and generally less than the target torque [1012]. While accuracy of new and used electronic torque controllers has been reported without significant difference, unpredictable torque level with great range of errors (1% to 165% from the target torque) mechanical torque limiting devices (mtlds), eliminating operator variability, are recommended as a common device to ensure consistency in tightening implant components to specific target torque . These two types are toggle type or friction - style and beam type or spring - style . Beam type or spring - style mtlds apply a torque to the screw with spring action . This type does not have a release mechanism and mtlds (f - s mtlds) refer to those wrenches that use a ball detent system to disengage the lever arm at the desired torque and then by flipping the head of the wrench to the side, limit the torque applied . Sterilization and aging seems to affect the accuracy of mtlds [1517]. Despite the evaluation of independent influence of steam sterilization on the accuracy of friction - style and spring - style mtlds, the independent effect of aging has not been evaluated . The accuracy of new mtlds has been examined in few studies [2022]. By comparing peak torque values of different mtlds, standlee and colleagues (2002) concluded that the mean torque values of new spring - style and friction - style mtlds were within 10% of their respective torque levels . Cehreli (2004) examined the accuracy of fifteen new and used spring - style mtlds, and showed that new devices applied higher torque values than used devices (sterilized up to 1000 times) for the 35 ncm torque target (p<0.05). Vallee and colleagues (2008) assessed the accuracy of new mtlds and showed that spring - style mtlds were more accurate than friction - style mtlds in delivering the target torque (p<0.001). The accuracy of mtlds were dependent not only on the wrench style, but also on the manufacturer . Some f - s mtlds in clinical service delivered unacceptably high torque values . Due to the high variability in peak torque delivery, the purpose of this study was to assess the accuracy (10% of the target torque) of new f - s mtld, before and after aging . The null hypothesis was that there would be no significant difference in the absolute value of errors (absolute difference between peak torque and target torque) after aging in each group of f - s mtld . The second null hypothesis was that there would be no significant difference in the absolute value of errors after aging among the three groups of f - s mtlds . New f - s mtlds from three different implant manufacturers were collected to determine their accuracy by comparing their peak torque with their target torque values (figure 1). Five mtlds from each of the three types were selected and their respective drivers were tested: astra tech (25 ncm, hader sa, la chaux - de - fonds, switzerland)biohorizon (30 ncm, dynatorq itl, irvine, california, usa)dr idhe (1560 ncm, dr idhe dental, eching / munich, germany). Astra tech (25 ncm, hader sa, la chaux - de - fonds, switzerland) biohorizon (30 ncm, dynatorq itl, irvine, california, usa) dr idhe (1560 ncm, dr idhe dental, eching / munich, germany). Target torque was 25ncm for astra tech mtlds and 30 ncm for biohorizon and dr idhe mtlds . The total specimen size of fifteen mtlds were selected according to other studies and considering the minimum effect size of 0.41ncm, sd=0.13 and = 0.2, using 2-level factorial design and using minitab 14 . Each manufacturer was given an abbreviation and each mtld was randomly assigned a number from 1 to 5 and then labeled accordingly: astra tech (x1 to x5), biohorizon (y1 to y5), dr idhe (z1 to z5). The tohnichi torque gauge (tohnichi, 6-btg (-s), japan) was used to measure the peak torque values of each mtld . The torque gauge was new and calibrated by the manufacturer to be accurate within 2% of the full scale . After connection of the mtld to the driver, torque indicator on the gauge was set to zero . Force was applied to the mtlds until release and flipping of the head of the wrench to the side at a precalibrated torque value . The torque was applied by one operator that was blind to the measured values and the other operator registered the peak torque values presented on the torque indicator of the gauge for each mtld using a magnifier (figure 2). The procedure of peak torque measurement was performed in ten sequences before aging . In each sequence, ten repetitions of peak torque values was registered . Totally, the procedure was repeated 100 times for each mtld in three selected types . For the aging procedure, 1000 applications of target torque in 100 sequences, with at least 3 hours of delay between each sequence were performed using tohnichi torque gauge . This 3 hours of delay was considered for simulating sterilization procedures in the clinical situation . The mean and range of difference between the measured torque value and the target torque values were evaluated before and after aging . Descriptive statistical analysis was used and a 2 way repeated- measure anova was performed to assess the difference in accuracy among the three groups of friction - style mtlds after aging . For pair wise comparison, bonferroni post hoc test was used . The distribution of absolute difference was not normal, though to compare the absolute difference values after aging in each group, the non parametric wilcoxon signed rank test was used . Repeated measures anova before aging demonstrated that peak torque values decrease with the number of repetitions and this decrease was significant until twenty times repetition of torque value measurements (p<0.05) and then did not show any significant difference until the end of the measurements (1000 times in 100 sequences) (figure 3). For pair wise comparison bonferroni post hoc test was used (table 13). According to this test, biohorizon and dr idhe mtlds, showed significant differences in the fifteen first repetitions of torque value measurements (p<0.05) and then they were comparable but showed significant difference with astra tech mtlds (p<0.05). The mean difference of astra tech mtlds was always lower than the target torque compared to the two other groups . Repeated measures anova after aging did not show any significant difference of peak torque values with the increased number of peak torque measurement . A significant difference was seen between raw error values (absolute value of difference between peak torque and target torque) of the three groups studied (p<0.05). Peak torque values were almost always higher than the target torque for dr idhe group and always lower than the target torque for astra tech and biohorizon groups (figure 4). For pair wise comparison, bonferroni post hoc test was used . According to this test, biohorizon and astra tech mtlds did not show significant difference after aging (p<0.001). According to this test, biohorizon and astra tech mtlds did not show significant difference, but showed significant difference with dr idhe group (p<0.05). To compare the absolute difference values (error) after aging in each group, the non parametric wilcoxon signed rank test was used . The results demonstrated significant difference of error in astra tech and biohorizon mtlds after aging . The data support rejection of the first null hypothesis, as there was significant difference between the absolute difference of peak torque and target torque values (error) after aging in astra tech and biohorizon mtlds (p <0.001). The data also support rejection of the second null hypothesis, as there was a statistically significant difference of absolute error values after aging between the three groups (p<0.05). Before aging, statistically significant differences were found among the three types of f - s mtlds (p<0.05), but all the devices tested in this study delivered fairly consistent torque output within 10% of their preset target values . Vallee s findings on the accuracy of new f - s mtlds showed all the recorded torque values lower than the target torque (in 50 consecutive measurements). However, our results showed that only recorded values of astra tech mtlds always demonstrated a lower peak torque range than target torque values . Peak torque values decreased until twenty times repetition of torque value measurements in all the three study groups (p<0.05) before aging . Evaluation of the range of this difference demonstrated the decrease of peak torque values for astra tech (3.5,1 ncm) and biohorizon (4,-1ncm) torque devices . This decrease demonstrated a predictable pattern for these two groups (always-2 ncm lower than target torque). After aging, the increase of torque output was seen for dr idhe torque wrenches in the range of (1, 5ncm), with no predictable pattern . More than 50% of all torque wrenches in this study demonstrated more than 10% difference from their torque values . Maximum difference values were 16% for astra tech in 64% of peak torque measurements, 15% for biohorizon in 52.7% of peak torque measurements and 16.7% for dr idhe torque limiting devices in 34% of peak torque measurements . In this study, instead of using aggregate measures, the range of error was evaluated, which can demonstrate the torque values measured in 10% suitable torque range, with more confidence . Peak torque values within 10% of the target torque were proposed as a clinically suitable torque . Standlee demonstrated the ability of new f - s mtlds in producing consistent torque values that did not vary more than 10% from target torque values . This might confirm the present findings that demonstrated that all the devices tested in this study delivered fairly consistent torque output within 10% of their target values before aging . As yet, a gold standard for clinically suitable torque has not been defined and the safety zone for inevitable errors of mechanical torque limiting devices has not been determined . Measuring the variability of f - s mtlds in clinical service, showed their capability of producing accurate torque values within 10% of their target torque, but high variability was seen and frequent calibration recommended . After aging, peak torque values of mechanical torque limiting devices did not show any significant difference with the increased number of peak torque measurements . Gutierrez et al . Evaluated the peak torque delivery of friction - style torque wrenches used routinely in dental practice . They did not find any correlation between the age of torque wrenches and peak torque delivery . They concluded that the number of uses producing wear is probably not the major factor of inaccurate torque delivery . This finding supports our results that demonstrated a lower variability of peak torque values in comparison with other studies evaluating the accuracy of torque devices in clinical services . Therefore, corrosion of the moving parts was found to be the reason of inaccurate values, as high as 455% of the stated target torque . Congealation of the lubricant inside the friction - style torque wrench is proposed to increase the applied torque . Separate and combined effect of steam sterilization and aging must be determined before consideration of other methods of sterilization . The number of peak torque measurements for testing accuracy vary from five, seven, ten to fifty and finally 100 times in different studies . In this study, torque values were measured 100 times in 10 sequences . In each sequence, 10 consecutive measurements, with 3 hours delay between each sequence were made, simulating the clinical procedure of sequential screw tightening . Mtlds of the current in vitro study were new and had not been exposed to clinical or sterilization procedures . Considering the independent influence of the number of use as aging, is a new point of view . Generally, aging in the clinical situation is stated as a combined effect of the number of use and sterilization cycles without defining the exact number of use or sterilization cycles [16, 17, 21]. While other studies used torque wrenches in clinical services to investigate their accuracy, their results will not apply to every clinical situation, because too many confounding variables associated with sterilization, maintenance and use exist that cannot be checked and defined precisely . Future observations may be needed to simulate clinical situation and control the confounding variables such as sterilization, aging and maintenance . Considering the independent and combined effect of sterilization methods and aging will give a clinical guideline to determine the calibration frequency needs of these devices . Within the limitation of this study, aging as an independent factor affects the accuracy of f - s mtlds.
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We used a mean - field version of a biophysical cortical attractor network model to derive predictions of the temporal dynamics of activity in a cortical region that selects between inputs reflecting the value of two options . The model comprises two populations of excitatory pyramidal cells selective for each option, with strong recurrent excitation between cells of similar selectivity, and effective inhibition between the two pools mediated by inhibitory interneurons (see online methods). This effective inhibition mediates a competition between the two excitatory pools, with one pool ending up in a high firing attractor state (chosen option), and the other pool staying in a low firing attractor state (unchosen option). Neurons selective for option o receive inputs ro at firing rates proportional to the subjective value of that option, sevo . The neurons further receive background noise inputs and currents from other cells in the network . Importantly, the network has very few free parameters that are not otherwise constrained by their biophysical plausibility . The behavior of single units in the network has been described elsewhere; here, we focus on predictions suited to investigation with meg, namely behavior of the summed input currents to all pyramidal cells . We simulated network behavior using a set of trials with varying sev0 (as used in the human experiment, below). The network attracted faster to a decision when overall value or value difference were higher, yielding the prediction of decreased reaction times (rts) under these conditions . We tested this prediction more formally using a multiple regression in which model rts were predicted as a function of both overall value and value difference; both variables were found to have a negative effect on rt (fig 1b). Intuitively, the model reaches an asymmetric attractor state when the basin of attraction for this option is larger due to larger value difference (which determines the difference between the two inputs). An increase in overall value causes the network activity to rise faster and diverge faster, also resulting in faster rt . We then performed a time - frequency analysis of network responses, which aided our subsequent comparison of model predictions with meg data . We used morlet wavelets to decompose network activity on each trial, and regressed the decomposed data onto overall value and value difference . Network transitions typically took several hundred milliseconds to occur, and so most key model predictions were limited to frequencies ranging from approximately 210hz (fig . 1c). Overall value had a broadband effect on model activity in the 39 hz frequency range, soon after selective inputs were delivered to the network (fig . 1c, top), whereas value difference had a later and slightly lower - frequency effect, predominantly in the 24.5 hz range (fig . The different frequencies reflect the fact that overall value affects the population synaptic input earlier and over a shorter time period than value difference . The effect of the two regressors on network responses is a reflection of the fact that network transitions occur at different speeds depending upon the input presented; thus, the network does not explicitly represent such quantities, but these effects are a manifestation of trial - to - trial variability in the speed of the different network transitions . If we collapsed across the relevant frequencies, the temporal progression from an overall value signal to a value difference signal could be clearly seen (fig . It was also found that on trials where the network model made an error (i.e. Sevchosen <sevunchosen), there was an effect of overall value on the model s activity, but no clear effect of value difference (fig . (however, it should be borne in mind that error trials inherently covered a smaller range of value differences than correct trials, which may have caused the absence of any effect). The key predictions derived from the model were therefore: (i) the temporal evolution from an difference value signal; (ii) a difference in the frequency of the response, with value difference dominating responses at lower frequencies than overall value; (iii) the presence of an overall, but little or no difference signal, on error trials . Each option had a certain number of points available, represented by the width of an onscreen bar, and a probability of obtaining those points, represented by a percentage underneath the bar . The aim was to accumulate points (displayed on a progress bar) in order to reach a gold target, at which point monetary reward was delivered and the progress bar was reset to its initial position . To accumulate maximal returns, subjects should compute the objective pascalian value (bar width multiplied by probability of winning, denoted evo for option o), and select the option with the higher value on each trial . In fact, most subjects tended to overweight low probabilities of winning and underweight high probabilities, and exhibited a concave utility function, in accordance with predictions from prospect theory (supplementary fig . Subject rts correlated negatively with both the difference in subjective option values (= sevchosensevunchosen) and with the overall value of the decision (= sev1+sev2), in line with model predictions (fig . 3b shows the group results of a multiple linear regression of value difference (t(29)=7.98,p<0.0005) and overall value (t(29)=2.36,p<0.05) on reaction times across all subjects (see also supplementary fig . We also included some trials in which both reward magnitude and probability were higher on one option than the other . There was an additional bonus in speed beyond that related to value for these no brainer trials (t(29)=8.32, p<0.0005; fig . Subjects were therefore faster on average on these trials than on those where probability and magnitude advocated opposing choices, and so needed to be translated into a there was a steady decrease in reaction time as subjects progressed through the task (fig . 3c), without any coincident change in parameters describing choice behavior (supplementary table s2), suggesting subjects became less deliberative and more automated in their choices as they became familiar with the task . We used linearly constrained minimum variance beamforming to spatially filter meg data to locations in source space . We epoched data with respect to both stimulus onset and subject response, and focused our analyses on responses in the 210 hz frequency range, in accordance with model predictions . We first used a whole - brain statistical parametric mapping analysis to look for areas showing a main effect of performing the task relative to a pre - stimulus (300ms to 100ms) or post - response (+ 100ms to + 300ms) baseline . We hypothesized that, in addition to areas important to stimulus valuation such as ventromedial prefrontal cortex, the stimulus - locked analysis would reveal early visual areas involved in basic stimulus processing, and the response - locked analysis would reveal areas involved in visually guided manual movements in parietal and premotor cortices, in addition to primary motor areas . A distributed network of areas was found to be task - sensitive at these frequencies (fig . F; supplementary movie s1, s2). Stimulus - locked, early visual cortex activation (fig . 4a) was followed by slowly ramping bilateral activation at the frontal pole and ventromedial prefrontal cortex (fig . Whilst 210hz activity in these frontal regions peaked relatively late in the trial (1000ms after stimulus onset), it ramped from a much earlier point in the trial (see fig . 5b and supplementary fig . Response - locked, a prolonged activation spread from a mid - posterior portion of the superior parietal lobule, which extended medially into the marginal ramus of the posterior cingulate sulcus (fig . 4c), to a bilateral medial portion of the mid - intraparietal sulcus (ips) (fig . 4f) were activated at the time of the response . Having isolated areas that showed changes in activity relative to baseline, we then examined whether activity within these regions co - varied with decision values, and where this activity matched with predictions derived from the biophysical decision model . Importantly, by selecting regions based on the main effect of task versus baseline, we ensured we would not be subject to a selection bias when examining these regions for value - related activity . We also investigated activity in several a priori defined areas commonly found to be important in functional mri studies of decision making, bearing in mind that value correlates might not be restricted to regions showing a main effect of task versus baseline . Crucially, we applied exactly the same analysis to the timeseries from the source reconstructed meg data as we had applied to the biophysical model (fig . 1). We found that activity in the right posterior superior parietal lobule (pspl) bore several hallmarks of the biophysical model (fig . 5a). On trials where subjects chose the option with higher subjective value (correct trials), activity in pspl showed a broad correlate of overall value between 3 and 10 hz (p<0.0005, permutation test, cluster corrected for multiple comparisons across time), followed by a lower frequency (24hz) correlate of value difference (fig . 5a(ii)) (p<0.01, corrected), as predicted by the model (cf . 5a(iii)), activity on these correct trials differed from error trials; error trials showed a positive correlate of overall value (dashed black line, fig . 5a(iii)) (p<0.05, corrected), but no such positive correlate of value difference (dashed grey line in fig . 1d) (p>0.5). Finally, we tested an additional model prediction: that across subjects there would be a behavioral speed - accuracy tradeoff, elicited by varying the degree of recurrent excitation in the network model, and that this would predict cross - subject variance in neural data . This prediction was also found to hold in pspl (see supplementary information; supplementary figs . We also investigated whether the main effects of task performance in this region was affected by factors shown behaviorally to modulate reaction time independently of value . We looked for changes in activity in early trials relative to late trials (where reaction time was speeded, fig . 3c), and also compared activity in trials where reward magnitude and probability advocated opposing choices with activity on no brainer trials (where an additional bonus to reaction time was present beyond that explained by overall value or value difference, fig . There was some difference between the patterns of activity in pspl on these trials; an increase in 25 hz power relative to baseline that was present on the first half of trials (fig . 5a(i) top left) was largely absent on the second half of trials (fig . A similar distinction could be seen between activity on trials where reward magnitude and probability advocated opposing choices, and a we also investigated value - related activity in ventromedial prefrontal cortex (vmpfc), focusing our analyses on a subregion that has often been shown to signal value - related metrics during decision tasks . Notably, there has been debate over the precise role of this region in value - guided choice, perhaps triggered by the heterogeneity of responses observed there; in some functional magnetic resonance imaging (fmri) studies it has been found to signal a difference between chosen and unchosen values, whilst in others it has appeared to signal the overall value of available reward, or the value of just the chosen option . In vmpfc, there was an even more striking distinction between those situations where subjects would be more deliberative and exhibit slower rts (fig . Vmpfc recruitment steadily decreased through the task, as could be seen more clearly when trials were further subdivided into separate quartiles of the experiment (supplementary fig . S5). We found that this region transitioned from signaling overall value (p<0.05, corrected) to value difference (p<0.05, corrected) (fig . 5b(ii)(iii)) specifically if we restricted our analysis to the first half of trials in which it was task active (fig . When we directly contrasted the effect of overall value and value difference on early and late trials, we found that only the value difference signal was significantly stronger on earlier trials in this region (supplementary fig . There was not a significant effect of either overall value or value difference on error trials (fig . 5b(iii), dashed lines), although the somewhat weaker signals in this region relative to pspl may result from the relative insensitivity of meg to deep, anterior sources, as opposed to posterior, superficial ones, and from the analysis including only half the number of trials . One possible concern with the differences between the first and second halves of the experiment is that it might reflect more trivial cognitive differences, such as subject fatigue, rather than a change in the cortical networks underlying choice behavior . To address these concerns, we performed an additional whole - brain analysis in which we searched for regions coding more strongly for value difference in the second than in the first half of the experiment - that is, the opposite pattern of activity witnessed in vmpfc . A bilateral portion of the anterolateral intraparietal sulcus - more lateral than the main effect pspl activation described above - selectively reflected value difference in the second half of trials (supplementary fig ., there were also no clear differences between the main effect of task performance on early versus late trials, or on harder trials versus no brainers (supplementary fig . Lastly, we also searched for effects of value in other regions identified in the main effect contrast of task vs. baseline (fig . 4), and in several regions defined a priori from previous fmri studies of value - based choice . In these analyses, we found that several areas exhibited value - dependent activity, but none of these regions matched well with predictions from the biophysical decision model (supplementary fig . We hypothesize that the value correlates in these regions might be better described by appealing to their role in other computational processes that are likely to covary with value, such as attention or response preparation . Alternatively, it may be the case that these other regions are involved in value comparison, but do so in a manner that is different to that proposed using the biophysical modeling approach . We used a mean - field version of a biophysical cortical attractor network model to derive predictions of the temporal dynamics of activity in a cortical region that selects between inputs reflecting the value of two options . The model comprises two populations of excitatory pyramidal cells selective for each option, with strong recurrent excitation between cells of similar selectivity, and effective inhibition between the two pools mediated by inhibitory interneurons (see online methods). This effective inhibition mediates a competition between the two excitatory pools, with one pool ending up in a high firing attractor state (chosen option), and the other pool staying in a low firing attractor state (unchosen option). Neurons selective for option o receive inputs ro at firing rates proportional to the subjective value of that option, sevo . The neurons further receive background noise inputs and currents from other cells in the network . Importantly, the network has very few free parameters that are not otherwise constrained by their biophysical plausibility . The behavior of single units in the network has been described elsewhere; here, we focus on predictions suited to investigation with meg, namely behavior of the summed input currents to all pyramidal cells . We simulated network behavior using a set of trials with varying sev0 (as used in the human experiment, below). The network attracted faster to a decision when overall value or value difference were higher, yielding the prediction of decreased reaction times (rts) under these conditions . We tested this prediction more formally using a multiple regression in which model rts were predicted as a function of both overall value and value difference; both variables were found to have a negative effect on rt (fig 1b). Intuitively, the model reaches an asymmetric attractor state when the basin of attraction for this option is larger due to larger value difference (which determines the difference between the two inputs). An increase in overall value causes the network activity to rise faster and diverge faster, also resulting in faster rt . We then performed a time - frequency analysis of network responses, which aided our subsequent comparison of model predictions with meg data . We used morlet wavelets to decompose network activity on each trial, and regressed the decomposed data onto overall value and value difference . Network transitions typically took several hundred milliseconds to occur, and so most key model predictions were limited to frequencies ranging from approximately 210hz (fig . 1c). Overall value had a broadband effect on model activity in the 39 hz frequency range, soon after selective inputs were delivered to the network (fig . 1c, top), whereas value difference had a later and slightly lower - frequency effect, predominantly in the 24.5 hz range (fig . The different frequencies reflect the fact that overall value affects the population synaptic input earlier and over a shorter time period than value difference . The effect of the two regressors on network responses is a reflection of the fact that network transitions occur at different speeds depending upon the input presented; thus, the network does not explicitly represent such quantities, but these effects are a manifestation of trial - to - trial variability in the speed of the different network transitions . If we collapsed across the relevant frequencies, the temporal progression from an overall value signal to a value difference signal could be clearly seen (fig . It was also found that on trials where the network model made an error (i.e. Sevchosen <sevunchosen), there was an effect of overall value on the model s activity, but no clear effect of value difference (fig . (however, it should be borne in mind that error trials inherently covered a smaller range of value differences than correct trials, which may have caused the absence of any effect). The key predictions derived from the model were therefore: (i) the temporal evolution from an difference value signal; (ii) a difference in the frequency of the response, with value difference dominating responses at lower frequencies than overall value; (iii) the presence of an overall, but little or no difference signal, on error trials . Each option had a certain number of points available, represented by the width of an onscreen bar, and a probability of obtaining those points, represented by a percentage underneath the bar . The aim was to accumulate points (displayed on a progress bar) in order to reach a gold target, at which point monetary reward was delivered and the progress bar was reset to its initial position . To accumulate maximal returns, subjects should compute the objective pascalian value (bar width multiplied by probability of winning, denoted evo for option o), and select the option with the higher value on each trial . In fact, most subjects tended to overweight low probabilities of winning and underweight high probabilities, and exhibited a concave utility function, in accordance with predictions from prospect theory (supplementary fig . Subject rts correlated negatively with both the difference in subjective option values (= sevchosensevunchosen) and with the overall value of the decision (= sev1+sev2), in line with model predictions (fig . 3b shows the group results of a multiple linear regression of value difference (t(29)=7.98,p<0.0005) and overall value (t(29)=2.36,p<0.05) on reaction times across all subjects (see also supplementary fig . We also included some trials in which both reward magnitude and probability were higher on one option than the other . There was an additional bonus in speed beyond that related to value for these no brainer trials (t(29)=8.32, p<0.0005; fig . Subjects were therefore faster on average on these trials than on those where probability and magnitude advocated opposing choices, and so needed to be translated into a common currency in which the two stimulus features could be equated . There was a steady decrease in reaction time as subjects progressed through the task (fig . 3c), without any coincident change in parameters describing choice behavior (supplementary table s2), suggesting subjects became less deliberative and more automated in their choices as they became familiar with the task . We used linearly constrained minimum variance beamforming to spatially filter meg data to locations in source space . We epoched data with respect to both stimulus onset and subject response, and focused our analyses on responses in the 210 hz frequency range, in accordance with model predictions . We first used a whole - brain statistical parametric mapping analysis to look for areas showing a main effect of performing the task relative to a pre - stimulus (300ms to 100ms) or post - response (+ 100ms to + 300ms) baseline . We hypothesized that, in addition to areas important to stimulus valuation such as ventromedial prefrontal cortex, the stimulus - locked analysis would reveal early visual areas involved in basic stimulus processing, and the response - locked analysis would reveal areas involved in visually guided manual movements in parietal and premotor cortices, in addition to primary motor areas . A distributed network of areas was found to be task - sensitive at these frequencies (fig . F; supplementary movie s1, s2). Stimulus - locked, early visual cortex activation (fig . 4a) was followed by slowly ramping bilateral activation at the frontal pole and ventromedial prefrontal cortex (fig . 4b). Whilst 210hz activity in these frontal regions peaked relatively late in the trial (1000ms after stimulus onset), it ramped from a much earlier point in the trial (see fig . 5b and supplementary fig . Response - locked, a prolonged activation spread from a mid - posterior portion of the superior parietal lobule, which extended medially into the marginal ramus of the posterior cingulate sulcus (fig . 4c), to a bilateral medial portion of the mid - intraparietal sulcus (ips) (fig . Having isolated areas that showed changes in activity relative to baseline, we then examined whether activity within these regions co - varied with decision values, and where this activity matched with predictions derived from the biophysical decision model . Importantly, by selecting regions based on the main effect of task versus baseline, we ensured we would not be subject to a selection bias when examining these regions for value - related activity . We also investigated activity in several a priori defined areas commonly found to be important in functional mri studies of decision making, bearing in mind that value correlates might not be restricted to regions showing a main effect of task versus baseline . Crucially, we applied exactly the same analysis to the timeseries from the source reconstructed meg data as we had applied to the biophysical model (fig . 1). We found that activity in the right posterior superior parietal lobule (pspl) bore several hallmarks of the biophysical model (fig . 5a). On trials where subjects chose the option with higher subjective value (correct trials), activity in pspl showed a broad correlate of overall value between 3 and 10 hz (p<0.0005, permutation test, cluster corrected for multiple comparisons across time), followed by a lower frequency (24hz) correlate of value difference (fig . 5a(ii)) (p<0.01, corrected), as predicted by the model (cf . 5a(iii)), activity on these correct trials differed from error trials; error trials showed a positive correlate of overall value (dashed black line, fig . 5a(iii)) (p<0.05, corrected), but no such positive correlate of value difference (dashed grey line in fig . 1d) (p>0.5). Finally, we tested an additional model prediction: that across subjects there would be a behavioral speed - accuracy tradeoff, elicited by varying the degree of recurrent excitation in the network model, and that this would predict cross - subject variance in neural data . This prediction was also found to hold in pspl (see supplementary information; supplementary figs . We also investigated whether the main effects of task performance in this region was affected by factors shown behaviorally to modulate reaction time independently of value . We looked for changes in activity in early trials relative to late trials (where reaction time was speeded, fig . 3c), and also compared activity in trials where reward magnitude and probability advocated opposing choices with activity on no brainer trials (where an additional bonus to reaction time was present beyond that explained by overall value or value difference, fig . There was some difference between the patterns of activity in pspl on these trials; an increase in 25 hz power relative to baseline that was present on the first half of trials (fig . 5a(i) top left) was largely absent on the second half of trials (fig . A similar distinction could be seen between activity on trials where reward magnitude and probability advocated opposing choices, and a we also investigated value - related activity in ventromedial prefrontal cortex (vmpfc), focusing our analyses on a subregion that has often been shown to signal value - related metrics during decision tasks . Notably, there has been debate over the precise role of this region in value - guided choice, perhaps triggered by the heterogeneity of responses observed there; in some functional magnetic resonance imaging (fmri) studies it has been found to signal a difference between chosen and unchosen values, whilst in others it has appeared to signal the overall value of available reward, or the value of just the chosen option . In vmpfc, there was an even more striking distinction between those situations where subjects would be more deliberative and exhibit slower rts (fig . Vmpfc recruitment steadily decreased through the task, as could be seen more clearly when trials were further subdivided into separate quartiles of the experiment (supplementary fig . S5). We found that this region transitioned from signaling overall value (p<0.05, corrected) to value difference (p<0.05, corrected) (fig . 5b(ii)(iii)) specifically if we restricted our analysis to the first half of trials in which it was task active (fig . When we directly contrasted the effect of overall value and value difference on early and late trials, we found that only the value difference signal was significantly stronger on earlier trials in this region (supplementary fig . There was not a significant effect of either overall value or value difference on error trials (fig . 5b(iii), dashed lines), although the somewhat weaker signals in this region relative to pspl may result from the relative insensitivity of meg to deep, anterior sources, as opposed to posterior, superficial ones, and from the analysis including only half the number of trials . One possible concern with the differences between the first and second halves of the experiment is that it might reflect more trivial cognitive differences, such as subject fatigue, rather than a change in the cortical networks underlying choice behavior . To address these concerns, we performed an additional whole - brain analysis in which we searched for regions coding more strongly for value difference in the second than in the first half of the experiment - that is, the opposite pattern of activity witnessed in vmpfc . A bilateral portion of the anterolateral intraparietal sulcus - more lateral than the main effect pspl activation described above - selectively reflected value difference in the second half of trials (supplementary fig ., there were also no clear differences between the main effect of task performance on early versus late trials, or on harder trials versus no brainers (supplementary fig . Lastly, we also searched for effects of value in other regions identified in the main effect contrast of task vs. baseline (fig . 4), and in several regions defined a priori from previous fmri studies of value - based choice . In these analyses, we found that several areas exhibited value - dependent activity, but none of these regions matched well with predictions from the biophysical decision model (supplementary fig . We hypothesize that the value correlates in these regions might be better described by appealing to their role in other computational processes that are likely to covary with value, such as attention or response preparation . Alternatively, it may be the case that these other regions are involved in value comparison, but do so in a manner that is different to that proposed using the biophysical modeling approach . The cortical correlates of value during decision under risk are typically spread over a distributed network of areas, but the unique contribution of each of these areas to choice is unclear . A region involved in value comparison should receive inputs relating to the value of available options, and transform these inputs into a categorical choice . We used a biophysically plausible model that exhibits this property to derive novel predictions of the temporal dynamics of cortical activity . We applied linear regression to investigate at which timepoints and in which frequency bands value correlates could be found in network activity . These responses typically occurred in low frequencies (<10 hz), consistent with a slow integrative process . We then applied the same analysis to source - reconstructed meg data, in order to identify regions involved in value comparison . A distributed network of areas were task - sensitive at the relevant frequencies, but only pspl and vmpfc closely matched predictions of the biophysical model, with the latter doing so selectively in trials early in the experiment . Other regions were found to show value correlates, but did not match closely with predictions from the biophysical model; this suggests that extensions to the model are necessary to fully capture the role of different brain regions in the task . Furthermore, meg will be limited in its ability to resolve sources from deep brain structures that do not possess an open field layout, such as in striatum; thus, the role of alternative mechanisms for selection (dependent upon cortico - basal ganglia loops) cannot be addressed in the current study . A key feature of the biophysical model is the ability to slowly integrate value - related inputs, afforded by its recurrent excitatory structure and long synaptic time constants mediated by nmda receptors . It is not immediately obvious that value comparison should be subject to a process of integration in the same way as a noisy sensory stimulus . However, the observed distribution of reaction times fits well with a process of integration, as has been investigated more closely in previous studies that used a drift diffusion model to predict reaction times . The drift diffusion model was originally designed to make predictions of behavioral data, and has often been used to make predictions of single unit activity during perceptual choice . However, because it essentially describes differences in activity between different populations of selective cells and also ignores any non - selective activity, it is not transparent how to make predictions of imaging measures such as meg or fmri . In this study, we elected to use a biophysical implementation of a competition model, which makes clear and explicit predictions of the measurable data . Unsurprisingly, when we used the pseudo - variable in the ddm as a marker for integrated brain activity, we found differences between the predictions (supplementary information; supplementary fig . The predictions from the model also form a striking example of the distinction between two types of representation - content and functional representations - in cortical circuits . To the external observer, recording with an imaging technique (or an electrode), the content of the network first appears to represent the overall value and later the value difference between the two options . By contrast, the functional representations in the network - those used by the brain - are quite different . There is a representation of option values on the input to the network, and a representation of choice on the outputs of the network, as should be decoded by a suitable downstream observer . The reason that the network shows value - related activity is simply that the same network transitions occur faster on high value and high value difference trials . Hence, whilst neural activity in the network may covary with the overall value and value difference, this content need never be decoded by another brain region . Thus, the extent to which the network can be said to functionally represent these two quantities in a meaningful way is questionable . The region in pspl isolated as matching with model predictions is close to the cytoarchitectonic region hip3, which may be the human homologue of the medial intraparietal area (mip). It is also referred to as ips4 and dipsa, which resembles macaque mip . In the macaque, this region is often implicated in visually - guided movements of the forelimbs . It may therefore have a role in integrating information in order to guide limb movements that is analogous to the role of lip in generating saccades . This process of saccade generation is closely linked to the tracking of value associated with generating a saccade in a particular direction . The region in vmpfc is often found using fmri to be responsive to the value of stimuli during decision tasks, but its precise role has been debated, perhaps as a result of the relative absence of published single - unit recording data in comparison to the nearby lateral orbitofrontal cortex . In early trials, this region strikingly, this same transition was also recently found in single - unit recordings from the most ventral portion of the striatum, which receives a particularly dense projection from vmpfc, and also in prefrontal cortex . Similarly to the present study, this task required monkeys to combine two stimulus properties to form their decision, namely the reward magnitude and the delay to reward delivery . In our task, vmpfc was selectively activated in trials where subjects had to combine probability and magnitude information to choose accurately . This is also consistent with the finding that lesions to this area, but not nearby lateral orbitofrontal cortex, produce impairments in value comparison, and more specifically produce changes in tasks where multiple dimensions have to be considered in forming a choice . Some previous studies have attempted to apply a modeling approach to capture signals from distributed cortical regions during choice, measured using fmri . These studies have made predictions based either on drift diffusion models or biophysically plausible networks but the predictions of these models are heavily dependent upon whether fmri signal is assumed to reflect activity from all timepoints including after a decision has been formed, or whether it only reflects activity until the decision threshold is reached . Moreover, several key predictions of these models also relate to how their activity evolves over time as a decision is made, and the slow hemodynamic response means fmri is limited in how well it can tease apart these predictions of temporal dynamics . We argue that it is important to use a time - resolved technique, such as meg, to test these predictions . Biophysically - inspired models have also been used to infer the structure of connections between or within different cortical areas from m / eeg data . However, these studies have not inferred the specific neuronal mechanism underlying a particular cognitive process, as we have proposed here . The present model performs the critical computation of transforming value - related inputs into a choice, and does so in a way that has captured single - unit activity during perceptual decision tasks . The application of this computational biophysical modeling approach may not be limited to decision making paradigms . Novel predictions might, for instance, be derived from biophysical models already designed to capture single unit data in inhibitory control or working memory processes . In models of working memory, for instance, gamma - band (3070 hz) responses can be elicited, and parametric modulation of input to these models may explain variation in gamma - band frequencies seen during working memory tasks in frontal cortex . Alternatively, by varying internal parameters of a biophysical model, novel predictions might be derived of the effects of cross - subject variation on cortical responses measurable with m / eeg (see also supplementary discussion). Because these parameters relate to specific biophysical properties such as the density of network connectivity or the concentration of a specific neurotransmitter, it may be possible to directly relate these parameters to cross - subject variation in these properties, for instance via local measurements of neurotransmitter concentrations, or perhaps genetic polymorphism or pharmacological challenge.
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Force systems in most orthodontic treatments are considered indeterminate and the magnitude of forces and moments are, in practice, largely unknown . The physiological mechanism primarily responsible for tooth movement in response to a force is the periodontal ligament (pdl). Short - term tooth movement is regarded as primarily governed by pdl deformation because teeth are virtually rigid and are connected to an almost as rigid mandible by the pdl . From a biomaterials perspective, the pdl is a complex, fiber - reinforced substance that responds to force in a viscoelastic and nonlinear manner . The pdl consists of 5374% collagen fibers and 1 - 2% blood vessels and nerve endings that are embedded into an amorphous muccopolysaccharide matrix [6, 7]. Fibrous collagen elements resist tensile forces and the highly hydrated viscous ground substance into which fibrous proteins are embedded forms the extracellular matrix . The ground substance is responsible for the pdl's viscoelastic properties when subject to loading . Also, the pdl's cellular response to mechanical loading results in a metabolic response (remodeling of the ground substance and fibrous tissue). Orthodontic tooth movement is thought to require a minimum of four to eight hours duration of force application and has been shown to be optimal when approximately continuous forces are applied [911]. The pdl's response is characterized by instantaneous displacement, followed by a more gradual (creep) displacement that reaches a maximum after five hours, suggesting that fluid bound within the pdl may play an important role in the transmission, and damping of forces acting on teeth . Pdl steady - state elastic response is usually attained about five hours after the teeth are loaded when the flow of the pdl's interstitial fluid through the surrounding alveolar bone causes the pressure within the pdl to decrease and the solid phase to carry the load alone . Similar results were described in earlier experimental studies in which teeth were loaded laterally [13, 14], and teeth subjected to an intrusive [1517], and/or an extrusive load . Further, the tissue responds rigidly to rapid deformations (mastication) while deforming elastoplastically when subjected to low - grade continuous forces (orthodontic movements). Biomechanical analyses of not only pdl stress - strain responses, but also viscoelastic responses such as hysteresis, creep, and stress - relaxation, help elucidate the tooth support function of the tissue . Therefore, it seems highly probable that viscoelasticity is important for the tooth support function of the pdl, but little is known about the relation between the viscoelastic response and structure of the pdl . Thus, the pdl's mechanical properties are essential parameters for understanding the mechanical behavior of a tooth root and surrounding tissues . These properties are important to orthodontic biomechanics, since its main focus is to understand how forces, which are applied to the tooth crown by means of elastic deformation of metal wires, are transferred to a tooth root and surrounding tissues . Although much is known about the mechanical properties of teeth and alveolar bone, there is no similar definitive knowledge about the pdl . Unfortunately, quantitative experimental data describing the complete behavior of the pdl are unavailable . Determining stress levels in different areas of the pdl, which are the most important and least understood stress - strain levels in orthodontic biomechanics, may offer the best means of correlating the application of force on a tooth with the tooth's response [3, 30]. The objective of this paper is to highlight and discuss the discrepancies that exist in published literature regarding experimentally determined mechanical properties of the pdl . Articles were identified by searches of the following databases: compendex, embase, medline, pubmed, sciencedirect, and scopus, starting from the early 1900s through august 2010 in the english language (citations of publications in other languages were included, but these were sourced from english publications). Finite element analysis, finite element method, mechanical properties, nonlinear elasticity, orthodontic tooth movement, periodontal ligament (pdl), and viscoelastic . To date, there has been considerable research on the pdl given its crucial role in tooth movement and bone remodelling [31, 32]; much of which has been on determining pdl mechanical properties . Histological periodontal tissue changes during experimental tooth movement have been fervently investigated [3335]. The pdl's biological reaction is determined by stress - strain levels induced by mechanical forces applied to the tooth [3640]. Young's (elastic) modulus of the pdl appears to be the most important determinant for instantaneous tooth displacement . However, lack of pdl's mechanical properties consistency throughout the literature is a function of several, often interrelated, parameters . Difficulty lies in directly obtaining pdl data because tooth movement greatly depends on tooth size / shape and, likely as with similar tissues, on strain rate . Differences in pdl material properties and experimental approaches obviate the need for a consistent, standard protocol for testing and modeling the pdl . Elastic modulus was found to range from 0.01 to 1750 mpa, and poisson's ratio ranged from 0.28 to 0.49 . Table 1 summarizes reported pdl material properties (does not include some studies that reference previously reported values)highlighting the variability existing throughout the literature . Experimental approaches for determining pdl material properties are summarized in table 2 . Diversity in experimental approaches is a cause of the wide dispersion of results when investigating pdl behavior, obviating the need for a standardized testing protocol . However, extracting small samples with a regular geometry from a complex biological structure is an arduous task . The results show that regardless of the approach / methodology utilized (i.e., 2d - fem, experimental, 3d - fem, etc . ), there is no conclusive correlation or relationship between the reported material parameters of the pdl and the experimental method that was used to obtain them . In fact, there exists such variability in these parameters that it may be a possibility that each individual pdl (i.e., from every single tooth) has its own distinct biomechanical behaviour much like the uniqueness of a fingerprint . This could potentially be an area, not considered before, that calls for further work and investigation . It may be that a single, all - encompassing, pdl behavioural model is not the appropriate approach in understanding and predicting orthodontic tooth movement . The results, similar to those found in table 1, show that regardless of the methodology (i.e., in vivo or in vitro) numerous varying individual factors may impact the pdl's biomechanical response . Thus, a similar conclusion presents itself a single, all - encompassing, pdl behavioural model may not be the appropriate approach in understanding and predicting orthodontic tooth movement . In addition, for researchers performing further investigation into the pdl's material properties, the results raise an awareness of what may influence their results . The discrepancy and inconsistency of elastic constants for the pdl was evident from the literature . Experimental studies on tooth movement are difficult to interpret because the description of orthodontic forces is not uniform and is incomplete . . Indicated that the stress - strain curves from their experiments showed that the pdl is characterized by time - dependent, nonlinear mechanical behavior with the typical features of collagenous soft tissues . When applying an orthodontic force to a tooth, the generally accepted concept is that bone resorption occurs on the pdl's compressed side, and bone apposition occurs on the tensed side . A widening of the pdl space follows and then a tooth migration towards the compressed side . The external stresses and strains the pdl is subjected to are integral stimuli for alveolar bone remodeling . However, cattaneo et al . Showed that loading of the periodontium cannot be explained in simple terms of compression and tension along loading direction . It was observed that tension in the alveolar bone was far more predominant than compression . Furthermore, meikle indicated that firmly embedded in the orthodontic subconscious was the idea that pressure and tension sites were generated within the pdl, but he believed there were two major conceptual problems with this hypothesis; based on experimental evidence, it was unlikely that pdl principal fibers underwent significant tensile strain, or transferred forces directly to the alveolar bone . Conversely, he supported the idea that tooth movement experiments seemed to corroborate the hypothesis that differential pressures can be generated within the periodontium . . Indicated that reliable values of the pdl's material properties were lacking, specifically noting the significant variance in young's modulus . They also pointed out the dearth of experimental evidence to justify the common assumption of pdl incompressibility, since it is extremely complicated and difficult to conduct accurate and reproducible experiments on thin, soft, and delicate pdl tissue . Tanaka et al . Pointed out that some previous experiments [28, 90] examined the pdl's viscoelastic behavior with quasistatic experimental setups . However, the quasistatic model is only an abstraction as it was found to work reasonably well for soft tissues, but even so, only for certain ranges of stresses, (rates of) strains, and frequencies of oscillations in which the formula (model) does not represent a specific tissue accurately . A quasistatic viscoelastic response corresponds to stress relaxation and creep testing in terms of pdl experimentation . Damping is not modeled effectively and can be problematic when investigating the strain rate effect . Considerable variation in pdl tissue response to tooth movement has been reported, and it results from differences in biomechanical signals and also to specific host differences, such as diurnal rhythms, occlusion, systemic metabolism, age [74, 103, 104], or normal variation in bony trabeculation . Since the pdl is viscoelastic [27, 73, 85, 105108], its properties can vary with the mode of loading and species type . Iwasaki et al . Stated that the speed of orthodontic tooth movement was a function of environment, genotype, and genotype environment . Specific examples include: environment orthodontic treatment, plaque, smoking status, drug use, disease, and diet; genotype simple (single) or complex (multiple) gene interaction(s); genotype - environment ageing, behavior, lifestyle, education, and socioeconomic status . Various authors [16, 85, 86, 111113] have demonstrated the nonlinearity and time dependence of the relationship between load and tooth displacement through in vivo experiments . Other studies [28, 66, 90] described the pdl as nonlinear and segmentspecific (viscoelastic properties could be assumed to differ locally and be dependent on the magnitude and frequency of the loads applied). Genna et al . Pointed out that there was an effect associated with freezing collagen that must be accounted for in pdl models . Recently, bergomi et al . Concluded that (i) the pdl showed pseudo - plastic viscous features for cyclic compressive loading, and (ii) these viscous features essentially resulted from interactions between the porous matrix and the unbound fluid content of the tissue . A summary of the factors that may affect the mechanical properties of the pdl is shown in table 3 . However, a clear separation in the importance and a definitive measure of the degree of influence of these factors was absent from the literature . Most experimental research focused on determining single - factor effects on the pdl (i.e., differences in tooth movement based on the size / shape of the tooth) and, thus, no inference or distinction can be stated with a reasonable degree of confidence on the combined effect of several factors . The degree of importance or level of error of the factors could not be ascertained from the literature . Therefore, possible future research could be to examine the effect that various parameters / factors have on the pdl's biomechanical properties and then quantitatively determine the degree of influence of each factor on the pdl's response . Considering the gamut of data and the sporadic nature of experimental results, taken together with the uniqueness of the pdl's biomechanical properties, it can be concluded that the research focus should be placed on human pdl biomechanics and the impact of individual factors to the material properties . Future research should gravitate towards delineating the relative contributions of each individual factor in order to increase model accuracy and to tailor clinical treatment to be as patient specific as possible . In terms of modeling, it is believed that the most appropriate approach is to use a combined 3d fem and experimental methodology in order to ensure a reasonably accurate fit . As a first step, the research should, however, focus on the development of a preliminary model that allows for a better phenomenological description of pdl behaviour under static, near clinical, orthodontic loading conditions . The gamut of experimental approaches and simplistic / inaccurate assumptions highlight the need for continued research in ascertaining pdl properties under various and well - defined loading conditions, both clinical and experimental . These inconsistent properties underlying the pdl knowledge base are due, in part, to difficulties in examining this thin tissue and variations in experimentation approaches . It was shown that there were significant variations, some on the order of six orders of magnitude, of the elastic constants and mechanical properties of the pdl . A better understanding of the pdl's biomechanical behavior under physiologic and traumatic loading conditions might enhance the understanding of its biologic reaction in health and disease . By providing a greater insight into human dental tissue response, we can help orthodontists improve people's health, appearance, and self - confidence.
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Bloodstream infection (bsi) is a life - threatening condition caused by the presence of microorganisms, generally bacteria or fungi, in the blood . Bacterial bloodstream infection (bbsi), caused by a range of bacteria, can be distinguished as either community acquired or hospital acquired and lead to high morbidity and mortality rates all over the world . Bacterial virulence factors gain access to the blood circulation and are thereafter presumed to cause target organ damage . Culture - based techniques are still of considerable interest for the detection and identification of pathogens causing bbsi . The presence of bacteria and bacterial products in circulating blood has been known for decades . Thus, detection and identification of bacteria based on detection of circulating nucleic acids has been a constant and ongoing challenge . Polymerase chain reactions (pcr) assays which can be done on blood collected in an anticoagulant (edta) tube are highly promising . In the absence of specific clinical certainty, broad - range pcr, using primers targeting the 16s rrna gene, the 23s rrna gene, and the rpob gene, are particularly suitable as they are ubiquitous to all bacteria . In addition, matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof ms) is highly utilized for high - throughput identification of bacteria from agar plates . The current gold standard method of bloodstream microbial detection and identification is the blood culture (bc). The latter is currently based on an automatic and continuous manipulation of liquid culture, followed by gram staining, subculture, and the use of phenotypic methods to identify the bacteria and their associated antibiotic susceptibility . A major disadvantage to culture is the time required to complete the entire described process, which normally ranges from 1 to 5 days or more . Results from traditional bc are usually not available before 24 to 72 hours after the initial patient presentation to the clinic . In resource poor healthcare settings, as bc runs slowly, this can sometimes oblige the physicians to prescribe nonspecific antibiotic treatment to patients necessitating initial use of empirical therapy [7, 8]. Therefore, a quick detection of bacterial infection is one of the most crucial and foreseen functions of most of laboratory bacteriology units . Therefore, the discovery and application of rapid and reliable diagnostics for bbsi would represent a major unattainable need in curing seriously ill patients . In this regard, we conducted a systematic review on currently available molecular diagnostics for bbsi with the main aim of presenting their clinical significance in healthcare settings . Published papers related to our review topic were carefully searched through google scholar and pubmed searching tools . Google scholar has been of our interest because it covers a broad range of scientific papers in different research areas; the same is for pubmed which covers around 26 million of biomedical papers from medline and life science journals . The online paper search was conducted on two different dates (february 12 and 27, 2016). We used the following keywords: bacterial bloodstream infections, bbsis, thus, we considered and reviewed all papers published on clinical significance of molecular diagnostic tools for bbsi . The papers were examined, extracted, and considered based on different inclusion and exclusion criteria we set . All these criteria were applied to paper titles we got for our first time of searching; therefore all chosen papers for our review have met and satisfied the following criteria: being published in english, providing information on development of molecular diagnostic tools for bbsi and their clinical significance, or being published in 2000 to 2016 . However, other retrieved papers were rejected based on the following exclusion criteria: being published before 2000, published in a language other than english, or published as a book . After examining all these aforementioned criteria, papers were considered for review if they were available in full text through pubmed . A complete description of our review strategy is found on the flowchart presented in figure 1 . Analysis of the total 18 papers included in this review was done based on the following aspects: year of publication, research country, sample size, patients' setting, type of diagnostic tool, research design, performance, and findings of the paper (table 1). The first category of the reviewed papers underlined the development and benefits of pcr - based assays . Dark et al ., 2011, reported the usefulness of pcr techniques mostly by using universal probes, followed by sequencing, and highlighted their high sensitivity and specificity . Liesenfeld et al ., 2014, added that the pcr technique seems to be superior to bc given its accuracy of detecting bacteria and fungi . Tennant et al ., 2015, investigated the development of quantitative pcr (q - pcr) and its application in detecting salmonella species . Carrara et al ., 2013, reported that the diagnosis challenges of bsis could be decreased by use of pcr method, especially multiplex pcr, which can improve patient lives . Lecuit and eloit, 2014, advised that blood culture has to be supplemented with nucleic acid - based tests and pcr . Lehmann et al ., 2008, added that pcr - based diagnostic techniques are more accurate in terms of their sensitivity and specificity towards detection of target pathogen . Chang et al . Discussed the available molecular techniques by emphasizing more real - time pcr which is accurate and quick in detecting infection, thus reducing mortality and morbidity . The second category of the reviewed papers compared the diagnostic significance of bc to pcr . Some reviewed publications reported on bc as a good technique in diagnosis of bbsi but also presented its various disadvantages such as long turnaround time, easy contamination, and false negative and positive results [8, 13, 1517, 19]. A total of 12 out of 18 reviewed papers explored the bc's performance and recommended different and improved molecular techniques to detect bsis with a particular emphasis on bacteria . 2011, mentioned the interest in the use of bc but also notified that pcr technology is more crucial as it enables detecting even minute organisms by using short turnaround time with emphasis on bacteria . Jordana - lluch et al ., 2014, confirmed the limitations of bc, mainly centered in biochemical identification, and recommended a need for bc to be replaced by molecular techniques such as pcr . This was also emphasized by jordana - lluch and his coauthors who reported that bc is the gold standard diagnostic tool for bbsi, but as it suffers from low sensitivity, it must be supplemented with no cultivable methods such as pcr . Underscored some pitfalls of bc such as a need for a long turnaround time and risk for contamination and concluded that affording molecular techniques, especially real - time multiplex pcr, would improve diagnosis of bbsi . In the same line of overcoming bc's disadvantages, chang with his colleagues proposed molecular tools other than conventional pcr such as dna microarrays, rna - based fluorescence, in situ hybridization probes, and real - time pcr . Most of the works by faria et al ., 2015, evaluated the illumina sequencing of pcr amplified 16s rdna samples collected from intensive care unit (icu). As part of their findings, they suggested that a molecular approach may enable improved detection of polymicrobial infections . The application of sensitive molecular methods to clinical samples can identify more organisms in samples when compared to bc clinical diagnostics, which is selective for specific organisms . By working on patients' samples from icu using real - time pcr, dark et al ., 2011, revealed a high diagnostic specificity and a 3- to 10-fold higher sensitivity for real - time pcr compared to conventional bc . Jordana - lluch et al ., 2015, compared sensitivity, specificity, and positive and negative predictive values of blood culture to the ones of pcr coupled with electrospray ionization mass spectrometry (pcr / esi - ms) evaluated on clinical samples and concluded that molecular techniques are performing far better than bc . Wallet et al ., 2010, by examining the icu patients' samples using both bc and lightcycler - septifast (lc - sf), obtained the following results: the positivity rate of bcs for bacteremia was 10%, whereas the lc - sf test allowed detection of dna in 15% of cases . The lc - sf performance, based on its clinical relevance, was as follows: sensitivity, 78%; specificity, 99%; positive predictive value, 93%; and negative predictive value, 95% . Management was positively changed for four of eight (50%) of the patients because organisms were detected by the lc - sf test but not by bc . Therefore, their results suggest that the lc - sf test may be a valuable complementary tool in the management of patients with clinically suspected sepsis . Lehmann et al ., 2008, by comparing multiplex real - time pcr identification results with conventional bc for 1,548 clinical isolates, reported an overall specificity of 98.8% for pcr; this specificity is significantly higher than the one of bc . This clearly shows how multiplex real - time pcr holds a promise for more rapid bacterial identification in clinical sepsis . The same multiplex pcr was evaluated by boyd and his coauthors on samples retrieved in the hospital and their results showed that, compared to bc, pcr is more sensitive to bacterial infections . Tennant et al ., 2015, have examined the sensitivity of q - pcr in endemic region of typhoid, and samples were taken from hospitals and healthy volunteers . In the field trials, however, when q - pcr positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity and a specificity of 90% for all comparisons . The q - pcr was significantly faster than blood culture in terms of detection of typhoid and paratyphoid infections . All the 18 reviewed papers emphasize the need of using molecular techniques for the diagnosis of bbsis . Warhurst et al ., 2015, reported that septifast real - time pcr is more rapid in the detection of bsis though it has some limitations that must be handled over time; this was emphasized by wang et al ., 2014, who mentioned that sepsis is one of the main causes of mortality due to therapy delay . To overcome this challenge, molecular technique has to be used for rapid screening of bacteria . Tennant et al ., 2015, investigated the use of gold standard method for diagnosis of enteric fever caused by salmonella typhi or salmonella paratyphi a or b in bone marrow culture . However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead . Among other molecular techniques, 2014, worked on sepsis and considered a race to the death between the pathogens and the host immune system . In order to increase the speed of diagnosis, to improve sensitivity and the clinical benefit of detection of pathogens in the blood, molecular detection techniques for bacterial dna molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients than bc . Here, multiplex real - time pcr - based assay for rapid detection of 25 clinically important pathogens directly from whole blood in less than 6 hours is presented . Lecuit and eloit, 2014, reported that gold standard technique suffers a number of limitations, including the need for a dedicated specialized staff and its intrinsic inefficiency to detect propagated fastidious bacteria such as treponema pallidum and mycobacterium leprae . Bc has been progressively complemented and sometimes replaced by nucleic acid - based tests like pcr or nucleic acid sequence based amplification (nasba). The advantages of pcr are numerous: speed, low cost, automation, sensitivity, and specificity ., 2015, remarked that rapid identification of the etiological agent in bsi is of vital importance for the early administration of the most appropriate antibiotic therapy; thus, molecular methods may offer an advantage to current culture - based microbiological diagnosis . The rapid administration of the most appropriate antimicrobial treatment is of interest for the survival of septic patients; therefore, a rapid method that enables direct diagnosis from analysis of a blood sample without culture is needed . A recently developed platform that couples broad - range pcr amplification of pathogen dna with electrospray ionization mass spectrometry (pcr / esi - ms) identifies any microorganism that might be present in whole clinical blood specimens . The pcr / esi - ms assay presents an advantage over the matrix - assisted laser desorption / ionization time - of - flight (maldi - tof) mass assay as it has been optimized to achieve a rapid diagnosis from direct clinical blood specimens . Pcr / esi - ms is a robust tool that offers an alternative for the diagnosis of bsi as it can be used alone and reliable results are provided following its new version that has been released recently . The same molecular tool shows high specificity with a rate of positivity which is similar to that of bc; therefore, changes in its design would be needed to increase bacterial detection and to develop its automated version in clinical laboratories . 2015, indicated that each delay in antibiotic administration decreases the survival chance of the patient; then rapid diagnostic tools are needed and nucleic acid - based technologies and proteomic approaches are taking part in a more accurate diagnosis of bbsi . The lc - sf test is the first dna based test developed to detect microorganisms directly from blood sample without the need for prior incubation . Such a test has great potential to optimize the management of patients with suspected sepsis . In one paper, authors revealed that the rate of recovery from bacteremia was apparently better with the lc - sf and this has confirmed the ability of this test to improve the life of clinically ill patients . Lightcycler - septifast (lc - sf), which is a real - time multiplex pcr test, can detect 25 common pathogens that cause bsi within few hours; as a matter of fact, lc - sf test can still provide valuable information for identifying the disease . Carrara et al ., 2013, reported that mortality from bsi is related to diagnostic delay and the use of empirical antibiotic therapy; and pcr - based diagnostic assays decrease empirical treatment and improve patient outcome . According to bacconi et al ., 2014, developing a more automated, rapid, and sensitive molecular tool capable of detecting the diverse agents of bbsi at low titers has been challenging but would contribute enormously to the reduction of inappropriate treatment . Globally, bbsis are the most common cause of sepsis and characterized by high mortality rates . Incidence of bbsis is still high in developing countries; for instance, in africa, bbsis have been reported among 10.7% of the children and among 13.9% of the adult patients with severe febrile illness admitted to hospitals . Rapid, accurate diagnosis and treatment of bbsis are crucial for the survival of the patient . Reported a strong relationship between delay in appropriate antibiotic treatment and survival of patients with severe bacteremia . Correct treatment within the first hour was reported to be associated with a survival rate of 79.9% and each hour of delay associated with an average decrease in survival of 7.6% . The spread of antibiotic resistant bacteria is considered to be one of the most important threats to the global public health . In most settings, diagnosis of bbsis is still based on conventional blood culture followed by the identification and antibiotic susceptibility testing of the grown bacteria . However, blood culture shows a sensitivity rate of only 60% and is not only time - consuming, but also laborious . In addition, it possesses serious biosafety risks since the bacteria are grown in vitro for subsequent microbiological analysis . In the last decades, there have been improvements in enriched growth media towards automated blood culture systems such as bactec and bact / alert . This automated system uses software allowing a quicker detection of grown bacteria in culture; and this has significantly decreased contamination rates [28, 29]. In spite of this automated bc system, the technique still remains slow (up to 3 days) and not sensitive enough for accurate diagnosis of bbsis . Molecular diagnostic methods are an interesting alternative to bc since they lead to a sensitive, specific, and rapid (<3 hours) detection of the bacterial genetic materials in blood samples . This technique amplifies a specific region in the bacterial genome to levels sufficient for detection . However, these molecular tools have not been implemented in clinical settings of developing countries yet because they require specific laboratory facilities and skills . In this review, it is clearly noticed that pcr - based techniques increase the sensitivity and specificity in the detection of bbsi . In addition, the use of such molecular techniques in diagnosing bbsi has reduced associated risks such as long turnaround time and false negative and positive results and has contributed to easy identification of fastidious bacteria and prevention of empirical therapy . Reviewed techniques are mainly based on automated dna extraction, pcr set - up, pcr amplification, amplicons purification, and pcr / esi - ms . They overall lead to microbial identification from whole blood in not more than 6 hours . Among other promising molecular tools, fluorescent in situ hybridization (fish) has also the ability to detect pathogen within a shorter time, 2 to 3 hours . Perfect diagnostic technology is able to identify the infecting organism and also the determinants of antibiotic resistance in a timely fashion so that the administration of appropriate therapy could start soon after diagnostic results . The ideal molecular method would analyze a patient's blood sample and provide all the information needed to immediately direct the optimal antimicrobial therapy for bbsi . Therefore, the potential of molecular tools such as real - time pcr technology is to address this problem based on their ability to detect minute amounts of pathogenic dna in patient blood samples and generate results in less than 6 hours of the test . From a theoretical point of view, pcr - based diagnostic techniques hold promise for sensitive and specific detection of target pathogen within a short time . In contrast, for a good and accurate identification of a pathogen in bbsi, several parallel or serial specific pcr analyses or a more universal pcr assay followed by specific probe hybridization or sequencing of the targeted bacteria would bring more promise . The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and exact knowledge about causative microbial agent . For instance, when it comes to targeting bacteremia, the accuracy of the lc - sf is high enough to reach 80% and its specificity reaches 95% . This has also revealed the improved discrimination for the specific bacteremia outcome as compared to multiple target bacteremia . Multiplex pcr has the potential to rapidly identify bsi, compensating for the loss of blood culture sensitivity . For instance, in all italian hospitals, multiplex pcr (the lightcycler - septifast, lc - sf, test) was compared to routine blood culture with samples obtained from 803 patients with suspected sepsis . In this study, excluding results attributable to contaminants, septifast showed a sensitivity of 85.0% and a specificity of 93.5% compared to blood culture; and the rate of positive results was significantly higher with septifast, 14.6%, than blood culture, 10.3% . In a related study conducted in pakistan, real - time pcr was significantly faster at detecting and identifying salmonella typhi or salmonella paratyphi a than classical microbiological techniques; though this technique is more sensitive it has missed some microbes detected by bc . Pcr of bacterial dna seems to be the most sensitive molecular technique nowadays; even though more has to be done to improve its sensitivity, pcr stands to be the future direction tool in bbsi diagnosis . Broad - range assays, with primers targeting variable regions in the16s rrna or 18s/23s rrna gene, present clinical applicability for diagnosis of bbsi due to their short turnaround time and ability to directly detect any noncultivable or cultivable pathogens in patients' blood sample . For instance, liesenfeld et al ., 2014, described that fluorescent in situ hybridization (fish) is among the available molecular techniques and has ability to detect pathogen within 2 - 3 h; another technique is based on chemiluminescent dna probes (rrna) and works like normal pcr assays . Furthermore, another novel approach in molecular diagnosis of bbsis, 16s metagenomics, has been recently developed . 16s metagenomics consists in parallel sequencing of the bacterial 16s ribosomal rna (rrna) gene using next generation sequencing (ngs) technologies . The same technique has shown a superior sensitivity compared to standard blood culture during its proof - of - concept which was conducted in 75 children with severe febrile illness in burkina faso . The use of microarrays and biomarkers has been also exploited and investigated for their inclusion in the diagnostic package of bbsi . Prove - it consists in multiplex pcr in combination with microarray and can detect 60 bacterial pathogens in a positive culture sample . It mainly detects two antibiotic resistant genes, meca and vana / b, with a total assay time of 3.5 hours . Verigene, a bacterial nucleic acid - based microarray assay, can detect meca and vana / b resistant genes in addition to 13 gram - positive bacteria in a total assay time of 2.5 hours [12, 32]. Next to microarray - based tools, there is an emergence of various assays targeting biomarkers . Most of these techniques target endotoxins; acute - phase protein biomarkers such as c - reactive protein (crp), lipopolysaccharide - binding protein (lbp), procalcitonin (pct), pentraxin, serum amyloid a, ceruloplasmin, and alpha 1 acid glycoprotein; cytokines and chemokines; coagulation biomarkers; soluble receptor and cell surfaces . The detection of endotoxins produced by gram - negative bacteria that might be circulating in the blood of septic patients was reported to be inhibited by other various products such as fungal cell wall components and plasma proteins [33, 34]. The crp, usually released by liver upon inflammation during infection, has been exploited in sepsis diagnosis, especially when it comes to the assessment of the occurrence of bbsi . Lipopolysaccharide - binding protein (lbp), as an acute - phase reactant binding to the lipopolysaccharide of gram - negative bacteria, levels increase during the acute - phase stage up to 200 g / ml; thus, it is a good marker for the severity or outcome of the infection . However, this biomarker is not recommended for use in clinical settings as it failed to distinguish between gram - negative and gram - positive bacteremia . The pct is the mostly used protein marker in most of the parts of the world; it has the potential to distinguish between sepsis and system inflammatory respiratory syndrome (sirs) and can determine the bacterial load or also guide the antibiotic therapy in icu . Pct is produced in response to bacterial endotoxin or immune mediators such as interleukin-1, tumor necrosis factor-, and interleukin-6 . Pentraxin, a superfamily of immune system proteins, is still being investigated on its potential to differentiate among sepsis, septic shock, and sirs . For the rest of other acute - phase proteins, amyloid a, ceruloplasmin, alpha 1 acid glycoprotein, and hepcidin are reported to be elevated in septic patients . The secretion of cytokines is simultaneously done in both proinflammatory and anti - inflammatory forms from the initial stage of infection; the rate of cytokines is higher in septic patients compared to nonseptic ones [40, 41]. However, cytokines present a limited usefulness as sepsis biomarkers because they can sometimes be linked to other noninfectious diseases as well . Chemokines such as macrophage migration inhibitory factor (mif) and high mobility - group box 1 provide value in the assessment of the immunological response . However, they also fail to distinguish between infectious and noninfectious systemic inflammation . Among other markers, we can mention the triggering receptor expressed on myeloid cells 1 (strem-1), soluble urokinase - type plasminogen activator (supar), proadrenomedullin (proadm), and polymorphonuclear cd64 index . They are all promising markers for the diagnosis and prognosis of septic patients [43, 44]., the use of combinatorial biomarkers could definitely lead to an improved diagnostic power and follow - up of septic patients . Following the results of this systematic review, we believe that next generation molecular tools constitute a new and powerful approach that could identify main species causing bbsis and detect their respective genetic markers responsible for antibiotic resistance . Surveillance studies at all health system levels are important to know the causative agents of bbsi and devise appropriate interventions to control the spread of antibiotic resistance and guide physicians in deciding which adequate antibiotics to prescribe . In conclusion, it is noteworthy that molecular techniques are now emerging as another promising option for diagnosis of bbsi . In this review, pcr - based assays were highly reported to have significantly changed diagnostics of bbsi by increasing a bit sensitivity, specificity, and test accuracy overall . These techniques are generally good as they yield much better and reliable results in much shorter time than bc . In countries where trials have been conducted, reports have emphasized the accuracy of test results leading to a timely and right antibiotic administration . Although the cost of some of the newly developed techniques is still comparably high to be used in some poor endemic settings, we hope to get cheap, accurate, and fast methods requiring low training soon . This will be achieved through the advances in genomics, metagenomics, transcriptomics, metatranscriptomics, and proteomics together with much collaboration in international health services . Thus, the use of these sophisticated tools will soon shift from research settings and developed world to clinical settings and developing world . This will obviously tackle the challenge of usual delay in test results deliverance when using conventional bc . We are all convinced that elaboration of a quick and affordable tool for detecting bacterial pathogens in patients' blood sample is of great interest in global public health.
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The provision of antiretroviral therapy (art) in sub - saharan africa (ssa) has resulted in reduced mortality, morbidity, and an increase in life expectancy.1,2 despite these successes, approximately 59% of all people in africa remain without treatment despite being considered eligible.3 also, for those in care, improvements are required along every step of the care cascade to improve health outcomes for both individuals and the population.4 the first entry point to care is hiv testing, which is often accessed through provider - initiated testing at the health center . In many settings, provider - initiated testing has favored women and children, usually in the antenatal care (anc) period.5,6 there has been recent growing recognition that men are disadvantaged because no equivalent routine testing opportunity exists for them,7 and this is considered at least partly to account for the fact that men initiate art later than women,8 and have worse outcomes on antiretroviral therapy compared to women.913 in this study, we aimed to determine mortality between sexes after initiation of art in the greater kampala area of uganda . We further sought to determine whether differences in disease progression across the sexes could be explained by differential access to care via anc services, and estimated how much of the increased risk of mortality in men can be explained by later art initiation . All patient data for this study is from the mildmay uganda observational cohort (mug).14 mildmay is a medical service organization providing art and other healthcare services to the hiv - positive community in uganda . The organization opened in 1998 in order to provide palliative outpatient care for people living with hiv / aids, and to be a teaching and training center for hiv / aids healthcare personnel . This study was restricted to patients who initiated art between january 2004 and april 2011, were aged 14 years or older at baseline, and had completed the selection form . Selection forms contain baseline data including whether a woman was pregnant or eligible for art for prevention of mother to child transmission (pmtct). To the best of our knowledge, selection forms were missing completely at random (ie events leading to missing selection forms are independent of variables of interest). The primary outcome was all - cause mortality throughout the study period that ended in october 2011 to ensure that patients had the possibility of at least 6 months of follow - up time . Time was measured beginning at first art date and ending at either date of death, 6 months after last visit, or at end of study period, respectively . Follow - up time beyond the last visit is justified by the fact that death is externally observed . The principal explanatory variables of interest were sex, baseline cd4-cell counts and world health organization disease stage . Baseline cd4 cell counts were obtained within 6 months before starting art . During the observation period, the ugandan national guidelines stipulated that patients were considered eligible for art if they had a cd4 count 200 cells / mm (this was changed to 350 cells / mm in 2012). We also measured demographic (age, marital status, and education), behavioral (sexual activity, disclosure to partner, and testing), and clinical variables (hepatitis b and c, syphilis, malaria, and anemia). Women were deemed to be in antenatal care if they reported either being pregnant at baseline or being pmtct eligible . To determine the existence of any important differences between men and women with respect to demographic, behavioral, and clinical variables in the full cohort, we used fisher s exact and wilcoxon rank sum tests . Logistic regression and confidence intervals for difference in means were used to estimate the differences in disease progression, as defined by who disease stage and baseline cd4 cell counts, in men and women . Meier survival curves to demonstrate mortality over time in both the full and restricted cohorts . We applied a weighted analysis whereby 30% of patients lost to follow - up were assumed to be dead, weighted by baseline cd4 and age, as suggested by egger et al.15 mediational modeling was conducted to estimate the proportion of increased risk of mortality in men attributable to later art initiation16 using recently developed methods for survival analysis.1719 mediator variables are distinguished from confounders in that they lie on the causal pathway . The effect of the initial variable (gender) summed across all pathways is its total effect . As shown in figure 1, the total effect can be partitioned into the direct effect and the indirect effect . As with any model, the model assumptions must be met and, particularly for models considering causal pathways, it is important that confounding be fully controlled . The ratio of indirect to total effects is the proportion of the effect attributable to the mediator . In this analysis we considered age as a confounder, gender as the initial variable, baseline cd4 as the mediator, and survival as the outcome . We opted for the product method16 of estimating the indirect effect which required the construction of a linear regression model for baseline cd4 and an accelerated failure time model with a weibull distribution using gender, baseline cd4 cell count, and age . Full details on the indirect effect estimation and the assumptions required for this model are available in the appendix . Confidence intervals for the indirect and total effects as well as their quotient (the proportion of effect explained by cd4) were obtained by bootstrapping.1922 all analyses were done in sas version 9.3 (sas institute inc, cary, nc, usa) and r version 2.15 (vienna, austria). This study received ethical approval from the institutional review boards of the university of ottawa and mildmay uganda . All patient data for this study is from the mildmay uganda observational cohort (mug).14 mildmay is a medical service organization providing art and other healthcare services to the hiv - positive community in uganda . The organization opened in 1998 in order to provide palliative outpatient care for people living with hiv / aids, and to be a teaching and training center for hiv / aids healthcare personnel . This study was restricted to patients who initiated art between january 2004 and april 2011, were aged 14 years or older at baseline, and had completed the selection form . Selection forms contain baseline data including whether a woman was pregnant or eligible for art for prevention of mother to child transmission (pmtct). To the best of our knowledge, selection forms were missing completely at random (ie events leading to missing selection forms are independent of variables of interest). The primary outcome was all - cause mortality throughout the study period that ended in october 2011 to ensure that patients had the possibility of at least 6 months of follow - up time . Time was measured beginning at first art date and ending at either date of death, 6 months after last visit, or at end of study period, respectively . Follow - up time beyond the last visit is justified by the fact that death is externally observed . The principal explanatory variables of interest were sex, baseline cd4-cell counts and world health organization disease stage . Baseline cd4 cell counts were obtained within 6 months before starting art . During the observation period, the ugandan national guidelines stipulated that patients were considered eligible for art if they had a cd4 count 200 cells / mm (this was changed to 350 cells / mm in 2012). We also measured demographic (age, marital status, and education), behavioral (sexual activity, disclosure to partner, and testing), and clinical variables (hepatitis b and c, syphilis, malaria, and anemia). Women were deemed to be in antenatal care if they reported either being pregnant at baseline or being pmtct eligible . To determine the existence of any important differences between men and women with respect to demographic, behavioral, and clinical variables in the full cohort, we used fisher s exact and wilcoxon rank sum tests . Logistic regression and confidence intervals for difference in means were used to estimate the differences in disease progression, as defined by who disease stage and baseline cd4 cell counts, in men and women . Meier survival curves to demonstrate mortality over time in both the full and restricted cohorts . We applied a weighted analysis whereby 30% of patients lost to follow - up were assumed to be dead, weighted by baseline cd4 and age, as suggested by egger et al.15 mediational modeling was conducted to estimate the proportion of increased risk of mortality in men attributable to later art initiation16 using recently developed methods for survival analysis.1719 mediator variables are distinguished from confounders in that they lie on the causal pathway . The effect of the initial variable (gender) summed across all pathways is its total effect . As shown in figure 1, the total effect can be partitioned into the direct effect and the indirect effect . As with any model, the model assumptions must be met and, particularly for models considering causal pathways, it is important that confounding be fully controlled . The ratio of indirect to total effects is the proportion of the effect attributable to the mediator . In this analysis we considered age as a confounder, gender as the initial variable, baseline cd4 as the mediator, and survival as the outcome . We opted for the product method16 of estimating the indirect effect which required the construction of a linear regression model for baseline cd4 and an accelerated failure time model with a weibull distribution using gender, baseline cd4 cell count, and age . Full details on the indirect effect estimation and the assumptions required for this model are available in the appendix . Confidence intervals for the indirect and total effects as well as their quotient (the proportion of effect explained by cd4) were obtained by bootstrapping.1922 all analyses were done in sas version 9.3 (sas institute inc, cary, nc, usa) and r version 2.15 (vienna, austria). This study received ethical approval from the institutional review boards of the university of ottawa and mildmay uganda . In total, 4775 patients aged 14 years or older initiated art between january 2004 and april 2011 . The median age was 33 (interquartile range [iqr] 2739), and 1676 (35.1%) were men . During this time period there were 200 deaths over a median follow - up time of 3.25 years (iqr 2.504.25); 135 patients were lost to follow - up (42 males and 93 females). Men tended to be older, were more likely to be married and sexually active, tended to be better educated, more likely to disclose their status to their partners, and more likely to be hepatitis b positive (odds ratio [or]: 1.42, 95% confidence interval [ci]: 1.10 1.85). There were 84/1676 (5.0%) deaths among men and 116/3099 (3.7%) among women . When analyzing all patients, the p - value for the log - rank test was 0.02 indicating a higher rate of death among men (figure 2, panel a). When we restricted this to the cohort involving only women who accessed care outside anc services, the p - value for the log - rank test was similar at 0.04 (figure 2, panel b). Similarly, 42% of men compared to 32% of women presented for art at who stage 3 or 4 (or: 1.46; 95% ci: 1.291.66) and this did not change much when anc cases were removed (or: 1.54; 95% ci: 1.371.75). As shown in table 2, the difference in cd4 cell count was only slightly larger, 23 versus 25, when anc cases were included . The weibull survival model shows that in univariate analysis, gender has a significant effect on survival (hazard ratio [hr]: 1.38; 95% ci: 1.031.83), but in the presence of the mediators and confounders its effect is no longer significant (adjusted hazard ratio: 1.20; 95% ci: 0.951.64; table 3). The scale coefficient of 1.87 indicates that the hazard of death is declining with time, in keeping with the fact that the majority of the deaths occur within months of initiating art.10 table 4 presents the partitioning of the effect of gender into direct and indirect effects . Approximately 43% (95% ci: 22%113%) of the additional deaths seen in men are attributable to their lower cd4 cell counts at baseline (results for reduced cohort shown in table a2). A sensitivity analysis restricted to the sample of those not accessing care through anc, is presented in table a3 in the appendix . When we examined whether single men had differing baseline risk, according to baseline cd4, compared with other groups, we found that single men had significantly lower cd4 (median 99, iqr 30190) than married men (120, iqr 48199) [p = 0.03] and compared to single women (144, iqr 68209) [p <0.0001]. Although the international hiv / aids community has placed a particular emphasis on targeted art of female patients, there is emerging evidence that less men engage in care for hiv / aids in ssa, and have less favorable outcomes . A proposed reason for this inconsistency is that antenatal care provides a platform through which women can access care more frequently and earlier in their disease progression.23 our study found that men had an increased risk of death and nearly half the additional deaths appear to be attributable to delayed engagement in care . In the multivariable model, we divided the effect of gender into the direct effect and the indirect effect (effect due to cd4 at baseline). Therefore, the fact that gender is not significant in the multivariable model, implies that the increased risk observed in men is not due to their gender, but mostly due to their lower baseline cd4 cell counts . Anc accounted for only a small fraction of this inconsistency . A sustained effort to reduce this discrepancy our study had low rates of death and low rates of loss to follow - up . The mildmay program puts emphasis on community care that may explain the high retention to care, an observation mirrored in other community - based care programs.24 although there is accumulating evidence that men across ssa are at increased risk of morbidity and mortality in comparison to women,25 our study was restricted to a single cohort in uganda . Our bivariate analyses reveal that our cohort has a relatively higher number of married men, suggesting that the engagement of women has led them to bring their husbands into care . Consequently the differences in disease progression reported in this cohort may underestimate the discrepancy in the community . The role of antenatal care in the discrepancy between how men and women seek access to healthcare is often acknowledged, but seldom analyzed in detail . Our study clearly shows the existence of other, more important factors at the source of the divide . It is well known that initiating care at a lower cd4 cell count increases the risk of mortality.26 therefore it is not surprising that our results show that the increased mortality in men can in part be explained by later initiation . Nonetheless, by quantifying the proportion of increased mortality attributable to later initiation we have highlighted the urgency of increasing engagement to care . Men are seldom targeted because they do not fall into our conventional definitions of vulnerability and marginality.27 on the contrary, in general, men are recognized for having poor health - seeking behavior, so that efforts to engage them are even more critical ., the ministry of health has initiated men s health days.28 studies have shown that home - based testing programs are allowing men to test earlier, giving them a chance to engage in a timely fashion.29 encouraging men to accompany women in antenatal care, as recommended by who in uganda, has had some success in rwanda and may have a role in other settings,30 although this would only target couples with active relationships . The implementation of programs to encourage further participation by men in care is a difficult task, but earlier recruitment into care remains the best method to align the health outcomes of men with those of women . A growing number of studies are reporting that among hiv - positive individuals on art in ssa, men tend to be at higher risk of death as was the case in our cohort . Little has been done to determine the underlying causes that are crucial to making changes to the health systems that create them . Our study found that among the leading causes, and quite possibly the leading cause, is the lower cd4 cell counts at which men on average present themselves for art initiation . Targeted campaigns need to continue to be evaluated, to reduce the gender disparity and improve testing, linkage to care, and ultimately health outcomes for men . This study can contribute to the growing body of evidence designed to inform evidence - based policy, to reduce barriers to healthcare experienced by men . Finally, antenatal care programs are a key difference in how men and women access care and are often cited as a plausible cause for observed differences in health outcomes, yet this difference did not appear to have a large impact in our cohort . Our findings suggest that further investigation is needed as access to care through antenatal care may play a more muted role than believed in explaining the differences in art uptake between men and women . Data were acquired by sk, mn, dm, mo, mk, wm, sy and af . The analyses were conducted by sk, af, and ejm . The canadian institutes of health research (cihr) provided funding for this analysis . No funding agency has seen this study.
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For the first half of the twentieth century the clandestine abortion industry seems to have been tacitly accepted by south african law enforcement officials . Generally abortionists, medical and untrained alike, were prosecuted only in the aftermath of a woman s death when the police could no longer look the other way . Similar situations had existed elsewhere; in the united states (usa), for example, during the first half of the twentieth century an unwritten agreement existed that there was no prosecution unless there was a death. The impression that criminal abortion was tolerated by the police is reinforced by watts who says he performed abortions on the girlfriends of police officers (see below), as well as by events surrounding an equally sensational abortion trial that occurred twenty years prior to the crichton dr gerhardus buchner, dr edward blumberg and a nurse named susanna pieterse were arrested for providing abortions in an extremely busy and lucrative abortion clinic located at castle mansions in eloff street in downtown johannesburg . Buchner, a south african doctor who received his medical degree at the sorbonne, was in charge of the clinic that had been open since at least 1942 . The clinic s services were in high demand; white women came from all four provinces as well as from outside south africa . A nurse who once worked for buchner testified that she looked after up to nine women at a time at the clinic . The clinic operated discreetly and the need for secrecy meant that abortions performed at night were lit only by flash lights because it was feared electric lights would attract unwanted attention . Yet the johannesburg police knew about buchner s clinic and for years tolerated its existence, probably because of bribes . In fact it was an official complaint made to police in pretoria, located 60 km (37 miles) from johannesburg, by a man determined to have the clinic exposed that spurred the investigation . During the trial the attorney - general for the transvaal, f.e . Lutge, accused the police of colluding in buchner s gigantic practice because for years he had operated on the main thoroughfare of johannesburg apparently abortionist in an official report by police, and, in 1946, an afrikaans newspaper, the weekblad, published a story about his practice in an article entitled aborsie in johannesburg (abortion in johannesburg). Lutge said police officials had even wined and dined at buchner s premises in castle mansions . The lack of official action led the attorney - general to surmise the police must have connived at the practice of abortion in castle mansions, and the case of this well - known clinic suggests that police for decades must have turned a blind eye to clandestine abortion elsewhere . After the convictions of buchner, blumberg and pieterse the clandestine abortion industry once again receded to the shadows until the late 1960s when the changed cultural climate made abortion a highly visible and important political issue, to the degree that the government found it expedient to destroy the career of another medical abortionist, dr derk crichton . Derk crichton qualified as a doctor in 1944 at the university of cape town (uct), where his father e.c . Crichton held the chair in the department of obstetrics and gynaecology for many years . He earned a first class in surgery at uct, obtained a doctor of philosophy in radiological gynaecology and obstetrics in 1952 from oxford, and became in 1954 one of the first clinical professors at the medical school at the university of natal in durban . At the age of 32 he took up the position of chair of the university of natal s department of gynaecology and obstetrics . At the time of his arrest in 1972 crichton was managing the gynaecology and obstetrics ward located at the king edward viii hospital, built in 1936 and the teaching hospital of the university s medical school for non - europeans. The hospital managed the delivery of 23 000 african babies a year and, with 200 beds, was at the time the third largest such ward in the world . He became a fellow of the royal college of surgeons of edinburgh in 1953 and a fellow of the royal college of obstetricians and gynaecologists in 1957 . He was awarded the blair - bell lectureship by the latter in 1965 for outstanding research and in 1967 won the hamilton - maynard medal for the best south african medical publication of the year, an article describing his method of repairing ureteric fistulae . In 1969 he became the external examiner for the royal college of obstetricians and gynaecologists; this was in addition to the english - speaking south african universities and the university of salisbury in rhodesia (zimbabwe). From 1967 until he left south africa in 1974 he performed all sex - realignment operations (male to female) in natal . At the time of his arrest in 1972 he was president of the regional council of the royal college of gynaecologists and obstetricians in south africa . As an indian patient embarking on the process of sex reassignment said about crichton, he had an unshakeable confidence in what he was doing which seemed to boost the spirits of those he came in contact with. An indian doctor, formerly a student of crichton s, recalled, derk crichton was like a god to us, we would queue for his lectures which for us were like the ten commandments . He was so sure of his medicine that if he told us to treat a patient a certain way, regardless of what the textbooks said, we did it . He was also infamous for his temper and inability to suffer fools. Bongiwe bolani, who worked as a nurse at king edward viii hospital in the mid-1960s, remembers crichton as arrogant and terrifying: he was a horrible man . He screamed, he shouted, he humiliated us, made you feel like imbeciles . Other people, older white males, terrified me from that time for many years . One of crichton s former students is dr sam mokgokong, south africa s first african gynaecologist and instructor in a medical school, and he recalls crichton with huge affection, saying he s like a father to me, he took me up, he made me. He says crichton was colour - blind and even - handed with his students, meaning he treated his white and black students the same . Even bolani recalls crichton urging black nurses to obtain further training so they could be promoted to higher positions . She took up this educational challenge and eventually became a matron . At both addington hospital (for whites and coloureds in segregated wards) and king edward viii hospital (for africans), as well as in private practice, crichton was constantly confronted with the effects of clandestine abortion, telling an audience in 1970, were i a member of the clergy i might say that abortion is my i know more about criminal abortions in durban than anyone else it would be extraordinary if i didnt. He estimated that during his eighteen years at king edward viii and mccord zulu hospital between 1954 and 1972 he had treated an astounding 40 000 african women suffering the effects of illegal abortion at least one died every month . Some girls and women he treated had procured abortions on themselves but most had gone to untrained abortionists, the vast majority of which were what he called soap injectors because they injected liquid soap into women s uteri, a dangerous method that often caused infection or death as a result of air or soap entering the blood stream . The method was popular with clandestine abortionists because it was relatively easy and could be employed in one visit . He estimated the soap - injection method killed one in every 200 women and left another 5% sterile . In court in november 1972 he said that even in the last two or three days, we ve had to do two hysterectomies for gangrenous uteri as a result of soap injections. Complications from abortion were the number one issue facing his unit: in the first eight months of 1973, 934 cases of complicated abortion were admitted to king edward viii, of which 354 had been septic . He said, we can only admit the hard core of complicated abortion cases because of restricted bed space. As a consequence of this experience crichton concluded that the law on abortion was unrealistic and outmoded . In july 1970 he gave a speech on abortion at the university of natal where he declared: [t]he struggle for emancipation from this bondage (of legal and religious condemnation), to have a fertility franchise for women constitutes one of the major challenges of our time . My crusade for this goal is inspired not only ideologically, but also as a result of my personal contact with the human tragedies which result from our present system, especially the consequences of illegal abortion . To crichton this problem assumes proportions which render a fight for its eradication an inescapable duty. True to form, therefore, he did not keep his opinion to himself . Beginning in the late 1960s he began calling for law reform and very quickly became the country s most visible and outspoken medical advocate of abortion on demand, telling an audience in 1970 we must remain dauntless that there can be no compromise in this struggle to achieve the ultimate ideal that every woman should be the captain of her womb. A radical in a conservative profession, he became a self - described just a few months before his arrest crichton called for abortion law reform at the annual meeting of the south african society of obstetricians and gynaecologists (sasog). James watts, aged 41 at the time of his arrest in 1972, was a complex man with a vastly different background from crichton s . Born in cape town in 1931, watts grew up on a farm in kraaifontein . His mother had once been a schoolteacher in namibia but was a stay - at - home mother when he grew up; his father had lost his business making refrigerator cabinets during the depression after which his parents managed a convenience store on their farm . Watts had eight years of public schooling and moved to durban in 1953 where he worked for five years as a fire fighter on the railways . In 1961 watts says that while working on the railways a co - worker urged him to do more with his life . He said to me you must educate yourself, read reader s digest from front to back. An extremely intelligent man, watts would undoubtedly have been a professional of some kind, probably a doctor or a veterinarian, if he had been able to pursue an education . His motivation for becoming an abortionist appears very similar to that of other excellent clandestine abortionists, such as the american ruth barnett who had an incredibly successful and lucrative abortion clinic in oregon from 1918 to 1968 . Barnett s biographer rickie solinger, writes that her restlessness, her intellect, and her belief in her own capabilities fuelled her desire to become a first - rate abortionist . Watts was an independent thinker and seems to have been instinctively subversive (though never interested in politics), and his contempt for apartheid morality combined with his sharp intellect, fascination with biology, and ambitiousness made it easy for him to live a double life as an employee of shell and a criminal abortionist . A friend whose girlfriend was pregnant was looking for an abortionist and watts told him he should just do the abortion himself . I did it myself. He studied as much as he could beforehand, looking for relevant medical literature in the library, bookstalls and at cna (a south african chain of bookstores), and claims there were mountains of literature available, there have been mountains of articles and books on abortion dating back to plato. After performing his first abortion his friend s sister came to him for the procedure . After that it became easier and thus began his career as an abortionist . Watts became a highly skilled and meticulous abortionist, which is why crichton referred the teenagers and young women needing abortions to him (see below); during his trial doctors said they were i was very sterile conscious, he said, knowing there was the possibility of infection: while growing up in the cape he knew abortionists who used the syringe method, mostly retired white nurses, and girls who became ill afterwards . He said that, like his clients, he was always nervous when performing abortions because he was intensely aware of what could go wrong and knew there was always the possibility that he could be arrested . Determined to avoid medical and legal trouble, from the beginning he only used the far safer method of inserting sterilised catheters . He used urinary catheters, bought from a contact who supplied pharmacies, and always shaved girls anywhere where hair could touch the catheter to prevent infection, or else told the girls to shave themselves. He had his own supply of antibiotics for patients who could not obtain any (obtained from the same contact), sterilised his equipment beforehand, and sealed off catheters after insertion with sterile cotton wool dipped in hibitane (a disinfectant). He also told girls to obtain a medical follow - up, instructing them to wait until there was significant bleeding (so that the miscarriage could not be stopped) and then go to a doctor for a dilation and curettage (d&c) to ensure the abortion was complete and avoid infection . Watts cut - off point for performing abortions was four months of pregnancy because after that abortions are more dangerous. Watts is an intriguing figure . He was a self - taught abortionist wholly unlike the stereotype of the unscrupulous and dangerous operator often invoked by doctors and liberal feminists furious about the horrific consequences of criminalising abortion, a negative image reinforced by the occasional prosecution of untrained abortionists, usually women, after the deaths of their clients led to discovery . The abortion business for money and added that he saw no difference in that regard between himself and trained medical doctors like crichton . Indeed, watts took pride in his professionalism and considered himself an ethical, non - judgmental practitioner; even crichton referred to watts patients at one point during the trial (see below). As watts told the court, what motivated me was to protect girls i have helped from going to other abortionists that use syringes and knitting needles as i have often seen terrible consequences from their work . I knew somebody had to help them. He says he performed abortions to help good girls who were in trouble, he said, bad girls knew how to prevent pregnancy . (watts himself was a father; at the time of his arrest he was married with two sons aged 13 and 15 .) Watts performed abortions on all kinds of women. Most clients were white, about 60% afrikaans and 40% english . Although he did occasionally do abortions on indian and coloured girls pregnancy knew no colour barrier, they were all girls in need segregation under apartheid and his acceptance of it ensured he mainly came into contact with other whites . His clients generally paid with cash, although he accepted cheques, and he consulted his own version of a sliding scale that reflected his moral worldview when charging fees . For most girls and women he charged the same as crichton, i.e. Usually between r60 and r80, which he considered fair because in addition to being an expert who deserved to make the same wage as a doctor he was operating entirely outside the law . He says he charged nurses and prostitutes less and sometimes nothing at all because as working women their jobs were at stake . He also charged less, if anything, to help police officers girlfriends, undoubtedly because it was in his interest to have friendly relations with police . Watts reputation reached women and doctors beyond durban, and word - of - mouth, as was so commonly the case with abortionists willing to break the law, was the means by which information about him circulated . He says himself, the more i did, the more my reputation spread by word of mouth, and several doctors, some as far away as cape town, referred some patients in need to me. The number of clients varied from a few per month to a few per week; requests often came after holidays . One teenager learned of watts from a woman she met at the local movie theatre . Another client, from east london, said a friend - of - a - friend from durban told her about him . He also had a contact in south west africa (namibia) who flew in a few girls . One was an afrikaans girl whose father was a preacher; she said her dad would kill her if he found out she was pregnant . She threatened to kill herself by jumping off a building if i did nt help her so i did not have much choice. He never asked clients for personal information and kept no records but recalls a host of dramatic stories . One afrikaans woman flew up from cape town and we met in a car park as arranged and she was wearing a veil . I never interrogated or questioned their identity [sic], i did not wish to embarrass them . I could only speculate, but (she was) definitely upper bracket. He reports that sometimes it was more affordable or convenient for women to bring him to them . He was flown to cape town and johannesburg by clients and went twice to kruger national park to perform abortions on women ostensibly on holiday . There was no feminist movement in south africa in the 1960s and watts had no contacts outside south africa . However, evidence given by teenagers and young women conveys the image of a diligent and sympathetic practitioner . For example, one teenager testified that she came to durban alone for an abortion and watts met her at her hotel to perform the procedure . However, the hotel did not have hot water, so he said, oh well then he would have to take me to his house as we had nowhere else to go, and drove her to his home where he sterilised his equipment, took her to his bedroom and inserted the catheter . Afterwards his wife gave her a cup of tea before he drove her back to her hotel . The next day he picked her up and drove her to a clinic for a d&c where he did all the talking for me. After her procedure she took a taxi to the hotel where she was met by watts who stopped by to see how i was, just as i arrived back . He came upstairs with me to my bedroom, and he had a drink, and he stayed for a little while and then he left. He would commonly allow girls to stay overnight at his downtown flat purchased for his practice while they waited to miscarry and sometimes had drinks with the girls and/or their boyfriends after inserting the catheter . One boyfriend said the atmosphere in watts flat was very friendly. A young woman described how watts prepared his instruments and inserted a catheter, then watts had another drink together while i did exercises in the room. (she did not explain what kind of exercises .) Occasionally watts impersonated girls fathers or boyfriends when calling doctors to schedule d&cs, and at times he adopted the role of moral guide, strongly advising one young woman, for example, against marrying a young man who admitted impregnating another girl . He said that i should nt marry a man like that, she testified in court . In another case a boyfriend reported returning to watts flat after the catheter had been inserted and found watts waiting for him . Then, seemingly empathising with the girl and expressing what he imagined to be her feelings, watts made an extraordinary statement: what us poor women have to go through for you men. During the trial watts lawyer asked a mother who had arranged her daughter s abortion if she was satisfied with watts treatment and she replied, derk crichton qualified as a doctor in 1944 at the university of cape town (uct), where his father e.c . Crichton held the chair in the department of obstetrics and gynaecology for many years . He earned a first class in surgery at uct, obtained a doctor of philosophy in radiological gynaecology and obstetrics in 1952 from oxford, and became in 1954 one of the first clinical professors at the medical school at the university of natal in durban . At the age of 32 he took up the position of chair of the university of natal s department of gynaecology and obstetrics . At the time of his arrest in 1972 crichton was managing the gynaecology and obstetrics ward located at the king edward viii hospital, built in 1936 and the teaching hospital of the university s medical school for non - europeans. The hospital managed the delivery of 23 000 african babies a year and, with 200 beds, was at the time the third largest such ward in the world . He became a fellow of the royal college of surgeons of edinburgh in 1953 and a fellow of the royal college of obstetricians and gynaecologists in 1957 . He was awarded the blair - bell lectureship by the latter in 1965 for outstanding research and in 1967 won the hamilton - maynard medal for the best south african medical publication of the year, an article describing his method of repairing ureteric fistulae . In 1969 he became the external examiner for the royal college of obstetricians and gynaecologists; this was in addition to the english - speaking south african universities and the university of salisbury in rhodesia (zimbabwe). From 1967 until he left south africa in 1974 he performed all sex - realignment operations (male to female) in natal . At the time of his arrest in 1972 he was president of the regional council of the royal college of gynaecologists and obstetricians in south africa . As an indian patient embarking on the process of sex reassignment said about crichton, he had an unshakeable confidence in what he was doing which seemed to boost the spirits of those he came in contact with. An indian doctor, formerly a student of crichton s, recalled, derk crichton was like a god to us, we would queue for his lectures which for us were like the ten commandments . He was so sure of his medicine that if he told us to treat a patient a certain way, regardless of what the textbooks said, we did it . He was also infamous for his temper and inability to suffer fools. Bongiwe bolani, who worked as a nurse at king edward viii hospital in the mid-1960s, remembers crichton as arrogant and terrifying: he was a horrible man . He screamed, he shouted, he humiliated us, made you feel like imbeciles . Other people, older white males, terrified me from that time for many years . One of crichton s former students is dr sam mokgokong, south africa s first african gynaecologist and instructor in a medical school, and he recalls crichton with huge affection, saying he s like a father to me, he took me up, he made me. He says crichton was colour - blind and even - handed with his students, meaning he treated his white and black students the same . Even bolani recalls crichton urging black nurses to obtain further training so they could be promoted to higher positions . She took up this educational challenge and eventually became a matron . At both addington hospital (for whites and coloureds in segregated wards) and king edward viii hospital (for africans), as well as in private practice, crichton was constantly confronted with the effects of clandestine abortion, telling an audience in 1970, were i a member of the clergy i might say that abortion is my i know more about criminal abortions in durban than anyone else it would be extraordinary if i didnt. He estimated that during his eighteen years at king edward viii and mccord zulu hospital between 1954 and 1972 he had treated an astounding 40 000 african women suffering the effects of illegal abortion at least one died every month . Some girls and women he treated had procured abortions on themselves but most had gone to untrained abortionists, the vast majority of which were what he called soap injectors because they injected liquid soap into women s uteri, a dangerous method that often caused infection or death as a result of air or soap entering the blood stream . The method was popular with clandestine abortionists because it was relatively easy and could be employed in one visit . He estimated the soap - injection method killed one in every 200 women and left another 5% sterile . In court in november 1972 he said that even in the last two or three days, we ve had to do two hysterectomies for gangrenous uteri as a result of soap injections. Complications from abortion were the number one issue facing his unit: in the first eight months of 1973, 934 cases of complicated abortion were admitted to king edward viii, of which 354 had been septic . He said, we can only admit the hard core of complicated abortion cases because of restricted bed space. As a consequence of this experience crichton concluded that the law on abortion was unrealistic and outmoded . In july 1970 he gave a speech on abortion at the university of natal where he declared: [t]he struggle for emancipation from this bondage (of legal and religious condemnation), to have a fertility franchise for women constitutes one of the major challenges of our time . My crusade for this goal is inspired not only ideologically, but also as a result of my personal contact with the human tragedies which result from our present system, especially the consequences of illegal abortion . To crichton this problem assumes proportions which render a fight for its eradication an inescapable duty. True to form, therefore, he did not keep his opinion to himself . Beginning in the late 1960s he began calling for law reform and very quickly became the country s most visible and outspoken medical advocate of abortion on demand, telling an audience in 1970 we must remain dauntless that there can be no compromise in this struggle to achieve the ultimate ideal that every woman should be the captain of her womb. A radical in a conservative profession, he became a self - described just a few months before his arrest crichton called for abortion law reform at the annual meeting of the south african society of obstetricians and gynaecologists (sasog). James watts, aged 41 at the time of his arrest in 1972, was a complex man with a vastly different background from crichton s . Born in cape town in 1931 his mother had once been a schoolteacher in namibia but was a stay - at - home mother when he grew up; his father had lost his business making refrigerator cabinets during the depression after which his parents managed a convenience store on their farm . Watts had eight years of public schooling and moved to durban in 1953 where he worked for five years as a fire fighter on the railways . In 1961 watts says that while working on the railways a co - worker urged him to do more with his life . He said to me you must educate yourself, read reader s digest from front to back. An extremely intelligent man, watts would undoubtedly have been a professional of some kind, probably a doctor or a veterinarian, if he had been able to pursue an education . His motivation for becoming an abortionist appears very similar to that of other excellent clandestine abortionists, such as the american ruth barnett who had an incredibly successful and lucrative abortion clinic in oregon from 1918 to 1968 . Barnett s biographer rickie solinger, writes that her restlessness, her intellect, and her belief in her own capabilities fuelled her desire to become a first - rate abortionist . Watts was an independent thinker and seems to have been instinctively subversive (though never interested in politics), and his contempt for apartheid morality combined with his sharp intellect, fascination with biology, and ambitiousness made it easy for him to live a double life as an employee of shell and a criminal abortionist . A friend whose girlfriend was pregnant was looking for an abortionist and watts told him he should just do the abortion himself . I did it myself. He studied as much as he could beforehand, looking for relevant medical literature in the library, bookstalls and at cna (a south african chain of bookstores), and claims there were mountains of literature available, there have been mountains of articles and books on abortion dating back to plato. After performing his first abortion his friend s sister came to him for the procedure . After that it became easier and thus began his career as an abortionist . Watts became a highly skilled and meticulous abortionist, which is why crichton referred the teenagers and young women needing abortions to him (see below); during his trial doctors said they were i was very sterile conscious, he said, knowing there was the possibility of infection: while growing up in the cape he knew abortionists who used the syringe method, mostly retired white nurses, and girls who became ill afterwards . He said that, like his clients, he was always nervous when performing abortions because he was intensely aware of what could go wrong and knew there was always the possibility that he could be arrested . Determined to avoid medical and legal trouble, from the beginning he only used the far safer method of inserting sterilised catheters . He used urinary catheters, bought from a contact who supplied pharmacies, and always shaved girls anywhere where hair could touch the catheter to prevent infection, or else told the girls to shave themselves. He had his own supply of antibiotics for patients who could not obtain any (obtained from the same contact), sterilised his equipment beforehand, and sealed off catheters after insertion with sterile cotton wool dipped in hibitane (a disinfectant). He also told girls to obtain a medical follow - up, instructing them to wait until there was significant bleeding (so that the miscarriage could not be stopped) and then go to a doctor for a dilation and curettage (d&c) to ensure the abortion was complete and avoid infection . Watts cut - off point for performing abortions was four months of pregnancy because after that abortions he was a self - taught abortionist wholly unlike the stereotype of the unscrupulous and dangerous operator often invoked by doctors and liberal feminists furious about the horrific consequences of criminalising abortion, a negative image reinforced by the occasional prosecution of untrained abortionists, usually women, after the deaths of their clients led to discovery . The abortion business for money and added that he saw no difference in that regard between himself and trained medical doctors like crichton . Indeed, watts took pride in his professionalism and considered himself an ethical, non - judgmental practitioner; even crichton referred to watts patients at one point during the trial (see below). As watts told the court, what motivated me was to protect girls i have helped from going to other abortionists that use syringes and knitting needles as i have often seen terrible consequences from their work . I knew somebody had to help them. He says he performed abortions to help good girls who were in trouble, he said, bad girls knew how to prevent pregnancy . (watts himself was a father; at the time of his arrest he was married with two sons aged 13 and 15 .) Watts performed abortions on all kinds of women. Most clients were white, about 60% afrikaans and 40% english . Although he did occasionally do abortions on indian and coloured girls pregnancy knew no colour barrier, they were all girls in need segregation under apartheid and his acceptance of it ensured he mainly came into contact with other whites . His clients generally paid with cash, although he accepted cheques, and he consulted his own version of a sliding scale that reflected his moral worldview when charging fees . For most girls and women he charged the same as crichton, i.e. Usually between r60 and r80, which he considered fair because in addition to being an expert who deserved to make the same wage as a doctor he was operating entirely outside the law . He says he charged nurses and prostitutes less and sometimes nothing at all because as working women their jobs were at stake . He also charged less, if anything, to help police officers girlfriends, undoubtedly because it was in his interest to have friendly relations with police . Watts reputation reached women and doctors beyond durban, and word - of - mouth, as was so commonly the case with abortionists willing to break the law, was the means by which information about him circulated . He says himself, the more i did, the more my reputation spread by word of mouth, and several doctors, some as far away as cape town, referred some patients in need to me. The number of clients varied from a few per month to a few per week; requests often came after holidays . One teenager learned of watts from a woman she met at the local movie theatre . Another client, from east london, said a friend - of - a - friend from durban told her about him . He also had a contact in south west africa (namibia) who flew in a few girls . One was an afrikaans girl whose father was a preacher; she said her dad would kill her if he found out she was pregnant . She threatened to kill herself by jumping off a building if i did nt help her so i did not have much choice. He never asked clients for personal information and kept no records but recalls a host of dramatic stories . One afrikaans woman flew up from cape town and we met in a car park as arranged and she was wearing a veil . I never interrogated or questioned their identity [sic], i did not wish to embarrass them . I could only speculate, but (she was) definitely upper bracket. He reports that sometimes it was more affordable or convenient for women to bring him to them . He was flown to cape town and johannesburg by clients and went twice to kruger national park to perform abortions on women ostensibly on holiday . There was no feminist movement in south africa in the 1960s and watts had no contacts outside south africa . However, evidence given by teenagers and young women conveys the image of a diligent and sympathetic practitioner . For example, one teenager testified that she came to durban alone for an abortion and watts met her at her hotel to perform the procedure . However, the hotel did not have hot water, so he said, oh well then he would have to take me to his house as we had nowhere else to go, and drove her to his home where he sterilised his equipment, took her to his bedroom and inserted the catheter . Afterwards his wife gave her a cup of tea before he drove her back to her hotel . The next day he picked her up and drove her to a clinic for a d&c where he did all the talking for me. After her procedure she took a taxi to the hotel where she was met by watts who stopped by to see how i was, just as i arrived back . He came upstairs with me to my bedroom, and he had a drink, and he stayed for a little while and then he left. He would commonly allow girls to stay overnight at his downtown flat purchased for his practice while they waited to miscarry and sometimes had drinks with the girls and/or their boyfriends after inserting the catheter . One boyfriend said the atmosphere in watts flat was very friendly. A young woman described how watts prepared his instruments and inserted a catheter, then watts had another drink together while i did exercises in the room. (she did not explain what kind of exercises .) Occasionally watts impersonated girls fathers or boyfriends when calling doctors to schedule d&cs, and at times he adopted the role of moral guide, strongly advising one young woman, for example, against marrying a young man who admitted impregnating another girl . He said that i should nt marry a man like that, she testified in court . In another case a boyfriend reported returning to watts flat after the catheter had been inserted and found watts waiting for him . Then, seemingly empathising with the girl and expressing what he imagined to be her feelings, watts made an extraordinary statement: what us poor women have to go through for you men. During the trial watts lawyer asked a mother who had arranged her daughter s abortion if she was satisfied with watts treatment and she replied, as a rare outspoken liberal on the controversial issue of abortion crichton attracted a great deal of publicity for his radical views . Consequently teenagers and young women from around the country approached him for abortions . As he put it, his public comments accounted for the flocks of young women coming to him for help . Since i have started lecturing on the subject, he said, the numbers have swelled tremendously. By the early 1970s he was confronted almost daily with the unmarried girl with the unwanted pregnancy pleading for abortion. In addition, doctors unwilling to perform abortions referred their pregnant patients to him, and he often helped young women when other doctors have refused to do so . I have often had a great deal of trouble getting other doctors to agree that an abortion is necessary. (two of the teenagers who testified said they were told about crichton by their family doctors .) Crichton did not relish the flood of young women seeking abortions for he was already he therefore repeatedly told girls that doing abortions was out of the question regardless of the amount of money they offered . Yet he realized girls were determined to have an abortion no matter the risks, as many subsequently confirmed in court . Many of them were in tears and averred that they would commit suicide if a solution could not be found . I usually experienced frustration in finding that most of what i was saying to the patients was passing in one ear and out of the other. Knowing full well from grim experience that girls would have no difficulty in finding abortionists i was terrified that they would go to a soap injector he felt it my duty to safeguard them to the best of my capabilities. He devised a system that would assist the girls and, he believed, shield him from prosecution . The common law on abortion prohibited a doctor s induction of a miscarriage but allowed for treatment in cases of miscarriages already begun . So he started to unofficially refer girls to abortionists whom he knew used sterilised catheters, what he called the tube inserters, to initiate a miscarriage, along with firm instruction that they should visit a doctor immediately afterwards for a d&c in order to ensure that the abortion was complete . Most tube inserters were indian but, much to his irritation, the majority of white patients preferred white abortionists because they thought indians were unclean. Regardless of his assurances, most white girls wanted a white abortionist, so he referred them to watts . They told him that a man named watts was a careful and effective abortionist who took precautions to avoid infection, such as shaving pubic hair, and whose patients never fell ill . Watts patients told me that he inserted a sterile soft rubber catheter with extreme gentleness and great regard to sterility. He said i ve never seen a case (of watts) go wrong, in fact his cases seem to be doing a jolly side [sight] better than the others . And we noted this over the months that these patients did not seem to have the complications the others had. In spite of never meeting and their vast differences in class and social status, the two men developed a kind of working relationship . Crichton gave girls watts telephone number, usually a prescription for antibiotics to take in advance of the procedure to minimise the risk of infection, and advice to return to him or another doctor for a d&c after watts had induced bleeding . Conversely, girls who first contacted watts were sent by him to crichton for a prescription for antibiotics before returning to him for the miscarriage, after which he insisted they return to crichton for a d&c . Crichton was certain this method protected him; in court he said confidently, indeed, he was so assured of his safety that he kept case records with detailed notes on patients that, ironically, would ultimately help the prosecution convict him . His confidence was a combination of his naturally optimistic outlook, navet, and arrogance derived from his privileged status in his profession and society . When warned by a colleague that he would get into trouble if he continued i ve got nothing to worry about. His wife reported that he was thoroughly shocked when the police arrived at his home early one morning to arrest him . For girls and women, the crichton watts system was safe, rapid and relatively respectful of their privacy and dignity . In contrast, staff at hospital casualty departments were often prying, judgmental, and sometimes openly hostile to girls and women wanting help with incomplete or botched abortions . For example, women who went to the addington hospital casualty department after abortionists had induced miscarriages, would spend many hours waiting for a d&c, during which time they were frequently questioned in detail about their abortions . Cross examined there, and crichton said girls complained that the nursing staff and doctors embarrassed them by gossiping amongst themselves about their private lives . In fact, he said girls who were miscarrying refused with increasing frequency to go directly to casualty at addington hospital and would instead contact him directly to arrange their d&cs . At the beginning of 1972 the police targeted crichton for prosecution; ultimately watts became significant to authorities only because of his association with crichton . Alleged country - wide abortion network began in 1969 . At some point the natal provincial council conducted an investigation into abortions being performed in provincial hospitals and handed their findings to the attorney - general, who in turn notified the durban police who apparently did not get very far with their investigation . Kobus visser, chief of the criminal investigation department (cid) in durban, was determined to uncover clandestine abortion, however, so he sent out a call around the country for an officer to take charge of the investigation and sergeant dan matthee (b. 1925) was hired . In a recent interview matthee claimed the minister of health himself was responsible for targeting crichton (though he would not say which one). He said the minister had been at an international conference where crichton had spoken out against south africa and as a consequence he wanted something done. The story is plausible because the np was intensely conscious of international criticism of south africa . Since 1960, the year of the sharpeville massacre when police shot at least sixty - nine unarmed african protesters (many in the back as they were fleeing from police attack), the united nations had repeatedly censured south africa for its racist policies and called for cultural and economic sanctions . If crichton was openly criticising south africa overseas it is entirely believable, given the regime s vicious retaliation against previous critics, the np would take revenge upon him at home . What is certain is that convicting a highly respected doctor like crichton for providing abortions would be a means by which to instil fear into the hearts of other doctors and thereby stem white young women s access to medical abortionists (discussed further below). Carte blanche and apparently unlimited funds, for ultimately he and his staff took thousands of witness statements . He proved to be as effective as visser (and his political superiors?) Had hoped, and as a consequence of his investigation he earned the nickname according to crichton s allies matthee was obsessed with convicting crichton, a contention that watts strengthened in a statement to the trial judge in which he said matthee threatened to put the screws on me unless i agreed to incriminate prof . I have been terrorised by major mathee [sic] that he will ensure that i receive a long jail sentence unless i give him a statement according to his instructions . Consequently i have lost confidence in the impartiality of the justice of the court which is to try me . In addition to his extreme dedication to his job as a police officer, matthee s fixation on crichton was likely fuelled by class and ethnic resentments that have genealogies dating back to the south africa war (18991902) and the subsequent enmity between english and afrikaans south africans . Poor white afrikaans family in de poort, a government settlement town in the cape, whereas crichton came from a distinguished english - speaking family of means . I like challenges, especially when it comes to people with high qualifications and many letters after their last names. He acknowledged that crichton had a reputation for being an excellent doctor and said even his own sister had visited him (not for an abortion) but he found the man he directed hospitals and nursing homes in durban to check their records and report procedures that could have been abortions, and then interviewed doctors and operating theatre nurses who assisted or witnessed crichton s operations . At least one nurse who worked at st augustine s hospital described a number of crichton s cases to the matron as crichton told his lawyer that the nurse probably did so out of annoyance at my ticking her off by calling her in to work in order to attend to a young woman in need of a d&c . He also admitted berating the nurse for her inefficiency and obstructionism, which probably annoyed her further; in hindsight crichton would have been wise to avoid alienating nurses, the very people who knew better than most about his abortion - related activities . (according to watts, it was also a nurse who alerted matthee to his involvement with crichton .) Next matthee travelled around the country locating and interviewing girls and women . In total, the police contacted at least 2000 suspected cases whose names and addresses were found in hospital records, and drew up a list of 400 women to be shocked to receive telephone calls from local police asking them to come to the station for an interview with him . One said she was afraid of matthee and that he had not allowed her to read over her statement . Matthee had threatened that unless she testified her father would lose his (government) job, a claim also made in court by crichton s defence . The boyfriend of the girl caught leaving watts building on the evening of his arrest said matthee threatened to lock him up unless he gave a statement . When called in for an interview he brought a friend who was an articled law clerk with him to the police station . Matthee told the boyfriend he considered him an accomplice and wanted him to make a statement but his friend advised him not to unless matthee gave him an indemnity from prosecution . This angered matthee who allegedly told the boyfriend, i will lock you up and i will keep you locked up until you make a statement. After he threatened the young man that the attorney - general might charge him as an accomplice he admitted that he helped his girlfriend obtain an abortion and signed a statement . Matthee admitted in court that he took 98% of witnesses statements himself and even re - interviewed witnesses who had been interviewed by other officers . Watts was arrested first . On 9 june, 1972 three plain clothes officers, one of which was matthee, watched a young couple enter watts building at about 7:00 p.m. when they emerged an hour later the police arrested them and took the couple back to watts flat, which was searched, and watts was arrested . (the young woman was taken to a district surgeon who removed the catheter watts had just inserted, which was then taken into evidence .) Crichton was arrested on the morning of 21 june 1972, at the same time that a colleague of his, dr angini maharaj, was also arrested on suspicion of procuring abortions . The police seized their medical records and then compared the names found in crichton s and maharaj s records to those listed in hospital and nursing home records as having had d&cs . In june 1972 i thought i had a very strong case, and masters decided to hold not one, but two trials for crichton, the first to include the easy witnesses who had given affidavits; the second trial the less reliable witnesses and some (who) would nt talk. The first trial included watts and took place in november and december 1972 . The second trial, at which he was tried alongside maharaj, took place in durban regional court in march 1973, an event i discuss elsewhere . (during the second trial dr maharaj reported that she had refused an offer of indemnity from prosecution if she gave evidence against prof . Crichton because of her loyalty and high regard for him.) Crichton s lawyers and friends believed at the time, and still do, that the reason for holding two trials was to drag out legal proceedings for as long as possible in order to cause him maximum financial and professional hardship; a reasonable conclusion in light of the significant time and money it cost him to mount two legal defences while attempting to maintain his practice and teach at the university of natal . The state charged crichton and watts, with the following: having been approached to procure an abortion, accused no . 1 recommended that someone else had to start the bleeding in the private parts of each female concerned, for which purpose he referred her to accused no . A dilatation and curettage was performed by accused no . 1 subsequently to complete the abortion . It is unknown to the state when the common purpose was formed or whether there was an express agreement between the two accused, but it can, however, be inferred from their conduct that there was a common purpose and that they acted in concert . The girls and young women who had procured abortions were considered accomplices along with the people who had helped them boyfriends, brothers, girlfriends, parents and all were pressed into testifying in exchange for indemnity from prosecution . The twenty - six young women called to testify had been between the ages of 17 and 23 at the time of their abortions, all unmarried . About half were english - speaking south africans and half were afrikaans - speaking . Crucially, all were white and it was their whiteness that provoked authorities to intervene in the clandestine abortion industry in the first place . Major problem in his unit at the king edward viii hospital, which served africans and treated the greatest number of septic abortions in south africa at the time . Yet there was never an attempt by police to investigate the extensive, deadly abortion industry in relation to black women . The np was concerned only with white girls, suggesting that, as signifiers of whites supposedly superior morality, they were expected to be chaste and obedient . Every day both the white and non - white courtroom galleries were packed with spectators, and all the national newspapers as well as the local press covered in voyeuristic terms what was called south africa s most sensational abortion trial. The young women were objects of fascination and the press tried to read their appearance for signs of immorality; one newspaper described many of the girls as attractive mini - skirted young women. Indeed, the courtroom had what solinger, in her study of the prosecution of american abortionists, calls a cryptoporno atmosphere, for two reasons . First, the spectacle was made sexually tantalising by crichton himself a handsome, athletic, dapper man of 52 whose stylish attire was regularly noted by reporters . Furthermore, crichton, already twice divorced, was regularly accompanied to court by two much younger, unmarried women, his attractive secretary linda redman and his live - in girlfriend susan pohl, age 26 . The young women s presence, photographed and commented upon in the press, heightened the fascination with crichton and the trial generally . The visibility of middle - aged, respectable crichton, a divorcee and father of three children, living with a very beautiful woman half his age added an exotic cache of sin, pleasure and glamour to the situation . Moreover, crichton s defiant, thoroughly unrepentant attitude during the trial enhanced his presence and audience fascination . For example, when asked by the prosecutor, haasbroek, why he did not inform the police of the names of criminal abortionists he knew of, he said, i do not think it is my duty to act as a policeman . Haasbroek: yes but you must have known that the police were investigating matters of this type . Haasbroek: but out of your own accord you would nt give them the names? Crichton: i do not regard it my duty as a doctor to report to the police every time i find that a patient has had an abortion done which is illegal . Second, the prosecution humiliated the girls with embarrassing questions about their sex lives, bodies and abortions . Of an 18-year - old the judge asked whether she had had intercourse with her boyfriend (she made the unsurprising admission that she had). The state s lawyer schutte asked a girl if there was there anything irregular about your breasts before she saw watts, and continued: now what did mr . Schutte: where did he insert this thing, you need nt be afraid . Q: judge: did he insert it between your legs into your private parts? A: yes. Of another young woman the state prosecutor, haasbroek, asked questions about the size of her breasts prior to the abortion . Answer: he did nt shave me, he just cut cut the pubic hair with scissors . Haasbroek: yes? Answer: and then he but just then justice james intervened and asked, i m sorry, what did he cut with the scissors? And haasbroek replied, oh your pubic hair. Of a teenager who was 17 at the time of her abortion haasbroek asked, what did he (watts) actually do with the tube and the wire? Answer: into my virginia [sic]. Many answers were so similar in phrasing that the defence accused the prosecution of coaching the young women in order to illustrate that a system had been devised by crichton and watts . The magistrate presiding over the second trial noted that the girls testified under extreme stress and strain and were understandably nervous . They were subjected to lengthy, embarrassing, repetitive and sometimes torrid cross - examination. Some young women were so distressed by the questions they buckled on the stand . For example, one 19-year old girl at the first trial, broke down in the witness box, sobbing loudly when asked to describe the abortion and, with her head bowed was led from the packed court to compose herself. There was an exception . One young woman was impervious to intimidation, and rebellious, even surly while giving testimony . She was unforthcoming and repeatedly claimed she could not remember details about the abortion, in the process angering crichton s lawyer, shaw, who subjected her to increasingly aggressive questioning . Clearly unafraid, her tussle with shaw was entertaining to observers who sometimes laughed in court as they witnessed the exchange between the two . Significantly, in previous as well as contemporaneous trials of medical abortionists, white girls and women were spared public shaming . In 1949, during the preparatory inquiry in advance of the prosecution of buchner and his employees working at the castle mansions abortion clinic (mentioned above), the identity of girls and women had been protected lest they be embarrassed or even ruined by public exposure . Displaying great concern for the women s welfare the prosecutor had explained why the inquiry should be held in camera: the crown will be calling women to give evidence, some of them unmarried, some married, but their husbands do not know of any operation . In certain cases these women would be seriously prejudiced, and perhaps their whole lives would be ruined if the facts were made public . These women would be required to testify about intimate sexual details, and they have told the inquiring officer of their extreme reluctance to testify in open court . The magistrate agreed, saying with apparent sympathy that otherwise witnesses might not give evidence with necessary frankness . During the actual trial, at which an astonishing fifty - eight women testified, there had been a publication ban placed on their names and personal details (although this time the press could attend court on condition they protect the women s identity); the court even provided female shorthand writers to transcribe evidence to spare the women discomfort . And in july 1972, just four months before crichton was prosecuted, an afrikaans doctor named jurgen backeberg was prosecuted on sixteen counts of procuring abortion and one charge of culpable homicide after the death of a 14-year old girl following an abortion, and during his trial teenage girls and young women gave testimony behind closed doors to protect their privacy . Watts trial was exceptional, a show trial aimed at buttressing apartheid culture by disciplining both wayward young white women and liberal members of the medical profession . On judgment day, december 7, the court was sardine - packed hours before the judgment was due to come down . Dozens of fashionably - dressed women ranging from teenage to elderly spilled over the galleries and stood in the aisles to hear justice james verdict . One newspaper described how spectators, white and black, crammed every available space in the galleries and overflowed into the aisles . City workers in safari suits and overalls rubbed shoulders with elegantly dressed elderly women some of whom have been present throughout the trial. Crichton and watts bore the stress of waiting for the verdict very differently; indeed they were a study in contrasts, with each man s demeanour reflective of his class position and status in society . Crichton appeared cheerful and confident: smartly dressed in an olive green suit, an orange shirt and matching tie, [he] chatted happily with his lawyers and smilingly acknowledged the many nods of encouragement he received from spectators. Another newspaper reported, easily the most smartly dressed man present was professor derk crichton, in vivid orange shirt and tie and an avocado green suit. Conversely, watts, who had no faith whatsoever in the justice system, was extremely nervous . While awaiting the judgment he requested permission to go outside and smoke a cigarette . A journalist who interviewed him just prior to the judgment observed, his hands trembled . The strain of ten days in court was clear in his gestures, inability to concentrate and sudden emotional outbursts. He said if he were jailed his family would be destitute. Justice james found both men guilty, crichton on sixteen counts and watts on twenty - four . The judge declared he found the duty to pass sentences on the men one of agonizing difficulty, for on the one hand the law had to be upheld but on the other hand the court must take into account the tremendous pressures placed on medical men and those of lesser skill by desperate women who feel their whole social and economic future is bound up in getting rid of an unwanted pregnancy. Bringing additional pressure to bear upon the government, james also observed that the situation regarding illegal abortions in this part of the country appears to have become most serious, and appears to require some sort of official action and it is the function of the legislature to amend the law . The sunday express headline of 10 december 1972 read: judge favours law reform on illegal abortions. In his judgment james s opinion about the two men could not have been more different; a clear reflection of his class - biased interpretation of their actions . For the uneducated watts he had no sympathy . Though doctors in durban had expressed being amazed at the skill displayed by watts in inducing abortions and witnesses had testified to his diligence and kindness, the judge decided watts was a mercenary opportunist . James did acknowledge watts took pains about avoiding infection and treated the women with consideration, but nevertheless sentenced him to two years in prison, with one year conditionally suspended . In the 1950s, for example, american judges and prosecutors found the economic motive for performing abortions particularly disgusting; in sexist societies it is seen as immoral for anyone to benefit from the irresponsible sexual behaviour of a woman. The lack of respect was mutual: watts later referred to james as a pompous old fart. In sharp contrast the judge was greatly impressed by crichton, describing him as a noble man with a history of tremendous service to the community . He believed crichton was compassionate, not greedy, and had acted only in the best interest of his patients . Nevertheless, he said, given the existing law, his hands were tied and knowing a criminal conviction would inevitably bring harm to crichton s stellar career he fined him only r500 . Ironically, for watts, the untrained abortionist justice james dismissed as simply greedy, imprisonment merely imposed a temporary halt to his abortion business . He served six months at durban central prison and after his release immediately resumed performing abortions . In fact, watts was an abortionist for another 20 years . Because of the near - impossibility of procuring a legal abortion during the apartheid era, south african women of all races continued seeking the services of clandestine abortionists . As for crichton, a few months after the conviction he once again stood trial, this time alongside dr angini maharaj . Both were found guilty and subsequently struck off the medical roll, which barred them from practicing and teaching medicine in south africa . Crichton left south africa for swaziland in 1974, one of few jurisdictions that would allow him to practice medicine, and repeatedly applied for reinstatement to the medical roll . He returned to south africa in 1977 after reinstatement and opened a private practice, but his academic career was finished . The prosecution of crichton and watts revealed with supreme clarity the contested nature of the terrain of sex and reproduction in south africa during apartheid . The trial heightened the visibility of young white women s errant sexuality, which intensified patriarchal anxiety about the disintegration of the traditional family . Clearly, many ordinary girls were choosing to have pre - marital sex and avoid motherhood, and with the help of doctors like crichton were finding ways to do so . In subsequent discussion and debate about abortion leading up to the passage of the act the theme of the wayward white daughter was a leitmotif . At an official level the trial played a role, albeit indirectly, in the production of south africa s first statutory law on abortion the extremely restrictive abortion and sterilisation act (1975). Reflecting the regime s double determination to police white girls sexuality and the medical profession, the act contained so many byzantine limitations and bureaucratic requirements for both women and doctors that obtaining a legal abortion became all but impossible for the remainder of apartheid . Among the many new regulatory requirements put in place the law stipulated that a doctor could perform an abortion only in a state - controlled institution after receiving written permission from the official in charge, who then must submit the name of the doctor to the minister of health within twenty - one days after the procedure . Doctors who performed a suspiciously high number of abortions could then be investigated . During the debate in parliament preceding the passage of the act, the minister of health, dr s.w . Van der merwe, claimed with smugness this would help expose medical snakes in the grass. To abortionists, medical and non - medical alike, it must have seemed as if almost overnight abortion transmogrified from a nominally criminal practice largely tolerated by police to a serious criminal act . Yet two weeks after crichton s arrest the sunday tribune found an abortionist in durban after just three hours of searching, and in the two months after crichton and watts were convicted five women in the city died and at least another 250 were admitted to hospital because of botched abortions . The chilling effect on the medical profession, however, was immediate and considerable . Doctors previously willing to perform abortions had learned the lesson that the government wanted to teach them by seriously damaging the career of so eminent a doctor as crichton: the state was powerful and relentless, able to spend seemingly unlimited funds to expose and prosecute them . As a result they became extremely wary of performing even legal therapeutic abortions, never mind illegal ones . Complaints were soon lodged that a medical abortion for what previously would have been considered a legitimate reason was next to impossible to obtain . In may 1974 an anonymous gynaecologist told a newspaper the police clampdown had ended sympathetic doctors willingness to perform abortions, which had the dual effect of eliminating from the field those qualified medical practitioners who were prepared to perform abortions safely and encouraging the proliferation of backstreet operators. Some doctors did continue to perform abortions clandestinely but if caught were also harshly treated . In 1977, for example, dr philippus lodewyk jacobus smith, aged 50, of ottosdal was sentenced to a prison term for performing abortions, free of charge, on three white university students . Convicted in february 1976, he was already in prison when the disciplinary committee of the medical and dental council held a hearing about his case, and he was brought manacled before the committee . He pleaded guilty to the charge of disgraceful conduct and said in mitigation he gave in to pleadings from the three girls to do the abortions . He felt very sorry for the girls, who were facing scandal and an end to their university careers. He was struck off the medical register and returned to prison . Afterwards, a gynaecologist who for forty years had believed in abortion law reform, dr e.w . Bischoff, told a meeting of members of the feminist group abortion reform action group, a doctor has no rights today. This had little impact on women of other races who had little to no access to medical abortion in the first place . But in the aftermath of the downfall of crichton and until the demise of apartheid, white girls and women in south africa found it harder to have safe medical abortions . Which is precisely what the np government wanted.
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Functional bowel disorders, including irritable bowel syndrome (ibs), are common gastrointestinal disorders all over the world . However, in the last few decades, advances in our understanding of the pathophysiology of ibs have revealed several factors, including alterations in the microbiota, as potentially relevant to the cause of this syndrome and the precipitation of its symptoms . Indeed, alterations in the gut microbiota are being increasingly implicated in the pathogenesis of several gastrointestinal and systemic diseases . We wish, therefore, to review the gut microbiota and its alterations in, and relationships to, ibs . The human gut harbours a huge microbial ecosystem, which is equipped to perform a variety of functions such as the digestion of food, metabolism of the drugs, detoxification of toxic compounds, production of essential vitamins, prevention of attachment of pathogenic bacteria to the gut wall and maintenance of homeostasis in the gastrointestinal tract (git) [13]. The human gut is first colonized at birth; this microbiota gradually increases in size and diversity up to the end of the first year of life; by that time, the gut microbiota has come to resemble that of the adult and remains relatively stable thereafter . The composition of the gut microbiota varies according to age, sex, diet, geographical origin of the individual and is also influenced by certain environmental factors, such as administration of antibiotics . There are 10 times more microbial cells (10) in the gut than cells in the entire body (10). A recent study has suggested that the human gut microbiota consists of more than 35,000 bacterial species and that 70% of all of microbes in the human body reside in the colon . The small intestine consists of mainly gram - positive and aerobic bacteria, whereas the large intestine consists largely of gram - negative and strictly anaerobic bacteria [5, 6]. Alterations in the normal gut microbiota have been suggested as etiologic factors in the development of functional gastrointestinal disorders such as ibs and functional dyspepsia, common gi disorders of unknown etiology [79]. Quantitative alterations in the gut microbiota in the small bowel may result in the clinical syndrome recognized as small intestinal bacterial overgrowth (sibo). In sibo, such is the change in the number and type of bacteria in the upper small intestine that diarrhea, abdominal bloating, malabsorption, abdominal pain, and excessive gas production result; severe motor dysfunction may be an underlying cause [1113]. Quantitative changes in the colonic microbiota may lead to the proliferation and development of specific species that produce more short - chain fatty acids (scfas) and gases, such as methane, hydrogen, and carbon dioxide, potentially resulting in abdominal bloating and distension . An increase in the concentration of scfas (acetate, butyrate, and propionate) leads to acidification of the colon and deconjugation of bile acid . This in turn may cause significant changes in water and electrolyte transport in the colon which result in diarrhea [8, 14, 15]. Malabsorption of carbohydrates may cause increased production of hydrogen gas, which is associated mainly with diarrhea - predominant ibs (ibs - d) whilst excess methane gas production is associated with constipation - predominant ibs (ibs - c). Ibs is a functional gastrointestinal disorder associated with abdominal discomfort or pain, distension and bloating, diarrhea, constipation, or mixed bowel habits (i.e., both constipation and diarrhea; ibs - m). Ibs subjects may also experience greater levels of stress, anxiety, and depression compared to healthy subjects [16, 17]. All of these co - morbidities are associated with impaired quality of life in ibs patients [1820]. Several diagnostic criteria (kruis, manning, rome) have been used to distinguish ibs patients from those with organic bowel disease in daily clinical practice [11, 21]. The prevalence of ibs varies from 9% to 22% in the united states and europe [22, 23]. In asian countries, the lowest prevalence has been reported from india, at 4.2%, and the highest from japan and singapore [2325]. Several hypotheses have been proposed which include alteration in the gut microbiota, dysregulation of the brain - gut axis and autonomic nervous system, visceral hypersensitivity, and altered levels of gastrointestinal neuropeptides and hormones [11, 22, 26]. Furthermore, abnormal gastrointestinal motility, as well as genetic, environmental, and psychological factors, may also play important roles in the development of ibs . Recent studies have also shown that ibs is associated with low - grade intestinal inflammation resulting from an activated immune system, in response to a normal or abnormal gut microbiota [1, 27, 28]. There is a growing interest in investigating the role of an altered gut microbiota in the pathogenesis of ibs [14, 29]. Normal gut microbiota have either direct bactericidal effects or can prevent the adherence of pathogenic bacteria to the wall of the gastrointestinal tract [7, 30, 31]. Dysbiosis in the gut may facilitate the adhesion of enteric pathogens in the human gut which can be associated with ibs symptoms [30, 32]. Alteration in the composition of the normal microbiota and disturbed colonic fermentation in ibs patients may play an important role in development of ibs symptoms . Firmicutes and bacteroidetes are the major beneficial gut microbiota in the normal human and changes in their relative numbers have been reported in ibs patients . Significantly, a two - fold increase in the ratio of firmicutes to bacteroidetes has been reported in ibs patients . This results from an increase in the quantity of ruminococcus, clostridium, dorea species and a decrease in the quantity of bifidobacterium and faecalibacterium species . Significantly greater abundance of classes of gammaproteobacteria and enterobacteriaceae in healthy controls were positively correlated with the inflammatory markers il-6 and il-8 . Higher levels of these cytokines have been reported in ibs patients [34, 35]. Due to a different microenvironment in the intestinal epithelium and lumen, the composition of microbiota is not the same at the level of the gut epithelium and lumen . Culture - based analysis had shown that there were significant differences between the mucosa - associated microbiota and fecal samples in both healthy control and ibs patients . For example, a significantly increased quantity of aerobic bacteria and lactobacillus was noted in ibs - d in feces but not in mucosal samples . Several studies using different methodologies to define changes in the microbiota have been published, including classic culture - based techniques, pcr - based molecular technologies such as real - time pcr (q pcr), microarray, dgge (denaturation gradient gel electrophoresis), gc profiling, and high - throughput sequencing based on 16 s rrna [33, 36] (see table 1). Bacterial fermentation of undigested, unabsorbed food causes production of scfas, which are bacteriostatic for a group of species, either directly or by reducing ph [2, 7, 30, 31, 37]. A previous study reported that the prevalence of sibo in ibs subjects detected by lactulose hydrogen breath test was about 78% . However, this study used the lactulose hydrogen breath test which has now been shown to be prone to a high rate of false positive result and may have led to an overestimation of its frequency . Ibs develops in a subgroup of individuals following an episode of acute gastrointestinal infection, known as pi - ibs . Acute enteric infections are characterized by abdominal discomfort, fever, vomiting, bloating, and diarrhea, [14, 39, 40]. Fever and vomiting generally improve after a few days while abdominal discomfort, diarrhea and bloating persist in those who develop pi - ibs . Risk factors for the development of pi - ibs include younger age, female gender, prolonged duration of diarrhea, the presence of bacterial pathogens, and psychological morbidities including anxiety, frustration, and depression [4143]. The risk of ibs increased sixfold after an acute gastrointestinal infection and persists for up to 3 years [44, 45]. Recent studies have shown that pi - ibs develops in 330% of individuals with bacterial gastroenteritis caused by a number of enteric pathogens such as campylobacter species, clostridium perfringens, staphylococcus aureus, bacillus cereus, shigella and viruses [7, 30, 32, 39]. The prevalence of pi - ibs in subjects who had suffered from diarrhea whilst travelling in developing countries has been estimated to be 714% . The diagnosis of pi - ibs was made according to rome criteria for ibs among individuals who did not have ibs previously but developed it after the episode of acute gastroenteritis [14, 39]. Recent studies have suggested that pi - ibs is associated with dysbiosis in the gut (induced either by enteric infection or by the use of antibiotics), psychological factors (such as stress, anxiety, and depression), genetic susceptibility of the host, persistent activation of the host immune system, increased intestinal permeability, and an increased number of enterochromaffin cells in the gut [22, 39, 47]. Potentially harmful microorganisms and their metabolites cause the disruption of tight junctions between epithelial cells leading to increased mucosal permeability during the acute phase of infection [48, 49]. The permeability of the proximal small intestine in pi - ibs was shown to be significantly increased compared to controls . A lesser expression of zo-1 has been observed in biopsy samples of ibs subjects . As a result of increased permeability of intestinal epithelial cells, an influx of immune cells including mast cells, t lymphocytes, macrophages, and inflammatory mediators such as il-2, il-1, il-6, il10, tgf- occurs after an acute enteric infection [48, 49, 52, 53]. A growing body of evidence indicates that mast cells and lymphocytes are increased in the mucosa of both pi - ibs and ibs patients . Il-1 is significantly increased in pi - ibs patients during and after the infection compared with those subjects who do not develop pi - ibs . These inflammatory mediators (il-1, il-1, il-6, ifn-) damage the intestinal epithelial barrier and cause inflammation . Furthermore, the administration of antibiotics altered the composition of the gut microbiota and inhibited the expression of antimicrobial peptide reg iii produced by commensal bacteria . Reg iii strengthens the intestinal epithelial barrier by inhibiting gram - negative pathogens but has no effect on gram - positive bacteria; its inhibition results, therefore, in a favourable environment for enteric pathogens to proliferate [4, 7, 55]. Normally, the density of bacteria is much lower in the small intestine than in the large intestine . There are 10 colony - forming units (cfu) per ml, 10 cfu / ml, 10 cfu / ml and 10 cfu / ml bacteria in the cecum, terminal ileum, proximal ileum, and jejunum / duodenum, respectively [54, 56]. The major families of bacteria in the small intestine include bacilli, streptococcaceae, actinobacteria, actinomycinaea, and corynebacteriaceae . Qualitative or quantitative changes in the microbiota of small intestine may lead to the clinical features of sibo [56, 57]. In healthy individuals, the normal bacterial count in the proximal small intestine is 10 cfu / ml; sibo is traditionally defined by a bacterial count of 10 cfu per ml in jejunal aspirate [2, 56]. Several techniques have been used for the diagnosis of sibo, which include the lactulose hydrogen (lhbt), c xylose and glucose hydrogen (ghbt) breath tests and culture of jejunal aspirate [13, 56, 58]. Hydrogen and methane are normally produced in the large intestine but in case of sibo these gases are produced in the small bowel also . Methanobrevibacter smithii, methanobrevibacter stadtmanae, and, possibly, coliform bacteria produce methane gas [13, 14]. The jejunal aspirate culture has, traditionally, been used as the gold standard to diagnose sibo . The limitations of this test however include invasiveness and the challenges posed by attempting to culture all strains and species [54, 57]; therefore, hydrogen breath tests (ghbt or lhbt) are most commonly used . In a study where endoscopic jejunal biopsy culture was used to diagnose sibo, the prevalence of sibo in ibs patients was 4% (based on the definition of 10cfu / ml of bacteria in jejunal aspirate) and no different from that seen in healthy individuals . In contrast, in a study of 111 ibs subjects using lhbt, pimentel et al . Reported a prevalence of sibo of 84% in ibs (in comparison to 20% in healthy individuals). Recent published data have shown that, using jejunal aspirate as the gold standard, double - peak in lhbt only diagnoses one - third of sibo patients; the sensitivity and specificity of lhbt were 31% and 86% and those of ghbt were 44% and 80%, respectively . In a separate study by berthold, the sensitivity and specificity of lactose-(13c) ureide breath test (lubt) were 66.7% and 100% and those of ghbt 41.7% and 44.4%, respectively . The sensitivity and specificity after glucose were 62.5% and 82% and after lactulose were 52% and 86%, respectively . The variation in lhbt and ghbt may be due to differences in the diagnostic criteria used for selection of ibs patients, ecological origin, nature of the substrate used, and diagnostic methods . The administration of antibiotics and probiotics reduced not only gas - related problems but also ibs - like symptoms [54, 56, 61, 64]. Sibo may arise due to hypochlorhydria, altered intestinal motility [2, 56], altered bacteriostatic properties of pancreatic and biliary secretion, and a dysregulated immune response . Proton pump inhibitors (ppis) may predispose to sibo by decreasing acid secretion in the stomach . Theisen reported that the inhibition of acid secretion related to the administration of omeprazole led to an increased concentration of unconjugated bile acids . Thus, the potential side effects of ppi include constipation, diarrhea, bloating, and abdominal pain, symptoms which resemble those of ibs (see table 2). Commensal bacteria provide a favourable environment, prevent the adherence of pathogenic bacteria, and modulate innate and adaptive immune responses [55, 69]. The intestinal epithelium contains a large number of lymphocytes that can remove infected epithelial cells . Paneth cells found at the base of the intestinal gland (crypt of lieberkuhn) throughout the small intestine can prevent the penetration of intestinal epithelium by commensal and pathogenic bacteria . Iecs are protected by a glycocalyx layer of mucus, the epithelial junction adhesion complex, secretory iga, chloride secretion, and other glycoproteins [30, 55]. The intestinal barrier consists of the tight junction complex, adherin junctions, gap junctions, and desmosomes . There are more than 40 types of proteins in the tight junction complex, which play crucial role in maintaining the permeability of the intestinal epithelium . A leaky intestinal epithelium has been reported in subjects with ibs, ulcerative colitis, crohn's disease, celiac disease, and food - borne infections [7275]. A recent study has shown that microrna-29a regulates the permeability of the intestine through the generation of glutamine synthetase in patients with ibs . Decreased concentration of glutamine leads to an increase in the permeability of intestinal epithelial cells (iec), whilst the permeability of iecs can be recovered by the supplementation of glutamine in patients with ibs . The administration of probiotics, fermented milk (streptococcus thermophilus, lactobacillus bulagaris lactobacillus acidophilus, and bifidobacterium longum) and l. plantarum has been shown to strengthen the intestinal barrier [7274]. Paneth cells and enterocytes in the gut secrete antimicrobial molecules such as angiogenin 4, defensins, iga antibodies, and regiii . These antimicrobial peptides destroy pathogenic bacteria by forming a pore in the bacterial cell wall [7, 55, 76]. These data suggest that a symbiotic relationship is present between commensal bacteria and the host . The composition of the gut microbiota influences the development of the immune system . Any alterations in the gut microbiota due to enteric infections, antibiotic therapy or acid suppressive treatment lead to activation of both the innate and adaptive immune responses . Commensal bacteria from the phylum of bacteroidetes and firmicutes have been shown to induce t regulatory cells and inhibit th17-mediated inflammation . In contrast, the administration of bifidobacterium animalis subspecies inhibited intestinal inflammation via a reduction of the commensal bacteria enterobacteriace [55, 77, 78]. Thus, the gut microbiota plays an important role in the maintenance of homeostasis of various subpopulations of t cells: regulatory t cells (tregs), t helper 1 (th1), and t 17 (th17) cells in the gut . Low - grade inflammation in the intestine in ibs patients is associated with the activation of t lymphocytes and mast cells, increased expression of proinflammatory cytokines such as il-6 and il-8, and elevated levels of il-1, tnf-, and il-8 in peripheral blood mononuclear cells [80, 81]. Significantly increased levels of tnf-, il-1, and il-6, stimulated by lipopolysaccharide (lps), have been reported in an ibs - d subgroup while increased levels of lps - stimulated il-1 were described in an ibs - c subgroup (figure 1). In pi - ibs, lps - induced cytokines (tnf-, il-1, and il-6) are significantly increased when compared with controls . Increased concentrations of il-1 are associated with the development of ibs symptoms such as alteration in bowel habits . Thymus - derived t regulatory cells (treg) are involved in the suppression of inflammation in ibs, ulcerative colitis, and crohn's disease through the inhibition of t effector cells . It has been shown that t cells express high levels of cd25 tregs in the colon in ibs patients . Therefore, any alteration in the frequency of tregs may lead to recruitment of immune effectors which, consequently, results in inflammation [83, 84]. Intestinal epithelial cells recognize pathogens by way of pattern recognition receptors including toll - like receptors (tlrs) and nucleotide - oligomerization - domain-(nod) like receptors . They induce innate immune responses by the transcription and translation of antimicrobial proteins and the induction of proinflammatory cytokines and chemokines through the nf-b pathway [30, 70]. Ten tlrs have been reported in man so far, which recognized various microbial pathogens, including viruses, bacteria, fungi, and protozoa . A recent study has reported upregulation of tlr 4 and tlr 5 and down - regulation of tlr 7 and tlr 8 in ibs patients . Increased levels of tlr 4 and tlr 5 indicate that their cognate ligands, lps and flagellin, are also increased in ibs patients . Since the ligand for tlr 7 and tlr 8 is single - stranded rna, decreased level of tlr 7/8 suggested that viral infection may also play an important role in the development of ibs - like symptoms . These data further support that increased permeability is present in at least a subgroup of ibs patients [28, 85]. -defensin 2 is an antimicrobial protein secreted by intestinal epithelial cells and induced by tlr 4 . Increased levels of -defensin 2 have been reported in the intestine of patients with either ibs or ulcerative colitis [84, 85]. Several studies have shown that commensal bacteria may reduce inflammation, in part, by directly acting on dendritic cells to stimulate the induction of il-10 and regulatory t cells (treg). With an increase in the number of commensal bacteria, dendritic cells provide signals to lymph nodes to stimulate adaptive immune responses leading to induction of iga antibodies that wrap the luminal antigens and, thus, prevent them from breaching the intestinal barrier and the inhibition of the systemic immune response . Dendritic cells can directly sample luminal pathogens without disruption of tight junctions [30, 55, 86]. Degranulation of mast cells releases histamine and other potent mediators that can influence the function of the enteric nervous system and smooth muscles, causing ibs - like symptoms . In addition, a study has shown that mast cells are significantly increased in the caecum in patients with ibs . Tryptase, a protease released by mast cells, has been reported to be significantly increased in the colonic mucosa of patients with ibs . Increased concentrations of serine protease the observation of dysbiosis in the gut microbiota, altered mucosal barrier function, activated immune responses, and sibo support a role for bacteria in the pathogenesis of ibs . Probiotics are live or attenuated microorganisms which, when administered in sufficient quantities, have been shown to improve gut epithelial integrity, as well as alleviate the symptoms of ibs [30, 8890]. Previous studies have shown that the administration of adequate amounts of probiotics (live microorganisms) may alleviate the symptoms of ibs, suppress proinflammatory cytokines, and promote the integrity of the intestinal barrier [3, 31]. One study showed that the consumption of bifidobacterium infantis 35624 was associated with proliferation of t regulatory cells, reduction of proinflammatory cytokines, down regulation of t cells, reduced expression of co - stimulatory molecules, and attenuation of nf-b . In vitro, increased levels of proinflammatory cytokines (il-12) and decreased concentrations of the anti - inflammatory cytokine (il-10) by pbmcs have been reported in ibs patients . The ratio of il-10/il-12 was altered in ibs patients compared to healthy volunteers and the administration of bifidobacterium infantis normalised this ratio . Probiotics should have the following characteristics: (1) they must survive in the gastrointestinal tract following passage and eventually reside in the colon, (2) they must not have a major adverse effect on other beneficial bacteria in the gut, (3) they should be hostile to mutagenic or pathogenic organisms in the gut, and (4) they must be stable genetically . In clinical studies, probiotics have been shown to improve infectious or secretory diarrhea, traveller's diarrhea, and antibiotic - induced diarrhea via a number of mechanisms that may include direct effects on gastrointestinal motility and the enteric nervous system . In a separate study, patients with ibs were treated with bifidobacterium infantis or lactobacillus salivarius (1e10) in malted milk or malted milk alone (as a placebo) for 8 weeks; there was a significant reduction in abdominal pain, discomfort, bloating, distension, and bowel movement difficulty in patients who received bifidobacterium infantis compared with those who had placebo . Vsl#3 contains a mixture of different bacterial species including lactobacillus species (l. casei, l. plantarum, l. acidophilus, and l. delbrueckii), bifidobacterium species (b. longum, b. breve, and b. infantis), and streptococcus thermophilus . A randomized controlled trial showed that the oral administration of vsl#3, twice daily for 8 weeks, significantly reduced abdominal bloating, but not other parameters (colonic transit time, bowel dysfunction, abdominal pain, flatulence, or urgency) in a subgroup of diarrhea predominant ibs patients, when compared with placebo . In a second study targeting 48 ibs patients with bloating, vsl#3 significantly reduced flatulence and colonic transit compared with the placebo group . In summary, many clinical trials have investigated the therapeutic benefits of probiotics in patients with ibs . However, differences in duration of therapy, heterogeneity in species or strains of selected bacteria, and differences in characteristics of the enrolled patients have resulted in inconsistent results . Prebiotics are nondigestible dietary supplements that affect the host by stimulating the growth of beneficial bacteria in the colon . Prebiotics have the capability to stimulate only microbes which are already residing in the gut . Prebiotics are fermented by host bacteria and have been associated with a reduction in the level of triglyceride, improvement of the postprandial glucose level and a reduction in intestinal permeability [3, 95]. The fermentation of prebiotics leads to the production of scfas such as butyric acids, which can serve as energy source for intestinal epithelial cells . When galactooligosaccharides are used as prebiotics, they are known to stimulate gut bifidobacteria in ibs patients and, thereby, reduce the symptoms of ibs . A potential limitation of prebiotic treatment is that prebiotics undergo fermentation and could produce bloating and flatulence . One study has shown that a combination of bifidobacterium spp . And a prebiotic, inulin, significantly increased the quantity of bifidobacteria . Furthermore, prebiotics also help passage of probiotics through the upper gastrointestinal tract and facilitate their establishment in the colon . Is associated with dysbiosis and sibo, prokinetics could benefit ibs patients through an effect on the microbiota . However, studies reporting the use of erythromycin for the treatment of ibs have shown limited efficacy . Furthermore, domperidone and cisapride were not always effective for the treatment of ibs . In any event, cisapride has been withdrawn from the market due to adverse cardiac effect . As discussed in previous sections, accumulating data support the role of bacteria in the etiology of ibs [102104], and studies using antibiotics to target the intestinal microbiota to treat ibs are now emerging . In a double blind, randomized placebo - controlled study, neomycin was more effective than placebo in reducing ibs symptoms . However, the use of neomycin in the treatment of ibs has been limited by a marginal degree of efficacy above placebo and side effects . Rifaximin, derived from rifamycin, is highly concentrated in the gut lumen and has little systemic absorption . It has been used in the treatment of traveller's diarrhea and sibo . In a recent, large, double - blind, placebo - controlled trial, in which subjects were administered 550 mg rifaximin 3 times daily for 2 weeks and followed up for 10 weeks, there was a significant reduction in global ibs symptoms in the rifaximin group in comparison to placebo (40.8% versus 31.2%). In addition, there was a significantly greater reduction in bloating in those who received rifaximin compared to placebo (40.2% versus 30.3%) [89, 103, 105]. A combination of probiotics and antibiotics may play a beneficial role in the treatment of ibs symptoms . Probiotics may increase the efficiency of antibiotics and reduce gastrointestinal pathogens by the production of antibacterial molecules including bacteriocins . The literature on pi - ibs, sibo, the relationship between gut microbiota and gi sensorimotor functions, and the potential for probiotics and antibiotics to alter these functions and to improve some of the symptoms of ibs, taken together, provide strong evidence in support of a major role for the gut microbiota in the pathogenesis of ibs . This concept represents a potential paradigm shift in our understanding of the underlying mechanism (for at least a subset of patients with ibs) from that of ibs as an entirely psychosomatic disorder to that of a more organic disorder related to an altered gut microbiota and low - grade inflammation . This could, ultimately, lead to a potential change in the management of ibs to strategies that alter the gut microbiota and inflammation.
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High - heeled shoes (hhss) are defined as shoes in which the heel is higher than the forepart . Hhss often also include a narrow toe box, rigid heel cap, and curved plantar region, all of which interfere with natural foot motion . Hhss have been worn for several centuries1, and despite numerous cautions against their use, they remain extremely popular . Surveys suggest that 37% and 69% of women wear them in their daily lives, representing a huge proportion of the female population2 . The use of hhss cause abnormal postural alignment, particularly in the lower limbs and spine3 . Thus, increased forward head posture, lumbar hyperlordosis, pelvic anteversion, and valgus knees . Standing in hhss causes immediate and temporary postural changes because of forward shifting of the center of gravity3 . When hhss are removed, the body goes back to its original alignment . However, previous studies4 indicated that the permanence of postural changes with excessive use of hhss . Additionally, wearing hhss has been associated with an increased potential for slips and falls because the consequent changes in local sensation around the ankle may affect posture balance in women5 . If the heel height is> 5 cm, women tire easily because it is more difficult for the feet to balance the body weight5 . They attempt to provide a more normal physiologic standing posture and static balance than general hhss by making use of tunnel technology with excellent shock absorption and a rearward decrease in the wedge angle . The purpose of this study was to investigate the effects of revised hhss on the foot pressure ratio and static balance during standing . The subjects were 15 young women (21.7 2.1 years; 161.2 5.8 cm; 50.4 5.6 kg) in one of two conditions: (1) wearing general hhss with a heel height 7 cm, or (2) wearing revised hhss with a heel height 7 cm . Exclusion criteria were past or present neurological or musculoskeletal diseases, contractures of the lower limbs, and significant weakness of the quadriceps muscles that would preclude full knee extension while sitting . In addition, pregnant women or women with any psychological problems were excluded . In order to get familiar with the test shoes, subjects were allowed to wear several different sizes, and were allowed to familiarize themselves with the walk around . Foot pressure ratio and static balance were measured during standing tasks using a spacebalance 3d system (cybermedic corp ., the spacebalance 3d system was equipped with 2 wireless force plates (foot placement: 20 cm from heel to heel and 24 cm from toe to toe) that recorded the weight distribution of 4 zones (front, back, left, and right), and a sensor located in the front measured plate inclination . During measurements the foot pressure ratio was calculated as the ratio between the front and back pressures and, expressed as a percentage (%). Static balance was evaluated under 3 conditions (eyes open, eyes closed, screen blocked), and the balance posture ratio (bpr) score was calculated by multiplying by a weight (a= 100%, b= 80%, c= 60%, d= 40%, and e=20%) to zone (a e) ratio . Data analysis was performed using spss version 21.0 (ibm corporation, armonk, ny, usa). The kolmogorov - smirnov test was used to assess the normality assumption . After verifying that the data were normally distributed the independent t - test was used to compare the data between the revised hhss and general hhss . The forefoot pressure ratio was 51.5% for the revised hhss and 62.0% for the general hhss, and the differences between the hhs conditions were significant . The rearfoot pressure ratio was 48.5% for the revised hhss and 38.0% for the general hhss, and the differences between the hhs conditions were significant . The static balance score was higher for the revised hhss than for the general hhss under all 3 conditions . However, the differences between the two types of hhs were not statistically significant (table 1table 1.comparison between the general and revised high heeled shoes (hhss)variablerevised hhssgeneral hhssforefoot (%) 51.5 4.962.0 7.3*rearfoot (%) 48.5 4.438.0 5.8*eyes open (score)90.7 5.288.7 4.9eyes closed (score)88.2 6.885.1 6.2screen blocked (score)88.2 7.187.0 6.0*p<0.05). Standing while wearing hhss causes forward weight shifting due to the change in the plantar pressure and in the location of the center of gravity (cg)3 . Wearing hhss first provoke elevation of the calcaneus bone6 . Changing the forward displacement of the cg, resulting in postural imbalance and promoting adaptive postural adjustments for balance recovery3, 6 this occurs because of the adaptability of the postural system, which meets the demands of the excessive or attenuated postual muscle activities against gravity . So that, either by increasing the heel height, or decreasing the base of support, results in joint pain, muscle shortening, and forefoot deformities3 . Revised hhss were developed to address the weaknesses of general hhss, and the purpose of this study was to investigate the effects of revised hhss on the foot pressure ratio and static balance during standing . When wearing general hhss, the pronounced misalignment of the ankle in plantar flexion and increased plantar pressure in the forefoot area promote overload of the forefoot7 . Schwartz et al . Demonstrated that at a 5-cm heel height, pressure under the forefoot increases and heel pressure decreases8 . Consequently, the ankle joint axis moves anteriorly, and the line of gravity moves posteriorly toward the ankle joint9, 10 . Moreover, van der leeden et al . Established a relationship between joint damage and increased forefoot pressure11 . In the same way, a reduction in the weight bearing area in hhss can result in pain12 . Furthermore, the thinner the shoe heels, the greater the instability of the ankle, the overall imbalance, and the postural impairment3 . . However, habitual wearing of hhss seems to lead to greater strength through the range of ankle joint plantar flexion, compensating for the loss of control13,14,15 . Our study demonstrated that revised hhss produce significantly higher rearfoot pressure and lower forefoot pressure than general hhss . Additionally, revised hhss have a greater positive effect than general hhss on static balance . Revised hhss seem to normalize physiologic standing posture and static balance by making use of tunnel technology with excellent shock absorption and a rearward decrease in the wedge angle . In conclusion, this study showed that in comparison with general hhss, revised hhss produce higher pressure on the rearfoot, lower pressure on the forefoot, and improved balance control . The only limitation of this study is that the alteration during standing for the revised hhss was not based on a sufficient variety of methods . Further research should be conducted to determine the effects of the use of revised hhss on posture with a larger number of subjects and a variety of methods.
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Herries ([1, page 11]) reanalyses the published data from the most recent excavations at twin rivers and argues that there is considerable uncertainty about the age of the deposits and as a consequence about the significance of the site . He concludes that the sequence may be significantly younger than claimed and makes the general observation that excavators need to be extremely careful when relating fragments of flowstone to wider archaeological deposits in caves due to their complex depositional history . Often dates are presented without any information regarding their reliability or context ([1, page 11]). As the archaeologist responsible for the most recent research at twin rivers, herries, however, in the case of twin rivers has misunderstood the stratigraphic sequence of the site and unwittingly conflated dates from unrelated deposits . He has also overlooked our critical assessment of the reliability of the association of the dated material with the archaeological deposits . His resulting reinterpretation throws considerable doubt on the integrity of the published association of dates with the deposits excavated in 1999 . According to herries his concerns .many researchers are sceptical over the association of the flowstone to the msa bearing deposits . If this is indeed the case [no sources are cited] then this uncertainty needs to be discussed in detail so that the reader can assess the validity of the critique . A brief history of the excavations provides the context for understanding the limitations of the site and the rationale for the most recent research in the 1990s . The location of the dated samples in relation to the excavated deposits is made clear which in turn reveals the source of herries' confusion . Twin rivers hill is located near the capital city of lusaka and was extensively excavated in 1954 and 1956 by clark . The excavated deposits were bone and artefact bearing breccias found in karst features (fissures and former cave passages) on the top of the hill and down its western flank . The largest area excavated was labelled f block (on the hill top) and below it the breccias were sampled in a series of excavation blocks labelled a the breccias were removed by controlled blasting using dynamite, and the blocks were reduced manually to extract artefacts . The artefacts were attributed to the lupemban industry of the middle stone age based on similarities with lupemban material excavated at kalambo falls to the northeast . Radiocarbon dating was the only relevant radiometric technique available in the 1950s, and unsurprisingly the lateral extent of flowstone deposits was not recorded as that information was not considered significant at the time . (the development of uranium - series dating of carbonates in the 1970s postdated the excavations at twin rivers .) It was clear from clark's published sections that flowstone was interlayered with the f block breccia . The lack of information on the lateral extent of flowstone remains problematical for reconstructing the association of the dated flowstone with the breccia deposits . The roofs of the cave passages (f and a block) had collapsed in the past and so it was not possible to identify points of entry for water that formed the flowstone deposits . The hill surface had also undergone extensive lowering by dissolution that meant the original height and size of these two separate caves could not be reconstructed (see, pages 169172 for site formation details). These far from ideal conditions were the starting point for the research undertaken in the 1990s . The site was reexamined in 1995 to collect flowstone from f block for uranium - series dating (th / u). A band of flowstone illustrated in clark's section drawing was sampled from near the top of the 1956 excavation, but only isolated patches of breccia remained on the passage surfaces as recorded in our figure 10.15 ([4, page 182]). The middle pleistocene age that resulted (230 35/28 ka, barham and smart 1996) and subsequently redated using higher precision thermal ionisation mass spectrometry [tims] to 195 19 ka, indicated that the underlying lupemban was probably of comparable age if not older . Although no breccia was sampled, we found a small tufa and sediment filled dip tube off the passage, and that material was excavated in 1999 . These deposits could not be associated directly with dated flowstone from the passage . In 1999, a flowstone was found that extended from near the base of the passage to the top and it was sampled at its thickest point (overlying a limestone shelf) for dating . The resulting two ages from different layers within the sample were younger (~178 ka and ~139 ka, see below) than the flowstone from near the top of clark's section . As we reported, the irregular morphology of the f block passage had made the stratification of speleothem more complex than in a block ([4, page 181]). Down slope in a block sufficient breccia deposits survived along the western wall of this passage to justify making this area the focus of excavation in 1999 . A series of flowstone deposits also survived adhering to the walls and floor of the a block passage, and one sample was interlayered with the artefact bearing breccia . The underlying breccia contained a fragment of flowstone dated to beyond the age limit of the technique (> 400 ka), and a flowstone on the floor of the cave passage was also beyond the age range (> 400 ka). Above the breccia were two separate flowstones that formed in either a passage or fissure now destroyed by the erosion of the hill slope ([4, page 178]). These flowstones form part of the concordant sequence of dates from this western wall (below), and more significantly they provided a minimum age for the underlying archaeological deposit of 172 ka/225 ka . The a block dates are discussed in more detail below as they are critical to unravelling the source of herries' confusion, but there is one more area of the site to be described in this history as it too features in his reinterpretation of the site's chronology . G block forms a part of the hilltop platform and is separate from f and a block and is not a cave or fissure but a lag deposit resulting from the dissolution of the limestone . Though not part of the original project plan for 1999, the sampling of g block was initiated to give a better understanding of the archaeological record on the surface of the hill top and to assess its formation . Clark found later stone age and iron age material in these surface deposits, and our excavation confirmed his observations but also added middle stone age tools to the record . The densest concentrations were found in a shallow linear root - filled solution feature (grike) from which burnt quartz and sediments were collected for thermoluminescence (tl) dating ([4, pages 181183]). As the excavation of g block had not been planned in advance, there was no opportunity to incorporate in situ dosimetry as part of the sampling procedure . The resulting tl dates were all late pleistocene in age (ranging from ~101 ka to ~13 ka), but they showed no consistency with depth with the youngest dates associated with the base of the grike ([4, page 183]). Given the extent of mixing, no quantitative analysis of the archaeological deposit was undertaken . Our report included illustrations of middle and later stone age retouched tools to demonstrate their presence, and the suggestion was made that middle stone age might be associated with the ~101 ka date . Herries unfortunately incorporated the g block tl dates in his analysis of the f and a block speleothem dates without making reference to our published assessment of the deposits as being mixed . He inserted the misleading comment that the g block dates, being younger than the speleothem ages, lend further suspicion to an extremely complicated stratigraphy and infill (the full quote is presented below). The g block sediments are of course part of twin river's archaeological record, but they are unrelated to the formation or age - range of the cave passages and should be treated with great caution generally . The primary area of excavation in 1999 was the surviving breccia and decalcified sediments adhering to the western wall of the a block passage . The main aim of the excavation was to recover artefacts from contexts that could be dated by association with speleothem (using u - series tims). Six flowstone samples were collected from a block of which five came from the western wall of the passage and the sixth (tra5a) was from the eastern side of the passage and unrelated to the surviving deposits as was stated clearly and illustrated in our report ([4, pages 178, 181 and figure 10.5]). With the exception of one sample (tra14a) all the flowstone samples were collected from lenses overlying and within the breccia ([4, page 172]). Tra14a was unusual in that it was a fragment of flowstone that had become incorporated into the breccia . We included a photograph (figure 10.14) showing the stratigraphic context of this one dislocated piece of flowstone as part of our critical discussion of the dating of a block ([4, page 178]). Photographs of the intact flowstones were not included as they were the norm in both a and f block, but herries' point is well taken that photographs should have been included for each speleothem . He also suggests that micromorphological analysis of the contact between the speleothem and archaeological deposit would help assess the depositional history of each sample . This is considered good practice now, but as far as i am aware it was not the norm in 1999 . Our section drawings (10.13 for a block, 10.15 for f block) show the vertical location of the dated samples associated with the surviving breccias and the plan of a block (figure 10.5) shows the horizontal location . In a block, only two samples were accepted by us as being in contact with the archaeological deposits, traa1 and the fragment enclosed in breccia, tra14a . Traa1 is a flowstone lens at the base of breccia wedged between two limestone blocks . The breccia grades into a decalcified deposit (red sandy earth, figure 10.13) that is preserved between the passage wall and the adhering breccia that forms a continuous deposit along the western wall . The detritally corrected age of the flowstone (266 ka) was interpreted as representative of the breccia deposits at approximately this depth in the absence of other dating controls . (the one tl date on calcite from the decalcified deposit lacks in situ dosimetry, and as herries' correctly observes it cannot be considered to be reliable .) The remaining flowstone samples provided maximum and minimum ages for the existence of the cave and for the archaeological deposits excavated in 1999 ([4, page 172]). Samples tra4a and tra3a both formed in the passage or fissure above the breccia (figure 10.5) and each was sub - sampled for dating as two distinct layers could be seen (, page 175). Below the 266 ka age, the flowstone fragment incorporated into the breccia (tra14a) exceeds the age range of the u - series technique used and a sample of flowstone adhering to the bedrock below (tra2a) also has an open date of> 400 ka . We speculated that this basal flowstone might have been the source of the redeposited fragment but concluded that these two samples only provide a potential maximum age for the archaeological deposits ([4, page 178]). We do not know how long after the formation of the basal flowstone that sediments began to fill the passage as slurry flows . The deposits may all be 266 ka and younger or perhaps slightly older given the depth of deposit below traa1 [7, 8]. Block follow and include the uncorrected age, sample identifier, depth below datum, and the corrected age (for further analytical detail see barham et al . 2000 [4, table 10.1]). The thick flowstone sample from f block (figure 10.15, east wall) was subsampled and the results are presented for each layer as is the case for tra4a and tra3a in a block . The corrected ages are included as these have been used in other publications [9, 10]. A block, western wall: 173 3 ka (tra4a, 243 cm, layer 1, corrected age = 173 ka),166 3 ka (tra4a, 243 cm, layer 2, corrected age = 160 ka), 225 4 ka (tra3a, 320 cm, layer 1, corrected age = 225 ka),178 2 ka (tra3a, 320 cm, layer 2, corrected age = 172 ka), 275 6 ka (traa1, 340 cm, corrected age = 266 ka),> 400 ka (tra14a, 383 cm),> 400 ka (tra2a, 390 cm). 173 3 ka (tra4a, 243 cm, layer 1, corrected age = 173 ka),166 3 ka (tra4a, 243 cm, layer 2, corrected age = 160 ka), 225 4 ka (tra3a, 320 cm, layer 1, corrected age = 225 ka),178 2 ka (tra3a, 320 cm, layer 2, corrected age = 172 ka), 275 6 ka (traa1, 340 cm, corrected age = 266 ka),> 400 ka (tra14a, 383 cm),> 400 ka (tra2a, 390 cm). 173 3 ka (tra4a, 243 cm, layer 1, corrected age = 173 ka), 166 3 ka (tra4a, 243 cm, layer 2, corrected age = 160 ka), 225 4 ka (tra3a, 320 cm, layer 1, corrected age = 225 ka), 178 2 ka (tra3a, 320 cm, layer 2, corrected age = 172 ka), 275 6 ka (traa1, 340 cm, corrected age = 266 ka),> 400 ka (tra14a, 383 cm),> 400 ka (tra2a, 390 cm). A block, eastern wall: 199 2 ka (tra5a, 220 cm, corrected age = 192 ka). 199 2 ka (tra5a, 220 cm, corrected age = 192 ka). 199 2 ka (tra5a, 220 cm, corrected age = 192 ka). F block, eastern wall: 181 6 ka (trf layer 1, 163 cm, corrected age = 178 ka),141 2 ka (trf layer 2, 163 cm, corrected age = 139 ka). 181 6 ka (trf layer 1, 163 cm, corrected age = 178 ka),141 2 ka (trf layer 2, 163 cm, corrected age = 139 ka). 181 6 ka (trf layer 1, 163 cm, corrected age = 178 ka), 141 2 ka (trf layer 2, 163 cm, corrected age = 139 ka). The above history of the excavation has been presented in some detail to address herries' claim that in many instances, flowstone is sampled from the wall or edges of a cave cavity without definitive evidence for its association to the archaeology . I repeat, only two sample of flowstone were considered by us as being in contact with the archaeological deposits, traa1 and tra14a, and both were from a block . The near absence of continuous layers of flowstone that could be linked directly to the deposits is an artefact of the history of excavation . The following extended quote from herries ([1, page 11]) contains the primary source of confusion in his account, and the points of misunderstanding are labelled with capital letters for referencing in the discussion that follows . (a) the fact that younger speleothem dated to between 184172 ka (178 6 ka) and 141137 ka (139 2 ka) occurs under a speleothem dated to between 200190 ka (195 5 ka; 131) in block a. [sic] all tl dates from g block are younger than 117 ka (101 16 ka) and lend further suspicion to an extremely complicated stratigraphy and infill . (b) the speleothem dates to between 266 6 ka (272260 ka) and 172 2 ka (174170 ka) may also have been eroded out from earlier deposits when the msa in - filled the cavity . (c) again younger speleothem samples occur with depth with the 172 ka sample being deeper (3.2 m) than the 192 2 ka sample at the top of a block at 2.2 m. (d) all the msa in the top 1 m of a block is, therefore, younger than 174 ka, as the speleothem must have formed before it was eroded and incorporated into the breccia and so provides a maximum age . There is no speleothem dated to 141137 ka (139 2 ka) in a block . The cited age range most closely matches sample trf (layer 2, 141 2 ka uncorrected, 139 ka corrected) from f block (see above). The sample that dated to 195 5 ka (tra5a) occurs on the eastern wall of the a block passage, and unless herries assumes that it was once continuous across the passage, it cannot be related to the west wall speleothem this spatial separation was made clear in the published report and seems to have been overlooked . The speleothem is flowstone deposits that are in situ as described in the report (above) and not redeposited fragments . Only sample tra14a (> 400 ka, 383 cm) is a redeposited fragment of speleothem as was stated clearly in the report ([4, page 178]). Herries expands this line of reasoning later to suggest the if the majority of speleothem represents material eroded into the deposit then the lupemban from block a would be younger than 141 ka, significantly younger than the 266170 ka cited by barham et al . The 141 ka date is erroneous in relation to a block as is the suggestion that the majority of the speleothem samples are redeposited . Again, the sample dated to 195 5 ka (tra5a) cannot be related to any other speleothem on the western wall of a block . There is no extant middle stone age material in the upper 1 m of a block, only from below sample tra3a (320 cm) (see [4, page 178 and figure 10.13]). Herries continues this line of reasoning later in stating that the sample with a date of 160 3 ka also occurs at 2.4 m depth and is the youngest age from block a. this suggests that all the msa in block a may in fact be younger than 163 ka . This sample (tra4a, layer) overlies tra43 and as described clearly in our report is related (along with tra3a) to a separate passage or fissure above the archaeological deposits and unrelated to the material excavated in 1999 . Science - based dating underpins our understanding of the process of human evolution and its variability in place and time . The past 20 years or so have seen a revolution in the development of radiometric methods of dating that have opened the middle pleistocene to closer and more informed scrutiny by palaeoanthropologists . We can now see more clearly the tempo of changes in human anatomy and behaviour that were previously obscured by poor chronological controls . It is in this context of an improving database that herries' provides a much needed review of the archaeological and dating evidence of the transition from the acheulean to the middle stone age in southern africa . Thoughtful reviews play an important role in the development of a discipline by distilling what is known about a subject and highlighting issues for further investigation . Reviews are also often the first port of call for non - specialists and advanced students in need of a clear statement of the state of the art of a subject . Herries cavalier treatment of the twin rivers published data, however, undermines my confidence in what appears otherwise to be a comprehensive treatment of the acheulean - middle stone age transition . Twin rivers remains an important middle pleistocene site despite its history of excavation and complex stratigraphic sequence . I stand by the published interpretation of the age range of the limited a block deposits and am actively searching for a new this is the way forward in a region for which we still know so little about the behaviour of hominins before the evolution of homo sapiens.
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In october 2013, university of north carolina hospitals, an 853-bed facility, implemented a new hand hygiene program (clean in, clean out; http://news.unchealthcare.org/empnews/handhygiene) in all inpatient areas, after a successful pilot implementation of the program in the pediatric intensive care unit (2). Key features were that the focus for observation was simply on cleaning hands upon entering and leaving patient rooms and that all healthcare personnel (including physicians, advanced practice providers, nurses, nursing assistants, hospital unit coordinators, housekeeping, radiology, occupational / physical / recreational therapists, nutrition and food services staff, phlebotomists, and respiratory therapists) were asked to make observations and provide immediate feedback to each other (3). Previously, infection preventionists and designated nursing staff on each inpatient unit performed covert observations of hand hygiene compliance according to centers for disease control and prevention (cdc; atlanta, ga, usa) indications for hand hygiene (1), and compliance reports by location were disseminated quarterly . Comprehensive surveillance for device - associated and non device - associated hai was assessed by 4 infection preventionists according to cdc national healthcare safety network case definitions and included all hospital locations and all infections . We compared hand hygiene compliance data from the last quarter of the covert observations by infection preventionists and designated nursing staff to compliance data from the first month of the new program by using a test . Hand hygiene compliance data were collected at the unit level, and hospital - wide estimates were obtained by averaging all reporting units, weighted by patient - days for each respective unit . We also used a to compare the average historical hai rate from january 2013 until the implementation of the new program in october 2013 to the average hai rate during the study period of october 2013february 2015, after implementation of the new program . We examined overall longitudinal hand hygiene compliance rates and hai rates during the new program by using generalized linear models to describe overall trends . To examine the association between hai and hand hygiene compliance, we used poisson regression using generalized estimating equations with an exchangeable working correlation matrix using sas version 9.3 (sas institute, inc ., cary, nc, usa). We used data for hand hygiene compliance and number of overall hai, hai with multidrug - resistant organisms (mdro), and healthcare - associated clostridium difficile infection (ha - cdi) from each nursing unit to estimate the overall association between hand hygiene and hai rates . An offset of patient - days was used to account for varying levels of time at risk for each unit and month . During the 17-month study period, we noted a significant increase in overall hand hygiene compliance rate (p<0.001) and a significantly decreased overall hai rate (p = 0.0066), supported by 197 fewer infections (figure) and an estimated 22 fewer deaths (4). Overall healthcare - associated infection (hai) rate and hand hygiene compliance by month, october 2013february 2015 . The association between hand hygiene compliance and hai, adjusting for unit - level data, showed a 10% improvement in hand hygiene, associated with a 6% reduction in overall hai (p = 0.086). The association between hand hygiene compliance and ha - cdi, adjusting for unit - level data, showed a 10% improvement in hand hygiene, associated with a 14% reduction in ha - cdi (p = 0.070). No association was noted between hand hygiene compliance and mdro infections (p = 0.7492). Hospital - wide hand hygiene compliance measurements by using the previous method (covert observation by designated staff) in the final measurement quarter were not statistically different than in the first month of compliance data measured by all staff in the new program (p = 0.7503). In addition, the average hai rate in the 9 months before implementation of the new program was not statistically different (p = 0.542) from the average hai rate during the 17-month study period after implementation . When the cdc hand hygiene guideline was published in 2002, hand hygiene compliance was summarized on the basis of then - current studies to be very low (average 40%, range 5%81%) (1). Investigators have demonstrated reductions in hai and mdro infections when compliance increased from low to medium levels (48% to 66%) (6). More recently, hospital epidemiologists and infection preventionists have worked to achieve and sustain higher compliance by using shared accountability, incentives, and feedback strategies (7), but until now, no analysis has demonstrated whether an improvement in hand hygiene from a baseline high level (> 80%) to an even higher level (> 95%) would lead to hospital - wide decreases in hai (8). Demonstrating the importance of continuously improving hand hygiene compliance is critical for staff and hospital leaders who may underestimate the impact on hai . Hand hygiene compliance measurements have been studied and methods have been proposed to alleviate concerns associated with interobserver variation, sampling bias, and the hawthorne effect (9). We overcame these concerns by simplifying the compliance measurement to only evaluate the opportunities that cover most (87%) of the world health organization defined five moments on the basis of a 24-hour validation video surveillance of activity in patient rooms; that is, 21% of episodes before patient contact, 22% of episodes after touching a patient, and 44% of episodes after touching patient surroundings (10). Furthermore, by engaging all hospital staff in measuring hand hygiene compliance, all opportunities of the hygiene program were eligible opportunities for measurement . In this way, the hawthorne effect was a consistent presence that became the main intervention for achieving improvement . Finally, the finding that our previous hand hygiene compliance rates measured by trained, designated staff was not statistically different than the compliance rates from the beginning of the new program further supports that the new compliance metric was not affected by any new, unanticipated measurement bias . Although we cannot eliminate the possibility that other infection prevention factors were also associated with a decreased hai rate, no other specific hospital - wide infection prevention goals were adopted during the time period of this analysis . The associations (and absence thereof) we found with hand hygiene and specific types of infections are biologically plausible . Absence of association between mdro hai and hand hygiene is understandable because many mdro infections occur in patients who may be colonized before admission, have invasive devices, and are at increased risk for becoming infected with their own flora . However, c. difficile infections in healthcare facilities are predominantly spread through contact with infected patients or a contaminated environment, then carried on the hands of healthcare personnel . Therefore, a weak association between ha - cdi reduction and hand hygiene improvement is plausible . Although we adjusted the hand hygiene compliance data by patient - days, some units had patients at much lower risk for infections (e.g., psychiatric units). Despite including units of varying risk for hai, we demonstrated that increased hand hygiene compliance improvements from already high rates can be an important strategy for achieving infection reductions, particularly for healthcare - associated c. difficile infections . A program designed to improve hand hygiene compliance among hospital staff successfully engaged all healthcare personnel in monitoring and improving their own hand hygiene compliance . This pursuit of excellence for hand hygiene compliance led to substantial hai reductions hospital wide.
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World stroke incidence studies have shown that there has been a 42% decrease in stroke incidence in high - income countries and more than a 100% increase in low - to middle - income countries . Early stroke case fatality has decreased in both high - income and low - to middle - income countries1 . Despite the increase in stroke incidence, the decrease in mortality rate leads to an increase in people living with disabilities . Therefore, stroke constitutes the leading cause of serious, complex, and long - term adult disability2, 3 . Although 1530% of the stroke survivors become permanently disabled, 5070% of stroke patients regains functional independence and mostly regains walking ability4, 5 . Due to residual functional disabilities such as impaired balance and motor weakness a study by jorgensen et al . Showed that 73% stroke patients fell in the six months after a stroke, that 21% fell after 6 months, and that over half of all reported falls occurred during walking activities due to problems such as loss of balance, misjudgement, and foot dragging6,7,8 . Balance deficits like reduced postural stability during quiet standing, delayed and less coordinated responses to internal and external balance perturbations, and increased weight bearing on the unaffected limb are one of the main risk factors for falls after stroke9,10,11 . Together with the increased risk of falling, fear of falling increases, self - confidence decreases, and individual autonomy and participation in social activities reduces . Therefore, patients become more dependent on caregivers, and as a result, quality of life decreases . The rehabilitation approaches aiming to increase the balance functions of patients promotes reintegration into the community, recreational and daily life activities, positive behavioral changes, and social acceptance of the patients12, 13 . Balance rehabilitation programs after stroke were found to be effective in enhancing functional recovery; on the other hand, the limited resources of health - care systems hamper the implementation of intensive practice14, 15 . Therefore, innovative rehabilitation technologies including virtual reality systems are currently being used in stroke rehabilitation mainly because they provide an enriched environment, task - specific goals, and repetitive practice16, 17 . These systems may decrease the amount of time spent for one - to - one sessions with physiotherapists and may provide patients with the opportunity to use the systems at home . Currently, nintendo wii fit, video game technology using virtual reality, has become a very popular rehabilitation approach in balance treatment . In the wii fit games, patients are directed to shift their weight anteriorly, posteriorly, and laterally within a center of pressure (cop) range in order to reach a target and to enhance their limits of stability . The wii fit games have been used in the literature due to their effects in improving motor skills and postural control by using visual - perceptual systems, providing information about the performance, showing the direction, speed and acceleration changes, and being interactive, motivating, useful and cheap18,19,20 . Few studies have used the wii fit method for balance treatment in chronic stroke patients . Two case report studies have demonstrated that static and dynamic balance can be improved with the wii fit system in patients with stroke21, 22 . In a previous randomized controlled study in which standard rehabilitation plus virtual reality balance training with wii fit was compared with standard rehabilitation only greater improvement was observed in dynamic balance23 . This may be because of the disparity in the duration (length of session) and intensity (massed practice) of the treatment in the two groups . Therefore, it was expecting that more improvement would be obtained in the experimental group due to more intensive treatment . The aim of this study was to compare the effectiveness of game technology based wii fit balance training (wbt) and progressive balance training (pbt) in addition to a neurodevelopmental treatment program on static and dynamic balance functions, activity specific balance confidence, and activities of daily living in patients with chronic stroke . Individuals with stroke admitted to a physiotherapy and rehabilitation department over a period of 12 months were considered for inclusion in this study . The study was approved by the local ethics committee for medical, surgery and drug research (lut 11/01), and written informed consent was obtained from all patients prior to data collection . The criterion for inclusion was first ever stroke with hemiparesis (6 months). Patients were excluded if they had any other physical problem or epilepsy, moderate to severe cognitive deficits as evaluated by the mini mental state examination test (mmse) (20 points), severe depression as measured by the beck depression inventory (bdi) (30 points), and were unable to walk independently according to the modified rankin scale (mrs) (> 3 points). Participants were randomly allocated to one of two different treatment groups: pbt and wbt . A simple randomization method was used to allocate patients in each group according to registration number (even numbers, pbt group; odd numbers, wbt group). A neurodevelopmental treatment, a problem solving and analytical approach, was applied to each group . The treatment programs were planned considering the functional level, individual needs, and demands of the patients based on selective movement as a basis of functional activity and successful goal acquisition24 . Patients assigned to the wbt group received a 1 hour treatment program consisting of a 30-minute neurodevelopmental training program and 30-minute wii balance training program for a total of 12 sessions, which were held 3 days a week for 4 weeks . Patients assigned to the pbt group received a 1 hour treatment program consisting of a 30-minute neurodevelopmental training program and 30-minute progressive balance training program for a total of 12 sessions, which were held 3 days a week for 4 weeks . The descriptive variables of the patients including age, gender, body mass index, duration of stroke, and affected side were recorded before the treatment . All participants were assessed at baseline (week 0), after the treatment (week 4), and one month post treatment (week 8). The static balance, that is the mediolateral weight distribution on the paretic and nonparetic extremities, was measured with a wii balance board (wbb), which has characteristics similar to laboratory - based force platform (fp) systems that contain four transducers used to assess weight distribution and cop25 . Dynamic balance was measured with four balance tests reflecting different aspects of balance: the berg balance scale, timed up and go test, dynamic gait index, and functional reach test . The berg balance scale (bbs), which is a psychometrically sound measure of balance impairment post stroke, was used to assess functional balance of the patients26, 27 . The test is a 14-item objective measure used to assess the balance performance of patients during functional tasks ranging from sitting to standing on one foot28 . The timed up and go test (tug) was used to measure the functional mobility level of the patients . The time taken to complete the task (standing up from a chair, walking 3 meters, turning around, walking back, and sitting down) the dynamic gait index (dgi), which assesses an individual s ability to modify balance while walking in the presence of external demands, was used to assess balance and gait ability of the patients31, 32 . The functional reach test (frt), which consists of measurement of the maximal distance one can reach forward beyond arm s length while maintaining a fixed base of support in the standing position, was used to measure the stability limits of the patients33 . The balance confidence of the patients was measured with the activities - specific balance confidence (abc) scale developed to measure the confidence of subjects when performing various ambulatory activities without falling34 . The activities of daily living of the patients were evaluated with the frenchay activity index (fai), which contains 3 factors (indoor domestic activities, outdoor domestic activities and outdoor social activities) related to the self care and mobility of patients35 . We calculated that the required sample size was 30 subjects for 90% power with a 5% significance level . The variables are presented as the mean standard deviation (x sd). The differences within groups were tested using the wilcoxon signed - rank test, and those between groups were tested using the mann - whitney nonparametric test . The friedman test was used for repeated measurements, and bonferroni correction was used to assess changes in balance functions from week 0 to week 8 between the groups . The 95% level of confidence (= 0.05, or the margin of error) was used for the assumptions to identify differences in the variance analysis . Forty patients were assessed during the study period . Of these, 33 patients were recruited and randomly allocated into the wbt (n=17) and pbt (n=16) groups . Reasons for exclusions and drop - outs are shown in the study profile (fig . 1). Fifteen patients in the pbt group and fifteen patients in the wbt group completed the 4-week treatment program and the follow - up assessment at 8 weeks . Before treatment, there were no significant differences between the wbt and pbt groups with respect to demographic characteristics, mental state, and level of depression . However, disease severity measured by the modified rankin scale was found to be higher in the pbt group (table 1table 1.demographical and physical characteristics of patientswbt(n=15)(xsd)pbt(n=15)(xsd)gendermale67female98affected sideright87left 78age (years)mean62.8056.60sd10.8716.42body mass index (kg / m)mean29.1428.30sd4.524.56duration (years)mean3.704.23sd4.424.86modified rankin scale (06)mean1.802.40*sd0.770.82mini mental test (030)mean22.8622.26sd2.382.05beck depression inventory (063)mean14.0014.00sd6.147.58*p0.05). The functional balance performance scores of the patients in the wbt group were higher for the bbs and tug tests performed before treatment (week 0). The results of the four different functional balance tests (bbs, tug, dgi, and frt) showed that both groups improved significantly after treatment (week 4). However, there was no significant difference between the groups after treatment (week 4). The patients in the pbt group showed a significantly lower results of the bbs and tug tests in their follow - up measurements (week 8) (table 2table 2.within-and between - group comparisons of all outcome measuresweek 0xsdweek 4xsdweek 8xsdweek 0 vs. 4(wilcoxon test)week 4 vs. 8(wilcoxon test)weight distribution (%) wbtparetic44.047.4748.002.8046.644.40*nonparetic55.887.4552.002.8053.364.41*pbtparetic 47.5410.1648.598.3848.148.94nonparetic52.4610.1651.278.3651.868.94wbt vs pbt(mann - whitney u test) (friedman test) bbs (056)wbt45.605.2650.334.0950.334.16*pbt39.609.3144.807.4844.207.77**wbt vs. pbt(mann - whitney u test)***(friedman test)*tug (sec)wbt 17.967.7716.178.2315.957.93*pbt26.3611.6022.1111.8822.7311.95**wbt vs pbt(mann - whitney u test)*(friedman test) * dgi (024)wbt14.864.1216.863.3516.863.33*pbt12.603.9614.463.4814.864.06*wbt vs. pbt(mann - whitney u test)(friedman test) * frt (cm)wbt 25.265.9229.405.1630.065.16*pbt24.206.6627.136.1926.806.58*wbt vs. pbt(mann - whitney u test) (friedman test) * asbct (0100)wbt 59.6217.2668.3617.2270.1016.92*pbt53.1017.4759.3718.0859.0418.27*wbt vs. pbt(mann - whitney u test) (friedman test) * fai (1560)wbt 14.466.5416.267.1816.206.79*pbt10.406.5212.536.5813.267.37**wbt vs. pbt(mann - whitney u test)*(friedman test) * * statistically significant values (p0.05) are indicated with an asterisk . Bbs: berg balance scale; tug: timed up and go test; dgi: dynamic gait index; frt: functional reach test; asbct: activity specific balance confidence test; fai: frenchay activity index). Bbs: berg balance scale; tug: timed up and go test; dgi: dynamic gait index; frt: functional reach test; asbct: activity specific balance confidence test; fai: frenchay activity index both groups showed significant improvement in terms of the balance confidence (abc) scale from baseline to after the treatment period (week 4). No significant difference was recorded between the two groups after treatment (weeks 4 and 8) (table 2). The activities of daily living measured with the fai were found to be increased in both groups after treatment (week 4). When comparing the groups, no significant differences were found between them after treatment (weeks 4 and 8). The results at follow - up showed that adl continued to improve in the pbt group despite cessation of treatment (week 8) (table 2). The main objective of this study was to compare the effects of balance training with an increasingly popular virtual reality based game technology, nintendo wii fit (wbt), and progressive balance training (pbt) on balance functions, activity specific balance confidence, and activities of daily living in patients with chronic stroke . Although the two approaches tend to be more effective on different parameters of functional balance measures, there were no remarkable superiorities of either treatment approach over the other . On the basis of the idea that visual feedback, which was used more intensely in the wii fit group, would have additional effects on symmetrical weight bearing on the paretic and nonparetic extremities36, the effects of the two treatment approaches on symmetrical weight distribution were compared in our study . Although the weight bearing on the paretic extremity was found to be improved and was more symmetrical in the wbt group, no significant superiority of either treatment method over the other was observed for the weight distribution of the patients . We speculate that the results might be due to the baseline weight distribution of the patients in our study . The average weight distribution of the patients on the nonparetic extremity at the beginning of this study was relatively symmetrical (5255%) compared with the values specified for stroke patients in the literature (6180%)11 . Moreover, the weight distribution was more symmetrical in the pbt group (52%) than in the wbt group (55%); thus the effects of the treatment methods might not have emerged exactly . The balance exercises used in both treatment methods included weight shifting activities closely related to static balance, i.e., the ability to maintain a chosen posture with minimal postural sway10 . These exercises aims to improve postural control by increasing anteroposterior and mediolateral weight transfer and reducing postural sway, but do not especially focus on improving dynamic balance . As postural control is a prerequisite for most functional activities, weight shifting exercises aimed at improving postural control, may have significant effects on balance during functional activities . All of the functional balance measurements (bbs, tug, dgi, and frt) applied to the patients for that purpose showed that functional performance was improved in both groups . We supposed that training approaches emphasizing weight shifting abilities, whether using visual feedback (wbt) or not (pbt), provides benefits to both static and dynamic aspects of balance . The measurements after 8 weeks showed that there was a decline in the bbs and tug test results in the pbt group . In a case - control report of deutsch et al ., two individuals in the chronic phase post stroke received balance training with either a nintendo wii and wii fit program or standard of care balance and mobility program for 12 training (60-minute) sessions, and it was found that the patient who received the standard therapy maintained his improvements after treatment . It was propounded that the activities used for the treatment (balance and coordination exercises and outdoor activities e.g. Crossing the road, walking in the busy areas, and market shopping) were transferred to real life activities due to the similarity between them, and thus the patient had used these activities after treatment22 . Considering the activities of the study mentioned above, we did not include outdoor activities in addition to the balance exercises in our study; this may constitute a reason why the improvements were not maintained after treatment in our study . Balance confidence, defined as the perceived ability or confidence to perform an activity or action without falling37, was found in our study to be improved in both groups after treatment . Bandura s theory identifies four factors affecting self - efficacy, that is, one s belief in one s ability to succeed in specific situations: performance accomplishments (physical practice), vicarious experience (mental practice), verbal persuasion (encouragement), and emotional sources (motivation)38 . Associating these factors with falls - related self - efficacy, it can be said that both the pbt and wbt approaches may involve adequate physical practice, mental practice, encouragement, and motivation, which lead to improved balance confidence in patients . The results for activities of daily living results indicate that the patients in both groups improved after treatment . In addition to this result, improvements in the pbt group proceeded after the cessation of treatment (week 8). It is known that the wii fit balance training method includes task - specific activities, though wii fit exercises are not very similar to daily life activities in many ways . Games, therefore, even though there are improvements in functional balance, these activities do not contribute to the transfer of improvements to daily life activities . In a further aspect, progressive balance training is more similar to daily life activities, and patients may perceive pbt activities as games, therefore, pbt may result in more improvement in patients daily lives . The limitations of this study include the fact that, the feedback given with wii fit may not be sufficient for patients to use normal movement strategies; therefore, physiotherapists should prevent abnormal patterns during activities . In another way, during the wii fit training, the need for a physiotherapist to help with symmetrical weight shifting poses a problem with respect to use of this technology at home . The main aim of this study was to use wii fit technology in order to reduce the need for one - to - one interaction between physiotherapist and patients; however, if there is a necessity for physiotherapists to correct the abnormal patters used by patients, it should be questioned whether this technology can be used at home or not . This study found that physiotherapist - assisted wii fit balance training is an effective method, but there is a need to compare the effects of wii fit balance training with and without any help from physiotherapists in future studies . Another limitation of this study was the disease severity, which was found to be higher in the pbt group . This limitation was due to the randomization procedure; therefore it could not be eliminated . Some studies have asserted that although there are beneficial effects of using wii fit in rehabilitation, this technology may have some safety problems . Like the other commercial game systems, the wii fit system is a black box; thus various parameters of the system cannot be controlled . In this sense, not being a rehabilitation - specific tool, being difficult for people with physical problems, allowing the use of abnormal movement patterns, having an incomprehensible scoring system, giving feedback that is not rehabilitation specific, and making degrading comments sometimes are some problems preventing the wii fit system from becoming widely used in rehabilitation22, 39 . There are also reports of injuries arising from the use of nintendo wii game systems (metacarpal and metatarsal fractures, wrist and ankle traumas, clavicular fracture, patellar dislocation, muscle and tendon injuries, c7 fracture, stroke, and hemothorax) in the literature40,41,42,43,44,45,46 . In this sense, there were no negative effects and/or any injuries attributable to use of wii fit in our study . To our knowledge, this is the first study comparing the effectiveness of wii fit balance training (wbt) and progressive balance training (pbt) in addition to neurodevelopmental treatment approaches in patients with chronic stroke . The two treatment programs used in the current study were both found to be effective on balance functions, activity - specific balance confidence, and activities of daily living . When comparing the two treatment approaches, they seem to be effective on different parameters of balance performance, with no superiorities of either treatment approach over the other . Wii fit balance treatment can be used as an effective method for patients with difficulty accessing treatment or for physiotherapists who require alternative methods of treatment.
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The cytoskeleton has a well - organized and dynamically regulated subcellular architecture composed of core filaments and various accessory proteins . A variety of cellular signaling events are supported by cytoskeletal / scaffold components, which in some cases constitute structural hubs for signaling and/or in other cases contribute directly or indirectly to signal transduction . Such roles of cytoskeletal elements are not limited to purely cytoplasmic events but extend to signals ultimately targeted to the nucleus . Nuclear signaling is fundamentally important for cells to properly orchestrate adaptive responses to various environmental changes and functional requirements . The spatio - temporal steps involved in transducing a signal from the plasma membrane to the nucleus occur in different subcellular locations: signal reception / transduction at the plasma membrane, signal amplification and cascade in the cytoplasm, shuttling / signaling through the nuclear pore complex (npc) at the nuclear envelope, and modulation of nuclear functions . Cytoskeletal involvement in signal transduction is well documented in signal recognition at the plasma membrane and the signal cascade in the cytoplasm . Many membrane receptors and transporters interact with the cytoskeleton (e.g., ankyrin and spectrin) and transmit their specific signals to their respective target sites . Thus, the cytoskeleton often functions as a platform for signal transduction in the cytoplasm and has been assumed to only indirectly contribute to the subsequent steps of nuclear signaling, that is, signal transmission to the nucleus and regulation of nuclear functions . There is increasing evidence, however, that many types of cytoskeletal proteins are localized to the nucleus, suggestive of their direct involvement in the transmission of nuclear signaling and the regulation of nuclear functions (table 1). Some of these are predominantly localized to the cytoplasmic side and nearly undetectable in the nucleus by using fluorescence techniques under normal conditions; even so, a small subpopulation of this class of proteins shuttles between the cytoplasm and the nucleoplasm [1, 2]. In another case, homologues, variants or fragments of several cytoskeletal proteins are predominantly found in the nucleus [3, 4]. Such cytoskeletal proteins are strong candidates for components of the nucleoskeleton, the structural basis for nuclear functions . In addition to the linc complex, which links the cytoskeleton and nucleoskeleton by sun - kash protein interactions penetrating through the nuclear membrane, the two compartments may be directly connected via the exchange of structural molecules . To fully understand the biological significance of the cytoskeletal proteins in the nucleus, the molecular mechanisms of their nuclear translocation should be clarified . We will review the studies of cytoskeletal proteins in the nucleus and propose possible mechanisms that regulate their nuclear localization . Actin is well known as a component of actin filaments and is one of the most abundant proteins in cells . The first report of actin in the nucleus was published in 1969, and the association of nuclear actin with rna polymerase ii was found subsequently [3335]. A report in 1998 revealed the nuclear export signal- (nes-) dependent active nuclear export of actin through the npc . Thereafter, multiple roles of nuclear actin have been revealed, including transcription [3639], chromatin remodeling, and mrna transport [41, 42]. Furthermore, varieties of other cytoskeletal proteins are localized to the nucleus and are involved in the regulation of nuclear functions (table 1). The notion that localization of cytoskeletal proteins in the nucleus is indispensable for proper nuclear functions is now gaining widespread acceptance . Nuclear actin has been implicated in a variety of nuclear functions, such as transcription [3639], chromatin remodeling, and molecular transport of mrna [41, 42] and proteins . It associates with an swi / snf - like chromatin remodeling complex, hnrnps [39, 42, 45], and rna polymerases i, ii, and iii [4650]. These findings of nuclear actin in various fundamental biological processes substantiate the constitutive requirement for actin in the nucleus . One of the most famous actin - interacting proteins in the cytoplasm, myosin, is also found in the nucleus . Nuclear myosin i (nmi) is a monomeric and single - headed myosin, which possesses an additional n - terminal 16 amino - acid domain that directs the molecule into the nucleus [7, 8]. Unlike nuclear actin, nmi is a nuclear - specific isoform, which is almost exclusively localized to the nucleus . Nmi functions cooperatively with nuclear actin and especially participates in the association with the chromatin remodeling complex and rna polymerases [47, 51]. The precise molecular states of actin in the nucleus, the monomeric or polymeric form, have not yet been fully resolved . A role of nuclear actin in the serum - induced gene activation pathway favors monomeric actin in the nucleus . Mal, a coactivator of the serum response transcription factor (srf), is known to interact with monomeric actin . Under normal conditions, mal - actin complexes are actively exported from the nucleus, possibly due to the nes of the actin molecule . When serum - induced signaling is triggered, the mal - actin interaction is disrupted, resulting in the accumulation of mal in the nucleus and the activation of srf target genes . On the other hand, polymerized actin is required for the transcription of ribosomal rna by rna polymerase i . Nuclear actin and nmi cooperate in the activation of rrna transcription, and actin - dependent motor activity of nmi is required for transcription elongation . Coupled with the observation of actin - containing filamentous structures around the npc, it is likely that nuclear actin exists in polymerized states, at least to some extent . Considering the cellular concentration of actin, which is estimated to be 100 m, remarkably higher than the critical concentration for polymerization, and also the high abundance of actin in xenopus oocyte nuclei, it is reasonable to assume that both monomeric and polymeric forms of actin exist in the nucleus . Mobility measurements of nuclear actin using fluorescence recovery after photobleaching (frap) provided important supporting evidence . Recovery of the fluorescence of gfp--actin demonstrated that nuclear actin contains several different kinetic populations, suggesting that approximately 16% of the nuclear actin is in a polymeric form . In a recent study demonstrating the role of actin and actin - interacting protein in xenopus oocytes, these findings suggest that dynamic conformational changes of the nuclear actin in association with its interacting partners play significant roles in various cellular events, both for general biological processes and for specific signal responses . Several different isoforms of myosins, other than nmi, were also found to localize to the nucleus . Myosin ii (smooth muscle myosin) is localized to the nucleus in smooth muscle cells and regulates transcription by binding to the promoter regions of target genes and interacting with the rna polymerase ii complex . Myosin vi is also detected in the rna polymerase ii complex, and it enhances transcription much like myosin ii . Myosin va and vb were recently found in nuclear speckles and nucleoli, and are involved in transcription by rna polymerase i [10, 11]. One of the cytoplasmic actin - depolymerizing factors cofilin-1 was also found to function in the nucleus . Cofilin-1 associates with a complex containing nuclear actin and phosphorylated rna polymerase ii, and it plays a key role in transcriptional elongation, presumably by regulating polymerization states of nuclear actin along target genes . Several different mechanisms were found to target neuronal wiskott - aldrich syndrome protein (n - wasp), a primary cytoplasmic regulator for cortical actin filaments, to the nucleus . N - wasp phosphorylated by focal adhesion kinase at a tyrosine residue y256 is predominantly localized to cytoplasm while wild - type and kinase - insensitive mutant y256f are localized both in cytoplasm and in the nucleus . Coprecipitation assays demonstrated that n - wasp interacts with a multifunctional large nuclear protein complex, psf - nono (polypyrimidine - tract - binding - protein - associated splicing factor - non - pou - domain octamer - binding protein / p54nrb), that functions to couple n - wasp with rna polymerase ii to regulate transcription . Since n - wasp modulates actin polymerization in nuclear extracts in vitro, it is suggested that nuclear n - wasp promotes polymerization of nuclear actin to regulate transcription . It is likely that the regulation of actin polymerization is a critical factor for nuclear transcription, and there are various factors that cooperatively modulate the process, just as in the cytoplasm . There are at least ten actin family proteins that are conserved among eukaryotes, termed actin - related proteins (arps), which show 3070% similarity with actin and possess a similar atp binding motif . In spite of their well - conserved molecular structures six of them, including arp4, 5, 6, 7, 8, and 9, displayed predominant nuclear localization in s. cerevisiae, and it is also the case for at least four human arps, arp4, 5, 6, and 8 [3, 16]. In addition to nuclear actin, these nuclear arps are known to serve as a functional subunit of evolutionally conserved chromatin remodeling complexes, such as ino80, swr1, rsc, and adcr [6062]. Taking into account the atp - dependent mechanical sliding or displacement of nucleosomes by a chromatin remodeling complex, nuclear actin and arps may function as a structural element for chromatin dynamics, just as the cytoplasmic actin cytoskeleton serves as a platform for various molecular dynamics . Several nuclear arps are known to participate in chromosomal organization and mitotic chromosomal segregation without associating with chromatin remodeling complexes . Arp6 binds to several chromatin regions including centromeres and the promoters of ribosomal protein genes . This association is required to anchor a specific chromatin region to the nuclear periphery, suggesting a role of arp6 in organizing proper chromatin regions in the nucleus . When the endogenous arp8 was depleted by sirna transfection, mitotic cells exhibited abnormal chromosome alignment . Some of the arps are expressed in a tissue - specific manner . Human arpt1 and mouse arpm1 are exclusively expressed in testis [66, 67]. Arpm1 interacts with profilin iii, a testis - specific nucleotide exchange factor for actin, suggesting that the arpm1-profilin complex functions in spermiogenesis - specific chromosomal organization . It is expected that nuclear arps may participate in specific steps of development or differentiation, while widely expressed nuclear actin is involved in the regulation of general nuclear functions . The spectrin - repeat (sr) is a widely conserved domain from bacteria to humans, found in several cytoskeletal crosslinking proteins such as spectrin, actinin, dystrophin, and nesprin . A single sr is a rod - shaped structure consisting of a bundle of three amphiphilic -helices facing each other via hydrophobic interactions, forming a hydrophobic inner surface with a hydrophilic outer surface . Sr - containing cytoskeletal crosslinkers possess multiple srs and crosslink their target filaments into variously arranged forms, such as a contractile bundle, parallel array, or meshwork . In the last few years, several sr proteins have been found in the nucleus . -actinin contains four srs at the center of the molecule and forms a homodimer to crosslink actin filaments . One of the nonmuscle -actinin isoforms, actinin-4, was found to be localized to the nucleus in some cancer cell lines . Actinin-4 does not contain an nls and possesses at least one functional nes, which regulates the nucleocytoplasmic shuttling of this molecule in a crm1-dependent manner . Nuclear actinin-4 is coprecipitated with the ino80 chromatin remodeling complex, which contains nuclear actin and several arps, and it has also been revealed that a splicing variant of actinin-4 that lacks the amino acids 89478 is predominantly localized to the nucleus and mediates the transcription of the taf55 gene in association with myocite enhancer factor-2 . It is likely that actinin-4 maintains cooperative relationships with actin in the nucleus, just as in the cytoplasm, though the detailed mechanisms of the molecular interaction have not yet been fully revealed . Spectrins are large proteins (over 200 kda) containing a tandem array of sixteen to twenty srs . As a crosslinker of the actin cytoskeleton in the cytoplasm, spectrins form a tetramer consisting of two each of - and -spectrins . It has been reported that an isoform of nonerythroid -spectrin (iisp) is required for recruitment of the dna - repair proteins fanca and xpf to the sites of dna interstrand crosslinks . The iisp isoform from patients of fanconi anemia exhibits reduced stability, which correlates with a decreased level of dna repair activity . This deficiency results in mitotic chromosomal aberrations, suggesting a role of nuclear iisp as a scaffold for dna repair . One of the truncated isoforms of -spectrin, ivsp5 (72 kda), is localized to the promyelocytic leukemia (pml) bodies . This protein is tightly bound to the highly insoluble nuclear scaffold and overexpression of gfp-ivsp5 results in an increase in the number of pml bodies, suggesting that spectrin iv may be involved in the genesis of pml bodies . Interestingly, a screen for proteins that co - immunopreciptated with iisp identified several plausible candidate interacting proteins, including ivsp5, actin, lamin a, emerin, and pml . In addition, the screen identified proteins associated with dna repair (e.g., rad51, rad50, ku70, ku80, fanca, fancg, fancd2, xpf, xpg, rpa70), chromatin remodeling (e.g., brg1, brm), and rna processing (hnrnp a2/b1, ribosomal - associated protein p40). Many of these interacting proteins associate in their own complexes; so it is not surprising that immunoprecipitation of iisp pulled down so many candidates . The results are consistent with a role of nuclear spectrins as dynamic scaffolds for coordinating diverse nuclear complexes . Several other sr proteins such as nesprins and bpag1 have also been found in the nucleus [2022]. These proteins are expected to play a variety of roles in the nucleus, such as organization of nuclear lamina and regulation of mapk signaling by tethering erk1/2 at pml bodies . Since most of these sr proteins contain actin binding domains, nuclear sr proteins may have cooperative functions with nuclear actin and arps, possibly serving as a structural base or platform for their functions . -catenin, one of the cell adhesion molecules, is also known to be a signal transduction molecule for the canonical wnt signaling pathway . Wnt signaling is one of the key signaling pathways in embryonic development and other processes, including bone formation and homeostasis, as well as adult tissue maintenance; dysregulation of wnt signaling is implicated in some cancer and degenerative diseases [23, 7476]. The canonical wnt pathway regulates cell fate by modulating gene expression directed by the nuclear accumulation of -catenin . As the number of reports on wnt signaling is rapidly increasing, we will mainly focus on the molecular interactions related to the nuclear translocation of -catenin . The primary subcellular localization of -catenin is in the adherence junctions . At the junction sites, -catenin is bound to e - cadherin and -catenin and functions to link the actin cytoskeleton to the plasma membrane . The adherence junction complex is dynamically regulated by the association of actin, - and -catenins, cadherin, and also actin - bundling proteins such as actinin and vinculin, which was demonstrated in vitro and on isolated cadherin - containing membrane patches . At the resting stage, these free -catenins are captured by adenomatous polyposis coli (apc) and are phosphorylated by glycogen synthase kinase 3 (gsk3) and then ubiquitinated and consigned to proteasomal degradation . When the canonical wnt pathway is activated by the binding of wnt to the membrane receptor, this degradation pathway is inactivated due to the inhibition of -catenin phosphorylation . This results in a high level of the free molecules in the cytoplasm and leads to their nuclear accumulation . In the nucleus, -catenin interacts with the transcription factor lef-1, which activates transcription of the target genes . Thus, the key event in wnt signal transduction is the molecular transport of -catenin from the cytoskeleton to the nucleus . This finding is a remarkable example of cytoskeleton - modulated regulation of nuclear functions, which is mediated by the direct translocation of a cytoskeletal component into the nucleus . Zyxin, a 61 kda integrin - associated focal adhesion protein, possesses a functional nes and shuttles between the cytoplasm and the nucleoplasm [25, 80]. Zyxin promotes nuclear translocation of active akt and participates in antiapoptotic cell survival of cardiomyocytes . Zyxin is also translocated into the nucleus in response to mechanical stress and regulates mechanosensitive gene expression . One of the zyxin - family proteins lipoma - preferred partner (lpp), which is predominantly localized to cell adhesion sites and interacts with the tumor suppressor protein scrib, was shown to shuttle into the nucleus in a similar fashion as zyxin . Lpp interacts with, and functions as a coactivator of, the transcription factor pea3, which plays important roles in development and oncogenesis . While many reports describe nuclear localization and functions of actin - related cytoskeletal proteins, several other types of cytoskeletal proteins may also shuttle into the nucleus . In many organisms, including yeast and most fungi, the nuclear membrane does not disassemble during mitosis . In yeast, the -tubulin homologue tub4p is actively imported into the nucleus to form a spindle pole body on the nucleoplasmic side . This nuclear transport is mediated by spc98p, an nls - containing component of the yeast -tubulin complex . A study on fungi has demonstrated that tubulin is excluded from interphase nuclei but is present in mitotic nuclei . Live observation of gfp - coupled tubulin revealed that tubulin enters the nucleus seconds before the formation of the mitotic spindle and is removed from the nucleus at the m - to - g1 transition . Immunofluorescence analyses of several normal and cancer - derived human cells have revealed ii - tubulin, but not other isotypes, in the nucleoplasm as well as the cytoplasm only in the cancer cell lines . Ii - tubulin accumulates in the nucleus after taxol treatment, and it associates with the notch 1 receptor intracellular domain (n1ic), the activated form of notch 1 receptor . It has also been reported that nuclear -tubulin coprecipitated with rad51 and colocalized with rad51 in dna repair foci after treatments with various dna - damaging reagents . Though it has not been fully revealed whether nuclear tubulins are a general phenomenon for normal cells, these findings suggest roles for nuclear tubulins in various nuclear events . Vimentin, one of the building blocks of type - iii intermediate filaments (ifs), has also been found in the nucleus . Vimentin directly interacts with dna through its arg - rich n - terminal head domain . Interestingly, coinjection of fitc - labeled vimentin with various single - stranded oligodeoxyribonucleotides or double - stranded circular dna resulted in the nuclear accumulation of fluorescently labeled vimentin . This n - terminal dna binding domain is responsible for the morphological changes of the nucleus induced by the human immunodeficiency virus (hiv) type-1 protease, suggestive of the role of nuclear vimentin as a scaffold for chromatin organization . More recently it was reported that vimentin upregulates the expression of p21waf1, a cyclin - dependent kinase inhibitor in neuroblastoma, and inhibits osteocalcin gene expression in association with activating transcription factor-4 (atf4) in osteoblasts . Thus, the direct dna binding property of vimentin suggests that its role in chromatin organization extends to transcriptional regulation . There is also emerging evidence that other cytoplasmic if proteins, such as keratins, localize to the nucleus . Considering their conserved molecular structure and polymerization mechanisms, it is conceivable that not only vimentin but also various if components and their accessory proteins can pass through the npc . Cytoplasmic ifs are known to cooperatively function to provide mechanical strength to the cells, raising the intriguing possibility that those if factors in the nucleus interact and cooperatively function with nuclear lamins, the nuclear - specific if . The nucleoplasm is separated from the cytoplasm by the nuclear envelope, which harbors the npc as a selective channel / barrier for macromolecules . The npc is an octameric structure composed of ~30 different subunits termed nucleoporins (nups). The nups facing the inner surface of the pore are rich in hydrophobic phenylalanine - glycine repeat motifs (fg - nups) that are expected to form a meshwork of hydrophobic barriers inside the pore [91, 92]. Water, ions, and molecules smaller than ~40 kda seem to pass through the pore by passive diffusion . Transportation of larger proteins often requires the help of karyopherins, a family of transport mediators . Karyopherins interact with their specific cargos to be transported; for example, the importin and complex recognizes and imports nls - containing proteins, and crm1 exports nes - containing proteins . These two pathways, passive diffusion and karyopherin - mediated transport, are considered to be the two major mechanisms for molecular transport through the npc . Nuclear transport serves as a critical regulatory step for signal transduction from the cytoplasm to the nucleus in some cases . For example, nf-b, a transcriptional factor involved in multiple signaling events related to cell differentiation, apoptosis, and immune responses, is rapidly imported into the nucleus in response to various stimuli . In the tnf-stimulated transport of nf-b p50/p65 heterodimer into the nucleus, importin recognizes the nf-b nls only when nf-b is released from its negative regulatory binding partner, ib . In another case, phosphorylation of the ser385 residue of epstein - barr virus nuclear antigen 1 (ebna-1) upregulates the nls - dependent nuclear import of this molecule, while phosphorylation of ser386 and ser383 downregulates it . These findings illustrate diverse ways with which the nuclear transport of functional molecules is precisely regulated . It may also be reasonable to predict that in cells that reform the nuclear membrane upon completion of mitosis, some cytoskeletal molecules can bind to nuclear material (e.g., chromatin) during mitosis and thereby be passively localized to the interphase nucleus . This may partially contribute to the nuclear localization of some cytoskeletal proteins, especially the proteins with many nuclear interactors such as actin . However, we believe that there should be a transport pathway through the npc to regulate their nuclear populations . Considering the case of yeast cells, in which the nuclear membrane does not break down during mitosis, and of terminally differentiated somatic cells that have exited the cell cycle, the nuclear population cannot be maintained without transport through the nuclear envelope . Though it is not proven, it is posited that nuclear transport is a significant, practical mechanism to regulate nuclear populations of cytoskeletal proteins, even for the constitutively - nuclear proteins . In the following we focus on the molecular mechanisms underlying nucleocytoplasmic shuttling of cytoskeletal proteins through the npc and propose possible regulatory factors determining their nuclear localization (figure 1). While it is certainly true that karyopherin - dependent active transport contributes to the nuclear localization of many nuclear proteins, the nuclear transport mechanisms of cytoskeletal proteins, in particular, have not yet been fully elucidated . Nuclear export, rather than import, is shown to play a central role for regulating the nuclear localization of several cytoskeletal proteins, such as actin, actinin-4, arp5, and zyxin [1, 2, 25, 98]. In some cases karyopherins there is strong evidence that exportin 6 (exp6) critically functions in the nuclear export of actin . Interestingly, exp6 recognition of actin is significantly enhanced by the formation of an actin - profilin complex, suggesting a novel role of profilin as a cofactor for the nuclear export of actin . Apc itself shuttles between the cytoplasm and the nucleoplasm and enhances active export of -catenin to the cytoplasm in the absence of wnt stimulation . Leucine zipper tumor suppressor 2 (lzts2) and ran - binding protein 3 (ranbp3) were also found to function as nuclear exporters of -catenin [100, 101]. Taken together, nuclear localization of several representative cytoskeletal proteins is largely determined by controlling their nuclear export . There is increasing evidence of karyopherin - independent nuclear transport of several cytoskeletal proteins . Whereas it is generally accepted that molecules smaller than the size limit for passive diffusion through the npc (conventionally around 40 kda), such as monomeric actin and some arps, may freely migrate into the nucleus, some of the nuclear - localizing cytoskeletal proteins are substantially larger than the size limitation and are nevertheless able to pass through the npc in a karyopherin - independent manner . For example, in vitro nuclear transport assays using semipermeabilized cells demonstrated that -catenin and actinin-4 can pass through the npc in a karyopherin - independent manner [2, 102, 103]. These proteins possess an amphiphilic structural domain in common: an armadillo repeat for -catenin and sr for actinin-4, which both play an essential role in their nuclear migration . Although the precise mechanism of the molecular transport through the npc is the subject of continuing debate, surface hydrophobicity, promoting interactions with hydrophobic fg - nups, is a critical factor . This was demonstrated with bovine serum albumin (bsa), a protein not normally found in the nucleus . Chemical modification of bsa using several hydrophobic moieties on the surface significantly facilitated nuclear transport of the modified protein, demonstrating the role of surface hydrophobicity in the protein's ability to traverse the npc . By inference, cytoskeletal proteins with amphiphilic domains may adapt to the hydrophilic environment inside the npc and spontaneously migrate into the nucleus . We speculate that this spontaneous migration will be the third mode of molecular transport through the npc, which acts through the amphiphilic property of the molecule . We suggest three possible mechanisms for the nucleocytoplasmic transport of cytoskeletal proteins through the npc: passive diffusion, karyopherin - dependent active transport, and karyopherin - independent spontaneous transport . In vivo, the nucleocytoplasmic distribution of a molecule is determined by the combination of these three factors (figure 1(a)). It is reasonable to assume that passive diffusion and spontaneous transport permit bidirectional molecular translocation, and karyopherin - dependent directional transport drives the differential distribution of the molecule . For example, in the case of actinin-4, the amphiphilic sr enables bidirectional spontaneous transport of this molecule through the npc in addition to crm1-dependent nuclear export . This combination of two driving forces not only results in the predominant localization of actinin-4 in the cytoplasm but also allows a small population of the protein in the nucleus . It may be possible to predict similar covert nuclear localizations of proteins by focusing on the size, shape, and hydrophobicity of the molecules . To fully understand the nuclear localization of cytoskeletal proteins, it is also important to consider their accessibility to the npc . In light of the studies we have mentioned in this review, we propose the following regulatory steps of nucleocytoplasmic shuttling of cytoskeletal proteins: (1) change in the amount of npc - accessible molecules in the cytoplasm, (2) translocation through the npc, and (3) retention in the nucleus (figure 1(b)). The first step includes the changes in the equilibrium of soluble / polymeric molecules, active release from the cytoskeleton, and the inhibition of the degradation system in the case of -catenin under wnt stimulation, which results in the increase of npc - accessible free molecules in the cytoplasm . If the molecule can passively diffuse or spontaneously migrate through the npc, molecules in the cytoplasm and nucleoplasm reach a dynamic equilibrium via bidirectional transport, whereas the association with karyopherins strongly influences the differentially localized steady - state concentrations . Retention or release rate of the molecule from a nuclear binding partner may serve as another factor regulating the number of npc - accessible molecules in the nucleoplasm . These three steps can independently modulate the ratio of molecules in the nucleus versus the cytoskeleton . In the case of arp4 in yeast, associations with nuclear factors seem to play a central role in the dominant nuclear localization of this protein . Mutation in the putative nls in arp4 did not affect its nuclear targeting, suggesting that arp4 can passively diffuse through the npc, and the binding of arp4 with nuclear proteins prevents its escape from the nucleus . If the concentration of cytoplasmic free molecules is lowered by incorporation into the newly organized cytoskeleton, for example, the result may be the loss of npc - accessible free molecules in the cytoplasm, rebalance of molecular distribution between nucleoplasm and cytoplasm by the molecular transport through the npc, and a subsequent decrease in the population of molecules in the nucleus . We expect that this molecular dynamics, linking cytoplasmic and nuclear functions, may be especially relevant to the regulation of nuclear functions . Transmission of environmental signals to the nucleus is fundamentally important for cells in various states to properly organize a response . In contrast to the normal nuclear proteins that are consistently retained in the nucleus, transnuclear signaling molecules should dynamically change their nucleocytoplasmic localization in response to the specific signals . We expect that nuclear shuttling and reorganization of nuclear cytoskeletal proteins will emerge as central to nuclear responses to external signals, just as they are central to transduction of those cues from the plasma membrane . Here we propose a model of cytoskeleton - modulated regulation of nuclear functions that are mediated by translocation of cytoskeletal components to the nucleus through the npc (figure 1). We predict that the dynamic distribution of many of these proteins will depend predominantly on their association with nuclear complexes and on active export by karyopherins . Ultrastructural mapping by immunoelectron microscopy revealed the nuclear localization of various cytoskeletal proteins in several foci or territories and also showed somewhat different localization patterns between hela cells and lymphocytes . Such structural analyses will broaden our understanding of the role of cytoskeletal proteins in the nucleus . At the same time, a computational approach will help to specify the criteria for their passage through the npc, which, especially for karyopherin - independent transit, remains largely a mystery . Silencing of arp4 in arabidopsis results in strong pleiotropic phenotypes such as altered organization of plant organs, early flowering, delayed flower senescence, and high levels of sterility . Together with the studies on yeast and mammalian cells, these findings suggest that cytoskeletal proteins are generally involved in various nuclear events in virtually all eukaryotes . Considering the roles of the bacterial actin homologue mreb in a wide variety of biological events such as cytoskeletal organization, cell shape maintenance, cell wall biosynthesis, viral dna replication, and gene expression [111115], it is no wonder that actin and other associated proteins play roles in both the nuclear and cytoplasmic compartments in eukaryotic cells . Furthermore, we suggest that such cytoskeletal proteins may act to functionally link the nucleoplasm and cytoplasm to organize integrated cellular activities that are required for both basal cellular events and for responses to specific environmental changes . While the focus of this review has been on the presence and regulation of cytoskeletal proteins in the nucleus, we would like to extend the discussion to nuclear proteins in general . Since all proteins are synthesized in the cytoplasm, all nuclear proteins should pass through the npc, at least once . Bioinformatic analysis on whole yeast proteins revealed that only about 57% of steady - state nuclear proteins are predicted to use the classical nuclear import pathway, whereas the remaining 43% may use other mechanisms to enter the nucleus . It also became apparent in that analysis that 40% of cytoplasmic proteins possess nls consensus sequences, suggesting a limited influence of the classical nls in the nuclear localization of molecules in vivo . The idea of export - based regulation of nuclear proteins may extend to other nuclear proteins, and to a general concept for understanding nuclear localization of proteins . This active and selective nuclear export system may also act to remove unneeded / harmful proteins from the nucleus . In contrast to the well - known cytoplasmic ubiquitin - proteasomal system, quality control of proteins in the nucleus is not well understood . Recently, a nuclear - specific proteolytic pathway has been identified, and enzymes catalyzing the steps of the ubiquitin - proteosomal degradation in the nucleus are being characterized in both yeast and humans [117, 118]. Thus, the nuclear border may be leakier than thought, and the nucleus appears to have two options for the clearance of unwanted proteins: export or degradation . Currently, nuclear actin and -catenin are two leading examples of constitutively required and specific signal - dependent cytoskeletal molecules, respectively, that shuttle and function in the nucleus . Considering the structural and dynamic features of cytoskeletal organizations, it is plausible that various types of cytoskeletal proteins shuttle through the npc and cooperatively function in the nucleus . It is becoming apparent that cytoskeletal proteins in the nucleus play indispensable roles in a variety of nuclear events, even though their nuclear population is quite small . We suggest that there is a need to reconsider the potential functions of apparently cytoplasmic proteins in the nucleus, which may result in the discovery of their hidden talents.
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While drugs can cause acute rhabdomyolysis individually, they can also interact with other drugs by cytochrome p450 and p - glycoprotein mechanisms to bring on the syndrome.clinicians should evaluate drug interactions in patients taking two or more medications . Acute rhabdomyolysis (ar) is a clinical syndrome that occurs when the electrolytes and myoglobin that are released upon skeletal muscle damage enter into the circulation system . There are several ways in which ar may occur, one of which is drugs (fig . 1etiology of acute rhabdomyolysis and its complications etiology of acute rhabdomyolysis and its complications a comprehensive study was carried out in 2011 using 20042009 data of the us food and drug administration (fda) to reveal which drugs may be associated with rhabdomyloysis . The etiologies of 16,435 drug - induced cases of rhabdomyloysis involved hmg - coa reductase inhibitors or statins (n = 4325, 26.3%) as the first suspect . A total of 100 cases were reported for diclofenac and 99 for omeprazole, a proton pump inhibitor, while there were no reports of cases associated with pantoprazole . The cases of drug - induced rhabdomyolysis in previous literature were also associated with statins, and the frequency is similar to that reported in the abovementioned report . That said, drug interactions could not be assessed because the fda data did not include that level of systematic information . The gastric side effects of non - steroidal anti - inflammatory drugs (nsaids) are well known, and their concomitant or subsequent combination with proton pump inhibitors is common . A review of adverse drug reactions, creatinine kinase elevations, and muscle events, including muscle weakness, myopathy, myositis, and polymyositis, analyzed such multiple drug regimens and found that the addition of pantoprazole to a treatment regimen was the cause of all muscle events, aside from myalgia . A 45-year - old man was referred to hospital complaining of fatigue and extensive body pain . His medical history included stress - induced headaches that had been present for almost the entire previous year, for which he began using diclofenac at 50 mg / day as the frequency of headaches had increased during the prior month . He was referred to a physician for pyrosis, 1 week prior to presenting at the hospital, and was prescribed pantoprazole at 40 mg / day . He started to experience pain in his leg muscles on the fourth day of the diclofenac and pantoprazole combination regimen and used two 50-mg doses of diclofenac to control the pain . The muscle pain became more widespread on the fifth day, and the patient also developed fatigue . An examination revealed a blood pressure of 120/80 mmhg and a pulse of 76 bpm, full and rhythmic . Muscular strength was good; however, the patient complained of muscle tenderness upon palpation . Neurological and general system examinations were normal; no abnormal findings were seen in an electrocardiogram; and an abdomen ultrasound revealed no pathology, aside from an abdominal gas distention . A laboratory analysis revealed high levels of muscle destruction products, the foremost of which was creatinine kinase (table 1). Glucose, urea, creatinine, cholesterol levels, amylase, direct and indirect bilirubin, electrolytes, anti - streptolysin o, c - reactive protein, rheumatoid factor, and thyroid hormones were all within their normal ranges.table 1laboratory parameters of patientadmission7 days afterreference rangeglucose (mg / dl)96n / a70105alp (alkaline phosphatase) (iu / l)565164306sgpt (iu / l)4512042sgot (iu / l)8814534ck (iu / l)31146724190creatinine (mg / dl)0.760.670.751.25crp (mg / dl)0.880.5905ldh (iu / l)230168125220 alp alkaline phosphatase, ck creatine kinase, crp c - reactive protein, ldh lactate dehydrogenase, n / a not available, sgot serum glutamic oxaloacetic transaminase, sgpt serum glutamic pyruvic transaminase laboratory parameters of patient alp alkaline phosphatase, ck creatine kinase, crp c - reactive protein, ldh lactate dehydrogenase, n / a not available, sgot serum glutamic oxaloacetic transaminase, sgpt serum glutamic pyruvic transaminase a cardiac pathology was excluded because the electrocardiographic assessments and cardiac sounds were normal, and because the patient did not describe any pain that would indicate an infarct, and test results and findings were stable on the following days . Ar was considered as a pre - diagnosis, as there was a chronologic alignment between the patient s clinical representation and the medications he used, and this was supported by the laboratory parameters . Both medications were discontinued and the patient was followed up for 1 week, during which no medical interventions were carried out, aside from a recommendation of general hydration . The ar diagnosis was confirmed because his complaints disappeared, with his laboratory values returning to the normal range, a stable electrocardiogram, and other systemic examinations being found within normal ranges . To date, among the cases involving nsaids, two cases were reported that include diclofenac - induced rhabdomyloysis [4, 5]. The patient detailed in this study was referred with extensive pain and fatigue, and represents the third case in which rhabdomyloysis developed while using 100-mg / day diclofenac tablets . The other two cases were induced by diclofenac alone, with no interaction with other drugs . Of these, one patient was admitted with pain that increased after the patient took 200 mg of diclofenac within 2 days (50 mg tablet / twice a day) for joint pain . The other case involved a patient who used diclofenac to ease an acute exacerbation of gout . The patient was referred to a physician on the 13th day of intramuscularly self - administered diclofenac at 75 mg per day for 6 days, followed by 75 mg orally three times a day for 7 days . Unlike the previous case, this patient was referred to the hospital because of erythematous pruritic eruptions that developed on various parts of his body . While drugs can cause ar individually, they can also interact with other drugs to bring on the syndrome . The cyp1, cyp2, and cyp3 gene families are overall responsible for drug metabolism, and the similarities between the amino acid sequences within a gene family are at least 40%, although the level of similarity may increase to 85% between subgroups . The genetic polymorphisms in these genes alter the biotransformation of the drug . While fast metabolizers have increased enzyme activity, slow metabolizers show decreased enzyme activity . Among these genes, theophylline, which is metabolized through this pathway, when used with erythromycin, returns to the cyp1a2 pathway and cannot reach toxic concentrations [79]. That said, to date, no such theory has been put forward for diclofenac and pantoprazole in the cyp enzyme system . The fact that they are metabolized through the same subgroup (2c) can be associated with a possible drug interaction . This possibility of interaction between diclofenac and pantoprazole has been investigated in a randomized crossover trial of 24 subjects, and the results showed that the peak plasma concentrations of diclofenac are sustained in a stable range, and that diclofenac does not affect the pharmacokinetics of pantoprazole, meaning that this combination is completely safe . However, the results of an in - vitro study using diclofenac as a substrate marker for human liver microsomes suggested the contrary . According to this study, proton pump inhibitors, the strongest one being pantoprazole, carry a strong potential for drug interaction by inhibiting the activity of cyp enzymes, and it was underlined that drug interactions are significantly more pronounced in weak metabolizers with an enzyme deficiency . P - glycoprotein (p - gp) is an adenosine triphosphate - dependent efflux transprotein that is found in the membranes of the enterocytes . It is expressed by the mdr1 gene in humans and can be found in various organs, including the liver, kidneys, intestinal region, and blood brain barrier . P - gp actively pumps its substrates xenobiotics to the extracellular space and decreases their intracellular concentrations . Proton pump inhibitors, including pantoprazole, anti - arrythmic drugs, anticancer drugs, antibiotics, and antidepressant drugs, inhibit p - gp, and therefore the modulation of this pump plays a crucial role in drug drug interactions [12, 13]. While drugs can cause ar individually, they can also interact with other drugs to bring on the syndrome . The cyp1, cyp2, and cyp3 gene families are overall responsible for drug metabolism, and the similarities between the amino acid sequences within a gene family are at least 40%, although the level of similarity may increase to 85% between subgroups . The genetic polymorphisms in these genes alter the biotransformation of the drug . While fast metabolizers have increased enzyme activity, slow metabolizers show decreased enzyme activity . Among these genes, theophylline, which is metabolized through this pathway, when used with erythromycin, returns to the cyp1a2 pathway and cannot reach toxic concentrations [79]. That said, to date, no such theory has been put forward for diclofenac and pantoprazole in the cyp enzyme system . The fact that they are metabolized through the same subgroup (2c) can be associated with a possible drug interaction . This possibility of interaction between diclofenac and pantoprazole has been investigated in a randomized crossover trial of 24 subjects, and the results showed that the peak plasma concentrations of diclofenac are sustained in a stable range, and that diclofenac does not affect the pharmacokinetics of pantoprazole, meaning that this combination is completely safe . However, the results of an in - vitro study using diclofenac as a substrate marker for human liver microsomes suggested the contrary . According to this study, proton pump inhibitors, the strongest one being pantoprazole, carry a strong potential for drug interaction by inhibiting the activity of cyp enzymes, and it was underlined that drug interactions are significantly more pronounced in weak metabolizers with an enzyme deficiency . P - glycoprotein (p - gp) is an adenosine triphosphate - dependent efflux transprotein that is found in the membranes of the enterocytes . It is expressed by the mdr1 gene in humans and can be found in various organs, including the liver, kidneys, intestinal region, and blood brain barrier . P - gp actively pumps its substrates xenobiotics to the extracellular space and decreases their intracellular concentrations . Proton pump inhibitors, including pantoprazole, anti - arrythmic drugs, anticancer drugs, antibiotics, and antidepressant drugs, inhibit p - gp, and therefore the modulation of this pump plays a crucial role in drug drug interactions [12, 13]. The fda recommends that clinicians check cyp - mediated potential drug interactions routinely . In this case report, a case of diclofenac- and pantoprazole - induced ar is evaluated for drug - interaction mechanisms, and different perspectives are presented . While evaluating the patients with side effects of drugs, the cyp and p - gp mechanisms that play roles in drug absorption and distribution should be considered . Written informed consent of the participant was obtained after the nature of the procedures had been fully explained . The case report was carried out in agreement with the latest version of the declaration of helsinki.
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Several decades ago, treatments for benign prostatic hyperplasia (bph) were almost entirely surgical . During the 1990s, various minimally invasive surgical procedures (mis) were developed, including transurethral microwave thermotherapy, transurethral needle ablation, and transurethral laser vaporization . Although techniques have advanced, transurethral resection of the prostate (turp) remains the gold standard surgical intervention for bph . The introduction of -antagonists in the early 1990s resulted in significant improvements in the medical treatment of bph . After the early 2000s, combination therapy with -antagonists and 5--reductase inhibitors has resulted in significant changes in the management of lower urinary tract symptoms (luts) secondary to bph . In the proscar long - term efficacy and safety and medical therapy of prostate symptoms (mtops) studies, some patients cannot or will not tolerate medical therapy, whereas others experience symptomatic deterioration despite long - term medical therapy . Despite the advances in medical and surgical intervention, many men continue to suffer from bph . The incidence of bph is 50% in men between 51 and 60 years of age and as high as 88% in men up to 80 years of age . In an aging population, it would be advantageous to determine the roles of medical and surgical intervention for bph and the effect of changes in medical therapy trends on the indications and outcomes of surgical intervention for bph . The aim of this study was to compare the outcomes in patients who underwent surgery in 1985 to 1989 (before the widespread use of medical therapy for bph), in 1995 to 1999 (when medical therapy was developed and began to be prescribed as alternative treatment to surgery), and in 2005 to 2009 (when medical therapy superseded surgical intervention to become first - line treatment and when combination therapy became widely adopted). Medical records were retrospectively reviewed for all patients who underwent surgery for bph from 1 january 1985 to 31 december 1989 (group 1), from 1 january 1995 to 31 december 1999 (group 2), and from 1 january 2005 to 31 december 2009 (group 3). University staff urologists .with more than 1 year of surgical experience with bph performed all the operations . Preoperatively, we evaluated age, body mass index (bmi), prostate - specific antigen (psa) level, prostate volume, prostate transitional zone volume, maximum urinary flow rate, postvoid residual urine volume, international prostate symptom score (ipss), qol score, bph medication use, and chief complaints . Bph medication history included -blocker or 5--reductase inhibitor therapy for bph more than 3 months before surgery . Perioperative and postoperative parameters were investigated representatively in patients who underwent turp, who were divided into 3 subgroups according to time period: 1985 to 1989 (subgroup 1), 1995 to 1999 (subgroup 2), and 2005 to 2009 (subgroup 3). Transfusion and the weight of the resected tissue were evaluated as operative parameters . The postoperative course was evaluated on the basis of length of hospital stay, the postoperative day on which the catheter was removed, and postoperative complications . A postoperative complication was defined as any event requiring additional treatment within 1 year of bph surgery: secondary hemorrhage that required transfusion or hospital admission within 4 weeks postoperatively, impaired detrusor contractility that required suprapubic cystostomy as a result of recurrent acute urinary retention (aur) or failure to void, repeat surgery for bph, urethral stricture that required surgical intervention, long - term stress incontinence, and irritative luts that required medication use until 1 year postoperatively . For the statistical analysis, one - way analysis of variance was used for continuous variables and a chi - square test was used for categorical variables to assess differences between means . A p value <0.05 was considered to be statistically significant . Spss ver . 18.0 (ibm, new york, ny, usa) was used for the statistical analysis . A total of 190 men underwent surgery for bph in group 1, 484 in group 2, and 278 in group 3 . The mean ages of the men were 65.4, 65.9, and 69.3 years in group 1, group 2, and group 3, respectively . Over time, the mean age increased and was significantly different between group 2 and group 3 (p<0.001). The prevalences of hypertension, a history of surgery, and " others " (e.g., stroke, cancer, and cardiac conditions) increased significantly over time . The mean bmis were 21.6, 22.9, and 24.0 in group 1, group 2, and group 3, respectively (p<0.001). The most common indication for surgery in all groups was worsening symptoms, followed by aur . The incidence of symptomatic deterioration continued to increase, but the rate of aur kept decreasing ., only one psa level was found in the chart review; therefore, group 1 was excluded from the psa comparison . The mean psa levels of group 2 and group 3 were 6.8 and 5.8, respectively; however, they were not significantly different . There was a slight increasing trend from 34.4 ml to 38.4 ml between group 1 and group 2 . However, there was a sharp increase between group 2 and group 3 (p<0.001). We could not elucidate sufficient data to analyze prostate transitional zone volume, maximum urinary flow rate, residual urine volume, total ipss score, and qol score in group 1 . Therefore, we compared groups 2 and 3, and a significant difference was detected in prostate transitional zone volume (p<0.001) (table 1). Only two patients (1.1%) in group 1 required bph medication; the remaining patients in this group did not receive medical therapy . However, 33.5% of the patients in group 2 used -blockers . A marked improvement was observed in group 3 . -blockers were taken by 47.8% of patients, and 25.9% of patients required combined therapy with an -blocker and a 5--reductase inhibitor (fig . On the other hand, mis had only begun to be performed with group 2 (fig . The basic characteristics of these patients were similar to those of patients who underwent surgery for bph (table 1); however, one difference was that the mean prostate volume gradually and significantly increased over the 20-year study period (p<0.001). We analyzed perioperative and postoperative parameters in subgroups who underwent turp for direct comparison in one type of surgery: 131 patients in subgroup 1,339 patients in subgroup 2, and 194 patients in subgroup 3 underwent turp for bph . Perioperative parameters and postoperative variables for the three patient subgroups are illustrated in table 2 . The mean weight of resected tissue was significantly greater in subgroup 3 than in subgroup 1 and subgroup 2 . The number of patients who required transfusion because of perioperative hemorrhage continuously deceased over time . Over time secondary hemorrhage within four postoperative weeks and the need for repeat turp within 1 year after surgery decreased significantly (p=0.03 and p=0.003, respectively). The number of patients who required suprapubic cystostomy gradually increased over time, albeit not significantly so (p=0.523). No significant changes in any of the other complications were found in the three studied periods . Over the past two decades, medical therapy has been the standard first - line therapy for bph . Long - term combination therapy reduced the incidence of aur and bph surgery . In our study, aur decreased significantly, but worsening symptoms increased significantly . Our results confirmed an increasing trend in -blocker monotherapy and combination therapy with 5--reductase inhibitors after the 1990s, when medical therapy became widespread . However, surgery for bph is still required and turp became the mainstay of surgical intervention for bph . Although medical therapy is appropriate for patients with symptomatic bph, it has obvious limitations . Failure of medical therapy results in worsening luts and recurrent aur . In this study, the most common chief complaint was worsening symptoms followed by aur . In a study by izard and nickel, patients who finally required surgery were inevitably older, had worse health conditions, and had more advanced disease . Therefore, late surgical intervention could result in poorer outcomes associated with worse postoperative complications . In this study, the mean age of patients who underwent surgery was greater in the 1990s than in the 1980s, and the increase was larger after 10 years . This trend was also shown in another study . According to vela - navarrete et al ., patients who underwent surgery for bph in 2002 were significantly older than those who underwent surgery in 1992, by a mean of 3.1 years . A considerable number of patients had hypertension and previous surgical and other medical histories in the 2000s . We concluded that the increasing comorbidity was due to aging, advancement in diagnostic techniques, and increasing health concerns . The decreased aur and the increased frequency of symptomatic deterioration were due to combination therapy that reduced aur . According to our results, the mean prostate volume increased during the 20 year period in patients who underwent surgical intervention for bph . A review by han et al . Showed that the mean prostate volume in surgically managed patients over 5 successive years increased significantly, from 37.1 ml to 53.6 ml, between 1998 and 2002 . They attributed these findings to the priority given to medical therapy by patients who required surgery in the past and to development of the endoscopic technique, which extended the indication of turp . Because of larger prostate volumes, it is natural that the weight of the resected tissue increased correspondingly . This study showed that the mean prostate volume was significantly greater in group 3 than in group 1; simultaneously, the weight of the resected tissue was also significantly greater in subgroup 3 than in subgroup 1 . Shin and park reported that the mean resection volume increased with both turp and open prostatectomy between 1999 and 2010 . Reich et al . Evaluated 10,654 patients who underwent turp and showed that mortality and morbidity were closely associated with the weight of tissue resected . According to mebust et al ., increased morbidity was seen in patients with a gland size greater than 45 g and an age greater than 80 years . According to our survey of perioperative and postoperative variables, the length of hospital stay, the number of days to catheter removal, and the number of transfusions, secondary hemorrhages, and repeat turps deceased with time . These findings were due to advancements in surgical techniques, experience, and improved instruments . We believed that critical outcomes such as impaired detrusor contractility would be poor and hypothesized that long - term bladder outlet obstruction would impair bladder detrusor contractility . Experimented with rat bladders and found that whole - bladder pressure was significantly lower in rats subjected to long - term outflow obstruction than in controls . However, the number of suprapubic cystostomies required after turp did not increase significantly, because new techniques and equipment reduced the number of complications and overcame the risks associated with aging . In addition, combination therapy resolved bladder outlet obstruction and protected progress to impaired detrusor contractility . The results showed that surgical intervention conferred benefits to patients, despite their older ages and larger prostate volumes at the time of surgery . Following the widespread adoption of medical therapy for the treatment of bph, the rate of medical therapy increased and the number of surgeries decreased . Although this study did not represent all bph patients, it analyzed data spanning two decades to determine the changing trends in surgery for bph . However, because of its retrospective nature, ipss and transitional zone volume data were not available in the 1980s . An additional limitation was the lack of pre- and postoperative data, such as ipss data and maximum urinary flow rates, with which to compare the direct results of surgery . Furthermore, we were unable to determine the length of time required to complete the surgeries - a variable that is useful in determining the skill level of surgeons when resection weights are identical . Instead, we used transfusion history in the perioperative period to indirectly determine the level of surgical skill and care . The length of hospital stay appeared to be longer in the current study than in other studies, which was due to korean health insurance policies and patient tendencies . The mean number of hospitalizations in subgroup 3 in the current study was similar to that in other korean publications . Several studies reported that these technologies have led to shorter hospital stays, shorter catheterization periods, and fewer complications . Even in patients with large prostate volumes, in whom turp was inappropriate in the past, these procedures have shown excellent results, especially concerning perioperative blood loss . Continued advancement in surgical intervention the ages and comorbidities of patients who underwent surgery for bph increased over the two decades studied . Our study showed that the mean prostate volume at the time of surgery was significantly greater than it was 20 years ago, as was the weight of the resected tissue . Although these conditions can increase the risk of complications, the prevalence of secondary hemorrhage within four postoperative weeks and the need for repeat turp within 1 year after surgery decreased significantly . Although patients who underwent surgery were older after widespread use of medical therapy for bph, advancements in surgical techniques have benefitted these patients.
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Root - end filling materials play an important role due to their physical, chemical and biological properties . Several materials have been suggested for root - end filling, including amalgam1, zinc oxide19, irm9,13, composite resin15,22, eba1,14,20, polycarboxylate cement16, sealer 2624, sealapex24, hydroxyapatite14, verrm8 and, more recently, mta1,2,6,13,17,23,25,26 . Lysanda is a zinc oxide - eugenol material presented in two pastes; it is widely used in prosthodontics . Dutra and horta11 (1994), custodio and costa7 (1994) and bernardineli3 (1995) used this materials for root - end filling with good results and excellent adaptation to the cavity walls . Morais18 (2003) studied the reaction of rat subcutaneous connective tissue to lysanda paste, mta and portland cement containing iodoform and observed that lysanda paste caused significantly lower inflammatory reaction than mta and portland cement, including formation of a more organized fibrous capsule compared to the other two groups . Bismuth oxide, barium sulfate, zinc oxide and iodoform are radiopacifiers used in endodontics and root - end filling materials4,6,10,21 . Since lysanda paste does not show this characteristic, it is important to determine whether the presence of these substances (zinc oxide or iodoform) would alter the properties of this paste and influence the apical marginal sealing of root - end fillings . The root canals of 50 single - rooted teeth from the tooth bank of the department of restorative dentistry, endodontics and dental materials, dental school of bauru were instrumented by the stepback technique, irrigated with saline, dried with absorbent paper points and filled with gutta - percha, zinc oxide and eugenol by the lateral condensation technique . The apices were cut into approximately 2 mm at 45 with a #699 tapered bur in a buccal direction . The teeth were rendered impermeable by application of fast - setting araldite and nail varnish . Apical cavities were prepared to a depth of 2 mm with #2 round bur, complemented by irrigation with saline . The teeth were randomly divided into five groups of 10 teeth each and submitted to root - end filling with the following test materials: group i lysanda paste with iodoform; group ii lysanda paste with iodoform and zinc oxide; group iv -lysanda paste with zinc oxide; and group v mta . In group i, 0.62 g of red lysanda paste and 0.46 g of white lysanda paste were placed on a plate, with addition of 0.22 g of iodoform, and were mixed for 30 s. the material was then inserted in the root - end cavity with a blunt - tip probe until complete filling . Procedures for group ii were the same as performed for group i, with addition of 0.22 g of iodoform and 0.11 g of calcium hydroxide . In group iii, the procedures were the same as those for group i, with addition of 0.11 g of iodoform and 0.11 g of zinc oxide . In group iv, the procedures were the same as for group i, with addition of 0.22 g of zinc oxide . In group v, mta was prepared by mixing the power with distilled water until the ideal consistency for root - end filling was obtained . After root - end filling, the teeth were immediately immersed in 2% aqueous methylene blue solution and incubated for 24 hours in an oven at 37c in humid environment, with a small container with water . Then, the teeth were removed from the dye and sectioned buccolingually with a diamond disc, until half of root canal filling and consequently the root - end filling were exposed . This area was analyzed by reflected light microscopy for determination of leakage at buccolingual interfaces, in millimeters . Data were statistically analyzed by one - way analysis of variance to investigate the possible significant differences between groups . Table 1 shows the means and standard deviations of apical leakage observed for the experimental groups, namely 0.44 mm for group i (lysanda+iodoform), 0.46 mm for group 11 (lysanda + iodoform + calcium hydroxide), 0.48 mm for group v (mta), 0.68 mm for group iv (lysanda+zinc oxide), and 0.77 mm for group iii (lysanda + iodoform + zinc oxide). One - way analysis of variance showed no significant differences in apical leakage between the different experimental groups (table 2). Lysanda paste yielded similar results as the other groups, regardless of the substances added (table 1). Dutra and horta11 (1994) analyzing root - end fillings in dog's teeth, confirmed the high effectiveness of lysanda paste compared to amalgam . Custdio and costa7 (1994) investigated the marginal leakage of root - end fillings with lysanda, amalgam, ionomer and resin, and observed significantly better results for lysanda . This is in agreement with the present results, including the observation of excellent adaptation of lysanda paste with its components and of mta . The addition of other materials to lysanda paste also provided satisfactory results, as shown by the numerical differences that yet were not statistically significant . The highest leakage rates, without statistical difference, were observed for groups iii and iv, which contained zinc oxide, probably due to lack of affinity between its components . Analysis of table 1 shows that the lowest leakage occurred in group i (lysanda with iodoform), without statistically significant difference compared to mta . According to bramante and berbert5 (2000), four aspects are important for the success of root - end filling: the employed material, including its adequate physical, chemical and biological characteristics, is probably the most important factor . As mentioned earlier, numerous materials have been tested for root - end filling, all of which have advantages and disadvantages, yet certainly without meeting all or most requirements for good sealing . With respect to lysanda paste, one of the first studies using this material for root - end filling was conducted by bernardineli3 (1995), who demonstrated its high sealing ability and excellent adaptation to the cavity walls, especially when compared to other materials . These data indicate the possible application of lysanda paste as a new root - end filling material . However, the addition of radiopaque substances was needed to better test this material . In this respect, zinc oxide, calcium hydroxide and iodoform, either alone or in combination, seemed to be the ideal substances for this purpose, due to their good biocompatibility and broad clinical applicability . The advantages of iodoform as an intracanal medication include the formation of granulation tissue, new bone formation and lesion repair, leading to indication of its use . Zinc oxide and calcium hydroxide also show broad clinical applicability and their biological effects are widely known . Another important aspect is the biocompatibility of lysanda paste; its good acceptance is mainly related to the clinical indications of the material itself . Morais18 (2003), evaluating the reaction of rat subcutaneous connective tissue to mta, portland cement with iodoform and lysanda paste, observed no difference between materials at 7 and 30 days, while at 60 days lysanda paste showed significantly less inflammatory reaction than the other two cements . An interesting aspect reported by morais18 (2003) is that the use of iodoform with portland cement, whose combination with other cements is poorly known, yielded satisfactory results when compared to lysanda and even to mta, somehow agreeing with the present results observed for addition of iodoform to lysanda paste . Conversely, in our opinion, the use of lysanda paste is encouraging, even though its indication in dentistry has always been limited . In this context, a very important aspect is its low cost, in contrast to mta, which has a relatively high cost . The combination of lysanda with other substances was necessary to complement some of its properties, as required for clinical application . All these observations suggest that mta may still be indicated for root - end filling . Lysanda paste with iodoform yielded the best result and thus can also be considered a good option as root - end filling material, as demonstrated by its positive aspects observed in the present experiment and in previous studies . Studies using lysanda paste are limited, but the definitely promising results encourage further investigations, especially on its biological properties, to confirm its qualities as a root - end filling material and as a possible paste or cement for root canal filling in the future . Lysanda paste with iodoform showed the lowest leakage, without statistically significant difference compared to other groups; 3.
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Although cardiovascular disease has been decreased in developed countries, it is increasing in developing countries without noticeable public health responses (1). In iran, like other developing countries, cardiovascular disease is the main reason of death and disability (2). It has been indicated that this disease causes about a half of all deaths (3). Furthermore, it was revealed that this increased rate has been due to increased major risk factors in iran (4). The major risk factors like hypertension, hypercholesterolemia, smoking and diabetes are increased in this country (5). Recognition of patients with vascular disease and intervention with advice about life style, cigarette smoking, alcohol, nutrition and physical activity are among the preventing methods of cardiovascular disease (6). Disease preventing methods focus mostly on lifestyle factors such as physical activity, healthy diet and not smoking (7). Confederacy of international health education organizations (cnheo) declared that theory and technology for the purpose of educational research should be used in methodology designing, performance and evaluation of health education programs . Health belief model is one of the preventive models used for public health problem such as cardiovascular disease . This model was developed to explain lack of public participation in health screening and prevention programs in the 1950s (10). In addition, this model is used to examine patients' motivations for adopting a health - related behavior as well as assessing health - behavior interventions . It includes six key domains of perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action and self - efficacy (11). Self - efficacy domain refers to one's beliefs in one's capability to organize and execute the courses of action required to achieve given results (12). In many evidences, self - efficacy has been associated with healthy nutrition (13) and physical activity behavior (14). In previous evidence, it was focused that higher levels of self - efficacy contributed to physical activity and lower dietary (15). Previous studies showed that health belief model is a useful framework for describing the healthy nutrition behavior (16 - 19). Moreover, this model has been used in risk reduction of cardiovascular disease in college students (20). Considering the importance of behavioral determinants of nutrition and physical activity behaviors due to cardiovascular disease, this study aimed to identify the important predictors of nutrition and physical activity in relation to cardiovascular disease in students of tehran university . This was a cross - sectional study performed on students of tehran university from october to december 2012 . A proportional quota sampling was used to select students studying in majors of humanity, basic sciences and technical - engineering in different grades of bs, msc and phd . Inclusion criteria were being student of tehran university and aged between 18 and 55 years . Exclusion criteria were unwillingness to participate in the study and guest student from other universities . The sample size was calculated as 323 subjects considering = 5%, statistical power of 90%, p = 0.5, d = 0.09 and the following equation: considering 10% attrition rate, we added 45 samples, so we totally considered 368 samples . In total, 36% were from engineering field, 11.3% basic sciences field and 52.7% humanity field . A self - administrated questionnaire consisted of four sections the first section was about demographic characteristics like age, gender, height, weight, level of education and information sources . The second part consisted of 16 questions regarding knowledge about causes, diagnosis and risk factors of cardiovascular disease, healthy diet and physical activity due to the disease prevention . The answering options of these questions were as " correct " with score of 1, " incorrect " and " i do not know " with score of zero . In this scale, scores below 7 were considered as " weak ", scores 8 - 11 as " average " and scores 12 as " good " . The third section of the questionnaire consisted of questions regarding health belief model (hbm) constructs such as perceived severity (4 items), perceived benefits (5 items), perceived barriers (11 items), self - efficacy (4 items) and cues to action (3 questions) for healthy nutrition and physical activity behaviors due to cardiovascular disease prevention . These questions were answered through 5-point likert options from totally agree to totally disagree ranged from 1 to 5 . In this the fourth section of the questionnaire was about nutrition and physical activity behavior ranged from 1 to 4 . Ten specialists, three cardiologists and others in health education field assessed the questionnaire content . In content validity, changing the format of questions and omitting irrelevant questions were performed . Afterward, mean content validity index (cvi) and content validity rate (cvr) of the questionnaire calculated as 0.83 and 0.77, respectively . Reliability of the scale was calculated through test - retest on 25 homogeneous students with two weeks interval . The correlation score was 0.73 for knowledge question, 0.71 for perceived susceptibility questions, 0.61 for perceived severity questions, 0.73 for self - efficacy questions, 0.65 for perceived benefits and barriers questions and 0.80 for cues to action questions . The cronbach s alpha of knowledge questions were 0.66, 0.62, 0.51, 0.54, 0.68, 0.50, 0.70 for perceived severity, perceived susceptibility, perceived benefits, perceived barriers, perceived self efficacy and perceived cues to action, respectively . Continuous variables were presented as mean standard division (sd), whereas categorical data were presented as frequency and percentages . Kolmogorov - simirnov test was used to check normal distribution of data . Also linear regression with enter method and logistic regression method were used to study association between independent and response variables . This was a cross - sectional study performed on students of tehran university from october to december 2012 . A proportional quota sampling was used to select students studying in majors of humanity, basic sciences and technical - engineering in different grades of bs, msc and phd . Inclusion criteria were being student of tehran university and aged between 18 and 55 years . Exclusion criteria were unwillingness to participate in the study and guest student from other universities . The sample size was calculated as 323 subjects considering = 5%, statistical power of 90%, p = 0.5, d = 0.09 and the following equation: considering 10% attrition rate, we added 45 samples, so we totally considered 368 samples . In total, 36% were from engineering field, 11.3% basic sciences field and 52.7% humanity field . The first section was about demographic characteristics like age, gender, height, weight, level of education and information sources . The second part consisted of 16 questions regarding knowledge about causes, diagnosis and risk factors of cardiovascular disease, healthy diet and physical activity due to the disease prevention . The answering options of these questions were as " correct " with score of 1, " incorrect " and " i do not know " with score of zero . In this scale, scores below 7 were considered as " weak ", scores 8 - 11 as " average " and scores 12 as " good " . The third section of the questionnaire consisted of questions regarding health belief model (hbm) constructs such as perceived severity (4 items), perceived benefits (5 items), perceived barriers (11 items), self - efficacy (4 items) and cues to action (3 questions) for healthy nutrition and physical activity behaviors due to cardiovascular disease prevention . These questions were answered through 5-point likert options from totally agree to totally disagree ranged from 1 to 5 . In this the fourth section of the questionnaire was about nutrition and physical activity behavior ranged from 1 to 4 . Ten specialists, three cardiologists and others in health education field assessed the questionnaire content . In content validity, changing the format of questions and omitting irrelevant questions were performed . Afterward, mean content validity index (cvi) and content validity rate (cvr) of the questionnaire calculated as 0.83 and 0.77, respectively . Reliability of the scale was calculated through test - retest on 25 homogeneous students with two weeks interval . The correlation score was 0.73 for knowledge question, 0.71 for perceived susceptibility questions, 0.61 for perceived severity questions, 0.73 for self - efficacy questions, 0.65 for perceived benefits and barriers questions and 0.80 for cues to action questions . The cronbach s alpha of knowledge questions were 0.66, 0.62, 0.51, 0.54, 0.68, 0.50, 0.70 for perceived severity, perceived susceptibility, perceived benefits, perceived barriers, perceived self efficacy and perceived cues to action, respectively . Data was analyzed using spss version 18 (spss inc ., chicago, ill, usa). Continuous variables were presented as mean standard division (sd), whereas categorical data were presented as frequency and percentages . Kolmogorov - simirnov test was used to check normal distribution of data . Also linear regression with enter method and logistic regression method were used to study association between independent and response variables . Totally, 368 subjects including 318 female students (86.4%) and 50 male students (13.6%) took part in the study . The mean age of participants was 24.9 years (sd = 4.55) and the mean weight was 59.58 kg (sd = 11.3). The mean score of health belief model constructs and cardiovascular disease preventive behaviors are shown in table 2 . Data are presented as mean standard deviation . To explore the predictor variables of nutrition and physical activity behaviors, the main independent variables such as knowledge and all health belief model constructs entered the regression model analysis . Accordingly, there was a positive association between knowledge and nutrition behavior (p = 0.023) and a negative association between perceived barriers and this behavior (p = 0.004). Also in this model, male subjects showed lower mean nutrition behavior compared to female subjects (p <0.001). Abbreviations: b, standardized beta; s.e ., standard error; ci, confidence interval . Furthermore, predictor variables of physical activity behavior are shown in table 4 . According to this table, knowledge (p = 0.011), perceived severity (p = 0.009) and self - efficacy (p = 0.033) showed positive associations with physical activity behavior, but perceived barriers had a negative association with this behavior (p = 0.019). This study investigated how hbm constructs could predict nutrition and physical activity behaviors due to nutrition and physical activity in tehran university students . Regarding nutrition behavior, this study found that gender, knowledge and perceived barrier could contribute to behavior . Accordingly, this study verified that female students had a better nutrition compared to male students . In consistent with this result, unhealthier nutrition behaviors in male students and lack of physical activity in female students were reported in previous study (21). Contrary to this finding, bahreynian and coworkers found that healthy pattern of nutrition behavior in boys was better than girls, so that girls obtained higher body mass index (bmi) than boys (22). Furthermore, previous evidence verified that determinants of food quality in students of islands university were physical activity behavior, gender, age and the number of meals consumed per day (23). Another study demonstrated that there were differences between two genders regarding the receipt dietary fat (24). In another study, the dietary knowledge and behavior among female students were better than males (25, 26). Thus, it could be argued that in most studies, the important role of gender regarding nutrition behavior has been noticed . However, there are differences between studies to determine the role of gender in nutrition behavior . In the present study, therefore, further studies with more male and female students should be performed . In the present study, in the study of vassallo, it was revealed that perceived benefits and barriers could improve dietary regime (27). Furthermore, oconnell demonstrated that perceived benefit is a strong predictor of dietary behavior among obese teenagers (28). However, in non - obese teenagers, the perceived susceptibility was the important factor predicting nutrition behavior . It seems that in healthy and unhealthy people, the role of perceived barriers is different so that in healthy people these variables are more effective . The present study showed that knowledge has less effect on nutrition than the perceived barrier . However, previous evidence showed a significant relation between knowledge and food intake, so that knowledge has been an effective factor in defining food choice (29). Moreover, a previous evidence showed that consumers nutrition knowledge was related to label use behavior (30). However, some studies showed no significant associations between knowledge and dietary behavior (31). The present study showed that for physical activity behavior, perceived severity of cardiovascular disease could affect this behavior more than any other factors . Furthermore, in this study it was revealed that perceived barriers could predict physical activity . In consistent with this result, a previous study pointed out that physical activity levels of students were influenced by perceived barriers such as fear of strangers, bad weather and many school assignments (32). Therefore, motivation for walking should be affected by stressing on perceived benefits of physical activity such as positive health benefits, recreational benefits and a normal weight . In these motivational interviews, personal limitations such as lack of time, getting sunburn because of walking outdoors, non - supportive climate, traffic threat, crimes, etc garza and coworkers indicated that self - efficacy as a stronger predictor of the health belief model constructs could affect cardiovascular disease prevention behaviors (34). A previous study on patients with multiple sclerosis, self - efficacy and perceived benefits were the main determinants of physical activity (35). Furthermore, self - efficacy also predicted oral health behavior in other study (36). Parcel and coworkers indicated that self - efficacy plays a significant role in choosing the diets in students (37). This study indicated that health belief model contrasts could predict the risky behavior of university students regarding cardiovascular disease . However, more researches are needed to verify other predictors of high risky behaviors in students . Although, this study had some strong points, there were some limitations such as self - reporting of questions responded by the participants . Furthermore, the study was performed through a cross sectional method in which the relations between the variables were not assessed . Although, this study had some strong points, there were some limitations such as self - reporting of questions responded by the participants . Furthermore, the study was performed through a cross sectional method in which the relations between the variables were not assessed.
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Airbags protect the passengers against a crash by providing a padding device, which allows the impacting and impacted surface to deform, thereby extending the duration of the impact and reducing its severity.1 although airbags have substantially reduced the rate of mortality and morbidity, those who have survived may suffer from various fatal and nonfatal injuries to the head, eyes, neck, chest, or arms.2,3 ocular traumas are among the most severe airbag - induced injuries due to a high risk for detrimental vision impairment after an impact.4 airbag - induced ocular trauma includes corneal abrasion, corneoscleral laceration, subluxated lens, endothelial cell loss, cyclodialysis, choroidal rupture, globe rupture, retinal detachment, and periorbital fracture.511 given the initiatives in cataract or corneal surgeries for better vision, an increase in the population achieving the visual acuity test criteria for operating a vehicle would be assumed.4 any driver could be at risk of airbag - induced ocular trauma . In a 19911998 review of 97 patients with airbag - associated eye trauma, 50% of traumas were limited to the anterior segment, 6% to the posterior segment, and 44% to both.12 postcataract surgery patients with implantation of an intraocular lens (iol) may be at a higher risk with impact for wound rupture, subluxation of a posterior chamber (pc) iol, anterior capsule rupture, and dislocation of the lens.11,13,14 unlike with human bones and ribcages, the injury biomechanics of soft organs, such as human eyes, are difficult to simulate due to limited available mechanical information . Cadavers and dummies have been used for research purposes; however, the physiological and biological properties of these eyes do not resemble living eyes, making the trauma research more difficult and results only marginally reliable . Therefore, creating a humanlike eye with raw data from the human eye for biomechanical simulations using finite element analysis (fea) would help to investigate and better explain the physical and physiological responses to impact injuries.1 we have previously developed a simulation model resembling a human eye based on the information obtained from cadaver eyes and applied three - dimensional fea to determine the physical and mechanical conditions of impacting foreign bodies that cause an intraocular foreign body.15 this model human eye was also used in our studies on airbag impact in a postradial keratotomy eye and on a post - transsclerally fixated pc iol eye.4,16 in general, the capsular bag is considered the best position for iol implantation . When the capsular support is insufficient or absent, ciliary sulcus fixation or, more recently, scleral fixation, is recommended.4 in this study, we extended the simulation model after renovation to further determine the physical and mechanical response of an impacting airbag deploying at additional velocities on transsclerally fixated pc iol eyes with different axial lengths, especially surveying the mechanical threshold in a highly myopic eye . The model human eye was created and used in simulations with a computer using an fea program, pam - crash (nihon esi, tokyo, japan), described elsewhere.15 the material properties and geometry of the model were obtained from past experiments with three pairs of human cadaver eyes.15 poisson ratios of the cornea at 0.420 kg / mm and the sclera at 0.470 kg / mm were used to determine the standard stress strain curves for the cornea and sclera.1719 the reference point for globe rupture was then calculated to be at a strain of 18.0% and stress of 9.45 mpa for the cornea, and at a strain of 6.8% and stress of 9.49 mpa for the sclera.15 the cornea was assumed to be spherical, with a central thickness of 0.5 mm and a central radius of curvature of 7.8 mm . A transsclerally fixated polymethyl methacrylate pc iol element was integrated at the original physiological position of the crystal lens . The vitreous length was assumed to be 18.6 mm, and the posterior curvature of the retina was assumed to be 12.0 mm . The mass densities of ocular tissues from past reports were applied as follows:15,20 cornea, 1.149 kg / mm; sclera, 1.243 kg / mm; and vitreous and aqueous humor, 1.002 kg / mm and 1.000 kg / mm, respectively . A new approach in this study was the addition of model human eyes with different axial lengths, representing a normal eye with a normal axial length of 23.85 mm, a hyperopic eye with a shorter axial length of 21.85 mm, and a myopic eye with a longer axial length of 25.85 mm . Eyes with different axial lengths were created by setting the mass density of the cornea and sclera as constants, and the element types including the three layers of the model eye (outer, middle, and inner) as variables for meshing principles.15 a threshold for the thickness reduction ratio was 0.5 (50%). The elastic properties and meshing principles of the model human eye were similar to those in previous reports.15,16 the vitreous model as a solid mass was also assigned with a hydrostatic pressure of 20 mmhg (2.7 kpa). The changes in the deformity of the eye and the strain induced were calculated by the virtual performance solver (vps) (nihon esi kk) and evaluated by color mapping . In this study, mapping properties were renovated for more finite displaying due to the development of computer technology following the previous study.4 the simulation of pc iol implantation was based on a corneoscleral incision size of 6.0 mm and wound closure with 10 - 0 polypropylene sutures in five places . A detailed description of a single - piece polymethyl methacrylate iol and its elasticity and mass density of 2.85 gpa and 1.185 kg / mm, respectively, are described elsewhere.4 the suturing method was done the same way as reported, with a 10 - 0 polypropylene looped suture (by an alcon surgical pc 91/4 circular needle; alcon, inc ., hnenberg, switzerland) and the loop of each suture was tied with the girth hitch method.4 two tying needles were placed at the 4 and 10 oclock positions, and the sutures exited the sclera 1.0 mm posterior to the limbus and were tied directly to the superficial sclera . The limit of tensile force (n) of a 10 - 0 polypropylene suture was specified to be 0.16 n by the united states pharmacopeia xxii.21,22 breakage of the fixating suture is assumed to occur in the middle of the suture when the strain becomes intolerable due to deformity of the eye caused by airbag impact . Element deletion was incorporated in the vps, with rupture of the eye and the breakage of the suture defined as the strain exceeding the corneoscleral and fixation suture threshold, respectively . A biomechanical head of a dummy was created, assuming that everything excluding the eye was a solid element, to reduce the computing time . The hybrid iii model was modified4 by replacing the head of the dummy with a biomechanical model of the head in which the iol - fixated model eye was inserted.4,23 for simplification of calculation, the angular momentum of the impact missile was ignored in this study . The impact missile for the airbag simulation was set up as described.15 the impact missile was a decahedron that was composed of a cuspidal hexahedron and a rectangular parallelepiped, so as to be blunt in shape.15 in addition to our previous study, the airbag was deployed at five different velocities (20 m / second, 30 m / second, 40 m / second,50 m / second, and 60 m / second) and it impacted eyes of three different axial lengths . The simulation program was similar to that of a previous report by digital equipment corporation alphastation4 using the fea program, pam - crash version 1998 (nihon esi kk).4 similarly, suture strain was recorded sequentially in all velocities and the deformity of the eye was displayed sequentially in milliseconds in slow motion . Breakage of the suture was defined as the point when the strain exceeded the tensile tolerance . The postoperative pc iol model human eye with an axial length of 23.85 mm created by the vps is shown in figure 1 . The skull surrounding the eye was assumed to be a solid element to minimize the computing time for impact simulation (figure 2). The thickness of the eye wall depicted by the color maps showed correlation with the axial lengths of the eyes (figure 3). The eye with a longer axial length is shown in orange, representing a thinner eye wall compared to eyes with normal and shorter axial lengths in red . The deformity of the three eyes with transsclerally sutured iol at five impact velocities (20 m / second, 30 m / second, 40 m / second, 50 m / second, and 60 m / second) is captured in slow motion (shown in figures 4a, 5a, and 6a). The deformity of the eye was small at 20 m / second and 30 m / second . Deformity reached the threshold of corneoscleral strain (0.068) at 40 m / second, 50 m / second, and 60 m / second, causing the model eye to rupture . The tensile force on the 10 - 0 polypropylene suture of the three eyes is shown in figures 4b, 5b, and 6b . At 0.3 ms after the impact, the eye with the longest axial length had the greatest extent of deformity at any given impact velocity (figure 7). The tensile force on the suture exceeded the critical value at only the velocity of 60 m / second in all eyes . This breakage of the suture occurred in all eyes after 0.3 ms impact, with the longest axial length exceeding the breaking force initially between 0.2 ms and 0.3 ms . There was no breakage of the scleral fixation suture at 40 m / second and 50 m / second, even when the force on the corneoscleral incision suture was continuously greater with increasing velocity . At 60 m / second impact, the suture tensile force was exceeded at the sites of the scleral fixation suture, leading to both ocular rupture and breakage of the suture (figures 4b, 5b, and 6b). In a frontal crash automobile accident, drivers move forward rapidly and may be struck forcefully by a rearward - expanding bag at high velocities, causing the impacted eye to deform.1,12 despite its overall protective effects, this forceful inflation of the airbag itself could be the major contributor of ocular injuries, even in minor accidents such as bumper - to - bumper contact or even a flat tire.14 according to the us federal motor vehicle safety standards, airbags take only 20 m / second to fully inflate, which is about four times faster than blinking.25 airbag deployment occurs 15 ms after an impact with complete expansion at 50 ms and deflation by 100 ms.8 automobile airbags are reported to deploy at a speed ranging from 81322 kph (2289 m / seconds).8,2527 considering that some situations exceeded the impact velocities in the previous report,4 we added 50 m / second and 60 m / second velocities to this study . From our results, ocular rupture and breakage of the sutures occurred at 60 m / second . Therefore, no further velocities were performed for the simulation . Since eyeballs are viscoelastic, their response to impact injury is rate sensitive.1 therefore, we simulated the impacts of various velocities within the range of speed of deployment in this study to see the biomechanical response for different axial lengths . Our present study demonstrated that eyes remained intact up to a 30 m / second impact speed . Corneoscleral opening occurred at 40 m / second, 50 m / second, and 60 m / second, and eventually the ocular and fixation suture ruptured at 60 m / second . The results suggested that ocular rupture and fixating suture breakage could happen even at mid - speed of airbag deployment . Furthermore, at the fastest velocity of 60 m / second, corneoscleral rupture was observed as early as 0.1~0.2 ms after the airbag impact, suggesting that all eyes regardless of axial length would be vulnerable to a higher speed of impact . In the era of refractive surgeries, the effect on the ocular integrity of cataract surgical techniques may depend on the physical properties of the eye . To our knowledge, no published data on the mechanical properties, especially on the tensile strength, of the zonule of zinn, which is essential for the simulation of airbag impact on a phakic or a pseudophakic eye, are available . Transsclerally fixating the pc iol with a 10 - 0 polypropylene suture in contrast, since the tensile strain of the polypropylene suture is available from the manufacturer, the simulation of airbag impact on the transsclerally - iol - sutured eye can be established by modification of our previous simulation model.4 we have already demonstrated and assessed the mechanical properties of an eye with a transsclerally fixated pc iol in a past report showing iol subluxation with an impact velocity of 40 m / second.4 however, individual variations regarding axial lengths, age, or ethnicity were not considered in previous studies.4 then, we carried out the current simulated study using the pc iol transsclerally fixing model with several modifications . It is known that there has been a global increase in the prevalence of myopia over the past half - century, particularly in affluent, industrialized areas of east asia.24 higher myopia, which means a longer axial length, is associated with comorbidities that increase the risks of severe and irreversible loss of vision, such as retinal detachment, subretinal neovascularization, dense cataract, and glaucoma.24 therefore, longer eyes with thinner walls would be much more vulnerable to any kind of impact trauma . This study demonstrated that these eyes with a longer axial length are prone to a more extensive ocular deformity, causing irreversible strain on the eye, leading to ocular and fixation suture rupture if present . Longer eyes showed breakage of the fixation suture as early as 0.1 ms after the airbag impact . Other possible factors affecting the integrity of the driver s eye upon airbag impact would be age, sex, weight, usage of spectacles, and ethnicity.2 some data indicated that women of small stature and shallower eye sockets, common in asians, are more likely to be injured by airbag impacts because they are likely to sit too close to the wheel, which could cause fatal or nonfatal injuries when the airbag is deployed.2,3 elderly women with smaller statures would be physiologically characterized with normal or smaller eye axial lengths.3 this physiological factor would make this population at high risk for blunt ocular injuries . Our data showed that even at the medium velocity of 30 m / second, corneoscleral incisions and iol would be at risk in normal to shorter axial length eyes . Sitting closer to the steering wheel would decrease the distance to the driver s face and, therefore, increase the impact velocity and its force on these individuals . The study of trauma could provide insights on how the eye responds to impact injury, and would thus help to establish preventative surgical measures such as revising the surgical wound, performing iol fixation methods, improving suture material, or inserting iol implants . At the same time, blunt airbag impact studies could also help automobile industries make more adaptable airbags customized for different ethnic backgrounds and physical characteristics . Despite our careful calculation of the simulation model based on cadaver eyes, there are still limitations to our study.15 first, since studies are based on only three pairs of cadaver eyes, the results do not completely resemble a living humanlike eye response, or represent the normal population . Second, in order to reduce the time for computer calculation, the impact object was placed almost adjacent to the surface of the eyeball for the impact simulation . Therefore, further study on the distance of the deploying airbag to the driver in regard to the change in velocity would be necessary to determine the extent of impact that is likely to happen in real situations . Finally, the vitreous model was a solid mass with a physiological intraocular pressure of 20 mmhg.15 the age factor for vitreous viscosity or other physical characteristics were not taken into account as variables in our simulation . However, we believe that our goal to assess the mechanical properties of eyes with different axial lengths with a fixated iol suture was achieved by demonstrating that the corneoscleral incision would open at a medium velocity followed by iol subluxation, with the eye with the longest axial length being more susceptible to impact injuries . Furthermore, at a maximum velocity of 60 m / second, the impact force exceeded the fixated suture force, causing suture breakage . In conclusion, even with improvements in automobile industry technology, there is still need for the conscientious reporting of vision - threatening airbag impact injuries . The lack of basic research data on the biomechanical response has retarded the development of more sophisticated models or dummies . Therefore, this current method of injury biomechanics research for assessing eye injuries would help advance our knowledge on the mechanism of ocular traumas and reduce the need for animal experiments and cadavers, which are difficult to obtain and could pose ethical issues . Finally, an ability to predict anatomical or structural injury upon airbag impact would be critical to safety . Continuous injury biomechanics research would be helpful not only in developing an adequate evaluation system for airbag safety, but also in further understanding ocular preventative measures . These may include the use of protective goggles, the revision of pc iol fixations sutures, minimal surgical incisions, or placing the pc iol without sutures.
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Isolated left ventricular noncompaction (lvnc) is a congenital cardiomyopathy caused by a defect in endomyocardial morphogenesis.1) it is characterized by prominent trabeculation and deep intertrabecular recesses, resulting in thickened myocardium consisting of 2 layers - compacted and noncompacted myocardium.2) lvnc is occasionally combined with other congenital cardiac diseases3 - 6) and genetic cardiomyopathies.7) its clinical manifestations include heart failure, thromboembolism, ventricular tachyarrhythmia, and sudden death.8) the risk of embolic complications was mostly associated with impaired systolic function.9) there were only a few cases about coexisting left ventricular aneurysm.10 - 12) we report a rare case of lvnc associated with left ventricular (lv) aneurysm, presenting with recurrent embolism . A 40-year - old woman was admitted to our hospital for abdominal and lower back pain lasting for 10 days . In 1999, she had an embolic stroke of the right middle cerebral artery territory; however, after 2007, she was lost to follow - up and discontinued all medications, including antiplatelet and anticoagulant therapy she had none of the classic cardiovascular risk factors such as diabetes or hypertension nor any family history of cardiomyopathy or heart failure . Her chest radiography and laboratory data, including b - type natriuretic peptide and cardiac enzymes levels, were unremarkable . Abdominal computed tomography, performed because of her abdominal pain, revealed left renal infarction . To evaluate for an embolic source, transthoracic and transesophageal echocardiography were performed . Two - dimensional echocardiography showed prominent trabeculations at the interventricular septum and the lateral wall . Color doppler imaging revealed deep intertrabecular recesses filled by blood flowing from the ventricular cavity (fig . The ratio of maximal thickness of the noncompacted to compacted layers in the lateral wall was greater than 2 at end - systole . The 4-chamber and 2-chamber views defined the lvnc in the mid - septal and inferior walls and apical wall thinning (fig . Because wall motion of the apex was akinetic at end - systole and end - diastole, we confirmed the diagnosis as an apical aneurysm . The lv ejection fraction by modified simpson's rule was 48% (lv end - diastolic volume 89 ml and lv end - systolic volume 49 ml) and the ratio of early diastolic mitral inflow velocity to early diastolic mitral septal annular velocity (e / e') was 7.62 . We performed coronary computed tomography angiography and cardiac magnetic resonance imaging (mri) to differentiate the aneurysm from post - infarction aneurysms . On computed tomography 4). Cardiac mri showed two - layered appearance of trabeculated and compacted myocardium, it revealed a thin compacted layer preserving contractility and an apical aneurysm with akinetic motion at end - diastole and end - systole (fig . We therefore diagnosed her with left renal infarction caused by lvnc, coexisting with an lv aneurysm . Lvnc is a rare congenital cardiomyopathy characterized by multiple prominent trabeculations with deep intertrabecular recesses.1) an arrest of compaction of the developing myocardium is strongly suggested as the probable mechanism of lvnc.1)9) recently, the american heart association classified lvnc as a primary genetic cardiomyopathy.13) in contrast, the european society of cardiology considers lvnc to be an " unclassified cardiomyopathy".14) multiple diagnostic criteria for lvnc have been proposed on the basis of echocardiography and cardiac mri findings . They are as follows: 1) thickened myocardium with a 2-layered structure consisting of a thin compacted epicardial layer [c] and a much thicker, noncompacted endocardial layer [n] or trabecular meshwork with deep endomyocardial spaces (n / c ratio> 2.0 at end - systole); 2) predominant location of the pathology in the mid - lateral, mid - inferior, and apical areas; 3) color doppler evidence of deep intertrabecular recesses filled with blood from the lv cavity; and 4) absence of coexisting cardiac abnormalities (in isolated lvnc). There have been many reports of coexistent congenital cardiac disorders, including atrial septal defect, ventricular septal defect, pulmonary stenosis, anomalous pulmonary venous connection, ebstein's anomaly, and a bicuspid aortic valve.3 - 6) however, only a few cases of lvnc with lv aneurysm have been reported.10 - 12) the mechanism of aneurysm is uncertain . Sato et al.10) proposed impaired microcirculation of noncompacted and compacted layers as the mechanism of aneurysm formation in lvnc . However, in our patient, the epicardial coronary arteries appeared normal on coronary computed tomography angiography and neither perfusion defects nor delayed enhancement were seen on cardiac mri . We therefore thought that our patient's aneurysm might be congenital rather than degenerative change of lvnc . The classical triad of complications with lvnc is heart failure, ventricular arrhythmia, and systemic embolic events.8) because there are limited data regarding treatment of this condition, it is recommended that clinical complications be managed according to the current guidelines for each clinical complication . The prevalence of systemic embolic events in patients with lvnc varied in reports . Based on the high rate of embolic events reported in long - term follow - up data, oechslin et al.8) recommended anticoagulant therapy for these patients, independent of ventricular systolic function . However, oechslin and jenni9) recently recommended anticoagulation therapy for patients with impaired systolic function (lv ejection fraction <40%) because deep intertrabecular recesses and slow blood flow might increase the risk of thrombus formation . We believed that the apical thrombus was the embolic source of her presentation with renal infarction and that the apical aneurysm with slow blood flow was a risk factor for recurrent embolic events . Therefore, we suggest that anticoagulation therapy might be needed in patients with lvnc with coexisting lv aneurysm, even in the absence of systolic dysfunction or atrial fibrillation . In conclusion, we described a rare case of lvnc with lv aneurysm presenting as recurrent thromboembolic events . We believe that careful evaluation of lvnc patients for coexisting heart abnormalities such as aneurysms is essential for making the best clinical decisions for their management.
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The fungal kingdom encompasses an enormous diversity of taxa with varied ecological niches, life - cycle strategies, and morphologies . Of the 1.5 million species estimated to belong to this kingdom, only about 5% were formally classified . Plant pathogenic fungi are able to cause extensive damage and losses to agriculture and forestry including the rice blast fungus, dutch elm disease, and chestnut blight . Some other fungi can cause serious diseases in humans, several of which may be fatal if left untreated . These include aspergillosis, candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetomas, mucormycosis, and paracoccidioidomycosis . The so - called dermatophytic and keratinophilic fungi can attack eyes, nails, hair, and especially skin and cause local infections such as ringworm and athlete's foot . Fungal spores are also a cause of allergies, and fungi from different taxonomic groups can provoke allergic reactions . In this paper, after a brief presentation of the medical impact of fungal infections at the global level and a summary of clinical treatments available today for clinicians, we will review the mechanisms underlying in vitro resistance to antifungals in fungal species of major importance in human medicine . Lastly, an overview of ongoing research undertaken to develop new therapeutic strategies to fight against fungal infections will be exposed . At the beginning of the 20th century, bacterial epidemics were a global and important cause of mortality . Since the late 1960s when antibiotic therapies were developed, a drastic rise in fungal infections was observed, and they currently represent a global health threat . This increasing incidence of infection is influenced by the growing number of immunodeficient cases related to aids, cancer, old age, diabetes, cystic fibrosis, and organ transplants and other invasive surgical procedures . Primary pathogens are naturally able to establish an infection in the healthy population . In contrast, opportunistic pathogens, among them commensal microorganisms of the healthy population, are able to develop infectious colonization of the human body when particular criteria, such as immunosuppression, are met . Most of the primary pathogens are filamentous fungi, while most of the opportunistic pathogens are yeasts and some species of filamentous fungi are increasingly identified as opportunistic pathogens . It is also important to note that fungal infections can be classified in function of the tissue infected (see table 1). Superficial mycoses, such as tinea versicolor, are limited to the most external part of the skin and hair . These infections are most frequently caused by the species malassezia globosa and m. furfur, which are estimated to be carried by 2% to 8% of the healthy population worldwide but could lead to tinea versicolor in some conditions that are still unclear . The most frequently involved dermatophyte genera are trichophyton, epidermophyton, and microsporum . In most cases, cutaneous fungal infections require a challenge of immune system, and their incidence varies depending on the site of infection . For example, onychomycoses are very frequent in the global population, with an incidence varying from 5 to 25% . Mucosal infections are mostly caused by opportunistic yeasts, and those belonging to the candida genus are by far the most frequent . Vaginal, esophageal, oropharyngal, and urinary tract candidiasis are very frequent in immunocompromised patients . For example, esophageal candidiasis is associated with the entry into the clinical phase of aids and during the 1980s more than 80% of seropositive patients developed such an infection . Fungal infections, of the eye are also classified as mucosal fungal infections, but are caused more frequently by fusarium or aspergillus species rather than candida species . Theoretically systemic mycoses may involve any part of the body, and a lot of species formerly considered as nonpathogenic are now recognized opportunistic pathogens responsible for deep - seated mycoses . These infections, with symptoms ranging from a simple fever to a severe and rapid septic shock, are very common in immunocompromised patients and are frequently associated with an elevated mortality rate . The most frequent pathogens involved in systemic fungal infections are candida, pneumocystis, histoplasma, aspergillus, cryptococcus, mucor, rhizopus, and coccidioidomyces [46]. Despite extensive research dedicated to the development of new therapeutic strategies, there are only a limited number of available drugs to fight against invasive fungal infections . Indeed, only four molecular classes that target three distinct fungal metabolic pathways are currently used in clinical practice to treat essentially systemic fungal infections: fluoropyrimidine analogs, polyenes, azoles, and echinocandins . Several other classes, such as morpholines and allylamines are only used as topical agents due to either poor efficacy, or severe adverse effects when administered systemically . Fluoropyrimidines, of which only 5-fluorocytosine (5-fc) and 5-fluorouracil (5-fu) are used in human medicine, are synthetic structural analogs of the dna nucleotide cytosine (figure 1)., initially as an antitumor therapy . In 1963, grunberg and coworkers discovered its antifungal potential by means of murine models of cryptococcosis and candidiasis . Several years later 5-fc was successfully used for the treatment of systemic candidiasis and of cryptococcal meningitis . Due to its high hydrosolubility and small size, 5-fc possesses interesting pharmacokinetic properties, since it diffuses rapidly throughout body even when orally administered . Generally, it produces negligible side effects, despite some severe adverse effects, such as hepatotoxicity or bone marrow lesions [11, 1315], occurring in rare cases . Surprisingly, these side effects are identical to those observed with 5-fu treatment, despite the fact that humans do not possess a cytosine deaminase enzyme that is responsible for the conversion from 5-fc into 5-fu in fungal cells [17, 18]. Some data suggest that the intestinal microbiome could be responsible for the 5-fu production and the observed side effects . Despite its numerous pharmacological advantages, the use of 5-fc in clinical practice is decreasing because of the frequent occurrence of innate or acquired resistance to this drug in fungal pathogens . Thus, with few exceptions, 5-fc is never used as monotherapy but always in combination with another antifungal, such as amphotericin b [21, 22]. However, the elevated renal and liver toxicities of amphotericin b, that further increase 5-fc hepatotoxicity, has led to combination therapy of 5-fc more frequently with azole drugs . 5-fc itself has no antifungal activity, and its fungistatic properties are dependent upon the conversion into 5-fu [16, 20, 23]. The drug rapidly enters the fungal cell through specific transporters, such as cytosine permeases or pyrimidine transporters, before it is converted into 5-fu by the cytosine deaminase . 5-fu itself is converted into 5-fluorouracil monophosphate (5-fump) by another enzyme, uridine phosphoribosyltransferase (uprt). 5-fump can then be either converted into 5-fluorouracil triphosphate, which incorporates into rna in place of utp and inhibits protein synthesis, or converted into 5-fluorodeoxyuridine monophosphate, which inhibits a key enzyme of dna synthesis, the thymidylate synthase, thus inhibiting cell replication (figure 2) [16, 25, 26]. More than 200 molecules belonging to the chemical class of polyenes have an antifungal activity, most of them being produced by streptomyces bacteria . However, only three possess a toxicity allowing their use in clinical practice: amphotericin b (amb), nystatin, and natamycine . Streptomyces bacteria synthesize polyenes through a gene cluster phylogenetically conserved within these species . This cluster contains genes coding for several polyketide synthases, abc (atp - binding cassette) transporters, cytochrome p450-dependent enzymes, and enzymes responsible for the synthesis and the binding of the mycosamine group . Although it is possible to synthesize polyenes chemically, they are still produced from streptomyces cultures for economic reasons . Most of them consist of a 20 to 40 carbons macrolactone ring conjugated with a d - mycosamine group . Their amphiphilic properties are due to the presence of several conjugated doublebounds on the hydrophobic side of the macrolactone ring, and to the presence of several hydroxyl residues on the opposite, hydrophilic side (figure 3). Magnetic nuclear resonance data suggest that eight amb molecules bind eight ergosterol molecules through their hydrophobic moieties, with their hydrophilic sides forming a central channel of 70100 nm in diameter (figure 4). Pore formation promotes plasma membrane destabilization, and channels allow leakage of intracellular components such as k ions, responsible for cell lysis . While structural data suggest that polyenes target ergosterol, and despite the fact that their binding to ergosterol was experimentally demonstrated [2931], controversy remains over a possible intracellular mode of action . Some research has suggested that polyene drugs are able to induce an oxidative stress (particularly in c. albicans [32, 33]) as well as their activity seems to be reduced in hypoxic conditions . Polyenes possess a lower but non - negligible affinity for cholesterol, the human counterpart of ergosterol . This slight affinity for cholesterol explains the high toxicity associated with antifungals and is responsible for several side effects . For this reason, only amb is given systemically, while nystatin and natamycin are only used locally or orally . These two last molecules fortunately possess a very limited systemic activity, since their absorption trough gastrointestinal mucosa is almost nonexistent [35, 36]. For these reasons, amb is the most used polyene antifungal for systemic infections . Due to its high hydrophobicity and poor absorption through the gastrointestinal tract, however, amb administration is accompanied with adverse effects, mostly at the level of kidneys and liver . New amb formulations, such as liposomal amb or lipid amb complexes, minimize such side effects . For more than 40 years, amb was one of the goldstandards for the treatment of systemic fungal infections due to the low occurrence of acquired or innate resistance to this drug and also because of its broad range of activity . It is recommended for the treatment of infections caused by candida, aspergillus, fusarium, mucor, rhizopus, scedosporium, trichosporon, cryptococcus, and so on . Natamycin and nystatin are active against fungi belonging to the genera cryptococcus, candida, aspergillus, and fusarium . If nystatin is not used to treat molds infections, this drug is frequently used for the treatment of cutaneous, vaginal, and esophageal candidiasis, and natamycin can be used for the treatment of fungal keratosis or corneal infections . Azoles are by far the most commonly used antifungals in clinical practice, and consequently, they are also the mostly studied by the scientific community regarding their mode of action, their pharmacological properties, and the resistance mechanisms developed by microorganisms . Azole antifungals are also largely studied by pharmaceutical companies, who seek to enhance their efficacy and to develop the perfect antifungal . Azoles are cyclic organic molecules which can be divided into two groups on the basis of the number of nitrogen atoms in the azole ring: the imidazoles contain two nitrogen atoms, and the triazoles contain three nitrogen atoms (figure 5). Azole drugs target the ergosterol biosynthetic pathway by inhibition of a key enzyme, the lanosterol 14alpha demethylase, encoded be the erg11 gene . This inhibition occurs through the binding of the free nitrogen atom of the azole ring to the iron atom of the heme group of the enzyme . The resulting accumulation and metabolism of 14alpha methylated sterol species leads to the synthesis of toxic compounds, which are unable to successfully replace ergosterol . The first azole was synthesized in 1944 by woolley, but it was not until 1958 that scientific community began to consider azoles as potential antifungal agents . In late 1960s, however, their use was restricted to external application due to their high toxicity when administered orally [43, 44]. In 1968, miconazole became the first antifungal available for parenteral injection, but due to its toxicity and relatively limited range among fungal species, its use decreased until it was no longer commercialized . In 1981, the food and drug administration (fda) approved a new antifungal, ketoconazole, developed by heeres and his coworkers . This drug was the only antifungal available for treatment of systemic fungal infections caused by yeasts for the following ten years . It is poorly absorbed when administered orally, and no ketoconazole form has ever been developed for intravenous injection . Moreover, it cannot cross the cerebrospinal barrier and is less active in immunosuppressed patients [42, 4749]. It causes some severe side effects such as a decrease in testosterone or glucocorticoids production and liver and gastrointestinal complications [5052]. Fluconazole became available for use by clinicians in 1990 and provided many advantages over the use of imidazoles . It is almost completely absorbed through the gastrointestinal tract, and it diffuses throughout the whole body, including cerebrospinal fluid [53, 54]. Fluconazole is suitable for the treatment of superficial candidiasis (oropharyngal, esophageal, or vaginal), disseminated candidiasis, cryptococcal meningitis, coccidioidomycosis, and cutaneous candidiasis . Due to its good pharmacokinetic properties as well as its broad spectrum of activity, unfortunately, the overprescription of this drug by physicians for prophylaxis or treatment led to an increase in resistance to azole drugs . Moreover, fluconazole is almost ineffective against most molds . This triazole possesses a broad spectrum of activity across fungal species comparable to this of ketoconazole and wider than fluconazole . Moreover, it is less toxic than ketoconazole and replaced it for treatment of histoplasmosis, blastomycosis, and paracoccidioidomycosis . Contrary to fluconazole, it is also used for the treatment of infections due to species belonging to the genera aspergillus and sporothrix . However, itraconazole is hydrophobic and is thus more toxic than fluconazole . Itraconazole is only indicated for the treatment of onychomycosis, of superficial infections, and in some cases for systemic aspergillosis . A new itraconazole formulation with an enhanced absorption and a decreased toxicity was approved by fda in 1997 . Fluconazole and itraconazole are still not the perfect antifungals, since they have some nonnegligible drug interactions with such drugs that are used in chemotherapy or with aids treatment . These interactions can result in a decrease in azole concentration or even to an increase in toxicity . Furthermore itraconazole and fluconazole are ineffective against some emerging pathogens like scedosporium, fusarium, and mucorales, and resistance to azoles is increasingly reported . They possess a wide range of activity, since they are active against candida, aspergillus, fusarium, penicillium, scedosporium, acremonium, and trichosporon, and dimorphic fungi, dermatophytes, and cryptococcus neoformans [61, 62]. While new generation triazoles were shown to be more effective against candida and aspergillus, compared to classical triazoles their side effects and drug interactions are similar to those observed with fluconazole and itraconazole . Likewise, fungal isolates resistant to classical triazoles are also cross - resistant to new generation triazoles . Echinocandins constitute the only new class of antifungals made available for clinicians to fight invasive fungal infections within the past 15 years . Three echinocandins were currently approved for clinical use by the fda in united states and later by the european agency for the evaluation of medicinal products (emea): caspofungin in 2001 by the fda and in 2002 by the emea, micafungin in 2005, and lastly anidulafungin in 2006 . These lipopeptides are naturally produced by several fungal species: aspergillus rugulovalvus synthesizes caspofungin b, zalerion arboricola synthesizes pneumocandin b, and papularia sphaerosperma synthesizes papulacandin . Echinocandins are noncompetitive inhibitors of (1 - 3)-glucan synthase, an enzyme that catalyzes the polymerization of uridine diphosphate - glucose into (1 - 3) glucan, one of the structural components responsible for the maintenance of fungal cell - wall integrity and rigidity [65, 66]. (1 - 3)-glucan synthase consists of an activating and a catalytic subunit encoded by fks genes . In most fungi, it has been shown in the model organism saccharomyces cerevisiae that fks1 is expressed during the vegetative growth phase and fks2 during sporulation . Inhibition of (1 - 3)-glucan synthase leads to cell wall destabilization and to the leakage of intracellular components, resulting in fungal cell lysis . These drugs are poorly absorbed in the gastrointestinal tract because of their high molecular weights and are therefore only used intravenously . Their pharmacologic properties are one of the reasons responsible for the approval of echinocandins by the fda and the emea . These molecules possess a low toxicity (very rare side effects were reported) and are slowly degraded, and a daily injection is sufficient, and contrary to other antifungals, interactions between echinocandins and other drugs are rare . Combined therapy between echinocandins and amb or another azole often leads to a synergistic effect or at least to an additive effect [70, 71]. Indeed, echinocandins are active against most fungal species, including candida and aspergillus . For still unclear reasons, these molecules are fungicidal in candida but only fungistatic in aspergillus [72, 73]. Moreover, fungicidal activity of echinocandins is species and isolate dependent within the candida genus . Other species have an intermediate susceptibility to echinocandins, such as scedosporium apiospermum, s. prolificans, and cladophialophora bantiana . However, echinocandins constitute a good alternative to fight against fungal infections and most of treatment of infections for which classical therapy with azoles or polyenes failed are successfully managed with echinocandins . Therefore, caspofungin is indicated for the treatment of candidemia and invasive candidiasis, for fungal infection prophylaxis, and for the treatment of invasive aspergillosis for which itraconazole, voriconazole, or amb is ineffective . Micafungin is used for treatment of candidemia and is particularly indicated for fungal infection prophylaxis in bone - marrow transplant patients . Anidulafungin has no particular indications, but its main advantage is its slow degradation in the body without liver or kidney involvement, thus it can be used in patients with liver and/or kidney insufficiencies ., the fungal cell wall was considered to be a promising target for the development of new antifungal molecules . Its integrity is necessary for the fungal survival, since it provides a physical barrier against the host immune cells or against other microorganisms . Cell wall integrity is also responsible for osmolarity homeostasis and the maintenance of cell shape and size . Cell wall is also indispensable to essential enzymatic reactions and as an important role in cell - cell communication . The internal layer of the cell wall is composed of a (1 - 3)-glucans and chitin web, in which are included some mannoproteins, while external layer is composed of mannoproteins (figure 7). Considering that the ergosterol biosynthetic pathway requires several enzymes that are unique to fungi, they constitute good targets for antifungal therapy, and three minor ergosterol biosynthesis inhibitors are used as topical antifungals . The allylamines and thiocarbamates, such as terbinafine and tolnaftate, both inhibit the erg1-encoded enzyme, squalene epoxidase . The morpholines such as amorolfine act by inhibiting two different enzymes of the pathway, the 7,8-isomerase (encoded by erg24) and the c14-reductase (encoded by erg2). Despite their wide spectrum of activity, these antifungal agents are essentially used to treat dermatophyte infections such as tinea capitis, tinea pedis, and onychomycosis, because they do present numerous side effects . Ciclopirox is also used as a topical antifungal agent, but its mode of action remains poorly understood in fungi [78, 79]. The incidence of fungal infections has drastically increased over the past three decades and was simultaneously accompanied by increased acquired and innate resistance to antifungal drugs . However, antifungal resistance occurrence has to be considered independently for each antifungal class and for each fungal genus . Moreover, epidemiological data regarding incidence of resistance among fungal species is not identically distributed worldwide [8183]. Lastly, clinical resistance, defined as the treatment failure in the patient, does not always correlate with in vitro resistance, measured as an increase in minimal inhibitory concentration of a drug . In this paper, only in vitro resistance incidence will be described . The development of resistance can be intrinsic, as is the case for c. tropicalis, or acquired through the selection of resistant mutants after antifungal exposure . Within the candida genus, 7% to 8% of clinical isolates are resistant to 5fc, and this frequency increases to 22% when only nonalbicans candida species are considered . Filamentous fungi such as aspergillus and dermatophytes are not susceptible to 5fc . Despite the reported increase of polyene resistance, it remains a relatively rare event in clinical isolates of fungal pathogens, probably in relation with their mode of action, and the absence of systematic and standardized determination of susceptibility of clinical isolates . Most fungal species are considered as susceptible to polyene drugs . However, some of them are intrinsically poorly susceptible to these antifungals, such as c. glabrata, scedosporium prolificans, or aspergillus terreus one may cite c. lusitaniae [88, 89], c. guilliermondii, c. krusei, and trichosporon beigelii and among filamentous fungi scedosporium apiospermum and sporothrix schenckii [90, 91]. The early 1990s was the start of a drastic increase in resistance among fungal clinical isolates . However, the improvement of antifungal therapeutic strategies throughout the last several years has helped to stabilize resistance frequencies . Increase in azoles use selected less susceptible species as well as those able to develop resistance . For example, it is estimated that more than 97% of clinical isolates belonging to the candida genus are susceptible to these drugs [93, 94]. Contrary to acquired resistance in other fungi, intrinsic echinocandin resistance in cryptococcus neoformans is not linked with a fks1 or fks2 mutation . Indeed, c. neoformans (13)-glucan synthase is inhibited by echinocandins, but this yeast is able to grow in the presence of high concentrations of these drugs . C. neoformans resistance to echinocandins seems to be due to a particular cell - wall polysaccharides composition in this species . As antifungal in vitro resistance poorly correlates with clinical outcome, better attention was needed to define parameters that produced reproducible and reliable intra- and interlaboratory results . For this purpose, two standardized methods for the testing of yeast and mould isolates (clsi and eucast) are recognized as the gold standards for drug susceptibility testing [9698]. These standardized approaches produce susceptibility results comparable between laboratories, which may help to establish breakpoints for antifungal agents (see [9698] for details). These breakpoints, defined as susceptibility ranges, together with pharmacokinetic and pharmacodynamic analyses and identification of resistance mechanisms, help to assess the in vivo activity of antifungal agents in invasive disease and therefore clinical outcome [99, 100]. Microorganisms develop mechanisms to counteract the fungicidal or fungistatic effects of all antifungals classes that are based on three major mechanisms, namely, (i) reducing the accumulation of the drug within the fungal cell, (ii) decreasing the affinity of the drug for its target, and (iii) modifications of metabolism to counterbalance the drug effect (table 2). The molecular mechanisms leading to azole resistance have been most studied in yeast, and taking them as an example, such mechanisms are divided into four categories (figure 8): (i) decrease in azole affinity for their target, (ii) increase in azole target copy number, (iii) alteration of ergosterol biosynthetic pathway after azoles action, and (iv) decrease in intracellular azole accumulation . In some highly resistant clinical isolates, sampled from long - term treated patients, this increase in resistance along antifungal treatment is due to the sequential acquisition of different mechanisms [104106]. In the following section, cdr1 and cdr2 (candida drug resistance 1 and 2) from c. albicans are the two major abc transporters involved in azole resistance in this species . Cdr1 and cdr2 can be coordinately upregulated in some azole - resistant strains or by exposure to a wide variety of chemically unrelated inducers such as terbinafine, amorolfine, fluphenazine, or steroid hormones . Several cis - acting regulatory elements responsible for the regulation of these two genes were identified by several investigators [107111]. Promoter deletion studies have revealed 5 different regulatory elements in the cdr1 promoter including a bee (basal expression element), a dre (drug responsive element), two sres (steroid responsive element), and a nre (negative regulatory element) (see table 3 for details). Internal deletions of the bee and dre motifs in the cdr1 promoter affect basal cdr1 expression and drug - induced expression, respectively . Sre1 and sre2 were reported to be involved in the response to steroid hormones: with sre1 responding only to progesterone and sre2 to both progesterone and -oestradiol . Finally, the deletion of the nre motif leads to an increase in the basal expression of cdr1 [110, 111]. In contrast to cdr1, the cdr2 promoter contains only a dre motif (table 3). Among these different cis - acting elements, dre was the only element involved in constitutive high expression and in transient upregulation of both cdr1 and cdr2 . This dre sequence was functionally analyzed by systematic mutation each base of the initially described dre sequence [107, 112]. The data obtained from systematic mutational studies are in agreement with chip - chip assays performed with the trans - acting factor binding to the dre . In other candida species, functional homologues to cdr1 and cdr2 were described as involved in drug resistance . In c. glabrata, cgcdr1 and cgcdr2 (formerly denoted pdh1) as well as snq2 (another abc transporter coregulated with cgcdr1 and cgcdr2) are upregulated in azole - resistant clinical isolates and participate in azole resistance [114118]. All the three genes contain cis - acting elements in their promoters, so - called pdre . These elements are similar to those described in s. cerevisiae for pdr5, an abc transporter involved in drug resistance of s. cerevisiae [119, 120]. Disruptions of cgcdr1 and cgcdr2 lead to hypersusceptibility to fluconazole, cycloheximide, and chloramphenicol [115, 117]. In both c. albicans and c. glabrata, cdr1 was shown to be the main contributor in azole - resistance among the abc - transporters [121123]. Other abc - transporters from c. dubliniensis (cdcdr1 and cdcdr2) [124, 125], c. krusei (abc1 and 2) [126, 127], c. tropicalis (cdr1-homologue), and c. neoformans (cnafr1, antifungal resistance 1) were reportedly upregulated in azole - resistant isolates . In a. fumigatus, atrf, and afumdr4 are upregulated in itraconazole - resistant strains [128130]. The cis - acting regulatory elements of these genes are still awaiting in - depth dissection analysis . The overexpression of abc - transporters have also been identified as a resistance mechanism to azole in aspergillus nidulans [131, 132]. The identification of trans - acting factors regulating abc - transporters in pathogenic fungi relied first on the well - described s. cerevisiae pdr network as a model [138142]. Since the zn2-cys6 transcription factors pdr1/pdr3 are master regulators of this network in s. cerevisiae, an in silico search for pdr1/pdr3 homologues in fungal genomes was performed . Cgpdr1p has 40% and 35% identity with pdr1p and pdr3p, respectively, and was able to complement a pdr1 s. cerevisiae mutant strain . Likewise, pdr1 deletion in c. glabrata leads to a loss of cgcdr1 and cgcdr2 regulation and to a sharp decrease in azole mics . . Three studies have identified separate gain - of - function mutations in cgpdr1 alleles of azole - resistant strains which are responsible for constitutive high expression of cgcdr1, cgcdr2, snq2, and cgpdr1 itself (figure 9) [120, 145, 146]. Attempts to identify c. albicans pdr1/3 functional homologues were undertaken to complement the absence of pdr1/pdr3 in s. cerevisiae by genetic screens . Several genes were identified including fcr1 and fcr3 (fluconazol resistance) [147149] and shy1 - 3 (suppressor of hypersusceptibility) (formerly, resp . Fcr1, cta4, asg1, and atf1 encode zn2-cys6 transcription factors, while fcr3 encodes a bzip transcription factor . Even though fcr1 was able to restore pdr5 expression in a pdr1/pdr3 s. cerevisiae mutant strain, its disruption in c. albicans resulted in decreased susceptibility to fluconazole, suggesting that fcr1 acts as a negative regulator of fluconazole susceptibility . Nevertheless, the target genes of fcr1 in c. albicans are not yet known . Up to now, the relevance of fcr3 in azole resistance has not been addressed in c. albicans . Cta4, asg1, and atf1 expression in s. cerevisiae could restore pdr1/pdr3 functions in s. cerevisiae; however, their disruption in c. albicans did not affect azole susceptibility and expression of cdr1 and cdr2 . An additional regulator of cdr1 was identified by a genetic screen in s. cerevisiae with a lacz reporter system under the control of the cdr1 promoter . A c. albicans gene was subsequently identified that encodes for a protein candt80p similar to the s. cerevisiae meiosis specific transcription factor ndt80p . Disruption of candt80 in c. albicans was shown to affect basal expression levels of cdr1 in c. albicans and reduce the ability of this gene to be upregulated in the presence of miconazole [151, 152]. More recently, ndt80p was shown to have a global effect on azole - resistance through is regulon which includes many genes involved in ergosterol metabolism . The release of the entire data from the c. albicans genome sequence has encouraged other approaches for identifying trans - regulators of cdr1 and cdr2 . Since the dre motifs present in the promoter of cdr genes contains two cgg triplets that are potentially recognized by zn2-cys6 transcription factors (tf) [154157], it was likely that one of the 78 orfs encoding proteins with zn2-cys6 signatures could be involved in the regulation of cdr1 and cdr2 . Interestingly, genome data revealed that three of these orf (the so - called zinc cluster) were located in tandem close to the mating type locus (mtl) at a distance of 14 kb . Homozygosity at the mtl locus is associated with the development of azole resistance in c. albicans, thus indicating that one the genes of the zinc cluster could control cdr1 and cdr2 expression . As a matter of fact, deletion of one of these zn2-cys6 tf - encoding genes in an azole - susceptible strain led to increased drug susceptibility and loss of transient cdr1 and cdr2 upregulation in the presence of inducers . Consistent with the mutant phenotype, tac1p can bind in vitro and in vivo to the dre [112, 158]. However, tac1 is not involved is the basal expression of cdr1 controlled at least by the bee . Hyperactive alleles that confer constitutive high cdr1 and cdr2 expression, and therefore drug resistance to a tac1/ mutant strain of tac1, were isolated from azole - resistant strains . Wild - type and hyperactive alleles differed by point mutations defined as gain - of - function mutations (gof). Up to now, at least 15 gof were described in tac1 at 12 different positions [112, 158, 160, 161] (figure 9). Wild - type and hyperactive alleles are co - dominant for the expression of their phenotypes [112, 158, 160, 161], and because of this property, high drug resistance levels correlate with homozygosity of hyperactive alleles . Interestingly, the tac1 locus and the associated mtl are rendered homozygous in the development of azole resistance . Such events are accomplished by rearrangements on chromosome 5 including mitotic recombinations on one chromosome 5 arm or the loss of one chromosome 5 homologue followed by duplication . Increase of resistance can still be obtained by isochromosome formation with the left arm of the chromosome 5 . This allows for the increase of drug resistance genes present on this chromosome (among which tac1 and erg11) and thus can contribute to drug resistance increase [160164]. Up to this date, regulation of tac1p activity remains unknown . In c. albicans, mdr1 (multidrug resistance 1, previously named benfor benomyl resistance) is a transporter currently shown to be the only mfs transporter involved in azole resistance of clinical isolates [165, 166]. Mdr1 is not usually expressed at detectable levels in fluconazole - susceptible isolates, but is constitutively upregulated in some fluconazole - resistant isolates . As for cdr1 and cdr2, mdr1 can be specifically transiently upregulated by drugs such as benomyl, cycloheximide, methotrexate, and several oxidizing agents . Homologues of mdr1 in c. dubliniensis and c. tropicalis, named cdmdr1 and ctmdr1, respectively, are upregulated in azole - resistant strains [167170]. In a. fumigatus, in vitro - generated itraconazole - resistant isolates show constitutive high expression level of the mfs transporter, afumdr3 . The role of cis - acting regulatory elements in resistance was investigated in the c. albicans mdr1 gene by separate studies . And riggle and kumamoto identified a similar region, called bre (benomyl response element) or mdre (mdr1 drug resistance element) respectively . This region is responsible for the constitutive high expression of mdr1 in fluconazole - resistant isolates [171, 172] and was also shown to be responsible for the response to benomyl . A second regulatory element involved in the response of mdr1 to oxidative stress this region contains two yre (yap1 response element) motifs, one perfectly conserved (-532 ttagtaa-526) and the other with two mismatches (-549 taactat-543). Interestingly, the hre is not required for constitutive upregulation of mdr1 in azole - resistant isolates . A separate study undertaken by hiller et al . Described three distinct cis - activating regions (1, 2, and 3) in mdr1 . Region 2, which overlaps with encompassing the hre, was implicated in benomyl - dependent mdr1 response . Region 1 and 3, close to the bre / mdre region, were required for a constitutive high expression of mdr1 in an azole - resistant isolate . Mdr1 expression was shown to be regulated by at least four trans - acting factors: cap1p [175177], mrr1p [178, 179], upc2p [180, 181], and mcm1p . Nucleotide sequence data of cis - acting elements has provided some clues to their identification . When comparing the mdr1 cis - acting elements with existing transcription factor binding site databases, the bzip transcription factor cap1 was shown to directly interact with the cis - acting domains and to be involved in drug resistance . The bre / mdre motif contains a perfect match for the mads - box transcription factor mcm1p in its sequence . Showed that mcm1p acts as a coregulator for cap1 and mrr1p and is not required for mdr1 upregulation by h2o2 but is required for full mdr1 induction by benomyl . Genome - wide transcriptional analyses of clinical isolates that exhibit mdr1upregulation permitted the identification of a zn2-cys6 transcription factor that is coregulated with mdr1 . Deletion of mrr1 in azole - resistant strains abolishes the constitutive overexpression of mdr1, therefore identifying mrr1p as a central regulator of mdr1 . Like for tac1 wild - type alleles are needed for a transient upregulation of mdr1 by drug exposure . In contrast, hyperactive alleles confer constitutive overexpression of mdr1 and therefore also confer increased resistance to fluconazole . Wild - type and hyperactive alleles differ by gof mutations and until now, 14 gof mutations at 13 positions were described in hyperactive mrr1 (figure 9). Interestingly, hyperactive mrr1 proteins were also shown to be able to confer mdr1p - independent drug resistance probably through the regulation of oxydoreductases implicated in the detoxification of yeast cells after fluconazole exposure . A blast analysis in c. dubliniensis allowed for the identification of a gene encoding for a protein that shares 91% of identity with mrr1 of c. albicans and able to complement a camrr1 mutant strain . The properties of cdmrr1 are similar to those of camrr1 and two types of alleles can also be distinguished . Until now another mechanism by which fungal pathogens are able to develop resistance is a decrease in antifungal affinity for their respective targets, without a major decrease in target activity . Such is the case for azole drugs, in which a decreased affinity between azole and a mutated lanosterol 14-demethylase, can lead to resistance . A point mutation in the erg11 gene that codes for lanosterol 14-demethylase leads to the complete inhibition of the binding capacity of the azole drug to its target [184, 185]. Numerous of these point mutations identified in erg11 were previously described, and their involvement in azole resistance was experimentally demonstrated for fungi such as cryptococcus neoformans, c. albicans, (see also figure 9), and c. tropicalis . In aspergillus fumigates, cyp51a and cyp51b encode two distinct forms of 14-demethylase and mutations in the first of these two genes seem to be the most frequent mechanism responsible for azole resistance in clinical isolates . In this species, it was demonstrated that the nature of the nucleotide mutation, and therefore, the nature of the amino acid substitution, influences the development of resistance to different azole agents [188192]. Interestingly, it was demonstrated that some clinical isolates share common mutations in cyp51a with environmental azole - resistant strains, suggesting that some clinical azole resistant isolates might originate from the environment [193195]. While target site alteration is far from being the most significant mechanism of resistance to azole drugs, it is the only known mechanism by which fungal pathogens are able to develop resistance to echinocandin drugs . Echinocandin resistance is systematically associated with point mutations in either fks1 or fks2 [196, 197]. Analysis of the location of these mutations within the fks genes led to the characterization of two regions, the so - called hot spots, integrity of which seems to be essential for enzyme activity . In contrast to azoles and erg11, mutations in fks1 did not alter the -glucane synthase affinity for its target but decreased only the enzyme processivity . Hot - spot mutations have also been identified in other species, such as c. glabrata [196, 199], c. krusei, scedosporium apiospermum, and a. fumigatus [202, 203] (figure 9). Numerous enzymes of the pyrimidine salvage pathway are involved in 5fc mode of action and thus numerous molecular mechanisms could lead to resistance to this drug [16, 204]. The most frequently found mechanism in clinical isolates of pathogenic fungi is a point mutation in the fur1 gene that encodes the enzyme responsible for the conversion of 5fu into metabolites able to enter the cytosine metabolism (figure 9). This second mutation is a point mutation in the fcy1 gene that codes for cytosine deaminase, the enzyme responsible for the conversion from 5fc into 5fu . Several such point mutations that lead to decreased activity of the cytosine deaminase were identified, essentially in candida yeast species such as c. glabrata [205, 206] and in s. cerevisiae . A third mechanism of drug resistance is the deregulation of the drug target expression . For drugs targeting, the biosynthesis of ergosterol, such as azoles, terbinafine, or fenpropimorph, even relative short exposures of two to three hours lead to the transient upregulation of the erg gene family in c. albicans, glabrata, tropicalis, and krusei . These data suggest a common regulation of the ergosterol biosynthetic pathway in the presence of inhibitors . Longer azoles in vitro exposures (minimum 24 h) lead to constitutive upregulation of erg genes, including erg11, and decrease drug susceptibility . In clinical isolates of c. albicans and c. dubliniensis resistant to azoles isolated from hiv patients, upregulation of erg11 was described as a minor mechanism often combined with other more major mechanisms of resistance such as pump overexpression or erg11 mutations [103, 169, 210]. The overexpression of erg11 originates either by gene dosage effect through duplication of the gene or by upregulation of the gene by a trans - acting factors, both hypotheses were verified . In c. albicans and c. glabrata, it was shown that increased azole resistance due to erg11 upregulation was in fact due to genome rearrangement via formation of an isochromosome in c. albicans and duplication of a chromosome in c. glabrata, and therefore amplification of erg11 [163, 211]. In cryptococcus neoformans, the well - known sre1 gene was shown to regulate the ergosterol biosynthesis pathway and also to be involved with virulence of the fungus . In s. cerevisiae, the erg gene family was shown to be regulated by two zinc cluster transcription factors encoded by scupc2 and scecm22 . Two homologues of scupc2 were found in the genome of c. glabrata: cgupc2a and cgupc2b . It appears that while both transcription factors regulate sterol biosynthesis and exogenous uptake, only cgupc2a is responsible for the regulation of the transcription of the erg gene family in response to sterol inhibitors . In c. albicans, only one gene homologue of scupc2/scecm22 it was shown that caupc2 is necessary for the upregulation of erg genes in the presence of ergosterol synthesis inhibitors . Moreover, the upc2 / mutant shows increased susceptibility to most drugs and a decrease in sterol uptake as compared to the wild - type strain [214, 215]. Further studies demonstrated the ability of caupc2 to bind to the are motif in the promoter of c. albicans erg11 (table 3) [215, 216]. Genome - wide location analysis of caupc2 confirmed the sre motif as the dna binding site, and also confirmed the erg gene family as a caupc2 target as well as cdr1, mdr1, and upc2 itself as new target genes . Analysis of clinical strains resistant to fluconazole with upregulated erg11 expression, demonstrated the existence of a hyperactive allele of caupc2 that confers intrinsic upregulation of erg genes . Currently, two gof mutations were described for caupc2 (figure 9) [134, 180]. Some yeasts and filamentous fungi are able to grow in elevated echinocandin concentrations much higher than the mics [136, 217]. This phenomenon, called paradoxical effect, is due to the metabolic adaptation of microorganism and is mediated by the cell wall integrity signalization pathway . This response is the direct consequence of the (1 - 3)-glucans synthesis inhibition and the subsequent cell wall composition modifications, upon echinocandin administration [218, 219]. Several studies suggest that the magnitude of the paradoxical effect is variable depending on the microorganism itself as well as on the echinocandin nature . Therefore, the paradoxical effect would be more pronounced in the presence of caspofungin as compared to anidulafungin or micafungin . However, the clinical significance of paradoxical effect has never been studied nor has it ever been observed in echinocandin - treated patients . It is possible that 5fc resistance could be the consequence of an overall induction of the de novo pyrimidine biosynthetic pathway . In this case, the antifungal drug competes with the regular pyrimidine intermediate metabolites for incorporation into nucleic acids . This increase in activity of the de novo pyrimidine biosynthetic pathway is reflected by an increased expression of the cdc21 gene, whose product is inhibited by 5fc . However, its downregulation has also been demonstrated to be involved in 5fc decreased susceptibility . A 4-fold decreased expression of this gene of high importance in 5fc mode of action is sufficient to lead to a total resistance to this pyrimidine fluorinated analog in c. glabrata . Modifications of main metabolic pathways could also lead to azole drugs resistance . For example, alteration of the late steps of the ergosterol biosynthetic pathway through inactivation of the erg3 gene gives rise to cross - resistance to all azole drugs . Indeed, the antifungal activity of azole drugs relies on the synthesis of toxic 14 methylated sterols by the late enzymes of this pathway . A point mutation that occurs in the erg3 gene can lead to the total inactivation of c5 sterol desaturase . In this case, toxic 14 methylated sterols are no longer synthesized and even in the presence of azole drugs sterols species able to replace ergosterol are generated . While very uncommon, this mechanism was identified in several clinical isolates of c. albicans [221223]. Polyene drugs do not require internalization into fungal cells in order to exert their antifungal activity, since they incorporate into the plasma membrane from the external side . Thus, they escape metabolizing enzymes and efflux systems, and the only possibility for fungi to develop resistance to polyene is to modify their target, ergosterol . However, ergosterol is responsible for the integrity and fluidity of the plasma membrane, and therefore, possibilities to compensate for its absence are very limited . Although rarely described, resistance mechanisms responsible for acquired or innate resistance to polyene drugs were studied in several fungal species . In each case, resistance to polyenes results from a decrease or total absence of ergosterol in the plasma membrane through mutations in nonessential genes of the ergosterol biosynthetic pathway . Molecular polyene resistance mechanisms were described in laboratory mutants of yeasts belonging to the candida genus and in s. cerevisiae . Thus, both erg11 deletion in c. albicans and erg3 deletion in s. cerevisiae lead to mutants with cross - resistance to azole and polyene drugs . Likewise, erg6 inactivation in c. lusitaniae and s. cerevisiae results in polyene resistance . Only a few studies have associated polyene resistance to an erg3 mutation in clinical isolates of c. albicans [221, 229] and to an erg6 mutation in c. glabrata [230, 231]. It is well characterized that microbial communities engulfed in a polysaccharides - rich extracellular matrix, also known as biofilm, are by far more resistant to antifungal drugs than isolated cells . The mostly known and widely studied of those species able to form biofilms are the species of the candida genus . Another yeast frequently responsible for biofilm - associated infections is cryptococcus neoformans [233, 234]. Some clinical cases have also reported the involvement of other yeast species, such as pichia fabianii or trichosporon asahii . Additionally, it is now accepted that filamentous fungi, and particularly those of the aspergillus genus, can grow as biofilms in humans [237239]. Fungal biofilms are frequently polymicrobial biofilms, meaning that bacterial species frequently associate with one or several fungi [240, 241]. In medical mycology they constitute a nonnegligible source of nosocomial fungal infections, essentially through the use medical devices . Moreover fungal biofilms are resistant to almost all the currently used antifungals, with the exceptions of echinocandins and lipid formulations of amb . The molecular mechanisms underlying the persistence of the fungal biofilms despite antifungal treatment remain unclear . It is likely that biofilm resistance is the result of a combination a multiple factors, among them an increased expression of efflux pumps, a modification of plasma membrane composition, and the biofilm - produced extracellular matrix itself [232, 243]. Current antifungal treatments are limited in their capacity to treat infections, especially systemic infections and no considerable advancements in antifungal therapies were developed recently . Several approaches were developed during the last several years in order to find new solutions . Researchers aim to discover new antifungal drugs either by testing already existing medical compounds, compounds from natural sources such as plants, sea, microorganisms or by systematic screens of chemical compound libraries . Researchers also strive to elucidate the underlying biology of fungal microorganism both in vitro and in vivo . Host - fungal interactions play a critical role for all fungal pathogens . Targeting this interaction provides novel therapies, which could be used alone or in combination with existing antifungal drugs . Much effort has gone towards analyzing the antifungal properties of what is called natural compounds (np) or natural bioactive compounds isolated from plants, other microorganisms, or marine organisms [244246]. Some such compounds are investigated because their known triggering mechanisms important for fungi, while other compounds are tested blindly for their antifungal properties . Currently, none of these studies have produced a compound suitable for the clinical trial stage although interesting results were obtained . Other studies focused on in vitro screens of several drugs currently used in clinical practice for their potentiation of the antifungal effect of the fungistatic agent fluconazole (flc) on candida albicans . This facilitated the discovery of several compounds, such as inhibitors of the calcineurin [247, 248] or tor pathways [249251], efflux pump inhibitors (derived compounds of milbemycin) [252254], and more recently, antibodies against heat - shock 90 protein (hsp90). In particular, inhibitors of the calcineurin pathway were shown to be fully active in vivo in the potentiation of fluconazole, and they also led to a dramatic decrease in fungi virulence [256260]. Systematic screening of chemical compounds libraries was also undertaken, essentially by industrial laboratories as an attempt to discover new antifungal compounds . High throughput screening of the legacy schering - plough compound collection has recently lead to the discovery of a new glucan synthase inhibitor effective again c. albicans and c. glabrata [261263]. Some analysis used reverse genetic assay in which, c. albicans heterozygous deletion or transposon disruption mutants collection were screened for growth under treatment with collections of chemical compounds [264, 265]. This approach allowed identification of both antifungal drugs and the genes related to the mechanism of action of the related compounds . Another type of high - throughput screens of chemical libraries was achieved measuring the viability of drug - treated caenorhabditis elegans infected with c. albicans . Compounds can be simultaneously screened for antifungal efficacy and host toxicity, which overcomes one of the main obstacles in current antimicrobial discovery . A pilot screen for antifungal compounds using this novel c. elegans system identified 15 compounds that prolonged survival of nematodes infected with the medically important human pathogen c. albicans . One of these compounds, caffeic acid phenethyl ester (cape), had effective antifungal activity in a murine model of systemic candidiasis and had in vitro activity against several other fungal species . In addition, this whole - animal system may enable the identification of compounds that modulate immune responses and/or affect fungal virulence factors that are only expressed during infection . The improvement of already existing antifungal drugs and the limitation of drugs resistance apparition has helped to elucidate the basic biology of the fungal pathogen . For this purpose, several groups made efforts to develop collection of systematic mutants essentially for c. albicans . An important difficulty for antifungal therapy is to develop drugs that exploit factors unique to fungi, which can be challenging considering that organism are eukaryotic and share many conserved biological pathways . Genes that are essential to fungal survival are possible targets for drug development . Using the grace (gene replacement and conditional expression) or cpr (conditional promoter replacement) technologies, some research groups have assessed the essentiality of c. albicans and aspergillus fumigatus genes [267, 268]. One study identified 567 essential genes in c. albicans . And another study screened 54 genes of a. fumigatus based on ortholog functions and essentiality in c. albicans and s. cerevisiae, of which 35 were defined as essential in a. fumigatus . Authors were able to show that while the erg11 gene family (cyp51a and erg11b) is essential in a. fumigatus, the individual genes themselves are not . These analyses provide interesting and fully informative data for antifungal drug design and improve upon previous in silico analyses that when using s. cerevisiae data were only able to identify 61% of homologous genes reported in the genes found in the roemer et al the diploid state of the genome presents a major problem to the development of a mutant collection . Therefore, some collections consist of heterozygous deletion or transposon disruption mutants [264, 265]. Other collections contain homozygous transposon disruption mutants based on the random insertion thanks to the tn7 transposon to a uau cassette [269, 270]. These collection were first restricted to the transcription factors of c. albicans [269, 271, 272] but continue to be enlarged for the entire genome [270, 273]. Other collections consist of deletion mutants constructed with pcr generated deletion cassettes, with two different markers for each allele in the case of c. albicans [274, 275]. They aimed a better understanding of the modifications occurring in the fungi submitted to antifungal treatments or of the relationship developed between the fungus and its host all along the infectious process . Such knowledge might improve the actual therapy to avoid resistance development or might allow playing on the host - fungus equilibrium to improve recovery of patients . First of all, treating strains with already known antifungal drugs and analyzing for example, growth modification and later transcriptional rewiring, some authors try to better understand drugs mechanisms of action and/or to find synergistic effect between them [272, 274]. Gene encoding the transcription factor cas5 was found to be involved in the response to caspofungin . Other studies showed that age3, which encodes an adp - ribosylation factor gtpase activating effector protein, if deleted, abrogates fluconazole tolerance in c. albicans . Interestingly, brefeldin a, an inhibitor of adp - ribosylation factor, resulted in a synergistic effect with other drugs for c. albicans as well as for aspergillus . Finally, homann et al . Screened a collection of 143 transcription factor mutants under 55 distinct conditions among which exposition to fluconazole and 5fc, and they conclude in their analysis that nearly a quarter of the knockout strains affected sensitivity to commonly used antifungal drugs . Other studies were geared better understand the biology of fungal species . For this purpose, mutant collections were subjected to a wide range of environmental conditions, modifying elements such as ph, salt concentration, carbon sources, oxidative conditions, temperature, and availability of essential elements such as metals (iron, copper, zinc, etc .) Understanding the relationship between fungus and host during infection may provide further information useful for the improvement of antifungal treatment . In order to analyze the cross - talk occurring between fungus and host during the infectious process one study that was performed with 1201 gene knockout mutants of cryptococcus neoformans analyzed their in vivo proliferation profile in the murine lung, and they were able to identify 40 infectivity mutants . Gene deletions in these mutants were previously uncharacterized and did not show any defect in traits known to be linked to virulence (polysaccharide capsule formation, melanization, and growth at body temperature). Two of them were done in invertebrate host models such as c. elegans or d. melanogaster [266, 278]. Interestingly, the cas5 / mutant, which has already been shown to be important for caspofungin response, was shown to be less virulent in both invertebrate models of infection [278, 279]. Finally, this transcription factor was demonstrated as crucial for cell wall integrity, and its importance in virulence was confirmed in the mice intravenous model of infection . Two other studies screened collections of c. albicans mutants directly in mice by pools of mutants that were previously tagged [280, 281]. One collection was restricted to zn2-cys6 transcription factors (tf) mutants and the other was composed of mutants affecting about 11% of the entire c. albicans genome with no respect to a gene class . In both cases, mutants were also screened for traits known to be linked to virulence, such as the ability to filament and proliferate as well as the ability to grow at 42c, at high and low ph, and in oxidative conditions . Identified 115 mutants among the 674 screened with attenuated infectivity, but normal morphological switching and proliferation . More precisely, they identified glycolipid and glucosylceramide as the first small molecules synthesized by c. albicans that are specifically required for virulence . These mutants displayed attenuated infectivity in their pool test, which was also confirmed in independent single strains infection of mice zcf13 and zcf18 . Zcf18 showed a slight growth defect in contrast to zcf13 which grew normally at body temperature, but slightly less at 42c . Zcf13 mutant displayed an abnormal morphology, producing strongly filamentous colonies on ypd medium at 35c and displaying high invasion ability . Unfortunately, whenever promising, up to now, no new compound and/or new target have been selected for further development from these approaches . These last years were very rich in better knowledge of molecular basis of antifungal resistance and more generally of the metabolism of pathogenic fungi . Antifungal drug resistance appears to essentially be due to point mutations in either drug targets or transcription factors regulating actors of the resistance . In the near future, high throughput diagnostic tools could be used in the course of treatment of fungal infections in order to detect resistance and adjust therapeutic strategies accordingly before any clinical evidence and therefore allow a rapid adjustment of the antifungal treatment . One of the challenges of finding new antifungal targets in c. albicans was the lack of sophisticated screening technologies often employed with other fungal species such as saccharomyces cerevisiae . The recent application of genome - wide studies to pathogenic fungi for both host - pathogen interactions and the biological study will hopefully encourage and facilitate the development of new effective therapeutic strategies.
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As an abrupt or rapid decline in renal filtration function, acute kidney injury (aki) is a common condition associated with significant morbidity and mortality . In critically ill patients, those with aki usually present a worse clinical outcome than their non - aki counterparts . Although regarded as the standard indicators of kidney function loss, serum creatinine (scr) level and urine output are recognized as having limitations . On the one hand, scr cannot accurately reflect the glomerular filtration rate (gfr) in a patient with unsteady state, and urine output is easily affected by water intake and the primary water load of the body . On the other hand, scr and urine output have limited sensitivity and specificity, and a delayed response to kidney impairment . All these factors suggest that scr and urine output are not appropriate markers in the early detection of aki . Thus, an accurate and timely biomarker to predict aki onset or progression after renal insult is urgently needed . Interleukin-18 (il-18), a member of the il-1 family of cytokines, is synthesized as an inactive 23-kda precursor by several tissues including monocytes, macrophages, and proximal tubular epithelial cells, and is processed into an active 18.3 kda cytokine by caspase-1 . It has been demonstrated in some animal studies as a mediator of renal ischemia reperfusion injury, inducing acute tubular necrosis, and neutrophil and monocyte infiltration of the renal parenchyma . More recently, numerous clinical studies have focused on the diagnostic accuracy of il-18 level in predicting aki [57]. With the evidence accumulating, contradictory results have raised concerns about the predictive value of aki across various settings . Thus, we performed a systematic review and meta - analysis to investigate the diagnostic accuracy of il-18 level for predicting aki . Since there is no clear consensus about the appropriate cutoff level of il-18 to predict aki and different thresholds have been reported by different studies, the summary receiver operating characteristic (sroc) curve was delineated for the meta - analysis . A computer - based search was performed in medline, embase, cochrane library, ovid, and springerlink databases, from inception until november 15, 2013, to identify potentially relevant articles . The search strategy consisted of terms related to aki (acute kidney injury and acute renal failure) combined with the term 18/il-18 . To ensure all articles were located, a secondary search of the articles already retrieved was undertaken, reviewing their reference lists . The preferred reporting items for systematic reviews and meta - analysis (prisma) guidelines for the conduct of meta - analyses were followed . The titles and abstracts were examined independently by two of the authors (x. l. and j. Y.) To ascertain their relevance and inclusion for further analysis . Inclusion criteria were that studies investigated the diagnostic accuracy of urinary il-18 to predict aki and had data which could be extracted into a 2 2 table or complete data which could be obtained from the corresponding author by e - mail . The following variables were recorded or recalculated: study population, sample size, age, sex, baseline il-18 level, cutoff value for urine il-18, aki definition, number of patients who developed aki, timing of obtaining the specimen, specificity, sensitivity, and area under the roc curve (auroc) with 95% confidence interval (ci). Two reviewers independently excluded articles on the basis of the title and abstract following a custom - made standardized table . The true - positive, true - negative, false - positive, and false - negative results of each included study were quantified . Quality assessment was performed using the quality assessment of diagnostic accuracy studies (quadas) tool and independently by the authors (x. l. and j. Y.). The meta - analysis was conducted using sroc curve as described by rosman and the computation was performed using meta - disc1.4 . If multiple cutoff points were adopted in any study, the cutoff point (test threshold), sensitivity and specificity in this analysis were selected according to the youden index . Random effects models (fem or rem) were used on the basis of heterogeneity . We performed subgroup analysis to explore the remarkable heterogeneity in the light of different clinical settings . A computer - based search was performed in medline, embase, cochrane library, ovid, and springerlink databases, from inception until november 15, 2013, to identify potentially relevant articles . The search strategy consisted of terms related to aki (acute kidney injury and acute renal failure) combined with the term 18/il-18 . To ensure all articles were located, a secondary search of the articles already retrieved was undertaken, reviewing their reference lists . The preferred reporting items for systematic reviews and meta - analysis (prisma) guidelines for the conduct of meta - analyses were followed . The titles and abstracts were examined independently by two of the authors (x. l. and j. Y.) To ascertain their relevance and inclusion for further analysis . Inclusion criteria were that studies investigated the diagnostic accuracy of urinary il-18 to predict aki and had data which could be extracted into a 2 2 table or complete data which could be obtained from the corresponding author by e - mail . The following variables were recorded or recalculated: study population, sample size, age, sex, baseline il-18 level, cutoff value for urine il-18, aki definition, number of patients who developed aki, timing of obtaining the specimen, specificity, sensitivity, and area under the roc curve (auroc) with 95% confidence interval (ci). Two reviewers independently excluded articles on the basis of the title and abstract following a custom - made standardized table . The true - positive, true - negative, false - positive, and false - negative results of each included study were quantified . Quality assessment was performed using the quality assessment of diagnostic accuracy studies (quadas) tool and independently by the authors (x. l. and j. Y.). The meta - analysis was conducted using sroc curve as described by rosman and the computation was performed using meta - disc1.4 . If multiple cutoff points were adopted in any study, the cutoff point (test threshold), sensitivity and specificity in this analysis were selected according to the youden index . Random effects models (fem or rem) were used on the basis of heterogeneity . We performed subgroup analysis to explore the remarkable heterogeneity in the light of different clinical settings . Of 179 primary articles searched, 156 were excluded on the basis of abstract and/or title because they were laboratory studies, genetic studies, review / comment articles, animal studies, or irrelevant to the current analysis; 3 were excluded because non - english articles . Of the 20 remaining articles, 5 studies with accurate diagnostic values of il-18 missing and 3 studies lacking effective data (though we had contacted the corresponding authors to request the missing information) were excluded . As a result, 11 studies [6, 7, 1119] met the inclusion criteria and were selected for the analysis (fig . 1flowchart representing study selection for systematic review of urine il-18 as a diagnostic marker for aki . Il interleukin, aki acute kidney injury flowchart representing study selection for systematic review of urine il-18 as a diagnostic marker for aki . Il interleukin, aki acute kidney injury characteristics of the included trials are shown in table 1 . Two come from china [6, 11], seven from the usa [7, 13, 14, 1619], one from taiwan, china, and one from australia . Most studies were conducted on critical patients, including those with cardiac surgery (cardiopulmonary bypass) and transplantation, as well as various intensive care patients . Five studies [6, 11, 13, 17, 18] focused on pediatric patients . Eight studies defined aki using acute kidney injury network (akin) and/or risk, injury, failure, loss and end - stage renal disease (rifle) criteria, two [11, 17] using modified pediatric rifle, and one using scr level which, however, rose to 50% of baseline in the first 72 h. ten studies harnessed urine il-18 concentration as the cutoff value and two [11, 15] the ratio of urine il-18 to urine - creatinine concentration.table 1characteristics of included studiesstudy (first author + year)country of originstudy populationmean age (years)men (%) sample sizenumber (%) of akimean baseline il-18 (pg / ml)definition of akiil-18 assaysample storage (c)zheng et al . Chinachildren with chd or undergoing cpb surgerynon - aki 11.4 (2.247.0)aki 5.9 (0.644.5) 39 (67.2)5829 (50)non - aki 7.9 (3.823.1)aki 17.6 (7.241.5)scr 0.3 mg / dl or 50% baseline, urine 0.5 ml / kg/6 h akin criteriaelisa80sirota et al . 6.827 (67.5)407 (17.5)aki 0 (018.57)non - aki 0 (0200.10)scr 50% baseline, rifle and akin criteriaelisa80li et al . Chinanon - septic critically ill neonates34.1 3.2 34 (54.8)6211 (17.7)/scr> 1.5 mg / dl on the first 3 days of life, after the first 3 days of life, a 25% decrease in eccl . Taiwan, chinaccu patients66 1113 (75)15043 (28.7)71 5akin criteriaelisa80parikh et al . Usapediatric patients with congenital cardiac lesions surgery3.8 4.5171 (55)31153 (17.0)/receipt of acute dialysis during the entire hospital stay or scr double baseline . Usacardiac surgery adults at high risk for aki71 10826 (68)121960 (4.9)/receipt of acute dialysis during the entire hospital stay or scr double baseline . Australiaicu admission60 17367 (70.2)523147 (28.1)73 340 scr 0.3 mg / dl or 50% baselineakin48 or rifle 24 criteriaelisa80liangos et al . Usapatients undergoing on - pump cardiac surgery68 1174 (72)10313 (12.6)/scr 50% baseline in the first 72 h following termination of cpbelisa80washburn et al . Usapicu children who received mechanical ventilation6.5 6.473 (53)137103 (75.2)179.0 337.9paediatric modified rifleelisa80parikh et al . 5.330 (54.5)5520 (36.4)1.65 1.01scr 50% baselineelisa80parikh et al . Usaards and ali patient50.2 17.072 (52.2)13852 (37.7)aki 104 (0955)control 0 (0173)scr 50% baselineelisa80 aki acute kidney injury, akin acute kidney injury network, elisa enzyme - linked immunosorbent assay, chd congenital heart disease, cpb cardiopulmonary bypass, scr serum creatinine, rifle risk, injury, failure, loss and end - stage renal disease, eccl estimated cr clearance, ccu coronary care unit, icu intensive care unit, picu pediatric intensive care unit, ards acute respiratory distress syndrome, ali acute lung injury months; gestational age, weeks; (pg / ml)/mmol / l cr characteristics of included studies aki acute kidney injury, akin acute kidney injury network, elisa enzyme - linked immunosorbent assay, chd congenital heart disease, cpb cardiopulmonary bypass, scr serum creatinine, rifle risk, injury, failure, loss and end - stage renal disease, eccl estimated cr clearance, ccu coronary care unit, icu intensive care unit, picu pediatric intensive care unit, ards acute respiratory distress syndrome, ali acute lung injury months; gestational age, weeks; (pg / ml)/mmol / l cr table 2 lists the methodological quality assessment of the included 11 studies . Ten studies enrolled single or multicenter consecutive patients prospectively, one was retrospective and prospective in design . The study by chen et al . Did not define inclusion and exclusion criteria clearly . The reference standards used in the afore - mentioned studies were creatinine based, which currently is the most widely used standard for evaluating kidney function . The conditions of follow - up were reported in the 10 prospective cohort studies; there were no selective losses in them . Overall, the quality of the studies was suboptimal, without sufficient information to assess all the bias, and uninterpretable, indeterminate or intermediate index test results were not reported . The quality assessment was performed independently by the authors (x. l. and j. Y. ).table 2quality assessment of individual studiesstudystudy designspectrum bias eligibility criteria clearly definedappropriate reference standarddifferential verification bias index test and reference standard sufficiently describedselective loss during followup important confounders identifiedzheng et al . Prospective studynoyesyesyesyesnoyes this item is labeled no if the spectrum of patients was representative of patients who received the test in practice, otherwise it is labeled yes this item is labeled no when all patients received the same reference standard, otherwise it is labeled yes this item is labeled yes if patients who were lost to follow - up differed systematically from those who remained, otherwise it is labeled no this item is not applicable if the study was retrospective in design quality assessment of individual studies this item is labeled no if the spectrum of patients was representative of patients who received the test in practice, otherwise it is labeled yes this item is labeled no when all patients received the same reference standard, otherwise it is labeled yes if patients who were lost to follow - up differed systematically from those who remained, otherwise it is labeled no this item is not applicable if the study was retrospective in design as the respective number of true - positive, false - positive, true - negative, and false - negative results was not provided by the studies, these indexes were calculated from available sensitivity, specificity, and sample size values . Results are reported in table 3 which enumerates specimen obtaining times and aki predictive times of each study . Cutoff and auroc values are also included in the table . As can be seen, cutoff values for urine il-18 varied across studies.table 3sensitivity and specificity of individual studies for urine il-18 to predict akistudytime of obtaining specimentpfpfntncutoff value (pg / ml)sensitivity (%) specificity (%) auroc (95% ci)assess time (h)zheng et al . 0, 4, 6, 12, and 24 h after the initiation of cpb28111184996.6062.100.835 (0.7290.940)4sirota1 et al admission50839729336 34780.62 (0.560.67)0liangos et al . 2 h post - cardiopulmonary bypass10313599275660.66 (0.490.83)2washburn et al . 2 pm each day39764277538780.54 (0.310.77)24551048247553710.61 (0.43 0.78)48parikh et al . Every 2 h for the first 12 h and then once every 12 h5115345025970.61410210335050940.75121248312060890.7324parikh et al . Days 0, 1, and 3382914572574660.73124 il interleukin, aki acute kidney injury, auroc area under the receiver operating characteristic curve, ci confidence interval, cpb cardiopulmonary bypass, fn false - negative, fp false - positive, tn true - negative, tp true - positive pg / mg ucr (pg / ml)/mmol / l cr sensitivity and specificity of individual studies for urine il-18 to predict aki il interleukin, aki acute kidney injury, auroc area under the receiver operating characteristic curve, ci confidence interval, cpb cardiopulmonary bypass, fn false - negative, fp false - positive, tn true - negative, tp true - positive the distribution of accurate estimator in sroc curve floor plan and the spearman correlation coefficient (r = 0.764, p = 0.006) of logarithmic sensitivity and logarithm (1-specificity) showed that there was a threshold effect in different studies (fig . 2; table 4).fig . 2the distribution of accurate estimator in sroc curve floor plan . Sroc summary receiver operating characteristictable 4analysis of diagnostic thresholdspearman correlation coefficient: 0.764, p value = 0.006logit (tpr) vs. logit (fpr)moses model (d = a + bs)weighted regression (inverse variance)varcoeff.std . Errort p valuea1.7910.2547.0610.0001b(1)0.2240.1571.4280.1870tau - squared estimate = 0.1904 (convergence is achieved after 6 iterations)restricted maximum likelihood estimation (reml)no . Studies = 11 add 1/2 to all cells of the studies with zero the distribution of accurate estimator in sroc curve floor plan . Sroc summary receiver operating characteristic analysis of diagnostic threshold tau - squared estimate = 0.1904 (convergence is achieved after 6 iterations) restricted maximum likelihood estimation (reml) no . Studies = 11 add 1/2 to all cells of the studies with zero figure 3 shows the forest plots and pooled estimates of sensitivity, specificity and diagnostic odds ratios (ors) respectively . We found a diagnostic or of 5.11 (95% ci 3.228.12) for urine il-18 level to predict aki (cochran - q = 28.19, p = 0.0017) with a sensitivity and specificity respectively of 0.51 (heterogeneity chi - squared 84.53, p <0.001) and 0.79 (heterogeneity chi - squared 62.84, p <as there was no statistically significant between b and 0 (p = 0.1870), the mantel haenszel model was utilized to draw the fitting curve of sroc (fig . The pooled auroc was 0.77 (95% ci 0.710.83, q = 0.71).fig . 3forest plots and pooled estimates of a diagnostic odds ratio (or), b sensitivity, and c specificityfig . 4the fitting curve of sroc forest plots and pooled estimates of a diagnostic odds ratio (or), b sensitivity, and c specificity the fitting curve of sroc subgroup analysis (table 5) showed that urine il-18 level in pediatric patients (<18 years) was more effective in predicting aki, with a diagnostic or of 7.510 (95% ci 2.98818.875) compared to 4.652 (2.7107.987) for the adult group (p = 0.334); the early aki predictive time (<12 h) subgroup displayed the highest diagnostic or of 8.176 (95% ci 2.19130.507) among the three subgroups (<12, 24 and 48 h, p = 0.037). The pooled estimate diagnostic or of the admission subgroup was 3.81 (95% ci 2.086.99) and that of the subgroup for other times 7.16 (3.6214.18). There were no significant differences between the two (p = 0.388). There were significant differences (p = 0.008) between cardiac surgery patients and patients in intensive care unit or coronary care unit patients in the subgroup analysis, with diagnostic ors of 5.28 (3.597.76) and 5.31 (2.5910.87), respectively.table 5pooled diagnostic accuracy of il-18 in various settingssubgroup factorssubgroup criteriareference numbersq valuep valuei (%) hierarchical summary or (95% ci)p value for between subgroupsage<18 years510.230.03760.97.51 (2.9918.88)0.33418 years615.540.00867.84.32 (2.487.51)predictive time12 h518.270.00178.18.18 (2.1930.51)0.03724 h44.480.21433.05.07 (2.589.96)48 h55.040.28320.74.95 (3.397.24)obtaining specimenadmission413.40.00477.63.81 (2.086.99)0.388other times710.380.10942.47.16 (3.6214.18)patientscardiac surgery40.590.900.015.28 (3.5937.762)0.008other patients723.280.00174.25.31 (2.5910.87) or odds ratio, ci confidence interval pooled diagnostic accuracy of il-18 in various settings or odds ratio, ci confidence interval of 179 primary articles searched, 156 were excluded on the basis of abstract and/or title because they were laboratory studies, genetic studies, review / comment articles, animal studies, or irrelevant to the current analysis; 3 were excluded because non - english articles . Of the 20 remaining articles, 5 studies with accurate diagnostic values of il-18 missing and 3 studies lacking effective data (though we had contacted the corresponding authors to request the missing information) were excluded . As a result, 11 studies [6, 7, 1119] met the inclusion criteria and were selected for the analysis (fig . 1flowchart representing study selection for systematic review of urine il-18 as a diagnostic marker for aki . Il interleukin, aki acute kidney injury flowchart representing study selection for systematic review of urine il-18 as a diagnostic marker for aki . Il interleukin, aki acute kidney injury characteristics of the included trials are shown in table 1 . Two come from china [6, 11], seven from the usa [7, 13, 14, 1619], one from taiwan, china, and one from australia . Most studies were conducted on critical patients, including those with cardiac surgery (cardiopulmonary bypass) and transplantation, as well as various intensive care patients . Five studies [6, 11, 13, 17, 18] focused on pediatric patients . Eight studies defined aki using acute kidney injury network (akin) and/or risk, injury, failure, loss and end - stage renal disease (rifle) criteria, two [11, 17] using modified pediatric rifle, and one using scr level which, however, rose to 50% of baseline in the first 72 h. ten studies harnessed urine il-18 concentration as the cutoff value and two [11, 15] the ratio of urine il-18 to urine - creatinine concentration.table 1characteristics of included studiesstudy (first author + year)country of originstudy populationmean age (years)men (%) sample sizenumber (%) of akimean baseline il-18 (pg / ml)definition of akiil-18 assaysample storage (c)zheng et al . Chinachildren with chd or undergoing cpb surgerynon - aki 11.4 (2.247.0)aki 5.9 (0.644.5) 39 (67.2)5829 (50)non - aki 7.9 (3.823.1)aki 17.6 (7.241.5)scr 0.3 mg / dl or 50% baseline, urine 0.5 ml / kg/6 h akin criteriaelisa80sirota et al . 6.827 (67.5)407 (17.5)aki 0 (018.57)non - aki 0 (0200.10)scr 50% baseline, rifle and akin criteriaelisa80li et al . Chinanon - septic critically ill neonates34.1 3.2 34 (54.8)6211 (17.7)/scr> 1.5 mg / dl on the first 3 days of life, after the first 3 days of life, a 25% decrease in eccl . Taiwan, chinaccu patients66 1113 (75)15043 (28.7)71 5akin criteriaelisa80parikh et al . Usapediatric patients with congenital cardiac lesions surgery3.8 4.5171 (55)31153 (17.0)/receipt of acute dialysis during the entire hospital stay or scr double baseline . Usacardiac surgery adults at high risk for aki71 10826 (68)121960 (4.9)/receipt of acute dialysis during the entire hospital stay or scr double baseline . Australiaicu admission60 17367 (70.2)523147 (28.1)73 340 scr 0.3 mg / dl or 50% baselineakin48 or rifle 24 criteriaelisa80liangos et al . Usapatients undergoing on - pump cardiac surgery68 1174 (72)10313 (12.6)/scr 50% baseline in the first 72 h following termination of cpbelisa80washburn et al . Usapicu children who received mechanical ventilation6.5 6.473 (53)137103 (75.2)179.0 337.9paediatric modified rifleelisa80parikh et al . 5.330 (54.5)5520 (36.4)1.65 1.01scr 50% baselineelisa80parikh et al . Usaards and ali patient50.2 17.072 (52.2)13852 (37.7)aki 104 (0955)control 0 (0173)scr 50% baselineelisa80 aki acute kidney injury, akin acute kidney injury network, elisa enzyme - linked immunosorbent assay, chd congenital heart disease, cpb cardiopulmonary bypass, scr serum creatinine, rifle risk, injury, failure, loss and end - stage renal disease, eccl estimated cr clearance, ccu coronary care unit, icu intensive care unit, picu pediatric intensive care unit, ards acute respiratory distress syndrome, ali acute lung injury months; gestational age, weeks; (pg / ml)/mmol / l cr characteristics of included studies aki acute kidney injury, akin acute kidney injury network, elisa enzyme - linked immunosorbent assay, chd congenital heart disease, cpb cardiopulmonary bypass, scr serum creatinine, rifle risk, injury, failure, loss and end - stage renal disease, eccl estimated cr clearance, ccu coronary care unit, icu intensive care unit, picu pediatric intensive care unit, ards acute respiratory distress syndrome, ali acute lung injury months; gestational age, weeks; (pg / ml)/mmol / l cr ten studies enrolled single or multicenter consecutive patients prospectively, one was retrospective and prospective in design . The study by chen et al . Did not define inclusion and exclusion criteria clearly . The reference standards used in the afore - mentioned studies were creatinine based, which currently is the most widely used standard for evaluating kidney function . The conditions of follow - up were reported in the 10 prospective cohort studies; there were no selective losses in them . Overall, the quality of the studies was suboptimal, without sufficient information to assess all the bias, and uninterpretable, indeterminate or intermediate index test results were not reported . The quality assessment was performed independently by the authors (x. l. and j. Y. ).table 2quality assessment of individual studiesstudystudy designspectrum bias eligibility criteria clearly definedappropriate reference standarddifferential verification bias index test and reference standard sufficiently describedselective loss during followup important confounders identifiedzheng et al . Prospective studynoyesyesyesyesnoyes this item is labeled no if the spectrum of patients was representative of patients who received the test in practice, otherwise it is labeled yes this item is labeled no when all patients received the same reference standard, otherwise it is labeled yes this item is labeled yes if patients who were lost to follow - up differed systematically from those who remained, otherwise it is labeled no this item is not applicable if the study was retrospective in design quality assessment of individual studies this item is labeled no if the spectrum of patients was representative of patients who received the test in practice, otherwise it is labeled yes this item is labeled no when all patients received the same reference standard, otherwise it is labeled yes if patients who were lost to follow - up differed systematically from those who remained, otherwise it is labeled no this item is not applicable if the study was retrospective in design as the respective number of true - positive, false - positive, true - negative, and false - negative results was not provided by the studies, these indexes were calculated from available sensitivity, specificity, and sample size values . Results are reported in table 3 which enumerates specimen obtaining times and aki predictive times of each study . Cutoff and auroc values are also included in the table . As can be seen, cutoff values for urine il-18 varied across studies.table 3sensitivity and specificity of individual studies for urine il-18 to predict akistudytime of obtaining specimentpfpfntncutoff value (pg / ml)sensitivity (%) specificity (%) auroc (95% ci)assess time (h)zheng et al . 0, 4, 6, 12, and 24 h after the initiation of cpb28111184996.6062.100.835 (0.7290.940)4sirota1 et al . 24 h after orthotopic liver transplantation57226/72790.74924li et al . 48 h admitted744471,800 64920.72 (0.520.93)48chen et al . Admission50839729336 34780.62 (0.560.67)0liangos et al . 2 h post - cardiopulmonary bypass10313599275660.66 (0.490.83)2washburn et al . 2 pm each day39764277538780.54 (0.310.77)24551048247553710.61 (0.43 0.78)48parikh et al . Every 2 h for the first 12 h and then once every 12 h5115345025970.61410210335050940.75121248312060890.7324parikh et al . Days 0, 1, and 3382914572574660.73124 il interleukin, aki acute kidney injury, auroc area under the receiver operating characteristic curve, ci confidence interval, cpb cardiopulmonary bypass, fn false - negative, fp false - positive, tn true - negative, tp true - positive pg / mg ucr (pg / ml)/mmol / l cr sensitivity and specificity of individual studies for urine il-18 to predict aki il interleukin, aki acute kidney injury, auroc area under the receiver operating characteristic curve, ci confidence interval, cpb cardiopulmonary bypass, fn false - negative, fp false - positive, tn true - negative, tp true - positive the distribution of accurate estimator in sroc curve floor plan and the spearman correlation coefficient (r = 0.764, p = 0.006) of logarithmic sensitivity and logarithm (1-specificity) showed that there was a threshold effect in different studies (fig . 2; table 4).fig . Sroc summary receiver operating characteristictable 4analysis of diagnostic thresholdspearman correlation coefficient: 0.764, p value = 0.006logit (tpr) vs. logit (fpr)moses model (d = a + bs)weighted regression (inverse variance)varcoeff.std . Errort p valuea1.7910.2547.0610.0001b(1)0.2240.1571.4280.1870tau - squared estimate = 0.1904 (convergence is achieved after 6 iterations)restricted maximum likelihood estimation (reml)no . Studies = 11 add 1/2 to all cells of the studies with zero the distribution of accurate estimator in sroc curve floor plan . Sroc summary receiver operating characteristic analysis of diagnostic threshold tau - squared estimate = 0.1904 (convergence is achieved after 6 iterations) restricted maximum likelihood estimation (reml) no . Studies = 11 add 1/2 to all cells of the studies with zero figure 3 shows the forest plots and pooled estimates of sensitivity, specificity and diagnostic odds ratios (ors) respectively . We found a diagnostic or of 5.11 (95% ci 3.228.12) for urine il-18 level to predict aki (cochran - q = 28.19, p = 0.0017) with a sensitivity and specificity respectively of 0.51 (heterogeneity chi - squared 84.53, p <0.001) and 0.79 (heterogeneity chi - squared 62.84, p <as there was no statistically significant between b and 0 (p = 0.1870), the mantel haenszel model was utilized to draw the fitting curve of sroc (fig . The pooled auroc was 0.77 (95% ci 0.710.83, q = 0.71).fig . 3forest plots and pooled estimates of a diagnostic odds ratio (or), b sensitivity, and c specificityfig . 4the fitting curve of sroc forest plots and pooled estimates of a diagnostic odds ratio (or), b sensitivity, and c specificity the fitting curve of sroc subgroup analysis (table 5) showed that urine il-18 level in pediatric patients (<18 years) was more effective in predicting aki, with a diagnostic or of 7.510 (95% ci 2.98818.875) compared to 4.652 (2.7107.987) for the adult group (p = 0.334); the early aki predictive time (<12 h) subgroup displayed the highest diagnostic or of 8.176 (95% ci 2.19130.507) among the three subgroups (<12, 24 and 48 h, p = 0.037). The pooled estimate diagnostic or of the admission subgroup was 3.81 (95% ci 2.086.99) and that of the subgroup for other times 7.16 (3.6214.18). There were no significant differences between the two (p = 0.388). There were significant differences (p = 0.008) between cardiac surgery patients and patients in intensive care unit or coronary care unit patients in the subgroup analysis, with diagnostic ors of 5.28 (3.597.76) and 5.31 (2.5910.87), respectively.table 5pooled diagnostic accuracy of il-18 in various settingssubgroup factorssubgroup criteriareference numbersq valuep valuei (%) hierarchical summary or (95% ci)p value for between subgroupsage<18 years510.230.03760.97.51 (2.9918.88)0.33418 years615.540.00867.84.32 (2.487.51)predictive time12 h518.270.00178.18.18 (2.1930.51)0.03724 h44.480.21433.05.07 (2.589.96)48 h55.040.28320.74.95 (3.397.24)obtaining specimenadmission413.40.00477.63.81 (2.086.99)0.388other times710.380.10942.47.16 (3.6214.18)patientscardiac surgery40.590.900.015.28 (3.5937.762)0.008other patients723.280.00174.25.31 (2.5910.87) or odds ratio, ci confidence interval pooled diagnostic accuracy of il-18 in various settings or odds ratio, ci confidence interval the current study presents a meta - analysis of urine il-18 for predicting aki development via the sroc analysis approach, both overall and across a range of subgroups . The finding derived is consistent with previous studies, which lends more force to the use of urine il-18 as a marker of aki in clinical practice . The results of this meta - analysis indicate that urine il-18 had a pooled diagnostic or of 5.11 and the estimated area under the curve (auc) of the mean roc plot was 0.77 (q = 0.71), with a high heterogeneity in pooled sensitivity and specificity . This suggests that urine il-18 has a higher chance of early detecting an aki compared to scr, but the application of this biomarker in the diagnosis of aki should be limited to a certain range in the light of limitations intrinsic to such studies . Since pooled results may increase statistical power and lead to more precise estimates of a treatment effect and the pooled random or fixed effect models reflect the between - study variance, this may shed light on larger populations . Due to the presence of a threshold effect, we used sroc curve fitting, the area under roc and q index, to merge the data . In the studies included, cutoff values were expressed in two types, urinary concentration or ratio of urine il-18 and creatinine concentration, and identified as varying across studies (11.25125 pg / ml). Such differences can be explained by: (1) use of different reagents in these studies though the assay methods were enzyme - linked immunosorbent assay (elisa); and (2) differences in clinical settings and study population . Therefore, it might be necessary for each center using urine il-18 level for early aki diagnosis to define a specific reference range and cutoff value for each clinical setting . In addition, a significant non - threshold effect heterogeneity exists across these studies . Since these studies were based on different institutions across the world, heterogeneity was inevitable concerning differences in aki definitions, aki settings, times for obtaining specimen, and experimental groups admitted to assess the predictive value of urine il-18 . We attempted to use meta - regression to explore the sources of heterogeneity, but failed . So subgroup analysis was performed and proved that urine il-18 level in pediatric patients and the early aki predictive time group (<12 h) was more effective in predicting aki, which might principally account for the heterogeneity . Nevertheless, as the kidney undergoes growth and maturation in neonates who manifest different physiological states due to the abrupt changes at birth, many risk factors are correlated with aki and the timing of kidney injury remains unknown . The metabolic capacity and compensatory ability of neonates and children are also different from those of adults . Subgroup analysis of age should be more specific if more clinical trials are conducted . Regarding aki definition, akin, rifle, akin and/or rifle, and modified pediatric rifle criteria the pooled diagnostic or of aki within 12 h was greater than that of 24 or 48 h in subgroup analysis . This is useful in clinical processing since preventive strategies can be formulated if aki can be predicted in advance . Using scr level as the gold standard of diagnosis of aki is another limitation of such studies because scr is not an ideal marker of early loss of glomerular filtration or kidney injury . However, its use in routine clinical practice is restrained because it is invasive, time - consuming and radioactive . Cross - sectional studies indicate that urinary il-18 levels are markedly elevated in patients with acute tubular necrosis compared with healthy controls and a variety of other renal pathologies, including urinary tract infection, chronic renal insufficiency, and pre - renal azotemia . Therefore, the pathophysiology of il-18 still remains incompletely understood and the true role of il-18 may be as a mediator of specific injury subtypes rather than as a marker of injury . Though il-18 can be induced in the proximal tubule after aki, and released into urine after cleavage by caspase-1 [25, 26], it can also be derived from lung injury and myocardial ischemia, etc . The cost of a single creatinine test is less than a dollar, while that of il-18 is five times higher . In the course of aki, biochemical indices need to be monitored and detected many times; given the modest clinical value of il-18 and its high cost, serum creatinine concentration and change might still be a good indicator rather than il-18 . In conclusion, this meta - analysis included more than 2,700 patients from 11 studies . The diagnostic accuracy for aki tends to be more effective in pediatric patients and early aki predictive time . To improve the diagnostic value of urine il-18, more appropriately designed investigations (e.g. With randomized design and eliminating potential confounders)
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An abundance of literature documents an association between childhood trauma and the later development of a broad list of physical and mental illnesses including; eating disorders, substance abuse, phobias, personality disorder, irritable bowel syndrome, rheumatoid arthritis, and autoimmune disorders.15 more recently, evidence has accumulated to link the childhood adversity to the development of adult psychosis . A meta - analysis showed a significant association between adversity and psychosis across three research designs that were systematically reviewed (prospective cohort studies, cross - sectional studies, and case - control studies) with an overall effect of odds ratio 2.78 (95% confidence interval 2.343.31).6 a causal link between childhood trauma and psychosis has been suggested and it is postulated that this is mediated via a neuropathological stress response that involves dysregulation of the hypothalamic pituitary axis, disruption of dopamine and neurotransmitter systems, and hippocampal damage.7 the existing evidence highlights the need for a routine, systematic trauma history to be taken from every patient presenting with significant mental health problems in order for the most effective care to be provided.8 this includes the provision of evidence based psychosocial therapies such as those recommended in the uk by the national institute for health and care excellence schizophrenia guidelines.9 failure to assess whether past trauma can impact on current problems means a failure to fully determine the potential benefits of referral to therapy, thus hindering a patient s recovery from psychosis . Furthermore, a patient bias to underreport experiences of trauma has been documented, further obviating the need for direct questioning from mental health care professionals.10,11 in this study we aimed to determine the proportion of patients in an early intervention service with a history of trauma and to describe the types of trauma most commonly experienced . The data for this paper were derived from a retrospective case note audit, and an evaluation of an uk early intervention service . The audit aimed to assess service accessibility, quality of assessments, and to gain a better understanding of the characteristics of patients within the service in order to assist future service development . The study was approved by the lancashire care research and development department as a retrospective case note audit . All the data was anonymized and patients were not contacted at anytime for any information, hence consent was not obtained . The early intervention service is located in the north of england and criteria for entry include; aged between 16 and 35 years and within 3 years of commencing treatment for a psychotic disorder . Data were collected from all patients currently in the service at the time (n=296). Every patient entering the service was assessed by a member of the early intervention team . The assessing health care professional gathered a range of data on the patients history and mental state in a systematic fashion and recorded this on a health and social needs assessment proforma, which also acted as a prompt and guide during the assessment . Information was collected on fifteen categories of childhood trauma including physical abuse, physical neglect, emotional abuse or neglect, sexual abuse, witnessing domestic abuse (emotional, physical, or sexual), death of one or both parents, absence of one or both parents, parental mental illness, family suicide, forced marriage, bullying, and severe or repeated disruption . Categories were not exclusive ie, a patient could be recorded as experiencing one of the categories of trauma, or any number of them . The parents included biological and non - biological parents, and legal or non - legal guardians . Severe or repeated disruption included frequent, repeated, or traumatic house or school moves, including suspension or expulsion from a number of schools, repeated custodial sentences, placement in state or foster care, and separation from parents or siblings . An other category allowed qualitative information to be collected and documentation of trauma not fitting into the other fifteen categories . Results were analyzed using crosstab and frequency analysis to show trauma correlates and to compare different groups of patients . In conducting the audit we recorded in what proportion of cases the early intervention service is located in the north of england and criteria for entry include; aged between 16 and 35 years and within 3 years of commencing treatment for a psychotic disorder . Data were collected from all patients currently in the service at the time (n=296). Every patient entering the service was assessed by a member of the early intervention team . The assessing health care professional gathered a range of data on the patients history and mental state in a systematic fashion and recorded this on a health and social needs assessment proforma, which also acted as a prompt and guide during the assessment . Information was collected on fifteen categories of childhood trauma including physical abuse, physical neglect, emotional abuse or neglect, sexual abuse, witnessing domestic abuse (emotional, physical, or sexual), death of one or both parents, absence of one or both parents, parental mental illness, family suicide, forced marriage, bullying, and severe or repeated disruption . Categories were not exclusive ie, a patient could be recorded as experiencing one of the categories of trauma, or any number of them . The parents included biological and non - biological parents, and legal or non - legal guardians . Severe or repeated disruption included frequent, repeated, or traumatic house or school moves, including suspension or expulsion from a number of schools, repeated custodial sentences, placement in state or foster care, and separation from parents or siblings . An other category allowed qualitative information to be collected and documentation of trauma not fitting into the other fifteen categories . Results were analyzed using crosstab and frequency analysis to show trauma correlates and to compare different groups of patients . In conducting the audit we recorded in what proportion of cases the most predominant ethnicity was white (66%, n=195), followed by asian (23%, n=68), and mixed race (0.7%, n=2). Of the 296 patients studied, 178 (60%) had experienced some sort of childhood trauma (figure 1). Fifty - five patients (19%) had insufficiently completed health and social needs assessments documented to determine whether or not childhood trauma had occurred, and 63 patients (21%) had no evidence of childhood trauma . As shown in figure 2, the most common types of trauma reported were: 1) other trauma (24%); 2) severe or repeated disruption (21%); 3) parental mental illness (19%); 4) bullying (18%); and 5) absence of a parent (13%). It is interesting to note that three out of five of the most common traumas relate to factors within the family unit . Qualitative analysis of the other trauma category revealed victimization events to be the most common, with isolated sexual assaults making up the majority (29%). Traumatic parental divorce was second to this with 17% describing this as contributing to their current problems . Interestingly, 22% of patients in this category stated they had a difficult relationship with one or both parents . Of the 178 patients who reported childhood trauma, 118 (66%) this is in keeping with the finding that severe or repeated disruption was the second most common trauma type (figure 3). The most predominant ethnicity was white (66%, n=195), followed by asian (23%, n=68), and mixed race (0.7%, n=2). Of the 296 patients studied, 178 (60%) had experienced some sort of childhood trauma (figure 1). Fifty - five patients (19%) had insufficiently completed health and social needs assessments documented to determine whether or not childhood trauma had occurred, and 63 patients (21%) had no evidence of childhood trauma . As shown in figure 2, the most common types of trauma reported were: 1) other trauma (24%); 2) severe or repeated disruption (21%); 3) parental mental illness (19%); 4) bullying (18%); and 5) absence of a parent (13%). It is interesting to note that three out of five of the most common traumas relate to factors within the family unit . Qualitative analysis of the other trauma category revealed victimization events to be the most common, with isolated sexual assaults making up the majority (29%). Traumatic parental divorce was second to this with 17% describing this as contributing to their current problems . Interestingly, 22% of patients in this category stated they had a difficult relationship with one or both parents . Of the 178 patients who reported childhood trauma, 118 (66%) had experienced multiple forms . This is in keeping with the finding that severe or repeated disruption was the second most common trauma type (figure 3). Our finding that the majority (60%) of patients presenting to the early intervention service had experienced some form of childhood trauma is consistent with other studies that have reported a high prevalence of childhood trauma in those with adult psychosis.57 we must also take into account previous evidence that patients are biased to underreport trauma . This, and the high proportion of insufficient notes, might have resulted in misleadingly lower rates of trauma.10,11 based on extrapolation from our data, another 32 patients in this sample could reasonably be expected to have experienced unreported trauma . The number of patients with incomplete notes is concerning we cannot provide sufficient help for patients coping with trauma sequelae if we are not aware of the trauma in the first place . The ideology behind the most commonly offered psychological therapies in early intervention services cognitive behavioral therapy (cbt) is to understand how one s past experiences affect current thoughts and behaviors and to try and break unhelpful cycles . Failure to document or explore trauma prevents us fully assessing whether a patient is likely to benefit from therapy and potentially hinders referral rates and ultimately, the patient s recovery . As suggested by read et al,8 by failing to provide timely psychological intervention to patients we could be unnecessarily exposing them to the side - effects of neuroleptic medication. Metabolic side effects of antipsychotic medication are a particular concern in first episode psychosis patients.12 possible reasons for failing to explore trauma may be suboptimal staff training, resulting in a lack of confidence to approach the subject with patients, fear of jeopardizing staff - patient relationship, and uncertainty of what to do should a patient respond positively when asked about trauma . Lardinois et al13 proposed that those who have experienced childhood trauma develop an acquired vulnerability to psychosis through previous trauma exposure, creating increased emotional reactivity to daily stressors . Essentially, it appears that the patient is able to cope with their trauma but they are less able to cope with daily stressors in addition to their trauma and it is this that ultimately leads to their deterioration in mental health.13 neria et al14 also found an increased rate of psychosis in those exposed to repeated or ongoing trauma . Although evidence suggests that generally patients do not become distressed when asked about trauma, there are also patients who are not ready to or able to come to terms with or discuss their trauma.15 for individuals who decline cbt on this basis, an appropriate alternative may be offered which focuses on helping patients cope with daily stressors . Safeguarding issues are also raised here as those children exposed to trauma appear more likely to be exposed again . Similarly, those who have already been exposed to multiple trauma have obviously not been safeguarded appropriately . In this study, 80% of the most common traumas reported were related to factors within the family home (severe or repeated disruption, parental mental illness, absence of a parent, and traumatic parental divorce). This echoes janssen et als15 finding that parental rearing styles predicted the onset of psychosis and suggests factors within the family unit may have more clinical significance than previously thought . In particular, they appear to have more relevance than victimization events, such as sexual abuse, which have historically been the mainstay of research into trauma and psychosis.15,16 increased mindfulness of this inference and efforts to strengthen family relationships may serve as protective factors in the development or relapse of psychosis . Family therapy may be especially beneficial and these findings warrant its increased role in the management of psychosis . We also did not include a control group to establish the prevalence of trauma in people who never develop psychosis or display early signs of psychosis . The definition of trauma is open to researcher bias for some categories of trauma . Severe or repeated the design of this study assumes only association between the childhood trauma and subsequent psychotic illness; it does not establish causality and therefore, other confounding factors have not been accounted for . We are also unable to rule out that more recent life events were causal factors in the development of patients psychosis . Furthermore, due to an apparent lack of direct questioning from health care professionals within the service or shortage of appropriately documented history taking, it is likely that a substantial amount of trauma has not been included in our results . Lardinois et al13 proposed that those who have experienced childhood trauma develop an acquired vulnerability to psychosis through previous trauma exposure, creating increased emotional reactivity to daily stressors . Essentially, it appears that the patient is able to cope with their trauma but they are less able to cope with daily stressors in addition to their trauma and it is this that ultimately leads to their deterioration in mental health.13 neria et al14 also found an increased rate of psychosis in those exposed to repeated or ongoing trauma . Although evidence suggests that generally patients do not become distressed when asked about trauma, there are also patients who are not ready to or able to come to terms with or discuss their trauma.15 for individuals who decline cbt on this basis, an appropriate alternative may be offered which focuses on helping patients cope with daily stressors . Safeguarding issues are also raised here as those children exposed to trauma appear more likely to be exposed again . Similarly, those who have already been exposed to multiple trauma have obviously not been safeguarded appropriately . In this study, 80% of the most common traumas reported were related to factors within the family home (severe or repeated disruption, parental mental illness, absence of a parent, and traumatic parental divorce). This echoes janssen et als15 finding that parental rearing styles predicted the onset of psychosis and suggests factors within the family unit may have more clinical significance than previously thought . In particular, they appear to have more relevance than victimization events, such as sexual abuse, which have historically been the mainstay of research into trauma and psychosis.15,16 increased mindfulness of this inference and efforts to strengthen family relationships may serve as protective factors in the development or relapse of psychosis . Family therapy may be especially beneficial and these findings warrant its increased role in the management of psychosis . We also did not include a control group to establish the prevalence of trauma in people who never develop psychosis or display early signs of psychosis . The definition of trauma is open to researcher bias for some categories of trauma . Severe or repeated the design of this study assumes only association between the childhood trauma and subsequent psychotic illness; it does not establish causality and therefore, other confounding factors have not been accounted for . We are also unable to rule out that more recent life events were causal factors in the development of patients psychosis . Furthermore, due to an apparent lack of direct questioning from health care professionals within the service or shortage of appropriately documented history taking, it is likely that a substantial amount of trauma has not been included in our results . We found a high prevalence of childhood trauma in patients either with established psychosis or displaying signs of early psychosis . Irrespective of this, good psychiatric practice requires a comprehensive understanding of an individual s problems . As such, mental health care professionals should ensure a full trauma history is taken from all patients presenting with psychosis in order that the most optimal mental health care can be delivered . This high prevalence of trauma reported within the family home raises the possibility that patients may benefit from family therapy or trauma focused cbt in the management of psychosis.
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Corticobasal degeneration (cbd) is an uncommon, sporadic, neurodegenerative disease described for the first time by rebeiz et al . . It can be associated with an extraordinary variety of motor, sensory, behavioural, and cognitive symptoms . It is an asymmetrical parkinsonism affecting a limb, typically an arm: rigidity is the most common manifestation of the parkinsonian syndrome followed by bradykinesia, gait disorder (postural instability and falls), and tremor; asymmetrical limb dystonia is common as well . Other cardinal signs include higher cortical dysfunctions such as apraxia (limb more common than orofacial, eyelid - opening). Dementia, progressive nonfluent aphasia, speech apraxia, progressive - supranuclear - palsy- (psp-) like syndrome and posterior cortical atrophy syndrome are other presentations of cbd [3, 4]. Cbd is a tauopathy (characterised by abnormal deposition of the microtubule - associated protein tau), similar to frontotemporal dementia and progressive supranuclear palsy (psp). The typical pathological findings in cbd include focal asymmetric cortical atrophy, nigral degeneration, taupositive neuronal, and glial lesions in both gray and white matters . To achieve more accurate clinical diagnosis, neuropsychological, electrophysiological, and imaging methods could be applied to differentiate this disease from the other parkinsonism syndromes [3, 4]. In comparison with other neurological diseases, the symptoms of cbd are particularly difficult to understand and the patients have considerable difficulties in describing their experience . A better understanding of the disease may help clinicians to make diagnosis, providing patients with comprehensive information about prognosis and difficulties they will encounter during the course of the disease, improving their quality of life as well as their careers . A 71-year - old woman, primary school graduate, formerly farmhand, was referred to our department of neurology with left rigid - akinetic syndrome and cognitive dysfunction . She had no previous medical or family neurological history; she just reported in the previous months frequent falls and postural instability . Cognitive symptoms included impairment of spoken - language production and attention / concentration deficits . Her main complaint was difficulty in using her left arm and hand, which gradually progressed . The limb became severely rigid and adopted a dystonic posture associated with pain and functional disability . Her clinical features slowly deteriorated and, 1 year later, involved also the left lower limb with gait disorder associated to postural instability and falls . Subsequently, the patient underwent brain mri and 18f - fluorodeoxyglucose - positron emission tomography (pet). She has been previously diagnosed as parkinson's disease but with no response to levodopa or dopaminergic medications . Her clinical symptoms gradually progressed and, four months later, blepharospasm and mild dysphagia appeared . When she was admitted to our neurological centre, the neurological examination revealed: blepharospasm; hypomimia and asmmetric bradykinesia; left upper - arm - fixed - dystonia; spasticity in left lower limb and pyramidal signs (babinski, hoffmann, and grasp reflex); moderate disturbance of gait with short steps, tendency to drag her left leg, bradykinesia, and propulsion requiring assistance . The other tests revealed impairment in visuospatial abilities, severe visuospatial neglect (figure 1), constructional and ideomotor apraxia, poor word fluency with mild visual confrontation anomia, and nonverbal oral apraxia . Brain mri showed asymmetric cortical atrophy in the right frontotemporal cortex (figure 2). No abnormalities were found on eeg examination, even if, a deeper analysis showed a focal slow wave activity in the right parietotemporal area . The diagnosis was based on the gradual onset of a parkinsonian disorder associated with cortical dysfunctions and other supportive features such as cognitive dysfunction, asymmetric atrophy on mri imaging, and asymmetric hypoperfusion on pet . Clinically cbd begins in the sixth, seventh, or eighth decade, with slight predilection for women [8, 9]. Typically the primary symptoms develop in a profoundly asymmetric way, affecting either one arm or, less frequently, a leg, which appears to be rigid, dystonic, akinetic, or apraxic . Clinical features include a series of motor, cognitive and neuropsychiatric symptoms, that can be explained by impairment of the cortical and subcortical structures . Motor symptoms include progressive asymmetric rigid - akinetic parkinsonism usually involving the upper limbs, without resting tremor, focal stimulus - sensitive or action myoclonus [4, 11], blepharospasm, speech abnormalities, gait disorder with postural instability and falls, and asymmetric limb dystonia, generally of the upper limbs, sometimes evolving towards the development of a dystonic clenched fist [3, 12]. Eye movements are usually preserved, although a delay in the initiation of saccades may occur, in absence of pursuit and optokinetic nystagmus impairment [13, 14]. Involvement of higher cortical functions results in often symmetric ideomotor apraxia, firstly affecting the limb, then, as the disease progresses, eyelid - opening, tongue, lips . The alien - limb phenomenon, that is seen in 50% of the cases, can be defined as a circumstance in which one of the patient's hands behaves in a way which the patient finds foreign, alien or at least uncooperative; it commonly co - occurs with cortical sensory loss [11, 16]. Cognitive decline is a common feature of the disorder, occasionally the presenting feature of the disease . The prominent characteristics are impairment of spoken - language production (typically nonfluent aphasia), frontal executive impairment, calculation and visuospatial skills impairment, whereas semantic and episodic memory may be spared . Neuropsychiatric symptoms may include depression, apathy, anxiety, irritability, disinhibition, delusions, and obsessive compulsive disorder . Abnormalities on magnetoencephalography, an exaggerated electromyographic - electromyographic (emg - emg) coherence, and an alteration in cortical excitability evaluated by means of transcranial magnetic stimulation (tms) were noted in patients with cbd . On the other hand, conventional electroencephalography (eeg) may be normal when the first clinical symptoms appear, and often remains unchanged as the disease progresses . Nevertheless, an unilateral slowing may be evident in some patients, which may occasionally generalise to the whole cortex as the disease evolves [24, 25]. In a study involving six patients, vion - dury and coworkers, using a quantitative standard eeg (eegq) with spectral analysis, found indeed the occurrence of several eeg abnormalities (generally enhanced by hyperventilation or intermittent photic stimulation), such as an increase of slow rhythms (delta or theta frequency range) and occasionally the occurrence of sharp waves . These abnormalities were lateralised in five patients (more often after hyperventilation) and were bilateral in one, confirming the asymmetrical features of cbd . Moreover, huang et al . Showed that the eeg recordings with jerk - locked back average do not present any jerk - locked cortical potentials . In cbd, the cortical sensory evoked potentials (seps) are not enlarged as in cortical reflex myoclonus, and backaveraged cortical potentials do not precede each myoclonic jerk [2628]. Clinical and imaging evidence suggests that the localized parietal cortical damage is a pivotal factor for the absence of a giant sep in these patients . An asymmetric alternation of inhibitory and excitatory balance at the level of cortical neurons leading to a particularly enhanced cortical excitability may moreover play an important role in the generation of myoclonus [27, 28]. The loss of the inhibitory input from the somatosensory cortex to the relatively intact motor cortex, which results from the prominent asymmetric parietal atrophy, may give rise to the asymmetric hyperexcitable motor cortex without giant sep [30, 31], even - though the existence of an alternative hyper - excitable thalamo - cortical pathway cannot be excluded . In effect, motor cortex disinhibition has been clearly demonstrated in cbd by means of tms applied in several paradigms in different neurophysiological studies [3234]. By applying single pulse - tms, lu and coworkers discovered a relatively higher motor evoked potential (mep) amplitude and a significantly shorter cortical silent period in the affected hand of cbd patients . They therefore supposed that the relatively enlarged mep may be explained postulating that an increased number of motorneurons are being recruited by the descending volleys from the motor cortex, while the shorter silent periods may reflect mainly defective inhibitory processes . The result from paired pulse - tms studies also supported the last hypothesis [22, 23, 35]; for example, frasson et al . Showed that, in patients with cbd, paired magnetic stimuli delivered at short (inhibitory) interstimulus intervals (isis) invariably elicited enlarged meps; moreover, asymmetric corticocortical disinhibition [22, 36], as well as asymmetric tms maps organization [32, 37], has been observed in patients with cbd . In conclusion, several mechanisms could explain this abnormal motor cortical excitability, namely, loss of inhibitory neurons in the cortex or thalamus, effect of morphological changes in cortical neurons mainly in the somatosensory cortices, disruption of some neuronal circuits, or the existence of alternative cortical - subcortical pathways [33, 38]. Morphologic imaging of the brain, although normal in the early phases of the disease, may demonstrate asymmetrical cortical atrophy, in particular of the frontal and parietal lobe, more evident contralaterally to the side most severely clinically affected [39, 40]. Asimmetrical atrophy in the basal ganglia, corpus callosum, lateral ventricles, and cerebral peduncles may be present . Functional imaging studies might be useful in the differential diagnosis of patients with suspected cbd, showing asymmetrical hypoperfusion on spect and asymmetrical hypometabolism on pet involving the parietal - frontal cortex and basal ganglia . All criteria stress the combination of an akinetic - rigid syndrome with apraxia, alien limb syndrome, and cortical sensory deficits . A universally recognized feature is the asymmetry of clinical presentation, further corroborated by a contralateral asymmetrical atrophy on the structural and hypometabolism on the functional neuroimaging . Neuropathologically cbd presents as asymmetrical focal atrophy of the cerebral cortex focused on the peri - rolandic posterior frontal and parietal cortex, especially the motor and sensory areas . There is a relative sparing of temporal and occipital cortex, except in some forms presenting with dementia or primary progressive aphasia, which are characterised by a more symmetric and more severe involvement of the frontal and temporal lobes . Basal ganglia are also involved with substantial atrophy in the lateral two - thirds of the substantia nigra, and, to a lesser extent, of putamen, pallidum, thalamus, and hypothalamus . Histologically cbd is characterised by large pale ballooned neurons (neuronal achromasia), with tau - positive cytoplasmatic inclusions and astrocytic plaques (annular clusters of tau - positive deposits within the distal processes of astrocytes), typically distributed in atrophic cortices [5, 6, 42]. Molecularly cbd is a taupathy, characterised by accumulation of abnormal filamentous inclusions of hyperphosphorylated tau - protein in neurons and glia, similarly to progressive supranuclear palsy (psp), and some forms of frontotemporal dementia with parkinsonism (ftd) [5, 44]. This molecular overlap, especially with psp is very argued, and whether they are the extremities of the spectrum of a single disorder or two different disorders with a similar genetic predisposition is not clear [3, 18]. The symptoms can be gathered in four categories: natural history and presentation, motor, sensory motor, and cognitive symptoms . The first three categories include characteristics which have been taken into account in almost all the previous diagnostic criteria . Up to 1994, dementia was an exclusion criterion of cbd; from 2003, on the base of new criteria, cognitive impairments support diagnosis, so the inclusion of the cognitive criteria reflects the growing recognition of the importance of cognitive assessment in the diagnosis of cbd [3, 45]. The core features of disease are insidious onset and progressive course, no identifiable cause (tumor, infarct) of symptomatology, cortical dysfunction includes at east one of the following: (i) focal or asymmetric ideomotor apraxia, (ii) alien - limb phenomena, (iii) cortical sensory loss, (iv) visual or sensory hemineglect, constructional apraxia, (v) focal or asymmetric myoclonus, (vi) apraxia of speech / nonfluent aphasia: extrapyramidal dysfunction as reflected by one of the following: (i) focal or asymmetrical appendicular rigidity lacking prominent and sustained l - dopa response, (ii) focal or asymmetrical appendicular dystonia . The supportive investigations are variable degrees of focal or lateralized cognitive dysfunction, with relative preservation of learning and memory, on neuropsychometric testing, focal or asymmetric atrophy on computed tomography or magnetic resonance imaging, typically maximal in parietofrontal cortex, focal or asymmetric hypoperfusion on single - photon emission computed tomography and positron emission tomography, typically maximal in parietofrontal cortex / basal ganglia / thalamus . Predominant parkinsonian features might not be easy to distinguish from idiopathic pd and atypical parkinsonian syndromes (e.g., psp and multiple system atrophy, msa). Akinetic - rigid syndrome, early inbalance and poor response to dopaminergic treatment are typical symptoms for cbp, psp, and msa, in contrast with pd . Psp is clinically and pathologically related to cbd, but with differences in symmetrical versus asymmetrical parkinsonism, and in the pattern of rigidity, which tends to be more nuchal / axial in psp and more limb - accentuated in cbd . Also, while the superanuclear gaze palsy can occur in both conditions, in cbd it tends to affect the horizontal more than the vertical gaze and the saccade latency (delay in initiating saccades) more than saccade velocity (the speed of saccades). In patients with cognitive presentation, the diagnosis depends on the specific case: pronounced visuoperceptual deficits related to dementia with lewy bodies (dlb), language involvement to primary progressive aphasia (ppa), and prominent behavioral features to frontotemporal dementia (ftd). It is important to explain the nature of the motor as well as the cognitive deficits to the patients as well as to all people involved in their care . As a motor disorder, cbd has been recognized for over 30 years . However, more recent findings suggest that there is a neuropsychological syndrome associated with cbd . The most prominent neuropsychological features of cbd include limb apraxia, ideomotor and ideational apraxias . Executive function and language impairments are also reported . The characterization of the natural history of patients with the cbd (clinical, laboratory, neuropsychological, radiological features) is important to improve the accuracy of diagnosis.
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Fibrosarcoma is a neoplasm of mesenchymal cell origin that is composed of malignant fibroblasts in a collagenous background . Kulander and bolen defined it as a neoplasm of fibroblasts capable of producing metastatic spread . Fibrosarcoma is a very rare malignancy with possible occurrence in the whole head and neck region . Occurrence of fibrosarcoma in the maxillofacial region is extremely rare . If present, the mandibular region is more often involved than the maxillary one . This report describes a patient with soft tissue fibrosarcoma of the anterior maxillary region with clinical, radiographic, pathological and management aspects . A 71-year - old male patient visited pacific dental college and hospital, udaipur, rajasthan, with complaints of pain and swelling of upper front region of jaw for the past 12 days (figure 1). He gave the history of dull, but continuous pain, which aggravated during night time and gradually increasing swelling . Clinically, the lesion was thought to have developed following extraction of an upper anterior tooth 15 days earlier . The tooth had been extracted due to mobility and intense pain . However, the pain persisted even after extraction . The antibiotics and analgesics provided no relief and the tissue continued to grow, so rapidly in fact, that 10 days later he could no longer close his mouth without traumatizing the mass . Clinically swelling extended from philtrum to outer canthous of eye on the left side of the face . The orthopantomograph revealed well - defined unilocular radiolucency apical to the extraction socket of maxillary left lateral incisor (figure 2). Plain and post contrast computed tomography scan of facial bone performed using 3 mm thickness cuts in axial planes . It showed soft tissue density mass with well - defined margins in midline of face overlying maxillary alveolus causing mild erosions more involving left alar nobule of nose . Mass was extending along the left lateral surface of nose to reach up to medial canthus of left orbit (figure 3). A linear defect was seen involving right nasal bone and frontal process of maxillary bone . After the provisional diagnosis a representative portion of target lesion was biopsied by incisional technique under local anesthesia and sent for histopathological examination (figure 4). Figure 1clinical picture of the patient revealing the mass . Clinical picture of the patient revealing the mass . Figure 2orthopantomograph revealing well - defined unilocular radiolucency apical to the extraction socket of maxillary left lateral incisor generalized bone loss and retained root stumps . Orthopantomograph revealing well - defined unilocular radiolucency apical to the extraction socket of maxillary left lateral incisor generalized bone loss and retained root stumps . Figure 3plain and post contrast computed tomography scan pictures revealing soft tissue density mass with well defined margins in midline of face overlying maxillary alveolus causing mild erosions more involving left alar lobule of nose . Plain and post contrast computed tomography scan pictures revealing soft tissue density mass with well defined margins in midline of face overlying maxillary alveolus causing mild erosions more involving left alar lobule of nose . Histopathological examination revealed a fibrocellular stroma with spindle cells arranged in herring bone pattern (figure 5). The tumour cells showed strong cytoplasmic positivity for vimentin, focal positivity for s-100 protein (figures 6 and 7) and negative for pancytokeratin epithelial membrane antigen smooth muscle actin and desmin . Based on the clinical, radiographic and immuno - histo - pathological findings a diagnosis of fibrosarcoma, grade 2/3 (coindre grading system) was made . After the final diagnosis of fibrosarcoma the whole mass was excised (figure 8) and along with that left side partial maxillectomy was done (figure 9). In order to aid in feeding and deglutition figure 5photomicrograph showing fibro cellular stroma and have the herringbone pattern (h & e, 10x). Photomicrograph showing fibro cellular stroma and have the herringbone pattern (h & e, 10x). Figure 10 in order to aid in feeding and deglutition an obturator was fabricated . In order to aid in feeding and deglutition chemotherapy with etoposide (75 mg / m), ifosfamide (1.2 g / m), and cisplatin (20 mg / m) was given intravenously daily for five days every four weeks . In addition, the patient was subjected to postoperative radiotherapy for a period of six weeks . Due to the poor prognosis after one year and two months of the treatment the patient displayed multiple metastases . They are believed to have arisen either from the periosteum, periodontal membrane, enclaved embryonic mesenchymal cells of developing teeth or cells of the connective tissues surrounding nerves and vessels within the jaw . Although fibrosarcomas may develop in any mesenchymal tissue in which fibroblasts are present, they are most frequently located in the extremities and the trunk . Fibrosarcoma can arise in soft tissues or within bone . In the head and neck region, soft tissue sarcomas fibrosarcomas of soft tissues usually affects a wider age spectrum of patients with an age range of 3555 years . The present case is a soft tissue fibrosarcoma of premaxillary region in a 71-year - old adult male . Typically, the tumour presents with swelling, pain, parasthesia, occasionally losing of teeth and ulceration of the overlying mucosa . Facial asymmetry, and in advanced cases, considerable displacement of the eye with resultant diplopia often occurs . Because these lesions often arise deep in the muscular fascia, they may become extremely large tumors prior to diagnosis . In our case, patient had an asymmetrical face and complained of a painful, gradually increasing swelling which continued to grow so rapidly in fact that, ten days later he could no longer close his mouth without traumatizing the mass . Radiological imaging of fibrosarcomas revealed radiolucent lesions with geographical, moth - eaten or permeative pattern of bone destruction . The absence of tumoural calcification or ossification can be of importance in differentiating fibrosarcomas from other malignancies such as chondrosarcomas and osteosarcomas . Histologically, the diagnosis of fibrosarcoma is made much less frequently today because of the recognition and separate classification of other spindle cell lesions that have similar microscopic features . The degree of differentiation is variable, being comparable to a benign fibroma or an anaplastic tumor, becomes a challenging diagnosis . The present case was diagnosed as fibrosarcoma based on the immunohistochermical features . The histological similarity and positive immunostaining for vimentin and s100 protein, together with negativity for pancytokeratin and epithelial membrane antigen helped to confirm the diagnosis of high - grade fibrosarcoma . The treatment choice to fibrosarcoma is radical surgery with 3 to 5 cm clearance margins . This is not possible in the oral cavity and sinonasal region because of anatomy and mutilation effect . This restriction leads to failure of local control regardless of the histologic grade and is responsible for their high mortality . The need for adjuvant radiotherapy and/or chemotherapy is still unclear but there is normally an indication in high grade tumours because these tumours may present with subclinical or microscopic metastases at the time of diagnosis . Prognosis is directly related to histological grade, tumor size and adequate surgery treatment with margins free . Fibrosarcoma is a rare malignancy in the oral cavity; local recurrence is frequent, but metastases are rare . Fibrosarcoma spreads by both local invasion and haematogenous dissemination . A latent period of 10 years before metastasis is quite possible and the commonest site for metastases is lungs . The present case reaffirms the importance of early investigation of symptoms that are chronic in nature . Any one complaining of pain, swelling, loosening of teeth, parasthesia, lesions changing in character, or progressively ill - fitting dentures should be scrutinized carefully . The dentist must be suspicious of an underlying disease process possibly responsible for these symptoms.
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Autism spectrum disorder (asd) is a highly heterogeneous disorder, diagnosed on the basis of behavioral criteria . From the neurobiological perspective, asd can be considered an umbrella term that may encompass multiple distinct neurodevelopmental etiologies (geschwind and levitt, 2007). Since any given cohort is thus likely composed of ill - understood subtypes (whose brain features may vary subtly or even dramatically), it is not surprising that brain markers with perfect sensitivity and specificity remain unavailable . Nonetheless, given the specificity of diagnostic criteria (american psychiatric association, 2013), the hope that some (potentially complex) patterns of brain features may be unique to the disorder is not unreasonable and worthy of pursuit . Issues of heterogeneity and cohort effects can be partially addressed through the use of large samples, as provided by the recent autism brain imaging data exchange (abide) (di martino et al ., 2014), which incorporates over 1100 resting state functional mri (rs - fmri) datasets from 17 sites . The use of these data for examining functional connectivity matrices for large numbers of rois across the entire brain is further promising, as there is growing consensus about asd being characterized by aberrant connectivity in numerous functional brain networks (schipul et al . However, the functional connectivity literature in asd is complex and often inconsistent (mller et al ., 2011; nair et al ., 2014), and data - driven machine learning (ml) techniques provide valuable exploratory tools for uncovering potentially unexpected patterns of aberrant connectivity that may characterize the disorder . A few previous asd studies have used intrinsic functional connectivity mri (fcmri) (van dijk et al ., 2010) for diagnostic classification, i.e., for determining whether a dataset is from an asd or typically developing participant solely based on functional connectivity . Anderson and colleagues (2011), using a large fcmri connectivity matrix, reached an overall diagnostic classification accuracy of 79%, which was however lower in a separate small replication sample . 2013a) used a logistic regression classifier for 10 rs - fmri based features identified by ica, which corresponded to previously described functional networks . The classifier achieved accuracies about 6070% for all but one component identified as salience network, for which accuracy reached 77% . Imperfect accuracy in these studies may be attributed to moderate sample sizes (n 80). However, in a recent classification study using the much larger abide dataset, nielsen et al . (2013) reported an overall accuracy of only 60%, suggesting that the approach selected, a leave - one - out classifier using a general linear model, may not be sufficiently powerful . In the present study, we implemented several multivariate learning methods, including random forest (rf), which is an ensemble learning method that operates by constructing many individual decision trees, known in the literature as classification and regression trees (cart). Each decision tree in the forest makes a classification based on a bootstrap sample of the data and a random subset of the input features . The forest as a whole makes a prediction based on the majority vote of the trees . One desirable feature of the random forest algorithm is the bootstrapping of the sample to have a built - in training and validation mechanism, generating an unbiased out - of - bag error that measures the predictive power of the forest . Features were intrinsic functional connectivities (van dijk et al ., 2010) between a standard set of regions of interest using only highest quality (low motion) datasets from abide . Data were selected from the autism brain imaging data exchange (abide, http://fcon_1000.projects.nitrc.org / indi / abide/) (di martino et al ., 2013), a collection of over 1100 resting - state scans from 17 different sites . In view of the sensitivity of intrinsic fcmri analyses to motion artifacts and noise (as described below), we prioritized data quality over sample size . We excluded any datasets exhibiting artifacts, signal dropout, suboptimal registration or standardization, or excessive motion (see details below). Sites acquiring fewer than 150 time points were further excluded . Based on these criteria, we selected a subsample of 252 participants with low head motion (see details below). Groups were matched on age and motion to yield a final sample of 126 td and 126 asd participants, ranging in age from 6 to 36 years (see table 1 for summary and see inline supplementary table s1 for fully detailed participant and site information). Data were selected from the autism brain imaging data exchange (abide, http://fcon_1000.projects.nitrc.org / indi / abide/) (di martino et al ., 2013), a collection of over 1100 resting - state scans from 17 different sites . In view of the sensitivity of intrinsic fcmri analyses to motion artifacts and noise (as described below), we prioritized data quality over sample size . We excluded any datasets exhibiting artifacts, signal dropout, suboptimal registration or standardization, or excessive motion (see details below). Sites acquiring fewer than 150 time points were further excluded . Based on these criteria, we selected a subsample of 252 participants with low head motion (see details below). Groups were matched on age and motion to yield a final sample of 126 td and 126 asd participants, ranging in age from 6 to 36 years (see table 1 for summary and see inline supplementary table s1 for fully detailed participant and site information). Inline supplementary table s1table s1abide demographic information.sitessample sizeagermsdnviqgenderados_totalhandednessasdtdasdtdasdtdasdtdmalefemalerangenaleftrightmean (sd)mean (sd)mean (sd)[range][range][range][range]full samplenyu (n=70)363415.6 (6)16.96 (6.17).02 (.02).02 (.02)108 (15.1)103 (14.9)5416[5 - 22]0nana[8.5 - 30][6.4 - 30.8][.03-.09][.03-.09][72 - 149][67 - 137]um_1 (n=29)151414.7 (2.1)15.5 (2.8).06 (.02).05 (.02)112 (19.6)102.2 (9.4)1712na15521[11.5 - 18.6][11 - 19][.02-.09][.02-.08][81 - 148][82 - 113]usm (n=29)192021.9 (6.4)6.4 (7.2).05 (.02).06 (.02)105.7 (13)111 (12.5)290[6 - 21]0nana[11.3 - 35.7][13.8 - 34][.02-.09][.03-.09][75 - 133][90 - 129]sdsu (n=25)101514.6 (2.3)13.4 (2.3).03 (.01).04 (.02)112 (16)111 (11)214[4 - 17]0619[9.6 - 17.7][8 - 16.8][.02-.05][.02-.07][86 - 114][92 - 137]leuven_1 (n=17)9822.2 (4.9)23.75 (2.9).061 (.02).065 (.02)103 (16.4)114 (18.9)170na9116[19 - 32][21 - 29][.04-.09][.04-.09][74 - 120][93 - 155]leuven_2 (n=17)8913.8 (1.1)14.3 (1.4).07 (.01).08 (.02)97.5 (11.8)101.4 (6.5)116na8413[12.2 - 15.3][12.3 - 16.6][.05-.1][.05-.1][83 - 117][89 - 109]pitt (n=14)8621.2 (8.4)20.2 (4.1).07 (.02).09 (.02)11.7 (14.2)115 (5.3)122[7 - 19]1113[12.6 - 35.2][12.8 - 24.6][.04-.1][.07-.1][93 - 128][108 - 121]trinity (n=12)4817.2 (3.4)17.9 (4).06 (.010).07 (.01)121 (7.4)111 (6.2)120[7 - 15]0012[13.6 - 20.8][12.7 - 24.8][.05-.07][.06-.07][114 - 131][103 - 121]yale (n=12)6614.6 (1.9)13.2 (3.4).06 (.02).05 (.01)85 (29)97 (11.2)84na5111[11.8 - 17.2][8.7 - 16.7][.03-.09][.03-.06][37 - 120][84 - 115]kkin (n=8)359.9 (1.51)9.9 (1.7).01 (.004).01 (.02)nana62[10 - 16]017[8.2 - 11.1][8.5 - 12.8][.06-.07][.05-.1]olin (n=8)4418.3 (4.03)19.5 (2.4).09 (.01).09 (.3)nana62[7 - 19]408[15 - 24][16 - 21][.07-.1][.06-.1]um_2 (n=6)1517.4 (na)18.4 (5.9).08 (na).05 (.01)80 (na)102 (6.8)60na106[17.4 - 17.4][14.5 - 28.9][.08-.08][.03-.06][80 - 80][92 - 109]cmu (n=5)3226.7 (4.5)26 (.07).06 (.02).08 (.01)113.7 (10.8)109.5 (0.7)41[8 - 16]005[22 - 31][21 - 31][.04-.07][.07-.08][106 - 126][109 - 110] trainingnyu (n=60)312915.96 (6.22)16.96 (6.50).05 (.02)0.05 (0.02)108.03 (15.80)102.93 (15.17)4614[6 - 22]29nana[8.51 - 29.98][6.47 - 30.78][.03 -.09][0.03 - 0.09][72.00 - 149.00][67.00 - 137.00]um_1 (n=25)141114.64 (2.11)15.28 (2.85)0.06 (0.02)0.05 (0.02)111.43 (19.97)105.11 (6.72)1510na14517[11.50 - 18.60][11.00 - 19.20][0.02 - 0.09][0.02 - 0.08][81.00 - 148.00][92.00 - 113.00]leuven_1 (n=17)9822.22 (4.87)23.75 (2.92)0.06 (0.02)0.07 (0.02)103.00 (16.42)113.88] (18.88)170na9116[19.00 - 32.00][21.00 - 29.00][0.04 - 0.09][0.04 - 0.09][74.00 - 120.00][93.00 - 155.00sdsu (n=17)9815.19 (1.65)13.43 (3.35)0.03 (0.01)0.04 (0.01)112.67 (16.77)110.25 (13.61)170[4 - 17]0512[12.88 - 17.67][8.02 - 16.59][0.02 - 0.05][0.02 - 0.07][86.00 - 140.00][92.00 - 137.00]usm (n=16)8823.67 (7.35)21.60 (6.84)0.05 (0.02)0.06 (0.02)101.75 (6.82)111.38 (13.22)160[9 - 17]0nana[15.85 - 35.71][13.81 - 34.01][0.02 - 0.07][0.03 - 0.09][87.00 - 106.00][90.00 - 129.00]leuven_2 (n=14)6813.53 (1.23)14.59 (1.32)0.07 (0.01)0.07 (0.01)96.17 (9.54)102 (6.70)86na6311[12.20 - 15.30][12.30 - 16.60][0.05 - 0.08][0.05 - 0.10][83.00 - 106.00][89.00 - 109.00]pitt (n=11)6523.03 (9.07)20.01 (4.56)0.08 (0.02)0.09 (0.01)114.67 (14.90)114.20 (5.07)101[7 - 19]1110[12.62 - 35.20][12.83 - 24.60][0.06 - 0.11][0.07 - 0.11][93.00 - 128.00][108.00 - 119.00]yale (n=9)4514.96 (2.39)12.53 (3.30)0.07 (0.02)0.04 (0.01)84.75 (37.03)98.20 (12.32)81na318[11.83 - 17.17][8.67 - 16.66][0.04 - 0.09][0.03 - 0.06][37.00 - 120.00][84.00 - 115.00]kki (n=8)359.88 (1.51)9.91 (1.69)0.07 (0.00)0.07 (0.02)nana62[10 - 16]017[8.20 - 11.14][8.46 - 12.76][0.06 - 0.07][0.05 - 0.10]olin (n=7)3418.3 (4.93)19.50 (2.38)0.09 (0.01)0.09 (0.03)nana52[9 - 19]0073[15.00 - 24.00][16.00 - 21.00][0.08 - 0.10][0.06 - 0.13]trinity (n=7)3416.46 (3.79)19.48 (4.53)0.06 (0.01)0.06 (0.01)123.00 (7.21)112.00 (6.38)70[7 - 15]007[13.58 - 20.75][13.75 - 24.83][0.05 - 0.07][0.06 - 0.08][117.00 - 131.00][107.00 - 121.00]cmu (n=5)3226.67 (4.51)26.00 (7.07)0.06 (0.02)0.08 (0.01)113.67 (10.79)109.50 (0.71)41[8 - 16]005[22.00 - 31.00][21.00 - 31.00][0.04 - 0.08][0.07 - 0.08][106.00 - 126.00][109.00 - 110.00]um_2 (n=4)1317.40 (0.00)15.23 (0.67)0.08 (0.00)0.04 (0.02)80.00 (0.00)99.33 (7.02)40na104[17.40 - 17.40][14.50 - 15.80][0.08 - 0.08][0.03 - 0.06][80.00 - 80.00][92.00 - 106.00] validationusm (n=13)11220.7 (5.7)28.2 (5.6).05 (.02).06 (.005)105.9 (17.9)109.5 (11.3)121[6 - 21]1nana[11.4 - 32.8][22 - 34][.03-.09][.05-.07][75 - 133][100 - 119]nyu (n=10)5513.1 (3.99)16.4 (4.3).07 (.02).05 (.01)38.8 (11.5)102.8 (14.7)28[5 - 13]0nana[8.9 - 17.9][9.8 - 20.0][.04-.09][.03-.06][92 - 119][83 - 117]sdsu (n=8)179.6413.5 (2.9)0.025.04 (.018)112111.7 (8.3)4411017[9.6 - 16.8][.02-.07][103 - 124]trinity (n=5)1419.416.3 (3.2)0.057.066 (.0087)114109.8 (6.7)501305[12.7 - 20.1][.05-.076][103 - 116]um_1 (n=4)1316.116.2 (2.7)0.048.05 (.01)125178 (9.9)22na104[13.1 - 18.2][.03-.06][82 - 96]leuven_2 (n=3)2114.5 (.29)12.4.08 (.02)0.11101.5 (21.9)9730nan312[14.3 - 14.7][.07-.09][86 - 117]pitt (n=3)2115.6 (1.9)21.22.05 (.03)0.11103 (9.9)12121[12 - 16]003[14.2 - 16.9][.03-.07][96 - 110]yale (n=3)2114 (0.53)16.66.049 (.02)0.05785 (1.4)9303na203[13.66 - 14.4][.03-.06][84 - 86]um_2 (n=2)02na23.2 (7.9)na.06 (.003)na107.5 (2.2)20nana02[17.6 - 28.8][.058-.06][106 - 109]olin (n=1)1018na0.07nanana107001inline supplementary table s1 table s1abide demographic information.sitessample sizeagermsdnviqgenderados_totalhandednessasdtdasdtdasdtdasdtdmalefemalerangenaleftrightmean (sd)mean (sd)mean (sd)[range][range][range][range]full samplenyu (n=70)363415.6 (6)16.96 (6.17).02 (.02).02 (.02)108 (15.1)103 (14.9)5416[5 - 22]0nana[8.5 - 30][6.4 - 30.8][.03-.09][.03-.09][72 - 149][67 - 137]um_1 (n=29)151414.7 (2.1)15.5 (2.8).06 (.02).05 (.02)112 (19.6)102.2 (9.4)1712na15521[11.5 - 18.6][11 - 19][.02-.09][.02-.08][81 - 148][82 - 113]usm (n=29)192021.9 (6.4)6.4 (7.2).05 (.02).06 (.02)105.7 (13)111 (12.5)290[6 - 21]0nana[11.3 - 35.7][13.8 - 34][.02-.09][.03-.09][75 - 133][90 - 129]sdsu (n=25)101514.6 (2.3)13.4 (2.3).03 (.01).04 (.02)112 (16)111 (11)214[4 - 17]0619[9.6 - 17.7][8 - 16.8][.02-.05][.02-.07][86 - 114][92 - 137]leuven_1 (n=17)9822.2 (4.9)23.75 (2.9).061 (.02).065 (.02)103 (16.4)114 (18.9)170na9116[19 - 32][21 - 29][.04-.09][.04-.09][74 - 120][93 - 155]leuven_2 (n=17)8913.8 (1.1)14.3 (1.4).07 (.01).08 (.02)97.5 (11.8)101.4 (6.5)116na8413[12.2 - 15.3][12.3 - 16.6][.05-.1][.05-.1][83 - 117][89 - 109]pitt (n=14)8621.2 (8.4)20.2 (4.1).07 (.02).09 (.02)11.7 (14.2)115 (5.3)122[7 - 19]1113[12.6 - 35.2][12.8 - 24.6][.04-.1][.07-.1][93 - 128][108 - 121]trinity (n=12)4817.2 (3.4)17.9 (4).06 (.010).07 (.01)121 (7.4)111 (6.2)120[7 - 15]0012[13.6 - 20.8][12.7 - 24.8][.05-.07][.06-.07][114 - 131][103 - 121]yale (n=12)6614.6 (1.9)13.2 (3.4).06 (.02).05 (.01)85 (29)97 (11.2)84na5111[11.8 - 17.2][8.7 - 16.7][.03-.09][.03-.06][37 - 120][84 - 115]kkin (n=8)359.9 (1.51)9.9 (1.7).01 (.004).01 (.02)nana62[10 - 16]017[8.2 - 11.1][8.5 - 12.8][.06-.07][.05-.1]olin (n=8)4418.3 (4.03)19.5 (2.4).09 (.01).09 (.3)nana62[7 - 19]408[15 - 24][16 - 21][.07-.1][.06-.1]um_2 (n=6)1517.4 (na)18.4 (5.9).08 (na).05 (.01)80 (na)102 (6.8)60na106[17.4 - 17.4][14.5 - 28.9][.08-.08][.03-.06][80 - 80][92 - 109]cmu (n=5)3226.7 (4.5)26 (.07).06 (.02).08 (.01)113.7 (10.8)109.5 (0.7)41[8 - 16]005[22 - 31][21 - 31][.04-.07][.07-.08][106 - 126][109 - 110] trainingnyu (n=60)312915.96 (6.22)16.96 (6.50).05 (.02)0.05 (0.02)108.03 (15.80)102.93 (15.17)4614[6 - 22]29nana[8.51 - 29.98][6.47 - 30.78][.03 -.09][0.03 - 0.09][72.00 - 149.00][67.00 - 137.00]um_1 (n=25)141114.64 (2.11)15.28 (2.85)0.06 (0.02)0.05 (0.02)111.43 (19.97)105.11 (6.72)1510na14517[11.50 - 18.60][11.00 - 19.20][0.02 - 0.09][0.02 - 0.08][81.00 - 148.00][92.00 - 113.00]leuven_1 (n=17)9822.22 (4.87)23.75 (2.92)0.06 (0.02)0.07 (0.02)103.00 (16.42)113.88] (18.88)170na9116[19.00 - 32.00][21.00 - 29.00][0.04 - 0.09][0.04 - 0.09][74.00 - 120.00][93.00 - 155.00sdsu (n=17)9815.19 (1.65)13.43 (3.35)0.03 (0.01)0.04 (0.01)112.67 (16.77)110.25 (13.61)170[4 - 17]0512[12.88 - 17.67][8.02 - 16.59][0.02 - 0.05][0.02 - 0.07][86.00 - 140.00][92.00 - 137.00]usm (n=16)8823.67 (7.35)21.60 (6.84)0.05 (0.02)0.06 (0.02)101.75 (6.82)111.38 (13.22)160[9 - 17]0nana[15.85 - 35.71][13.81 - 34.01][0.02 - 0.07][0.03 - 0.09][87.00 - 106.00][90.00 - 129.00]leuven_2 (n=14)6813.53 (1.23)14.59 (1.32)0.07 (0.01)0.07 (0.01)96.17 (9.54)102 (6.70)86na6311[12.20 - 15.30][12.30 - 16.60][0.05 - 0.08][0.05 - 0.10][83.00 - 106.00][89.00 - 109.00]pitt (n=11)6523.03 (9.07)20.01 (4.56)0.08 (0.02)0.09 (0.01)114.67 (14.90)114.20 (5.07)101[7 - 19]1110[12.62 - 35.20][12.83 - 24.60][0.06 - 0.11][0.07 - 0.11][93.00 - 128.00][108.00 - 119.00]yale (n=9)4514.96 (2.39)12.53 (3.30)0.07 (0.02)0.04 (0.01)84.75 (37.03)98.20 (12.32)81na318[11.83 - 17.17][8.67 - 16.66][0.04 - 0.09][0.03 - 0.06][37.00 - 120.00][84.00 - 115.00]kki (n=8)359.88 (1.51)9.91 (1.69)0.07 (0.00)0.07 (0.02)nana62[10 - 16]017[8.20 - 11.14][8.46 - 12.76][0.06 - 0.07][0.05 - 0.10]olin (n=7)3418.3 (4.93)19.50 (2.38)0.09 (0.01)0.09 (0.03)nana52[9 - 19]0073[15.00 - 24.00][16.00 - 21.00][0.08 - 0.10][0.06 - 0.13]trinity (n=7)3416.46 (3.79)19.48 (4.53)0.06 (0.01)0.06 (0.01)123.00 (7.21)112.00 (6.38)70[7 - 15]007[13.58 - 20.75][13.75 - 24.83][0.05 - 0.07][0.06 - 0.08][117.00 - 131.00][107.00 - 121.00]cmu (n=5)3226.67 (4.51)26.00 (7.07)0.06 (0.02)0.08 (0.01)113.67 (10.79)109.50 (0.71)41[8 - 16]005[22.00 - 31.00][21.00 - 31.00][0.04 - 0.08][0.07 - 0.08][106.00 - 126.00][109.00 - 110.00]um_2 (n=4)1317.40 (0.00)15.23 (0.67)0.08 (0.00)0.04 (0.02)80.00 (0.00)99.33 (7.02)40na104[17.40 - 17.40][14.50 - 15.80][0.08 - 0.08][0.03 - 0.06][80.00 - 80.00][92.00 - 106.00] validationusm (n=13)11220.7 (5.7)28.2 (5.6).05 (.02).06 (.005)105.9 (17.9)109.5 (11.3)121[6 - 21]1nana[11.4 - 32.8][22 - 34][.03-.09][.05-.07][75 - 133][100 - 119]nyu (n=10)5513.1 (3.99)16.4 (4.3).07 (.02).05 (.01)38.8 (11.5)102.8 (14.7)28[5 - 13]0nana[8.9 - 17.9][9.8 - 20.0][.04-.09][.03-.06][92 - 119][83 - 117]sdsu (n=8)179.6413.5 (2.9)0.025.04 (.018)112111.7 (8.3)4411017[9.6 - 16.8][.02-.07][103 - 124]trinity (n=5)1419.416.3 (3.2)0.057.066 (.0087)114109.8 (6.7)501305[12.7 - 20.1][.05-.076][103 - 116]um_1 (n=4)1316.116.2 (2.7)0.048.05 (.01)125178 (9.9)22na104[13.1 - 18.2][.03-.06][82 - 96]leuven_2 (n=3)2114.5 (.29)12.4.08 (.02)0.11101.5 (21.9)9730nan312[14.3 - 14.7][.07-.09][86 - 117]pitt (n=3)2115.6 (1.9)21.22.05 (.03)0.11103 (9.9)12121[12 - 16]003[14.2 - 16.9][.03-.07][96 - 110]yale (n=3)2114 (0.53)16.66.049 (.02)0.05785 (1.4)9303na203[13.66 - 14.4][.03-.06][84 - 86]um_2 (n=2)02na23.2 (7.9)na.06 (.003)na107.5 (2.2)20nana02[17.6 - 28.8][.058-.06][106 - 109]olin (n=1)1018na0.07nanana107001 abide demographic information . Data were processed using the analysis of functional neuroimages software (cox, 1996) (http://afni.nimh.nih.gov) and fsl 5.0 (smith et al ., 2004) (http://www.fmrib.ox.ac.uk/fsl). Functional images were slice - time corrected, motion corrected to align to the middle time point, field - map corrected and aligned to the anatomical images using flirt with six degrees of freedom . Fsl's nonlinear registration tool (fnirt) was used to standardize images to the mni152 standard image (3 mm isotropic) using sinc interpolation . The outputs were blurred to a global full - width - at - half - maximum of 6 mm . Given recent concerns that traditional filtering approaches can cause rippling of motion confounds to neighboring time points (carp, 2013), we used a second - order band - pass butterworth filter (power et al ., 2013; satterthwaite et al ., 2013) to isolate low - frequency bold fluctuations (.008 <f <.08 hz) (cordes et al ., 2001). Regression of 17 nuisance variables was performed to improve data quality (satterthwaite et al ., 2013). Nuisance regressors included six rigid - body motion parameters derived from motion correction and their derivatives . White matter and ventricular masks were created at the participant level using fsl's image segmentation (zhang et al ., 2001) and trimmed to avoid partial - volume effects . An average time series was extracted from each mask and was removed using regression, along with its corresponding derivative . Whole - brain global signal was also included as a regressor to mitigate cross - site variability (power et al ., 2014). All nuisance regressors were band - pass filtered using the second - order butterworth filter (.008 <f <.08 hz) (power et al ., 2013; satterthwaite et al ., 2013) motion was quantified as the euclidean distance between consecutive time points (based on detected six rigid - body motion parameters). For any instance greater than 0.25 mm, considered excessive motion, the time point as well as the preceding and following time points were censored, or scrubbed if two censored time points occurred within ten time points of each other, all time points between them were also censored . Datasets with fewer than 90% of time points or less than 150 total time points remaining after censoring were excluded from the analysis . Motion over the truncated run was summarized for each participant as the average euclidean distance moved between time points (including areas that were censored) and was well matched between groups (p = 0.92). We used 220 rois (10 mm spheres) adopted from a meta - analysis of functional imaging studies by power et al . (2011), excluding 44 of their 264 rois because of missing signal in> 2 participants . Mean time courses were extracted from each roi and a 220 220 connectivity matrix of fisher - transformed pearson correlation coefficients was created for each subject . We then concatenated each subject's functional connectivities to construct a group level data matrix . For each roi pair, we regressed out age (as numerical) and site (as categorical) covariates from data matrix . For interpretation of findings the other was anatomical, using each roi's center mni coordinate and a surface based atlas (fischl et al ., 2004) included in freesurfer . These anatomical labels were also used to generate connectograms (irimia et al ., 2012) for complementary visualization of informative connectivities (inline supplementary table s2). We used 220 rois (10 mm spheres) adopted from a meta - analysis of functional imaging studies by power et al . (2011), excluding 44 of their 264 rois because of missing signal in> 2 participants . Mean time courses were extracted from each roi and a 220 220 connectivity matrix of fisher - transformed pearson correlation coefficients was created for each subject . We then concatenated each subject's functional connectivities to construct a group level data matrix . For each roi pair, we regressed out age (as numerical) and site (as categorical) covariates from data matrix . For interpretation of findings the other was anatomical, using each roi's center mni coordinate and a surface based atlas (fischl et al ., 2004) included in freesurfer . These anatomical labels were also used to generate connectograms (irimia et al ., 2012) for complementary visualization of informative connectivities (inline supplementary table s2). Inline supplementary table s2table s2anatomical labels used in connectogram.frontal lobetrfpog / sfmarg / smfslorssborsorsrginffgorpmfgorginffgtripinffsmedorssupfgsupfsinffgoppinfprcssupprcsprcgsbcg / scstransverse frontopolar gyri and sulcifronto - marginal gyrus (of wernicke) and sulcusmiddle frontal sulcus lateral orbital sulcussuborbital sulcus orbital sulcistraight gyrus (gyrus rectus)orbital part of the inferior frontal gyrus middle frontal gyrus orbital gyritriangular part of the inferior frontal gyrus inferior frontal sulcus medial orbital sulcus (olfactory sulcus)superior frontal gyrussuperior frontal sulcus opercular part of the inferior frontal gyrusinferior part of the precentral sulcussuperior part of the precentral sulcus precentral gyrus subcentral gyrus (central operculum) and sulcicentral sulcus (rolando s fissure)insular lobealshorpacirinsalsverpshoingsupcirinsloing / cinsinfcirinsposlshorizontal ramus of the anterior lateral sulcusanterior circular sulcus the insulavertical ramus of the anterior lateral sulcusshort insular gyrisuperior segment of circular sulcus of the insulalong insular gyrus and central insular sulcusinferior segment of circular sulcus of the insulaposterior ramus of lateral sulcuslimbic lobeacgg / smacgg / ssbcagpercasmposcgg / scgsmarpposdcggposvcgganterior part of the cingulate gyrus and sulcus middle - anterior part of the cingulate gyrus / sulcussubcallosal area, subcallosal gyruspericallosal sulcus (s of corpus callosum)middle - posterior part of cingulate gyrus / sulcusmarginal branch of the cingulate sulcusposterior - dorsal part of the cingulate gyrusposterior - ventral part of the cingulate gyrustemporal lobetpopoplsuptglphgatrcostrtsmtgtplinftginftssuptstemporal polepolar plane of the superior temporal gyruslateral aspect of the superior temporal gyrusheschl s gyrus (anterior transverse temporal gyrus)anterior transverse collateral sulcus transverse temporal sulcus middle temporal gyrustemporal plane of the superior temporal gyrusinferior temporal gyrus inferior temporal sulcus superior temporal sulcusparietal lobeposcgsumargpacl / sposcsjssbpsintps / trpssupplprcunanggpocspostcentral gyrussupramarginal gyrusparacentral lobule and sulcus postcentral sulcussulcus intermedius primus (of jensen)subparietal sulcusintraparietal sulcus and transverse parietal sulcisuperior parietal lobuleprecuneus angular gyrusparietal - occipital sulcusoccipital lobepahipgcos / linsloctsfugccslingaocsinfocg / ssupocs / trocspostrcoscunmocgmocs / lussupocgocpoparahippocampal gyrusmedial occipito - temporal sulcus and lingual sulcuslateral occipito - temporal sulcusfusiform gyrus calcarine sulcus lingual gyrusanterior occipital sulcusinterior occipital gyrus / sulcus superior occipital sulcus / transverse occipital sulcusposterior transverse collateral sulcuscuneusmiddle occipital gyrus middle occipital sulcus and lunatus sulcus superior occipital gyrusoccipital polesubcorticalpupalcannaccamgthahipputamen pallidumcaudate nucleusnucleus accumbensamygdalathalamushippocampuscerebellumcebcerebellumbrain stembstbrain steminline supplementary table s2 table s2anatomical labels used in connectogram.frontal lobetrfpog / sfmarg / smfslorssborsorsrginffgorpmfgorginffgtripinffsmedorssupfgsupfsinffgoppinfprcssupprcsprcgsbcg / scstransverse frontopolar gyri and sulcifronto - marginal gyrus (of wernicke) and sulcusmiddle frontal sulcus lateral orbital sulcussuborbital sulcus orbital sulcistraight gyrus (gyrus rectus)orbital part of the inferior frontal gyrus middle frontal gyrus orbital gyritriangular part of the inferior frontal gyrus inferior frontal sulcus medial orbital sulcus (olfactory sulcus)superior frontal gyrussuperior frontal sulcus opercular part of the inferior frontal gyrusinferior part of the precentral sulcussuperior part of the precentral sulcus precentral gyrus subcentral gyrus (central operculum) and sulcicentral sulcus (rolando s fissure)insular lobealshorpacirinsalsverpshoingsupcirinsloing / cinsinfcirinsposlshorizontal ramus of the anterior lateral sulcusanterior circular sulcus the insulavertical ramus of the anterior lateral sulcusshort insular gyrisuperior segment of circular sulcus of the insulalong insular gyrus and central insular sulcusinferior segment of circular sulcus of the insulaposterior ramus of lateral sulcuslimbic lobeacgg / smacgg / ssbcagpercasmposcgg / scgsmarpposdcggposvcgganterior part of the cingulate gyrus and sulcus middle - anterior part of the cingulate gyrus / sulcussubcallosal area, subcallosal gyruspericallosal sulcus (s of corpus callosum)middle - posterior part of cingulate gyrus / sulcusmarginal branch of the cingulate sulcusposterior - dorsal part of the cingulate gyrusposterior - ventral part of the cingulate gyrustemporal lobetpopoplsuptglphgatrcostrtsmtgtplinftginftssuptstemporal polepolar plane of the superior temporal gyruslateral aspect of the superior temporal gyrusheschl s gyrus (anterior transverse temporal gyrus)anterior transverse collateral sulcus transverse temporal sulcus middle temporal gyrustemporal plane of the superior temporal gyrusinferior temporal gyrus inferior temporal sulcus superior temporal sulcusparietal lobeposcgsumargpacl / sposcsjssbpsintps / trpssupplprcunanggpocspostcentral gyrussupramarginal gyrusparacentral lobule and sulcus postcentral sulcussulcus intermedius primus (of jensen)subparietal sulcusintraparietal sulcus and transverse parietal sulcisuperior parietal lobuleprecuneus angular gyrusparietal - occipital sulcusoccipital lobepahipgcos / linsloctsfugccslingaocsinfocg / ssupocs / trocspostrcoscunmocgmocs / lussupocgocpoparahippocampal gyrusmedial occipito - temporal sulcus and lingual sulcuslateral occipito - temporal sulcusfusiform gyrus calcarine sulcus lingual gyrusanterior occipital sulcusinterior occipital gyrus / sulcus superior occipital sulcus / transverse occipital sulcusposterior transverse collateral sulcuscuneusmiddle occipital gyrus middle occipital sulcus and lunatus sulcus superior occipital gyrusoccipital polesubcorticalpupalcannaccamgthahipputamen pallidumcaudate nucleusnucleus accumbensamygdalathalamushippocampuscerebellumcebcerebellumbrain stembstbrain stem anatomical labels used in connectogram . Three ml algorithms (see supplemental methods for technical details) were implemented in this study to perform a binary classification (asd vs. td) using rs - fmri data: (i) support vector machines (svm) in combination with particle swarm optimization (pso) for feature selection (pso - svm); (ii) support vector machines with recursive feature elimination (rfe - svm) for feature ranking; and (iii) a nonparametric ensemble learning method called random forest (rf). We first used pso (kennedy, 1995) in combination with a base classifier, a linear support vector machine . Pso is a biologically inspired, stochastic optimization algorithm that models the behavior of swarming particles . The pso algorithm was utilized as a feature selection tool to obtain a compact and discriminative feature subset for improved accuracy and robustness of the subsequent classifiers . In a second approach, we used recursive feature elimination, a pruning technique that eliminates original input features by using feature ranking coefficients as classifier weights, and retains a minimum subset of features that yield best classification performance (guyon, 2002). The output obtained from this algorithm is a list of all features ranked in the order of most informative to least . We used rfe to reduce the feature space dimensionality and select the top informative features . In rf, the basic unit is a classification tree and the ensemble of trees (or forest) is used to classify participants using features (e.g., connectivity measures from fmri; see inline supplementary fig . Each tree classifies or predicts a diagnostic status, and the forest's final prediction is the classification having the most votes based on all the trees in the forest . In random forests, it is not necessary to perform cross - validation or a separate test set to obtain an unbiased estimate of the test set error because validation is intrinsic to rf . Out - of - bag (oob) error is estimated internally, during the rf run (http://www.stat.berkeley.edu/~breiman/randomforests). Each tree in the forest is constructed using a bootstrap sample from the original data . Each bootstrap sample will contain approximately two - thirds of the original observations, which results in one - third of the subjects being left out (i.e., not used in the construction of a particular tree). These out - of - bag samples are used to attain an unbiased estimate of the classification error (breiman, 2001). The oob error estimate can be shown to be almost identical to the error obtained by cross - validation (hastie et al ., 2009). Rf is an example of bootstrap aggregation or bagging and is relatively protected from overfitting (breiman, 2001). As an additional improvement of the bagging process, rf reduces the correlation between trees by using a subset of the variables at each split in the growing of each tree . The oob data are used to determine variable importance, based on mean decrease in accuracy . This is done for each tree by randomly permuting a variable in the oob data and recording the change in accuracy or misclassification using the oob data . For the ensemble of trees, the permuted predictions are aggregated and compared against the unpermuted predictions to determine the importance of the permuted variable by the magnitude of the decrease in accuracy of that variable . When the number of variables is very large (in the order of 2 10), rf can be run once with all the variables, then again using only the most important variables selected from the first run . In rf, the basic unit is a classification tree and the ensemble of trees (or forest) is used to classify participants using features (e.g., connectivity measures from fmri; see inline supplementary fig . Each tree classifies or predicts a diagnostic status, and the forest's final prediction is the classification having the most votes based on all the trees in the forest . In random forests, it is not necessary to perform cross - validation or a separate test set to obtain an unbiased estimate of the test set error because validation is intrinsic to rf . Out - of - bag (oob) error is estimated internally, during the rf run (http://www.stat.berkeley.edu/~breiman/randomforests). Each tree in the forest is constructed using a bootstrap sample from the original data . Each bootstrap sample will contain approximately two - thirds of the original observations, which results in one - third of the subjects being left out (i.e., not used in the construction of a particular tree). These out - of - bag samples are used to attain an unbiased estimate of the classification error (breiman, 2001). The oob error estimate can be shown to be almost identical to the error obtained by cross - validation (hastie et al ., 2009). Rf is an example of bootstrap aggregation or bagging and is relatively protected from overfitting (breiman, 2001). As an additional improvement of the bagging process, rf reduces the correlation between trees by using a subset of the variables at each split in the growing of each tree . The oob data are used to determine variable importance, based on mean decrease in accuracy . This is done for each tree by randomly permuting a variable in the oob data and recording the change in accuracy or misclassification using the oob data . For the ensemble of trees, the permuted predictions are aggregated and compared against the unpermuted predictions to determine the importance of the permuted variable by the magnitude of the decrease in accuracy of that variable . When the number of variables is very large (in the order of 2 10), rf can be run once with all the variables, then again using only the most important variables selected from the first run ., data are bootstrapped into in - bag and out - of - bag (for testing of the model). The tree starts with the in - bag data at the root node, with a random subset of features, and begins the process of splitting . The splitting criteria at each node are determined by a type of greedy algorithm that recursively partitions the data into daughter nodes, until reaching a terminal node . (greedy recursive partitioning algorithms make the partitioning choice that performs best at each split, with the goal of globally optimal classification). The out - of - bag data are then sent down the tree as a test of model fit . Once all 10,000 trees in the forest have finished this process, the forest takes the majority vote from the trees, and obtains variable importance measures and oob error . Fig ., data are bootstrapped into in - bag and out - of - bag (for testing of the model). The tree starts with the in - bag data at the root node, with a random subset of features, and begins the process of splitting . The splitting criteria at each node are determined by a type of greedy algorithm that recursively partitions the data into daughter nodes, until reaching a terminal node . (greedy recursive partitioning algorithms make the partitioning choice that performs best at each split, with the goal of globally optimal classification). The out - of - bag data are then sent down the tree as a test of model fit . Once all 10,000 trees in the forest have finished this process, the forest takes the majority vote from the trees, and obtains variable importance measures and oob error . Random forest procedure . For each classification tree (rf s single unit), data are bootstrapped into in - bag and out - of - bag (for testing of the model). The tree starts with the in - bag data at the root node, with a random subset of features, and begins the process of splitting . The splitting criteria at each node are determined by a type of greedy algorithm that recursively partitions the data into daughter nodes, until reaching a terminal node . (greedy recursive partitioning algorithms make the partitioning choice that performs best at each split, with the goal of globally optimal classification). The out - of - bag data are then sent down the tree as a test of model fit . Once all 10,000 trees in the forest have finished this process, the forest takes the majority vote from the trees, and obtains variable importance measures and oob error ., we used the r package randomforest (liaw and wiener, 2002) for feature selection and classification analysis . As importance scores, proximity measures, and error rates vary because of the random components in rf (out of bag sampling, node - level permutation testing), we created a forest with a sufficient number of trees to ensure stability . To optimize the most important rf parameters, we fine - tuned the number of trees per forest to 10,000 as the plotted error rate was observed to stabilize (see inline supplementary fig . S2), and set the number of variables randomly selected per node to 150, after tuning for this parameter . The rf algorithm ranked all features based on variable importance given by the mean decrease in accuracy . After the initial run with all features, we used the variable importance measures to select for 100 top informative features that were then used to build a classifier and obtain oob error (see inline supplementary fig ., we obtained the oob error to assess how well the model predicted new data (see inline supplementary fig ., we used the r package randomforest (liaw and wiener, 2002) for feature selection and classification analysis . As importance scores, proximity measures, and error rates vary because of the random components in rf (out of bag sampling, node - level permutation testing), we created a forest with a sufficient number of trees to ensure stability . To optimize the most important rf parameters, we fine - tuned the number of trees per forest to 10,000 as the plotted error rate s2), and set the number of variables randomly selected per node to 150, after tuning for this parameter . The rf algorithm ranked all features based on variable importance given by the mean decrease in accuracy . After the initial run with all features, we used the variable importance measures to select for 100 top informative features that were then used to build a classifier and obtain oob error (see inline supplementary fig ., we obtained the oob error to assess how well the model predicted new data (see inline supplementary fig . Initially, the error fluctuates greatly, then eventually stabilizes with a large enough forest with 10,000 trees . The number of trees in the forest is an important parameter for fine - tuning to ensure stability of the algorithm . The red, green, and black lines represent the oob errors for asd, td, and out - of - bag participants, respectively . Initially, the error fluctuates greatly, then eventually stabilizes with a large enough forest with 10,000 trees . The number of trees in the forest is an important parameter for fine - tuning to ensure stability of the algorithm . The red, green, and black lines represent the oob errors for asd, td, and out - of - bag participants, respectively . Initially, the error fluctuates greatly, then eventually stabilizes with a large enough forest with 10,000 trees . The number of trees in the forest is an important parameter for fine - tuning to ensure stability of the algorithm . The red, green, and black lines represent the oob errors for asd, td, and out - of - bag participants, respectively . This is done for each tree by randomly permuting a variable in the oob data and recording the change in accuracy . For the ensemble of trees, the permuted predictions are aggregated and compared against the unpermuted predictions to determine the importance of the permuted variable by the magnitude of the decrease in accuracy of that variable . We selected the top features using this mda plot, where we observed stabilized rate of change of the mda curve . Here, 100 features were selected using the mda measures . This is done for each tree by randomly permuting a variable in the oob data and recording the change in accuracy . For the ensemble of trees, the permuted predictions are aggregated and compared against the unpermuted predictions to determine the importance of the permuted variable by the magnitude of the decrease in accuracy of that variable . We selected the top features using this mda plot, where we observed stabilized rate of change of the mda curve . Here, 100 features were selected using the mda measures . This is done for each tree by randomly permuting a variable in the oob data and recording the change in accuracy . For the ensemble of trees, the permuted predictions are aggregated and compared against the unpermuted predictions to determine the importance of the permuted variable by the magnitude of the decrease in accuracy of that variable . We selected the top features using this mda plot, where we observed stabilized rate of change of the mda curve . Here, 100 features were selected using the mda measures . S4fine tuning the mtry parameter for rf to select the optimal number of features to split at each node . Using the top 100 features, we fine tuned rf again on the parameter mtry, the number of features used at each split, to 5 . S4fine tuning the mtry parameter for rf to select the optimal number of features to split at each node . Using the top 100 features, we fine tuned rf again on the parameter mtry, the number of features used at each split, to 5 . Fine tuning the mtry parameter for rf to select the optimal number of features to split at each node . Using the top 100 features, we fine tuned rf again on the parameter mtry, the number of features used at each split, to 5 . Inline supplementary figs . S2, s3, s4 can be found online at http://dx.doi.org/10.1016/j.nicl.2015.04.002 . The pso - svm achieved an accuracy of 81% on the training, and of 58% on the validation dataset, with 44% sensitivity and 72% specificity (for matching details, see inline supplementary table s3). Accuracy for rfe - svm was 100% on the training dataset and 66% on the validation dataset, with 60% sensitivity and 72% specificity . . However, using the top 100 features with the highest variable importance (as defined above), rf achieved 90.8% accuracy (oob error rate 9.2%), with sensitivity at 89% and specificity at 93% . In fact, using only the 10 most informative features, rf still attained an accuracy of 75%, with 75% sensitivity and 75% specificity . Note that the rf methodology does not have an external validation set, but the bootstrap sample of the data for each tree acts as an internal validation dataset . Using an external 20% validation set, accuracy from rf was at similar levels as for pso and rfe (inline supplementary table s4). However, since rf is an ensemble learning method, such external validation is not usually part of the rf procedure (see the discussion section). The pso - svm achieved an accuracy of 81% on the training, and of 58% on the validation dataset, with 44% sensitivity and 72% specificity (for matching details, see inline supplementary table s3). Accuracy for rfe - svm was 100% on the training dataset and 66% on the validation dataset, with 60% sensitivity and 72% specificity . However, using the top 100 features with the highest variable importance (as defined above), rf achieved 90.8% accuracy (oob error rate 9.2%), with sensitivity at 89% and specificity at 93% . In fact, using only the 10 most informative features, rf still attained an accuracy of 75%, with 75% sensitivity and 75% specificity . Note that the rf methodology does not have an external validation set, but the bootstrap sample of the data for each tree acts as an internal validation dataset . Using an external 20% validation set, accuracy from rf was at similar levels as for pso and rfe (inline supplementary table s4). However, since rf is an ensemble learning method, such external validation is not usually part of the rf procedure (see the discussion section). Inline supplementary table s3table s3abide cohort matching (p - values from independent two - sample t - tests).full sampletrainingvalidationtraining <=> validationtraining <=> validationn = 252n=200n=52asd <=> tdasd <=> tdasd <=> tdasd <=> asdtd <=> tdmotion0.9230.9220.9840.9850.984age0.7930.7800.9840.8400.962nviq0.7710.7530.9240.9340.986ados0.824inline supplementary table s3 table s3abide cohort matching (p - values from independent two - sample t - tests).full sampletrainingvalidationtraining <=> validationtraining <=> validationn = 252n=200n=52asd <=> tdasd <=> tdasd <=> tdasd <=> asdtd <=> tdmotion0.9230.9220.9840.9850.984age0.7930.7800.9840.8400.962nviq0.7710.7530.9240.9340.986ados0.824 abide cohort matching (p - values from independent two - sample t - tests). Inline supplementary table s3 inline supplementary table s4table s4training and predictive (validation) accuracies for pso - svm, rfe - svm, and rf, using external validation sets.pso-svmrfe-svmrftraining (n=200)81%100%91%validation (n=52)58%66%62%number of features10040150inline supplementary table s4 table s4training and predictive (validation) accuracies for pso - svm, rfe - svm, and rf, using external validation sets.pso-svmrfe-svmrftraining (n=200)81%100%91%validation (n=52)58%66%62%number of features10040150 training and predictive (validation) accuracies for pso - svm, rfe - svm, and rf, using external validation sets . Inline supplementary table s4 inline supplementary tables s3, s4 can be found online at http://dx.doi.org/10.1016/j.nicl.2015.04.002 . Given the overall better performance from rf (compared to the other ml techniques), we proceeded to examine the most informative features selected by rf . When selecting only the 10 top features, several regions were noted to participate in two or more informative connections: left anterior cingulate gyrus, postcentral gyrus bilaterally, right precuneus, and left calcarine sulcus (see inline supplementary fig . S5a). The pattern for the top 100 features, which yielded the peak accuracy of 91%, involved connections between 124 rois (see inline supplementary fig . The left paracentral lobule and the right postcentral gyrus participated in 6 informative connections, which was 2sd above the mean participation among the 124 informative rois . Given the overall better performance from rf (compared to the other ml techniques), we proceeded to examine the most informative features selected by rf . When selecting only the 10 top features, several regions were noted to participate in two or more informative connections: left anterior cingulate gyrus, postcentral gyrus bilaterally, right precuneus, and left calcarine sulcus (see inline supplementary fig . The pattern for the top 100 features, which yielded the peak accuracy of 91%, involved connections between 124 rois (see inline supplementary fig . S5b). The left paracentral lobule and the right postcentral gyrus participated in 6 informative connections, which was 2sd above the mean participation among the 124 informative rois . The labels of the connectograms can be found in inline supplementary table s1, in the order in which they appear in the figure . The labels of the connectograms can be found in inline supplementary table s1, in the order in which they appear in the figure . The labels of the connectograms can be found in inline supplementary table s1, in the order in which they appear in the figure . We further examined these top 100 features by functional networks, as defined in power et al . We first determined the number of times rois from a specific network participated in an informative connection (fig . The distribution differed significantly from the overall distribution of rois across different networks in power et al . Default mode, somatosensory / motor (hand region), and visual networks ranked at the top, accounting for half of all informative connections . We further examined normalized network importance (i.e., the number of times network rois participated in an informative connection divided by the total number of rois in the given network; fig . This ratio highlighted the importance of somatosensory / motor networks (both mouth and hand regions), ahead of subcortical, memory retrieval, and visual networks . Within the two top networks, the somatosensory cortex (postcentral gyrus) participated in 23 of the top 100 connections, whereas the primary motor cortex (precentral gyrus) participated in only 9 . Finally, we depicted the 100 top connections in a heat map matrix (fig . 1c), which highlighted the importance of connections between dmn and visual rois (14 informative features). Informative features for the somatosensory / motor hand regions were dominated by connections with subcortical rois . Examining the 100 top features by broad anatomical subdivisions (fischl et al ., 2004), the distribution did not differ significantly from expected (= 5.225, p = 0.98). We found 60 participations by parietal lobes bilaterally, followed by 45 for occipital lobes bilaterally . The right frontal lobe also had heightened density of informative connections (with 21 participations; fig . 1d). When normalizing participations by the number of rois within each subdivision (analogous to the procedure described above), the bilateral parietal lobes again stood out, followed by the right temporal lobe (fig . 1f), the highest concentration of the most informative connections was found to involve the parietal lobes bilaterally, with six informative connections each within the left and right parietal lobes, and an additional six between the right parietal and left occipital lobes and five between the left parietal and right frontal lobes . They were almost evenly divided with respect to the direction of mean fc differences (45% over-, 55% underconnected in the asd group) and type of connection (49% inter-, 51% intra - hemispheric). 95% of the top connections were between networks, 5% were within networks, 87% were mid- to long - distance (> 40 mm euclidean distance), and 11% were short - distance (<40 mm). However, these percentages of euclidean distance (= 0.228, p - value = 0.63) and of network connection (= 2.24, p - value = 0.13) did not differ significantly from those expected based on the totality of 24,090 features examined in the entire connectivity matrix . In this study, we used intrinsic functional connectivities between a set of functionally defined rois (power et al ., 2011) for machine learning diagnostic classification of asd . While accuracy remained overall modest for pso - svm and rfe - svm approaches, it was high (91%) for random forest . The rf algorithm is well - suited for classification of fcmri data for several reasons: it (i) is applicable when there are more features than observations, (ii) performs embedded feature selection and is relatively insensitive to any large number of irrelevant features, (iii) incorporates interactions between features, (iv) is based on the theory of ensemble learning that allows the algorithm to learn accurately both simple and complex classification functions, and (v) is applicable for both binary and multi - category classification tasks (breiman, 2001). We examined in two steps which single regions and functional networks were most informative, contributing most heavily to diagnostic classification . First, focusing on only the ten top features, for which an accuracy of 75% was achieved, we found that limbic (left anterior cingulate), somatosensory (postcentral gyri bilaterally), visual (calcarine sulcus), and default mode (right precuneus) regions stood out (supplementary fig . This was largely supported by subsequent examination of the 100 top features, for which the peak classification accuracy of 91% was reached . Half of all rois involved in these connections belonged to three (out of 14) networks, which were default mode, somatosensory / motor (hand) and visual (fig . . The informative role of the dmn in diagnostic classification was not surprising, given extensive evidence of dmn anomalies in asd . These include fmri findings indicating failure to enter the default mode in asd (kennedy et al ., 2006; murdaugh et al ., 2012), as well as numerous fcmri reports of atypical connectivity of the dmn (assaf et al ., 2010; courchesne et al ., 2005;, 2013b; von dem hagen et al ., 2013; washington et al ., 2014; zielinski et al ., functionally, the dmn is considered to relate to self - referential cognition, including domains of known impairment in asd, such as theory of mind and affective decision making (andrews - hanna et al ., 2010). The visual system appears to be overall relatively spared in asd (simmons et al ., 2009), and atypically enhanced participation of the visual cortex has been observed across many fmri (samson et al ., 2012) and fcmri studies (shen et al ., 2012), with the added recent finding of atypically increased local functional connectivity in asd being associated with symptom severity (keown et al ., 2013). The relative importance of visual regions and occipital lobes probably reflects atypical function of the visual system in asd (dakin and frith, 2005) rather than integrity . The role of somatosensory regions was particularly prominent when considering a normalized ratio of informative rois (fig . Although only a total of 26 somatosensory / motor rois (hand and mouth regions combined) were included in the analysis, these participated in 44 out of the 100 most informative connections . This effect was distinctly driven by the primary somatosensory cortex in the postcentral gyrus (rather than motor cortex). Our finding may be related to impaired emotional recognition of facial expressions in patients with focal damage to the right somatosensory cortices (si and sii) observed by adolphs and colleagues (2000), given that perception of facial expressions is also frequently impaired in individuals with asd (nuske et al ., 2013). Although somatosensory anomalies have been described in a number of asd studies (puts et al ., 2014; tomchek and dunn, 2007; tommerdahl et al ., 2007), the highly prominent role of somatosensory regions in diagnostic classification was remarkable . Cascio and colleagues (2012) found atypical activation patterns in adults with asd for pleasant and unpleasant tactile stimuli in the pcc and insula, which could reflect altered functional connectivity with the somatosensory cortex (not tested in the cited study). Atypical postcentral responses to somatosensory stimuli have also been observed in one evoked potential study in young children with asd (miyazaki et al ., 2007). Although little is known about differential responses between asd and td groups to resting state instructions or confined conditions within bore and head coil during scanning, it is possible that tactile or proprioceptive processing had some effect on the role of somatosensory rois in diagnostic classification . However, focus on low frequencies in our study would be expected to emphasize intrinsic fluctuations (thought to reflect history of co - activation (lewis et al ., 2009)) over online processing effects that tend to occur at higher frequencies . Diagnostic classification accuracy achieved using random forest machine learning was distinctly above accuracies reported from previous studies using rs - fcmri (anderson et al ., 2011), including a recent publication (nielsen et al ., rf implements a bootstrapping out - of - bag validation process (randomly selected subsamples), contrary to the other machine learning tools (pso - svm, rfe - svm) used here, which required external validation datasets . This latter procedure may provide stringent protection from overfitting to the idiosyncrasies of any given training dataset, as may be seen with conventional leave - one - out validation (ecker et al ., 2010a; ecker et al ., 2010b; iidaka, 2015; ingalhalikar et al ., 2011; nielsen et al ., 2013) the procedure depends, however, on the assumption that training and validation samples can be adequately matched . Such matching may be difficult, if many variables (e.g., age, sex, motion, site, eye status, scanner, imaging protocol) are potentially relevant (cf . Some potentially crucial variables, such as iq, symptom severity, and handedness, were incomplete in abide (missing for some sites) and could thus not be well matched . Other possibly important variables, some of which are hard to operationalize (e.g., history of interventions, medication) were entirely unavailable . Even if matching on all those variables were possible, a more intractable problem would remain . There is general consensus that asd is an umbrella term for several maybe many underlying biological subtypes (jeste and geschwind, 2014), which are currently unknown . Any substantial difference between training and validation datasets in the composition of these subtypes, which cannot be detected and cannot therefore be avoided, is likely to result in poor prediction in a validation dataset . In asd machine learning classification, accuracy will tend to be overestimated in training datasets (with common leave - one - out procedure), but underestimated in external validation datasets because of the problems described above . Indeed, when using such a split into training and external validation sets, rf yielded accuracies similar to pso and rfe - svm . However, such split is inconsistent with rf as an ensemble learning technique, which instead implements out - of - bag validation . In our view, this approach presents a solution to the matching problems described above, as the bootstrapping procedure itself ensures matching of training and validation datasets . This solution may be considered ideal, given the currently available sample limitations even from consortium datasets . As described in section 2.2, the mandatory (power et al ., 2015; satterthwaite et al ., 2013) selection of low - motion data reduced the initially large abide sample to only 252 participants . External 20% validation sets included only 26 participants per group, highlighting the matching issues described above . Distinctly larger high - quality datasets may in the future allow investigators to go beyond the pragmatic bootstrapping solution offered by rf and return to even more stringent external validation procedures because the probability of adequate matching for unknown subtypes by chance alone will increase with much larger numbers . Our findings suggest that intrinsic functional connectivity may identify complex biomarkers of asd, which is not unexpected given the extensive literature on functional connectivity anomalies in this disorder (e.g., vissers et al ., 2012). Aside from the use of rf machine learning and its virtues described above, the main difference between our study and the relatively unsuccessful classification study by nielsen et al . (2013) was our careful selection of high - quality (low - motion) datasets, resulting in the inclusion of only 252 (out of a total of 1112) participants, equally divided between two tightly motion - matched diagnostic groups . Our procedure was informed by the recent flurry of studies suggesting that even small amounts of head motion well below 1 mm affect interregional fmri signal correlations (carp, 2011; jo et al ., 2013; power et al ., 2013b; power et al ., 2014; satterthwaite et al ., 2013; van dijk et al ., although we modeled effects of site, the use of multisite data with the numerous factors of variability it entails remains a challenge that needs to be accepted as high - quality rs - fmri data for comparable sample sizes from a single site are not currently available . Inclusion of 126 asd participants may provide relative protection from cohort effects that are probably at work in many small - sample imaging studies . However, the need for high levels of compliance in fmri studies, even when there is no explicit task, inevitably results in inclusionary biases because low - functioning people with asd are unable to participate . The methodologically indispensable step of selecting low - motion data probably added to this issue, given that higher - functioning participants were likely to move less during scanning . Indeed, our diagnostic groups were matched for nonverbal iq, and relatively few asd participants with intellectual disability and iqs below 70 were included . The pattern of informative connections observed in our study may therefore not apply to the lower - functioning end of the autism spectrum . The pattern of these connections was highly complex, involving numerous brain regions . While some networks (somatosensory, default mode, visual) stood out, many others contributed to the classification this may be disappointing to anyone harboring the hope that the neurobiology of asd might ultimately be pinned down to simple and localizable causes . However, the literature on functional and anatomical brain anomalies in asd is fully consistent with the concept of a disorder with highly distributed, rather than localized, patterns (akshoomoff et al ., 2002; mller, 2007; philip et al ., 2012; vissers et al ., 2012; wass, 2011). The complex pattern (cf . Supplementary fig 5b) should, however, not be viewed as definitive for the asd population as a whole (in particular lower - functioning segments). Instead, it reflects a distinctive connectivity pattern for a large cohort of children and young adults with asd . Sample size, careful data denoising, and refinement of machine learning approaches in the present study thus present a distinct advance in the search for functional connectivity biomarkers of asd, but given the challenges described above, much further work remains to be done . Although fcmri is generally accepted as a powerful technique for identifying network abnormalities, the addition of complementary techniques (e.g., diffusion weighted mri for assays of anatomical connectivity; magnetoencephalography for detection of neuronal coherence in high - frequency bands) will likely enhance diagnostic classification and the detection of complex biomarkers (see zhou et al . (2014) for a recent multimodal attempt with moderate success). However, it is also important to recognize that machine learning diagnostic classification can only be performed at the level permitted by the diagnostic procedure itself, which is fundamentally imperfect . It cannot be expected that a disorder recognized as neurobiological in nature would be detected by means of purely behavioral and observational criteria currently used for diagnosis . There the contribution of machine learning to the discovery of biomarkers, which can ultimately replace behavioral criteria, is therefore important.
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Recent studies have suggested that memory might benefit from physical exercise as well [36]. These behavioral results are in accord with neuroanatomical observations showing that the volume of the human hippocampus, a key structure for the consolidation of long - term memories, increased in humans who had exercised for one year [7, 8]. Not only chronic effects of exercise interventions lasting for months up to years have been reported, but also a single bout of exercise has been shown to increase performance on a large variety of cognitive tasks [9, 10]. Compared to other cognitive domains, the number of studies on the effects of a single bout of exercise on memory is rather limited and their results are inconsistent so far with some studies showing beneficial effects, no effects, or even detrimental effects . The results seem to depend on the exercise intensity, the type of memory being tested, and the timing of exercise relative to the memory task . Models of memory consolidation emphasize the dynamic nature of memory representations by proposing two main memory stages: a label state in which memories are susceptible to enhancements or improvements and a stable state in which they are rather insensitive to any treatment [1215]. The transient label state is seen shortly after learning and after the reactivation of memory traces . Thus, memory might especially be modified by physical exercise when performed during early phases of memory consolidation . In most studies reporting beneficial effects of acute physical exercise on long - term memory, however, based on the results of these studies it is not possible to distinguish whether exercising facilitates memory encoding, consolidation, or both processes . For example, labban and etnier did not find a significant memory benefit in participants exercising after a learning session compared to a resting condition, while memory improved in participants who had exercised before learning . This finding contrasts with results of mcnerney and radvansky and segal et al . Who showed that aerobic exercise immediately after encoding enhanced memory . These studies differed with regard to the duration of the exercise intervention, the type of memory being tested, and the delay between exercising and recall . While participants in the study by segal et al . Exercised for only six minutes and had to recall their memories 60 minutes later, participants in the study by labban and etnier exercised for 30 minutes and were asked to recall their memories immediately after exercise . It might be speculated that the high arousal induced by the exercise intervention might have impaired retrieval in the study of labban and etnier . Mcnerney and radvansky, however, reported a positive effect of a short bout of high intense exercise after learning on immediate tests of procedural and declarative memory . The superior performance of the exercise group compared to a control group sustained over a one - week delay . This is in line with a further report on the effects of exercise on procedural memories . In this study, participants learned a visuomotor task either before exercising for 15 minutes, after exercising, or after the same time of relaxing . The physical exercise did not affect the acquisition of the task nor the retention one hour after the intervention . However, participants who exercised after the learning task showed better long - term retention seven days later compared to participants who exercised before learning . The authors concluded that positive effects of exercise might be maximized when performed during early stages of memory consolidation . In sum, results so far suggest that the timing of exercise relative to memory encoding or retrieval as well as exercise intensity might influence the effects of acute exercise on memory . However, only a few studies have applied exercise interventions after learning new material and, to our knowledge, none of these studies has varied exercise intensity . The brain - derived neurotrophic factor (bdnf) and cortisol bdnf is involved in the cellular and subcellular mechanisms of learning and memory, for instance, in the induction and maintenance of long - term potentiation and in structural remodeling of dendritic spines and neurogenesis [2527]. Exercise has been shown to increase the expression of bdnf in the hippocampus and perirhinal cortex, which correlated with better learning and memory [28, 29]. Bdnf is synthesized both in the central nervous system and in somatic cells outside the cns . Therefore, changes in peripheral blood serum or plasma levels have been used as an estimate of central bdnf in humans . Correlations between bdnf levels in the periphery and the brain have been reported in animals [30, 31]; however, other results questioned a direct association between central and peripheral bdnf responses . Acute physical exercise induces a reliable transient increase in peripheral bdnf [20, 3340]. There is evidence that this effect depends on the exercise intensity with an increase in bdnf only after high - intensity exercise protocols [19, 34, 37, 39]. Exercise can be considered as a physical stressor, which activates the hypothalamic - pituitary - adrenal axis . Accordingly, an elevation of cortisol has been observed after acute exercise with at least moderate intensity, but not after light exercise . It is known that glucocorticoids enhance memory consolidation; however, depending on the timing of the stressor, a stress - related increase in glucocorticoids might impair memory retrieval and reconsolidation . Thus, stress hormones released during exercise might mediate exercise related memory effects . The aim of the present study was to assess the effects of a single bout of physical exercise on memory consolidation and the underlying neuroendocrinological mechanisms . Participants learned new vocabulary before exercising for 30 minutes with either high intensity or low intensity or before a relaxing phase . Vocabulary learning was chosen as memory task because of its high ecological validity . Moreover, we expected a dose - dependent effect of physical exercise on memory consolidation with better retention of the newly learned vocabulary with increasing exercise intensity . Based on previous reports of bdnf and cortisol enhancements, especially after intense exercise protocols, increases in bdnf and cortisol were expected for the high - intensity group . Moreover, we hypothesized a direct relation between changes in bdnf and cortisol and memory consolidation . Eighty - one young and healthy university students participated in the experimental session (40 female, 41 male, mean age: 22 years, sd: 2.36, age range 1829 years, mean bmi: 21.7 kg / m, sd: 1.85). Exclusion criteria were a history of psychiatric or neurological disorders, cardiovascular diseases, smoking, medication (except contraceptives), obesity, competitive sports, and any knowledge of polish or other slavic languages (since participants were asked to learn polish vocabulary). It was conducted in accordance with the principles laid down in the declaration of helsinki (2013). The second session comprised the experimental learning phase and the exercise intervention . During this experimental session, three blood samples were collected for the bndf analysis and four saliva samples for the cortisol analysis . Moreover, participants filled in an online test of vocabulary retention 24 hours after the experimental session . During the preexperimental screening session participants completed several questionnaires to assess demographic variables, their physical activity level, and medical contraindications for a cardiovascular fitness test and physical exercise . The physical activity level was measured with the freiburg questionnaire of physical activity (fqpa). Furthermore, participants had to indicate the number of foreign languages they had learned and the number of musical instruments they played as foreign language processing and musical expertise are known to interact [4648]. We assessed the state of health during the last five years using a short questionnaire asking for specific disorders, such as vertigo, impaired vision, chest pain, tachycardia, or dyspnea, which could potentially result from cardiovascular diseases . We also checked for other chronic diseases such as diabetes, asthma, epilepsy, respiratory disorders, and disorders of the musculoskeletal system, as well as surgical interventions within the last five years, pregnancy, and acute infections . During the screening session after a delay period of 20 minutes, only the pseudowords were presented auditorily and participants had to write down the associated german words . To conform with our previous studies [1719], participants who were able to memorize more than 20 pseudowords were excluded from the main experiment . Finally, five participants were rejected since they were bilinguals and one due to smoking . To assess the participants' cardiovascular fitness and to determine an individually adjusted intensity for the exercise intervention, they performed an incremental exercise test on a cycle ergometer (conditronic 100 pv / zr - ns, dynavit, germany). Heart rate was measured continuously with a chest strap (polar s810, polar, bttelborn, germany). At the end of each step this scale has previously been shown to be a reliable and valid tool for measuring perceived exertion . The exercise test was terminated when participants reported subjective exhaustion or when their cadence was below 60 rpm for more than 10 sec . Vo2max was estimated as described in . At the beginning of the study, all participants were instructed to avoid changes in their daily life activity, in particular, their physical activity, until the second vocabulary test had been completed at the day after the main experiment . Participants were pseudorandomly assigned to one of the three experimental groups, a high - intensity physical exercise group (n = 26), a low - intensity physical exercise group (n = 27), and a relaxing group (n = 28), matched according to gender, maximum watt rate in the incremental exercise test, and their performance in the pseudoword learning task during the screening session . That is, we formed triplets which were controlled for the above - mentioned parameters and assigned them randomly to the respective experimental groups . An overview of the experimental procedure with the timing of the learning phase, memory tests, exercise intervention, and the blood and saliva collections is depicted in figure 1 . The experimental session started between 2:30 pm and 5:00 pm . During the learning phase, participants listened to 20 polish - german word pairs (10 nouns and 10 verbs) presented via headphones (philips shp 1900) while sitting on a desk chair . Participants were told that their memory for the vocabulary would be tested on the same day and the day after . Two blocks were run with each polish - german pair presented once in each block . Initially, all stimuli were normalized to an intensity level of 75 db(a). At the beginning of the experimental session polish and german items were spoken by two females, native speakers of polish and german, respectively . The stimulus onset asynchrony (soa) of polish - german vocabulary pairs was 2 sec within a vocabulary pair and 6 sec between successive vocabulary pairs . During this pause, participants were asked to repeat the just heard vocabulary pair aloud . After they encoded the polish - german vocabulary, participants of the physical exercise groups were asked to exercise on a cycling ergometer (conditronic 100 pv / zr - ns, dynavit, germany) for 30 minutes . The heart rate was constantly monitored with a chest strap (polar s810, polar, bttelborn, germany). For the low - intensity group, the intensity was set to <57% of their individual maximal heart rate (hr max) as determined during the exercise test (see preexperimental screening session), while it was set to 80% of their hr max 5 beats / min for the high - intensity group . This corresponded to very light and vigorous physical exercise according to . During the initial five minutes of the physical intervention, resistance was increased until participants' heart rate reached their prescribed target heart rate . Additionally, participants had to indicate their perceived exertion level on the borg scale after 15 min and 30 minutes, respectively . Members of the relaxing group were resting in a canvas chair for thirty minutes after they had learned the new vocabulary . After the intervention, all participants watched a silent movie called shaun the sheep - abracadabra for 20 minutes . During this period arousal they listened to 40 polish words (20 old items and 20 new items) and had to respond, if possible, with the german translation using a standard computer keyboard . The reason for adding new items in the recall phase is that we have planned to measure additional eeg during the recall phase in a follow - up experiment to study the old / new effect . Response time for the vocabulary test was limited to 8 s for each item . At the end of the experimental session, all participants were asked to indicate their motivation and perceived difficulty to learn the new vocabulary, their perceived exertion during exercising, their current daily stress level and workload at the university, and the quality of their last night's sleep . Answers were given on a 5-point likert scale ranging from low to high . Furthermore, we asked the participants about their caffeine and alcohol consumption and the hours they slept during the last 24 hours . Twenty - four hours after the learning session participants took part in an additional customized online vocabulary test . The access was temporally limited to make sure that participants adhered to their individual test time (1 hour). Participants were asked to listen again to the polish words encoded during the experimental learning session and were asked to enter the correct german counterpart . For bdnf analysis, 4.5 ml of venous blood from the antecubital vein was collected with a clotted blood tube at three time points . The second blood sample was collected immediately after the learning phase and the third one was taken immediately after the intervention (exercising or relaxing). After the clotting period, samples were centrifuged for 10 minutes with 4800 rounds per minute using the heraeus labofuge 200 (thermo fisher scientific, germany). Serum was immediately pipetted into separate safeseal microtubes (sarstedt, nrnberg, germany). Samples were stored at 23c for a mean of 13.5 days and then transferred to a 80c freezer until the analysis started . Bdnf levels in serum were measured using the quantikine human bdnf immunoassay from r&d systems (wiesbaden, germany) with intra- and interassay coefficients of variation in the range between 8.8 and 11.4 for values between 7.24 and 41.6 ng / ml . The minimum detectable bdnf dose was less than 20 pg / ml, according to the manufacturer's information . Bdnf analysis was performed by the institute of laboratory medicine, clinical chemistry and molecular diagnostics in leipzig . The first three samples were collected immediately before the blood sampling, that is, at the beginning of the experimental session, after the learning phase, and after exercising / relaxing . The fourth saliva sample was taken 20 minutes after exercising, that is, after participants had watched the silent movie . (sarstedt ag & co., nmbrecht) collection devices were used for collecting saliva . They consist of a cotton swab in a suspended insert which itself is placed in a centrifuge vessel . Participants were instructed to gently chew on the cotton swabs for at least one minute . Afterwards they return the saturated swab to the suspended insert without touching it . To reduce errors, participants were not allowed to eat or drink anything else than water for half an hour before the testing session started and during the session . Salivary samples were stored at 23c until the biochemical analysis was conducted, which was performed by the dresden lab service gmbh . The concentration of free salivary cortisol was analyzed using a luminescence immunoassay (ibl, hamburg, germany) with intra- and interassay precision of 4.5% and 4.3%, respectively . Changes in memory scores, bdnf, and cortisol after the exercise intervention were compared between experimental groups by a univariate analysis of variance (anova) with the factors group (high - intensity exercise versus low - intensity exercise versus relaxing) and time (day 1 versus day 2 for the memory score, t0, t1, and t2 for bdnf, and t0, t1, t2, and t3 for cortisol). As we had a specific hypothesis about the direction of the exercise effect, differences between groups were further analyzed with planned contrasts or a linear trend analysis whenever the main effect of group or the group time interaction was at least marginally significant (p <0.1). In the contrast analyses, results of the relaxing group were compared to those of the high - intensity group (first contrast) and to those of the low - intensity exercise group (second contrast). For the trend analysis, we expected a linear trend with increasing scores of the dependent variables with increasing exercise intensity (1: relaxing, 0: low intensity, and 1: high intensity). Possible correlations between vocabulary retention (vocabulary score day 2 minus day 1) and changes in bdnf (t2 minus t0) and cortisol (t2 minus t0, t3 minus t0) after exercising / relaxing were explored by nonparametric correlations using kendall's tau because the data were not normally distributed . Groups were pseudorandomly assigned to a high - intensity exercise group, a low - intensity exercise group, or a relaxing group . Groups did not differ with regard to age, sex, body mass index, daily physical activities, physical fitness, the number of foreign languages learned, number of participants playing a musical instrument, or memory scores during preexperimental screening (see table 1). The number of women taking oral contraceptives differed between groups, x(2) = 7.10, p = 0.029, and was highest in the high - intensity exercise group . As contraceptives might influence memory performance and neuroendocrinological parameters [5456], we ran additional analyses excluding women without contraceptives . The pattern of results remained the same as in the complete sample suggesting that differences in the number of women taking contraceptives did not account for the group differences reported in the following (see supplementary material available online at http://dx.doi.org/10.1155/2016/6860573). The number of women not taking contraceptives was too small in this sample to analyze their data separately or to analyze the interaction between the use of contraceptives and exercise . As expected, the heart rate during the experimental intervention differed between groups, f(2,75) = 533.18, p <0.001, with a significantly higher heart rate for the exercise groups compared to the relaxing group (planned contrast t(75) = 27.00, p <0.001) and a significant difference between the high - intensity group and the low - intensity group (planned contrast t(75) = 18.08, p <0.001). The latter additionally differed in the rating of the perceived exhaustion after exercising, t(51) = 13.03, p <0.001 . One participant assigned to the relaxing group was excluded from the analyses of memory scores, as he did not recall any word correctly, neither at day one nor at day two . Exercising after learning did not enhance the absolute number of recalled vocabulary, main effect of group f(2, 77) = 1.99, p = 0.144, = 0.049 (figure 2(a)). The time group interaction was marginally significant, f(2, 77) = 2.84, p = 0.064, = 0.069, indicating that group differences in vocabulary retention depended on the time of testing . The contrast analysis for the vocabulary score at day one revealed that the relaxing group initially recalled more words than both the high - intensity exercise group, t(77) = 2.10, p = 0.039, and the low - intensity exercise group, t(77) = 2.12, p = 0.038 . Planned contrasts for day two did not reveal any significant difference between the relaxing group and the exercise groups, all p> 0.16 . To explore the changes in vocabulary retention after 24 hours, the difference score recalled words day two minus recalled words day one was compared between the three groups (figure 2(b)). There was a significant linear trend, f(1, 77) = 5.51, p = 0.022, indicating that vocabulary retention increased proportionately with increasing exercise level . Moreover, planned contrasts for these difference scores showed a significant lower loss of memory for the high - intensity exercise group compared to the relaxing group, t(77) = 2.35, p = 0.022 . Blood samples to assess bdnf serum level were taken at baseline (t0), after learning (t1), and after exercising / relaxing (t2), respectively . Furthermore, data of two participants with bdnf values more than three standard deviations above the sample mean at at least one time point were excluded . Thus, the bdnf analyses were based on n = 77 participants (n = 26 high - intensity exercise group, n = 23 low - intensity exercise group, and n = 28 relaxing group). Serum bdnf only increased after exercising in the high - intensity exercise group, time group f(4,148) = 17.99, p <0.001, = 0.327 (figure 3(a)). The analysis per time point showed that groups did not differ with respect to bdnf at t0 and at t1 (all f <0.50, all p> 0.60) but that there was a significant effect of group after exercising at t2, f(2,74) = 8.68, p <0.001 . Planned contrasts for t2 confirmed a significant difference between the high - intensity exercise group and the relaxing group, t(74) = 3.58, p = 0.001, while the low - intensity exercise group and the relaxing group did not differ, t(74) = 0.22, p = 0.824 . When comparing the change from baseline to postintervention (t2 minus t0), the linear trend indicating an increase in bdnf with increasing exercise intensity was significant, f(1,74) = 40.08, p <0.001 . As seen in figure 3(b), this effect was mainly driven by the large increase in the high - intensity exercise group . Saliva samples to assess cortisol levels were taken at baseline (t0), after learning (t1), immediately after exercising / relaxing (t2), respectively, and again 20 minutes after t2 (t3). Furthermore, five participants with cortisol levels more than three standard deviations above the sample mean at at least one time point were excluded from the analysis . Thus, cortisol analyses were based on n = 74 participants (n = 26 high - intensity exercise group, n = 23 low - intensity exercise group, and n = 25 relaxing group). Cortisol levels changed differentially across time for the three experimental groups, time group f(6,213) = 6.53, p <0.001, = 0.155 (figure 4(a)). Comparisons between groups for each time point showed no significant group differences at t0, t1, and t2 (all f <2.35, all p> 0.10). Twenty minutes after exercising (t3), however, there was a significant main effect of group f(2,71) = 3.16, p = 0.049, with the high - intensity group showing a larger cortisol level compared to the resting group (contrast analysis high - intensity group versus relaxing group t(71) = 2.41, p = 0.019; low - intense exercise group versus relaxing group t(71) = 0.57, p = 0.569). Furthermore, the change in cortisol levels from t0 to t3 increased with increasing exercise intensity (linear trend analysis for the difference score of cortisol t3 minus t1: f(1,71) = 15.34, p <0.001, figure 4(b)). As for bdnf, however, this was mainly driven by the increase in the high - intensity group . When analyzing data of all participants, there was a significant positive correlation between the increase in bdnf after exercise / relaxing (bdnf t2 minus t0) and vocabulary retention (day 2 minus day 1), = 0.17, p = 0.046 . However, this correlation was not significant when analyzing the high - intensity group only, = 0.11, p = 0.463 . There was no positive relationship between the increase in bdnf after exercising / relaxing and the absolute number of recalled words, neither at day one nor at day two (<0.15, p> 0.05). An analysis including all participants revealed a significant positive correlation between the increase in cortisol immediately after exercise / relaxation (cortisol t2 minus t0) and vocabulary retention (day 2 minus day 1), = 0.19, p = 0.027, and a marginally significant positive correlation between the increase in cortisol 20 minutes after exercise / relaxation (cortisol t3 minus t0) and vocabulary retention (day 2 minus day 1), = 0.15, p = 0.076 . There was no positive relationship between these variables when analyzing the high - intensity group only, = 0.04, p = 0.819 for cortisol t2 minus t0 and = 0.17, p = 0.271 for cortisol t3 minus t0 . The increase in cortisol after exercising / relaxing did not correlate with the absolute number of recalled words (<0.11, p> 0.22). The larger the increase in bdnf immediately after exercising (bdnf t2 minus t0), the larger the increase in cortisol 20 min later (cortisol t3 minus t0), = 0.23, p = 0.005 . The correlation between bdnf and cortisol increase was also significant when analyzing the high - intensity exercise group only, = 0.32, p = 0.023 . The aim of the present study was to assess the effects of a single bout of exercise on memory consolidation and the release of the neuroendocrinological parameters bdnf and cortisol . Exercise after learning did not enhance the recall of newly learned vocabulary at the day of learning and exercising . On the contrary, at that time point, participants of a relaxing group recalled more words than participants of a high - intensity exercise group and participants of a low - intensity exercise group . However, participants who engaged in high - intensity exercise for 30 minutes after learning showed better memory consolidation compared to the relaxing group assessed 24 hours after the learning session; that is, they retained more of the initially learned words . Moreover, high - intensity physical exercise led to a significant increase in peripheral bdnf and saliva cortisol . In contrast to most previous studies assessing the effects of acute exercise on memory [16, 19, 20], participants in the present study did not exercise before or during the learning phase but after learning . Thus, we were able to study the effects of physical exercise during early phases of memory consolidation while holding learning conditions constant across groups . Experimental interventions to enhance (e.g., strychnine administration) or impair memories (e.g., electroconvulsive shocks, administration of protein synthesis inhibitors, and behavioral distractors) have an impact on later recall especially when administered within a short time window after learning when synaptic consolidation processes take place [12, 57]. In parallel, or as a consequence, mechanisms of system consolidation are initiated which take days up to years and are important to stabilize long - term memories . Therefore, one might speculate that physical exercise enhances long - term memory by facilitating early stages of memory consolidation . The timing of the observed behavioral effects fits well with this explanation: 20 minutes after the exercise intervention, there was no beneficial effect of exercise on memory . On the contrary, at that time point, participants of the relaxing group recalled more words than participants of the exercise groups . However, participants in the high - intensity group did not show any forgetting of the learned words after 24 hours while there was a significant decrease in the relaxing group . Thus, the immediate memory was better in the relaxing group, but the high - intensity exercise group showed better long - term consolidation of the vocabulary . The difference between the immediate memory test and the 24-hour test for the low - intensity group was between the same difference for the high - intensity group and the relaxing group, suggesting a dose - response relationship between exercise intensity and memory consolidation . The results are in line with reports of previous studies showing that exercising after learning did not improve immediate memory, but improvements were only seen after a delay of at least 24 hours . Argued that memory tests administered too early after encoding, when memory traces are still undergoing consolidation, are not able to detect exercise induced memory gains and might even interrupt the consolidation process . As the timing of memory tests relative to the exercise interventions differ between studies, this variability might explain inconsistent results in previous studies . One reason for a better memory of the relaxing group at day one, compared to the exercising groups, might be exercise induced arousal and exhaustion . Null effects as well as detrimental effects of physical exercise on cognitive performance were mostly seen in studies with high - intensity exercise protocols, leading to dehydration and exhaustion [5961]. In the present study, however, participants had the opportunity to drink water during and after the exercise session, making it unlikely that dehydration impaired cognitive processes . Moreover, the intensity of the exercise was individually adjusted to approximately 80% of their maximal heart rate and below 60% for the low - intensity exercise group . Both the high- and low - intensity groups showed worse immediate memory compared to the relaxing group, without any difference between the two making it very unlikely that exhaustion could account for the immediate memory effect . While the exercise groups cycled on a stationary bicycle, the relaxing group sat quietly in a chair . Thus, it is possible that they silently rehearsed the newly learned vocabulary more than the exercise groups which might have improved their immediate memory . However, the relaxing group showed more forgetting after 24 hours compared to the exercise group, suggesting that immediate memory and long - term consolidation were differentially affected by the experimental interventions . Further studies should introduce a task that interferes with memory rehearsal to rule out these effects on memory . Participants were not asked to recall the vocabulary immediately after learning to avoid any interference effects before exercising . Thus, it is possible that groups differed in the number of words they encoded successfully before exercising . Participants did a word - pseudo - word learning task in the preexperimental screening session which was very similar to the learning task in the main experiment . Groups did not differ with regard to their learning success in this preexperimental task suggesting that there were no baseline differences in learning abilities between groups . However, future studies should include an immediate memory test to control for possible baseline differences in learning success . Furthermore, we argue that in future studies a within - subject design would be more appropriate to assess the effect of exercise on memory to increase the statistical power compared to the current between - subject design . Given the low performance of subjects we expect to find even clearer memory effects by applying a within - subject design . Serum bdnf was significantly increased after high - intensity exercise, but not after low - intensity exercise . This is in agreement with previous studies reporting an increase of bdnf after acute exercise for high - intensity exercise protocols only [34, 37, 39, 62]. With regard to memory, bdnf is essential for the formation and storage of long - term memories . For instance, results in animals demonstrated that interfering with bdnf expression in the early phase of memory consolidation selectively impairs long - term memory, while recognition memory at the day of acquisition was unaffected . Accordingly, an intracerebroventricular injection of bdnf in rats was found to enhance hippocampus - dependent learning . As acute exercise in humans leads to a transient increase in bdnf, it might be speculated that bdnf mediates the beneficial effects of acute exercise on memory processes . Data on the relationship between an acute exercise induced increase in bdnf and cognitive variables in humans are rare so far . Changes in cognitive tasks like executive functions and attention after acute exercise were found to be unrelated to changes in bdnf . With regard to memory, winter et al . Found a positive correlation between the increase in bdnf after exercise and immediate learning success in a declarative memory task, but no relationship to long - term memory . Reported an increase in bdnf after acute exercise and an increase in a hippocampus - dependent declarative memory task; however, they did not show a correlation between these two variables . For procedural memory, skriver et al . Showed a positive correlation between skill retention up to seven days after learning and serum bdnf, but no relationship to skill acquisition . In the present study, there was a positive correlation between memory consolidation and bdnf increase when analyzing data of all participants . However, this relationship could not be confirmed when only participants of the high - intensity group were included into the analysis . This might be due to a lack of power and smaller variances in this sample . Furthermore, the less consistent findings on the relationship between an exercise induced increase in bdnf and memory in humans, compared to animal studies, might be due to methodological differences in bdnf assessment . In animals, the local bdnf release in the hippocampus and neocortex is measured, while in human studies bdnf is assessed in the peripheral blood serum . It is disputed whether an increase in central bdnf is accompanied by an increase in serum bdnf . While some results provide evidence for a strong correlation between cortical and serum bdnf, other data speak against such a relationship . Nevertheless, bdnf in the serum does not reflect a local measurement of bdnf in specific subregions of the central nervous system, making it harder to detect a correlation with cognitive variables . Thus, a direct link between an acute increase in bdnf and long - term memory consolidation in humans has still to be established . Psychosocial stress, as well as the application of moderate doses of corticosterone immediately after learning, are known to facilitate memory consolidation [65, 66]. Therefore, it has been hypothesized that an exercise induced increase in cortisol might contribute to beneficial effects of a single bout of exercise on memory performance . Saliva cortisol levels were significantly elevated after high - intensity physical exercise in the present study . As in previous studies, we did not observe a cortisol response after low - intensity physical exercise [19, 68]. The increase in cortisol after exercise correlated positively with vocabulary retention after 24 hours suggesting that glucocorticoids release after learning might contribute to the consolidation of newly learned vocabulary . However, the relationship disappeared when only participants of the high - intensity group were considered . Reasons for the lack of a substantial relationship between cortisol and memory could be due to the low variance in this subgroup and the rather small increase in cortisol after exercising in the present study . An inverted u - shaped dose - response function has been described for glucocorticoid effects on memory consolidation . Studies using the cold water test or an aversive psychosocial situation to induce stress reported increases in saliva cortisol around 30% up to 100% [7073]. The mean increase in cortisol in the present study was 17% in the high - intensity group and, therefore, might have been too low to induce beneficial effects on memory . Alternatively, the lack of a substantial relationship between neuroendocrinological parameters and memory in the present study might suggest that other mechanisms except an increase in bdnf and cortisol mediate exercise induced changes in cognitive variables . For instance, an exercise induced increase in dopamine and norepinephrine has been linked to better memory performance [20, 22, 64]. Furthermore, acute exercise induces alterations in cerebral blood flow, glucose and lactate levels which modulate learning and memory [64, 7476]. The present results provide evidence that high - intensity but not low - intensity physical exercise led to less forgetting of newly learned vocabulary compared to a relaxing group . Furthermore, high - intensity exercise increased the release of bdnf and cortisol in humans . However, it remains an open question whether bdnf and cortisol are mediators of exercise induced benefits on memory . Acute exercise protocols varying the timing of exercise relative to memory encoding and retrieval and the assessment of additional neuroendocrinological parameters might be a promising experimental approach for future studies exploring the factors underlying memory consolidation.
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Committing a crime and its legal penalties in mental patients has always been a controversial issue among psychiatrists and lawyers . Commission of crime and hostility and their forensic consequences can worsen patient's condition and disturb his family and society and even make legal engagements for the psychiatrist . Based on previous studies, these patients have a high risk for non - violent crimes and have a very higher risk for violent crimes; and being single, unemployed, having active symptoms of psychosis, and not accepting the treatment could boost the condition. [14] however, recently forensic psychiatrists have wondered whether the nature and the course of schizophrenia would increase the prevalence and severity of crimes among these patients or its comorbidity with antisocial and aggressive traits . These include antisocial personality traits and determining the probability of hostility based on psychopathy checklist - revise (pcl - r); its sensitivity has been confirmed in many studies and currently is being used widely . In a study by hodgins et al . In 1996 on schizophrenic criminals, it was revealed that due to reasons of committing a crime and their association with antisocial personality disorder, patients with schizophrenia receive legal penalties for 28.6% of the crimes they commit; this is a considerable number . In that study, the prevalence of antisocial personality disorder in criminal patients with schizophrenia was 62%, which was significantly higher than its prevalence among non - criminal patients (23%). Conducted a study in 2001 about the age prevalence of crime in patients with schizophrenia . The study revealed that the prevalence of antisocial personality disorder and score of pcl - r were significantly higher in patients who had committed crime under 18 years of age . In a study by moran et al . In 2004, it was mentioned that the first hospitalized male patients with schizophrenia and antisocial personality disorder had longer history of antisocial behaviors including non - violent crimes, drug abuse, and impulsive disorder, which all increase the risk of hostility . Hodgins et al . In 2005 studied the outcomes of conduct disorder in 248 male patients, aged about 39 years old, with schizophrenia or schizoaffective disorder . Results showed that these patients had higher risk for committing crime than the normal population . Evidence showed a relation between personality traits and committing crime among patients with schizophrenia . In this regard, fresan et al . In 2007 studied 102 patients with schizophrenia . Results showed that high novelty seeking in temperament and low cooperativeness would increase the risk of hostility . In 2006 conducted a study on 35 patients with schizophrenia in which 19 had violent and 16 had non - violent behaviors . They used pcl - r for evaluating psychopathic characteristics and brief psychopathy rating scale (bprs) for evaluating symptoms . In violent patients, 3 (16%) had a pcl - r score of> 30 . High hostility rate in bprs and behavioral part (factor 2) in pcl - r can be significant predictors of violent behaviors in male patients with schizophrenia . High psychopathic scores showed the existence of violence . In men with high score of psychopathy, even treating the symptoms of the disease cannot eliminate the probability of violence . In a study in 2008, swanson et al . Investigated the relation between antisocial behaviors in childhood and aggression in adults . Aggression was more common among those who had history of conduct disorder; this aggression was associated with drug abuse . In the group with no history of conduct disorder, positive signs of psychosis were related with violence . In general, they had two reasons for violence in patients with schizophrenia; one is the history of conduct disorder and antisocial personality disorder and the other is the psychopathology of acute schizophrenia . In a study by maghsoodloo et al . In 2002 on criminal mental patients, it was accidentally revealed that most of the schizophrenic criminal patients had antisocial personality disorder . The aim of this study was to determine whether the existence of antisocial personality disorder and history of conduct disorder among patients with schizophrenia increase the risk for violence . In this prospective case - control study, characteristics of antisocial personality disorder, history of conduct disorder, and the score of pcl - r were compared between criminal and non - criminal patients with schizophrenia . Case group included patients who had committed a crime and were referred to the court . Being referred to forensic psychiatry unit and were diagnosed with schizophrenia based on diagnostic and statistical manual of mental disorders dsm - iv - tr standards.being at least 18 years old.not having seizure disorder and mental retardation.not having mood disorder with psychotic feature, organic psychosis, or psychotic disorder (not any other specified by reviewing the medical history); in doubtful cases, organic evaluations like eeg and/or brain computer tomography were used . Being referred to forensic psychiatry unit and were diagnosed with schizophrenia based on diagnostic and statistical manual of mental disorders dsm - iv - tr standards . Not having mood disorder with psychotic feature, organic psychosis, or psychotic disorder (not any other specified by reviewing the medical history); in doubtful cases, organic evaluations like eeg and/or brain computer tomography were used . After reviewing medical and legal history of patients, those who were selected as control but had criminal records all the patients who were referred to forensic psychiatry unit of forensic medicine center in isfahan between 2006 and 2009 were selected . After semi - structure clinical interview, studying evidences and medical documents of patients, if the unit's psychiatrist diagnosed the patient with schizophrenia, the patient would be included in the study . If the patient was referred to the unit after committing a crime, he would enter the case group; otherwise, he would enter the control group . This was continued until 30 patients were assigned to each group and informed written consent was taken from all patients and controls . Then, during structured clinical interviews, symptoms of antisocial personality disorder and conduct disorder were asked from the patients and their escorts, preferably their parents or older siblings, based on dsm - iv - tr criteria, and then diagnosis of these disorders was made . This questionnaire included 20 items; each could get a score of 0 - 2: 0 meant never, 1 meant sometimes, and 2 meant most of the times . Questions were divided into two groups of factor 1 and factor 2; factor 1 evaluated interpersonal and emotional aspects and factor 2 evaluated lifestyle and social deviations . All of the pcl - r's items were asked openly from the patients by a psychiatrist (executer and the main colleague of the study who was totally familiar with the questionnaire) and then they were confirmed by one of the patient's escorts who had been with him all the time; then, the forensic psychiatrist chose one of the scores from 0 to 2 based on the answers . Since it was the first time that this questionnaire was used in iran, its standardization was carried out as follows: the accuracy of questionnaire's translation was evaluated by five psychiatrists using back translation method . A couple of psychiatrists studied the content of the questionnaire to evaluate its content validity . To evaluate its reliability, a pilot study was conducted on 40 patients with schizophrenia; it led to some changes on item 1 of the pcl - r questionnaire . Then, the cronbach's of 87% was reached and the study was continued . Also, another questionnaire was completed for each patient, including demographic data, criminal records of the patient and his family, starting time of the crimes according to the onset of the disease, type of the committed crimes, alcohol and drug abuse, age at the onset of crimes, age at the onset of the disease, hospitalization experience, total hospital admission, treatment adherence, etc . The research protocol was approved by the ethics committee of isfahan university of medical sciences (research project number: 184090). The research protocol was approved by the research and ethics committee of legal medicine organization of iran . Chi - square test was used to evaluate the difference of the frequency distribution of antisocial personality disorder and conduct disorder between the schizophrenic patients with crime and those with no crime . Student's t - test was used to compare the mean score of factor 1 and 2 between criminal and non - criminal patients . Also, to compare criminal and non - criminal patients regarding the score of 25 or more in pcl - r questionnaire, chi - square test was used . Being referred to forensic psychiatry unit and were diagnosed with schizophrenia based on diagnostic and statistical manual of mental disorders dsm - iv - tr standards.being at least 18 years old.not having seizure disorder and mental retardation.not having mood disorder with psychotic feature, organic psychosis, or psychotic disorder (not any other specified by reviewing the medical history); in doubtful cases, organic evaluations like eeg and/or brain computer tomography were used . Being referred to forensic psychiatry unit and were diagnosed with schizophrenia based on diagnostic and statistical manual of mental disorders dsm - iv - tr standards . Not having mood disorder with psychotic feature, organic psychosis, or psychotic disorder (not any other specified by reviewing the medical history); in doubtful cases, organic evaluations like eeg and/or brain computer tomography were used . After reviewing medical and legal history of patients, those who were selected as control but had criminal records were excluded from the study . All the patients who were referred to forensic psychiatry unit of forensic medicine center in isfahan between 2006 and 2009 were selected . After semi - structure clinical interview, studying evidences and medical documents of patients, if the unit's psychiatrist diagnosed the patient with schizophrenia, the patient would be included in the study . If the patient was referred to the unit after committing a crime, he would enter the case group; otherwise, he would enter the control group . This was continued until 30 patients were assigned to each group and informed written consent was taken from all patients and controls . Then, during structured clinical interviews, symptoms of antisocial personality disorder and conduct disorder were asked from the patients and their escorts, preferably their parents or older siblings, based on dsm - iv - tr criteria, and then diagnosis of these disorders was made . This questionnaire included 20 items; each could get a score of 0 - 2: 0 meant never, 1 meant sometimes, and 2 meant most of the times . Questions were divided into two groups of factor 1 and factor 2; factor 1 evaluated interpersonal and emotional aspects and factor 2 evaluated lifestyle and social deviations . All of the pcl - r's items were asked openly from the patients by a psychiatrist (executer and the main colleague of the study who was totally familiar with the questionnaire) and then they were confirmed by one of the patient's escorts who had been with him all the time; then, the forensic psychiatrist chose one of the scores from 0 to 2 based on the answers . Since it was the first time that this questionnaire was used in iran, its standardization was carried out as follows: the accuracy of questionnaire's translation was evaluated by five psychiatrists using back translation method . A couple of psychiatrists studied the content of the questionnaire to evaluate its content validity . To evaluate its reliability, a pilot study was conducted on 40 patients with schizophrenia; it led to some changes on item 1 of the pcl - r questionnaire . Then, the cronbach's of 87% was reached and the study was continued . Also, another questionnaire was completed for each patient, including demographic data, criminal records of the patient and his family, starting time of the crimes according to the onset of the disease, type of the committed crimes, alcohol and drug abuse, age at the onset of crimes, age at the onset of the disease, hospitalization experience, total hospital admission, treatment adherence, etc . The research protocol was approved by the ethics committee of isfahan university of medical sciences (research project number: 184090). The research protocol was approved by the research and ethics committee of legal medicine organization of iran . Chi - square test was used to evaluate the difference of the frequency distribution of antisocial personality disorder and conduct disorder between the schizophrenic patients with crime and those with no crime . Student's t - test was used to compare the mean score of factor 1 and 2 between criminal and non - criminal patients . Also, to compare criminal and non - criminal patients regarding the score of 25 or more in pcl - r questionnaire, chi - square test was used . In this study, 30 criminal and 30 non - criminal patients with schizophrenia who were referred to the forensic center were investigated . Mean age of the case group was 34 years and mean age of the control group was 41 years . 70% in the case group and 73.2% in the control group had high school diploma . 86.7% of the case group and 76.7% of the control group were unemployed . In both groups, being single had the highest frequency (66.7% in the case group and 53.3% in the control group). Frequency of committing a crime according to the onset of the disease revealed that 73.3% of criminal patients (22 cases) had committed their first crime after the onset of the disease, 23.2% (7 cases) before the onset, and 3.3% (1 case) at the onset . Eleven criminal patients (36%) had committed a severe crime, mostly murder, 50% had committed tolerable crimes, and 13% had committed mild crimes . Frequency of committing a crime among criminal patients showed a repetitive pattern; some had even 10 criminal records . Of the 30 criminal patients in the case group, 26 had a history of conduct disorder while this number was 9 in the control group; the difference was significant [table 1]. Twenty - two patients of the case group had antisocial personality disorder traits, while this number was two in the control group, and the difference was also significant [table 1]. Frequency distribution of history of conduct disorder, antisocial personality disorder, and high score of pcl - r between criminal and non - criminal schizophrenic patients 40% of criminal patients had a score of 25 or more on pcl - r questionnaire . This was significantly higher than that of the control group in which no one had a score of 25 or more . This also meant that the probability of committing a crime again existed in 40% of cases [table 1]. The mean of this score in the case group was not only higher than the total mean score (which was 8), but also was significantly higher than the mean score of the control group [table 2]., it was higher than the total mean (which was 10) and also significantly higher than the mean score of the control group (which was 7.3) [table 2]. Comparing the mean scores of factor 1 (interpersonal and emotional aspects) and factor 2 between the case and control groups the present study was conducted on 30 criminal and 30 non - criminal patients with schizophrenia to evaluate the relation between committing a crime and the history of conduct disorder, antisocial personality disorder, and the score of pcl - r . The mean age of the criminal group was 34 years and that of non - criminal group was 41.1 years . This result was in agreement with the results of another study about prevalence and severity of crimes in mental patients; in that study, criminal patients with schizophrenia were mostly between 25 and 40 years old . About committing murder by mental patients, the most common age of committing a crime, especially violent crimes, in schizophrenic patients with axis ii disorder (all types of cluster b personality disorders) was 31.64 years, which was in agreement with the results of the present study . Most of the patients in both groups were single (66.7% in the case group and 53.2% in the control group), unemployed (86.7% in the case group and 76.7% in the control group), and did not have high school diploma (70% in the case group and 73.3% in the control group); these can be justified by the effect of this disease on social, individual, and professional operation of the patient . Prevalence of drug abuse was 66.7% in the criminal group and 36.7% in the non - criminal group . This was also mentioned in the results of erkiran et al . That drug abuse would increase the risk of violent behavior in patients with schizophrenia . Also, valenca et al . Revealed that there is a significant relation between homicide and mental disorders, especially disorders related to drugs, alcohol, and personality traits . Another important issue was to know in which period, the prevalence of committing a crime was higher in patients with schizophrenia . This topic is important because sometimes, after committing a crime, criminals try to mimic psychosis symptoms to flee from legal punishments . But this study revealed that in 73.3% of cases, the crime occurred after the onset of the disease, and the criminals had hospitalization and treatment records before committing a crime; only in one case, the diagnosis was made right after committing a crime . They mentioned that 77.8% of the patients who were released from legal units had previous records of hospitalization in psychiatric units . 's study that the disease of 56% of criminal schizophrenic patients was diagnosed 12 months before they committed a crime; the time interval was 1 month for murdering . 28% had no previous contact with psychiatrist (it was 23% in the present study). One aim of this study was to investigate the relation between committing a crime and conduct disorder . In this study, 6.7% of criminal patients had history of conduct disorder in their childhood, which was significantly higher than 30% in the control group (p <0.001). In a study by swanson et al . About the relation between antisocial traits in childhood and violence in adulthood in patients with schizophrenia using national institute of mental health (nimh) clinical antipsychotic trials of intervention effectiveness (catie) criteria, it was revealed that the prevalence of violence was higher among those patients who had a history of conduct disorder . History of conduct disorder was accompanied by drug abuse and not having a history of conduct disorder was accompanied by positive symptoms of psychosis . They mentioned conduct disorder as one of the most important factors of showing violence in patients with schizophrenia.- hodgins et al . Also confirmed that the association of conduct disorder with major psychiatric disorders would increase the risk of aggressive behavior and violent crimes . Also, in another study by hodgins et al . Which investigated the outcomes of conduct disorder in 248 male patients with schizophrenia, it is mentioned that conduct disorder increases the risk of committing violent and non - violent crimes, but it has no relation with murdering . Also, there was a relationship between conduct disorder and early onset of schizophrenia, the first hospitalization, and its duration; in general, it is considered as a parallel comorbidity with schizophrenia . Another controversial matter is to know whether schizophrenia increases the prevalence of committing a crime by its nature or its simultaneous traits like its comorbidity with antisocial personality disorder would lead to this increase . In the present study, 22 patients (73.3%) of the criminal group had antisocial personality traits, while only 3 patients (10%) in the control group showed these traits; the difference was significant (p <0.001). Schug et al . In a study showed that comorbidity of antisocial personality disorder with schizophrenia personality disorder's spectrum would increase the criminal behaviors significantly . Studied 125 schizophrenic murderers and mentioned that positive symptoms of psychosis had no significant relation with severe hostile behaviors and other reasons for hostility should be studied here, besides clinical reasons, like different comorbidities . Fresan et al . In a study on 102 patients with schizophrenia showed a significant relation between personality traits and committing a crime . Their results revealed that high novelty seeking in temperament and low cooperativeness in personality traits increased the risk for violent behaviors in the patients . Van damme et al . In their study investigated the relation between personality traits and committing a crime in four groups of patients with schizophrenia using cloninger's temperament and character questionnaire . These groups were as follows: patients with schizophrenia who committed a murder.patients with schizophrenia who had no history of hostile behaviors.paranoid murderers.murderer prisoners who did not provide any psychiatric records . Patients with schizophrenia who committed a murder . Patients with schizophrenia who had no history of hostile behaviors . Results showed that the score of self - transcendence was significantly higher in murderer schizophrenic patients; this could mean that aggressive and violent behaviors of these patients could be predicted . Moran and hodgins mentioned in a study that the prevalence of antisocial personality disorder is higher in patients with schizophrenia than in normal population . In their study on 232 male schizophrenic patients, 75% who had committed a crime those patients who had longer history of antisocial behaviors including non - violent crimes, drug abuse, and impulsive disorder, all had increased risk for violence . These results were in line with the results of the present study . One of the important objectives of this study was to evaluate the score of hare psychopathy checklist in patients with schizophrenia . This checklist is used to predict re - offense risk and the probability of criminals rehabilitation . In the present study, 12 criminal patients with schizophrenia (40%) had pcl - r score of 25 or more, which is considered as a psychopathic characteristic; no one in the control group had a score of 25 or more and the difference was significant (p <0.001). Also, comparing the mean score of factor 1, which evaluates individual and emotional aspects of pcl - r, it was significantly higher in the case group than in the control group (8.9 vs. 4.8, p <0.001). Moreover, the mean score of factor 2, which evaluates lifestyle and social deviations of pcl - r, in the case group was significantly higher than in the control group (11.8 vs. 7.2, p <0.001). In a study by carmen et al . On 120 male prisoners, it was revealed that the high score in factor 2 was significantly related to antisocial personality disorder and conduct disorder, while the score of factor 1 was just significantly related to antisocial personality disorder . High score of factor 2 means being impulsive and irresponsible, and having no long - term goal . In a study on 35 hospitalized male patients with schizophrenia observed that the score of pcl - r was significantly higher in those who had committed violent crimes . They also revealed that high score of factor 2 was a significant predictor of violent behaviors in male patients with schizophrenia . In male patients with high score of psychopathy, sometimes, even treating the symptoms cannot reduce the risk for violence . Pay special attention to criminal schizophrenic patients . Only hospitalizing them for a while to treat psychotic symptoms or imprisoning them cannot prevent future crimes . Addressing the treatment, psychotherapy, and rehabilitation of comorbid personal disorders are important too . Associations with substance abuse and treatment compliance may be the focus of these interventions.it is recommended that in a cohort study on patients with schizophrenia, the effect of rehabilitation, psychotherapy, and treating comorbid disorders (conduct disorder, antisocial personality disorder, and high score of pcl - r) on preventing crimes in high - risk patients needs to be investigated . A while to treat psychotic symptoms or imprisoning them cannot prevent future crimes . Addressing the treatment, psychotherapy, and rehabilitation of comorbid it is recommended that in a cohort study on patients with schizophrenia, the effect of rehabilitation, psychotherapy, and treating comorbid disorders (conduct disorder, antisocial personality disorder, and high score of pcl - r) on preventing crimes in high - risk patients needs to be investigated . Since this study had a case - control design, strong relationship was not possible to be found between the variables and outcome . A while to treat psychotic symptoms or imprisoning them cannot prevent future crimes . Addressing the treatment, psychotherapy, and rehabilitation of comorbid associations with substance abuse and treatment compliance may be the focus of these interventions.it is recommended that in a cohort study on patients with schizophrenia, the effect of rehabilitation, psychotherapy, and treating comorbid disorders (conduct disorder, antisocial personality disorder, and high score of pcl - r) on preventing crimes in high - risk patients needs to be investigated . Addressing the treatment, psychotherapy, and rehabilitation of comorbid personal disorders are important too . It is recommended that in a cohort study on patients with schizophrenia, the effect of rehabilitation, psychotherapy, and treating comorbid disorders (conduct disorder, antisocial personality disorder, and high score of pcl - r) on preventing crimes in high - risk patients needs to be investigated . Since this study had a case - control design, strong relationship was not possible to be found between the variables and outcome . Results of the present study showed that history of conduct disorder in childhood, antisocial personality disorder, and high score of psychopathy in pcl - r were significantly more prevalent in schizophrenic patients with criminal history compared with non - criminal schizophrenic patients . This means that besides the type of clinical symptoms and their severity, which can lead to hostile behaviors and homicidal reactions, other factors must also be considered to prevent criminal behaviors and worsening patient's condition . This has a significant effect on schizophrenic patients rehabilitation, predicting crimes, and preventing re - offense.
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The obvious questions i asked were what was happening? And, why were we seeing these improvements? We asked other parents of children with autism spectrum disorder (asd) and many had observed similar changes while taking antibiotics . In fact, we discovered, much to our surprise, that many parents of autistic children routinely give their children antibiotics to improve their symptoms . It should also be noted, however, that we later also heard from some parents that felt that their children's autism symptoms became worse when they received antibiotics, whereas still others felt that that their children's autism only arose after repeated or long - term antibiotic use . In my view, these stories are not contradictory but rather reinforce the notion that an antibiotic can create an effect in autism . I was determined to better understand this phenomenon because i believed that if we could understand the biological basis of his improvements, we might gain insight into how autism works and be able to help him . To try and understand the link between autism and antibiotics further, i reviewed the medical literature and found a single relevant paper that described a clinical trial that gave vancomycin, an antibiotic that is not systemically absorbed when taken orally, to a small group of severely autistic children as part of a clinical research study at chicago rush children's hospital (2). Eight out of ten of these children experienced a marked improvement in their autism symptoms . The authors speculated that the improvements were likely triggered by changes in the gut microbiome resulting from the antibiotic . I remain confused by the lack of follow - up studies on this commonly observed phenomenon . Despite the alarming rise in autism (discussed below), yet, i personally witnessed evidence that this seemingly intractable condition could be rapidly and dramatically ameliorated in response to an antibiotic, at least for some children, but surprisingly there were no attempts to repeat this study, even as a means to study it mechanistically or to subtype patients . The latest numbers from the us centers for disease control and prevention (based on data for 2010) estimate the prevalence of asd to be 1:68 (and 1:42 for boys) (3). It is difficult to think of another medical condition with such a wide and growing prevalence that science cannot say what it is: neurological, genetic, infectious, autoimmune, metabolic? All of the above? Although it is almost certainly a heterogeneous condition, for years the conventional wisdom has been that autism is a developmental disorder, largely driven by genetics where the associated behaviors and symptoms are a by - product of abnormal neuronal development and connectivity . When our child was diagnosed we were told, like so many other parents, we don't know what it is, but he will not just outgrow it. The conventional view suggests it is a wiring problem, but the evidence we observed on thanksgiving of 2012 suggested something very different . After speaking with some of the original authors of the sandler et al . Paper (2), as well as other key researchers in the field, i found that there was substantial interest in continuing the investigation into this the consistent message i heard from researchers, however, was that it was almost impossible to get funding for projects like these from the handful of public and private organizations that fund autism research because most in those organizations simply do not believe that there is enough evidence that a link exists - it does not fit the current paradigm of a genetically driven cause for asd . If the history of science has taught us anything, it is that real breakthroughs do not occur until paradigms shift, and that that process can be messy and full of controversy (5). In reviewing the literature, i have come to rely on a handful of papers to serve as a starting point for what may be going on (2, 69). The good news is that this area seems to be gaining momentum, though not because of an improvement in the funding picture, but because the scientific evidence of a link seems to be accumulating . The evidence suggesting a link between autism and alterations in the microbiome,1 at least for some children, is strong and continues to build in momentum: several studies have now shown that, compared to neurotypical children, children with autism as a group have compositional changes in their gut microbiomes, as well as less species diversity (6, 815). Some papers have found species in children with autism that they did not find in controls, for example (6, 8). A recent meta - analysis of all autism and gut microbiome studies concluded that there were consistent differences in the gut bacteria of groups of asd patients compared to healthy controls, although the compositional differences were inconsistent (16). This is not surprising given the great variation in our diets (a powerful driver of gut microbiome composition) (17), or the restrictive food interests and specialized diets of many asd children . The lack of standardized approaches, including what type of sample is used (stool vs. biopsy), collection techniques, and how the samples are analyzed, could contribute to inter - study variances . However, the point remains that regardless of experimental conditions, when examined, children with autism have consistently been found to have differential gut bacterial compositions, compared to controls . If one accepts the premise that there are differences in the gut microbiome of asd vs. neurotypical children (and more research needs to be done on this alone), a logical question is how and when during development might this come about? Intriguing evidence has come from a very recent study showing gut microbiome variations in the maternal immune activation (mia) mouse model of autism (7). In this well - studied model, pregnant mice the male offspring of those pregnant immune - activated mice develop autism - like symptoms (7, 1821). More recently, the offspring were found to have different gut bacterial compositions compared to controls . What is striking about this finding is that from birth these mice were grown under identical lab conditions and received identical food (7) something that could not be done with human subjects . Regardless of whether this particular mouse model is representative of autism, the fact that a prenatal, immunological event results in a different gut microbiome under highly controlled conditions is a fascinating finding . This finding suggests that the differences seen in the gut bacteria of the mice were not a result of chance but perhaps allowed or enabled by the changes provoked by the mother's simulated infection . The question of how this process occurs and what it means becomes critical here, and it is an important one to investigate thoroughly as we may be flirting with a core mechanism of the development of autism . Do the emergent autistic - like behaviors exhibited by the mice arise from changes provoked by the maternal infection or the shifts in the gut microbiota that develop? Interestingly, many of the young mice's autistic behaviors were corrected by the administration of a bacterial probiotic (7). Could there be microbes that are required for normal development? As mentioned earlier, some parents feel that repeated or long - term antibiotic use was a trigger for their child's autism . The gut microbiome of newborns and babies change and mature dramatically in the first few years of life (22, 23). Is it possible that in some cases disrupting the development of gut microbiome through repeated antibiotic use early in life could set the stage for the development of autism in some children? (2), as well as anecdotally according to parents who have seen a similar antibiotic effect, when the drug is withdrawn, the children lose their gains or at least stop making them . Is this a sign that the source of improvement is a result of some yet unknown action of the antibiotics or a result of the gut flora returning to its previous state? It is possible that the mechanism could be due to the antibiotic working in some non - antimicrobial way we are not familiar with . However, the fact that some lose their gains quickly (days) following removal of the antibiotic, whereas others lose them slowly (weeks and months) and still others keep their improvements, seems more consistent to me with changes in gut microbiome brought about by the antibiotic rather than some non - antimicrobial action of the antibiotic . Although it is known that most antibiotics have dramatic effects on the gut microbiome (24), the degree to which it recovers following withdrawal of an antibiotic is variable from person to person (25). Does this return to a previous state coincide with the return of asd symptoms in those antibiotic - responders? Any study of these responding children should closely examine the relationship between the return of autism symptoms (or loss of gains) and the return of the gut microbiome to a stable state . What might cause differences in the gut microbiome in children with asd to begin with? Hsiao et al . Showed one possible trigger, in the animal model used, was an early immunological event; but, by what mechanism might this occur? Is it possible that in some cases of autism there is an immunological shift that permits some potentially problematic species to thrive or that blocks colonization by other beneficial species? Or could the altered microbiome simply be a by - product of an asd - induced alteration in the physiological conditions of the gastrointestinal tract (e.g. Altered nutritional adsorption, ph, redox states) that favor a different ecological landscape in the guts of these children, much like different climates on earth favor different plants and animals? Is there such a thing as an autism - driven ecology that is a by - product of autism but that contributes to some of its symptoms? Many people think they know why some children with autism have a positive response to antibiotics . Many parents and even clinicians strongly believe that the benefits seen from antibiotics are a result of killing a specific, problematic bacterium . This view has the benefit of simplicity and fits the limited data well; however, the problem is that despite some extensive searching, a indeed, the current approach to the identification of bacterial species using 16s ribosomal rna gene libraries necessitates that we know what we are looking for in advance; however, the bacterial species it is also theoretically possible that a so - called bad species could have a 16s region so similar to that of another known species that it could be practically invisible using this particular characterization technique . Importantly, knowing which species are present does little to tell us about what they are capable of doing . Although the concept of a bad species in asd is seductive, other hypotheses need to be considered and studied in parallel . Instead of the presence of a bad species, perhaps it is the absence of certain beneficial species that is important . Bacteria frequently share dna in ways that are foreign to our human way of thinking . A more beneficial construct may be to think about the functional genome or even metabolic capability or output of the microbial community (26). Such a paradigm shift may put the disparate findings of those early studies that have looked at gut bacterial composition in asd in a whole new light or may even render that way of thinking an impediment to what is really going on . Thinking about species may be a case of not being able to see the forest for the trees . Recent advances in the field of metabolomics have dramatically improved the number of metabolites that can be detected and characterized . A meaningful percentage of those metabolites are by - products of microbial ecology (27), any number of which could be interfering with core cellular metabolism, gene regulation, neuronal transmission, or other perturbations commonly observed in autism . Showed that the metabolite 4-ethly - phenyl sulfate (4eps) was found at levels 46 times higher in the affected mice compared with controls . When 4eps was injected into the bloodstream of healthy mice, they developed some of the abnormalities seen in the mia mice (7). Macfabe et al . Have reported similar findings for propionic acid, a short - chain fatty acid that is a known fermentative by - product of certain gut microbial species and that is also used as a preservative by the food industry (2830). These findings argue for further investigations into the possible role of microbial metabolites in asd . Much exciting autism research in recent years has been devoted to the metabolic abnormalities commonly observed in autism, in particular those related to mitochondrial pathways (20, 3133). Historically this work was not linked to the microbiome, however, this is beginning to change (21, 28, 34). Indeed the metabolic and catabolic processes of our mammalian cells and the microbiome may be an intricate biochemical dance although it is possible that metabolic by - products of the microbiome such as 4eps are directly responsible for some behaviors associated with autism, it is also possible that alterations of the microbiome may somehow dysregulate immunity and lead to the formation of autoantibodies . The proposed molecular mimicry mechanism of pandas (pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections), which sometimes co - occurs with autism, serves as one example of where the presence of certain bacteria is thought to create antibodies reactive with the basal ganglia resulting in behavioral abnormalities (35). In summary, there is no shortage of proposed mechanisms: presence or absence of certain species or functional genes, metabolic by - product or toxin, molecular mimicry, provocation of cellular or immune hyper - responsiveness, and others . Each proposed mechanism has its own intriguing strands of evidence and plausibility worth consideration and further inquiry . There is compelling evidence of a link between autism and the microbiome in at least a subset of children . Are the microbial abnormalities observed in autism merely a superfluous by - product of autism, or are they at the root of its cause or symptomology? Science will eventually tell us, but for me the simple fact that my child and many others like him improved dramatically while taking an antibiotic suggests the latter . The true devil, however, will be in the details: long - term use of antibiotics does not seem like a good option or treatment approach because of its potential to further disrupt the microbiome and promote resistance . To tease apart what may be going on will take a tremendous commitment of resources and can only be done in a highly collaborative, multidisciplinary way . To even begin to address some of the questions posed in this paper requires expertise in disparate fields of medicine and science (fig . 1), many of which have historically had little reason to collaborate . Such cooperation will require a departure from the typically siloed nature of medical research and (perhaps more importantly) how grants are made . I formed the n of one: autism research foundation, with the mission of encouraging, sponsoring, and communicating breakthrough autism research . Our focus is to garner support for promising research areas that we believe are not receiving sufficient funding relative to their scientific merit . Additionally, we favor research that treats the observations of parents and doctors as clues to the mechanism of autism . Research into the microbiome's role in asd fits both of those conditions well . In february of 2014, working with dr . Richard frye at arkansas children's hospital, we came up with the idea of assembling an interdisciplinary group of experts to address what could be done to further investigate the antibiotic effect first documented in 2000 (2). The group considered investigations into the microbiome and autism that might provide insight into mechanism or therapeutic benefit . An in - person meeting was held on june 27, 2014, in little rock, arkansas, to discuss further the development of a clinical research trial . An additional day was set aside to conduct a conference, free and open to the public, dedicated to topics surrounding autism and the microbiome . The point of the conference was to increase awareness of possible links between autism and the microbiome and to bring together interested members of the scientific community as well as parents to discuss, collaborate, and exchange ideas on this subject . That same group of researchers decided to publish this special issue, and i am proud and honored to be a part of that group . I am appreciative that scientists and clinicians would ask an ordinary parent to contribute to this journal . At the time of his diagnosis, we had been led to believe that our son's autism was a hard - wired neurological condition from which he would not emerge, but during the fall and winter of 2012 and subsequent periods since then we have seen our son with the veil of autism partially lifted . I love him unconditionally regardless of his autism or how he is doing on any given day, but because i have seen what is possible, i will endeavor to promote research that benefits all children with autism and to remove all impediments from him becoming the fullest embodiment of who he can be and until it is definitively proven otherwise, i will strive to foster research consistent with the evidence of the microbiome's involvement in autism . The author has not received any funding or benefits from industry or elsewhere to conduct this study.
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Aesthetic facial concerns have been the main reason for dermatological consults in the last few years . Periorbital hyperpigmentation is defined as bilateral, homogeneous hyperchromic macules and patches primarily involving the lower eyelids but also sometimes extending towards the upper eyelids, eyebrows, malar regions, temporal regions and lateral nasal root . The age of onset is usually after puberty or in early adulthood (16 - 25 years). It is more pronounced in certain ethnic groups and is also frequently seen in multiple members of the same family . Although considered a phenomenon within the limit of physiology, patients, especially women are really bothered and concerned about it, even relating the presence of dark circles with significant impairment on their quality of life . Although not rare in males, females dc are caused by multiple etiologic factors that include genetic / constitutional, dermal melanin deposition, post inflammatory hyperpigmentation, secondary to atopic or allergic contact dermatitis, anemia, stress, faulty habits, periorbital edema, superficial location of vasculature, and shadowing due to skin laxity, etc . While there are not much statistics giving the frequency of its occurrence, judging from the amount of advertising of cosmetics marketed to treat it, dark circles are a cosmetic concern for large number of individuals . Our aims of the study were to determine the prevalence and type of periorbital hyperpigmentation within various age and sex groups in patients attending dermatology opd at tertiary care hospital and to find out etiology of periorbital hyperpigmentation and its association with habits and other disorders . This was single centre, cross - sectional, descriptive, anonymous study conducted in patients attending the skin opd of the teaching hospital from november 2009 to july 2011 with approval of institutional ethical committee (human). Two hundred new patients from skin opd having age between 16 - 45 years and fitzpatrick skin type i to iv were included in study . Detailed history was taken including duration of the condition, family history, history of atopy or drug intake, associated faulty habit or lifestyle, use of cosmetics, precipitating factor such as photosensitivity, allergies, seasonal variations, presence of associated pigmentation in other areas of the face and the body and presence of any concomitant illness such as anemia, gastrointestinal diseases, hepato - biliary diseases, renal diseases, thyroid diseases, etc . This was followed by careful physical examination to detect involvement of upper or lower or both eyelids and extension beyond the periorbital region, colour of hyperpigmented areas (light brown / dark brown / red / blue), presence of any dermatological disease or scar in periorbital region, presence of any visible bulging, shadow effect, superficial visible vasculature (i.e., capillaries or veins) in the infraorbital region, pallor in palpebral conjunctiva, face, nails and palms; any cosmetics applied over face, presence of pigmentation in other areas of face e.g., melasma, freckles, etc . Diagnosis of periorbital hyperpigmentation was done clinically and the patients were classified according to the classification proposed by ranu et al . As follows: constitutional the presence of a curved band of brownish to black pigmentation on the skin of the lower eyelids approximating the shape of the orbital rim with frequent involvement of upper eyelids.post inflammatory presence of irregular patches of brownish or grey pigmentation on the skin on the upper, lower or both eyelids with features of lichenification, accentuation of skin creases, and eczematous papules or patches in the surrounding areas . Personal and/or family history of atopy may or may not be present.vascular presence of erythema predominantly involving the inner aspect of lower eyelids, with prominent capillaries or telangiectasia (capillaries) or the presence of bluish discoloration of the lower eyelid and visible bluish veins that becomes more prominent when the overlying skin is stretched . This type of dark circle appears to be due to a combination of transparency of the overlying skin and dermal vascularity.shadow effect presence of a dark shadow under an overhanging tarsal muscle, eye bags, or the presence of a deep tear trough over the medial aspect of inferior orbital rim that disappear with direct lighting.others poh from other causes including anemia, hormonal disturbances, nutritional deficiencies, acanthosis nigricans, skin laxity, associated chronic illness, habits, etc . Constitutional the presence of a curved band of brownish to black pigmentation on the skin of the lower eyelids approximating the shape of the orbital rim with frequent involvement of upper eyelids . Post inflammatory presence of irregular patches of brownish or grey pigmentation on the skin on the upper, lower or both eyelids with features of lichenification, accentuation of skin creases, and eczematous papules or patches in the surrounding areas . Vascular presence of erythema predominantly involving the inner aspect of lower eyelids, with prominent capillaries or telangiectasia (capillaries) or the presence of bluish discoloration of the lower eyelid and visible bluish veins that becomes more prominent when the overlying skin is stretched . This type of dark circle appears to be due to a combination of transparency of the overlying skin and dermal vascularity . Shadow effect presence of a dark shadow under an overhanging tarsal muscle, eye bags, or the presence of a deep tear trough over the medial aspect of inferior orbital rim that disappear with direct lighting . Poh from other causes including anemia, hormonal disturbances, nutritional deficiencies, acanthosis nigricans, skin laxity, associated chronic illness, habits, etc . Grading of poh was done in comparison to surrounding skin as follows: 0 skin colour comparable to other facial skin areas, 1 faint pigmentation of infraorbital fold, 2 pigmentation more pronounced, 3 deep dark colour, all four lids involved, 4 grade 3 + pigmentation spreading beyond infraorbital fold . Wood's lamp examination was done to determine whether pigmentation is epidermal or dermal . If there is no accentuation of pigment, then it is due to dermal pigmentation or vascularity . Eyelid stretch test was done in which if the dark circles improves, then it is because of skin laxity . If there is no change in dark circles, then it is due to epidermal and/or dermal pigmentation . Were carried out in each patient: complete blood count, serum tsh, sgpt, serum cholesterol, random blood sugar . While history and physical examination are usually sufficient to determine the primary cause of periorbital hyperpigmentation, confirmation by histologic examination may rarely be necessary . Data were statistically described in frequencies (number of cases) and percentages (%) when appropriate . All statistical calculations were done using computer programs microsoft excel 2010 and ibm spss (statistical package for the social science) version 20 for microsoft windows . Data were statistically described in frequencies (number of cases) and percentages (%) when appropriate . All statistical calculations were done using computer programs microsoft excel 2010 and ibm spss (statistical package for the social science) version 20 for microsoft windows . Majority of the patient were housewives (n = 91, 45.5%) (p = 0.0001). Demographic features of the patients the commonest form of poh observed was constitutional type (n = 103, 51.5%) followed by post inflammatory type (n = 45, 22.5%) [table 2]. Various types of poh observed in our study are shown in figures 1 to 4 . In our study, the most common site involved were lower eyelids (n = 134, 67%) followed by involvement of both upper and lower eyelids (n = 41, 20.5%). Patients having grade 1 to grade 4 type of poh as observed in our study are shown in figure 5 to 8 . Majority of the patients were having poh of grade 2 (n = 115, 57.5%). One hundred twenty six (63%) patients had a positive family history of poh (p = 0.03). On wood's lamp examination, 121 (60.50%) classification of poh constitutional type of periorbital hyperpigmentation post inflammatory type of periorbital hyperpigmentation vascular type of periorbital hyperpigmentation shadow effect type of periorbital hyperpigmentation grade 1 type of periorbital hyperpigmentation grade 2 type of periorbital hyperpigmentation grade 3 type of periorbital hyperpigmentation grade 4 type of periorbital hyperpigmentation distribution of patients according to various parameters multinomial logistic regression shows that the model showing association of poh with different variables is very good (chi square value 419.515, df = 57, p = 0.0001 and cox and snell pseudo r- square value = 0.877). As regards with habits, the multinomial logistic regression of poh with variables like lack of correction for myopia (p = 0.02) and lack of adequate sleep i.e., 6 - 8 hours per night regularly (p = 0.01) were statistically significant, both of which signifying strain on eyelid muscles . Association of poh with other habits and eye diseases was not statistically significant (p> 0.05). Multinomial logistic regression of poh with atopy (p = 0.001) and irregular menstruation (p = 0.009), premenstrual aggravation (p = 0.024), stress (p = 0.010) and anemia (p = 0.006) was highly significant . Laboratory investigations revealed anemia in 100 (50%) patients, raised blood sugar in 1 (0.5%) patient, serum tsh was raised in 3 (1.5%) patients, while it was decreased in 2 (1%) patients and serum cholesterol was raised in 1 (0.5%) patient . Association of poh with personal habits and lifestyle association of poh with ophthalmic and other disorders present study shows that most common age group was 16 - 25 years and that poh has higher preponderance for females over males (1:4.2). The genotype is fixed at conception, but the phenotype may not manifest until adult life . In such cases the penetrance is age - related, which supports that age of onset of poh in majority of the patients in our study was in early adulthood, i.e., 16 - 25 years . Seasonal variation was seen in only 15% patients having aggravation of their condition in winter showing that seasonal change has got a little impact on poh (p> 0.05). Our results are in agreement with findings of ranu et al . Regarding the commonest type of poh in indian patients to be of constitutional type . Present study shows that 51.50% of the patients were having constitutional type of poh, while ranu et al . Had reported that in their study on chinese, malay and indian patients, the commonest form of poh observed was vascular (41.8%), followed by constitutional (38.6%), post inflammatory type (12%), and shadow effect (11.4%). The constitutional type of poh was more commonly seen in malays (65%) and indians (94%). In pih type of poh, 33% patients had history of atopy in our study while it was 55.4% in study conducted by ranu et al . Sarma et al . Have proposed that all the described pdls on face including pdl - f, g, h actually represent the margins of pigmentary patches which closely conforms with blaschko's lines on face and have proposed that mosaicism may play a role in development of pdl . Have defined periorbital melanosis as an extension of pigmentary demarcation line f of the face which was observed in 22 (11%) patients in our study [figure 9]. Only one patient (0.5%) was having pigmentary demarcation line g. periorbital hyperpigmentation with pigmentary demarcation line f in our study, 126 (63%) patients had positive family history of poh . Has reported poh to be an autosomal dominant trait which usually runs in the families, which is reflected in our study, as out of 103 patients of constitutional type of poh, 79 (76.69%) patients had positive family history of poh, which reflects its genetic transmission . Out of 16 patients of vascular type, 10 (62.5%) patients had positive family history of poh, which may be due to thin skin of periorbital region which is an inherited trait . Watanabe et al . Reported that out of 12 patients of dark circles included in their study; all the 12 patients (100%) revealed dermal melanin deposition by histopathological examination . In contrast to that our study showed 60.50% patients had dermal pigmentation, 38% patients had epidermal pigmentation, while 1.5% patients had mixed type of pigmentation on wood's lamp examination . In the present study, 40% patients reported lack of adequate sleep . In a study by ranu et al ., 94 (51.1%) and 83 (41.5%) patients reported sleep deprivation and insomnia respectively . All the patients who were using different cosmetics in periorbital area (n = 65, 36.5%) gave history of frequent eye rubbing and scratching in periorbital area which implies that there may be some ingredient in it that causes allergic contact dermatitis and on resolution leaves post inflammatory hyperpigmentation in periorbital area . However, this needs further evaluation by a separate scientific study as the results were not statistically significant in our study . Thirty percent patients had error of refraction i.e., myopia, out of which only 12% patients were regularly using the spectacles or contact lenses thereby causing exhaustion of periorbital muscles . According to gathers exhaustion of periorbital muscles may play a significant role in causation of poh . . It may be due to effect of increased msh secretion via hpa axis in response to stress which creates a vicious cycle and it is very difficult to differentiate whether stress increases poh or vice versa but once dark circles appear, it definitely increases stress regarding their aesthetic appearance . In our study, 71% patients of gave positive history of stress due to dark circles under the eyes which shows that it is a major esthetic concern and having significant impact on quality of life . According to gathers fatigue, stress, emotional liability and aging all may play a significant role in development of poh . In the present study, poh may be either because enough oxygen is not reaching the periorbital tissues or due to facial pallor which makes the periorbital region look comparatively darker . Thirty percent patients of poh had menstrual disturbances like irregular menstruation for longer periods (> 1.5 - 2 years). Eighteen percent patients were taking oral contraceptive pills which reflects the hormones to be one of the factors involved in causation or exacerbation of poh . According to gathers, chronic use of some drugs including oral contraceptives, hormone - replacement therapy, antipsychotics, gold, chemotherapeutic compounds can lead to periorbital hyperpigmentation . In our study, patients of poh having either present or past positive history of systemic diseases like diabetes, hyperthyroidism, hypothyroidism, high cholesterol, hypertension, seizures constituted only 9% which was not statistically significant (p> 0.05). Gendler et al . Stated that some medical problems that may contribute to dark circles include disorders of heart, thyroid, kidney or liver, vitamin k deficiency, addison's disease, etc . Systemic conditions that can lead to pigmentation of periorbital area include metabolic and endocrine disorders, but no statistical data have been shown . Considering its multifactorial etiology and increasing cosmetic concerns, we look forward to larger studies to further enlighten our knowledge over this condition . Assessment of pigment depth by wood's lamp in indian skin may be unreliable.clinico-histopathological correlation was not done . Assessment of pigment depth by wood's lamp in indian skin may be unreliable.clinico-histopathological correlation was not done . Periorbital hyperpigmentation is a multi - factorial entity . It is absolutely essential to classify the type of poh, determine underlying causative factors and correction of faulty habits in order to direct the appropriate treatment for better and successful outcome in future this is a unique study showing prevalence, classification and association of periorbital hyperpigmentation with habits and other disorders in indian patients, which will help to explore and direct appropriate measures for treatment of this aesthetic condition.
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Positron emission tomography (pet) is a non - invasive imaging technique used to diagnose neurological and neuropsychiatric disorders . This method facilitates longitudinal studies of various animal models of alzheimer's disease, cerebral ischemia, depression, epilepsy, parkinson's disease, and schizophrenia . This compound is a radioactive analogue of glucose and altered fdg uptake indicates metabolic changes of neuronal activity in the brain . Application of fdg - pet in rodents for neuroscience research may be limited by unwanted extracranial accumulation of fdg in the harderian gland . Unlike humans, small laboratory rodents have harderian glands that show strong fdg uptake within the orbit near the frontal region of the brain . However, one major secretory product of the gland is porphyrin, which affects photoreception and lubrication of the eyeball . Fdg uptake in the harderian gland varies greatly depending on study conditions prior to pet imaging such as the use of anesthesia during the uptake period . Moreover, other radioactive tracers like c - pib and f - fallypride reportedly accumulate in the harderian gland as well . This characteristic of the harderian gland makes it difficult to obtain exact measurements of fdg uptake near the frontal brain region because it is impossible to account for the radioactive spillover coming from the adjacent harderian gland . To resolve these problems, surgical procedures for harderian adenectomy (ha) since the mouse brain is significantly smaller compared to rat brain, the additive effect of the harderian gland on fdg uptake measurement is much greater . The purposes of the present study were to develop a surgical procedure for ha in mice and to assess the effectiveness of this procedure for improving fdg - pet . Adult balb / c mice (22.0 1.9 g; orientbio, korea) were used for all experiments . Two mice were used for unilateral ha, and six mice underwent bilateral ha and control operations, respectively . The experimental procedure was approved by the institutional animal care and use committee of konkuk university, korea . Ha was performed in mice anesthetized intraperitoneally with zoletil (90 mg / kg; virbac, france) and xylazine (10 mg / kg; bayer, germany). The whiskers were clipped, and then the conjunctival sac and periocular skin were disinfected with povidone - iodine diluted (1: 50) in saline . Mice were placed in a lateral recumbent position and the nictitating membrane was pulled toward the lateral canthus using serrated micro - dissecting forceps (fig . At the base of the nictitating membrane, the medially protruding, cream - colored harderian gland could be seen under the thin conjunctival tissue (fig . The conjunctival tissue that covered the gland was incised using the tip of a surgical blade . Once removed, the space between the eye and orbital rim was examined for residual glandular tissue . During the resection procedure, a small amount of bleeding occurred but stopped once slight pressure was applied with a cotton swab . Postoperative care was immediately provided and performed once a day for the next 3 days . Ophthalmic antibiotic ointment (terramycin; pfizer, usa) and solcoseryl eye gel (solcorin; hanlim pharm, korea) were applied for protection against infection and to lubricate the eyes . An analgesic (ketoprofen, 5 mg / kg; unibiotech, korea) was also administered subcutaneously daily for 3 days . The adenectomized mice were subjected to fdg - pet scans on postoperative days 5, 10, 15, and 30 . Fdg (300 ci; korea institute of radiological and medical sciences, korea) was injected via the lateral tail vein while the mice were anesthetized with 2% isoflurane (jw pharmaceutical, korea). The mice were positioned on the scanning bed, and pet was performed for 20 min in an inveon pet / ct system (siemens healthcare, usa). The acquired pet emission data were reconstructed using a fourier re - binning algorithm with three span and 31 ring difference, and ordered subsets expectation maximization 2d algorithm with 16 subsets and four iterations . In order to improve the anatomical localization of fdg uptake in the brain, computed tomography (ct) the ct images were acquired with 70 kvp, 500 a, and 200 msec exposure times for each step (inveon; siemens healthcare). Cone - beam reconstruction was performed with four down - sample factors using bilinear interpolation and shepp.logan filters as previously described . In order to demonstrate the influence of the harderian gland on fdg uptake in specific brain regions, statistical parametric mapping (spm5; wellcome trust centre for neuroimaging institute of neurology, uk) pet images were realigned to a template image so that the static bounding box could be used to tightly crop the region of interest . The template image was created from the mean image, which was realigned and provided an average representation of all images . The normalize function determines the transformation that minimizes the differences between two images by minimizing the sum of squares of intensity differences . During normalization, 16 nonlinear warping iterations and trilinear interpolation of the images were performed . To improve normalization accuracy, we used a full affine transformation and nonlinear deformations, which facilitate the correction of subtle differences between subjects . Global calculation was performed using standard mean voxel values . Using this method, voxels with values less than 1/8 of the overall mean value were deemed extracranial . For the statistical analysis, we defined t contrast and p value adjustments to the control associated with significantly active regions and rest regions (p <0.005). In order to compare regional standard uptake values (suvs) affected by ha, suv from brain regions including areas of removed harderian glands, frontal brain and the cerebellum were measured . Then the changes of suvs of the regions in relation to postoperative time were statistically analyzed using one - way anova and post - hoc analysis (prism ver . When the last pet / ct scan was completed on postoperative day 30, a visual limb - placing test was performed to evaluate visual acuity and determine if any complication had arisen following the ha . The whisker - clipped mice were lifted by the tail over the wire bar lid of a mouse cage to a height of 20 cm and then lowered onto the lid within a second . Mice with normal vision extended their forelimbs toward the lid before their noses reached it . The ha mice recovered from surgery without any postoperative complications and the incision site healed without suturing . All adenectomized mice were found to have normal visual acuity according to the visual placing test, indicating there were no surgical complications . Strong fdg uptake of the harderian gland was observed in the intact side and radioactive spillover into the brain was also apparent (fig . This result suggests that excess signals emitted from the harderian gland interfere with the net measurement of brain fdg uptake, especially in the frontal brain region . Bilateral ha efficiently eliminated extracranial accumulation of fdg in the harderian gland and subsequent radioactive spillover into the brain (fig . 3). In particular, pet images of the ha mice showed more defined frontal regions of the brain than those of mice that did not undergo ha . Analyses of suvs revealed significant reductions in the regions of harderian gland and frontal brain (fig . After bilateral ha, fdg uptake in the regions of the removed harderian glands was significantly reduced starting on postoperative day 5 (figs . The reduction in fdg uptake was approximately 55%, and the left and right harderian glands both showed similar levels of reduction . Suvs for the frontal brain region decreased significantly from postoperative day 10 since harderian gland fdg accumulation was eliminated (fig . We also measured suvs for the cerebellum and the entire brain region but no statistical differences were found (fig . These results indicate that the effect of the harderian gland on fdg - pet analysis was limited to the frontal brain region . To localize the brain regions affected by harderian gland fdg uptake, we carried out a statistical analysis using the spm tool . We were able to obtain parametric maps for mice with intact harderian glands and the adenectomized animals . When parametric maps were overlaid on a pet template, regions that had significantly high fdg uptake (fig . Results of the spm analysis concur with those obtained for manually measured suvs (figs . Our study demonstrates that ha in mice is a simple and effective method which enables accurate measurement of fdg uptake in the frontal brain region . We found that the surgical procedure used for rat ha was equally effective in mice although more caution is required for successful surgeries in the latter species . For instance, bleeding must be strictly controlled because blood obstructs views of the limited surgical area that is available in mice . The shape of the resected harderian glands should be carefully checked to avoid potential tearing during resection . This is because any residual harderian gland left behind may result in an inaccurate fdg uptake reading along with the additional concern that the gland will regenerate . Moreover, the remaining tissue may cause inflammation that in turn could increase fdg uptake over a short - term period . The simple surgical procedure performed in our study can be carried out by non - veterinarians and should take no more than 5 min . If the surgery is successful, the animal should recover readily without any postsurgical complications including vision impairment . A limitation associated with fdg - pet scanning is known as the " partial volume effect ", which stems from the finite spatial resolution of the images during sampling and reconstruction . A partial volume effect can distort both the contour and signal intensity of images for an actual subject . Due to the partial volume effect, small pieces of tissues that accumulate high level of fdg such as harderian glands could appear larger than the actual size and obscure images of the brain during pet imaging . Interference attributed to harderian gland radioactive spillover in the rodent brain has previously been described in rats and mice . Thus, the adverse effects of harderian gland fdg uptake are an unavoidable issue since anesthesia is essential for obtaining pet scans of rodents . We found that ha in mice was feasible and had the same effect as it did in rats . After ha, strong fdg uptake signals were excluded from the regions of the gland and distorted fdg - pet images were corrected . It should be pointed out that it took at least 10 days before significant differences in suvs were achieved . We were also interested in assessing the range of signal spillover in the brain due to harderian gland fdg uptake . Using spm analysis, we were able to demonstrate the additive effect that the harderian gland has on measuring fdg uptake in the frontal brain region . Our results support a previous finding that fdg uptake by the harderian gland results in a slight over - estimation of fdg uptake in the frontal lobe of rats . We found that the level of fdg uptake stabilized starting 10 days after the surgery and was maintained for the remainder of the study . This result suggests that the glandular tissue did not regenerate, which is important for long - term experiments . In conclusion when performed prior to pet scanning of mice brains, ha may be useful for visualizing the frontal lobe with 18f - fdg micro pet / ct imaging.
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A network is generated by determining the first- and second - order interactions of cd proteins associated with a given morphological subgroup in a human protein interaction network consisting of refined experimental proteomics data . This network is described in high detail in lage et al (2007, 2008), and online (http://www.cbs.dtu.dk/suppl/dgf/). Interactions of the cd proteins are integrated into a network by always including direct interactions between cd proteins, and only including indirect interactions mediated through proteins with q percent of its interactions to the cd set . Various thresholds for q are iteratively tested and value of q for the final network is chosen based on which value gives the optimal network significance, this procedure is described in detail in bergholdt et al (2007) and d'hertog et al (2007). The method for determining network significances can be seen below . Detailed views of the networks can be seen in supplementary figures s1, s2, s3 and s4 . The significance of each of the generated 19 networks was determined by randomization testing as described in detail earlier (bergholdt et al, 2007; d'hertog et al, 2007). Specifically, for an input set of ninput proteins yielding an interaction network (connected component) with g input proteins and t total proteins, a network score (nsinput) was determined . This network score is the fraction of input proteins of all proteins in the network (g / t). We then determined the significance of the network score by empirically estimating the probability of observing a similar or better network score in networks generated by using 10 000 random input sets of size ninput . The random gene sets were chosen so the degree distribution of proteins in the random sets approximate the input set . As each query generates a varying number of networks (connected components) the probability estimates can be calculated from the total amount of networks produced by all 10 000 randomizations that have a network score> nsinput . For this reason, network p - values can be lower than the amount of random queries . All network p - values can be seen in supplementary figures s1, s2, s3 and s4 below the title of the network . To rule out the chance of functional bias in the cd set a set of raw candidates were determined by querying all proteins in our interaction network for the amount of interactions to the cd set and determining the hypergeometric probability of this interaction profile . Out of all proteins in the proteome, 49 novel candidates had a significant interaction profile to the cd proteins after adjustment for multiple testing (described in detail in supplementary information and supplementary figure s5). We then used the functional networks assigned to each morphological subgroup to determine the most likely developmental function of the candidates . This was done by identifying the specific subnetworks to which the interactions of the candidates were most significant (as described in supplementary information and supplementary figure s6). In all, 12 of the 49 candidates were chosen for ih analysis based on the overlap between morphological subgroups and the developmental stages present in our panel of embryonic hearts available for validation experiments . Human embryonic tissues were collected from legal abortions, according to the helsinki declaration ii, and their use was approved by the local science ethics committee . Embryonic or fetal age was based on measurement of crown - rump length . Immediately after dissection, the samples were snap frozen in liquid nitrogen or treated with rna later according to the manufacturer's instructions (ambion, austin, tx). Samples for ih were dissected into appropriate tissue blocks and fixed for 1224 h at 4 c in either 10% neutral - buffered formalin, 4% formol - calcium, lillie's or bouin's fixatives . Serial sections, 35 m thick, were cut in transverse, sagittal or horizontal planes and placed on silanized slides . Sections were deparaffinized and rehydrated in xylene followed by a series of graded alcohols according to the established procedures . The sections were treated with a fresh 0.5% solution of hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase and then rinsed in tris - buffered saline (tbs, 5 mm tris hcl, 146 mm nacl, ph 7.6). Non - specific binding was inhibited by incubation for 30 min with blocking buffer (chemmate antibody diluent s2022, dakocytomation, glostrup, denmark) at room temperature . The sections were then incubated overnight at 4c with the primary antibody in blocking buffer (chemmate antibody diluent s2022, dakocytomation). The sections were washed with tbs and then incubated for 30 min with a peroxidase - labeled secondary antibody . The sections were washed with tbs, followed by incubation for 10 min with 3,3-diamino - benzidine chromogen solution . The sections were dehydrated in graded alcohols followed by xylene and coverslipped with dpx mounting media . Specificity of the antibodies were determined by their ability to stain specific cell populations in the tissue sections (examples are shown in supplementary figures s7s12). The following primary antibodies were used: anti - ptgs2 (35 - 8200, invitrogen), anti - mapk8 (sc-6254, santa cruz biotechnology, santa cruz, ca), anti - cav3 (610421, bd transduction laboratories, franklin lakes, nj), anti - mapk3 (sc-7383, santa cruz biotechnology), anti - src (at-7016, mbl international, woborn, ma), anti - jag2 (sc-8157, santa cruz biotechnology), anti - dll1 (sc-9102, santa cruz biotechnology), anti - notch3 (sc-7474, santa cruz biotechnology), anti - notch4 (sc-5594, santa cruz biotechnology), anti - bmx (ab73887, abcam, cambridge, uk), anti - ptk2b (ab78119, abcam), anti - bmp4 (ab31165, abcam), anti - egfr (#2232, cell signaling technology, boston, ma). Pcr for this analysis because it is considered to be the most accurate and sensitive method for detecting rna differences also at very small amounts (lang et al, 2009). Total rna was isolated from tissues using trizol reagent (invitrogen, taastrup, denmark) and cdna synthesized with superscript ii (rnase h) reverse transcriptase (invitrogen) according to the manufacturer's instructions . Qpcr analysis was carried out on an abi 7500 fast real - time pcr system using a lightcycler faststart dna master sybr greeni kit (roche, hvidovre, denmark). Primer sequences used for qpcr analysis are available on request . To exclude that polymorphic gene expression between the individual developing hearts could account for the observed differential expression trends reported by qpcr, we also used polony multiplex analysis of gene expression (kim et al, 2007) to measure the expression of the 49 candidates in right ventricular outflow tract from tof patients at the time of primary surgical repair and left ventricle collected from patients with either heart failure or diabetic cardiomyopathy . The expression levels of the candidates were compared with the expression levels of a different set of 49 randomly chosen controls after normalizing both gene sets against gene expression in glioblastoma tissue . Here, the heart developmental candidates were significantly higher expressed in heart tissue than the controls (p=0.016) (see supplementary information and supplementary figure s13). A network is generated by determining the first- and second - order interactions of cd proteins associated with a given morphological subgroup in a human protein interaction network consisting of refined experimental proteomics data . This network is described in high detail in lage et al (2007, 2008), and online (http://www.cbs.dtu.dk/suppl/dgf/). Interactions of the cd proteins are integrated into a network by always including direct interactions between cd proteins, and only including indirect interactions mediated through proteins with q percent of its interactions to the cd set . Various thresholds for q are iteratively tested and value of q for the final network is chosen based on which value gives the optimal network significance, this procedure is described in detail in bergholdt et al (2007) and d'hertog et al (2007). The method for determining network significances can be seen below . Detailed views of the networks can be seen in supplementary figures s1, s2, s3 and s4 . The significance of each of the generated 19 networks was determined by randomization testing as described in detail earlier (bergholdt et al, 2007; d'hertog et al, 2007). Specifically, for an input set of ninput proteins yielding an interaction network (connected component) with g input proteins and t total proteins, a network score (nsinput) was determined . This network score is the fraction of input proteins of all proteins in the network (g / t). We then determined the significance of the network score by empirically estimating the probability of observing a similar or better network score in networks generated by using 10 000 random input sets of size ninput . The random gene sets were chosen so the degree distribution of proteins in the random sets approximate the input set . As each query generates a varying number of networks (connected components) the probability estimates can be calculated from the total amount of networks produced by all 10 000 randomizations that have a network score> nsinput . For this reason, network p - values can be lower than the amount of random queries . All network p - values can be seen in supplementary figures s1, s2, s3 and s4 below the title of the network . To rule out the chance of functional bias in the cd set a set of raw candidates were determined by querying all proteins in our interaction network for the amount of interactions to the cd set and determining the hypergeometric probability of this interaction profile . Out of all proteins in the proteome, 49 novel candidates had a significant interaction profile to the cd proteins after adjustment for multiple testing (described in detail in supplementary information and supplementary figure s5). We then used the functional networks assigned to each morphological subgroup to determine the most likely developmental function of the candidates . This was done by identifying the specific subnetworks to which the interactions of the candidates were most significant (as described in supplementary information and supplementary figure s6). In all, 12 of the 49 candidates were chosen for ih analysis based on the overlap between morphological subgroups and the developmental stages present in our panel of embryonic hearts available for validation experiments . Human embryonic tissues were collected from legal abortions, according to the helsinki declaration ii, and their use was approved by the local science ethics committee . Embryonic or fetal age was based on measurement of crown - rump length . Immediately after dissection, the samples were snap frozen in liquid nitrogen or treated with rna later according to the manufacturer's instructions (ambion, austin, tx). Samples for ih were dissected into appropriate tissue blocks and fixed for 1224 h at 4 c in either 10% neutral - buffered formalin, 4% formol - calcium, lillie's or bouin's fixatives . Serial sections, 35 m thick, were cut in transverse, sagittal or horizontal planes and placed on silanized slides . Sections were deparaffinized and rehydrated in xylene followed by a series of graded alcohols according to the established procedures . The sections were treated with a fresh 0.5% solution of hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase and then rinsed in tris - buffered saline (tbs, 5 mm tris hcl, 146 mm nacl, ph 7.6). Non - specific binding was inhibited by incubation for 30 min with blocking buffer (chemmate antibody diluent s2022, dakocytomation, glostrup, denmark) at room temperature . The sections were then incubated overnight at 4c with the primary antibody in blocking buffer (chemmate antibody diluent s2022, dakocytomation). The sections were washed with tbs and then incubated for 30 min with a peroxidase - labeled secondary antibody . The sections were washed with tbs, followed by incubation for 10 min with 3,3-diamino - benzidine chromogen solution . The sections were dehydrated in graded alcohols followed by xylene and coverslipped with dpx mounting media . Specificity of the antibodies were determined by their ability to stain specific cell populations in the tissue sections (examples are shown in supplementary figures s7s12). The following primary antibodies were used: anti - ptgs2 (35 - 8200, invitrogen), anti - mapk8 (sc-6254, santa cruz biotechnology, santa cruz, ca), anti - cav3 (610421, bd transduction laboratories, franklin lakes, nj), anti - mapk3 (sc-7383, santa cruz biotechnology), anti - src (at-7016, mbl international, woborn, ma), anti - jag2 (sc-8157, santa cruz biotechnology), anti - dll1 (sc-9102, santa cruz biotechnology), anti - notch3 (sc-7474, santa cruz biotechnology), anti - notch4 (sc-5594, santa cruz biotechnology), anti - bmx (ab73887, abcam, cambridge, uk), anti - ptk2b (ab78119, abcam), anti - bmp4 (ab31165, abcam), anti - egfr (#2232, cell signaling technology, boston, ma). We chose quantitative real - time quantitative rt pcr for this analysis because it is considered to be the most accurate and sensitive method for detecting rna differences also at very small amounts (lang et al, 2009). Total rna was isolated from tissues using trizol reagent (invitrogen, taastrup, denmark) and cdna synthesized with superscript ii (rnase h) reverse transcriptase (invitrogen) according to the manufacturer's instructions . Qpcr analysis was carried out on an abi 7500 fast real - time pcr system using a lightcycler faststart dna master sybr greeni kit (roche, hvidovre, denmark). Primer sequences used for qpcr analysis are available on request . To exclude that polymorphic gene expression between the individual developing hearts could account for the observed differential expression trends reported by qpcr, we also used polony multiplex analysis of gene expression (kim et al, 2007) to measure the expression of the 49 candidates in right ventricular outflow tract from tof patients at the time of primary surgical repair and left ventricle collected from patients with either heart failure or diabetic cardiomyopathy . The expression levels of the candidates were compared with the expression levels of a different set of 49 randomly chosen controls after normalizing both gene sets against gene expression in glioblastoma tissue . Here, the heart developmental candidates were significantly higher expressed in heart tissue than the controls (p=0.016) (see supplementary information and supplementary figure s13).
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Greater than 80% of pavms are congenital, and approximately 50% of are associated with hereditary hemorrhagic telangiectasia (hht). In addition to common complaints, such as dyspnea and epistaxis, a pavm can cause hemoptysis, hemothorax, and serious neurologic complications, such as stroke, seizures, and brain abscesses . Ebstein's anomaly (ea) is a congenital cardiac malformation characterized by downward displacement of the attachment of the septal and posterior leaflets of the tricuspid valve . Ea is also a rare disorder, and patients with ea may have various additional cardiovascular anomalies . Although the causes of a pavm and ea have not been established, embryogenic or genetic factors might give some contributions . However, there are no reports concerning the co - existence of pavms or hht with ea . We present a case of a pavm with ea that is suspected to have hht . She had experienced spontaneous recurrent epistaxis for the last several years, with no other remarkable medical history . A chest radiograph showed cardiomegaly and a well - demarcated, 12-mm nodular opacity in the left lung field (fig . The electrocardiogram showed tall and broad p waves, as well as an incomplete right bundle - branch block . On transthoracic echocardiography, the displacement index (distance between the mitral annulus and tricuspid annulus, divided by the body surface area) was 15.6 mm / m, and it fulfilled the displacement index criteria for the diagnosis of ea (8 mm / m). Moreover, tethering of the septal and posterior leaflets of the tricuspid valve was observed with central coaptation failure and severe regurgitation . A portion of the right ventricle was atrialized because of apical displacement of the tricuspid valve . She was diagnosed with ea, and agreed to undergo surgical treatment . To evaluate the nodule in the left lung, a 12-mm serpiginous vascular structure connecting the left interlobar pulmonary artery to the left inferior pulmonary vein was detected in the superior segment of the left lower lobe (fig . No part of her medical history could explain the causes of secondary pavm, such as chest trauma, thoracic surgery, long - standing hepatic cirrhosis, metastatic carcinoma, mitral stenosis, or infections . Three - phase contrast ct scans of the liver and mr angiography of the brain were performed to identify other visceral arteriovenous malformations . A liver ct showed diffuse dilatation of the hepatic arteries and veins with multifocal arteriovenous malformations, suggesting hht (fig . The brain mr showed no evidence of vascular malformation . On the 7th day of admission, after discharge, she has remained stable and comfortable, and will undergo percutaneous embolotherapy of the pavm in the near future . A pavm is a rare pulmonary vascular anomaly with an incidence of 2 - 3 per 100,000 population.1) pavms may be single or multiple, and the left lower lobe of the lung is the most common location of solitary pavm.2) greater than 80% of pavms are congenital, and approximately 50% of these are associated with hht, also known as rendu - osler - weber disease.2)3) a single pavm <2 cm in diameter on chest radiography usually does not cause symptoms.4) the most common complaint is epistaxis . A pavm can cause dyspnea, cyanosis, hemoptysis, hemothorax, and serious neurologic complications, such as strokes, seizures, and brain abscesses.4)5) patients with co - existing hht tend to have multiple arteriovenous malformations, rapid disease progression, and a higher complication rate.2) the brain, lungs, and liver are the most frequently involved organs in patients with hht.6) ea is a congenital cardiac malformation characterized by downward displacement of the attachment of the septal and posterior leaflets of the tricuspid valve.7) ea is also a rare disorder occurring in 1 per 200,000 live births.7) most cases are sporadic, and the embryologic and genetic contributors are unclear,8) but the ea gene might be located on chromosome 9.9) the genetic linkages to hht are located on chromosome 9 or 12.10) patients with ea may have various additional cardiovascular anomalies.8)11) however, there are no reports concerning the co - existence of a pavm or hht with ea . Although it is unclear whether or not a concurrent pavm and ea has an embryologic or genetic relationship, we report a patient with a pavm and ea who was suspected to have hht according to the curaao criteria.12) further genetic and embryonic studies are needed to detect a possible relationship between the two medical conditions.
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Cassava (manihot esculenta crantz) is one of the most important crops worldwide in terms of production and it is considered the most important food source for people living in tropical regions with arid soils . World production of cassava was estimated to be 276 millions of tons in 2013 and therefore it is considered to be the eighth most important product . Cassava is euphorbiaceae and belongs to the genus manihot, which contains 90 species approximately . Cassava is a high - yielding crop and its roots constitute a major food source for over 800 million people [46] mainly from africa, asia, and south america . This starch is transformed to be used in different industrial processes, in derivates such as alcohol and fructose - glucose syrups . This crop has high conversion rates of solar energy into carbohydrates and shows high tolerance to adverse abiotic stress . Cassava plants can survive after long periods of drought in arid and low fertility soils . Microsatellites (also known as short tandem repeats (strs), simple sequence repeats (ssrs) [11, 12], simple sequence length polymorphism (sslp), and sequence tagged microsatellite site (stms)) are sequence motifs of one to six bp, repeated in tandem . Ssrs have been employed for studies of diversity [10, 1618], phylogeny, and evolution, as molecular markers in marker - assisted selection and have contributed significantly to the construction of genetic linkage maps [15, 22]. Ssrs are considered allele specific, highly polymorphic, codominant, heterozygous, reproducible, economic, and multiallelic molecular markers . The ssrs offer the opportunity to be employed in different studies given that they are under neutral selection when located in noncoding regions . Ssrs can be classified in two classes: class i is composed of those with 20 bp repeats and class ii grouped ssrs from 12 to 20 bp . This classification is based on the observation that larger ssrs (class i) are demonstrated as more polymorphic than the shorter ssrs (class ii). Changes in length are due to a replication phenomenon known as slippage, although the unequal crossover in recombination also has a significant influence . These markers are relatively easy to automate and are generally considered more informative than other markers such as the single nucleotide polymorphisms (snps) due to the number of alleles that can be detected . Ssrs have the advantage of being pcr - based markers because the flanking sequences are suitable for primer design . Ssrs could be functionally implicated in chromatin organization, gene expression, and recombination hotspots and could affect dna replication . Furthermore, in some cases position and changes in ssrs are associated with phenotypic changes . Despite their importance and wide usefulness, ssrs genomic distribution studies in plant species are relatively scarce . With the advent and new advances in sequencing technologies, it is possible to analyze whole plant genomes for ssr discovery . Genomic studies of ssrs distribution have been conducted on arabidopsis where it was first found that coding regions have a low frequency of ssrs and that these regions are highly rich in trinucleotides and hexanucleotides . These analyses also led to the conclusion that 5 sequences had higher frequencies of ssrs than other genome sequences [20, 33] and that selective pressure acts differentially across genomic regions . An important feature is that in arabidopsis there is high prevalence of a - rich repeats . Molecular markers have been of paramount importance in cassava for genetic diversity [17, 3439], evolution, and molecular systematic studies . Ssrs in cassava have been favored over darts (diversity arrays technology) due to their codominant and multiallelic nature . Strategies for ssrs identification in cassava have included enriched dna libraries [25, 34] and the pursuit in ests sequences [10, 23]. Given that multiple groups have identified ssrs markers independently, it is highly probable that the same markers have been found several times and named differently . Genomic analyses of cassava ssrs would contribute to the understanding of cassava genome architecture and evolution and possibly correlate ssr's frequency, distribution, and sequence motifs, with genomic localization and function . We searched the cassava genome near - complete sequence (http://www.phytozome.com/) to gain an insight into genomic composition of cassava's ssrs . We carried out ssrs identification and characterization on the cassava's genome and their distribution in exons, introns, and utr (untranslated regions). A gene ontology (go) annotation was conducted for the ssrs present in the gene regions . Cassava whole genome sequence (version cassava4) was obtained from the phytozome database available at http://www.phytozome.net/cassava . This 532.5 mbp cassava genome sequence belongs to the genotype am560 - 2, an inbred lined derived from the cultivar mcol1505 . Ssrs identification was made with the pearl script misa (microsatellite identification tool, http://pgrc.ipk-gatersleben.de/misa). The parameters established for misa were adjusted for the identification of class i ssrs (length 20 bp) of di-, tri-, tetra-, penta-, and hexanucleotides . Class i ssrs were chosen because they have proven to be more polymorphic than ssrs of 12 to 20 bp . Mononucleotides were not considered because of the possibility of sequencing or assembly errors . For compound ssrs (distinct and adjacent ssrs), the maximum difference between two ssrs was set as 100 bp or less . For comparative purposes, a genomic identification of ssrs in other species the genomes of the related species selected were: populus trichocarpa (poplar) and linum usitatissimum (flax) which belongs to the order malpighiales; ricinus communis (ricinus) and jatropha curcas (jatropha) that belong to the euphorbiaceae family . Coding, 3 utr, and 5 utr cassava sequences were extracted using the biomart tool and introns were extracted using a pearl script . Altogether we obtained ~40 mbp of coding sequences, ~50 mbp of intron sequences, and ~2 mbp and ~4 mbp of 5 and 3 utr sequences, respectively . Ssrs density, ssr types, and motif distribution in cassava were assessed, analyzed, and compared through information stored in excel files . With the aim of obtaining the codon usage in cassava coding sequences we used the cusp program of emboss (the european molecular biology open software suite, cambridge, uk; http://emboss.sourceforge.net/). For the purpose of assigning functional categories to the sequences from the different gene regions containing ssrs, we searched for the classes to which each sequence belonged and were grouped according to go categories . The functional classes for each gene were obtained using the biomart data mining tool hosted in phytozome . The categorizer tool (http://www.animalgenome.org/bioinfo/tools/countgo/) was used to count go classes and group them into functional categories . The go_root classification method and single counting method were set as parameters for the go terms counting, to obtain a classification based on the three main categories: molecular function, biological process, and cellular component . Plant_goslim classification method and single counting method were used as parameters to group the sequences in the different subcategories . In order to conduct an exploratory analysis of the ssrs present in the complete cassava genome and to make comparisons with genomes of related species, we detect 26.579 class i ssrs in the cassava genome, using the misa tool . Considering the whole genome sequence length (536 mbp), the density of ssrs present in cassava was estimated to be ~50 ssrs per mbp (figure 1). In ricinus a density of 71,7 ssrs / mbp was identified, while in poplar we found 99 ssrs / mbp . The ssrs density was 30 ssrs / mbp for flax and 87,7 ssrs / mbp in jatropha . In general, the ssrs density in cassava was less than the average found in the assessed species (67,7 ssrs / mbp). Based on the type of repetition we found that 37,4% of all ssrs found in cassava correspond to dinucleotides, 24% are trinucleotides, 8,6% are tetranucleotides, 24,2% pentanucleotides, and 5,8 correspond to hexanucleotides (figure 2). Most ssrs in cassava genome are dinucleotides as have been observed in most species . Indeed in most of the evaluated species we observed that the most common ssr type is dinucleotide with the exception of flax, which has a higher number of trinucleotides . According to this, in flax dinucleotides just accounted for 24%, while trinucleotide accounted for 47,3% of all ssrs identified . A high proportion of pentanucleotides was found in cassava (24,2%) in contrast to the other species, which have an average of 9% of this type of ssr . To determine the distribution of ssrs in the cassava genome, we carried out an ssr search in coding, utrs, and intron sequences . We obtained coding sequences corresponding to 34.151 annotated genes, 3 utr sequences from 15.420 genes and 5 utr sequences from 14.111 genes . A pearl script allowed the extraction of 122.806 intron sequences corresponding to 24.309 genes . Following the ssrs search on each of these regions, as we expected, coding regions were found to have the lowest density of ssrs (figure 3). We found that the average density of ssrs in the whole genome is higher than in coding regions . For example we identified 49,9 ssrs / mbp ssrs in the whole genome while only 15,5 ssrs / mbp were found in coding sequences . Of 34.151 coding sequences analyzed, we found that 587 contained at least one ssr and 32 had more than one ssr . The density of ssrs in the whole genome was lower than in noncoding regions (introns and utrs) (figure 3). According to a previous report, which have indicated that utr regions are ssr rich, we observed that in cassava 5 utrs contain the greatest amount of ssrs; we identified 434 (196,1 ssrs / mbp) ssrs in 5 utrs and 202 (50,5 ssrs / mbp) ssrs in 3 utrs . We expected to find more ssrs in the 3 utr than in the 5 utrs as it has been reported previously . However, we observed that 5 utr sequences have between 2,4- and 12,6-fold higher ssr density than other regions and almost fourfold higher density than in the whole genome . Higher ssrs densities in 5 utrs were also observed in arabidopsis and a similar proportion to that we found was identified in rice . The existence of more than one ssr in a single sequence was found to be scarce . Only 0,09% of coding sequences, 0,39% of introns, 0,16% of 5 utr, and 0,02% of 3 utr have more than one ssr in a single sequence . A comparison of the motifs in different genomic regions was done because the motifs proportion changes across the genome in a similar manner to the ssrs number . As a result of selection pressure it has been noted that most of the ssrs found in coding regions are tri- or hexanucleotides avoiding frame shifts in this way [20, 49, 50]. This situation was also found in cassava coding sequences where tri- and hexanucleotides account for 95,6% of the ssrs and almost no tetra- and pentanucleotides were identified on these regions . The results also suggest that noncoding sequences, as observed in the whole genome, have a high proportion of pentanucleotides (figure 4). This type of ssr may have evolved from polya stretches and could generate important secondary structures . In the entire cassava genome there is prevalence of the at / at motif represented by approximately 22% of the ssrs identified (figure 5(a)). This is the most abundant motif found in several plant genomes [12, 15, 26, 48, 51]. In cassava coding sequences we found a prevalence of aag / ctt and agc / ctg (figure 5(b)). Ssrs in coding regions could give an indication of codon usage preference . To determine if the ssrs identified in coding regions correspond to the most used triplets in cassava the motif aag which is the most commonly found in coding sequences is in fact the third most used codon in cassava with a percentage of 3,2% among all the nucleotide combination triplets (table 1). The ssr motifs agc / ctg, agg / cct, atc / atg, and acc / ggt that were also found in high frequencies are used in percentages between 0,9 and 1,4% indicating that these codons are not used frequently . In noncoding sequences the most common ssr motif is ag / ct (figures 5(c), 5(d), and 5(e)) similar to previous reports for several plant species [27, 48, 53]. In 5 utr sequences, most of the ssrs were of the ag / ct and aag / ctt type . Similar observations were reported in other dicotyledonous species like arabidopsis and soybean, but not in monocot plants such as rice or maize . These differences in motif distribution in upstream gene sequences often lead to differences in genomic structure and gene regulation on both groups of plants [20, 49, 54]. The gc type was not identified in any of the cassava sequences groups . In order to gain some insight into the putative function of the genes containing ssrs, we classified those genes according to go categories (figure 6). Putative molecular function was attributed to 55,4% of gene coding sequences that contain ssrs, 51,3% of intronic sequences, and 54,7% and 44,3% of 3 and 5 utr sequences, respectively . About 35,7% of the sequences containing ssrs belonged to genes classed in biological processes, while 10,2%, 12,5%, 9,9%, and 18,7% of the coding, intronic, and 3 and 5 utr sequences, respectively, corresponded to genes grouped in the cellular component category . A detailed categorization for each different go category was made (supplementary figures 1, 2, 3, and 4 in supplementary material available online at http://dx.doi.org/10.1155/2014/471461). When the subcategories comprised in biological process were compared, we observed that although there were some evident differences, the majority of genes containing ssrs belonged to the cellular process subcategory . The metabolic, biosynthetic, and protein metabolic processes were the categories with more ssr - containing genes . In terms of the cellular component, a common feature was that sequences containing ssrs belonged to genes that encoded proteins located frequently inside cell, cell membrane, and nucleus . Regarding the molecular function, the ssrs - containing genes were mostly related to catalytic, binding, hidrolase, transferase, and transporter activity . Interestingly we found that cassava genes coding for proteins located in thylakoid, vacuole, and golgi apparatus contain ssrs exclusively on intron sequences . A similar situation was observed for genes related to carbohydrate binding, pollen - pistil interaction, pollination, regulation of gene expression, epigenetic process, and reproduction . We also observed that there is just one gene with ssr associated with embryonic development and its ssr is located on the 5 utr of the gene . In addition some genes contain ssrs in their coding and intron sequences but not in the utr regions (related to carbohydrate metabolic processes, lipid binding, motor activity, and genes that encode proteins located on the cell wall, external encapsulating structures, and peroxisomes). In a similar manner, genes related to response to biotic stimulus have ssrs exclusively on the coding and intron sequences . On the other hand genes related to response to abiotic stimulus had ssrs exclusively on their utr sequences . For genes associated with response to endogenous stimuli and stress response, ssrs were detected in all their regions (intron, coding, and utrs sequences). We found that, as expected, cassava has high frequencies of dinucleotides and that a unique feature of this plant was its unusual high frequency of pentanucleotides . The predominance of a specific ssr class has been observed in rice . In terms of gene analysis, coding sequences are the regions with the lowest density of ssrs while the 5 utrs are the counterpart with the highest content . In general we identified 621 ssrs (15.5 ssrs / mbp) in coding sequences, 4.120 ssrs (82,3 ssrs / mbp) in introns, and 434 (196,1 ssrs / mbp) and 202 (50,5 ssrs / mbp) ssrs in 5 and 3 utr sequences, respectively . It is estimated that in cassava there are approximately 1.000 ssrs previously identified . After following the methodology proposed here we identified 26.579 ssrs in the cassava genome . Previous studies on ssr detection in cassava genomic libraries reported the identification of 12, 32, 545, or 1.576 ssrs . Previous efforts in searching cassava ssrs in sequences yielded the identification of 531, 49, 836, 1.889, 431, 7.270, or 163 ssrs in cassava ests . The low number of ssrs previously found could be explained for the low number of genome sequences reported at that moment . With the recent cassava genome release a global and genomic analysis of ssrs is possible . Additionally, no study had focused earlier on the distribution of ssrs along different gene regions in cassava . The identification of ssrs in several gene sequences is not only informative but also useful to develop makers to map the genes in which they reside . However it is important to note that some of these could correspond to anonymous, with an unknown function, type of markers and although they have been useful for developing genetic maps and for diversity studies most of them have no specific known function . Through ssrs data comparison we determined that cassava has only 49,9 ssrs / mbp being one of the species with lower ssrs densities in its genome compared with phylogenetically closer species . Based on cassava nuclear dna quantity it has been estimated that the cassava genome is 772 mbp and nonetheless the sequenced genome is 533 mbp . The lacking sequences of about ~240 mb could consist of repetitive dna which has not been assembled . Although it has been observed that ssrs are preferentially found in nonrepetitive dna, the low quantity of ssrs in cassava could be associated with the possibility that the nonassembled sequences in the genome would have a considerable amount of ssrs . It is important to note that ssrs identified in gene sequences are potential powerful molecular markers for use in breeding programs . Due to their location inside genes, these markers save effort and resources in the early stages of searching for markers closely linked to particular genes . The ssr markers identified in this work would be an important resource for genetic mapping analysis of the genes in which they are located . In addition, they could help to make phylogenetic analysis to understand the diversity of those genes . Owing to their mutation properties, these ssrs would give hints about evolutionary changes on the cassava genome . The prevalence of dinucleotide ag / ct in cassava has been observed in previous studies where the search was made through the development of ssrs libraries [25, 34] or by in silico ssr search in ests [10, 56]. Here we observed that this is the second most observed motif in the genome and it is the most frequent in noncoding sequences . The latest result is consistent with the statement that the ag / ct is in fact the most common dinucleotide in vascular plant ests [52, 53] and in coding regions according to morgante et al . . We observed that in cassava the dinucleotide ac / gt or ga / ct accounted only for 1,5% of the ssrs in all the evaluated sequences, while the other dinucleotides added up to 38% . Compared to the genomes of animals previous studies highlighted the absence of this motif in sequences of several plant species [51, 53] and are indeed the least frequent ssr in almost every assessed organism with the exception of escherichia coli . The availability of cassava genome sequences enabled a more effective assessment of ssr marker distribution in this study . . This will be quite useful in cassava since the genome sequence is highly fragmented . Finally the go categories assignment of the genes where the ssrs were identified can be useful in studies where the objective is to map a specific group of genes corresponding to a functional category such as abiotic or biotic stress.
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The treatment of lymphoma depends on the type, location and stage of the disease . The majority (50%90%) of oal is low - grade mucosa associated lymphoid tissue (malt) lymphoma . According to the world health organization modification of the real classification, the other 4 main types of oal are follicular cell, mantle cell, diffuse large b cell (dlbcl) and lymphoplasmacytic lymphoma . They are all b cell, non - hodgkin s lymphoma, the subtype derived from various stages of b cell maturation . The new classification of lymphoma is dependent on histological, immunophenotypic and molecular genetic analysis . Hodgkin s lymphoma does not affect the ocular adnexal structures unless there is widespread systemic involvement or a history of previous systemic hodgkin s lymphoma . According to the ann - arbor staging system lymphoma confined to the orbit is designated as stage i, involvement of adjacent structures (sinuses, tonsil and nose) is stage ii . Stage iii is abdominal nodal disease below the diaphragm and iv by definition refers to disseminated involvement of one or more extra - nodal sites (eg, liver, bone) and e is used when there is a local extra - nodal extent (eg, ie, iie, iiie and i ve). The grading system is similar to nodal lymphomas . Broadly speaking, oal with systemic involvement requires systemic treatments whereas low - grade (emzl or follicular cell) lymphoma localized to the eye can be very successfully managed with local treatments such as cryotherapy, surgical excision or external beam radiotherapy . When considering local treatment it is important to exclude bilateral ocular involvement . Even in the absence of clinical signs staging investigations should include full blood count, hepatic enzymes including ldh, ct abdomen, chest and pelvis, mri or ct of the orbits, and a bone marrow biopsy and aspirate . This is only appropriate for low - grade lymphoma localized to the conjunctiva or orbit with no systemic involvement . Surgical excision or cryotherapy is most appropriate for localized stage 1 conjunctival emzl / malt lymphoma.1 this is not curative treatment but can control local disease and relieve local symptoms . Tanimoto et al2 reported very good local control of stage 1 malt lymphoma following biopsy or surgical resection alone . Patients were followed for a median of 7.1 years, 69% of patients did not require any further treatment . The estimated overall survival rates were 94% at 10 years and 71% at 15 years.2 although, patients should be closely followed for recurrence, histological transformation of the tumor and long - term systemic manifestations of lymphoma . This has been the mainstay of treatment for localized oal for many years with excellent control rates . Central nervous system prophylactic radiotherapy is not required as metastatic intraocular or brain involvement is rare.3 the dose of radiation is tapered according to the grade of the lymphoma, 28 to 36 gy for low - grade lymphoma and 30 to 40 gy for high - grade . Emzl, lymphoplasmacytic and follicular cell lymphoma are considered low grade, whereas dlbcl and mantle cell are high - grade lymphomas . Five - year local control rates for emzl and follicular lymphoma after a dose of more than 30 gy reach 100%.4 however, high rates of delayed systemic recurrence suggest long - term follow up studies are required to assess the true benefit of radiotherapy.5 there is ongoing debate about use of lens shielding radiotherapy to protect the lens from cataract formation . Some studies show no effect on local recurrence, whereas in others all recurrences have occurred in patients whose lenses were shielded.6,7 partial orbit volume irradiation leads to unacceptable incidence of intraorbital recurrence and is presently not recommended . Ocular complications, including dry eye and cataract are frequently reported in patients that have long - term follow up.8 these localized complications need to be kept in mind by the treating oncologist . Ifn - alpha has been used for the treatment of systemic lymphoma for more than 20 years . One study treated 5 patients with intralesional ifn - alpha using an intensive protocol.9 an initial complete response was seen in all patients with stage i oal, one patient with more advanced disease at presentation (stage iia) died from systemic lymphoma . Long - term follow up studies are required to assess the value of this treatment . Rituximab is a monoclonal antibiotic against cd20 positive b cells, which is being considered as an alternative first line treatment for localized cd20 positive oal to avoid the ocular complications of radiotherapy . Antibody - induced destruction of cd20 positive b cells can occur by apoptosis, complement mediated cytolysis and antibody - dependent, cell - mediated cytoxicity . Rituximab has also been used as monotherapy for localized emzl / malt, in some cases complete regression can be achieved . Initial responses are encouraging but the efficacy of rituximab is lower than that reported for gastric malt lymphoma,10 as long - term distal relapse is seen in 50% of cases . Very few case series are published on the effect of single agent rituximab on ocular malt / emzl . Ferreri and associates achieved regression of malt lymphoma using first line single agent rituximab in all 5 newly diagnosed patients; however early relapse was seen in 4 of the 5 patients.10 therefore, close follow up intervals are required to detect early relapse when rituximab is used alone . Rituximab was not successful for the treatment of local relapse.10 greater success is achieved when rituximab is used in combination with chemotherapy . Rituximab and chlorambucil were used as first - line therapy for 9 patients with newly diagnosed emzl . Treatment achieved complete remission in 89% with a median follow up of 2 years.11 first - line chemotherapy is often treatment of choice for mantle cell oal, as a high proportion of patients have systemic involvement at presentation . Encouragingly, rituximab containing chemotherapy for mantle cell oal had a significantly better 5-year survival than patients receiving chemotherapy alone (83% overall 5-year survival verses 8%).12 combination treatment with rituximab and chemotherapy or radioimmunotherapy may achieve a more durable local response and improve patient survival.1113 chemotherapy is reserved for treatment of oal when there is systemic involvement, except for the high - grade dlbcl when systemic chemotherapy is first - line treatment even for stage i disease . More recently chlorambucil and the purine analogs fludarabine, clardarabine and pentostatin have also been used . The extranodal lymphoma study group (ielsg) is currently recruiting patients to a clinical trial investigating the role of rituximab alone or in combination with chlorambucil for malt lymphoma (ielsg #19).14 novel mechanisms of tumor pathogenesis have related lymphoma to infection leading to new approaches to diagnosis and treatment . Chronic antigen stimulation may result in an abnormal lymphoproliferative response that ultimately develops into lymphoma . This association is most profound for gastric malt cell lymphoma, which is related to chronic gastritis from helicobacter pylori infection in more than 90% of cases.15 h. pylori has been isolated from ocular lymphoma specimens . However, h. pylori eradication is not effective treatment for oa malt lymphoma.16 chlamydia psittaci infection has also been linked to oal . Ferreri and associates17 identified c. psittaci in 80% of italian oa malt lymphoma specimens . In korea, c. psittaci was isolated in 78% of oa malt lymphoma specimens.18 subsequent reports from south florida, new york state, chicago, netherlands, japan and france failed to identify dna for c. psittaci in any of their oa malt lymphoma specimens.1924 there is wide geographical variation in the prevalence of c. psittaci infection in oa malt lymphoma specimens.25 of the european countries studied, the lowest rate of infection (12%) was present in the uk . Results following first line antibiotic treatment are variable.26 abramson and coworkers27 documented a clinical response following antibiotic treatment in 3 patients with oa malt lymphoma . Ferreri and associates28 recorded an objective response following oral doxycycline in 4 out of 9 patients with c. psittaci positive oa malt lymphoma . Although, grunberger and associates29 were unable to replicate these results . In a multicenter prospective trial, 27 patients with ocular adnexal malt lymphoma were treated with oral doxycycline . C. psittaci dna positive cases had an overall response rate of 64%, the response was of short duration.30 a clinical response was also seen in 38% in c. psittaci dna negative cases.30 lack of response to antibiotic therapy may be a reflection of the biological and genetic heterogeneity of this disease . The extranodal lymphoma study group (ielsg) has activated a prospective trial (ielsg #27) into oal to explore the role of antibiotic treatment and to identify potential infectious agents.14 in summary, advances in our knowledge of the immunophenotypic and genetic alterations in lymphoma cell lines have led to a better understanding of the pathogenesis of the disease . The interplay between a possible infective agent and the immune response has allowed a new wave of treatment possibilities . Staging the patient at diagnosis guides subsequent management and local treatments should be reserved for localized disease . However, the practical approach to the patient remains first do no harm and with this in mind ocular oncologists are tempted to administer an initial trial of oral antibiotics for stage 1 malt lymphoma . Although, general blanket treatment with antibiotics should be discouraged unless there is proof of an infective agent . We recommend a diagnostic biopsy with immunophenotyping and genetic analysis to determine the grade of the lymphoma combined with pcr analysis to determine the presence of an infective agent . If h. pylori or c. psittaci infection is found a trial of oral doxycycline (100 mg twice daily for 3 weeks) we have also seen some early impressive responses to systemic rituximab (weekly intravenous infusion over 6 weeks). However, close follow up is required as others have reported early local relapse with rituximab alone.10 at present, despite the ocular side effects, localized radiotherapy still remains the standard first - line treatment for stage i low - grade oal.
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For well over a decade, the association of connective tissue growth factor (ccn2, also known as ctgf) with fibrotic diseases has been the driving force behind many investigations designed to understand its biology and biochemistry . There are now countless studies that attest to the importance of this molecule in fibrotic pathology . Indeed, the initial recognition of ccn2 as a downstream mediator of the pro - fibrogenic effects of transforming growth factor beta (tgf-) has resulted in elegant investigations of the transcriptional regulation of the ccn2 gene as well as to its recognition as a potentially superior anti - fibrotic target (leask et al . Extensive investigations have shown convincingly that ccn2 expression is up - regulated in fibrotic disorders, that it is produced temporally and spatially in close proximity to fibrotic areas in vivo, and that it is often localized to and a target for fibrotic (pro - collagenic) cells (leask et al . Antagonists of ccn2 have proven effective in blocking pro - fibrogenic ccn2 signaling pathways in vitro and have yielded promising data with respect to preventing or reversing fibrosis in several animal models in vivo (brigstock 2009). What more, then, do we need to know? Unfortunately, as with many other areas of ccn2 research, the recognition of ccn2 as a matricellular protein rather than a conventional growth factor (rachfal and brigstock 2005; leask and abraham 2006; chen and lau 2009) requires us to think differently as to how this molecule is involved in fibrosis . Collectively, its multi - domain structure, association with the extracellular matrix, utilization of multiple receptors on the cell surface (integrins, heparan sulfate proteoglycans, low density lipoprotein receptor - related protein, nerve growth factor tyrosine kinase receptor), binding interactions with other cell - regulatory molecules (tgf-, insulin - like growth factor, vascular endothelial growth factor), and propensity for proteolytic cleavage into various bioactive fragments adds many layers of complexity to its mode of action and we have either a very rudimentary understanding or no knowledge at all as to the importance of these or other factors in determining the fibrotic response to ccn2 . Data reported over the last few months regarding some new and very interesting ccn2 transgenic models have now begun to shed light on this critical issue . These studies have revealed that the consequences of ccn2 overexpression are not necessarily predictable and that the manifestation of ccn2 fibrotic activity in vivo involves crucial interactions with co - stimulatory signals in the microenvironment . Four different systems have recently been described in which ccn2 transgenes were individually expressed in podocytes (yokoi et al . 2009), cardiomyocytes (panek et al . 2009) or respiratory epithelial cells (wu et al . 2009) to achieve overexpression in, respectively, the kidney, liver, heart, or lung . Kidney - overexpressing transgenic c57bl/6 j mice constitutively expressed a mouse ccn2 transgene that was under the control of a human nephrin promoter; mice were examined up to 24 weeks of age (yokoi et al . Liver - overexpressing transgenic fvb / n mice were produced in this laboratory and constitutively expressed a human ccn2 transgene that was under the control of a mouse albumin promoter enhancer; mice were examined up to 28 weeks of age (tong et al . Heart - overexpressing fvb / n mice or sprague - dawley rats constitutively expressed rat ccn2 under the control of a mouse myosin light chain 2 promoter; animals were examined up to 7 months of age (panek et al . Lung - overexpressing transgenic c57bl/6 j mice conditionally expressed human ccn2 under the control of the clara cell secretary protein promoter using a deoxycycline - inducible system; transgene expression was induced over the first 2 weeks of post - natal life at which time the mice were examined (wu et al . Directed overexpression of the ccn2 transgene was successfully documented in all of these systems . However, the most surprising observation was that ccn2 overexpression in the kidney, liver, or heart consistently failed to cause any kind of fibrotic reaction whatsoever . Kidney transgenics exhibited no glomerular abnormalities or proteinuria while liver transgenics exhibited no hepatic histological abnormalities or elevated liver function tests . In contrast, the heart transgenics exhibited cardiomyocyte hypertrophy and age - dependent heart disease which progressed from compensatory hypertrophy to ventricular dilation and systolic heart failure but even so these dysfunctional hearts were not fibrotic . On the other hand, the lung transgenic mice provided clear evidence that ccn2 overexpression during the first 2 weeks of post - natal life causes abnormal alveolarization and impaired formation of the alveolar vascular network and that these deficiencies were associated with fibrosis in and around the alveolar septa, bronchi, bronchioles, and vessels . One possibility is that the pro - fibrotic effects of ccn2 may be dependent on the developmental stage of the target organ . Perhaps mere overexpression of ccn2 in the kidney, liver or heart was insufficient to drive fibrosis because these organs were examined in mature animals whereas the lung transgenics were analyzed at an early and critical time of lung development when, possibly, the transgenic ccn2 protein was able to interact with and somehow impair essential growth, differentiation or maturation signaling pathways . If this is the case, one might speculate that the pathological sequelae of pulmonary ccn2 overexpression might be less severe in more mature adult animals, a question that is highly suitable to the inducible transgenic system developed for these mice . Likewise, it also would be interesting to assess fibrosis during critical stages of organ development in the kidney, liver and heart transgenics, although if any such abnormality was present during pre- or early post - natal life it appears to be resolved by adulthood . Interesting additional data were reported for the kidney and liver transgenics that provide important clues as to the role of ccn2 in fibrotic pathways . When the kidney transgenic mice were challenged with streptozotocin to induce type i diabetes, they exhibited a more pronounced glomerular pathology than wild - type littermates in as much as they demonstrated mesangial expansion, podocyte loss, and a reduction in the activity of matrix - metalloprotease 2, a matrix - degrading enzyme that is often suppressed in fibrotic conditions . Thus the effect of the ccn2 transgene was to exacerbate some aspects of a fibrotic phenotype in a setting of diabetic nephropathy, although expression of extracellular matrix components such as fibronectin or collagen was unchanged and no fibrosis per se was observed (yokoi et al . A somewhat similar but more compelling scenario was observed in our liver transgenic mice after they were subjected to liver injury by carbon tetrachloride administration or ligation of the bile duct (tong et al . Both of these interventions are well - characterized methods of inducing hepatic fibrosis yet the transgenic mice fared worse than their wild - type littermates as shown by the presence of more -smooth muscle actin - positive cells (presumptive activated hepatic stellate cells, a principal pro - fibrogenic cell type in the liver), increased deposition of collagen, and elevated mrna expression of key fibrotic markers including -smooth muscle actin, collagen 1(i), and tissue inhibitor of matrix - metalloprotease-1 (the latter of which blocks matrix breakdown and favors collagen accumulation). This phenomenon was highly reproducible as further exemplified by the additional data shown in fig . 1 which illustrates that the liver transgenic mice treated chronically with the hepatotoxin thioacetamide show a greatly exaggerated fibrotic response in their livers as compared to their wild - type counterparts . We have further shown that homozygous liver transgenic mice exhibit more severe hepatic fibrosis than heterozygous liver transgenic mice in response to carbon tetrachloride (tong et al . This finding is important because it suggests that ccn2 transgene dose may also account for some of the phenotypic variability reported between the four transgenic systems, a question that will require clarification through further experimental analysis . 2009). Four - week old wild - type (a, b) or homozygous transgenic (c, d) mice (n = 4 per group) were treated three times a week with thioacetamide (200 mg / kg i.p .) For four weeks . Liver sections were stained immunohistochemically for -smooth muscle actin (a, c; brown staining) or with sirius red for collagen deposition (b, d; red staining). Staining is shown at 10 enhanced thioacetamide - induced hepatic fibrosis in ccn2 transgenic livers . 2009). Four - week old wild - type (a, b) or homozygous transgenic (c, d) mice (n = 4 per group) were treated three times a week with thioacetamide (200 mg / kg i.p .) For four weeks . Liver sections were stained immunohistochemically for -smooth muscle actin (a, c; brown staining) or with sirius red for collagen deposition (b, d; red staining). Staining is shown at 10 collectively, the observations to date support the notion that the pro - fibrogenic properties of ccn2 are highly contextual . Whereas normal adult tissues are seemingly refractory to elevated ccn2 levels (at least in terms of the fibrotic response), the pro - fibrotic properties of ccn2 are manifested in conjunction with other changes in the microenvironment, such as those induced by tissue damage or during wound healing . The co - stimulatory signals that contribute to this phenomenon may include known synergistic factors such as tgf- or fibronectin (tong et al . 2009), changes in the level or nature of signaling through ccn2 receptors, components of additional signaling pathways in injured tissues such as those associated with inflammation, necrosis or wound healing, or modifications to the structure or composition of the extracellular matrix which is a reservoir of many bioactive molecules including ccn2 itself . In addition, injured tissues contain infiltrating immune cells and macrophages as well as specialized cells that promote wound healing, and the presence of these cells may be important either directly or indirectly for ccn2 fibrotic action . However, data from the heart transgenic animals show that pathways of tissue damage or pathology that are themselves ccn2-dependent do not necessarily cause co - stimulatory pro - fibrogenic pathways to be triggered . Further, since different strains of mice show variable responses to common fibrotic agents (schrier et al . 2004) as well as in production of ccn2 itself (cardoso et al . 2009), genetic background likely plays an important role in influencing the fibrotic action of ccn2 . The concept of a requirement for co - stimulants may well prove to be the unifying feature in these animal models in that elevated ccn2 levels are not fibrotic (e.g. Kidney, liver, heart) unless there is a background of other signaling events produced by processes such as growth and development (e.g. Lung) or injury and wound healing (e.g. Kidney, liver). Indeed, the data obtained from the ccn2 transgenic animals reinforce previous in vivo studies which have pointed to a modifying rather than causative role for ccn2 in fibrosis . For example, subcutaneous co - administration of tgf- and ccn2 resulted in protracted and sustained dermal fibrosis whereas this effect was not achieved by either tgf- or ccn2 individually (mori et al . Additionally, balb / c mice are genetically resistant to bleomycin - induced lung fibrosis but nonetheless developed fibrosis after administration of bleomycin and a ccn2-overexpressing adenovirus (bonniaud et al . 2004). The behavior of ccn2 in all of these studies is likely illustrative of its role as a matricellular protein whereby it resides in the matrix or is tethered to the cell surface and modifies cellular function by sensing changes in the environment through its engagement of a myriad of molecular binding partners . Ccn2 has been likened to a molecular hub or adapter (leask and abraham 2006), capable of integrating inputs from multiple signaling pathways and regulating cellular responses accordingly . Readouts for ccn2 are highly dependent on a plethora of other molecular cues within the microenvironment and conclusions about its activity including its role in fibrosis cannot be made without careful attention to contextual considerations . In turn, this raises broader questions such as how to best design meaningful mechanistic studies to probe ccn2 action (especially those that rely on in vitro approaches) and whether components of its co - stimulatory pathways are useful targets for fibrotic therapy in vivo . In conclusion, ccn2 transgenic systems have begun to provide valuable information about the fibrotic pathways that may be triggered by ccn2 overexpression in vivo . On the one hand, overexpression of ccn2 does not necessarily lead directly to fibrotic pathology (e.g. Kidney, liver, heart) but can cause severe non - fibrotic tissue damage due to other deleterious effects on cell function (e.g. Heart). On the other hand, overexpression of ccn2 in concert with signaling pathways associated with growth and development (e.g. Lung) or fibrosing injuries (e.g. Kidney, liver) can lead to the initiation or exacerbation of fibrosis.
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The hippocampus is a major component of the brain of humans and other mammals located bilaterally in the medial temporal lobe, underneath the cortical surface . It belongs to the limbic system and plays important roles in long - term memory and spatial navigation particularly the ca3 subregion of the hippocampus . Our earlier studies have shown that glycyrrhiza glabra (gg) aqueous root extract treatment (2, 4 and 6 weeks duration of the treatment) in different age grouped (1 and 3 months old) wistar albino rats enhances both spatial learning ability and retention of learned tasks accordingly, the present study was designed to study the effects of gg root extract on rat hippocampal neurons particularly the ca3 subregion of the hippocampus . The roots of gg were purchased from a local ayuredic store in udupi, karnataka, india during 2/4/2012 . The crude aqueous extract of gg was prepared by macerating dried powdered root with respective solvent for 24 h. the macerated powdered roots were then extracted by using soxhlet extractor for 36 h, 1 - 2 cycles / h . The extract was dried and weighed . A brownish black waxy residue with 16% yield was obtained . This aqueous extract of gg was administrated orally to separate groups of 1-month old male wistar albino rats in four different doses 75, 150, 225 and 300 mg / kg respectively . The experimental protocol was approved during september 2011 by the institutional animals ethics committee, yenepoya university and care of laboratory animals was taken as per committee for the purpose of control and supervision of experiments on animals (cpcsea) guidelines . Rats were housed individually (animal house, yenepoya university, reg.no 347/cpcsea) in polypropylene cages of standard dimensions (22.5 cm 35.5 cm 15 cm) and maintained at temperature (25 c 2 c) and light (light period, 08.00 - 20.00) in a controlled room with relative humidity of 50 - 55% . Food and water were provided ad libitum . Experiments were carried out between 09:00h and 14:00 h. rats were randomly divided into eight groups . Group i-: control (n = 6): a known volume of distilled water was administrated orally each day for 4 weeksgroup ii-: diazepam control (n = 6): diazepam 7 mg / kg was injected i.p . 20 min before the test sessiongroup iii (n = 6): received 75 mg / kg aqueous extract of gg orally every day for 4 weeksgroup iv (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeksgroup v (n = 6): received 225 mg / kg aqueous extract of gg orally every day for 4 weeksgroup vi (n = 6): received 300 mg / kg aqueous extract of gg orally every day for 4 weeksgroup vii-: gg 150 mg + diazepam (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks . N = number of animals.group viii-: gg 225 mg + diazepam (n = 6):received 225 mg / kg aqueous extract of gg orally every day for 4 weeks . Group i-: control (n = 6): a known volume of distilled water was administrated orally each day for 4 weeks group ii-: diazepam control (n = 6): diazepam 7 mg / kg was injected i.p . 20 min before the test session group iii (n = 6): received 75 mg / kg aqueous extract of gg orally every day for 4 weeks group iv (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks group v (n = 6): received 225 mg / kg aqueous extract of gg orally every day for 4 weeks group vi (n = 6): received 300 mg / kg aqueous extract of gg orally every day for 4 weeks group vii-: gg 150 mg + diazepam (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks . Group viii-: gg 225 mg + diazepam (n = 6):received 225 mg / kg aqueous extract of gg orally every day for 4 weeks . After the treatment period, all experimental animals were subjected to spatial learning (morris water maze, and elevated plus maze) tests . At the end of the spatial memory tests, the rats were deeply anesthetized with pentobarbitone and killed; their brains were removed rapidly and fixed in rapid golgi fixative . Briefly, tissues were fixed for 5 days in golgi fixative and impregnated with a 1.5% aqueous silver nitrate solution for 48 h. sledge microtome sections of 120-m thickness were excised, dehydrated, cleared and mounted with distrin plasticizer xylene mounting media . From each rat, hippocampal ca3 pyramidal neurons were traced using camera lucida and their dendritic branching points (a measure of dendritic arborization) and dendritic intersections (a measure dendritic length) were quantified . Concentric circles with a distance of 20 m between 2 adjacent concentric circles were drawn on a transparent sheet for quantification of dendritic branching points and dendritic intersections . The number of branching points between the two concentric circles that is each successive 20 m concentric zone (circle) was counted . The dendritic intersections point (a dendrite intersected a given concentric circle) at each concentric circle were counted . Both branching points and intersections were counted up to a radial distance of 140 m from the center of the cell body of the ca3 neuron . Mean number of apical and basal dendritic quantification (dendritic branching points and dendritic intersections) in each concentric zone were calculated . The roots of gg were purchased from a local ayuredic store in udupi, karnataka, india during 2/4/2012 . The crude aqueous extract of gg was prepared by macerating dried powdered root with respective solvent for 24 h. the macerated powdered roots were then extracted by using soxhlet extractor for 36 h, 1 - 2 cycles / h . The extract was dried and weighed . A brownish black waxy residue with 16% yield was obtained . This aqueous extract of gg was administrated orally to separate groups of 1-month old male wistar albino rats in four different doses 75, 150, 225 and 300 mg / kg respectively . The experimental protocol was approved during september 2011 by the institutional animals ethics committee, yenepoya university and care of laboratory animals was taken as per committee for the purpose of control and supervision of experiments on animals (cpcsea) guidelines . Rats were housed individually (animal house, yenepoya university, reg.no 347/cpcsea) in polypropylene cages of standard dimensions (22.5 cm 35.5 cm 15 cm) and maintained at temperature (25 c 2 c) and light (light period, 08.00 - 20.00) in a controlled room with relative humidity of 50 - 55% . Food and water were provided ad libitum . Group i-: control (n = 6): a known volume of distilled water was administrated orally each day for 4 weeksgroup ii-: diazepam control (n = 6): diazepam 7 mg / kg was injected i.p . 20 min before the test sessiongroup iii (n = 6): received 75 mg / kg aqueous extract of gg orally every day for 4 weeksgroup iv (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeksgroup v (n = 6): received 225 mg / kg aqueous extract of gg orally every day for 4 weeksgroup vi (n = 6): received 300 mg / kg aqueous extract of gg orally every day for 4 weeksgroup vii-: gg 150 mg + diazepam (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks . N = number of animals.group viii-: gg 225 mg + diazepam (n = 6):received 225 mg / kg aqueous extract of gg orally every day for 4 weeks . Group i-: control (n = 6): a known volume of distilled water was administrated orally each day for 4 weeks group ii-: diazepam control (n = 6): diazepam 7 mg / kg was injected i.p . 20 min before the test session group iii (n = 6): received 75 mg / kg aqueous extract of gg orally every day for 4 weeks group iv (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks group v (n = 6): received 225 mg / kg aqueous extract of gg orally every day for 4 weeks group vi (n = 6): received 300 mg / kg aqueous extract of gg orally every day for 4 weeks group vii-: gg 150 mg + diazepam (n = 6): received 150 mg / kg aqueous extract of gg orally every day for 4 weeks . Group viii-: gg 225 mg + diazepam (n = 6):received 225 mg / kg aqueous extract of gg orally every day for 4 weeks . After the treatment period, all experimental animals were subjected to spatial learning (morris water maze, and elevated plus maze) tests . At the end of the spatial memory tests, the rats were deeply anesthetized with pentobarbitone and killed; their brains were removed rapidly and fixed in rapid golgi fixative . Briefly, tissues were fixed for 5 days in golgi fixative and impregnated with a 1.5% aqueous silver nitrate solution for 48 h. sledge microtome sections of 120-m thickness were excised, dehydrated, cleared and mounted with distrin plasticizer xylene mounting media . From each rat, hippocampal ca3 pyramidal neurons were traced using camera lucida and their dendritic branching points (a measure of dendritic arborization) and dendritic intersections (a measure dendritic length) were quantified . Concentric circles with a distance of 20 m between 2 adjacent concentric circles were drawn on a transparent sheet for quantification of dendritic branching points and dendritic intersections . The number of branching points between the two concentric circles that is each successive 20 m concentric zone (circle) was counted . The dendritic intersections point (a dendrite intersected a given concentric circle) at each concentric circle were counted . Both branching points and intersections were counted up to a radial distance of 140 m from the center of the cell body of the ca3 neuron . Mean number of apical and basal dendritic quantification (dendritic branching points and dendritic intersections) in each concentric zone were calculated . Figures 14 illustrates camera lucida tracings (a1, b1, c1, d1, e1, f1, g1 and h1) of golgi - stained (silver nitrate impregnated) hippocampal ca3 pyramidal neurons (a, b, c, d, e, f, g and h) of control and different doses of the aqueous root extract of gg treated rats for 4-weeks . Representative photomicrographs (a - b) and camera lucida tracings (a1-b1) of golgi - stained hippocampal ca3 neurons . A and a1-control (group i); b and b1- diazepam control (group ii); black arrow- basal dendrites of hippocampal ca3 neurons; red arrow- apical dendrites of hippocampal ca3 neurons representative photomicrographs (c - d) and camera lucida tracings (c1-d1) of golgi - stained hippocampal ca3 neurons . C and c1- hippocampal ca3 neurons of rats treated with 75 mg / kg aqueous extract of gg orally every day for 4 weeks (group iii); d and d1- hippocampal ca3 neurons of rats treated with 150 mg / kg aqueous extract of gg orally every day for 4 weeks (group iv) representative photomicrographs (e - f) and camera lucida tracings (e1- f1) of golgi - stained hippocampal ca3 neurons . E and e1- hippocampal ca3 neurons of rats treated with 225 mg / kg aqueous extract of gg orally every day for 4 weeks (group v); f and f1- hippocampal ca3 neurons of rats treated with 300 mg / kg aqueous extract of gg orally every day for 4 weeks (group vi) representative photomicrographs (g - h) and camera lucida tracings (g1-h1) of golgi - stained hippocampal ca3 neurons of rats treated with aqueous extract of glycyrrhiza glabra (gg) for 4 weeks . G and g1- hippocampal ca3 neurons of rats treated with gg150 mg / kg / p.o + diazepam 7 mg / kg / i.p (group vii); h and h1- hippocampal ca3 neurons of rats treated with gg225 mg / kg / p.o + diazepam 7 mg / kg / i.p (group viii) the aqueous root extract of gg in the dose of 150 and 225 mg / kg / p.o showed significantly (p <0.01) increased numbers of dendritic branching points and dendritic length along the length of both apical and basal dendrites in all the (0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120, 120 - 140 m) concentric zones is comparable to control rats [tables 14]. Basal dendritic branching points of hippocampal ca3 neurons at different concentric zones in 1-month old male wistar albino rats (four weeks duration) basal dendritic intersections of hippocampal ca3 neurons at different concentric zones in 1- month old male wistar albino rats (four weeks duration) apical dendritic branching points of hippocampal ca3 neurons at different concentric zones in 1-month old male wistar albino rats (four weeks duration) apical dendritic intersections of hippocampal ca3 neurons at different concentric zones in1- month old male wistar albino rats (four weeks duration) furthermore diazepam induced amnesia reversed by the aqueous root extract of gg (150 and 225 mg / kg, p.o) has shown a significant (p <0.01) increased numbers of both apical and basal dendritic branching points and dendritic intersections in all the (0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120, 120 - 140 m) concentric zones . In addition, the aqueous root extract of gg in the dose of 300 mg / kg / p.o has shown a significant (p <0.05) increased basal dendritic arborization in the 0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120 m concentric zones and increased basal dendritic intersections (p <0.01) in the 60 - 80, 80 - 100, 100 - 120, 120 - 140 m concentric zones . This dose also shown a significant (p <0.01) increased apical dendritic arborization in the 20 - 40, 40 - 60, 60 - 80, 80 - 100 m concentric zones and increased (p <0.01) apical dendritic intersections in the 80 - 100 and 100 - 120 m concentric zones . The aqueous root extract of gg in the dose of 150 and 225 mg / kg / p.o showed significantly (p <0.01) increased numbers of dendritic branching points and dendritic length along the length of both apical and basal dendrites in all the (0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120, 120 - 140 m) concentric zones is comparable to control rats [tables 14]. Basal dendritic branching points of hippocampal ca3 neurons at different concentric zones in 1-month old male wistar albino rats (four weeks duration) basal dendritic intersections of hippocampal ca3 neurons at different concentric zones in 1- month old male wistar albino rats (four weeks duration) apical dendritic branching points of hippocampal ca3 neurons at different concentric zones in 1-month old male wistar albino rats (four weeks duration) apical dendritic intersections of hippocampal ca3 neurons at different concentric zones in1- month old male wistar albino rats (four weeks duration) furthermore diazepam induced amnesia reversed by the aqueous root extract of gg (150 and 225 mg / kg, p.o) has shown a significant (p <0.01) increased numbers of both apical and basal dendritic branching points and dendritic intersections in all the (0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120, 120 - 140 m) concentric zones . In addition, the aqueous root extract of gg in the dose of 300 mg / kg / p.o has shown a significant (p <0.05) increased basal dendritic arborization in the 0 - 20, 20 - 40, 40 - 60, 60 - 80, 80 - 100, 100 - 120 m concentric zones and increased basal dendritic intersections (p <0.01) in the 60 - 80, 80 - 100, 100 - 120, 120 - 140 m concentric zones . This dose also shown a significant (p <0.01) increased apical dendritic arborization in the 20 - 40, 40 - 60, 60 - 80, 80 - 100 m concentric zones and increased (p <0.01) apical dendritic intersections in the 80 - 100 and 100 - 120 m concentric zones . The dendrites of hippocampal ca3 pyramidal neurons receive inputs from entorhinal cortex, septal area, mamillary body, dentate granule cells and the contralateral ca3 regions and play an important role in the encoding of new spatial information within short - term memory with duration of seconds and minutes . It is believed that some areas of the brain particularly the hippocampus vulnerable to glutamate, ischemia, inflammatory processes, repeated psychosocial or oxidative stress, may leads to dendritic atrophy in ca3 pyramidal neurons of the hippocampus, accompanied by specific cognitive deficits in spatial learning and memory . Alzheimer's disease and schizophrenia are progressive neurodegenerative disorders associated with loss of neurons in distinct brain areas particularly the hippocampus . Such areas of brain structures has been shown to significantly increase the density of spines and dendritic complexity due to repeated exposure to enriched environments . Increase in the dendritic arborization and dendritic intersections in hippocampal ca3 pyramidal neurons may result in alterations in synaptic connectivity . It may result in alteration in learning and memory the present study showed that the aqueous root extract of gg in the dose of 150 and 225 mg / kg / p.o significant (p <0.01) enhancement of dendritic arborization and dendritic intersections in hippocampal ca3 pyramidal neurons . Increase in the dendritic arborization and dendritic intersections in hippocampal ca3 pyramidal neurons may result in alterations in synaptic connectivity, which probably is one reason for the enhanced learning and memory in same rats has been reported previously . Thus the aqueous root extract of gg may stimulate the release of neuromodulators or neuronal dendritic growth stimulating factors that alter the activity of neurotransmitters that are involved in learning and memory, which thereby contributes to enhanced learning and memory . In conclusion, the aqueous root extract of gg in the dose of 150 and 225 mg / kg / p.o showed a significant (p <0.01) enhancement of dendritic arborization and dendritic intersections in hippocampal ca3 pyramidal neurons is comparable to the control . Based on our results obtained, we conclude that constituents present in aqueous extract of root of gg have neuronal dendritic growth stimulating properties.
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Syncope is a common problem accounting for 3% of emergency department (ed) visits, 16% of hospital admissions and affecting 6 per 1,000 people per year . The aetiologies of syncope vary from benign conditions like neurogenic (vasovagal) syncope to serious life - threatening cardiac arrhythmias . In addition, falls as a result of syncope lead to 16 - 35% of hip fractures in elderly patients [4, 5]. Evaluation of syncope and its appropriate management is imperative, but the exact cause remains unclear in many cases even after administering extensive work - up [6, 7]. In pakistan as the burden of diseases in pakistan differs from western countries, there might be a possibility of difference in major causes of syncope and factors associated with it . The objective of the study was to determine the characteristics of patients admitted to the hospital for syncope to a tertiary care hospital in karachi, pakistan . This single - centre study was conducted in the department of medicine at aga khan university hospital (akuh), karachi . It is a private, fee for service, urban tertiary care teaching hospital with 500 inpatient beds . The ed at akuh has an annual census of approximately 40,000 patients from both adult and paediatric populations . (made by the attending physician) were selected by a computerized data - based system from all hospital admissions during the period starting from january 2000 to december 2005 . We determined the specific causes of syncope by the final diagnosis made by the attending physician and categorized them into (1) neurogenic (vasovagal), (2) cardiogenic, (3) unknown origin, (4) orthostatic and (5) psychogenic syncope . Anyone who presented to the ed with transient loss of consciousness (tloc) underwent evaluation for syncope . Initial evaluation included history, physical exam, orthostatic blood pressure and heart rate measurement and a 12-lead ecg with or without echocardiogram . If the initial evaluation identified the tloc as syncope, specific tests were carried out to identify the underlying cause in the next step . We considered patients with age> 60 years, prior history of coronary heart disease, hypertension, diabetes mellitus and abnormal ecg at presentation to have high risk factors . During chart review we recorded the demographic details, presenting symptoms, precipitating factors, past medical history, drug history and results of investigations . For analysis, these variables were entered into the statistical package for social sciences (spss) version 14 . A cross - tab was applied to compare different variables and p values were calculated using the chi - square test . This single - centre study was conducted in the department of medicine at aga khan university hospital (akuh), karachi . It is a private, fee for service, urban tertiary care teaching hospital with 500 inpatient beds . The ed at akuh has an annual census of approximately 40,000 patients from both adult and paediatric populations . (made by the attending physician) were selected by a computerized data - based system from all hospital admissions during the period starting from january 2000 to december 2005 . We determined the specific causes of syncope by the final diagnosis made by the attending physician and categorized them into (1) neurogenic (vasovagal), (2) cardiogenic, (3) unknown origin, (4) orthostatic and (5) psychogenic syncope . Anyone who presented to the ed with transient loss of consciousness (tloc) underwent evaluation for syncope . Initial evaluation included history, physical exam, orthostatic blood pressure and heart rate measurement and a 12-lead ecg with or without echocardiogram . If the initial evaluation identified the tloc as syncope, specific tests were carried out to identify the underlying cause in the next step . We considered patients with age> 60 years, prior history of coronary heart disease, hypertension, diabetes mellitus and abnormal ecg at presentation to have high risk factors . During chart review we recorded the demographic details, presenting symptoms, precipitating factors, past medical history, drug history and results of investigations . For analysis, these variables were entered into the statistical package for social sciences (spss) version 14 . A cross - tab was applied to compare different variables and p values were calculated using the chi - square test . There were 358 patients during the study period who were admitted with the diagnosis of syncope or collapse . Records of 317 (89%) patients were found, of which 48 (13%) patients were excluded as they were found to have a as per the nature of the problem most of the patients (93%) were admitted to the hospital through the ed . Forty - six percent (n = 124) of patients were admitted to the general medical ward and 145 patients were admitted to the high dependency unit (monitored / telemetry beds). The median length of hospital stay was 2.0 days [interquartile ranges: 1.25 (25), 2.0 (50), 3.0 (75)]. The mean length of stay for patients with cardiogenic syncope was double (4 days) that of patients with neurogenic (vasovagal) syncope (2 days). Eighty - three percent (n = 224) of patients were admitted under the care of the cardiology service, 17% (n = 45) under general medicine while one patient was admitted under the neurology service . The most commonly associated comorbid condition of our study population was hypertension (58%), followed by coronary artery disease (44%), hyperlipidaemia (33%) and diabetes mellitus (27%). The frequencies of commonly associated prodromal symptoms in our study population were: dizziness (61%), sweating (25%), palpitation (19%), nausea / vomiting (19%), visual symptoms (transient blackouts / blurred vision, haloes around light) (17%) and breathlessness (11%). The frequencies of these symptoms according to cause of syncope and precipitating factor are described in table 1 . Regarding the position of the patient at the time of onset of syncope, 82% developed it while standing and sitting, less than 5% developed it while lying down and in 13% of the cases, the patient s position could not be determined from the records . The most common investigation done was an electrocardiogram (96%), which was abnormal in 13% . Table 1distributions of symptoms, precipitating factors, age and previous history of syncope across various causes of syncope causes of syncopevasovagal (n = 126)unknown (n = 64)cardiogenic (n = 50)orthostatic (n = 26)psychogenic (n = 3)n (%) n (%) n (%) n (%) n (%) symptoms dizziness87 (69)40 (62)23 (46)12 (48)3 (100) visual symptoms33 (26)9 (14)4 (8)10 (20)1 (33) nausea27 (21)12 (18)10 (20)9 (33)1 (33) palpitation23 (18)12 (18)10 (20)9 (33)1 (33) sweating26 (21)11 (17)6 (12)6 (24)1 (33) chest pain11 (9)11 (09)4 (08)3 (12)0 (0) shortness of breath13 (10)5 (08)14 (28)1 (4)0 (0)precipitating factors defecation / micturition28 (22)11 (17)9 (18)3 (12)1 (33) exertion18 (14)12 (19)7 (14)3 (12)1 (33) office work6 (5)5 (8)8 (16)4 (15)0 (0) standing after sleep14 (11)6 (9)7 (14)6 (23)0 (0) eating9 (7)10 (16)5 (10)1 (4)0 (0) unknown51 (40)20 (31)14 (28)9 (35)1 (33)age groups 1640 years (33/269)23 (18)5 (8)1 (2)3 (12)1 (33) 4165 years (146/269)71 (56)37 (58)22 (44)14 (54)2 (67) 66 and above (90/269)32 (25)22 (34)27 (54)9 (34)0 (0)previous history of syncope yes28 (22)16 (25)18 (36)4 (15)1 (33) no98 (78)48 (75)32 (64)22 (85)2 (66)table 2non - invasive tests along with their diagnostic yield in the study populationdiagnostic testn / n (%) electrocardiogram34/258 (13)troponin i4/180 (2)echocardiogram13/92 (7)holter monitoring6/46 (13)head - up tilt test15/38 (39)stress test4/28 (14)eeg0/22 (0)magnetic resonance imaging0/18 (0)myocardial perfusion scan3/16 (19)dobutamine echo1/11 (9)head computed tomography scan0/10 (0)carotid doppler0/7 (0)number of patients with positive diagnostic testnumber of patients who underwent the test distributions of symptoms, precipitating factors, age and previous history of syncope across various causes of syncope non - invasive tests along with their diagnostic yield in the study population number of patients with positive diagnostic test number of patients who underwent the test neurogenic (vasovagal) syncope was the most common type (46.8%) regardless of the age, followed by cardiogenic (18%), orthostatic (9%) and psychogenic syncope (1%). Twenty - four percent were discharged with the diagnosis of syncope of unknown origin . Most of our patients had syncope for the first time in their life while one fourth (n = 67) of our patients presented with recurrence . Among those who had recurrence, 36% of patients were diagnosed with cardiogenic syncope, 25% with syncope of unknown origin, 22% with neurogenic syncope and 15% with orthostatic hypotension . Among 145 patients who were in monitored beds, only 19% had the diagnosis of cardiogenic syncope, and 18% of patients admitted to a general medical ward were diagnosed with cardiogenic syncope . We also noticed that 48% (n = 60) of patients with neurogenic syncope were admitted to the telemetry unit and more than half (58%) of them had no high risk factors . Sixty - nine percent had troponin i measured, 46% had echocardiography, 20% exercise tolerance test, 11% holter monitoring, 9% electroencephalogram, 6% magnetic resonance imaging of the brain, 6% computed tomography scan of the brain, 6% myocardial perfusion scan and 3% dobutamine stress echocardiography . Syncope of unknown origin were admitted to monitored beds and 50% of them had no significant risk factors; and they had multiple investigations that were negative . The average cost of evaluation and stay was estimated to be pakistani rupees 20,000 (us $350) per patient including emergency department charges, hospitalization charges, subspecialty consultations, baseline investigations and specific tests . We also found that patients with cardiogenic syncope had a significantly higher frequency of coronary artery disease (72%, p <0.001), abnormal ecg at presentation (92%, p <0.001) and age above 40 (98%, . The risk factor profile of different types of syncope is shown in table 3 . Table 3association of risk factors with different causes of syncoperisk factorsn / n (% & p values)history of coronary artery disease (cad) cardiogenic syncope36/50 (72 & <0.001) vasovagal syncope40/126 (32 & = 0.707) syncope of unknown origin32/64 (50 & = 0.247) orthostatic syncope11/26 (42 & = 0.500)abnormal ecg at time of presentation cardiogenic syncope46/50 (92 & <0.001) vasovagal syncope57/126 (45 & = 0.186) syncope of unknown origin32/64 (50 & = 0.632) orthostatic syncope18/26 (69 & = 0.245)number of patients found in each categorytotal number of patients with the same type of syncope association of risk factors with different causes of syncope number of patients found in each category total number of patients with the same type of syncope most of the published reports of syncope evaluation have been from developed countries . In this regard the current manuscript provides important information that adds to knowledge about patients presenting with syncope in a developing country in south asia, with limited health care resources . Our study highlights the features of syncope and their frequencies in hospitalized patients in pakistan . We had a higher proportion of males presenting with syncope; the possible cause of this observation is either a cultural norm of our society that prefers to bring sick men to the hospital earlier than sick women . Other studies have not demonstrated such a marked gender difference [911]. According to the literature, prodromal symptoms of nausea / vomiting are more common with neurogenic syncope [10, 12]; however, we did not observe such a difference in our patients . Similarly chest pain, sweating and palpitation, which point towards a cardiac aetiology, were found to be equally distributed in both neurogenic and cardiac aetiologies . Calkins et al . Also observed that symptoms such as palpitation, nausea and diaphoresis are not significantly helpful in ascertaining the diagnosis of syncope . Similarly de graf et al . Concluded that specific symptoms cannot predict the cause of neurogenic syncope, rather it is the number of prodromal symptoms . Dizziness was prevalent in our patients, and more common in patients with neurogenic and syncope of unknown aetiology . Because of its vague nature found that 70% of their subjects had dizziness, light - headedness or a sensation of impending syncope, but dizziness alone was observed in 66% of their patients . Said dizziness is relatively less common and better defined than light - headedness . In our study, shortness of breath was the only symptom that was significantly (p <0.05) associated with cardiogenic syncope . Of note, shortness of breath is also one of the prognostic factors in the san francisco syncope study . We conclude that presenting symptoms per se do not help in ascertaining the cause of syncope except shortness of breath . Neurogenic syncope was the most common cause of syncope in our patients regardless of their age, but its incidence decreased with age . In contrast, cardiogenic, orthostatic and syncope of unknown aetiology follow the opposite trend . . Stated that age could be one of the predictors of the cause of syncope . Increasing incidence of cardiogenic syncope with age may be because of the higher incidence of ischaemic and structural heart diseases in the elderly . Likewise increasing frequency of orthostatic syncope with advancing age is possibly explained by increased intake of multiple drugs that either affect the autonomic nervous system or deplete intravascular volume . An important problem that emergency physicians face while dealing with patients with syncope is the decision regarding their disposition . And kapoor concluded that patients with cardiac syncope are at higher risk of death from any cause while vasovagal syncope pursues a benign course . This emphasized that patients with cardiogenic syncope need hospitalization, but ascertaining the cause of syncope in most of the cases at the time of presentation is very difficult . This led investigators to find out the prognostic risk factors that can help in selection of those patients who require hospital admission . . Showed that mortality in patients with syncope increases with increase in number of comorbid conditions and age . Also demonstrated that older age, lack of prodromal symptoms, history of cardiovascular disease (especially heart failure and ventricular arrhythmias) and abnormal ecg at presentation in patients with syncope are associated with an increase in 1-year mortality . Patients with cardiogenic syncope in our study possessed many of these prognostic risk factors in a significantly higher proportion than patients with other types of syncope . Therefore, if the diagnosis about specific type of syncope is not clear in the ed, these risk factors will help to decide who needs hospital admission . Syncope of unknown origin had been admitted to beds with cardiac monitoring and 50% of them had no risk factors, i.e. Age below 60, no history of coronary artery disease and normal ecg at presentation . Similarly half of the patients with neurogenic (vasovagal) syncope were admitted to specialized units and 75% of these had no history of coronary artery disease or abnormal ecg at presentation and many were aged below 60 but still underwent extensive evaluation . In addition table 2 also shows that many patients had low - yield neurological tests . First, this study only includes patients who were admitted to the hospital for syncope, which is why there were only 269 in the final cohort . No data on those who were discharged from the emergency department or left against medical advice are presented . Second, our hospital is a private, fee for service, urban, tertiary care centre, leading to selection bias towards urban and middle and upper income groups . Finally, some individuals were admitted for evaluation of sudden and transient loss of consciousness, under the diagnosis of some established cause rather than syncope and thus escaped our inclusion criteria and never showed up in analysis . First, this study only includes patients who were admitted to the hospital for syncope, which is why there were only 269 in the final cohort . No data on those who were discharged from the emergency department or left against medical advice are presented . Second, our hospital is a private, fee for service, urban, tertiary care centre, leading to selection bias towards urban and middle and upper income groups . Finally, some individuals were admitted for evaluation of sudden and transient loss of consciousness, under the diagnosis of some established cause rather than syncope and thus escaped our inclusion criteria and never showed up in analysis . It poses risk to the patients, hassle to the attendants, stress to the medical practitioner and burden to the health care system in general . It is essential to diagnose it right and to initiate the treatment as soon as possible . By the same token, the diagnostic approach should be cost - effective especially in a third - world country like ours . Patients with age above 40, prior history of coronary artery disease, associated shortness of breath and abnormal ecg at presentation were more prone to have the cardiogenic type . Further studies are needed to develop a protocol to expedite the evaluation and limit the work - up and admission in low - risk patients.
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Cervical cancer is the second major cause of cancer - related mortality in women worldwide and accounts for 250,000 deaths each year (1). It is well established that the infection with high - risk types of human papillomavirus (hpv) plays a central role in the pathogenesis of invasive cervical cancer (2). Although many women are infected with high - risk types of hpv, only a subset of infected women develops cervical cancer, suggesting that other cofactors including host genetic factors must be present for the development of malignancy . Chronic inflammation has been shown to be an important risk factor for a variety of epithelial cancers (3). Cytokines, as the products of host response to inflammation, play an important role in the defense against viral infections . In cervical cancers, a number of previous reports suggested that chronic inflammation is associated with the precancerous intraepithelial lesion and cancer of uterine cervix (4 - 6). The interleukin-1 family of cytokines consists of several members including ineterleukin-1 alpha, interleukin-1 beta and interleukin-1 receptor antagonist . The genes for these cytokines are clustered within a 430-kb segment on human chromosome 2 . Interleukin-1 beta is a pro - inflammatory cytokine mainly produced by blood monocytes and tissue macrophages and has been implicated in mediating both acute and chronic inflammation (7). Recently, a common polymorphic allele of the regulatory region of the il1b gene was found to be associated with increased il-1 production (8). Also, the polymorphism in il1b was associated with various human cancers (8 - 11). Since there were several reports supporting the positive association with increased il1b secretion and cervical cancer risk (12, 13), we hypothesized that an individual with a il1b genotype producing more il1b might have an increased risk of cervical cancer . The c> t polymorphism in il1b -511 site has been correlated with increased intracellular il1b levels in the previous reports (14). Here we report results from a hospital based case - control study examining the association of il1b -511 c> t polymorphisms with the risk of cervical cancer . Case subjects were selected from among cervical cancer patients treated between april 1996 and july 2002 at the seoul national university hospital . A total of 182 patients with confirmed cervical squamous cell carcinoma consented to participate in the study and provided a blood specimen . Age - matched (1:2) control subjects were comprised of 364 healthy, unrelated, cancer - free subjects recruited from visitors who attended a comprehensive screening clinic at the same institution and agreed to participate in this study . All case and control subjects were korean, and the institutional review board of seoul national university hospital approved the protocol used in this study . All genotyping of 182 cases and 364 control samples was performed by polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp). In brief, il1b -511 c> t polymorphism was distinguished by pcr - rflp, using the known primer pairs (forward primers 5'-gcctgaaccctgcataccgt; reverse primers 5'gccaatagccctccctgtct-3') and restriction enzyme avai . Amplification was performed in a volume of 25 l, containing 2.5 l of 10pcr buffer (100 mm tris - hci, 15 mm mgcl2, and 500 mm kcl, ph 8.3), 200 nm each dntp (roche diagnostics korea, seoul, korea), 1 m each primer (bioneer, daejeon, korea), 1 u taq dna polymerase (roche diagnostics korea, or takara shuzo, otsu, japan), and 100 ng of genomic dna . The thermocycling conditions were as follows: 95 for 5 min; then 35 cycles of 95 for 30 sec, 58 - 60 for 30 sec, and 72 for 1 min; then 72 for 10 min . Fifteen microliters of the reaction mixture was treated with 5 u of avai (ne biolabs, beverly, ma, u.s.a .) At 37 for 12 hr and subsequently analyzed on 3% agarose (2% nusieve [bio - whittaker molecular applications, rockland, me, u.s.a .] And 1% agarose) gel . Hardy - weinberg equilibrium analyses were performed to compare observed and expected genotype frequencies using the chi - square test (d.f.=1). Allele frequency differences between cases and controls were analyzed using the pearson chi - square test . Cervical cancer risk was estimated by odds ratios (ors) and 95% confidence intervals (cis) using conditional logistic regression model after adjustment for age . Case subjects were selected from among cervical cancer patients treated between april 1996 and july 2002 at the seoul national university hospital . A total of 182 patients with confirmed cervical squamous cell carcinoma consented to participate in the study and provided a blood specimen . Age - matched (1:2) control subjects were comprised of 364 healthy, unrelated, cancer - free subjects recruited from visitors who attended a comprehensive screening clinic at the same institution and agreed to participate in this study . All case and control subjects were korean, and the institutional review board of seoul national university hospital approved the protocol used in this study . All genotyping of 182 cases and 364 control samples was performed by polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp). In brief, il1b -511 c> t polymorphism was distinguished by pcr - rflp, using the known primer pairs (forward primers 5'-gcctgaaccctgcataccgt; reverse primers 5'gccaatagccctccctgtct-3') and restriction enzyme avai . Amplification was performed in a volume of 25 l, containing 2.5 l of 10pcr buffer (100 mm tris - hci, 15 mm mgcl2, and 500 mm kcl, ph 8.3), 200 nm each dntp (roche diagnostics korea, seoul, korea), 1 m each primer (bioneer, daejeon, korea), 1 u taq dna polymerase (roche diagnostics korea, or takara shuzo, otsu, japan), and 100 ng of genomic dna . The thermocycling conditions were as follows: 95 for 5 min; then 35 cycles of 95 for 30 sec, 58 - 60 for 30 sec, and 72 for 1 min; then 72 for 10 min . Fifteen microliters of the reaction mixture was treated with 5 u of avai (ne biolabs, beverly, ma, u.s.a .) At 37 for 12 hr and subsequently analyzed on 3% agarose (2% nusieve [bio - whittaker molecular applications, rockland, me, u.s.a .] And 1% agarose) gel . Hardy - weinberg equilibrium analyses were performed to compare observed and expected genotype frequencies using the chi - square test (d.f.=1). Allele frequency differences between cases and controls were analyzed using the pearson chi - square test . Cervical cancer risk was estimated by odds ratios (ors) and 95% confidence intervals (cis) using conditional logistic regression model after adjustment for age . In korean population, we observed that the allelic frequencies of the 182 case subjects (c, 0.42; t, 0.58) were not significantly different from those of the 364 control subjects (c, 0.43; t, 0.57). The allelic frequencies of control subjects were not statistically different from those reported for korean populations by other investigators (9). We found that carriers of the t allele had a significantly increased risk of cervical cancer . The frequency distribution of the different genotypes for the il1b -511 c> t polymorphism is shown in table 1 . The cc genotype was less frequent among the case subjects than among the control subjects (7.7% and 16.8%, respectively). With the use of the chi - square test, we found the significant difference in genotype frequencies between case and control subjects (p<0.001). Using the cc genotype as the reference genotype, we performed logistic regression analysis with adjustment for age variable . The ct genotype was associated with significantly elevated or of 2.83 (95% ci=1.52 to 5.28, p<0.001). Also, the tt genotype was associated with elevated or of 1.68 (95% ci=0.85 to 3.32, p=0.136). When we combined ct and tt genotypes together, we found that ct and tt genotypes were associated with or of 2.42 (95% ci=1.31 to 4.46, p<0.005). Here we reported that -511 polymorphism in il1b gene was associated with the risk of cervical cancer . Our findings in this hospital - based case - control study suggest that the carriers of -511 t allele may be at increased risk of developing cervical cancer . Our results support the previous hypothesis that -511 t allele is associated with increased production of il-1, and that il-1 may play a role as host factors promoting cervical carcinogenesis . Since it is assumed that the host immune system is important in the surveillance of hpv - related cervical neoplasia, cytokines, including il-1, have been frequently correlated to a risk of cervical cancer . The level of the il-1 was increased in the cervicovaginal washings of patients with cervical cancer (13). Individuals with high and intermediate il-1 secretor phenotypes may be more susceptible to lower grade lesions rather than high grade lesion or cervical carcinoma (12). Elevated vaginal lavage il-1 these evidences, including the results of this study, suggest that il-1 may be involved in early step of cervical carcinogenesis and that individual difference of il-1 secretion may affect individual susceptibility to cervical cancer progression . The polymorphism of il1b gene was reported to be associated with various diseases including cancer, but it is most intensively studied in gastric cancer . El - omar et al . Have recently reported that proinflammatory genotypes of the interleukin-1 gene cluster (il1b -511/-31 and il-1rn*2/*2) were associated with increased risk of gastric cancer and its presumptive precursors, gastric atrophy and hypochlorhydria, in white populations from poland and scotland (8). Their results contained data that il1b -511 c> t polymorphism was associated with risk of gastric cancer . While explaining this, they addressed that there were no differences in binding activity between il1b -511 genotypes, indicating that the effect of il1b -511 polymorphism may be mediated by linkage disequilibrium with the tata box polymorphism . This may be possible explanation of our finding, no genedosage relationship between -511 genotypes . In consistence with our data, another recent study reported that il1b -511 ct heterozygous genotype was the main risk of intestinal type gastric cancer in korean population (9). Since our results is the first report about the association between il1b -511 c> t polymorphism and the risk of cervical cancer, we could not compare our results with other data . However, there were several previous reports suggesting the possible association between cervical cancer risk and the polymorphism of other cytokines or cytokine receptors, such as tnf - alpha, interleukin 10, and interleukin 1 receptor antagonist (16 - 21). Therefore, it can be speculated that functional variation of inflammatory cytokine may influence on the individual susceptibility of cervical cancer . Degradation of the p53 gene by oncogenic hpv e6 protein is the most well known carcinogenic mechanism in human cervical cancer . Recent findings indicate an increased p53 mutation load or altered p53 protein function in a number of inflammatory diseases (22). It has been shown that, in rats, intratracheal instillation of il-1 caused hydrogen peroxide production in lung tissue, initiated neutrophil influx and stimulated their release of reactive oxygen species (23). Therefore, it can be speculated that reactive free radicals produced by inflammatory cells may cause dna damage in epithelial cells . Inflammatory cytokines have also been shown to induce dna damage and inhibit dna repair in vitro (24). In addition, il-1 has been shown to reduce apoptosis by changing the ratio of bcl-2/bax proteins (25). Therefore, a higher production of il-1 may lead to increased p53 mutation load, and the increased level of il-1 may play a role not only in hpv - related cervical carcinogenesis but also in hpv - non - related cervical carcinogenesis . The limitations of the present study are as follows . Second, our sample size is so limited that it has not enough statistical power to exclude the existence of gene - dosage relationship . Third, the various epidemiologic risk factors of cervical cancer, such as smoking, alcohol intake, diet, or sexual behavior, were not included for analysis . Fourth, since our study did not include all clustered polymorphic site of il1b and associated genes, the haplotype analysis could not be done . So, it would be worthwhile to perform further large - scale population - based study including the analysis of various clustered polymorphisms . It also should be noted that the risk of cancer caused by foreign pathogen such as virus or bacteria was repeatedly reported by numerous study (8, 26). Therefore, it can be speculated that increased il-1 production may be associated with host genetic factor defending from foreign carcinogenic pathogen . In conclusion, in korean population, il1b -511 cc genotype was significantly associated with decreased risk of cervical cancer . This relationship supports the idea that polymorphism of inflammatory response genes may be host genetic susceptibility to cervical cancer . Il1b polymorphism should be considered as candidate genetic factor in future study elucidating the genetic risk of cervical cancer.
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An unprecedented opportunity for identification of disease biomarker candidates has been provided by the advent of high throughput technologies in the past decade [1, 2]. The explosive growth of large data sets has been overwhelming in terms of the number, size, format, and complexity [3, 4]. While diversified data sets have led to numerous opportunities and studies for discovering new disease marker candidates, the success of those efforts has been largely disappointing in terms of validating the results across populations . Current strategies for biomarker discovery tend to focus on one of two approaches: data - driven or expert knowledge - driven . A data - driven approach makes use of large data sets to unearth the underlying structures embedded in the data to facilitate identification of robust features . The value of this purely statistical approach has been evident in the successful identification of cancer biomarkers, for instance, using an artificial neural network (ann) model for detecting early stage epithelial ovarian cancer with a panel of five serum markers . In contrast, an expert knowledge - driven approach takes advantage of constantly increased understanding in pathophysiological mechanisms of diseases at both molecular and systems levels to extract discriminating features of diseases . Despite many intense efforts devoted to the field of biomarker discovery, no robust yet generalizable framework has been widely accepted by the community . Recent studies have suggested that integrating data - driven and knowledge - driven approaches rather than exclusive reliance on either can potentially improve the robustness of selected biomarker candidates and their performances across populations . For instance, an empirical bayes method has been used to combine the information on pathways and networks into the experimental results of cancer biology . However, many challenges can be recognized immediately and need to be addressed properly, such as optimal knowledge databases to use, suitable formats of expert knowledge, reasonable ways to integrate these disparate kinds of data, and appropriate selection strategies . As one of the leading causes of death worldwide, chronic obstructive pulmonary disease (copd) is a prevalent condition that is characterized by progressive and not fully reversible airflow limitations [9, 10]. No new classes of drugs for copd treatment have been approved for use in the united states in more than twenty years . Despite associations with multiple pathological components, one hallmark of copd is a persistent inflammatory state that contributes to a progressive decline in lung function . A mouse model with adenosine deaminase (ada) deficiency has been established to develop a rapid pulmonary inflammation and progressive destruction of lung tissue that closely mimics many aspects of human copd and other chronic lung diseases [13, 14]. It is a key signaling molecule involved in multiple intracellular signaling pathways related to the modulation of inflammatory responses [15, 16]. Ada is the purine catabolic enzyme responsible for converting adenosine to inosine, which is frequently induced in response to cell stress or damage and involved in anti - inflammatory, tissue - protective pathways [16, 17]. Accumulating evidence has suggested that elevated adenosine levels in lung are associated with chronic lung diseases in both human and animal models [12, 17]. An in - depth understanding of the biological relevance of ada in lung will benefit our general understanding of copd . In this paper, we describe a semiautomated framework, identification of signatures from integrated clustering (isic), for merging data - driven and knowledge - driven approaches into a biomarker selection scheme in an iterative manner, with a defined metric provided for performance evaluation . To demonstrate isic, we applied it to proteomics data sets of bronchoalveolar lavage fluid (balf) and plasma from a mouse model of copd, the ada - deficient mouse model [13, 14], to identify marker candidates of copd . The resulting candidates were subsequently validated in a human plasma data set from a cohort of low body mass index (bmi) smokers with copd and healthy controls . We believe that isic is a novel and powerful tool for integrating data types in the context of biosignature discovery and show that it produces robust results between a model system and human disease . Bronchial secretions and blood plasma from the ada / and ada + / mice were individually collected and processed as described previously . Human plasma samples were selected from a large cohort (n = 467) of the genetics of addiction program at the university of utah medical school . Plasma samples from 7 low bmi smokers with copd and 7 low bmi never smokers (copd free) were used for the patient and control samples . All balf and plasma (mouse and human) samples were processed, tryptic digested, separated, and analyzed using liquid chromatography - mass spectrometry (lc - ms). Detailed information on animal and human sample collections and data collection is provided in supplemental information, which includes supplemental methods, brief descriptions and rationales of the framework, discussion on copd data sets used in the current study, supplemental figures, supplemental tables, and references . The peptides were identified and quantified using a collection of in - house developed tools that are freely available at http://omics.pnl.gov/. For the mouse data, the peak intensity values of the final identified peptides were obtained from the analyses of lc - ltq - orbitrap spectral data . The raw peak intensity values were processed in the matlab environment, including quality control, normalization, protein quantification, and comparative statistical analyses [1921]. The final peptide abundances were transformed into the log10 scale for the subsequent data analyses . Quality control was a process performed to identify and remove the peptides with an insufficient amount of data across the set of samples, as well as to identify and remove the lc - ms data sets that showed significant deviations from the standard behaviors of all lc - ms analyses . The outlier lc - ms data sets were identified at a significance level of 0.0001 . The peptides were normalized across all technical replicates to ensure the least amount of bias introduced into the data sets . Specifically, the balf data were normalized using a linear combination of order statistics to determine a subset of peptides followed by mean centering, and the plasma data were normalized using a rank invariant peptide subset followed by median centering . The normalized log10 abundance values were averaged across the technical replicates within each biological sample . The subsequent protein quantifications were performed using the most abundant reference peptide through an r - rollup method [19, 24]. The human plasma data were processed using the same protocols for the balf data, as described above . The quantified proteins in balf and plasma were compared quantitatively and qualitatively between the time - matched ada + / and ada / mice (three in each group) at each of the five time points, respectively . Quantitative comparison was performed using a dunnett adjusted t - test to assess the numeric change in the average abundance of a protein between the two phenotypes at the individual time points . Qualitative comparison (the presence or absence of a protein) was implemented by a g - test, a modified test of independence, which assessed the associations between the presence / absence of proteins and the phenotypes of the mice . A significantly altered protein was defined if at least one of the five t - tests or five g - tests was statistically significant (p <0.05) after bonferroni multiple hypothesis correction . All the protein abundances were compared with their time - matched counterparts . To provide sufficient replicates for the subsequent analyses, we combined the significantly altered proteins from all five time points and grouped them into either disease (ada /) or control (ada + /) categories for the balf and plasma samples, that is, a sample size of 15 mice in each of the groups . All clustering analyses using the mouse data were performed on the complete data set of significantly altered proteins . Missing values were imputed at the protein abundance level using a regularized expectation - maximization algorithm . The first was based on the protein expression profiles and was calculated as euclidean distance of the protein abundances in the log10 scale . The second was based on the functional relationships between the proteins and was determined by the semantic dissimilarities in the biological process subontology from the gene ontology (go). The semantic dissimilarity was defined as 1-semantic similarity between protein pairs and calculated using the cross - ontological analysis (xoa) tool or the gosemsim package in the r statistical language . It was calculated as the weighted average of the other two distances with weighting factors of 0.25, 0.50, 0.75, or 1 or the average of their individual logistic functions . A logistic function is a common sigmoidal function with equation: (1)f(x)=11+ex, where x is associated with a distance matrix, that is, the first or second distance . Where ai, aj is a distance or dissimilarity between two proteins, is a smooth threshold (chosen as the mean of the distance matrix), and 6/ is a heuristically chosen parameter of the slope in our calculation . The averaged logistic function of the two distance matrices was used as a candidate of the joint distance measures . The numbers of clusters were empirically determined as 6 and 12 for the balf and 6 for the plasma data sets based on their sample sizes . A biological function - centric approach was used to determine the functionally enriched biological processes in the balf and plasma samples of mice . The biological processes are referred to the terms included in the biological process hierarchy in the go . The significantly changed proteins in the ada / mice (relative to their controls) were mapped to their corresponding genes and compared with a list of all genes in the mouse genome in order to determine the levels of significance for individual biological processes in the data using a hypergeometric test . The go terms with the enrichment p values smaller than 10 (in both balf and plasma) for each enriched go term, we determined its own level (how specific the term is) and its top - level ancestor (broadest category that includes the term) within the biological process subontology . The level of a go term is the number of steps taken to reach the top - most node when ascending the go tree starting from the term of interest . The top - level ancestor of a go term is the ancestor term that is directly below the top - most node (biological process, go i d: 8150). The enriched go terms were then grouped according to their top - level ancestors and resulted in a number of biological process groups that were enriched in the ada / mice . An expert knowledge - driven disease selection was subsequently implemented on the enriched go terms selected above . Specifically, a subset of the enriched terms was further selected based on expert knowledge on the ada - deficient model, the p values of enrichments, and the levels of the go terms within individual functional groups, that is, the go terms sharing the same top - level ancestor(s). Those functional clusters with their corresponding proteins were the final results of this expert knowledge - driven disease - model - related annotation selection . Each of the clusters was represented by either all differentially expressed proteins or, alternatively, the top three most differentially altered proteins in the cluster between the two phenotypes for the subsequent analyses in isic . A bayesian integration approach was applied on clusters to derive the optimal probability models for the data sets . Four standard statistical algorithms were applied to the individual clusters (or subsets) generated from the hierarchical clustering or expert knowledge - driven functional annotation to build likelihood probability models: linear discriminant analysis, fuzzy k - nearest neighbor, multinomial logistic regression, and nave bayes . Classification accuracy (ca) was used to evaluate the performances of the individual subsets and the integrations of the multiple subsets of each round of the analyses . Our approach predicts the disease state of each animal (ada / or ada + /), and so true positives (tp; correct predictions of disease) and negatives (tn) are compared to incorrect predictions (false positives and negatives; fp and fn). Ca is expressed as (3)ca = number of correctly classified samplestotal number of samples = tp+tntp+fp+tn+fn . The optimal algorithm for a specific cluster was the one providing the best ca among the four probability models . The posterior probabilities were integrated through a bayesian approach using the different combinations of the subsets along with their optimal algorithms determined [33, 38]. Within either of the approaches, the cas from all possible combinations of clusters were calculated, and the highest value was reported as the optimal integrated ca for the data set under that setting . Five - fold cross - validations were performed in all analyses to assure that the probabilities were independent from the training data . The cluster membership and the corresponding proteins of each cluster from the optimal integration were recorded for the comparisons . Once the cluster membership of the optimal integration for a data set was determined, the integration and determination of ca from the selected clusters were repeated 100 times with five - fold cross - validations . The average ca was reported as the final optimal integrated ca of each round of the analyses . Additionally, the integrated cas were reported from the use of a full data set and a partial data set . The full data set refers to the entire data set that was divided into the numbers of clusters indicated . The partial data set refers to the subset of the clusters that provided the best integrated ca for each combination of parameters (overall number of clusters, distance matrix used, and weight for expert versus data matrix integration . ). Biomarker candidate selection was conducted separately in the clustering approach versus the expert - driven functional selection . In the clustering approach, we selected the biomarker candidates as the set of protein clusters that gave the best integrated cas . In the expert - driven functional selection, the biomarker candidates were selected as the several most differentially altered proteins belonging to the functional clusters providing the best integrated cas . Validation was performed on a proteomics data set of human plasma at both the cluster (six cluster optimization) and individual protein levels . For the validation on clusters, we used the clusters identified from the mouse balf and plasma proteins using their joint distance matrices . In each cluster, we filtered to only include proteins that were detected in both human and mouse . The cas for individual clusters and the integrations from all six clusters were calculated . For the validation on the individual proteins, the biomarker candidates selected from mouse balf, which were also detected in the plasma data sets, were evaluated in the human plasma data set . The objective of this study was to develop a semiautomated framework for integrating expert knowledge into disease marker selection scheme in an iterative manner guided by the use of a defined metric providing the evaluation of performances . The framework, named isic, was designed to serve as a conceptual pipeline rather than a collection of detailed protocols; the basic flow is illustrated in figure 1 . The overall process consists of five components, that is, data reduction, distance - based hierarchical clustering, bayesian integration and classification, selection of biomarker candidates, and validation . In addition, an expert knowledge - driven disease model - related functional selection was provided as a parallel approach, the results of which can be compared to those from isic using ca as a discriminator between the two approaches . Biomarker candidates were selected based on their ca performances in the individual approaches, which were subsequently validated in a human data set (see supplemental information for additional explanation). To demonstrate isic, we applied it to three proteomics data sets associated with copd . First, we identified the potential biomarkers in data obtained in the balf and plasma samples from the ada - deficient mouse model of copd . This model system has a clear distinction between diseased and nondiseased samples and therefore is well - suited to developing and testing our classification approach . To validate the candidates identified from the development phase, we chose to examine plasma data from smokers with copd along with their corresponding controls . This data set, derived from the actual patient samples, allowed us to test whether the signatures identified from mouse would be robust in their ability to classify diseased and nondiseased human samples with complex and varied genetic and environmental backgrounds . A special effort was focused on how to appropriately handle the missing values in the data sets . It is not uncommon to have 30% or more missing values, that is, measurements for a specific protein that are missing from individual samples but present in others, even from a carefully designed and implemented proteomics data set . In the mouse demonstration data sets, the missing rates were 26% in balf and 17% in plasma . Dunnett adjusted t - tests were performed on the proteins having adequate abundance values in both types of mice, and g - tests, a modified test of independence, were implemented on the proteins without adequate abundance values, respectively . The former assesses the quantitative changes, and the latter evaluates the qualitative changes in the individual protein abundances . A quantitative change is self - explanatory, while a qualitative change here refers to a real biological absence or presence of a protein between the two groups of mice . Numbers of changed proteins as well as the direction of change are indicated in figure s1 . The collections of the proteins that are quantitatively (a numerical change in the abundance) and qualitatively (absence / presence) different at individual time points were considered as our initial biomarker searching spaces in balf (396 out of 532) and plasma (150 out of 351). Heat maps of protein expression from both data sets are depicted in figure s2, and no distinct patterns are observed in either . We calculated distances between all proteins based on their abundance levels across all observations or their functional similarity based on their annotations in go . The clusters derived from three different dissimilarity matrices were used in the bayesian integration and classification step to obtain ca scores for each combination of parameters . No significant differences in the ca scores were observed between different weighted averages or logistic functions (data not shown). The integrated ca scores from using the full and partial data sets are listed in the table 1(a), and the information from individual clusters is provided in table s1 . The optimal ca scores from the use of the entire data set as a single cluster provided the baseline performances of the approach . In balf, using the 396 proteins, the classification performances were approximately 80% ca or higher, and their ca counterparts from plasma (using the 150 proteins) were lower at about 60% . Interestingly, the optimal integrated ca scores in balf and plasma were both derived from using a subset of the clusters rather than a full data set . A total of 303 go terms (data not shown) were determined as enriched (p <10) in the biological process hierarchy from the mouse balf samples using a hypergeometric test . Intermediate level go terms were selected based on knowledge of the disease model and then grouped into 13 groups of go annotations (the rightmost column in table 2). These groups were summarized into their top - level go - based biological processes (center column, table 2) into ten general enriched functional groups for the ada - deficient mouse model of copd, which was the final result of the expert knowledge - driven analysis . The ca performances of the ten functional groups were assessed in a similar way as those in the clustering approach . Specifically, the optimal individual ca scores for functional clusters from using all and the top three differentially expressed proteins within individual clusters were calculated and are summarized in table s2 . The ca results using the entire data set as a single cluster are also provided as the reference points . The integrated ca results are listed in table 1(b). To our surprise, for both individual and integrated results, the cas calculated using the top three proteins outperformed the cas using all proteins in the majority of the cases . In addition, the best integrated ca scores were derived from the partial instead of the full data sets, similar to what we observed in the clustering approach . This similar pattern implies that collecting more data from the same sample source may not guarantee gaining better performances . In the distance - based clustering approach, the biomarker candidates were the protein clusters . Specifically, 215 (out of 396) proteins in two clusters (with the total number of clusters set as six) or 129 proteins in two clusters (with the total number of clusters set as twelve) in balf from the best performing combination of clusters were considered to be the biosignatures . Similarly, a group of 13 proteins from the best performing cluster were selected as a narrowed set of biomarker candidates in plasma . Because our approach combines patterns in abundance with functional relationships, we hypothesized that these signatures would be more robust relative to the individual candidates with top performances . In the disease model - related functional selection driven by expert knowledge, cas from the top three most differential proteins of each cluster generally outperformed the cas from all differential proteins of the cluster . Therefore, we examined the biomarker candidates in the top three proteins from each go term instead of all proteins under it . The information on the five best ca performances is listed in table s3, including the ca scores, the cluster names, and the protein lists . A single functional cluster, carbohydrate derivative metabolic process, yielded the best ca score of 0.99, which includes complement c3 (co3/c3, uniprot / gene symbol), prothrombin (thrb / f2), vitamin d - binding protein (vtdb / gc), and v - type proton atpase 16 kda proteolipid subunit (vatl / atp6v0c). These proteins were also present in some of the top - performing clusters from our cluster - based approach . Note that most proteins have multiple annotations and the six proteins that consistently recurred in the top - performing functional clusters were considered as a list of biomarker candidates of copd (using the balf data set) from the expert knowledge - driven approach . The panel of six biomarker candidates includes thrb, vtdb, co3, vatl, adiponectin (adipo / adipoq), and liver fatty acid - binding protein (fabpl / fabp1). To compare the robustness of signatures derived using different approaches and to validate our findings, we chose to use a data set of human plasma samples . These samples were taken from subjects with low bmi (<25) who smoke and have been diagnosed with copd and their corresponding healthy controls . A total of 44 proteins in human data were differentially expressed in the mouse plasma, which was used in validation . The optimal cas from using the 44 common proteins in the six clusters that were defined by the mouse plasma are listed in table 3 . The integrated ca from using all six clusters was 0.93 in the human and 0.83 in the mouse plasma . Though the best - performing cluster (the fourth cluster) in mouse did not provide a better performance in human, the top integrated ca (using the first and fourth clusters) gave comparable classification result in human plasma . Validation using the six clusters defined by the mouse balf showed similar outcomes (table 3). We also calculated the individual cas of the 44 common plasma proteins in mouse and human data sets, respectively . The results show that the top - performing individual proteins in mouse do not provide consistent classification performances using human data and only marginally discriminate the patients from their controls (table s4). This is in contrast to our findings in the cluster - based biomarker candidates in the mouse plasma, which could also classify human patients quite well . The receiver operating characteristic (roc) curves and the areas under the curves (auc) were also performed for validation, which obtained comparable results relative to the cas (figure s3). The roc curves provide the estimates for sensitivity and specificity, which is a commonly used evaluation metric in clinical studies, of the tested biomarker candidates . At the level of individual proteins, four out of the six candidates selected by the expert - driven functional selection were also identified in human plasma samples . The ca scores from individual marker candidates and several top integrations of both mice and human samples are summarized in table 4 . This panel of balf - based marker candidates, that is, thrb, vtdb, co3, and adipo, consistently showed better performances in human plasma relative to those in mouse plasma . Reasonable results were observed in all three specimens: the ca score of 0.93 in the mouse balf, 0.70 in the mouse plasma, and 0.93 in the human plasma . Detailed information on the four candidates is illustrated in figure 2, including the average fold changes and their regulation directions in the ada / group relative to the controls . The significance levels of the protein abundance changes are also provided, which were determined by the p values from the corresponding t - tests or g - tests . With the data sets available for the time - matched diseased and controlled animals, we were able to compare the individual and integrated optimal cas at the different time points during the developmental course of copd from the ada - deficient mouse model . In particular, we compared the optimal cas derived from the proteins that were individually and cumulatively significantly changed at the five time points in both fluids (figure 3). The individually changed proteins at a specific time point refer to the proteins that showed significant alterations in their abundances at this single time point, while the cumulatively changed proteins at the same time point refer to a collection of individually changed proteins from day 26 up to this time point . Both the individual and cumulative cas from balf (solid lines) consistently outperformed their counterparts from plasma (dashed lines) in terms of discriminating between diseased and nondiseased animals . Not surprisingly, the ability of this discrimination shows an increasing trend as the ada / mice get sicker in both plasma and balf . It is also interesting that these four candidates are able to classify mice fairly well at very early time points, even before outward manifestations of disease . In the field of biomedical science, the primary challenge has been shifting from data generation to data interpretation . The explosive growth of high dimensional data sets has demanded the development of semiautomated or automated tools as a necessity for knowledge discovery [4, 40]. In this study, we introduced the isic as a framework that is designed for integrating experimental measurements and expert knowledge into disease marker identification in a semiautomated manner . One main merit of the isic lies in the manner of integration, which simultaneously combines data- and knowledge - driven information into a quantitative format . A pipeline was assembled accordingly and demonstrated on several proteomics data sets of copd to identify biomarker candidates of copd . The focus of the semiautomated clustering approach is to separate the initial marker searching candidates, that is, the differentially expressed proteins in the data sets, into several different groups that contain features with similar expression patterns and functionalities within groups . In contrast, the expert - driven functional selection may be somewhat subjective; however, it can be an efficient way to extract a handful of biomarker candidates with the incorporation of proper knowledge . The classification performances of this demonstration of copd data sets on both approaches obtained comparable results that were both quite good . It is also worth mentioning that our intention here is to illustrate the individual merits and weaknesses of both approaches in the biomarker selection schemes in order to gain insight on how to comprehensively and efficiently extract valuable information from data sets . Biomarker identification is a process to select a limited number of biomolecules that convey the essential biological information distinguishing a disease state from a nondisease state . In the clustering approach, our results show that our cluster - based biomarkers are much more robust in their ability to classify human patients than the individual proteins . A possible explanation is that features in clusters may capture more consistent and comprehensive information from data relative to the individual proteins . We are currently working to include an extra step of feature selection, which will rigorously identify subsets of proteins with optimal cas, to focus smaller biomarker sets . We found that small sets of proteins could be selected with good performance using our expert knowledge - driven approach . The biomarker candidates selected in this way have a subjective component but also can potentially filter out the low quality markers identified from pure statistical processes . Another limitation of expert - driven strategy is that not all gene or gene products have annotations, which eliminates the possibilities for exploring the functional relationships among them in the currently available knowledge databases . One noticeable consistency of the two approaches is that all the optimal classification performances, indicated by the optimal integrated cas, resulted from using partial instead of the full data sets (table 2). This trend was particularly striking when using the top - three most differentially changed proteins to represent the individual go terms in clusters defined by the expert - knowledge driven functional selection (table s2). This indicates that simply using more data collected from the same data source does not guarantee improved performance and that redundant information most likely is included in the additional data . Each component serves its distinct functionality and is implemented at a different stage in the biomarker discovery process . The independence between them makes it easy to tailor individual compartments for specific data sets or to substitute using other methods with similar functions . The data reduction largely is a data - dependent process . As a means to group similar data into clusters based on a similarity criterion, the distance - based hierarchical clustering can also be achieved by other grouping mechanisms, such as k - means, self - organized maps, and fuzzy clustering . In the model integration portion, bayesian integration can be replaced with a support vector machine [42, 43], decision trees and random forests [44, 45], and artificial neural networks, which have been applied in many types of data integration . In terms of performance evaluation, ca was chosen mainly due to its suitability in cases with more than two categories . An roc curve and the measurement of auc as well as recall and precision are both reasonable substitutions for the ca but mostly limited to cases with binary responses . In conclusion, we describe a generalizable framework for integrating expert knowledge into processes of disease biomarker discovery . Our framework, isic, consists of several independent and collaborative components and is flexible enough to accommodate addition, subtraction, and modification of analyses . The integration of data - driven and knowledge - driven information is used in a distance - based clustering approach in a semiautomated manner . An expert - driven functional selection approach was also performed to select individual proteins for comparison to our automated approach . We identified signatures in a mouse model of copd and subsequently validated them in a human cohort, where they demonstrated a comparable accuracy in discriminating patients with copd from those without copd . This was in contrast to standard approaches to identify biomarkers in the mouse model, which were not robust in the human cohort . We believe that isic represents a generalizable platform for identification of robust biosignatures from integrated data sources.
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Leukemia is a hematological disorder which is caused by proliferating white blood cell - forming tissues resulting in a marked increase in circulating immature or abnormal white blood cells . Leukemia arises from a hematopoitetic stem cell characterized by a disordered differentiation and proliferation of neoplastic cells.1,2 leukemia is characterized into acute or chronic forms, according to its clinical behavior, and characterized as lymphocytic and myelocytic, according to its histogenetic origin . Acute myeloblastic leukemia (aml), which is also known as; acute myelocytic leukemia, acute granulocytic leukemia or acute nonlymphocytic leukemia, is commonly classified under 8 subgroups according to the french - american - british (fab) classification system . However in 1997, the world health organization proposed 4 groups in the aml category.25 patient with aml generally present with symptoms related to complications of pancytopenia (anemia, neutropenia, and thrombocytopenia), including weakness, and easy fatigue, infections of variable severity, and/or hemorrhagic findings such as gingival bleeding, ecchymoses, epistaxis, or menorrhagia . Combinations of these symptoms are also common.13 gingival overgrowth is defined as the excessive overgrowth of the gingival tissue . Gingival overgrowth has several causes, including poor oral hygiene, medications, serious systemic illnesses, hematological disorders, genetic conditions and it even can be idiopathic.610 oral manifestation in patient with leukemia have been described in all subtypes of aml, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.7 gingival infiltration represents a 5% frequency as the initial presenting complication of aml.4,5 gingival infiltration of leukemic cells is most commonly seen in acute monocytic leukemia (m5) and acute myelomonocytic leukemia (m4).11,12 dreizen et al13 evaluated 1076 leukemic patients and found gingival involvement in 66.7% of m5 patients and 18.5% of m4 patients.8,11,1317 this report defines an aml patient with rapid gingival hyperplasia . The dentist was the first medical examiner who diagnosed the disease, referred her to hematology clinics, and after a successful chemotherapy patient s gingival hyperplasia relapsed together with the remission of the disease . A 17-year - old caucasian woman applied to cumhuriyet university, faculty of dentistry, periodontology clinic with the chief complaint of severe gingival hyperplasia with rapid development in two weeks time . Dental examination showed a prominent gingival hyperplasia, in both jaws, especially in the anterior region . The gingival was bulbous, pale and lacked stippling with local erythematic areas (figure 1). The dentist avoided from using periodontal probe and any periodontal treatment due to the unusual patient history . In the same day, the patient referred to cumhuriyet university, faculty of medicine, hematology clinic with the preliminary diagnosis of leukemia . In medical examination, she suffered from systemic manifestations such as fatigue, nausea, vomit, anorexia, and weight loss in a month . Complete blood count, peripheral blood smear and bone marrow biopsy were taken from the patient . Complete blood count displayed a decrease in red blood cells with lowered hematocrit and hemoglobin levels (anemia); and a low platelet count (thrombocytopenia), although white blood cell levels were in normal values, a decrease in neutrophil levels (neutropenia) (table 1). Hematology clinic diagnosed the case as aml m5 according to peripheral blood smear and bone marrow biopsy in the several days . After the diagnosis of aml m5, the patient was only advised brushing her teeth, during the chemotherapy . The patient was given chemotherapy consisting of intravenous administration of cytosine arabinoside (ara - c, 100 mg / m2/day (200 mg) x 7 days) and idarubicin hydrochloride (idamycin, 12 mg / m/ day (12 mg) x 3 days). Eight weeks following the initial therapy, complete blood count values indicated remission (table 1). Regression of the gingival hyperplasia without performing periodontal therapy was also prominent (figure 2). One month later following initial therapy, monoblast cells were decreased from 80% to 5% . The patient was taken into maintenance therapy after initial therapy two times for two month . The medical administration was included; cytosine arabinoside (ara - c, 100 mg / m/ day (200 mg) x 5 days) and idarubicin hydrochloride (idamycin, 12 mg / m2/ day (12 mg) x 3 days). Although we had recalled the patient after chemotherapy for periodontal therapy, the patient did not come to our clinic . The patient was given chemotherapy consisting of intravenous administration of cytosine arabinoside (ara - c, 100 mg / m2/day (200 mg) x 7 days) and idarubicin hydrochloride (idamycin, 12 mg / m/ day (12 mg) x 3 days). Eight weeks following the initial therapy, complete blood count values indicated remission (table 1). Regression of the gingival hyperplasia without performing periodontal therapy was also prominent (figure 2). One month later following initial therapy, monoblast cells were decreased from 80% to 5% . The patient was taken into maintenance therapy after initial therapy two times for two month . The medical administration was included; cytosine arabinoside (ara - c, 100 mg / m/ day (200 mg) x 5 days) and idarubicin hydrochloride (idamycin, 12 mg / m2/ day (12 mg) x 3 days). Although we had recalled the patient after chemotherapy for periodontal therapy, the patient did not come to our clinic . Even there are several etiologies for gingival overgrowth; usually each etiology has its own overgrowth characteristics . For example, while genetically induced gingival overgrowth show firm, normal coloured (pink), non - inflamed gingival tissues, gingival overgrowth due to blood dyscrasias are edematous, soft, tender to touch and show tendency to bleed.4,5,9,10,18 gingival hyperplasia is most commonly seen with the aml subtypes acute monocytic leukemia (m5) (66.7%), acute myelomonocytic leukemia (m4) (18.5%), and acute myelocytic leukemia (m1, m2) (3.7%).19 in this case report, a rapid gingival hyperplasia together with gingival bleeding was the main reason of the patient to seek therapy . The dentist who examined the patient suspected from the duration of the overgrowth, and from the spontaneous gingival bleeding, without a prominent microbial dental plaque or calculus accumulation . A systemic disease such as probably one of the hematologic diseases was the dentist s early diagnosis, and a hematology consultation was asked . Gingival involvement is common in aml and found in 66.7% of the 1076 leukemic m5 patients, in a study by dreizen et al.13 in these kinds of patients, a dental therapy driven without hematological consultation could be fatal . The fact that gingival hyperplasia are sometimes the first manifestation of the disease implies that dental professionals must be sufficiently familiarized with the clinical manifestations of systemic diseases.6,8,11 also a prominent remission was observed in gingival hyperplasia after chemotherapy together with peripheral blood values . This shows that, in cases like aml, elimination of the factor initiating the gingival hyperplasia or maintaining the ideal systemic condition might give a full success in periodontal healing . In this case, our patient s medical condition forced us to do the medical therapy first, because of that the dental therapy of the patients was delayed to see the effect of medical therapy . So, for totally remission of gingival hyperplasia, necessary periodontal therapy must be applied after systemic status was controlled by chemotherapy . As a result, this case reminds that dentists and physicians should be aware of the importance of recognizing oral manifestations of systemic diseases . Although physicians most commonly diagnose leukemia, dentists can also diagnose patients with acute myelogenous leukemia . The dental practitioner should have an awareness of diagnostic signs and complications associated with leukemia to better aid in diagnosis and subsequent treatment and management . This case also shows that gingival overgrowth due to acute leukemia is commonly improves by chemotherapy without any periodontal treatment.
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Endometriosis is a chronic, benign, oestrogen - dependent inflammatory disease affecting approximately 10% of reproductive age women and 3550% of women with pelvic pain and infertility . It can be a debilitating disease with symptoms of dysmenorrhoea, dyspareunia, and chronic pelvic pain . The definition of endometriosis is histological and it requires the identification of the presence of endometrial gland and stroma - like tissue outside (ectopic) the uterus . Occasionally, ectopic endometriotic lesions can be found in other parts of the body such as kidney, bladder, lungs, and even in the brain . There has been efforts to standardize the surgical staging of endometriosis and histopathological changes with updated modified american fertility society scoring . Furthermore, there is a severe lack of knowledge on the natural progression of the disease in women since the severity measurement will require repeated invasive surgery . There are reports of endometriosis associated with spontaneous regression, no progression, and progressing to ovarian carcinomas [9, 10]. At the present time no methods exist to predict future prognosis of the disease stage from initial surgical diagnosis . Endometriosis has estimated annual costs of us $12 419 per woman (approximately 9579), comprising one - third of the direct health care costs with two - thirds attributed to loss of productivity . For obvious and above mentioned reasons, despite being the causal basis for over 30% of new referrals to gynaecology clinics (local data), the management of endometriosis remains difficult . Currently, there is no curative treatment for endometriosis and clinical management of symptoms such as pain is through medical and/or surgical measures . Medical management follows the basic principle of reducing inflammation, suppressing ovarian cycles and inhibiting the effect of oestrogen . Surgical management attempts to either remove only the identified endometriotic lesions or complete excision of pelvic organs . Controversies exist regarding the best method of treatment; for example, some authors have suggested that surgical excision promotes disease recurrence whilst others consider surgical excision as a way to reduce the risk of progression to severe disease or future ovarian cancer [10, 12]. Neither medical nor surgical options provide long term or universally acceptable relief for patients . Improving our current knowledge on the pathogenesis of endometriosis therefore helps the clinical and basic science researchers to identify novel more suitable targets for formulating more effective therapeutic and diagnostic means . Many theories have been proposed to explain the pathogenesis of endometriosis and to date they all remain to be conclusively confirmed . In this review, the predisposing factors in developing endometriosis, as well as the interplay between the pathological mechanisms involved in the initiation and propagation of different endometriotic lesions, will be discussed . We initially searched pubmed for relevant literature using the terms endometriosis and pathogenesis or although those papers provided the basis for this review, for detailed understanding of the topic we extended our search to much older yet frequently referred articles . Studies that were deemed suitable by the authors included those that examined the pathophysiology of human endometriosis: from in vitro basic science (molecular, genetical and functional) studies, studies employing animal (rodent / primate) models, gene expression, and epidemiological studies . Interrogation of pathogenesis of endometriosis highlights the current drawbacks associated with the classification of this disease . The revised american fertility society classifies endometriosis according to multiple criteria including histopathological as well as anatomical features, distinguishing superficial endometriosis from deep lesions of the peritoneum and ovaries . Deep endometriosis is defined arbitrarily as adenomyosis externa, infiltrating the peritoneum by> 5 mm . It is noteworthy that the current classification system is limited by observer error as well as reproducibility and this may explain the poor correlation between extent of the disease and its clinical presentation . Furthermore, histological information of endometriosis is limited by the technical efficiency in endometriotic biopsy sampling and processing, particularly when the lesions are located close to organs such as ureters, bowel and bladder . A separate classification system (enzian score) it is a helpful aid in describing this type of endometriosis but it needs further refinement . Clinical differences between superficial and deep endometriosis have been described, where severe pain is associated with> 95% of deep endometriosis as compared with superficial endometriosis . Progression of superficial endometriosis has been compared to that of a benign tumour, whereas the recurrence and progression of deep endometriosis has been reported to be rare [8, 17]. Superficial and deep endometriosis has been categorised by some authors as two different diseases with different pathogeneses whereas others regard them as different manifestations of the same disease . Naturally this lack of consensus with disease classification creates another ambiguity around much of the available literature on pathogenesis . This theory proposes that endometriosis occurs due to the retrograde flow of sloughed endometrial cells / debris via the fallopian tubes into the pelvic cavity during menstruation . However, retrograde menstruation occurs in 76%90% of women with patent fallopian tubes and not all of these women have endometriosis . The larger volume of retrograde menstrual fluid found in the pelvises of patients with endometriosis as compared with healthy women may increase the risk of endometriotic lesions implantation . In non - human primate models, it is possible to induce endometriosis by inoculating autologous menstrual products simulating retrograde menstruation in the peritoneal cavity of baboons and macaques . With a single inoculation of menstrual endometrial tissue directly in to the pelvic cavity, up to 46% of the animals have shown development of endometriotic lesions in the pelvic cavity, whereas 100% of animals developed peritoneal endometriotic lesions after two consecutive cycles of inoculations of curetted menstrual endometrium . These lesions were histologically and clinically similar to human ectopic endometriotic lesions . Furthermore, in a recent study deep nodular endometriosis was generated by ectopic implantation of full thickness endometrium including the basalis layer, highlighting the involvement of the endometrial basalis layer in development of ectopic lesions . However, only the well - differentiated cells from the superficial functionalis layer are shed normally with the menstrual flow, the deep endometrial basalis layer remains intact throughout the woman's life . The regeneration of endometrial functionalis after menstrual shedding is thought to originate from this basalis . Therefore by placing this basalis tissue with the ability to generate endometrial functional layer in the pelvis, the non - human primate models may not completely mimic the events of spontaneous retrograde menstruation . Further evidence to support sampson's theory come from the observation that factors obstructing menstruation, such as congenital abnormalities including imperforate hymen and iatrogenic cervical stenosis, increase retrograde menstruation and the risk of developing of endometriosis . Increased retrograde menstruation through experimentally induced cervical stenosis also caused endometriosis in non - human primate models . The location of superficial endometriotic lesions in the posterior aspect and left side of the pelvis may be due to the effects of gravity on regurgitated menstrual product and the anatomical position of the sigmoid colon . However, this theory has been disputed in the past since it cannot explain the occurrence of endometriosis in pre - pubertal girls, newborns, or males . Neonatal uterine bleeding, occurs in the immediate postnatal period in most girls following the withdrawal of (maternal) ovarian hormones, similar to menstrual bleeding and retrograde flow of this uterine bleeding has been proposed as the reason for prepubertal endometriosis . Other theories have proposed that endometriosis originates from extrauterine cells that abnormally transdifferentiate or transform into endometrial cells . The coelomic metaplasia theory postulates that endometriosis originates from the metaplasia of specialised cells that are present in the mesothelial lining of the visceral and abdominal peritoneum . Hormonal or immunological factors are thought to stimulate the transformation of normal peritoneal tissue / cells into endometrium - like tissue . However, the usual driving force for endometrial growth, oestrogen, is not present in the pre - pubertal girls and therefore this condition may be different from endometriosis that is found in women of reproductive age . Ectopic endometrial tissue has also been detected in female foetuses and it has been suggested that endometriosis may be the result of defective embryogenesis . According to this theory, residual embryonic cells of the wolffian or mullerian ducts persist and develop into endometriotic lesions that respond to oestrogen . Furthermore, recent theories that are put forward suggest coelomic metaplasia to be the origin of adolescent variant of severe and progressive form of endometriosis . However, this theory is imperfect due to endometriotic lesions being found in areas outside of the course of mullerian duct . Others have also proposed that endogenous biochemical or immunological factors induce resident undifferentiated cells to differentiate into endometrial - like tissue in ectopic sites resulting in endometriosis . This suggestion is supported by the studies describing hormone - dependent transformation of peritoneal cells into mullerian - type cells . Steroid hormones should play a central role in the aetiology of endometriosis since it is a disease of women in reproductive age and not usually seen in postmenopausal women who are not on hormonal treatment . Similar to the eutopic endometrium, the growth of ectopic lesions are thought to be regulated by ovarian steroid hormones . Oestrogen is the driving force of endometrial proliferation and ectopic lesions may have an increased responsiveness to oestrogen, thus enhancing the development of endometriosis . Environmental toxins, such as dioxin, are implicated in the aetiology of endometriosis, which may mimic oestrogen via interacting with oestrogen receptors . Furthermore, there may be a higher bioavailability of oestradiol in endometriotic tissue due to the local aromatisation of circulating androgens to oestradiol by endometriotic stromal cells and also there may be reduced conversion of oestradiol to the less potent oestrone due to the ectopic endometriotic tissue expressing decreased 17-hydroxysteroid enzymes . Many authors believe that endometriosis is associated with resistance of the endometrium to progesterone which plays a pivotal role in the pathogenesis [33, 34]. The harnessing of the oestrogen - driven mitotic / proliferative action on the endometrium by progesterone during the secretory phase of the cycle does not occur in the endometriotic lesions and sustained proliferative activity is seen in the eutopic endometrium of women with endometriosis in the secretory phase [35, 36]. The progesterone resistance may be due to the endometriotic lesion having a lower expression of progesterone receptors or as a result of a functional abnormality of the existing progesterone receptors . Increased oxidation of lipoproteins has been associated with the pathogenesis of endometriosis, where reactive oxygen species (ros) cause lipid peroxidation that leads to dna damage in endometrial cells . The presence of water and electrolytes in the increased peritoneal fluid volume in patients with endometriosis harbours the source of ros . These patients also have iron overload in their peritoneal cavities from the breakdown of haemoglobin, which in turn causes redox reactions . The release of the proinflammatory heam products and the oxidative stress signals generated from the ros cause inflammation which leads to the recruitment of lymphocytes and activated macrophages producing cytokines that induce oxidizing of enzymes and promotes endothelial growth . The excess production of ros is also accompanied by a decreased level of antioxidants that usually eliminates these molecules [38, 41]. Resulting accumulation of ros may contribute to the propagation and maintenance of endometriosis and associated symptoms . The observation that autoimmune diseases to be more common in women with endometriosis support the possibility that pathogenesis of endometriosis may involve a defective immune response in these patients . Women with endometriosis have a higher concentration of activated macrophages, decreased cellular immunity, and a repressed nk cell function [43, 44]. The regurgitation of endometrial cells into the peritoneum triggers an inflammatory response, recruiting activated macrophages and leukocytes locally . This inflammatory response may cause a defective immune - surveillance that prevents elimination of the menstrual debris and promotes the implantation and growth of endometrial cells in the ectopic sites . Furthermore, there are suggestions that during the evolutionary process the peritoneal immune clearance that occurs in non - human primates has been lost in humans, and this may contribute to the persistence of the menstrual debris in the pelvic cavity and subsequent development of endometriosis in women . The survival and resistance to immune - cell - mediated lyses of endometriotic cells are ensured by masking these ectopic cells to the immune system, where, for example, ectopic endometrial cells modulate the expression of hla class i molecules [43, 47]. Both immune and endometrial cells secrete cytokines and growth factors, which induce cell proliferation and angiogenesis; thereby promoting implantation and growth of ectopic lesions . Possibly as a consequence, women with endometriosis have higher expression of cytokines and vascular endothelial growth factors in their peritoneal fluid, which promote proliferation of endometrial cells and angiogenesis [49, 50]. Alteration of the endometrial cell fate to favour antiapoptotic and proproliferative phenotype is paramount for the survival of the endometrial cells in the peritoneal cavity to initiate ectopic deposits and for the maintenance of the established lesions . By examining matched eutopic endometrium and ectopic lesions from women with endometriosis and in baboon with induced disease, we have recently shown that telomerase enzyme may play a central role in this altered endometrial cell phenotype [36, 51]. There is plethora of evidence suggesting an upregulation of antiapoptotic and prosurvival genes and reciprocal downregulation of the genes regulating the apoptosis pathway in ectopic endometrial cells . In addition to the decreased scavenger activity, the endometrium in patients with endometriosis expresses higher levels of antiapoptotic factors . The inhibition of the apoptosis of endometrial cells may also be mediated by the transcriptional activation of genes that normally promotes inflammation, angiogenesis, and cell proliferation . A genetic basis for the development of endometriosis is suggested by the reports of familial aggregation, the high risk of endometriosis in those with an affected first - degree relative, and the observations of concordance of endometriosis in twins . A great number of studies have related genetic polymorphisms as a factor that contributes to the development of endometriosis . Endometriosis has a polygenic mode of inheritance that is likely to involve multiple loci and some chromosomal regions were reported to be associated with the corresponding endometriosis phenotype . Inherited as well as acquired genetic factors may predispose women to the attachment of ectopic endometrial cells to the peritoneal epithelium and the evasion of these lesions from immune clearance . Differences in genes and protein expression between patients with and without endometriosis have been reported . Genes that have been implicated in the pathogenesis of endometriosis include those encoding detoxification enzymes, polymorphism in oestrogen receptor, and genes involved in the innate immune system . Genetic mutations that cause cell damage are implemented in the progression of endometriosis, since women with endometriosis show altered endometrial cell behaviour, favouring extrauterine adhesion and growth . Over the past decade using laser capture microdissection and high throughput and high resolution comparative genomic hybridization (cgh) arrays, considerable genomic alterations in both eutopic and ectopic endometria of women with endometriosis have been identified [59, 60]. Collectively this data suggests that different types of endometriosis may be associated with altering different gene clusters that regulate specific cellular functional aberrations . The monthly regeneration of the endometrium after menstrual shedding, reepithelialisation of the endometrium after parturition or surgical curettage, supports the existence of a stem cell pool . Since the basalis layer of the endometrium is not shed with the monthly menstrual shedding of the functional layer, the stem cells are thought to reside in the basalis layer of the endometrium . Recently, clonogenic cells, which are thought to represent the stem cell population in the human endometrium have been identified and proposed to be involved in the formation of ectopic endometrial lesions . Stem cells are undifferentiated cells, characterized by their ability to self - renew and differentiate into one or several types of specialized cells . Differentiation is defined as a change in cell phenotype secondary to alteration in the cell's gene expression, enabling the cell to have a specific function . Endometrial self - generation may occur through stem cells in specific niches of the endometrium . The undifferentiated endometrial stem cells may be less responsive to ovarian steroids than the terminally differentiated progeny due to lack of expression of hormone receptor . In addition to the resident endometrial stem cells, incorporation of circulating bone marrow - derived stem cells may contribute to the cyclic regeneration of the endometrium . The involvement of stem cells in the formation of endometriotic deposits could be as a result of abnormal translocation of normal endometrial basalis via retrograde menstruation . Brosens et al . Postulated that the uterine bleeding in neonatal girls contains a high amount of endometrial progenitor cells . Some of these cells may deposit and survive in the peritoneal cavity after retrograde flow and may reactivate in the adolescents in response to ovarian hormones . However, there is no current data on the amount of endometrial stem / progenitor cells in neonatal period when compared to the adult endometrium . Furthermore, since even the aging postmenopausal endometrium seem to have adequate amount of progenitor cells to generate a competent normal functionalis with the essential hormonal stimulation, it seems unlikely that there are significant differences in the progenitor activity between the premenopausal and postmenopausal endometrium . Proposed that women with endometriosis abnormally shed the endometrial basalis tissue, which initiate endometriotic deposits after retrograde menstruation . The observation in the baboon model of endometriosis induction, where placement of the stem cell rich endometrial basalis in the pelvic cavity resulting in 100% induction of endometriosis in all animals, may further support leyendeckers theory . If the basalis contains the stem / progenitor cells, they are likely to survive and initiate endometriotic deposits in the pelvis than the differentiated endometrial cells from the functionalis . Due to their natural ability to regenerate, these stem cells may give rise to new endometriotic deposits . The fact that women with endometriosis possibly shed significantly more of the stem - cell rich basalis layer as compared to healthy women, together with the similarity observed between ectopic lesions and the basalis layer, may support the possibility of retrograde menstruation providing an access for the endometrial stem cells to extrauterine structures [63, 69]. Alternatively, these stem cells may be transported via the lymphatic or vascular pathways to ectopic sites . The fact that some of the endometrial stem cells have bone marrow origin further supports the haematogenous dissemination theory of these cells . Recent studies have further suggested that mobile stem cells may be involved in endometriosis progression, where cells derived from ectopic lesions in induced endometriosis migrated to the eutopic endometrium . However, since stem cells are normally expected to differentiate into mature cells in concordance with the environmental niche, the supposedly multipotential endometrial stem cells in the peritoneal cavity should differentiate in to peritoneal - type cells . It is possible that the deposition of endometrial tissue fragments containing both endometrial stem cells and their niche cells in the peritoneal cavity promote regeneration of endometrium - like tissue, due to the signal received by the stem cells from the surrounding endometrial niche cells . On the other hand, the relocation of an aberrant or committed stem cell from the endometrium to an ectopic site may also generate endometrium - like lesions . Endometrial tissue produces several chemokines and angiogenic cytokines; therefore, neovascularisation in the ectopic sites can presumably follow, thus ensuring the establishment of these lesions . A further possibility of stem cell involvement in endometriosis is the transdifferentiation of the peritoneal, haematopoietic, or ovarian stem cells into endometrium like tissue . Peritoneal cavity connects directly with the uterine cavity and there is a free flow of the cytokine / chemokine rich fluid between the two environments . This direct connection may regulate the endometrium - like differentiation of resident stem cell population in the peritoneal cavity . Although possible, the reasons for such specific differentiation of the peritoneal stem cells in to endometrium - like tissue in only up to 10% of the female population remain unexplained . The different theories implicated in the pathogenesis of endometriosis indicate that the aetiology of endometriosis is complex and multifactorial, involving hormonal, genetic, immune, and environmental components . While retrograde menstruation may be one of the initiating steps in the pathogenesis of superficial endometriosis, genetic and microenvironmental factors that prevent clearance of ectopic lesions and allows remodelling of peritoneum are essential for the propagation of endometriotic lesions [73, 74]. Pathogenesis of endometriosis is propagated by an altered peritoneal fluid composition as a result of genetic, hormonal, and environmental factors [75, 76]. Figure 1 depicts the interplay between the different factors that may be involved in the pathogenesis of endometriosis . Retrograde menstruation may not explain the pathogenesis of deep endometriosis, where no deep endometrial lesions could be induced in animal models through peritoneal instillation after endocervical removal of menstrual endometrium . However, deep nodular lesions, which is not usually shed at menstruation, could be readily induced with the transplantation of endometrial basalis tissue in a baboon model . Other theories such as the coelomic metaplasia, induction of cellular transformation into endometrial cells, and the embryonic remnant theory may better explain the aetiology behind deep endometriosis . Ectopically placed stem cells that are of endometrial or haematopoietic origin or abnormal endometrial differentiation of a resident tissue stem cell may be the first step in the establishment of an ectopic endometrial lesion . The subsequent proliferation and propagation of such lesions may also be dependent on mobile, endometrial progenitor - type cells in these ectopic lesions that are involved in initiating further lesion and also in maintaining the disease . A dysfunctional immune clearance and a genetic predisposition that allow these ectopic lesions to grow in an aberrant microenvironment may also contribute to the development of the disease . The current therapeutic regimens for endometriosis are usually based on manipulating the ovarian steroid hormones that may preferentially target terminally - differentiated ectopic endometriotic cells which would normally die off via apoptosis, while the stem cells that propagate the disease may not be affected . Improving our understanding of the pathogenesis of endometriosis will direct further future work on more appropriate therapeutic targets that can provide the much needed curative and universally acceptable treatments for endometriosis.
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Bloc excision is mandatory but can represents a real technical challenge, expecially if tumor involves both the skull and the dura . The term primary intraosseous meningioma (pim) is used for an extradural meningioma that arises from the cap cells staying in the bone . They are mostly met at the periorbital and frontoparietal regions and they are, generally, devoid of neurological findings . Other tumors that can involve the skull are hemangiomas, metastases, many type of carcinoma and sarcoma arising directly by bone or by contiguous tissue (cutis, muscle, dura) and invading secondarily the skull . The degree of resection is the most important factor correlating with recurrence and clinical long - term outcome . The aggressive surgery that is needed in these cases could bring a large bone defects to fill . The removal of a tumor involving the skull and the intracranial space could present a technical challenge, especially if the tumor has a dural and/or brain attachment . With a standard craniotomy, the maneuver of separating the intraosseus portion of the tumor from the intracranial portion might produce hemorrhage, traction and injury to the underlying brain . We describe a technique where the tumor involving the bone is before left in situ, normal dura is exposed around the tumor, and the tumor can be afterwards removed en bloc with direct vision and minimally traction of the brain surface . 8 patients, with different tumors involving both skull and dura, were treated with these technique . The histological examination of these patient s specimens showed: atypical meningioma, pim and anaplastic meningioma . Below, we describe this technique used in the last patient treated, a man aged 45 with no neurological signs . Patient underwent dcc, total surgical excision of pim and cranioplasty with peek custom made prothesis (synthes). A 45 years old man, b.v ., presented to our department of neurosurgery with a mass arising from the right frontoparietal bone . He had not neurological signs and symptoms . Computed tomography (ct) scan showed a homogeneous expansive bone lesion at the right fronto - parietal region, without contrast enhancement . On magnetic resonance imaging (mri), the lesion was hypointense on t1- and t2-weighted images with no contrast enhancement after intravenous gadolinium administration, but contrast enhancement was seen at the adjacent dura . In the differential diagnosis, were first considered a fibrous dysplasia and primary bone tumors . Patient undergoes tumor removal and cranioplasty with peek custom made prothesis (synthes). Under general anesthesia, the patient was positioned with the head turned approximately 30 toward the contralateral side, to expose the skull mass, with a pillow under the ipsilateral shoulder . The temporal muscle was splitted, the muscolar part that covering the tumor was left in situ for a completely radical resection and the remaining part was overturned . One only burr hole was performed laterally to the midline and we performed the first circle craniotomy around the lesion . Then, using the same burr hole, a second and more enlarged circular concentric craniotomy was performed . There was no tumor in the bone ring and the dura beneath the ring was not involved . The inner surface of the dura was amply involved by the tumor, and it was dissected by the underlying brain surface under direct vision thanks to the space guaranteed by bone ring removal . The dural defect was repaired with pericardium bovinum patch and a peek custom made cranioplasty was performed (fig 3). Tumor involving many tissues should be resected en bloc for minimizing risk of seeding and recurrency . In fact, meningioma with calvarial hyperostosis is commonly associated with an increased rate of recurrence if limited bone resection is performed . Although radical excision of tumor involving the skull is the gold standard of oncological treatment, elevating a bone flap when the tumor involves both the skull and the dura could be very hard and represents a technical challenge . With a standard craniotomy this maneuver, done without direct vision, could produce hemorrhage and unwanted traction on the underlying brain surface . We have described a technique, a dcc, that permit to expose the normal dura surrounding the tumor and permit the resection of entire specimen with a direct vision of the underlying brain . In our opinion also have described a similar procedure for the removal of nail that was hammered in both the skull and the brain . Sekhar et al . Have utilized the concentric craniotomy technique for a fronto - orbital approach and ibarra - de la torre et al . For a posterior fossa craniotomy to protect the underlying dura and vascular structures . We believe that dcc is an optimal alternative on standard technique because permits the total removal of tumor involving both skull and dura structures, minimizing tractions on the underlying brain and permitting the direct vision during the dissection.
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In this article, we describe a new computational tool to determine nearly optimal folding pathways between two given secondary structures of an rna sequence . Our tool, rnatabupath, and related web server, rnapathfinder, have potential applications in synthetic biology; in particular, our work can be used to help engineer bistable conformational switches with reasonable folding kinetics [see abfalter et al . (1) and flamm et al . (2) for methods to computationally design bistable switches . ). Folding pathways play an important role in various biological processes, including the hok / sok (host killing / suppression of killing] system (3) and transition between two metastable structures, as in the conformational switch in spliced leader (sl) rna from leptomonas collosoma (4). In the hok / sok system, the hok gene of escherichia coli codes a small (52 amino acids) toxin causing irreversible damage to the cell membrane . While the very stable hok - mrna is constitutively expressed from a weak promoter, the highly unstable (rapidly degraded) sok - rna is constitutively expressed from a strong promoter . Dalgarno sequence; however, slow exonucleolytic processing digests the last 40 nt of the 3-end of hok - mrna, thus transforming the molecule into its active form in which the shine dalgarno sequence is no longer sequestered . If r1 plasmids of e. coli are present in sufficient copy number, then a portion of the 64 nt sok - rna, which is complementary to hok - mrna leader region, binds to the active conformation of hok - mrna, thus causing degradation of the complex by rnaseiii (3). If plasmids are not present in sufficient copy number, then the cell is killed by hok toxin . In this fashion, efficient plasmid stabilization is ensured in the population . [see (3) for a review of the hok / sok system .] In the case of sl rna from certain trypanosomes and nematodes, a portion of the 5 exon is donated to another mrna by trans splicing . Intermediate structures may be important for the process of splicing, as shown by lecuyer and crothers (4), who performed stopped - flow rapid - mixing and temperature - jump measurements of the kinetics for the structural transition between two low energy structures of sl rna from l. collosoma . Conformational switches are thought not only to play a role in such trans splicing but also in transcriptional and translational regulation, protein synthesis and mrna splicing . As indicated by the examples of hok / sok and sl rna, for that reason, this problem has been considered by a number of authors, both in the context of rna secondary and tertiary structure . Nudge elastic band (neb) method in amber to sample low energy paths for rna conformational changes at the three - dimensional atomic scale . Crick / hoogsteen gg non - canonical pairs, where one g is syn around the glycosidic bond while the other g is anti . Since prior nmr - constrained modeling had demonstrated that the gg pairs change from (syn)g-(anti)g to (anti)g-(syn)g on the millisecond timescale, such atomic - level simulations using amber were feasible . Due to large structural transitions between metastable structures in conformational switches, it seems clear that three - dimensional atomic scale simulations using molecular dynamics cannot adequately address the general problem posed in this article . For that reason, it is important to develop efficient algorithms to determine optimal and suboptimal folding pathways between rna secondary structures . Intermediate structures from such low energy pathways can then be further investigated using atomic scale methods such as neb . Morgan and higgs (6) appear to be among the first to have considered the problem of determining an optimal folding pathway between two given secondary structures a, b of a given rna sequence . If a, b are secondary structures for a given rna sequence s, then a folding pathway from a to b is a sequence a=0, 1,, n = b such that each intermediate structure i differs from the next structure i+1 by exactly one base pair . Direct if every intermediate structure i+1 is obtained from the preceding structure i by either adding a base pair that belongs to b but not a or by removing a base pair that belongs to a but not b. if a pathway is not direct, then it is indirect. The saddle point in a pathway a = 0, 1,, n = b is the intermediate structure i of highest energy (in case there is more than one intermediate structure having maximum energy along the path, we define the saddle point to be the first such structure, having smallest index . ). Barrier energy of a pathway from a to b is the energy difference e() e(a), where is the saddle point of the pathway . Clearly, the barrier energy is of fundamental importance in folding kinetics . Morgan and higgs describe both a greedy algorithm to construct a direct pathway, as described in the materials and methods section, as well as an algorithm to construct an indirect pathway by gluing together greedy direct pathways between low energy structures sampled from the partition function (in (6), morgan and higgs compute the partition function z = s exp(e(s)/rt), where the sum is over all secondary structures of a given rna sequence, and e(s) is the nussinov energy model (7). Since the partition function is inductively computed, it is simple to the stochastically sampled structures from the low energy boltzmann ensemble . Later, ding and lawrence (8) describe the same stochastic sampling algorithm, sfold, with the exception that turner energy model (9) is used in the place of the nussinov energy model . ). While morgan and higgs had worked with the nussinov energy model (7), which ascribes 1 per base pair, with no energetic contribution due to base stacking or loop entropies, our implementation of the direct and indirect pathway algorithms of morgan and higgs uses the turner nearest neighbor energy model (914), whose parameters have been obtained by uv absorption (optical melting) experiments . Since the pioneering work of morgan and higgs, other groups have developed methods to compute folding pathways between secondary structures . Flamm et al . (2,15) describe an exact algorithm, barriers (barriers is available at http://www.tbi.univie.ac.at/ivo / rna / barriers/. ), that computes optimal (possibly indirect) folding pathways between any two locally optimal secondary structures (a locally optimal secondary structure is one in which the energy is not lowered if a single base pair is either added or removed . Sometimes such structures are called metastable). While most biologically important examples of pathway computation concern metastable or locally optimal structures, there are nevertheless important exceptions, such as conformational switches (incompletely) determined by experimental methods; the adenine riboswitch from vibrio vulnificus (rb2) (16) is indeed one such example . Barriers relies on the vienna rna package program rnasubopt (17) that exhaustively generates all secondary structures within a user - specified energy upper bound . For this reason, although barriers is the only exact algorithm, it is generally limited to relatively small rna sequences or those for which the energy of the saddle point between a and b is not too large . In (18), flamm et al . Describe a breadth - first search algorithm with bounded look - ahead, to compute nearly optimal the algorithm is implemented in the program findpath.c, now part of the vienna rna package . Finally, as part of the method parnass, voss et al . Our new algorithm, rnatabupath, produces (possibly indirect) almost optimal folding pathways by using a heuristic from combinatorial optimization theory known as tabu search. Tabu search, described in the text by f. w. glover and m. laguna (20), is a meta - heuristic to avoid being trapped in local optima in local search algorithms . One of its key components, which we use in this article, is a short memory, often called the tabu search then selects the best configuration in the neighborhood which is not in the tabu list . This neighbor may in fact degrade the value of the objective function tabu search has been a very effective technique in combinatorial optimization for a wide variety of problems and is an integral part of the repertoire of optimization techniques . To fix ideas, figure 1 depicts three folding pathways for a toy 12 nt rna sequence ggggggcccccc, with structures a = . ((.....)).. having free energy of 1.40 kcal / mol and b = .. (((...))). Having free energy of 1.70 kcal / mol . Structure b is not locally optimal, since by adding the base pair (1, 11) to a and by adding base pair (2, 12), one obtains structures (((.....))). And . ((((...)))) having free energies 4.70 and 4.20 kcal / mol, respectively . It follows that barriers cannot be applied (in such cases, following the suggestion of an anonymous referee, one could first determine locally optimal structures, that, respectively, contain a, b, apply barriers to find an optimal path between, . This yields a near - optimal path between a, b.). The left, middle and right panels displays the path computed by our implementation of the morgan figure 1.three folding pathways for the (toy) rna sequence s = ggggggcccccc, between the secondary structure a = . ((.....)).. with free energy 1.40 kcal / mol and the structure b = .. (((...))). With 1.70 kcal / mol . The left panel of this figure depicts a (direct) folding pathway from a to b produced by our implementation of the morgan the middle panel depicts the indirect folding pathway produced by our implementation of the extension of morgan (.........) contains the base pair (2, 12) which is present in neither a nor b. the right panel depicts a folding pathway from a to b produced by our rnatabupath algorithm . Although rnatabupath often yields indirect pathways, in this case, the pathway returned by rnatabupath is direct . Note that the last three structures proposed by rnatabupath are...(.....).., .. ((.....)). Loop is energetically more favorable than the alternative (not proposed by rnatabupath), given by structures .... (...)...,...((...)).., .. (((...)))., respectively, having free energy of 4.90, 1.60 and 1.70 kcal / mol . Secondary structures are indicated in the familiar (vienna) dot bracket notation, while free energy in kcal / mol appears to the right of each structure . Free energies are determined by the program rnaeval from the vienna rna package (27). Three folding pathways for the (toy) rna sequence s = ggggggcccccc, between the secondary structure a = . ((.....)).. with free energy 1.40 kcal / mol and the structure b = .. (((...))). With 1.70 kcal / mol . The left panel of this figure depicts a (direct) folding pathway from a to b produced by our implementation of the morgan the middle panel depicts the indirect folding pathway produced by our implementation of the extension of morgan (.........) contains the base pair (2, 12) which is present in neither a nor b. the right panel depicts a folding pathway from a to b produced by our rnatabupath algorithm . Although rnatabupath often yields indirect pathways, in this case, the pathway returned by rnatabupath is direct . Note that the last three structures proposed by rnatabupath are...(.....).., .. ((.....)). Loop is energetically more favorable than the alternative (not proposed by rnatabupath), given by structures .... (...)...,...((...)).., .. (((...)))., respectively, having free energy of 4.90, 1.60 and 1.70 kcal / mol . Secondary structures are indicated in the familiar (vienna) dot bracket notation, while free energy in kcal / mol appears to the right of each structure . Free energies are determined by the program rnaeval from the vienna rna package (27). Figures 24 depict examples where indirect pathways may have (provably) lower barrier energies than every direct pathway, while figure 5 displays the two meta - stable structures of host killing (hok) rna . Pseudobase (21), furnish canonical examples where direct pathways are likely to have greater barrier energies than even naive indirect pathways . Type - h pseudoknots admit a planar representation where certain base pairs are depicted above the horizontal line corresponding to the rna sequence, while others are depicted below the line see figure 2 for illustration . Define structure a [respectively b] to consist of those base pairs above [respectively below] the line . Clearly any direct path from a to b must proceed by removal of all base pairs from a, resulting in the empty structures, followed by addition of all base pairs from b. it follows that e(a) is a lower bound for the barrier energy of every direct path from a to b, where a, b are indicated in figure 2 . Figure 2.consider the 46 nt rna sequence cgcgacggcu acgcgacggc aaugccguug cgaagccguc gcgauc, with secondary structures a = (((((((((..............))))))))).............. having free energy 16.04 kcal / mol and b = ........... (((((((((..............)))))))))... having free energy 18.14 kcal / mol . The structure a consists of the base pairs lying above the line in this figure, while the structure b consists of the base pairs lying below the line . Program barriers cannot be used, since neither a nor b is locally optimal . Figure 3.a manually designed indirect folding pathway for the 46 nt rna sequence cgcgacggcu acgcgacggc aaugccguug cgaagccguc gcgauc, proceeding from locally optimal secondary structure a = (((((((((..............))))))))).............. having free energy 16.04 kcal / mol to locally optimal structure b = ........... (((((((((..............)))))))))... having free energy 18.14 kcal / mol . Intuitively, this pathway can be visualized as repeatedly moving the remaining rightmost right - parenthesis to the right, then repeatedly moving the rightmost left - parenthesis to the right . In this manner, the barrier energy of this indirect path is 13.68 kcal / mol, while every direct path must have a barrier energy of at least 16.04 kcal / mol, since the empty structure must be an intermediate structure in every direct path in this example . Indeed, due to nucleation energy required to start a hairpin in the empty structure, the barrier energy of every direct path must properly exceed 16.04 . In this case, vienna package program findpath.c with look - ahead 100 returns a barrier energy of 18.27, while rnatabupath returns a barrier energy of 16.84 . Figure 4.this figure depicts a folding intermediate in a low energy indirect path from a to b (unexplained notation taken from figure 2). Clearly, every direct path from a to b must have the empty structure as an intermediate structure, hence the lowest barrier energy of a direct folding pathway must be at least 16.04 kcal / mol (in fact even larger due to nucleation energy). However the indirect folding pathway depicted in table 3 has a barrier energy of 13.68 kcal / mol . Figure 5.two secondary structures of host killing (hok) rna, taken from figure 8 of shapiro et al . The left panel depicts the secondary structure of 396 nt hok - rna, presumably based on figure 1b of franch et al . The right panel depicts the secondary structure of the 361 nt truncated hok - rna, after 3 processing . Since rnatabupath requires two structures a, b, of the same length for a given rna sequence, we have extended the secondary structure of truncated hok - rna to consist of unpaired nucleotides . Free energy of tructure a is 186.1 kcal / mol, while that of structure b is 142.8 kcal / mol . In this case, findpath (18) obtained the best barrier energy . Consider the 46 nt rna sequence cgcgacggcu acgcgacggc aaugccguug cgaagccguc gcgauc, with secondary structures a = (((((((((..............))))))))).............. having free energy 16.04 kcal / mol and b = ........... (((((((((..............)))))))))... having free energy 18.14 kcal / mol . The structure a consists of the base pairs lying above the line in this figure, while the structure b consists of the base pairs lying below the line . Program barriers cannot be used, since neither a nor b is locally optimal . A manually designed indirect folding pathway for the 46 nt rna sequence cgcgacggcu acgcgacggc aaugccguug cgaagccguc gcgauc, proceeding from locally optimal secondary structure a = (((((((((..............))))))))).............. having free energy 16.04 kcal / mol to locally optimal structure b = ........... (((((((((..............)))))))))... having free energy 18.14 kcal / mol . Intuitively, this pathway can be visualized as repeatedly moving the remaining rightmost right - parenthesis to the right, then repeatedly moving the rightmost left - parenthesis to the right . In this manner, the barrier energy of this indirect path is 13.68 kcal / mol, while every direct path must have a barrier energy of at least 16.04 kcal / mol, since the empty structure must be an intermediate structure in every direct path in this example . Indeed, due to nucleation energy required to start a hairpin in the empty structure, the barrier energy of every direct path must properly exceed 16.04 . In this case, vienna package program findpath.c with look - ahead 100 returns a barrier energy of 18.27, while rnatabupath returns a barrier energy of 16.84 . Clearly, every direct path from a to b must have the empty structure as an intermediate structure, hence the lowest barrier energy of a direct folding pathway must be at least 16.04 kcal / mol (in fact even larger due to nucleation energy). However the indirect folding pathway depicted in table 3 has a barrier energy of 13.68 kcal / mol . Two secondary structures of host killing (hok) rna, taken from figure 8 of shapiro et al . The left panel depicts the secondary structure of 396 nt hok - rna, presumably based on figure 1b of franch et al . The right panel depicts the secondary structure of the 361 nt truncated hok - rna, after 3 processing . Since rnatabupath requires two structures a, b, of the same length for a given rna sequence, we have extended the secondary structure of truncated hok - rna to consist of unpaired nucleotides . Free energy of tructure a is 186.1 kcal / mol, while that of structure b is 142.8 kcal / mol . In this case, findpath (18) obtained the best barrier energy . Given the combinatorial difficulty of determining optimal pathways for the turner energy model and the inherent exponential time complexity of the program barriers, it is perhaps not surprising that the problem of computing the minimum energy path between two given rna structures has recently been announced to be np - complete . The np - completeness of computing an optimal pathway is proven in the pre - print, manuch, j ., (2009) np - completeness of the direct energy barrier problem without pseudoknots, in 15th international meeting on dna computing and molecular programming, june 811, fayetteville, arkansas . In this section, we survey several known heuristics for determining folding pathways between two secondary structures, as well as present our novel semi - greedy and rnatabupath methods . To explain the morgan higgs greedy direct pathway algorithm, we first define the notion of a base pair clashing with another base pair base pair (i, j) is said to clash with base pair (x, y) if either x i y j or i x j y. more generally, a base pair (i, j) clashes with a secondary structure a if there exists (x, y) a such that (i, j) clashes with (x, y). The set of base pairs (x, y) a such that (i, j) clashes with (x, y) is denoted clash(i, j, a); i.e. With this definition, the morgan higgs greedy algorithm repeatedly performs the following steps: (i) determine the base pair (i, j) belonging to b but not a which has minimum size clash set c, (ii) remove base pairs from c, and (iii) add base pairs in b that do not induce any new clashes . Figure 6.morganhiggs greedy algorithm (6) to construct a greedy direct pathway from secondary structure a to b. morgan higgs greedy algorithm (6) to construct a greedy direct pathway from secondary structure a to b. the morgan higgs algorithm to compute a nearly optimal (possibly) indirect pathway between secondary structures a, b proceeds as follows . By sampling, if either a or b does not belong to, then add the missing structure to . Define a complete, weighted, undirected graph g = (v, e), where the set v of vertices consists of all structures in, and the edge weight between any two structures, is defined to be energy barrier max{e(i) e(): 1 i n}, where = 0,, n = is the greedy direct pathway from to, as determined by the morgan morgan and higgs then apply single link cluster (slc) algorithm, as described in (22), in order to determine an optimal pathway, starting from structure a, proceeding by hopping from one low energy structure in to another via a greedy direct pathway, and terminating by the structure b. in our implementation of the morgan higgs indirect algorithm, we sample low energy structures with respect to the turner energy model by applying the ding lawrence algorithm (8), as implemented in rnasubopt - p from the vienna rna package (step 2 of figure 7). In place of the slc algorithm, we apply a modified form of dijkstra's single source shortest path algorithm (23), in order to determine a sequence a=0, 1,, n = b of structures, where each i, then concatenate the direct pathways between successive i to i+1, as determined by the morgan figure 7.morganhiggs algorithm (6) to construct an indirect pathway from secondary structure a to b. in line 2 of this algorithm, we use stochastic sampling of ding and lawrence (8), as implemented in rnasubopt - p, and applied a modified version of dijkstra's single source shortest path algorithm to determine low energy structures, whose greedy direct paths can be glued together for a pathway from a to b. morgan higgs algorithm (6) to construct an indirect pathway from secondary structure a to b. in line 2 of this algorithm, we use stochastic sampling of ding and lawrence (8), as implemented in rnasubopt - p, and applied a modified version of dijkstra's single source shortest path algorithm to determine low energy structures, whose greedy direct paths can be glued together for a pathway from a to b. perhaps the simplest possible algorithm to find a nearly optimal (direct) pathway between a and b is to apply a greedy approach, where at each step we choose to remove a base pair belonging to a but not b, or add a base pair belonging to b but not a, where the choice of base pair to be removed or added is made so as to ensure the lowest energy next structure . Pseudocode for this method, described by voss et al . (19), is depicted in figure 8 . Figure 8.greedy method to determine direct pathway between a and b, as described by voss et al . Secondary structures a, b can be considered to be sets of base pairs, so the requirement that a b means that every base pair of belongs to either a or b. this condition ensures that the pathway produced is direct . The notation dbp(,) = 1 means that the base pair distance (30) between, is 1; i.e., differ by one base pair . Moreover, since dbp(, b) = dbp(, b) + 1, each iteration in the while loop ensures advancement by one base pair to the target structure b. it follows that the while loop involves dbp(a, b) iterations . Greedy method to determine direct pathway between a and b, as described by voss et al . Secondary structures a, b can be considered to be sets of base pairs, so the requirement that a b means that every base pair of belongs to either a or b. this condition ensures that the pathway produced is direct . The notation dbp(,) = 1 means that the base pair distance (30) between, is 1; i.e., differ by one base pair . Moreover, since dbp(, b) = dbp(, b) + 1, each iteration in the while loop ensures advancement by one base pair to the target structure b. it follows that the while loop involves dbp(a, b) iterations . However, we can benefit from a more randomized approach in which we randomly add or remove a valid base pair that yields a structure that is among the k lowest energy structures since the result is clearly dependent on a parameter k we can iterate the same approach for several values of k and return the route with the lowest energy barrier . Figure 9.semi-greedy method to determine direct pathway between a and b. the only difference between the greedy and semi - greedy method is that the latter randomly selects one of the k lowest energy neighbors (step 5) rather than the minimum energy neighbor . Benchmarking indicates that the semi - greedy method generally outperforms the greedy method when determining low energy pathways between conformers of a riboswitch . Semi - greedy method to determine direct pathway between a and b. the only difference between the greedy and semi - greedy method is that the latter randomly selects one of the k lowest energy neighbors (step 5) rather than the minimum energy neighbor . Benchmarking indicates that the semi - greedy method generally outperforms the greedy method when determining low energy pathways between conformers of a riboswitch . Indirect routes present more opportunities and challenges, since the space of possible routes increases considerably . Also, a purely greedy approach is not possible since the algorithm would not be able to escape from cycles . Indeed, suppose that the structure a is the minimum free energy structure for the given rna sequence; then the first step would add or remove a base pair, yielding a structure that is no longer the minimum free energy structure . In the next step, the added (respectively removed) base pair would then be removed (respectively added), in order to return to the minimum free energy structure . For that reason tabu search (20) is a well - studied combinatorial optimization method that entails a greedy strategy where a list of recently taken moves is placed temporarily on a tabu list, and cannot be applied until removed from the tabu list . In figure 10, we present pseudocode for a tabu semi - greedy algorithm (rnatabupath) to find nearly optimal, possibly indirect pathways between designated secondary structures a and b. the algorithm starts with the initial structure . At each successive step in the execution of the algorithm, we choose to add or remove that base pair resulting in the lowest energy (greedy), after which the base pair is placed in the tabu list, hence cannot be added or removed for a certain number of steps . Figure 10.tabu semi - greedy algorithm to compute near - optimal folding pathway between two designated structures a, b for a given rna sequence . In line 11, we assume that is obtained from without using a base pair in the tabu list . The tabu list contains base pairs that were recently added or removed from an intermediate structure . When added to the tabu list, a base pair fitness f of a structure is defined by f = e + w bp, where e is energy of current structures, w is weight defined in line 2 and bp is incremental distance toward the target, i.e. 1 . Tabu semi - greedy algorithm to compute near - optimal folding pathway between two designated structures a, b for a given rna sequence . In line 11, we assume that is obtained from without using a base pair in the tabu list . The tabu list contains base pairs that were recently added or removed from an intermediate structure . When added to the tabu list, a base pair fitness f of a structure is defined by f = e + w bp, where e is energy of current structures, w is weight defined in line 2 and bp is incremental distance toward the target, i.e. 1 . As in every optimization algorithm we need to define the fitness function, f. the fitness function is a measure of quality of each state . In the case at hand, a state is a secondary structure, and the fitness function must account for the free energy e() as well as the distance dbp(, b) from to the target structure b. hence, the fitness f() of secondary structure is defined by where w represents a weight that regulates the importance of reaching the target structure versus choosing a low energy structure . A low weight has the potential of driving the algorithm to structures that are too far away from the target, b, while a higher weight can quickly converge to the target structure at the expense of including higher energy intermediate structures in the path produced . An intermediate value for weight w will tend to cause the algorithm to behave in a manner similar to that of the greedy algorithm for direct pathways . In order to avoid the latter, we have developed a weight oscillation strategy that can be explained in the following steps: start with a given initial weight w0.increase the value of w when the distance to the target has not been improved for a number of iterations and restart from the structure found to be closest to the target.decrease the weight when the distance to the target is improved.if the weight reaches a certain value wmax, increase the value of w0 and restart the search (with w = w0). The distance to the target has not been improved for a number of iterations and restart from the structure found to be closest to the target . If the weight reaches a certain value wmax, increase the value of w0 and restart the search (with w = w0). Our tabu strategy starts with the initial structure a, and in each step either adds or removes the base pair that minimizes the fitness function f. the base pair that has just been added or removed will be kept in a tabu list for a certain number of steps during which time it cannot be added or removed to any structure in the pathway being constructed . The fitness function f is adaptive, since it depends on the weight oscillation scheme . Additionally, the algorithm introduces an aspiration criterion for which a base pair can be changed (even if it is tabu) when the resulting structure reduces the best distance to the target found so far, provided that its free energy does not exceed that of the maximum energy of a structure in the pathway constructed so far . Additionally, we introduce two stochastic aspects to the tabu algorithm: the time a base pair remains on the tabu list, and the way to break ties when choosing the best base pair consequently, we can start with a given value and iterate the algorithm using different values while maintaining the best pathway so far found . Note that we assume that in line 10 of the pseudocode of figure 10, we assume that is obtained from without using a base pair in the tabu list unless the aspiration criterion just mentioned has been applied, and that the tabu list is updated ., we found that by adding a semi - greedy component to tabu search, the resulting algorithm was substantially improved . (19), described in figure 8, is improved by adding a semi - greedy component for the search . Clearly, one could apply monte carlo and simulated annealing strategies to sample low energy folding pathways, as well as envision a genetic algorithm, that permits the crossover between folding pathways having a common source a and target b. nevertheless, the tabu semi - global approach of rnatabupath appears to be a very fast method to quickly determine near - optimal folding pathways . The web site for rnapathfinder includes additional tools to determine the frequency of occurrence of secondary startures in (say) 1000 low energy folding pathways, and to determine the similarity between two pathways . In this section, we survey several known heuristics for determining folding pathways between two secondary structures, as well as present our novel semi - greedy and rnatabupath methods . To explain the morgan higgs greedy direct pathway algorithm, we first define the notion of a base pair clashing with another base pair base pair (i, j) is said to clash with base pair (x, y) if either x i y j or i x j y. more generally, a base pair (i, j) clashes with a secondary structure a if there exists (x, y) a such that (i, j) clashes with (x, y). The set of base pairs (x, y) a such that (i, j) clashes with (x, y) is denoted clash(i, j, a); i.e. With this definition, the morgan higgs greedy algorithm repeatedly performs the following steps: (i) determine the base pair (i, j) belonging to b but not a which has minimum size clash set c, (ii) remove base pairs from c, and (iii) add base pairs in b that do not induce any new clashes . Figure 6.morganhiggs greedy algorithm (6) to construct a greedy direct pathway from secondary structure a to b. morgan higgs greedy algorithm (6) to construct a greedy direct pathway from secondary structure a to b. the morgan higgs algorithm to compute a nearly optimal (possibly) indirect pathway between secondary structures a, b proceeds as follows . By sampling, if either a or b does not belong to, then add the missing structure to . Define a complete, weighted, undirected graph g = (v, e), where the set v of vertices consists of all structures in, and the edge weight between any two structures, is defined to be energy barrier max{e(i) e(): 1 i n}, where = 0,, n = is the greedy direct pathway from to, as determined by the morgan morgan and higgs then apply single link cluster (slc) algorithm, as described in (22), in order to determine an optimal pathway, starting from structure a, proceeding by hopping from one low energy structure in to another via a greedy direct pathway, and terminating by the structure b. in our implementation of the morgan higgs indirect algorithm, we sample low energy structures with respect to the turner energy model by applying the ding lawrence algorithm (8), as implemented in rnasubopt - p from the vienna rna package (step 2 of figure 7). In place of the slc algorithm, we apply a modified form of dijkstra's single source shortest path algorithm (23), in order to determine a sequence a=0, 1,, n = b of structures, where each i, then concatenate the direct pathways between successive i to i+1, as determined by the morgan figure 7.morganhiggs algorithm (6) to construct an indirect pathway from secondary structure a to b. in line 2 of this algorithm, we use stochastic sampling of ding and lawrence (8), as implemented in rnasubopt - p, and applied a modified version of dijkstra's single source shortest path algorithm to determine low energy structures, whose greedy direct paths can be glued together for a pathway from a to b. morgan higgs algorithm (6) to construct an indirect pathway from secondary structure a to b. in line 2 of this algorithm, we use stochastic sampling of ding and lawrence (8), as implemented in rnasubopt - p, and applied a modified version of dijkstra's single source shortest path algorithm to determine low energy structures, whose greedy direct paths can be glued together for a pathway from a to b. perhaps the simplest possible algorithm to find a nearly optimal (direct) pathway between a and b is to apply a greedy approach, where at each step we choose to remove a base pair belonging to a but not b, or add a base pair belonging to b but not a, where the choice of base pair to be removed or added is made so as to ensure the lowest energy next structure . Figure 8.greedy method to determine direct pathway between a and b, as described by voss et al . Secondary structures a, b can be considered to be sets of base pairs, so the requirement that a b means that every base pair of belongs to either a or b. this condition ensures that the pathway produced is direct . The notation dbp(,) = 1 means that the base pair distance (30) between, is 1; i.e., differ by one base pair . Moreover, since dbp(, b) = dbp(, b) + 1, each iteration in the while loop ensures advancement by one base pair to the target structure b. it follows that the while loop involves dbp(a, b) iterations . Greedy method to determine direct pathway between a and b, as described by voss et al . Secondary structures a, b can be considered to be sets of base pairs, so the requirement that a b means that every base pair of belongs to either a or b. this condition ensures that the pathway produced is direct . The notation dbp(,) = 1 means that the base pair distance (30) between, is 1; i.e., differ by one base pair . Moreover, since dbp(, b) = dbp(, b) + 1, each iteration in the while loop ensures advancement by one base pair to the target structure b. it follows that the while loop involves dbp(a, b) iterations . However, we can benefit from a more randomized approach in which we randomly add or remove a valid base pair that yields a structure that is among the k lowest energy structures . Since the result is clearly dependent on a parameter k we can iterate the same approach for several values of k and return the route with the lowest energy barrier . Figure 9.semi-greedy method to determine direct pathway between a and b. the only difference between the greedy and semi - greedy method is that the latter randomly selects one of the k lowest energy neighbors (step 5) rather than the minimum energy neighbor . Benchmarking indicates that the semi - greedy method generally outperforms the greedy method when determining low energy pathways between conformers of a riboswitch . Semi - greedy method to determine direct pathway between a and b. the only difference between the greedy and semi - greedy method is that the latter randomly selects one of the k lowest energy neighbors (step 5) rather than the minimum energy neighbor . Benchmarking indicates that the semi - greedy method generally outperforms the greedy method when determining low energy pathways between conformers of a riboswitch . Indirect routes present more opportunities and challenges, since the space of possible routes increases considerably . Also, a purely greedy approach is not possible since the algorithm would not be able to escape from cycles . Indeed, suppose that the structure a is the minimum free energy structure for the given rna sequence; then the first step would add or remove a base pair, yielding a structure that is no longer the minimum free energy structure . In the next step, the added (respectively removed) base pair would then be removed (respectively added), in order to return to the minimum free energy structure . For that reason, it makes sense to exclude certain moves at certain times during the search . Tabu search (20) is a well - studied combinatorial optimization method that entails a greedy strategy where a list of recently taken moves is placed temporarily on a tabu list, and cannot be applied until removed from the tabu list . In figure 10, we present pseudocode for a tabu semi - greedy algorithm (rnatabupath) to find nearly optimal, possibly indirect pathways between designated secondary structures a and b. the algorithm starts with the initial structure . At each successive step in the execution of the algorithm, we choose to add or remove that base pair resulting in the lowest energy (greedy), after which the base pair is placed in the tabu list, hence cannot be added or removed for a certain number of steps . Figure 10.tabu semi - greedy algorithm to compute near - optimal folding pathway between two designated structures a, b for a given rna sequence . In line 11, we assume that is obtained from without using a base pair in the tabu list . The tabu list contains base pairs that were recently added or removed from an intermediate structure . When added to the tabu list, a base pair is given a time stamp . Fitness f of a structure is defined by f = e + w bp, where e is energy of current structures, w is weight defined in line 2 and bp is incremental distance toward the target, i.e. 1 . Tabu semi - greedy algorithm to compute near - optimal folding pathway between two designated structures a, b for a given rna sequence . In line 11, we assume that is obtained from without using a base pair in the tabu list . The tabu list contains base pairs that were recently added or removed from an intermediate structure . When added to the tabu list, a base pair fitness f of a structure is defined by f = e + w bp, where e is energy of current structures, w is weight defined in line 2 and bp is incremental distance toward the target, i.e. 1 . As in every optimization algorithm we need to define the fitness function, f. the fitness function is a measure of quality of each state . In the case at hand, a state is a secondary structure, and the fitness function must account for the free energy e() as well as the distance dbp(, b) from to the target structure b. hence, the fitness f() of secondary structure is defined by where w represents a weight that regulates the importance of reaching the target structure versus choosing a low energy structure . A low weight has the potential of driving the algorithm to structures that are too far away from the target, b, while a higher weight can quickly converge to the target structure at the expense of including higher energy intermediate structures in the path produced . An intermediate value for weight w will tend to cause the algorithm to behave in a manner similar to that of the greedy algorithm for direct pathways . In order to avoid the latter, we have developed a weight oscillation strategy that can be explained in the following steps: start with a given initial weight w0.increase the value of w when the distance to the target has not been improved for a number of iterations and restart from the structure found to be closest to the target.decrease the weight when the distance to the target is improved.if the weight reaches a certain value wmax, increase the value of w0 and restart the search (with w = w0). Start with a given initial weight w0 . Increase the value of w when the distance to the target has not been improved for a number of iterations and restart from the structure found to be closest to the target . Decrease the weight when if the weight reaches a certain value wmax, increase the value of w0 and restart the search (with w = w0). Our tabu strategy starts with the initial structure a, and in each step either adds or removes the base pair that minimizes the fitness function f. the base pair that has just been added or removed will be kept in a tabu list for a certain number of steps during which time it cannot be added or removed to any structure in the pathway being constructed . The fitness function f is adaptive, since it depends on the weight oscillation scheme . Additionally, the algorithm introduces an aspiration criterion for which a base pair can be changed (even if it is tabu) when the resulting structure reduces the best distance to the target found so far, provided that its free energy does not exceed that of the maximum energy of a structure in the pathway constructed so far . Additionally, we introduce two stochastic aspects to the tabu algorithm: the time a base pair remains on the tabu list, and the way to break ties when choosing the best base pair . Consequently, we can start with a given value and iterate the algorithm using different values while maintaining the best pathway so far found . Note that we assume that in line 10 of the pseudocode of figure 10, we assume that is obtained from without using a base pair in the tabu list unless the aspiration criterion just mentioned has been applied, and that the tabu list is updated ., we found that by adding a semi - greedy component to tabu search, the resulting algorithm was substantially improved . (19), described in figure 8, is improved by adding a semi - greedy component for the search . Clearly, one could apply monte carlo and simulated annealing strategies to sample low energy folding pathways, as well as envision a genetic algorithm, that permits the crossover between folding pathways having a common source a and target b. nevertheless, the tabu semi - global approach of rnatabupath appears to be a very fast method to quickly determine near - optimal folding pathways . The web site for rnapathfinder includes additional tools to determine the frequency of occurrence of secondary startures in (say) 1000 low energy folding pathways, and to determine the similarity between two pathways . In this section, we survey several known heuristics for determining folding pathways between two secondary structures, as well as present our novel semi - greedy and rnatabupath methods . In this section, we present summary results on folding pathways and energy barriers computed for each of the algorithms: greedy (19), semi - greedy, rnatabupath, morgan higgs direct (6), morgan higgs indirect (6), findpath (18) and barriers (2,15). Due to the stochastic nature of the semi - greedy method and rnatabupath, we report the best results found over 1000 runs . In the case of rnatabupath, fitness of the current structure is defined by f = e + w bp, where e is the free energy of the current structure, and bp is the rnatabupath allows the user to input parameters wmin, wmax that confine the weight w [wmin, wmax]. Reported values were for wmin = 1 and wmax = 7, which are the default values on the web server . Table 1 presents the energy barrier in the pathway a=0, 1,, n = b between low energy structures a and b of known conformational switches, where energy barrier is defined to be max{e(i) e(a): i = 1,, n}. Structures a and b are two metastable states of five riboswitches, guanine riboswitch from bacillus subtilis (rb1), adenine riboswitch from v. vulnificus (rb2), s - adenylmethionine riboswitch from thermoanaerobacter tecongensis (rb3), thymine pyrophosphate riboswitch from t. tecongensis (rb4), and xpt - pbux riboswitch from b. subtilis (rb5), whose metastable secondary structures were found by in - line probing experiments of various groups . Table 1 also contains results for some conformational switches found on the parnass web site, http://bibiserv.techfak.uni-bielefeld.de/parnass/examples.html; however, since the metastable structures for the latter conformational switches have not been experimentally determined, we ran the software rnabor, which determines for each integer value of, the minimum free energy structure mfe() and partition function z() over all -neighbors of the minimum free energy structure . Here a structure is said to be a -neighbor of structure if base pair distance between, is . [see (25) for details on rnabor .] For the conformational switches taken from the parnass web site, we defined a to be the minimum free energy structure and b to be that structure which is the minimum free energy structure over all -neighbors of a, where 10 <and the output of rnabor indicated a second peak at the value . Table 1.algorithm benchmarks for computing folding pathways between two low energy secondary structuresinstancegreedysemi - greedyrnatabupathgreedymhindirectmhfindpathbarriersrb132.8025.2424.0426.2423.9924.04rb214.649.207.2510.0010.008.20*rb324.8022.7017.9028.4020.0022.40rb416.9016.9016.9016.9016.9016.90rb533.3025.6724.5426.7426.7424.54hok36.3733.7029.6636.3036.3028.5sl14.0914.0912.9018.2016.2013.0011.80attenuator11.509.008.6012.6014.708.708.30s157.107.106.609.709.707.106.60s - box leader7.105.305.2010.209.305.20*thim leader21.4416.6714.8420.5731.0016.13ms28.306.606.6011.7011.706.60*hdv25.5021.7017.0023.5322.5017.4dsra8.308.308.2014.6010.778.308.00ribd leader13.8411.709.5018.1116.9010.71amv10.006.405.8015.610.45.80*alpha operon6.506.506.509.906.506.50*hiv-1 leader14.2813.4911.3017.9018.509.30greedy refers to our implementation of voss et al . (19), where a direct path is constructed by choosing the lowest energy base pair to remove or add at each step; semi - greedy refers to to our modification of voss et al . (19), where a direct path is constructed by choosing one of the k lowest energy base pairs to remove or add at each step; rnatabupath refers to our semi - greedy tabu search method described in the text; indirectmh refers to our implementation of morgan higgs method (6) to produce a possibly indirect path; 18) with look - ahead parameter k = 10; barriers refers to the exact method of flamm et al . In each case, near - optimal low energy pathways between two low energy secondary structures of five different riboswitches: guanine riboswitch from b. subtilis (rb1), adenine riboswitch from v. vulnificus (rb2), s - adenylmethionine riboswitch from t. tecongensis (rb3), thymine pyrophosphate riboswitch from t. tecongensis (rb4) and xpt - pbux riboswitch from b. subtilis (rb5). Secondary structures for rb1rb5 were experimentally determined; see wakeman et al . (16) for rb1rb4 and mandal et al . Sequences of additional conformational switches were taken from the parnass web site http://bibiserv.techfak.uni-bielefeld.de/parnass/examples.html, courtesy of the giegerich lab . For the latter, the two low energy structures were taken to be the minimum free energy structure a and the structure b determined by rnabor (25) to be the minimum free energy structure over all structures having base pair distance k with a, where 10 k and a second peak was found at position k in the output of rnabor . Energy barrier in the pathway a = 0, 1,, n = b from a to b is here defined to be max{e(i) e(a)}: i = 1,, n}, where free energy is measured in kcal / mol, as computed by rnaeval from vienna rna package . Notation used in last column given as follows: means barriers could not converge; * means that either structure a or b is not locally optimal, hence barriers could not be directly applied . However, one could apply barriers in the following manner, as suggested by an anonymous referee . Given non - locally optimal structures a, b, one can first determine locally optimal structures, that, respectively, contain a, b, then apply barriers to find an optimal path between, . This will yield a near - optimal path from a to b. boldface numbers indicate the minimal barrier energy found by one of the heuristic algorithms, while underlined numbers indicate the minimum barrier energy found by the exact algorithm, barriers . (19), where a direct path is constructed by choosing the lowest energy base pair to remove or add at each step; semi - greedy refers to to our modification of voss et al . (19), where a direct path is constructed by choosing one of the k lowest energy base pairs to remove or add at each step; rnatabupath refers to our semi - greedy tabu search method described in the text; indirectmh refers to our implementation of morgan higgs method (6) to produce a possibly indirect path; 18) with look - ahead parameter k = 10; barriers refers to the exact method of flamm et al . Near - optimal low energy pathways between two low energy secondary structures of five different riboswitches: guanine riboswitch from b. subtilis (rb1), adenine riboswitch from v. vulnificus (rb2), s - adenylmethionine riboswitch from t. tecongensis (rb3), thymine pyrophosphate riboswitch from t. tecongensis (rb4) and xpt - pbux riboswitch from b. subtilis (rb5). Sequences of additional conformational switches were taken from the parnass web site http://bibiserv.techfak.uni-bielefeld.de/parnass/examples.html, courtesy of the giegerich lab . For the latter, the two low energy structures were taken to be the minimum free energy structure a and the structure b determined by rnabor (25) to be the minimum free energy structure over all structures having base pair distance k with a, where 10 k and a second peak was found at position k in the output of rnabor . Energy barrier in the pathway a = 0, 1,, n = b from a to b is here defined to be max{e(i) e(a)}: i = 1,, n}, where free energy is measured in kcal / mol, as computed by rnaeval from vienna rna package . Notation used in last column given as follows: means barriers could not converge; * means that either structure a or b is not locally optimal, hence barriers could not be directly applied . However, one could apply barriers in the following manner, as suggested by an anonymous referee . Given non - locally optimal structures a, b, one can first determine locally optimal structures, that, respectively, contain a, b, then apply barriers to find an optimal path between, . This will yield a near - optimal path from a to b. boldface numbers indicate the minimal barrier energy found by one of the heuristic algorithms, while underlined numbers indicate the minimum barrier energy found by the exact algorithm, barriers . For technical reasons having to do with computation of the partition function, the treatment of dangles in rnabor is identical to that of vienna rna package rnafold with option d2 . In some instances, the metastable structure we chose using rnabor was no longer locally optimal under the d1 treatment of dangle, which latter is used in all the algorithms appearing in table 1 . In particular, we should mention that one must explicitly use d1 option with rnasubopt, to ensure that rnafold, rnaeval and barriers all use the same treatment of dangles . Due to the energy model differences (d2 versus d1) in using rnabor to choose one of the metastable structures, barriers could not be used in some instances rb2, s - box leader, ms2, amv and alpha operon . We see that the greedy approach is simple, but yields considerably poorer results than other methods tested . However, a small change such as a semi - greedy component yields great improvements . Tabu search for indirect routes outperforms both greedy and semi - greedy approaches (data for the tabu greedy method is not shown). In the semi - greedy algorithm and rnatabupath, we experimented with different choices of the value k, where randomly one of the best k neighbors is chosen . After computational experiments over the range of lengths typical for conformational switches, we fixed the value k = 8 for semi - greedy algorithm and k = 5 for rnatabupath . The initial weight w0 in rnatabupath ranges from 1 to 7, the default setting for the web server, although best results for this range depend on the input sequence . In general, w0 [4, 7] works better for larger sequences . Higgs direct and indirect algorithms did not perform well in all but one instance; morgan higgs indirect algorithm curiously outperformed all algorithms for rb1 . In general, findpath is a very fast algorithm that produces excellent quality direct pathways, with barrier energies often equal or close to those of rnatabupath . In the case of hok - rna and hiv-1 leader, findpath outperformed all other approaches . In our benchmarking, we set the look - ahead of findpath to be 10; often increasing the look - ahead to 100 did not change the results . However, in the 396 nt hok - rna, findpath improved dramatically with increased look - ahead k: barrier energy of 28.5 for k = 10, 28.17 for k = 20, 23.5 for k = 100, 22.7 for k = 200, 21.4 for k = 500 and k = 1000 . Figures 2 and 3 demonstrate cases where a well - chosen indirect pathway necessarily has lower barrier energy than that of any direct pathway . Applying this principle to 304 examples derived from pseudoknotted structures in pseudobase (21), we found that in roughly half the examples, rnatabupath and findpath produced the same barrier energy, while in all other instances, rnatabupath produced a lower barrier energy barrier than did findpath; indeed, the maximum difference in barrier energy was 6.51, while the average was 1.93 kcal / mol with standard deviation of 1.45 . Figure 11 depicts a folding pathway computed by rnatabupath between the two meta - stable secondary structures of the adenine riboswitch from v. vulnificus (rb2) (16). Figure 12 depicts the free energy of intermediate structures in this pathway as a function of step number . Figure 11.comparison of best direct pathway (above) and best indirect pathway (below), as found by rnatabupath between the two metastable secondary structures of the adenine riboswitch from v. vulnificus (rb2), as reviewed in wakeman et al . Figure 12.graph of free energy of intermediate structure as a function of step number or index in the rnatabupath folding pathway between two metastable secondary structures of the adenine riboswitch from v. vulnificus (rb2), corresponding to data from figure 11 . Dotted lines depict a similar graph for a folding pathway computed by the semi - global method . Comparison of best direct pathway (above) and best indirect pathway (below), as found by rnatabupath between the two metastable secondary structures of the adenine riboswitch from v. vulnificus (rb2), as reviewed in wakeman et al . Graph of free energy of intermediate structure as a function of step number or index in the rnatabupath folding pathway between two metastable secondary structures of the adenine riboswitch from v. vulnificus (rb2), corresponding to data from figure 11 . Dotted lines depict a similar graph for a folding pathway computed by the semi - global method . One useful application of rnatabupath is to provide an energy upper bound for subsequent application of barriers, an observation pointed out by an anonymous referee . Specifically, given rna sequence s and two metastable structures a, b, let e0 denote the minimum free energy of s and let e(a) denote the free energy of structure a. if e is the barrier energy computed by rnatabupath (or another method) for a folding pathway from a to b, then barriers with bound e + (e(a) e0) will compute an optimal pathway, provided it converges . The barrier energies obtained by barriers in table 1 since barriers is the only exact algorithm, when it converges, a provably optimal pathway is produced . In the cases of rb1, rb3, rb4, rb5, thim leader, ribd leader and hiv-1 leader, barriers did not converge, even when started with the energy bound obtained by rnatabupath . Molecular folding pathways are low energy routes taken along an energy surface . As previously noticed by morgan and higgs (6), this is clear from the toy example presented in figures 2 and 3 . In other data, we see how the creation of a base pair in a region with no base pairs leads to the stabilization of other secondary structures along the folding pathway . Since barriers is an exact algorithm, it should be used whenever possible; i.e. One should first apply findpath or rnatabupath to obtain an energy upper bound for subsequent application of barriers . In other cases, findpath and rnatabupath if large type - h pseudoknots appear in the structure obtained by adjoining two metastable structures, then rnatabupath is likely to be the best algorithm, since indirect pathways will have lower barrier energy in this case . To assist those interested in computing near - optimal folding pathways, we have created the web server rnapathfinder, located at http://bioinformatics.bc.edu/clotelab/rnapathfinder . In addition to supporting rnatabupath computations, source code can be downloaded for several algorithms discussed in this article . Fundacion caja madrid (to i.d . ); national science foundation (grants dbi-0543506 and dms-0817971 to p.c . And w.a.l . ); rna ontology consortium (to i.d . ; deutscher akademischer austauschdienst (to p.c . For funding a visit to martin vingron's; group in the max planck institute of molecular genetics); digiteo foundation (to p.c . ). Funding for open access charge: national science foundation (grant dbi-0543506).
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After introduction of the acid etch bonding method by buonocore in 1955, direct bonding of orthodontic brackets was first performed by newman in 1965 [1, 2]. This method expanded so rapidly that nowadays, utilization of the acid etch technique along with light cure composites is the most common bonding system in orthodontics . Since re - bonding of brackets could be a time consuming and challenging process, achieving an appropriate bond strength is an important clinical objective . While various types of composites such as microfilled, microhybrid and flowable are available, the latest development in this field has been the introduction of nano - filled composites that are claimed to achieve higher wear resistance and appropriate mechanical properties . They also enhance the hybrid layer, increase marginal seal and reduce polymerization shrinkage due to their higher filler content . Furthermore, nano - filler bondings have shown satisfactory bond strength to enamel and dentin, and can be utilized for direct and indirect restorations [6, 7, 8, 9]. Therefore, it is likely that nano - filled composites may replace other types of composites in the near future . Despite the extensive applications of nano - composites in restorative dentistry, there is inadequate data regarding the possibility of using them for bonding orthodontic brackets . The aim of this study was to evaluate the shear bond strength and failure sites of two types of nano - composites in comparison to the conventional orthodontic composite adhesive system, and evaluate the possibility of their clinical application in bonding orthodontic brackets . Sixty human first premolars were randomly divided into three groups . In order to prevent dehydration and inhibit bacterial growth, the teeth were preserved in 0.1% aqueous thymol solutionimmediately after extraction . Only teeth with intact buccal surfaces were selected, and any teeth with evidence of cracks, caries, hypoplastic areas or other enamel abnormalities were excluded . After thorough rinsing with water, the buccal surfaces were etched via 35% phosphoric acid gel (ultradent, usa) for 30 seconds and then dried until a chalky appearance was visible . Standard metal premolar brackets (dentarum, inspringon, germany) with surface area of 10.23 mm2 were bonded in this study . Three groups of brackets were bonded using the following systems: the first group was bonded using a conventional orthodontic composite: transbond xt (3 m unitek, usa)and transbond xt primer . For the second group, a nano - filled bonding and composite; adpertm single bond and filtektm supreme xt (3 m espe, usa) was used . In the third group, bonding was performed via another nano - filled composite bonding system; aelite aesthetic enamel (bisco, usa)and one - step plus . The bonding process was initiated by placing a thin layer of bonding on the enamel surface and light curing for 10 seconds . After that, the main bulk of composite was placed onto the bracket bases and pressed with a force of 300 g using a force gauge . After that, the samples were light cured for 40 seconds (10 seconds on each side) using a standard halogen light - curing unit (optilux, 3 m unitek). The samples were then placed in 37c distilled water for 24 hours, followed by thermocycling for 2000 times at temperatures between 5c and 55c . A mounting jig was used to facilitate mounting each tooth onto an acrylic block . In order to prevent deformation of brackets during the debonding process, a stainless steel wire (0.0180.025 mm) was inserted into the brackets ` slots during mounting . After completion of acrylic setting, the samples were placed in a universal testing machine (zwick gmbh 8 co, germany) in a manner that the labial surfaces of the teeth were parallel to the shearing force . A wire loop was connected to the machine and an occlusogingival force with a crosshead speed of 1mm / min was exerted onto each sample . The sbs value for each sample was determined by dividing the maximum force by the surface area of the brackets . In order to investigate the sites of bond failure, adhesive remnant index (ari) was determined for each sample by observation of the tooth surface under a stereomicroscope at 10 magnification and based on the following scale: 0: no adhesive is remained on the tooth surface1: less than 50% of the adhesive is remained on the tooth surface2: more than 50% of the adhesive is remained on the tooth surface3: 100% of adhesive is remained on the tooth surface 0: no adhesive is remained on the tooth surface 1: less than 50% of the adhesive is remained on the tooth surface 2: more than 50% of the adhesive is remained on the tooth surface 3: 100% of adhesive is remained on the tooth surface mean and standard deviation (sd) values for sbs in each group were determined . One way analysis of variance and tukey hsd multiple comparisons was used for statistical analysis of sbs . Descriptive values of the shear bond strength for the three groups are shown in table 1 . The results of this study revealed that aelite aesthetic enamel had the highest sbs value (8.442.1), whilst transbond xt (6.922.13) and filtek tm supreme xt (6.052.02) were in the next ranks . One - way anova test showed statistically significant differences between the shear bond strengths of the three groups(p= 0.019). Multiple comparisons revealed that the shear bond strength of aelite aesthetic enamel was significantly higher than the other two groups (p<0.001), whilst there was not a statistically significant difference between transbond xt and filtek tm supreme xt (p=0.453). There was not any statistically significant difference between ari indices of the three groups(p=0.142). The results of the present study revealed that aelite aesthetic enamel nano - composite showed the highest sbs value, followed by transbond xt and filtek supreme xt respectively; however, these differences were not statistically significant (p>0.05). Therefore, it can be concluded that there is no significant difference in the shear bond strengths of transbond xt and the nano - composites . According to a study by reynolds et al ., an appropriate adhesive for orthodontic purposes should meet a sbs value of at least 5.97.8 mpa . Therefore, in addition to transbond xt, both supreme xt and aelite aesthetic enamel are appropriate for orthodontic purposes and could be utilized for bonding, despite not displaying any additional advantages compared to transbond xt . Since various studies regarding bond strength of composites have utilized adhesives with different size / concentration of filler, it is difficult to compare their results accurately . The results of this study are consistent with that of bishara et al ., whereby there was no significant difference between the sbs value of transbond xt and a restorative nano - composite, and both materials were considered applicable in orthodontics . On the other hand,, whereby comparison of sbs of a nano - composite (filtek supreme plus universal) and a nano - ionomer (ketac n100 light curing nano - ionomer) with transbond xt revealed a significantly higher sbs value of transbond xt . However, such different findings may be due to a number of factors; in the study by uysal et al ., the teeth were polished with nano - fluoridated pumice, which may have interfered with the entrance of nano - fillers into the etched enamel surface, and led to decreased bond strength . A further significant difference in addition to the use of a qth light source was their use of porcelain based brackets compared to the use of metal brackets in our study . The penetrance of the bonding agent in all of the groups was not matched, as transbond xt paste was applied onto uncured bonding while in the case of the nano - composites; the bonding was cured prior to application of the paste . For purpose of testing orthodontic shear bond strength, a previous study has shown that the wire loop method has been found to be superior to the shear blade method that was used in the study by uysal et al . . The results of the present study revealed that in the majority of samples of transbond xt and supreme xt, bond failure occurred in a manner that less than 50% of the original composite had remained on the enamel surfaces (grade 1). Meanwhile, in aelite aesthetic enamel group (displaying highest sbs value), more than 50% of the original composite had remained on the enamel surfaces (grade 2). . However, uysal et al . Revealed that the ari was grade 1 in all of the groups, which is consistent with our results for transbond xtand supreme xt . On the other hand, in our study, the majority of the samples in aelite aesthetic group were in grade 2, which was the second rank amongst the composites in our study . Overall, it can be concluded that the failure sites of transbond xt and nano - composites is mainly at the tooth - adhesive interface . Considering the results of this study and bishara`s study, successful application of nano - composites
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It is estimated that there will be> 234,000 new cases and 41,000 deaths from breast cancer in the united states in 2015, with 6% of women with breast cancer having metastatic disease at the time of diagnosis.1,2 for patients who progress to metastatic disease, the 5-year survival rate is 25%.1 data are unclear as to how many women progress to metastatic disease following diagnosis and treatment of a primary tumor . It is estimated, however, that up to 30% of node - negative and 70% of node - positive breast cancers eventually relapse.3 once breast cancer metastasizes, the goal of treatment changes from being curative to prolonging the survival of patients and preserving the quality of life.3,4 the approach to treatment for metastatic disease is often driven by the status of important markers such as hormone receptors or human epidermal growth factor receptor 2 (her2). A recent retrospective study of women with metastatic breast cancer (mbc) found that 33% were her2-positive and 71% were hormone receptor - positive; 19% of patients in a recent phase iii trial were both hormone receptor - negative and her2-negative, a status often referred to as triple - negative disease.5,6 patients with her2-positive and/or hormone receptor - positive disease have improved survival due to recent novel therapy options, whereas survival rates remain poor for patients with triple - negative mbc.5,7 as a result, there is a dire need to find novel treatments to manage patients with mbc, including those with triple - negative disease . Treatment guidelines recommend endocrine therapy in this setting when possible, due to its lower toxicity profile compared with that for chemotherapy . For hormone receptor - negative patients and hormone - refractory disease, chemotherapy is recommended; however, there is no standard of care.8 combination chemotherapy is associated with higher objective response rates and longer time to progression,911 while single - agent chemotherapy is associated with lower toxicity and fewer dose reductions.4,10 given that overall survival (os) outcomes are equivalent for single - agent and combination chemotherapy regimens, guidelines from both the national comprehensive cancer network4 and the european society for medical oncology)3 recommend single - agent treatment in patients without directly life - threatening or severely symptomatic disease . This article describes two patients with mbc who had hormone - refractory disease and who were candidates for single - agent chemotherapy . In both patients, eribulin mesylate (halaven, eisai inc), a nontaxane microtubule dynamics inhibitor, was administered . A woman in her 60s was diagnosed with node - positive invasive ductal breast cancer in october 2009 . After a lumpectomy, she received adjuvant docetaxel, doxorubicin, and cyclophosphamide, followed by radiotherapy and adjuvant oral endocrine therapy with anastrozole 1 mg / day . A complete time line of the patient s treatment history is summarized in figure 1 . Recurrent metastatic bone disease was discovered following a fall in 2012, and the patient was treated with fulvestrant and zoledronic acid . Upon disease progression in june 2012, she was started on weekly paclitaxel (80 mg / m) every 34 weeks . This treatment continued for almost 1 year until may 2013, when therapy had to be interrupted so she could undergo surgery to address osteonecrosis of the jaw . Treatment continued for 1 month, but she became cachectic and performance status declined to eastern cooperative oncology group (ecog) grade 2; cancer antigen 15 - 3 (ca 153) had increased from 6070 u / ml to 296.3 u / ml . By july 2013, her performance status had declined further to ecog grade 3, and the patient had alopecia, grade 2 anemia, and grade 1 elevation in serum glutamic while still an inpatient, she was started on a trial of eribulin mesylate, dosed at 1.4 mg / m intravenously on days 1 and 8 of a 21-day cycle . Eribulin therapy was chosen due to the good tolerance and efficacy previously seen in frail and elderly patients, minimal toxicities that can be managed with dose modifications, and ease of administration . She was released to rehabilitation, and in september 2013, her performance status, calcium levels, and ca 15 - 3 were all improving . One month later, the patient fell, again requiring surgery to the left hip and an interruption in eribulin treatment of 45 weeks . Following the surgery and palliative radiation, her condition worsened and she started taking hydrocodone and oral morphine for generalized bone pain . The patient is now independent, with eribulin continuing at full dose, despite national cancer institute (nci) grade 1 neuropathy in the hands and feet . The patient no longer takes morphine and takes hydrocodone only intermittently for occasional hip and back pain . Alopecia and grade 2 anemia continue, her white blood cell count is normal, and there have been no changes in liver function tests . In january 2014, ca 15 - 3 was 162 u / ml, which further reduced to 101.7 u / ml in april . A woman in her 60s was diagnosed with stage iib hormone - positive, her2-negative invasive ductal carcinoma in march 2008 . She was treated with dose - dense adjuvant doxorubicin plus cyclophosphamide and weekly paclitaxel, followed by adjuvant endocrine therapy with anastrozole . In february 2010, she presented with recurrent hormone receptor - positive / her2-negative mbc, with metastases identified in bone and liver . She was started on fulvestrant, but after 6 months, in august 2010, she developed progressive disease in liver and bone, with additional metastatic sites in bone . At this time, the patient was switched to single - agent chemotherapy with nab - paclitaxel, but after a short interval, she again presented with progressive disease in october 2010 . Following disease progression on nab - paclitaxel, in october 2010, she was switched to single - agent eribulin mesylate, 1.4 mg / m intravenously on days 1 and 8 of a 21-day cycle . The rationale for choosing eribulin therapy for this patient was its favorable tolerability profile and the failure of prior taxane therapy . Although she had some baseline nci grade 1 neuropathy at the time eribulin therapy was initiated, she tolerated the drug relatively well without any nausea or significant fatigue . There was a mild increase in neuropathy with eribulin, but this did not exceed grade 1 or require dose modification . As shown in figure 2, she experienced a partial response with eribulin that was sustained until february 2011, when she again developed progressive disease in bone and liver . She subsequently underwent two additional lines of chemotherapy with vinorelbine and capecitabine, but progressed on both and elected to pursue palliative treatment alone . The two patients in the cases described, both with extensive treatment experience in the metastatic setting and few new treatment options, were able to derive clinical benefit from eribulin . Both patients met the entry criteria for eisai metastatic breast cancer study assessing physician s choice versus e3789 (embrace), an open - label, randomized, controlled phase iii trial comparing eribulin with treatment of physician s choice (tpc) in patients with locally recurrent or mbc who had received between two and five prior chemotherapy regimens and two or more regimens for locally recurrent or mbc.6 patients in embrace had few viable treatment options, with 99% having received prior taxane, 99% prior anthracyclines, and 73% prior capecitabine; 84% had also received at least one prior course of hormonal therapy.6 most patients in the trial had metastatic disease, with 84% having metastases in two or more organs.6 the patients in the cases presented here were therefore typical of the population included in the embrace trial . Median os with eribulin was 13.1 months in embrace, significantly longer than 10.6 months for the tpc (hazard ratio [hr] = 0.81; 95% confidence interval [ci]: 0.66, 0.91; p = 0.041).6 this was analyzed with a two - sided stratified log - rank test at a significance level of 0.049 and a cox regression model to calculate the hr . The median duration of response was 4.2 months (95% ci: 3.8, 5.0) for eribulin and 6.7 months (95% ci: 6.7, 7.0) for tpc (p = 0.159). Although not reaching statistical significance, median progression - free survival (pfs) was 3.7 months vs 2.2 months by independent review for eribulin and tpc, respectively (hr = 0.76; 95% ci: 0.71, 1.05; p = 0.137).6 based on these data, the patients discussed in these cases appear to have had a generally typical response . A second phase iii study, conducted in less treatment - experienced patients (3 prior chemotherapy regimens with 2 for advanced disease in this study) showed that eribulin was similar to capecitabine in terms of os (hr = 0.88; 95% ci: 0.77, 1.00; p = 0.056) and pfs (hr = 1.08; 95% ci: 0.93, 1.25; p = 0.30).12 in this study, patients who were triple - negative (os = 14.4 vs 9.4 months; hr = 0.70; 95% ci: 0.55, 0.91; p = 0.01), estrogen receptor - negative (os = 14.4 vs 10.5 months; hr = 0.78; 95% ci: 0.64, 0.96; p = 0.02), or her2-negative (os = 15.9 vs 13.5 months; hr = 0.84; 95% ci: 0.72, 0.98; p = 0.03) also showed improved responses to eribulin compared to capecitabine treatment, respectively.13 the two patients in the cases reported here were her2-negative, thus their responses to eribulin are consistent with these results . However, serious adverse events (aes) were reported in 25% of patients on eribulin and 26% receiving the tpc in embrace.6 the most common aes of any grade in both arms were asthenia, or fatigue, and neutropenia.6 neutropenia, leukopenia, and peripheral neuropathy were the most common grade 34 aes seen with eribulin, and peripheral neuropathy was the most common ae leading to discontinuation, with 5% of patients stopping treatment.6 similar results were seen in the second phase iii study, with a similar incidence of overall toxicity and serious aes for both eribulin and capecitabine.13 patients should be monitored closely for signs of peripheral motor and sensory neuropathy . This was of particular importance for the patient in case 2, because she had grade 1 neuropathy following treatment with nab - paclitaxel at the time eribulin was initiated . The patients in both cases were monitored consistent with guidelines, and grade 1 neuropathy not requiring intervention was noted . In the event that grade 34 peripheral neuropathy occurs, eribulin should be withheld until resolution to grade 2 or below.14 other aes of concern are neutropenia and febrile neutropenia . In trials, these were more common in patients with alanine aminotransferase (sgpt) or aspartate aminotransferase (sgot) 3 times the upper limit of normal (uln) or bilirubin 1.5 times the uln . Consequently, complete blood counts should be monitored prior to each dose, with increased frequency in patients who develop grade 3 or 4 cytopenias . When necessary, neutropenia, peripheral neuropathy, and other serious aes can be managed by following the recommendations for eribulin dose delay and dose reduction listed in table 1.14 it is important for clinicians to become familiar with additional treatment options for patients with triple - negative or hormone - refractory mbc . It is also essential to recognize potential toxicities that could arise from eribulin therapy and how to manage them . The two cases described here build on the data from the embrace study6 by confirming the benefit of eribulin therapy in the clinical setting . These cases demonstrate the favorable tolerability profile, efficacy, and ease of administration of eribulin for triple - negative and hormone - refractory mbc . Toxicities are usually mild and can be managed through dose modifications, making this agent ideal for elderly patients who fail previous lines of therapy . The two cases described here are typical of those in phase iii clinical trials of eribulin, as well as in clinical practice, and highlight the role of this new agent . For management of advanced and mbc in pretreated patients, eribulin has been shown to be more effective than the tpc, a choice that equates to the current usual standard of care . Importantly, eribulin has been shown to be effective in those with triple - negative disease, few treatment options, and poor prognosis . The ae profile of eribulin is similar to that of other commonly used agents in this setting, as demonstrated by a similar rate of aes as with the standard - of - care treatment in clinical trials.6,13 the most serious aes seen with eribulin, namely, neutropenia and peripheral neuropathy, can be readily managed by following recommendations for dose delays and dose reductions . Eribulin therefore offers a treatment option for patients with advanced and mbc who have few alternatives.
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In the early 1960s ross and barratt - boyes introduced the use of human allograft cardiac heart valves, or homografts, into clinical practice [1, 2]. In 2012 the 50th anniversary of the first so - called ross operation was celebrated . The ross operation encompasses implantation of a pulmonary autograft in the aortic position, while an allograft is transplanted in the pulmonary position . Ever since, there has been a need to store available donor grafts, so that they can be prepared, stored in a tissue bank, and used for implantation, either in elective or in emergency patients . From the end of the sixties and into the eighties tissue banks were founded all over europe . In the same period studies about the techniques and successes of homograft implantation in larger series of patients were published, followed in the nineties by studies which covered more than a decade . Because, over time, they were the only successful biological heart valve prostheses beside the mechanical ones, the results were very satisfactory . The advantages were clear: a low rate of thromboembolic events, thus avoiding a lifetime of anticoagulation therapy . In addition, their hemodynamic properties were superior to those of mechanical valves, especially those available in the early 1960s and 1970s . As time went by, it became clear that the availability and cardiectomy techniques to obtain cardiovascular tissues were a problem as suitable donors were recipients of heart transplants, organ donors whose hearts were not accepted, or donors who were autopsied and their relatives had agreed to their tissues being used . In the last 20 years, the european cardiovascular tissue banks have invested a great deal of finances and effort in improving the safety and quality of their tissue banking methods and facilities . Issues such as donor selection, validation of testing methods, the improvement of sterility systems and clean rooms were addressed . Regulations based on directives of the european union became law in all member states . The foundation of european tissue banks initiated a survey to obtain an assessment and quantification of the situation in the field of cardiovascular tissue banks, after implementation of the european directives into national legislation . In 2011, questionnaires were sent out to 30 cardiovascular tissue banks, 18 of which completed and returned them . One cardiovascular tissue bank had started its activities in early 2011; hence no data could be reported as yet . Three additional questionnaires were received after the statistical analysis was closed, and these data are not included . Ranges and means were calculated and tabulated giving insight into the level of activities of these cardiovascular tissue banks . Percentages of detected positive serology were assembled, and a break - down of microbiological contamination as the reason for discarding tissue should yield information on the reasons for tissues being discarded during the process . The statistics in table 1 are based on the assumption that every heart received in the cardiovascular tissue banks provided two grafts . Out of 18 tissue banks, 11 had registered the number of donor reports rather than the hearts actually received in the bank . In these 11 tissue banks, 67% of the donors reported resulted in the receipt of a heart in the bank . Table 1 shows that from the total of 1640 hearts received by 18 tissue banks in 2010, only 46.9% provided suitable grafts; hence the discard rate is 53.1% . The cardiovascular tissue banks show a considerable difference in their activities: while in 2010 the highest number of grafts received was 262, the smallest bank processed only 17 grafts . When it comes to issuing grafts, similar differences are observed . As shown in table 2, the statistics in table 2 confirm that the demand for pulmonary grafts is about twice as high as the demand for aortic valves: 67% of all grafts issued were pulmonary valves . The data provided by the 18 cardiovascular banks show that, in 2010, exporting of tissues to other countries was done by 7 banks, with the proportion varying from 1% to 72% of the annual number of processed grafts . The average donor age ranges from 40 (in 2007) to 42 in 2010 . Fifty - seven percent of the hearts originated from organ donors of whom the heart could not be transplanted, 28% from non - organ donors (those who become donors after an extended period of cardiac arrest, and are thus unsuitable as organ donors) and 15% were retrieved from so called domino donors . Domino donors are people who undergo a heart transplantation, and whose native heart may still have valves that are transplantable as tissue grafts . The criteria for the time between cardiac arrest and cardiectomy, as observed by the tissue banks in this study, ranged from 2 to 48 hours . In reality, the average time until cardiectomy was between 8 hours in 2007, and 11 hours in 2010 . After receipt in the tissue bank, the valvular grafts are excised from the heart and decontaminated . Also here, the criteria differed greatly between the banks and the time varied from 18 hours to 72 hours, while the average number of hours in practice was 24 . Table 4 shows the reasons for discarding donor tissue . In 2010, 45.3% of the tissue grafts had to be discarded . In many cases there was more than one reason for not accepting the heart, or its tissue grafts, for transplantation . Heart valve discards in 2010, average% of all cardiovascular banks . In 32.7% of the cases the reason for discard was that there were contraindications for transplantation of the tissue in the donor s medical history . During processing 35.8% of the cardiovascular tissue was found to be unsuitable because of its morphology . In 17.65% and 4.2% of the cases, respectively, microbiology or serology test results were a reason not to accept the grafts for transplantation . Technical and unknown reasons were responsible for 7.3% and 7.8%, respectively, of the discards . Substantial differences can be observed in the number of hours during which the tissue banks culture the tissue to detect and/or eliminate micro - organisms; the range is 5 - 72 hrs . Also, the temperature under which incubation takes place shows a large variety: from 4o c to 37o c. the banks use 25 different antibiotics in many different concentrations . In table 6 a breakdown of other tissues provided by the banks in this study shows that pericardium, arteries and veins are processed alongside valvular allografts . The level of activity in cardiovascular tissue banks is determined by the numbers of donors . This study shows that the range of donor hearts received in 18 banks varied from 1640 in 2010 to 1700 in 2007 . As the number of hearts received represents only 67% of the number of donors referred, it may be worthwhile to analyze the reasons why the hearts of 33% of the reported donors were eventually not allocated to the tissue bank . By eliminating factors preventing the donation from materializing on the other hand the statistics document that in 2010 45.3% were not suitable for transplantation and had to be discarded . Better donor screening beforehand, and a more effective process from cardiectomy to excision and for decontamination in the bank are three factors which could decrease this high number of discards . This study shows in statistics what cardiovascular tissue bankers have known for a long time, that the demand for pulmonary valves is about twice as high as the demand for aortic valves: 66% of all grafts are pulmonary valves . Although this study does not extend to the use of grafts, the literature shows that for many centers the pulmonary valve is the allograft of choice in congenital as well as in acquired cardiac diseases . The activity of the banks varies from processing less than 20 to 262 donor hearts in 2010 . One has to wonder about the routine capabilities of personnel as well as about the optimal use of the investment and costs of maintenance of a class a laboratory . The donor age (table 3) has gradually increased from an average of 40 in 2007 to 42 years in 2010 . As the average age in the european population increases, the donor age increases accordingly . The reason is twofold: some authorities forbid the use of non - organ donors;to set up a cardiectomy team on a 24/365 basis requires additional organizational constraints and investments which some banks wish to avoid . Some authorities forbid the use of non - organ donors; to set up a cardiectomy team on a 24/365 basis requires additional organizational constraints and investments which some banks wish to avoid . The dependency on the receipt of organ donor and domino donor hearts brings them into a vulnerable position . The need for additional cardiovascular grafts could be compensated by an effort to set up a non - organ donor program . However, the differences in decontamination methods, use of antibiotics and their concentrations, as well as temperature should be a subject to cause concern in the cardiovascular tissue banks participating in this study . In 2010, a conference of these tissue bankers and their microbiologists was organized by the foundation of european tissue banks . Substructuring and validation methods were exchanged, and some arguments were proven to be right . At that conference, and from the questionnaire in this study, no adverse events were reported by any of the participating tissue banks . While most of the cardiovascular tissue banks in this study concentrate on the processing and distribution of the classic homograft heart valves, nine banks showed activities with respect to processing tissues such as arteries, veins and pericardium . Correspondence with different tissue bank representatives revealed that the demand for arterial grafts is growing throughout europe . While veins are used in access surgery (shunts), pericardium serves as patching material to bridge larger gaps of deficient tissue during cardiothoracic operations . The numbers of these tissues issued over the period 2007 - 2010 also show a considerable increase . For the first time since the start of the clinical use of human allogeneic heart valves, data from a number of european cardiovascular tissue banks could be accumulated . Statistics with respect to numbers, discard and use of cardiovascular tissue provide insight into the magnitude of their activities as well as into some of the parameters they use . First of all, looking at the number of tissue grafts issued for transplantation, one can conclude that the demand for tissues has not decreased during the period of 4 years encompassed in this study . Apparently the demand the results show that cardiovascular tissue bank activities have remained relatively stable over the years, though the number of donors has somewhat decreased (3.5%). While the demand for pulmonary grafts still increased from 810 to 981 (21.1%), only 505 aortic grafts were issued in 2010 . What happens with all the aortic grafts which are not issued is a logistical as well as an ethical question . In order to cope with the persistently high demand for pulmonary grafts and arteries, those cardiovascular tissue banks which do not retrieve hearts from non - organ donors should seriously consider initiating such a donor program . Although not clinically proven, studies show that stem cell techniques may eventually contribute to the quality and availability of human heart valves, yet none of the cardiovascular tissue banks indicated that they are in any way involved in stem cell research . The differences in accepted time lapses from death to cardiectomy, and from cardiectomy until excision of the valves and further processing find their origin in view - points with respect to quality and safety . A consensus between the tissue banks contributing to this study should be based on data with respect to the potential loss of tissue quality starting at cardiac arrest and measured over time . As there are very large methodological differences with respect to microbiology testing, incubation and decontamination of cardiovascular tissue between the 17 contributing tissue banks, there is a necessity to validate procedures and room for improvement . This survey shows an increased demand for other tissues, which may be worth further exploration . After all, where alternatives seem to fail or are absent, it is the task of tissue banks to satisfy the clinical demand for tissue grafts.
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We also classified patients based on age, sex, performance status (according to the eastern cooperative oncology group), international prognostic index (ipi) score, presence of b symptoms, clinical stage, serum lactate dehydrogenase (ldh) level, primary site and treatment protocol . Clinical staging was determined according to the revised american joint committee on cancer (ajcc) for lymphoid neoplasm.9 the patients had the following laboratory work ups: complete blood count (cbc), serum lactate de - hydrogenase (ldh) level, alkaline phosphatase (alp), liver function tests, renal function tests, and x - ray or computed tomography (ct) of the bone lesion, chest x - ray (cxr), abdominopelvic ultrasonography (us) or ct, and bone marrow biopsies (bmb) in 9 patients . All patients were evaluated for presence of b symptoms (fever38c, night sweating, and weight loss10 kg in 6 months). Overall survival (os) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . Disease free survival (dfs) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . Differences were considered significant if the p value was 0.05 (two tailed). All survival analyses were performed using the spss (ibm company, united states), version 14 . We retrospectively analyzed all patients (28 ones) who were diagnosed with pbl and referred to our center, (omid hospital), between march 2001 and february 2009 . All pathological paraffin blocks were reviewed by an experienced pathologist in our center and the pbl diagnosis was confirmed . We also classified patients based on age, sex, performance status (according to the eastern cooperative oncology group), international prognostic index (ipi) score, presence of b symptoms, clinical stage, serum lactate dehydrogenase (ldh) level, primary site and treatment protocol . Clinical staging was determined according to the revised american joint committee on cancer (ajcc) for lymphoid neoplasm.9 the patients had the following laboratory work ups: complete blood count (cbc), serum lactate de - hydrogenase (ldh) level, alkaline phosphatase (alp), liver function tests, renal function tests, and x - ray or computed tomography (ct) of the bone lesion, chest x - ray (cxr), abdominopelvic ultrasonography (us) or ct, and bone marrow biopsies (bmb) in 9 patients . All patients were evaluated for presence of b symptoms (fever38c, night sweating, and weight loss10 kg in 6 months). Overall survival (os) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . Disease free survival (dfs) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . Differences were considered significant if the p value was 0.05 (two tailed). All survival analyses were performed using the spss (ibm company, united states), version 14 . We retrospectively analyzed all patients (28 ones) who were diagnosed with pbl and referred to our center, (omid hospital), between march 2001 and february 2009 . All pathological paraffin blocks were reviewed by an experienced pathologist in our center and the pbl diagnosis was confirmed . We also classified patients based on age, sex, performance status (according to the eastern cooperative oncology group), international prognostic index (ipi) score, presence of b symptoms, clinical stage, serum lactate dehydrogenase (ldh) level, primary site and treatment protocol . Clinical staging was determined according to the revised american joint committee on cancer (ajcc) for lymphoid neoplasm.9 the patients had the following laboratory work ups: complete blood count (cbc), serum lactate de - hydrogenase (ldh) level, alkaline phosphatase (alp), liver function tests, renal function tests, and x - ray or computed tomography (ct) of the bone lesion, chest x - ray (cxr), abdominopelvic ultrasonography (us) or ct, and bone marrow biopsies (bmb) in 9 patients . All patients were evaluated for presence of b symptoms (fever38c, night sweating, and weight loss10 kg in 6 months). Overall survival (os) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . Disease free survival (dfs) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . Differences were considered significant if the p value was 0.05 (two tailed). All survival analyses were performed using the spss (ibm company, united states), version 14 . Patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement (71%) including humerus 7 (25%), femur 7 (25%), and tibia 4 (14.3%). The pelvis was the second most common site of involvement in 7 (25%) cases . Mandible was involved in 1 (3.5%), radius in 1 (3.5%) and clavicle in 1 (3.5%) patient . 26 (93%) patients had dlbcl and 2 (7%) patients had small lymphoblastic lymphoma . In 15 (53.6%), patients b symptoms were reported . Serum ldh level was recorded in 20 patients; in 6 (30%) patients was 500 and in 14 (70%)> 500 . According to eastern cooperative oncology group (ecog) performance status scoring, 5 patients (17.9%) had ps of -1, 9 (32.1%) had ps of -2, 12 (42.9%) had ps of -3 and 2 (7.1%) ones had ps of -4 . We did nt have any patient with stage 3 (based on ajcc staging system). A stage 4 was defined as multiple bone sites lesions (2 cases), diffuses involvement of single bone (3 cases) or bm involvement (1 case). Because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma, we excluded all patients with bone lymphoma and non - regional lymph node involvement . Chemotherapy with cyclophosphamide (ctx), vincristin (vcr), doxorubicin (adr) and prednisolone (chop regimen) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 (range: 1 - 8). One patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy, on cobalt 60 conventional protocol . We excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . At the end of follow up, 16 (61.5%) dfs did nt reach the median because of small number of events (death or recurrence) but the mean dfs was 51 months (range: 37 - 66). Overall survival was 54 months (range: 40 - 68, figure 2). Overall survival curve 4 (15%) cases recurred . The mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies, 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients (29%) died because of disease progression in 5 cases, recurrence in 3 cases and treatment complication in one . We also compared chemotherapy alone with chemoradiotherapy based on dfs and os (figures 3 and 4). Dfs curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) os curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) 19 (73%) patients received chemoradiotherapy and 7(27%) patients received chemotherapy alone . Os was significantly better in the chemoradiotherapy group compared with other two groups (64 vs. 27 months, respectively, p=0.014). Dfs was also significantly better in combined modality arm compared with other two groups (64 versus 21 months, respectively, p=0.003). Patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement (71%) including humerus 7 (25%), femur 7 (25%), and tibia 4 (14.3%). The pelvis was the second most common site of involvement in 7 (25%) cases . Mandible was involved in 1 (3.5%), radius in 1 (3.5%) and clavicle in 1 (3.5%) patient . 26 (93%) patients had dlbcl and 2 (7%) patients had small lymphoblastic lymphoma . In 15 (53.6%), patients b symptoms were reported . Serum ldh level was recorded in 20 patients; in 6 (30%) patients was 500 and in 14 (70%)> 500 . According to eastern cooperative oncology group (ecog) performance status scoring, 5 patients (17.9%) had ps of -1, 9 (32.1%) had ps of -2, 12 (42.9%) had ps of -3 and 2 (7.1%) ones had ps of -4 . We did nt have any patient with stage 3 (based on ajcc staging system). A stage 4 was defined as multiple bone sites lesions (2 cases), diffuses involvement of single bone (3 cases) or bm involvement (1 case). Because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma, we excluded all patients with bone lymphoma and non - regional lymph node involvement . Chemotherapy with cyclophosphamide (ctx), vincristin (vcr), doxorubicin (adr) and prednisolone (chop regimen) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 (range: 1 - 8). One patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy, on cobalt 60 conventional protocol . We excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . At the end of follow up, 16 (61.5%) dfs did nt reach the median because of small number of events (death or recurrence) but the mean dfs was 51 months (range: 37 - 66). Overall survival was 54 months (range: 40 - 68, figure 2). Overall survival curve 4 (15%) cases recurred . The mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies, 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients (29%) died because of disease progression in 5 cases, recurrence in 3 cases and treatment complication in one . We also compared chemotherapy alone with chemoradiotherapy based on dfs and os (figures 3 and 4). Dfs curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) os curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) 19 (73%) patients received chemoradiotherapy and 7(27%) patients received chemotherapy alone . Os was significantly better in the chemoradiotherapy group compared with other two groups (64 vs. 27 months, respectively, p=0.014). Dfs was also significantly better in combined modality arm compared with other two groups (64 versus 21 months, respectively, p=0.003). Patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement (71%) including humerus 7 (25%), femur 7 (25%), and tibia 4 (14.3%). The pelvis was the second most common site of involvement in 7 (25%) cases . Mandible was involved in 1 (3.5%), radius in 1 (3.5%) and clavicle in 1 (3.5%) patient . 26 (93%) patients had dlbcl and 2 (7%) patients had small lymphoblastic lymphoma . In 15 (53.6%), patients b symptoms were reported . Serum ldh level was recorded in 20 patients; in 6 (30%) patients was 500 and in 14 (70%)> 500 . According to eastern cooperative oncology group (ecog) performance status scoring, 5 patients (17.9%) had ps of -1, 9 (32.1%) had ps of -2, 12 (42.9%) had ps of -3 and 2 (7.1%) ones had ps of -4 . We did nt have any patient with stage 3 (based on ajcc staging system). A stage 4 was defined as multiple bone sites lesions (2 cases), diffuses involvement of single bone (3 cases) or bm involvement (1 case). Because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma, we excluded all patients with bone lymphoma and non - regional lymph node involvement . Chemotherapy with cyclophosphamide (ctx), vincristin (vcr), doxorubicin (adr) and prednisolone (chop regimen) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 (range: 1 - 8). One patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy, on cobalt 60 conventional protocol . We excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . At the end of follow up, 16 (61.5%) dfs did nt reach the median because of small number of events (death or recurrence) but the mean dfs was 51 months (range: 37 - 66). Overall survival was 54 months (range: 40 - 68, figure 2). Overall survival curve 4 (15%) cases recurred . The mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies, 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients (29%) died because of disease progression in 5 cases, recurrence in 3 cases and treatment complication in one . We also compared chemotherapy alone with chemoradiotherapy based on dfs and os (figures 3 and 4). Dfs curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) os curves comparing chemoradiotherapy (crt) (1) versus chemotherapy alone (2) 19 (73%) patients received chemoradiotherapy and 7(27%) patients received chemotherapy alone . Os was significantly better in the chemoradiotherapy group compared with other two groups (64 vs. 27 months, respectively, p=0.014). Dfs was also significantly better in combined modality arm compared with other two groups (64 versus 21 months, respectively, p=0.003). It was first described as a distinct clinicopathological entity in 1939 by parker and jackson.11 the real prevalence of pbl is unclear because of the considerable difficulty distinguishing primary from secondary bone lymphoma . In our study pbl did constitute 5.3% of all dlbcls like many previous study results.312 long bone was the primarily affected site in our case series, the same as western country reports in which femur was the most affected site.36713 but in ramadan et al . Results, spine involvement had the same prevalence as the long bones (33%).4 in dai maruyama et al . Report, the pelvis was the common involved site (54%).5 horsman et al . Reported that the most commonly presented site was the pelvis, although they described that the femur was the most frequently involved bone.13 most of our patients were younger than 60 years which was similar to other reports.3 according to the who classification, lymphoma involving the bone can be classified in four groups: group 1, lymphoma in a single bone site with or without regional lymph node involvement; group 2, lymphoma in multiple bones, but no visceral or lymph node involvement; group 3, bone tumor with involvement of other visceral sites or lymph nodes at multiple sites; and group 4, lymphoma involving any other site and found by bone biopsy which was done to rule out possible involvement . We defined pbl in our study according to who classification groups 1 and 2 . Who classification and some previous reports have indicated that groups 1 and 2 should be considered as pbl, but group 3 should be excluded from pbl and be considered as systemic lymphoma regardless of the bone lesion . Histopathologically, the previous studies reported that the majority of patients with pbl had dlbcl.3615 in our study, 26 (93%) patients had dlbcl and 2 (7%) patients had small lymphoblastic lymphoma . Several studies have suggested that the combined modality (chemotherapy and radiotherapy) was the best treatment for patients with pbl.36 beal et al . Concluded that pbl patients treated with combination chemotherapy and irradiation had significantly better survival than the patients treated with single modality (chemotherapy or radiotherapy alone), but the 5-year os rate between the two groups was not significantly different.16 in our study, os and dfs was significantly better in combined modality group (os was 64 vs. 27 months, respectively, p=0.014, and dfs was 64 vs. 21 months, p=0.003). Ramadan et al . Reported patients with advanced - stage disease who received chemotherapy plus irradiation with a poor outcome when compared with those who received chemotherapy alone (10-years os were 25% and 56%, respectively).4 however, this difference must be very cautiously interpreted because the decision to use radiotherapy was individualized . Because of small number of patients in our study, further studies are necessary to clarify the characteristics of pbl and its optimal treatment strategy . We will continue our study on pbl and report our results with more patients and longer follow ups . All the authors have carried out the study, participated in the design of the study and acquisition of data performed the statistical analysis and wrote the manuscript.
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Diabetic kidney disease (dkd) can be seen in approximately 30 to 40% of both type 1 and type 2 diabetic patients . According to american diabetes association (ada) data, there have been 17.5 million diagnosed and 6.6 million undiagnosed diabetics in the united states, and this situation was found to be closely associated with both cardiovascular morbidity and mortality in this population . Despite improvements in their chemical structure, contrast dye agents are still the third leading cause of hospital - acquired acute kidney injury . Reduction in the incidence of contrast - induced nephropathy (cin) can lead to a decrease in morbidity, mortality (up to 36%), and length of hospital stay . Diabetic patients are found to be more prone to cin, which might further enhance the progression of dkd . The exact pathophysiologic mechanisms of cin are not fully understood; however, hypoxia and oxidative stress (oxs) constitute the main responsible pathways of this situation, especially in dkd . Other important molecules that are closely associated with dkd and cin are inflammatory cells such as leukocytes, monocytes, and macrophages - derived inflammatory cytokines, including interleukin- (il-) 1, il-6, and il-18 . Recently, a new member of the il-1 family, il-33, is presented as an alarmin molecule that invites inflammatory cells to the kidney in cisplatin - induced nephropathy . Accumulating evidence suggests that il-33 is one of the most important cytokines in the pathogenesis of various disorders, including autoimmune diseases, myocardial infarction, heart failure, and allergic pulmonary diseases; however, in the literature, the data regarding the role of il-33 in dkd and cin are scant . If a causative role of il-33 is confirmed, preventing such inflammation and/or the immediate consequences of such inflammation may represent the key target to prevent dkd . Although melatonin (mt) is one of the attempted molecules in dkd, the role of this drug in diabetic patients with cin remains unclear . Hence, we sought to determine the levels of il-33 along with other inflammatory cytokines and oxs . The second aim of the present study was to determine whether mt has a preventive effect on kidneys in diabetic rats (drs) with cin treatment . The study protocol was approved by the medical ethics committee of erzincan university (mengucek gazi training and research hospital, erzincan, turkey). Thirty male sprague - dawley rats were obtained from ataturk university medical experimental application and research center . The rats were housed in a standard environment with a room temperature of 22 2c, humidity level of 4060%, and 12/12 h of light - dark cycle . Before starting the experimental procedure, after completion of the acclimation period, the rats were randomly allocated into five groups . The first group was comprised of healthy rats (hrs), whereas the other four groups were made up of drs, diabetic rats with contrast - induced nephropathy (cin + drs), melatonin - treated diabetic rats (mtdrs), and melatonin - treated cin + diabetic rats (mtcin + drs). All groups except the hrs received 50 mg / kg / day streptozotocin (stz) (sigma, st . Louis, usa) within 0.1 m sodium citrate solution (ph: 4.50). Daily blood sugar measurements were performed through tail - end puncture using a glucomax ultra td 4227 (germany) blood sugar measurement device . In all stz - administered groups, the blood sugar level was> 400 mg / dl at the end of the 5th day . Drs did not receive any other treatment, whereas cin + drs and mtcin + drs were given 1.5 mg / kg of intravenous radiocontrast dye (omnipaque, amersham, ireland) on the 35th day . In addition, mtdrs and mtcin + drs were given 20 mg / kg / day of intraperitoneal injection of mt (sigma, st . Louis, usa) starting on the 28th day for seven days as described previously by gazi et al . . All rats were sacrificed under thiopental sodium (5 mg / kg) anesthesia on the 36th day using blood evacuation via cardiac puncture and cervical dislocation . Each kidney was fixed in 10% formalin solution for 4855 h, dehydrated in a graded alcohol series, embedded in paraffin wax, and serially sectioned using a microtome (leica rm2125rt). For light microscope histological examination, thin sections of 5 m thickness were taken from the same paraffin blocks . Slides were covered, and photographs were taken using a light microscope with a camera attachment (nikon eclipse e600, japan). For electron microscopy, kidneys were fixed in buffered 3% glutaraldehyde in 0.1 m phosphate solution, postfixed in 1% osmium tetroxide, dehydrated in a graded acetone series, and transferred to propylene oxide . Sections were cut, using an ultratome (nova lkb bromma, sweden), into 7080 nm thin sections for histological evaluation at the ultrastructural level . After staining in 2% uranyl acetate and 0.4% lead citrate, sections were examined using a transmission electron microscope (jeol 100 sx, tokyo, japan). Also, semithin sections (1 m) obtained with an ultramicrotome from the same araldite blocks were stained with toluidine blue and histopathologically examined using a light microscope . The kidneys were examined by light microscopy for histological evaluation of the following parameters: h&e and toluidine staining for inflammatory cell infiltrates, armanni - ebstein lesions, apoptotic cell death, glomerular changes and ultrastructural evaluation by electron microscopy for changes in glomerular basement membrane, podocytes and their pedicels, capillary structure, and mesangial cells . Blood - containing tubes (vacutainer sst, becton dickinson, usa) were centrifuged for 7 min at 3,000 rpm . Serum samples were placed in 2 ml eppendorf tubes (eppendorf, germany) and stored at 80c until the time of analysis . Il-6 (ebioscience, bms625, usa), il-33 (cusabio, csb - e14077r, china), and il-1 (ebioscience, bms630, usa) levels were measured using elisa kits appropriate to the species in both kidney tissue and serum samples . Serum blood - urea nitrogen, creatinine, glucose, sodium, potassium, and calcium levels were measured in an autoanalyzer (au 5800, beckman coulter, japan) using original kits . The level of lipid peroxidation (lpo) in kidney tissues was determined by estimating malondialdehyde (mda) using the thiobarbituric acid test . The kidney tissues were dissected, weighed, and homogenized in 10 ml of 100 g / l kcl . The homogenate (0.5 ml) was added to a solution containing 0.2 ml of 80 g / l sodium lauryl sulphate, 1.5 ml of 200 g / l acetic acid, 1.5 ml of 8 g / l 2-thiobarbiturate, and 0.3 ml of distilled water . The mixture was incubated at 98c for 1 h. upon cooling, 5 ml of n - butanol: pyridine (15: 1) was added . The mixture was vortexed for 1 min and centrifuged at 4,000 g for 30 min . Myeloperoxidase (mpo) activity was measured according to the modified method of bradley et al . . The homogenized samples were frozen and thawed three times and then centrifuged at 1,500 g for 10 min at 4c . Mpo activity in the supernatant was determined by adding 100 ml of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer (ph 6.0) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% (wt / vol) hydrogen peroxide . The changes in absorbance at 450 nm of each sample were recorded on a uv - vis spectrophotometer . Mpo activity in tissues was expressed as micromoles per min per milligram of tissue (mol / min / mg tissue). Sod estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with nitroblue tetrazolium (nbt) to form formazan . Sod activity was then measured at 560 nm by the degree of inhibition of this reaction and was expressed as mmol / min / mg of tissue . Decomposition of h2o2 in the presence of catalase (cat) was measured at 240 nm according to aebi . The cat activity was defined as the amount of enzymes required to decompose 1 mmol of h2o2 per min at 25c at ph 7.8 . The amount of total glutathione (gsh) in kidney tissues was measured according to the method described by sedlak and lindsay, with some modifications . The kidney tissues were homogenized in 2 ml of 50 mmtris - hcl buffer containing 20 mm edta at ph 7.5 . After adding 2 ml ethanol (to precipitate the proteins), the supernatant was used to determine the gsh level using 5,5-dithiobis(2-nitrobenzoic acid) (dtnb). Subsequently, the gsh level of the kidney was expressed as nmol / g of tissue . All analyses were performed with the statistica software program version 7.0 (statsoft, tulsa, ok, usa). In the present study, an lpo level which is accepted as an indicator of tissue damage as well as oxs was studied . In addition, the activities of sod, cat, and myeloperoxidase (mpx) enzymes and gsh levels were also measured to understand how the defense mechanisms were affected in kidney tissues . Kidney lpo levels of drs were significantly increased when compared with hrs (40.1 0.6 versus 34.7 0.6, p <0.05, resp .) (figure 1(a)). When contrast dye was administered to drs, kidney lpo levels were further increased when compared with hrs and drs (44.1 2.6 versus 40.1 0.6 and 34.7 0.6, p <0.05 for both, resp .) (figure 1(a)). In the present study, the kidney lpo levels were significantly decreased in both mtdrs and mtcin + drs when compared to drs without mt treatment (37.7 1.3 and 37.2 0.3 versus 40.1 0.6, p <0.05, resp .) (figure 1(a)). However, there was no statistically significant difference between mtdrs and mtcin + drs (37.7 1.3 versus 37.2 0.3, p> 0.05, resp .) Mpx uses hydrogen peroxide and chloride ions for production of hypochlorous acid that is a defense of neutrophil against bacteria and pathogens . In this study, the kidney tissue mpx activity was heightened in drs and cin + drs when compared with hrs (0.0080 001 and 0.015 0.001 versus 0.003 0.0006, p mpx levels of mtdrs and mtcin + drs were markedly decreased when compared with drs and cin + drs (0.0034 0.0014 and 0.012 0.001 versus 0.0083 0.001 and 0.015 0.001, p <0.05 for both, resp .) (figure 1(a)). The sod enzyme forms hydrogen peroxide by using superoxide radicals for elimination of these harmful radicals . Formed hydrogen peroxide is converted to water by the gsh peroxidase enzyme, which uses the gsh as a substrate . The increments or decrements of sod activity were found to be closely related to the intensity of superoxide radicals . In the present study, the kidney levels of sod activity were significantly raised in both drs and cin + drs when compared with hrs (1.35 0.02 and 1.44 0.02 versus 1.28 0.01, p <0.05, resp . ). Kidney levels of gsh were significantly diminished in drs and cin + drs when compared with hrs (0.81 0.01 and 0.78 0.06 versus 097 0.04, p <0.05, resp . ). Gsh levels of mtdrs and mtcin + drs were markedly increased when compared with drs and cin + drs (0.89 0.01 and 0.85 0.03 versus 0.81 0.01 and 0.78 0.06, p <0.05 for both, resp . ). Kidney tissue cat activity was increased in drs and cin + drs when compared with hrs (0.008 0.001 and 0.015 0.001 versus 0.003 0.0006, p <0.05 for both, resp . ). However, cat activities of mtdrs and mtcin + drs significantly decreased compared with drs and cin + drs (81.7 3.6 and 84.6 3.8 versus 90.9 3.7 and 94.2 3.3, p <0.05 for all, resp . ). Kidney tissue il-33 levels as well as il-6 and il-1 levels were significantly increased in drs and cin + drs when compared with hrs (table 1, figure 1(b)) (p <0.05, for all). Il-6 and il-33 levels were found to be diminished in mtdrs and mtcin + drs when compared with drs and cin + drs (p <0.05, for all) (table 1, figure 1(b)). In accordance with kidney tissue levels of ils, serum levels of il-33, il-6, and il-1 were found to be increased in drs; moreover, contrast dye markedly boosted this rise (table 1) (p <0.05, for all). Mt treatment significantly decreased serum il-33 levels in both drs and cin + drs (p <0.05, for all) (table 1). Serum creatinine (scr) levels were observed to increase significantly in drs and cin + drs compared to hrs (0.35 0.05 and 0.43 0.05 versus 0.20 0.06, resp . Mt treatment significantly diminished scr in both drs and cin + drs (0.33 0.05 and 0.37 0.05, resp ., p <0.05, for both). In the ultrastructural and histopathological sections of kidney tissues, mesangial cells and capillaries of the glomerulus, podocytes (visceral epithelial cells) and their pedicels in bowman's capsule, and cortical and medullary tubules appeared normal (figures 2 and 3). Renal tissues of the drs showed histopathological changes as demolition in some glomeruli, inflammatory cell infiltration, transparent tubules that show glucogenic vacuolization (armanni - ebstein lesions), apoptotic cell death of renal tubules (figures 2 and 3) and ultrastructural changes as closure of the filtration slits with atypic pedicels and basement membrane thickening (figures 2(a)2(d)), and mesangial hypertrophy in some glomeruli . In addition, electron - dense vesicle accumulation within endothelial cells was also observed (figure 2(b)). Apoptosis, necrotic changes, and armanni - ebstein lesions were found to be diminished in mtdrs (figures 4(a)4(d)). Also, basement membranes, pedicel, and endothelial cells generally appeared normal (figures 2(a)2(d)). However, local interstitial inflammatory cells were observed in mtdrs, although they were found to be less than drs (figure 4). In contrast, we observed less regenerative changes in mtcin + drs (figures 2 and 4). First, kidney tissue and serum levels of il-33 as well as other inflammatory cytokines and oxs markers were found to be significantly increased in drs, and further increase was also determined in contrast - applied drs . Second, both kidney tissue and serum levels of oxs markers, inflammatory cytokines, and il-33 were found to be significantly diminished in mtcin + drs compared with the groups without mt treatment . Third, these findings are also supported by the histopathological findings determined by electron and light microscope . This study is the first to demonstrate the possible role of il-33 in the pathogenesis of drs with cin and the preventive effect of mt in this situation . Increased serum glucose levels along with chronic low grade inflammation, oxs, advanced glycation end products (age), sorbitol accumulation, hexosamine, and protein kinase c (pkc) pathway activation are well - known features of diabetes mellitus . Diabetic patients might need further investigations with contrast using methods such as computed tomography imaging for clarifying the situations that are commonly faced . Contrast agents have direct toxic effects on renal tubular cells and renal hemodynamics, and based on previous studies, oxygen radicals might play a major role as the primary physiological insult [16, 17]. Infusion of contrast agents increases in osmotic load and viscosity, and thus hypoxemia of the renal medulla and renal free radical production through postischemic oxs occur . In the present study, we found that markers of oxs are increased and antioxidants are decreased in drs and drs with cin, in accordance with the current literature [7, 18, 19]. Previous clinical studies have established an association between a rise in inflammatory markers, including il-6, il-18, and tnf-, and dkd . Among several cytokines that may be relevant to the development of dkd, it has been demonstrated that serum il-18 levels are markedly elevated in patients with type 2 diabetes with microalbuminuria when compared to those with normoalbuminuria, while il-6 rises at a later time, when clinical macroalbuminuria is present . According to a hypothesis adopted by many authors, oxs might be the main trigger system for the initiation and progression of dkd . Another novel concept is that chronic inflammation may result in local disruption of insulin signaling, leading to the initial development of both micro- and macrovascular complications of diabetes . Hence, further experimental data are needed to characterize the causative role of cytokines in the development of dkd . In this regard, we hypothesized that il-33, also named alarmin, might be a candidate as a trigger cytokine in drs with cin . Il-33 is a proinflammatory cytokine that can be released from necrotic cells, binds the st2r on immune cells, and increases secretion of cytokines, with resultant inflammation [23, 24]. For instance, balb / c mice were found to be resistant to the induction of t cell - mediated diabetes when multiple low doses of stz were administered . Additionally, balb / c mice with the st2 gene deleted were also found to be more prone to stz - induced diabetes . Recently, karatas et al . Showed that il-33 expression was typically located in the peritubular and intraglomerular capillaries . They also found 6-fold (diabetic mice at the 4th month versus healthy mice, p <0.01) and 5.8-fold (diabetic mice at 6th month versus healthy mice, p <0.01) increases in il-33 protein expression in western blots of whole kidney tissue, and assessment of il-33 with elisa confirmed their results (il-33 levels 0.409 pg/g diabetic mice at the 6th month versus 0.164 pg/g healthy mice, p <0.01). These results also support our hypothesis that il-33 may participate in the initiation and progression of dkd . The exact function of il-33 in the nucleus has not been established; however, il-33 can activate the transcription factor ap-1 as well as nuclear factor - kappa b (nf-b). Recently, akcay et al . Clearly demonstrated that il-33 plays a central role in the pathogenesis of cisplatin - induced acute kidney injury via invoking cd4 t lymphocyte to the kidney . The major proinflammatory effect of il-33 is to induce cd4 th2 cell responses via the st2r . The present study is also unique in that it shows an effect of a nonimmune stimulus, contrast dye, on il-33 . In this regard, our study demonstrates that il-33 is expressed in the kidneys of both drs and contrast - applied drs . Inflammatory mediators, including cytokines, have important roles during the recruitment of inflammatory cells; these drive the fibrogenic process . Recent studies also demonstrated that inhibition of inflammatory cell infiltration might contribute to the control of renal tubulointerstitial fibrosis . For instance, melatonin was found to be beneficial in terms of leukocyte infiltration, via reduction in the expression of nf-b in a rat model of unilateral ureteral obstruction . In another rat model of carbon tetrachloride nephrotoxicity, interstitial mononuclear cell infiltration and fibrosis of kidney were ameliorated by melatonin treatment . In this context, according to the present study results, it is wise to consider that melatonin treatment might reduce tubulointerstitial fibrosis via il-33 related pathways . Previous studies have reported that mt can prevent drug - induced nephropathy including stz and contrast dye [10, 18]. Since mt has both highly lipophilic and hydrophilic properties, it passes rapidly through all biological membranes and enters the cells and their subcellular compartments when administered intraperitoneally or intravenously . The widespread subcellular distribution of mt may allow it to reduce oxidative damage in both the lipid and aqueous environments of the cell . This is an advantage of mt over some other antioxidants, which penetrate cells more slowly . However, there has been no data regarding whether mt is preventive against both dkd and cin situations together . In our daily practice, the physicians use diagnostic tools such as computed tomography with contrast dye or do angiography when needed in patients with dkd . The risk of morbidity and mortality was found to be higher in patients with dkd when exposed to contrast dye . Our study is unique in that it has demonstrated that pretreatment with mt significantly improved cin in patients with dkd . Structural features of mt, including lipophilicity, hydrophilicity, and electron - richness, provide additional beneficial effects of this molecule in terms of antioxidant capacity and insulin secretion effect via the mt1 receptor . Mt activates several antioxidative enzymes, including gsh peroxidase, sod, and lipid peroxidase . Mt also easily crosses cellular membranes and provides on - site protection against free radical - mediated damage to biomolecules . Although the exact mechanism of cin is not very well known, deleterious factors such as vasoconstriction, tubular obstruction, mitochondrial injury, and plasma membrane toxicity have been implicated . Hence, mt might be a promising agent to protect renal functions in dkd and cin via its antioxidant and antihyperglycemic effect . Between 1990 and 2000, many studies have demonstrated the possible interaction between mt and the immune system [35, 36]. Mt's immunoenhancing effect depends upon its ability to enhance the production of cytokines as well as its antiapoptotic and antioxidant action . Mt has been proposed to regulate the immune system by affecting cytokine release from immunocompetent cells . Additionally, mt was found to augment cd4 + lymphocytes and decrease cd8 + lymphocytes in rat submaxillary lymph nodes . Mt enhances the production of il2, ifn-, and il-6 by cultured human mononuclear cells . Mt might also increase the production of il-1, il-6, and il-12 via activating monocytes . Repetitive stimulation of t helper (th) cells via mt might force th cells to switch to th1 lymphocytes that enhance the release of il-2 and ifn-. These latter cytokines can particularly invite macrophages and other phagocytes to the inflammation site . In addition, mt supplementation was found to increase cd4 th production . Besides the release of proinflammatory th-1 cytokines, raghavendra et al . Also demonstrated that mt might activate an anti - inflammatory th-2-like immune response . Collectively, these data indicate that mt possesses important immunoenhancing properties and suggest that mt may favor both th-1 and th-2 responses . Il-33 stimulation was found to be crucial for the transformation of cd4 t cells into the th-2 subgroup . We did not measure the levels of cd4 + lymphocytes before and after mt treatment . However, the beneficial effect that we show in the mtdrs and mtcin + drs might depend on a cd4 + lymphocytes - il-33-mt circle . In summary inhibition of il-33 with mt provides functional and histologic protection against cin, demonstrating that il-33 might be a candidate for mediating cin in dkd . Inhibition of il-33 and oxidants with mt pretreatment offers therapeutic potential in contrast dye - applied diabetic patients.
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Multiple myeloma (mm) is a common disease, accounting for 10% of hematologic malignancies . Pleural effusions occur in up to 6% of patients with mm [1, 2] but rarely as a result of primary myelomatous infiltration or pleural involvement [2, 3, 4]. Most pleural effusions in mm are secondary to heart failure from amyloidosis, nephrotic or nephritic syndrome, pulmonary embolism, lymphatic obstruction, hypoalbuminemia or infections [3, 5, 6]. Myelomatous pleural effusion (mpe) is a rare entity, occurring in only about 1% of mm pleural effusions [1, 2, 3, 4, 5] with <100 having been reported . Mpe is usually a sign of advanced disease [1, 2, 7] and the reported median survival time is <4 months . Diagnosis is based on criteria proposed by the international myeloma working group, and includes monoclonal protein detection by pleural fluid protein electrophoresis, presence of atypical plasma cells in the effusion or histological confirmation by pleural biopsy [3, 7]. The most frequently used study is pleural cytology [1, 2, 9, 10, 11], which establishes diagnosis in> 90% of cases and has been considered the best method, although electrophoresis confirmation is sometimes advocated for [7, 12]. A general male predominance has been found [1, 2], and associated pleural or chest wall plasmacytomas are usually present [1, 4]. Although its development in advanced disease is more common [2, 3, 4, 6, 7, 9], less frequently it is the initial presenting feature of mm [1, 5]. A higher prevalence of igg and iga was found in different series [1, 2, 4], while light chain type is uncommon [2, 4] except in mpe as initial presentation . Ferritin is irregularly used as a tumor marker and has recently been explored as a prognostic factor in mm, as it can reliably represent the inflammatory and oxidant pathways that play a significant role in plasma cell malignancies . We describe a case in which mpe presented with extremely high ferritin levels, reflecting the potential value of ferritin as an mm marker . A 40-year - old male was admitted to our institution for acute hypoxic respiratory failure for 3 days . Seven months prior to admission, he had been diagnosed with mm stage iii (according to the international staging system). At that time, records showed a bone marrow (bm) biopsy consistent with plasma cell myeloma: hypercellular marrow (90%) with residual trilineage hematopoiesis and increased immature morphology plasma cell infiltrate involving 6070% of cellularity . Flow cytometry of bm specimen showed a clonal population of plasma cells (27%) with an immunophenotype positive for cd138, cd38 and cd56 markers . Cytogenetics displayed complex abnormalities consisting of monosomy 13, trisomy 3 and 9 and 1p deletion . Serum protein electrophoresis showed a monoclonal iga lambda paraprotein and urine protein electrophoresis revealed a monoclonal lambda light chain (bence - jones protein). Imaging studies revealed widespread lytic lesions in the calvarium, bilateral humerus, pelvis and spine, as well as a 5.8 3.8 cm right 5th lateral rib plasmacytoma . At the time of the initial diagnosis, he showed high disease activity with a baseline 2-microglobulin level of 6.2 mg / l and a serum ferritin level of 728 ng / ml . He was also found to have compromised kidney function consistent with stage iii chronic kidney disease from light chain nephropathy, but no major hepatic, pulmonary or coagulation abnormalities were found . He failed treatment with vincristine, cyclophosphamide, and methylprednisolone as well as bortezomib, cyclophosphamide, and solu - medrol, but remarkably responded to a modified phase ii study regimen with bortezomid, bendamustine, and dexamethasone for refractory mm . After ruling out pulmonary embolism with a ventilation / perfusion scan and heart failure with a normal brain natriuretic peptide level and left ventricular function on the echocardiogram, infectious causes were considered, despite normal body temperature, as chest imaging showed bilateral ground - glass opacities with superimposed interlobular septal thickening and intralobular reticular thickening (fig . He received empiric broad - spectrum antibiotics, including coverage for atypical bacteria and pneumocystis jiroveci . Bronchoalveolar lavage with methenamine silver stain for fungal and p. jiroveci - like organisms was negative, as were immunohistochemical studies for cytomegalovirus and herpes simplex virus . Special stains including congo red and periodic acid - schiff stain were negative for amyloidosis and pulmonary alveolar proteinosis, respectively . After a brief initial response to supportive respiratory measures and empiric antimicrobial therapy, he developed multisystemic organ failure, including lactic acidosis, acute liver failure with metabolic encephalopathy, acute kidney injury and worsening pancytopenia dependent on blood and platelet transfusions . The workup revealed active myelomatous disease with rising serum iga and 2-microglobulin levels to 8.6 mg / l . Remarkably, a serum ferritin level of 11,598.5 ng / ml was found and concern for hemophagocytic lymphohistiocytosis (hlh) led to empiric treatment with high doses of dexamethasone and an urgent bm biopsy, which showed atypical plasma cells comprising 90% of the marrow cellularity but no evidence of hemophagocytosis or increased blasts . Sequential chest imaging showed a timely correlation between his acute clinical decline and the acute development of a large right pleural effusion . Thoracentesis revealed a heavily blood stain exudate (ph 7.38, protein 6.9 g / dl, lactate dehydrogenase 1,543 iu / l, white blood cell count of 4,610/l with 94% lymphoid cells), with negative fungal and bacterial cultures . 2) and immunostaining confirmed mm involving pleural effusion [cd138- and cd38-positive (fig . High - dose steroids were continued and the effusion was drained; however, multisystemic organ failure prevented the initiation of intravenous chemotherapy . Despite aggressive supportive measures, he developed disseminated intravascular coagulation, required endotracheal intubation, vasopressor support, and finally perished soon after the diagnosis . Mpe is extremely rare, with a recent series reporting only 64 cases from relevant sources in the english literature since 2000 . We present a case of mpe associated with hyperferritinemia and severe multiorgan failure, a clinical course resembling catastrophic inflammatory conditions such as hlh that has not been described in mpe before . Given its infrequency, a high suspicion is needed for diagnosis . Only about 1% of pleural effusions in mm patients will be attributed to myelomatous involvement [1, 2, 3, 4, 5], but unless the clinical presentation is obvious and rules it out, we believe mpe should always be considered in mm patients presenting with pleural effusions . This is important because mpe represents an aggressive clinical progression of mm, and a recent series reported a median survival of 2.8 months despite aggressive local and systemic treatment . As in our case, the diagnosis is most likely revealed by pleural cytology [1, 2, 5]. Protein electrophoresis is another option, by itself or as a confirmation to cytology, but is unreliable since most effusions are hemorrhagic . Pleural biopsies are less commonly used due to their inconvenience and a lower diagnostic yield given the patchy pleural involvement in mm . Some risk factors for developing mpe in mm were found in our patient and are consistent with the review of other reported cases and series . These include its occurrence in the late stage of mm [2, 4, 7], association with high - risk cytogenetics, such as deletion 13 chromosomal abnormality [1, 2] and usual coexistence of chest wall plasmacytomas [1, 4]. The mm subtype does not seem to be a risk factor given that igg was most common in 2 series with a total of 28 patients [1, 4], while iga was predominant in a 19-patient single - institution series and overall all types have been reported in at least 1 case [7, 8, 9]. Except in mpe as initial presentation of mm, where it constituted 24% of the cases, light chain type seems to be very uncommon [2, 4]. As the majority of the literature on this topic is from single reported cases and small series, the relationship between tests or treatment and prognosis is not known; however, as prognosis is known to be poor, palliation is considered the mainstay of treatment and includes chemotherapy and pleurodesis . There is no chemotherapeutic regimen of choice since a wide variety has been used over time and no meaningful comparisons can be made [1, 2, 4]. Recent studies suggest that the inflammatory and oxidant pathways play a significant role in the pathogenesis of plasma cell malignancies, and ferritin levels can be a reliable indicator of these processes in carcinogenesis . Therefore, ferritin has been used as a marker in mm and other hematologic malignancies [10, 11]. Studies show that ferritin levels are an independent predictor of mortality in newly diagnosed mm, that higher ferritin levels are present in recurrent mm compared to patients in remission and that ferritin has a prognostic value in mm patients undergoing autologous transplantation [13, 14]. This evidence has been proposed to reflect a correlation between ferritin levels and disease activity, tumor load and malignant potential . Our case is the first report that links mpe to extreme levels of hyperferritinemia, which are usually present only in medical conditions associated with severe inflammatory states such as hlh and septic shock where ferritin is thought to have a pathogenic role leading to a cytokine storm and catastrophic clinical outcomes known as the hyperferritinemic syndrome . It is known that hematologic malignancies and sepsis can lead to hlh, but this was ruled out in our patient . Unfortunately, ferritin levels have not been reported in previous mpe cases, and it is unknown if this finding is an isolated coincidence or if it has been missed in previous reports . We believe mpe could represent or be part of an extreme manifestation of myeloma disease activity leading to a severe inflammatory cascade with subsequent multiorgan failure . We recommend investigating ferritin levels in patients with mpe to better determine its relationship with pathogenic inflammatory states such as hlh . This relationship could certainly explain the poor prognosis and severe clinical complications seen with mpe . Written informed consent for personal clinical and imaging information was obtained from the patient before publication of this case report and its accompanying images . A copy of the written consent is available for review from the editor - in - chief of this journal . The authors declare that there is no conflict of interest regarding the publication of this paper.
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Blood - borne pathogens first encounter the adaptive immune system in the marginal zone region of the spleen where the convergence of innate and adaptive immune mechanisms insures an early and effective response to pathogen antigens (1, 2). Both thymic - independent and -dependent responses are elicited in response to infection (1, 3). The thymic - independent response involves the targeting and activation of marginal zone b cells (mzbs)*through their interaction with the repetitive antigenic determinants of pathogens with complement and b cell antigen receptors (4, 5). In contrast, the thymic - dependent ab response is driven by the interaction and reciprocal stimulation of apcs, t lymphocytes, and b cells . The organization of the splenic white pulp nodule into discrete zones enriched for either b cells, t cells, or apcs provide a spatial microenvironment that facilitates an efficient interaction of pathogens with the various cellular populations required for insuring an efficient immune response (68). Antigen presentation and stimulation of t and b cells ultimately results in the formation of germinal centers, high affinity neutralizing abs, and memory cells . Recent reports have begun to define the cellular components and molecular signals that are necessary to establish the marginal zone . B cell intrinsic pathways have been described involving specific chemokines and their receptors, molecules involved in b cell activation, as well as adhesion molecules and their ligands (9, 10). Apart from the mzb, the other predominant cell of the marginal zone is the marginal zone macrophage (mzmo), which is distinct from the metallophilic macrophage, defined by the marker moma-1, located at the border of the marginal and follicular zone (11). The mzmo is defined by its location, interspersed in several layers within the marginal zone, and by its expression of the markers marco and er - tr9 (12, 13). The former molecule is a scavenger receptor belonging structurally to the class a receptor family whereas the latter is identical to the c - type lectin sign - ri (1417). Marco has been shown to bind a range of microbial ags including staphylococcus aureus and escherichia coli whereas sign - ri is the predominant receptor for uptake of polysaccharide dextran by mzmos . Even though both mzbs and mzmos are implicated in both thymus - dependent and -independent immune responses, the exact roles of the two cell types in initiation of the response to blood - borne pathogens is not known . We now define a unique role for the mzmo in regulation of mzb retention and activation and show that movement of this subset of macrophages to the red pulp of the spleen involves signaling via sh2-containing inositol-5-phosphatase 1 (ship) and bruton's tyrosine kinase (btk). In addition, we show a direct interaction between mzmos and mzbs via the marco receptor on mzmos and a ligand on mzbs . C57bl/6 mice obtained from the jackson laboratory were used as wt mice and controls unless otherwise stated . Founders of ship - deficient mice were provided by g. krystal (terry fox laboratory, bc cancer agency, vancouver, canada; reference 18) and btk - deficient mice were purchased from the jackson laboratory . Op / op mice were provided by j. pollard (albert einstein college of medicine, new york, ny) and lysmcre transgenic mice (19) were provided by i. forster (technical university of munich, germany). All experiments involving mice were performed in accordance with national institutes of health (nih) guidelines . For histological examination 6-m frozen sections were stained, and for facs analysis erythrocyte - depleted spleen cells were used . Macrophages were detected using moma-1, marco abs from serotec, and er - tr9 from accurate chemical & scientific corp . Abs to cd1d, b220, cd19, cd21/cd35 (cri / ii), cd23, mac-1, anti rat alkaline phosphatase, and anti secondary abs for immunohistochemistry, anti - biotin, anti - fitc f(ab) horseradish peroxidase, or alkaline phosphatase were from dakocytomation and rabbit anti vector blue alkaline phosphatase substrate from vector laboratories and dab peroxidase substrate from sigma - aldrich were used for development of immunohistochemistry stains . Soluble marco receptor was provided by t. pikkarainen (the karolinska institute, stockholm, sweden; reference 20) and was biotinylated using the ez - link kit from pierce chemical co. the biotinylated soluble marco was detected using streptavidin - cychrome from bd biosciences . S. aureus fluorescent bioparticles were purchased from molecular probes, inc . And macs anti - fitc and anti - biotin beads were from miltenyi biotec . Floxed ship mice were created by insertion of loxp sites flanking the 10th and 11th exons (see fig . The targeting vector was introduced into embryonic stem (es) cells by electroporation and clones were selected with neomycin and ganciclovir and verified by southern blot and pcr . Clones were subsequently selected for a conditional floxed allele (ship) or null allele (ship) using southern blot and pcr . These mice were subsequently crossed with mice expressing cre in the myeloid compartment (lysmcre; reference 19) to generate cre / null / flox mice . Mice were screened for respective genotype by pcr and ship protein expression using western blot (21) on equal numbers of spleen cells purified by macs (miltenyi biotec) sorting according to protocol from the manufacturer . Relative expression of ship in macrophage and b cell populations (comparing wt / null with flox / null / cre) were estimated using alpha imager software from alpha innotech corp . C57bl/6 mice obtained from the jackson laboratory were used as wt mice and controls unless otherwise stated . Founders of ship - deficient mice were provided by g. krystal (terry fox laboratory, bc cancer agency, vancouver, canada; reference 18) and btk - deficient mice were purchased from the jackson laboratory . Op / op mice were provided by j. pollard (albert einstein college of medicine, new york, ny) and lysmcre transgenic mice (19) were provided by i. forster (technical university of munich, germany). All experiments involving mice were performed in accordance with national institutes of health (nih) guidelines . For histological examination 6-m frozen sections were stained, and for facs analysis erythrocyte - depleted spleen cells were used . Macrophages were detected using moma-1, marco abs from serotec, and er - tr9 from accurate chemical & scientific corp . Abs to cd1d, b220, cd19, cd21/cd35 (cri / ii), cd23, mac-1, anti rat alkaline phosphatase, and anti secondary abs for immunohistochemistry, anti - biotin, anti - fitc f(ab) horseradish peroxidase, or alkaline phosphatase were from dakocytomation and rabbit anti vector blue alkaline phosphatase substrate from vector laboratories and dab peroxidase substrate from sigma - aldrich were used for development of immunohistochemistry stains . Soluble marco receptor was provided by t. pikkarainen (the karolinska institute, stockholm, sweden; reference 20) and was biotinylated using the ez - link kit from pierce chemical co. the biotinylated soluble marco was detected using streptavidin - cychrome from bd biosciences . S. aureus fluorescent bioparticles were purchased from molecular probes, inc . And macs anti - fitc and anti - biotin beads were from miltenyi biotec . Floxed ship mice were created by insertion of loxp sites flanking the 10th and 11th exons (see fig . The targeting vector was introduced into embryonic stem (es) cells by electroporation and clones were selected with neomycin and ganciclovir and verified by southern blot and pcr . Clones were subsequently selected for a conditional floxed allele (ship) or null allele (ship) using southern blot and pcr . These mice were subsequently crossed with mice expressing cre in the myeloid compartment (lysmcre; reference 19) to generate cre / null / flox mice . Mice were screened for respective genotype by pcr and ship protein expression using western blot (21) on equal numbers of spleen cells purified by macs (miltenyi biotec) sorting according to protocol from the manufacturer . Relative expression of ship in macrophage and b cell populations (comparing wt / null with flox / null / cre) were estimated using alpha imager software from alpha innotech corp . Mice deficient in the inhibitory signaling molecule ship display pleiotropic defects in macrophages, nk cells, and lymphocytes (18, 22). A prominent feature of these mice is their splenomegaly resulting from dysregulation of myeloid proliferation . As seen in fig . 1, ship - deficient mice also display a specific defect in the organization of the splenic follicle with the loss of mzbs measured as the cd21/cd23 population in facs and in sections as the b220 cells localizing peripherally to the moma-1 cells (fig . The marco mzmo cells are no longer organized within the marginal zone and adjacent to the moma-1 macrophages but are redistributed to the red pulp, whereas moma-1 metallophils remain unaffected (fig . Because ship is expressed in most hematopoietic cells, including lymphoid and myeloid subsets, we determined if this marginal zone phenotype in ship - deficient mice was the result of primary macrophage dysregulation . A conditional disruption of ship was generated in which macrophages displayed an approximate> 90% reduction in ship expression whereas b cell expression was reduced by <10% (fig . 2, a and b). This is consistent with the expression patterns of cre recombinase, driven by the lysosyme promoter used (19). 2 b), similar to that of complete ship deletion, thus implicating a primary macrophage defect as the cause for splenomegaly in ship mice (18). In addition, the mice displayed essentially the same marginal zone phenotype with significantly reduced mzbs as defined by flow cytometry and reorganization of the mzmos as observed by histological staining (fig . 2 c). To confirm that the ship phenotype is b cell nonautonomous and that ship - deficient b cells can give rise to mzb populations when wt mzmos are available, we produced bm chimeras using ship - deficient bm combined with wt bm and injected these cells into irradiated wt recipients . In the resulting chimeric mice the ship - deficient and wt bms contributed equally to the mzb population (unpublished data). (a) facs profiles of single cell suspensions from the spleen of ship - heterozygous (ship) and -deficient (ship) mice . The numbers shown represent percent of cd19 cells for the depicted gates as an average of five mice . At least four serial sections from each mouse were stained for moma-1 (blue, top) metallophilic macrophages or marco mzmos (blue, bottom). Sections were also stained for b220 (brown) to show the positioning of the follicle . 10 . Conditional targeting of ship in macrophages results in mzmo displacement and reduced numbers of mzbs . (a) a targeting construct covering exons 10 to 13 of ship, from ecori (e) to hindiii (h), was made . Boxes represent exons and triangles represent loxp sites flanking exons 10 to 11 and a neomycin resistance gene (neo). Properly integrated es cell clones were transiently transfected with cre recombinase to create conditional floxed (ship) or null (ship) clones . These cells were subsequently used to create floxed (flox) and null mice, which were crossed to mice expressing cre from a macrophage - specific lysosomal promoter (cre). (b) western blot analysis of mac1 and cd19 spleen cells (spc) from wt, wt / null, null / null, lysm floxed (flox / null / cre), and relative spleen size of 6-wk - old wt / null and flox / null / cre ship mice . (c) facs and histological profiles of single cell suspensions from the spleen of the conditionally targeted ship ko mice . The numbers shown represent percent of cd19 cells for the depicted gates as an average of five mice and the numbers for the follicular b cells are shown for comparison . For representative immunohistochemical analysis, at least four serial sections were stained for moma-1 (blue, top) metallophilic macrophages or marco mzmos (blue, bottom). Sections were also stained for b220 (brown) to show the positioning of the follicle . It has been shown that ship functions as a negative regulator of cellular activation by regulating the association of the positive signaling kinase btk with the membrane, thus raising the threshold required for stimulation (23). It does so by hydrolyzing pip3, the substrate for btk association with the membrane, thereby reducing the ability of btk to become membrane associated and activated (24). Because both ship and btk are expressed in macrophages and a link between these molecules had been suggested, we reasoned that the myeloid proliferation and mzmo phenotype leading to the loss of mzbs might be the result of inappropriate activation of btk in macrophages of ship - deficient animals (25, 26). Disruption of btk in macrophages may thus be sufficient to restore normal signaling thresholds in ship - deficient mice . Combining the ship deficiency with a btk deficiency resulted in the restoration of both the normal marginal zone structure (fig . 3 a) and spleen size (fig . 3 b) indicating that btk is an important target of ship in myeloid cells in vivo . Similarly, btk deficiency counteracted the over responsiveness of myeloid progenitors to gm - csf and m - csf in ship - deficient mice (unpublished data). Both the dysregulation of myeloid proliferation and follicular architecture likely result from enhanced signaling through the btk pathway in myeloid cells . Reversion of the mzb and myeloid phenotypes in ship mice by deletion of btk suggests that btk is the dominant tec family member regulated by ship in these cells . The observation that other members of the family are expressed in macrophages and have been shown to be able to substitute for btk both in vivo and in ko mice indicates a surprising degree of specificity to the ship inhibitory pathway (2729). (a) facs and histological profiles of single cell suspensions from the spleen of ship and btk double ko mice (ship / btk). The numbers shown represent percent of cd19 cells for the depicted gates as an average of four mice and the numbers for the follicular b cells are shown for comparison . For representative immunohistochemical analysis, at least four serial sections from were stained for moma-1 (blue, top) metallophilic macrophages or marco mzmos (blue, bottom). Sections were also stained for b220 (brown) to show the positioning of the follicle . 10 . (b) relative spleen size of 5-wk - old heterozygous ko or double ko mice . These results suggested that mzmos might be critical to the organization of the white pulp nodule and localization of mzbs in this structure . To test this directly we exploited the observation that mzmos can be ablated by their preferential ingestion of macrophage - depleting liposomes (30). At a low concentration of these liposomes we could see preferential depletion of marco mzmos as opposed to the adjacent moma-1 macrophages (fig . Other phagocytic cells in the spleen, such as red pulp macrophages and dendritic cells were largely unaffected by this treatment (not depicted). When mzmos were depleted in this fashion, we observe a specific reduction in the mzbs by both flow cytometry and histological staining . In contrast, moma-1 macrophages are specifically absent in the csf-1deficient strain op / op but these mice retain marco / er - tr9 mzmos (31, 32). The absence of the moma-1 cells and the er - tr9 marker did not result in reduction in mzbs, but rather, an expansion of these cells is observed, indicating that the macrophage population that is required for mzb retention are the marco mzmos . Representative immunohistochemical analysis and facs profiles of spleens from at least four wt mice treated with liposomes or untreated op / op mice . Wt mice were injected i.v . With 100 l pbs containing liposomes or with liposomes containing clodronate at a 1:24 dilution where mzmos were preferentially depleted . 48 h later serial spleen sections were stained for moma-1 (blue, top) metallophilic macrophages or marco (blue, middle) mzmos . The sections were also stained for b220 (brown) to see the positioning of these populations in relation to the b cell follicle . 10 . Spleen cells were analyzed by facs analysis for detection of mzbs as measured by the cd19, cri, and cd23 population . The identity of the retention signal expressed by marco mzmo cells was next determined by investigating the role of specific surface receptors on the mzmo in maintaining the marginal zone structure . The marco receptor, in addition to binding to bacteria (33), contains an srcr domain that has been implicated in binding to cd19 lymphocytes (34, 35). To determine if marco itself is capable of binding to mzbs, we expressed the extracellular domains of marco as a soluble molecule (20) and used it to stain splenic populations (fig . Three populations of cells were distinguished by flow cytometry when stained with cd21 and cd23 . Maximal binding to soluble marco was observed for the mzbs (cd21 cd23), whereas the follicular b cells (cd21 cd23) displayed reduced binding . None of the other splenic populations (t cells, macrophages, or dendritic cells) were capable of binding to soluble marco . This binding was specific for the marco srcr domain, as determined by the ability of a monoclonal ab to this domain (ed31; reference 33) to block the binding of soluble marco to mzbs . When the marco - specific ab was injected i.v . To wt mice it resulted in disruption of the marginal zone structure in which mzbs, identified by cd1d staining, were found in the follicular region whereas mzmos, identified by er - tr9 staining, were retained in the marginal zone (fig . These results suggest that a direct interaction between mzmo and mzbs is mediated by marco mzb binding, through a marco ligand expressed on these b cells, and provides a mechanism for the retention of mzbs by marco - expressing mzmo cells . Perturbation of this interaction either by disruption of adhesion and/or induction of macrophage activation by marco cross - linking results in the appearance of cells expressing a mzb surface phenotype in the follicular zone . Representative facs analysis of spleen cells from wt mice stained with cri, cd23, and biotinylated smarco . Binding of smarco to different spleen cell populations was based on gates set on the cri versus cd23 stain . Red, mzbs; blue, follicular b cells; black, non - b cells . The histogram (bottom) shows the mean fluorescence index (mfi) and sd (n = 5) for the different populations as well as the avidin (av) control and block using the marco - specific ed31 ab . Data shown are representative of three independent experiments . In vivo disruption of marco and mzb interactions wt mice were given 100 g control rat igg or anti - marco (ed31) igg i.v . 3 h later the mice were killed and the spleens were stained for macrophage and b cell populations . Mzmos were detected with anti - marco (blue, top) or er - tr9 (blue, middle) antibodies whereas metallophilic macrophages were stained with moma-1 (brown, bottom). B220 b cells (brown) were stained for positioning of the follicle and mzbs as the cd1 (blue, bottom) population . 10 . Part of the spleen was used for flow cytometric analysis to determine the cd19, cri, and cd23 populations . The percent of cd19 cells for either mzbs or follicular b cells is shown for comparison . Data shown are representative of two independent experiments . To address the relevance of the marco mzmo and its retention of mzbs to its contribution to the development of an immune response to pathogens, we injected mice i.v . With rhodamine - conjugated s. aureus, which is a known ligand for the marco receptor (12). Within 30 min of injection bacteria were visualized exclusively bound to the mzmo cells, a role consistent with the phagocytic property of these scavenger receptor expressing cells (fig . 18 h after injection the microbes and the mzmo were found to have comigrated into the red pulp and cells with a mzb phenotype (cd1d) were mostly found in the follicular region . These results are consistent with a model in which interaction of s. aureus with marco on mzmos results in their migration into the red pulp and the concomitant migration of mzbs into the follicular region as has been reported for lps and e. coli (8, 9). The deletion of the inhibitory signaling molecule ship results in a similar mzmo migration response, suggesting that mzmo activation can trigger migration into the red pulp . We presume that the likely explanation for the migration seen in response to s. aureus ingestion is the activation of mzmos by their encounter with these bacteria as has been described (36, 37). A similar result was observed for e. coli suggesting a more general migratory response by mzmo cells to microbial challenge (unpublished data). The migratory response of the mzmo, carrying ag to the red pulp, could simply be a method of clearance of particulate ags or alternatively mzmos could function as ag transporters / presenters and supporters of plasmablast formation shown to take place in the red pulp (fig . This has previously been reported to be a function of dendritic cells in the t / b cell border of the follicle and by macrophages supporting b1 b cells in the peritoneum (10). Interestingly, kang et al . (14) recently showed that phagosomes in mzmos, after uptake of dextran polysaccarides via sign - ri did not stain positive for the endosomal markers lamp-1 and transferrin . This suggests that ags taken up by mzmos may not necessarily take the route of normal phagosome maturation (41) resulting in destruction or ag presentation and thus could provide a mechanism to transport intact ag to the red pulp by mzmos . Wt mice were injected i.v . With 250 g heat - killed and rhodamine - conjugated s. aureus in pbs . 0.5 or 18 h later the mice were killed and the spleens were sectioned and stained . Marco mzmos (left) are stained blue and b220 b cells are stained brown . The middle shows the same stains as in the left, merged with the fluorescent stain of s. aureus . The right shows stains for the cd1 mzb population (blue) and moma-1 metallophilic macrophages (brown). 10 . The data shown are representative of two independent experiments . Proposed model for interactions between mzmo and mzb and the response of these cells to blood - borne pathogens . In the marginal zone (mz), mzbs interact with the mzmo via the marco receptor (a) and with stromal elements via the icam / vcam and their respective ligands lfa-1 and 41 (b). Upon phagocytosis of particulate ags, the marco mzmos migrate to the red pulp (c) and the majority of the mzbs migrate to the follicle where they interact with cells such as dendritic and follicular dendritic (d, dc and fdc). In the early response to t cell independent ags, the mzb also has the capacity to migrate to the red pulp to take part in plasma cell formation (e), where a possible interaction with mzmos and mzbs may take place . These results suggest that the interaction of mzmo cells with mzbs is required to maintain the marginal zone structure and that this association is perturbed upon mzmo binding and activation by microbial pathogens . It is likely that the mzbs migrate into the follicular zone in response to cxcl13 (9) in the absence of retention signals from the marco mzmo . This pathway is likely to be independent of the integrin pathway involving stromal vcam / icam and b cell lfa-1/41 because disruption of that pathway with antibodies to lfa-1 and 41 results in the release of mzbs to the blood stream (9), not their migration into the follicle, in contrast to the results presented here (fig . In addition, we see no effect on the localization of mzmo cells using antibodies to the stromal integrins, nor do we observe effects on their ligand expression when mzmo cells are triggered to migrate (unpublished data). These pathways are thus likely to serve different functions in the organization of the marginal zone, with the mzmo pathway specific for the antimicrobial response, leading to internalization of the organism and trafficking of b cells into the follicular zone to propagate the immune responses . Mzbs have the capacity to bind polysaccharide ags through complement - mediated pathways and transport these to the follicular area of the spleen (6, 8, 42). The events we have described appear to be another mechanism for delivery of mzbs and ag to the t cell rich follicular region . Mzbs have mostly been implicated in the response to t cell independent ags, however, they are also capable of presenting ags (43) and may thus be important both for the t cell dependent and independent phase of the earliest defense against a pathogen.
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Pulmonary artery aneurysms and pseudoaneurysms are uncommon; most are caused by infections, like tuberculosis, vasculitis (behet's syndrome) or trauma, often iatrogenic; less common causes include pulmonary hypertension, congenital heart disease, lung carcinoma, and connective tissue disease (1 - 3). Because of potential risk of high mortality, secondary to pseudoaneurysm enlargement and rupture, prompt we, hereby, describe a case of ascending branch of the left pulmonary artery pseudoaneurysm, following a left upper lobectomy, for which a transcatheter endovascular embolisation of the aneurysm with intra - aneurysmal n - butyl 2-cyanoacrylate (glue) injection was performed . A 30-year - lady had been presented to us 3 months ago with multiple episodes of hemoptysis and one major episode of 400 cc, and gave us a history of intermittent fever, cough, breathlessness and chest pain for a period of 2 - 3 months . She was a known case of pulmonary tuberculosis with left upper lobe fungal ball (pulmonary aspergillosis) for the past 7 years . She had had complete a course of anti - tuberculosis therapy and was asymptomatic until this period when she was presented to us with hemoptysis . At this time the patient visited an emergency department again a month ago for exacerbation of the above symptoms . Because of the recurrent symptoms and deteriorating pulmonary function test results, the left upper lobectomy for pulmonary aspergilloma was performed . The inferior pulmonary ligament was excised in order to enable the lower lobe to occupy the entire left thoracic cavity . The early postoperative period was uneventful . In the third post - operative week, she started having streaky hemoptysis . Fibre optic bronchoscopy detected a normal tracheobronchial tree, however, the left upper lobe surgical closure site was congested and inflamed . Though the white blood cell count was normal, the patient was treated for a presumed chest infection . The patient had spotty hemoptysis for the next two weeks, until she developed 2 - 3 episodes of massive hemoptysis of about 400 - 500 cc of blood . Patient was intubated and emergency computed tomography (ct) pulmonary angiography was done, which revealed a large pseudoaneurysm of the ascending branch of the left pulmonary artery (fig . Because of the patient's severe pulmonary insufficiency, she was not a surgical candidate and was referred to us for endovascular embolisation . Under local anesthesia, with anesthetists standing by for emergency resuscitation if need arise, pulmonary angiography with 5 fr, 110-cm pigtail catheter (cordis, johnson & johnson, new jersey) was introduced via the right femoral venous approach, which showed approximately 3.5 cm aneurysm arising from the ascending branch of the left pulmonary artery, shortly after its origin (fig . Then, a 5 fr, 100-cm head hunter catheter (cordis, johnson & johnson, new jersey) was introduced into the feeding artery of the aneurysm, just short of the aneurysm sac . Initially, we tried to occlude the feeding vessel with 35 - 5 - 5 and 35 - 8 - 8 coils (cook, bloomington, in, usa), but both of these coils got misplaced proximally in the lateral basal and lower lobe trunk arteries, respectively . Because of larger diameter of the coils than the caliber of these arteries, the misplaced coils got stuck in a proximal part of the arteries . On an angiogram, we could see the uninterrupted rapid blood flow through the coils to the lower branches . Further, with digital road mapping, 5 fr head hunter catheter in the feeding artery, and acting as a guiding catheter, the aneurysm sac was catheterized with a 2.7 fr microcatheter (terumo progreat; terumo deutschland gmbh, germary). Now we decided to occlude the aneurismal sac by completely filling the sac with n - butyl 2-cyanoacrylate (glue) injection . We calculated the volume of the glue mixture required in order to fill the aneurysm cavity by slowly calculating the volume of contrast injected, without causing the reflux of the contrast into the normal pulmonary circulation proximally . The volume required was approximately 4 cc . To prevent a reflux, 5 cc of higher concentration i.e. 75% glue mixture of histoacryl (braun, melsungen, germany) and iodized oil (lipiodol, andre guerbert, aulnay sous bois, france) was taken in a 5-ml luer - lock syringe, however we could only inject approximately 1.5 cc of the glue, as the microcatheter got blocked . Catheter sticking or breakage did not occur during the process of catheter retrieval and was immediately flushed vigorously with 5% dextrose to reopen the lumen . Check angiogram showed the glue cast in the central part of the aneurysm sac with aneurysm still filling in the periphery (fig . We now attempted to occlude the feeding artery with coil, but this time, the coil got dislodged in the aneurysmal sac . Now, the angiographic picture was much clearer, and on correlating with ct saggital reconstructed images, we got to know that the length of the feeding artery to the pulmonary artery aneurysm (pap) sac was very short . So now, not to compromise the pulmonary circulation, we did not have any other option except to fill the sac either with coils or liquid materials . In order to be economical on the cost, as well as our past experience with the glue, we decided to occlude pap with intra - aneurysmal injection of the glue . We introduced the previously used microcatheter into the sac, and this time, we used 50% glue mixture and were able to fill the sac completely with calculated volume of 3 cc of liquid mixture without spillage into the proximal normal circulation . Post embolisation check angiogram showed a complete obliteration of the aneurysm sac with anterior ascending branch, and other branches of the left pulmonary artery being patent (fig . There was no alteration in the blood flow through the coils into the lower lobe branches, and therefore, we did not try to retrieve the coils . Ct performed on the 6th day showed glue cast with no filling of the aneurysm . However, after being asymptomatic for 2 months, she presented again to our hospital with a single episode of 300 cc of hemoptysis . Emergency ct was performed, which did not show the new aneurysm or refilling of the previously embolised aneurysm (fig . 1 g); however, at this time, we could see a new aspergilloma in lateral basal segment of the lower lobe, and rib osteomyelitis (fig . At present, she is still admitted and is on a conservative management and being further investigated for these new ct findings . Pulmonary artery aneurysms and pseudoaneurysms (paps) are rare; most are caused by infections, like tuberculosis, vasculitis (behet's syndrome) or trauma, often iatrogenic, especially after swan ganz catheter insertion; and less common causes include pulmonary hypertension, congenital heart disease, lung carcinoma, and connective tissue disease (1 - 3). Thus, any destructive process of the lung, whatever its pathogenesis, can erode the vessels in its vicinity, be it a pulmonary or systemic vessel . The etiology in this case can be attributed to inadvertent insult to the pulmonary artery during surgery for pulmonary aspergilloma . Also, after lobectomy, significant increase in the pulmonary vascular resistance index has been described in the literature (4), and this could have had a possible contributory role for the development of the pseudoaneurysm . Another cause could be a low grade infection of the already weakened upper lobe pulmonary arterial wall, though the blood investigations were normal in our patient . The most common cause for intrapulmonary bleedings is the hemoptysis, due to bronchial artery erosion seen in as many as 95% of all cases . In contrast, bleeding from the pulmonary arteries is very rare, usually massive, accounting for less than 5% of all cases (5), and is usually due to pseudoaneurysm rupture . Because of the risk of pap enlargement and rupture, which leads to death in approximately 50% of patients, prompt therapy is required . The available treatment modalities for pap are medical therapy, surgery and percutaneous catheter embolisation of the pseudoaneurysm . Medical treatment by the means of immunosuppressive drugs and steroids has been found to cure or decrease the size of aneurysms in behcets disease (6, 7). Several surgical techniques, such as lobectomy, pneumonectomy, hilar clamping with direct arterial repair and ligation, have been used . However, in our case, due to the patient's severe pulmonary insufficiency, she was not a surgical candidate . There are number of reports in the literature addressing the treatment of paps - most of them using metallic coils (8) or silicon balloon (9) to occlude the arterial feeders . These placements can also occlude the perfusion to aerated lung, distal to the embolisation site, and are associated with complications, like coil migration and damage to vessel wall . Other authors have occluded the pap by intraneurysmal placement of coils (10); however it carries a potential risk of mass effect and aneurysm rupture . Still, other authors have described the use of covered stents (11), and recently, hovis and zeni (12) have used thrombin percutaneously for the paa refractory to coil the embolisation . In our case, initially, we attempted unsuccessfully to occlude the feeding artery with the coils . However, the coils migrated proximally to enter into the lower lobe circulation without compromising the blood circulation to these segments . We later realized that this was due to a short length and wide neck of the feeding artery, that we were not able to keep the catheter stable in this branch . We did not want to occlude the aneurysmal sac with coils, firstly because of the large size of the lesion, use of multiple coils could result in a mass effect after embolisation, and secondly it would have been very expensive; also it might be further complicated by coil migration, resulting from the wide neck of the aneurysm . In our case, because of regular and comfortable experience at tackling abdominal visceral aneurysms with glue embolization, we decided to use this liquid material as embolic material . (7) embolised the feeding branch to paa, using the glue with a " bubble technique "; however, this was not possible in our case because of the short length in the feeding artery . Therefore, we considered the alternative option of intraaneurysmal injection of liquid embolic (glue). Glue (n - butyl 2-cyanoacrylate) is a rapidly hardening liquid adhesive, and has been used as an effective embolic agent for brain vascular malformations; however, necessity of operator experience with the use of liquid embolic material is a major limitation in its application to aneurysms . There are some studies showing the feasibility of intraaneurysmal injection of the glue in intracranial aneurysms (13), thereby keeping the antegrade flow in the aneurysm bearing artery patent . We report the first case, in which the n - butyl 2-cyanoacrylate was successfully injected, intraaneurysmally, through the transcatheter route for the occlusion of a pulmonary artery pseudoaneurysm, after an upper lobe lobectomy for aspergillosis . Glue offers the advantage of permanent occlusion of the vessel, and because of its low viscosity, it can be injected through a microcatheter into small and tortuous arteries . It is admixed with ethiodized oil in various ratios to achieve radiopacity and to adjust the polymerization time allowing for more controlled embolisation . The use of higher concentrations of glue results in quicker solidification; however, on the contrary, the longer the polymerization time, the greater the risk of non - target embolisation because there is the possibility of embolic material being washed away before it solidifies . In our case we initially used 75% glue to prevent the reflux into the parent artery; however there was early polymerisation of the glue with the sac preventing an evenly distribution . Therefore, we had to dilute the glue to make it 50% concentration, and this time, there was complete filling of the aneurysm . In conclusion intraaneurysmal injection of the liquid embolic materials is feasible, safe, and effective trancatheter treatment option for pulmonary artery aneurysms and is possibly not associated with the risk of rupture seen with packing of aneurysms with coils . However, appropriate concentration of the glue, long term results, and etc . Will require further experience to confirm its safety and efficacy in pulmonary circulation.
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This phenomenon is common on soils which have developed on naturally enriched rock substrates . Another source of soil pollution can be attributed to human activities, including mining, metal and ore processing, and industrial and agricultural activities (padmavathiamma and li, 2007). During recent years several plant species have been identified that extract, tolerate and/or hyperaccumulate pollutants from soils in their above - ground tissues (shah and nongkynrih, 2007; verbruggen et al . The accumulation of metals and the final concentration in tissues often exceed one hundred - times the concentration of common plant species (maestri et al ., 2010). Major disadvantages of these species (e.g. Thlaspi caerulescens, arabidopsis halleri) are their slow growth, low biomass production and short life cycle . Therefore, researchers over the world are interested in how to make the process of phytoextraction more effective (zhao and mcgrath, 2009). One of the possible solutions is the utilization of fast growing woody plant species with high biomass production and high genetic variability (pulford and watson, 2003). Several studies about different woody species have been published in recent years dealing with their uptake, translocation and accumulation capacity, as well as tolerance to various metals . Trees from the salicaceae family, especially the genera salix and populus, might be suitable candidates for these purposes (e.g. Greger and landberg, 1999; sebastiani et al ., 2004; vyslouilov et al ., 2006; dos santos utmazian and wenzel, 2007;, 2007; wieshammer et al ., 2007; jensen et al ., 2009; vollenweider et al .,, salix caprea represents a suitable candidate for remediation because of fast colonization of mineral substrates, high biomass production and landscape restoration . Variability in the uptake capacity of several elements has been reported between populations or clones of the same species, sometimes originating from different environmental conditions . Metallicolous and non - metallicolous populations of the heavy metal tolerant plant t. caerulescens have shown different responses to soil cd and zn contamination (escarr et al ., 2000; lombi et al ., 2000; jimnez - ambriz et al ., 2007 (2009) compared characteristics important for phytoextraction such as metal tolerance, translocation and accumulation of cd in poplar hybrids (populus x canadensis, p. x deltoides, p. x generosa, p. x nigra, p. x alba, p. x trichocarpa) and willow (s. alba) clones . A similar study was published by dos santos utmazian and wenzel (2007) on six different willows (s. babylonica, s. caprea, s. dasyclados, s. matsudana x alba, s. purpurea, s. smithiana) and two poplar species (populus tremula, p. nigra) and discovered that s. smithiana leaves accumulated the highest cd concentration . Differences in the tolerance and accumulation capacity of cd, cu and zn between clones of five salix species (s. viminalis, s. dasyclados, s. daphnoides, s. purpurea and s. triandra) grown in metal polluted and unpolluted areas were compared by landberg and greger (1996). Clones from polluted areas accumulated more metals in roots while the transport to shoots was decreased when compared with clones from unpolluted areas . However, these authors found no difference in the concentration of metals in stems between these two groups of salix species . (2010) analyzed 170 s. caprea isolates of four metal polluted and three uncontaminated middle european sites to reveal potential selective effects of long - term heavy metal contaminations on the genetic structure and zn / cd accumulation capacity . They found differentiation of metallicolous and non - metallicolous s. caprea populations based on phenotypic characteristics and nuclear microsatellite (ssr) markers (puschenreiter et al ., 2010). The root is the first plant part to encounter soil heavy metal pollution; therefore the molecular uptake mechanisms at the root soil interface are the subject of intensive study in hyperaccumulating species (lombi et al ., 2000; concerning the uptake and translocation of heavy metals, there is very limited knowledge about their relation to root tissue organization and development in the woody plants used for phytoremediation . (2004) described differences in the anatomy and apoplastic barrier development of adventitious roots of willows (s. viminalis, s. viminalis x schwerinii, s. daphnoides) with contrasting characteristics of cd accumulation and cd sensitivity . They show that both the casparian strips and suberin lamellae developed more distantly from root tips in high cd than in low accumulating and translocating cuttings . However, the influence of cd and/or zn on the development of root tissues of genetically distinct s. caprea plants with different zn and cd translocation capabilities has not been evaluated (puschenreiter et al ., 2010). Therefore the present study focuses on anatomical differences and metal accumulation of root tissues of cuttings from s. caprea plants originating from polluted and uncontaminated areas . The development of apoplastic barriers affecting radial transport of elements across the root (casparian strips and suberin lamellae), changes in tissue proportions, and metal localization within root tissues were compared . The aim of this work was to get a better understanding of the involvement of root tissues in the uptake, translocation and accumulation of cd and zn in a woody species promising for phytoextraction, s. caprea . All experiments were performed with two genetically distinct s. caprea isolates which differed in the ability to tolerate and accumulate zn and cd in above - ground tissues in perlite cultures . The term isolate refers to an individual plant collected in a specific area as part of a group of 2025 individuals representing the population at this site (puschenreiter et al ., 2010). Isolate kh21 originated from an old mining area (kutn hora, czech republic) isolate f20 originated from a non - polluted control area (forchtenstein, austria); it accumulated less zn and cd than kh21 but had a higher biomass production in perlite - based hydroponics (puschenreiter et al ., 2010). All cuttings of both s. caprea isolates used in our experiments were obtained from a stock cutting cultivated in non - polluted control soil at the ages (vienna, austria). Green cuttings of approx . 10 cm length were rooted in a mixture of perlite and coarse sand (20:80) in a greenhouse (ages and boku, vienna, austria). Rooting was initiated in tap water for 60 days at 24/18 c day / night, a 12 h photoperiod, 60% relative humidity and 200 m m s par . For the effect of zn and cd exposure on root anatomy and the distribution of minerals in root tissues, each pre - rooted cutting with new shoot of 1520 cm length and 2 5 adventitious roots of an average total length of 250 mm the plants were watered twice a week with a nutrient solution containing (in m) 1000 ca(no3)2, 500 mg(so4)2, 50 kh2po4, 100 kcl, 5 h3bo3, 0.2 h24mo7n6o24, 10 mnso4, 2.5 cuso4, 0.25 niso4, 2.5 znso4 and 50 fe (iii)-eddha (ethylenediamine - di(o - hydroxyphenylacetic acid)). Solution ph was adjusted to 6.0 with 1 mm mes as potassium salt (shen et al ., 1997). Four different treatments were used: 1) standard nutrient solution, 2) nutrient solution with 0.5 mg cd / l in the form of cdno34 h2o (cd), 3) nutrient solution with 5 mg / l zn in the form of znso47 h2o (zn), and 4) nutrient solution with 0.5 mg cd / l and 5 mg / l zn (cd + zn). Plants were cultivated for 14 weeks . For the analysis of the development of casparian strips and suberin lamellae, rooted cuttings were transferred to 0.5 l pots and watered twice a week for three months with the solutions described above . Plants for apoplastic barrier analysis were cultivated for 6 weeks . For the analysis of zn and cd concentration in below- and above - ground tissues, plant material was divided into roots, shoots and leaves and washed with distilled water before drying at 80 c . After recording the dw, samples were ground in a metal - free mill (ika werke, mf 10) and digested in a mixture of hno3 (puriss . (4:1, v / v) using an automated heating block (digester dk 42/26, velp scientifica, milano, italy). Zinc and cd concentrations were measured with a flame atomic absorption spectrometer (aas, perkin elmer 2100). For quality assurance, six biological replicate samples, blanks and standardized reference materials were included in all analyses . To determine if anatomical changes of the main root tissues correlate with the different origin and metal translocation behavior of the two s. caprea isolates, the areas of different tissues were quantified on transverse roots sections at a distance of 1.21.5 cm from the apex . Root cells at this distance are already developed and well differentiated and this part of the root mostly contributes to the uptake of elements . Approximately 2 m thick semi - thin sections were prepared on microtome microm hm36 and stained with toluidine blue and basic fuchsine according to lux (1981). Sections from eight different roots were analyzed with a zeiss axioskop 2 + microscope (zeiss, germany), equipped with olympus dp72 camera . The tissue areas (total root area, area of the central cylinder, area of xylem elements) were analyzed with the image analysis software lucia g 4.80 (lim, czech republic). To determine the developmental point where casparian strips and suberin lamellae start to differentiate in roots, and if the developmental program of casparian strips and suberin lamellae differentiation varies between isolates from an unpolluted (f20) and a heavy metal polluted site (kh21), six different roots from each isolate and each treatment were stained with fluorescent dyes for casparian strips and suberin lamellae visualization . Free hand sections were made at regular distances from the apex to the base of the root (in 1 mm distance for the first 10 mm from the root apex and later in 1 cm distance for the rest of the root). Casparian strips were visualized by staining with 0.2% berberine hemisulphate and post - staining with 0.1% toluidine blue . Suberin lamellae were stained with 0.2% fluorol yellow 088 according to brundrett et al . All sections were observed with a zeiss axioskop 2 + epifluorescence microscope (zeiss, germany). Approximately 2 mm long root segments were cut from the basal part of roots and immediately frozen in liquid nitrogen according to markhart and luchli (1982) and mccully et al . Dried segments were coated with carbon and fixed on aluminum stubs covered with a carbon sticker . Surface conductivity was increased by coating with carbon; sputter coating was stopped as soon as the carbon film showed a golden to bluish color, resulting in a uniform thickness of the coating (approx . 60 nm). Cross sections of root tissues and the distribution of eight elements (zn, cd, ca, k, s, si, mg, na) were observed and analyzed with the phillips xl 20 scanning electron microscope equipped with an edx analyzer . All samples were observed at 1.200 magnification at 30 kv, working distance 12.0 mm . Data were collected for 100 live seconds and areas of 50 50 m were analyzed . As uneven surfaces may heavily distort edx measurements (goldstein, 2003), only flat parts of the samples were selected for analysis . The element distribution was analyzed from three different roots of each treatment . In total, 24 different roots were investigated by measuring thirty different spots on each root . Raw data were processed with the genesis spectrum 5.11 (edax, usa) and all the values are expressed as weight% of total analyzed elements . Zinc and cd were selected because of the main interest of this paper and the other investigated elements share chemical and physical similarities with zn and cd or are important macro- and micronutrients and beneficial elements . Significances according to student's t - tests and pearson correlations were calculated using the statgraphics (statgraphics centurion xv v. 15.2.05, statpoint, inc .) And excel (microsoft office 2003) programs . The principal component analysis was performed in simca - p 11 (umetrics, ume, sweden) on log - transformed weight% data of all analyzed elements (zn, cd, ca, k, s, si, mg, na). Loadings plots are based on a correlation matrix, scores scatter plots on a covariance matrix . Ellipses in scores scatter plots represent the 95% confidence interval according to hotelling's t distribution . All experiments have been performed on six biological replicates / clones with an adequate number of repetitions (see above) for statistical analysis . This was observed in zn and also in the combined cd + zn treatment (fig . Similarly, the zn concentration was not different in leaves and roots of both isolates when grown in control conditions or excess cd (fig . However, significantly more cd was detected in leaves of isolate kh21 from the polluted site compared to isolate f20 from the control site . The same tendency was observed when plants were treated with both metals (cd + zn) together, but the difference was not significant (fig . 2b, d) start to differentiate, the exo- and endodermis of roots were stained with fluorescent dyes . In the roots of both a striking difference between isolates f20 and kh21 could be detected in the casparian strips development already in control conditions (fig . 2e, f; table s1); in f20, the casparian strips differentiated first in the endodermis and later in the exodermis . In contrast, the casparian strip development in kh21 followed an opposite pattern in these two tissues and differentiated first in the exodermis and later in the endodermis . Another difference was in the starting point of the casparian strip development from the root apex; in f20 the zone of exo- and endodermal casparian strip development was between 2.5 and 3.5 mm from the root apex while in kh21 this distance was shorter, between 1.0 and 2.0 mm from the root apex (fig . The formation of casparian strips in the exodermis and endodermis of roots after exposure to zn or / and cd also varied between the isolates; while the casparian strips differentiated in the exo- and endodermis closer to the apex in f20 roots exposed to zn or cd, this was not the case in kh21 . Exposure to zn or cd, as well as the combined treatment, had the same effect indicating that the development of apoplastic barriers relates to the presence of these metals . The unresponsiveness of kh21 could indicate a limit in the root apex beyond which the differentiation cannot take place . Similar to the casparian strips, the suberin lamellae developed differently in exo- and endodermis of the two isolates . For both isolates, the suberin lamellae form earlier in the exodermis than in the endodermis . However, the distance of suberin lamellae development from the root apex was different between the two isolates . While the exodermal suberin lamellae of f20 started approximately 15 mm from the apex, the distance was only 10 mm in kh21 . The endodermal suberin lamellae were detectable 30 mm from the root apex in f20 under control conditions, while in kh21 the distance was only 15 mm (fig . The exodermal suberin lamellae started at around 10 mm from the root apex in both isolates . Metal treatment affected the development of the endodermal suberin lamellae in f20 and kh21 in an opposite manner . While for f20 the onset of endodermal suberin lamellae differentiation shifted towards the root apex in roots exposed to heavy metals, in kh21 the onset of endodermal suberin lamellae development was at about 25 mm and shifted further from the apex in roots exposed to heavy metals . Interestingly, the same effects were observed in zn, cd, and the combined treatment (fig . If anatomical changes of the main root tissues correlate with the different origin and metal translocation behavior of the two s. caprea isolates, the areas of different tissues were quantified on transverse roots sections . 3a), vascular bundles (fig . 3b) and xylem elements (fig . 3c) were significantly larger in f20 than kh21 under control conditions, indicating that all tissues contribute to the thicker roots of f20 . While in f20 a significant decrease was evident for the total root area, the area of vascular bundles and the area of xylem elements after metal exposure, only the total area of kh21 roots decreased in the zn and cd + zn treatments (fig . The area of the vascular bundles remained constant while the area of xylem elements slightly increased with zn or cd treatment, but decreased if zn and cd were offered together (fig . Three different zones could be clearly distinguished: the central part of the root consists of secondary xylem (fig . The outer part of the root, here called outer bark or rhytidome (fig . 4a, b), is highly heterogeneous and includes secondary (periderm) and primary vascular bundles, the endodermis, the primary cortex and the exodermis . The distribution of zn, cd, ca, k, s, si, mg and na in this zone was analyzed by edx (fig . 4b, table s2). To determine which parameter (tissues, treatment and/or isolate) affected the variation in the edx data most, the weight% values were subjected to a principal component analysis (pca) with all elements as variables . The first principal component (pc1) (45.5% of the total variance) was interpreted as responsiveness to metal treatment . The main descriptors of pc2 are cd and si, correlating oppositely with this axis (fig . 1b) along with its generally higher si weight% compared to kh21 (fig . The third pc, contributing 11.5% of the total variance, was most influenced by zn (fig . The scores scatter plots show that tissue - specific clusters form along all three pcs (fig . Pc3 is able to separate the effects of the cd from the zn and zn / cd treatments (fig . The pc1/pc3 and pc2/pc3 plot also shows that the cd treatment largely overlaps with the xylem while the zn treatment correlates with the bark values and the cd / zn treatment with the phloem (fig . S1a f). In this way a weak separation can be visualized in the zn and cd + zn treatment of plot pc1/pc2 . Pca of all measured elements revealed a grouping in treatment-, isolate- and tissue - specific clusters . But since the pca cannot visualize the effect of the individual elements, we analyzed their weight% distribution relative to the tissues and the isolates separately (fig . 7). As already noticeable in the pca the weight% of zn was highest in the peripheral bark zone in both isolates however, an isolate - specific response was revealed since kh21 had less weight% of zn in the combined treatment while f20 had an increased zn weight% . Cd had a slightly higher weight% in the xylem tissues except in f20 upon the combined zn + cd treatment where the cd weight% was increased in peripheral bark zone . This different pattern of the cd distribution is consistent with the finding that the isolate from the unpolluted site (f20) accumulated less cd in the above - ground tissues (fig . 1). For calcium (ca), a descending trend was observed from the peripheral bark zone towards the xylem in both isolates . However, the isolates differed with a higher weight% distribution in f20 upon cd exposure and a dramatic increase in the peripheral bark zone for f20 upon the combined treatment with zn + cd . Pearson correlation analyses showed that the distribution of ca and zn is positively correlated and stronger for zn uptake in f20 than in kh21 isolate (fig . Addition of cd even increased this correlation in root tissues of f20 (fig . The zn / ca correlation is lost in the presence of cd (fig . Weight% distributions had a tendency of descending from the peripheral bark zone to the xylem in kh21 while in f20 this tendency was not detectable in zn treatment . A similar general gradient was detected for sodium (na) but the weight% levels were different between the isolates for the zn and the cd treatment (fig . 7). The weight% of potassium (k) was generally highest in the secondary phloem and was increased upon cd treatment in both isolates and in f20 upon cd + zn treatment (fig . Furthermore, a positive correlation existed between k and cd but not zn (fig . S2e, f). In both isolates the silicon (si) weight% was highest in the peripheral bark zone (fig . 7), but with a clear difference between the isolates . Upon cd or zn treatment the values of si however, this effect was lost upon exposure of cd + zn together . Sulfur (s) distribution was elevated in the phloem and most prominent in kh21 upon zn treatment . While for most treatments and in both isolates s was higher in the peripheral bark zone than in the xylem, this tendency was exaggerated in f20 upon zn + cd and shifted upon zn treatment (fig ., the analyses of the individual elements in different tissues and isolates showed that their distributions are altered upon cd, zn and cd + zn treatment in most cases in an isolate - specific manner . To better understand uptake and accumulation of elements into shoots, knowledge of root anatomy and physiology is essential . Elements are transported radially from the rhizodermis through the apoplast or symplast across the cortex to the xylem and the shoot . Uptake is controlled by apoplastic barriers in the endo- and exodermis (white, 2001; ma and peterson, 2003; baxter et al ., 2009; schreiber, 2010). First, a mixture of lignin and suberin is deposited into the radial and transversal cell walls (casparian strips), which is usually followed by lamellar suberin deposition on the inner surface of exo- and endodermal cell walls (suberin lamellae) and, in some species, also by secondary thickening of the walls (von guttenberg, 1968). The development of these apoplastic barriers is variable and often differs between plant species and environmental conditions (zimmerman and steudle, 1998; seago et al ., 1999; enstone and peterson, 2005; redjala et al ., 2011). We observed that the initiation of the casparian strip and suberin lamellae formation varied between two s. caprea isolates originating from different environments . The kh21 isolate, collected from metal polluted soil and previously characterized by high cd and zn accumulation capacities (puschenreiter et al ., 2010), develops casparian strips and suberin lamellae closer to the root apex compared to the genetically distinct f20 isolate which originated from unpolluted soil and showed phenotypic differences to kh21 . Furthermore, f20 first developed casparian strips in endodermis and later in exodermis, oppositely to kh21 . Similarly, differences in the development of apoplastic barriers between isolates of s. viminalis differing in metal tolerance and accumulation were also observed by lux et al . Therefore we propose that apoplastic barrier development is an adaptive trait and can vary between different isolates of the same species . Similarly, we have observed that anatomical reactions to heavy metal exposure differed between isolates . In f20, exposure to cd, zn or cd + zn resulted in earlier development of both barriers while the development in kh21 was not influenced by metal exposure . The behavior of f20 is consistent with several studies showing that roots exposed to cd develop earlier apoplastic barriers (schreiber et al ., 1999; martinka and lux, 2004; zelko and lux, 2004; vaculk et al ., 2009). Accelerated apoplastic barrier development was also induced by higher salinity (reinhardt and rost, 1995; karahara et al ., 2004). Thus the typical reaction is to minimize the uptake of pollutants by developing the exo- and endodermis closer to the root apex . This is consistent with the finding that the concentration of cd in leaves was lower in f20 than in kh21 . On the other hand, delayed development of the endodermal suberin lamellae of kh21 upon exposure to heavy metals could be the reason of the higher accumulation capacity of cd in shoots . However, the understanding of the delay in apoplastic barriers development after metal stress in isolate kh21 originating from metal polluted soil remains still poorly understood . Changes in root tissue areas might also be the consequence of continuous exposure to metal polluted soils . (2004) showed that willow clones characterized by high tolerance to cd had a higher proportion of epi-, exo- and endodermal tissues when compared with the sensitive clones, which had a higher proportion of mid - cortex . We found that kh21 produced thinner roots with a smaller area of vascular bundles and xylem elements when compared with f20 . However, when the same isolates were exposed to elevated metal concentrations, the reaction of roots was different . In roots of f20 the total area and areas of central cylinder and xylem elements decreased in comparison to control conditions . We suppose that the differences in the root anatomy of the salix isolates treated with metals reflect an adaptive predisposition and depend on the different natural origins . Cd - induced differences in the proportions of specific root tissues (rhizodermis cortex vascular bundles) or in the size and shape of individual cells have been reviewed recently by lux et al . (2011). Described syndromes of cd toxicity on roots are a decrease of root number, length and dry mass as well as enlargement of the root diameter . (2003) reported that roots of s. alba and populus x euramericana became shorter and thicker upon cd treatment . However, the reactions of root tissues to cd mostly differ among plant species and used cd concentrations (e.g. Lux et al ., 2011). Conversely, cd, zn and cd + zn treatment in our experiments decreased the total area of the s. caprea roots in f20 . The total root area of kh21 was unaffected when cd was applied, although after zn and zn + cd application the total area of root decreased . (1992) reported that the root diameter of bean (phaseolus vulgaris) remained unaffected, although the number of cortical parenchyma cells decreased when cd was applied . (1993) did not find any changes in the root diameter, length or surface area in two different maize inbred lines exposed to cd . We expected that unresponsiveness of kh21 tissues to elevated cd concentration probably related with higher cd accumulation capacity . This pattern of root tissue development probably disappeared when plants were exposed to zn or cd + zn . (2005) found that the total area of the root, the diameter of the vascular bundles, the number of xylem strands and the area they occupied in stele were higher in coffee plants grown without zn in the medium than in plants exposed to zn . A similar decrease of tissue areas after zn application was observed in our experiments in f20 while kh21 differed only in the total root area . Balanced zn concentration and distribution is essential for optimal growth and function of metabolic processes (broadley et al ., 2007). (2009) for poplar, macfarlane and burchett (2000) for gray mangrove and brunner et al . (2008) for spruce exposed to elevated zn, in our experiments most of the zn accumulated in the peripheral tissues of the secondary thickened root . Therefore we assume that the peripheral bark zone of secondary thickened roots serves as an effective barrier for radial zn transport to vascular bundles . Freeze drying of the samples prevented leakage of elements during fixation, allowing us to detect tissue - specific distribution of elements with low abundance such as cd (fig . In contrast to zn, most of the cd was detected in the xylem in both isolates treated with cd . However, when both metals cd + zn were applied to kh21, no significant differences in the cd distribution between the peripheral zone and vascular bundles were observed . (2008) found that in poplar roots exposed to higher doses of cd this element accumulated predominantly in cells surrounding the central cylinder . Nevertheless, in the combined cd + zn treatment, double values of cd in each investigated tissue of f20 were detected when compared with kh21 . Together with the observations that the cd weight% in kh21 tissues is lower as in f20 (table s2) we assume that kh21 is more efficiently translocating cd to the shoot, especially when excess of zn is present . These differences in cd localization within root tissues probably relate with the uptake and translocation of cd and zn, and might reflect the situation on the site of kh21's origin in natural conditions, which is adapted to a soil containing up to 84 mg total cd kg and 8.6 g total zn kg (puschenreiter et al . The levels of zn and ca are positively correlated in both isolates when treated with excess zn . In the combined cd + zn treatment, this correlation is lost only in kh21 . Cadmium is among others transported into the root via ca uptake and translocation pathways (lux et al ., 2011). Kh21 accumulates more cd in the shoot than f20, therefore it is possible that higher amounts of cd are taken up via ca transport mechanisms . Cadmium competes with ca uptake, which might explain the lower ca concentration in the combined zn + cd treatment . Furthermore, a positive correlation exists between k and cd, but not zn, distribution in root tissues . It was recently found that k supplement alleviated cd toxicity in soybean (shamsi et al ., 2010), which might be also one possible tolerance mechanism in s. caprea . Silicon repressed root - to - shoot translocation of cd in brassica chinensis and increased cd tolerance (song et al ., consistently, f20, which translocates less cd into shoots, accumulates significantly more si than kh21 in all root tissues . Proposed mechanisms which reduce translocation to the shoot are si co - precipitation with cd and zn in roots or physical blockage of metal flow across cell walls (neumann and zur nieden, 2001; shi et al ., 2005). Edx analyses show that cd and zn interfere differently with the element uptake systems in s. caprea, and that for some elements this effect is isolate - specific . Variation in the organization of root tissues, development of apoplastic barriers and the distribution pattern of zn and cd and other elements in s. caprea isolates originating from polluted and unpolluted sites support the hypothesis that these features are results of environmental adaptation . Understanding the molecular basis of these adaptive traits might help to increase the tolerance and heavy metal accumulation capacities of s. caprea for phytoremediation technologies.
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The term nonconvulsive status epilepticus (ncse) is defined as a range of conditions in which electrographic seizure activity is prolonged and results in nonconvulsive clinical symptoms, . The clinical features of ncse may be subtle and diverse, and confirmation relies on the electroencephalogram (eeg). Several approaches to establishing criteria for ncse in adults have been published (table 1),,, and some clinical states may lie along an ictal interictal continuum . Periodic discharges are often regarded on the interictal end of the spectrum, but when accompanied by clinical features including impaired vigilance and when signs resolve proximate to treatment, then this lends evidence to the ictal nature of periodic discharges along this spectrum . Periodic lateralized epileptiform discharges (pleds), in particular, have sparked controversy with regard to being part of the ictal vs. interictal spectrum . Periodic lateralized epileptiform discharges can occur in the temporal vicinity of an overt seizure, thus reflecting an ictal interictal continuum phenomenon that can continue for weeks (chronic pleds) in conscious patients with retained alpha or basic rhythm . However, when they occur in a patient with altered level of consciousness, some authors regard it as ictal,,,,,,,, . Since changes in mental status are common in epileptic encephalopathies and developmental delay such as in patients with cerebral palsy, it can be difficult or impossible to determine whether they are in status epilepticus, unless there is a discernible clinical improvement with treatment . We report the case of a patient with chronic epilepsy and cerebral palsy (cp) who presented with deterioration from an already impaired baseline . Although the eeg revealed slow pleds which are not typical of ncse, the patient showed a good clinical and eeg response to benzodiazepines with pled regression . This 49-year - old man with cerebral palsy, mental retardation, and epilepsy residing in a long - term care facility for the prior 13 years had been awake, noncommunicative, but responsive to auditory and visual stimuli . He was treated with phenytoin and levetiracetam and had yearly breakthrough seizures over the prior 10 years . He had recently developed fever and dyspnea with a drop in oxygen saturation and was suspected of having aspiration pneumonia complicated by sepsis . He was given intravenous antibiotics, fluids, and supplemental oxygen and was transferred to our facility . Following a mild generalized tonic clonic seizure, his examination remained persistently unchanged but he was now unresponsive to auditory, visual, and noxious stimuli, with a fluctuating level of alertness . Plasma phenytoin level was low at 34.8 mol / l (normal: 4080 mol / l). 1) showed intrusions of excess slow left - sided periodic lateralized epileptiform discharges (pleds) on a background of normal alpha pattern . Administration of 1 mg of iv lorazepam resulted in resolution of the slow pleds (fig . 2) and improvement in the patient's neurological status, which now became responsive to auditory, noxious, and visual stimuli that occurred in a matter of 15 min after administration of lorazepam . By the next morning, his condition had greatly improved and he was near his baseline status . He received additional intravenous loading doses of phenytoin and levetiracetam on the same day, and his maintenance doses were adjusted . Brain ct showed a large central meningioma as well as a small posterior fossa meningioma (fig . Review of his old brain ct films (done seven years ago) showed no such lesions . Because of his poor quality of life and the risks involved, neurosurgery was not considered an option, and the patient was managed medically with antiepileptic medication . 4), and the patient was discharged to a long - term care facility for further care . This patient with chronic epilepsy and cerebral palsy (cp) showed deterioration from a baseline that was already impaired, a finding described in ncse in those with developmental disability, . The eeg findings were not typical of ncse in that slow pleds lie in the gray zone along the ictal interictal continuum . Cerebral palsy is a complex, heterogeneous lifelong condition with significant disability and long - term challenges that persist into adult life . Epilepsy is reported in 35% to over 60% of patients with cp, and is more frequent in patients with more severe disability . Sixteen percent of children with cp and epilepsy may develop nonconvulsive and/or convulsive status epilepticus . Brain tumors, particularly primary ones, are associated more frequently with both convulsive and nonconvulsive status epilepticus, . Electroencephalographic changes in mass lesions reflect irritability around the penumbra of the lesion, in this case, on the left . One can speculate that the focal structural lesion in the setting of chronic diffuse brain dysfunction, along with intercurrent infection and low aed levels, represented the multiple risk factors seen in the recurrence of seizures and appearance of ncse, . Periodic lateralized epileptiform discharges (fig . 1) consist of periodic spike - and - slow wave or sharp - and - slow wave complexes, typically with a frequency of 12 hz . In our patient, the frequency was ~ 0.5 hz . Periodic lateralized epileptiform discharges occur in a variety of conditions such as strokes, tumors, and infections; these together comprise around 50% of the underlying etiologies . Slow growing extraaxial tumors such as meningiomas usually produce little eeg disturbance unless they compress and displace brain tissue or cause hydrocephalus such as in our case . Periodic lateralized epileptiform discharges are reported to support a diagnosis of ncse if they meet certain conditions, in particular, good proximate clinical and eeg response to benzodiazepines with pled regression (table 1). When eeg and clinical regression are not clear, spect or pet scanning can guide the clinician in the possible ictal (versus interictal) nature of the periodic discharges, . There are diagnostic and management challenges in patients with long - term disability whose cognitive and neurological baselines are impaired and not always known to the clinical caregivers . Additionally, a diagnosis of ncse may be difficult to make when this baseline is unknown and when the eeg findings represent a pattern that lies along the ictal interictal continuum . Electroencephalogram can establish where along this continuum the patient resides, following which treatment that balances the risk benefit needs of the patient, and the needs of chronic caregivers, can then be provided and monitored so as to offer the patient the optimal chance for regaining lost function with minimal risk.
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The authors declare that there is no conflict of interests regarding the publication of this paper . This is a case report, patient written consent has been obtained and will we available on request . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Asp and kh performed the documentation of the patient and follow up care, assisted in operation and draft writing.
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Tissue valves are considered as ideal human cardiac valve substitutes because they have excellent hemodynamic properties and chronic anticoagulants are not necessary . However, the structural failure after prolonged usage prevents widespread adaption of the tissue valves, especially for young age groups (1). The reasons for the structural failure of the implanted tissue valves include repetitive mechanical wear, chronic inflammatory response by glutaraldehyde fixation, and pannus overgrowth . Recently, it has been found that the mammalian cell surface xenoantigen called -gal epitopes are still present on the commercially available, glutaraldehyde fixed tissue valves (2). Additionally it has been reported that patients who received these tissue valves exhibited increased levels of natural anti - gal antibody titers against -gal epitopes (2). Therefore, it is believed that the animal immune response may play an important role in structural damage of the commercially available, glutaraldehyde fixed tissue valves . The -gal epitopes on the mammalian cell surface trigger a strong immune response, especially on endothelial cells, resulting in a hyperacute rejection when we transplant mammalian organs to the primates (3). On the other hand, xenoantigen mediated immune response of the tissue valves were not seriously considered until now because the valvular tissue and pericardium of mammals were treated with glutaraldehyde to eradicate xenoantigenicity and potential risk of xenozoonosis before using them to make tissue valves . However, after anti - gal immune response in glutaraldehyde treated, commercially available tissue valves was recently observed, there is a heightened concern in the immune response as a cause of tissue valve structural failure . In porcine aortic valvular tissue and pericardial tissue, it became possible to stain -gal epitopes on the cell surface with griffonia simplicifolia isolectin b4 (gs - ib4) immunohistochemical stain (4 - 6). And as green coffee bean -galactosidase is known to degrade -gal epitopes at the site of gal 1 - 3gal chain (7 - 9), it is possible that it could remove -gal epitopes from those tissues . Previously we examined this possibility and reported that all -gal epitopes could be removed from porcine valvular and pericardial cell surfaces using green coffee bean -galactosidase (10). Meanwhile, there are limitations in cost effectiveness using green coffee bean -galactosidase in the commercial valve manufacturing process . We decided to investigate whether recently made, easily available recombinant human -galactosidase a has same enzymatic activity as green coffee bean -galactosidase, and whether the recombinant enzyme can effectively eradicate -gal epitopes on the cell surface of porcine aortic valve and pericardium . Additionally, we used standard indirect immunoperoxidase avidin - biotin technique in detecting -gals on cell surface instead of previously used immunofluorescent method . The heart and pericardium of pigs, aged 6 - 12 months, were obtained from the local slaughter house and transported in 4 normal saline bag to our facility . After removing the aortic valve and pericardial tissue, samples of the valve and pericardial tissue of 55 mm size were excised and washed with pbs 3 times, 5 min each . The samples were then immersed into 30% sucrose solution for preventing cell rupture during freezing process . Next, the frozen sample were sectioned and were mounted on the slides and fixed with cold acetone for 10 min . After thorough washing in pbs 3 times, for 5 min each, the sections were incubated on the slides in 1/500 diluted 500 mg / ml gs - ib4/biotin conjugates (invitrogen, carlsbad, ca, u.s.a .) At 37 for 1 hr . Again sections were washed as above . After blocking with 100 ml of 1% bovine serum albumin (bsa)/pbs at 37 for 1 100 ml of 5 mg / ml horseradishperoxidase (hrp) conjugated streptavidin (hrp - sa) (pierce biotechnology, rockford, il, u.s.a .) Were applied to the slides and incubated at 37 for 1 hr . Finally 3,3'-diaminobenzidine (dab) substrate (dab kit, vector lab ., burlingame, ca, u.s.a .) Was applied on the tissue slide for 5 min and the slides were observed under mounting media . In this indirect immunoperoxidase avidinbiotin technique using dab as a substrate, brown staining spots on the cell surface indicate gs - ib4 bound -gal epitopes . Recombinant human -galactosidase a (isu abxis, seoul, korea) made from chinese hamster ovary mammalian cells were used in our experiment . 100 mm hepes buffer solution at ph 5.0 was prepared, and used to make concentrations of recombinant -galactosidase a of 1.0, 5.0, 10.0 unit / ml . The sliced aortic valve and pericardial tissue of pig of 55 mm size were incubated with each of the solution for 24 hr under 4. after 24 hr, the tissues were washed with pbs solution for 5 min 3 times, then immersed into 30% sucrose solution . After then -gal epitopes on the enzyme treated aortic valve and pericardial tissue were stained as described above . The heart and pericardium of pigs, aged 6 - 12 months, were obtained from the local slaughter house and transported in 4 normal saline bag to our facility . After removing the aortic valve and pericardial tissue, samples of the valve and pericardial tissue of 55 mm size were excised and washed with pbs 3 times, 5 min each . The samples were then immersed into 30% sucrose solution for preventing cell rupture during freezing process . Next, the frozen sample were sectioned and were mounted on the slides and fixed with cold acetone for 10 min . After thorough washing in pbs 3 times, for 5 min each, the sections were incubated on the slides in 1/500 diluted 500 mg / ml gs - ib4/biotin conjugates (invitrogen, carlsbad, ca, u.s.a .) At 37 for 1 hr . Again sections were washed as above . After blocking with 100 ml of 1% bovine serum albumin (bsa)/pbs at 37 for 1 100 ml of 5 mg / ml horseradishperoxidase (hrp) conjugated streptavidin (hrp - sa) (pierce biotechnology, rockford, il, u.s.a .) Were applied to the slides and incubated at 37 for 1 hr . Finally 3,3'-diaminobenzidine (dab) substrate (dab kit, vector lab ., burlingame, ca, u.s.a .) Was applied on the tissue slide for 5 min and the slides were observed under mounting media . In this indirect immunoperoxidase avidinbiotin technique using dab as a substrate, brown staining spots on the cell surface indicate gs - ib4 bound -gal epitopes . Recombinant human -galactosidase a (isu abxis, seoul, korea) made from chinese hamster ovary mammalian cells were used in our experiment . 100 mm hepes buffer solution at ph 5.0 was prepared, and used to make concentrations of recombinant -galactosidase a of 1.0, 5.0, 10.0 unit / ml . The sliced aortic valve and pericardial tissue of pig of 55 mm size were incubated with each of the solution for 24 hr under 4. after 24 hr, the tissues were washed with pbs solution for 5 min 3 times, then immersed into 30% sucrose solution . After then -gal epitopes on the enzyme treated aortic valve and pericardial tissue were stained as described above . The images representing the distribution of -gal epitopes on porcine aortic valve and pericardial tissue before and after treatment with recombinant -galactosidase a were obtained via gs - ib4 conjugated indirect immunoperoxidase avidinbiotin staining technique . After removing -gal epitopes with consecutive incubations in 1.0, 5.0, 10.0 unit / ml of recombinant -galactosidase a, we noted that different degrees of -gal epitopes were removed from the tissues according to tissue type and enzyme concentration . An enzyme concentration of 1.0 unit / ml was not enough to completely eradicate the -gal epitopes from cell surface of the aortic valve (fig . / ml, nearly all -gal epitopes were removed from cell surface according to gs - ib4 staining (fig . 2). There were no differences between 5.0 unit / ml to 10.0 unit / ml enzyme concentrations in removing -gal epitopes from the cell surface of the porcine aortic valve (fig ., we could conclude that about 5.0 unit / ml concentration of recombinant -galactosidase a with reaction conditions of ph 5.0, temperature 4, 24 hr of incubation was enough to remove all -gal epitopes from cell surface of porcine aortic valve . On the other hand, in porcine pericardial tissue, enzyme concentration of 1.0 unit / ml (fig . 5) were not enough to completely eradicate -gal epitopes from cell surface . At 10.0 unit / ml, all the -gal epitopes from the cell surface of pericardium were removed according to gs - ib4 staining (fig . Therefore, in porcine pericardial tissue, the more recombinant -galactosidase a was needed to remove all -gal epitopes from cell surface than that needed in aortic valve . This finding is in accordance with the result of our previous study (10). Improving the durability of the tissue valves are crucial, not only for the children - adolescent patients but also for the increasing number of elderly patients receiving tissue valves in the aortic valve position . Recently, nearly 70% of aortic valve replacement in the patients older than 70 yr were done using tissue valves (11, 12). The tissue valve degeneration have been caused by degenerative calcification, glutaraldehyde related calcification (13), mechanical wear, and pannus overgrowth . Recently it was shown that -gal epitopes, responsible for hyperacute rejection in xenotransplantation, were still present on glutaraldehyde fixed, commercially available tissue valves . Also an increased number of anti - gal antibodies in the patients who received those tissue valves was noted (2). Therefore, it is possible that the -gal - anti - gal immune reaction may play a certain role in valve calcification and degradation after tissue valve replacement and removing -gals from the surface of the tissue valves can improve the durability of the tissue valves . Until now two methods of removing -gal epitopes from porcine tissues are available . The other is creating an -1,3 galactosyltransferase deficient pigs (-gal knockout pigs) with the recently well established animal cloning methods (14, 15). Using -gal knockout pigs is theoretically ideal and we can use organs and tissues without a fear of hyperacute rejection . However, in the enzyme treated organs -gal reappears after a certain time (9). To remove -gal epitopes from cardiac valvular and pericardial tissues, enzymatic removal is feasible so, we used enzymatic method to remove -gals from cell surface of the tissues . Green coffee bean -galactosidase is a plant enzyme which eradicates the xenoantigenicity of -gal by cleaving gal1 - 3gal chain of -gal(gal1 - 3gal1 - 4glcnac) (7 - 9). Previously we reported that -gal epitopes could be removed from porcine aortic valve and pericardial tissue using green coffee bean -galactosidase (sigma, st . But green coffee bean -galactosidase is not cost effective for the commercial valve manufacturing process . Fabry disease, which exhibits x - linked genetic trait, is a congenital human -galactosidase a deficiency representing early peripheral neuropathy, late chronic renal failure, coronary artery disease and cerebral vascular disease . It is well known that enzyme replacement therapy (ert) using recombinant human -galactosidase a is helpful for those patients (16, 17). Recently, recombinant human -galactosidase a with chinese hamster ovary mammalian cell expression system was developed for ert in fabry disease . This enzyme cleaves exactly the same site of -gal as green coffee bean -galactosidase (10), which can also applied in removing -gal epitopes from cell surface of porcine aortic valve and pericardial tissue . In aortic valve, the recombinant enzyme of 5.0 unit / ml concentration could remove almost all -gals under reaction condition of ph 5.0, temperature 4, 24 hr of incubation . In pericardial tissue first and most popular is detecting -gals with griffonia simplicifolia type i isolectin b4 which highly specifically bind to cell surface -gals (4 - 6). Others are using monoclonal antibody m86 made by galili (18), using fitc labeled anti - immunoglobulin m, anti - immunoglobulin g which are species specific secondary antibodies derived from anti - gal immunoglobulin m and anti - gal immunoglobulin g, respectively (7). We previously reported that all three methods of -gal detection technique must be simultaneously used to prove complete removal of -gals from cell surface (10). But in this study we intended to prove only that recombinant human -galactosidase a can just as effectively remove -gal epitopes from porcine aortic valve and pericardial tissue as green coffee bean -galactosidase . In other words, we did not intend to prove the completeness of removal of -gals from cell surface of those tissues . We used standard indirect immunoperoxidase avidin - biotin technique instead of previously used fitc labeled immunofluorescent method in detecting gs - ib4 bound to -gals on cell surface ., burlingame, ca, u.s.a .) As a substrate for the indirect immunoperoxidase avidin - biotin staining method and as a result, the cell surface -gals exhibit brown colors . In this way we could examine the distribution of -gals on cell surface more precisely and avoid autofluorescence issues of fitc . In this study, we conclude that -gal epitopes can be removed from the porcine aortic valve and pericardial tissue using the recombinant human -galactosidase a as effectively as using green coffee bean -galactosidase . Thus with this recombinant enzyme, -gal deficient, more durable tissue valves can be made more economically in the future.
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Experimental animals: male c57bl/6ncrslc mice (8 weeks old) were purchased from japan slc (hamamatsu, japan). All mice were maintained in 40.5 20.5 18.5 cm individual ventilated cages (sealsafe plus mouse; tecniplast, buguggiate, italy) under controlled temperature (23 2c) and humidity (50 10%) on a 12-hr light / dark cycle at the kobe university life - science laboratory with ad libitum access to a pellet diet (dc-8; clea japan, tokyo, japan) and filtered water . This study was approved by the institutional animal care and use committee (permission #24 - 10 - 03) and carried out according to the kobe university animal experimental regulation . Ctd purification and hplc analysis: water - soluble dantotsu (involving 16% of ctd; sumitomo chemical co., tokyo, japan), donated by sado city (niigata, japan), was washed with 10 times the amount of distilled water to remove the surfactant activating and granulating agents . After being left to stand for at least 48 hr, this step was repeated five times, and then, the white precipitate was collected and air - dried naturally for a week . The content rate of ctd in the white precipitate was measured by a lachrom high - performance liquid chromatography (hplc) system (interface l-7000, pump l-7100, auto sampler l-7200, column oven l-7350 and uv - vis detector l-7420; hitachi, tokyo, japan) using a capcell pak c18 ug120 column (5 m particles, 4.6 250 mm; shiseido, tokyo, japan). Ctd standard (> 99.8%; wako chemical, osaka, japan) and the obtained white powder were completely dissolved in dimethyl sulfoxide (dmso) and then serial - diluted with a mobile phase consisting of 55% acetonitrile in 50 mm potassium phosphate buffer (ph 3.0), followed by filtration with a 0.20 m syringe driven filter unit (millex - lg; millipore, billerica, ma, u.s.a . ). The column maintained at 40c was eluted with the mobile phase at a flow rate of 1.0 ml / min . After the column was equilibrated, 10 l of the samples was injected into the hplc system . We monitored the resultant chromatograph at the wavelength of 260 nm and then calculated the ctd content of the obtained white powder from the calibration curve created by the peak areas and heights of the ctd standard . A single peak was observed in the samples from the white precipitate with the same retention time as that of the ctd standard . A linear calibration curve (r=0.999) created from samples serial - diluted with the ctd standard showed that our purification makes the content rate of ctd increased from 1416% to 9397% by weight . Ctd administration and stress exposure: all mice were allowed to acclimate to their home cages for a week prior to the initiation of experiments . We divided the mice into eight groups (n=5 mice in each cage): ctd-0 (control), ctd-10 (10 mg / kg / day), ctd-50 (50 mg / kg / day) and ctd-250 (250 mg / kg / day) with the presence or absence of stress exposure . As a substitute for filtered water for the mice, we used the medigel sucrarose 2 oz cup (clearh2o, portland, me, u.s.a . ), which is a flavored thermoreversible hydration gel matrix . The amounts of the purified ctd for the respective administration groups were calculated from the ctd purity (95%), daily gel intake (5 g / day / mouse), total gel weight (60 g; excluding the package weight) and average mouse weight (24 g; weighed at initiation of experiments). These amounts of ctd were completely dissolved in 600 l dmso (1% volume of a gel) and injected into the gels and then double - boiled at 60c followed by shaking to ensure that the ctd was diffused well . For the ctd-0 group, the same volume of dmso without the purified ctd all gels were strapped with cable ties under the grate of the cage lid to prevent contamination with the beddings and the excretions . All gels were weighed daily to estimate the amounts of the putative ctd exposure . In the four stress groups, the mice were subjected to an unpredictable chronic stress procedure as described in our earlier report with some modifications . Briefly, the following six stressors were used: 5 min forced swimming in water at room temperature (rt), 24 hr food and water deprivation, continuous overnight illumination, 30 min horizontal cage shaking (80 rpm), 24 hr switching of cagemates (being housed with another mouse) and 24 hr wet bedding . To maximize the unpredictability of this paradigm, the mice were randomly exposed to two mild stressors per day at varying times for 4 weeks . Behavioral analysis: on the last day of the 4 week experimental period, an open field test was conducted during the light phase to evaluate the locomotor activity and the anxiety - like behavior of the mice . Briefly, the mouse was placed on the corner of an open field (60 60 30 cm) with led illumination . All of the mouse s activities were recorded by a video camera for the subsequent 10 min, and we then analyzed the total distance traveled and the time spent in the center zone (30 30 cm) using smart video tracking software v3.0 (san diego instruments, san diego, ca, u.s.a . ). Tissue preparation: on the day after the completion of the 4 weeks of combined exposure to ctd and stress, all mice were deeply anesthetized with diethyl ether and transcardially perfused with 0.9% normal saline, followed by perfusion with ice - cold 4% paraformaldehyde in phosphate buffer . The testes were excised, weighed and postfixed with the same fixative overnight at 4c . The testes were dehydrated through a graded series of ethanol followed by xylene and embedded in paraffin . Serial sections of testes were then cut at 4 m thickness on a sliding microtome (sm2000r; leica microsystems, wetzlar, germany) and mounted on slide glasses (platinum pro; matsunami glass ind . Histological and immunohistochemical analyses: for the general histological analysis, testis sections were stained with hematoxylin and eosin (he; merck kgaa, darmstadt, germany) after their deparaffinization and hydration, following the manufacturer s instructions . To detect antioxidant enzymes in the testes the sections were immersed in absolute methanol and 0.5% h2o2 for 30 min, respectively, at rt to quench the endogenous peroxidase activity . Kyoto, japan) for 1 hr at rt for protein blocking and then incubated with the rabbit polyclonal anti - gpx4 antibody (item no . 10005258; cayman chemicals, ann arbor, mi, u.s.a .) Diluted 1:8,000 in phosphate buffered saline with 0.05% tween-20 (pbst; ph 7.4) for 18 hr at 4c . After being washed with pbst, the sections were reacted with goat anti - rabbit immunoglobulins conjugated to peroxidase - labeled dextran polymer in tris (hydroxymethyl) aminomethane - hcl buffer (envision+; dako, glostrup, denmark) for 1 hr at rt . Immunoreactivity was then detected by incubation with 3,3-diaminobenzidine solution (envision+ kit / hrp[dab], dako). The sections were then rinsed with distilled water and counterstained lightly with hematoxylin solution for 1 min . Next, the sections were placed in a graded series of ethanol, dehydrated with absolute ethanol, cleared by xylene and coverslipped with eukitt (o. kindler gmbh, freiburg, germany). Statistical analysis: statistical analyses were performed with excel statistics 2012 (ssri version 1.00, tokyo, japan). In the behavioral analyses, outliers more distant than 1.5 interquartile ranges from the upper or lower quartile were omitted . All data were analyzed by two - way anova (ctd stress) followed by the tukey - kramer s post hoc test . The results were considered significant when the p - value was less than 0.05 . Gel intake, body and testis weights: the daily gel consumption, body weights and testes weights are shown in fig . 1fig . 1.effects of combined exposure on the daily gel intake (a), body weights (b) and testis weights (c) in the non - stressed (open column) and stressed groups (closed column). The two - way anova showed significant main effects for the ctd and stress (p<0.01), but the interaction effects were not significant in gel intake and body weights . (a) ctd dose - dependently inhibited the gel intake, and the inhibition was significant in all ctd - administration groups compared to the ctd-0 groups . The stress procedure significantly inhibited the gel intake in each ctd - administration group . (b) the body weights of the ctd-250 stress and non - stressed groups were significantly lower than those of the other dose groups, in addition to the significant difference between the ctd-0 and ctd-10 groups . The post hoc analyses also revealed that there were significant differences between the ctd-0 and ctd-250 stress and non - stressed groups . (c) the two - way anova showed a significant main effect for stress (p<0.01), but the interaction effect was not significant in the absolute weights of testes . The stress procedure significantly suppressed the absolute testes weights in all stress groups, except the ctd-10 group . Ctd significantly suppressed the daily gel intake in all ctd administration groups compared to the non - ctd administration groups (fig . Although the daily intake was significantly inhibited in the stress groups, there was little difference if the values in the days of food deprivation in our stress protocol were omitted (table 1table 1.body weight, gel intake, putative ctd exposure and testis weightsgroupsctd-0ctd-0 + stressctd-10ctd-10 + stressctd-50ctd-50 + stressctd-250ctd-250 + stressbody weight (g)28.62 1.6626.48 0.7226.98 0.9925.36 1.2227.50 0.8225.24 0.7825.82 0.9923.76 0.40gel intake / day (g)6.13 1.375.64 2.804.41 0.842.85 1.774.60 1.593.55 2.093.64 0.642.38 1.42 [6.66 2.56] [3.73 1.59] [4.65 1.79] [3.16 1.09] putative exposure (mg / kg / day)008.82 1.685.70 3.5446.0 15.935.5 20.9182 32119 71 [7.46 3.18] [46.5 17.9] [158 54.5] testis weight (mg)99.6 5.588.2 11.895.7 9.690.5 9.7100.3 9.881.1 8.297.5 7.887.5 the values in square brackets are the averages after the removal of values in the deprivation days from the calculation . ). As a result, the body weights of mice reflected the same tendency of the gel intake . The body weights were suppressed in all stress groups relative to the non - stressed groups at the same dose of ctd (fig . The body weights of the ctd-250 groups were significantly lower than those of the other three dose groups . On the other hand, the absolute weights of the testes were decreased by the stress procedure in the ctd-0, ctd-50 and ctd-250 groups (fig . Effects of combined exposure on the daily gel intake (a), body weights (b) and testis weights (c) in the non - stressed (open column) and stressed groups (closed column). The two - way anova showed significant main effects for the ctd and stress (p<0.01), but the interaction effects were not significant in gel intake and body weights . (a) ctd dose - dependently inhibited the gel intake, and the inhibition was significant in all ctd - administration groups compared to the ctd-0 groups . The stress procedure significantly inhibited the gel intake in each ctd - administration group . (b) the body weights of the ctd-250 stress and non - stressed groups were significantly lower than those of the other dose groups, in addition to the significant difference between the ctd-0 and ctd-10 groups . The post hoc analyses also revealed that there were significant differences between the ctd-0 and ctd-250 stress and non - stressed groups . (c) the two - way anova showed a significant main effect for stress (p<0.01), but the interaction effect was not significant in the absolute weights of testes . The stress procedure significantly suppressed the absolute testes weights in all stress groups, except the ctd-10 group . The values in square brackets are the averages after the removal of values in the deprivation days from the calculation . 2.behavioral effect of combined exposure of ctd and stress in the open field activity in the non - stressed and stressed groups . (a) a representative trajectory map of the mice as illustrated by the video tracking software . The exploratory behaviors in the center zone (30 30 cm) of the open field (60 60 cm) were dose - dependently suppressed by ctd compared to the non - administration groups . (b) total distances traveled in the open field of the non - stressed (open columns) and stressed groups (closed columns). No marked difference was detected by two - way anova in the total distances traveled . (c) time spent in the center zone in the open field of the non - stressed (open columns) and stressed groups (closed columns). Two - way anova showed significant main effects for ctd (p<0.05) and stress (p<0.01), but the interaction effect was not significant . Ctd significantly inhibited the times spent in the center zone in the ctd-250 groups compared to the non - ctd administration groups . A significant anxiogenic effect of the stress procedure was observed between the ctd-0 groups . In the non - stressed condition, there were significant differences in the ctd-10 and ctd-50 groups compared to the ctd-0 group . * p<0.05, * * p<0.01 .) Revealed that the mice with the combined ctd exposure and stress procedure tended to walk alongside the walls . Ctd and the stress procedure had insignificant impacts on the locomotor activities measured by the total distances traveled (fig . By contrast, the anxiety - like behaviors that avoid being in the center zone were elevated by the stress procedure and were significantly different between the ctd-0 groups (fig . Ctd also enhanced the anxiety - like behaviors in a dose - dependent manner, and in the non - stressed condition, the difference was significant in the ctd-10 and ctd-250 groups compared to the ctd-0 group . Behavioral effect of combined exposure of ctd and stress in the open field activity in the non - stressed and stressed groups . (a) a representative trajectory map of the mice as illustrated by the video tracking software . The exploratory behaviors in the center zone (30 30 cm) of the open field (60 60 cm) were dose - dependently suppressed by ctd compared to the non - administration groups . (b) total distances traveled in the open field of the non - stressed (open columns) and stressed groups (closed columns). No marked difference was detected by two - way anova in the total distances traveled . (c) time spent in the center zone in the open field of the non - stressed (open columns) and stressed groups (closed columns). Two - way anova showed significant main effects for ctd (p<0.05) and stress (p<0.01), but the interaction effect was not significant . Ctd significantly inhibited the times spent in the center zone in the ctd-250 groups compared to the non - ctd administration groups . A significant anxiogenic effect of the stress procedure was observed between the ctd-0 groups . In the non - stressed condition, there were significant differences in the ctd-10 and ctd-50 groups compared to the ctd-0 group . Histological and immunohistochemical findings: the general histological analyses of the testes by he staining are shown in fig the seminiferous tubules of the control group showed robust spermatogenesis with densely stacked germ cells . Multinucleated giant cells (arrow) were occasionally observed in the testes of the ctd-0+stress groups . In the ctd groups, dose - dependent vacuoled degeneration (arrowhead) of seminiferous tubules was observed . Degenerated seminiferous tubules only composed of sertoli cells were present in the ctd-250 groups . Bar=100 m .. although there were seminiferous tubules densely arranged with germ cells and sperms in the testes of the control group, several testicular signs of toxicity were observed in combined exposure groups . Multinucleated giant cells were occasionally observed in the seminiferous tubules of the mice in the stress groups . In addition, variably sized vacuolizations were scattered at the bottom of seminiferous tubules of the ctd - administered groups . In particular, seriously degenerated seminiferous tubules, from which almost all germ cells had dropped off, were found in the ctd-250 groups . There was no marked histological difference between the leydig cells in the testicular interstitial tissues of any of the experimental groups . The seminiferous tubules of the control group showed robust spermatogenesis with densely stacked germ cells . Multinucleated giant cells (arrow) were occasionally observed in the testes of the ctd-0+stress groups . In the ctd groups, dose - dependent vacuoled degeneration (arrowhead) of seminiferous tubules was observed . Degenerated seminiferous tubules only composed of sertoli cells were present in the ctd-250 groups . Bar=100 m . The results of the immunohistochemical analyses of the testes visualizing gpx4 are shown in fig . 4.representative immunohistochemistry for gpx4 of the testis in the non - stressed and stressed groups . Gpx4 immunoreactivities were detected in sperms and spermatids in the testes of the control group . In the testes of the stressed groups, reduced immunoreactivity of gpx4 in sperm was observed . In the ctd groups, spermatids showed weakened immunoreactivity of gpx4 . Sertoli cells ectopically expressed gpx4 in degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . Bar=100 m .. in the control group, strong gpx4 immunoreactivity was detected in the sperm, and diffuse gpx4 immunoreactivity was seen in the cytoplasm of spermatids . The intensity of gpx4 immunoreactivity in the sperm was decreased by stress, and the cytoplasmic immunoreactivity in the spermatids was dose - dependently decreased by ctd . In addition, abnormal immunoreactivity of gpx4 in sertoli cells was detected in the combined exposure groups, and it became strong in degenerated seminiferous tubules in the ctd-50+stress group and the ctd-250 groups . Representative immunohistochemistry for gpx4 of the testis in the non - stressed and stressed groups . Gpx4 immunoreactivities were detected in sperms and spermatids in the testes of the control group . In the testes of the stressed groups, reduced immunoreactivity of gpx4 in sperm was observed . In the ctd groups sertoli cells ectopically expressed gpx4 in degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . We verified the combined effects of ctd and stress on the reproductive and behavioral function in mature male mice by a non - invasive administration method . There are several advantages of the gels we used; compared to baking pellets, the gels can uniformly deliver the test articles to animals without exposing them to high temperature . The gels could not completely cloak the bad palatability of ctd as reported earlier using similar gels mixed with an aromatase inhibitor, anastrozole; however, the gel consumption observed in combined exposure groups was enough for maintaining the health of the mice . In our preliminary experiments, we initially suspected that the ctd avoidance effect was attributable to any accessory constituents of the commercial pesticide containing ctd, but ctd itself appeared to have some repellent effects on mice . In agricultural fields, ctd - containing pesticides are used at dilutions from 1:20 to 1:4,000 for treating seeds and plants . Our purification protocol and the water solubility of ctd imply that the white precipitate resulting from the recommended concentration of ctd products is particularly dangerous for pesticide users . In the present study, the unpredictable stress model, which is often used in the assessment of antidepressant medications, evidently raised the anxiety level in the mice . Ctd dose - dependently induced anxiety alone, but there was little difference in this measure among the combined exposure groups . To the best of our knowledge, there are only a few reports about the behavioral effects of neonicotinoids on mammals . Several parameters of behavioral developmental tests that evaluated the sense of equilibrium, coordinated movement and muscular power of offspring were changed by maternal exposure to low - dose ctd, with a sex difference [34, 35]. Thiamethoxam (tmx, 100 mg / kg / day) significantly reduced the locomotor activity of rats in an open field test and induced anxiety in an elevated plus - maze test while suppressing acetylcholinesterase (ache) activity in three brain regions . Considering that tmx is metabolized to ctd in mammals, our findings regarding the anxiogenic effects of ctd sufficiently agree with the findings of these prior studies . Although the mechanisms of the behavioral effects of neonicotinoids remain unclear, cholinergic systems and downstream neurotransmitters are undoubtedly involved . An in vivo study revealed that ctd directly injected into the rat brain evoked a striatal dopamine surge through nachrs . Early types of neonicotinoids caused a subsequent desensitization of nachrs as well as immediate neuronal excitation . The behavioral consequences of chronic nicotine intake are also attributed to both the upregulation and desensitization of nachrs . The anxiety - like behaviors in several behavioral tests are part of the phenotype of nachrs subunit 4 knockout mice, implying that the anxiogenic effect of ctd observed in the present study was presumably triggered by the chronic desensitization of nachrs . The chrna7 coding 7 subunit of nachrs contains a glucocorticoid response element, which may explain the decreasing nachrs 7 expression by chronic immobilization stress . These observations may support the hypothesis that the behavioral effects of ctd and stress partly share a common mechanism mediating weakened cholinergic activities, such as the desensitization of nachrs, the suppressed level of ache and the disturbance of the release of downstream neurotransmitters . In the ctd-250+stress groups, the stress procedure and the ctd - induced feeding suppression presumably fully suppressed the cholinergic activities, which made the anxiogenic effect of ctd invisible . On the other hand, we found that the chronic exposure to ctd and the stress influenced the testes in different ways . The stress procedure significantly decreased the testicular weight in addition to causing the emergence of the multinucleated giant cells, and both of these are common toxicant observations in testes produced by an abnormal meiotic division of germ cells . Ctd did not show an impact on the testicular weight, whereas it dose - dependently degenerated the seminiferous epithelia . In particular, the sertoli cell - only seminiferous tubules found in the ctd-250 groups were unexplainable by ctd - induced chronic nutritional deprivation . In the clinical field, sertoli cell - only syndrome is known to result from partial deletions of the azoospermia factor region on the y - chromosome, which is one of the markers of male infertility . From the toxicological perspective, germ cells are vulnerable to damage from exogenous chemicals, and they are easily phagocytized by sertoli cells . Several endocrine - disrupting compounds (edcs) including diethylstilbestrol and flutamide induce such a destruction of seminiferous tubules [1, 11, 40]. An increasing number of vacuolizations in seminiferous epithelia are also caused by the absence of germ cells, suggesting that they are the first target of ctd in the testes through the blood - testis barrier . This corresponds to the ctd dose - dependent increase of single - stranded dna immunopositive germ cells observed in the testes of quails [15, 36]. Gpx4 (also called phospholipid - hydroperoxide glutathione peroxidase as a member of the glutathione peroxidase family) is an antioxidant enzyme expressed in every mammalian organ . There are three types of transcripts from the same gpx4 gene: mitochondrial, non - mitochondrial and nuclear types were identified . The mitochondrial and non - mitochondrial types in particular are expressed in spermatocytes, and gpx4 is the principal antioxidant enzymes in the testes with 30 times greater expression in the testes compared to other organs . Notably, in sperm, gpx4 is responsible for the abilities to scavenge approx . Testis - specific gpx4 knockout mice showed a phenotype of infertility with degenerated testicular tissue and morphological aberration of sperm . The antigen of the polyclonal antibody used in this study is a common sequence among the three types of mouse gpx4, and thus, we could not classify the types of gpx4 immunoreactivity . The weakened gpx4 immunoreactivity in the lumen of seminiferous tubules presumably resulted from the decreases in the number and quality of the sperm . Neonicotinoids are known to have strong oxidizing properties that affect the liver and the reproductive and central nervous systems [7, 19]. The present report is the first to describe the localized decrease of antioxidant enzymes by ctd in the testes of a mammal . Similarly, oxidative effects of psychological stress via stress hormones have been reported [23, 42]. Although a constant level of reactive oxygen species is necessary for the proliferation of spermatogonial stem cells, the present results also showed that oxidative mechanisms partly explain how stress impacts the reproductive function cooperatively with ctd . Although there are no reports of plentiful expressions of gpx4 in sertoli cells, strong ectopic expression of antioxidant enzymes was observed in sertoli cells in degenerated seminiferous tubules in the ctd-250 groups . Because gpx4 mrna was not expressed in sertoli cell in rat, investigated whether gpx4 mrna in the testes was altered by several edcs, implying the measurements of oxidative conditions provide an indication of the testicular toxicity by environmental factors . In the present study, although no remarkable interaction effect was detected, we investigated whether ctd and stress affected the behavioral and reproductive functions additively rather than synergistically in adult mice . The endogenous nachrs agonist lynx1 was recently reported to serve as a molecular break system for brain plasticity . The developmental effects of neonicotinoids in juvenile and fetal periods remain to be determined . Neonicotinoids and their metabolites are detected at high frequency in urine samples from japanese people . Human exposure to ctd at a high concentration as high as that of the present ctd-250 groups does not occur naturally, but the precipitates that result from the low water solubility of ctd are very dangerous . In addition, a low concentration of ctd could become harmful under a stressed condition, such as fasting . The finding that one of the metabolites of imidacloprid (imi) seriously disturbs mapk / erk signaling compared to imi itself suggests that the safety coefficient (100) for calculating the acceptable daily intake is not satisfactory . Taking the results of the present study as an example, it is clear that further research is needed to identify the toxicant mechanisms of environmental chemicals in order to protect humans and animals with high sensitivities under stressed conditions.
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The hippocampus is one of the first and most affected brain regions impacted by both alzheimer's disease (ad) and mild cognitive impairment (mci; arnold et al ., 1991; bobinski et al ., 1995;, 2004; schonheit et al ., 2004). In mild - to - moderate ad patients, it has been shown that hippocampal volumes are 27% smaller than in normal elderly controls (callen et al ., 2001; du et al ., 2001), whereas patients with mci show a volume reduction of 11% (du et al ., so far, from a neuropathological point of view, the progression of disease from early or very early mci to later stages seems to follow a linear course . Nevertheless, there is some evidence from functional (gold et al ., 2000; della maggiore et al ., 2002; hamalainen et al ., 2006) and biochemical studies (lavenex and amaral, 2000) that the process of conversion from non - demented to clinically evident demented state is not so linear . Recent fmri studies have suggested increased medial temporal lobe (mtl) activations in mci subjects vs controls, during the performance of memory tasks (dickerson et al ., 2004, 2005). Nonetheless, fmri findings in mci are discrepant, as mtl hypoactivation similar to that seen in ad patients (pariente et al ., 2005) recent postmortem data from subjects who had been prospectively followed and clinically characterized up to immediately before their death indicate that hippocampal choline acetyltransferase levels are reduced in alzheimer's dementia, but in fact they are upregulated in mci (lavenex and amaral, 2000), presumably because of reactive upregulations of the enzyme activity in the unaffected hippocampal cholinergic axons . Quantitative electroencephalography (eeg) have been demonstrated a reliable tool in identifying specific patterns in dementia research (john and prichep, 1990; prichep et al ., 1994; coburn et al ., 2006; john and prichep, 2006). Previous eeg studies (jelic et al ., 1996, 2000; 2003) have shown a decrease ranging from 8 to 10.5 hz (low alpha) of the alpha frequency power band in mci subjects, when compared to normal elderly controls (zappoli et al ., 1995; huang et al . However, a recent study has shown an increase ranging from 10.5 to 13 hz (high alpha) of the alpha frequency power band, on the occipital region in mci subjects, when compared to normal elderly and ad patients (babiloni et al ., 2006). These somewhat contradictory findings may be explained by the possibility that mci subjects have different patterns of plastic organization during the disease, and that the activation (or hypoactivation) of different cerebral areas is based on various degrees of hippocampal atrophy . If this hypothesis is true, then eeg changes of rhythmicity have to occur non - proportionally to the hippocampal atrophy, as previously demonstrated in a study of auditory evoked potentials (golob et al ., 2007). A recent study (moretti et al ., 2007), has confirmed the hypothesis that the relationship between hippocampal volume and eeg rhythmicity is not proportional to the hippocampal atrophy, as revealed by the analyses of both the relative band powers and the individual alpha markers . Such a pattern seems to emerge because, rather than a classification based on clinical parameters, discrete hippocampal volume differences (about 1 cm) are analyzed . Indeed, the group with moderate hippocampal atrophy showed the highest increase in the theta power band on frontal regions, and of the alpha2 and alpha3 power bands on frontal and temporo - parietal areas (figures 13). Statistical anova interaction among group factors, and relative band powers (delta, theta, alpha1, alpha2, alpha3), on the full scalp region . Group 1, no hippocampal atrophy; group 2, mild hippocampal atrophy; group 3, moderate hippocampal atrophy; group 4 severe hippocampal atrophy . Post hoc results are indicated in the diagram (see moretti et al ., 2007). Schematic diagrams of the possible structures involved in the thalamo - cortical arrhythmia in groups without hippocampal atrophy and with mild hippocampal atrophy . The eeg changes in the mild hippocampal - atrophy group are probably due to a prevalent depolarizing effect of the brainstem cholinergic system on the thalamus, because of initial neuronal loss in the cholinergic basal forebrain . On the other hand, the initial hippocampal atrophy gives rise to an increase in the theta activity, but it is not able to trigger thalamo - cortical synchronization activity (see moretti et al ., 2007). Black thick arrow, excitatory activity; blue arrow, synchronization effect; red arrow, desynchronization effect . Schematic diagrams of the possible structures involved in the thalamo - cortical arrhythmia in groups with moderate and severe hippocampal atrophy . The progressive hippocampal atrophy, as in the moderate hippocampal - atrophy group, triggers thalamo - cortical synchronization, with increase in the alpha power band . This is likely to happen because the return pathway from the cortex to the hippocampus is not direct, but it mainly relays to the midline and mediodorsal thalamic nuclei . The decrease in the values for the alpha frequency markers in the moderate hippocampal - atrophy group also suggests a greater hyperpolarization state of thalamo - cortical pathways . As the hippocampal atrophy progresses, like in the group with severe hippocampal atrophy, the thalamo - cortical activity is sustained not by cortical activation, but by the prevailing cholinergic desynchronizing activity of the brainstem, with a decrease in the relative alpha power but the highest values for the alpha indices (see moretti et al ., 2007). Black thick arrow, excitatory activity; blue arrow, synchronization effect; red arrow, desynchronization effect . Recently, two specific eeg markers, theta / gamma and alpha3/alpha2 frequency ratio have been reliable associated to the atrophy of amygdalo hippocampal complex (ahc; moretti et al ., 2009a, b), as well as with memory deficits, that are a major risk for the development of ad in mci subjects (figure 4). Based on the tertile values of decreasing ahc volume, ahc atrophy is associated with memory deficits as well as with increase of theta / gamma and alpha3/alpha2 ratio . Moreover, when the amygdala and hippocampal volume are separately considered, within ahc, the increase of theta / gamma ratio is best associated with amygdala atrophy whereas alpha3/alpha2 ratio is best associated with hippocampal atrophy . A large body of literature has previously demonstrated that in subjects with cognitive decline is present an increase of theta relative power (moretti et al ., 2007, 2008a, b), a decrease of gamma relative power (stam et al ., 2003; moretti et al ., 2008a, b) as well as an increase of high alpha as compared to low alpha band (moretti et al ., 2008a, b). On the whole theta / gamma ratio and alpha3/alpha2 ratio the amygdalo hippocampal network is a key structure in the generation of theta rhythm . More specifically, theta synchronization is increased between lateral amygdala and ca1 region of hippocampus during long - term memory retrieval, but not during short - term or remote memory retrieval (seidenbecher et al ., 2003; narayanan et al ., 2007). In particular, the ahc is critically involved in the formation and retention of fear memories (narayanan et al ., 2007). Theta synchronization in ahc appears to be a neural correlate of fear, apt to improve the neural communication during memory retrieval (narayanan et al ., 2007). On the other hand, the retrieval of hippocampus - dependent memory is provided by the integrity of ca3ca1 interplay coordinated by gamma oscillations (montgomery and buzsaki, 2007). The brain oscillatory activity of this mci state is characterized by an increase of theta / gamma and alpha3/alpha2 relative power ratio, confirming the overall reliability of these eeg markers in cognitive decline . Previous results suggest that theta synchronization is mainly due to the amygdala activation or as a subsequent final net effect within the ahc, driven by the amygdala excitation . The increase in theta activities in ahc, is considered as an increase in neuronal communication apt to promote or stabilize synaptic plasticity (montgomery and buzsaki, 2007). This is in relation to the effort to retention associative memories (sauseng et al ., 2004), and could be active also during an ongoing degenerative process . The excitation mechanism could be facilitated by the loss of gaba inhibitory process, determining the decrease of gamma rhythm generation in ad (bragin et al . The functional coupling of brain areas is also modulated by hippocampal atrophy . In the mci subjects, hippocampal atrophy is linked to an increase of interhemispheric coherence seen on frontal and temporal regions whereas subcortical cerebrovascular disease (cvd) is linked to a decrease of coherence in fronto - parietal regions . Moreover, significant differences of eeg functional coupling were present in the fronto - temporal network in mci patients with severe cvd and severe hippocampal atrophy, but with a different pattern . In high cvd, the eeg coherence in low frequencies was increased (with the exception of alpha1 band) while coherence in the fast frequencies was decreased in a way directly proportional to increasing damage . In high hippocampal atrophy a change of coherence was present in the delta and alpha2 frequency bands that was not proportional to the hippocampal damage, fast frequencies being unaffected . Moreover, our results show a lateralization (right hemisphere for cvd and left hemisphere for hippocampal atrophy) of the pathological modifications of functional coupling (moretti et al . An increase of neuronal excitability could explain the pathological modifications of the functional coupling (moretti et al . When mci subgroups are considered, based on different pathological substrate, the results show that the increase of interhemispheric coherence seen in frontal (delta frequency) and temporal regions (delta, theta, alpha1, and, in a tendential way, alpha2 and alpha3 frequency bands) is exclusively linked to the hippocampal atrophy (figure 5). Previous studies show that the increase of coherence between temporal regions is determined by an increase of excitability (ferreri et al ., 2003; this hypothesis could receive a support from studies demonstrating a dysregulation of inhibitory gaba - ergic system following the hippocampal atrophy (de curtis and par, 2004; gloveli et al ., 2005). Through the hippocampal commissure, the increase of excitability could spread over the two hemispheres . Our results confirm this view because the increase of coherence between temporal regions is present only in the mci subjects with the greater hippocampal atrophy . Precedent literature (jiang, 2005; jiang and zheng, 2006) showed an increase of coherence in mci subjects as compared to normal controls during a working memory task, in which prefrontal and medial temporal areas are involved (johnson, 2006). Our results suggest that it could be due to an increase of excitability specifically due to the hippocampal atrophy . Given the absence of either a significative cvd or cholinergic vascular damage in this group of mci compared to normal old subjects, a possible explanations is that the increase of excitability in medial temporal areas subsequent to the hippocampal atrophy creates a sort of temporal areas wall impairing the long - range, fronto - parietal functional connections within each hemispheres . A recent study (zheng et al ., 2007) has demonstrated that mci subjects have a decrease in intrahemispheric and an increase in interhemispheric coherence in alpha1 and alpha2 frequency bands during an experimental condition needing a memory demand increase in order to maintain a good level of cognitive processing . Our results suggest that this compensatory effort is more frequently observed in subjects with hippocampal atrophy . Moreover, it could be argued that this activity (pijnenburg et al ., 2004) emerges as a new default mode of brain activity (moretti et al ., 2007) characterized by an hyperexcitability of the cortex, even at a rest state . No difference was found on frontal and parietal interhemispheric coherence between normal old and mci subjects with hippocampal atrophy . This could suggest that, in the initial stage of disease, interhemispheric connections are damaged only on the temporal lobes . Another possible explanation is that the hippocampal functional connections are mostly branched within each hemisphere (de curtis and par, 2004; gloveli et al ., 2005). Anova statistical results . On the left part of the figure statistical anova results of the first session analysis (normal - mci whole group); on the right part of the figure statistical anova results of the second session analysis (mci subgroups - matched normal old group; (mci - chol, mci with greater cholinergic damage, mci - cvd, mci with greater cerebrovascular damage; mci - hipp, mci with greater hippocampal atrophy; see moretti et al ., 2008a). The vulnerability and damage of the connections of hippocampus with amygdala could affect reconsolidation of long - term memory and give rise to memory deficits and behavioral symptoms . Several experiments shows that amygdala activity is prominent during period of intense arousal, e.g., the anticipation of a noxious stimulus (par et al ., 2002) or the maintenance of vigilance to negative stimuli (garolera et al ., 2007). So far, the theta synchronization induced by the amygdala is deeply involved in endogenous attentional mechanism . Interestingly, the increase of high alpha synchronization has been found in internally cued mechanisms of attention, associated with inhibitory top - down processes (klimesch et al ., 2007). Of note, the amygdala is intimately involved in the anatomo - physiological anterior pathways of attention through its connections with anterior cingulated cortex, anteroventral, anteromedial, and pulvinar thalamic nuclei (young et al ., 2007). The particular role of amygdala in negative human emotions could indicate that ahc atrophy is associated with excessive level of subcortical inputs not adequately filtered by attentive processing, determining fear and anxiety, and generating cognitive interference in memory performance . Of note, an altered emotional response is very frequent in mci patients (ellison et al ., 2008; rozzini et al ., 2008) in a feed - back process, this alteration could determine a general state of hyperattention during which top - down internal processes prevail on the bottom - up phase, altering attention mechanism, and preventing a correct processing of sensory stimuli . Focused attention has been found impaired in mci patients in particular when they have to benefit from a cue stimulus (johannsen et al ., 1999; berardi et al ., 2005; levinoff et al ., 2005; tales et al ., 2005a, b). This particular state could be useful for maintain a relatively spared global cognitive performance, whereas it could fail when a detailed analysis of a sensory stimulus is required . This hyperattentive state could represent the attempt to recollect memory and/or spatial traces from hippocampus and to combine them within associative areas connected with hippocampus itself . The increase of alpha3/alpha2 frequency ratio in our results support the concomitance of anterior attentive mechanism impairment in subject with mci, even though there are not overt clinical deficits . The mayor association of the increase of alpha3/alpha2 frequency ratio with the hippocampal formation within the ahc, suggest that this filter activity is carried out by hippocampus and its input output connections along anterior attentive circuit and ahc . Interestingly, a recent work has demonstrated that the mossy fiber (mf) pathway of the hippocampus connects the dentate gyrus to the auto - associative ca3 network, and the information it carries is controlled by a feedforward circuit combining disynaptic inhibition with monosynaptic excitation . Analysis of the mf associated circuit revealed that this circuit could act as a highpass filter (zalay and bardakjian, 2006). There are two main pathways of the hippocampal connections: the polysynaptic hippocampal pathway encompassing posterior cingulate / retrosplenial and medial temporal cortex and the direct hippocampal pathway encompassing the temporal pole, temporo - parietal association cortex, and dorsal prefrontal cortex . Recent studies have demonstrated that the polysynaptic hippocampal pathway is early affected in mci whereas the direct pathway is affected later, in patients with ad (frisoni et al ., 2009). Indeed, corticopetal cholinergic pathways, originating in the nucleus basalis of meynert are affected early in the decourse of ad . On the contrary, the damage of the cholinergic pathways that innervate directly the hippocampal formation the possible different roles of the cholinergic pathways are supported by the different effects of increasing cholinergic damage on alpha3 power band (moretti et al ., 2008b). Indeed, our results show that, when cholinergic damage becomes larger (i.e., in the group with greater cholinergic impairment) alpha3 power band increases in patients with damage in the perisylvian and medial cholinergic pathway and decreases in patients with damage in the capsular cholinergic pathway (figure 6). The cholinergic system is thought to exert two mean functions: (1) fronto - parietal, top - down, attentional inhibitory control and (2) signal - driven, bottom - up, excitatory effect when external stimuli (via - thalamus) have to be processed (sarter et al . For anatomical reason, being the largest tract connecting the opposite cerebral lobes, each capsular pathway is more suitable for the antero - posterior inhibitory effects and modulation of attentive functions whereas the perisylvian and medial pathways could subserve the integration of thalamus - driven stimuli in higher order cortical areas providing a common substrate for development of superior cognitive functions such as language and memory (mesulam, 2004; sarter et al ., 2005). For anatomical reasons, perisylvian pathway is suitable to subserve language function through language areas and the medial pathway to subserve retrieval of episodic memory through medial temporal cingulate cortex (nestor et al ., 2004). The physiological basis of alpha synchronization relies on inhibitory mechanisms (klimesch et al ., 2007). Moreover, previous works proposed that alpha3 is more involved in cortical processes than other alpha sub - bands (klimesch, 1999; moretti et al ., 2007). Taken together, the results suggest that in the long - range connection pathway (lateral capsular) a decrease in cortical, inhibitory attentive top - down control occurs, related to a decrease in alpha3 power band . By contrast, in local, smaller, neural assemblies, like those subserved by perisylvian and medial cholinergic pathways, an increase of cortical inhibitory mechanism occurs, related to alpha3 power band synchronization . A reasonable explanation is that there is a removal of excitatory, synaptic inputs in intracortical networks which would produce a decrease in synaptic efficacy and functional disconnection of cortical circuits (steriade, 2006). The dysregulation of cholinergic system could result in specific clinical symptoms according to the cholinergic pathway involved: attention for the capsular pathway, language for the perisylvian pathway, and memory for the medial pathway (figure 5). Theoretical, neurophysiological and clinical effects of damage to the cholinergic pathways (see moretti et al ., 2008b). In this view, such process could be imagined: (1) damage of corticopetal cholinergic pathways determining an impairment of the multisynaptic hippocampal pathway . The damage of this posterior pathway induces an interruption of large brain networks and a reorganization in smaller loco - regional areas . Alpha2 rhythm is the expression of functional coupling of large brain areas whereas alpha3 suggests synchronization of smaller areas . So, the increase of alpha3/alpha2 ratio is a reasonable consequence; (2) the damage of corticopetal cholinergic pathways could induce an excitation / disinhibition of the cholinergic pathways that innervates the ahc . This cholinergic action could stimulate the direct hippocampal pathway relatively spared in the initial stage of ad . Together with the loss of inhibition mechanism, an increase of theta / gamma ratio is produced together with and increase of medial temporal functional coupling; given that the theta rhythm generation seems more functionally linked with amygdala (moretti et al ., 2009b), with the progression of disease, and degeneration of the hippocampal direct pathway, an increase of theta / gamma ratio could be expected whereas the alpha3/alpha2 ratio is more suitable as eeg predictor of conversions from mci state in ad (figure 4; moretti et al ., 2009c); (3) in the later stages of ad, with the progressive atrophy of the neural networks, both alpha3/alpha2 and theta / gamma ratio will modify according to the progression of atrophy in amygdala and/or hippocampus . Moreover, a recent study shows a correlation between brain electrical activity collected by scalp eeg and discrete mapped hippocampal areas in subjects with ad (moretti et al . In particular, in ad patients the increase of both alpha3 rhythm spectral power and alpha3/alpha2 power ratio is correlated with the decrease of left hippocampal gray matter volumes . In particular, hippocampal areas involved in correlation are: presubiculum, dorsal and ventral subiculum, ca2ca3 sectors of the body, ca1 mesial and lateral portion of the head . These findings confirms previous results obtained in a large cohort of patient with mci who convert in ad . Obviously, this physiological model needs further evidence supporting the relationship with the clinical symptoms . Anyway, although speculative, it could be an interesting starting point for future works . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Tweak (tnfsf12) is a cell surface - associated type ii transmembrane protein (249 amino acids) belonging to the tumor necrosis factor (tnf) superfamily . Transmembrane tweak is processed into a secreted 156-amino - acid form, which adopts a homotrimeric conformation . Tweak mrna has been reported to be expressed in several tissue types, such as heart, brain [3, 4], kidney [5, 6], and also in mononuclear blood cells . Its protein product has multiple biological activities, including stimulation of cell growth and angiogenesis, induction of inflammatory cytokines [9, 10] and stimulation of apoptosis [11, 12]. It has been shown to be involved in the induction of cellular proliferation in liver cells, osteoblasts, astrocytes, synoviocytes, kidney cells [17, 18], and skeletal muscle . Tweak may also play a role in the cellular differentiation of osteoclasts; however it remains controversial whether this effect is direct or indirect, via effects on the osteoblastic stromal cell expression of rankl (tnfsf11). Tweak also plays a role in inducing glioma cell survival via imparting resistance to cytotoxic agents [3, 22]. It induces the endothelial cell survival and can be a potential proangiogenic or antiangiogenic agent based upon the presence of angiogenic promoting cytokines [8, 23]. Additionally, an apoptotic effect of tweak has been observed in endometrial cancers and peripheral blood monocytes [25, 26]. The apoptotic function of tweak appears to be mediated via the induced secretion of tnf, with the tnf-tnf receptor complex, thereafter inducing autocrine cellular apoptosis by activating the ripk1-fadd - caspase-8 complex [11, 27]. Tweak was first described as an apoptotic factor by interacting with dr3 (tnfrsf25). However, there were conflicting reports to the tweak - dr3 interaction [28, 29]. In addition, tweak has been reported to interact with cd163; however, the downstream effect of this interaction remains to be explored . Tnfrsf12a (tumor necrosis factor receptor super - family, member 12a), also known as fgf - inducible 14 (fibroblast growth factor - inducible-14/fn14), has been established to date to be the major, if not sole, receptor for tweak [12, 31, 32]. Fn14 is the smallest member of the tnfr superfamily described so far, and it appears to signal via recruitment of several different tnfr - associated factors . This molecule has been reported to be expressed in variety of organs including the heart [34, 35], kidney [6, 36], and lung . The cytoplasmic domain of fn14, like other members of the tnfr superfamily, does not contain consensus amino acid sequences characteristic of domains with enzymatic activity . This interaction can stimulate a variety of biological responses, depending on the cell type analyzed . (2008) hypothesized two modes of tweak - fn14 (ligand - receptor) interaction: (i) the ligand - dependent interaction, which involves the higher concentration of homotrimeric tweak, that binds to low concentration of fn14 in a heterohexameric complex [38, 39], and (ii) ligand - independent interaction when the ligand concentration is lower than the receptor concentration . Here they are the canonical and noncanonical nf-b pathways [21, 33, 34, 40] and the mapk pathway [4143] with possible binding to traf proteins . These diseases include autoimmune disorders [16, 21, 44, 45], neurological disorders [46, 47], periodontal disease, and cancers [3, 22, 24, 4850]. Because of its multifunctional properties, tweak is also being considered for use in therapeutics . It is also being considered as a potential early and prognostic biomarker for conditions such as kidney injury [52, 53], sle, atherosclerosis [55, 56], cardiovascular disorders [5759], immune preconception marker, and abdominal aortic aneurysms . Although the results obtained to date are captivating, it is clear that additional studies are required to determine whether tweak, and/or fn14 could be novel molecular targets for developing anticancer and antiautoimmune therapeutic agents in humans . Thus, given its importance in the field of biomedical research, we carried out an extensive and iterative compilation of tweak - fn14 signaling pathway by literature mining . Information gathered on protein - protein interactions, posttranslational modifications, protein transportation events, and regulation of gene expression, which are stimulated by tweak were compiled into a signaling pathway using a visualization tool, pathvisio . Our compiled data will be useful for the scientific community to explore, further, the role of tweak in differential disease pathogenesis, in biomarker development . Using similar approach, we have also developed signaling pathways on leptin receptor activator for nuclear factor b ligand (rankl) and follicle stimulating hormone (fsh). In the current study, we have generated a reaction map of tweak signaling pathway, which is available for visualization at netslim (http://www.netpath.org/netslim/), an accessory resource for visualization of netpath pathways . Pubmed searches were performed using tweak or fn14 and their alternate names as keywords to retrieve relevant articles pertaining to tweak signaling . The articles were screened to capture molecular reactions stimulated by tweak in mammalian cells as compared to the corresponding unstimulated state . Thereafter, with the use of an in - house developed software, pathbuilder that enables conversion of pathway data into standard community formats, namely, psi - mi, biopax, and sbml formats, we annotated biological information and reactions pertaining to tweak signaling . These included protein - protein interactions, enzyme - substrate reactions, gene regulation events, and also various activation / inhibition reactions under a tweak stimulus . These data after manual revision were exported to the netpath database, (http://www.netpath.org/), a manually assembled resource for signaling pathways generated by our group which provides the criteria for data compilation . The protein - protein interactions gathered from several experimental platforms were cataloged from literature into either binary or complex interactions . A binary interaction represents the interaction of two proteins either in homomeric or heteromeric form . A complex protein interaction comprises reactions involving more than two proteins, which again can be either homomeric or in heteromeric . For every protein - protein interaction, we documented information on subcellular localization, the experimental method used, the name and species of cell models, and finally, the hyperlinked pubmed identifier for the corresponding publication . We compiled the posttranslational modifications under tweak stimulus and mapped them to their corresponding protein sequences in the refseq database . Further, activation or inhibition of the substrate in response to the stimulus was also compiled . The notable modifications chosen were phosphorylation, acetylation, ubiquitination, sumoylation, protein degradation, and methylation . Direct included those reactions where the enzyme has been reported for the specific type of protein (substrate) modification . Indirect reactions include those where the type of modification is experimentally proved; however no information exists about its immediate upstream enzyme . The features added for the enzyme - substrate reactions include the type of posttranslational modification, the site and residue of each modification, the source of protein, the species used, and cellular localizations of the respective reaction . Additionally, we have incorporated a pubmed identifier as a hyperlink pertaining to the reaction . Several molecules, including the caspases-3, -7, and -8 (casp3, casp7 and casp8), jun [20, 29, 69, 70], and nik (map3k14) [52, 71], were activated, whereas stat1 was inhibited under tweak stimulus . These molecules do not abide by the enzyme - substrate reactions and protein - protein interaction parameters as described previously and thus cannot be connected directly to the main frame of the tweak pathway and are referred to as orphan molecules . We have provided the source of protein, subcellular localization, species, and cell line in which the activation or inhibition event was reported . Subcellular transportation events of proteins under the influence of tweak reported to date, with appropriate gene ontology terms, were added into the pathbuilder tool . These events were selected on the basis of the posttranslational modifications, physical interaction or regulatory events . A tweak stimulus resulting in subcellular relocalization of proteins was evidenced by fluorescent microscopy and immunohistochemical studies . In addition to a particular protein's altered localization, we have also documented the source of protein and cell lines used . We have documented genes whose expressions are regulated by the tweak - fn14 signaling in humans . Such genes that have been identified by various groups at the mrna level were catalogued from dna microarray and nonarray - based experiments such as northern blotting, quantitative rt - pcr, or sage . Further, we have included transcription regulators (transcription factors, or their coactivators / corepressors) downstream of tweak - fn14 stimulus . Some of these transcription regulators are involved in the regulation of the genes (mentioned above) upon tweak signaling . Such transcriptional regulators have been identified by approaches such as chromatin immunoprecipitation assays, electrophoretic mobility shift assays, gene silencing, and promoter activity assays in tweak - fn14 signaling . Data for protein - protein interactions, catalytic reactions, and transportation events were collected from diseased or normal mammalian sources that include humans and their orthologs . The manually assembled data in pathbuilder were compiled and imported into netpath (explained under methodology). A composite map of pathway reactions pertaining to tweak signaling were generated using pathvisio by following the netslim parameters as have been employed earlier by our group . Netslim (http://www.netpath.org/netslim/) is a tributary of netpath, which projects or summarizes only stringent reactions pertaining to the specific receptor - ligand complex compiled in a particular study, for example, tweak in this case . The criteria for selecting high confidence reactions for tweak pathway are provided in the netslim database (http://www.netpath.org/netslim/criteria.html). We show here for the first time in any scientific repository a pathway illustration under tweak stimulus . Given the multifunctional properties of tweak, we carried out a comprehensive literature search under tweak stimulus followed by manual amassment, thereafter reviewing and adding the data into netpath database . Fifty - eight articles were found relevant to our amassment criteria from amongst 357 articles published between 1997 and 2011 . This study led to the documentation of 46 unique proteins amid which 17 were associated with protein - protein interactions, 20 involved in enzyme - substrate reactions, 13 involved in activation - inhibition reactions, and 8 were identified to be translocated from cytoplasm to nucleus . There were 28 genes identified to be differentially regulated under tweak stimulus in human systems . The data for visualization of tweak signaling pathways were obtained after filtering netpath data using netslim parameters . A total of 36 molecules involved in 42 reactions are visually depicted in the tweak pathway in netslim . The tweak data in netpath are available freely and can be used by the scientific community . The data are represented in various standard exchange formats that include biological pathway exchange (biopax), systems biology markup language (sbml) and proteomics standards initiative molecular interaction (psi - mi) language formats . The psi - mi is a community standard language for molecular interaction data used for data comparison and exchange . Biopax is another standard language that has features compatible with sbml and psi - mi formats . The tweak signaling representation can be downloaded from the netslim database in various formats, such as gpml, genmapp, png, and pdf . The gene regulation data are made available in tab - limited and microsoft excel formats . A pathway module is defined as an established cascade of events that takes place inside a cell that has no defined boundaries and is part of a generic network . Some well - known modules are the nf-b, mapk, the jnk pathways and the pi3k / akt pathway modules . A schematic model of the tweak pathway with identified pathway modules is represented in figure 3 . The tweak - fn14 complex binds to the traf molecules, traf 1, 2, 3, and 5 . However, the downstream signaling cascade(s) that proceeds upon the association of tweak - fn14 complex and traf 1/3/5 (traf1, traf3, traf5) is unavailable due to the lack of published studies to date . It was possible to decipher the downstream events following the formation of the traf2-ciap1 (birc2) complex . The degradation of the traf2-ciap1 complex leads to the stabilization of nik and activation of the noncanonical nf-b pathway as represented in the model . The degradation of the traf2-ciap1 complex also leads to the activation of the caspase pathway resulting in the apoptosis of tumor cells [11, 27]. Ikner and ashkenazi have shown that tweak activates apoptosis through the formation of a rip1-fadd - caspase8 complex by tnf mediated signaling, wherein ciap1 plays a crucial role . A possible role of tweak has been reported in bone and cartilage damage . In fibroblast - like synoviocytes, experimental evidence indicates that tweak - fn14 complex formation leads to the activation of p38 (mapk14), erk1/2 (mapk3/mapk1), jnk1/2 (mapk8/mapk9), and tak1 (map3k7). No evidence has been obtained from existing literature for further direct downstream targets of p38 and erk1/2 . However, the activation of tak1 leads further to the downstream activation of the nf-b / p65/p50 pathway . Also, rac1 has been reported to interact directly with the tweak - fn14 complex leading to activation of the nf-b pathway . Activation of akt via phosphorylation has been observed under tweak stimulus with an exception in the case of skeletal muscle . Akt phosphorylation leads further to the inactivation of gsk3 resulting in an increase in levels of phospho - gsk3 and active (dephosphorylated) -catenin1 (ctnnb1). The cytoplasmic accumulation of active -catenin1 results in its nuclear translocation . In addition to binding of tweak with fn14, we have also documented the binding of cd163 and dr3 with tweak . Since the interaction between tweak and dr3 remains controversial [28, 29] and the downstream consequences of a possible tweak - cd163 interaction remain to be explored, the pathway illustration does not elaborate on the downstream events for these interactions . The ever increasing experimental data on the various molecular events taking place following ligand - receptor interactions, in this case between tweak and fn14, make it essential to have a repository for the data and also to create a signaling pathway summary . Our current work, which incorporates the tweak - signaling pathway data into netpath, would open avenues for further studies of tweak - associated proteins and related disorders, such as cancers and autoimmune diseases . To our understanding, this study compiles for the first time tweak induced signaling events; these include (i) the inactivation of gsk3 followed by dissociation of -catenin1, (ii) the proapoptotic nature of tweak mediated through the expression of tnf, which further leads to the activation of caspase8, and (iii) the association between tweak and ciap proteins (1 and 2) [11, 76]. We believe that our data will be informative in therapeutic studies, in selecting / pathological events and the simultaneous production of blocking agents . Repository is dynamic and will allow a progressive update of relevant data, as more published literature is introduced . In addition to the direct usage of the data stored in the netpath database, data can also be exported to other databases, enabling comparison and sharing amongst multiple databases, especially those which have compatible language formats, such as biopax . Despite the minimal amount of data, ours can also be used in the overlay of various high - throughput data enabling pathway analysis and can be accessed by any pathway resource to generate a customized pathway . We are currently working on the features in netpath, which are incompatible with biopax, especially the ontology hierarchy that has been proposed by the biopax group . To our knowledge, our compilation of data in netpath will allow, for the first time for any available scientific repository, a comprehensive study of the tweak pathway and its potential biomedical applications.
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Colorectal cancer (crc) remains the second leading cause of cancer deaths in the united states (us) (1), despite the high survival rate from early treatment (2). It is estimated that 136830 new cases of colorectal cancer will be recorded in 2014 in the us and 50310 of these cases will die from the disease (3). Cancer of the colon and rectum affects both males and females equally, and the risk increases with age (3, 4). The survival rate for colorectal cancer is very low at late diagnosis of the disease . Five - year survival rates as high as about 90% have been reported for tumors detected and removed before extension (2). Evidence also indicates a reduction in the survival rate to about 70% for tumors which have already extended, and as low as 13% when metastasis has already occurred (2). Decline in crc mortality rates in the us has been attributed to early detection and surgical removal of the tumor before metastasis (5). Early detection and early treatment are possible due to the availability of effective and relatively inexpensive crc screening tests (6). A clinical trial showed 33 and 43% reductions in incidence and mortality of crc respectively as result of a single sigmoidoscopy screening of adults between 55 and 64 years (7). In the us, crc screening is covered by most health plans and there is a published guideline for crc screening (8). However, uptake of crc screening is relatively low, and it is about 50% of those for whom the test is highly recommended (9 - 11). This calls for public health efforts to increase awareness, acceptance and uptake of crc screening, especially for those with increased risk . Reported predictors of crc screening include age, educational level, income level, and health insurance status (9, 12). It therefore raises questions about the low uptake of crc compared to screening programs for other cancers . This calls for more investigation into predictors of crc screening uptake to inform policy and intervention planning . The link between depression, alcohol and tobacco use; and screening uptake have also been investigated . A study investigated the influence of depression on other cancer screening among breast cancer survivors in latino . A recent survey in washington state in the us identified depression as a significant barrier to cervical cancer screening uptake (14). However, kaida and co - workers (15) observed that age played an important role in the relationship between depression and cervical cancer screening . In contrast, kodl and co - workers observed a significant increase in crc uptake among those with mental health diagnosis in a bivariate analysis (16). However, inverse association was observed when they controlled for timing of diagnosis and outpatient visits ., it was observed that lower depression indirectly increases uptake of crc screening through a better self - rated health (17). These findings call for more investigation into the relationship between mental health and crc screening to inform policy and intervention . However, to the best of our knowledge, the relationship between insomnia and crc screening uptake has not yet been evaluated . Again, racial differences in the influence of depression on crc screening have not yet been evaluated . We examined the influence of and racial differences in depression, insomnia, alcohol use, and tobacco use on crc screening uptake in the usa . The national health interview survey (nhis) is a multi - purpose health survey which is conducted by the national center for health statistics (nchs), center for disease control and prevention (cdc). It is a principal source of health information of the civilian noninstitutionalized household population of the us . Public - use data files are released annually and can be accessed from the internet . From each family in the nhis, one adult aged 18 years or older is randomly selected to respond to sample adult core questionnaires . The 2012 nhis sample is the largest sample size since the current sampling strategy came into force in 2006 . Analysis of 2012 data from the national health information survey (nhis) was done to determine associations between depression, insomnia, alcohol use and tobacco use, and colorectal cancer screening in participants . Information on depression, insomnia, alcohol use and tobacco use was self - reported . Participants were considered to have had a crc screening (case) if they responded yes to the question test for colon cancer, past 12 months (table 1) demographic variables collected and used in this analysis included age, classified as young (18 - 44 years), middle aged (45 - 64 years), and elderly (65 years or older); gender; race / ethnicity (white, african american (aa), asian and other). Marital status was classified into married / living with partner; widowed / divorced / separated; and single . Alcohol consumption was classified as never, current light or moderate drinking, and past drinking . Income level was categorized into low income (0 - 34999 usd), middle income (35000 - 74999 usd) and high income (75000 usd). Population proportions in cases and controls of independent variables and demographic factors were estimated by proc surveyfreq procedure in sas . Proc surveymeans procedure was also used to estimate the overall prevalence of crc screening; while sas proc surveyfreq was used to estimate the prevalence in potential factors . Odds ratios (ors) and their 95% confidence intervals (cis) were estimated for each of the suspected predictors of crc screening by prc surveylogistic procedure . A multiple logistic regression was performed for all races combined and then stratified by race (white and aa). All the analyses were conducted with sas statistical software, version 9.2 (sas institute, cary, nc, usa). The national health interview survey (nhis) is a multi - purpose health survey which is conducted by the national center for health statistics (nchs), center for disease control and prevention (cdc). It is a principal source of health information of the civilian noninstitutionalized household population of the us . Public - use data files are released annually and can be accessed from the internet . From each family in the nhis, one adult aged 18 years or older is randomly selected to respond to sample adult core questionnaires . The 2012 nhis sample is the largest sample size since the current sampling strategy came into force in 2006 . Analysis of 2012 data from the national health information survey (nhis) was done to determine associations between depression, insomnia, alcohol use and tobacco use, and colorectal cancer screening in participants . Information on depression, insomnia, alcohol use and tobacco use was self - reported . Participants were considered to have had a crc screening (case) if they responded yes to the question test for colon cancer, past 12 months (table 1). Subjects who answered no to the question served as controls . Demographic variables collected and used in this analysis included age, classified as young (18 - 44 years), middle aged (45 - 64 years), and elderly (65 years or older); gender; race / ethnicity (white, african american (aa), asian and other). Marital status was classified into married / living with partner; widowed / divorced / separated; and single . Alcohol consumption was classified as never, current light or moderate drinking, and past drinking . Income level was categorized into low income (0 - 34999 usd), middle income (35000 - 74999 usd) and high income (75000 usd). Abbreviations: aa, african american . Population proportions in cases and controls of independent variables and demographic factors were estimated by proc surveyfreq procedure in sas . Proc surveymeans procedure was also used to estimate the overall prevalence of crc screening; while sas proc surveyfreq was used to estimate the prevalence in potential factors . Odds ratios (ors) and their 95% confidence intervals (cis) were estimated for each of the suspected predictors of crc screening by prc surveylogistic procedure . A multiple logistic regression was performed for all races combined and then stratified by race (white and aa). All the analyses were conducted with sas statistical software, version 9.2 (sas institute, cary, nc, usa). There were 21511 participants included in the analysis, comprising 9542 (44%) males and 11966 (56%) females . A higher percentage of males reported having crc screening in the last 12 months (54% vs. 46%). Proportion of older adults was higher in cases than in controls (36% vs. 27%). African - american (aa) adults and married also formed higher proportions in cases than in controls (13% vs. 10%, 70% vs. 66%, respectively). A higher proportion of cases had higher income than controls (41% vs. 36%). Table 2 illustrates the prevalence of crc screening by demographic factors and other independent variables . The prevalence of crc screening in adults with cancer, depression and insomnia was significantly higher than those without the conditions (26% vs.18%; 21% vs.18%; and 21% vs.18%, respectively). Prevalence of crc screening was highest in past smokers compared to current smokers and never smokers (23% vs. 16% vs. 18%). Participants with past alcohol use recorded the highest prevalence of crc screening compared to current and never alcohol users (20% vs.19% vs. 16%). Results of both univariate and multiple logistic regression analyses of the association between crc screening and all factors included in the model are shown in table 3 . There were significant associations between crc screening and all factors examined in the unadjusted analysis (p <0.05). Adjusting for all other factors, cancer history (or = 1.51, 95%ci = 1.32 - 1.73); moderate alcohol drinking (or = 1.16, 95%ci = 1.01 - 1.30); past smoking (or = 1.17, 95%ci = 1.04 - 1.32); depression (or = 1.37, 95%ci = 1.18 - 1.58); aa race (or = 1.74, 95% ci = 1.53 - 1.98) and insomnia (or = 1.18, 95%ci = 1.06 - 1.32) were all positively associated with crc screening . Females (or = 0.73; 95% ci = 0.66 - 0.82) and single (or = 0.78, 95% ci = 0.66 - 0.91) were inversely associated with crc screening prevalence . Compared to young adults (18 - 44 years), screening was significantly higher in middle - aged and the older adults (65) (or = 2.91, 95%ci = 2.35 - 3.62 and or = 3.76, 95%ci = 2.99 - 4.74, respectively). Compared to low income earners (0 - 34999 usd), crc screening was significantly higher in middle income (35000 - 74999 usd) and high income (75000) earners (or = 1.32, 95% ci = 1.17 - 1.48; and or = 1.50, 95% ci = 1.31 - 1.73, respectively). Abbreviations: aa, african american; or, odds ratio; ci, confidence interval . Adjusting for all other factors, age, income and depression showed significant positive association with crc screening in both whites and aas . In both races, females were less likely to screen for crc . While the single were significantly less likely to receive crc screening among whites (or = 0.75, 95%ci = 0.61 - 0.92), marital status was not significantly associated with crc screening among aa (p> 0.4). Cancer history was positively associated with crc screening in whites (or = 1.55, 95%ci = 1.33 - 1.79) but not significant in aas (p> 0.1). There was a 47% significant increase in crc screening in past alcohol users among aas (95% ci = 1.06 - 2.09) but alcohol use was not associated with crc screening in whites (p> 0.1). In both races, crc screening was slightly but not significantly reduced in current smokers, however, past smoking was positively associated with crc screening in whites (or = 1.29, 95% ci = 1.09 - 1.53) but not in aas . History of insomnia also increased crc screening in whites (or = 1.17, 95% ci = 1.03 - 1.34) but it was not significantly associated with crc screening in aas . Abbreviations: aa, african american; or, odds ratio; ci, confidence interval . There were 21511 participants included in the analysis, comprising 9542 (44%) males and 11966 (56%) females . A higher percentage of males reported having crc screening in the last 12 months (54% vs. 46%). Proportion of older adults was higher in cases than in controls (36% vs. 27%). African - american (aa) adults and married also formed higher proportions in cases than in controls (13% vs. 10%, 70% vs. 66%, respectively). A higher proportion of cases had higher income than controls (41% vs. 36%). Table 2 illustrates the prevalence of crc screening by demographic factors and other independent variables . The prevalence of crc screening in adults with cancer, depression and insomnia was significantly higher than those without the conditions (26% vs.18%; 21% vs.18%; and 21% vs.18%, respectively). Prevalence of crc screening was highest in past smokers compared to current smokers and never smokers (23% vs. 16% vs. 18%). Participants with past alcohol use recorded the highest prevalence of crc screening compared to current and never alcohol users (20% vs.19% vs. 16%). Results of both univariate and multiple logistic regression analyses of the association between crc screening and all factors included in the model are shown in table 3 . There were significant associations between crc screening and all factors examined in the unadjusted analysis (p <0.05). Adjusting for all other factors, cancer history (or = 1.51, 95%ci = 1.32 - 1.73); moderate alcohol drinking (or = 1.16, 95%ci = 1.01 - 1.30); past smoking (or = 1.17, 95%ci = 1.04 - 1.32); depression (or = 1.37, 95%ci = 1.18 - 1.58); aa race (or = 1.74, 95% ci = 1.53 - 1.98) and insomnia (or = 1.18, females (or = 0.73; 95% ci = 0.66 - 0.82) and single (or = 0.78, 95% ci = 0.66 - 0.91) were inversely associated with crc screening prevalence . Compared to young adults (18 - 44 years), screening was significantly higher in middle - aged and the older adults (65) (or = 2.91, 95%ci = 2.35 - 3.62 and or = 3.76, 95%ci = 2.99 - 4.74, respectively). Compared to low income earners (0 - 34999 usd), crc screening was significantly higher in middle income (35000 - 74999 usd) and high income (75000) earners (or = 1.32, 95% ci = 1.17 - 1.48; and or = 1.50, 95% ci = 1.31 - 1.73, respectively). Abbreviations: aa, african american; or, odds ratio; ci, confidence interval . Adjusting for all other factors, age, income and depression showed significant positive association with crc screening in both whites and aas . In both races, females were less likely to screen for crc . While the single were significantly less likely to receive crc screening among whites (or = 0.75, 95%ci = 0.61 - 0.92), marital status was not significantly associated with crc screening among aa (p> 0.4). Cancer history was positively associated with crc screening in whites (or = 1.55, 95%ci = 1.33 - 1.79) but not significant in aas (p> 0.1). There was a 47% significant increase in crc screening in past alcohol users among aas (95% ci = 1.06 - 2.09) but alcohol use was not associated with crc screening in whites (p> 0.1). In both races, crc screening was slightly but not significantly reduced in current smokers, however, past smoking was positively associated with crc screening in whites (or = 1.29, 95% ci = 1.09 - 1.53) but not in aas . History of insomnia also increased crc screening in whites (or = 1.17, 95% ci = 1.03 - 1.34) but it was not significantly associated with crc screening in aas . Abbreviations: aa, african american; or, odds ratio; ci, confidence interval . In the combined analysis, increasing age, high income, being male, depression, insomnia, past smoking, moderate alcohol drinking, and aa race were all associated with increased crc screening . Crc screening prevalence was significantly reduced in the single compared to the married or living with partner . In a stratified analysis by race groups, significant differences in crc screening predictors were observed between whites and aas . There was a significant increase in crc screening with increasing age, increasing income level, and depression in both races . Similar to the combined analysis, females were less likely to screen for crc in both whites and aas . Cancer history, marital status, and insomnia were associated with crc screening in whites but not in aas . Past alcohol drinking increased screening in aas but alcohol was not associated with screening in whites . The relationship between age, gender, income level and crc screening have been widely reported in the literature, and our findings are in line with those already reported (9, 12). However, in contrast with findings in the literature (9, 12), our analysis found a significant increase in crc screening among aas compared to whites . This increase may stem from perception of risk . Though our analysis cannot fully explain why crc screening is higher in aas than whites, it is important to note that incidence of and mortality from crc have been reported to be higher in aas than all other racial / ethnic groups in the usa (20 - 24). Awareness of this statistics can trigger uptake of screening as has been shown previously (25). The observation that moderate alcohol drinking was associated with increase crc screening is similar to previously reported influence of alcohol consumption on mammography (26). However, our results indicate that the relationship between moderate alcohol drinking and cancer screening may be race - dependent . When stratified by race, the observed 16% increase in crc screening in moderate alcohol consumption completely attenuated . It resulted in 47% significant increase in crc screening among past alcohol users but only in aas . We have found no significant difference in crc screening between current and never smokers in contrast to earlier finding that smoking reduces cancer screening (27, 28). In line with a previous report (10), we found past smoking to increase crc screening in the combine analysis . It suggests that campaigns to increase smoking cessation may also lead to improvement in healthy behaviors . Our findings on depression contrast most of the evidence reported earlier (13 - 15, 29). Number of outpatient visits has been found to mediate mental illness and health behavior (16). We are unable to attribute the observed influence of depression to physician visit based on our data, however, it is more plausible for depressed individuals to have increased contact with care providers (16) and the result may reflect the importance of health care provider contacts and screening uptake . This may also apply to those who reported symptoms of insomnia in the last 12 months . Insomnia may affect up to 50% of patients with cancer and increases cancer symptom burden and impairs quality of life (18, 30 - 33). However, to the best of our knowledge, this study is the first to examine the relationship between insomnia and crc screening uptake . We found that insomnia was positively associated with crc screening and the associations showed race difference . Of significant important though our data is limited to explain these differences, it suggests that race specific interventions may be important in improving uptake of crc screening . However, further studies on these variations may be important to explain the difference . Since information from participants was self - reported, our study may suffer from a recall bias as well as social desirability bias . However, recall bias is likely to be minimal since information collected was within one year . In conclusion, cancer history, smoking status, insomnia and alcohol intake status may influence crc screening uptake differently between whites and aas . To improve overall crc screening uptake in the us, it is important to consider racial differences in predictors and tailor appropriate interventions to each racial / ethnic group.
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Medulloblastoma is the most common pediatric brain tumor, with an incidence of ~0.5/100,000 children <15 years old (1). It is classified as grade iv according to the world health organization (who) (2). Current treatment consisting of surgery, radiotherapy and adjuvant chemotherapy has a limited role in improving the prognosis of patients (3,4). Novel therapeutic methods based on the specific mechanism of medulloblastoma carcinogenesis an increasing number of studies have revealed that micrornas (mirnas / mirs) serve a role in tumorigenesis (59). Gene expression is regulated by thousands of known mirnas, which bind to imperfect complementary sites within the 3 untranslated regions of their target protein - coding mrnas, and repressing the expression of these genes at the level of mrna stability and translation (10). Previous research has revealed that mirnas are involved in the regulation of the majority of physiological and pathological process, including development, life span, and metabolism, and their dysregulation contributes to a number of types of disease, notably cancer (11). These small rnas may function as oncogenes or tumor suppressors by modulating the levels of critical proteins, and their relevance in human disease and therapy is currently under investigation (12). An increasing number of studies have demonstrated abnormal mirna expression levels in the central nervous system (cns) of cancer patients, suggesting that mirnas may serve as key regulatory components in cancer of the cns (5,10,13). It was observed that 22 mirnas were upregulated and 26 mirnas were downregulated in the tumor tissue . Ingenuity pathway analysis (ipa) was used to analyze the regulatory network of the differentially expressed mirna in medulloblastoma . It was proposed that the pathways exhibited in the network may have an important role in the tumorigenesis of medulloblastoma . The data were downloaded from the ncbi gene expression omnibus (geo) database (gse42657, www.ncbi.nlm.nih.gov/geo). The data include 7 control samples (consisting of 3 cerebellum and 4 frontal lobe controls) and 9 medulloblastoma samples . The data from all samples was normalized using the limma package in r software version 3.3.0 (https://www.r-project.org/). The selected samples of this profile are exhibited in table i. the differentially expressed genes (degs) were obtained with the thresholds of |logfc|>1.0 and p<0.01, using linear models and empirical bayes methods for assessing differential expression in microarray experiments in the limma package (table ii). A heat map of the different groups was produced to visualize the degs and cluster the corresponding groups with the differentially expressed mirnas from the tumor tissue, using the r package gplots (fig . The dataset (48 differentially expressed mirnas) was uploaded to the ingenuity pathway analysis software version 2016 (qiagen, inc ., valencia, ca, usa), using the microrna target filter to find the targeting information . A total of 40 micrornas targeting 11,757 mrnas were identified by the filter . The core analysis function (rapid assessment of the signaling and metabolic pathways, molecular networks, and biological processes that are most markedly perturbed in the dataset of interest) to analyze the associated network functions of the mirnas . Using mirna microarray data from the geo database, 48 mirnas which are differentially expressed in medulloblastoma were identified, compared with the control group (table ii). The core analysis demonstrated the molecular network interactions and signaling pathways associated with 28 differentially expressed mirnas of medulloblastoma, and their predicted molecular targets were rebuilt using ipa . The network organismal injury and abnormalities, reproductive system disease, cancer had an ipa score of 41, focusing on 17 mirna molecules, and the network cancer, organismal injury and abnormalities, cell death and survival had an ipa score of 23, focusing on 11 mirna molecules (fig . The most impacted biological processes and diseases regulated by the analyzed mirnas included organismal injury and abnormalities, reproductive system disease and cancer . The molecular network maps demonstrated that three primary components were identified to be at the central hub of the most significant network with a score of 41; these were tumor protein p53 (tp53), argonaute 2 (ago2) and mitogen activated kinases 3 and 1 (erk1/2) (fig . Tp53, sirtuin 1 (sirt1) and y box protein 1 (ybx1) were located in the center of the second network with a core analysis score of 23 (fig . The first network included mir-17 - 5p, mir-130a-3p, mir-182 - 5p, mir-18a-5p, mir-19b-3p, mir-4288, mir-96 - 5p, which were upregulated in the medulloblastoma samples, and mir-128 - 3p, mir-132 - 3p, mir-133a-3p, mir-299a-5p, mir-409, mir-668 - 3p, which were downregulated . The second notable network (fig . 2b) involved upregulated mir-217 - 5p and mir-216a-5p, and downregulated mir-329, mir-330 and mir-584, which are associated with the regulation of key regulatory genes in medulloblastoma development, including tp53, sirt1 and ybx1 . However, when the cerebellum samples were used as the control, it was observed that 4 mirnas were overexpressed and 5 mirnas were underexpressed in the medulloblastoma (table iii). The network cell death and survival, gastrointestinal disease, hepatic system disease had an ipa score of 9, focusing on 4 mirna molecules (fig . Mirnas function as master regulators and signal modulators by fine - tuning gene expression in multiple complex pathways . A number of the mirnas which exhibit dysregulated expression in medulloepithelioma have been reported to serve various roles in carcinogenesis (6,7,9). Through analysis of a geo mirna expression profiling dataset, mirnas which are over- and underexpressed in medulloblastoma were identified, including mir-217, mir-216, mir-183, mir-182 and mir-96, which are upregulated in tumor tissue . Previous research demonstrates that mir-183 - 96 - 182 regulates cell survival, proliferation and migration in medulloblastoma, and increased mir-183 - 96 - 182 expression is associated with aggressive clinical course, high rates of metastasis and poor overall survival (8,9,14). Mir-217 and mir-216 were overexpressed in medulloblastoma . However, the role of mir-217 was controversial . Mir-217 is an oncogene that is overexpressed in aggressive human b cell lymphomas (15). However, it may be a tumor suppressor in hepatocellular carcinoma (16). In pancreatic intraepithelial neoplasm, pancreatic ductal adenocarcinomas and further studies are required to determine whether increased expression of mir-217 in medulloblastoma represents an oncogenic effect, or if the dysregulation functions as a potential tumor suppressor . While mir-216 was underexpressed in various types of cancer, including breast cancer, pancreatic cancer and nasopharyngeal carcinoma (1921), it may serve as a tumor suppressor to inhibit cell proliferation, invasion and tumor growth . Further studies are required to determine whether increased expression of mir-216 in medulloblastoma represents an oncogenic effect or is a potential tumor suppressor . In the present study, marked downregulation of mir-383, mir-206, mir-138, mir-128a / b and mir-133b was identified . Mir-383, repressing peroxiredoxin 3 at transcriptional and translational levels, suppressed the cell growth of medulloblastoma (6). Additionally, mir-206 was downregulated in all the four molecular subgroups of medulloblastoma as well as in cell lines . Orthodenticle homeobox 2 (otx2), a target gene of mir-206 which is overexpressed in all non - sonic - hedgehog - driven medulloblastomas, is an oncogene in medulloblastoma . Therefore, underexpression of mir-206 contributed to the upregulation of otx2 expression and enhanced growth of medulloblastoma cell lines (7). Studies have demonstrated a reduction in the mirnas mir-383, mir-138, mir-128a / b, mir-133, mir-124 and let-7 g in medulloblastoma . Nevertheless, mir-328, mir-133b, mir-149, mir-181b and mir-154 were downregulated in the study of ferretti et al (5) and the present study . In addition, the present study revealed that mir-433, mir-488, mir-584, mir-329, mir-299 - 5p, mir-330, mir-770 - 5p, mir-656 and mir-642 were underexpressed in medulloblastoma, and further research is required to identify whether these mirnas have a specific role in the tumorigenesis of medulloblastoma . Based on microarray analysis, mirnas that distinguish normal brain from medulloblastoma tissue were identified . As presented in the heat map, the dysregulated mirnas are more closely associated with the medulloblastoma samples compared with the normal control samples . Additionally, by uploading the mirnas from the first network to the ipa, it was identified that the dysregulated mirnas were associated with key regulatory genes in tumorigenesis, including tp53, insulin - like growth factor 1 receptor, ago2 and erk1/2 . The overexpressed mirnas mir-17 - 5p and mir-182 (log fold change> 2) have interactions with tp53; studies have demonstrated an association between these mirnas and tp53 in tumorigenesis (22,23). Agos are associated with mirnas, due to their function in the rna induced silencing complex (risc), wherein they use small rna guides to recognize targets . Ago2 had an interaction with mirnas that are dysregulated in medulloblastoma, including mir-17 - 5p, mir-128, mir-96, mir-433, mir-383 and mir-138 . The second network, including mir-217 and mir-216, which are upregulated in medulloblastoma, exhibits associations with key regulatory genes in medulloblastoma development, including tp53, sirt1 and ybx1 . Sirt1 is a well understood member of the sirtuin protein class, preventing cell aging and apoptosis of normal cells (24,25). The effects of sirt1 are controversial; the protein was considered to be a tumor promoter in neuroblastoma, prostate and skin cancer (2628), whereas it was considered to be a tumor suppressor in breast and colon cancer (29,30). Therefore, sirt1 may serve a role in medulloblastoma and correlate with the formation and prognosis of human medulloblastomas (31). Recently, dey et al (32) proved that ybx1 (yb1) is upregulated across human medulloblastoma subclasses and is required for medulloblastoma cell proliferation . Furthermore, the study identified that the shh: yap: yb1:igf2 axis may be a powerful target for therapeutic intervention in medulloblastoma . When the medulloblastoma samples were compared with the normal cerebellum, 9 dysregulated mirnas were identified . The 9 mirnas were uploaded to the ipa in order to perform the core analysis . The network involved 4 downregulated mirnas and was associated with important regulators of tumorigenesis, including met proto - oncogene (met), akt serine - threonine kinase 1 and sterol regulatory element - binding factor 1 (srebf1). Previous evidence has demonstrated the association between aberrant met signaling and medulloblastoma development (33,34). Previous studies have revealed that overexpressed mir-206 may inhibit met in lung cancer (35,36), however the function of downregulated mir-206 in medulloblastoma is unknown . Srebf1 is a transcription factor, regulating lipogenesis, which is also involved in tumorigenesis (37). These network components alone are not sufficient to initiate medulloblastoma formation; the interactions between these dysregulated factors and additional genetic insults promote tumorigenesis . In conclusion, based on the mirna array data in the geo database, mirnas which are differentially expressed in medulloblastoma samples were identified . Certain dysregulated mirnas have been confirmed; however, further research is required to verify the mirnas which have not been previously identified . Additionally, the ipa core analysis, presenting the interaction of mirnas and counterpart targets, provides a method to understand the tumorigenesis of medulloblastoma . The results of the present study may provide a potent target for therapeutic intervention or diagnosis in medulloblastomas.
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The study is a retrospective, single - center, observational study approved by the institutional review board . There were 1872 patients that underwent ffr assessment between october 2002 and july 2010 at our institution . From 2002 to 2008, an ffr value 0.75 was used to defer revascularization based on the defer (ffr to determine appropriateness of angioplasty in moderate coronary stenoses) study.3 in 2008, an ffr value> 0.80 was used to defer lesions based on the fame (fractional flow reserve versus angiography for multivessel evaluation) trial.4 of these 1872 patients, 742 patients with 906 coronary lesions were deferred revascularization based on ffr . Of the 742 patients, 21 patients or 24 lesions were excluded because of loss of follow - up after ffr assessment . Furthermore, 47 patients or 68 lesions were excluded because of ffr less than or equal to 0.80 . Thus, the final population for this study consisted of 674 patients with 816 lesions that were deferred revascularization based on ffr> 0.80 (figure 1). Ffr was performed according to standard techniques as detailed in a previous observational study using this study population.18 ffr was measured at maximal hyperemia after administering intracoronary (n=812 lesions) or intravenous adenosine (n=4 lesions), with the dose determined by the operator . Ffr was not performed on lesions with less than thrombolysis in myocardial infarction (timi) 3 flow . Every patient included in the study was followed from the date of index stenting until march 12, 2013 by reviewing the medical records and/or telephone interview . All angiograms were reviewed independently by at least 2 authors . For patients with follow - up or hospitalizations outside our institution, the medical records, including angiograms, were obtained for review . The primary outcome was a composite of cardiovascular (cv) death, mi, or deferred lesion intervention (dli). Secondary outcomes included a composite of cv death or mi and mi or dli, as well as the individual endpoints of cv death, mi, mi - lesion, deferred lesion failure (dlf), and dli . Cv death and mi were defined by the academic research consortium guidelines.19 dli was defined as any percutaneous coronary intervention (pci) performed within 5-mm proximal or distal to, or any coronary artery bypass graft (cabg) placed distal to, a lesion deferred revascularization based on the index ffr assessment.18 mi lesion was defined as any mi that was directly attributable to a lesion deferred revascularization at the index ffr . The study population was divided into acs and non - acs groups based on clinical diagnosis at the time of index ffr assessment . The acs group included unstable angina (n=257), non - stemi (nstemi; n=70), and stemi (n=7) patients . Patient characteristics were compared between the acs and non - acs groups using the student 2-sample t test for continuous variables and fisher s exact test for categorical variables . Non - normal and ordinal variables were reported as median (25th, 75th percentiles) and compared using the kruskal lesion characteristics were compared using models developed from generalized estimating equation (gee) methods to account for patients having multiple lesions (clustering). Lesion characteristics that were continuous were evaluated by gee methods using a normal probability distribution . Owing to the inherent risk for subsequent adverse cardiac events post - acs that persist despite statistical adjustment, the clinical outcomes between acs and non - acs groups were not compared directly . Instead, the association between ffr and clinical outcomes was evaluated within each group using cox proportional hazards modeling . A marginal cox model was used to account for correlated data for patients with multiple lesions.20 tests of interaction were performed to determine whether the ffr association with outcome was different between acs and non - acs groups . Furthermore, for the outcomes of a composite of cv death, mi or dli, composite mi or dli, and dlf, a multivariable analysis was performed adjusting for age, diabetes, smoking status, previous cad (including pci or cabg), congestive heart failure, chronic kidney disease, and myocardial jeopardy index.21 for the association between ffr and clinical outcomes as well as tests of interaction, a value of p<0.05 was considered statistically significant.22 all statistical analyses were performed using sas software (version 9.4; sas institute inc ., cary, nc). The study is a retrospective, single - center, observational study approved by the institutional review board . There were 1872 patients that underwent ffr assessment between october 2002 and july 2010 at our institution . From 2002 to 2008, an ffr value 0.75 was used to defer revascularization based on the defer (ffr to determine appropriateness of angioplasty in moderate coronary stenoses) study.3 in 2008, an ffr value> 0.80 was used to defer lesions based on the fame (fractional flow reserve versus angiography for multivessel evaluation) trial.4 of these 1872 patients, 742 patients with 906 coronary lesions were deferred revascularization based on ffr . Of the 742 patients, 21 patients or 24 lesions were excluded because of loss of follow - up after ffr assessment . Furthermore, 47 patients or 68 lesions were excluded because of ffr less than or equal to 0.80 . Thus, the final population for this study consisted of 674 patients with 816 lesions that were deferred revascularization based on ffr> 0.80 (figure 1). Ffr was performed according to standard techniques as detailed in a previous observational study using this study population.18 ffr was measured at maximal hyperemia after administering intracoronary (n=812 lesions) or intravenous adenosine (n=4 lesions), with the dose determined by the operator . Ffr was not performed on lesions with less than thrombolysis in myocardial infarction (timi) 3 flow . Every patient included in the study was followed from the date of index stenting until march 12, 2013 by reviewing the medical records and/or telephone interview . All angiograms were reviewed independently by at least 2 authors . For patients with follow - up or hospitalizations outside our institution, the medical records, including angiograms, the primary outcome was a composite of cardiovascular (cv) death, mi, or deferred lesion intervention (dli). Secondary outcomes included a composite of cv death or mi and mi or dli, as well as the individual endpoints of cv death, mi, mi - lesion, deferred lesion failure (dlf), and dli . Cv death and mi were defined by the academic research consortium guidelines.19 dli was defined as any percutaneous coronary intervention (pci) performed within 5-mm proximal or distal to, or any coronary artery bypass graft (cabg) placed distal to, a lesion deferred revascularization based on the index ffr assessment.18 mi lesion was defined as any mi that was directly attributable to a lesion deferred revascularization at the index ffr . The study population was divided into acs and non - acs groups based on clinical diagnosis at the time of index ffr assessment . The acs group included unstable angina (n=257), non - stemi (nstemi; n=70), and stemi (n=7) patients . Patient characteristics were compared between the acs and non - acs groups using the student 2-sample t test for continuous variables and fisher s exact test for categorical variables . Non - normal and ordinal variables were reported as median (25th, 75th percentiles) and compared using the kruskal lesion characteristics were compared using models developed from generalized estimating equation (gee) methods to account for patients having multiple lesions (clustering). Lesion characteristics that were continuous were evaluated by gee methods using a normal probability distribution . Owing to the inherent risk for subsequent adverse cardiac events post - acs that persist despite statistical adjustment, the clinical outcomes between acs and non - acs groups were not compared directly . Instead, the association between ffr and clinical outcomes was evaluated within each group using cox proportional hazards modeling . A marginal cox model was used to account for correlated data for patients with multiple lesions.20 tests of interaction were performed to determine whether the ffr association with outcome was different between acs and non - acs groups . Furthermore, for the outcomes of a composite of cv death, mi or dli, composite mi or dli, and dlf, a multivariable analysis was performed adjusting for age, diabetes, smoking status, previous cad (including pci or cabg), congestive heart failure, chronic kidney disease, and myocardial jeopardy index.21 for the association between ffr and clinical outcomes as well as tests of interaction, a value of p<0.05 was considered statistically significant.22 all statistical analyses were performed using sas software (version 9.4; sas institute inc ., cary, nc). Baseline patient and lesion characteristics of the study population (674 patients with 816 coronary lesions) are shown in tables1 and 2 . Approximately half of the study population was deferred revascularization post - ffr assessment in the setting of non - acs (n=340 patients). The remaining patients (n=334) underwent ffr assessment during an acs, where 77% of these patients presented with unstable angina, 21% with nstemi, and 2% during stemi . The acs group patients had a higher rate of congestive heart failure and were discharged more frequently with angiotensin - converting enzyme inhibitors (aceis) or angiotensin receptor blockers (arbs) post - ffr assessment, compared to the non - acs group . However, both groups were discharged on a similar median number of optimal medical therapy medications.23 baseline patient - level characteristics values are shown as absolute numbers (percentages), meansd, or median (1st, 3rd quartile). Acei / arb indicates angiotensin - converting enzyme inhibitor / angiotensin receptor blocker; acs, acute coronary syndrome; cabg, coronary artery bypass graft; cad, coronary artery disease; ffr, fractional flow reserve; omt, optimal medical therapy; pci, percutaneous coronary intervention . All remaining comparisons between the acs and non - acs groups were not statistically significant . Baseline lesion - level and procedural characteristics values are shown as absolute numbers (percentages), meansd, or median (1st, 3rd quartile). Acs indicates acute coronary syndrome; cad, coronary artery disease; ffr, fractional flow reserve; ic, intracoronary; lad, left anterior descending artery; pci, percutaneous coronary intervention; rca, right coronary artery . All remaining comparisons between the acs and non - acs groups were not statistically significant . Multivessel cad was defined as 2 or more significant lesions (angiographic percent stenosis 50% at the time of ffr assessment . Maximum ic dose was the highest dose of adenosine given to induce hyperemia during ffr assessment . The left anterior descending artery (lad) was the predominant vessel location (42%) for deferred lesions . The acs group had more ostial lesions (25% vs. 18%) and fewer bifurcation lesions, compared to the non - acs group . The mean ffr value for the study population was 0.880.05 and was not different between the acs and non - acs groups . Maximal hyperemia for ffr assessment was induced predominantly using intracoronary adenosine with similar maximum doses for both groups (120 g). There were no significant differences between patients who were lost to follow - up and those who were included in the analysis except for the history of cad (38% vs. 68%; p=0.008), proximal circumflex lesions (27% vs. 13%; p=0.044), ffr value (0.86 vs. 0.88; p=0.033), and adenosine dose (180 vs. 120; p=0.025). Patients were followed for a mean of 4.52.1 years . Subsequent mi in the lesion that was initially deferred (ie, mi lesion) accounted for 30% of the total mis that occurred during follow - up . Acs indicates acute coronary syndrome; dlf, deferred lesion failure; dli, deferred lesion intervention; mi, myocardial infarction . Table4 stratifies outcomes based on ffr value ranges and demonstrates a numerically higher absolute event rate for each composite outcome within the 0.81 to 0.85 and 0.86 to 0.90 ranges in the acs group, compared to the non - acs group . The associations between ffr values obtained during maximal hyperemia at the time of index ffr assessment and clinical outcome within each group assessed by marginal cox proportional hazard modeling are shown in table5 . In the acs group, lower ffr values were associated with a significantly higher rate of cv death, mi or dli (hazard ratio [hr], 1.08 per 0.01 decrease; 95% confidence interval [ci], 1.03 to 1.12), mi or dli (hr, 1.09; 95% ci, 1.04 to 1.14), dlf (hr, 1.12; 95% ci, 1.06 to 1.18), mi (hr, 1.07; 95% ci, 1.00 to 1.14), and dli (hr, 1.12; 95% ci, 1.06 to 1.18). A trend toward a lower ffr value being associated with mi lesion in the acs group was also observed (hr, 1.12; 95% ci, 0.996 to 1.26). The relationship between ffr and each of the clinical outcomes was found to differ significantly between acs and non - acs groups (p<0.05 for each). There was also a trend toward lower ffr being more likely associated with mi in the acs group (p=0.05). In the non - acs group, lower ffr values were not associated with an increase in the rate of any of the clinical outcomes . Using cox proportional hazard modeling adjusting for covariates (figure 2), lower ffr values remained an independent predictor in the acs group, but not the non - acs group of cv death, mi, or dli (hr, 1.07; 95% ci, 1.02 to 1.11; interaction, p=0.08), mi or dli (hr, 1.08; 95% ci, 1.03 to 1.13; interaction, p=0.026), and dlf (hr, 1.11; 95% ci, 1.05 to 1.17; interaction, p=0.005). A sensitivity analysis was performed including the 21 patients lost to follow - up and did not show any difference in adjusted hrs . Acs indicates acute coronary syndrome; cv, cardiovascular; dli, deferred lesion intervention; ffr, fractional flow reserve; mi, myocardial infarction . Cox proportional hr per 0.01 unit decrease in ffr acs indicates acute coronary syndrome; dlf, deferred lesion failure; dli, deferred lesion intervention; ffr, fractional flow reserve; hr, hazard ratios; mi, myocardial infarction . For comparing hrs between acs and non - acs groups a marginal cox proportional hazard model was performed to adjust for age, diabetes, smoking status, previous coronary artery disease (including pci or cabg), congestive heart failure, chronic kidney disease, and myocardial jeopardy index . Acs indicates acute coronary syndrome; cabg, coronary artery bypass graft; dlf, deferred lesion failure; dli, deferred lesion intervention; ffr, fractional flow reserve; hr, hazard ratios; mi, myocardial infarction; pci, percutaneous coronary intervention . Baseline patient and lesion characteristics of the study population (674 patients with 816 coronary lesions) are shown in tables1 and 2 . Approximately half of the study population was deferred revascularization post - ffr assessment in the setting of non - acs (n=340 patients). The remaining patients (n=334) underwent ffr assessment during an acs, where 77% of these patients presented with unstable angina, 21% with nstemi, and 2% during stemi . The acs group patients had a higher rate of congestive heart failure and were discharged more frequently with angiotensin - converting enzyme inhibitors (aceis) or angiotensin receptor blockers (arbs) post - ffr assessment, compared to the non - acs group . However, both groups were discharged on a similar median number of optimal medical therapy medications.23 baseline patient - level characteristics values are shown as absolute numbers (percentages), meansd, or median (1st, 3rd quartile). Acei / arb indicates angiotensin - converting enzyme inhibitor / angiotensin receptor blocker; acs, acute coronary syndrome; cabg, coronary artery bypass graft; cad, coronary artery disease; ffr, fractional flow reserve; omt, optimal medical therapy; pci, percutaneous coronary intervention . All remaining comparisons between the acs and non - acs groups were not statistically significant . Baseline lesion - level and procedural characteristics values are shown as absolute numbers (percentages), meansd, or median (1st, 3rd quartile). Acs indicates acute coronary syndrome; cad, coronary artery disease; ffr, fractional flow reserve; ic, intracoronary; lad, left anterior descending artery; pci, percutaneous coronary intervention; rca, right coronary artery . All remaining comparisons between the acs and non - acs groups were not statistically significant . Multivessel cad was defined as 2 or more significant lesions (angiographic percent stenosis 50% at the time of ffr assessment . Maximum ic dose was the highest dose of adenosine given to induce hyperemia during ffr assessment . The left anterior descending artery (lad) was the predominant vessel location (42%) for deferred lesions . The acs group had more ostial lesions (25% vs. 18%) and fewer bifurcation lesions, compared to the non - acs group . The mean ffr value for the study population was 0.880.05 and was not different between the acs and non - acs groups . Maximal hyperemia for ffr assessment was induced predominantly using intracoronary adenosine with similar maximum doses for both groups (120 g). There were no significant differences between patients who were lost to follow - up and those who were included in the analysis except for the history of cad (38% vs. 68%; p=0.008), proximal circumflex lesions (27% vs. 13%; p=0.044), ffr value (0.86 vs. 0.88; p=0.033), and adenosine dose (180 vs. 120; p=0.025). Patients were followed for a mean of 4.52.1 years . Clinical outcomes after deferred revascularization based on ffr are shown in table3 . Subsequent mi in the lesion that was initially deferred (ie, mi lesion) accounted for 30% of the total mis that occurred during follow - up . Acs indicates acute coronary syndrome; dlf, deferred lesion failure; dli, deferred lesion intervention; mi, myocardial infarction . Table4 stratifies outcomes based on ffr value ranges and demonstrates a numerically higher absolute event rate for each composite outcome within the 0.81 to 0.85 and 0.86 to 0.90 ranges in the acs group, compared to the non - acs group . The associations between ffr values obtained during maximal hyperemia at the time of index ffr assessment and clinical outcome within each group assessed by marginal cox proportional hazard modeling are shown in table5 . In the acs group, lower ffr values were associated with a significantly higher rate of cv death, mi or dli (hazard ratio [hr], 1.08 per 0.01 decrease; 95% confidence interval [ci], 1.03 to 1.12), mi or dli (hr, 1.09; 95% ci, 1.04 to 1.14), dlf (hr, 1.12; 95% ci, 1.06 to 1.18), mi (hr, 1.07; 95% ci, 1.00 to 1.14), and dli (hr, 1.12; 95% ci, 1.06 to 1.18). A trend toward a lower ffr value being associated with mi lesion in the acs group was also observed (hr, 1.12; 95% ci, 0.996 to 1.26). The relationship between ffr and each of the clinical outcomes was found to differ significantly between acs and non - acs groups (p<0.05 for each). There was also a trend toward lower ffr being more likely associated with mi in the acs group (p=0.05). In the non - acs group, lower ffr values were not associated with an increase in the rate of any of the clinical outcomes . Using cox proportional hazard modeling adjusting for covariates (figure 2), lower ffr values remained an independent predictor in the acs group, but not the non - acs group of cv death, mi, or dli (hr, 1.07; 95% ci, 1.02 to 1.11; interaction, p=0.08), mi or dli (hr, 1.08; 95% ci, 1.03 to 1.13; interaction, p=0.026), and dlf (hr, 1.11; 95% ci, 1.05 to 1.17; interaction, p=0.005). A sensitivity analysis was performed including the 21 patients lost to follow - up and did not show any difference in adjusted hrs . Acs indicates acute coronary syndrome; cv, cardiovascular; dli, deferred lesion intervention; ffr, fractional flow reserve; mi, myocardial infarction . Cox proportional hr per 0.01 unit decrease in ffr acs indicates acute coronary syndrome; dlf, deferred lesion failure; dli, deferred lesion intervention; ffr, fractional flow reserve; hr, hazard ratios; mi, myocardial infarction . For comparing hrs between acs and non - acs groups . A marginal cox proportional hazard model was performed to adjust for age, diabetes, smoking status, previous coronary artery disease (including pci or cabg), congestive heart failure, chronic kidney disease, and myocardial jeopardy index . Acs indicates acute coronary syndrome; cabg, coronary artery bypass graft; dlf, deferred lesion failure; dli, deferred lesion intervention; ffr, fractional flow reserve; hr, hazard ratios; mi, myocardial infarction; pci, percutaneous coronary intervention . This study demonstrates that, for patients with coronary lesions deferred revascularization in the setting of acs based on non - significant ffr values (ie, ffr> 0.80), lower ffr values were associated with a significantly higher rate of adverse cardiac events . After adjustment, lower ffr values remained an independent predictor of cv death, mi, or dli, cv death or mi, and dlf among acs patients . The association between lower ffr values and adverse clinical outcomes was not observed for any of the composite or individual endpoints among non - acs patients deferred revascularization based on ffr . One third of the subsequent mis that occurred during follow - up were attributable to the lesion initially deferred based on ffr assessment . Our study provides new evidence that lesions with lower ffr values deferred in the setting of acs are at higher risk for subsequent adverse cardiac events . In the defer and fame trials, when compared with angiographic guidance, ffr - guided selection of lesions for the deferral or performance of pci improved clinical outcomes, although the majority of patients included in those trials had stable cad.3,4 ffr can improve the diagnostic efficiency over angiography alone in the setting of acs,24 especially in the presence of multivessel cad . However, the reliability of ffr assessment of lesions during acs has been controversial.6 ffr assessment is performed by inducing maximal hyperemia to determine the physiological significance of a coronary stenosis.25 compared with stable cad, acs is a dynamic process with the potential for lesion instability and microvascular dysfunction that has been found in patients with mi to involve both infarct - related and non - infarct - related myocardial segments, and that persists for a poorly defined period, possibly up to 6 months.7 microvascular dysfunction has also been demonstrated in patients with unstable angina, where vasoconstriction and increased distal microvascular resistance reduces coronary blood flow.26 theoretically, when contrasted with intact microvascular function, an inability to induce maximal coronary hyperemia could lead to a smaller gradient and a higher ffr value for a given stenosis.8,27 beyond microvascular dysfunction, lower oxygen consumption in an infarcted territory, abnormal vasomotion of the epicardial and resistance vessels distal to a coronary thrombosis, and/or obstruction of the microvasculature, could limit myocardial blood flow and affect ffr assessment.9 therefore, there are multiple potential mechanisms that could adversely influence the reliability of ffr in the setting of acs . Given these considerations and the fact that the decision to revascularize is largely based on an accepted ischemic threshold of 0.80,17 it appears possible that a nonsignificant ffr value that is close to the threshold in a patient with acs could underestimate lesion physiological significance and potential for causing ischemia.4,5 not inconsistent with our data, a recent meta - analysis suggested that ffr has been shown to have a continuous and independent relationship with clinical outcomes, with the suggestion that the ffr threshold for composite major adverse cv events in acs patients may exceed the current threshold of 0.80.28 several studies have previously examined the reliability of ffr for lesion assessment in the setting of acs.911 a study of 57 patients who suffered an mi 6 days before ffr assessment determined that an ffr <0.75 had an 82% sensitivity and 87% specificity for detecting inducible ischemia determined by stress single - photon emission computed tomography (spect) imaging.9 despite concluding that an ffr value of 0.75 was valid 6 days after acute mi, the investigators cautioned that the results should not be extrapolated to ffr measurements obtained during the acute phase of an mi.9 a prospective study performed spect, ffr, and pci of the infarct - related artery 3.71.3 days after an mi (73% stemi; 27% nstemi) in 48 patients and then repeated spect imaging 11 weeks later to assess for reversibility after revascularization.10 the sensitivity and specificity of an ffr value 0.75 to accurately identify reversible ischemia on spect were 88% and 50%, respectively.10 the findings of these studies provided information regarding ffr post - acs, but they may not be generalizable to our study, given that ffr measurements in the acs group in our study were obtained during the acute phase of a coronary event . To examine the reproducibility of an ffr value measured acutely to one measured after recovery from acs, a prospective study measured ffr in 112 nonculprit lesions in ami patients (75% stemi) at the time of clinical presentation and 354 days later.11 only 2 patients with an ffr value> 0.80 at the time of their mi had an ffr value <0.75 at follow - up.11 given that microvascular dysfunction may persist for an unknown interval and possibly up to 6 months after an mi,7 the follow - up time between ffr measurements in that study may not have been sufficient for full recovery and restoration of microcirculatory function . Observational studies and post - hoc analyses of randomized trials have suggested that ffr - guided revascularization is safe in acs.1216 in fame, 33% of patients (n=328) were studied in the setting of acs (unstable angina or nstemi), where 178 and 150 patients were randomized to angiographic- or ffr - guided pci, respectively.12 the benefit of ffr - guided pci, compared with angiography alone, appeared similar for the acs and stable cad (n=677) groups.12 despite a higher rate of adverse cardiac events in acs patients, compared to stable cad patients, none of the mis during follow - up reportedly occurred in the index lesion deferred pci based on ffr.4,12 nevertheless, it is notable that the number of acs patients in fame undergoing ffr - guided revascularization was relatively small with shorter follow - up, which, as acknowledged by the investigators, may have limited the power of the study to show differences among subgroups . Several additional smaller observational studies have assessed the clinical outcomes for lesions that were deferred revascularization in the setting of acs.1316 three of these studies, including a total of less than 250 acs patients, concluded that there were similar outcomes for patients deferred pci based on ffr in the setting of acs or non - acs.14,15 a more recent study of 162 patients deferred pci based on an ffr 0.75 in the setting of acs (60%) and non - acs (40%) demonstrated that patients with an ffr value of 0.75 to 0.85 and microvascular dysfunction (measured by an abnormal corrected thrombolysis in myocardial infarction frame count [ctfc]) had a significantly higher rate of adverse cardiac events, compared with patients with an ffr value> 0.85 and normal ctfc.16 similar to our study, lower ffr values in their analysis were an independent predictor of later adverse cardiac events (hr, 1.09; 95% ci, 1.01 to 1.19; p=0.03).16 previous studies assessing the clinical safety and reliability of ffr for deferring revascularization among patients with acs were limited by small sample sizes1316 and by lesion assessment by ffr beyond the acute phase of acs.12 our study represents one of the largest studies on the clinical outcomes of lesions deferred pci based on ffr, where half of the study population underwent assessment during acs . Compared with previous studies,1216 our study has a significantly higher rate of adverse cardiac events, which may be secondary to the nonselected, real - world study population and the longer follow - up (mean 4.52.1 years). Our study population also included patients with multivessel disease (64%) and left main lesions (7%), many of which were excluded from other studies.12,13 the recent prospective randomized fractional flow reserve versus angiography in guiding management to optimize outcomes in nstemi (famous - nstemi) trial demonstrated that routine ffr measurement is feasible in nstemi patients, but was not powered to assess for differences in clinical outcomes compared to angiographic - guided care.6 further prospective evidence is needed to fully elucidate the reliability, reproducibility, and safety of ffr measurements in acs . This is a retrospective, observational study conducted at a large, single urban tertiary referral center . The possibility of reporting bias and the absence of an independent clinical events committee are potential limitations . A small number of patients (n=21) were excluded from the study population because of loss to follow - up . The results may not be generalizable to different populations and hospitals of different sizes and/or level of acuity . In the acs group, culprit versus nonculprit lesions were distinguished based on the operator s judgment and this determination was therefore subjective, similar to common clinical practice . However, ffr was not performed on any lesion with less than timi 3 flow . Moreover, there may be unmeasured confounders or selection bias that contributed to differences in clinical outcomes between the acs and non - acs groups . Although ffr has been well validated as a measure of ischemia, it may have limited relevance for predicting the stability of a plaque . The study cannot determine the safety of deferring lesions based on ffr versus angiographic - guidance in the setting of acs . Although our study demonstrated an association between lower ffr values and worse clinical outcomes in acs patients, the clinical benefits of ffr - guided pci over angiography alone, as demonstrated in fame,12 may still persist in an acs population . This is a retrospective, observational study conducted at a large, single urban tertiary referral center . The possibility of reporting bias and the absence of an independent clinical events committee are potential limitations . A small number of patients (n=21) the results may not be generalizable to different populations and hospitals of different sizes and/or level of acuity . In the acs group, culprit versus nonculprit lesions were distinguished based on the operator s judgment and this determination was therefore subjective, similar to common clinical practice . However, ffr was not performed on any lesion with less than timi 3 flow . Moreover, there may be unmeasured confounders or selection bias that contributed to differences in clinical outcomes between the acs and non - acs groups . Although ffr has been well validated as a measure of ischemia, it may have limited relevance for predicting the stability of a plaque . The study cannot determine the safety of deferring lesions based on ffr versus angiographic - guidance in the setting of acs . Although our study demonstrated an association between lower ffr values and worse clinical outcomes in acs patients, the clinical benefits of ffr - guided pci over angiography alone, as demonstrated in fame,12 may still persist in an acs population . For patients with intermediate severity coronary lesions that are deferred revascularization in the setting of acs based on ffr> 0.80, lower ffr values are associated with a significantly higher rate of adverse cardiac events . The association between lower ffr values and adverse clinical outcomes was not observed among deferred lesions in non - acs patients . Further study is needed to determine the reliability and safety of deferring lesions with relatively lower ffr values in the setting of acs based on the currently accepted ffr threshold validated in predominantly non - acs patients . Dr singh is a consultant for abbott vascular, boston scientific, and volcano corp and is on the speakers bureaus of, or receives honoraria from, the medicines company, medtronic vascular, volcano corp, and st . Dr bach receives research grants from bristol - myers squibb, eli lilly, glaxosmithkline, merck, and schering - plough and is a consultant (clinical event committee activity only) for eli lilly, novo nordisk, and pfizer.
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The saint louis university androgen deficiency in the aging male (adam) questionnaire is widely used to screen late - onset hypogonadism,1 it has a sensitivity of 72%88%, and has a specificity of 20%32% for androgen deficiency in chinese populations.2,3 due to its lack of specificity, it is not appropriate to be used as a surrogate to serum testosterone testing . However, self - tests on the adam questionnaire are available online . It is not uncommon in daily practice to see middle - aged or older male patients seek help for andropause, which is indicated by the adam questionnaire . Nonetheless, the positive response to the adam questionnaire (positive adam) has been associated with depression and poorer health - related quality of life in a number of studies.47 the samples of the aforementioned studies include community - dwelling men and urological outpatients . To our knowledge, the adam questionnaire has not been investigated in psychiatric populations . Like women, men experience an age - related decline of physical and mental capcity.8 aging male symptoms are of multifactorial origin, such as medical problems, mental disorders, testosterone levels, and androgen receptor polymorphism.9 a number of studies suggest that high aging male symptoms may be associated with depressive and anxiety disorders, alexithymia,911 and poor quality of life.12,13 in the present study, we aimed to determine the prevalence of positive adam among male psychiatric outpatients and to examine the association between positive adam and depression / anxiety as well as aging male symptoms, health - related quality of life (hqol). We reanalyzed the data from the validation study of the chinese version of the aging males symptoms (ams) scale.14 in 2006, we conducted a cross - sectional survey in the psychiatric clinic of chang gung memorial hospital, linkou, taiwan . This study was approved by the local institutional review board of chang gung memorial hospital . We recruited a convenience sample of 176 men (mean age: 54.3 years; standard deviation: 10.7 years; range: 4080 years). The inclusion criteria were: 1) age 40 years or above; 2) taiwan resident; and 3) able to speak and understand mandarin . The exclusion criteria were: 1) history of psychotic, bipolar, and cognitive disorders; 2) active substance use disorder; 3) blindness, language or communication disorders; and 4) receiving psychotropic agents . We recorded demographic information such as patients age, their spouse s age, education attainment, employment status, and marital status . Both height and weight of the participants were measured to derive the body mass index (bmi) as weight (in kg) divided by the square of height (in m). The adam questionnaire is a ten - item self - administered dichotomous scale.1 its ten items include decreased libido; lack of energy; reduced strength and/or endurance; loss of height; decreased enjoyment of life; sadness / grumpiness; erectile dysfunction; deterioration of performance in sports and work; and falling asleep after dinner . A positive adam is defined as a yes to decreased libido or erectile dysfunction, or to any three other symptoms . The validated chinese version has good internal consistency (cronbach s = 0.74) and test retest reliability (pearson s correlation coefficient r=0.86).15 the hospital anxiety and depression scale (hads) is a 14-item polychotomous scale that is widely used for assessment of anxiety and depression among medical patients and the general population.16,17 respondents rate each item in a four - point response category (03: none = 0; mild = 1; moderate = 2; and severe = 3), so the possible scores ranged from 0 to 21 for the anxiety subscale (hads - a) and 0 to 21 for the depression subscale (hads - d). For hads - a varied from 0.68 to 0.93 (mean 0.83), and for hads - d from 0.67 to 0.90 (mean 0.82).18 with a threshold of 8,16 the sensitivity and specificity of hads - a and hads - d for anxiety disorders and depression are in the range of 0.700.90.18 the area under a receiver operating characteristic curve of hads for anxiety disorders and depression is in the range of 0.840.96.18 when compared with other questionnaires for anxiety and depression such as general health questionnaire and beck depression inventory, the correlations to hads - a and hads - d are in the range of 0.60 to 0.80.18 we used the validated chinese version of hads19 in this study . We used a threshold of 8 of hads - a and hads - d for presence of clinically significant anxiety and depression, respectively . The ams scale is a 17-item polychotomous scale to measure the health - related quality of life among aging men.8 respondents rate each item in a five - point response category (15: none = 1; mild = 2; moderate =3; severe = 4; and extremely severe = 5), so the possible scores ranges from 17 to 85 for the ams scale . Its 17 items include decline of one s feeling of general well - being; joint pain and muscular ache; excessive sweating; sleep problems; increased need for sleep; irritability; nervousness; anxiety; physical exhaustion; decrease in muscular strength; depressive mood; feelings of having passed one s peak; decrease in beard growth; decrease in ability / frequency to perform sexually; decrease in the number of morning erections; and decrease in sexual desire / libido . The severity of symptoms according to the total score was classified as none / little (1726), mild (2736), moderate (3749), and severe (50 or more). The cronbach s was 0.90, and the 3-week retest reliability was 0.72.14 we used a threshold of 37 for presence of moderate / severe impairment of hqol . All analyses were conducted in r version 3.1.1 with packages of hmisc and stargazer (r foundation for statistical computing, vienna, austria). We used two sample t - tests with equal variance for continuous or ordinal variables, and used pearson s test or fisher s exact test for categorical variables . For analysis of the 17 ams items by the response to the adam questionnaire, we used bonferroni correction to control the familywise error rate . We used logistic regression analysis to model the moderate / severe impairment of hqol by the response to adam questionnaire, as well as by anxiety and depression . The p - values were two - tailed, and the -level was set at 0.05 . We reanalyzed the data from the validation study of the chinese version of the aging males symptoms (ams) scale.14 in 2006, we conducted a cross - sectional survey in the psychiatric clinic of chang gung memorial hospital, linkou, taiwan . This study was approved by the local institutional review board of chang gung memorial hospital . We recruited a convenience sample of 176 men (mean age: 54.3 years; standard deviation: 10.7 years; range: 4080 years). The inclusion criteria were: 1) age 40 years or above; 2) taiwan resident; and 3) able to speak and understand mandarin . The exclusion criteria were: 1) history of psychotic, bipolar, and cognitive disorders; 2) active substance use disorder; 3) blindness, language or communication disorders; and 4) receiving psychotropic agents . We recorded demographic information such as patients age, their spouse s age, education attainment, employment status, and marital status . Both height and weight of the participants were measured to derive the body mass index (bmi) as weight (in kg) divided by the square of height (in m). The adam questionnaire is a ten - item self - administered dichotomous scale.1 its ten items include decreased libido; lack of energy; reduced strength and/or endurance; loss of height; decreased enjoyment of life; sadness / grumpiness; erectile dysfunction; deterioration of performance in sports and work; and falling asleep after dinner . A positive adam is defined as a yes to decreased libido or erectile dysfunction, or to any three other symptoms . The validated chinese version has good internal consistency (cronbach s = 0.74) and test retest reliability (pearson s correlation coefficient r=0.86).15 the hospital anxiety and depression scale (hads) is a 14-item polychotomous scale that is widely used for assessment of anxiety and depression among medical patients and the general population.16,17 respondents rate each item in a four - point response category (03: none = 0; mild = 1; moderate = 2; and severe = 3), so the possible scores ranged from 0 to 21 for the anxiety subscale (hads - a) and 0 to 21 for the depression subscale (hads - d). For hads - a varied from 0.68 to 0.93 (mean 0.83), and for hads - d from 0.67 to 0.90 (mean 0.82).18 with a threshold of 8,16 the sensitivity and specificity of hads - a and hads - d for anxiety disorders and depression are in the range of 0.700.90.18 the area under a receiver operating characteristic curve of hads for anxiety disorders and depression is in the range of 0.840.96.18 when compared with other questionnaires for anxiety and depression such as general health questionnaire and beck depression inventory, the correlations to hads - a and hads - d are in the range of 0.60 to 0.80.18 we used the validated chinese version of hads19 in this study . We used a threshold of 8 of hads - a and hads - d for presence of clinically significant anxiety and depression, respectively . The ams scale is a 17-item polychotomous scale to measure the health - related quality of life among aging men.8 respondents rate each item in a five - point response category (15: none = 1; mild = 2; moderate =3; severe = 4; and extremely severe = 5), so the possible scores ranges from 17 to 85 for the ams scale . Its 17 items include decline of one s feeling of general well - being; joint pain and muscular ache; excessive sweating; sleep problems; increased need for sleep; irritability; nervousness; anxiety; physical exhaustion; decrease in muscular strength; depressive mood; feelings of having passed one s peak; decrease in beard growth; decrease in ability / frequency to perform sexually; decrease in the number of morning erections; and decrease in sexual desire / libido . The severity of symptoms according to the total score was classified as none / little (1726), mild (2736), moderate (3749), and severe (50 or more). The cronbach s was 0.90, and the 3-week retest reliability was 0.72.14 we used a threshold of 37 for presence of moderate / severe impairment of hqol . The adam questionnaire is a ten - item self - administered dichotomous scale.1 its ten items include decreased libido; lack of energy; reduced strength and/or endurance; loss of height; decreased enjoyment of life; sadness / grumpiness; erectile dysfunction; deterioration of performance in sports and work; and falling asleep after dinner . A positive adam is defined as a yes to decreased libido or erectile dysfunction, or to any three other symptoms . The validated chinese version has good internal consistency (cronbach s = 0.74) and test retest reliability (pearson s correlation coefficient r=0.86).15 the hospital anxiety and depression scale (hads) is a 14-item polychotomous scale that is widely used for assessment of anxiety and depression among medical patients and the general population.16,17 respondents rate each item in a four - point response category (03: none = 0; mild = 1; moderate = 2; and severe = 3), so the possible scores ranged from 0 to 21 for the anxiety subscale (hads - a) and 0 to 21 for the depression subscale (hads - d). For hads - a varied from 0.68 to 0.93 (mean 0.83), and for hads - d from 0.67 to 0.90 (mean 0.82).18 with a threshold of 8,16 the sensitivity and specificity of hads - a and hads - d for anxiety disorders and depression are in the range of 0.700.90.18 the area under a receiver operating characteristic curve of hads for anxiety disorders and depression is in the range of 0.840.96.18 when compared with other questionnaires for anxiety and depression such as general health questionnaire and beck depression inventory, the correlations to hads - a and hads - d are in the range of 0.60 to 0.80.18 we used the validated chinese version of hads19 in this study . We used a threshold of 8 of hads - a and hads - d for presence of clinically significant anxiety and depression, respectively . The ams scale is a 17-item polychotomous scale to measure the health - related quality of life among aging men.8 respondents rate each item in a five - point response category (15: none = 1; mild = 2; moderate =3; severe = 4; and extremely severe = 5), so the possible scores ranges from 17 to 85 for the ams scale . Its 17 items include decline of one s feeling of general well - being; joint pain and muscular ache; excessive sweating; sleep problems; increased need for sleep; irritability; nervousness; anxiety; physical exhaustion; decrease in muscular strength; depressive mood; feelings of having passed one s peak; decrease in beard growth; decrease in ability / frequency to perform sexually; decrease in the number of morning erections; and decrease in sexual desire / libido . The severity of symptoms according to the total score was classified as none / little (1726), mild (2736), moderate (3749), and severe (50 or more). The cronbach s was 0.90, and the 3-week retest reliability was 0.72.14 we used a threshold of 37 for presence of moderate / severe impairment of hqol . All analyses were conducted in r version 3.1.1 with packages of hmisc and stargazer (r foundation for statistical computing, vienna, austria). We used two sample t - tests with equal variance for continuous or ordinal variables, and used pearson s test or fisher s exact test for categorical variables . For analysis of the 17 ams items by the response to the adam questionnaire, we used bonferroni correction to control the familywise error rate . We used logistic regression analysis to model the moderate / severe impairment of hqol by the response to adam questionnaire, as well as by anxiety and depression . The p - values were two - tailed, and the -level was set at 0.05 . Table 1 shows the demographic characteristics as well as the responses to the hads and ams scales by response to the adam questionnaire . Patients with positive adam had a lower bmi and scored higher in hads - a, hads - d, and ams than those with negative adam . Positive adam was not associated with anxiety or depression, but was associated with moderate / severe impairment of hqol . Table 2 shows the results of two sample t - tests with equal variance of the 17 ams items by the response to the adam questionnaire . With bonferroni correction, positive adam was associated with five items of the ams scale: ams1 (decline of one s feeling of general well - being), ams11 (depressive mood), ams15 (decrease in ability / frequency to perform sexually), ams16 (decrease in the number of morning erections), and ams17 (decrease in sexual desire / libido). Table 3 shows the results of logistic regression analysis of the moderate / severe impairment of hqol by the response to adam questionnaire, as well as by anxiety and depression . Positive adam was associated with an unadjusted 20-fold risk and an adjusted 15-fold risk for moderate / severe impairment of hqol, independent of anxiety and depression . In this study, we showed a 93.2% prevalence of positive adam among male psychiatric outpatients of age 4080 years, which was comparable to73%80% in two multicenter studies in taiwan,2,3 87.8% in hong kong,20 77.6% in saudi arabia,21 and 81.3% in chile.22 with regard to the low specificity of the adam questionnaire, our results support that the adam questionnaire would be of little value for screening late - onset hypogonadism, particularly in samples with high prevalence of psychological symptoms.22 we showed an association of positive adam and a lower bmi, echoing pastuszak et al7 but liu et al showed no such association.23 as we did not record other indicators of obesity such as waist circumference, the association of bmi and the response to the adam questionnaire requires further validation . As was the case with lee et al20 positive adam was associated with a higher score on the ams and hads scales, indicating more symptomatic and poorer health - related quality of life . However, the adam questionnaire might not be suitable for screening of anxiety or depressive disorders . By using the ams scale, we showed that the symptomatology of positive adam was characterized by impaired well - being, depressed mood, and sexual complaints among psychiatric outpatients, consistent with the initial construct of the adam questionnaire.1 although the adam questionnaire contains items of decreased enjoyment of life and sadness / grumpiness, which may be present in anxiety or depressive disorders, our results support that the adam questionnaire would measure different phenomena compared with the hads . As the adam questionnaire has only ten dichotomous items and is easier to be completed than the ams scale, it might be suitable for screening sexual symptoms among psychiatric outpatients . We showed that positive adam was associated with moderate / severe impairment of hqol independently of clinically significant anxiety and depression . As shown in our previous study,24 assessment of anxiety and depression by the ams scale might be similar to assessment of anxiety and depression by the hads . Clinicians would have to rule out anxiety and depressive disorders in patients with high ams scores . In this respect, the adam questionnaire would help identify cases who have a poor health - related quality of life and need intervention in addition to treatment for anxiety and depression.20 in particular, clinicians should explore sexual symptoms among patients who report positive adam . Several limitations of the present study should be recognized . Firstly, our results were cross - sectional and cannot indicate change over time or causality . Nonetheless, all three questionnaires used in our studies have good retest reliability.14,15,18 secondly, our study was based on a convenience sample comprised of outpatients with neurotic complaints, which was of course bound to be highly selective, and hence limited generalization from our data . Our results also cannot be applied to severely ill psychiatric patients, and it would not be appropriate to use questionnaires alone for assessment of severely ill patients . We did not perform diagnostic interviews such as the structured clinical interview for dsm - iv (scid)25 to determine psychiatric diagnoses in the sample . Nonetheless, as the hads has good sensivity and specificity for anxiety and depressive disorders,18 our results would not be considerably different whether using scid or not . Furthermore, a dimensional approach of anxiety and depression has been suggested as more powerful and homogeneous than the diagnostic and statistical manual of mental disorders (dsm - iv - tr) category of anxiety and depression.26 fourthly, we did not record confounding factors of depression and anxiety such as perceived stress and suicidal ideation, which might also explain the association between positive adam and minor anxiety and depressive symptoms . But the association between positive adam and moderate / severe impairment of hqol was robust and independent of anxiety and depression . Lastly, we did not perform hormone and medical workups as physical illness might be responsible for affective and somatic symptoms . Nonetheless, the economic and technical constraints are common to clinical settings . The adam questionnaire can be used to screen sexual symptoms but not depression / anxiety in male psychiatric outpatients . Positive adam may indicate moderate / severe impairment of hqol independent of anxiety and depression.
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The idea that the nervous system is a network of interconnected neurons has a long and illustrious history in neuroscience . Anatomical studies of the brain's cytoarchitecture, cellular circuits, and long - range fiber systems have yielded an extraordinary amount of detailed information about the brain's structural organization . The ongoing quest to map the intricate networks of the human brain with ever - increasing accuracy and resolution technological developments in noninvasive neuroimaging have opened up new avenues towards studying the structure and function of the human brain . These advances are increasingly combined with powerful network modeling tools developed in the course of a broader research effort to understand the structure and dynamics of complex systems . This recent confluence of neuroscience and network science opens up a number of new opportunities for approaching brain function from a complex systems perspective . This review is intended as a primer on current research efforts to map and model the networks of the human brain, with the long - term aim of understanding how the functioning of the brain depends on its network architecture . Modern noninvasive imaging techniques applied to the human brain allow the mapping of anatomical regions and their interconnecting pathways at near - millimeter resolution . The resulting large - scale networks provide a comprehensive description of the brain's structural connectivity, also called the human connectome . The connectome essentially comprises a complete map of the brain's structural connections . These structural connections shape large - scale neuronal dynamics which can be captured as patterns of functional and effective connectivity . Functional connectivity describes statistical patterns of dynamic interactions among regions, also called functional networks, while effective connectivity attempts to discern networks of causal influences . Functional networks can be measured with a variety of neuroimaging or electrophysiological recording methods, and they unfold within the structural networks of the connectome while the brain is endogenously active (or at rest) as well as in the course of stimulus- or task - evoked perturbations . Creating maps of structural or functional connections brings the challenge of extracting relevant or significant aspects of network organization, and this challenge can be met by applying modern network modeling and analysis tools . How these modern network approaches have enriched our understanding of brain function is the main topic of this article . The first section will provide an overview of major quantitative methods for analyzing brain network data . The following section will focus on current efforts directed at mapping networks of the human brain, with a focus on structural networks delivered by diffusion imaging and tractography . The article then turns to the important problem of linking structural networks to ongoing and evoked brain dynamics . Finally, the article examines the state of the art in using network approaches directed at uncovering the role of connectivity in brain and mental disorders . The article concludes with a brief reflection on the future promise of network approaches for understanding the function of the healthy and diseased brain . Brain networks can be derived from anatomical or physiological observations, resulting in structural and functional networks, respectively . When interpreting brain network data sets, it is important to respect this fundamental distinction . Structural connectivity describes anatomical connections linking a set of neural elements . At the scale of the human brain structural connectivity of this kind is thought to be relatively stable on shorter time scales (seconds to minutes) but may be subject to plastic experience - dependent changes at longer time scales (hours to days). In human neuroimaging studies, structural brain connectivity is commonly measured as a set of undirected links, since the directionality of projections currently cannot be discerned . Functional connectivity is generally derived from time series observations, and describes patterns of statistical dependence among neural elements . Time series data may be derived with a variety of techniques, including electroencephalography (eeg), magnetoencephalography (meg), and functional magnetic resonance imaging (fmri), and can be computed in a number of ways, including as cross - correlation, mutual information, or spectral coherence . While the presence of a statistical relationship between two neural elements is often taken as a sign of functional coupling, it must be noted that the presence of such coupling does not imply a causal relationship . Functional connectivity is highly time - dependent, often changing in a matter of tens or hundreds of milliseconds as functional connections are continually modulated by sensory stimuli and task context . Even when measured with techniques that operate with a slow sampling rate such as fmri, functional connectivity may exhibit non - stationary fluctuations (see below). Effective connectivity attempts to capture a network of directed causal effects between neural elements . As such it represents a generative and mechanistic model that accounts for the observed data, selected from a range of possible models using objective criteria like the model evidence . Recent developments in this area include approaches towards network discovery involving the identification of graph models for effective connectivity that best explain empirical data . While effective connectivity bears much promise for the future, most current studies of brain networks are still carried out on either structural or functional connectivity data sets, and hence these two modes of connectivity will form the main focus of this review . Within the formal framework of graph theory, a graph or network comprises a set of nodes (neural elements) and edges (their mutual connections). Structural and/or functional brain connectivity data recorded from the human brain can be processed into network form by following several steps, starting with the definition of the network's nodes and edges (figure 1). Nodes are generally derived by parcellating cortical and subcortical gray matter regions according to anatomical borders or landmarks, or by defining a random parcellation into evenly spaced and sized voxel clusters . Once nodes are defined, their structural or functional couplings can be estimated, and the full set of all pairwise couplings can then be aggregated into a connection matrix . To remove inconsistent or weak interactions, connection matrices can be subjected to averaging across imaging runs or individuals, or to thresholding . One approach is based on graph theory and offers a particularly large set of tools for detecting, analyzing, and visualizing network architecture . A number of surveys on the application of graph theory methods in neuroscience are available . An important part of any graph - theoretical analysis is the comparison of measures obtained from empirical networks to appropriately configured populations of networks representing a a commonly used random null model is generated by randomizing the global topology of a network while preserving local node statistics, most importantly the graph's degree sequence . Figure 2 illustrates a selection of graph measures that are widely used in studies of human brain networks . Based on the insights they deliver, they can be classified into measures reporting on aspects of segregation, integration, and influence . Segregation (or specialization) refers to the degree to which a network's elements form separate cliques or clusters . Integration refers to the capacity of the network as a whole to become interconnected and exchange information . Influence measures report on how individual nodes or edges are embedded in the network and the extent to which they contribute to the network's structural integrity and information flow . An important measure of segregation is the clustering coefficient of a given node, essentially measuring the density of connections among a node's topological neighbors . If these neighbors are densely interconnected they can be said to form a cluster or clique, and they are likely to share specialized information . The average of clustering coefficients over all nodes is the clustering coefficient of the network, often used as a global metric of the network's level of segregation . Another aspect of connectivity within local (ie, topologically connected) sets of network nodes is provided by the analysis of network motifs, constituting subgraphs or building blocks of the network as a whole . Every network can be uniquely decomposed into a set of motifs of a given size, and the distribution of different motifs can reveal which subgraphs occur more frequently than expected, relative to an appropriate null model . Measures of integration are generally based on the concept of communication paths and their path lengths . A path is any unique sequence of edges that connects two nodes with one another, and its length is given by the number of steps (in a binary graph) or the sum of the edge lengths (in a weighted graph). The length of the shortest path between each pair of nodes corresponds to their distance (also often referred to as the shortest path length), and the global average of all distances across the entire network is called the network's characteristic path length . Closely related to this measure is the global network efficiency, which is computed as the average of the inverse of all distances . One can see easily that the global efficiency of a fully connected network would be maximal (equal to one) while the global efficiency of a completely disconnected network would be minimal (equal to zero). Short path lengths promote functional integration since they allow communication with few intermediate steps, and thus minimize effects of noise or signal degradation . Measures of influence attempt to quantify the importance of a given node or edge for the structural integrity or functional performance of a network . The simplest index of influence is the node degree, and in many (but not all) cases the degree of a node will be highly correlated with other more complex influence measures . Many of these measures capture the centrality of network elements, for example expressed as the number of short communication paths that travel through each node or edge . This measure of betweenness centrality is related to communication processes, but is also often found to be highly correlated with the related measure of closeness, quantifying the proximity of each node to the rest of the network . Another class of influence measures is based on the effect of node or edge deletion on short communication paths or network dynamics . For example, vulnerability measures the decrease (or, in some cases, the increase) in global efficiency due to the deletion of a single node or edge . Hubs, but it should be noted that there is no unique way of detecting these hubs with graph theory tools . Instead, a conjunction of multiple influence measures (eg, degree, betweenness, vulnerability) should be used when attempting to identify hub nodes . While measures of segregation, integration, and influence can express structural characteristics of a network from different perspectives, recent developments in characterizing network communities or modules can potentially unify these different perspectives into a more coherent account of how a given network can be decomposed into modules (segregation), how these modules are interconnected (integration), and which nodes or edges are important for linking modules together (influence). A number of new community detection techniques have found applications in the analysis of structural and functional brain networks . One of the most commonly- used community detection algorithms is based on newman's q - metric coupled with an efficient optimization approach . Another approach called infomap identifies communities on the basis of a model of a diffusive random walk, essentially utilizing the fact that a modular network restricts diffusion between communities . In contrast, the q - metric essentially captures the difference between the actually encountered within - module density of connections compared with what is expected based on a corresponding random model, given a particular partitioning of the network into modules . Since combinatorics makes it impractical to examine all possible module partitions, an optimization algorithm is needed to identify the single partition for which the q - metric is maximized . Several methodological issues have arisen in recent years that impact the way community detection is carried out in brain networks, particularly in networks describing functional connectivity (figure 3). The first issue concerns the widespread practice of thresholding functional networks to retain only a small percentage (often less than 10%) of the strongest functional connections . In addition, the remaining connections are then set to unit strength, resulting in a greatly sparsified binary network which is then subjected to standard graph analysis . Since the appropriate value of the threshold is a free and completely undetermined parameter, most practitioners vary the threshold across a broad range and then compute and compare graph metrics for the resulting networks . The practice of thresholding functional networks has two immediate consequences, a much sparser topology which then tends to result in more and more separate clusters or modules, and a topology that discards all (even strong) negative correlations . While the status of negative correlations in resting fmri remains controversial, it could be argued that the presence of an anticorrelation between two nodes does contribute information about their community membership . Building on this idea, variants of the q - metric and other related measures that take into account the full weight distribution of a network have been proposed . These new metrics can also be applied to functional networks regardless of their density (including fully connected networks), thus eliminating the need for thresholding entirely . The second issue relates to the optimization of the module partition given a cost or quality metric like newman's q. studies of various real - world networks have shown that identifying the single optimal partition cannot only be computationally difficult, but that many real networks can be partitioned at near - optimal levels in a number of different or degenerate ways; aggregating these degenerate solutions can provide additional information about the robustness with which a given node pair is affiliated with the same or a different module . This idea has been developed further into a quantitative approach called consensus clustering . Consensus clustering has not yet been widely applied to brain networks, but it may soon become a useful tool since it provides information about the strength with which individual neural elements affiliate with their home community . An attractive hypothesis is that elements with generally weak affiliation are good candidates to assume functional roles as hub nodes that crosslink diverse communities . The next three sections of the article will review our current knowledge about the network architecture of structural brain networks, how structural networks relate to functional networks in both rest and task conditions, and what we can learn by applying network approaches to clinical problems . Due to the invasive nature of most classical anatomical methods like tract tracing, these methods cannot be applied to large samples of individual brains and they cannot be deployed in vivo, hence rendering tract tracing studies in human populations and relating structural network features to brain dynamics or behavior virtually impossible . Tract tracing has an important role to play for the study of anatomical connections in animal models, particularly in non - human primates, and it is of vital importance for validating anatomical data derived from noninvasive imaging technology . To the extent that such validation has been carried out, indications are that most projections identified by noninvasive imaging have counterparts in white matter fascicles described by classical anatomy . Most studies on human brain connectomics have been carried out by charting structural connections on the basis of data coming from diffusion mri and tractography (figure 4). Diffusion mri and traclography infer the spatial orientations and trajectories of bundles of myelinated axons traversing the brain's white matter, on the basis of measurements of the diffusion anisotropy of water or other small molecules within biological tissue . Importantly, diffusion imaging and tractography deliver inferential and statistical models of fiber anatomy but cannot directly trace or visualize anatomical connections . Methods for signal acquisition and fiber reconstruction are under continual development, with important recent advances in imaging complex (eg, intersecting) fiber architecture, and new algorithms for improved accuracy in inferring fiber pathways, including estimates of their uncertainty and evidence . Another area of important methodological development concerns the biological interpretation of connection weights resulting from aggregating fiber counts or probabilities into a connection matrix . New approaches for obtaining additional measures of white matter microstructure, eg, axonal diameters and packing densities, will likely help to refine estimates of the weight, strength, and conduction velocity of individual long - distance projections . The node degree (the number of connections attached at each node) is one of the most easily accessible graph measures and it is also highly informative, as is the distribution of node degrees across the whole network . Most, if not all, complex networks found in natural, especially biological, systems have been shown to have a broad degree distribution, with a small but important admixture of nodes that maintain considerably higher numbers of connections than most other nodes . In the human brain, node degrees appear to be distributed broadly, with some studies reporting exponential or exponentially truncated power - law distributions for node degree . Such broad (non - gaussian, log - normal) degree distributions are also seen in tract tracing studies in cortex of nonhuman primates . Virtually all studies of human brain networks have found evidence of small - world attributes, generally measured as high clustering and a short path length, or alternatively as high local and global efficiency . The presence of small - world organization is indicative of a balance between anatomical and functional segregation on the one side (indexed by clustering and local efficiency) and the capacity for global integration on the other side (indexed by the prevalence of short communication paths and global efficiency). The brain appears to be one among many examples of small - world networks encountered in many different contexts, from social to technological to biological systems . However, it should be noted that small - world attributes are not uniquely diagnostic of particular network architectures and can appear in a variety of connectivity models, including randomly rewired lattices, modular and even scale - free networks . Closer analysis of brain networks has shown that high clustering is often due to the presence of modules, or network communities of densely interconnected neural elements . Such modules are collectives of elements that share common input and output projections, exhibit similar physiological responses and form coherent functional systems . More recent studies have suggested that modularity of structural and functional brain networks extends across multiple scales, resulting in a hierarchy of nested modules - within - modules, a mode of organization encountered in other networks specialized for information - processing . In functional terms, modules allow for rapid and efficient sharing of information among brain regions that tend to contribute to a common set of tasks or responses, while promoting their functional specialization by creating boundaries that restrict the spread of information across the entire network . To ensure functional integration across modules requires specialized hub regions, generally identified by their high degree, high centrality, and diverse connection profiles that straddle the boundaries between modules . Several studies of human structural brain networks have attempted to identify hubs, and most studies have converged on a set of regions including portions of the medial and superior parietal cortex as well as selected regions in orbitofrontal, superior frontal, and lateral prefrontal cortex . Many of these regions have been previously described as multi- or transmodal association areas and exhibit complex physiological responses, diverse activation patterns across tasks, and widespread functional connectivity . These physiological characterizations are consonant with their high centrality and structural embedding as connector hubs interlinking multiple modules . Hubs perform important integrative roles in structural networks, but until fairly recently it has been unclear how they connect and interact with each other . Several early studies carried out in humans and other species had suggested a tendency for hubs to be densely interconnected in a hub complex, or a structural core (figure 5). Network studies in other disciplines have pointed to the existence of a rich club, a set of hub regions that are more densely interconnected than predicted by chance alone . Rich clubs may be significant features of network architecture as they provide a structural substrate for integrating and disseminating information across the entire network . The first report on rich club organization came from a study of cat cerebral cortex, where a rich club of hub regions was found to form a densely interconnected core circuit cross - linking all major functional subsystems . A detailed analysis of the topology of human brain structural connectivity acquired with diffusion imaging and tractography revealed a rich club of highly interconnected hub regions including portions of the superior frontal cortex, superior parietal cortex, and the precuneus, in addition to several subcortical regions including the thalamus, hippocampus, and part of the basal ganglia . Graph analysis showed that 89% of all short communication paths among non - rich club regions across the network pass through the rich club, and that damage to pathways linking rich club regions to each other had a larger disruptive effect on network communication than equal amount of damage to connections among non - rich club regions . Rich club organization has been confirmed and extended in subsequent studies focusing on the role of the rich club in brain communication, its disruption in a mental disorder, and its presence in the cerebral cortex of a non - human primate, the macaque monkey . The latter study not only demonstrated rich club organization in a directed network of inter - regional projections derived from classical tract tracing studies, but also showed again that the rich club is interspersed between structural and functional communities . The macaque rich club contains several regions of association cortex that are homologues to rich club regions found in the human brain . The emerging picture of the organization of the human connectome is one of a modular small world network, with network communities that are interlinked by a coherent sub - network or core of hub regions whose position within the overall network is strongly suggestive of a central role in global information flow and integration . The implications of such a structural core or rich club for cognition and behavior have only begun to be explored . An emerging trend in network analyses of human structural networks is to interpret network attributes not only in reference to network topology (which only considers the link structure of the network) but also in reference to the network's spatial embedding (which additionally considers the spatial positions of nodes and the lengths and trajectories of edges). This trend is fueled by the realization that many aspects of network topology are driven by the brain's spatial embedding which places tight constraints on the cost of building and maintaining networks, including wiring length and volume, metabolic energy used for signaling, and developmental mechanisms . For example, a propensity of the network to exhibit high clustering may be due to greater network economy that is conferred by mostly short projections . Indeed, modules of structural brain networks are often spatially compact with member regions located in close physical proximity and linked by relatively short projections . But long - distance projections have not only been evolutionarily conserved, they have been expanded in cases where their expansion has promoted increased network performance . These findings suggest that the benefits brought by conserved network cost are balanced in a closely negotiated trade - off with the demands of network efficiency . Interesting questions for the future concern how this trade - off is instantiated in the healthy human brain and how its disturbance might contribute to brain and mental disorders (see below). Ever since hans berger's first electrophysiological recordings, it has been recognized that the brain is never silent, but always engaged in apparently spontaneous and endogenously driven neural activity . While the investigation of endogenous neural dynamics has a long and illustrious history in the study of human eeg and meg recordings, ongoing fluctuations in the brain's blood - oxygenation - level - dependent (bold) signal acquired with fmri were long regarded as background noise, to be filtered and averaged away as an undesirable source of variability that obscured stimulus- and task - evoked neural responses . The discovery of structured correlations in spontaneous bold signals, together with the realization that many attentiondemanding tasks were accompanied not only by regional activations but also by a consistent pattern of regional deactivations, paved the way for a reconsideration of spontaneous brain activity as anatomically structured and physiologically meaningful . This reconsideration has ushered in a fundamental paradigm shift in human neuroimaging, away from thinking of the brain as a reflexive organ, whose responses are driven primarily by the momentary demands of the environment, and towards a new view that regards the brain's intrinsic dynamics as a dominant feature of its functional activity . Task - dependent deactivations in regions including the posterior cingulate / precuneus and medial prefrontal cortex led to the notion that increased activity among these regions during rest constituted the brain's default mode, soon followed by the observation that resting bold fluctuations in these regions exhibited coherent inter - regional patterns of functional connectivity constituting a default mode network . Other coherent resting - state networks were found to be associated with attention and cognitive control, and some of these networks were found to engage in anticorrelations . In parallel with studies that primarily examined specific networks revealed by seed - based patterns of whole - brain functional connectivity, an increasing number of studies attempted to decompose whole - brain resting - state fmri recordings into independent components or communities, drawing on a variety of clustering, dimension reduction and network analysis techniques . Recent comprehensive surveys have shown that resting - brain dynamics can be broken down into a relatively small set of resting - state networks (rsns). Some of these networks are primarily composed of regions that, on the basis of their task - evoked responses, can be regarded as either sensory or motor, while others such as the default mode network, the dorsal / ventral attention network and the frontoparietal network comprise sets of regions that exhibit a wide range of responses to more complex multimodal stimuli and tasks . Despite the cognitively unconstrained nature of the resting state (an issue that once gave rise to contentious discussion about its relevance for studying brain function), resting brain fluctuations and resting - state networks form largely consistent topographical patterns across individual subjects as well as scanning sessions and imaging centers . While the global arrangement of these patterns remains largely unchanged during global state transitions such as waking and sleeping or other states of consciousness, some functional connections exhibit experience - dependent modifications for example in response to specific sensorimotor training . Rsns are not unique to humans, and have also been described in macaque monkey as well as in the rodent brain . The reproducibility of rsn topography strongly suggests an anatomical basis in the brain's structural connection patterns, the connectome . Hiis idea was explored in neurocomputational models that pointed to a relationship between an anatomical coupling matrix of inter - regional projections and emergent patterns of functional connectivity resulting from spontaneous neural dynamics unfolding within this coupling matrix . Empirical studies in nonhuman primates showed significant overlap between anatomical projections mapped by tract tracing studies and resting - state functional connections . Studies carried out in humans combining diffusion imaging /tractography and resting - state fmri recordings obtained within the same cohort of participants also documented a robust statistical relationship between structural and functional connectivity (figure 6). Other studies examined anatomical connections in relation to functional rsns demonstrating that functionally coherent networks were linked by anatomical projections . More detailed network analyses of structural and functional connectivity revealed that while functional connectivity reflected the underlying structural networks, the relation between structural and functional connections was non - trivial and complex . For example, many strong functional connections were observed among pairs of regions that were not linked by a direct structural projection . Network models strongly suggest that all functional connections reflect a combination of numerous dynamic influences traveling through the network along many paths, most of them indirect paths that take multiple intermediate steps . An anatomical basis for the organization of dynamic brain fluctuations into rsns is compatible with the related idea that rsns represent the result of strengthening and weakening of connections due to a history of co - activation and common recruitment during task - evoked activity . This proposal suggests that patterns of functional connectivity expressed during rest recapitulate co - activation patterns expressed across many tasks, with the strengths among regions modified through a mechanism akin to hebbian plasticity . This mechanism of plasticity further sculpts and shapes the efficiency of the underlying anatomical substrate, essentially tailoring rsn configurations to reflect individual history and experience . Indeed, task - evoked patterns of coactivation are robustly related to functional connectivity observed in the resting brain, lending support to the notion that the resting brain cycles through or rehearses patterns of co - activation that are at other times purposefully deployed in response to varying stimulus and task contexts . This idea of rehearsal or recapitulation implies that spontaneous brain activity displays fine - grained temporal structure on time scales that are considerably shorter than the several minutes typically required for sampling stable and consistent patterns of resting - state functional connectivity . The idea that the resting state is less a state, but rather a dynamic spatiotemporal pattern, was first encountered and explored in computational models of resting brain activity . The inherently irregular and chaotic dynamics generated at each cortical region gave rise to fluctuations in their inter - regional coupling that spanned several time scales, including slow variations in coupling strength over the course of seconds to minutes . These findings have been confirmed and extended in a series of computational studies revealing the critical role of noise and dynamic instability in inducing spontaneous fluctuations of resting brain activity . An emerging theoretical idea is that of a functional repertoire of network states that is continually revisited and rehearsed in the course of noise - driven endogenous neural activity . In line with these computational observations, recent empirical studies carried out in human, macaque, and rat brain have shown that functional couplings among remote brain regions can indeed exhibit non - stationarities in coupling strength, manifesting as slow variations in functional connectivity and hence in the topology of functional networks across time . The relation of these slow network dynamics to cognitive processes, their relation to much faster non - stationarities in synchronization patterns measured with eeg and meg, and their potential significance for clinical studies remain to be explored . Over the past few years, network studies of the brain's structural connections as well as resting or task - evoked functional connectivity increasingly, network measures are deployed to characterize patterns of development and individual differences within cohorts of healthy participants . The mapping of individual network differences is a principal goal of the human connectome project which aims at drawing relations between network structure and dynamics on the one side, and patterns of heritability, behavior, and genomic variations on the other . These studies will allow, for the first time, to construct an overview of the range of variability in network organization across the human population . An important additional step, which is already pursued in a growing number of recent and ongoing studies of brain networks, involves identifying network correlates of brain and mental disorders . So far, this review has focused on how network approaches can become useful tools for understanding and characterizing the structure and function of the intact, healthy brain . However, a major promise of human connectomics is that it will lead to a deeper understanding of the biological substrates underlying brain and mental disorders, including their genetic bases . The primary aim of human connectomics is to map patterns of structural brain connectivity and uncover their relationship to emerging patterns of brain dynamics . Disturbed interactions among brain regions have been shown to be associated with virtually all brain and mental disorders, as well as with brain injury and recovery . A comprehensive treatment of disturbances of network organization in the diseased and damaged brain is beyond the scope of the present review . An instructive example of how studies of connectivity have begun to illuminate disease processes is provided by recent studies of schizophrenia . Schizophrenia is a severe and partly heritable psychiatric disorder characterized by a number of symptoms generally leading to a loss of integration across several domains of cognition and mental function, and impacting social interactions, emotional and thought processes . Ever since eugen bleuler coined the term schizophrenia noting that the disorder seems to interrupt the thousands of associative threads which guide our thinking, the condition has been thought to involve the disturbance or disconnection of connectivity in the brain . Rather than involving a net loss of connections, dysconnectivity, an abnormal pattern of connections among distinct brain regions that may involve both the strengthening and weakening of pathways and result in altered functional integration . In recent years, numerous studies deploying the full range of electrophysiological and imaging techniques have documented system - wide as well as topographically specific disruptions of structural and functional brain connections . Among the structural pathways that are consistently found to be disturbed are connections linking portions of the frontal and temporal lobes . Studies of effective connectivity in controls and patients with schizophrenia conducted in the course of a working memory task have additionally revealed a selective impairment of effective connections between parietal and prefrontal regions . Going beyond studies of single regions or pathways, a number of whole - brain connectivity analyses have demonstrated that schizophrenia is associated with the disruption of extended brain networks . Resting - state fmri analyses in patients with schizophrenia have shown that functional connectivity within the default mode network is selectively disturbed in patients with schizophrenia . Other studies have shown regionally specific and yet widespread patterns of functional dysconnectivity, eg, involving both stronger and weaker couplings of the dorsolateral prefrontal cortex with other regions across the brain, as well as selectively impaired functional connectivity between components of rsns involved in cognitive control . Diffusion mri and tractography have shown that connectivity deficits involving frontal and temporal brain regions result in reduced centrality of prominent brain hubs and a less centrally integrated network architecture . A different study also found reduced structural connectivity in patients with schizophrenia, as well as disturbances in the centrality of hub regions, for example in the medial frontal and left temporal lobe, both studies pointed to a reduction in global network efficiency, a potential index of impaired functional integration . More recently, network analysis of structural brain connectivity has shown a selective disturbance of pathways cross - linking regions forming the brain's rich club, a collective of highly connected and densely linked nodes . Given its central role in brain communication, an impairment of rich club connections is likely to manifest in functional disturbances of integrative neural processing . The complexity of the genetic basis for most common brain and mental diseases in conjunction with their pronounced phenotypic heterogeneity greatly complicates any systematic attempts at mapping genetic risk factors to clinical disorders, and even hinders their objective characterization on the basis of biologically based criteria . It has been suggested that the study of intermediate phenotypes, occupying positions that are intermediate between genetics and clinical phenotypes, may represent a promising way forward (figure 7). Intermediate phenotypes may allow for an objective classification of heterogeneous phenotypes into more coherent subgroups, and thus allow a better understanding of which genetic or other biological factors participate in each subgroup's disease mechanisms . The connectome and its endogenous and task - driven dynamics is an attractive candidate for an intermediate phenotype as it represents a point of convergence for a multitude of genetic and environmental factors, while also offering a plethora of potential biomarkers or probes that have proven to be of value in characterizing disease states of the brain . As brain network approaches continue to mature, it is to be expected that much work will focus on developing network measures that can characterize healthy and abnormal variations in brain structure and function . Such measures may help to identify factors that are associated with genetic and environmental disease mechanisms, and they may also serve as potential biomarkers for more objective diagnosis and prediction of effective treatment options . There is great potential for learning about disease states by mapping variations in network architecture in large cohorts of healthy participants, a chief goal of the human connectome project . Understanding the normal range of variability will provide insight into how disease phenotypes differ . It has been suggested that brain and mental disorders (indeed many common human diseases) represent quantitative rather than qualitative deviations from health . Rather than being caused by the presence or absence of single genetic factors, it appears that many common diseases, including those affecting brain and mind, manifest through the accumulation of small effects contributed by numerous genetic variants and thus represent quantitative traits that form the extremes of otherwise continuous phenotypic distributions . How various measures of brain networks relate to such phenotypic traits is still largely unknown . As more data on the network architecture of healthy and diseased brains becomes available, it will become possible to test the intriguing idea that metrics of connectivity can define new ways of classifying and inter - relating common mental disorders the development of new analytic techniques and modeling approaches, in parallel with continued methodological refinements in the area of human neuroimaging, continue to allow ever more detailed analyses of human structural and functional networks . Graph methods have proven useful for capturing how networks vary across individuals, how they change with experience, how they evolve across the human life span, and why they fail in a variety of brain and mental disorders . In the immediate future, some important areas of research will likely focus on structure function relationships, leading to a better understanding of how structural networks give rise to rich and flexible neural dynamics . Another promising area involves the analysis of network dynamics, the ever - changing topology of brain networks that are endogenously generated and modulated by stimuli and the environment . Studies of how networks change in the course of early development will likely stimulate new approaches towards mapping individual cognitive and behavioral developmental trajectories . Studies of networks may also prove important in the context of neurodegenerative disorders such as alzheimer's disease, contributing to the as - yet elusive goal of developing better diagnoses and treatment options . Whatever the future may bring the convergence of new technologies for observing the structure and function of the human brain with new analysis and modeling methods for the study of complex networks will almost certainly continue to bring change to the field . The new science of networks may provide a much needed theoretical framework for uniting empirical and computational studies of the nervous systems at all scales, from neurons to systems . Along the way, we may finally uncover the principles of network organization that account for the human brain's astonishing computational power, flexibility, and robustness.
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The group of pattern recognition receptors (prrs) includes families of toll - like receptors (tlrs), nod - like receptors, c - type lectin receptors (clrs), and rig - i - like receptors (rlrs). A summary of the most modern conceptual data about members of these families and their structure and functions can be obtained from recent comprehensive papers of kawai and akira,1 elinav et al,2 osorio and reis e sousa,3 and loo and gale.4 receptors constituting these families are united by two general features . Firstly, they directly recognize common antigen determinants of virtually all classes of pathogens (so - called pathogen - associated molecular patterns) and initiate immune response against them via specific intracellular signaling pathways.14 secondly, they recognize endogenous ligands (since they are usually released during cell stress, they are called damage - associated molecular patterns), and, consequently, prr - mediated immune response can be activated without the influence of infectious agents.14 therefore, prrs may also initiate development of aseptic inflammation caused by physical factors such as mechanical pressure, thermal damage, ionizing and nonionizing radiation, or chemical factors (eg, acidic damage, alkaline damage, exposure to chemical war gases, croton oil or turpentine, exposure to allergens, liberation of toxic substances during tumor disintegration, aseptic necrosis, internal bleeding, hemolysis, and autoimmune processes).14 it may promote further progression of inflammation or, on the contrary, prevent hazardous infectious complications (the combination of these two effects may also be true).14 the final outcome of prr working is enhanced production of many proinflammatory cytokines participating in plenty of the immune system s processes.14 expression of prrs on different levels (transcriptomic or proteomic) was detected in a lot of cells and organs,15 providing evidence that these receptors control many elements of the complex machinery of the human immune system: they allow epithelium and endothelium to defend against infectious agents on their own, they mediate the activation of adaptive immune response by antigen - presenting cells and t - helpers, they stimulate expression of cell adhesion molecules for leukocyte rolling and for other processes of inflammation development, and, finally, they contribute to phagocytosis efficacy.5 as a consequence of all mentioned above, prrs play a key role in the realization of innate and adaptive immune response . In addition, many prrs have a number of other vital functions apart from participation in immune response realization: they may regulate various aspects of cell proliferation, survival, apoptosis, autophagy, reactive oxygen species generation, pyroptosis, angiogenesis, and, consequently, of tissue remodeling and repair.69 the fundamental characteristics and diversity of prr functions have led to amazingly rapid research in this field, and such investigations are very promising for medicine as the immune system plays a key role in the vast majority, if not all, human diseases, and the process of discovering new aspects of immune system functioning is rapidly ongoing . One of the most actively explored fields in prr biology is their role in cancer etiopathogenesis . Not surprisingly, it is (as well as tumor immunology in general) a hot spot in cancer biology as well . Since prrs mediate the immune response induced by many immunoadjuvants,10,11 and many of them regulate the immune response against potentially carcinogenic infectious agents12,13 (helicobacter pylori,1417 epstein barr virus (ebv),18,19 human papillomavirus,18,19 human herpesvirus-8/ kaposi sarcoma - associated herpesvirus,18,19 mycobacterium tuberculosis,1517,20 streptococcus pneumoniae,1517,21 enteropathogenic escherichia coli,1517,22 shigella flexneri,1517,23 salmonella typhimurium,1517,24 borrelia burgdorferi,1517,25 chlamydophila pneumoniae,1517 chlamydia trachomatis,1517,26 chlamydophila psittaci,1517 and campylobacter jejuni1517,27), it seems to be possible to stimulate antitumor immunity through their enhanced activation.28,29 this hypothesis, originally developed for tlrs, should be also true for all prrs as well.28,29 according to this suggestion, reinforced prr activation may protect from infectious agents and prevent, inhibit, or block carcinogenesis whilst disrupted functioning of these prrs may allow infectious agents or tumor cells to avoid recognition by the immune system and, consequently, not be eliminated.28,29 at the same time, such prr activation may promote carcinogenesis, creating a proinflammatory microenvironment (via action of respective cytokines) that is favorable for tumor progression and chemoresistance development.30 it may also result in immunosuppression caused by chronic inflammation . 28 chronic inflammation may promote the development of cervical, endometrial, ovarian, breast, prostate, testicular, nasopharyngeal, lung, esophageal, gastric, colorectal, liver, pancreatic, gallbladder, kidney, bladder, lymphatic malignancies, and feasibly several other cancer types.11,31 in this case, on the contrary, lower prr activity should minimize effects of chronic inflammation such as enhancement of cancer initiation and promotion/ progression and, consequently, decrease probability of tumor development.30 so, the situation resembles a double - edged sword . The ideal variant, possibly, is the golden mean the balance between low and high prr activity . This hypothesis, initially developed for prrs,29 may also be successfully projected on prr intracellular signaling pathways; if their elements are overexpressed / constantly activated, it may lead to similar consequences as enhanced prr activation . On the other hand, if members of prr pathways are underexpressed / inactivated / unable to do their work, it may result in the same effects that arise after decreased prr activity, and the analogical golden mean in functioning of all genes encoding proteins constituting prr signaling pathways will be the optimal variant . The completion of the human genome project and widespread distribution of genotyping technologies have led to an enormous number of studies devoted to the association of inherited gene polymorphisms with various diseases . Single nucleotide polymorphisms (snps) may result in amino acid substitutions altering protein function or splicing, and they can also change the structure of enhancer sequences during splicing32 or affect messenger ribonucleic acid (rna) stability . 33 snps may alter transcription factor binding motifs, changing the efficacy of enhancer or repressor elements,34 and can alter the structure of translation initiation codons that may lead to downregulation of wild - type transcript.35 gene polymorphisms located in leucine - rich repeats constituting ectodomain of prrs may affect the ability of the receptor to bind pathogens they normally recognize,36 snps in transmembrane domain can lead to defects of intracellular receptor transport that do not allow location of a receptor on the membrane,37 and, finally, polymorphisms in the internal domain may result in altered interaction with adaptor proteins or in disrupted dimerization . So, inherited snps of genes encoding prrs may alter prr expression and activity, modulating cancer risk and, possibly, influencing various features of cancer progression . Based on the plethora of fundamental and epidemiological studies carried out, it is possible to specify two fundamental mechanisms for modulation of cancer risk by polymorphisms of genes encoding prrs and proteins of prr pathways . The first mechanism is impairment of the immune response to certain pathogens (it can be bacteria, viruses, fungi, protozoan, and helminths), which increases the risk of potentially carcinogenic infection and promotes its development along with further chronical persistence . The second mechanism is an increase in production of proinflammatory cytokines after binding of the ligand (exogenous or endogenous), which creates a condition of carcinogenic chronic inflammation . The group of pattern recognition receptors (prrs) includes families of toll - like receptors (tlrs), nod - like receptors, c - type lectin receptors (clrs), and rig - i - like receptors (rlrs). A summary of the most modern conceptual data about members of these families and their structure and functions can be obtained from recent comprehensive papers of kawai and akira,1 elinav et al,2 osorio and reis e sousa,3 and loo and gale.4 receptors constituting these families are united by two general features . Firstly, they directly recognize common antigen determinants of virtually all classes of pathogens (so - called pathogen - associated molecular patterns) and initiate immune response against them via specific intracellular signaling pathways.14 secondly, they recognize endogenous ligands (since they are usually released during cell stress, they are called damage - associated molecular patterns), and, consequently, prr - mediated immune response can be activated without the influence of infectious agents.14 therefore, prrs may also initiate development of aseptic inflammation caused by physical factors such as mechanical pressure, thermal damage, ionizing and nonionizing radiation, or chemical factors (eg, acidic damage, alkaline damage, exposure to chemical war gases, croton oil or turpentine, exposure to allergens, liberation of toxic substances during tumor disintegration, aseptic necrosis, internal bleeding, hemolysis, and autoimmune processes).14 it may promote further progression of inflammation or, on the contrary, prevent hazardous infectious complications (the combination of these two effects may also be true).14 the final outcome of prr working is enhanced production of many proinflammatory cytokines participating in plenty of the immune system s processes.14 expression of prrs on different levels (transcriptomic or proteomic) was detected in a lot of cells and organs,15 providing evidence that these receptors control many elements of the complex machinery of the human immune system: they allow epithelium and endothelium to defend against infectious agents on their own, they mediate the activation of adaptive immune response by antigen - presenting cells and t - helpers, they stimulate expression of cell adhesion molecules for leukocyte rolling and for other processes of inflammation development, and, finally, they contribute to phagocytosis efficacy.5 as a consequence of all mentioned above, prrs play a key role in the realization of innate and adaptive immune response . In addition, many prrs have a number of other vital functions apart from participation in immune response realization: they may regulate various aspects of cell proliferation, survival, apoptosis, autophagy, reactive oxygen species generation, pyroptosis, angiogenesis, and, consequently, of tissue remodeling and repair.69 the fundamental characteristics and diversity of prr functions have led to amazingly rapid research in this field, and such investigations are very promising for medicine as the immune system plays a key role in the vast majority, if not all, human diseases, and the process of discovering new aspects of immune system functioning is rapidly ongoing . One of the most actively explored fields in prr biology is their role in cancer etiopathogenesis . Not surprisingly, it is (as well as tumor immunology in general) a hot spot in cancer biology as well . Since prrs mediate the immune response induced by many immunoadjuvants,10,11 and many of them regulate the immune response against potentially carcinogenic infectious agents12,13 (helicobacter pylori,1417 epstein barr virus (ebv),18,19 human papillomavirus,18,19 human herpesvirus-8/ kaposi sarcoma - associated herpesvirus,18,19 mycobacterium tuberculosis,1517,20 streptococcus pneumoniae,1517,21 enteropathogenic escherichia coli,1517,22 shigella flexneri,1517,23 salmonella typhimurium,1517,24 borrelia burgdorferi,1517,25 chlamydophila pneumoniae,1517 chlamydia trachomatis,1517,26 chlamydophila psittaci,1517 and campylobacter jejuni1517,27), it seems to be possible to stimulate antitumor immunity through their enhanced activation.28,29 this hypothesis, originally developed for tlrs, should be also true for all prrs as well.28,29 according to this suggestion, reinforced prr activation may protect from infectious agents and prevent, inhibit, or block carcinogenesis whilst disrupted functioning of these prrs may allow infectious agents or tumor cells to avoid recognition by the immune system and, consequently, not be eliminated.28,29 at the same time, such prr activation may promote carcinogenesis, creating a proinflammatory microenvironment (via action of respective cytokines) that is favorable for tumor progression and chemoresistance development.30 it may also result in immunosuppression caused by chronic inflammation . 28 chronic inflammation may promote the development of cervical, endometrial, ovarian, breast, prostate, testicular, nasopharyngeal, lung, esophageal, gastric, colorectal, liver, pancreatic, gallbladder, kidney, bladder, lymphatic malignancies, and feasibly several other cancer types.11,31 in this case, on the contrary, lower prr activity should minimize effects of chronic inflammation such as enhancement of cancer initiation and promotion/ progression and, consequently, decrease probability of tumor development.30 so, the situation resembles a double - edged sword . The ideal variant, possibly, is the golden mean the balance between low and high prr activity . This hypothesis, initially developed for prrs,29 may also be successfully projected on prr intracellular signaling pathways; if their elements are overexpressed / constantly activated, it may lead to similar consequences as enhanced prr activation . On the other hand, if members of prr pathways are underexpressed / inactivated / unable to do their work, it may result in the same effects that arise after decreased prr activity, and the analogical golden mean in functioning of all genes encoding proteins constituting prr signaling pathways will be the optimal variant . The completion of the human genome project and widespread distribution of genotyping technologies have led to an enormous number of studies devoted to the association of inherited gene polymorphisms with various diseases . Single nucleotide polymorphisms (snps) may result in amino acid substitutions altering protein function or splicing, and they can also change the structure of enhancer sequences during splicing32 or affect messenger ribonucleic acid (rna) stability . 33 snps may alter transcription factor binding motifs, changing the efficacy of enhancer or repressor elements,34 and can alter the structure of translation initiation codons that may lead to downregulation of wild - type transcript.35 gene polymorphisms located in leucine - rich repeats constituting ectodomain of prrs may affect the ability of the receptor to bind pathogens they normally recognize,36 snps in transmembrane domain can lead to defects of intracellular receptor transport that do not allow location of a receptor on the membrane,37 and, finally, polymorphisms in the internal domain may result in altered interaction with adaptor proteins or in disrupted dimerization . So, inherited snps of genes encoding prrs may alter prr expression and activity, modulating cancer risk and, possibly, influencing various features of cancer progression . The same statement should be true for genes encoding proteins of prr signaling pathways . Based on the plethora of fundamental and epidemiological studies carried out, it is possible to specify two fundamental mechanisms for modulation of cancer risk by polymorphisms of genes encoding prrs and proteins of prr pathways . The first mechanism is impairment of the immune response to certain pathogens (it can be bacteria, viruses, fungi, protozoan, and helminths), which increases the risk of potentially carcinogenic infection and promotes its development along with further chronical persistence . The second mechanism is an increase in production of proinflammatory cytokines after binding of the ligand (exogenous or endogenous), which creates a condition of carcinogenic chronic inflammation . There is a variety of cancer types definitely or possibly having infectious etiology12,13 that can be associated with inherited alterations in genes encoding prrs and proteins of prr signaling pathways: esophageal cancer (variation in immune response to pathogens infecting esophagus)38 gastric cancer (on the basis of modulation of immune response to h. pylori)39,40 cancer of the small bowel (modulation of immune response to c. jejuni)41 colorectal cancer (alteration of immune response to many, mostly undefined, infectious agents inhabiting the colon and rectum)4244 liver cancer (variation in immune response to hepatitis b virus, hepatitis c virus, helicobacter hepaticus, or liver flukes)45,46 gallbladder cancer (modulation of immune response to infectious agents found in bile)47 pancreatic cancer (alteration of immune response to pathogens inhabiting the pancreas)48 endometrial cancer (modification of immune response to several kinds of infectious agents colonizing the endometrium)49 cervical cancer (alteration of immune response to human papillomavirus and some infectious agents colonizing the cervix)50 ovarian cancer (variation in immune response to c. trachomatis)51,52 breast cancer (modulation of immune response to some viruses infecting the breast)52,53 prostate cancer (variation in immune response to propionibacterium acnes and other uncertain pathogens found in prostate tissue)54 testicular cancer (modification of immune response to ebv)55 kidney cancer (variation in immune response to bacteria and viruses infecting the kidneys)56 bladder cancer (modulation of immune response to certain viruses or schistosoma spp. )57 nasopharyngeal carcinoma (alteration of immune response to ebv)58 lung cancer (variation in immune response to m. tuberculosis, s. pneumoniae, c. pneumoniae, and, possibly, to other infectious agents causing chronic inflammatory lung diseases)52,59 lymphoma (modification of immune response to ebv and many other infectious agents such as b. burgdorferi or h. pylori)60,61 kaposi sarcoma (variation in immune response to human herpesvirus-8/kaposi sarcoma - associated herpesvirus infection).62 it is important to remember that there are two main components determining the importance of the snp in programs of cancer prevention based on genomic risk markers: the odds ratio value between cases and controls (as in the whole population and subgroups) and the prevalence of the polymorphism in the population, and they both may vary in different geographic regions . It is desirable to develop not one general program, but a number of individual programs for different countries / populations . At the moment, it is possible only to recommend a list of polymorphisms for further investigation since only a small number of studies with perfect design were carried out . The list of relevant polymorphisms that can be admitted as the most promising for further oncogenomic investigations may be created according to the following rules . A gene polymorphism may be included into the short list for further oncogenomic studies if: the snp leads to substantial functional consequences on the molecular level (for instance, it strongly affects transcription, splicing, translation, stability and transport of pre - messenger rna, messenger rna, noncoding rna, or protein encoding by the gene, or it noticeably influences signaling of synthesized protein) it is associated with the risk of cancer in conducted studies it has any functional consequences on the molecular level and it is strongly (threshold odds ratio value may be individual for each cancer type) associated with conditions that significantly increase the risk of cancer . A gene polymorphism can be also included into the extended list if: it is characterized by more subtle functional alterations in the gene that, however, still result in qualitative or quantitative alterations of the encoding protein (or noncoding rna) it is associated only with conditions that substantially increase the risk of cancer (ie, not associated with the risk of cancer). In concordance with this conception, the following snps of genes encoding prrs and proteins of prr signaling pathways may be accepted as the most valuable for further oncogenomic investigations based on the analysis of relevant published literature:6365 tlr1-tlr6-tlr10 gene cluster: rs10008492, rs4833103, rs5743815, rs11466657 tlr2 gene: rs3804100, rs4696480, 196 to 174 del (delta22), gt - microsatellite polymorphism tlr4 gene: rs4986790, rs4986791, rs16906079, rs11536891, rs7873784, rs1927911, rs10759932, rs10116253, rs11536889, rs11536858 tlr9 gene: rs5743836, rs352140 tirap / mal gene: rs8177400, rs8177399, rs8177374, rs7932766 myd88 gene: rs1319438, rs199396 irak1 gene: rs1059703, rs3027898, rs10127175 traf3 gene: rs7143468, rs12147254, rs11160707 traf6 gene: rs331455, rs331457 tollip gene: rs5743867 irf5 gene: rs2004640, rs2280714, rs10954213, 5 bp indel (cgggg) polymorphism nod1 gene: rs2075820, nd(1) + 32656 nod2 gene: rs2066842, rs2066844, rs2066845, rs2006847 mrc1 gene: rs1926736, rs2478577, rs2437257, rs691005 cd209 gene: rs2287886, rs735239, rs735240, rs4804803 clec7a gene: rs16910526 rig - i gene: rs36055726, rs11795404, rs10813831 . Although gene polymorphisms of genes encoding rlrs, clrs, and specific proteins of their signaling pathways are investigated relatively less than snps of tlrs and nod - like receptors, it is possible to conclude that since they recognize bacterial, viral, fungal, protozoan, and helminth pathogen - associated molecular patterns as tlrs and nod - like receptors, inherited structural variation in them may influence cancer risk and progression as well . For instance, some human clrs (mrc1, cd207, ly75, cd209, clec7a, clec1b, clec6a, clec4e, clec4a) recognize ligands3 of potentially carcinogenic infectious agents,12,13 such as m. tuberculosis,66 s. pneumoniae,67 klebsiella pneumoniae,68 human immunodeficiency virus-1,69 cytomegalovirus,70 candida albicans,71 c. neoformans,72 pneumocystis carinii,73 paracoccidioides brasiliensis,74 histoplasma capsulatum,74 and schistosoma mansoni.75 many polymorphisms of genes encoding these receptors may alter immune response to indicated ligands, possibly, modulating etiopathogenesis of certain cancer types such as lung cancer (m. tuberculosis, s. pneumoniae, and k. pneumoniae),12,13 glioblastoma (cytomegalovirus),76 oral cancer (fungi),77 colorectal cancer, hepatocellular carcinoma, prostate cancer, or cervical cancer (s. mansoni).78 one rlr, rig - i, also recognizes ligands of hepatitis c virus and ebv,4 and thus structural inherited variation in this receptor may alter risk of hepatocellular carcinoma, nasopharyngeal carcinoma, and lymphoma . There are certain disparities in different population studies investigating the association of polymorphisms of genes encoding prrs and proteins of their signaling pathways with various aspects of cancer development.6365 general reasons for these discrepancies may include confounding host, bacterial, or environmental factors in different ethnicities modulating penetrance of variant allele and affecting risk of conditions increasing cancer risk (such as autoimmune diseases, precancerous gastric lesions, tuberculosis, and recurrent pneumonia), different bacterial impact in etiology of such conditions in different populations (that will be reflected in different features of prr - mediated immune response because of specific prr - ligand interaction), differences in sample size, differences in age / gender/ body mass index / ethnicity / tumor, node, metastasis stage/ other clinicopathological characteristics between study samples, differences in prevalence of infectious agent (eg, helicobacter pylori or ebv) in case and control groups, and differences in diagnostics, stratification, genotyping methods, and chance . In addition, certain studies in which negative results were obtained could never have been published (so - called file drawer effect), which may create a significant bias and distort a picture that cannot be observed at the moment . Unfortunately, although some genome - wide association studies relevant to the discussed problem were performed, it is usually not possible to compare them with non - genome - wide association studies on the same cancer type since there are no non - genome - wide association studies investigating association of the same snps with similar malignancies . It may be feasible in the future when the number of studies devoted to this issue will be enough for correct comparative analysis . The most intriguing aspects of the problem of the association of inherited structural variation in genes encoding prrs and proteins of prr signaling pathways with features of cancer development are: are snps in genes encoding prrs or proteins of prr signaling pathways associated with features of cancer progression or only with cancer risk? Existing studies show controversial results, and most of the results suggest that there is no, or weak, correlation between such polymorphisms and peculiarities of cancer progression . Are polymorphisms of genes encoding clrs, rlrs, or specific proteins of their signaling pathways connected with risk or progression of cancer? If yes, would it be appropriate to include them in the list of polymorphisms used in programs of cancer risk determination and further cancer prevention? As shown above, there is some premise to the thought that these snps may be associated with cancer risk . Do polymorphisms of genes encoding prrs or proteins of prr signaling pathways (particularly tlrs and tlr pathway) correlate with altered prostate cancer risk or progression? Almost all large studies devoted to this issue showed that there is no association between inherited variation in indicated genes and features of prostate cancer development . Are polymorphisms of genes of prr signaling pathways associated with cancer risk or progression to the same extent as polymorphisms of genes encoding prrs? It is logical that if snp of gene encoding specific prr is associated with risk or progression features of certain malignancies, polymorphisms in genes encoding specific signaling molecules constituting pathways of these receptors should correlate with similar neoplasms, if they have substantial functional consequences on the molecular level . How do polymorphisms of genes encoding prrs and proteins of prr signaling pathways interact with each other in relation to determination of cancer risk and progression? Particularly, how do snps of positive and negative regulators of prr activity (especially, micro rna) influence cancer risk or progression if they are inherited together? Answers to these questions remain elusive at the present time and should be obtained from fundamental and population studies in the future . Which snps of genes encoding prrs and proteins of prr pathways have independent significance, and which are just in the linkage disequilibrium? Knowledge of this may help in creating the list of polymorphisms useful in programs of cancer risk determination and further prevention . Which snps of genes encoding prrs and proteins of prr pathways should be included in such a list? Which of them have universal effect for each cancer type, and which influence risk and progression of one cancer type but have no effect in relation to another malignancy? Differences in association of the same snp with different malignancies should be explained by features of specific pathogen - associated molecular pattern prr interaction (probably certain characteristics of ligand binding), or, possibly, on peculiarities of damage - associated molecular pattern prr interaction . Lists of prospective snps for further oncogenomic investigations may be created according to the concept suggested above . How do snps of genes encoding prrs and proteins of prr pathways affect cancer risk or progression in different populations and subgroups of such populations? How can this information be adjusted for application in the creation of programs of cancer risk determination and further prevention? Only large, comprehensive, well - designed population studies may provide an answer to these questions . Do polymorphisms of genes encoding prrs and proteins of prr pathways influence cancer risk only through increase in risk of chronic inflammatory conditions, or can they affect it through other mechanisms as well? How can this information be used in programs of cancer risk determination and further prevention? To answer these questions, control groups in population studies should include not only healthy controls, but also controls with chronic inflammatory conditions predisposed to investigating cancer type . Which infectious agents recognized by various prrs are carcinogenic, and which are not? It may help to define cancer types associated with snps of genes encoding specific prrs and proteins constituting prr signaling pathways . Fundamental studies devoted to the investigation of infectious agent prr interactions, to the investigation of carcinogenicity of known infectious agents and to the discovery of new, possibly carcinogenic, infectious agents should answer this question . No doubt, determination of the role of snps in genes encoding prrs and proteins of prr signaling pathways in fields of tumor immunology and molecular epidemiology of cancer may open new pages in cancer biology and cancer prevention.
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Mucinous carcinoma (mc) of the breast is not a common disease and the incidence of mc was reported to range from 1% to 6% of all primary breast cancers (1 - 4). Mc of the breast was characterized by a large amount of mucin production and in general, defined as having a mucinous component of 50% or more (5, 6). Those lesions had different features from invasive ductal carcinoma - not otherwise specified (idc - nos). Median age at diagnosis was older than 60 and older than that of idc - nos (4, 6). Furthermore, mc showed more favorable clinicopathological characteristics, such as lower incidence of nodal metastasis, higher expression of estrogen and progesterone receptors, and differentiated grade (2, 6, 7). Therefore, prognosis of mc is better than that of idc - nos with 10-yr survival rates better than 80% (1, 8). Histologically mc is classified into 2 subgroups based on the degree of cellularity, which are pure type and mixed mucinous - ductal type (9). However, cut - off values of the mucinous component for defining pure type mc was not standardized, and later, others divided mc into type a and type b (10). The former is the classical type with large amounts of mucins and the latter is a variant type with neuroendocrine differentiation or feature of signet ring cells (5, 10). Prognostic significance of clinicopathological characteristics in mc patients is not well established, because of its low incidence rate and lack of a standard definition . Treatment guidelines for mc are mostly extrapolated from data based on invasive ductal carcinoma (idc) without clear validation . As incidence of breast cancer was significantly increasing in korea (11, 12), it was necessary to investigate incidence rates, clinicopathological characteristics, and prognosis of mc to prepare an adequate treatment guideline . But there are few studies on mc and information is limited . In this study, we investigated the clinicopathological characteristics and treatment patterns for mc and compared survivals of mc with those of idc - nos . We retrospectively reviewed the data of 4,932 breast cancer patients who were treated at the department of surgery, yonsei university college of medicine in seoul, korea between january 1986 and december 2006 . These patients were compared with 3,936 patients with idc - nos who were treated during the same period . However, slides of the remaining 30 patients were not available and we reviewed these cases through previous pathologic reports . Pure type mc was defined as having a mucinous component of more than 90% and specialized pathologists with extensive experience in breast pathology performed a pathologic slide review . Sixty - one (82.4%) of 74 patients were categorized as having pure type mc . Data regarding patient demographics, histopathology of primary tumor, treatment patterns, and survivals were obtained by reviewing medical records . Patients were treated with either mastectomy or lumpectomy and axillary lymph node dissection or sentinel lymph node biopsy with local radiotherapy . After completion of surgery, adjuvant treatments were administered as indicated based on international guidelines . Tumor stage was based on the 6th american joint committee on cancer (ajcc) criteria . Ten percent or more of positively stained cells was used as the cut - off for estrogen receptor (er) and progesterone receptor (pr) positivity . Her-2/neu staining was scored from 0 to 3 + according to the guideline suggested for herceptest (dako, glostrup, denmark) (13). Her-2/neu immunohistochemical staining was considered positive when strong (3 +) membranous staining was observed, whereas cases with 0 to 1 + were regarded as negative . Because fluorescent in - situ hybridization test had not been performed routinely, cases with equivocal (2 +) staining were excluded in statistical analyses . Clinical follow - up included history taking, physical examination, laboratory tests and radiologic imaging every 6 - 12 months . Survival curves were determined and plotted using the kaplan - meier method and group differences in survival time were investigated by log - rank test . Median age at diagnosis was 47 yr in all patients (range, 20 - 93 yr), and age at diagnosis was much younger in mc patients than in idc - nos patients with statistical significance (p=0.022); 45 yr old for mc patients and 47 yr old for idc - nos patients . Among 74 patients whose slides were available for reviewing, 61 patients (82.4%) were pure mc and 13 (17.6%) were mixed mucinous - ductal carcinoma . Mammographic and sonographic findings according to breast imaging reporting and data system (bi - rads) category were investigated . Mammographic and sonographic findings were available in 21 and 38 pure mc patients, respectively and 5 and 5 mixed type mc, respectively (table 2). In pure type mc, sensitivity of mammography and sonography was 76.5% and 94.7%, respectively . However, in mixed mc, the sensitivity of mammography and sonography was 100% in both modalities . Eighty - five (81.7%) of 104 mc patients had no axillary lymph node involvement and more mc patients had lower tumor stage than idc - nos patients (p=0.000 and 0.005, respectively). Expressions of er and pr were higher in mc than in idc - nos; 77.3% and 63.2%, respectively, but er only showed a statistical significance (p=0.008). Of the 70 patients whose status of her-2/neu was available, only 10 (14.3%) showed her-2/neu overexpression . Tumor size and treatments patterns including operation methods, adjuvant chemotherapy, hormonal therapy, and radiation therapy were not statistically different from idc - nos, but less chemotherapy and more hormone therapy were applied to mc patients due probably to more favorable histologic features associated with higher er expression in mc . Clinicopathological characteristics and treatment patterns of pure mc patients are similar to those with the mixed type except tumor size, histologic grade, and radiation therapy (table 3). More pure mc patients had smaller in size and differentiated histologic grade than mixed type . More radiation therapy was applied to pure mc because more pure mc patients received breast - conserving surgery . The median follow - up duration was 63.5 months in all patients (range, 0 - 262 months). There were 9 disease relapses and 6 deaths during follow - up in mc and 5- and 10-yr disease - free survival (dfs) rates were 93.3% and 86.1%, respectively (fig . Five- and 10-yr overall survival (os) rates were 93.5% and 86.3%, respectively (fig . Ten - year dfs and os rates of idc - nos were 74.7% and 74.9%, respectively . Mc showed better survival outcomes than idc - nos with statistical significance (fig . 1, p=0.012 for dfs and 0.005 for os). In pure mc, the 10-yr dfs and os rates were 90.2% and 91.3%, respectively and 10-yr dfs and os rates of the mixed type were 68.8% and 100%, respectively . Dfs and os rates were not statistically different between pure and mixed types (fig . 2, p=0.360 and 0.374, respectively) due probably to the small sample size . Univariate analysis for dfs and os according to clinicopathological characteristics in mc revealed that lymph node status and tumor stage were statistically significant factors for survival (table 4). In stage - matched analysis for os this was probably due to the small sample size and short follow - up duration . In age - matched analysis for os, mc showed a better survival rate than idc - nos in the patient group whose age was 50 or younger (fig . Patients whose pathology slide was available for reviewing, survivals of mc according to the subtype were compared with idc - nos (fig . Pure mc showed a better dfs than idc - nos (p=0.037) and there was similar dfs patterns between mixed type mc and idc - nos (p=0.795). Pure mc showed a better os than idc - nos (p=0.049) but differences of os between the mixed type and idc - nos did not reach statistical significance in our study (p=0.143). It accounted for 16.8% of all female cancers in 2002 and incidence of breast cancer in korean women is anticipated to increase (11, 14, 15). Besides incidence, the percentage of early - stage breast cancer has steadily increased (16). Screening mammography and use of ultrasonography play an important role in detection of breast cancers in earlier stages (17). Since 1996, it was recommended that all korean women over 40 yr old underwent a mammography every 1 or 2 yr (18). From 1996 to 2004, the incidence of stage 0 and i breast cancers increased remarkably by 128.6% and 81.6%, respectively (16). However, because of the low incidence rate and small number of studies on specific type of breast cancer including mc, limited information is available regarding epidemiology, clinical, radiological and pathological features, risk factors, and prognosis . It is necessary to investigate incidence rate, clinicopathological characteristics, and prognosis of mc to prepare an adequate treatment guideline . Most mc patients complained palpable mass in their breast, and radiologically it frequently mimics a benign lesion (19). Mc especially appears as a well - defined mass lesion but the mixed type shows an indistinct or microlobulated pattern (20, 21). Although image study findings were only available in a small number of patients, 23.5% of pure mc cases had false negative findings in mammography but ultrasonography showed much better sensitivity at 94.7% . Among patients available in ultrasonographic findings, these radiologically benign lesion - like characteristics of mc sometimes cause a delay in diagnosis (19). Physicians must understand the limits of screening mammography because the peak age of incidence in korean breast cancer patients is in the 40s (16) and the sensitivity of mammography for premenopausal women is quite low (22 - 24). Middle - aged women presenting breast symptoms should be examined carefully and ultrasonography may be helpful . Many western studies reported that mc had more favorable pathological characteristics than idc - nos (1 - 4, 6, 7). They were smaller in tumor size, less axillary lymph node involvement, more expression rates of er and pr, lower grade, lower proliferation rates and less overexpression of her-2/neu . Another characteristic of mc was that it usually occurred in postmenopausal women (2, 4, 6), and their age at diagnosis ranged from 65 to 71 yr and older than that of idc - nos ranged from 59 to 61 yr (3, 6). The 10-yr os of mc was reported at more than 80% (4, 6, 7). In our study, the median age of mc patients was younger than idc - nos patients . The median age of our study population was not different from that of korean breast cancer patients and another study reported the mean age of mc and idc - nos at 45.5 and 46.9 yr, respectively, which was similar to our results (12, 25). Therefore, the age of onset is quite different from that of western mc cases . The reason for the younger age of onset in korean women is not clear, but it might be partly due to wide use of ultrasonography, genetic or environmental factors, a cohort effect of high incidence in the younger generation or relatively easier accessibility to detect breast cancer among middle - aged women (15). However, histological features and survival rates of mc patients were similar to those of western patients . Mc of the breast is not a homogeneous entity and was divided into pure and mixed types (5, 10). Pure mc was associated with better survival rates than the mixed type, but survival rates of mixed type were not different from those found in idc - nos (2, 26, 27). Among 74 available patients with histologic subtype, there were no differences in dfs and os between pure and mixed mc . This was probably due to the small sample size and short follow - up duration, and larger data samples with longer follow - up would be necessary to confirm these differences . Because age distribution was different between mc and idc - nos, we compared survival matched by age . In patients whose age was 50 yr or less, mc showed better survival than idc - nos (p=0.008). There was a similar trend in patient groups older than 50 yr, although it was not statistically significant (p=0.349). Some western studies reported that age at diagnosis of mc was a significant prognostic factor (4, 6). This may be related to the older onset age in western mc patients . In summary, mucinous carcinoma shows more favorable histologic features and better prognosis than idc - nos . However, korean mc developed at younger ages than idc - nos . Since only pure mc showed better prognosis than idc - nos, it is important to differentiate mixed mc from pure mc . Patients with mixed mc might be treated as guidelines for idc . In pure mc patients with favorable risk factors, however, adjuvant chemotherapy might be more saved . Mc clinically and radiologically mimics a benign lesion and this may lead to a delay in diagnosis . Therefore, middle - aged korean women presenting a palpable mass should be examined carefully and the appropriate diagnostic procedures such as ultrasonography are warranted for early detection of malignant tumors.
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Coronary artery disease (cad) is the leading cause of death in western countries [1, 2]. Multi detector computed tomography (mdct), a non - invasive imaging modality featuring large scan coverage up to 320 detector system rows, sub - millimetre spatial resolution up to 0.23 mm, and high temporal resolution up to 135 ms for a single source ct and 75 ms for a dual source ct (dsct) system (with options for further increase using multi - segmental reconstruction techniques), is the current preferred method for cad assessment [36]. Because of patient movement, irregular heart rate, and insufficient temporal resolution for high heart rate, cardiac mdct images are often hampered by motion artefacts . Although identification of motion artefacts in large structures such as the myocardium can be relatively easy, it is not always possible to identify motion artefacts in smaller structures like the coronary vessels . These artefacts could lead to misinterpretation in the coronary computed tomography angiography (cta) analysis . Motion artefacts are commonly evaluated qualitatively, either by visually determining their presence / absence or by assigning a severity rating [9, 10]. However although qualitative analysis is not necessarily insufficient, a quantitative analysis can give more precise and objective information; and make the user aware of suspicious regions . Therefore, the purpose of this study was to develop an algorithm for quantitative image analysis for the detection of motion artefacts in coronary artery computed tomography as an added value to the qualitative analysis and test it in phantom scans of two different ct devices . An anthropomorphic moving heart phantom (limbs & things, bristol, uk), with an artificial coronary vessel was used . The movement of the heart phantom and the artificial coronary vessel have been shown to be comparable to the clinical setting . The artificial coronary vessel was filled with a contrast agent (ultravist-300, schering, switzerland) diluted to a concentration of about 250 hu . The phantom was scanned on a 64-row mdct (64ct; somatom sensation 64, siemens medical solutions, forchheim, germany) at 120 kv, 770 mas and a dsct (somatom definition, siemens medical solutions, forchheim, germany) at 120 kv, 300 mas / rot; both at 330 ms rotation speed in cranio - caudal direction . The field of view (fov) was set at 200 mm 200 mm . The heart phantom was placed in supine position with its apex facing away from the bore hole . A respiratory device was connected to the phantom, which inflated and deflated the phantom at a programmed rate to simulate a beating heart and produced an ecg signal which was connected to the ct scanner . The phantom was scanned at rest and at 50110 beats per minute (bpm) with 10 bpm intervals, without changing the phantom position . R interval was chosen as the optimal phase and datasets were reconstructed at 0.6/0.4 mm slice thickness / increment using kernel b25f and b26f for 64ct and dsct, respectively . Figure 1 shows the scanned heart phantom, where motion artefacts were absent (left images) and present (right images).fig . 1the phantom used for experiment . Images without motion artefacts (left) and with motion artefacts (right) are shown both in a volume reconstruction (top) and a sagittal reconstruction (bottom). 1 and 2 denote start and end location of vessel analysis, respectively . Dotted white lines on the top row indicate the location of the sagittal slices the phantom used for experiment . Images without motion artefacts (left) and with motion artefacts (right) are shown both in a volume reconstruction (top) and a sagittal reconstruction (bottom). Arrows no . 1 and 2 denote start and end location of vessel analysis, respectively . Dotted white lines on the top row indicate the location of the sagittal slices two plexiglas tubes with reference / stenosis diameters of 6/4 and 4/2 mm (resulting in area stenosis of 56 and 75%, respectively) were also used . The lumen was filled with contrast agent (visipaque 320, general electric healthcare) diluted to a concentration of about 250 hu . The vessel phantoms were scanned on the 64ct without motion at 120 kv and 107 mas . The images were reconstructed at 0.75/0.4 mm slice thickness / increment using kernel b35f . A siemens syngo workstation (siemens medical solutions, erlangen, germany) was used for visual three - dimensional observation . Software for quantitative mathematical analysis was developed using matlab software (mathworks inc, usa). Quantitative analysis was performed in the myocardium and the coronary for each heart rate and both modalities . The analysis was performed as follows: (see fig . 2 for the overview diagram)fig . 2diagram of the quantitative image analysis methods for the detection of motion artefacts in coronary artery computed tomographymyocardium analysisdue to the nature of the phantom s movement, motion artefacts in the myocardium were most apparent in the sagittal plane, especially in the anterior part of the myocardium . The sagittal cross - section images were taken at approximately the centre of the phantom (dotted line in fig . 1). The anterior inner lining of the myocardium (green line at fig . 3, left) was semi automatically extracted using a gradient vector flow (gvf) snake algorithm by first placing several seed points for the initial contour which were then allowed to grow to match the inner lining . From the extracted line, the following parameter was determined: smoothness of the inner - lining of the myocardiumsmoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3, a gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . 3illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference linevisual observations by two independent observers were performed . Each observer was asked to score the sagittal images for the presence of no, mild or severe motion artefacts resulting in scores of 0, 1 and 2, respectively . Diagram of the quantitative image analysis methods for the detection of motion artefacts in coronary artery computed tomography due to the nature of the phantom s movement, motion artefacts in the myocardium were most apparent in the sagittal plane, especially in the anterior part of the myocardium . The sagittal cross - section images were taken at approximately the centre of the phantom (dotted line in fig . 1). The anterior inner lining of the myocardium (green line at fig . 3, left) was semi automatically extracted using a gradient vector flow (gvf) snake algorithm by first placing several seed points for the initial contour which were then allowed to grow to match the inner lining . From the extracted line, the following parameter was determined: smoothness of the inner - lining of the myocardiumsmoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3, a gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . 3illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line smoothness of the inner - lining of the myocardium smoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3, right). A gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . 3illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line visual observations by two independent observers were performed . Each observer was asked to score the sagittal images for the presence of no, mild or severe motion artefacts resulting in scores of 0, 1 and 2, respectively . Vessel analysisbecause of the occurrence of motion artefacts along z - axis, the analysis was limited to the vessel segment which was relatively parallel to the z - axis . The vessel segment was predetermined and set equal for all heart rates (segment from arrow 1 to arrow 2 at fig . 1, top). Started by manual selection of the vessel lumen in the axial view at location 1 of fig . 1, a small (50 by 50 pixels) region of interest (roi) was determined around the vessel lumen . The image inside the roi was thresholded at level 41% of the lumen peak value . Afterwards, using gvf snake algorithm, the lumen boundary was extracted and its centre of mass was determined as centre point . The detection was continued to the next slice without further user interaction, and repeated until the last slice (fig . 4, top).fig . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzedthree parameters were determined from the extracted vessel: smoothness of the vessel centreline pathway along the z - axisthe vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Comparison to 0 bpm dataset was made.consistency of vessel lumen areas along the z - axisblurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set.consistency of vessel lumen value along the z - axisblurring can also change the lumen intensity value . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Because of the occurrence of motion artefacts along z - axis, the analysis was limited to the vessel segment which was relatively parallel to the z - axis . The vessel segment was predetermined and set equal for all heart rates (segment from arrow 1 to arrow 2 at fig . 1, top). Started by manual selection of the vessel lumen in the axial view at location 1 of fig . 1, a small (50 by 50 pixels) region of interest (roi) was determined around the vessel lumen . The image inside the roi was thresholded at level 41% of the lumen peak value . Afterwards, using gvf snake algorithm, the lumen boundary was extracted and its centre of mass was determined as centre point . The detection was continued to the next slice without further user interaction, and repeated until the last slice (fig . 4, top).fig . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzed illustration on vessel analysis . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzed three parameters were determined from the extracted vessel: smoothness of the vessel centreline pathway along the z - axisthe vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Comparison to 0 bpm dataset was made.consistency of vessel lumen areas along the z - axisblurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set.consistency of vessel lumen value along the z - axisblurring can also change the lumen intensity value . Therefore the consistency of the lumen value along the vessel was examined . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Smoothness of the vessel centreline pathway along the z - axis the vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Consistency of vessel lumen areas along the z - axis blurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Consistency of vessel lumen value along the z - axis blurring can also change the lumen intensity value . Therefore the consistency of the lumen value along the vessel was examined . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . A mean - shift algorithm was performed to suppress noise while preserving large changes . The quantitative vessel lumen area and value consistency analysis was also applied to the second vessel phantom to see whether real stenosis would give any difference the results of myocardium (i) and vessel (iia c) analysis were combined by correlating them side - by - side at the corresponding locations on z - axis, to determine whether there is coincidence of findings between the analysis results . Quantitative analysis was performed in the myocardium and the coronary for each heart rate and both modalities . The analysis was performed as follows: (see fig . 2 for the overview diagram)fig . 2diagram of the quantitative image analysis methods for the detection of motion artefacts in coronary artery computed tomographymyocardium analysisdue to the nature of the phantom s movement, motion artefacts in the myocardium were most apparent in the sagittal plane, especially in the anterior part of the myocardium . The sagittal cross - section images were taken at approximately the centre of the phantom (dotted line in fig . 1). The anterior inner lining of the myocardium (green line at fig . 3, left) was semi automatically extracted using a gradient vector flow (gvf) snake algorithm by first placing several seed points for the initial contour which were then allowed to grow to match the inner lining . From the extracted line, the following parameter was determined: smoothness of the inner - lining of the myocardiumsmoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3, . A gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . 3illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium the inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference linevisual observations by two independent observers were performed . Each observer was asked to score the sagittal images for the presence of no, mild or severe motion artefacts resulting in scores of 0, 1 and 2, respectively . Diagram of the quantitative image analysis methods for the detection of motion artefacts in coronary artery computed tomography due to the nature of the phantom s movement, motion artefacts in the myocardium were most apparent in the sagittal plane, especially in the anterior part of the myocardium . The sagittal cross - section images were taken at approximately the centre of the phantom (dotted line in fig . 1). The anterior inner lining of the myocardium (green line at fig . 3, left) was semi automatically extracted using a gradient vector flow (gvf) snake algorithm by first placing several seed points for the initial contour which were then allowed to grow to match the inner lining . From the extracted line, the following parameter was determined: smoothness of the inner - lining of the myocardiumsmoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3 a gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line smoothness of the inner - lining of the myocardium smoothness of the inner - lining is determined by the presence of discontinuities, which was examined from its gradient . A second order polynomial line was fitted to the gradient to act as reference line (fig . 3, a gaussian smoothing filter (width = 5; = 4) was applied to the gradient plot to remove possible noise . Locations with gradient deviating more than a preset threshold to the reference line were marked as motion artefacts . 3illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line illustration of myocardium analysis . From the sagittal cross section image of the phantom (left), the inner - lining of the myocardium was extracted (green line). The inner - lining was then analyzed for any discontinuities (right) by plotting the gradient along z - axis . Discontinuities were found at locations whose gradient deviates more than a certain threshold from the reference line visual observations by two independent observers were performed . Each observer was asked to score the sagittal images for the presence of no, mild or severe motion artefacts resulting in scores of 0, 1 and 2, respectively . Vessel analysisbecause of the occurrence of motion artefacts along z - axis, the analysis was limited to the vessel segment which was relatively parallel to the z - axis . The vessel segment was predetermined and set equal for all heart rates (segment from arrow 1 to arrow 2 at fig . 1, top). Started by manual selection of the vessel lumen in the axial view at location 1 of fig . 1, a small (50 by 50 pixels) region of interest (roi) was determined around the vessel lumen . The image inside the roi was thresholded at level 41% of the lumen peak value . Afterwards, using gvf snake algorithm, the lumen boundary was extracted and its centre of mass was determined as centre point . The detection was continued to the next slice without further user interaction, and repeated until the last slice (fig . 4, top).fig . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzedthree parameters were determined from the extracted vessel: smoothness of the vessel centreline pathway along the z - axisthe vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Comparison to 0 bpm dataset was made.consistency of vessel lumen areas along the z - axisblurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set.consistency of vessel lumen value along the z - axisblurring can also change the lumen intensity value . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Because of the occurrence of motion artefacts along z - axis, the analysis was limited to the vessel segment which was relatively parallel to the z - axis . The vessel segment was predetermined and set equal for all heart rates (segment from arrow 1 to arrow 2 at fig . 1, top). Started by manual selection of the vessel lumen in the axial view at location 1 of fig . 1, a small (50 by 50 pixels) region of interest (roi) the image inside the roi was thresholded at level 41% of the lumen peak value . Afterwards, using gvf snake algorithm, the lumen boundary was extracted and its centre of mass was determined as centre point . The detection was continued to the next slice without further user interaction, and repeated until the last slice (fig . 4, top).fig . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzed illustration on vessel analysis . The vessel extraction algorithm (top images) was started by manual selection of the starting point (white arrow top left) inside the vessel lumen at location 1 depicted at fig . 1, from where an roi (blue rectangle) was selected . Inside the roi (top centre), the lumen boundary (bold blue line with centre point at blue circle) was detected using gvf snake algorithm . The vessel was constructed from the detected vessel boundaries and centre points (top right) along z - axis . Afterwards, the smoothness of vessel centreline (bottom left), the consistency of vessel lumen area (bottom centre) and the consistency of vessel lumen mean value (bottom right) along z - axis were analyzed three parameters were determined from the extracted vessel: smoothness of the vessel centreline pathway along the z - axisthe vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Comparison to 0 bpm dataset was made.consistency of vessel lumen areas along the z - axisblurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set.consistency of vessel lumen value along the z - axisblurring can also change the lumen intensity value . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Smoothness of the vessel centreline pathway along the z - axis the vessel centreline was constructed using the detected centre points . The smoothness of the centreline is also determined by the presence of discontinuities, which were analyzed from its second derivative in the y - direction (direction of phantom movement; see direction legends at fig . 1, bottom) at each heart rate . Consistency of vessel lumen areas along the z - axis blurring can smear out the vessel lumen pixels, which consequently changes the amount of pixels considered to be lumen . The axial lumen area on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . Consistency of vessel lumen value along the z - axis blurring can also change the lumen intensity value . The lumen mean value inside the detected lumen boundary on each position along the detected centreline points from each heart rate was analyzed and compared to the 0 bpm data set . A mean - shift algorithm was performed to suppress noise while preserving large changes . The quantitative vessel lumen area and value consistency analysis was also applied to the second vessel phantom to see whether real stenosis would give any difference . The results of myocardium (i) and vessel (iia c) analysis were combined by correlating them side - by - side at the corresponding locations on z - axis, to determine whether there is coincidence of findings between the analysis results . The association between the qualitative and the developed quantitative analysis is listed on tables 1 and 2, for 64ct and dsct, respectively . The definition of quality scores are given by table 3 .smoothness of the inner - lining of the myocardiumthe discontinuities threshold was set to 0.2 . The visual observation of the two observers resulted in 38 individual motion artefacts, where 30 (79%) were identified by both observers, and eight (21%) by only one of both observers . 22 of 30 (73%) motion artefacts found by both observers were graded equal . From the eight artefacts found only by either one of the observer, four were discarded after consensus . In total, the consensus resulted in 34 motion artefacts: 21 on 64ct (six found to be severe) and 13 on dsct (one found to be severe). The quantitative analysis managed to find 29 out of the consented 34 motion artefacts (85%), of which all 7 (100%) severe artefacts and 22 out of 27 (81%) mild artefacts were found . None of the four consensus - discarded artefacts were found to be artefacts by the quantitative analysis.tables 1 and 2 list the comparison of the true positive quantitative findings of myocardium inner - lining discontinuities artefacts versus the qualitative analysis . The qualitative findings scored dsct with higher quality than 64ct, and the developed quantitative analysis concurred by finding more severe myocardium artefacts at 64ct . Therefore, only the severe myocardium artefacts can be related to the qualitative analysis.smoothness of vessel centreline pathway along z - axisthe second derivatives of all heart rates have small absolute values below 1.5 indicating that no large discontinuities at the vessel pathway occurred and a student s t test comparing the second derivatives of all heart rates to 0 bpm showed no significant differences (p <0.05). The regular heart rate of the phantom and fixed selection of reconstruction phase in the r r interval most probably caused the vessel to be always at the same position along the scan direction.consistency of vessel lumen areas along the z - axiscomparing the lumen area of all heart rates to 0 bpm on each modality, consistent vessel volume (cumulative sum of lumen areas along the vessel) reduction was observed at all dataset, except at 60 bpm on dsct (see tables 1, 2 under field cumulative area differences for 64ct and dsct, respectively). For the rest of this article, this lumen area reduction will be called stenosis (as opposed to the conventional definition of a stenosis, i.e. Reduction of lumen area at certain location compared to the normal vessel proximal to it; which will be written in italic for the rest of the article). Although relatively small (<10%), this consistent stenosis implies that ct will always underestimate the vessel size and could thus underestimate stenosis severity in clinical settings . The stenoses occurred in segments, classified as medium (1020%) and large (> 20%) (see tables 1, 2 under field artefact - induced lumen area stenoses segments for 64ct and dsct, respectively). Small (<10%) lumen area stenoses segments were ignored because of their small significance.applying the algorithm to the vessel phantom, stenoses of 48 2% and 73 3% were detected for the designed stenoses of 56 and 75%, respectively.comparing to the qualitative results, the quantitative analysis concurred by finding larger overall cumulative stenosis on 64ct than on dsct (4.9 vs. 3.5%). Moreover, the largest cumulative stenosis and the presence of large stenoses segments concurred with the lowest qualitative score at 100 bpm on 64ct . However, in the dsct datasets qualitatively scored as ~4, medium stenoses segments were also found.consistency of vessel lumen value along the z - axisfluctuations on vessel lumen value along z - axis were observed both at 64ct and dsct . The (absolute) changes were classified as medium (2040 hu), and large (> 40 hu) (see tables 1, 2 under field lumen mean value changes, for 64ct and dsct, respectively). Small (<20 hu) changes were ignored because of their small significance.applying the algorithm to the vessel phantom, a small (1020 hu) lumen - mean value decrease and a medium (40 hu) decrease were detected at the designed stenoses of 56 and 75%, respectively.comparing to the qualitative results, the quantitative analysis appears to show the opposite by finding more lumen value changes on dsct than on 64ct . It is possible that these changes are not noticeable on the 3d vrt and curved mpr views used by the qualitative analysis.table 1qualitative and quantitative motion artefact analysis on 64ctheart rate (bpm)64ctqualitative analysisquantitative analysismyocardium inner - lining discontinuitieslumen arealumen valuecumulative area differences (%) artefact - induced lumen area stenoses segmentslumen mean value change(s)mediumlargemediumlarge04.0 03503.7 0.83.112603.5 0.53.414702.3 0.534.9111 (1)803.8 0.43.512903.0 1.34.1111001.3 0.57 (4)10.31 (1)2 (2)2 (1)1 (1)1102.0 0.67 (2)5.13 (3)1 (1)overall3.0 1.117 (6)4.99 (4)2 (2)15 (1)3 (3)the value was taken from previous publication . The value was given based on criteria listed in table 3amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observationamount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefactsamount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefactstable 2qualitative and quantitative motion artefact analysis on dsctheart rate (bpm)dsctqualitative analysisquantitative analysismyocardium inner - lining discontinuitieslumen arealumen valuecumulative area differences (%) artefact - induced lumen area stenoses segmentslumen mean value change(s)mediumlargemediumlarge04.3 0.53504.0 0.623.1141604.5 0.510.43703.8 0.42 (1)3.92 (1)31804.3 0.54.812904.5 0.53.411004.3 0.532.71 (1)3 (1)11103.8 0.846.82 (2)43 (2)overall4.2 0.612 (1)3.57 (4)23 (1)6 (2)the value was taken from previous publication . The value was given based on criteria listed in table 3amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observationamount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefactsamount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefactstable 3definition of image quality scores scoredefinition of image quality1image with step artefacts and/or stripes throughout the image limiting evaluation of the coronary artery and pericardium2image with step artefacts and/or stripes in part of the image that result in limited evaluation of the coronary artery and pericardium3image with step artefacts and/or stripes which have minor implication on the evaluation of the coronary artery and pericardium4image with minor motion artefacts not hampering the evaluation of the coronary artery and pericardium5excellent image quality without motion artefacts smoothness of the inner - lining of the myocardium the discontinuities threshold was set to 0.2 . The visual observation of the two observers resulted in 38 individual motion artefacts, where 30 (79%) were identified by both observers, and eight (21%) by only one of both observers . 22 of 30 (73%) motion artefacts found by both observers were graded equal . From the eight artefacts found only by either one of the observer, four were discarded after consensus . In total, the consensus resulted in 34 motion artefacts: 21 on 64ct (six found to be severe) and 13 on dsct (one found to be severe). The quantitative analysis managed to find 29 out of the consented 34 motion artefacts (85%), of which all 7 (100%) severe artefacts and 22 out of 27 (81%) mild artefacts were found . None of the four consensus - discarded artefacts were found to be artefacts by the quantitative analysis . Tables 1 and 2 list the comparison of the true positive quantitative findings of myocardium inner - lining discontinuities artefacts versus the qualitative analysis . The qualitative findings scored dsct with higher quality than 64ct, and the developed quantitative analysis concurred by finding more severe myocardium artefacts at 64ct . Smoothness of vessel centreline pathway along z - axis the second derivatives of all heart rates have small absolute values below 1.5 indicating that no large discontinuities at the vessel pathway occurred and a student s t test comparing the second derivatives of all heart rates to 0 bpm showed no significant differences (p <0.05). The regular heart rate of the phantom and fixed selection of reconstruction phase in the r r interval most probably caused the vessel to be always at the same position along the scan direction . Consistency of vessel lumen areas along the z - axis comparing the lumen area of all heart rates to 0 bpm on each modality, consistent vessel volume (cumulative sum of lumen areas along the vessel) reduction was observed at all dataset, except at 60 bpm on dsct (see tables 1, 2 under field cumulative area differences for 64ct and dsct, respectively). For the rest of this article, this lumen area reduction will be called stenosis (as opposed to the conventional definition of a stenosis, i.e. Reduction of lumen area at certain location compared to the normal vessel proximal to it; which will be written in italic for the rest of the article). Although relatively small (<10%), this consistent stenosis implies that ct will always underestimate the vessel size and could thus underestimate stenosis severity in clinical settings . The stenoses occurred in segments, classified as medium (1020%) and large (> 20%) (see tables 1, 2 under field artefact - induced lumen area stenoses segments for 64ct and dsct, respectively). Small (<10%) lumen area stenoses segments were ignored because of their small significance . Applying the algorithm to the vessel phantom, stenoses of 48 2% and 73 3% were detected for the designed stenoses of 56 and 75%, respectively . Comparing to the qualitative results, the quantitative analysis concurred by finding larger overall cumulative stenosis on 64ct than on dsct (4.9 vs. 3.5%). Moreover, the largest cumulative stenosis and the presence of large stenoses segments concurred with the lowest qualitative score at 100 bpm on 64ct . However, in the dsct datasets qualitatively scored as ~4, medium stenoses segments were also found . Consistency of vessel lumen value along the z - axis fluctuations on vessel lumen value along z - axis were observed both at 64ct and dsct . The (absolute) changes were classified as medium (2040 hu), and large (> 40 hu) (see tables 1, 2 under field lumen mean value changes, for 64ct and dsct, respectively). Small (<20 hu) changes were ignored because of their small significance . Applying the algorithm to the vessel phantom, a small (1020 hu) lumen - mean value decrease and a medium (40 hu) decrease were detected at the designed stenoses of 56 and 75%, respectively . Comparing to the qualitative results, the quantitative analysis appears to show the opposite by finding more lumen value changes on dsct than on 64ct . It is possible that these changes are not noticeable on the 3d vrt and curved mpr views used by the qualitative analysis . Qualitative and quantitative motion artefact analysis on 64ct the value was taken from previous publication . The value was given based on criteria listed in table 3 amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observation amount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefacts amount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefacts qualitative and quantitative motion artefact analysis on dsct the value was taken from previous publication . The value was given based on criteria listed in table 3 amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observation amount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefacts amount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefacts definition of image quality scores combining the vessel lumen value (iic) and area (iib) analysis, one large negative lumen value change (60 hu) at 100 bpm on 64ct was found to coincide with the large lumen area stenosis (30%). Combining the vessel lumen value (iic) and myocardium (ia) analysis, two out of 38 medium (5.3%) and five out of nine (55.6%) large lumen value changes were found to coincide with the myocardium artefacts (see tables 1, 2 under field, we can derive that motion could blur the vessel, reducing the attenuation value . From the experiment with the second vessel phantom, similar finding of a large lumen mean value decrease at the 75%-stenosis phantom this result indicated that a large stenosis decreases the amount of lumen pixels to be significantly influenced by partial volume effect . Therefore, it is hard to distinguish artefact - induced and real stenosis based on lumen mean value decrease alone . Combining the vessel lumen area (iib) and myocardium (ia) analysis, ten out of 29 myocardium artefacts were found to correlate with lumen area stenoses, of which two were severe stenoses at 100 bpm on 64ct . Figure 5 shows the combined analysis at this dataset . We can directly correlate the sharp change at point a with the qualitative step artefact observation, but not at point b. nevertheless, there is more than 20% lumen area reduction close to it . Without apparent step artefacts on the plot, this lumen area reduction could be regarded as a true stenosis . However, by finding a myocardial motion artefact at the corresponding location, this lumen area reduction could be marked as artefact - induced . Although, as can be seen also in point c and d in fig . 5, the presence of motion artefacts does not always have enough effect on the vessel visualization to result in apparent stenosis . Therefore, it is useful to check for the presence of motion artefacts in corresponding location in myocardium, if a stenosis is found . However, it might not be necessary if no stenosis is detected, although those areas will still be suspicious . The location of the detected myocardium artefacts are indicated by arrows a to d in the three - dimensional volume reconstruction view (left) and by red vertical lines a to d in the vessel lumen area consistency graph (right)table 4recommendation to interpret findingstype of findingsmeaningvessel stenosismyocardial artefactnormal vessel+true stenosis+suspicious area of motion artefact++possible artefact - induced stenosis combination of vessel (iib) and myocardium (ia) analysis . The location of the detected myocardium artefacts are indicated by arrows a to d in the three - dimensional volume reconstruction view (left) and by red vertical lines a to d in the vessel lumen area consistency graph (right) recommendation to interpret findings the association between the qualitative and the developed quantitative analysis is listed on tables 1 and 2, for 64ct and dsct, respectively . The definition of quality scores are given by table 3 .smoothness of the inner - lining of the myocardiumthe discontinuities threshold was set to 0.2 . The visual observation of the two observers resulted in 38 individual motion artefacts, where 30 (79%) were identified by both observers, and eight (21%) by only one of both observers . 22 of 30 (73%) motion artefacts found by both observers were graded equal . From the eight artefacts found only by either one of the observer, four were discarded after consensus . In total, the consensus resulted in 34 motion artefacts: 21 on 64ct (six found to be severe) and 13 on dsct (one found to be severe). The quantitative analysis managed to find 29 out of the consented 34 motion artefacts (85%), of which all 7 (100%) severe artefacts and 22 out of 27 (81%) mild artefacts were found . None of the four consensus - discarded artefacts were found to be artefacts by the quantitative analysis.tables 1 and 2 list the comparison of the true positive quantitative findings of myocardium inner - lining discontinuities artefacts versus the qualitative analysis . The qualitative findings scored dsct with higher quality than 64ct, and the developed quantitative analysis concurred by finding more severe myocardium artefacts at 64ct . Therefore, only the severe myocardium artefacts can be related to the qualitative analysis.smoothness of vessel centreline pathway along z - axisthe second derivatives of all heart rates have small absolute values below 1.5 indicating that no large discontinuities at the vessel pathway occurred and a student s t test comparing the second derivatives of all heart rates to 0 bpm showed no significant differences (p <0.05). The regular heart rate of the phantom and fixed selection of reconstruction phase in the r r interval most probably caused the vessel to be always at the same position along the scan direction.consistency of vessel lumen areas along the z - axiscomparing the lumen area of all heart rates to 0 bpm on each modality, consistent vessel volume (cumulative sum of lumen areas along the vessel) reduction was observed at all dataset, except at 60 bpm on dsct (see tables 1, 2 under field cumulative area differences for 64ct and dsct, respectively). For the rest of this article, this lumen area reduction will be called stenosis (as opposed to the conventional definition of a stenosis, i.e. Reduction of lumen area at certain location compared to the normal vessel proximal to it; which will be written in italic for the rest of the article). Although relatively small (<10%), this consistent stenosis implies that ct will always underestimate the vessel size and could thus underestimate stenosis severity in clinical settings . The stenoses occurred in segments, classified as medium (1020%) and large (> 20%) (see tables 1, 2 under field artefact - induced lumen area stenoses segments for 64ct and dsct, respectively). Small (<10%) lumen area stenoses segments were ignored because of their small significance.applying the algorithm to the vessel phantom, stenoses of 48 2% and 73 3% were detected for the designed stenoses of 56 and 75%, respectively.comparing to the qualitative results, the quantitative analysis concurred by finding larger overall cumulative stenosis on 64ct than on dsct (4.9 vs. 3.5%). Moreover, the largest cumulative stenosis and the presence of large stenoses segments concurred with the lowest qualitative score at 100 bpm on 64ct . However, in the dsct datasets qualitatively scored as ~4, medium stenoses segments were also found.consistency of vessel lumen value along the z - axisfluctuations on vessel lumen value along z - axis were observed both at 64ct and dsct . The (absolute) changes were classified as medium (2040 hu), and large (> 40 hu) (see tables 1, 2 under field lumen mean value changes, for 64ct and dsct, respectively). Small (<20 hu) changes were ignored because of their small significance.applying the algorithm to the vessel phantom, a small (1020 hu) lumen - mean value decrease and a medium (40 hu) decrease were detected at the designed stenoses of 56 and 75%, respectively.comparing to the qualitative results, the quantitative analysis appears to show the opposite by finding more lumen value changes on dsct than on 64ct . It is possible that these changes are not noticeable on the 3d vrt and curved mpr views used by the qualitative analysis.table 1qualitative and quantitative motion artefact analysis on 64ctheart rate (bpm)64ctqualitative analysisquantitative analysismyocardium inner - lining discontinuitieslumen arealumen valuecumulative area differences (%) artefact - induced lumen area stenoses segmentslumen mean value change(s)mediumlargemediumlarge04.0 03503.7 0.83.112603.5 0.53.414702.3 0.534.9111 (1)803.8 0.43.512903.0 1.34.1111001.3 0.57 (4)10.31 (1)2 (2)2 (1)1 (1)1102.0 0.67 (2)5.13 (3)1 (1)overall3.0 1.117 (6)4.99 (4)2 (2)15 (1)3 (3)the value was taken from previous publication . The value was given based on criteria listed in table 3amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observationamount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefactsamount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefactstable 2qualitative and quantitative motion artefact analysis on dsctheart rate (bpm)dsctqualitative analysisquantitative analysismyocardium inner - lining discontinuitieslumen arealumen valuecumulative area differences (%) artefact - induced lumen area stenoses segmentslumen mean value change(s)mediumlargemediumlarge04.3 0.53504.0 0.623.1141604.5 0.510.43703.8 0.42 (1)3.92 (1)31804.3 0.54.812904.5 0.53.411004.3 0.532.71 (1)3 (1)11103.8 0.846.82 (2)43 (2)overall4.2 0.612 (1)3.57 (4)23 (1)6 (2)the value was taken from previous publication . The value was given based on criteria listed in table 3amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observationamount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefactsamount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefactstable 3definition of image quality scores scoredefinition of image quality1image with step artefacts and/or stripes throughout the image limiting evaluation of the coronary artery and pericardium2image with step artefacts and/or stripes in part of the image that result in limited evaluation of the coronary artery and pericardium3image with step artefacts and/or stripes which have minor implication on the evaluation of the coronary artery and pericardium4image with minor motion artefacts not hampering the evaluation of the coronary artery and pericardium5excellent image quality without motion artefacts smoothness of the inner - lining of the myocardium the discontinuities threshold was set to 0.2 . The visual observation of the two observers resulted in 38 individual motion artefacts, where 30 (79%) were identified by both observers, and eight (21%) by only one of both observers . 22 of 30 (73%) motion artefacts found by both observers were graded equal . From the eight artefacts found only by either one of the observer, four were discarded after consensus . In total, the consensus resulted in 34 motion artefacts: 21 on 64ct (six found to be severe) and 13 on dsct (one found to be severe). The quantitative analysis managed to find 29 out of the consented 34 motion artefacts (85%), of which all 7 (100%) severe artefacts and 22 out of 27 (81%) mild artefacts were found . None of the four consensus - discarded artefacts were found to be artefacts by the quantitative analysis . Tables 1 and 2 list the comparison of the true positive quantitative findings of myocardium inner - lining discontinuities artefacts versus the qualitative analysis . The qualitative findings scored dsct with higher quality than 64ct, and the developed quantitative analysis concurred by finding more severe myocardium artefacts at 64ct . Smoothness of vessel centreline pathway along z - axis the second derivatives of all heart rates have small absolute values below 1.5 indicating that no large discontinuities at the vessel pathway occurred and a student s t test comparing the second derivatives of all heart rates to 0 bpm showed no significant differences (p <0.05). The regular heart rate of the phantom and fixed selection of reconstruction phase in the r r interval most probably caused the vessel to be always at the same position along the scan direction . Consistency of vessel lumen areas along the z - axis comparing the lumen area of all heart rates to 0 bpm on each modality, consistent vessel volume (cumulative sum of lumen areas along the vessel) reduction was observed at all dataset, except at 60 bpm on dsct (see tables 1, 2 under field cumulative area differences for 64ct and dsct, respectively). For the rest of this article, this lumen area reduction will be called stenosis (as opposed to the conventional definition of a stenosis, i.e. Reduction of lumen area at certain location compared to the normal vessel proximal to it; which will be written in italic for the rest of the article). Although relatively small (<10%), this consistent stenosis implies that ct will always underestimate the vessel size and could thus underestimate stenosis severity in clinical settings . The stenoses occurred in segments, classified as medium (1020%) and large (> 20%) (see tables 1, 2 under field artefact - induced lumen area stenoses segments for 64ct and dsct, respectively). Small (<10%) lumen area stenoses segments were ignored because of their small significance . Applying the algorithm to the vessel phantom, stenoses of 48 2% and 73 3% were detected for the designed stenoses of 56 and 75%, respectively . Comparing to the qualitative results, the quantitative analysis concurred by finding larger overall cumulative stenosis on 64ct than on dsct (4.9 vs. 3.5%). Moreover, the largest cumulative stenosis and the presence of large stenoses segments concurred with the lowest qualitative score at 100 bpm on 64ct . However, in the dsct datasets qualitatively scored as ~4, medium stenoses segments were also found . Consistency of vessel lumen value along the z - axis fluctuations on vessel lumen value along z - axis were observed both at 64ct and dsct . The (absolute) changes were classified as medium (2040 hu), and large (> 40 hu) (see tables 1, 2 under field lumen mean value changes, for 64ct and dsct, respectively). Small (<20 hu) changes were ignored because of their small significance . Applying the algorithm to the vessel phantom, a small (1020 hu) lumen - mean value decrease and a medium (40 hu) decrease were detected at the designed stenoses of 56 and 75%, respectively . Comparing to the qualitative results, the quantitative analysis appears to show the opposite by finding more lumen value changes on dsct than on 64ct . It is possible that these changes are not noticeable on the 3d vrt and curved mpr views used by the qualitative analysis . Qualitative and quantitative motion artefact analysis on 64ct the value was taken from previous publication . The value was given based on criteria listed in table 3 amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observation amount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefacts amount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefacts qualitative and quantitative motion artefact analysis on dsct the value was taken from previous publication . The value was given based on criteria listed in table 3 amount of myocardium inner - lining discontinuities found by the quantitative analysis . The values between brackets indicate findings categorized as severe by visual observation amount of vessel stenoses segments found by the quantitative analysis . The values between brackets indicate the amount of stenoses segments that coincide with myocardium artefacts amount of vessel lumen mean value changes found by the quantitative analysis . The values between brackets indicate the amount of the changes that coincide with myocardium artefacts definition of image quality scores combining the vessel lumen value (iic) and area (iib) analysis, one large negative lumen value change (60 hu) at 100 bpm on 64ct was found to coincide with the large lumen area stenosis (30%). Combining the vessel lumen value (iic) and myocardium (ia) analysis, two out of 38 medium (5.3%) and five out of nine (55.6%) large lumen value changes were found to coincide with the myocardium artefacts (see tables 1, 2 under field, we can derive that motion could blur the vessel, reducing the attenuation value . From the experiment with the second vessel phantom, similar finding of a large lumen mean value decrease at the 75%-stenosis phantom this result indicated that a large stenosis decreases the amount of lumen pixels to be significantly influenced by partial volume effect . Therefore, it is hard to distinguish artefact - induced and real stenosis based on lumen mean value decrease alone . Combining the vessel lumen area (iib) and myocardium (ia) analysis, ten out of 29 myocardium artefacts were found to correlate with lumen area stenoses, of which two were severe stenoses at 100 bpm on 64ct . Figure 5 shows the combined analysis at this dataset . We can directly correlate the sharp change at point a with the qualitative step artefact observation, but not at point b. nevertheless, there is more than 20% lumen area reduction close to it . Without apparent step artefacts on the plot, this lumen area reduction could be regarded as a true stenosis . However, by finding a myocardial motion artefact at the corresponding location, this lumen area reduction could be marked as artefact - induced . Although, as can be seen also in point c and d in fig . 5, the presence of motion artefacts does not always have enough effect on the vessel visualization to result in apparent stenosis . Therefore, it is useful to check for the presence of motion artefacts in corresponding location in myocardium, if a stenosis is found . However, it might not be necessary if no stenosis is detected, although those areas will still be suspicious . The location of the detected myocardium artefacts are indicated by arrows a to d in the three - dimensional volume reconstruction view (left) and by red vertical lines a to d in the vessel lumen area consistency graph (right)table 4recommendation to interpret findingstype of findingsmeaningvessel stenosismyocardial artefactnormal vessel+true stenosis+suspicious area of motion artefact++possible artefact - induced stenosis combination of vessel (iib) and myocardium (ia) analysis . The location of the detected myocardium artefacts are indicated by arrows a to d in the three - dimensional volume reconstruction view (left) and by red vertical lines a to d in the vessel lumen area consistency graph (right) recommendation to interpret findings the developed quantitative analysis managed to detect the motion artefacts in the phantoms scans at 64ct and dsct . Moreover, it explored into more details the effect of motion artefacts on vessel visualization, even the ones that were missed by qualitative analysis . When evaluating the coronary arteries, the proposed procedure could warn the radiologist for suspicious areas where motion artefacts are present that could hamper the evaluation of stenoses in the coronary arteries . This especially holds in the case that a radiologist is reviewing segmented and stretched views of the coronary arteries in which stenotic lesions could easily be misinterpreted . Detection of areas of motion artefacts could help to avoid false positive findings in coronary cta stenoses evaluation . A false positive finding could direct the patients into unnecessary treatment which could pose another risk such as the possible risks related to percutaneous transluminal coronary angioplasty (ptca). However, on the other side, patients with undetected coronary problems could live a long time without any problem, provided the patients were not subjected to excessive physical or emotional stress . This study used 64-mdct and dsct, two modalities with similar characteristics except for their respective temporal resolution . Dsct has twice the temporal resolution of 64-mdct, due to the two perpendicular x - ray tubes inside its gantry rotating simultaneously . The qualitative analysis had shown the superiority of dsct over 64-mdct in avoiding motion artefacts . However, the quantitative analysis managed to reveal some artefacts on both modalities that would otherwise be missed . . Attempted to quantitatively analyze motion artefacts in coronary arteries, using two variables . The first variable is the percentage of coronary - length that is imaged without artefact, which nicely described the effect of motion to the coronaries . In fact, our proposed method could be used for the motion artefact detection for this variable . The second variable is the contrast to noise ratio (cnr), which was calculated from the contrast of the vessel lumen mean attenuation value to the surrounding soft tissues, compared to the noise in the aorta . The consistency of vessel lumen value along z - axis measurement is similar to this variable, without comparison to surrounding soft tissue but with the advantage of location - specific depiction of motion artefacts . Reported their finding of lumen mean value decrease at stenoses larger than 20% based on patient study . This is consistent with our finding of lumen mean value decrease at large stenosis area . However, their study excluded dataset suffering from motion artefact which makes a direct comparison with our finding not possible . The limitation of this study is the use of phantom data instead of patients data . Lack of real myocardium and vessel tissue of the phantom, and of surrounding pericardial fat tissue and chest cavity environment are factors that separate our phantom study to those of clinical patient examinations . Some adjustments can be made to apply our proposed method to the clinical examinations, such as: the parameters controlling gvf snake to extract myocardium boundaries, as in clinical examination, the heart chambers will be filled with contrast - enhanced blood instead of air . Other algorithm can be directly applicable to clinical examinations, such as: the lumen peak value - dependent lumen thresholding as this method was taken from a clinical study . The pre - processing step of gvf snake should be able to handle the additional noises from scattering and attenuation inside the chest cavity . However, scans with different kv will affect hu values of materials, especially ones with high atomic number such as the contrast agent . The proposed method does not use a fixed hu threshold in any of the algorithms, which should make them also applicable to such examinations . In general, this phantom experiment has its advantage in the ability to adjust the heart rate in a controlled manner . The effects of heart rates in a large interval, from low until very high, can be individually studied . We conclude that the developed quantitative analysis adds to the diagnostic value of a qualitative analysis . The quantitative analysis allows for the detection of suspicious regions of the coronary arteries thus reducing the false positive stenosis rate . Several publications reported an almost perfect score of negative predictive value of mdct in detecting stenosis, but lower values were reported for positive predictive value [1719]. The quantitative analysis proposed in this study could improve the positive predictive value by reducing the number of false positive finding . . Such study would involve patients examined by mdct with x - ray angiography as stenosis reference . An adjusted version of the proposed method will be applied to the data to detect and quantify motion artefacts . The interpretation recommendation listed by table 4 will be used to examine the images with reference to x - ray angiography findings.
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From january 1998 through may 2005, a total of 2,421 fecal specimens were collected from children <10 years of age (median age 2.3 years) with acute diarrhea in rio de janeiro, brazil . Of these, 478 (19.7%) specimens were collected from hospitalized children (inpatients) and 1,943 from outpatient children (341 [14.1%] from the emergency department and 1,602 [66.2%] from the walk - in clinic). Of these samples, the median age was 12.5 months for rotavirus - positive patients and 12.2 months for adenovirus - positive patients . Overall, of the hospitalized, emergency department, and walk - in clinic patients, 11.7%, 6.2%, and 10.0%, respectively, had samples positive for rotavirus, and 4.8%, 4.1%, and 4.4%, respectively, had samples positive for adenovirus . Yersinia enterocolitica, campylobacter spp ., and shigella spp . Were found in 8% of the specimens ., 1 rotavirus and salmonella spp ., and 1 rotavirus and e. coli). We selected 289 specimens that represented a random subset of samples that had prior negative results for rotavirus and enteric adenovirus . Of these 289, 117 were collected from inpatients and 172 from outpatients (89 emergency department and 83 walk - in clinic). Suspensions of stool (10%) were prepared in diethylpyrocarbonate - treated water and vertrel xf (miller - stephenson, sylmar, ca, usa) and clarified by centrifugation at 2,100 g for 10 min . We used 200 l of suspension for rna extraction with the nuclisens extraction kit (organon tekninka, durham, nc, usa) according to the manufacturer s instructions . The rna was eluted in 50 l of elution buffer and stored at 70c until use . A total of 240 samples were tested for norovirus rna by light cycler pcr that used primers and probes for orf1/orf2 junction region specific for norovirus gi and gii, as described (3,12), and by the light cycler rna amplification kit hybridization probes (roche, basel, switzerland). The 49 remaining samples were tested only by conventional rt - pcr, as described (5). Conventional rt - pcr was performed with the qiagen onestep rt - pcr kit (qiagen, valencia, ca, usa). The rna samples were subjected to 1 cycle of reverse transcription (42c, 10 min) followed by 5 min at 95c . Pcr was performed for 40 cycles, each consisting of 1 min at 94c, 1 min at 40c (for gi detection) or 1 min at 44c for (for gii detection), 1 min at 72c, and a final extension cycle of 10 min at 72c . We selected 6 samples that were positive by real - time light cycler pcr (2 gi and 4 gii) for analysis by conventional rt - pcr with specific primers in capsid region d of norovirus gi and gii, as described above . The amplified cdna samples were purified from the gel by using qiaquick gel extraction kit (qiagen), and the sequences were determined with the bigdye terminator cycle sequencing kit and the abi prism 3100 automated dna sequencer (applied biosystems, foster city, ca, usa) by using the same primers as used for the conventional rt - pcr . The nucleotide sequences of the amplicons were aligned with corresponding sequences of selected norovirus strains available in the genbank database and analyzed by using the clustal v algorithm of the megalign program in the dnastar software package (madison, wi, usa). Dq496212, dq496213, dq496214, dq496215, dq496216, and dq496217 . Of the 289 fecal specimens tested, 42 (14.5%) were positive for norovirus: 36 (15%; n = 240) by real time light cycler pcr and 6 (12.2%; n = 49) by conventional rt - pcr . These percentages correspond only to single infections because we did not test samples already known to be positive for other pathogens such as rotavirus and adenovirus . Positive samples and genogroups varied by year with no obvious yearly pattern (table 1). The winter vomiting disease, we detected infection throughout the year, with no seasonal pattern (figure). Norovirus infections were equally common among outpatients and inpatients . Among 117 inpatients, 18 (15.4%) had positive norovirus test results compared with 24 (14%) of 172 outpatients (11 emergency department and 13 walk - in clinic). Although the disease caused by norovirus is described as mild (diarrhea, vomiting, abdominal pain, and fever) and generally does not lead to hospitalization (13,14), of 42 norovirus - infected children, 29 (69%) were either hospitalized or received medical care in the emergency department, which suggests that they had a more severe illness . Only 13 (31%) of the 42 norovirus - infected children attended the walk - in clinic, which suggests that they had mild disease (table 2). Other than diarrhea, fever was the most common symptom among the 42 norovirus - positive patients in this study, reported for 11 (26.2%) patients . Vomiting only was described for 8 (19.0%); vomiting and fever was described for 6 (14.3%). No mixed infection with bacteria seasonal distribution of norovirus (nov) infections in rio de janeiro, brazil, 19982005 . Although norovirus belonging to genogroup gii is considered the most prevalent strain worldwide (79,11,15), we found no important difference in the prevalence of the 2 genogroups detected in our study . Overall, 20 (48%) of the 42 samples were identified as genogroup gi and 22 (52%) as gii (table 1). No statistically significant difference in the prevalence of gi and gii was observed between inpatients and outpatients . Our study documents that noroviruses are a common cause of acute gastroenteritis in children who are inpatients or outpatients in brazil and are likely second only to rotavirus as a cause of severe childhood diarrhea . Nonetheless, it documents how common norovirus infections may be and indicates that further study will be necessary to assess their role among brazilian children, to understand the epidemiology of the disease, and to seek evidence of immunity in children, which might encourage development of a vaccine.
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Achieving emmetropia in eyes with full thickness corneal grafts is a target that has long been pursued . For decades, a successful keratoplasty was judged in terms of preservation of a clear surviving graft as a final outcome . However, the ultimate goal of vision restoration was often hampered by the frequent association with postoperative ametropia and/or anisometropia [1, 2]. Astigmatism, the main refractive error following penetrating keratoplasty (pkp), has been related to a variety of pre-, intra-, and postoperative factors, while the less commonly associated spherical errors as myopia and, rarely, hyperopia were attributed mainly to axial length abnormalities and postoperative suture manipulations [14]. A wide scope of therapeutic modalities has been proposed and employed for the correction of such refractive errors . These ranged from the very conservative spectacle prescription to the final possibility of repeating the whole grafting procedure . However, none of these techniques has proven itself as a sole ideal solution for the management of post - pkp ametropia and many are associated with graft survival and, even, vision threatening complications [1, 2, 5]. Visual rehabilitation using spectacles represents a good choice but their use is limited by significant anisometropia especially with astigmatism more than 3 d or the presence of irregular astigmatism . Contact lenses (especially rigid gas permeable) provide another conservative management option but dry - eye syndrome and fitting - related inconvenience as well as patient's age, dexterity, and lifestyle are major concerns that may affect contact lens tolerance . They may also induce peripheral corneal neovascularization which can result in graft rejection [1, 2, 69]. Initial surgical management options range from simple procedures like selective suture removal or suture adjustment [4, 10, 11] to the more sophisticated techniques of astigmatic keratotomy, relaxing incisions with or without compression sutures [1315], and wedge resection . The latter two have low predictability and are associated with a high incidence of recurrence of astigmatism . They also carry the risk of inducing overcorrection, perforation, wound dehiscence, and graft rejection . Moreover, they mainly aim for rectifying the astigmatic component of the refractive error but they do little, if any, when it comes to the spherical part of refraction [1, 2]. Intracorneal ring segment (icr) implantation has recently been reported in post - pkp eyes but with significant undercorrection and low predictability compared to their results in keratoconic eyes . Immune reaction induction leading to graft rejection and ring migration and perforation into the anterior chamber are possible complications that can violate the future integrity of the grafted cornea [1, 17, 18]. Excimer laser photorefractive keratectomy (prk) has also been proposed but was proven less predictable than when performed in previously nonoperated eyes . Limited astigmatic correction, irregular astigmatism induction, significant regression, corneal haze, and photoablation induced graft rejection are other important disadvantages of such a technique [19, 20]. The use of laser in situ keratomileusis (lasik) reduced haze and allowed for more refractive correction but showed an increased risk of flap complications compared to normal eyes . Moreover, the lamellar keratectomy step causes thinning of the graft - host interface as the flap usually has a larger diameter than the corneal button . This disruption of the healing scar can add to the risk of having wound dehiscence . Toric intraocular lens implantation (phakic or pseudophakic) provides a wider range of correction but lens rotation, increased endothelial cell loss, corneal decompensation, graft rejection, endophthalmitis, and secondary glaucoma are important drawbacks of such procedures [2732]. Small incision lenticule extraction (smile) is a novel, all - in - one, corneal laser refractive surgical procedure in which a lenticule of stromal tissue of planned thickness and diameter is isolated between two intracorneal planes created using a femtosecond laser platform . The lenticule is, then, manually removed from the cornea through a small incision to change the corneal curvature and exert its refractive effect . It was reported to be safe, predictable, and effective for treating myopia and myopic astigmatism in previously nonoperated eyes . It has the advantages of being flapless and less invasive than other intraocular procedures together with having the ability to tailor and center the whole procedure as required within a specific area of the cornea [3335]. This study was performed with the aim of evaluation of visual and refractive outcomes after smile for treating post - pkp myopia and myopic astigmatism . This interventional case - series included ten eyes of 10 patients with previous pkp and residual compound myopic astigmatism . Inclusion criteria consisted of patients who had had an 8.25 mm donor button transplanted to 8.00 mm trephination of the recipient cornea with a duration of at least 18 months since the time of keratoplasty and a residual myopic refractive error of up to 10.0 diopters (d) of spherical equivalent with astigmatism up to 6.0 d at the spectacle plane . A smooth postkeratoplasty course with no attacks of graft rejection or suture complications was mandatory . Sutures were completely removed prior to performing the preoperative examination by at least three months during which patients were followed up monthly to ensure a stable refraction . Only patients whose topography and anterior and posterior elevation maps' data within the acceptable range for laser vision correction and a thinnest graft location of 500 m or greater were enrolled in the study . Patients with graft apposition abnormalities (override or underride), severe dry eye, ocular surface disease, abnormal topographies, thin grafts, elevated intraocular pressure (iop), peripheral corneal neovascularization, nonsuture track related peripheral opacities, or central or paracentral opacities were excluded . Patients experiencing post - smile interface inflammation, cellular infiltration, or any other reported smile complication were planned to be excluded, as well . Also patients with other ocular or recorded eye - related systemic illnesses (e.g., diabetes mellitus) were not included in the study . The study protocol was based on the tenets of the declaration of helsinki and was approved by the ethics committee of the faculty of medicine of alexandria university . The risks and advantages of the procedure were explained to all patients and an informed consent was obtained from each of them . Full ophthalmic examination was performed including measurements of manifest refraction (mr), uncorrected distance visual acuity (udva), corrected distance visual acuity (cdva) (using snellen decimal notation), and intraocular pressure as well as biomicroscopic fundus examination . Keratometric data, corneal topography, thickness data, and height maps of the anterior and posterior corneal surfaces were obtained from the allegro oculyzer pentacam (wavelight, gmbh, germany). Following smile, postoperative follow - up visits were scheduled at 1 day, 1 week, and 1, 3, and 6 months postoperatively . During these visits, biomicroscopic examination of the anterior and posterior segments and udva, mr, and cdva testing and recording were performed . Efficacy was expressed in terms of the cumulative udva at 6 months postoperatively as well as the efficacy index calculated as the ratio of the postoperative udva to the preoperative cdva . Safety was judged by the change in the corrected distance visual acuity at 6 months postoperatively and also by the safety index calculated as the ratio of the postoperative cdva to the preoperative cdva . Cases were operated by two surgeons (osama ibrahim and tamer h. massoud) in roayah vision correction centre, alexandria, egypt . Preoperatively, refractive data was fed and revised on the computer system linked to the visumax femtosecond laser system (carl zeiss meditec ag, germany) with a 500 khz repetition rate . Data entered included the mr to be corrected (measured at 12 mm vertex distance), the mean corneal radius (mm), or mean k - reading (d) in addition to pachymetry of the thinnest corneal (graft) location (obtained from pentacam). For all cases, small suction cups were chosen as the patient - laser interface . The cap and the lenticule diameters were calculated to be smaller than those of the graft (8.0 mm) so that they are centered within its margins . Their diameters ranged from 6.9 to 7.5 mm depending on the clear area of the graft available for refractive correction . The width of the incision ranged from 3.4 to 3.6 mm and the side cut angle of the incision was set to 70 for all cases . The carved lenticules had optical zones ranging from 5.5 to 6.0 mm based on the residual stromal depth which was always kept above 300 m . Standardized lenticule data for all cases included a transition zone of 0.1 mm, a minimum lenticule edge thickness of 10 m, and a circumferential side cut angle of 130. during surgery, the laser suction cup was centered relative to the pupil and the graft . The patient was asked to keep looking at the flickering green fixation light during laser application to the cornea . After creation of the cuts, the lenticule and the cap were manipulated using the usual techniques described for smile [34, 36]. After extraction, the lenticule was spread on the corneal surface and stained with prednisolone acetate 1% eye drops to ensure its intactness as a complete disc and to detect the presence of any residual tissue remnants within the intrastromal pocket that can result in irregular astigmatism . The same postoperative treatment regimen consisting of topical prednisolone acetate 1%, gatifloxacin 0.3%, and nonpreserved artificial tears data analysis was performed using the software spss for windows version 20.0 (spss inc ., chicago, usa) and microsoft excel 2010 (microsoft corp ., redmond, wa, usa). Nonparametric tests were used as the sample size was less than that optimum for parametric analysis . The wilcoxon signed ranks test was used for comparison between the preoperative and postoperative data, and kruskal - wallis test was used for comparison between the postoperative data obtained from consecutive visits . Differences were considered to be statistically significant when the associated p value was <0.05 . Bivariate regression analysis was carried out to predict achieved sphere, cylinder, and seq accuracy using the preoperative attempted data . Standard graphs for reporting the outcomes in refractive surgery according to the waring protocol and its modifications [3840] were used for displaying and summarizing the outcomes of this study . For simplicity, only the preoperative and the 6-month follow - up data are demonstrated in the results . Demographic and pre- and postoperative clinical (refractive and visual) data of the ten patients are shown in table 1 . The cap and the lenticule were centered in relation to the pupil within the corneal graft except in two cases (n = 2, 5) in whom the graft itself was slightly decentered . Here the best centration in relation to the pupil was performed taking into consideration not to bisect the graft - host interface with the laser incisions . It is, however, worth mentioning that those two cases were the ones in whom astigmatic correction was unlikely, with almost the same amount of astigmatism remaining postoperatively . Intraoperatively, some resistance was met during dissection of both the cap and the lenticule at the sites of the suture related fibrous tracks . However, this has not led to any complication and cases were completed as planned . None of the cases enrolled in the study suffered from any of the reported post - smile complications . (ranging from 7.0 to 4.0 d) and the preoperative mean refractive spherical equivalent (mrse) was 6.84 1.38 d (ranging from 9.0 to 5.0). On the other hand, the mean postoperative spherical refraction was 0.8 0.97 d (ranging from 2.0 to + 1.0 d) while the mean postoperative mrse was 1.39 0.9 d (ranging from 2.63 to 0.0 d). The means of achieved correction values (calculated by subtracting the 6-month postoperative refraction from the preoperative target refraction) for sphere and mrse were 4.5 1.45 d and 5.45 1.59 d, respectively . On comparing these values to the values of the preoperative target refractive correction (preoperative manifest refraction), statistically significant differences existed for both sphere (p = 0.042) and mrse (p = 0.008). Vector analysis of the results of astigmatism correction revealed a mean target induced astigmatism (tia) magnitude of 2.61 1.06 d at axis 66 49.1 degrees and a mean surgically induced astigmatism (sia) magnitude of 2.06 0.76 d at axis 91.6 59.3 degrees, while the mean of the magnitude of the difference vector was 1.14 0.75 at axis 72.5 53.8 degrees . On comparing the magnitudes of tia versus sia, a statistically significant difference was found (p = 0.028). The mean correction ratio (induced / intended correction) of sphere was 0.84 0.19 d and mrse was 0.79 0.13 d. this means that for each diopter of sphere about 84.3% correction was achieved and for each diopter in mrse 79% correction was achieved . On the other hand, vector analysis of astigmatic results showed a mean percentage of astigmatic correction of 80.66 20.9% (correction index = 0.81 0.21) and a mean percentage of astigmatism reduction at the intended axis of 67 25.5% . The mean arithmetic angle of error was 10.4 15.4 degrees (p = 0.05) and the mean absolute angle of error was 12.4 13.7 degrees (p = 0.008). Assessment of the accuracy of the achieved correction values versus the attempted ones revealed a positive correlation for all three refraction elements with the sphere showing correlation values of p = 0.024, r = 0.702, and r = 0.4928 (figure 1), the cylinder (tia versus sia) of p = 0.002, r = 0.855, and r = 0.656 (figure 2), and the mrse of p = 0.004, r = 0.815, and r = 0.6642 (figure 3). As regards predictability, only one out of the ten eyes (10%) had a mrse between zero and 0.5 d, 4 eyes (40%) were between zero and 1.0 d, and 7 eyes (70%) were between zero and 1.5 (figure 4). Figure 5 displays the stability of the mrse values along the follow - up period with a fairly stable refraction . No statistically significant differences were found among the mrses measured at one week (1.26 0.9), one month (1.33 0.86), three months (1.35 0.85), and six months (1.39 0.9) (p = 0.937). On assessing the predictability of astigmatism correction, five out of the ten eyes (50%) had astigmatism above 3.0 d preoperatively, while six months after surgery 30% had astigmatism values within 0.5 d, 50% within 1.0 d, and 70% within 1.5 d (figure 6). Preoperatively, the mean cdva was 0.73 0.15 while, 6 months postoperatively, the mean udva was 0.68 0.14 and the mean cdva was 0.82 0.1 . This resulted in an efficacy index (e) of 0.93 and a safety index (s) of 1.12 . A more detailed evaluation of efficacy showed a cumulative snellen preoperative cdva of 0.9 or better in 20% of eyes, 0.8 or better in 60% of eyes, and 0.7 or better in 70% of eyes . On the other hand, the cumulative snellen postoperative udva was 0.8 or better in 40% of eyes and 0.7 or better in 70% of eyes (figure 7). Also, 90% of eyes had an udva within one line of the preoperative cdva . Figure 8 shows the safety data of the procedure with 40% of eyes experiencing no change from the preoperative cdva, 50% gaining one line, and 10% (one eye) gaining more than 2 lines . The quest for the best unaided visual performance following pkp has entailed the exploration of a variety of conventional and novel refractive correction procedures . Yet, none has proven enough refractive accuracy, predictability, efficacy, or safety to be adopted as the standard trustworthy technique . A wide variability as regards the obtained refractive and visual results in post - pkp eyes compared to results reported in healthy eyes with unoperated corneas has become a generally anticipated conclusion for all reports on such cases . In addition, the inherent complications of these refractive surgical correction techniques were found to have a higher incidence rate in grafted corneas adding an increased menace for the future viability of the graft and, rarely, the whole eye [132]. The introduction of the single step femtosecond laser small incision lenticule extraction (smile) for correction of myopia and astigmatism and its reported good results gave hope for a simple, fast, easily designed, readily centered, and theoretically safe refractive correction means that can be applied to corneal grafts while salvaging the circumferential graft - host interface scar as well as the endothelium from being violated [3335, 4143]. This study was performed with the aim of evaluating visual and refractive outcomes after correcting postkeratoplasty myopia and myopic astigmatism using small incision lenticule extraction (smile). To our knowledge, the only published data about smile after keratoplasty is a single case report in which the authors reported achieving the target refraction and an improved udva with a follow - up of 3 months . This study should, therefore, be one of the earliest clinical trials about the same subject . The timing of intervention has been a matter of debate among researchers; however, it is generally agreed upon that the corneal graft - host junction heals completely about one year following transplantation and that further surgical interventions should not be done until three to four months has passed since all the sutures have been removed . Our cases had a minimum of 18 months before complete suture removal and refractive stabilization were pursued for three months afterwards . As regards the refractive results, a statistically significant undercorrection was noted for sphere, cylinder, and mrse . Smile, in otherwise healthy nonoperated eyes, was reported to result in a slight undercorrection by about 0.25 d of mrse as reported by hjortdal et al . . Possible causes suggested to explain such an undercorrection after smile for treating myopia included a small difference in the achieved lenticule thickness of about 9 m (due to a hypothesized elastic recoil of the lamella between the cap and the residual stromal bed) and postoperative epithelial thickness changes . Smile undercorrection of astigmatism was also reported by ivarsen and hjortdal in unoperated eyes especially for higher degrees of astigmatism (up to 16% per diopter in highly astigmatic eyes). Proposed mechanisms included lenticule decentration, inappropriate energy and spot spacing settings, and again postoperative epithelial hyperplasia . In addition to the previous causes, corneal button decentration as well as the release of the tension within the graft caused by dissecting the planar incisions and the sutures - related fibrous tracts can also have a role in undercorrection or induction of lower and/or higher order astigmatism and coma . Therefore, centration of the treatment in relation to the graft and the pupil is of paramount importance for achieving the best possible astigmatic correction and reducing higher order coma induction . The statistically significant values of arithmetic and absolute angles of error of astigmatism correction denote the possibility cyclotorsion occurrence which can, also, aid the explanation of the relative imprecision of astigmatism correction at the intended angle as well as the induction of postoperative different axes cylindrical errors . Means for prevention of, compensating for, or correction of intraoperative cyclotorsion should be adopted . Compared to other techniques, undercorrection and, rarely, overcorrection have also been reported for almost all corneal refractive surgical methods of correcting post - pkp myopia and astigmatism including incisional surgeries, photoablation (prk or lasik) [1926], and icr implantation [17, 18]. Despite this undercorrection reported for smile, stability of the achieved refraction was statistically proven on comparing the achieved mrses at 1 week, 1 month, 3 months, and six months . The same was reported by other studies which investigated smile for myopia and myopic astigmatism [3335, 4143]. The early biomechanical stability status achieved after smile can aid the explanation of such a finding . Our results also showed that smile for correcting post - pkp myopia and astigmatism is of high efficacy and safety . The value of the efficacy index (e: 0.93) suggests that grafted patients undergoing smile can expect an udva of more than 90% of their preoperative cdva, while the value of the safety index (s: 1.12) indicates a potential improvement of the cdva postoperatively for such patients . (e: 0.9, s: 1.07) who assessed smile for correcting myopia in nonoperated eyes . The fact that none of the eyes enrolled in the study had lost any of its preoperative cdva postoperatively adds more to the safety profile of the technique . The feasibility of centering the whole treatment within the graft had the advantage of avoiding the violation of the graft - host interface, thus, preserving the structural integrity of this potentially weak spot . On the contrary, in other nonfemtosecond laser dependent techniques, moreover, the keratoplasty scar is recognized as a new limbus [1, 50] outside which any refractive correction should be almost worthless and unquestionably risky . Any extension of the treatment procedure into the recipient's possibly diseased corneal rim either through flap lifting or laser ablation is, therefore, considered undesirable and quite useless . Also, the facts that the endothelium is spared in femtosecond refractive lenticule extraction procedures compared to other intraocular procedures with no extra - stress added on the endothelial cells to achieve proper flap adhesion as in lasik add another advantage to smile when compared to those refractive correction techniques [21, 2732]. The drawbacks of this study, however, include the absence of controls, the lack of randomization, the few number of the enrolled eyes, and multiple surgeons . Since higher order aberrations evaluation and visual quality assessment were beyond the scope of this study, we strongly advocate them to be done in future similar studies to ascertain the nature and the amount of induced higher order aberrations as well as the quality of vision provided following smile in grafted eyes . The need for more prolonged follow - up, evaluation of induced graft biomechanical changes, and comparison to other techniques used for the same indication cannot be overlooked . To sum up, smile for correction of postkeratoplasty myopia and astigmatism can be considered a valuable addition to the armamentarium of procedures utilized to correct post - pkp myopia and astigmatism . The whole treatment can be centered within the graft preserving the viability of the healed graft - host interface . However, management of cyclotorsion as well as centration of both the graft initially and the lenticule afterwards is crucial for achieving the best refractive results.
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The epidemic of human immunodeficiency virus / acquired immunodeficiency syndrome (hiv / aids) is in its third decade and has reached to alarming proportions worldwide . According to the centers for disease control and prevention, more than one million people are living with hiv with an estimated 56,300 infections happening each year in the united states . Men who have sex with men (msm) population account for more than half (53%) of all the new hiv infections, and blacks represent almost half (46%) of people living with hiv in the united states.1 most of the hiv - related research is targeted towards high - risk groups such as prostitutes, gays and substance abusers but there is evidence that it is increasing in college students and adolescents as well.23 in 33 states with confidential name - based hiv reporting, of the 17,824 persons 13 - 24 years of age, 62% were males and 38% were females.2 an early step in preventive hiv counseling is behavioral risk assessment especially among high risk individuals in resource - limited settings.4 psychosocial stabilization as a means of coping among hiv infected individuals cannot be overemphasized . Besides the trained hiv counselors, family physicians, who are trained in psychosocial and community medicine, are equally poised to meet this demand.5 patient - provider relationships, when it comes to hiv positive patients, are ironically constrained as risk - reduction counseling falls woefully short of required percentage.6 si - milarly, counseling for hiv positive individuals who are on treatment for maintaining treatment adherence is quite poor.7 some of the barriers may be providercentric, such as dealing with emotional issues surrounding hiv and doubtfulness of providing good care.8 these barriers faced by counselors need to be addressed by techniques such as interactive counseling, motivational interviewing and application of stages of change.9 the usa preventive services task force made a recommendation few years back of counseling all adolescents and adults about risk reduction after they have been advised about risk factors for hiv infection and sexually transmitted diseases,10 and sexual history taking.11 some of the risk factors for engaging in unsafe sexual activities among the college students are use of alcohol,12 partner characteristics such as age,13 and substance abuse.14 the determinants of safer sex in adult population vary from whether they are hiv positive males, hiv positive females, gay or a bisexual population . The risk factors in a study which looked at hiv positive gays and bisexuals were mainly unprotected anal sex, which has a higher risk of hiv transmission . Some of the strategies tried in this population were problem solving exercises that attempt to increase perceived benefits of safer sex . Counselors also made the participants aware of alternative to regular use of male condoms.15 the reason for engaging in unsafe sexual practices especially in this gay population was pleasure seeking . Some of the barriers for attaining safer sex are inaccurate risk perception, and gender inequalities, like women being powerless to negotiate their sexual safety.16 risk perception can be improved by using social interactions . Social network formation which includes spousal communication related to aids risk has been proven to improve diffusion of behavioral change through the society.17 protection from getting hiv / aids consists of abstinence, consistent and correct condom usage and having just one sexual partner (monogamy). Several studies have found that pregnancy prevention rather than disease prevention was the impetus for condom use.1819 health education in the form of information about pregnancy prevention can unintentionally increase condom use . It was found that nigerian undergraduates, who had very good knowledge about hiv / aids, had low condom use to prevent its transmission.19 further evidence of knowledge - behavior gap comes from a study, which used a random sample survey of students in which the level of student knowledge was very high but did not lead to protective condom behaviors . However, knowledge was found to be an enabling factor in maintaining a comfort level when asking partners about their sexual histories and in requesting their partners to take an aids test.20 in a pilot project, which looked at the effect of hiv / aids prevention related messages in improving protective behaviors among indonesian participants, it was found that those exposed to the intervention were significantly more likely to use condoms to avoid aids . They also felt that these messages had a great impact on them to a point of wanting to change their lifestyle.21 this study did not show conclusively that there was actual behavior change on part of the participants . This helps to conclude that hiv / aids education related programs are not enough for generating a behavior change and can help in awareness of the disease to certain extent . Furthermore, from the point of view of clarifying risk perception, one needs to provide counseling which includes risk reduction or harm reduction as its integral part . The purpose of this review is to address a number of counseling strategies used for education and counseling of individuals at risk of getting hiv / aids . Moreover, later in this paper are discussed some of the counseling strategies used in individuals already diagnosed with hiv or aids and these strategies were used in preventing further transmission of the disease . In order to collect materials for this review, a detailed search of cinahl, medline, eric, academic search premier, scopus, web of science, and social sciences citation index databases was carried out for the time period 1995 - 2010 . A boolean search strategy where the key words entered for search were counseling and strategies and hiv / aids and hiv/ aids and counseling in differing orders was used to extract studies related to counseling skills and strategies for risk reduction as well as strategies in hiv positive people . Hiv prevention counseling is a very important mode of behavioral intervention especially in the absence of an effective vaccine or a curative treatment . It consists of dealing with a variety of issues such as medical, psychological and social . Client - centered counseling changed the focus of counseling from a sole educative one to one focusing on client's needs and circumstances . The word client - centered meant that counseling should be tailored to needs, circumstances and behaviors of a specific client which entailed active listening, to provide assistance and determining client's specific prevention needs.22 prevention counseling primarily consists of risk reduction counseling, pretestcounseling and post - test counseling . In terms of adolescent counseling, the risk reduction approach to hiv counseling can be divided into various phases such as, exploring clients feelings about sexual activity, using their existing hiv knowledge as an engaging tool, addressing the barriers they have for safer sex, focusing on perceptions that might affect risky behaviors, focus on safe sex planning and in the end, referral making.23 risk reduction counseling is used as a harm reduction technique quite effectively.24 other interesting approaches used were information - motivation - behavior change model . Results showed that men who received the full information motivation behavior (imb) model showed greater risk reduction skills and relatively lower rates of unprotected intercourse over 6 months of follow - up and had fewer sexually - transmitted infections.25 hierarchical counseling technique as opposed to single - method counseling in a group of women showed that there was a tendency for increased protective behavior among the group which received hierarchical counseling as compared to the other two groups.26 canadian counseling guidelines for counseling related to safer sex and contraception, mention using scaled questionnaire format . In this technique, the emphasis is on the clients or patients, as they are the best judge of what is important to them personally and how they would incorporate any change in their behaviors.27 counseling for safer sex is used in individuals who are hiv infected to prevent further transmission of the disease . Motivational interviewing has been used as an effective technique by some of the hiv care providers . In this counseling technique, the health care provider takes into account the readiness of the client to change his / her risky behavior and helps them resolve the ambivalence associated with changing the behavior.2829 one - on - one counseling was used in these studies . A meta - analytic review of effects of counseling and testing on sexual risk behaviors concluded that serodiscordant couples reduced unprotected intercourse and increased condom usage more than hiv - negative and untested participants.30 similarly, a multi - clinic safer sex behavioral counseling intervention using loss - frame intervention by emphasis on negative consequences of safe sex reduced the number of sexual partners among hiv positive patients compared with the control arm.31 hiv prevention counseling is a very important mode of behavioral intervention especially in the absence of an effective vaccine or a curative treatment . It consists of dealing with a variety of issues such as medical, psychological and social . Client - centered counseling changed the focus of counseling from a sole educative one to one focusing on client's needs and circumstances . The word client - centered meant that counseling should be tailored to needs, circumstances and behaviors of a specific client which entailed active listening, to provide assistance and determining client's specific prevention needs.22 prevention counseling primarily consists of risk reduction counseling, pretestcounseling and post - test counseling . In terms of adolescent counseling, the risk reduction approach to hiv counseling can be divided into various phases such as, exploring clients feelings about sexual activity, using their existing hiv knowledge as an engaging tool, addressing the barriers they have for safer sex, focusing on perceptions that might affect risky behaviors, focus on safe sex planning and in the end, referral making.23 risk reduction counseling is used as a harm reduction technique quite effectively.24 other interesting approaches used were information - motivation - behavior change model . Results showed that men who received the full information motivation behavior (imb) model showed greater risk reduction skills and relatively lower rates of unprotected intercourse over 6 months of follow - up and had fewer sexually - transmitted infections.25 hierarchical counseling technique as opposed to single - method counseling in a group of women showed that there was a tendency for increased protective behavior among the group which received hierarchical counseling as compared to the other two groups.26 canadian counseling guidelines for counseling related to safer sex and contraception, mention using scaled questionnaire format . In this technique, the emphasis is on the clients or patients, as they are the best judge of what is important to them personally and how they would incorporate any change in their behaviors.27 counseling for safer sex is used in individuals who are hiv infected to prevent further transmission of the disease . Motivational interviewing has been used as an effective technique by some of the hiv care providers . In this counseling technique, the health care provider takes into account the readiness of the client to change his / her risky behavior and helps them resolve the ambivalence associated with changing the behavior.2829 one - on - one counseling was used in these studies . A meta - analytic review of effects of counseling and testing on sexual risk behaviors concluded that serodiscordant couples reduced unprotected intercourse and increased condom usage more than hiv - negative and untested participants.30 similarly, a multi - clinic safer sex behavioral counseling intervention using loss - frame intervention by emphasis on negative consequences of safe sex reduced the number of sexual partners among hiv positive patients compared with the control arm.31 hiv prevention counseling is a very important mode of behavioral intervention especially in the absence of an effective vaccine or a curative treatment . It consists of dealing with a variety of issues such as medical, psychological and social . Client - centered counseling changed the focus of counseling from a sole educative one to one focusing on client's needs and circumstances . The word client - centered meant that counseling should be tailored to needs, circumstances and behaviors of a specific client which entailed active listening, to provide assistance and determining client's specific prevention needs.22 prevention counseling primarily consists of risk reduction counseling, pretestcounseling and post - test counseling . In terms of adolescent counseling, the risk reduction approach to hiv counseling can be divided into various phases such as, exploring clients feelings about sexual activity, using their existing hiv knowledge as an engaging tool, addressing the barriers they have for safer sex, focusing on perceptions that might affect risky behaviors, focus on safe sex planning and in the end, referral making.23 risk reduction counseling is used as a harm reduction technique quite effectively.24 other interesting approaches used were information - motivation - behavior change model . Results showed that men who received the full information motivation behavior (imb) model showed greater risk reduction skills and relatively lower rates of unprotected intercourse over 6 months of follow - up and had fewer sexually - transmitted infections.25 hierarchical counseling technique as opposed to single - method counseling in a group of women showed that there was a tendency for increased protective behavior among the group which received hierarchical counseling as compared to the other two groups.26 canadian counseling guidelines for counseling related to safer sex and contraception, mention using scaled questionnaire format . In this technique, the emphasis is on the clients or patients, as they are the best judge of what is important to them personally and how they would incorporate any change in their behaviors.27 counseling for safer sex is used in individuals who are hiv infected to prevent further transmission of the disease . Motivational interviewing has been used as an effective technique by some of the hiv care providers . In this counseling technique, the health care provider takes into account the readiness of the client to change his / her risky behavior and helps them resolve the ambivalence associated with changing the behavior.2829 one - on - one counseling was used in these studies . A meta - analytic review of effects of counseling and testing on sexual risk behaviors concluded that serodiscordant couples reduced unprotected intercourse and increased condom usage more than hiv - negative and untested participants.30 similarly, a multi - clinic safer sex behavioral counseling intervention using loss - frame intervention by emphasis on negative consequences of safe sex reduced the number of sexual partners among hiv positive patients compared with the control arm.31 there are several examples of one - on - one counseling methods used for risk reduction counseling among hiv positive individuals . The various dimensions touched upon were use of a negative approach,31 assessing readiness to change,32 and motivational enhancement therapy.33 hence, we see multiple approaches used by counselors in providing education and prevention counseling to at risk individuals and also individuals who have been infected with the virus . Internet - based counseling has gained some popularity over recent years but has to deal with issues such as lack of visual and verbal cues, confidentiality, accessibility problems by those in great need and increased client separation.34 counseling related to using spiritual coping techniques have been well established in cancer literature but has limited evidence in hiv related counseling.
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In india, influenza surveillance has been carried out in multisite regional centres located at different parts of india (pune and nagpur in west, delhi and lucknow in north, kolkata and dibrugarh in east, chennai, vellore and kerala in south)20 . The national institute of virology (niv) pune, monitors genetic variations and drug susceptibility in circulating influenza viruses received from regional centres . Influenza viruses, isolated from 2004 to 2011 were analyzed genetically for known resistance markers by m2 and na gene sequencing . Viruses: virus isolation and antigenic characterization was carried out at respective regional laboratories by inoculating nasal / throat swabs into madin darby canine kidney (mdck) cells21 . A total of a / h1n1 (n=206), a / h3n2 (n=371) viruses were studied for amantadine resistance and a / h1n1 (n=206), a / h3n2 (n=272), pandemic (h1n1) (n= 493) and type b (n=326) were studied for oseltamivir resistance . Fluorescent neuraminidase inhibition assay (ni): the half maximal inhibitory concentration (ic50) to oseltamivir carboxylate was determined by a fluorescence - based neuraminidase inhibition assay22 . Eleven sensitive and 50 resistant a / h1n1 virus isolates of 20072009 seasons, and 40 a / h3n2 sensitive viruses of 2009 - 2010 were tested along with reference viruses . The reference viruses were provided by who collaborating centre for reference and research on influenza, melbourne, australia . Molecular detection of m2 and na gene rna extraction and rt - pcr - viral rna was extracted from culture supernatant using the qiaamp viral rna mini kit (qiagen, usa). One step pcr [reverse transcription (rt)-pcr)] (invitrogen superscript iii platinum kit country) was performed to amplify m2 and na gene for detection of established molecular markers using published and gene - specific primers23242526 and ha1 gene using cdc (unpublished) for phylogenetic analysis . Sequencing - the amplicons for m2 (270 bp covering amino acid positions 26 to 31), n1 (253 bp covering 274 amino acid positions), n2 (1103 bp covering all known mutation sites from 118 to 294 amino acid position) and type b na (1146bp covering all known mutation sites from 119 to 371 amino acid position) genes were purified using pcr purification kits (qiagen). Dna sequencing was carried out using big dye terminator v 3.1 cycle sequencing ready reaction kit (abi, foster city, ca, usa) and unincorporated labelled ddntps (dideoxy nucleotides) were removed using dye - x removal column purification kit (qiagen). The sequencing was done on abi 3730 dna analyzer (abi, foster city, ca, usa) and pair wise sequence alignment and protein translation was performed with mega 4 program27 . Drug susceptibility markers: m2 gene sequencing of a / h1n1 (n=206) and a / h3n2 (n=371) and na gene sequencing of a / h1n1 (n=206), a / h3n2 (n=272), and type b (n=326) were performed to analyze the established molecular markers . Amantadine susceptibility of a / h1n1 and a / h3n2 viruses was examined for mutations at positions 26, 27, 30, 31 or 34 in m2 gene; for n1 subtype oseltamivir susceptibility was examined for amino acid position 274 (n2 numbering) and for n2 subtype amino acid positions 118, 119, 151, 152, 222, 224, 227, 274, 276, 292 and 294 and 244 - 247 were examined . Type b influenza positions 119, 152, 198, 222, 274, 371 (n2 numbering) were checked . Allelic real - time rt - pcr assay for detection of h274y in pandemic h1n1 - susceptibility to oseltamivir for pandemic (h1n1) viruses was evaluated by allelic discrimination real - time rt - pcr . The primers and probes used in this assay were designed by south - east asia regional influenza reference laboratory, thailand28 . Phylogenetic analysis of ha1 gene: phylogenetic analysis of a / h1n1 and a / h3n2 viruses used in this study along with who reference strains was done . Mega version 4 was used for constructing neiughbour - joining (nj) trees using the kimura's two - parameter distance model, with 1000 bootstrap replicates27 . Seasonal a (h3n2) viruses - as summarized in table i, a / h3n2 viruses circulating in 2004 were sensitive to amantadine . In 2005, resistant viruses (23%), were introduced and were co - circulating along with sensitive viruses in pune, delhi and kolkata . At the same time, the viruses from chennai remained sensitive to amantadine . In 2006, proportion of resistant viruses increased to 48 per cent in pune, delhi, chennai, kolkata and dibrugarh . In 2007, it reached to 90 per cent and 2008 onwards it became 100 per cent in india . The most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . Amantadine susceptibility testing of influenza a virus isolates (2004 - 2011) seasonal a (h1n1) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 (table i). A single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . In 2008, three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . Phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b, a / brisbane/59/2007 with signature amino acid changes d45n, k149r (h3 numbering). On the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y (table ii). Up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . In 2009, resistant viruses were detected from delhi (13/14), kolkata (7/7), chennai (5/5), pune (10/12) and similar findings were reported from kolkata . Neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k, e119q, d151e, r152k, i222v, r224k, e227d, h274y, e276d, r292k, n294s and a 244 - 247, and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . (accession nos: kf 314400-kf314591; kf 314609-kf314682; kf014004,6,8,10) (table ii). The analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . This mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . Its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain (0.6 - 3.0 nm) allowed to confirm 15/23 (65.22%) and 35/38(92.10%) a / h1n1 virus isolates from 2008 and 2009, respectively resistant to oseltamivir . The na sequence of oseltamivir - resistant viruses had t81n, d354 g when compared with a / brisbane/59/2007 . Majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain (48 to 180 nm). Table iii presents year - wise number of isolates tested along with mean ic50 (nm), standard deviation (nm), minimum ic50 (nm) and maximum ic50 (nm). Four isolates showed ic50 values 24.52, 55.24, 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . Though asn(n) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir; our study showed that the sensitive h3n2 viruses had either asp (d),/asn (n) or gly (g) at residue 151 . So we could not correlate the presence of d151 in four minor outliers with their ic50 values . Ic50 (half maximal inhibitory concentration) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a, r152k, d198n / e, i222 t, h274y and r371k indicated that all type b isolates were sensitive to oseltamivir (accession nos: kf314197- kf314399; kf364360-kf364482) (table ii). Pandemic (h1n1) viruses - pandemic (h1n1) isolates (n=493) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir seasonal a (h3n2) viruses - as summarized in table i, a / h3n2 viruses circulating in 2004 were sensitive to amantadine . In 2005, resistant viruses (23%), were introduced and were co - circulating along with sensitive viruses in pune, delhi and kolkata . At the same time, the viruses from chennai remained sensitive to amantadine . In 2006, proportion of resistant viruses increased to 48 per cent in pune, delhi, chennai, kolkata and dibrugarh . In 2007, it reached to 90 per cent and 2008 onwards it became 100 per cent in india . The most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . Amantadine susceptibility testing of influenza a virus isolates (2004 - 2011) seasonal a (h1n1) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 (table i). A single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . In 2008, three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . Phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b, a / brisbane/59/2007 with signature amino acid changes d45n, k149r (h3 numbering). On the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y (table ii). Up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . In 2009, resistant viruses were detected from delhi (13/14), kolkata (7/7), chennai (5/5), pune (10/12) and similar findings were reported from kolkata . Neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k, e119q, d151e, r152k, i222v, r224k, e227d, h274y, e276d, r292k, n294s and a 244 - 247, and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . (accession nos: kf 314400-kf314591; kf 314609-kf314682; kf014004,6,8,10) (table ii). The analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . This mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . Its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain (0.6 - 3.0 nm) allowed to confirm 15/23 (65.22%) and 35/38(92.10%) a / h1n1 virus isolates from 2008 and 2009, respectively resistant to oseltamivir . The na sequence of oseltamivir - resistant viruses had t81n, d354 g when compared with a / brisbane/59/2007 . Majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain (48 to 180 nm). Table iii presents year - wise number of isolates tested along with mean ic50 (nm), standard deviation (nm), minimum ic50 (nm) and maximum ic50 (nm). Four isolates showed ic50 values 24.52, 55.24, 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . Though asn(n) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir; our study showed that the sensitive h3n2 viruses had either asp (d),/asn (n) or gly (g) at residue 151 . So we could not correlate the presence of d151 in four minor outliers with their ic50 values . Ic50 (half maximal inhibitory concentration) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a, r152k, d198n / e, i222 t, h274y and r371k indicated that all type b isolates were sensitive to oseltamivir (accession nos: kf314197- kf314399; kf364360-kf364482) (table ii). Pandemic (h1n1) viruses - pandemic (h1n1) isolates (n=493) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir seasonal a (h3n2) viruses - as summarized in table i, a / h3n2 viruses circulating in 2004 were sensitive to amantadine . In 2005, resistant viruses (23%), were introduced and were co - circulating along with sensitive viruses in pune, delhi and kolkata . At the same time, the viruses from chennai remained sensitive to amantadine . In 2006, proportion of resistant viruses increased to 48 per cent in pune, delhi, chennai, kolkata and dibrugarh . In 2007, it reached to 90 per cent and 2008 onwards it became 100 per cent in india . The most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . Amantadine susceptibility testing of influenza a virus isolates (2004 - 2011) seasonal a (h1n1) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 (table i). A single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . In 2008, three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . Phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b, a / brisbane/59/2007 with signature amino acid changes d45n, k149r (h3 numbering). On the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y (table ii). Up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . In 2009, resistant viruses were detected from delhi (13/14), kolkata (7/7), chennai (5/5), pune (10/12) and similar findings were reported from kolkata . Neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k, e119q, d151e, r152k, i222v, r224k, e227d, h274y, e276d, r292k, n294s and a 244 - 247, and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . (accession nos: kf 314400-kf314591; kf 314609-kf314682; kf014004,6,8,10) (table ii). The analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . This mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . Its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain (0.6 - 3.0 nm) allowed to confirm 15/23 (65.22%) and 35/38(92.10%) a / h1n1 virus isolates from 2008 and 2009, respectively resistant to oseltamivir . The na sequence of oseltamivir - resistant viruses had t81n, d354 g when compared with a / brisbane/59/2007 . Majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain (48 to 180 nm). Table iii presents year - wise number of isolates tested along with mean ic50 (nm), standard deviation (nm), minimum ic50 (nm) and maximum ic50 (nm). Four isolates showed ic50 values 24.52, 55.24, 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . Though asn(n) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir; our study showed that the sensitive h3n2 viruses had either asp (d),/asn (n) or gly (g) at residue 151 . So we could not correlate the presence of d151 in four minor outliers with their ic50 values . Ic50 (half maximal inhibitory concentration) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a, r152k, d198n / e, i222 t, h274y and r371k indicated that all type b isolates were sensitive to oseltamivir (accession nos: kf314197- kf314399; kf364360-kf364482) (table ii). Pandemic (h1n1) viruses - pandemic (h1n1) isolates (n=493) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir the acquisition of a mutation in influenza a viruses conferring resistance to an antiviral agent may occur as a result of drug selection, spontaneous mutation or through genetic reassortment with another drug resistant influenza a virus . High prevalence of amantadine resistance in influenza a viruses was observed in countries, irrespective of its use56 . Globally, a marked increase in amantadine drug resistant virus isolates was reported from asian countries especially for a / h3n2 subtype4 . However, majority of a / h1n1 viruses remained sensitive till 2006 season and 2 per cent of a / h1n1 viruses worldwide showed resistance to amantadine29 . In the present study, resistance to amantadine was mainly observed for a / h3n2 subtype, with identification of common mutation s31n of m2 gene . In the present study, resistant a / h1n1 viruses detected in india were 3 per cent in 2007, 13 per cent in 2008 with s31n substitution and agreeing with global data4 . In 2009, a single isolate from kolkata had less common mutation l26f in m2 gene and h274y mutation in na gene which conferred dual resistance . Indian isolates with s31n mutation indicated that strains already harbouring drug resistant mutation were most likely introduced in the community . Since in india the use of amantadine for influenza treatment was extremely rare during the study period; the possibility of emergence of amantadine resistance due to drug pressure is remote . Before 2008, oseltamivir - resistance for any influenza type / subtype was not observed in india, due to negligible use of oseltamivir till may 2009 . Widespread use of oseltamivir for suspected h1n1 patients in india started after june 2009 when cases of pandemic a (h1n1) 09 were detected in india . Prior to this, use of antivirals for influenza was negligible indicating that there was no potential for drug resistance due to selective pressure . However, no evidence was found to suggest that increased access to oseltamivir had promoted resistance or lack of it . A similar study from japan, where oseltamivir is more widely used, reported no significant effect on the occurrence of resistance30 . In india, a probable importation or detection of global 2008 oseltamivir - resistant seasonal a / h1n1 virus was documented nine months after it was first reported in europe in january 2008910 . Oseltamivir resistance was observed for seasonal a / h1n1 influenza viruses with an increased frequency of 65.227 per cent in 2008 to 100 per cent in 2010 . These resistant viruses exhibited extremely high ic50 values, between 299.8 to 2513 nm and carried h274y mutation . It was well studied that oseltamivir susceptibility to influenza viruses differed significantly depending on the na subtypes . This was expected to occur due to minor structural differences between the na active sites of each na subtype viruses which could result in different oseltamivir binding affinities22 . The reduction in susceptibility to oseltamivir observed worldwide from 2005 - 2006 to 2007 - 2008 in a / h1n1 subtype was most probably a result of na antigenic drift from a / caledonia/20/1999 to a / brisbane/59/2007 . We detected oseltamivir resistance in n1 subtype from 2008 and it could be a result of same antigenic drift . Oseltamivir was important for controlling the transmission and dissemination of pandemic viruses before a vaccine became widely available . No vaccine was available in india until one year after the first case of influenza a(h1n1) pdm09 was detected . This study showed that all 493 influenza a(h1n1) pdm09 viruses from 2009 and 2011 were sensitive to oseltamivir . Even though indian isolates had permissive secondary mutations r194 g, e214 g, r222q and v234 m in na gene from 2005 - 2007; which were thought to have occurred before the emergence and spread of h274y nai resistant viruses31; a / h1n1 isolates remained sensitive to oseltamivir till 2007 . The role of observed additional mutations t81n and d354 g when compared with a / brisbane/59/2007 is still unclear . Our findings were in agreement with the widespread and sustained transmission of oseltamivir resistant a / h1n1 viruses observed worldwide since 2007/2008910 . Like influenza a viruses, influenza b viruses also undergo continuous evolution with accumulation of point mutations and reassortment events between co - circulating lineages . It was observed worldwide that the community acquired isolates of influenza b virus remained sensitive to nai drugs . However, sporadic cases of reduced nai susceptibility in the na inhibition assays have been reported from drug treated patients13 . Our results showed that the influenza b viruses were sensitive to oseltamivir with agreement to global data . Continued surveillance for antiviral drug resistance in influenza viruses is required to ensure that stockpiled neuraminidase inhibitors are effective and clinicians can be kept informed of the efficacy of neuraminidase inhibitors when treating patients for influenza.
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Modern drugs have made great contributions to better quality of life, less disabling symptoms, decreased demands of health care and a better prognosis . Despite this, one can nowadays notice an increasing proportion of negative side effects and adverse drug reactions due to extensive pharmacological treatment . To examine if patient - focused drug surveillances were associated with a higher quality of drug treatment at nursing homes? Physicians were told to systematically focus on the patient's basic health status as a fundamental starting point for further continuous medication, with the aim to obtain a rational drug usage . Mortality, health care consumption and number of drugs . Health status and evaluation of drug therapy . Evaluation of medications showed significant differences favouring the intervention group . Due to existing polypharmacy there was a significant reduction of number of drugs in the intervention group vs. a significant increase in the control group . The intervention seemed to have achieved significant positive results in quality of drug treatment, although it has shown immense lacks of monitoring the health status of frail elderly.
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More than four decades of research has demonstrated that although the brain remains plastic throughout life, continuously reorganizing its connections in the face of new experiences, childhood represents a specific phase in the development of the synaptic network that is characterized by overall remarkable plasticity . During this period of enhanced plasticity also called critical period, experience can produce permanent, large - scale changes in neural circuits . Studies on mechanisms that underlie activation and regulation of critical periods in the central nervous system (cns) are seminal in neuroscience, with the underlying motive being that manipulation of such mechanisms may potentially allow reactivation of neural circuit plasticity during times when the adult brain is less plastic, for example, to aid adaptive circuit rewiring following insult, such as stroke . Additionally, this line of inquiry may help us develop rational pharmacological approaches to correct alterations in the brain of children with neurodevelopmental disorders involving altered synapse formation and/or plasticity . Critical periods have been observed across sensory, motor, auditory, and also higher cognitive areas; however much of our knowledge of the cellular and molecular mechanisms of onset, maintenance, and termination of these periods derive from seminal studies by wiesel and hubel in the developing cat visual system . Electrophysiological recordings from neurons in the primary visual cortex show activation to different degrees by visual stimuli presented to one eye or the other, a property termed ocular dominance . Closing one eye during a specific postnatal time period starts a cascade of events leading to synaptic reorganization of neural circuits in visual cortex, resulting in lifelong, irreversible reduction of the ability of the deprived eye to drive neuronal responses in the cortex, and a dramatic increase in the number of neurons responsive to stimuli presented to the open eye . Such change in eye preference best able to elicit a response from cortical neurons in visual cortex following manipulation of visual inputs is called ocular dominance (od) plasticity . In marked contrast to what happen in young animals, prolonged eye closure in adults elicits no change in visual cortical neuron responsiveness . Further, monocular deprivation during critical period causes loss of visual acuity in the deprived eye, which is not ameliorated by subsequent experience . This is supported by human studies showing that treatment of amblyopia in children between 7 and 17 years of age was effective only in a fourth of the patients, and to a lesser degree than treatment in younger children . To date, ocular dominance plasticity remains the best - studied experimental model for experience - dependent refinement of neuronal circuits because of the ease of manipulating visual experience independently in the two eyes . One of the main players implicated in the onset of critical period plasticity is the development of inhibitory circuitry [5, 6]. Cortical inhibitory neurons, or interneurons, comprise 2030% of all cortical neurons and predominantly use gamma - aminobutyric acid (gaba) as neurotransmitter . Gabaergic interneurons control several aspects of neuronal circuit function from neuronal excitability and integration, to the generation of temporal synchrony and oscillation among networks of excitatory neurons . In addition, gabaergic interneurons also regulate key developmental steps, from cell migration and differentiation to experience - dependent refinement of neuronal connections [10, 11]. In the last years, many studies have started to elucidate the development and function of cortical gabaergic circuits . In this paper the focus is on the molecular mechanisms regulating postnatal gabaergic circuit development and the onset of critical period plasticity, followed by a brief discussion on how aberrations in inhibitory circuit development and alteration in the timing of critical period plasticity could be implicated in neurodevelopmental diseases . Recent studies indicate that the development of inhibitory circuitry in the cortex plays a pivotal role in controlling the onset and time course of critical periods [5, 10, 12]. In particular, two elegant studies envisage a direct role of gaba in the onset of od plasticity . In a first study, hensch and collaborators found that mice lacking the synaptic isoform of gaba - producing enzyme, glutamic acid decarboxylase (gad65), show no od plasticity . This deficit can be rescued by cortical infusion of the gabaa receptor agonist diazepam, demonstrating that a decrease in inhibition effectively abolished critical period and impaired plasticity mechanisms . In the second study, fagiolini and hensch showed that the early enhancement of gaba - mediated inhibition by diazepam application triggers the precocious onset of od plasticity . Further, precocious development of inhibitory circuitry via action of the brain derived neurotrophic factor (bdnf) accelerates the onset of the critical period for visual plasticity . Cortical gabaergic interneurons form a strikingly diverse and heterogenous group differing in morphology, physiological properties, and protein expression [14, 15]. The hypothesis that different interneuron subtypes play different roles in cortical development, function, and plasticity is therefore a tantalizing one . . Showed that gaba transmission mediated by the 1 subunit - containing gabaa receptors is required for the induction of critical period for od plasticity . Because different classes of inhibitory synapses preferentially signal through gabaa receptors with different subunit composition, these results suggest that maturation of specific subclasses of gaba interneurons is crucial to initiate critical period plasticity . More recent data indicate that site - specific optimization of gabaa receptor numbers on the soma - proximal dendritic compartment of pyramidal cells triggers the onset of od plasticity . The soma proximal dendritic compartment of pyramidal cells is preferentially innervated by parvalbumin (pv) positive basket interneurons . Taken altogether, these data suggest a critical role for basket cell interneuron maturation in the onset of critical period plasticity . A novel mechanism explaining how visual input is coupled to the onset of ocular dominance plasticity traditionally, the molecular signals linking visual experience to gaba interneuron maturation were thought to be recruited from within the cortex itself, such as the activity - dependent synthesis and release of bdnf by pyramidal neurons . Instead, sugiyama et al . Demonstrated that a retina - derived homeoprotein, otx2, is first transferred into the primary visual cortex via a visual experience - dependent mechanism . Once in the cortex, otx2 then nurtures gabaergic interneurons and promotes critical period plasticity . The investigation of the target genes and proteins of otx2 will reveal further insights into the mechanisms linking experience, gabaergic circuit maturation, and critical period plasticity . The gabaergic network comprises of diverse interneuron subtypes that have different morphological and physiological characteristics and localize their synapses onto distinct subcellular locations on the postsynaptic targets . Precisely how activity and molecular - driven mechanisms conspire to achieve the remarkable specificity of gabaergic synapse localization and formation is unknown . The functional maturation of gaba - mediated inhibition is a prolonged process that extends well into adolescence, both in rodents and primates [2023], and correlates with the time course of the critical period for od plasticity [21, 23]. Moreover, the inhibitory maturation process strongly depends on sensory experience, since sensory deprivation, induced either by dark rearing or by intraocular tetradotoxin (ttx) injection, significantly retards the morphological and functional maturation of gabaergic synapses [21, 23]. This dependence of gabaergic synapse maturation on sensory experience is not limited to visual cortex, indeed similar results have been found in the somatosensory cortex . What are the cellular and molecular mechanisms linking sensory experience to the maturation of gabaergic synapses? Brain - derived neurotrophic factor (bdnf), an activity - dependent molecule shown to be upregulated following light stimulation in the visual cortex [25, 26], is one of the first molecules implicated in the formation of gabaergic synapses in hippocampal and cortical cultures [27, 28]. Most importantly, in transgenic mice with precocious bdnf expression, a marked increase in perisomatic inhibitory innervation in the visual cortex is correlated with a premature onset and closure of ocular dominance plasticity, further supporting the link between gabaergic synapse maturation and onset of critical period plasticity [12, 29]. Since bdnf is produced only by pyramidal cells, it could work as an intercellular signaling factor that translates pyramidal cell activity to gabaergic synapse density . Another factor that has been shown to positively regulate gabaergic synapse maturation is gaba itself . Early in development, gaba has been shown to be a trophic factor, involved in cell proliferation, neuronal migration, and neurite growth . Since gad67 is the main isoform of gaba synthesizing enzyme, its deletion reduces gaba levels by 90% . Using transgenic mice to knockdown gad67 in single basket interneurons during the period of their maturation, recent studies show that intact gaba signaling is critical for the maturation of gabaergic synapses (figure 1). Intriguingly, even a partial reduction of gad67 was sufficient to cause aberrant perisomatic synapse maturation, underlying the importance of maintaining optimal gaba levels for normal synapse development . Basket cell perisomatic synapses have an exuberant innervation pattern; a single basket interneuron connects to hundreds of pyramidal cells in its vicinity, making numerous synapses onto each individual pyramidal cell soma . It is therefore important to appreciate that reduced gaba levels compromise not only the number of synapses that are made onto each pyramidal soma, but also drastically reduce the number of pyramidal soma it connects to, causing a potential circuit - wide disruption in connectivity . This study demonstrates that, in addition to mediating inhibitory transmission, gaba signaling also regulates interneuron axon arborization and synapse development in adolescent brain, which, in turn regulates critical period plasticity . Different aspects of this deficit were rescued by treatment with either gabaa or gabab agonists, suggesting a receptor - specific effect of gaba - mediated signaling during gabaergic synapse maturation . Since gabaa and gabab receptors are present on postsynaptic neurons, gaba terminal themselves, and surrounding glial processes, cell - autonomous activation of presynaptic gabab receptors, which modulate cachannels and gaba release, could influence growth cone motility and bouton stability, or gaba signaling through postsynaptic or glia receptors could trigger the release of retrograde factors, which promote axon branching and synapse formation . Modulation of gaba synthesis by the gad67 enzyme plays a central role in regulating gaba - mediated signaling . Gad67 itself is produced at a limiting level in the brain, since deletion of one copy of the gad1 gene results in a 40% reduction of enzyme activity and gaba content in many brain regions . Furthermore, the transcription of gad1, the key step in the physiological control of gad67 activity, is highly regulated during brain development, by neuronal activity, and experience [37, 38]. Activity - dependent production of gad67 thus results in online adjustment of intracellular pool for gaba release . Since alterations in gad67 and gaba levels profoundly influence interneuron axon growth, synapse formation and network connectivity during the establishment of inhibitory circuits, neuronal activity might regulate the strength and pattern of inhibitory synaptic innervation through gad67-mediated gaba synthesis and signaling . Such activity - dependent and cell - wide regulation of a transmitter resource implies a novel logic for the maturation and plasticity of gabaergic synapses and innervation . Since subtle variations in gaba levels can cause such dramatic effects on inhibitory circuits, and therefore overall network connectivity, it is critical to understand its implications in neuropsychiatric disorders and strive to regulate optimal gaba levels for proper circuit function . A recent study by fiorentino et al . Proposes that the interaction between bdnf and gaba signaling influences gabaergic synapse maturation . The authors demonstrate that activation of metabotropic gabab receptor triggers secretion of bdnf and promotes the development of gabaergic synapses, in particular, the perisomatic gabaergic synapses, onto ca3 pyramidal neurons in the hippocampus of newborn mice . Whether a similar mechanism is at play in the visual cortex is still unknown; however, the picture so far indicates a positive interplay between sensory experience, bdnf, and gaba signaling, to induce gabaergic synapse maturation and in turn promote the onset of ocular dominance plasticity . In addition to factors promoting gabaergic synapse maturation, recent studies have revealed inhibitory mechanisms that set the appropriate time course for establishment of mature gabaergic innervation patterns and the onset of critical period plasticity . In particular, polysialic acid (psa), linked to the neural cell adhesion molecule (ncam), acts as a negative signal to suppress the formation of inhibitory synapses and the onset of od plasticity in the developing visual cortex . In the mammalian brain, ncam is a predominant carrier of the unusual long - chain, polyanionic carbohydrate, psa, although outside the nervous system more carriers of psa are known, including neuropilin-2 . Psa is a long linear homopolymer of -2,8-linked sialic acid that is synthesized in the golgi by two polysialyltransferases, pst (also known as st8siaiv) and stx (also known as st8siaii), either of which is sufficient for the complete synthesis of psa chain on a standard asparaginyl - linked core carbohydrate attached to ncam [42, 43]. One of the most studied characteristics of psa is its ability to act as a de - adhesive factor, causing steric hindrance, between cellular membranes . Cell surface expression of psa constricts intercellular space between apposing cells, which in turn, decreases homophilic binding between ncam and other cells adhesion molecules including cadherins, l1 family, and integrins, therefore acting as a permissive regulating factor rather than a specific instructive cue . For example, in the developing nervous system psa creates conditions permissive for postmitotic migration of precursor cells . In the adult, migrating cells still retain psa, such as progenitor cells migrating along rostral migratory stream from the subventricular zone to the olfactory bulb and newborn granule cells in the hippocampus . Although psa expression is highest in the embryonic stages, it is expressed in the postnatal brain at different levels depending on brain region and age . In the mouse visual cortex, psa expression declines to almost undetectable levels shortly after eye opening, and this decline is attenuated by visual deprivation . Indeed, psa levels in visual cortex were higher in mice dark reared from birth compared to littermates reared in a normal light - dark cycle . This effect is echoed in the visual cortex contralateral to the eye that received daily intraocular injection of ttx compared to the ipsilateral cortex . Since the developmental and activity - regulated expression of psa inversely correlates with the maturation of gabaergic innervation, it is thus possible that psa decline might be sufficient for gabaergic synapse maturation . Indeed, premature enzymatic removal of psa in the developing visual cortex results in precocious maturation of perisomatic innervation by basket interneurons and enhanced inhibitory synaptic transmission . Most importantly, the same treatment causes an earlier onset of critical period plasticity in the visual cortex . Since psa removal promotes gabaergic synapse formation, and gaba signaling in turn further promotes the maturation of gabaergic innervation, together gaba signaling and psa removal may constitute a positive feedback mechanism to accelerate gabaergic synapse formation once sensory experience begins, and consequently to induce the onset of critical period plasticity in the visual cortex . Psa also regulates glutamatergic synapse formation [48, 49] and affects neuron - glia interactions thus the possibility of additional mechanisms by which psa influences ocular dominance plasticity cannot be excluded . What is the precise role of psa in gabaergic circuit maturation? One possibility is that developmental and activity - dependent removal of psa might coordinate the timing of axon and synapse morphogenesis during the maturation of gabaergic innervation; indeed precocious perisomatic synapse formation can be triggered by premature removal of psa . Excessive, premature synapse formation might constrain axon growth . Higher expression of psa during the early postnatal weeks might attenuate interactions between basket cell axons and pyramidal neurons, thereby holding off synapse formation and promoting the elaboration of axon arbors . Subsequent activity - dependent removal of psa might unmask mechanisms that are already in place along basket cell axon, allowing fast responses to local synaptogenic cues . A similar example of psa regulating the timing of a biological process comes from studies of migrating neuronal precursor . When psa is enzymatically removed from newly generated cells in the svz, they form neuronal processes and begin to express neuronal molecular markers . This premature developmental transition is dependent on cell contact and appears to involve signaling through ncam and p59fyn kinase . Why is such a mechanism in place and what could be its purpose? Interestingly, long polymers of sialic acid are not found in invertebrates, where neural circuits are to a large extent genetically determined . This raises the possibility that psa might have evolved to regulate vertebrate - specific developmental processes . An example is the role of psa in cell migration and differentiation . In invertebrates, the differentiation of neuronal precursors occurs close to the region of their birth and involves interactions with its immediate neighbor cells . On the other hand, in vertebrates, newly generated precursors often migrate long distances before acquiring their fate, and thus need to delay their differentiation till they reach their destination . Here, psa plays a dual role whereby it (a) promotes cell migration by reducing cell - cell adhesion and (b) blocks differentiation by interfering with contact - dependent signaling until the cells arrive at their final location . Such multifaceted roles for psa are well suited for the complex experience - dependent neural circuit fine - tuning that occurs in vertebrate cns . It is interesting to note that vision - dependent critical period plasticity does not start at the onset of eye opening . Instead, it is hypothesized that the critical period cannot start until the input to the circuit has developed reliability and precision . Thus, cellular mechanisms underlying critical period are not simply an activity - dependent process; instead, it is a sequence of timed events that appear to be important . Psa might then act as brake that holds off the onset of critical period plasticity until input information can be reliably relayed to the cortex . The challenge is to understand what happens if and when this timing is altered, whether onset of critical period before the appropriate time might lead to incorrect refinement of neural circuit based on unreliable, or nonoptimal inputs, and whether and how this would in turn affect behavior . Gabaergic circuit dysfunction has been implicated in various neurodevelopmental and psychiatric disorders such as autism and schizophrenia [22, 53, 54]. Therefore, our understanding of the mechanisms that control formation and plasticity of gabaergic circuits will likely yield molecular and cellular substrates that might be altered in neurodevelopmental disorders . Efforts to explore molecular mechanisms linking sensory experience to gabaergic circuit maturation have revealed several players that include both gabaergic synapse promoting factors (bdnf, otx2, and gaba itself) and gabaergic synapse inhibiting factors (psa). It has become increasingly clear that mechanisms are in place to tightly time events leading to the onset of critical period plasticity . This raises the question as to what maybe the correct or most permissible sequence of events and whether the onset of critical period at a time when circuits are not ready could lead to an altered developmental trajectory . Gaba synthesis and signaling has been shown to regulate the maturation of gabaergic innervation in visual cortex and the onset of critical period plasticity [5, 33]. These findings suggest that alteration of gaba synthesis and signalling, either due to genetic or environmental causes, can potentially affect nearly all stages of cortical circuit formation, thereby leading to impaired brain development . For instance, snps in the 5 regulatory region of the gad1 gene (coding for the gaba- synthesizing enzyme gad67) are associated with childhood onset schizophrenia . Moreover, allelic variations in gad1 have been shown to associate with schizophrenia and to influence multiple domains of cognition, including declarative memory, attention and working memory . This is interesting because reduction in the expression levels of gad67 in the dorsal lateral prefrontal cortex is one of the most consistent molecular pathological findings in individuals with schizophrenia . However, whether and how these genetic variants are directly involved in the regulation of gad1 expression levels is still unknown . In addition, the multifaceted role of gaba during cortical circuits development draws our attention to the possible deleterious effects of drugs acting on gaba receptors, notably benzodiazepines or certain antiepileptic agents, on brain development . Recent evidence from both clinical and animal studies suggests that certain antiepileptic drugs could interfere with normal cognitive development . Further studies are required to understand if gaba - targeting drugs could have long - term consequences in young children by interfering, between other things, with critical period plasticity . Gabaergic circuit dysfunction has also been implicated in autism spectrum disorders, including rett's syndrome [53, 54]. The homeodomain transcription factor dlx5, which regulates the differentiation and maturation of forebrain gabaergic interneurons, has been identified as a direct target of mecp2, which is linked to rett's syndrome . Critical period od plasticity is altered in mecp2 mutant mice, a well - recognized model for rett's syndrome . Recent studies using transgenic mice lacking mecp2 selectively in gabaergic neurons show that these mice behaviorally recapitulate many features of rett's syndrome, linking decreased gad levels and compromised mecp2 function in gabaergic neurons to the neuropsychiatric phenotype . Altered psa levels are associated with various neuropathological conditions including schizophrenia [61, 62] and temporal lobe epilepsy . In particular, a decrease in polysialylation of hippocampal neurons in schizophrenic brains correlates with early disease incidence [61, 64]. Recently, the chromosome where st8sia2, the human stx - encoding gene, is localized, 15q26, was reported as a common susceptibility region for both schizophrenia and bipolar disorder in a genome scan of eastern quebec families . Convergent evidence from the chinese han and japanese population [66, 67] strongly supports the possibility that developmental abnormalities associated with defective polysialylation may be involved in schizophrenia . In summary, multiple lines of evidence concur that alterations in molecular mechanisms of gabaergic synapse development and regulation of critical period plasticity are associated with neurodevelopmental disorders . Aberrant development of gabaergic circuits has been implicated in various dysfunctions such as autism, schizophrenia, rett syndrome, and epilepsy . Further research along these lines will help elucidate how and whether critical period plasticity is affected, which molecular pathway is critical, and whether therapeutic intervention is possible . Exciting recent evidence points to possible strategies to reopen plasticity in a mature brain [6870]. Altogether, increasing knowledge of such molecular mechanisms will further our understanding of the regulation of developmental plasticity in the brain and aid in designing strategies aimed to increase adaptive circuit rewiring following insult, such as stroke, and in developing rational pharmacological approaches to correct alterations in the brain of children with neurodevelopmental disorders.
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The uptake of anaesthetic agent is defined as the amount of vapours that is taken by the alveoli . It depends on the agent solubility, cardiac output (co) and alveolar to mixed venous partial pressure difference of the agent . Severinghaus (1954) postulated that the uptake of nitrous oxide is inversely proportional to the square root of time (sqrt). Lowe and ernst gave the sqrt, which was later utilized for predicting the uptake of commonly used volatile agents . It indicates that uptake is related to patient's weight and gradually decreases according to the sqrt . However, recent clinical observations have shown that the observed rate of isoflurane uptake differ from that predicted from the sqrt model . Addition of liquid volatile agent (ether) into the circuit was first used by william morton in 1847 . Subsequently, a series of dosage tables according to patient's body habitus have been predicted for use during closed circuit anaesthesia . The derivations of these complex formulae and mathematical models have deterred anaesthesiologists from using low flow anaesthesia . The anaesthesiologist is more interested in attaining a desired minimum alveolar concentration (mac) as the parameter and not the pharmacokinetics of the agent . By studying the rate of uptake of isoflurane during our pilot cases, we modified the dosages derived from lowe and ernst equation into a simplified form of rate of liquid isoflurane injection into the circle system using low flow anaesthesia . After approval by the hospital ethics committee, 10 patients of either sex undergoing elective surgeries for 1 h to 3 h duration of anaesthesia were enrolled . Patients with american society of anaesthesiologists (asa) physical status i, aged between 22 years and 55 years were included after informed consent . The surgeries performed were varicose vein stripping (n=3), modified mastectomies (n=4), inguinal hernia repair (n=1) and superficial parotidectomy (n=2). Patients with pregnancy, cardio - pulmonary pathology, any history of central nervous system disease, chronic use of psychoactive medication; with a body mass index (bmi) less than 15 and greater than 30 were excluded from the study . General anaesthesia was induced with iv fentanyl 2 g / kg, propofol 1 - 2 mg / kg followed by 150 g / kg / min infusion, and vecuronium 0.1 mg / kg . Bag and mask ventilation was performed for 4 min using the circle system with oxygen flows of 10 l / min (the circle system was flushed prior to this with 100% oxygen for 5 min for de - nitrogenation). The circle system was closed after ascertaining the correct position of the endotracheal tube with initiation of controlled ventilation and propofol infusion was discontinued . Ml / kg ideal body weight (ibw) h and later titrated to attain and maintain end - tidal isoflurane concentration (et - iso) of 1.5% (mac 95). The expiratory limb had a specialised connection with two luer lock ports, one for attachment to isoflurane infusion and the other for return of sampling gases (150 - 200 ml / min) by the multigas analyzer (datex - ohmeda s/5, instumentarium corp, helsinki, finland), which was calibrated before every case . Isoflurane was injected in 50 ml plastipak (polyethylene bd) syringe with polyethylene tubing using a syringe pump (fresenius vial sa, france). The dead space of the tubing (2 ml) was pre - filled with liquid isoflurane and the stated volumetric accuracy of the pump was 2% . The entire apparatus was kept at a lower level than the circle system to avoid accident spillage of liquid agent into the circuit [figure 1]. Ohmeda 7000 anaesthesia ventilator (ohmeda, boc health care division, madison, wi) with soda lime canister, corrugated rubber tubing and ascending bellows were used to provide pre - set volume ventilation . The volume of the circle system was determined individually for all the components and was 2.2 l, the volume of the connecting hose to the ventilator being 0.5 l. the amount of acceptable leak in the circuit was 50 ml / min determined during ventilation of a test lung at 30 cm peak inspiratory pressure . Oxygen was used as the sole carrier gas with fresh gas flow (fgf) to maintain a constant circuit volume . The incision was given after 30 min of the start of the isoflurane infusion to study isoflurane uptake in un - stimulated conditions . Intra - operative analgesia was maintained with fentanyl infusion of 1 g / kg / h iv . Patients requiring vasopressor or cardiac medication for haemodynamic changes of or 20% of baseline values were excluded from the study . Additional fentanyl (apart from infusion) and vecuronium boluses were given as clinically indicated . A oxygen flow meter; b and f unidirectional valves; c inspiratory limb; d expiratory limb; e specialised connector for isoflurane infusion and return of sampling gases; g adjustible pressure limiting valve; h ventilator bellows; i soda lime canister; j isoflurane infusion; k agent analyser; l computer for data recording rate of uptake and cumulative uptake of isoflurane was determined from the difference between the isoflurane administered and the system leaks (see appendix). Similarly, the expected rate of uptake and cumulative uptake was calculated using lowe and ernst's sqrt model for comparison with our study . Throughout the procedure bispectral (bis) index, non - invasive blood pressure, heart rate, haemoglobin oxygen saturation (spo2), temperature, end - tidal carbon dioxide (et - co2), inspired isoflurane (fi - iso), and et - iso were recorded continuously . A computer interphased to the syringe pump and the monitor recorded the data continuously . Pearson correlation coefficient was obtained between the cumulative uptake of isoflurane in the first 30 min of start of isoflurane infusion with the patients variables; body weight, ibw, height, body surface area (bsa), and (body weight). Bmi was correlated with cumulative uptake per kilogram body weight . The mean cumulative rate of uptake per kg derived from the lowe and ernst equation was compared with the observed rates at 10 min interval, from 10 min onward using repeated measured anova . The mean rate of uptake per kg per minute were averaged at 10 min intervals and also compared with that estimated from lowe and ernst equation using repeated measures anova . Pearson correlation coefficient was obtained between the cumulative uptake of isoflurane in the first 30 min of start of isoflurane infusion with the patients variables; body weight, ibw, height, body surface area (bsa), and (body weight). Bmi was correlated with cumulative uptake per kilogram body weight . The mean cumulative rate of uptake per kg derived from the lowe and ernst equation was compared with the observed rates at 10 min interval, from 10 min onward using repeated measured anova . The mean rate of uptake per kg per minute were averaged at 10 min intervals and also compared with that estimated from lowe and ernst equation using repeated measures anova . Patients characteristics (six male / four female) and anaesthesia details are given in table 1 . Perioperative haemodynamics were stable in all cases with no patient requiring cardiac medication, vasopressors and/or additional doses of fentanyl or vecuronium . Patient and anaesthesia details the mean fgf used was 25010 ml / min (equal to leaks and patients oxygen consumption) to maintain the circuit volume . After attainment of 1.5% et - iso, it was maintained between 1.4% and 1.6% . The rate of injection used did not result in liquid droplet formation in the circuit as seen by a glass window in the specialised connector . The observed cumulative uptake of isoflurane vapours by 30 min was 20.52.8 ml / kg / min . Since the target et - iso (1.5%) was achieved by 10 min in all the patients, we compared the predicted and the observed uptake after 10 min . Observed cumulative uptake was high as compared to lowe's equation, the difference gradually increased with time . This difference was statistically significant (p=0.042 from 60 min onwards from the start of isoflurane infusion [figure 2]. As compared to lowe's equation the mean observed uptake per kg per min was significantly higher after initial 10 min (p<0.05). Rate of observed uptake relatively stabilized after 30 - 40 min [figure 3] with a mean of 0.370.47 ml / kg / min vapour requirement for keeping a mean end - tidal isoflurane concentration of 1.510.1% . There was inter - individual variability in the uptake [figure 4] and it showed statistically significant correlation with ibw, wt, bsa, body weight from 30 min of start of isoflurane infusion (p<0.05) [table 2]. Cumulative uptake kg varied inversely with bmi (r=0.53), but the relation was not statistically significant (p>0.05). Cumulative rate of uptake of isoflurane (vapour) per kg body weight (meansd). * p<0.05; * * p<0.01 mean rate of uptake (sd) of isoflurane vapour per kg actual body weight per min . Also shown note the stability from 40 min onwards . * p<0.05; * * p<0.01 individual rates of infusion of isoflurane per kg ideal body weight per h. the different rates of infusion depict inter - individual variability for the same end tidal after functional residual capacity and circuit wash in correlation of cumulative isoflurane uptake (at 30 min) and measured patients variables . Value in parenthesis shows the actual p value bis index was in the range of 25 - 30 and et - co2 was maintained at 32.42.1 mm hg during the study . The mean expired oxygen concentration at the end for the longest duration of surgery (160 min) was 785% . We had derived isoflurane infusion rates [figure 4] from our pilot cases by modification of lowe and ernst's equation of uptake (1 ml of liquid isoflurane produces 205 ml vapours at 37c). Our aim was to provide enough vapours to wash - in the circuit volume and patients functional residual capacity (frc) to 1.5% vapour concentration and in addition 3 unit doses (according to sqrt principle) within 9 min . We had decided to achieve et - iso of 1.5% within 5 - 10 min for gradual build - up of concentration, avoiding excess over pressurization and hypotensions in anaesthetized patients thus try to maintain a stable co to study uptake . Though the rate of observed uptake in the initial period was lesser than predicted by the lowe's equation, we compared uptake after 10 min; by the time1.5% et - iso was attained in all patients . Sqrt and 4-compartment models have been compared before with a lot of discrepancies with actual clinical observational studies on volatile agent uptake . Changes in co with surgical stimulation were speculated as the main factor for the differences . However, even after substitution of measured co values in the formulae both models had differences between observed and calculated uptake . The same differences were observed when invasively measured fluctuating co was used to calculate and compare uptake . We tried to maintain stable haemodynamic to maintain a stable co as our fgf were near metabolic flows and did not fluctuate much . Moreover when using closed - loop feedback technique to maintain constant end expired concentration, the rate of increase and consequently the uptake becomes independent of co and ventilation; and depends on the infusion rate of the volatile agent . The other reasons to explain the difference of isoflurane requirement from the sqrt model are; isoflurane metabolism, loss from the surgical site, body surface, anaesthetic circuitry, and absorption into soda lime . Isoflurane is mainly exhaled unchanged from the body and only 0.2% of absorbed isoflurane undergoes oxidative metabolism . Anaesthetic loss into the circuitry depends on the rubber / gas (49) and plastic / gas (58) partition coefficients; however, this depot will be saturated with time and cannot explain the progressive increasing in the difference of cumulative uptake . Inter - individual variation is also a factor affecting uptake kinetics . On plotting the infusion requirements over time, we found that it varied for similar et - iso in different patients [figure 4]. This can be due to individual differences in the factors determining uptake i.e., co, blood / gas partition coefficient (b / g). The tissue and blood solubility coefficients have been derived from tissue homogenates and vary by as much as 150% . Solubility is also influenced by changes in body temperature, blood and tissue composition, genetic pre - disposition, and other unknown physiological variables . Brody's equation has been derived from a large normal sample population under resting conditions . It does not necessary predict the output for the individual patient and might be different from the measured co in clinical situations . Moreover, changes in co do not cause proportional changes in tissue perfusion to various compartments . Previous comparative studies state that isoflurane uptake is overestimated and later on underestimated by sqrt model . Our study shows similar results with a simpler methodology to calculate the rate of uptake . The constant difference after 30 min between the uptake curve [figure 3], imply that the difference of cumulative uptake between the observed and that from the sqrt model would gradually increase [figure 2]. Severinghaus had described the uptake of nitrous oxide in only 6 patients with primitive monitors . Arterial to end expired gradient due to ventilation - diffusion mismatch, different exponential organ uptake time constants, inter tissue diffusion of volatile agents and regional blood flow variability may cause inaccuracy in calculating uptake . Even current computer programs simulating anaesthetic uptake may not be correct because they are based on theoretical models, although they may be still useful to teach basic principles of uptake . Therefore, anaesthesia techniques with low flows continue to rely on anaesthetic monitoring . Nevertheless, the fact that authors have repeatedly come back to sqrt model as a benchmark suggests that it still might have some underlying validity . Different studies have shown weak correlations of uptake of the agent with all patient characteristics better with body weight, bsa and actual body weight . Thus, predicting anaesthetic uptake and its delivery at pre - determined rates derived from anthropometric measurements might not be accurate . We used ibw for calculating the infusion rate of isoflurane because it correlates better with lean body mass . Since, this was an open labelled study the observer was not blinded . Data were simultaneously being recorded intra - operatively in a computer logged on to the monitor . Isoflurane uptake was based on oxygen requirement, which were derived from nomograms (appendix). The decreased requirement under anaesthesia was taken care of by further decrease in fgf progressively to metabolic flows . Isoflurane infusion was not connected to the inspiratory limb to avoid any accidental exposure of liquid isoflurane to patient's airway . Moreover, at the expiratory limb warm expired gases assist to maintain near body temperature and ensure vaporization . Vapour loss due to system leaks and venting was evaluated based on the fgf and patient's oxygen consumption . Alveolar uptake proceeds simultaneously with wash in therefore the actual rate of uptake cannot be separated from that of the system . The rate of infusion stabilized by 15 - 20 min (0.15 - 0.10 ml ibw / kg / h) in all subjects and remained the same throughout . There is inter - individual variability in the pharmacokinetics of isoflurane in humans . During the initial period rate of uptake
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Innate immune cells are cells that participate in immune defense against microbes but lack the clonotypic receptors responsible for diverse antigen - specific recognition and immune memory . These cells have conventionally been viewed as having a constitutive distribution and being incapable of selective adaptation towards secondary encounters with the same microbe . However, the line between innate and adaptive immunity based on clonotypic receptors is blurred by t cells, natural killer t (nkt) cells, and b1b cells with strict partitioning further challenged by the behaviour of innate lymphoid cell (ilc) subsets, most notably that of nk cells . The missing self hypothesis of nk cell regulation was introduced 25 years ago, but the molecular details and a much deeper appreciation of the specialized degree of nk education towards self - tolerance and subsequent regulation of their effector functions have emerged over the last decade [13]. This has solidified the concept of substantial nk diversity based on subdivision into distinct subsets functionally specified by expression of a highly variable constellation of inhibitory and activating receptors . The first illustration of nk specificity and memory involved nk from c57bl/6 mice shown to directly recognize a murine cytomegalovirus (mcmv) glycoprotein (m157) through activating receptor ly49h [4, 5]. Expression of this receptor confers relative resistance of c57bl/6 mice to mcmv, primary infection with mcmv drives selective expansion of nk expressing ly49h, and an expanded population of ly49h nk that responds more effectively upon secondary mcmv exposure persists following primary infection [69]. This illustrates a clear exception to the rule that all immune memory depends on clonotypic receptors . Memory nk responses in the form of antigen - specific contact hypersensitivity mediated by murine nk that traffic to and from the liver have also been reported, but no mechanism for this specificity has been established . In human studies, associations between killer cell immunoglobulin - like receptor (kir) expression, human class i histocompatibility - linked antigen (hla) genotype, and outcomes of viral infection suggest that particular subsets of nk cells have greater antiviral activity depending on the presence or absence of corresponding class i hla ligands [1113]. Expansion of nk expressing particular kir occurs during acute human immunodeficiency virus (hiv) infection and there is evidence for nk escape mutations in chronic hiv infection [14, 15]. Recent studies show specific recognition of target cells pulsed with viral peptides or proteins by nk from peripheral blood of infected humans and by nk from the spleen and liver of simian immunodeficiency virus (siv) or shiv - infected macaques [16, 17]. Human (h)cmv infection leaves a distinct imprint on the nk repertoire involving expansion and accumulation of cells expressing the activating receptor nkg2c and maturation marker cd57 . These cells also tend to lack expression of nkg2a, while expressing activating kir and those inhibitory kir with corresponding class i hla ligands present [1921]. Expression of nkp30 and nkp46 is reduced on these nk, which is reflected in lesser natural cytotoxicity; however, antibody - dependent activation of nkg2ccd57 nk is enhanced relative to other nk [18, 22]. A majority of the nkg2ccd57 nk do not express fcr1 or the signaling kinase syk, suggesting an alternate form of intracellular signaling in association with antibody - mediated cd16 engagement [2326]. Although there is no direct evidence that nkg2c mediates specific recognition of hcmv or hcmv - infected cells, expression of cd57 and nkg2c clearly demarcates an nk subset selectively expanded following exposure to hcmv [1418]. As the relationship between nk and adaptive immunity against hcmv is largely unknown and likely to be important, we chose to investigate this relationship by comparing b and t cell responses against hcmv across a broad range of cd57nkg2c nk frequencies, excluding data from nkg2c individuals . We determined the frequency of cd57nkg2c nk, measured relative antibody levels against cmv - infected cell lysate, and characterized cd8 t cell responses against immunodominant cmv proteins in over 200 individuals . A large group of hiv - infected individuals were incorporated into the study because exaggerated responses against hcmv in this population produce a broader range of responses for comparison [27, 28]. Non - hiv - infected individuals were recruited from healthy memorial university of newfoundland faculty of medicine personnel . Individuals infected with hiv were recruited through the newfoundland and labrador provincial hiv clinic, st . All hiv - infected individuals were in the chronic stage of infection and most were receiving combination antiretroviral therapy at times of testing . Ethical approval for this study was granted by the newfoundland and labrador health ethics research authority and all participants provided informed consent for blood collection and immunological studies . Blood was collected by forearm venipuncture into acid - citrate - dextrose containing vacutainers and plasma for cmv antibody testing was collected by centrifuging whole blood at 400 g for 10 min, removing the upper acellular phase and immediately storing small aliquots at 80c until testing . Peripheral blood mononuclear cells (pbmc) were isolated by ficoll - hypaque (ge healthcare biosciences, mississauga, on, canada) density gradient centrifugation and suspended in lymphocyte medium consisting of rpmi 1640 supplemented with 10% fetal calf serum (fcs), 100 g / ml streptomycin, 100 iu / ml penicillin, 2 mm l - glutamine, 10 mm hepes buffer, and 2 10 m 2-mercaptoethanol (all from invitrogen, carlsbad, ca, usa), with fcs increased to 20% and dimethyl sulfoxide (sigma - aldrich, st . Louis, mo, usa) added to 10% for preservation in liquid n2 until testing . The percentage of nk in fresh pbmc expressing nkg2c and cd57 was assessed by multiparametric flow cytometry . Lymphocytes were identified by forward and side scatter, t cells excluded using anti - cd3peridinin chlorophyll protein (percp) from miltenyi biotec, san diego ca, usa (clone bw264/56), and nk cells identified with anti - cd56fluorescein isothiocyanate (fitc) from ebioscience, san diego, ca, usa (clone mem188). Anti - nkg2callophycocyanin (apc) from r&d systems, minneapolis, mn, usa (clone 134591), and anti - cd57phycoerythrin (pe), from biolegend, san diego ca, usa (clone hnk-1), were used to determine nkg2c and cd57 expression on cd56 nk cells . Samples were analyzed on a becton dickinson (san jose, ca, usa) facscalibur flow cytometer and acquired data processed with kaluza software (beckman coulter, brea, ca, usa). If nkg2c expression as measured by flow cytometry was below 1% of nk, sequence specific polymerase chain reaction was done as previously described to identify nkg2c subjects homozygous for deletion of a 16-kilobase segment on chromosome 12 encoding the nkg2c gene . Antibodies against hcmv were measured in plasma samples by elisa against cmv ad169-infected mrc-5 cell lysate . To generate lysate, 1 10 mrc-5 cells were infected with cmv ad169 at a multiplicity of infection of 0.5 and after 3 days were harvested by scraping, pelleted by centrifugation, and lysed in 1 ml lysis buffer . Lysate diluted 1/1000 in carbonate / bicarbonate coating buffer was added in 100 l overnight at 4c to wells of immulon-2 elisa plates (vwr scientific, mississauga, on, canada). Plasma samples diluted 1/500 were incubated on the plates for 90 min, washed, and developed with goat - anti - human igg - horseradish peroxidase conjugate (jackson immunoresearch labs, west grove pa, usa) followed by tetramethylbenzidine substrate (sigma - aldrich). Colour development ran for 30 min at room temperature, after which the reaction was stopped with 1 n h2so4 and optical density (od) read at 450 nm . Cmv ad169 was obtained through the nih aids reagent program, aids program, niaid, and nih from dr . K. hirasawa, division of biomedical sciences, faculty of medicine, memorial university of newfoundland . The in - house elsa was validated using samples diluted 1: 100 and tested against recombinant cmv pp150 and gb proteins (virogen, watertown, ma, usa) coated on elisa plates at 50 ng / well . Plasma from samples with the lowest and highest ods against cmv pp150 and gb were used to define a lysate coating concentration and plasma dilution producing the broadest signal range for the 2 samples over a short incubation period . Then plasma samples from 12 hiv - infected individuals and 12 uninfected controls with no detectable t cell response against either cmv pp65 or ie-1 and od <0.10 against cmv pp150 or gb were tested by elisa against the lysate as described above . For the hiv - infected and uninfected individuals, respectively, therefore, we set an od of> 0.10 as conservative threshold for cmv seropositivity by this assay . Subjects with an od between 0.10 and 0.20 and no detectable t cell response against cmv pp65 or ie-1 are not assigned as cmv - seronegative or seropositive . Only 1 of over 400 subjects tested falls into this category . On each plate, a positive sample initially producing an od = 2.27 and negative sample with an od = 0.034 are run as controls and the assay is repeated if there is more than 10% variation in the od of the positive control or> 50% variation in od of the negative control . To enumerate and phenotype both cmv - specific and bystander cd8 t cells, pbmc were thawed, cultured overnight, then counted, and resuspended in 3 aliquots of 2 10, each in 1 ml lymphocyte medium . One aliquot served as a negative control, while overlapping peptides from hcmv pp65 or hcmv immediate early- (ie-) 1 proteins (miltenyi biotec) were added to the other aliquots in 10 l rpmi 1640 to yield final individual peptide concentrations of 0.5 g / ml . The pbmc with peptides were placed in a 5% co2 37c humidity - controlled incubator for 1 hour, after which brefeldin a (sigma - aldrich) was added to 10 g / ml and incubation continued for another 4 hr . Following this, the cells were centrifuged at 400 g, washed with flow buffer (phosphate buffered saline [pbs] with 5 mm ethylenediaminetetraacetic acid and 0.1% bovine serum albumin [bsa, sigma - aldrich]), and surface stained with percpanti - cd8 (clone hit8a, biolegend, san diego, ca, usa), fitcanti - cd28 (clone cd28.2, biolegend), and peanti - cd57 (clone tb01, ebioscience). The cells were then fixed, permeabilized, and stained with apcanti - interferon - gamma (ifn-) from ebioscience (clone 4s.b3) or appropriate isotype controls . The processed cells were analyzed on a becton dickinson facscalibur flow cytometer with acquisition of 2 10 events / sample . Data for the groups were represented as medians with interquartile range as the distribution of values measured in most groups was not normal as determined by at least one of the kolmogorov - smirnov, d'agostino - pearson or shapiro - wilk normality tests . Spearman correlations were calculated and line of best fit was visualized by linear regression when a significant correlation was observed . All statistical analyses were carried out with the graphpad prism software program, version 5 . The percentage of nk cells expressing cd57 and nkg2c was measured in pbmc from 211 individuals in four different groups distinguished by hiv and hcmv infection status . Age and sex distribution within each group the group of cmv - seropositive, hiv - infected individuals had the highest percentage of cd57nkg2c nk (median with interquartile range [iqr] = 5.90, 2.3019.10) followed sequentially by cmv - seropositive controls (2.60, 1.257.70), the cmv - seronegative, hiv - infected group (1.55, 0.442.30), and cmv - seronegative controls (0.84, 0.341.45, figure 1). There was no significant difference in median age between any of the groups . In the cmv - seronegative, non - hiv - infected group, which had the lowest percentage of cd57nkg2c nk, there was a significant correlation between age and percentage of cd57nkg2c nk (correlation coefficient [r] = 0.468, p = 0.028). No significant correlation between age and percentage of cd57nkg2c nk was observed for any of the other groups . These data indicate that nk responses driven by cmv are exaggerated in hiv infection, as previously reported for cd8 t cell and antibody responses against cmv [27, 28] and that, in cmv- and hiv - infected groups, factors other than aging have a dominant influence on the extent to which the cd57nkg2c nk population expands . The median frequency of cd57nkg2c nk was not significantly higher in the cmv - seronegative hiv - infected group than the cmv - seronegative control group (p = 0.0877), indicating that, under most circumstances, hiv infection alone does not promote significant expansion of cd57nkg2c nk in the absence of hcmv infection (figure 1). To assess whether nk responses related to antibody responses against hcmv, we next measured relative levels of anti - cmv antibodies in plasma from each cmv - seropositive subject . Elisa od values ranged from 0.00 to 2.20 following background subtraction and values <0.10 were considered to indicate seronegative status for hcmv . Antibody levels were significantly higher in the hiv coinfected group than in the cmv - infected control group (median with iqr = 1.02, 0.791.33 versus 0.65, 0.26 to 1.04, p <0.0001, figure 2). We tested for significant relationships between age, nk responses, and antibody responses against cmv in hiv - infected and control cmv - seropositive groups by spearman correlation and found no significant correlations in either group . These data indicate that humoral immunity against cmv develops independently of nk responses against cmv and that factors other than aging have a dominant influence on the size of the response . To assess whether nk responses related to development or evolution of cd8 t cell responses against cmv, we next measured the frequency of cd8 t cells producing interferon - gamma (ifn-) in response to overlapping peptides from cmv ie-1 and pp65 proteins . We also characterized both the hcmv - specific and bystander cd8 t cell population for surface markers signifying t cell proliferative reserve (cd28) or senescence (cd57). As with expansion of the nkg2ccd57 nk population and anti - cmv antibody levels, median magnitude of the cmv - specific cd8 t cell response was significantly greater in the hiv coinfected group than in the cmv - infected control group (median with iqr = 2.30, 1.005.00 versus 1.00, 0.203.40, figure 3). While there was no significant direct correlation between cd8 t cell and nk response magnitudes against hcmv in either group, in the hiv - infected group there was a significant inverse correlation between the percentage of nkg2ccd57 nk and fraction of hcmv - specific cd8 t cells expressing cd28 (r = 0.193, p = 0.038, figure 4). As the cd28 fraction of hcmv - specific cd8 t cells also correlated inversely with the magnitude of the cd8 t cell response (r = 0.229, p = 0.013), this suggests some overlap between features driving hcmv - specific cd8 t cell and nk memory inflation . To address the impact of innate immune responses against hcmv on development of adaptive immunity against hcmv, we investigated relationships between expansion of nkg2ccd57 nk and the strength or character of adaptive immune responses against hcmv . Since adaptive immune responses against hcmv are often amplified in hiv infection, we included a large group of hiv - infected individuals in order to encompass a range of response strengths as broad as possible [27, 28]. Similar to adaptive immune responses, the innate immune response against hcmv, as indicated by expansion of nkg2ccd57 nk, was exaggerated in hiv infection, reaching levels> 70% of all circulating nk . We found no significant relationship between levels of antibodies against hcmv and nkg2ccd57 nk expansion, suggesting largely independent evolution of these responses . Only in the cmv - seronegative control group with the lowest frequencies of nkg2ccd57 nk there was a significant correlation between nkg2ccd57 nk expansion and age, indicating that features other than age or duration of hcmv infection have a dominant effect on expansion of the nkg2ccd57 nk population . In fact, despite the significant correlation, aging in the absence of hiv or cmv infection had only a very minor effect on nkg2ccd57 nk expansion . We did observe a significant inverse correlation between nkg2ccd57 nk expansion and the fraction of hcmv - specific cd8 t cells expressing cd28 . Loss of cd28 expression on antigen - specific memory t cells is associated with shortened telomeres, reduced proliferative potential, and progress towards senescence [31, 32]. As this occurs in parallel with nkg2ccd57 nk expansion, it raises the possibility that similar immunological or virological features prominent in hcmv / hiv coinfection drive both nkg2ccd57 nk expansion and hcmv - specific cd8 t cell proliferation underlying memory inflation and senescence . Expansion of nkg2ccd57 nk has been observed in primary hcmv infection and in hcmv reactivation following transplantation, suggesting that active hcmv replication is a major factor at least in initiation of the expansion [20, 33]. Periodic reactivation of hcmv is also thought to drive cd8 t cell memory inflation, and although overt hcmv reactivation with viremia is rare in well treated hiv infection, it is plausible that hcmv reactivation occurs more frequently in hiv - infected individuals . In fact, hcmv shedding in semen and saliva is relatively common in hiv - infected men who have sex with men [3436]. The parallel evolution of nk and cd8 t cell responses evoked by cmv in hiv - infected persons contrasts somewhat with a report describing general changes in the nk population and t cell repertoire of elderly individuals . In this study, expansion of the nkg2ccd57 nk population was independent of skewing of the cd4/cd8 t cell ratio towards a cmv - related immune risk profile . Another study reported that when cmv replication was well controlled by the immune system, as indicated by low titres of cmv - specific igg, cellular responses against cmv were dominated by either cd8 t cells or nkg2c nk, but not both . This suggests compartmentation of nk and t cell responses against cmv, with a reciprocal relationship between their magnitudes . Although we did not observe such a relationship, it may be more likely to occur in individuals who bring cmv under effective and stable immune control quickly and less likely to occur against the background of immune dysregulation associated with chronic hiv infection or unhealthy aging . The lack of any apparent direct relationships between nk and adaptive immune responses against hcmv observed in our study, despite their common trigger and shared responsibility for containing hcmv, likely reflects a complex, evolving relationship between chronic infection and the multiple immune effector arms raised against it . An inverse relationship between the magnitude of nk and adaptive immune responses against cmv should exist if either class of response alone was sufficient to effectively resolve primary infection and control cmv reactivation . However, since hcmv infection always persists and may continuously reactivate, all arms of the immune response may receive periodic stimulation with unequal reinforcement dependent upon the site and extent of reactivation and concurrent capacities of relevant immune system components to receive and deliver reinforcement signals . Over time, what began as a reciprocal relationship might, therefore, evolve towards a direct relationship or vice versa . We also speculated that a direct relationship between anti - cmv antibody levels and expansion of nkg2ccd57 nk might exist if activation and expansion of this nk subset, specialized to mediate adcc, was dependent upon interactions with antibody - coated target cells [2325]. The cumulative impact of inverse or direct relationships operating under different conditions and at different stages of evolution might obscure relevant shorter - term impacts of direct or reciprocal influences in retrospective cross - sectional studies such as this one . Therefore, longitudinal studies beginning in primary infection and investigating responses through primary and chronic stages of infection might be more revealing as to the coevolutionary nature of adaptive and innate immunity against hcmv . One longer term relationship between innate and adaptive immunity not overshadowed by cumulative effects was an inverse relationship between the extent of nkg2ccd57 nk expansion and the fraction of hcmv - specific cd8 t cells expressing cd28 in hiv infection . In fact, this relationship may represent the common cumulative effect of chronic hcmv infection and periodic reactivation on the t lymphocytes and nk cells that respond to it, at least in the context of hiv infection . Loss of cd28 expression on t cells reflects shortened telomeres and progression towards senescence, generally thought to be driven by persistent exposure to antigen [39, 40]. It also signifies t cell effector memory status and previous specialization to mediate cytotoxicity against infected host cells . The parallel between expansion of nkg2ccd57 nk, their differentiation towards enhanced adcc potential, and loss of cd28 on hcmv - specific t cells implies that both reflect increased hcmv reactivation to immunogenic levels and a possibly subtle and slow, but ultimately unrelenting, deterioration of immune containment of cmv replication . Just as failure to contain hcmv in the absence of memory inflation in the cd8 t cell response relates to late stage immune dysfunction in old elderly individuals, extensive hcmv - driven expansion of nkg2ccd57 nk may be indicative of a level of underlying immune degeneration in hiv infection . Restriction of nkg2ccd57 nk expansion to hcmv infection continues to raise questions as to a potential role for nkg2c in recognition of hcmv and as to the nature of the nk stimulus delivered in hcmv infection . While there was no statistically significant expansion of nkg2ccd57 nk in the cmv - seronegative, hiv - infected group compared to the cmv - seronegative, non - hiv - infected group, there was a trend towards an increased percentage of nkg2ccd57 nk . One possibility is that some hiv - infected, cmv - seronegative individuals were exposed to hcmv without seroconversion . There was also broad variability within each of the hcmv - infected groups illustrating the impact of secondary factors presumably related to the course of chronic hcmv infection in driving nkg2ccd57 nk expansion . The tempo or extent of periodic reactivation of hcmv is likely a contributing factor, but there was no significant relationship between anti - cmv antibody levels, often considered as a surrogate for exposure to extracellular hcmv and either nkg2ccd57 nk or hcmv - specific cd8 t cell expansion . Intracellular hcmv reactivation without release of new virions may be sufficient to drive both nkg2ccd57 nk and hcmv - specific cd8 t cell expansion in the absence of any boosting of the anti - hcmv antibody response . While both adaptive and nk immune responses against hcmv were stronger in hiv infection, we found no significant correlation between the strength of nk and adaptive immune responses against hcmv . Our data do suggest that nkg2ccd57 nk expansion to high levels reflects stress on the ability of the nk response to keep pace with hcmv reactivation or with other factors underlying this expansion . Thus, we expect adaptation in the character of nkg2ccd57 nk similar to that occurring in virus - specific cd8 t cells failing to contain replication . Accumulation of nkg2ccd57 nk may reflect an nk immune response homologous to the inflationary adaptive t cell response signified by accumulation of hcmv - specific effector memory cd8 t cells . Further functional and phenotypic analysis of nkg2ccd57 nk at different levels of expansion in different virological contexts may shed more light on their relationship to chronic hcmv infection and on the biology of nk responses in general.
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Male wistar rats weighing 220240 g and male c57bl/6j mice weighing 2025 g were from centre elevage janvier, le genest - st . The animals had free access to tap water and standard food and were treated in accordance with european community guidelines concerning care and use of laboratory animals . Gavages of conscious rats or mice with 1 g of glucose per kilogram of body weight were performed after a 16-h fast using a d - glucose solution without (control) or with relm-. The total bolus volume for mice and rats was 0.25 ml and 1 ml, respectively, and the amount of glucose in each bolus was adjusted for the animal weight . The recombinant murine relm- (18 kda; peprotech, neuilly - sur - seine, france) used was highly purified by high - performance liquid chromatography and was endotoxin - free . Relm-, at a final concentration of 0.1 and 1 nmol / l, results in dose range of 0.010.1 g / kg of body weight . Relm- is a stable molecule as high amounts of the homodimer form have been detected in mice and human stools (21). Preliminary experiments showed that the peptide given by gavage was found in proximal small intestine after 5 min with a recovery of 70% . Before starting the ogtt, blood samples were taken from the tail and fasting blood glucose levels (milligram per deciliter) were determined (accu - chek; roche diagnostic, meylan, france). The bleeds were further taken at 15, 30, 60, and 120 min after oral glucose administration . Rats were fasted for 16 h and were killed by pentobarbital overdose . The proximal jejunum was dissected out and four adjacent samples were mounted in ussing chambers as described previously (22). The tissues were bathed with 4 ml of krebs - ringer bicarbonate (krb) solution (ph 7.4) with 10 mmol / l glucose at 37c . In the solution bathing the mucosal side of the tissue, glucose electrogenic ion transport was monitored continuously as short - circuit current (isc) by using an automated voltage clamp apparatus (dvc 1000; wpi, aston, england) linked through a maclab 8 to a macintosh computer . Nmol / l) was added in the mucosal bath 2 min before a glucose challenge . Carbachol (100 mmol / l) was added at the end of each experiment as a control . Further, similar tests were performed with relm- incubated overnight at 4c with a rabbit polyclonal antibody raised against relm-. Results were expressed as the intensity of the isc (a / cm) or as the percentage of the peak isc obtained after glucose challenge (measured after 3 min) over basal isc (measured just before the addition of glucose). Jejunal loops (4 cm long) were prepared and 0.5 ml of krb solution without (control) or with 1 nmol / l relm- was inserted inside the jejunal lumen . The jejunal loops were incubated for 15 min in oxygenized krb at 37c and conditions were maintained during hexose transport assay . The corresponding jejunal loops were filled with 1 ml of krb solution without (control) or with 1 nmol / l relm- and containing 0.02 ci / ml of the isotopic tracer d-[1-c] glucose (49.5 mci / mmol) and glucose to obtain a final concentration of 10, 30, and 100 mmol / l . Similarly, we studied the paracellular transport with 30 mmol / l mannitol and the isotopic tracer d-[1-c] mannitol (59 mci / mmol) at 0.2 ci / ml . All the jejunal loops were incubated in 10 ml of krb solution during the indicated time . The radioactivity was measured in the collected samples of serosal krb solution and used to calculate glucose or mannitol transport as picomoles per milligram of jejunal protein . Five independent experiments were performed and significance is expressed as * p <0.05,**p <0.01 . Rats were anesthetized by pentobarbital and laparotomized for in situ experiments . Three jejunal segments (5 cm long) were tied and filled with 3 ml of krb without (control) or with 1 nmol / l relm-. After 3 min of in situ incubation, 3 ml of 60 mmol / l glucose solution were injected in the lumen to obtain a final concentration of 30 mmol / l . After a further 3 min, these loops were removed and opened along the mesenteric border and the mucosa was scraped off on ice with a glass blade . Total cell protein extracts and bbms were prepared from the scrapings as previously described (23), and enrichment was estimated by determination of alkaline phosphatase activity (20-fold increase of activity in bbms). Solubilized proteins were resolved by electrophoresis on 10% sds - page gels and transferred onto nitrocellulose membranes for immunoblot analysis . The following rabbit antibodies were used at a 1:1,000 dilution: sglt-1 (ab 1352; chemicon international, temecula, ca), glut2, pkc ii (sc-9117, sc-210; santa cruz biotechnology, tebu - bio, france), phospho - ampk- (thr172), ampk- (23a3), and phospho - pkc (pan (2531, 2603, 190d10; cell signaling technology, ozyme, france). The intensity of the specific immunoreactive bands was quantified using national institutes of health image (scion, frederick, md). Uptake experiments were performed using rat intestinal everted rings as previously described (24). Groups of eight intestinal rings were incubated at 37c for 15 min in oxygenized krb buffer in the absence (control) and the presence of relm- (1 nmol / l) and cytochalasin b as indicated . Then the rings were incubated for 2 min in an uptake solution corresponding to a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c] glucose . After adding the uptake solution, the rings were washed in ice - cold krb solution, and radioactivity incorporated in the tissue was quantified by liquid scintillation . Data were not corrected for extracellular substrate because relm- was found not to affect paracellular diffusion . Total protein from homogenized tissue with a dounce homogenizer was determined with bicinchoninic acid reagent from pierce (thermo scientific, brebires, france). Recombinant murine relm- was purchased from peprotech (neuilly - sur - seine, france). D-[1-c] mannitol was from ge healthcare amersham biosciences (les ulis, france), d-[1-c] glucose was from perkin elmer (boston, ma), and compound c from merck sharp & dohme - chibret (paris, france). All other chemical reagents were purchased from sigma (st . Louis, mo). One - way anova with turkey - kramer multiple comparisons post hoc test was performed using graphpad prism version 4.0 for windows (graphpad software, san diego, ca). Gavages of conscious rats or mice with 1 g of glucose per kilogram of body weight were performed after a 16-h fast using a d - glucose solution without (control) or with relm-. The total bolus volume for mice and rats was 0.25 ml and 1 ml, respectively, and the amount of glucose in each bolus was adjusted for the animal weight . The recombinant murine relm- (18 kda; peprotech, neuilly - sur - seine, france) used was highly purified by high - performance liquid chromatography and was endotoxin - free . Relm-, at a final concentration of 0.1 and 1 nmol / l, results in dose range of 0.010.1 g / kg of body weight . Relm- is a stable molecule as high amounts of the homodimer form have been detected in mice and human stools (21). Preliminary experiments showed that the peptide given by gavage was found in proximal small intestine after 5 min with a recovery of 70% . Before starting the ogtt, blood samples were taken from the tail and fasting blood glucose levels (milligram per deciliter) were determined (accu - chek; roche diagnostic, meylan, france). The bleeds were further taken at 15, 30, 60, and 120 min after oral glucose administration . Rats were fasted for 16 h and were killed by pentobarbital overdose . The proximal jejunum was dissected out and four adjacent samples were mounted in ussing chambers as described previously (22). The tissues were bathed with 4 ml of krebs - ringer bicarbonate (krb) solution (ph 7.4) with 10 mmol / l glucose at 37c . In the solution bathing the mucosal side of the tissue, glucose was replaced by mannitol . Both solutions electrogenic ion transport was monitored continuously as short - circuit current (isc) by using an automated voltage clamp apparatus (dvc 1000; wpi, aston, england) linked through a maclab 8 to a macintosh computer . Krb alone (vehicle) or containing relm- (0.0011 nmol / l) was added in the mucosal bath 2 min before a glucose challenge . Carbachol (100 mmol / l) was added at the end of each experiment as a control . Further, similar tests were performed with relm- incubated overnight at 4c with a rabbit polyclonal antibody raised against relm-. Results were expressed as the intensity of the isc (a / cm) or as the percentage of the peak isc obtained after glucose challenge (measured after 3 min) over basal isc (measured just before the addition of glucose). Animals were fasted for 16 h and were killed by pentobarbital overdose . The proximal jejunum was dissected out and rinsed in cold saline solution . Jejunal loops (4 cm long) were prepared and 0.5 ml of krb solution without (control) or with 1 nmol / l relm- was inserted inside the jejunal lumen . The jejunal loops were incubated for 15 min in oxygenized krb at 37c and conditions were maintained during hexose transport assay . The corresponding jejunal loops were filled with 1 ml of krb solution without (control) or with 1 nmol / l relm- and containing 0.02 ci / ml of the isotopic tracer d-[1-c] glucose (49.5 mci / mmol) and glucose to obtain a final concentration of 10, 30, and 100 mmol / l . Similarly, we studied the paracellular transport with 30 mmol / l mannitol and the isotopic tracer d-[1-c] mannitol (59 mci / mmol) at 0.2 ci / ml . All the jejunal loops were incubated in 10 ml of krb solution during the indicated time . The radioactivity was measured in the collected samples of serosal krb solution and used to calculate glucose or mannitol transport as picomoles per milligram of jejunal protein . Five independent experiments were performed and significance is expressed as * p <0.05,**p <0.01 . Three jejunal segments (5 cm long) were tied and filled with 3 ml of krb without (control) or with 1 nmol / l relm-. After 3 min of in situ incubation, 3 ml of 60 mmol / l glucose solution were injected in the lumen to obtain a final concentration of 30 mmol / l . After a further 3 min, these loops were removed and opened along the mesenteric border and the mucosa was scraped off on ice with a glass blade . Total cell protein extracts and bbms were prepared from the scrapings as previously described (23), and enrichment was estimated by determination of alkaline phosphatase activity (20-fold increase of activity in bbms). Solubilized proteins were resolved by electrophoresis on 10% sds - page gels and transferred onto nitrocellulose membranes for immunoblot analysis . The following rabbit antibodies were used at a 1:1,000 dilution: sglt-1 (ab 1352; chemicon international, temecula, ca), glut2, pkc ii (sc-9117, sc-210; santa cruz biotechnology, tebu - bio, france), phospho - ampk- (thr172), ampk- (23a3), and phospho - pkc (pan (2531, 2603, 190d10; cell signaling technology, ozyme, france). The intensity of the specific immunoreactive bands was quantified using national institutes of health image (scion, frederick, md). Uptake experiments were performed using rat intestinal everted rings as previously described (24). Groups of eight intestinal rings were incubated at 37c for 15 min in oxygenized krb buffer in the absence (control) and the presence of relm- (1 nmol / l) and cytochalasin b as indicated . Then the rings were incubated for 2 min in an uptake solution corresponding to a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c] glucose . After adding the uptake solution, the rings were washed in ice - cold krb solution, and radioactivity incorporated in the tissue was quantified by liquid scintillation . Data were not corrected for extracellular substrate because relm- was found not to affect paracellular diffusion . Total protein from homogenized tissue with a dounce homogenizer was determined with bicinchoninic acid reagent from pierce (thermo scientific, brebires, france). Recombinant murine relm- was purchased from peprotech (neuilly - sur - seine, france). D-[1-c] mannitol was from ge healthcare amersham biosciences (les ulis, france), d-[1-c] glucose was from perkin elmer (boston, ma), and compound c from merck sharp & dohme - chibret (paris, france). All other chemical reagents were purchased from sigma (st . Louis, mo). One - way anova with turkey - kramer multiple comparisons post hoc test was performed using graphpad prism version 4.0 for windows (graphpad software, san diego, ca). The area under the curve was significantly (p <0.05) increased in mice when relm- was used at 1 nmol / l and the two doses used (0.1 and 1 nmol / l) were effective in rats (fig . 1a and b, insets). A similar 15% increase in blood glucose as compared with control was observed in both mice and rats . These data show that luminal administration of exogenous relm- is active in vivo as previously described (6). Ogtt (1 g / kg) was performed in overnight - fasted mice (a) or rats (b) with a 15% d - glucose solution without (, dotted lines) or with relm- (, 0.1 nmol / l;, 1 nmol / l). Glucose concentration was determined in blood samples from the tail and is expressed as milligram per deciliter over the time points (minutes). Nmol / l () and 1 nmol / l () relm-. The route of glucose entry can involve active na - dependent glut (sglt-1) or diffusive transporter glut2 . We used the ussing chamber to characterize the effect of mucosal relm- on rat intestinal active glucose transport (sglt-1). Rat jejunal mucosa mounted in the chamber was allowed to reach a steady state (usually 40 min). Addition of 10 mmol / l glucose in the mucosal bath induced a rise in isc (maximum after 3 min) representing an increase in sglt-1 activity . Addition of relm- to the mucosal side 2 min before glucose challenge induced a marked and dose - dependent inhibition of glucose - induced isc . 2a, addition of relm- to the mucosal side inhibited glucose transport by jejunal mucosa in a concentration - dependent manner . Maximal inhibition was achieved with 0.1 nmol / l relm-. The concentration that produced a half - maximal inhibition (ic50) of glucose transport was 3 further, an overnight incubation of 0.1 nmol / l relm- with an antibody raised against relm- countered the inhibition of glucose - induced isc by relm- as shown in fig . Next, we investigated if the observed inhibition by relm- was associated with an altered abundance of sglt-1 in jejunal bbms . A typical immunoblot of sglt-1 protein in bbms after glucose challenge in the presence or the absence of relm- is shown in fig . The mean densitometry of three separate blots shows that glucose increased the amount of sglt-1 protein in bbms by 1.5-fold as compared with control . This increase is reduced by half in the presence of relm-. Effect of luminal relm- on glucose - induced isc . Rat jejunal mucosa was mounted in ussing chamber and the increase in isc was studied at steady state . Electrogenic (na) transport was followed as an index of the active glucose transport by cotransporter sglt-1 . Relm- was added in the mucosal bath 2 min before challenging the tissues with 10 mmol / l glucose . B: the effect of 0.1 nmol / l relm- after an overnight incubation with an antibody against relm-. C: representative sglt-1 immunoblot of solubilized bbms . Bbms were prepared from rat jejunum loops incubated in situ with and without luminal relm- during 6 min . Densitometric analysis of immunoreactive bands was performed using national institutes of health image analysis program . The densitometry represents the amount of sglt-1 relative to -actin and is representative of at least three separate experiments . Each point of the isc study represents the means se of four to eight noncumulative values from five separate experiments . Significant differences from control, * p <0.05, * * p <0.01 . We isolated rat jejunum and performed in vitro studies of transmural glucose transport to directly evaluate the effect of relm- on intestinal transport . As shown in fig . 3a, luminal glucose in jejunal loops significantly increased net mucosal to serosal glucose flux . A dose - dependent effect on glucose transport was observed with increasing glucose concentrations 10, 30, and 100 mmol / l . Further, relm-, at 1 nmol / l, significantly enhanced the jejunal transport of 10, 30, and 100 mmol / l glucose but not that of 30 mmol / l mannitol . Thus, increased jejunal glucose transport induced by relm- is unlikely to have been caused by changes in paracellular permeability . This is in line with histological studies of the jejunum tissues used in these experiments that did not show any visible mucosal deterioration (data not shown). Further, experiments were performed to identify if the effect of relm- implicated potential downstream kinases, pkc, and ampk, which are known as key effectors of intestinal glucose transport (6,20,23,25). The ampk inhibitor, compound c, and the pkc inhibitor, chelerythrine, inhibited the 30 mmol / l glucose - induced jejunal glucose transport as shown, respectively, in fig . 3b and c. the insets represent the area under the curve of the 30-min glucose transport kinetics . Relm- significantly increased glucose transport by approximately twofold and this effect was blunted by compound c and chelerythrine as shown, respectively, in fig . 3b and c. these results indicate a likely involvement of pkc and ampk in the luminal effect of relm- on glucose uptake . Transmural transport of glucose or mannitol was performed in jejunal sacs from adult wistar rats . / l relm- () or vehicle () in oxygenized krb buffer with glucose at 10, 30, and 100 mmol / l and the isotopic tracer d-[1-c] glucose . The radioactivity measured in the collected samples was used to calculate glucose transport as picomoles per milligram of jejunal protein per minute . The kinetics of glucose (30 mmol / l) transmural transport is shown in the absence (, dotted line) and in the presence () of 1 nmol / l relm- in b and c. b: the ampk inhibitor (compound c or cc) was incubated without () or with () relm-. C: similarly, the pkc inhibitor (chelerythrine chloride or ccl) is incubated without () and with () relm-. The insets show the corresponding area under the curve . The data are representative of the means se of at least four individual experiments, * p <0.05; * * p <0.01 vs. control . The above results prompted us to assess by in situ experiments the cellular effects of relm- on ampk and pkc . Jejunal segments were injected with a krb solution alone or containing 30 mmol / l glucose in the absence and the presence of 1 nmol / l relm-. The mucosal scrapings of jejunum were examined by western blot analysis . The results indicated that glucose as well as relm- stimulated ampk phosphorylation over control values as shown in fig . 4a . The corresponding mean densitometric analysis indicates that relm- induced a threefold increase in ampk phosphorylation . A further increase in the phosphorylation of ampk occurred when glucose and relm- were added together . Similarly, relm- induced a threefold increase in pkc phosphorylation in line with our data (6) in the mouse colon (data not shown). This prompted us to study the translocation of cytosolic pkc ii of enterocytes to bbms, a mechanism that is associated with intestinal glucose transport (20,23). We performed western blot analysis to determine the expression of pkc ii in bbms obtained from intestinal segments that were incubated with glucose (30 mmol / l) or relm- (1 nmol / l) as described above . 4b, relm- or glucose induced, respectively, 1.5- or 2-fold increase of pkc ii at the bbms as compared with control values . A further increase (2.7-fold compared with the control) of pkc ii at the bbms these results suggest that relm- stimulates the phosphorylation of ampk and pkc as well as an increased shift of pkc ii to the bbms of rat jejunal tissue . The loops were filled with krb buffer with or without 1 nmol / l relm-. After 3 min, glucose (30 mmol / l) was added in this mucosal bath . After a further 3 min, loops were excised and kept on ice before scraping off the mucosa . Total protein extraction and bbms preparation were performed immediately as described in research design and methods . Representative immunoblots for phospho - ampk (a) in mucosal extracts and pkc ii (b) in bbms are shown . We explored whether relm- could enhance the glucose - induced glut2 translocation to the apical membrane of enterocytes . To this end, we measured glucose uptake in rat everted jejunal rings in the presence of relm- and without or with cytochalasin b, a competitive inhibitor of glut2 (26). 5a, cytochalasin b inhibited glucose uptake by 50% in agreement with the implication of glut2 in glucose uptake (27,28). Relm- enhanced glucose uptake by twofold and this effect was also strongly inhibited by cytochalasin b. further, we performed western blot analysis to determine the expression of glut2 in bbms obtained from intestinal segments that had been incubated with relm- under the same conditions as above . We observed that glucose or relm- alone induced, respectively, a four- or threefold increase in the amount of glut2 found in the brush - border fraction, respectively (fig . 5b). When glucose and relm- were added together, a further increase in the amount of glut2 was observed (fivefold as compared with control). This suggests that relm- enhanced glucose uptake is a result of an increased insertion of glut2 into the bbms . A: rat jejunal everted rings were incubated in oxygenized krb buffer without (control) or with 1 the rings were incubated for 2 min in a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c] glucose). The radioactivity incorporated in the tissue b: rat jejunal loops were treated 6 min with a mucosal bath containing 30 mmol / l glucose with or without 1 nmol / l relm- and bbms were prepared as described in research design and methods . A representative western blot analysis of the corresponding protein lysates with glut2 antibody is shown . Densitometric analysis of immunoblots indicates an increase of glut2 in the presence of glucose or relm-. The data are expressed relative to -actin and is representative of at least three separate experiments . Significantly different from control, * p <0.05 . The area under the curve was significantly (p <0.05) increased in mice when relm- was used at 1 nmol / l and the two doses used (0.1 and 1 nmol / l) were effective in rats (fig . 1a and b, insets). A similar 15% increase in blood glucose as compared with control was observed in both mice and rats . These data show that luminal administration of exogenous relm- is active in vivo as previously described (6). Ogtt (1 g / kg) was performed in overnight - fasted mice (a) or rats (b) with a 15% d - glucose solution without (, dotted lines) or with relm- (, 0.1 nmol / l;, 1 nmol / l). Glucose concentration was determined in blood samples from the tail and is expressed as milligram per deciliter over the time points (minutes). The route of glucose entry can involve active na - dependent glut (sglt-1) or diffusive transporter glut2 . We used the ussing chamber to characterize the effect of mucosal relm- on rat intestinal active glucose transport (sglt-1). Rat jejunal mucosa mounted in the chamber was allowed to reach a steady state (usually 40 min). Addition of 10 mmol / l glucose in the mucosal bath induced a rise in isc (maximum after 3 min) representing an increase in sglt-1 activity . Addition of relm- to the mucosal side 2 min before glucose challenge induced a marked and dose - dependent inhibition of glucose - induced isc . 2a, addition of relm- to the mucosal side inhibited glucose transport by jejunal mucosa in a concentration - dependent manner . Maximal inhibition was achieved with 0.1 nmol / l relm-. The concentration that produced a half - maximal inhibition (ic50) of glucose transport was 3 further, an overnight incubation of 0.1 nmol / l relm- with an antibody raised against relm- countered the inhibition of glucose - induced isc by relm- as shown in fig . Next, we investigated if the observed inhibition by relm- was associated with an altered abundance of sglt-1 in jejunal bbms . A typical immunoblot of sglt-1 protein in bbms after glucose challenge in the presence or the absence of relm- is shown in fig . The mean densitometry of three separate blots shows that glucose increased the amount of sglt-1 protein in bbms by 1.5-fold as compared with control . This increase is reduced by half in the presence of relm-. Effect of luminal relm- on glucose - induced isc . Rat jejunal mucosa was mounted in ussing chamber and the increase in isc was studied at steady state . Electrogenic (na) transport was followed as an index of the active glucose transport by cotransporter sglt-1 . Relm- was added in the mucosal bath 2 min before challenging the tissues with 10 mmol / l glucose . Values for isc were standardized to control values and expressed as percentage of controls . A: dose - dependent inhibition . B: the effect of 0.1 nmol / l relm- after an overnight incubation with an antibody against relm-. C: representative sglt-1 immunoblot of solubilized bbms . Bbms were prepared from rat jejunum loops incubated in situ with and without luminal relm- during 6 min . Densitometric analysis of immunoreactive bands was performed using national institutes of health image analysis program . The densitometry represents the amount of sglt-1 relative to -actin and is representative of at least three separate experiments . Each point of the isc study represents the means se of four to eight noncumulative values from five separate experiments . Significant differences from control, * p <0.05, * * p <0.01 . We isolated rat jejunum and performed in vitro studies of transmural glucose transport to directly evaluate the effect of relm- on intestinal transport . As shown in fig . 3a, luminal glucose in jejunal loops significantly increased net mucosal to serosal glucose flux . A dose - dependent effect on glucose transport was observed with increasing glucose concentrations 10, 30, and 100 mmol / l . Further, relm-, at 1 nmol / l, significantly enhanced the jejunal transport of 10, 30, and 100 mmol / l glucose but not that of 30 mmol / l mannitol . Thus, increased jejunal glucose transport induced by relm- is unlikely to have been caused by changes in paracellular permeability . This is in line with histological studies of the jejunum tissues used in these experiments that did not show any visible mucosal deterioration (data not shown). Further, experiments were performed to identify if the effect of relm- implicated potential downstream kinases, pkc, and ampk, which are known as key effectors of intestinal glucose transport (6,20,23,25). The ampk inhibitor, compound c, and the pkc inhibitor, chelerythrine, inhibited the 30 mmol / l glucose - induced jejunal glucose transport as shown, respectively, in fig . 3b and c. the insets represent the area under the curve of the 30-min glucose transport kinetics . Relm- significantly increased glucose transport by approximately twofold and this effect was blunted by compound c and chelerythrine as shown, respectively, in fig . 3b and c. these results indicate a likely involvement of pkc and ampk in the luminal effect of relm- on glucose uptake . Transmural transport of glucose or mannitol was performed in jejunal sacs from adult wistar rats . / l relm- () or vehicle () in oxygenized krb buffer with glucose at 10, 30, and 100 mmol / l and the isotopic tracer d-[1-c] glucose . The radioactivity measured in the collected samples was used to calculate glucose transport as picomoles per milligram of jejunal protein per minute . The kinetics of glucose (30 mmol / l) transmural transport is shown in the absence (, dotted line) and in the presence () of 1 nmol / l relm- in b and c. b: the ampk inhibitor (compound c or cc) was incubated without () or with () relm-. C: similarly, the pkc inhibitor (chelerythrine chloride or ccl) is incubated without () and with () relm-. The insets show the corresponding area under the curve . The data are representative of the means se of at least four individual experiments, * p <0.05; * * p <0.01 vs. control . The above results prompted us to assess by in situ experiments the cellular effects of relm- on ampk and pkc . Jejunal segments were injected with a krb solution alone or containing 30 mmol / l glucose in the absence and the presence of 1 nmol / l relm-. The mucosal scrapings of jejunum were examined by western blot analysis . The results indicated that glucose as well as relm- stimulated ampk phosphorylation over control values as shown in fig . 4a . The corresponding mean densitometric analysis indicates that relm- induced a threefold increase in ampk phosphorylation . A further increase in the phosphorylation of ampk occurred when glucose and relm- were added together . Similarly, relm- induced a threefold increase in pkc phosphorylation in line with our data (6) in the mouse colon (data not shown). This prompted us to study the translocation of cytosolic pkc ii of enterocytes to bbms, a mechanism that is associated with intestinal glucose transport (20,23). We performed western blot analysis to determine the expression of pkc ii in bbms obtained from intestinal segments that were incubated with glucose (30 mmol / l) or relm- (1 nmol / l) as described above . 4b, relm- or glucose induced, respectively, 1.5- or 2-fold increase of pkc ii at the bbms as compared with control values . A further increase (2.7-fold compared with the control) of pkc ii at the bbms these results suggest that relm- stimulates the phosphorylation of ampk and pkc as well as an increased shift of pkc ii to the bbms of rat jejunal tissue . The loops were filled with krb buffer with or without 1 nmol / l relm-. After 3 min, glucose (30 mmol / l) was added in this mucosal bath . After a further 3 min total protein extraction and bbms preparation were performed immediately as described in research design and methods . Representative immunoblots for phospho - ampk (a) in mucosal extracts and pkc ii (b) in bbms are shown . The data are representative of three separate experiments . Significantly different from control, * p <0.05 . We explored whether relm- could enhance the glucose - induced glut2 translocation to the apical membrane of enterocytes . To this end, we measured glucose uptake in rat everted jejunal rings in the presence of relm- and without or with cytochalasin b, a competitive inhibitor of glut2 (26). 5a, cytochalasin b inhibited glucose uptake by 50% in agreement with the implication of glut2 in glucose uptake (27,28). Relm- enhanced glucose uptake by twofold and this effect was also strongly inhibited by cytochalasin b. further, we performed western blot analysis to determine the expression of glut2 in bbms obtained from intestinal segments that had been incubated with relm- under the same conditions as above . We observed that glucose or relm- alone induced, respectively, a four- or threefold increase in the amount of glut2 found in the brush - border fraction, respectively (fig . 5b). When glucose and relm- were added together, a further increase in the amount of glut2 was observed (fivefold as compared with control). This suggests that relm- enhanced glucose uptake is a result of an increased insertion of glut2 into the bbms . A: rat jejunal everted rings were incubated in oxygenized krb buffer without (control) or with 1 nmol / l relm-. The presence of cytochalasin b is indicated . The rings were incubated for 2 min in a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c] glucose). The radioactivity incorporated in the tissue b: rat jejunal loops were treated 6 min with a mucosal bath containing 30 mmol / l glucose with or without 1 nmol / l relm- and bbms were prepared as described in research design and methods . A representative western blot analysis of the corresponding protein lysates with glut2 antibody is shown . Densitometric analysis of immunoblots indicates an increase of glut2 in the presence of glucose or relm-. The data are expressed relative to -actin and is representative of at least three separate experiments . Significantly different from control, * p <0.05 . In the present study, we show that relm-, a resistin - like molecule, can directly increase jejunal absorption of glucose in the rat . Several lines of evidence suggest that transepithelial transport of glucose in the small intestine can be mediated by an active absorption through na / glucose cotransporter (sglt-1) as well as by a diffusive component glut2 at the apical membrane (27). We found that relm- inhibited the activity of sglt-1, whereas enhancing the presence of glut2 at the bbm of enterocytes . Moreover, the increased jejunal glucose transport induced by relm- was inhibited by cytochalasin b in agreement with a functional role of glut2 . The underlying molecular mechanism involves the activation of pkc ii and ampk kinases as described for such reciprocal regulation of glucose transporters (20). The acute treatment of rat jejunum with relm- had no significant effect on passive mannitol movement suggesting that relm- increases glucose transport by the use of transporters . We found that mucosal relm- inhibited the activity and the translocation of cytosolic sglt-1 to cell membranes . This effect was blunted by an antibody raised against relm-. Other peptides such as leptin, angiotensin ii, and cck-8 have also been shown to inhibit sglt-1 activity (14,16,28). We show that in contrast to ckk-8, which seems to have no effect on glut2 (28), relm- can induce glut2 translocation . Indeed, in response to relm- the amount of glut2 was increased in the bbms . Similarly, a rapid insertion of glut2 to apical membrane in response to another gut - peptide, glp-2, has been reported (29,30). Taken together, these findings suggest the involvement of glut2 in relm- stimulated glucose uptake . The mechanisms responsible for relm- effect may involve the activation of pkc (6) and ampk (25) that have been shown to regulate jejunal glucose transporters (20). This is sustained by the report that luminal epidermal growth factor increases jejunal glucose transport in rabbit through pkc activity (31). We found that chelerythrine, an inhibitor of pkc, blocked relm- stimulation of glucose uptake . The effect of relm- is accompanied by an increase in the amount of pkc ii at the bbms . The activation of pkc has been shown to inhibit sglt-1 mediated transport of hexoses (14,32). In oocytes expressing rat and rabbit sglt-1, the activation of pkc decreases the maximum rate of transport for both isoforms . This change is accompanied by proportional change in the number of sglt-1 molecules at the plasma membrane, indicating that pkc regulates endocytosis of the vesicles containing the transporter (33). Further, sglt-1 contains a consensus site of pkc phosphorylation and thus pkc phosphorylation of the transporter could control its activity (34). These data are in line with our results showing that relm- inhibits sglt-1 activity and its translocation to bbms in rat small intestine . The activation of ampk has been shown to downregulate sglt-1dependent glucose transport but also to enhance glut2 translocation to the apical membrane of the jejunum (20). This effect of ampk leads to an increased glucose uptake in jejunum (20) as well as in muscle (35). Interestingly, several hormones have been shown to regulate ampk in a strictly tissue - specific manner (36). We demonstrate that the compound c blocked relm- stimulation of glucose transport in rat intestine suggesting the involvement of ampk . Humans and rodents express two isoforms of the catalytic subunit (1, 2) that form the heterotrimeric complex ampk (,, and) known to serve as a regulator of energy balance (37). We showed that relm- could increase the phosphorylation of the conserved threonine residue (thr-172) of 1 in the jejunal mucosa that is crucial for the ampk activity . As evoked above, activated pkc decreases the number of sglt-1 transporters but can also activate the translocation of glut2 to bbms as described elsewhere (23). Thus, luminal relm- can directly enhance glucose transport by mustering glut2 at bbms through pkc and ampk activation . This inverse regulation of sglt-1 and glut2 by luminal relm- may be important when enterocytes require energy as shown in stress - induced pathology (38). The energy sensor molecule ampk as well as pkc can increase the glut2 energy - independent pathway to override that of sglt-1 that requires energy (20,32). In agreement with this concept, the sglt-1-mediated absorption of nutrients such as galactose and glucose is decreased during either systemic (39) or intestinal inflammation (40). Further, to meet the increased metabolic demand of inflamed tissue, it has been shown that proinflammatory cytokines (interleukin [il]-8, il-6, and il-1) can increase jejunal absorption of glucose without changes in bbm sglt-1 content (41). An increased expression of relm- has been described in the intestine during jejunal inflammation (19,42,43). The expression of relm- is increased by several proinflammatory cytokines and by lipopolysaccharide (9,21) and they may act together to modulate intestinal glucose absorption . Thus, the enhanced glucose absorption by relm- in response to inflammatory stimulus may contribute to the associated energy demand . Obese and insulin - resistant rodent models that are characterized by a low - grade inflammation are associated with an increased expression of the gut - derived relm- (8). Expression of relm- has been shown in rat (18) and mice (19) proximal intestine and the peptide may act locally in a paracrine manner or as a circulating hormone linking the gut to the liver . When given by gavage, it is possible that relm- could be partly absorbed by the small intestine to reach the blood as demonstrated for leptin (44). In favor of a hormonal effect, the infusion of intestinal relm- in mice has been shown to promote a marked increase in the rate of hepatic glucose production (7). We observed that acute relm- administration in the intestinal lumen of rodents resulted in an increased glycemia in ogtt . This could result from the observed increased intestinal glucose absorption and an acute hepatic insulin resistance (7). This is in line with a local intestinal and distant action of this gut peptide . As to whether relm- may also counteract the described insulin inhibition of intestinal sugar absorption remains to be established (45). There may be different relm- thresholds in insulin target tissues as described for resistin (46). A better understanding of relm- action on different tissues could emerge if putative relm- receptors were identified . In this context, the effect of relm- may even involve rapid neuronal activation because relm family members relm- (47) and relm- (4) can bind to neurons . Other than short - regulation, relm- can have a chronic effect as shown in diet - induced metabolic disorders (25). Transgenic mice over - expressing circulating relm- exhibited significant hyperglycemia, hyperlipedemia when fed on high - fat diet . In conclusion further studies may reveal if this process in conjunction with an adverse nutritional and inflammatory status, can participate in the onset of diabetes.
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Resections for pancreatic adenocarcinoma which whipple et al . Initiated after earlier attempts by codivilla and kausch have now been carried out for 80 years . Some authors claim a survival percentage of up to 22% and are widely quoted and extol the benefits and success of resections while others doubt that anyone survives pancreatic cancer . The incidence of pancreatic cancer has been estimated at 48,960 in 2015 in the us and is the fourth leading cause of death from cancer for both sexes . It is projected to increase to 62,000 in the year 2020 and to 88,000 for both sexes in 2030 and to surpass breast, prostate, and colorectal cancers to become the second and third leading causes of cancer - related deaths by 2030 . O'neill and colleagues studied the total direct medical cost of patients 66 years and older who were diagnosed from 2000 to 2007 in the us . The mean total direct cost was $65,500, for resectable locoregional disease cost was $134,700, and for unresectable locoregional or distant disease cost was $65,300 and $49,000, respectively . Lea and stahlgren had earlier pointed out the difference in the cost of resections versus bypass . Assuming 2.5% inflation over 8 years, the mean cost in 2015 would be $79,800, for resections $164,100, for unresectable (or bypasses) disease $79,500, and for distant disease $59,700 . With the estimated number of patients in 2015 there is a growing concern that reports of success in medical research are inflated . Here an attempt is made to evaluate the results over the last 80 years, the number of survivors, and the overall survival percentage . This author has continued to scrutinize the literature on surgery from the onset, initially using the index medicus and then ovid / pubmed until the end of 2014, with cross - references . Approximately 1230 papers have been found in 15 languages in approximately 200 journals from 44 countries which deal with resections and reveal at least some survival information . A total of 40.5% of the papers originated in the us, 19.3% from japan, 7.3% from germany, 5.3% from italy, and 4% from france and the uk . Papers on the surgical aspects of pancreatic cancer differ as to the approach and the composition of the patient group and the method of reporting . A few emphasize only the technical aspects and the mortality with limited or no survival information and indiscriminately cover patients with various malignant and benign pathologies which may require pancreatoduodenal resection, but without clearly separating each pathology group or presenting separate survival information . Only papers with separate pathologic information on patients were selected for analysis for this paper . Analysis of the database reveals that, of these 1230 papers, 609 do not report any 5-year survivors, some seem to be mainly technical, and some report only up to a 3-year survival rate . A total of 621 papers report 5-year survivors and will be examined further in detail in this paper . Special attention has been paid to the origin of each paper, the time period each study covered, patient composition, the subset of patients used for calculations, and the statistical method used . The first reservation about the effect of surgery on this disease was expressed by glenn and thorbjarnarson in 1964, again by gallitano et al . In 1968 whose only 5-year survivor was nonresected, and then strongly by crile jr . In 1970 he challenged the value of resection for pancreatic cancer, followed by shapiro in 1975 . 's criticism was directed at the then high mortality rate and the survival calculations which were carried out and might count only those who survived the operation and in ignorance of the nonresected survivors . The presence of nonresected survivors has been disputed, but it is a major issue in the debate on survival . It was first pointed out by cattell and young in 1957 and, as above, later by gallitano and crile jr . The data were summarized by the present author in a paper in 1995 and a letter in 1996 . In a review by the present author published in 1978 only 65 five - year survivors could be found in the literature, of whom 8 were nonresected . In a review published in 1987, 165 survivors could be found, but 12 of these were nonresected . In this review 41 reports have been found from 31 institutions in 12 countries, many from eminent institutions and renowned authors, thereof 17 from the us, two from yale [19, 20], two from the university texas md anderson [12, 21], and two from harvard mgh [22, 23], as well as from the university of chicago, the dana farber cancer clinic, and thomas jefferson, to mention a few . Nonresected survival is a fact and should be kept in mind in assessing overall therapeutic results . Initially reports detailed the course of all patients diagnosed at a particular institution but in recent decades reports have concentrated only on resected patients, completely ignoring any nonresected survivors . The survival percentage depends not only on the number of survivors but on the subset from which the number is calculated . A few earlier studies started by examining the respective tumor registries and disclosed that only about 3568% of patients in tumor registries had histologic confirmation . Survival calculations have been based on the original number of patients with histologic diagnoses at a particular institution, previously called the total number (tn), the approximately 80% of cases that were surgically explored, the cases that were resected, or location, size, or r status of the tumor, or even only those patients who survived the operation . Overall survival success must be based on the original group diagnosed with pancreatic cancer (the tn or total number) and the number of survivors and not only on a small subgroup of the cases . Different methods of calculation have been used to enumerate the results, that is, actual versus the actuarial, projected, or estimated percentage . Initially most papers revealed the tn, the number of resections, and simply the number of survivors, whereas later authors also presented actual percentage figures . In the late 1980s the papers started reporting only the number of resections and survival as actuarial percentages, usually calculated with the kaplan - meier method with or without the actual number of survivors being reported . Sir hill pointed out in his book in 1937 that when a large number of patients is lost sight of the outcome might be erroneously high . As indicated in table 1 the original number tn of patients studied in a report is only revealed in 90 or 14.5% of the papers and in these the actual number of survivors is stated in only 49 . In the remaining 41 with a documented tn and actuarial calculation the actual number of survivors is stated in 17, in addition to life table curves . Detailed information on the original tn group studied, number of resections, and actual number of survivors is therefore reported in only 66 or 10.6% of all papers on pancreatic cancer . In the remaining 89.4% some form of estimate or calculation there is no information on the original number of patients from which the number of resections was drawn, although in 102 of these the number of survivors is stated or confirmed by inquiry . In 424 of these 531 reports with survival calculations by actuarial methods 378 are by the kaplan - meier method and 48 by other or unclear methods, though km is also very likely . The number of survivors is stated in 147 of the reports or 34.6%, but not in the remaining 277 or 65.3% . A total of 240 inquiries were sent to authors where the actual number of patients was not reported and only 58 replies were obtained . The actual number of survivors with actuarial calculation is therefore known in 205 of the 424 reports or 48.3% . The actuarial and actual percentage figures can therefore be compared, as demonstrated previously [29, 30], and reveal that the actuarial percentage is on average 2.75 higher than the actual percentage . This figure has therefore been used to estimate the number of survivors and the survival percentage in the relevant studies where only the actuarial percentage has been published . The resection rate has been debatable and varies and can only be assessed accurately if the original group is large and well defined . Tertiary referral centers cannot know the size of the original group from which their resection group is drawn . Of the studies published in the last 5 years, 156 of 161 or 97% report only the number of resections and the percentages . In an earlier study by this author the resection rate was 10.8% . In earlier us studies in 2 european nationals [33, 34] the rate was from 8% to 12% over the last 5 years . In a recent report from the surgical service at a european university hospital the resection rate was 11.6% . It is therefore practical to assume that the resection rate is 10% in the studies where the original tn of the group is not reported in order to estimate the tn accordingly and divide the percentage by 2.53 where the actuarial km only has been published . After totaling the numbers in the 621 studies with survivors with the above correction, but without further adjustment, the tn comes to 1,731,834, the number of resected patients comes to 162,207, and the number of survivors comes to 11,300, for an apparent survival percentage of 0.77% . After totaling the number of patients in all the 1230 reports, the original tn comes to 3,188,543, the number of resected patients to 284,298, and the number of survivors to 11,330 . Repetition of reporting the same survivors in different papers was first pointed out in 1978 . It occurs in various ways, such as when papers include survivors from many different institutions or known databases in a specific country or even when a study includes patients from many countries . Thus 92 of the 620 studies with 5-year survivors are from many institutions in a specific country or 14.8% and 10 of these from many countries or 1.6% . Repetition occurs, though mainly when the patient population and survivors from a certain year are reported several times from the same institution . As can be seen in figures 1 5, repetition has occurred up to 68 times in germany, italy, and japan and up to 20 times in the us . Examination of reports from a single institution covering the entire study period and stating the number of survivors and then adding up the number of patients from all the studies, including those with an estimated number of survivors, reveals that the total number reported is over 10 times larger than the number reported in the studies with a documented number of survivors . Each paper may at times reveal some new information but only infrequently is it disclosed that the patients have been reported before . There is no scientific method to assess the number of repetitions accurately but each reported 5-year survivor and thereby respective resection and the tn seems to be reported 35 times . Dividing the number of reported survivors and respective resections and tn by 4, the overall number of 5-year survivors is hardly more than 2,800, the number of resections 40,500, and the original tn number of patients 433,000 . Repetitions occur also in the no - survivor group of reports, but not as frequently . It may be assumed that all published reports with or without survivors are drawn from a tn of approx . 1,000,000 patients and with fewer than 3,000 survivors, of whom a significant number were nonresected, meaning that the overall survival rate was no more than approximately 0.3% . Mortality during the first 20 years, 19451965, was on average 25.2% with a single report of 62.5% . During the next 20 years or up to 1984 was reduced to 6.8% and then 4.6% and during the last 5 years 4% with a high of 33% . Aside from the 33%, the average is now 3.7% . The majority of surgeons in recent decades have reported the number of positive margins and nodes and numbers over 6070% frequently quoted [3638]. It is of great interest that even in the most experienced hands only 16% of cases were both margins and nodes negative . Tumor cells can be found in the bone marrow in up to 50% of cases . Pancreatic cancer is thus both a costly and devastating disease and has usually spread beyond its boundaries at time of diagnosis and treatment and is thus a systemic disease . The literature on pancreatic surgery, the use of actuarial calculation methods exaggerates the percentage and thereby the number of presumed survivors in a particular study . Surgical skills are imperative for the care and palliation of pancreatic cancer patients including possible resections, but they have had only a minimal impact on the survival rate.
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This was a prospective non - randomized single - centre study of 66 eyes of 50 patients with progressive keratoconus seen at the cornea services of a tertiary eye care centre between january and december 2010 . Progression in keratoconus was defined as an increase of> 1.0 d of refractive astigmatism or an increase of> 1.5 d in average k by keratometry / topography or a decrease of> 5% in central corneal thickness within the previous 1 year . Patients with documented progression in keratoconus and with a minimum corneal thickness of 400 at the thinnest point were included in the study . Data collected during the 3 months to 6 months post - op follow - up visits were used for statistical analysis . Eyes with corneal thickness less than 400 at the thinnest point on the cornea, eyes with corneal scars or evidence of previous hydrops, pregnant women and lactating mothers were excluded from the study . Eyes with a minimum corneal thickness of more than 450, with clear mires on the topolyzer, which covered more than 50% of the corneal area were selected for the combined simultaneous t - cat + cxl procedure . This study received approval by the institutional review board and ethics committee and adhered to the tenets of the declaration of helsinki . All patients underwent retinoscopy, measurement of their best spectacle corrected visual acuity (bscva) on the early treatment diabetic retinopathy study (etdrs) chart, slit lamp biomicroscopy, goldmann applanation tonometry, dilated fundus examination, corneal topography with tms iv (tomey inc) and topolyzer (oculus inc), corneal tomography (pentacam hr, oculus inc) pre - operatively and again post - operatively at all follow - up visits . The keratoconus was classified as mild, moderate or severe, using the criteria adopted in the collaborative longitudinal evaluation of keratoconus study . The pattern of keratconus was classified as central / global, localized steepening and symmetric or asymmetric bowtie . It was conducted in sterile conditions under topical anaesthesia of 0.5% proparacaine (sunways [india] pvt . Ltd . ). A wire speculum was used to separate the lids and the central 9 mm of the cornea was debrided of its epithelium . Riboflavin 0.1% solution (10 mg riboflavin5-phosphate in 10 ml of 20% dextran - t 500) was instilled every 3 min for 30 min to photosensitize the cornea . After confirming the presence of riboflavin in the anterior chamber using the slit lamp with a blue filter, the cornea was exposed to ultraviolet - a (uva) irradiation (uv - x system, peschke meditrade gmbm, switzerland) of 370 nm wavelength and an irradiance of 3 mw / cm for 30 min . A trans - epithelial t - cat was performed in a manner similar to the athens protocol . In brief, it consists of importing the raw topographic data captured by the topolyzer . Only maps with clear mires, where> 50% of the corneal surface was analysed without extrapolated data and those which imaged the pupil fully were accepted . The allegretto excimer laser (400 hz eyeq, wavelight, erlangen) which has a customized platform for t - cat, has proprietary software that averages the imported topographic data and allows the surgeon to adjust the optical zone of treatment, the asphericity, tilt correction and refractive error . An optical zone of 5.5 mm with asphericity and refractive error kept at zero with no tilt correction was chosen in all cases in the study . Immediately following the t - cat, following cxl or t - cat with cxl, a bandage contact lens was placed on the cornea and the patient was put on topical ciprofloxacin eye drops 4 times a day for 1 week and prednisolone acetate 1% (pred forte, allergan) 4 times a day for 1 week and tapered over 1 month . The outcome measures in this study included retinoscopic and subjective refraction, bscva, the simulated k and steepest k from the corneal topography, the sag factor [the maximum dioptre difference between the peak of the cone and a point that was as equidistant and equiangular as the peak was from the centre of the cornea, depicted diagrammatically in fig 1], the area of the cone (using the mat lab version 7.03 a) the base of the cone (the dioptre power of the mean curvature of the cornea, determined by the mid - value in the normalized scale), the smolek - klyce and klyce - maeda indices of clinical similarity and pattern severity, the topographic indices of surface regularity and asymmetry (sri and sai) and keratoconus prediction index (kpi) and corneal aberrations at 6 mm . Diagrammatic representation of sag factor statistical analyses were performed with ms excel and spss14 . Non - parametric mean comparisons were done using mann - whitney u test . Patients with documented progression in keratoconus and with a minimum corneal thickness of 400 at the thinnest point were included in the study . Data collected during the 3 months to 6 months post - op follow - up visits were used for statistical analysis . Eyes with corneal thickness less than 400 at the thinnest point on the cornea, eyes with corneal scars or evidence of previous hydrops, pregnant women and lactating mothers were excluded from the study . Eyes with a minimum corneal thickness of more than 450, with clear mires on the topolyzer, which covered more than 50% of the corneal area were selected for the combined simultaneous t - cat + cxl procedure . This study received approval by the institutional review board and ethics committee and adhered to the tenets of the declaration of helsinki . All patients underwent retinoscopy, measurement of their best spectacle corrected visual acuity (bscva) on the early treatment diabetic retinopathy study (etdrs) chart, slit lamp biomicroscopy, goldmann applanation tonometry, dilated fundus examination, corneal topography with tms iv (tomey inc) and topolyzer (oculus inc), corneal tomography (pentacam hr, oculus inc) pre - operatively and again post - operatively at all follow - up visits . The keratoconus was classified as mild, moderate or severe, using the criteria adopted in the collaborative longitudinal evaluation of keratoconus study . The pattern of keratconus was classified as central / global, localized steepening and symmetric or asymmetric bowtie . It was conducted in sterile conditions under topical anaesthesia of 0.5% proparacaine (sunways [india] pvt . Ltd . ). A wire speculum was used to separate the lids and the central 9 mm of the cornea was debrided of its epithelium . Riboflavin 0.1% solution (10 mg riboflavin5-phosphate in 10 ml of 20% dextran - t 500) was instilled every 3 min for 30 min to photosensitize the cornea . After confirming the presence of riboflavin in the anterior chamber using the slit lamp with a blue filter, the cornea was exposed to ultraviolet - a (uva) irradiation (uv - x system, peschke meditrade gmbm, switzerland) of 370 nm wavelength and an irradiance of 3 mw / cm for 30 min . A trans - epithelial t - cat was performed in a manner similar to the athens protocol . In brief only maps with clear mires, where> 50% of the corneal surface was analysed without extrapolated data and those which imaged the pupil fully were accepted . The allegretto excimer laser (400 hz eyeq, wavelight, erlangen) which has a customized platform for t - cat, has proprietary software that averages the imported topographic data and allows the surgeon to adjust the optical zone of treatment, the asphericity, tilt correction and refractive error . An optical zone of 5.5 mm with asphericity and refractive error kept at zero with no tilt correction was chosen in all cases in the study . Immediately following the t - cat, following cxl or t - cat with cxl, a bandage contact lens was placed on the cornea and the patient was put on topical ciprofloxacin eye drops 4 times a day for 1 week and prednisolone acetate 1% (pred forte, allergan) 4 times a day for 1 week and tapered over 1 month . The outcome measures in this study included retinoscopic and subjective refraction, bscva, the simulated k and steepest k from the corneal topography, the sag factor [the maximum dioptre difference between the peak of the cone and a point that was as equidistant and equiangular as the peak was from the centre of the cornea, depicted diagrammatically in fig 1], the area of the cone (using the mat lab version 7.03 a) the base of the cone (the dioptre power of the mean curvature of the cornea, determined by the mid - value in the normalized scale), the smolek - klyce and klyce - maeda indices of clinical similarity and pattern severity, the topographic indices of surface regularity and asymmetry (sri and sai) and keratoconus prediction index (kpi) and corneal aberrations at 6 mm . A trans - epithelial t - cat was performed in a manner similar to the athens protocol . In brief, it consists of importing the raw topographic data captured by the topolyzer . Only maps with clear mires, where> 50% of the corneal surface was analysed without extrapolated data and those which imaged the pupil fully were accepted . The allegretto excimer laser (400 hz eyeq, wavelight, erlangen) which has a customized platform for t - cat, has proprietary software that averages the imported topographic data and allows the surgeon to adjust the optical zone of treatment, the asphericity, tilt correction and refractive error . An optical zone of 5.5 mm with asphericity and refractive error kept at zero with no tilt correction was chosen in all cases in the study . Immediately following the t - cat, following cxl or t - cat with cxl, a bandage contact lens was placed on the cornea and the patient was put on topical ciprofloxacin eye drops 4 times a day for 1 week and prednisolone acetate 1% (pred forte, allergan) 4 times a day for 1 week and tapered over 1 month . The outcome measures in this study included retinoscopic and subjective refraction, bscva, the simulated k and steepest k from the corneal topography, the sag factor [the maximum dioptre difference between the peak of the cone and a point that was as equidistant and equiangular as the peak was from the centre of the cornea, depicted diagrammatically in fig 1], the area of the cone (using the mat lab version 7.03 a) the base of the cone (the dioptre power of the mean curvature of the cornea, determined by the mid - value in the normalized scale), the smolek - klyce and klyce - maeda indices of clinical similarity and pattern severity, the topographic indices of surface regularity and asymmetry (sri and sai) and keratoconus prediction index (kpi) and corneal aberrations at 6 mm . A total of 66 eyes of 50 patients were included in this study . Of these, 40 eyes of 27 patients underwent cxl alone, while 26 eyes of 23 patients underwent simultaneous t - cat + cxl . The mean follow - up was 7.7 1.3 months (3 - 16.5 months). The pre - operative parameters in each group and the statistical analysis of their differences are given in tables 1 and 2 . The cxl group consisted of eyes with more severe grade of keratoconus, steeper mean k, higher mean posterior corneal elevation and thinner mean corneal pachymetry than the eyes in the t - cat + cxl group . The mean values of other parameters like retinoscopic cylinder, spherical equivalent, sag factor, area of cone, sai were comparable in both groups . Table 3 summarizes the changes in refractive, visual and topographic parameters in each group and the statistical difference between the two groups . Preoperative demographic and refractive parameters in each group and the statistical differences between them pre - operative topographic and tomographic parameters in each group and the statistical differences between them changes in refractive, visual and topographic parameters following treatment in each group and statistical differences within and between each group the retinoscopic cylinder decreased by 1.02 d 3.16 d in the cxl group (p = 0.11) and by 2.87 3.22 d in the t - cat + cxl group (p = 0.039). The difference between the 2 groups was also statistically significant (p = 0.026). The mean spherical equivalent (se) decreased by 0.44 + 2.16 d in the cxl group (p = 0.65) and by 2.17 2.68 d in the t - cat + cxl group (p = 0.21). The difference between the 2 groups was statistically significant (p 0.001) the bscva expressed in logarithm of minimum angle of resolution (log mar) improved by a mean of 0.06 0.23 in the cxl group and by a mean of 0.09 0.18 in the t - cat + cxl group . The change in bscva after treatment in each group and between the 2 groups were not statistically significant . However, 4 of 40 eyes (10%) in the cxl group and 6 of 26 eyes (23.3%) in the t - cat + cxl group showed an increase of 2 lines or more in bscva following treatment . The steepest k reduced by mean of 0.40 3.71 d (p = 0.77) in the cxl group and by a mean of 2.9 2.0 d (p 0.03) in the t - cat cxl group . The change in k between the 2 groups was also statistically significant (p = 0.005). The steepest k reduced by> 2 d in 17.5% of eyes in the cxl group and in 33.3% of eyes in the t - cat + cxl group . The base of the cone flattened by a mean of 1.39 13.41 d in the cxl group (p = 0.86) and by a mean of 4.94 11.46 d in the t - cat + cxl group (p = 0.05). The sag factor and surface asymmetry index showed no significant change in the cxl group but reduced significantly by 3.59 5.94 d (p = 0.01) and 0.72 1.18 (p = 0.02) respectively in the t - cat + cxl group . There was an increase in the area of the cone in both groups, more so in the t - cat + cxl group, although these changes were not statistically significant . Table 4 summarizes the changes in the keratoconus indices: the klyce maeda index for pattern similarly, the keratoconus index (kci), the keratoconus severity index and the kpi showed no significant changes in the cxl group but reduced significantly in the t - cat + cxl group (p <0.005 in all). Changes in topographic keratoconus indices before and after treatment and their statistical differences in each group the corneal pachymetry at the thinnest point, as measured by the pentacam, showed a reduction following treatment in both groups . Although the reduction from the pre - operative values was not statistically significant in either group, the change in thickness was significantly more in the t - cat + cxl group than in the cxl group (p = 0.007). There was a significant decrease in the highest anterior evaluation and a significant increase in highest posterior elevation following cxl (p <0.001 in both). There was a significant increase in highest anterior and posterior corneal elevation following t - cat + cxl (p <0.001 in both). Table 5 shows the pre - operative and post - operative mean corneal aberrations for coma, spherical aberration and the root mean square of total higher order aberrations (rmsh) in both groups . There was a significant reduction in the mean coma and rmsh and a significant increase in the mean spherical aberration in the t - cat + cxl group . There were no significant changes in the mean coma, spherical aberration or rmsh in the cxl group . Corneal aberrations (6 mm dia) in each group and the statistical differences between them fig . 2a and b show the change in the distribution of patterns of cones in each group . There was a marked tendency for greater symmetrization in the t - cat + cxl group . There was a perceptible trend for many of the eyes with an asymmetric bowtie prior to treatment to appear more symmetric following t - cat + cxl . In the cxl group, on the other hand, the pattern of cone did not appear to change appreciably . Fig 3 shows an example of two eyes with similar pre - operative pattern and severity of keratoconus . (a) bar diagram showing change in pattern of cone in collagen cross linking, (b) bar diagram showing change in pattern of cone in t - customized ablation treatment + collagen cross linking pre - operative and 6 months post - operative corneal topography of two topographically similar eyes, one of which underwent collagen cross linking (cxl) alone and the other t - customized ablation treatment + cxl parameters like age of the patient, duration of disease, severity and pattern of keratoconus were considered to calculate the odds ratio for symmetrization of the cone and flattening of the cornea . An asymmetric pattern had an odds ratio of 2.62 for symmetrization of the cone in the t - cat + cxl group . A mean k of <52 d (mild severity) had an odds ratio of 3.54 for flattening in the t - cat + cxl group, and an odds ratio of 1.25 for flattening in the cxl group . The retinoscopic cylinder decreased by 1.02 d 3.16 d in the cxl group (p = 0.11) and by 2.87 3.22 d in the t - cat + cxl group (p = 0.039). The difference between the 2 groups was also statistically significant (p = 0.026). The mean spherical equivalent (se) decreased by 0.44 + 2.16 d in the cxl group (p = 0.65) and by 2.17 2.68 d in the t - cat + cxl group (p = 0.21). The difference between the 2 groups was statistically significant (p 0.001) the bscva expressed in logarithm of minimum angle of resolution (log mar) improved by a mean of 0.06 0.23 in the cxl group and by a mean of 0.09 0.18 in the t - cat + cxl group . The change in bscva after treatment in each group and between the 2 groups were not statistically significant . However, 4 of 40 eyes (10%) in the cxl group and 6 of 26 eyes (23.3%) in the t - cat + cxl group showed an increase of 2 lines or more in bscva following treatment . The steepest k reduced by mean of 0.40 3.71 d (p = 0.77) in the cxl group and by a mean of 2.9 2.0 d (p 0.03) in the t - cat cxl group . The change in k between the 2 groups was also statistically significant (p = 0.005). The steepest k reduced by> 2 d in 17.5% of eyes in the cxl group and in 33.3% of eyes in the t - cat + cxl group . The base of the cone flattened by a mean of 1.39 13.41 d in the cxl group (p = 0.86) and by a mean of 4.94 11.46 d in the t - cat + cxl group (p = 0.05). The sag factor and surface asymmetry index showed no significant change in the cxl group but reduced significantly by 3.59 5.94 d (p = 0.01) and 0.72 1.18 (p = 0.02) respectively in the t - cat + cxl group . There was an increase in the area of the cone in both groups, more so in the t - cat + cxl group, although these changes were not statistically significant . Table 4 summarizes the changes in the keratoconus indices: the klyce maeda index for pattern similarly, the keratoconus index (kci), the keratoconus severity index and the kpi showed no significant changes in the cxl group but reduced significantly in the t - cat + cxl group (p <0.005 in all). Changes in topographic keratoconus indices before and after treatment and their statistical differences in each group the corneal pachymetry at the thinnest point, as measured by the pentacam, showed a reduction following treatment in both groups . Although the reduction from the pre - operative values was not statistically significant in either group, the change in thickness was significantly more in the t - cat + cxl group than in the cxl group (p = 0.007). There was a significant decrease in the highest anterior evaluation and a significant increase in highest posterior elevation following cxl (p <0.001 in both). There was a significant increase in highest anterior and posterior corneal elevation following t - cat + cxl (p <0.001 in both). Table 5 shows the pre - operative and post - operative mean corneal aberrations for coma, spherical aberration and the root mean square of total higher order aberrations (rmsh) in both groups . There was a significant reduction in the mean coma and rmsh and a significant increase in the mean spherical aberration in the t - cat + cxl group . There were no significant changes in the mean coma, spherical aberration or rmsh in the cxl group . Corneal aberrations (6 mm dia) in each group and the statistical differences between them fig . 2a and b show the change in the distribution of patterns of cones in each group . There was a marked tendency for greater symmetrization in the t - cat + cxl group . There was a perceptible trend for many of the eyes with an asymmetric bowtie prior to treatment to appear more symmetric following t - cat + cxl . In the cxl group, on the other hand, the pattern of cone did not appear to change appreciably . Fig 3 shows an example of two eyes with similar pre - operative pattern and severity of keratoconus . (a) bar diagram showing change in pattern of cone in collagen cross linking, (b) bar diagram showing change in pattern of cone in t - customized ablation treatment + collagen cross linking pre - operative and 6 months post - operative corneal topography of two topographically similar eyes, one of which underwent collagen cross linking (cxl) alone and the other t - customized ablation treatment + cxl parameters like age of the patient, duration of disease, severity and pattern of keratoconus were considered to calculate the odds ratio for symmetrization of the cone and flattening of the cornea . An asymmetric pattern had an odds ratio of 2.62 for symmetrization of the cone in the t - cat + cxl group . A mean k of <52 d (mild severity) had an odds ratio of 3.54 for flattening in the t - cat + cxl group, and an odds ratio of 1.25 for flattening in the cxl group . Over the last decade, several clinical studies have confirmed the efficacy of the cxl procedure in arresting the progression of keratoconus . However, cxl alone does not reduce the irregularity in corneal shape . The visually incapacitating symptoms due to irregular astigmatism and high corneal aberrations, are therefore likely to persist . A few attempts have been made to combine cxl with other procedures that could reduce irregular astigmatism . Intrastromal corneal ring segments have been proposed as one such technique, combined with cxl . This approach has been popularized by kanellopoulos et al ., who after initial attempts with a sequential technique, showed that a simultaneous t - cat + cxl was the preferred approach to regularize and stabilize the cornea in keratoconus . This simultaneous, combined procedure has been used in progressive keratoconus, pellucid marginal degeneration and post - lasik keratectasia with encouraging results . There has, however, been no published study that compares the results of cxl alone with those of simultaneous, combined t - cat + cxl . Our results clearly indicate that a combined t - cat + cxl resulted in better refractive, topographic and aberrometric outcomes than cxl alone . The reduction in refractive cylinder and in the steepest topographic k was significantly more in the t - cat + cxl group than in the cxl group . The change in topographic patterns in the 2 groups reflected the same results; the trend towards symmetrization of the asymmetric bowtie patterns in the t - cat + cxl group was significant in contrast to the relatively changeless topographic pattern in the cxl group . The significantly greater decrease in the sag factor is a quantitative indicator of the symmetrization being more effective in the t - cat + cxl group . The topographic indices of regularity and asymmetry (sri and sai), particularly the latter, were also significantly better after the combined procedure . The mechanism of topography - guided ablation is the fitting of a best - fit - sphere under the patients topography map with ablation of tissues in between . This would flatten some of the cone apex and explains why these eyes showed more flattening than eyes treated with cxl alone . In addition, the proprietary t - cat software of the wavelight allegretto excimer laser also incorporates a partial hyperopia - like ablation pattern that results in steepening of the cornea adjacent to the base of the cone . It is this combination of flattening of the steep portion and steepening of the flat portion of the cornea that results in the symmetrization seen in topography and a reduction of the refractive cylinder as well as a reduction in the hoa . The reduction in total corneal hoa, particularly in the coma (z3) was statistically significantly more in the t - cat + cxl group than in the cxl group . This would be expected to improve the quality of vision and reduce the annoying optical phenomena like haloes and shadowing of images often associated with irregular astigmatism and coma . Cxl alone showed a negligible reduction in rmsh and coma, which were not statistically significant, in agreement with the findings of vinciguerra et al . T - cat + cxl did, however, cause a significant increase in the mean spherical aberration . It is interesting to note that the zernike coefficient of spherical aberration is usually negative in keratoconic eyes, for it is the result of an increased prolateness shape factor in these eyes, especially in eyes with central cones . With localized paracentral cones, as also in eyes with asymmetric bowtie pattern of cones, there is an area of relative flattening in the sector opposite the cone that partially compensates for the increased prolateness due to the cone . In t - cat, there is a relative steepening of that flat portion, resulting in an overall increase in prolateness, and therefore in the negative zernike coefficient of spherical aberration . Our study also demonstrated a slight increase in the area occupied by the cone - more so in the t - cat + cxl group, where the increase approached statistical significance (p = 0.06). This broadening of the base of the cone may perhaps be the biomechanical response to the flattening, which was more in the t - cat + cxl group . A flatter and broader cone, as suggested by kanellopoulos may redistribute the biomechanical strain from forces like the intraocular pressure, or eye rubbing, etc . Although the change in corneal pachymetry was not statistically significant with either procedure, t - cat + cxl resulted in significantly more thinning than cxl alone . The combined t - cat + cxl procedure does involve some ablation over the already thin cone and could be a cause for concern . By choosing eyes with a minimum corneal thickness of 450 rather than 400 (which is recommended for cxl), by limiting the ablation to no more than 50 (arbitrarily chosen by kanellopoulos) and by performing a simultaneous cxl procedure, it is hoped that the combined procedure will not destabilize the biomechanical integrity of the cornea . This emphasizes the need for these eyes to be monitored closely in the post - operative period . Although all the eyes in both groups had a minimum corneal thickness of 400, the cxl group consisted of eyes with more severe forms of the disease, and had a significantly worse pre - operative bscva than the t - cat + cxl group . Had the two groups been more similar with respect to the severity of disease and had the treatment been randomized, the scientific credibility of the results would have been more convincing . A longer follow - up of both groups would also be necessary to confirm biomechanical stability . When programming the t - cat, the choice of keeping the target asphericity and refractive correction at zero and the maximum ablation to less than 50 was arbitrary . More data with variations in these parameters are required to understand the effect of those variations and to be able to make recommendations, if any, for suitable changes in the existing protocol . Although our study did suggest that the t - cat + cxl resulted in flatter, smoother and more symmetric corneal contour than cxl alone, with more number of eyes showing an increase of 2 lines or more in bscva we could not demonstrate any overall improvement in bscva . A questionnaire, a measurement of contrast sensitivity and the inclusion of psychophysical tests of quality of vision may have borne out any differences between the two groups that the high contrast snellen visual acuity may not have measured . When the tenets of the dresden protocol are not violated, the cxl procedure is believed to pose no threat to the corneal endothelium . Studies have also confirmed that the corneal endothelium cell loss is not significant at 12 months after cxl . Long - term studies on endothelial cell counts with both techniques are warranted, even though there are no published reports of irreversible endothelial failure complicating cxl so far . Our study suggests that a combined t - cat + cxl is an effective approach to not only biomechanically stabilize the cornea in keratoconus, but also to improve the corneal contour, reduce irregular astigmatism and offer a better quality of vision than cxl alone could do . However, long - term followup is needed to establish the safety of this combined procedure and to justify its rationale.
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Pregnancy and the postpartum can be times of joy and positive expectations but also of stress and difficulties . Pregnancy and delivery bring many physiological and psychosocial changes, and both mothers and fathers are required to face several new challenges during this period . Consequently, pregnancy and the post partum are times of increased vulnerability for the onset or relapse of a mental illness (smith et al ., 2011). Depression and anxiety are the most common psychiatric disorders during pregnancy and the post partum (alipour et al however, we still do not know why some women are more at risk of developing depression or anxiety symptoms while others remain resilient even in the face of adversity . The prevalence of antenatal depression is estimated to be between 7% and 20% in high- income countries (andersson et al ., 2003, evans et al ., 2001, lee et al ., 2007, marcus et al ., 2003, melville et al ., 2010), while rates of 20% or more have been reported in low- and middle - income countries, although less research has been conducted in these areas (faisal - cury et al ., 2009, golbasi et al ., 2010, husain et al ., 2012, husain et al ., 2011). Gavin et al . Found that the prevalence of antenatal depression in the first trimester is 11.0%, then drops to 8.5% in the second and third trimesters (gavin et al . In contrast, bennett et al . Found an opposite trend, with a prevalence of 7.4% during the first, 12.8% during the second, and 12% during the third trimester (bennett et al ., 2004). Postpartum depression prevalence is estimated to be between 7% and 30% across low-, middle- and high - income countries (beck, 2001, csatordai et al indeed, a recent review showed that, in 22 of 28 low- and middle - income countries, postnatal depression prevalence was higher than in high - income countries, with the highest values in vietnam (33%), zimbabwe (33%) and guyana (50%), and lowest in uganda (7.1%) and nepal (4.9%) (parsons et al ., 2012). Prevalence of postnatal depression in high - income countries begins to rise after delivery and reaches the highest value in the third month postpartum (12.9%), and then declines to 10.6% at month 7 and to 6.5% after month 7 (gavin et al ., 2005). The prevalence of both classes of disorders tends to be higher when symptoms, rather than disorders, are investigated, or when depression or anxiety is assessed by a self - report rating scale rather than a structured interview, or when operational criteria are not used for the diagnosis (bennett et al ., 2004). In general, the postpartum period has historically been the focus of far greater research attention than the antenatal period, despite the fact that some studies have shown a decrease, rather than an increase, in depression and anxiety after childbirth (heron et al ., 2004). A recent review (norhayati et al ., 2015) has shown that antenatal depression and anxiety are significant risk factors for postnatal depression in both developed and developing countries, together with a previous history of psychiatric illness, poor marital relationship, stressful life events, a negative attitude towards the pregnancy, and lack of social support . The present systematic review will focus on the risk factors for antenatal depression and anxiety . There are a number of reasons why mental health problems in the antenatal period have received much less attention than in the postpartum . For example, there is the misconception that women are hormonally protected from psychological disturbance during pregnancy (bennett et al ., moreover, women can themselves be reluctant to share symptoms of sadness and irritability owing to the stigma associated with depression and to the discrepancy between women's expectation of happiness during pregnancy (and the postpartum period) and their own experience (marcus, 2009). Furthermore, there is a tendency to focus on (maternal and foetal) physical health during pregnancy, rather than mental health, and to misattribute emotional complaints to the physical and hormonal changes that occur during pregnancy (bowen and muhajarine, 2006a). Indeed, these women often present with atypical symptoms of depression and unspecified somatic complaints (posternak and zimmerman, 2001), such as fatigue, loss of energy, appetite and sleep changes, rather than depressed mood . Therefore, it can be difficult to distinguish between normal pregnancy symptoms, which are common during pregnancy, and atypical somatic complaints, which may be related to depression or anxiety (lee et al . This obviously makes it more complicated to diagnose depression and anxiety without a standardized assessment (andersson et al ., for this reason, the most validated and widely used self - report screening tool for depression during the perinatal period, the edinburgh postnatal depression scale (epds), does not include questions about somatic complaints, fatigue and changes in appetite, as these complaints would not help to distinguish depressed from non - depressed women (murray and cox, 1990). Therefore, somatic complaints may lead to the overdiagnosis of depression during the perinatal period . However, it has also been argued that not considering somatic complaints may interfere with the measure of the severity of the illness (yonkers et al ., indeed, most of the women with higher epds scores also present a greater number of somatic complaints (apter et al ., 2013, zelkowitz et al ., 2004). Therefore, there is a risk that clinicians and patients may attribute somatic symptoms to the normal course of the pregnancy and the postpartum period rather than to a depressive disorder (klein and essex, 1994). Diagnosing antenatal depression can also be difficult if women are only screened once throughout pregnancy . In fact, multiple evaluations during pregnancy can show differences in the rates of depression and anxiety . To this end, some studies (bunevicius et al ., 2009, lee et al ., 2007, marchesi et al ., 2009, yanikkerem et al ., 2013) have shown that depressive episodes occur more frequently during the first and third trimester of pregnancy, compared with the second, possibly because the most vulnerable women are more likely to experience stress when they are coping with the new event of becoming mothers, and when they are about to deliver and start a new life (marchesi et al ., 2009). The fact that many women present anxiety or depressive symptoms at one or two time points implies that only one screening is not enough during pregnancy . These circumstances make antenatal depression among the most under - recognized and under - treated conditions (marcus, 2009). This lack of recognition has serious implications, as it is now widely recognized that maternal depression, anxiety and stress during pregnancy have powerful long - term effects on both mother and baby (dunkel schetter and tanner, 2012, glover, 2015). The underlying biological mechanisms have not been fully understood but it has been suggested that a decrease in blood flow to the foetus and/or an increased exposure of the foetus to cortisol may be some of the possible mediating factors . Maternal increased levels of cortisol that have been associated with depression, anxiety and stress, can cross the placenta and be transferred to the foetal environment and affect the developing foetus (glover, 2014). Antenatal depression and anxiety have also been associated with inadequate nutrition and weight gain, increased alcohol consumption, substance abuse and smoking (marcus, 2009); moreover, mothers with antenatal anxiety and depression are more likely to access perinatal services late, to attend antenatal appointments less frequently, and to fail to have regular scans (kim et al ., 2006, redshaw and henderson, 2013). Some studies have found that these women have more visits to the obstetrician, mainly related to the fear of childbirth, and show a preference for an elective caesarean section (andersson et al ., 2004, moreover, depression and anxiety have been linked to stillbirth, premature birth, low birth weight, low apgar scores, smaller head circumference and major congenital anomalies (marcus, 2009, raisanen et al ., 2014) as well as altered developmental trajectories as shown by a reduced score on the brazelton neonatal behavioural assessment scale, a more difficult temperament, and an increased risk of emotional problems (especially anxiety and depression), impaired cognitive development, and symptoms of attention deficit hyperactivity disorder and conduct disorder during childhood (glover, 2014, o'connor et al ., 2002, previti et al ., 2014). These children are also at increased risk of impulsivity and cognitive disorders at 14 and 15 years old (van den bergh et al ., 2005), and at increased risk of encountering adverse life events and of developing depression in adolescence and adulthood (pariante, 2014, pawlby et al ., 2011, plant et al ., 2015, stein et al ., antenatal depression has also been recognized as the strongest predictor of postnatal depression, and postnatal depression is the strongest predictor of parenting stress and difficulties in the mother infant relationship (leigh and milgrom, 2008). Taken together, these numerous lines of evidence point to the importance of focusing on the antenatal period in order to develop preventive and therapeutic interventions . A psychosocial assessment, in the sense of a comprehensive and multidimensional evaluation of a woman's psychosocial circumstances (e.g., sources of support, quality of her relationships, recent life stressors, past or current physical or sexual abuse) should be common practice for all women during the antenatal period . In fact, this assessment would help health professionals to identify women with a high - risk profile but not currently symptomatic and, therefore, to offer them preventive interventions (austin, 2014, milgrom and gemmill, 2014). Unfortunately, this assessment is not always conducted, and therefore many women are not identified as being at risk for, or as currently suffering from, antenatal anxiety and depression (andersson et al ., 2003, marcus et al ., 2003). However, in the perinatal period both parents are often highly motivated to seek help for their babies wellbeing and for the potential reduction in intergenerational family dysfunction, and hence this is a unique opportunity to provide preventive interventions for the mental health of the whole family (austin, 2014). A recent systematic review on this topic by lancaster et al . (2010) was focused on the identification of risk factors for antenatal depression that could be assessed during routine obstetric care . In fact, because most women use obstetric services during the course of their pregnancy, this is a great opportunity to identify women at risk, and eventually to treat them and to follow them up (lancaster et al ., 2010). This review identified 57 studies published between january 1980 and march 2008 in the united states, canada, europe, australia and new zealand and found that the main risk factors associated with depressive symptoms during pregnancy are maternal anxiety, life stress, a history of depression, lack of support, domestic violence and unintended pregnancy . The purpose of our systematic review is to describe the main psycho - social, obstetric and environmental risk factors involved in the development of antenatal anxiety and depression, including a wider range of low-, middle- and high - income countries compared with those considered by lancaster et al . A systematic literature analysis was performed with the aim of identifying the main psycho - social and environmental risk factors involved in the onset of antenatal anxiety and depression . We used databases for psychological and medical research (pubmed, psychinfo and the cochrane library), to select relevant studies and reviews, with the following key words, as single terms or in combination: antenatal depression; antenatal anxiety; risk factors for antenatal mental health; risk factors for depression / anxiety during pregnancy, risk factors for prenatal depression / anxiety; antenatal depression / anxiety risk factors; prenatal depression / anxiety risk factors; screening; assessments; evaluations . Relevant papers cited in the references of selected articles were further considered for inclusion in the review . Original papers were included if they were written in english and published between 1 january 2003 and 31 august 2015 . In addition, the following sources of grey literature were consulted: nice guidelines, reports from related charities (for example, tommy's) and scientific organizations (for example, marc society) and information on relevant websites (austin, 2014, nice, 2014, tommy's, 2013). Twelve literature reviews and meta - analyses regarding perinatal mental illness and relevant risk factors were consulted regardless of publication date (beck, 2001, bennett et al ., 2004, gavin et al ., 2005, howard et al ., 2013, lancaster et al ., 2010, norhayati et al ., 2015, after cross - referencing the different sources and excluding duplicates and irrelevant papers, 97 papers were selected (see table 1). Studies conducted on particular and high - risk populations, such as women exposed to major environmental catastrophes like earthquakes or tsunamis, women with pre - existing health conditions such as hiv or obesity, women with health conditions associated with pregnancy such as diabetes, with high risk pregnancies or with babies with congenital anomalies, were all excluded . Studies conducted with the aim of investigating risk factors for persistent and recurrent depressive episodes during pregnancy have been included, but studies describing risk factors for persistency of depression and anxiety from pregnancy into the postpartum period have been excluded . Moreover, studies were excluded if the measures used, the risk factors investigated, the times of the assessments and the statistical analysis were not clearly described, or if they had explored risk factors for depression and/or anxiety in the perinatal period (either pregnancy or postpartum) and it was not possible to determine if the risk factors were unequivocally related to the antenatal period . An additional 46 papers regarding the epidemiology of perinatal anxiety and depression, the bio - psycho - social mechanisms involved, child development outcomes, and preventive interventions, have been included in this review, either in the introduction and/or in the results . These papers have been identified either in the databases consulted or from the references of the studies included in the review . Depression and anxiety are highly comorbid during the antenatal period, and indeed high anxiety during pregnancy is one of the strongest risk factors for depression (lancaster et al ., 2010, verreault et al ., 2014). Women with feelings of anxiety are at increased risk of suffering from depression during pregnancy (edwards et al ., 2008); for example, a recent study (mohamad yusuff et al ., 2015) has found that women who had experienced antenatal anxiety were about three times more likely to suffer from depression during pregnancy . A previous history of mental illness, in particular a history of anxiety and depression and a history of psychiatric treatment during a previous pregnancy or at any time during the lifetime, is also a well - established risk factor in the development of antenatal anxiety and depression (akcal et al ., 2014, balestrieri et al ., 2012, bayrampour et al ., 2015, bowen et al ., 2009,, 2009, giardinelli et al ., 2012, jeong et al ., 2013, lydsdottir et al ., 2014, manikkam and burns, 2012, marchesi et al ., 2014, marchesi et al ., 2009, marcus et al ., 2003, martini et al ., 2015, nasreen et al ., 2011, redshaw and henderson, 2013, rich - edwards et al ., 2006, rubertsson et al ., 2014, shakeel et al ., 2015, verreault et al ., 2014). Many of these studies found that a history of previous anxiety or depression is the strongest risk factor for a new onset during the antenatal period . For example, one study (marcus et al ., 2003) found that almost half of the women depressed during pregnancy had a past history of major depressive disorder . In addition, another study (andersson et al ., 2006) found that absence of a previous psychiatric disorder was associated with a greater likelihood of recovery from antenatal anxiety and depression after the birth of the child . (2014) found that, despite a previous history of depression being the strongest risk factor for a new episode during pregnancy, half of the women depressed during pregnancy had never been depressed before . This suggests that it is not uncommon for the first episode of depression to occur during pregnancy . Some studies have also found a significant correlation between antenatal anxiety and depression and past (lee et al ., 2007) or current (marcus et al ., 2003) studies have also shown that smoking before or during pregnancy predicts anxiety and depression during pregnancy, even if it is not always clear whether smoking increases the risk of depression or if it is depression that is associated with less healthy behaviours, such as smoking (abuidhail and abujilban, 2014, bottomley and lancaster, 2008, bowen et al ., 2009, lydsdottir et al ., 2014, marcus et al ., 2003, raisanen et al ., 2014, rubertsson et al ., 2014) one recent research (smedberg et al ., 2015) found that women who continued to smoke during pregnancy were more likely to suffer from antenatal depression compared to the ones who quit smoking during pregnancy, with twice the prevalence of continuing smoking during pregnancy in women with depression than in healthy women . However, another study (jeong et al ., 2013) found that cigarette smoking increased the risk of antenatal depression even if women abstained from cigarette smoking during pregnancy . One study (fellenzer and cibula, 2014) found not only that smoking during pregnancy was associated with depression but also that a greater number of cigarettes per day was associated with higher levels of depression . Similarly, another study (bottomley and lancaster, 2008) found that there was a significant correlation between the number of cigarettes smoked during the 1st and 2nd trimesters and the depressive score on the epds in the 3rd trimester . However, one research found that smoking during pregnancy was not associated with antenatal depression (luke et al ., 2009). Substance use during pregnancy has also been found to be strongly associated with antenatal depression (fellenzer and cibula, 2014, holzman et al ., 2006). The quality of attachment and a woman's relationship with her own parents is another important risk factor . For example, childhood abuse has been recognized as a clear risk factor for depression and anxiety specifically during pregnancy (plant et al ., 2013, robertson - blackmore et al ., 2013), over and above the increased life - time risk for psychiatric disorders that childhood maltreatment carries (see section 3.4). However, despite considerable anecdotal clinical evidence, only a few studies have investigated the quality of parenting and attachment style in relation to depression and anxiety during the perinatal period . A study (grant et al ., 2012) found that the women reporting a style of parenting characterized by both low care and high control were six times more likely to have anxiety during pregnancy, and seven times more likely to have postnatal depression . Moreover, another study (simpson et al ., 2003) found that highly ambivalent women, who also perceived their husband as less supportive and angrier, were more likely to experience depressive symptoms in the perinatal period . The absence of a good caregiving experience makes it more challenging for the mother to cope with distressing feelings and difficult situations related to parenthood . We know from the literature that pregnant women go through a mental reorganization that allows the development of maternal identity . This process involves the elaboration and integration of mental representations of the unborn baby, of the woman as a mother, of non - maternal self - features and of other significant relationships (ammaniti and trentini, 2009). The nature and quality of these maternal representations are influenced by many factors, such as childhood experiences of being parented, actual relationship with the partner and the family, psychological state, and environmental risk factors (lara - carrasco et al . The quality of the childhood experiences of being parented, and especially the relationship with the mother figure, influences the nature of these representations during pregnancy (ammaniti and trentini, 2009). In fact, many researchers have shown that, at the time of the transition to parenthood, unresolved and unprocessed issues are reactivated, and fantasies and revitalized memories related to the past are transferred to the new situation, potentially leading to distorted responses towards the baby after the birth (raphael - leff, 2010). To this end, one study (jeong et al ., 2013) found that past experience of insufficient emotional support from the mother (but not from the father) was an independent predictor of antenatal depression . This suggests that positive internalized experiences with her own mother may help the pregnant women in managing negative emotions during pregnancy and in building a healthy mother this evidence highlights the importance of assessing the quality of childhood parenting experiences and the presence of a history of abuse, because these factors both increase the likelihood that mothers may experience difficulties in developing optimal relationships with their babies . A family history of psychiatric illness during the lifespan has been observed as another important risk factor for antenatal depression (jeong et al ., 2013, lydsdottir et al ., 2014). Nevertheless, this may sometimes be difficult to evaluate, because the woman may not be aware of mental problems that have affected relatives, or may be not willing to disclose this information (robertson et al . Lack of social support is another factor strongly associated with an increased risk of antenatal anxiety and depression . Social support is a multidimensional concept and includes informational support (information and advice), instrumental support (practical help) and emotional support (expression of caring and holding in esteem). The objective evaluation of the social support received may be challenging, because it has been noticed that depressed women tend to feel less supported than they objectively are (robertson et al ., 2004). Many studies report perceived lack of partner support and of social support as important risk factors for antenatal anxiety and depression (adewuya et al ., 2015, bowen and muhajarine, 2006b, bowen et al ., 2009, dibaba et al ., 2013, elsenbruch et al ., 2007,, 2010, groves et al ., 2012, hartley et al ., 2011, jeong et al ., 2013,, 2005, leigh and milgrom, 2008, lydsdottir et al ., 2014, marchesi et al ., 2014, martini et al ., 2015, nasreen et al ., 2011, ratcliff et al ., 2015, records and rice, 2007, rich - edwards et al ., 2006, rubertsson et al ., 2003,, 2014, waqas et al ., 2015, westdahl et al ., 2007, yanikkerem et al ., 2013, zelkowitz et al ., 2004) as well as social conflict (westdahl et al ., 2007) a study conducted with immigrants in canada (zelkowitz et al ., 2004) demonstrated that women with scores of 12 or more on the epds reported less satisfaction with, and greater need for, social support . Also, they had fewer women, fewer relatives and fewer people from their own ethnic group in their networks . Perceived support and marital satisfaction are protective factors against antenatal anxiety and depression (lee et al ., 2007, zeng et al ., 2015), while a problematic / conflictual or dissatisfied / poor relationship with partner have been identified as risk factors for the onset of anxiety and depression during pregnancy (giardinelli et al ., 2012, marchesi et al ., 2009, martini et al ., 2015, nasreen et al ., 2011, records and rice, 2007, srinivasan et al ., 2015, zelkowitz et al ., 2004). Good instrumental and emotional support, provided first of all by the partner but also by the family and the social environment, is crucial for the mother - to - be . The presence of a supportive partner acts as a buffer against the difficulties experienced in the transition to parenthood, protecting maternal mental health (bilszta et al ., 2008). A problematic relationship with the partner, instead, causes an additional stress to the woman, making it harder for her to adjust to pregnancy and motherhood (marchesi et al ., 2009). Other factors, such as marital status or length of relationship, may also influence the amount of support the new mother receives and could be considered to be risk factors for anxiety and depression during pregnancy . Some studies have identified that women experiencing depressive symptoms in the antenatal period are more likely to be not married, to be single or to have a partner not living in the same household (adewuya et al ., 2015, faisal - cury and rossi menezes, 2007, figueiredo et al ., 2013, manikkam and burns, 2012, marcus et al ., 2003, melville et al ., 2010, mezey et al ., 2005, raisanen et al ., 2014, rich - edwards et al ., 2006, rubertsson et al ., 2003, weobong et al ., 2014 a study found higher level of depression in women living with friends or in a community compared to the ones cohabitating with a partner (balestrieri et al ., 2012). However, some researchers (bilszta et al ., 2008) have observed that the evidence that single mothers report higher levels of depressive symptoms during pregnancy than women with supportive partners may be explained by a previous history of depression, current emotional problems, previous abuse, level of daily hassles, maternal perception of the infant and income level . Interestingly, single mothers have lower levels of depressive symptoms compared to women with unsupportive partners (bilszta et al ., 2008). Likewise, a few studies did not find that marital status was always a significant predictor of antenatal depression and anxiety (agostini et al . These findings highlight the importance of considering not only marital status but also the quality of the relationship . Given these results, we could conclude that being a single mother is in a way better than having a difficult and unsupportive relationship . Surprisingly, no studies regarding the psychopathology of the partner as potential risk factor for antenatal depression and anxiety have been identified in this systematic review . Research has been conducted regarding the possible impact of partner psychopathology on maternal depressive symptoms in the first postpartum year (d'anna - hernandez et al ., 2013), but no study has been found in relation to the antenatal period . One meta - analysis (paulson and bazemore, 2010) has found a positive and moderate in size correlation between maternal and paternal depression in the perinatal period (from the 1st trimester until the 1st year of the baby) but a direction of causal influence had not been reported in the studies included in the meta - analysis . Studies have examined many socio - demographic and economic risk factors in relation to antenatal anxiety and depression, but the results are equivocal . Many studies have found a significant correlation between young age and depression / anxiety during pregnancy (bodecs et al ., 2013, fellenzer and cibula, 2014, glazier et al ., 2004, hartley et al ., 2011, lee et al ., 2007, martini et al ., 2015, qiao et al ., 2009, rich - edwards et al ., 2006, rubertsson et al ., 2014, rubertsson et al ., 2003). Perhaps unsurprisingly, adolescents are at increased risk of become depressed during pregnancy (figueiredo et al ., 2007, lanzi et al ., 2009, raisanen et al ., 2014). This confirms what has been found in a systematic review about adolescence and mental health during pregnancy (siegel and brandon, 2014). Nevertheless, some studies found that an older age was positively associated with depression scores during pregnancy (ali et al ., 2012, faisal - cury et al ., 2009, fisher et al ., 2013, gavin et al ., 2011, golbasi et al ., 2010, luke et al ., 2009, nasreen et al ., 2011, raisanen et al ., 2014, weobong et al ., 2014, yanikkerem et al ., 2013) and other studies found that age was not associated with depression or anxiety during pregnancy (abuidhail and abujilban, 2014, agostini et al ., 2015, balestrieri et al ., 2012, faisal - cury and rossi menezes, 2007, husain et al ., 2012, husain et al ., 2011, karmaliani et al ., 2009, marcus et al ., 2003, ratcliff et al ., 2015, antenatal depression and anxiety also seem more common in women with low educational achievements (abuidhail and abujilban, 2014, abujilban et al ., 2014, bodecs et al ., 2013, ., 2014, faisal - cury and rossi menezes, 2007, fellenzer and cibula, 2014, gavin et al ., 2011, glazier et al ., 2004, husain et al ., 2011, jeong et al ., 2013, lydsdottir et al ., 2014, marcus et al ., 2003, martini et al ., 2015, qiao et al ., 2009, yanikkerem et al ., 2013). In particular, one study conducted in bangladesh (nasreen et al ., 2011) found that antenatal anxiety was inversely associated with literacy . Nevertheless, two studies conducted in malawi (stewart et al ., 2014) and pakistan (karmaliani et al ., 2009) found that women with more years of schooling were more likely to experiences symptoms of anxiety and depression . A few studies did not find education to be a significant predictor of antenatal depression (agostini et al ., 2015, husain et al ., 2012, lanzi et al ., 2009, luke et al ., 2009, ratcliff et al ., 2015, antenatal anxiety and depression have also been found to be more prevalent in unemployed women (bodecs et al ., 2013, dibaba et al ., 2013,, 2009, giardinelli et al ., 2012, lydsdottir et al ., 2014, marcus et al ., 2003, rubertsson et al ., 2014, rubertsson et al ., 2003) and housewives (balestrieri et al ., 2012, marchesi et al ., 2009, yanikkerem et al ., 2013), compared with employed women, even if a few studies did not find any significant association between employment and antenatal depression (agostini et al ., 2015, glazier et al ., 2004, husain et al ., 2011, karmaliani et al ., 2009, lanzi et al ., 2009, zelkowitz et al ., 2004) one study (cooklin et al ., 2007) analysed the role of workplace adversity on maternal wellbeing during pregnancy . It found that poor working conditions, in terms of discrimination and lack of key entitlements during pregnancy, are associated with higher levels of depression . Moreover, women whose partners were unemployed seem to be more likely to experience antenatal depression (akcal et al ., 2014, husain et al ., 2011, karmaliani et al ., 2009, studies that have examined the association with low income and financial hardships have also reported contradictory results . While some studies (bodecs et al ., 2013, faisal - cury and rossi menezes, 2007, fisher et al ., 2013, fisher et al ., 2010, glazier et al ., 2004, hartley et al ., 2011, prady et al ., 2013, rich - edwards et al ., 2006, weobong et al ., 2014, zeng et al ., 2015) found low income or financial difficulties to be relevant factors, others (abuidhail and abujilban, 2014, josefsson et al . Ethnicity and, in particular, belonging to a minority ethnic group, are other potential risk factors highlighted by some studies, even if the results are also contradictory . The prevalence of antenatal depression seems higher in black, latina and asian mothers compared to white mothers (faisal - cury and rossi menezes, 2007, fellenzer and cibula, 2014, gavin et al ., 2011, melville et al ., 2010, redshaw and henderson, 2013, shakeel et al ., 2015, verreault et al ., 2014),, 2008, jesse et al ., 2005, marcus et al ., 2003) and others show higher levels of depression among white mothers compared to black and hispanic mothers as well as women of other ethnicities (shen et al ., 2010). Belonging to a minority group could be a risk factor for antenatal depression because of the increased level of stress due to discrimination (prady et al ., 2013). However, research in the united kingdom has shown that some minority groups may have lower levels of mental illness than the majority population (edge, 2007). Specifically, in this study black caribbean mothers, despite belonging to a minority ethnic group, living in more deprived areas and being more likely to be lone mothers and to receive less support from their partners, did not show higher levels of depression during pregnancy and the postpartum period, and did not receive more treatment for perinatal depression, compared with white british mothers (edge, 2007). It is possible that social risk factors may have a different impact on the mental health of black caribbean and white women, as the prolonged and repeated exposure to adversity might have provided the black caribbean women with greater resilience and skills to manage psychological distress (edge, 2007, williams and healy, 2001, williams et al ., 1997). This may also be explained by the higher levels of resources (social support, spirituality and self - esteem) in black mothers than in white mothers (jesse et al ., 2005). These resources may act as a buffer against potential sources of risk and reduce the incidence of depression (jesse et al . Therefore, being part of a minority ethnic group may not be an independent factor in the relation to depressive symptoms, and the substantial risk in minority ethnic groups found in some studies may be the result of a higher prevalence of other risk factors . To this end, a study found that the higher prevalence of depression among black and hispanic mothers was mainly explained by lower income and financial hardship (rich - edwards et al ., another study found that financial concerns are significantly associated with mental health difficulties during pregnancy, rather than belonging to a specific ethnic group (prady et al ., 2013). Many studies have shown that adverse events in life and high perceived stress during pregnancy play an important role in the onset of antenatal depression (abujilban et al ., 2014, bayrampour et al ., 2015, bowen et al ., 2009, brittain et al ., 2015, bunevicius et al ., 2009, fisher et al ., 2013, fisher et al ., 2010, gavin et al ., 2011, glazier et al ., 2004,, 2005, leigh and milgrom, 2008, melville et al ., 2010, ratcliff et al ., 2015, rubertsson et al ., 2003, shakeel et al ., 2015, verreault et al ., 2014, woods et al ., 2010, zayas et al ., 2003, zelkowitz et al ., 2004). Stressful experiences may vary from mild to severe, also depending on stress perception and the ability to cope; nevertheless, highly stressful experiences, such as the death or illness of a relative, a relationship breakdown, losing a job, moving home, being assaulted or raped, can trigger depressive and anxiety symptoms or disorders in most individuals . Pregnancy is in itself a well - recognized time of stress because of the many obvious and potential changes and challenges . The occurrence of one or more stressful events can then lead to an increase in the probability that mothers and fathers experience psychological difficulties or mental health disorders . To this end, a study has highlighted the important role of social support in reducing the negative impact of adverse events in life: they have shown that women who experience stressful life events but have good social support are less likely to suffer from emotional distress compared with women without an available support network (glazier et al ., 2004). Therefore, the impact of adverse events in life on antenatal mental health may be mediated by social support . This sustains the stress buffering hypothesis, where supportive interactions among people protect them against the negative health consequences of stressful life events (cobb, 1976, cohen and wills, 1985). Nevertheless, in one study (groves et al ., 2012) social support did not attenuate the effects of partner violence on woman's mental health during pregnancy . Within adverse events in life, being exposed to intrusive life events, such as domestic violence or emotional, physical or sexual abuse, has a considerable impact on a mother's mental health in the perinatal period . A number of studies have found that having been exposed to domestic violence before or during pregnancy (particularly if perpetrated by the partner), having a history of abuse, or having experienced a sexual assault, are all risk factors for the development of antenatal anxiety, depression and post - traumatic stress symptoms (akcal et al ., 2014, ali et al ., 2012, brittain et al ., 2015, dibaba et al ., 2013, edwards et al ., 2008, fisher et al ., 2013, fisher et al ., 2010, fonseca - machado mde et al ., 2015, gavin et al ., 2011, groves et al ., 2012, hartley et al ., 2011, holzman et al ., 2006,, 2005, karmaliani et al ., 2009, leigh and milgrom, 2008, lydsdottir et al ., 2014, mahenge et al ., 2013, martini et al ., 2015, melville et al ., 2010, mezey et al ., 2005, miszkurka et al ., 2012, nasreen et al ., 2011, rich - edwards et al ., 2011, rodriguez et al, 2008, stewart et al ., 2014, verreault et al ., 2014, waqas et al ., 2015). In particular, a study found that having been exposed to a traumatic sexual experience is associated with antenatal anxiety but not depression (martini et al ., 2015). Another study found that women who are exposed to physical assault or sexual coercion by their intimate partners before or during pregnancy have higher levels of depressive symptoms during pregnancy (martin et al ., 2006). We know from the literature that women with a history of abuse often experience more than one traumatic event during their lives, and have higher life - time levels of depressive and post - traumatic symptoms than women who have only suffered a single trauma . Indeed, one study (holzman et al ., 2006) found a marked increase in depressive scores when adverse circumstances were present both in childhood and in adulthood . This is in line with the theory that accumulation of adversity is more damaging than single events (holzman et al ., 2006). As such, women who have been sexually abused as children are at significant risk of experiencing abuse as adults, including domestic violence (mezey et al ., 2005). In fact, childhood abuse has been identified as a particularly strong predictor of depression and anxiety during the antenatal period (brittain et al ., 2015, fisher et al ., 2013, groves et al ., 2012, jeong et al ., 2013, mezey et al ., 2005, plant et al ., 2013, robertson - blackmore et al ., 2013, seng et al ., 2014), with women with a history of childhood sexual abuse being twice as likely to develop antenatal depression (robertson - blackmore et al ., 2013 moreover, the relationship between childhood maltreatment and antenatal depression remains true even when accounting for a woman's antisocial characteristics, personal history of psychiatric illness outside pregnancy, and the availability of partner support during pregnancy, all of which have been identified as salient factors associated with maternal psychopathology (plant et al ., 2013). Many studies have found an increased risk of developing antenatal depression and anxiety in women with an unplanned or unwanted pregnancy (ajinkya et al ., 2013, akcal et al ., 2014, bayrampour et al ., 2015, brittain et al ., 2015,, 2009, dibaba et al ., 2013, fellenzer and cibula, 2014, giardinelli et al ., 2012, golbasi et al ., 2010, jeong et al ., 2013, karmaliani et al ., 2009, lee et al ., 2007, manikkam and burns, 2012, marchesi et al ., 2009, martini et al ., 2015, mohamad yusuff et al ., 2015, mohammad et al ., 2011, redshaw and henderson, 2013, rich - edwards et al ., 2006, waqas et al ., 2015, weobong et al ., 2014, yanikkerem et al ., 2013), although two studies did not find this (groves et al ., 2012, hartley et al ., 2011). Two studies (lee et al ., 2007, marchesi et al ., 2009) observed that an unwanted pregnancy was a significant predictor of depression only in the first trimester, with less importance over time . Some researchers (lee et al ., 2007) suggested that women initially find it more difficult to cope with an unexpected and undesired event and, therefore, are more likely to become depressed; however, as the pregnancy progresses, the shock reduces, and the relationship with the foetus grows stronger, the pregnancy becomes more accepted and the depressive symptoms decrease . Of note is also that fear of childbirth and negative thoughts about the upcoming delivery have been associated with increased risk of antenatal anxiety and depression (raisanen et al ., 2014, rubertsson et al ., 2014); indeed, a study found that a negative experience of pregnancy was significantly associated with antenatal depression (agostini et al ., 2015). The role of parity in increasing the risk of antenatal depression and anxiety is not clear . Multiparous women are at increased risk of developing antenatal anxiety and depression according to some studies (abuidhail and abujilban, 2014, golbasi et al ., 2010, lanzi et al ., 2009, redshaw and henderson, 2013, yanikkerem et al ., 2013) while other studies (ali et al ., 2012, fisher et al ., 2013, raisanen et al ., 2014) found nulliparous or primiparous women as more at risk than multiparous women, and others yet did not find any significant association between parity and antenatal mental health (abujilban et al . ., 2013, faisal - cury and rossi menezes, 2007, fisher et al ., 2010, glazier et al ., 2004, husain et al ., 2011, marcus et al ., 2003, ratcliff et al ., 2015, rubertsson et al ., 2003, zelkowitz et al ., 2004). Greater number of previous pregnancies (multigravida) has been found to be a predictor of antenatal depression in a few studies (ajinkya et al ., 2013, records and rice, 2007) while another study found that women in their first pregnancy (primigravida) were more likely to suffer from depression (karmaliani et al ., 2009), and other studies (fisher et al ., 2010, srinivasan et al ., women with current or past pregnancy / delivery complications, with a history of pregnancy loss, pregnancy terminations or stillbirth have been found to be more likely to experience antenatal depression, anxiety and pregnancy - specific anxieties (adewuya et al ., 2007, ajinkya et al ., 2013, ali et al ., 2012, armstrong, 2004, chojenta et al ., 2014,, 2009, faisal - cury and rossi menezes, 2007, fisher et al ., 2013, golbasi et al ., 2010, gong et al ., 2013, husain et al ., 2011, raisanen et al ., 2014, stewart et al ., 2014, waqas et al ., 2015, weobong et al ., 2014, zeng et al ., 2015) in particular, one study (bergner et al ., 2008) found that women with a history of miscarriage suffer more from trait anxiety and pregnancy - specific anxieties, and those who have had recurrent miscarriages also have higher levels of anxiety . However, some studies (bicking kinsey et al ., 2015, fisher et al ., 2010, jeong et al ., 2013, qiao et al ., 2009) did not find any significant association between previous obstetric history, including a history of either spontaneous or induced abortion, and antenatal anxiety or depression . A history of one or more episiotomies, caesarean section or a previous negative birth experience, have been found to be associated with a high incidence of antenatal anxiety and depression (rubertsson et al ., 2003, waqas et al ., 2015), but another study found no association between mode of delivery and antenatal depression (ajinkya et al ., 2013). In one study, women who had infertility treatments were more likely to suffer from antenatal anxiety (bayrampour et al ., 2015), but another study did not find this (rubertsson et al ., 2003). An unexpected loss of pregnancy can be a traumatic event and can lead to high level of anxiety and depression that can persist into subsequent pregnancies . One study found previous miscarriage to be associated with antenatal depression but no significant association was found with stillbirth (rubertsson et al ., 2003). The interval between a pregnancy that did not result in a live birth and a new pregnancy seems to play a crucial role in whether the woman will or will not experience anxiety and depression during a new pregnancy: the world health organization recommends an interval of at least 6 months to allow women time to recover physically and mentally (who, 2005). To this end, gong et al . Found that women with a history of miscarriage and inter - pregnancy interval of less than 6 months are at increased risk of developing anxiety and depression during the first trimester of pregnancy (gong et al ., 2013). Some studies have shown that some personality factors increase the risk of experiencing psychological difficulties in the antenatal period, and especially negative cognitive styles: pessimism, anger and rumination; a tendency to be nervous, worried or shy; low self - esteem and low self - efficacy; and high levels of neuroticism or psychoticism (bayrampour et al ., 2015, bunevicius et al ., 2009, ginsburg et al ., 2008, jesse et al ., 2005, lee et al ., 2007, leigh and milgrom, 2008, martini et al ., 2015, mohammad et al ., 2011, zeng et al ., 2015). On the contrary, active coping and high self - esteem / self - efficacy have been identified as protective factors (edwards et al ., 2008, zeng et al ., 2015). While more research has been conducted regarding general cognitive biases in relation to depression and anxiety, some studies have also pointed out that specific types of cognitive biases have an impact in the perinatal period . A study (sockol et al ., 2014) has found that maternal attitudes regarding motherhood have incremental predictive validity for symptoms of depression and anxiety during the perinatal period over more general cognitive biases . Moreover, maternal attitudes towards motherhood continue to be strongly associated with anxiety and depression even after controlling for interpersonal factors . Examples of maladaptive beliefs about motherhood are: it is wrong to have mixed feelings about my baby; i should feel more devoted to my baby; it is wrong to feel disappointed by motherhood; if my baby is crying, people will think i cannot care for him/ her properly . As such, women with maladaptive beliefs about motherhood are at increased risk of experiencing feelings of sadness, guilt and worthlessness, and to develop anxiety and depression during pregnancy and in the postpartum period, when their feelings do not match their attitudes and expectations . This means that specific beliefs about motherhood could be most strongly activated by the particular stressors of parenting and, therefore, they could mediate the relationship between the specific stressors of becoming a parent and the emotional response to these events (sockol et al ., 2014). Depression and anxiety are highly comorbid during the antenatal period, and indeed high anxiety during pregnancy is one of the strongest risk factors for depression (lancaster et al ., 2010, verreault et al ., 2014). Women with feelings of anxiety are at increased risk of suffering from depression during pregnancy (edwards et al ., 2008); for example, a recent study (mohamad yusuff et al ., 2015) has found that women who had experienced antenatal anxiety were about three times more likely to suffer from depression during pregnancy . A previous history of mental illness, in particular a history of anxiety and depression and a history of psychiatric treatment during a previous pregnancy or at any time during the lifetime, is also a well - established risk factor in the development of antenatal anxiety and depression (akcal et al ., 2014, balestrieri et al ., 2012, bayrampour et al ., 2015, bowen et al ., 2009,, 2009, giardinelli et al ., 2012, jeong et al ., 2013, lydsdottir et al ., 2014, manikkam and burns, 2012, marchesi et al ., 2014, marchesi et al ., 2009, marcus et al ., 2003, martini et al ., 2015, nasreen et al ., 2011, redshaw and henderson, 2013, rich - edwards et al ., 2006, rubertsson et al ., 2014, shakeel et al ., 2015, verreault et al ., 2014). Many of these studies found that a history of previous anxiety or depression is the strongest risk factor for a new onset during the antenatal period . For example, one study (marcus et al ., 2003) found that almost half of the women depressed during pregnancy had a past history of major depressive disorder . In addition, another study (andersson et al ., 2006) found that absence of a previous psychiatric disorder was associated with a greater likelihood of recovery from antenatal anxiety and depression after the birth of the child . (2014) found that, despite a previous history of depression being the strongest risk factor for a new episode during pregnancy, half of the women depressed during pregnancy had never been depressed before . This suggests that it is not uncommon for the first episode of depression to occur during pregnancy . Some studies have also found a significant correlation between antenatal anxiety and depression and past (lee et al ., 2007) or current (marcus et al ., 2003) studies have also shown that smoking before or during pregnancy predicts anxiety and depression during pregnancy, even if it is not always clear whether smoking increases the risk of depression or if it is depression that is associated with less healthy behaviours, such as smoking (abuidhail and abujilban, 2014, bottomley and lancaster, 2008, bowen et al ., 2009, lydsdottir et al ., 2014, marcus et al ., 2003, raisanen et al ., 2014, rubertsson et al ., 2014) one recent research (smedberg et al ., 2015) found that women who continued to smoke during pregnancy were more likely to suffer from antenatal depression compared to the ones who quit smoking during pregnancy, with twice the prevalence of continuing smoking during pregnancy in women with depression than in healthy women . However, another study (jeong et al ., 2013) found that cigarette smoking increased the risk of antenatal depression even if women abstained from cigarette smoking during pregnancy . One study (fellenzer and cibula, 2014) found not only that smoking during pregnancy was associated with depression but also that a greater number of cigarettes per day was associated with higher levels of depression . Similarly, another study (bottomley and lancaster, 2008) found that there was a significant correlation between the number of cigarettes smoked during the 1st and 2nd trimesters and the depressive score on the epds in the 3rd trimester . However, one research found that smoking during pregnancy was not associated with antenatal depression (luke et al ., 2009). Substance use during pregnancy has also been found to be strongly associated with antenatal depression (fellenzer and cibula, 2014, holzman et al ., 2006). The quality of attachment and a woman's relationship with her own parents is another important risk factor . For example, childhood abuse has been recognized as a clear risk factor for depression and anxiety specifically during pregnancy (plant et al ., 2013, robertson - blackmore et al ., 2013), over and above the increased life - time risk for psychiatric disorders that childhood maltreatment carries (see section 3.4). However, despite considerable anecdotal clinical evidence, only a few studies have investigated the quality of parenting and attachment style in relation to depression and anxiety during the perinatal period . A study (grant et al ., 2012) found that the women reporting a style of parenting characterized by both low care and high control were six times more likely to have anxiety during pregnancy, and seven times more likely to have postnatal depression . Moreover, another study (simpson et al ., 2003) found that highly ambivalent women, who also perceived their husband as less supportive and angrier, were more likely to experience depressive symptoms in the perinatal period . The absence of a good caregiving experience makes it more challenging for the mother to cope with distressing feelings and difficult situations related to parenthood . We know from the literature that pregnant women go through a mental reorganization that allows the development of maternal identity . This process involves the elaboration and integration of mental representations of the unborn baby, of the woman as a mother, of non - maternal self - features and of other significant relationships (ammaniti and trentini, 2009). The nature and quality of these maternal representations are influenced by many factors, such as childhood experiences of being parented, actual relationship with the partner and the family, psychological state, and environmental risk factors (lara - carrasco et al . The quality of the childhood experiences of being parented, and especially the relationship with the mother figure, influences the nature of these representations during pregnancy (ammaniti and trentini, 2009). In fact, many researchers have shown that, at the time of the transition to parenthood, unresolved and unprocessed issues are reactivated, and fantasies and revitalized memories related to the past are transferred to the new situation, potentially leading to distorted responses towards the baby after the birth (raphael - leff, 2010). To this end, one study (jeong et al ., 2013) found that past experience of insufficient emotional support from the mother (but not from the father) was an independent predictor of antenatal depression . This suggests that positive internalized experiences with her own mother may help the pregnant women in managing negative emotions during pregnancy and in building a healthy mother this evidence highlights the importance of assessing the quality of childhood parenting experiences and the presence of a history of abuse, because these factors both increase the likelihood that mothers may experience difficulties in developing optimal relationships with their babies . A family history of psychiatric illness during the lifespan has been observed as another important risk factor for antenatal depression (jeong et al ., 2013, lydsdottir et al ., 2014). Nevertheless, this may sometimes be difficult to evaluate, because the woman may not be aware of mental problems that have affected relatives, or may be not willing to disclose this information (robertson et al . Lack of social support is another factor strongly associated with an increased risk of antenatal anxiety and depression . Social support is a multidimensional concept and includes informational support (information and advice), instrumental support (practical help) and emotional support (expression of caring and holding in esteem). The objective evaluation of the social support received may be challenging, because it has been noticed that depressed women tend to feel less supported than they objectively are (robertson et al ., 2004). Many studies report perceived lack of partner support and of social support as important risk factors for antenatal anxiety and depression (adewuya et al ., 2007,, 2015, bayrampour et al ., 2015, bowen and muhajarine, 2006b, bowen et al ., 2009, dibaba et al ., 2013, elsenbruch et al ., 2007,, 2009, ginsburg et al ., 2008, golbasi et al ., 2010,, 2005, leigh and milgrom, 2008, lydsdottir et al ., 2014, marchesi et al ., 2014, martini et al ., 2015, nasreen et al ., 2011, ratcliff et al ., 2015, records and rice, 2007, rich - edwards et al ., 2006, rubertsson et al ., 2003,, 2015, stewart et al ., 2014, waqas et al ., 2015, westdahl et al ., 2007, yanikkerem et al ., 2013, zelkowitz et al ., 2004) as well as social conflict (westdahl et al ., 2007). A study conducted with immigrants in canada (zelkowitz et al ., 2004) demonstrated that women with scores of 12 or more on the epds reported less satisfaction with, and greater need for, social support . Also, they had fewer women, fewer relatives and fewer people from their own ethnic group in their networks . Perceived support and marital satisfaction are protective factors against antenatal anxiety and depression (lee et al ., 2007, zeng et al ., 2015), while a problematic / conflictual or dissatisfied / poor relationship with partner have been identified as risk factors for the onset of anxiety and depression during pregnancy (giardinelli et al ., 2012, marchesi et al ., 2009, martini et al ., 2015, nasreen et al ., 2011, records and rice, 2007, srinivasan et al ., 2015, zelkowitz et al ., 2004). Good instrumental and emotional support, provided first of all by the partner but also by the family and the social environment, is crucial for the mother - to - be . The presence of a supportive partner acts as a buffer against the difficulties experienced in the transition to parenthood, protecting maternal mental health (bilszta et al ., 2008). A problematic relationship with the partner, instead, causes an additional stress to the woman, making it harder for her to adjust to pregnancy and motherhood (marchesi et al ., 2009). Other factors, such as marital status or length of relationship, may also influence the amount of support the new mother receives and could be considered to be risk factors for anxiety and depression during pregnancy . Some studies have identified that women experiencing depressive symptoms in the antenatal period are more likely to be not married, to be single or to have a partner not living in the same household (adewuya et al ., 2007, brittain et al ., 2015, faisal - cury and rossi menezes, 2007, figueiredo et al ., 2007, jeong et al ., 2013, manikkam and burns, 2012, marcus et al ., 2003, melville et al ., 2010, mezey et al ., 2005, raisanen et al ., 2014, rich - edwards et al ., 2006, a study found higher level of depression in women living with friends or in a community compared to the ones cohabitating with a partner (balestrieri et al ., 2012). However, some researchers (bilszta et al ., 2008) have observed that the evidence that single mothers report higher levels of depressive symptoms during pregnancy than women with supportive partners may be explained by a previous history of depression, current emotional problems, previous abuse, level of daily hassles, maternal perception of the infant and income level . Interestingly, single mothers have lower levels of depressive symptoms compared to women with unsupportive partners (bilszta et al ., 2008). Likewise, a few studies did not find that marital status was always a significant predictor of antenatal depression and anxiety (agostini et al . These findings highlight the importance of considering not only marital status but also the quality of the relationship . Given these results, we could conclude that being a single mother is in a way better than having a difficult and unsupportive relationship . Surprisingly, no studies regarding the psychopathology of the partner as potential risk factor for antenatal depression and anxiety have been identified in this systematic review . Research has been conducted regarding the possible impact of partner psychopathology on maternal depressive symptoms in the first postpartum year (d'anna - hernandez et al ., 2013), but no study has been found in relation to the antenatal period . One meta - analysis (paulson and bazemore, 2010) has found a positive and moderate in size correlation between maternal and paternal depression in the perinatal period (from the 1st trimester until the 1st year of the baby) but a direction of causal influence had not been reported in the studies included in the meta - analysis . Studies have examined many socio - demographic and economic risk factors in relation to antenatal anxiety and depression, but the results are equivocal . Many studies have found a significant correlation between young age and depression / anxiety during pregnancy (bodecs et al ., 2013, fellenzer and cibula, 2014, glazier et al ., 2004, hartley et al ., 2011, lee et al ., 2007, martini et al ., 2015, qiao et al ., 2009, rich - edwards et al ., 2006, rubertsson et al ., 2014, rubertsson et al ., 2003). Perhaps unsurprisingly, adolescents are at increased risk of become depressed during pregnancy (figueiredo et al ., 2007, lanzi et al ., 2009, raisanen et al ., 2014 this confirms what has been found in a systematic review about adolescence and mental health during pregnancy (siegel and brandon, 2014). Nevertheless, some studies found that an older age was positively associated with depression scores during pregnancy (ali et al ., 2012, faisal - cury et al ., 2009, fisher et al ., 2013, gavin et al ., 2011, golbasi et al ., 2010, luke et al, 2009, nasreen et al ., 2011, raisanen et al ., 2014, weobong et al ., 2014, yanikkerem et al ., 2013) and other studies found that age was not associated with depression or anxiety during pregnancy (abuidhail and abujilban, 2014, agostini et al ., 2015, balestrieri et al ., 2012, faisal - cury and rossi menezes, 2007, husain et al ., 2012, husain et al ., 2011, karmaliani et al ., 2009, marcus et al ., 2003, ratcliff et al ., 2015, antenatal depression and anxiety also seem more common in women with low educational achievements (abuidhail and abujilban, 2014, abujilban et al ., 2014, bodecs et al ., 2013, ., 2014, faisal - cury and rossi menezes, 2007, fellenzer and cibula, 2014, gavin et al ., 2011, glazier et al ., 2004, husain et al ., 2011, jeong et al ., 2013, lydsdottir et al ., 2014, marcus et al ., 2003, martini et al ., 2015, qiao et al ., 2009, yanikkerem et al ., 2013). In particular, one study conducted in bangladesh (nasreen et al ., 2011) found that antenatal anxiety was inversely associated with literacy . Nevertheless, two studies conducted in malawi (stewart et al ., 2014) and pakistan (karmaliani et al ., 2009) found that women with more years of schooling were more likely to experiences symptoms of anxiety and depression . A few studies did not find education to be a significant predictor of antenatal depression (agostini et al ., 2015, husain et al ., 2012, lanzi et al ., 2009, luke et al ., 2009, ratcliff et al ., 2015, antenatal anxiety and depression have also been found to be more prevalent in unemployed women (bodecs et al ., 2013,, 2009, giardinelli et al ., 2012, lydsdottir et al ., 2014, marcus et al ., 2003, rubertsson et al ., 2014, rubertsson et al ., 2003) and housewives (balestrieri et al ., 2012, marchesi et al ., 2009, yanikkerem et al ., 2013), compared with employed women, even if a few studies did not find any significant association between employment and antenatal depression (agostini et al ., 2015, glazier et al, 2004, husain et al ., 2011, karmaliani et al ., 2009, lanzi et al ., 2009, zelkowitz et al ., 2004). One study (cooklin et al ., 2007) analysed the role of workplace adversity on maternal wellbeing during pregnancy . It found that poor working conditions, in terms of discrimination and lack of key entitlements during pregnancy, are associated with higher levels of depression . Moreover, women whose partners were unemployed seem to be more likely to experience antenatal depression (akcal et al ., 2014, husain et al ., 2011, karmaliani et al ., 2009, srinivasan et al ., 2015). Studies that have examined the association with low income and financial hardships have also reported contradictory results . While some studies (bodecs et al ., 2013, faisal - cury and rossi menezes, 2007, fisher et al ., 2013, fisher et al ., 2010, prady et al ., 2013, rich - edwards et al ., 2006, weobong et al ., 2014, zeng et al ., 2015) found low income or financial difficulties to be relevant factors, others (abuidhail and abujilban, 2014, josefsson et al . Ethnicity and, in particular, belonging to a minority ethnic group, are other potential risk factors highlighted by some studies, even if the results are also contradictory . The prevalence of antenatal depression seems higher in black, latina and asian mothers compared to white mothers (faisal - cury and rossi menezes, 2007, fellenzer and cibula, 2014, gavin et al ., 2011, melville et al ., 2010, redshaw and henderson, 2013, shakeel et al ., 2015, verreault et al ., 2014), but other studies do not show any significant difference (canady et al ., 2008, others show higher levels of depression among white mothers compared to black and hispanic mothers as well as women of other ethnicities (shen et al ., 2010). Belonging to a minority group could be a risk factor for antenatal depression because of the increased level of stress due to discrimination (prady et al . . However, research in the united kingdom has shown that some minority groups may have lower levels of mental illness than the majority population (edge, 2007). Specifically, in this study black caribbean mothers, despite belonging to a minority ethnic group, living in more deprived areas and being more likely to be lone mothers and to receive less support from their partners, did not show higher levels of depression during pregnancy and the postpartum period, and did not receive more treatment for perinatal depression, compared with white british mothers (edge, 2007). It is possible that social risk factors may have a different impact on the mental health of black caribbean and white women, as the prolonged and repeated exposure to adversity might have provided the black caribbean women with greater resilience and skills to manage psychological distress (edge, 2007, williams and healy, 2001, williams et al ., 1997). This may also be explained by the higher levels of resources (social support, spirituality and self - esteem) in black mothers than in white mothers (jesse et al ., 2005). These resources may act as a buffer against potential sources of risk and reduce the incidence of depression (jesse et al . Therefore, being part of a minority ethnic group may not be an independent factor in the relation to depressive symptoms, and the substantial risk in minority ethnic groups found in some studies may be the result of a higher prevalence of other risk factors . To this end, a study found that the higher prevalence of depression among black and hispanic mothers was mainly explained by lower income and financial hardship (rich - edwards et al ., 2006). Another study found that financial concerns are significantly associated with mental health difficulties during pregnancy, rather than belonging to a specific ethnic group (prady et al ., 2013). Many studies have shown that adverse events in life and high perceived stress during pregnancy play an important role in the onset of antenatal depression (abujilban et al ., 2014, bayrampour et al ., 2015, bowen et al ., 2009, brittain et al ., 2015,, 2009, fisher et al ., 2013, fisher et al ., 2010, gavin et al ., 2011, glazier et al ., 2004, holzman et al ., 2006, husain et al ., 2012,, 2005, leigh and milgrom, 2008, melville et al ., 2010, ratcliff et al ., 2015, rubertsson et al ., 2003, shakeel et al ., 2015, verreault et al ., 2014, woods et al ., 2010, zayas et al ., 2003, zelkowitz et al ., 2004). Stressful experiences may vary from mild to severe, also depending on stress perception and the ability to cope; nevertheless, highly stressful experiences, such as the death or illness of a relative, a relationship breakdown, losing a job, moving home, being assaulted or raped, can trigger depressive and anxiety symptoms or disorders in most individuals . Pregnancy is in itself a well - recognized time of stress because of the many obvious and potential changes and challenges . The occurrence of one or more stressful events can then lead to an increase in the probability that mothers and fathers experience psychological difficulties or mental health disorders . To this end, a study has highlighted the important role of social support in reducing the negative impact of adverse events in life: they have shown that women who experience stressful life events but have good social support are less likely to suffer from emotional distress compared with women without an available support network (glazier et al ., 2004). Therefore, the impact of adverse events in life on antenatal mental health may be mediated by social support . This sustains the stress buffering hypothesis, where supportive interactions among people protect them against the negative health consequences of stressful life events (cobb, 1976, cohen and wills, 1985). Nevertheless, in one study (groves et al ., 2012) social support did not attenuate the effects of partner violence on woman's mental health during pregnancy . Within adverse events in life, being exposed to intrusive life events, such as domestic violence or emotional, physical or sexual abuse, has a considerable impact on a mother's mental health in the perinatal period . A number of studies have found that having been exposed to domestic violence before or during pregnancy (particularly if perpetrated by the partner), having a history of abuse, or having experienced a sexual assault, are all risk factors for the development of antenatal anxiety, depression and post - traumatic stress symptoms (akcal et al ., 2014, ali et al ., 2012, brittain et al ., 2015, dibaba et al ., 2013, edwards et al ., 2008, fisher et al ., 2013, fisher et al ., 2010, fonseca - machado mde et al ., 2015, gavin et al ., 2011, groves et al ., 2012, hartley et al ., 2011, holzman et al ., 2006,, 2005, karmaliani et al ., 2009, leigh and milgrom, 2008, lydsdottir et al ., 2014, mahenge et al ., 2013, martini et al ., 2015, melville et al ., 2010, mezey et al ., 2005, miszkurka et al ., 2012, nasreen et al ., 2011, rich - edwards et al ., 2011, rodriguez et al, 2014, verreault et al ., 2014, waqas et al ., 2015). In particular, a study found that having been exposed to a traumatic sexual experience is associated with antenatal anxiety but not depression (martini et al ., 2015). Another study found that women who are exposed to physical assault or sexual coercion by their intimate partners before or during pregnancy have higher levels of depressive symptoms during pregnancy (martin et al ., 2006). We know from the literature that women with a history of abuse often experience more than one traumatic event during their lives, and have higher life - time levels of depressive and post - traumatic symptoms than women who have only suffered a single trauma . Indeed, one study (holzman et al ., 2006) found a marked increase in depressive scores when adverse circumstances were present both in childhood and in adulthood . This is in line with the theory that accumulation of adversity is more damaging than single events (holzman et al ., 2006). As such, women who have been sexually abused as children are at significant risk of experiencing abuse as adults, including domestic violence (mezey et al ., 2005). In fact, childhood abuse has been identified as a particularly strong predictor of depression and anxiety during the antenatal period (brittain et al ., 2015, fisher et al ., 2013, plant et al ., 2013, robertson - blackmore et al ., 2013, seng et al ., 2014), with women with a history of childhood sexual abuse being twice as likely to develop antenatal depression (robertson - blackmore et al ., 2013). Moreover, the relationship between childhood maltreatment and antenatal depression remains true even when accounting for a woman's antisocial characteristics, personal history of psychiatric illness outside pregnancy, and the availability of partner support during pregnancy, all of which have been identified as salient factors associated with maternal psychopathology (plant et al . Many studies have found an increased risk of developing antenatal depression and anxiety in women with an unplanned or unwanted pregnancy (ajinkya et al ., 2013, akcal et al ., 2014, bayrampour et al ., 2015, brittain et al ., 2015,, 2009, dibaba et al ., 2013, fellenzer and cibula, 2014, giardinelli et al ., 2012, golbasi et al ., 2010, jeong et al ., 2013, karmaliani et al ., 2009, lee et al ., 2007, manikkam and burns, 2012, marchesi et al ., 2009, martini et al ., 2015, mohamad yusuff et al ., 2015, mohammad et al ., 2011, redshaw and henderson, 2013, rich - edwards et al ., 2006, waqas et al ., 2015, weobong et al ., 2014, yanikkerem et al ., 2013), although two studies did not find this (groves et al ., 2012, hartley et al ., 2011) two studies (lee et al ., 2007, marchesi et al ., 2009) observed that an unwanted pregnancy was a significant predictor of depression only in the first trimester, with less importance over time . Some researchers (lee et al ., 2007) suggested that women initially find it more difficult to cope with an unexpected and undesired event and, therefore, are more likely to become depressed; however, as the pregnancy progresses, the shock reduces, and the relationship with the foetus grows stronger, the pregnancy becomes more accepted and the depressive symptoms decrease . Of note is also that fear of childbirth and negative thoughts about the upcoming delivery have been associated with increased risk of antenatal anxiety and depression (raisanen et al ., 2014, rubertsson et al ., 2014); indeed, a study found that a negative experience of pregnancy was significantly associated with antenatal depression (agostini et al ., 2015). The role of parity in increasing the risk of antenatal depression and anxiety is not clear . Multiparous women are at increased risk of developing antenatal anxiety and depression according to some studies (abuidhail and abujilban, 2014, golbasi et al ., 2010, lanzi et al ., 2009, redshaw and henderson, 2013, yanikkerem et al ., 2013) while other studies (ali et al ., 2012, fisher et al ., 2013, raisanen et al ., 2014) found nulliparous or primiparous women as more at risk than multiparous women, and others yet did not find any significant association between parity and antenatal mental health (abujilban et al ., 2013, faisal - cury and rossi menezes, 2007, fisher et al ., 2010, glazier et al ., 2004, husain et al ., 2011, marcus et al ., 2003, ratcliff et al ., 2015, rubertsson et al ., 2003, greater number of previous pregnancies (multigravida) has been found to be a predictor of antenatal depression in a few studies (ajinkya et al ., 2013, records and rice, 2007) while another study found that women in their first pregnancy (primigravida) were more likely to suffer from depression (karmaliani et al ., 2009), and other studies (fisher et al ., 2010, women with current or past pregnancy / delivery complications, with a history of pregnancy loss, pregnancy terminations or stillbirth have been found to be more likely to experience antenatal depression, anxiety and pregnancy - specific anxieties (adewuya et al ., 2014, faisal - cury et al ., 2009, faisal - cury and rossi menezes, 2007, fisher et al ., 2013, golbasi et al ., 2010, gong et al ., 2013, husain et al ., 2011, raisanen et al ., 2014, stewart et al ., 2014, waqas et al ., 2015, weobong et al ., 2014, zeng et al ., 2015) in particular, one study (bergner et al ., 2008) found that women with a history of miscarriage suffer more from trait anxiety and pregnancy - specific anxieties, and those who have had recurrent miscarriages also have higher levels of anxiety . However, some studies (bicking kinsey et al ., 2015, fisher et al ., 2010, jeong et al ., 2013, qiao et al ., 2009) did not find any significant association between previous obstetric history, including a history of either spontaneous or induced abortion, and antenatal anxiety or depression . A history of one or more episiotomies, caesarean section or a previous negative birth experience, have been found to be associated with a high incidence of antenatal anxiety and depression (rubertsson et al ., but another study found no association between mode of delivery and antenatal depression (ajinkya et al ., 2013). In one study, women who had infertility treatments were more likely to suffer from antenatal anxiety (bayrampour et al ., 2015), but another study did not find this (rubertsson et al ., 2003). An unexpected loss of pregnancy can be a traumatic event and can lead to high level of anxiety and depression that can persist into subsequent pregnancies . One study found previous miscarriage to be associated with antenatal depression but no significant association was found with stillbirth (rubertsson et al . The interval between a pregnancy that did not result in a live birth and a new pregnancy seems to play a crucial role in whether the woman will or will not experience anxiety and depression during a new pregnancy: the world health organization recommends an interval of at least 6 months to allow women time to recover physically and mentally (who, 2005). To this end, gong et al . Found that women with a history of miscarriage and inter - pregnancy interval of less than 6 months are at increased risk of developing anxiety and depression during the first trimester of pregnancy (gong et al ., 2013). Some studies have shown that some personality factors increase the risk of experiencing psychological difficulties in the antenatal period, and especially negative cognitive styles: pessimism, anger and rumination; a tendency to be nervous, worried or shy; low self - esteem and low self - efficacy; and high levels of neuroticism or psychoticism (bayrampour et al ., 2015, bunevicius et al ., 2009, ginsburg et al ., 2008, jesse et al ., 2005, lee et al ., 2015, mohammad et al ., 2011, zeng et al ., 2015). On the contrary, active coping and high self - esteem / self - efficacy have been identified as protective factors (edwards et al ., 2008, zeng et al ., 2015). While more research has been conducted regarding general cognitive biases in relation to depression and anxiety, some studies have also pointed out that specific types of cognitive biases have an impact in the perinatal period . A study (sockol et al ., 2014) has found that maternal attitudes regarding motherhood have incremental predictive validity for symptoms of depression and anxiety during the perinatal period over more general cognitive biases . Moreover, maternal attitudes towards motherhood continue to be strongly associated with anxiety and depression even after controlling for interpersonal factors . Examples of maladaptive beliefs about motherhood are: it is wrong to have mixed feelings about my baby; i should feel more devoted to my baby; it is wrong to feel disappointed by motherhood; if my baby is crying, people will think i cannot care for him/ her properly . As such, women with maladaptive beliefs about motherhood are at increased risk of experiencing feelings of sadness, guilt and worthlessness, and to develop anxiety and depression during pregnancy and in the postpartum period, when their feelings do not match their attitudes and expectations . This means that specific beliefs about motherhood could be most strongly activated by the particular stressors of parenting and, therefore, they could mediate the relationship between the specific stressors of becoming a parent and the emotional response to these events (sockol et al ., 2014). In this systematic review, psycho - social, environmental, obstetric and pregnancy - related risk factors for antenatal depression and anxiety have been investigated . Table 1 shows all the studies that have been included and discussed in this systematic review, while table 2 shows all the main risk factors that have been found to be associated with antenatal depression and anxiety . Depression and anxiety have been found to be highly comorbid during the antenatal period, and indeed high anxiety during pregnancy is a risk factor for antenatal depression (verreault et al ., 2014); therefore, most of the risk factors identified here are relevant to both conditions . The most relevant factors associated with antenatal depression or anxiety (ranked according to the number of studies that have found the factors to be significant predictors, and presenting also the number of studies who did not) are: lack of partner or of social support (29 vs. 0); history of abuse or of domestic violence (28 vs. 0); personal history of mental illness (23 vs. 0); unplanned or unwanted pregnancy (22 vs. 2); adverse events in life and high perceived stress (21 vs. 0); present/ past pregnancy complications or pregnancy loss (17 vs. 4); low education level (17 vs. 9); low income (15 vs. 3); single marital status (14 vs. 5); current or past smoking (11 vs. 1); negative cognitive style / low self - esteem and self - efficacy (10 vs. 0); problematic / dissatisfied relationship with partner (7 vs. 0); and childhood abuse (8 vs. 0). Inconsistent results have been found regarding unemployment (11 vs. 6) and black, asian and latina ethnic group (7 vs. 4). Also, 13 papers found young age to be significant associated with antenatal depression and anxiety, while 10 studies found old age to be a significant risk factor, and 10 studies did not find any association with age . Moreover, 5 studies found multiparous women to be more at risk of antenatal depression / anxiety, 3 studies found nulliparous and primiparous to be more at risk, and 10 studies did not find any association with parity . Similar to parity, 2 papers have found women with multiple pregnancies to be more likely to suffer from depression or anxiety during pregnancy, 1 paper found women at their first pregnancy to be more at risk, and 2 papers did not find any association with gravidity . Studies included in this systematic review have also shown the following risk factors to be associated with antenatal anxiety and depression but further studies are needed as only little research has been conducted: current / past alcohol abuse (2 vs. 0), substance use (2 vs. 0), family history of mental illness (2 vs. 0), quality / style of parenting, (3 vs. 0), mode of delivery (2 vs. 1), partner unemployed (4 vs. 0). Surprisingly, no studies regarding the psychopathology of the partner as potential risk factor for antenatal depression and anxiety have been identified in this systematic review . Only one meta - analysis (paulson and bazemore, 2010) has found a positive and moderate in size correlation between maternal and paternal depression in the perinatal period, including pregnancy . Clearly, more research is needed to explore the role of partner psychopathology in the onset of antenatal depression and anxiety . In fact, 29 studies have found lack of support, especially partner support, to be associated with antenatal depression and anxiety, with no studies reporting an absence of significant association . In turn, perceived support and marital satisfaction have been found to be protective factors for maternal mental health during pregnancy (lee et al ., 2007, zeng et al ., 2015) and to mediate the impact of adverse events in life on maternal wellbeing (glazier et al ., 2004 this is not surprising, since social support can help the woman to cope with negative emotions and stressors associated with pregnancy and to prepare positively for the birth and the postpartum period (jeong et al ., 2013). On the contrary, domestic violence and history of abuse have been shown to be some of the strongest independent predictors of antenatal depression and anxiety (dibaba et al ., 2013, lancaster et al ., 2010, melville et al ., 2010). In fact, 28 studies have found domestic violence and history of abuse as significant risk factors for depression and anxiety, and 8 studies have specifically investigated the role of childhood abuse and all found it to be highly associated . These results confirm and extend what has been reported in a recent systematic review on domestic violence and perinatal mental disorders (howard et al ., 2013); they found that women with probable depression during pregnancy had a 3-to-5-fold increased risk of having experienced domestic violence over the adult lifetime, during the past year and during pregnancy . The results suggest a two - way association between domestic violence and antenatal depression: domestic violence is associated with depression during pregnancy while, at the same time, depressive symptoms can increase the probability that the woman may experience violence (howard et al ., 2013). In fact, being exposed to a traumatic event leads to clear psychobiological changes that alter the ability to adjust and cope with subsequent stressful events . In particular, childhood maltreatment is a predisposing factor in the persistent activation of the two main biological systems involved in the stress response, the hypothalamic pituitary adrenal (hpa) axis and the inflammatory system (baumeister et al . The hyperactivity of these two systems is also present in depression, and it is therefore considered to be part of its pathogenesis . As both systems are over - stimulated during normal pregnancy, this may make mothers more susceptible to depression in the context of previous exposure to childhood maltreatment (danese et al ., 2007, heim et al ., 2008, pariante et al ., 2014, pariante and lightman, 2008, robertson - blackmore et al ., 2013). Psychological mechanisms, including reactivation of a previous sexual trauma during pregnancy, experiences and feelings of shame, and low self - esteem, are also very important (mezey et al . Together with partner / social support and satisfaction in the marital relationship, other potential protective factors that have been identified in this review are active coping, high self - esteem and high self - efficacy (edwards et al ., 2008, zeng et al ., 2015). In fact, these self - schema constructs are considered part of the phenomenology of depression and could possible mediate the relationship between adverse events in life and depression (ginsburg et al ., 2008). Education may also possibly function as a protective factor, via enhancing feelings of self - worth and reducing feelings of shame, which in turn may contribute to reduce depressive and anxiety symptoms (nasreen et al ., a previous review investigating risk factors for antenatal depression was conducted by lancaster et al . They found that maternal anxiety, life stress, history of depression, lack of support, domestic violence, unintended pregnancy, low income, lower education, single status, and poor relationship with partner were associated with depressive symptoms during pregnancy in bivariate analyses . Life stress, lack of social support and domestic violence continued to be associated with antenatal depression also in multivariate analyses . Inconsistent results were found for smoking, alcohol and drug use, parity, ethnic group and age, while no significant association was found with employment and obstetric history (lancaster et al ., 2010). Our review addressed a considerable limitation of this previous work, as they considered only studies performed in high - income countries . In fact, we have included studies conducted in low-, middle- and high- income countries, and this increases our power to generalize the results to a wider population . Similarly to lancaster at al ., our review has found that life - time adversities, history of depression, lack of support, domestic violence, unintended pregnancy, low education, lower income and poor relationship with partner are associated with antenatal depression and anxiety ., we also found no or inconsistent evidence for alcohol, substance abuse, age and employment ., we have found that obstetric history is a strong predictor of depression and anxiety during pregnancy . The results of this review confirm previous evidence that mood disorders during pregnancy and early parenthood have a complex and multi - factorial aetiology (bilszta et al ., 2008) where multiple factors are involved in the onset of depression and anxiety during pregnancy . Indeed, leigh and milgrom have proposed an interesting psychosocial model to help understand the diversity of these factors: they suggested that predisposing factors, antenatal stressors, and personal resources, all interact and concur in the development and maintenance of antenatal depression, and subsequently of postnatal depression and parenting stress (leigh and milgrom, 2008). According to this model, predisposing factors, such as a personal history of depression, a childhood history of abuse, low income, young age, and low education, make the woman vulnerable to the onset of antenatal depression . Poor personal resources, such as low self - esteem, negative cognitive style and low social support, make it more difficult for the mother - to - be to adjust positively to the changes in the transition to motherhood . Antenatal stressors, such as antenatal anxiety and stressful life events, can also work negatively to increase the risk of antenatal depression . Other factors involved in this complex process are biological (e.g., changes in the level of hormones) and socio - cultural (e.g., unrealistic beliefs and representation of motherhood as an exclusive time of joy) (milgrom et al ., 1999). The process whereby personal susceptibility is activated by stressful events and other kinds of current factors is also well described by the earthquake model of sichel and driscoll, developed with the aim of conceptualizing women's mental health and its treatment . Sichel and driscoll (1999) suggest that the genetic makeup of each woman, her hormonal and reproductive history, and experiences during her lifespan, all combine to predict her risk of an earthquake at critical times in life such as childbirth (sichel and driscoll, 1999). We did not conduct a meta - analysis of the findings, which may have added additional information about the differential impact of each risk factor . Nevertheless, most of the risk factors described in this review have been independently replicated by a number of studies . Moreover, this review does not specifically examine factors that may influence the severity of episodes of depression or anxiety, or the onset of different types of anxiety disorders . Furthermore, it has not been possible to assess the cumulative effects of different factors; however, this limitation is mainly due to the lack of studies addressing these questions . This systematic review has also excluded studies based on high - risk populations, such as women exposed to major environmental catastrophes, like earthquakes or tsunamis, studies conducted on women with pre - existing health conditions, such as hiv or obesity, or with health conditions associated with pregnancy, such as diabetes, and women with babies with congenital anomalies . Therefore, the generalizability of the findings to these populations may be limited, as studies in these high - risk populations may have possibly led to different risk factors . Moreover, studies that have examined potential risk factors related to physical health have been excluded, as the main aim of this review was to investigate the psycho - social, environmental and obstetric risk factors involved in the onset of antenatal depression and anxiety in the wider, physically healthy population of women . Finally, papers not written in english have been excluded, and therefore some interesting studies may have not been considered . Pregnancy is a time of increased vulnerability for the development of anxiety and mood disorders . Some women may experience their first depressive episode during pregnancy, while others are at risk of recurrence due to a previous history of depression and anxiety (raisanen et al ., 2014). Many studies have investigated the main risk factors involved in the onset of antenatal depression and anxiety and have highlighted a complex multi - factorial aetiology (lancaster et al . Different sources of psychosocial, environmental, obstetric and pregnancy related risk factors have been highlighted in this review . Correctly identifying the women at risk of suffering from antenatal anxiety and depression would give us the opportunity to target those women who would benefit from preventive and supportive interventions . Also, identifying the women at risk would allow us to follow them up during the course of their pregnancy and recognize earlier symptoms of depression and anxiety as they develop, and therefore implement therapeutic interventions if needed . This is now even more relevant, since most recent studies have shown a reassuring safety profile for antidepressant treatment in pregnancy (reviewed in pariante, 2015) and have identified effective non - pharmacological treatments (bowen et al ., 2014, su et al ., 2008, thomas et al ., 2014), although more research is still need to be done regarding the effectiveness of non - pharmacological interventions during pregnancy . The national society for the prevention of cruelty to children (nspcc) funded this review . Professor pariante is also funded by the uk national institute for health research (nihr) biomedical research centre for mental health at the south london and maudsley nhs trust and king's college london . Professor pariante s research is supported by grants mr / j002739/1 and mr / l014815/1 from the medical research council (uk). The corresponding author managed the literature searches and wrote the first draft of the manuscript.
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The structure of the human pelvis permits an upright posture and locomotion on two legs rather than on lore legs like other mammals . Although these structures are well adapted for bipedal locomotion, an upright posture may cause problem . For instance, lower extremity malalignment leading to overload of the femur and tibia has been considered to be the main cause of cartilage degeneration and osteoarthritis of the knee (aglietti et al ., 2008; the alignment of lower extremity maintains correct posture of the upper body and prevents the body from pains due to bad posture (eng and pierrynowski, 1993). Koreans, mostly aged people, are accustomed to sitting - on life style and are disposed to have larger external rotation of hip joint, genu varus of the knee, and inversion of the ankle . The difference of the life style might have effects on the anatomical alignment of the lower extremity and the function of the lower extremity during walking, and might act as the potential risk factors of the knee and the ankle injury . The purpose of the study is (a) to identify the incidence of each lower extremity alignment deformity and identify the relationship of the incidence between the deformities and (b) to identify the effects of physical characteristics (weight, height, age, and gender) and residence styles (sitting - on and standing - up life styles) on the development of 8 lower extremity deformities (genu valgus, genu varus, genu recurvatum, heel valgus, heel varus, tibial torsion, femoral torsion, and leg length discrepancy). 58 males were collected from standing - up life style and 57 from sitting - on life style . Sixty - seven females were collected from standing - up life style and 41 from sitting - on life style each . All subjects were screened and individuals were removed from the test if they had medical problems such as lumbago and previous lower extremity fracture . Each subject was asked wear pants and shirts only . The subjects were asked to answer each question on the questionnaire including age, gender, height, weight, dominant arm and leg . Also, questions about the residence area, years of residence, style of the house if the house is traditional or modern style, bedding style, table style for meal, toilet style, and sitting style for watching tv are included . The visual postural examination of the lower extremity was conducted from anterior, posterior, lateral, sitting, and prone positions . From the anterior view, any sign of genu valgus, genu varus, or genu recurvatum was observed . From the posterior view, the observations for heel valgus or heel varus were performed . From the sitting position, any sign of tibial torsion or femoral torsion was observed . From the prone position, the chi - square analysis was used to identify the difference between the residence styles for each dependent variable . 16.0 (spss inc ., chicago, il, usa) and statistical significance was set at p<0.05 for all tests . 58 males were collected from standing - up life style and 57 from sitting - on life style . Sixty - seven females were collected from standing - up life style and 41 from sitting - on life style each . All subjects were screened and individuals were removed from the test if they had medical problems such as lumbago and previous lower extremity fracture . Each subject was asked wear pants and shirts only . The subjects were asked to answer each question on the questionnaire including age, gender, height, weight, dominant arm and leg . Also, questions about the residence area, years of residence, style of the house if the house is traditional or modern style, bedding style, table style for meal, toilet style, and sitting style for watching tv are included . The visual postural examination of the lower extremity was conducted from anterior, posterior, lateral, sitting, and prone positions . From the anterior view, any sign of genu valgus, genu varus, or genu recurvatum was observed . From the posterior view, the observations for heel valgus or heel varus were performed . From the sitting position, any sign of tibial torsion or femoral torsion was observed . From the prone position, the chi - square analysis was used to identify the difference between the residence styles for each dependent variable . 16.0 (spss inc ., chicago, il, usa) and statistical significance was set at p<0.05 for all tests . Incidence of the lower extremity deviations for men and women according to the residence style was shown in table 2 . For men, the results of incidence of lower extremity deformity according to the life style showed that the incidence of genu varus was significantly high in standing - up life style compared to sitting - on life style (chi - square=8.28; p=0.004). However, the incidences of heel varus (chi - square=13.223; p=0.004) and femoral torsion (chi - square=19.347; p<0.0001) were significantly high in sitting - on life style than standing - up life style . For women, the incidences of genu varus (chi - square=24.18, p<0.0001), heel varus (chi - square=15.412, p<0.0001), and tibial torsion (chi - square = 6.285; p<0.012) were significantly high in sitting - on life style compared to standing - up life style (p<0.05). The odd ratio result for lower extremity deformities for men and women between the life styles are shown in table 3 . For men, the odd ratio result for sitting - on life style against standing - up life style showed 6.6 times significantly high relationship in femoral torsion (95% confidence range [ci], 1.6426.47) in men . For women, there were 3.8 times significantly high relationship in genu varus (95% ci, 1.0713.17), 8.8 times in genu recurvatum (95% ci, 1.5949.00), and 4.2 times in heel varus (95% ci, 1.4711.88) in women . The results of this study show that incidence of genu varus in men was significantly greater in standing - up life style compared to the counterpart . However, the incidence of heel varus and femoral torsion was significantly high in sitting - on life style . Genu varus is commonly observed postural disorders in adolescents (bell et al ., 2013) which is more frequent than genu valgus (cahuzac et al ., 1995). This deformity constitutes a risk factor biomechanically for knee osteoarthritis and aggravates it by increasing the load on the medial compartment (lim et al ., 2008; high incidence of genu varus in standing - up life style group might be explained that traditionally korean people are accustomed to the sitting - on life style rather than standing - up life style even though western life style is stabilized in their current daily life . Significantly high incidence of heel varus and femoral torsion might be due to large external rotation of the hip joint, large varus of the knee, and the large inversion of the ankle joint which is produced from sitting - on life style . Different from their counterpart, korean women experience high incidence of genu varus, heel varus, and tibial torsion in sitting - on life style . The sitting on the surface of the floor makes the knee joint externally rotated which brings heel varus and tibial torsion . According to the results, sitting - on life style has close relationship with the incidence of genu recurvatum in men while it has very high relationship with genu varus, genu recurvatum, and heel varus in women . Lack of femoral control might result in excessive adduction and external rotation of the knee (hewett et al.
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Internal anatomy of the mesiobuccal (mb) root of the maxillary first molar has been studied extensively for the past three decades because this is the root that exhibited the maximum number of variations not only in the presence of extra canals but also in their canal configuration . The literature showed 73.2% to 93% incidence of two canals and only 1.1% occurrence of three canals . Three canals in the mb root with a configuration of vertucci type 8 (3 - 3), sert and bairily type xv, or type xviii (3 - 2/3 - 1) have been reported . However, complex configurations of 3 - 2 - 1 and 2 - 3 - 2 - 1 - 2 canal types in the mb root have rarely been described in studies either in vitro or in vivo . The present case report describes one such atypical configuration of 3 - 2 - 1 in the mb root of maxillary first molar . A young indian male patient reported with the chief complaint of spontaneous pain of moderate intensity in the upper left back tooth region for the past two weeks . He gave a history of localized, mild, intermittent pain in the same region one month back that had increased in intensity in the past three days . On intraoral examination of the second quadrant, the first molar had a deep carious lesion and was mildly tender on vertical percussion . On radiographic examination, there was pulpal involvement with widening of periodontal ligament space [figure 1a]. Based on these findings, the tooth was diagnosed as a nonvital tooth with chronic apical periodontitis, and the patient was planned for endodontic therapy . The maxillary left first molar was first isolated using rubber dam and then access cavity preparation was initiated . After deroofing the pulp chamber, the mb, distobuccal (db), and palatal canals were located . Following along the dentinal map and slowly removing a shelf of dentin covering the dentinal map, a small groove connecting the mb and the palatal canal orifices was identified . Access cavity was further widened toward the mesial marginal ridge to facilitate easy exploration along this subpulpal groove, and by probing with dg 16 explorer two additional sticky canal entrances were identified . These extra canals were then negotiated using no . 08 k files . Based on their anatomic location on the pulpal groove, the canal orifice nearer to the palatal orifice was named as palatal mesiobuccal (p - mb) whereas the one nearer to the mb canal orifice was named as middle mesiobuccal (m - mb) [figure 1b] the three canals in the mb root and their working length were confirmed with multiple angled periapical radiographs using ingle's method as well as electronic apex locator (root zx; morita, tokyo, japan) [figure 1c]. A careful interpretation of working length radiograph revealed a complex canal configuration of 3 - 2 - 1 in the mb roots . A single canal with a single apical foramen was noted in both the distobuccal and palatal roots and the access was closed with a temporary restoration . (a) preoperative radiograph, (b) access opening showing three distinct orifices in the mb root, (c) working length radiograph, (d) postobturation radiograph the presence of this unusual 3 - 2 - 1 canal configuration in the mb root was confirmed using a three - dimensional imaging technique sct - dentascan (ge healthcare, usa) after explaining to the patient . The sct images showed buccolingually wide and bulbous mesiobuccal root with three separate canals at coronal third that merged into two canals in the middle and exited as a single canal at the apex [figure 2c]. Spiral ct images (a) coronal third, (b) middle third, (c) apical third at the next visit, shaping of the mb, db, and the palatal canals was done using ni - ti protaper rotary files (dentsply, maillefer, switzerland) in a passive crown - down manner under copious irrigation with 3% sodium hypochlorite solution . M - mb and p - mb canals were cautiously prepared to avoid stripping of the mb root using only hand instrumentation in step back technique . The canals were then obturated with gutta - percha in conventional lateral condensation technique followed by core build up with silver amalgam . After a week, the tooth was prepared for full veneer crown . At six month recall and evaluation, the tooth was functional and completely asymptomatic [figure 1d]. A clinician should always remember that the occurrence of a single canal with single exit is not always a rule because there is every possibility for the anatomic complexities to exist in any tooth and in any of the roots . Having the knowledge of anatomic variations and the clinician's skill helps in locating and negotiating these complexities . Endodontic success depends on thorough debridement and complete obturation of the entire root canal system . The present case is one such example of the presence of three canals in the mb root of maxillary first molar with unusual internal anatomy . The anatomic configuration of three canals as 3 - 2 - 1 was identified by scouting the canals with hand files . Among the three canals, the major canal was the mb; the second major canal, m - mb, joined the mb canal in the apical third, whereas p - mb is the smallest canal that joined mb canal in the middle third . This same configuration is also confirmed using multiple medially angulated radiographs taken with files in the mb root and further confirmed using three - dimensional imaging technique, sct . Although sct has the disadvantage of more radiation exposure than cone beam computed tomography (cbct), it is used due to the lack of availability of cbct . The axial scans in apical third showed buccopalatally elongated bean - seed shaped mb root, being wider buccally and with concavity toward the furcation that is on its distal side . Taking this shape into consideration, m - mb and p - mb were conservatively prepared so as to prevent stripping on the distal side . A literature search was conducted on pubmed data base to note the reported cases for the presence of three canals in the mb root of maxillary first molar and the results are tabulated in table 1 . The possible canal configurations reported were vertucci's type viii (3 - 3; 3 separate canals with separate foramina), sert and bairley's xv-3 - 2 configuration, xviii-3 - 1 configuration, and xxi-4 - 1 configuration . Case reports of three mesio buccal canals of maxillary first molar sert, in his in vitro study on turkish population, first reported the occurrence of 3 - 2 - 1 canal configuration in the mb root of maxillary first molar . Later, similar configuration was reported by arora et al . In the mb root of the maxillary second molar . Based on this literature review, the present article is one of the rarest configurations to be reported in the mb root of the first molar . An increased awareness of incidence of extra canals in the mb root of maxillary first molar with a better understanding of the complex canal configurations and use of advanced diagnostic aids, such as sct, helps the clinician in proper diagnosis and correct endodontic management of the rarest variations to achieve a successful endodontic outcome.
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Both chinese and american indian medical practices depend on many medicines, including plant medicines . The doctors who use these plants require training in how to identify the plants, prepare the medicines and use the medicines appropriately . Chinese and american indian therapy can also depend on healing touch that is used to ease pain and cure diseases . Both approaches to medicine are very practical, depend on the plants that are at hand and share some of the same elements of philosophy . The purpose of this writing is to put american indian (chumash) healing into a context that will make it more approachable to scientists . Chumash people believe that all life first started on santa cruz island, a channel island off the coast of ventura, california (1). There is scientific evidence that people came from asia to the american continent at several times in the distant past . Although the times of the various crossings remain a subject of research and debate, it is clear that humans have been in the americas for tens of thousands of years (2,3). These people brought with them tools, spiritual and medical knowledge, which could account for certain similarities between east - asian (chinese) and american indian health observances and practices . Chinese medicine is an array of ideas and practices that were developed in china, or adapted from abroad, over the past 3000 years or more (46). This historical dating does not necessary imply that the chinese did not have an oral tradition of health and safety practices prior to this . It only means that there are no known historical records prior to that date . Due to the predominately unwritten nature of american indian medicine, the historical dating of some traditions, such as the medical practices of the chumash people of the southern coast of california, has been difficult for scientists . Some anthropologists claim to find elements of spanish or other european traditions in chumash medical practices (1). Nonetheless, given that traditional narratives are passed down, usually from a person's grandparents, with the expressed understanding that they will be maintained unchanged, it is probably accurate to assume that much of the current medicinal knowledge of the chumash and other american indians is of ancient origin . There has been some speculation that chinese may have visited california in 1423 and had a direct influence on california indians before the arrival of europeans . However, at least one historian claims that a chinese visit to california was unlikely (7). If chinese did visit california, they had little impact, since no legends of such a visit exist among california indians . It is, however, very likely that polynesians visited california and other parts of the west coast of the american continent . Hawaiians brought poppies, cotton and sweet potatoes from america to hawaii prior to the arrival of europeans, and left hawaiian banana plants in mexico (8). Polynesians may have taught chumash people how to make plank canoes 1500 years ago (9). Chinese contact with polynesians is a possibility (7) and may be an indirect way that chinese could have influenced chumash people . The chinese tradition is that the chinese ancestors used plants as medicines since the beginning of human existence . Clearly, all human ancestors, all over the world, have evolved over the past several million years using plants as medicines . The core frame of reference in chinese medical practice is a combination of two key pattern categorization schemas (4): a combinatorial inhibiting - activating model (yin yang), and an associative five - parameter network (five - elements / agents). The yin yang model emanates from ancient chinese metaphysics that views ontogenesis (in the philosophical sense of the term) as the product of a wavering between an inhibiting and an activating force, called yin and yang, and symbolized by a broken (- -) and a solid () line, respectively . Binary qualities, such as cold and hot, female and male, heaven and earth, find their respective counterparts in the yin this universal inhibiting - activating model can be applied to any domain of knowledge, including modern natural sciences and chemistry (10). In chinese medicine, disease is predominantly perceived as a loss of an internal elemental / organic harmony (4,11). This internal state is conceptualized using the five - element theory (11,12). In this model, certain diseases are caused by excessive blood, lymph and qi stagnation that leads to what is known as heat (inflammation). The flow of qi may be increased by increased flow of lymph or blood, or decreased by decreased flow of blood or lymph . Other diseases are caused by a qi, lymph or blood deficiency known as cold, such as the common cold . Treatments consist of restoring the lost internal balance and returning the patient to the normal elemental state . However, for the practice of medicine, it is useful to describe qi as the source of life and to recognize that it belongs to the body . Qi can be described as life energy and is carried by channels throughout the body (11). The five elements contain a pair of yin and yang, which are water and fire . However, the five elements are described as coming from yin and yang (4). Other vital aspects of chinese diagnosis are the patient history and pulse (11). Several pulses are recognized and are used to assess the perfusion of yang and qi to various organs . The pulse may be described in several terms such as thin, deep, slippery, thready, slow and rapid . The pulse is used to assess the flow of qi, blood and lymph throughout the organs . Other diagnostic techniques for assessing the flow of yang and qi include the appearance of the tongue and swollen areas of the body . The appearance of the tongue is very important and may be described in many different ways such as pale, red, white greasy fur, thick yellow fur and thin white fur . Swollen areas such as lymph nodes, cysts, breasts and pimples indicate areas of local obstruction of the flow of blood, lymph, milk or qi . Although the treatment of a specific disease might be similar from one patient to another, there is considerable individualization . For instance, is a patient's fever caused by exposure to too much exopathic wind heat, or a reaction to too much exposure to exopathic wind cold? There is also considerable variation in the use of plants between doctors and between regions in china (13). A patient with a disease caused by cold is treated with a plant that augments heat . A hot treatment may have adrenergic properties, which are manifested by an increased blood perfusion and heart rate . A cold treatment, on the contrary, may have cholinergic properties and can shunt the blood to vital organs, produce a pale complexion and slow the heart rate . The chumash believe that life and death are decided by the sun and sky coyote, called snilemun in chumash (9). If the sun wins, the year will be hot and dry, food and medicine will be scarce and many people will die . If sky coyote wins, the year will be cool and wet, food and medicine will be abundant and people will survive . Sky coyote is beneficial to humans, and is seen in the sky as the north star . Knowing the position of sky coyote in the sky helps prevent people from becoming lost . He is a trickster according to legends and can sometimes cause floods and other problems . The chumash use ceremony and prayer to encourage the balance between the sun and sky coyote each year . Sweat lodges are heated with fire and steam to treat sickness, especially colds, flus and pulmonary infections . The antap (healer) takes a detailed history from the patient and relatives . The appearance of the patient's eyes, tongue, the antap may look intently into the patient's eyes since the antap's eyes are mirrors that make patients see themselves and see the truth . The antap may start with helping the patient to breathe more efficiently, by having the patient sing or suck on salvia apiana (white sage). Antap usually touches the patient to find the areas that are affected and the distant areas that may be causing the problem . For instance, if a patient complains of pain in the lower abdomen, the antap may touch the back to find out if an imbalance in the back is causing pain in the abdomen . The purpose of medical treatment is to restore balance to the patient, since balance is health . A sprain resulting from too much heavy lifting may be treated with rest and casting the area with scirpus acutus (tule). A cold from swimming in cold ocean water may be treated by heat treatments in the sweat lodge (1). Acupuncture consists of inserting one or more needles into specific points on the body surface for therapeutic purposes . This practice is generally believed to have originated in china, since sharpened stones and bones that date from about 8000 years ago have been interpreted as instruments for acupuncture treatment (1417). Nonetheless, tattoo marks seen on the ice man who died in the alps about 5000 years ago and whose body was revealed when a glacier melted (18), might indicate that acupuncture is not purely a chinese phenomenon, and that a form of it developed in europe quite independently of china but later disappeared (19,20). The earliest chinese texts to mention channels (i.e. Meridians) associated with diagnosis and treatment are dated around 150 bc (21,22). These channels and points are not related to nerves, blood vessels or other anatomical features, although recent anatomical research indicates that acupuncture channels follow fascia lines . Chinese believe that these meridians are energy lines that allow the flow of qi in the body . There are several methods and schools of thought regarding point selection: some apply needles directly to the painful site, some use distant non - painful sites and some use a combination of both sites . Acupressure, the application of pressure to points, has also been used in china as a means of relieving pain and treating diseases . It is clear that acupuncture can increase the release of endogenous pain relieving ligands in the body, such as enkephalins and endorphins (25). Healing touch is used by the chumash to comfort, cure and relieve pain (27,28). Usually, the treatment of pain involves manipulation of non - painful body areas to relieve pain in distant sites . Manipulation can involve the hands or deer antlers that are applied to sites to relieve pain . The areas that are treated to relieve distant pain are known to the healer from experience and from training by teachers . Manipulation with deer antlers can involve the rounded or pointed parts of the antlers, and may be applied with pressure, depending on the condition of the patient . The chumash have not devised formal channels of energy flow in the body, but recognize that a balance of power in the body is essential to health and that power is constantly decreasing (27). The chumash realize that water flows in the body . Where the flow of water is interrupted, an area of inflammation and pain results . Healing touch is used to manipulate non - painful areas since touch produces vortices in the body water that flow like waves to help establish flow in congested areas . Chumash view power as incorporeal, not restricted to a body or object (27). Healing power can be transmitted to an object, such as a healing rock (1). Antap through prayer and treating the rock with powdered eriodictyon crassifolium (yerba santa) leaves in eel oil (1). Once the rock has been given power, the rock is wrapped in white down and can be used to heal people . By touching a sick person with the rock, this is the inverse of acupuncture where qi comes from the sick person (14). Acupuncture sometimes relies on moxibustion with artemisia argyi to heat the area being treated (29). Chumash use artemisia douglasiana (formerly a. vulgaris) as a way of cauterizing wounds (1). Chinese ethnobotany is a highly structured practice, based on combining medicinal plants, minerals and animal parts according to their taste, hot or cold properties and channel - action . Plant medicine combination is based on the theory that natural medicines have complementary, synergistic and augmentative actions on each other . A chinese traditional prescription, is a highly structured, and hierarchical formula with elements named after the political positions of ancient china, and includes a chief (king or emperor) herb, a deputy (magistrate), an assistant (adjutant) and an envoy (messenger) that collectively potentiate the therapy (11). Deputy herbs might increase the action of the chief herb or treat its unwanted effects, such as dry mouth or blurred vision . Salvia apiana is added to almost any plant preparation in order to magnify the effects of the plant (28,30). For instance, dan shen (salvia miltiorrhiza) is a very popular chinese medicinal plant . A chumash plant called chia (salvia columbariae) is essentially equivalent to s. miltiorrhiza and contains tanshinones and a salvianolic acid (32). Both plants are used in the treatment of heart attack, stroke and other conditions in china and california . Table 1.california and chinese plant medicines a comparison of their usescalifornia plantcalifornia usechinese plantchinese usemajor chemical constituentsachillea millefoliumpainachillea alpinaabdominal discomfort, antibacterialflavonoidsaesculus californicatoothaches, hemorrhoid painaesculus chinensispain, digestion, increased strength and circulationescinsallium haematochitonstings and bitesallium sativumantibacterial, anticancer, stroke and heart attackalliin, allicin, ajoeneapocynum cannabiniumintestinal and lung diseasesapocynum venetumliver disease, antihypertensive, diureticglycosides, flavanols, cardenolidesaquilegia formosadiarrhea, stomach acheaquilegia ecalcaratatoxin elimination, muscle growthflavonoids, alkaloids and glycosidesartemisia douglasianawound cauterizationartemisia argyimoxibustionmonoterpenoidsastragalus purshiimenstrual painastragalus membranaceusdiuretic, antidiabetic, immunostimulantcycloartane glycosides, flavones, sitosterolbaccharis pilularispoison oak rash, skin diseasesbaccharis indicaskin diseases, stomach acheflavones, diterpenoidsberberis aquifoliumgonorrheaberberis soulienaantibacterialberberine alkaloidsclematis ligusticifoliasore throat, pain, coldsclematis chinensispain, diuretic, antibacterialanemonin saponinsdatura wrightiianesthesia, pain, asthmadatura metelpain, asthma, arthritisscopolamine, hyoscyamineephedra californicabladder infections, colds, painephedra sinicaasthma, colds, headache, painpseudoephedrine alkaloidsgnaphalium californicumpain, colds, gi problemsgnaphalium affineantitussive, expectorant, asthmaflavonoids and diterpenoidsiris missouriensispain, venereal diseaseiris pallasiicanceriris quinonejuniperus californicacolds, fevers, constipation, painjuniperus chinensisdigestion, circulation, arthritisamentoflavone, hinokiflavone, cedrollobelia cardinalispain, lung problemslobelia chinensisdiureticlobeline, lobelanineoenothera elatabladder and yeast infectionsoenothera odoratafevers, coldsmonoterpenes, flavonoids, sterolsopuntia littoralispoultice for wounds, painopuntia dilleniipromotes circulation, diarrhea, burnstriterpenoids, beta - sitosterolpaeonia californicadepression, lung disease, painpaeonia lactiflorafever, pain, liver diseasepaeoniflorin, paeonolpapaver californicumpainpapaver somniferumpain, diarrheamorphine and papaverine alkaloidsprunus ilicifoliumcolds, flus, headachesprunus armenicapain, antitussiveflavonoids, amygdalinquercus lobatadiarrhea, painquercus acutissimadiarrhea, hemorrhoidsstarch, tanninsrhamnus californicapurging, constipationrhamnus crenatafever, antifungal, constipationanthraquinone glycosidesrhus trilobatacolds, stomach ache, soresrhus chinensisdiarrhea, gi infections, skin problemsflavones, salicylates, tanninsrosa woodsiidiarrhea, coldsrosa laevigatadiarrhea, astringentvitamin c, flavonols, tanninssalix lasiolepisfever, colds, pain, malariasalix babylonicagoiter, tuberculosis, expectorantsalicin, saligenin, iodinesalvia columbariaestroke, heart attacksalvia miltiorrhizastroke, heart attacktanshinones, salvianolic acidssambucus mexicanacolds, flussambucus chinensisarthritis, edema, diarrhea, bronchitisflavonoids, sterols, tannins, alkaloidsscirpus acutuscastsscirpus dichotomadiureticdihydrocyper - aquinonesolanum douglasiianesthesiasolanum nigrumantibacterial, diureticalkaloids, steroidsstachys bullatastomach ache, inflammationstachys baicalensisinflammation, bleeding disorders, diarrheaflavonoid glycosidessuaeda esteroasoapsuaeda glaucafever, blood circulationtriterpenoids, sterolsurtica dioicaarthritis painurtica angustifoliadigestion, painhistamine, serotonin, leukotrienesthe data in this table is a compilation of published data (30,37,38). A comparison of their uses the data in this table is a compilation of published data (30,37,38). In china, rice porridge is used for any condition involving diarrhea, such as the flu, and is definitely effective . Rice porridge is frequently the first medicine given whenever a person becomes sick . The chumash use acorn porridge in the same way, to treat diarrhea (1). The most popular oak acorns are from quercus agrifolia, quercus douglasii and quercus lobata (30). Some plants of the same genus are medically valuable, but are not used for similar purposes in china and california . For instance, allium haematochiton from california is used to treat bites and stings, mostly from insects . In china, the chinese have imported many american plants and grow them in china for use as medicines . Agave sisalana, asclepias curassavica, cucurbita moschata, datura stramonium, helianthus annuus, hyptis suaveolens, nicotiana tabacum, panax quinquefolius and others from the american continent are now grown in china . Papaver somniferum, from turkey and the orient, is grown in the usa as a source of morphine . The chumash recognize that each person has a spirit that is eternal and not bound to the body since it can travel to heaven (similaqsa) after death (27). Spirit is what gives humans hope, the will to do good deeds, inner strength and tranquility . Sometimes the spirit forgets how to be well (30) and must be treated, usually with salvia apiana, in order to purify, calm and strengthen the spirit . When the spirit is rebalanced in this way, normal health can return . Some chinese recognize a non - corporeal, eternal spirit in humans also (33). The spirit has five components such as heart spirit that is involved in propriety and the ability to communicate properly, will that is involved in destiny and wisdom, non - corporeal soul that is involved in suffering and empathy and consciousness that is involved in motivation and creativity . The spirit can be treated by some chinese doctors in order to help a person regain purpose, will, empathy, creativity and other virtues . The chumash believe in an underworld that contains spirits called nunasis (pronounced nunassus). The nunasis come out at night to create problems for people, including illness (27). The doctor must be like the owl that flies at night with keen vision to see potential danger and prevent illness . Chinese believe in ghosts, gwei in chinese, that are never born and will never die . These ghosts come out mostly at night to cause disease and other problems for people (4,34). Feng shui (sometimes spelled feng swei) techniques are used to divert ghosts from entering houses where they might harm people . Of course there are dangers that fly at night, such as malaria - spreading mosquitoes . Malaria was introduced into california in 1833, killed 20 000 indians in california and was eradicated after the introduction of mosquito - killing ddt (30). Cold night winds can lead to exposure that may promote colds, flus, pneumonia and other diseases . Many chinese believe that exposing the head to cold night wind will lead to illness such as arthritis . I believe in nothing. This beautiful song could easily be from a chinese philosophy book . Chinese taoist philosophy teaches that excess wealth or knowledge is not good for people and may lead to disaster or illness (4). A person who possesses great knowledge, such as healing knowledge, will soon lose that knowledge . Therefore, it is best for the doctor to empty himself of knowledge and be ready to find a new remedy . I alone am drifting, not knowing where i am . Like a newborn baby before it learns to smile, i am alone without a place to go . Others have more than they need, but i alone have nothing. The quotation above is from the tao te ching (35) and was written> 2500 years ago by lao tzu, the founder of taoism . The antap must be a servant of the people, must be virtuous and must be spiritual . Although chumash healers are well paid for their work, they cannot be greedy . An each individual in a village was vital to the survival of the village . If an antap failed to heal three patients in succession, the healer might be killed by the village leaders . When a village felt the healer had lost his ability to heal, it was time for the antap to die (36). The chumash view of nothingness is that the antap and patient must empty themselves of preconceived ideas and be ready to accept spiritual guidance in the healing process . The antap must be in a detached state and must accept that his current knowledge is nothing, or useless . This allows the antap to create and the patient to accept the new treatment and possible healing . The chinese view of nothingness is that holding onto knowledge makes the doctor lose knowledge (4). It is better for the patient and doctor to accept the disease and to approach healing without preconceived ideas . The doctor and patient are encouraged to be like water that flows where it must . This prepares the patient and doctor to accept the results of therapy . Of course, chinese people used plants as medicines before taoism was conceptualized . The taoist approach to medicine is an old approach in china that is not as popular now as it was in the past . It is remarkable that two very divergent cultures, chinese and chumash, could have produced medical approaches that are similar in some ways . There is no question that chinese culture is based on a very strong central government and supports very large cities . However, both chinese and chumash medical practices are very practical and put patient treatment and cure as the foremost objectives . Could the chinese have come to california in 1423 and had a direct influence on chumash people? However, it is remarkable that artemisia fibers are used similarly by chinese and chumash people . It is possible that chumash people learned from the chinese how to use artemisia fibers . However, chinese medicine retains the traditional uses of plants, acupuncture, feng shui and other practices . . However, like the chinese, chumash people retain their use of traditional plant medicines, healing touch and other practices that have always been used by their ancestors . There has been some criticism of traditional chinese medicine from a scientific point of view (33). Is it a science? If we mean by science the relatively recent intellectual and technological development in the west, chinese medicine is not scientific. Chumash medicine could be criticized on the same basis . It is certainly true that chinese traditional medicine and chumash medicine are not limited to the narrow confines of current american scientific fads, such as knock out mice, polymerase chain reaction technology and other molecular biology techniques . Scientific observations are used to form theories that can be applied to the betterment of the human condition . The chinese have empirically and quantitatively observed patients and have formed theories that are useful in curing disease . There is no question that chinese medicine works, since chinese people still exist today . Chumash medicine is also a scientific approach to curing patients and returning them to productive lives . It is very fortunate that chinese and chumash healing have produced theories of disease causation that differ from western theories . This provides an opportunity for scientists to examine alternative approaches to disease causation and treatment that are not being currently used in western hospitals.
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Diabetic retinopathy is a major cause of visual loss worldwide, with vision - threatening diabetic retinopathy being present in 10% of people with diabetes . Diabetic macular edema (dme) is the main cause of visual impairment in diabetic retinopathy, and there is ample evidence for the role of vascular endothelial growth factor (vegf) in the pathogenesis of dme.16 several large clinical trials have established the important role of the anti - vegf agents, ranibizumab and bevacizumab, in the treatment of dme.712 aflibercept is a novel anti - vegf agent composed of the vegf - binding portions of the extracellular domains of human vegf receptors 1 and 2 fused to the fc portion of human immunoglobulin g1 . The da vinci, vista, and vivid studies have established that treatment with aflibercept yields greater visual gains than macular laser treatment, and the drug has since received us food and drug administration approval for this indication.1315 a recent trial has also demonstrated that aflibercept is superior to ranibizumab or bevacizumab for eyes with dme and poorer presenting acuity . Currently, many patients who have received ranibizumab or bevacizumab for the treatment of dme and who have failed to respond to these drugs are being switched to aflibercept . Differences in the pharmacodynamics of aflibercept compared with ranibizumab or bevacizumab have been the basis of this strategy . Unlike ranibizumab, aflibercept binds vegf with much greater affinity and also binds placental growth factor . Some patients may also be switched to aflibercept due to its reportedly longer duration of action . Retrospective data from subjects receiving this treatment for age - related macular degeneration have suggested that anatomical improvement with less significant visual improvement can be expected . The aim of this study was to evaluate the short - term outcomes following conversion from ranibizumab or bevacizumab therapy to aflibercept for the treatment of refractory dme . This was a retrospective series of patients treated at two tertiary eye care centers, ie, singapore national eye centre and massachusetts eye and ear infirmary . Subjects were identified from electronic medical records using the appropriate international classification of diseases and billing codes . Subjects were included if they had been treated for dme with ranibizumab and/or bevacizumab and were subsequently converted to aflibercept between september 2012 and september 2014 . Exclusion criteria included other visually significant ocular pathology and complications of diabetic retinopathy (eg, tractional retinal detachment, vitreous hemorrhage), loss to follow - up, fewer than three ranibizumab and/or bevacizumab injections prior to conversion to aflibercept, and incomplete imaging or clinical data . Patient demographics, systemic comorbidities, and patients diabetes control (hba1c levels) were recorded . The main outcomes of note were the best - corrected visual acuity, which was recorded as snellen visual acuity and converted to logmar equivalents for analysis, and the anatomical changes as documented on optical coherence tomography . Spectral - domain optical coherence tomography was performed using the cirrus (carl zeiss meditec, dublin, ca, usa) or spectralis (heidelberg engineering, heidelberg, germany) platforms . The main optical coherence tomography measures of note were central foveal thickness (cft) as well as the presence of intraretinal or subretinal fluid on the tomogram . The cft values were automatically generated by the imaging software and used in the analyses after the tomograms had been reviewed for segmentation errors . In cases where segmentation errors were seen, intraretinal or subretinal fluid was classified dichotomously as either present or absent, with cases in which only trace fluid was seen (very fine intraretinal cysts or very thin slivers of subretinal fluid) being classified as absent . The study complied with the health insurance portability and accountability act of 1996 and was performed in accordance with the tenets of the declaration of helsinki as revised in 1989 . This was an observational, retrospective, noninterventional study, and approval and waiver of consent were obtained from the institutional review boards of both institutions . The median number of ranibizumab or bevacizumab injections before switching to aflibercept was six, and the median number of aflibercept injections after switching was three . The mean interval between bevacizumab / ranibizumab injections prior to the switch was 2.42.2 months and the interval between aflibercept injections was 2.42.2 months (p=0.92). The mean cft at presentation was 385.65115.16 and 431.18147.79 m immediately prior to the switch (p=0.17). The mean cft after the first aflibercept injection reduced significantly to 362.5792.82 mm (wilcoxon signed - rank test; p<0.001). At the end of follow - up, the mean visual acuity was 0.420.23 logmar just prior to the switch, 0.390.31 logmar after one aflibercept injection, and 0.370.22 logmar at the end of follow - up (figure 2). The final visual acuity was significantly better compared with the visual acuity before the switch (p=0.04). Three of the 21 eyes (14%) had complete resolution of intraretinal cysts at the end of follow - up . A representative case is shown in figure 3 . A 78-year - old woman had long - standing diabetes with a recent hba1c of 7.5% . After four bevacizumab injections, the right eye had persistent center - involving dme with a cft of 516 mm and large cystoid spaces (figure 3a). After six aflibercept injections, the cft was 357 mm and the cystoid spaces had resolved, although a hyperreflective clump of hard exudates was still seen with disruption of the ellipsoid zone (figure 3c). In the course of dme treatment, physicians may choose to switch between anti - vegf agents for various reasons . Clinical reasons include the theoretically greater affinity of aflibercept for vegf as well as the fact that it also binds to and neutralizes placental growth factor . These pharmacodynamic differences may be particularly useful in situations unresponsive to ranibizumab or bevacizumab . In addition, physicians may choose to switch a patient to aflibercept to take advantage of its reportedly longer duration of action . Anti - vegf treatment with bevacizumab or ranibizumab has been clearly shown to be effective and superior to laser for the treatment of dme involving the foveal center . Pegaptanib was the first anti - vegf agent shown to be effective in the management of dme.16 several large trials, including the read-2 study,7 resolve,8 and the diabetic retinopathy clinical research (drcr) network group studies,9,10 have shown anti - vegf treatment with bevacizumab or ranibizumab to be superior to laser alone . Despite these encouraging results with anti - vegf agents, some cases either do not respond at all to ranibizumab or bevacizumab, or demonstrate an initial response to treatment followed by subsequent nonresponse to further treatment or during disease reactivation . Pharmacodynamic mechanisms include increased vegf expression from inflammatory cells and upregulation of vegf receptors, while pharmacokinetic tolerance represents an immune response to the anti - vegf antibodies injected leading to rapid clearance . In these cases, for example, an intravitreal dexamethasone implant (ozurdex) has been shown to be effective in eyes unresponsive to treatment with bevacizumab or ranibizumab.17 more recently, aflibercept has likewise been shown to be effective in the treatment of center - involving dme, producing results comparable with those for ranibizumab and bevacizumab . The da vinci trial evaluated different doses and dosing regimens of aflibercept with laser photocoagulation in eyes with dme.13,14 patients with center - involving dme were assigned to either laser treatment or one of four aflibercept dosing groups . At 24 weeks, all the aflibercept groups had greater visual gains than the laser group, with mean improvements in acuity ranging from 8.5 to 11.4 letters for the aflibercept groups compared with 2.5 letters for the laser group . The differences were even more distinct at 52 weeks, with letter mean gains of 9.712.0 in the aflibercept groups versus a mean letter loss of 1.3 in the laser group . All the aflibercept groups also demonstrated significantly greater reductions in cft compared with the laser group . The vista and vivid trials have shown similar results, with aflibercept being superior to laser.18 the drcr network has recently published the results of a seminal trial comparing the three anti - vegf agents for dme.19 over 1 year, eyes with presenting visual acuity worse than approximately 20/50 experienced greater visual improvement on treatment with aflibercept compared with bevacizumab or ranibizumab, with mean letter gains of 18.9 with aflibercept, 11.8 with bevacizumab, and 14.2 with ranibizumab . These studies suggest that cases unresponsive to ranibizumab and bevacizumab may respond instead to aflibercept . To our knowledge, there are limited data on the results of switching to aflibercept after treatment with ranibizumab or bevacizumab for dme . Our study is limited by its small sample size and the inherent limitations of a retrospective report . Different optical coherence tomography platforms were also used, although each patient was always imaged with the same platform for follow - up scans . Nevertheless, significant visual and anatomical improvement with cft reduction were seen after just one injection and at the end of follow - up . Our findings are consistent with those of the drcr network study,19 and support the notion that aflibercept may be more effective than ranibizumab or bevacizumab for more resistant cases . However, complete elimination of chronic cystoid spaces is difficult, and longer follow - up is needed to determine the effects of these lesions on vision . Patients with center - involving dme who are unresponsive to ranibizumab or bevacizumab demonstrate anatomical and visual improvements after conversion to aflibercept . While aflibercept injections are much more costly than bevacizumab, the improvement that can be anticipated in these challenging cases suggests that making the switch is justifiable.
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Most protein annotation and classification approaches depend heavily on the degree of observed amino acid sequence similarity to other related proteins . But even when sequence similarity between two proteins is low, structure similarity can be high . Thus, one of the most important improvements in protein classification would be protein homology / analogy identification at very low levels of sequence similarity (1). As redfern et al . (2) explain despite the advances in sequence comparison methods, remote homologs in the midnight zone of sequence similarity (<15% identity) described by rost, can still only be identified through protein structure comparison. Redfern et al . Also point out that structure - based classifications are becoming increasingly important resources for recognizing these distant relatives and providing datasets for more far - reaching analyses of protein family evolution. In our research and development we follow these observations, and in order to detect the benefits and limitations of using purely structure - based approaches, we curently concentrated on structure similarity analyses only . The protein data bank (pdb) (3) already contains more than 45 000 experimentally solved protein structures, and grows at a rate of more than 500 pdb entries per month . Among current entries, approximately 40% are multi - domain proteins (2) and, thus, there have been several attempts to classify individual domains of pdb protein structures into defined clusters (e.g. Classes, folds, superfamilies, families) based on structure similarity as measured by various criteria . The most commonly used protein classification databases are scop (4) and cath (5). The structural classification of proteins (scop) database, a manual classification of pdb structures, is recognized by many as the gold standard of protein classification . In scop clustering is based mainly on visual inspection of similarities between conformations of secondary structure elements and on sequence similarities . However, scop classification lags the insertion of new structures in pdb, and manual classification cannot scale to meet the demands of this rapidly growing dataset . For example, clustering can be done by selecting a single metric (e.g. Z - score (6) used in fssp (7) dali fold classification) or by combining different criteria to score the level of similarity, some examples of which are secondary structure content and orientation combined with calculated sequence similarities, and manual inspection, as used in the semiautomatic cath database . Depending on the algorithm, classification results may differ significantly if different criteria are used to assess the level of similarity between compared structures or if the clustering criteria are focused on different structural features . The same set of proteins could be grouped differently by automatic sequence or structure comparison tools based on minor modifications to cutoffs or classification parameters . The dali fold classification is based on exhaustive, all - against - all 3d structure comparisons of proteins from the pdb, and is constructed by average linkage clustering of the structural similarity score derived from calculated alignments of distance matrices . The tree (dendrogram) is cut at dali z - score levels 2, 4, 8, 16, 32 and 64, where the first level (z> 2) can be used as an operational definition of folds . A similar approach is used in ce (8) classification . After performing all - against - all comparisons of protein chains from the pdb, resulting ce z - score values of 4.5 and above are used to discriminate at the family level, values between 4.0 and 4.5 at the superfamily and/or fold levels, and values between 3.5 and 4.0 are presumed to indicate possible biologically interesting similarities . The authors of the struster (9) method explore the calculation of root mean square deviations (rmsd) and use their algorithm to cluster alternative structural models from the pdb (i.e. Models that correspond to different structure determination experiments). In addition to the traditional rmsd measure, the struster method uses two filters to define the final scoring metric called dissimilarity measure m (9). These two filters are introduced in order to identify both large and small (but significant) backbone conformational changes by reducing the influence in local large distances (only distances below 14.0 are considered) and also to restrict the analysis to significant structural differences (the distances above 1.0). An approach for structural comparisons, fundamentally different from those using rmsd, was proposed by rogen and fain (10). They introduced the sgm (scaled gauss metric), which is a metric derived from knot theoretical ideas to cluster proteins according to their structural topologies . They applied their method to predicting membership of proteins in cath and achieved 95% accuracy at all levels of the classification hierarchy . In order to achieve a high level of agreement with other clustering schemes, some algorithms that use a multi - criterion approach (weighted combination of different scoring schemes), are initially trained on labeled data from an existing structural hierarchy (scop or cath) and use cross - validation (or similar methods) to select the best parameters for their classifiers . For example, protclass (11) uses a nearest - neighbor - based classification scheme and several structural features to classify proteins at the fold level of the scop hierarchy . Their features include secondary structure elements predicted by the stride program (12), the sequence length, and the percentage of observed helices . Scopmap (13) is an approach that achieves roughly 95% accuracy when classifying proteins into the superfamily level of the scop hierarchy . This approach combines many existing protein sequence and structure comparison tools, including psi - blast (14), mammoth (15) and dali (6). The classification results depend on the accuracy of the individual tools, so the authors use a variety of cutoffs and parameters optimized by training schemes to apply these tools in a specific order . In ref . (16), the authors introduce a new structural representation of proteins to predict the family membership of proteins in the scop hierarchy . They define a graph theoretic representation of protein structures with nodes being residues and edges connecting residues when the distance separating them falls below a specified cutoff . Using these graphs as features, they train their support vector machine (svm) classifier with proteins from several scop families . The ultimate goal of the work presented here was to define criteria and to develop an algorithm that for a given set of protein structures would automatically identify structurally conserved regions and use them to create clustering results similar to those that would be obtained by manual inspection (e.g. Scop curators). In our novel approach, called stralcp (structure alignment - based clustering of proteins), for a given set of protein structures, we generate and combine detailed structural information about automatically detected global and local similarities between protein pairs, identify similar regions that are conserved within the set of proteins, report these regions, and use them to cluster the proteins according to their similarities in such identified structural frames . We use the local - global alignment (lga) algorithm (17) to perform all necessary structure comparison calculations . Our algorithm starts from structure alignment calculations performed by lga (with a default value of distance cutoff dist = 5) to determine de novo (no sequence information is used) residue residue correspondences between compared proteins . We use the lga_s measure as a scoring function to evaluate the overall level of structure similarity and to allow an initial grouping and structural clustering of proteins . In our stalcp approach, an optimal number of clusters is determined by grouping models according to their overall similarity (lga_s) combined with the information about local similarities in detected structurally conserved frames (we call them spans). To perform a particular clustering for a set of protein structures, a suitable scoring function or, in general, a scoring algorithm that takes into account a number of characteristics of the compared proteins must be defined . Depending on the goal of the clustering, this can be done by selecting one measure or by combining different criteria to score the level of similarity . The lga_s scoring function has two components, lcs (longest continuous segments) and gdt (global distance test), defined for the detection of regions of local and global structure similarities between analyzed structures . In comparing two protein structures the lcs procedure localizes and superimposes the longest segments of residues that can fit under a selected set of rmsd cutoffs . The gdt algorithm is designed to complement evaluations made with lcs searching for the largest (not necessary continuous) set of let: m the number of residues in m, t the number of residues in t, r(r, m, t) = 100/t * l(r, m, t) is the percentage of the target's (t's) residues that are involved in the maximal (longest) continuous segment that fits within an rmsd of r . L(r, m, t) is the length of such identified longest continuous segment of m: t residue pairs, x(m, t)the set of all m: t superpositions calculated by the lga algorithm, g(s, d, m, t)the number of m: t residue pairs for which the distance between ca (alpha carbon) atoms is not greater than d after the superposition s x(m, t) is applied, d(d, m, t)=100/t * max{g(s, d, m, t):s x(m, t)} is the maximal detected percentage of the ca atoms in t structure that are within a distance threshold of d from m structure upon calculated s x(m, t) superpositions . The number of residues in m, t the number of residues in t, r(r, m, t) = 100/t * l(r, m, t) is the percentage of the target's (t's) residues that are involved in the maximal (longest) continuous segment that fits within an rmsd of r . L(r, m, t) is the length of such identified longest continuous segment of m: t residue pairs, x(m, t)the set of all m: t superpositions calculated by the lga algorithm, g(s, d, m, t)the number of m: t residue pairs for which the distance between ca (alpha carbon) atoms is not greater than d after the superposition s x(m, t) is applied, d(d, m, t)=100/t * max{g(s, d, m, t):s x(m, t)} is the maximal detected percentage of the ca atoms in t structure that are within a distance threshold of d from m structure upon calculated s x(m, t) superpositions . Lga_s(m, t) structure similarity scoring function is defined as a function of two structures m and t calculated as a combination of r(r, m, t) results from lcs(m, t) calculations, and d(d, m, t) results from gdt(m, t): where and w = 0.75 is a parameter (0 w 1) representing a weighting factor between s(lcs) and s(gdt) results . S(lcs) is a weighted sum of r(r, m, t) values calculated for n different rmsd cutoffs r (e.g. N = 3; r = 1.0, 2.0, 5.0), and s(gdt) is a weighted sum of d(d, m, t) values calculated using k different distance cutoffs d (e.g. K = 20; d = 0.5, 1.0,, 10.0). In the formulae s(lcs) and s(gdt), the weighting schemes weight higher those r and d results that were calculated for smaller rmsd and distance cutoffs, respectively . The range of the lga_s values is 0100, and hierarchical clustering experiments performed on various folds from scop database showed that lga_s alone can serve as a good discriminator for the initial protein structure clustering (see the results shown in figure 5a). The essence of the stralcp algorithm is the ability to compare hundreds of protein structures in a single reference frame, identify similar fragments that are conserved within a set of analyzed proteins, and use this information to calculate the number of required clusters . Each calculated cluster is assigned with its structural fingerprint that can be defined by a representative structure and a set of spans that are shared among structures grouped together . Comparison of a new structure with a structural fingerprint determines whether the structure should be included to the particular cluster or whether it should be a member of another cluster . Our stralcp algorithm, which automatically clusters proteins and identifies representative structures, can be described as the following list of steps: lga is used to perform all - against - all comparisons in which, for a given set of structures, each structure is used as a frame of reference for comparisons with others.each frame of reference is assigned a set of sequential fragments, which are defined by splitting the corresponding amino acid sequence into consecutive n - residue - long sub - sequences (n = 10 is used as a default parameter; e.g. A 120-residue - long protein comprises 12 fragments).after performing all - against - all structure comparisons (step 1) the following information is assigned to each frame of reference: lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures).for each frame of reference, all structures having at least 80% (default parameter) of the non - empty fragments in common are identified . A list consisting of maximum number of such structures is created and assigned to each frame of reference.an optimal number of clusters is determined based on the following criteria: the minimum number of clusters that yields a complete set of proteins in the combined lists from (4),lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a).within each cluster, a representative structure is selected, which in comparison with other members of the cluster has the highest values determined in steps (iii.a), (iii.b) and (iii.c). Lga is used to perform all - against - all comparisons in which, for a given set of structures, each structure is used as a frame of reference for comparisons with others . Each frame of reference is assigned a set of sequential fragments, which are defined by splitting the corresponding amino acid sequence into consecutive n - residue - long sub - sequences (n = 10 is used as a default parameter; e.g. A 120-residue - long protein comprises 12 fragments). After performing all - against - all structure comparisons (step 1) the following information is assigned to each frame of reference: lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures). Lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures). For each frame of reference, all structures having at least 80% (default parameter) of the non - empty fragments in common are identified . A list consisting of maximum number of such structures is created and assigned to each frame of reference . An optimal number of clusters is determined based on the following criteria: the minimum number of clusters that yields a complete set of proteins in the combined lists from (4),lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a). The minimum number of clusters that yields a complete set of proteins in the combined lists from (4), lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a). Within each cluster, a representative structure is selected, which in comparison with other members of the cluster has the highest values determined in steps (iii.a), (iii.b) and (iii.c). Note: in step (v.a) a minimum number of clusters are defined based on local similarities in non - empty fragments along the protein sequence using initially selected representative frames of reference . Step (v.b) allows reassignment of less similar structures from one cluster to another . It also allows sub - division of clusters in order to satisfy the requirement that within each cluster any pair of proteins has at least 60% overall structure similarity . This way less similar structures are not grouped together even if they satisfy the requirement regarding a common set of non - empty fragments (step 4). To perform a particular clustering for a set of protein structures, a suitable scoring function or, in general, a scoring algorithm that takes into account a number of characteristics of the compared proteins must be defined . Depending on the goal of the clustering, this can be done by selecting one measure or by combining different criteria to score the level of similarity . The lga_s scoring function has two components, lcs (longest continuous segments) and gdt (global distance test), defined for the detection of regions of local and global structure similarities between analyzed structures . In comparing two protein structures the lcs procedure localizes and superimposes the longest segments of residues that can fit under a selected set of rmsd cutoffs . The gdt algorithm is designed to complement evaluations made with lcs searching for the largest (not necessary continuous) set of let: m the number of residues in m, t the number of residues in t, r(r, m, t) = 100/t * l(r, m, t) is the percentage of the target's (t's) residues that are involved in the maximal (longest) continuous segment that fits within an rmsd of r . L(r, m, t) is the length of such identified longest continuous segment of m: t residue pairs, x(m, t)the set of all m: t superpositions calculated by the lga algorithm, g(s, d, m, t)the number of m: t residue pairs for which the distance between ca (alpha carbon) atoms is not greater than d after the superposition s x(m, t) is applied, d(d, m, t)=100/t * max{g(s, d, m, t):s x(m, t)} is the maximal detected percentage of the ca atoms in t structure that are within a distance threshold of d from m structure upon calculated s x(m, t) superpositions . The number of residues in m, t the number of residues in t, r(r, m, t) = 100/t * l(r, m, t) is the percentage of the target's (t's) residues that are involved in the maximal (longest) continuous segment that fits within an rmsd of r . L(r, m, t) is the length of such identified longest continuous segment of m: t residue pairs, x(m, t)the set of all m: t superpositions calculated by the lga algorithm, g(s, d, m, t)the number of m: t residue pairs for which the distance between ca (alpha carbon) atoms is not greater than d after the superposition s x(m, t) is applied, d(d, m, t)=100/t * max{g(s, d, m, t):s x(m, t)} is the maximal detected percentage of the ca atoms in t structure that are within a distance threshold of d from m structure upon calculated s x(m, t) superpositions . Lga_s(m, t) structure similarity scoring function is defined as a function of two structures m and t calculated as a combination of r(r, m, t) results from lcs(m, t) calculations, and d(d, m, t) results from gdt(m, t): where and w = 0.75 is a parameter (0 w 1) representing a weighting factor between s(lcs) and s(gdt) results . S(lcs) is a weighted sum of r(r, m, t) values calculated for n different rmsd cutoffs r (e.g. N = 3; r = 1.0, 2.0, 5.0), and s(gdt) is a weighted sum of d(d, m, t) values calculated using k different distance cutoffs d (e.g. K = 20; d = 0.5, 1.0,, 10.0). In the formulae s(lcs) and s(gdt), the weighting schemes weight higher those r and d results that were calculated for smaller rmsd and distance cutoffs, respectively . The range of the lga_s values is 0100, and hierarchical clustering experiments performed on various folds from scop database showed that lga_s alone can serve as a good discriminator for the initial protein structure clustering (see the results shown in figure 5a). The essence of the stralcp algorithm is the ability to compare hundreds of protein structures in a single reference frame, identify similar fragments that are conserved within a set of analyzed proteins, and use this information to calculate the number of required clusters . Each calculated cluster is assigned with its structural fingerprint that can be defined by a representative structure and a set of spans that are shared among structures grouped together . Comparison of a new structure with a structural fingerprint determines whether the structure should be included to the particular cluster or whether it should be a member of another cluster . Our stralcp algorithm, which automatically clusters proteins and identifies representative structures, can be described as the following list of steps: lga is used to perform all - against - all comparisons in which, for a given set of structures, each structure is used as a frame of reference for comparisons with others.each frame of reference is assigned a set of sequential fragments, which are defined by splitting the corresponding amino acid sequence into consecutive n - residue - long sub - sequences (n = 10 is used as a default parameter; e.g. A 120-residue - long protein comprises 12 fragments).after performing all - against - all structure comparisons (step 1) the following information is assigned to each frame of reference: lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures).for each frame of reference, all structures having at least 80% (default parameter) of the non - empty fragments in common are identified . A list consisting of maximum number of such structures is created and assigned to each frame of reference.an optimal number of clusters is determined based on the following criteria: the minimum number of clusters that yields a complete set of proteins in the combined lists from (4),lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a).within each cluster, a representative structure is selected, which in comparison with other members of the cluster has the highest values determined in steps (iii.a), (iii.b) and (iii.c). Lga is used to perform all - against - all comparisons in which, for a given set of structures, each structure is used as a frame of reference for comparisons with others . Each frame of reference is assigned a set of sequential fragments, which are defined by splitting the corresponding amino acid sequence into consecutive n - residue - long sub - sequences (n = 10 is used as a default parameter; e.g. A 120-residue - long protein comprises 12 fragments). After performing all - against - all structure comparisons (step 1) the following information is assigned to each frame of reference: lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures). Lga_s values between the frame of reference and all other structures, the number of residue pairs that are superimposed locally within rmsd cutoff 0.5 (using 3-residue - long window). Continuous structural segments formed by such residue pairs that are at least five residues long are marked as candidate spans, the number of non - empty fragments (non - empty fragments are sequential fragments defined in step 2 that overlap by at least one residue with at least one detected span in compared structures). For each frame of reference, all structures having at least 80% (default parameter) of the non - empty fragments in common are identified . A list consisting of maximum number of such structures is created and assigned to each frame of reference . An optimal number of clusters is determined based on the following criteria: the minimum number of clusters that yields a complete set of proteins in the combined lists from (4),lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a). The minimum number of clusters that yields a complete set of proteins in the combined lists from (4), lga_s between any pair of proteins from the cluster is at least 60% (default parameter), minimum value from (iii.a). Within each cluster, a representative structure is selected, which in comparison with other members of the cluster has the highest values determined in steps (iii.a), (iii.b) and (iii.c). Note: in step (v.a) a minimum number of clusters are defined based on local similarities in non - empty fragments along the protein sequence using initially selected representative frames of reference . Step (v.b) allows reassignment of less similar structures from one cluster to another . It also allows sub - division of clusters in order to satisfy the requirement that within each cluster any pair of proteins has at least 60% overall structure similarity . This way less similar structures are not grouped together even if they satisfy the requirement regarding a common set of non - empty fragments (step 4). A proper protein classification is critical for better understanding and prediction of a protein's structure, function and interaction with other proteins . It is known that sequence similarities nearly always correspond to structure similarities, enabling structure and function prediction for uncharacterized proteins . Structural similarity, however, does not necessarily correspond to sequence similarity (figure 2). Through structural comparison and classification, we identified a family of crystal structures that failed to be detected (18) by sequence - based methods like psi - blast (14). Using a structure - based method (e.g. Dali, lga) it was found that three eap domains from staphylococcus aureus (18), which could not be properly classified by sequence - based methods, shared a previously unrecognized similarity to another class of bacterial toxins . For each of the eap domains [eap2 (pdb entry: 1yn3), eaph1 (1yn4), eaph2 (1yn5)], we have performed structural pdb searches using our lga server (19). As a result, 134 domains from the scop superfamily d.15.6 (superantigen toxins, c - terminal domain) were identified as most similar to eap structures (only 20 structures are shown in provided figures 1, 3, and 4; 3 eap domains and 17 domains from scop). Figure 1 shows that all 20 proteins are very similar in detected structurally conserved frames formed by 4 strands and 1 helix (figure 2). The superposition of 1yn4_a and d1m4va2 (1m4v_a in figure 2) corresponds to the fourth bar in figure 1 and shows that these two structures differ in several loop regions only (structural deviations above 2 are colored in yellow - red). Note that the level of sequence identity between these two proteins is only 14% (seq_id), whereas the level of structure similarity is 75% (lga_s). Figure 1.structure similarities between eap domains from s. aureus (pdb: 1yn3, 4 and 5) and 17 protein domains from the scop superfamily comprising superantigen toxins . All proteins were compared to the structure of eaph1 (1yn4_a), which serves as a frame of reference . Colored bars represent calpha calpha distance deviation between 1yn4_a [99 residues; from the left (n - terminal) to the right (c - terminal)] superimposed with 20 structures from pdb (first bar represents a 1yn4_a1yn4_a self - comparison). Colors represent distances between aligned residues and range from green (below 2) to red (above 6). The columns at the right contain information about the level of sequence identity (seq_id) and structure similarity (lga_s). Figure 2.a 3d plot of structural superposition between 1yn4_a and 1m4v_a (scop domain: d1m4va2) that corresponds to the fourth colored bar in figure 1 . The level of sequence identity between proteins seq_id: 14%, and the level of structure similarity lga_s: 75% . Figure 3.the results from the analysis of structure similarities between eap domains from s. aureus and proteins from the scop superfamily of superantigen toxins (same domains as in figure 1). Figure 4.stralcp clustering applied to the same set of 20 structures as in figures 1 and 3 . Stralcp calculations were performed using default parameters (lga_s = 60%, dist = 5). Each row begins from the cluster number, followed by the domain name, and the set of amino acids that are extracted from detected structurally conserved spans . Dots indicate regions that structurally deviate in at least one pairwise comparison between members of the cluster . Note: dots do not indicate the actual number of residue pairs between detected spans . Figure 5. (a) dendrogram showing the results of an lga_s - based (single measure) clustering of 24 scop domains from fold a.8 . Each code (entry_family) represents one protein from the scop classification: entry and family number . We used the r package (version 2.1.1; http://www.r-project.org/) for the hierarchical clustering and visualization of calculated lga_s results from all - against - all structure comparisons . Structure similarities between eap domains from s. aureus (pdb: 1yn3, 4 and 5) and 17 protein domains from the scop superfamily comprising superantigen toxins . All proteins were compared to the structure of eaph1 (1yn4_a), which serves as a frame of reference . Colored bars represent calpha calpha distance deviation between 1yn4_a [99 residues; from the left (n - terminal) to the right (c - terminal)] superimposed with 20 structures from pdb (colors represent distances between aligned residues and range from green (below 2) to red (above 6). The columns at the right contain information about the level of sequence identity (seq_id) and structure similarity (lga_s). A 3d plot of structural superposition between 1yn4_a and 1m4v_a (scop domain: d1m4va2) that corresponds to the fourth colored bar in figure 1 . The level of sequence identity between proteins seq_id: 14%, and the level of structure similarity lga_s: 75% . The results from the analysis of structure similarities between eap domains from s. aureus and proteins from the scop superfamily of superantigen toxins (same domains as in figure 1). Stralcp clustering applied to the same set of 20 structures as in figures 1 and 3 . Stralcp calculations were performed using default parameters (lga_s = 60%, dist = 5). Each row begins from the cluster number, followed by the domain name, and the set of amino acids that are extracted from detected structurally conserved spans . Dots indicate regions that structurally deviate in at least one pairwise comparison between members of the cluster . Note: dots do not indicate the actual number of residue pairs between detected spans . (a) dendrogram showing the results of an lga_s - based (single measure) clustering of 24 scop domains from fold a.8 . Each code (entry_family) represents one protein from the scop classification: entry and family number . We used the r package (version 2.1.1; http://www.r-project.org/) for the hierarchical clustering and visualization of calculated lga_s results from all - against - all structure comparisons . (b) clustering created using stralcp algorithm with default cutoff lga_s = 60% . In general, all eap domains have a high level of overall structure similarity (lga_s over 60%) to most of the other analyzed structures, whereas the level of sequence identity is very low (below 20%). In figure 1, we show the results from the structure comparisons of the set of selected 20 proteins when structure 1yn4_a was chosen as a frame of reference and in figure 3 the structure seh (pdb: 1f77; scop domain d1f77a2) (20) was selected as a frame of reference . From the comparison of these two plots we can conclude that d1f77a2 may serve as a better representation (average structure) for the analyzed set of 20 proteins (at least for the top 13 of them) than the structure 1yn4_a . The obtained results suggest that a given set of 20 structures can be structurally divided into at least two clusters . Our stralcp system creates such a clustering automatically (figure 4). By this clustering the eap structures: 1yn3 - 5 are grouped together (cluster2) with four other protein structures: set1 (pdb: 1v1p) (21), set3 (pdb: 1m4v) (22), and tsst1 (pdb: 1aw7, 2tss) (23,24). Additional tests showed that if we had introduced more strict structure similarity requirements [e.g. Lga_s cutoff 80% (see step 5.b in stralcp algorithm)], then cluster2 would have been split into two additional clusters (data not shown) where all three eap domains (1yn3 - 5) were separated from the set1, set3 and tsst1 structures . As described in the methods section the stralcp clustering approach consists of two steps: (i) calculating all - against - all structural alignments using lga program, and (ii) extracting from calculated alignments structurally conserved regions and using them to group proteins accordingly . For example, on a single linux workstation equipped with amd-64 5000 dual core processor, the calculations of all - against - all pairwise structural alignments for 20 discussed above structures (3 eap domains and 17 domains from scop) lasted about 10 min, while the clustering (step 2) was completed in less than 10 s. in order to estimate the accuracy of a stralcp clustering approach, we have performed comparisons with the scop (ver . We tested stralcp calculations on domains from 25 different scop folds: a.5, a.7, a.8, a.24, a.29, a.137, b.2, b.42, b.43, b.68, b.80, b.85, c.8, c.51, c.56, d.52, d.68, d.79, d.110, d.129, f.1, f.4, f.23, g.41, h.4 . We have selected these folds as a representative sample from all 7 scop classes with an additional requirement that each fold consists of at least four superfamilies . In the scop database ver . 1.71, there are only 63 such folds that satisfy this requirement . In total, our benchmark set consisted of nd = 4620 domains from nf = 343 families, and ns = 243 superfamilies . Complete sets of results from this experiment can be found at a server website: http://as2ts.llnl.gov / as2ts / stralcp/. In figure 5, we present the results from stralcp calculations applied to 24 domains from scop fold a.8 (immunoglobulin / albumin - binding domain - like). In figure 5a, we use scop fold a.8, as an example to show some of the details from the hierarchical dependencies among structures calculated using lga_s as a single measure for clustering . In figure 5b, we show how the structures from fold a.8 can be automatically separated using our stralcp (multi - criteria - based clustering) approach . This example shows that by using stralcp we can clearly separate proteins into appropriate clusters that correspond with a high agreement to the defined scop families (see figure 5b, right column; scop family codes). In order to assess the accuracy of our clustering approach, we estimated the differences between scop (ver . 1.71) and stralcp clustering (for example on the level of scop families) by introducing the following measure . Let: nc the number of created clusters, cf(i)the number of different families clustered together within the i cluster, nc the number of created clusters, cf(i)the number of different families clustered together within the i cluster, the score indicating the misclustering effect mc (when domains from different scop families are grouped together) can be calculated using the formula: the range of this measure is 0.0 mc <100.0, where 0.0 indicates no misclustering (i.e. Agreement with scop families separation). The mc measure allows the comparison of different clustering schemes by their agreement in separating proteins from different clusters . The goal of this measure is not to calculate how many domains are clustered differently, but rather how many of the created clusters are compromised (proteins that are separated in another clustering scheme being merged). The results from the evaluation of the differences between scop and stralcp clusters at the level of scop families showed that on average the level of misclustering (mc) is 3% . It suggests that the proposed strictly structure - based clustering method can be considered robust in that it detects relationships at the family level with a good agreement with the manually maintained scop database . As described in the methods section the stralcp clustering approach consists of two steps: (i) calculating all - against - all structural alignments using lga program, and (ii) extracting from calculated alignments structurally conserved regions and using them to group proteins accordingly . For example, on a single linux workstation equipped with amd-64 5000 dual core processor, the calculations of all - against - all pairwise structural alignments for 20 discussed above structures (3 eap domains and 17 domains from scop) lasted about 10 min, while the clustering (step 2) was completed in less than 10 s. in order to estimate the accuracy of a stralcp clustering approach, we have performed comparisons with the scop (ver . We tested stralcp calculations on domains from 25 different scop folds: a.5, a.7, a.8, a.24, a.29, a.137, b.2, b.42, b.43, b.68, b.80, b.85, c.8, c.51, c.56, d.52, d.68, d.79, d.110, d.129, f.1, f.4, f.23, g.41, h.4 . We have selected these folds as a representative sample from all 7 scop classes with an additional requirement that each fold consists of at least four superfamilies . In the scop database ver . 1.71, there are only 63 such folds that satisfy this requirement . In total, our benchmark set consisted of nd = 4620 domains from nf = 343 families, and ns = 243 superfamilies . Complete sets of results from this experiment can be found at a server website: http://as2ts.llnl.gov / as2ts / stralcp/. In figure 5, we present the results from stralcp calculations applied to 24 domains from scop fold a.8 (immunoglobulin / albumin - binding domain - like). In figure 5a, we use scop fold a.8, as an example to show some of the details from the hierarchical dependencies among structures calculated using lga_s as a single measure for clustering . In figure 5b, we show how the structures from fold a.8 can be automatically separated using our stralcp (multi - criteria - based clustering) approach . This example shows that by using stralcp we can clearly separate proteins into appropriate clusters that correspond with a high agreement to the defined scop families (see figure 5b, right column; scop family codes). In order to assess the accuracy of our clustering approach, we estimated the differences between scop (ver . 1.71) and stralcp clustering (for example on the level of scop families) by introducing the following measure . Let: nc the number of created clusters, cf(i)the number of different families clustered together within the i cluster, nc the number of created clusters, cf(i)the number of different families clustered together within the i cluster, the score indicating the misclustering effect mc (when domains from different scop families are grouped together) can be calculated using the formula: the range of this measure is 0.0 mc <100.0, where 0.0 indicates no misclustering (i.e. Agreement with scop families separation). The mc measure allows the comparison of different clustering schemes by their agreement in separating proteins from different clusters . The goal of this measure is not to calculate how many domains are clustered differently, but rather how many of the created clusters are compromised (proteins that are separated in another clustering scheme being merged). The results from the evaluation of the differences between scop and stralcp clusters at the level of scop families showed that on average the level of misclustering (mc) is 3% . It suggests that the proposed strictly structure - based clustering method can be considered robust in that it detects relationships at the family level with a good agreement with the manually maintained scop database . As discussed in ref . (13), a strategy combining information from both sequence and structure comparisons would be expected to perform better than either method alone . However the analysis of the clustering approach applied to the benchmark set of 25 scop folds leads to the encouraging conclusion that the stralcp algorithm, even if it is based purely on structure comparisons, exhibits a low (on average 3%) misclustering effect: domains from different scop families were clustered separately . It is important to keep in mind that a purely structure - based approach to clustering may result in two proteins that are identical in sequence being clustered separately if the two structures differ in conformation; we observed that the stralcp algorithm is able to detect the structural differences between domains from the same scop family and cluster them separately . It is for this reason that our clustering approach may produce more clusters than the number of scop families . For example the family a.8.6.1 (figure 5b) was separated by stralcp into two clusters: cluster3 (staphylocoagulase first domain) and cluster4 (staphylocoagulase second domain), and the family a.8.4.1 was divided into two clusters: cluster5 (dnak domain from escherichia coli) and cluster2 (dnak domain from rat). The stralcp algorithm will also group proteins in different clusters if they significantly differ in length or if multi - domain structures are in different conformations (e.g. Open and closed versions of the same protein). We also can observe additional sub - clustering of protein families when criteria for structure comparison are sufficiently stringent (e.g. A higher lga_s cutoff is introduced). It provides valuable information about the regions that are structurally in the same conformation, which could be useful in various studies and classification schemes . The separation of similar or identical proteins, but in different structural conformations, could be reduced by introducing a sequence similarity analysis into the stralcp algorithm . However, in this study, in order to detect the limits of purely structure - based approaches we do not include sequence information to the scoring and clustering algorithm . The sequence - based analysis may be considered as an option in future development efforts.
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The blood vessels, as part of the human circulatory system, transport the blood with nutrition and oxygen and remove the waste throughout the body . The development of the vascular system is not a random process but follows a set of optimization principles, such as the minimum friction between the blood flow and the vessel wall, the optimal heart rate to achieve proper blood supply, and the shortest transportation distance . In many diseases such as diabetes, glaucoma, hypertension, and other cardiovascular diseases, these optimal conditions are no longer maintained, leading topological abnormalities to appear in the vascular network . Vessels in organs like the brain, the lung, or the kidney can only be observed indirectly by certain image modalities, such as magnetic resonance angiograph, cta, and x - ray angiography . However, the vasculature in the nerve fiber layer of the retina can be observed directly and noninvasively by fundus cameras . Therefore, increasing attention has been drawn to the retinal images for the quantitative analysis of retinal blood vessels, which might provide useful information about the progress of systemic and cardiovascular diseases . One of the biomarkers that could describe changes in microvasculature due to the disease progression is the fractal dimension (fd). He proposed a set of mathematical definitions for a self - similar object and used a noninteger number to describe the dimension of this highly irregular shape . In 1989, after that, there has been a growing interest in studying the association between the fractal dimension of the retinal vasculature and the disease severity and progression [48]. In many clinical studies, the fractal dimension has shown its potential in characterizing the growth of neurons, tissues, and vessels . Firstly, the fractal describes growing progression of the neuron cells by quantifying their complex dendrites . Studied the morphology of the branching patterns in the cortical neuronal dendrites by fractal dimension and reichenbach et al . Used it as a discriminator for different mammalian astroglial cell types . In the case of tissue image analysis, li et al . Applied the fractal calculation on medical tissue images in order to detect the special texture of pathological tissues in addition, fractal dimension was used as a feature for parenchymal lung disease detection . Finally, as is the focus of this paper, the fractal dimensions have been applied widely on human retinal images for disease detection . Some literature reports a higher fd in images of a patient group with a late stage of proliferative diabetic retinopathy compared to a healthy control group [4, 8, 14]. Broe et al . Did a fractal analysis on optic disc centered images of 180 patients who had type 1 diabetes in a 16-year follow - up study . They compared the fractal dimension of the patients with their values that were recorded 16 years ago and found that the fractal dimension was generally decreased . Similarly, grauslund et al . Compared the box dimension of 94 type 1 diabetes patients without proliferative retinopathy to 79 patients with proliferative retinopathy (pr). Also papers report mixed results when comparing healthy and diabetic groups . In the study of aliahmad et al ., 189 optic disc centered retinal images of healthy and diabetic individuals were examined with box dimension . The statistical results showed that the healthy subjects had higher fractal dimension than the diabetic group . . Found higher fractal dimensions in the diabetic group with 498 patients compared to those in the normal group with 743 healthy subjects . . Showed that the longer the diabetic duration of one patient was, the higher his retinal fractal dimension was . Not only the number of patients but also the cameras used in data acquisition in each study were different . Therefore, the images' resolution, illumination, and quality varied across studies . Moreover, the computer software which semiautomatically does the optic disc detection, vessel segmentation, vessel skeletonization, and the fractal computation was also different in each study . Finally, the region of interest for fd calculation was not the same for all studies . These different experimental settings, therefore, may be the reasons of conflicting findings in each study . In that case, it is worth to investigate the reliability of the fd measurement, since the measurement itself might not be stable enough to provide reliable results . Wainwright et al . Studied the robustness of the fd measurement in terms of variation of brightness, focus, contrast, and image format and concluded that fd is highly sensitive to all these factors . . Found that the fd was highly dependent on both vessel segmentation and fd calculation methods . In our previous study, we have examined the stability of multiple fractal measurements in different cases . In this paper, we extend the previous work into 6 cases, in which we calculated the variation of the fractal dimension . (1) we calculated the fd values in groups of subjects with various diabetic retinopathy grades, where the intergroup and intragroup variations are compared . (2) we calculated the fractal dimension on the manual vessel segmentation annotated by different human observers . (3) we investigated the stability of fd using different vessel segmentation methods . (4) we explored the changes of fd in various regions of interest . (5) we tuned the segmentation threshold values to examine the influence of segmentation accuracy on the fractal measurements and (6) we compared the fractal dimensions that are calculated on the images acquired by different cameras . The paper is organized as follows: in section 2, we introduce the materials and datasets used in this study . In section 3, we explain the pipeline for computing the fractal dimension, including four state - of - the - art vessel segmentation methods, the region of interest determination, and three classic fractal dimension calculation methods that are widely used in clinical studies . In section 4 the results of different cases are compared, and the discussion is presented in section 5 . Finally, section 6 summarizes the conclusions . In this section, we introduce the public retinal image datasets and the test image dataset that were used in the stability studies . We used three datasets: messidor, drive, and a test dataset including images captured by five different cameras . The messidor public dataset includes 1200 eye color fundus images with diabetic retinopathy grades (r0, r1, r2, and r3). The grades are provided based on the number of microaneurysms and hemorrhages and the presence of neovascularization . The images were taken in 3 ophthalmologic departments in france by using the topcon trc nw6 (topcon, japan) with field of view (fov) of 45 degree . The images have three different sizes: 1440 960, 2240 1488, and 2304 1536 . In this paper the drive dataset contains 40 fovea centered color retinal images, which were captured on 33 nondiabetic retinopathy subjects and 7 with mild early diabetic retinopathy . The images were acquired by a canon cr5 nonmydriatic 3ccd camera (canon, japan) with a fov of 45 degree . The 40 images were randomly divided into a training set and a testing set of equal size . In the testing set, the images were manually annotated by 2 different well - trained ophthalmologists . These 20 test images were used for the fractal stability and robustness study . In order to investigate the variation of fd computed on the images acquired by different cameras, we established a new dataset which consists of the retinal images captured by 5 different fundus cameras on 12 young healthy volunteers . The 5 fundus cameras were installed in the ophthalmology department of the academic hospital maastricht (azm) in netherlands . The retinal photographs were taken on the left eye of every subject 5 times with each camera, both fovea centered and optic disc centered (120 images in total). The 5 cameras are 3nethra classic, canon cr-1 mark ii, nidek afc-230, topcon nw300, and easyscan . The 3nethra classic (forus, india) provides color fundus images with size of 2048 1536, and the fov is 40 degrees . The canon cr-1 mark ii (canon, japan) is a nonmydriatic retinal camera with fov of 45 degrees, and the image size is 3456 2304 . The nidek afc-230 (nidek, japan) is also a nonmydriatic autofundus camera with 45-degree fov and captures the fundus on a 3744 3744 color image . The topcon nw300 (topcon, japan) is a color fundus camera with picture angle of 45 degrees and its image size is 2048 1536 . Finally, easyscan (ioptics, netherlands) is a scanning laser ophthalmoscopy (slo) camera with fov of 45 degrees and the image size of 1024 1024 . In this section, we introduce the pipeline and methodologies, which are used to compute the fractal dimension from a fundus image . First of all, we import the raw color images from each dataset and rescale them to the same pixel size as the images in the drive dataset . As a result of the acquisition process, very often the retinal images are nonuniformly illuminated and exhibit local luminosity and contrast variability . In order to overcome this problem, each image is preprocessed using the method proposed by foracchia et al ., which normalizes both luminosity and contrast based on a model of the observed image . Luminosity and contrast variability in the background are estimated and then used for normalizing the whole image . After the image local normalization, we apply 3 state - of - the - art vessel segmentation methods on color retinal images and one particular segmentation method on the slo images to obtain the vessel probability maps (soft segmentation). Afterwards, a threshold value is applied to the obtained vessel probability maps in order to construct binary segmentations (hard segmentations). At the same time, we automatically determine the region of interest for fd calculation by detecting, segmenting, and parameterizing the optic disc and the fovea . Finally, the fractal dimension is calculated on the binary vessel segmented images within a circular roi using 3 classic fd measurements . In the following section, the fractal dimension is usually calculated on a vessel binary map, where pixel intensity of 1 is considered as vessel and 0 as background . Generally manual vessel annotations provided by the human observers have better quality than automatic vessel segmentation techniques . Additionally, for large volume clinical studies, an automatic vessel segmentation program is needed for the vessel detection . In our study, we investigated three vessel segmentation methods for extracting the vessels from rgb retinal images, frangi's vesselness method, soares' method, and zhang's method, and the bimso method for slo images . Frangi's vesselness is a multiscale vessel enhancement method proposed by frangi et al ., which uses the second - order derivatives to enhance elongated structures in the image . An important property for an elongated structure is a large change of gradient (principal curvature) in one direction but little gradient change in the direction perpendicular to the former . Therefore, the pixels of a vessel have 12, where 1 and 2 are the magnitudes of the local principle curvatures that can be calculated via the eigenvalues of the 2d gaussian hessian . Thus, the vessels can be enhanced by a normal probability distribution function:(1)expra2221exps222,where 1 and 2 are the eigenvalues of the 2d gaussian hessian, ra = 2/1 is an anisotropy (elongatedness) term, s = 1 + 2 is a structure term, and and are constant values that determine the sharpness of the filter . The vessel probability map generated by this method soares' segmentation is a supervised method for vessel enhancement proposed by soares et al . . First it extracts 5 features including the pixel intensity (the green channel) and 4 gabor filter responses from the images . By using a bank of gabor filters with multiscales, multifrequencies, and multiorientations, vessels with different sizes and orientations a supervised gaussian mixture model (gmm) classification method is used to classify the pixels into vessel or background using the obtained features . The output is a probability map indicating the likelihood for a pixel being a vessel (shown in figure 2(c)). Zhang's method is based on describing the image as a function on an extended space of positions and orientations . In the method, the image is lifted to the 3d space of positions and orientations via a wavelet - type transform . In the 3d domain, vessels are disentangled at crossings due to their difference in orientation . In the new space, left - invariant gaussian derivatives are used (exploiting a rotating coordinate system) in order to enhance the blood vessels . The method results in crossing preserving enhancement of blood vessels (shown in figure 2(d)). Bimso method is a brain - inspired multiscale and multiorientation technique proposed by abbasi - sureshjani et al ., which is mainly designed for the vessel segmentation in slo images . In this method, the image is lifted to the 3d orientation score using rotated anisotropic wavelets and then a nonlinear transform is applied to enhance the elongated structures (blood vessels) and to suppress the noise . Afterwards, several features for each pixel are extracted including the intensity, filter response to the oriented wavelets, and the multiscale left - invariant gaussian derivatives jet . The pixels are then classified by a neural network classifier into vessel or background using the obtained features . In this subtask, the fractal dimensions were calculated in different circular regions with various radii around the fovea and optic disc (od) centers . For fovea centered images, the regions of interest were centered at the fovea centralis with radii of 4, 5, and 6 times the optic disc radius (odr). These radii were set in accordance to the diameter of human optic discs and the average fovea - to - disc distance . According to the study of, the average diameter of the human optic disc is 1.83 mm and the distance from the fovea center to the optic disc center is 4.93 mm, which is about 5 times odr . Therefore, the circular roi with radius 4 odr covers the retina but excludes the optic disc, the 5 odr roi covers half of the optic disc, and the 6 odr roi covers the full optic disc . Throughout the studies, in this method, the od is detected via a cross - correlation based template matching in higher dimensional objects called orientation scores . An orientation score represents image data on the 3d space of positions and orientations, where the vessels with different orientations are lifted to different planes of the space . The templates are designed to detect the 3d pattern of vessels originating from the optic nerve head . Therefore, the global maximum of the correlated image reveals the position of the od . The segmentation is done within a small patch of an enhanced od to detect its circular boundary . On a regular rgb fundus image for instance, the tissue and vessels inside the disc have greater yellow - blue color difference than the background vessel and tissue (see figure 3). Therefore, the color derivatives of the red, green, and blue intensity can be used to enhance the od region and suppress the background tissue of the retina . The color derivatives of an rgb image are computed using the gaussian color model proposed in [27, 28], where the best linear transform from the rgb color domain to the gaussian color model is defined by(2)eee=0.060.630.310.190.180.370.220.440.06rgb, where e, e, and e represent the nonderivative, 1st - order derivative, and 2nd - order derivative with respect to the wavelength . The enhanced od image is obtained by combining invariant assemblies of e, e, and e. after the enhancement, the od boundary becomes stronger and the potential interferences caused by the edge of vessels are suppressed and a simple zero crossings of the laplace operator is used for od edge detection . After that, an ellipse is fitted to the detected boundary positions and the major and minor radius are obtained . Finally, the od radius (odr) is estimated as the average of the major and minor radii of the fitted ellipse . Fovea center detection is done within a ring area around the optic disc center . As mentioned earlier, the average distance between the fovea centralis and the optic disc centralis is about 5 odr, so the inner and outer radii of the ring of interest are selected as 4 odr and 6 odr, respectively . After determining the ring area, we reduced the interference of blood vessels by using the binary vessel segmentation obtained beforehand and an inpainting algorithm which replaces / paints the detected vessels by their neighbor background texture . Finally, the fovea center is detected as the global minimum at a large gaussian blurring scale . The fractal dimension is a measurement which quantifies the highly irregular shape of fractals or fractal objects . An important property of the fractal objects is their self - similarity over different scales or magnifications . This means that at different scales a same pattern with different sizes can be observed, such as trees, snowflakes, and river systems . This self - similar property can be described by the following formula:(3)nr = rd, where n(r) is some measurements applied on the complicated pattern of the object at a scale r; d is the fractal dimension that implies how many new similar patterns are observed as the resolution magnification (scale) decreases or increases . In order to solve for d we rewrite (3) into(4)d=lognrlogr.according to the definition, a fractal object is self - similar; therefore the comparison of two measurements in various scales should yield the same results . This implies that the fractal can also be calculated by comparing the measurements between any two scales:(5)dlognrnlognrn1logrnlogrn1 . Based on the above relation between measurements in different scales, a box - counting method is introduced to do a simple, fast estimation of the fractal dimension d. in this method, the full space is firstly covered by squared boxes with side - length rn . Finally, the size of the box and the corresponding measurement are plotted in a log - log plot . The estimated fractal dimension is the slope of the regression line that fits to these data points . In this paper, we are mainly interested in three fractal methods that are widely used in the literature: the box dimension db, information dimension di, and correlation dimension dc, which measure different properties (different n(r)) of the self - similar pattern of the object, respectively . Box dimension (db) is the most simple and popular method for estimating the fd of fractal objects proposed by . The box dimension is defined as the real number db, such that the number n(r) of balls with radius r that is needed to cover an object grows with (1/r) as r 0 . In other words, so, in the image domain, the measurement n(r) in (6) is the number of boxes with side - length r which overlap with the vessel segmentation . When dealing with discrete problems, taking the limit r 0 is not possible . Instead, as suggested by, db can be computed as the slope of n(r) plotted against r in a log - log plot . Information dimension (di) is inspired from information theory . In information theory, entropy is the measure of the uncertainty of a random event . The less likely a random event might happen, the more informative it is and thus the larger entropy it has . Conversely, if an event happens very often, it provides less information, implying lower entropy . The information dimension [31, 32] is defined as(7)di = lim0i=1npilogpilog1/,where n is the number of boxes with size overlapped with the object, the numerator i=1pilogpi is the first - order shannon entropy, pi = ni / m is the probability for finding a part of the object in the ith box, m is the total mass of it, and ni is the part of the object in the box . The limit of (7) is estimated as the slope of the regression line of the logarithmic points . Correlation dimension (dc) estimates the fd via the relationship between two pixels inside a region . A correlation integral is defined via the heaviside step function for counting the pair of points in a region with size rk and can be approximately expressed in terms of the probability density:(8)ck=1n2i=1,j=1,ijnrkxixjj=1nkpjk2,where (x) is the heaviside step function, xi is the ith pixel belonging to an object, and pjk = njk / m is the probability density of the object with mass m in the jth box with size rk . The correlation dimension dc is defined via the relationship between ck and rk as dc = limrk0(logck / logrk). In this section, we present our stability analysis of the fractal methods in terms of the choice of manual annotations, different segmentation methods, various regions of interest, the accuracy of the segmentation method, and different imaging modalities . To study the variation of fds, we use the relative error (re) with respect to the binary images annotated by observer 1 as the reference . Dr)|/dr, where dx is the obtained fd in different studies and dr is the reference fd . To test whether or not measurement methods are correlated, we use the pearson correlation coefficient test . Study 1: intergroup and intragroup fractal dimension variation . In order to show the significance of these relative errors in different experiments, the obtained fd values are compared with the coefficient of variation, also known as relative standard deviations (rsd) of all subjects in the drive dataset, which are 2.3%, 2.1%, and 2.0% for db, di, and dc, respectively . We also obtained the intergroup and intragroup fractal dimension (db) variations for the different groups of diabetic retinopathy in the messidor dataset . For all images with different dr grades, the box dimension is calculated once on the full image and once inside the region of interest around the fovea (5 odr). The averages and relative standard deviations of fd values for each separate dr group are shown in table 1 . As we can see in this table and in figure 4, the differences between the mean of fd values for different dr groups are small compared to the rsd of each dr group . The results of multiple one - way anova tests are shown in table 2 . With this test, we study whether a pair of subgroups have different distributions . In the case of using the full fov as roi, there are no significant mean differences between any two groups, except in group pairs r0r2 and r2r3 . For the circle roi around the fovea, the mean we compared the fd values that were calculated on the ground truth images annotated by two experts within the circular roi with 5 odr . Here we used the fds of observer 1 as reference as this is also considered as ground truth in . The main difference between the two manual annotations is the presence of the tiny vessels . The maximal differences of 7.11%, 6.70%, and 6.23% and mean relative errors of 1.97%, 1.88%, and 1.77% are obtained for db, di, and dc, respectively, which are noticeable compared to the calculated rsds . It means that even if the fds are calculated on vessel maps annotated by human observers, the methods cannot produce stable values for diagnosis, which makes fractal dimension measurement useless . The curves illustrate that the variations of fd for two observers in some subjects are too large which might cause wrong discrimination among subjects for clinical applications . For example, we see db of patient 5 is greater than patient 4 for observer 2, while the two patients have similar values obtained from the other observer ., we investigated the variation of fractal dimensions when using automatic vessel segmentation methods instead of human annotations ., soares et al ., and zhang et al . Were used as described previously . Each method produces a vessel probability map from the raw fundus image from which we obtain a binary map by setting an optimal threshold . The optimal threshold for each method is set to the value which maximizes the vessel segmentation accuracy for the whole dataset . The 2nd to 4th rows of table 3 show the res when using the binary images created by the segmentation methods instead of human observers . The maximum errors of the box dimension for the three segmentation techniques are 9.32%, 8.70%, and 7.37%, respectively . The average errors are 4.29%, 2.88%, and 3.97%, which are significantly compared to the rsd values . These values suggest that using an automatic segmentation would induce a large error in fractal calculation . In addition, the very high p values imply the weak association between the automatic methods and the manual . The variation among different segmentation methods is also large according to curves shown in figure 6, which shows the mean and standard deviation of db among the 3 methods . This suggests that the fractal measurement is very sensitive to the choice of vessel segmentation method . We calculate the fd in various circular regions around the fovea center of the drive ground truth images annotated by observer 1 . As mentioned previously, the roi radii are considered as 4 odr (roi1), 5 odr (roi2), and 6 odr (roi3), and roi3 is used as reference for the relative error calculation . When fds are calculated in roi1, the maximum error of the box dimension is 3.8%, and the average error is 2.4% . If we use roi2, the relative errors were smaller, with a maximum of 1.0% and average of 0.4% error . Figure 7 shows the plot of db calculated in roi1 (red), roi2 (green), and roi3 (blue) and also the mean and deviation of them (purple). According to the table and figure, changing roi causes a variation in fractal calculation; in particular the fds of roi1 are significantly lower compared to roi2 and roi3 . But, from another point of view, we see that p values are less than 0.01, which means the fds calculated in different rois are significantly associated . We studied the relation between the fd error and the quality of vessel segmentation methods . The fd is usually calculated on a vessel binary map, which is converted from the vessel probability map with a threshold value . The choice for threshold value changes the accuracy of vessel segmentation, and the accuracy of the segmentation method turns out to affect the fractal measurement significantly . The comparison is based on zhang's segmentation method in a fixed region of interest (roi2). Several threshold values t with range from 0.15 to 0.35 and step size 0.01 are applied to the vessel probability map for all test images in the drive database to obtain the vessel binary segmentations . Since there is a large difference between number of vessel pixels and nonvessel pixels in retinal images, we used the matthews correlation coefficient (mcc) instead of accuracy to evaluate the quality of binary images . The mcc is a balanced measure which can be used even if the classes are of very different sizes:(9)mcc = tptnfpfntp+fptp+fntn+fptn+fn, where tp, tn, fp, and fn are the true positive, true negative, false positive, and false negative parts of the segmentation with respect to the annotations by observer 1 . For each result of the binary segmentation, the mean relative errors for the 20 images with respect to the reference ones are shown in table 5 for 3 sample thresholds t = 0.15, 0.21, and 0.34 . As we can see in this table, using both 0.15 and 0.34 as threshold results in similar mcc values for the vessel segmentation, while one is the oversegmented (higher fd) and the other one is the undersegmented (lower fd), a threshold equal to 0.21 gives the highest mcc 78% as an average among 20 images . Note that no postprocessing was applied after the thresholding, so the segmentation accuracy in our studies might be lower than the proposed accuracy in the literature . From the table, we see that if the threshold is set properly (t = 0.21), the relative error is small . Moreover, figure 8 shows the plot of the mean mcc of vessel segmentation against the mean error of fd of 20 images . These results suggest that poor segmentation with improper selection of the threshold value leads to a large error for fractal dimension calculation . We investigated the variation of fractal dimension which is calculated on the images captured by different cameras described previously . The optic disc centered images of the 12 volunteers are used in this examination (see figure 9). The circular region of interest centered at the od center with radius 4 odr is used in all images . The vessel segmentation results of the rgb images captured by regular cameras are generated by zhang's method and those of the easyscan slo camera are generated by the bimso method . First we compare the variation among different cameras, where the box dimensions of 12 subjects are shown in figure 10 with different colors per camera . As we can see from this figure, the fractal dimension is very sensitive to image properties like resolution, amount of noise, quality, and imaging modality, which depend on the type of camera . For example, the mean relative difference between 3nethra (red dashed line) and nidek (purple dashed line) is 2.31% with respect to the average of two cameras . The slo images acquired by easyscan (green dashed line) in general have lower fds than the other color rgb cameras except for 3nethra . In addition, the average relative variation between the slo images and rgb images (by canon camera) is 1.95% . Finally, we investigate the repeatability of different cameras by comparing the fds of different acquisitions of one subject . The repeatability is measured as the standard deviation of the fractals calculated on 5 acquisitions of the same subject divided by the average of them . As we can see from table 6, the 5 cameras give an average of 1.11% variation on the same subject in different acquisition times . With canon and nidek cameras, this error is small (0.69% and 0.96% resp . ), which shows better stability compared to other cameras . In previous studies, fractal dimension is considered as a potential biomarker for disease detection . However, conflicting findings were found in different literature . Therefore, we examined the reliability of three classic fractal measurements for their use in clinical study applications . We divided our experiments into six studies, which we will discuss in the remainder of this section . In our first and second studies, we investigated intergroup and intragroup variability of fd methods using the messidor dataset . Also, we studied intraobserver variation using ground truth segmentation from the drive dataset . The experimental results show that, even with ground truth vessel maps, the fractal dimensions are not reliable . Moreover, the variation of fd between different human observers produces errors of 1.97%, 1.88%, and 1.77% on average on db, di, and dc . This significant variation makes the fd less informative and less reliable in discriminating dr patients in different severity levels from the healthy ones . No significant differences in fd were found between different dr groups of the messidor dataset . From figure 11, we see that the main difference between the vessel annotations of two observers is the presence or absence of small vessels . Therefore, the influence of small vessels on the fractal measurements cannot be neglected and should be considered seriously . In the third study we examined the fd on the vessel maps produced by three different vessel segmentation methods on the same imaging modality (rgb fundus images). The results show that the fds calculated with various segmentations have significant differences compared to the values calculated using the annotations by observer 1 . In addition, the statistical tests show that the fds were not associated with those computed from ground truth images . Therefore, the fd computed by automatic computer software might not be reliable, as was the case in the studies from [48]. In the fourth study, we investigated the variation of fd calculated within different regions of interest centered at the fovea centralis . This study is motivated by the fact that, in clinical retinal photography, the actual captured area on the retina is not always the same because of eye motion . The result shows that fds calculated in 3 different rois are associated with each other, with p values less than 0.01 . However, as we can see from figure 7, a smaller roi produces a lower fd in general, because fewer vessels are taken into account . Therefore, this study implies that a fixed region of interest is necessary in order to obtain comparable fd values . In the fifth study, we investigated the influence of the accuracy of vessel segmentation methods on the fractal measurements . Most vessel segmentation methods need a threshold value to convert the vessel probability map into a vessel binary map this threshold value also affects the accuracy of the segmentation . In this study, we computed the fd on vessel binary segmentations using different thresholds (mcc ranged from 61% to 78%). As expected, the computed fd values become closer to the ones obtained from manual segmentations when segmentation accuracies increase (with respect to manual segmentation). Finally, in the sixth study, we compared the fds calculated on images acquired by different fundus cameras . The result shows that the variations of fd are significant when different cameras are used . These five cameras use different flashing systems resulting in different contrast and tissue reflections . Finally, the image sizes and resolutions are different, so the details of retina captured by these cameras are also not identical . Moreover, some cameras were easier to operate (e.g., via autofocus), resulting in more consistent image quality . The comparison result shows that, in general, the fd of the same subject using different cameras has significant differences . The differences in terms of image properties cause significant variations as we see from the results . Besides the variation between cameras, we also investigated the repeatability of the fd measurement on the same subject using the same camera . The slight differences among multiple acquisitions on the same patient with the same camera are caused by variation in image quality, for example, caused by eye motions (blurry image), weak flashing / illumination, or incorrect focusing . Our experiments suggest that the classic fractal dimensions must be calculated under very strict conditions, and tiny changes on the images and vessel segmentation can cause significant variations . The vessel segmentation method must be very carefully chosen, the region of interest in all images must be equally set for the fd calculation, and an optimal threshold value for creating a high accuracy binary vessel segmentation map is required . For future studies, fd's high sensitivity to the segmentation methods and thresholding techniques will be addressed by measuring fd directly from the vessel probability maps.
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Klippel - feil syndrome (kfs) is a rare condition characterized by the congenital fusion of any two of the seventh cervical vertebrae . Sprengel's deformity (sd) is a congenital structural abnormality of the shoulder girdle; receive surgical treatment as a child or adolescence . The omovertebral bone is characterized as an abnormality in the musculoskeletal system . In this anomaly, however, some studies reported association between sd and omovertebral bone and introduced it as kfs . Shoulder girdle anomaly concomitant with muscle disorder such as hypoplasia or atrophy are called sd . Vertebral fusion of cervical spines is rare, but it is possible to occur in that spines . First symptoms that they explained were including short neck, restricted motion of the neck, and low hairline . After that time the prevalence of this syndrome is unclear, but some studies believe that its prevalence is about 1 in 40,000 of alive newborns . Furthermore, this syndrome affect female more than male . It should be considered in the setting of neurological symptoms characteristic of cervical myelopathy in patients with obvious skeletal dysmorphias of unknown etiology . Short neck, low hairline, and restricted neck motion are diagnostic triad is present in less than half of the patients . We report a 50-year - old woman who suffered from neck pain and ataxia for a long time . To the best of our knowledge she presented with a 2 years history of neck pain and ataxia for 1 year . She hadn't urinary incontinence and she was referred to a neurosurgeon by a neurologist because of her progressive gait ataxia . Neurological examination showed intact cranial nerves and no motor deficit, but we found impairment in pain sensory and light touch in both legs that was prominent on the right side of the body, hyperreflexia in the left knee, ankle jerk, and mild gait ataxia based on tandem and blind walking . Physical examination revealed a short neck, a low occipital hairline and diminished cervical range of motion . The patient also had an elevated left scapula and a bony prominence extending from the shoulder to the neck . Anteroposterior and lateral radiographs revealed fused vertebral body of c5-c6 without spina bifida [figures 1, 2a and b]. Multislice computed tomography, three dimensional (a) computed tomography (b) oblique view ct postroanterior view radiographs in flexion and extension position demonstrated neither vertebral instability nor narrowing of the retrodental distance in ante- and retroflection . Subsequent magnetic resonance (mr) and computed tomography imaging with three dimensional reconstructions of the neck and shoulder revealed an atypical bone configuration of the left shoulder with elevation and dysplasia of the scapula and an aberrant bony structure extending from the superomedial border of the scapula to the c5 transverse process leading to constriction of the spinal canal [figures 13]. Resection of the omovertebral bone and decompression of the spinal canal were indicated with respect to patient's clinical myelopathy . A curved incision was performed over the omovertebral bone from its cervical origin to its scapular termination . The intraspinal localized aberrant bony fragment, which we considered responsible for the neurological syndrome, was removed from the cleft in the posterior arch of c5, a laminectomy was performed at that level, and the omovertebral bone was partially resected . In terms of gait ataxia the patient was neurologically unchanged, but she experienced a significant reduction in her neck pain, improved range motion of the neck, and the cosmetic result was good immediately after the operation . She recovered well from the procedure and was discharged from the hospital 3 days post - operatively . Maurice klippel and andre feil explained a syndrome separately that is defined by a clinical triad include short neck, restricted range of motion in the neck and low posterior hairline . After that time, some patients with this syndrome found extra anomalies that can be accompanying to kfs . The prevalence of kfs has been reported about 1 of 40,000 or 1 of 42,000 persons and most of them are female . Present case was a woman who had mentioned triad . According to the level of vertebral fusion, some studies classified it into three groups: (1) many vertebras in cervical and thoracic zone are fused to each other, (2) fusion is seen in one or two vertebra like a fusion between c2-c3 or c5-c6, and (3) vertebras are associated with each other in cervical and thoracic or lumbar zone accompany with other anomalies . Our patient had fusion between c5-c6 with an elevated left scapula and a bony prominence extending from the shoulder to the neck . Torticollis and loss of symmetrical expression of the face has been reported in 10% and 11.5% of cases with kfs respectively . Our patient that was a rare presentation in that age had multiple anomalies including sd, omovertebral bone, and scoliosis . As it is mentioned, symptoms and signs that point to kfs are various and it can present as cosmetic problems or severe problem such as predisposing to severe neurologic damage . Thus, sometimes it is introduced as an emergent situation and if you misdiagnose it, it will remain serious neurologic sequel . Sd is another anomaly that is seen in 7 - 42% of cases with kfs . Muscular dysfunction resulted from some common defects in the trapezius, rhomboids and levator scapulae and some uncommon defects in muscles such as the pectoralis major / minor, latissimus dorsi, sternocleidomastoid, and serratus anterior . This process is started at levels of 4 - 6 cervical spines and then it descends to normal location opposite to second to seventh vertebra of thoracic . Events that happen in that time can cause sd omovertebral bone is seen in 25 - 35% (even 50% in some studies) of patients who are suffered from sd . In fact, it is a cartilaginous or bony and or fibrous tissue with a firm covers that fuse the scapula and processes of cervical spines . Conservative treatment is not acceptable in sd and surgery usually is useful for removing deformity . However, when physicians make a diagnosis and plan treatment, they should search the features of kfs and sd . The case of a unilateral sd and kfs as an etiology for cervical myelopathy is unique in the adults . Risk for brachial plexus injury because of compression or stretching by the clavicle accelerate with age . Therefore, the surgical approach of adults patients with sd can intend suitable surgical conclusions.
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Hormonal contraception is one of the most widely used contraceptive modalities and provides very good efficacy and low failure rates . Continued research has explored and introduced new formulations and clarified some of the concerns regarding the use of hormonal contraceptives, and indeed overall reductions in all - cause and heart disease mortality rates were recently reported by the uk royal college of general practitioners study . Drospirenone (drsp) represents the newest generation of progestogen used in oral contraceptive pills . A large multicenter prospective observational study assessed a combined oral contraceptive (coc) containing 30 g ethinylestradiol (ee) and 3 mg drsp (yasmin; bayer ag, leverkusen, germany), which improved water retention symptoms and bleeding pattern . An ultra - low - dose preparation containing 20 g ee and 3 mg drsp in a novel 24/4 regimen (yaz; bayer ag) has been marketed recently . Its efficacy is similar to, if not better than, that of older cocs and has an acceptable bleeding pattern . Yaz is currently the only coc with reported evidence for and approved indication in the treatment of emotional and physical symptoms of premenstrual dysphoric disorder and has shown improvement in productivity, social activities, and relationships [3 - 5]. Another new preparation (qlaira; bayer ag) containing 17-beta - oestradiol instead of ee as the oestrogen component has been marketed recently . It contains oestradiol valerate (e2v) and dienogest in a multiphasic regime that is optimised to provide good efficacy (adjusted pearl index of 0.34) and at the same time satisfactory cycle control . It is the first preparation using natural oestradiol, but clinical benefits over the older preparations remain to be explored in comparative studies ., ny, usa), a subcutaneous preparation of depot medroxyprogesterone acetate (dmpa) 104 mg in 0.65 ml, has been introduced in recent years . The slightly lower dosage, optimized for delivery by subcutaneous administration, was determined in pharmacokinetic studies on both caucasian and asian women to meet the minimum serum concentration required to provide consistent ovulation suppression over 3 months . Its efficacy is similar to, if not better than, that of the conventional intramuscular preparation of dmpa (dmpa - im) and allows self - administration by the user . A multicenter clinical study reported no pregnancies out of a total of 16,023 women - cycles . Bleeding disturbances are similar to dmpa - im, resulting in discontinuation in 3 - 4% of users in 1 year of use, and about 55% of women became amenorrhoeic with continuing use . A modest weight gain (of up to 4.5 kg) by 3 years the levonorgestrel (lng)-only regimen is now a standard for emergency contraception (ec). The effectiveness of the single - dose regime (lng 1.5 mg) is similar to that of split - dose lng and could minimize compliance problems and is currently the recommended regime approved for use up to 72 hours following unprotected sexual intercourse . Mifepristone is superior to lng in efficacy but is available only in china for ec . Doses of 25 - 50 mg are very effective, and lower doses (less than 25 mg) may be equally good . The progesterone receptor modulator, ulipristal acetate (cdb-2914), is a new option . Ulipristal acetate 30 mg (ellaone; hra pharma, paris, france) has been recently marketed in europe as an ec . A meta - analysis of two randomised controlled trials suggested that it is more effective than lng (failure rate 1.4% versus 2.2%), and it can be used up to 5 days after unprotected sexual intercourse . Multiple reports in the literature have suggested an association between dmpa use and a decreased bone mineral density (bmd), which is at least partially reversible upon discontinuation, but the clinical significance has remained unclear, particularly with regard to the long - term risk of clinical fracture . Recently, a population - wide case control analysis of contraceptive use in danish women who had clinical fractures was reported; this demonstrated a statistically significant increase in fracture risk (adjusted odds ratio 1.44, 95% confidence interval 1.01 - 2.06) in dmpa - users versus non - users, with the risk most pronounced in women more than 50 years old or those who used it for more than 4 years . However, there were only a small number of dmpa - users in the cohort, and due to the nature of the study design, not all potential confounders might have been addressed . A large - scale randomised controlled trial sufficiently powered to detect a difference in fracture risk would be extremely difficult practically . Another multicenter prospective randomised controlled trial compared subcutaneous dmpa with the conventional intramuscular dmpa over a 2-year period and found them to be very similar in regard to a small reversible bmd loss . On the other hand, it is increasingly recognised that combined hormonal contraceptives might also have a potential impact on bone mass accrual in adolescents and young adults . A systematic review found inconsistent data relating to effects of cocs on bone mass in adolescents and young women, and only one good quality study was identified; it concluded that coc - users did not gain as much bone mass as non - users . A 4-year non - randomised follow - up study found a significantly lower increment in the mean adjusted bone mineral content in young adolescent (12 - 19 years old) users of combined hormonal contraceptive for more than 2 years . A population - wide case control analysis of coc use in danish women who had clinical fractures suggested that there is no clear increase in fracture risk with cocs . Drospirenone (drsp) represents the newest generation of progestogen used in oral contraceptive pills . A large multicenter prospective observational study assessed a combined oral contraceptive (coc) containing 30 g ethinylestradiol (ee) and 3 mg drsp (yasmin; bayer ag, leverkusen, germany), which improved water retention symptoms and bleeding pattern . An ultra - low - dose preparation containing 20 g ee and 3 mg drsp in a novel 24/4 regimen (yaz; bayer ag) has been marketed recently . Its efficacy is similar to, if not better than, that of older cocs and has an acceptable bleeding pattern . Yaz is currently the only coc with reported evidence for and approved indication in the treatment of emotional and physical symptoms of premenstrual dysphoric disorder and has shown improvement in productivity, social activities, and relationships [3 - 5]. Another new preparation (qlaira; bayer ag) containing 17-beta - oestradiol instead of ee as the oestrogen component has been marketed recently . It contains oestradiol valerate (e2v) and dienogest in a multiphasic regime that is optimised to provide good efficacy (adjusted pearl index of 0.34) and at the same time satisfactory cycle control . It is the first preparation using natural oestradiol, but clinical benefits over the older preparations remain to be explored in comparative studies ., ny, usa), a subcutaneous preparation of depot medroxyprogesterone acetate (dmpa) 104 mg in 0.65 ml, has been introduced in recent years . The slightly lower dosage, optimized for delivery by subcutaneous administration, was determined in pharmacokinetic studies on both caucasian and asian women to meet the minimum serum concentration required to provide consistent ovulation suppression over 3 months . Its efficacy is similar to, if not better than, that of the conventional intramuscular preparation of dmpa (dmpa - im) and allows self - administration by the user . A multicenter clinical study reported no pregnancies out of a total of 16,023 women - cycles . Bleeding disturbances are similar to dmpa - im, resulting in discontinuation in 3 - 4% of users in 1 year of use, and about 55% of women became amenorrhoeic with continuing use . A modest weight gain (of up to 4.5 kg) by 3 years the levonorgestrel (lng)-only regimen is now a standard for emergency contraception (ec). The effectiveness of the single - dose regime (lng 1.5 mg) is similar to that of split - dose lng and could minimize compliance problems and is currently the recommended regime approved for use up to 72 hours following unprotected sexual intercourse . Mifepristone is superior to lng in efficacy but is available only in china for ec . Doses of 25 - 50 mg are very effective, and lower doses (less than 25 mg) may be equally good . The progesterone receptor modulator, ulipristal acetate (cdb-2914), is a new option . Ulipristal acetate 30 mg (ellaone; hra pharma, paris, france) has been recently marketed in europe as an ec . A meta - analysis of two randomised controlled trials suggested that it is more effective than lng (failure rate 1.4% versus 2.2%), and it can be used up to 5 days after unprotected sexual intercourse . Multiple reports in the literature have suggested an association between dmpa use and a decreased bone mineral density (bmd), which is at least partially reversible upon discontinuation, but the clinical significance has remained unclear, particularly with regard to the long - term risk of clinical fracture . Recently, a population - wide case control analysis of contraceptive use in danish women who had clinical fractures was reported; this demonstrated a statistically significant increase in fracture risk (adjusted odds ratio 1.44, 95% confidence interval 1.01 - 2.06) in dmpa - users versus non - users, with the risk most pronounced in women more than 50 years old or those who used it for more than 4 years . However, there were only a small number of dmpa - users in the cohort, and due to the nature of the study design, not all potential confounders might have been addressed . A large - scale randomised controlled trial sufficiently powered to detect a difference in fracture risk would be extremely difficult practically . Another multicenter prospective randomised controlled trial compared subcutaneous dmpa with the conventional intramuscular dmpa over a 2-year period and found them to be very similar in regard to a small reversible bmd loss . On the other hand, it is increasingly recognised that combined hormonal contraceptives might also have a potential impact on bone mass accrual in adolescents and young adults . A systematic review found inconsistent data relating to effects of cocs on bone mass in adolescents and young women, and only one good quality study was identified; it concluded that coc - users did not gain as much bone mass as non - users . A 4-year non - randomised follow - up study found a significantly lower increment in the mean adjusted bone mineral content in young adolescent (12 - 19 years old) users of combined hormonal contraceptive for more than 2 years . A population - wide case control analysis of coc use in danish women who had clinical fractures suggested that there is no clear increase in fracture risk with cocs . The drsp - containing combined hormonal contraceptives offer similar contraceptive efficacy with specific benefits of improved water retention symptoms and, for yaz, a licensed use for treatment of premenstrual dysphoric disorder . A new coc preparation that contains the natural 17-beta - oestradiol and that is similar to the conventional cocs in efficacy and acceptable cycle control has been marketed; however, its benefits over the older cocs are yet to be explored and it is markedly more expensive . A subcutaneous form of dmpa provides efficacy and side effect profiles that are similar to, if not more favorable than, those of the conventional intramuscular dmpa and provides a self - injectable option . In regard to ec, the single - dose lng - only regime is a recommended first - line option . A new product containing ulipristal acetate provides a more effective alternative to lng and can be used up to 5 days after unprotected sexual intercourse . Most reports in the current literature found a negative effect of both dmpa and combined hormonal contraceptives on bmd, but the clinical significance remains debatable . The evidence so far is not adequate to suggest any limit on their use or any additional monitoring in users who are otherwise healthy.
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Pertussis is an acute respiratory tract infection caused by bordetella pertussis; this infection is most severe in infants . There are still 60 million cases of pertussis each year worldwide, resulting in about 300,000 deaths1). However, pertussis cases are increasing in western countries with high vaccination coverage since the 1990s2). In korea, an average of approximately 11.5 cases of pertussis is reported to the korea center for disease control and prevention (kcdc) each year, and there have been no fatalities since the 1990s3). However, in the 2000s, the pertussis cases reported tended to increase, with up to 66 cases being reported in 20094, 5). Since the diagnosis of the reported cases was confirmed by pcr and culture without serology, this article was intended to review the latest knowledge about the pathogenesis, epidemiology, diagnosis, treatment, and prevention of pertussis . Pertussis is a mucosal infection whose pathogenesis is induced by the local and systemic effects of toxins . More than 95% of pertussis cases occur during epidemics and in endemic areas and are caused by b. pertussis infection; the remaining 5% of cases are caused by infection with bordetella parapertussis6). Possess the pertussis toxin (pt), which is the bacteria's major virulence protein that infects only humans . B. pertussis organisms are acquired through aerosols and have strict tropism for the ciliated epithelium of the respiratory tract . Attachment to target cells is mediated by filamentous hemagglutinin (fha), some agglutinogens (especially fim2 and fim3), a 69-kd nonfimbrial surface protein called pertactin (prn), and pt . Bacterial survival is aided by ciliostasis related to the tracheal cytotoxin (tct) and impaired leukocyte function due to pt and the adenylate cyclase (ac) toxin . Local epithelial damage and respiratory tract symptomatology pt plays a major role in the severity of pertussis cough, encephalopathy, and lymphocytosis by preventing migration of the organism from the circulating blood7). Pertussis is highly contagious, with attack rates as high as 100% in susceptible individuals who have been exposed to contaminated aerosol droplets at close range . However, neither the disease nor vaccination confer complete or lifelong immunity against the disease or protect against reinfection . Protection against the typical disease begins to wane 3 to 5 years after vaccination and is not measurable by 12 years . When exposed to pertussis infection, over 50% of vaccinated people develop asymptomatic, mild desease8) and become a source of infection for infants in whom pertussis may result in hospitalization and death . Pertussis is a significant cause of mortality in early infancy worldwide, despite widespread vaccination . It remains endemic in many industrialized countries, which have reported increases in incidence during the past several decades . This increase has been mainly among adults and adolescents, and chronic cough that persisted for over 2 weeks was the main cause (20 - 37.2%), although death and hospitalization from pertussis continue to occur predominantly in unvaccinated infants aged less than 6 months9, 10). In korea, the incidence of pertussis has dramatically decreased since dtwp vaccination started in 1958 and dtwp was changed to dtap in 1982 . The number of pertussis cases reported to the kcdc was approximately 11.5 cases each year until 1991, but the incidence of pertussis seems to be on the rise since 2000, although the vaccination rate continues to be over 94% . In 2009, 66 cases of pertussis were reported . The kcdc confirmed that these cases were pertussis by culture or pcr of pertussis from nasal specimens and a positive rate of culture and pcr is very low compared to serologic study . On the basis of the sensitivity of the test and reporting rate in 2009, it can be assumed that more than 2,000 pertussis occurred in 2009 (fig . Classical pertussis has 3 stages of clinical symptoms: catarrhal, paroxysmal, and convalescent . Nonspecific symptoms such as rhinorrhea, sneezing, mild fever, and eye infection without significant conjunctivitis occur after about 2 weeks in the catarrhal stage, and these symptoms are followed by characteristic paroxysmal stage symptoms such as a dry, intermittent, irritative hack which evolves into paroxysms . In the paroxysmal stage, a healthy - looking, playful toddler suddenly begins to express an anxious aura and experiences bouts of uninterrupted coughing while drawing a single breath . The coughs are followed by a loud whoop as inspired air passes through the still partially closed glottis (fig . 3 months, the catarrhal phase usually lasts for a few days or is not recognized at all; the paroxysmal and convalescent stages are protracted, with periods of spasmodic coughing throughout the first year of life . In the paroxysmal stage, young infants frequently present with gagging, gasping, choking, cyanosis, apnea, or an " apparent life - threatening event " instead of paroxysmal coughing; whooping is absent . In immunized children and adults, all stages of pertussis are shortened, and it is not uncommon for the disease to not have distinct stages and show chronic cough paroxysms that continue for more than 2 weeks . The principal complications of pertussis are apnea; secondary infections such as pneumonia and otitis media; respiratory failure because of apnea, pneumonia, or pulmonary hypertension; and physical sequelae because of forceful coughing . Pertussis - related complications and mortality are reported, especially in patients younger than 2 months, who have the highest reported rates of pertussis - associated hospitalization (> 90%), pneumonia (15 - 25%), seizures (2 - 4%), encephalopathy (0.5 - 1%), and death (0.5 - 1%)11 - 13). There are several reports on pertussis as a significant cause of sudden infant death syndrom14 - 16). Pertussis should be considered as a diagnosis in very young infants who have not completed 3 doses of vaccination and have classical symptoms or in children who display major symptoms such as chronic paroxysmal cough without significant fever, general weakness, and sore throat and do not have physical signs such as wheezing and rales . Chlamydia trachomatis infection in very young infants and adenovirus, mycoplasma pneumoniae, and chlamydia pneumoniae infections should be the differential diagnosis for children because despite the abovementioned differences, it is not easy to diagnose pertussis on the basis of only clinical symptoms and signs, especially if secondary infections such as pneumonia are present . The laboratory finding characteristic for pertussis is leukocytosis (leukocyte counts of 15,000 - 100,000 cells / mm due to absolute lymphocytosis in the late catarrhal and paroxysmal stages . The degree of leukocytosis parallels the severity of illness and is mainly observed in unvaccinated children; leukocytosis is not common in adolescents, adults, and partially immunized children with pertussis17, 18). Chest x - ray findings are normal in most cases, although chest x - ray images of hospitalized young infants may show hilar infiltration, interstitial edema, atelectasis, and, rarely, pneumothrax . There are several ways to confirm b. pertussis infection such as serology, culture, direct fluorescent antibody (dfa) testing, and pcr . The traditional gold standard for confirming pertussis is culture, but many factors that can influence the results, such as the stage of illness, use of antibiotics, cautious posterior nasopharyngeal sampling, adequate transport media (regan - lowe) and culture media (regan - lowe charcoal media with 10% horse blood or 5 - 40 g / ml cephalexin), and vaccination status of the patient . In adolescents and adults with pertussis, culture or pcr test results are positive in <10% of cases that have been confirmed as pertussis by serology . However, pcr is more rapid and sensitive than culture for confirming pertussis19). Serologic tests use enzyme immunoassay (eia) to detect antibodies to components of b. pertussis and are the most sensitive tests for diagnosis in distantly immunized individuals and those evaluated after the second week of experiencing coughs . A 4-fold increase in anti - pt igg with 4 - 6 week intervals appears to be the most reliable serologic test, and a single test showing an anti - pt igg level of> 94 - 110 eu / ml (over 2sd of the igg level in the community) has been proposed as the diagnostic cutoff point20). The goals of therapy are to limit the number, severity, and duration of paroxysms and prevent complications . Hospitalization is usually needed to treat infants with pertussis because they need supportive care such as hydration, nutrition, and monitoring for complications . If the patient experienced uncontrolled severe cough along with hypoxia, seizures, and apnea, ventilator care should be provided along with muscle paralysis to prevent bradycardia and cardiopulmonary complications . Antibiotics should be given to infants younger than 1 year old who have had the cough paroxysm for less than 6 weeks and to all the patients who began coughing within 3 weeks in order to improve symptoms, shorten their duration, and prevent further infection21). Erythromycin is the treatment of choice; 40 - 50 mg / kg erythromycin is prescribed for 10 - 14 days . Newer macrolides such as clarithromycin and azithromycin can be used instead of erythromycin and have the same effect22). Since pertussis is extremely contagious, there have been many secondary cases and outbreaks of pertussis in hospitals and chronic - care facilities . To prevent infection from the index patient, droplet precautions are recommended for at least 5 days after the initiation of macrolide therapy and those who have had close exposure should get the same macrolide treatment as pertussis patients . In addition, children younger than 7 years who did not complete the 5 rounds of pertussis vaccination should complete the schedule of vaccination . Adolescents older than 11 years and adults should receive the tdap (tetanus toxoid, diphtheria toxoid, acellular pertussis): booster vaccination . The current pertussis vaccination schedule in korea comprises a primary series of 3 vaccinations: the first dose is administered at 2 months of age, the second is a booster vaccination given at 18 months of age, and the third dose is also a booster vaccination given at 4 - 6 years of age . Despite longstanding and widespread vaccination programs, pertussis remains endemic in many industrialized countries, including australia, canada, italy, japan, the netherlands, switzerland, and the united states, all of which have reported recent increases in incidence . Factors contributing to pertussis resurgence remain unclear, but possible causes are waning immunity, improved surveillance and diagnosis, and adaptation of circulating b. pertussis strains23, 24). Although pertussis is classically a disease of infants and children, the main portions of the population showing an increased incidence of pertussis are adults and adolescents; these infected adolescents and adults become a source of infection for infants . The problem of waning immunity after pertussis vaccination and disease, tdap booster vaccination for adolescents older than 11 years and adults has been started since 2006 in the united states and was introduced in korea in 2009 . Pertussis was a leading cause of death of children before pertussis vaccination was started, and the disease continues to be a leading cause of child mortality . The incidence and mortality of pertussis has been increasing since 1990 in developed countries despite these countries having vaccination rates of 95% . After widespread vaccination, the diagnosis of pertussis is difficult because of lack of classical clinical manifestations in very young infants and vaccinated children . In korea, the prevalence of pertussis might be increasing since 2000 and there was a pertussis outbreak in 2009 . No epidemiological study based on serology has been done so far in korea, and the exact number of pertussis cases that occur and are undiagnosed remains unknown . Therefore, we do not have any weapons to fight against future outbreaks of pertussis, a significant cause of mortality of very young infants . Therefore, i suggest that a nationwide epidemiologic study of pertussis be undertaken immediately . The study should aim to develop methods for serologic diagnosis, new devise vaccination strategies to prevent waning immunity and reduce the number of very young unvaccinated individuals, and develop diagnosis criteria for atypical pertussis.
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Dental materials contain a great variety of different monomers and additives.1 because of the complex chemical composition and the incomplete monomer polymer conversion, several components are leached out from each resin - based restorative material into the oral environment.2,3 this in turn may cause some adverse effects.4 previous studies have used in vitro cytotoxicity tests to evaluate the biological risks of resin composites used in dentistry.1,5 cytotoxicity tests have primarily focused on restorative materials such as glass ionomers, dental adhesives and composite resins.68 however, fewer studies on prosthodontic materials have been published, and investigations regarding the cytotoxicity of provisional prosthodontic materials are even more limited.5,9 provisional restorations are used in the interim between tooth preparation and fitting a definitive restoration . The length of time between preparation of teeth and cementation of final restorations can vary from a few days for straightforward cases, to several weeks or even, in the case of complex reconstruction, several months . Provisional restorations are generally essential to cover freshly cut dentine, stabilize the position of the prepared tooth, regain chewing function and phonation, maintain esthetic appearance and evaluate the minimal thickness of the definitive restoration . They can also help stabilize the periodontal condition prior to definitive restoration.10 provisional materials can be classified by the type of resin . Acrylic polymethyl or polyethyl methacrylates belong to the oldest group of provisional materials . The latest class of materials is formed by bis - acryl composite resins, which are comparable to composite resins used for direct restoration therapy.10 they consist of an organic matrix and inorganic fillers . Bis - acryl composites produce less heat and shrinkage during polymerization than other resins, resulting in a better marginal fit.11 aesthetically they are reasonable and are more color stable than polymethyl or polyethyl methacrylates.12 most recently, visible light cured resins have been introduced based on urethane dimethacrylate . These resins have good mechanical properties, being light cured, the operator has some control over the material s working time and colour is relatively stable but marginal fit can be poor.10,13 acrylates and mainly methacrylates were found to cause cytotoxic effects.14 evaluation of the cytotoxicity of dental resin materials showed a relationship between their composition and the degree of cytotoxicity.15 continuous cell lines, like l929 mouse fibroblasts are being routinely used for the testing of cytotoxic properties of dental materials because of their reproducible growth rates and biological responses.1 the purpose of this in vitro study was to evaluate the effect of current bis - acryl and urethane dimethacrylate based provisional materials on the fibroblast cell viability . Two of the tested materials were bis - acryl based (tempofit duomix, detax, germany & protemp 3 garant, 3 m espe, germany) and one was urethane dimethacrylate based (revotek lc, gc corporation, japan) provisional restoration materials . Test specimens were prepared according to the manufacturers instructions in standard teflon discs, 5 mm in diameter and 2 mm of height . All specimens were prepared and handled under aseptic conditions to limit the influence of biological contamination on the cell culture tests . Specimens were prepared between mylor and glass slabs to minimize the oxygen inhibition and maximize the surface smoothness . Tempofit duomix is a two - part base / catalyst, hand - mix, self - curing and bis - acrylic composite based provisional restoration material . Base and catalyst were extruded equal amounts by pressing onto piston in the dispenser onto mixing pad . Protemp 3 garant is a two - part base / catalyst, auto - mix, self - curing and bis - acrylic composite based provisional restoration material . Using the garant dispenser, the base and catalyst were extruded directly into the teflon disc and after 2 min 30 sec curing completed . Revotek lc is a light cure single component sculptable composite resin for temporary restorations . Using a spatula required amount of material dispensed and applied into the teflon disc . The specimen was light - cured for 6 sec by led light curing unit (led, bluephase, ivoclar vivadent, liechtenstein, austria). The samples immersed in 7 ml culture medium for 24 hours at 37c to extract residual monomer or cytotoxic substances . The culture medium containing material extracts were sterile filtered to use on the cell cultures . L929 fibroblast cell line (atcc ccl 1) cultured in basal medium eagle (bme), biological industries, israel) containing 10% new born calf serum (biochrom ag, berlin, germany) and 100 mg / ml penicilin / streptomysin (biological industries, israel) at 37c in a humidified atmosphere of 95% air/5% co2 . Confluent cells were detached with 0.25% trypsin and seeded at a density of 510 well in 96-well plate at 37c under 5% co2 for 24h and . After 24 hours incubation, culture medium was replaced with 200 l of culture medium containing material extracts of provisional restoration materials . The succinic dehydrogenase activity has been shown to be reasonably representative of mitochondrial activity in the cells and reflects both cell number and activity.16 the old medium removed and cell cultures were rinsed with phosphate buffer saline (pbs) and 200 l aliquots of freshly prepared mtt [3-(4,5-dimethyl - thiazol-2-yl)-2,5-diphenyl - tetrazolium bromide, sigma aldrich, germany] solution (0.5 mg / ml in bme) were added to each well . After a 2h incubation period (37c, 5% co2) the supernatant was removed and the intracellulary stored mtt formazan was solubilized in 200 l dimethyl sulfoxide for 30 min at room temperature . Twelve replicate cell cultures were exposed to a constant concentration of a single material in at least two independent experiments . L929 fibroblast cell line (atcc ccl 1) cultured in basal medium eagle (bme), biological industries, israel) containing 10% new born calf serum (biochrom ag, berlin, germany) and 100 mg / ml penicilin / streptomysin (biological industries, israel) at 37c in a humidified atmosphere of 95% air/5% co2 . Confluent cells were detached with 0.25% trypsin and seeded at a density of 510 well in 96-well plate at 37c under 5% co2 for 24h and . After 24 hours incubation, culture medium was replaced with 200 l of culture medium containing material extracts of provisional restoration materials . The succinic dehydrogenase activity has been shown to be reasonably representative of mitochondrial activity in the cells and reflects both cell number and activity.16 the old medium removed and cell cultures were rinsed with phosphate buffer saline (pbs) and 200 l aliquots of freshly prepared mtt [3-(4,5-dimethyl - thiazol-2-yl)-2,5-diphenyl - tetrazolium bromide, sigma aldrich, germany] solution (0.5 mg / ml in bme) were added to each well . After a 2h incubation period (37c, 5% co2) the supernatant was removed and the intracellulary stored mtt formazan was solubilized in 200 l dimethyl sulfoxide for 30 min at room temperature . Twelve replicate cell cultures were exposed to a constant concentration of a single material in at least two independent experiments . In contrast, protemp 3 garant group demonstrate full cell density in figure 2(c). The results showed that, eluates of the revotek lc and protemp 3 garant lead to 99% and 101% cell survival . Statistically revotek lc and protemp 3 garant were not cytotoxic for cells when compared to control group (p>.05). Tempofit duomix was cytotoxic for cells when compared to control group and other tested materials (p<.05). The literature contains descriptions of cell - culture tests with various cell types to establish cell damage caused by dental materials.17 in the present study the effect of two bis - acryl and one urethane dimethacrylate based commercially available provisional restoration materials on fibroblast cells were investigated by mtt test . Fibroblasts are the targets of any chemical components that may be released from the dental restorative materials . L929 fibroblast cells were selected due to its availability, popularity and efficiency to grow in vitro.18 mtt assay is a well - established method for analyzing cell viability.16 the viability and proliferation of the cells are assessed by means of the functional state of the cell mitochondria.19 mitochondrial dehydrogenases in living cells reduce the yellow tetrazolium salt, mtt (3-(4,5-dimethyl) thiazol-2-yl) 2,5 diphenyltetrazolium bromide) to blue mtt formazan, which is then retained in the cell . Formation of the formazan product has been found to correlate well with number of viable cells.8,19,20 today, bis - acryl composites possess considerable amount of the market share for tooth colored provisional material . Main advantages of bis - acryl provisional materials include a lower curing temperature, reduced polymerization shrinkage (5%) with improved marginal fit, and minimal odour and taste.13,21 the low setting temperature of these materials allows them to be used directly with decreased risk of pulpal injury.22 in addition, bis - acryls are gaining in popularity, in part because of their cartridge delivery system . This dispensary method not only is convenient but also may allow for a more accurate and consistent mix.21 dental practitioners have clearly welcomed these products and very limited data can be available about their cytotoxicity and biocompatibility . In present study, two of the tested provisional restoration material was bis - acryl based which are chemically very similar to bisphenol - a - glycidyl methacrylate (bis - gma) composites . According to our results, eluates from tempofit duomix lead to 88% cell survival and when compared to control group and other tested materials it was cytotoxic for cells (figure 2a b). On the hand protemp 3 garant, the other bis - acryl based provisional material, was not cytotoxic for l929 fibroblast cells (figure 2c). The modifications include a newly developed monomer system, not with the rigid intermediate chain characteristic of some bis - gma homologues, but with a somewhat flexible chain in comparison to other synthetic resins (espe technical product profile). However, manufacturer of tempofit duomix do not state any difference in monomer formulation . Probably as most other bis - acryl based provisional materials, the organic polymer matrix of tempofit duomix is composed of traditional monomers such as bis - gma, triethylene glycol dimethacrylate (tegdma) or similar monomer systems . Patents may also hinder objective research.23 only available composition of the resin cements tested in this study . Current investigations reported the cytotoxic effects of some resin monomers, such as bis - gma, tegdma and urethane dimethacrylate (udma).24,25 these resin monomers are able to deplete intracellular glutathione as well as interfere with the expression of some proteins, such as collagen i, osteonectin, and dentin sialoprotein, which play a fundamental role in the pulp repair.26,27 among the tested materials, revotek lc is the only udma based and light cure provisional material . Geurtsen et al1 reported that udma is as cytotoxic as bis - gma and tegdma . Elution of residual monomers from resin materials related to degree of their polymerization, properties of resin composition, and chemistry of organic solvents in vitro situation.28 altntas et al29 demonstrated that leaching of udma was lower than bis - gma and tegdma from a resin cement . Consequently, in present study, eluates of the revotek lc showed similar cytotoxicity with control group . The results of this study demonstrated that cytotoxic potential may vary among provisional materials . Taking into consideration the limitations of this in vitro study, provisional restoration materials may have cytotoxic effects and should be selected carefully for clinical applications.
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Annually, an estimated 63000 new cases of primary nonmalignant and malignant central nervous system (cns) tumors are diagnosed in the united states with an estimated 13000 deaths . Most of the primary cns tumors are located within the frontal, temporal, parietal and occipital lobes of the brain . There appear to be some difference between the patterns of brain tumor epidemiology in iran and western countries . In the first report from iran by ameli et al ., the prevalence of glioma was estimated to be about 45% of all brain tumors, somewhat low in comparison to the western reports, but almost the same as southeast asian countries . In fact, in iranian reports of glial tumor subtypes, the majority of lesions are low grade astrocytoma and ependymomas [2, 3] which is very different from western countries, such as the us and france . Radiotherapy plays an important role in the management of most malignant and many benign primary cns tumors . With the goal of achieving uncomplicated loco - regional tumor control, balancing between benefits and side effects the most important challenge in radiotherapy is delivering prescribed dose to the tumor and minimize dose to the normal tissues . Emami et al . Obtained tolerance radiation dose of 28 critical organs; their findings showed the td5/5 for lens, optic nerve, retina, chiasma, parotid and thyroid as 10, 50, 45, 50, 32 and 45 gray (gy), respectively . An absorbed dose of 4 gy to the lens of eye in three weeks to three months leads to cataract . So in the frontal lobe irradiation, lenses will not save at all and cataract will be induced . In other organs, although organ dose is tolerated, but in order to minimize complications it seems necessary to develop new technique . Two major complications might occur after frontal lobe irradiation; necrosis and visual disturbances which might be due to high dose received by chiasma [13 - 15]. In this study, for frontal lobe brain tumors three routine conventional radiotherapy techniques were compared in terms of dose distribution and absorbed dose to critical organs using treatment planning system (tps) calculation and direct measurement in an anthropomorphic phantom . Then a new treatment technique was designed with same criteria as a mentioned above . Due to the high probability of visual disturbances in frontal lobe brain radiotherapy, we tried to reduce absorbed dose to the chiasma significantly considering new fields and bringing out the chiasma from radiation field with overall lower dose to other considered critical organs . Construction of phantom to construct the phantom, natural bone with paraffin wax with sodium chloride (nacl) as impurity was used; the effective atomic number and electron density of phantom soft tissue were 6.57 and 3.361023 (electron g-1), respectively . A hollow cavity and two hollow tubes were considered as the mouse cavity, trachea and esophagus, respectively . Six cylinders were made from phantom material which had cavities to insert thermoluminescent dosimeters (tlds) at several depths, one for parotid (a transverse one from the left to the right parotid), one for chiasma (perpendicularly inserted from top of brain), two for eyes and two for thyroids (figure 1). Tld measurement technique radiation dosimetry was done using cubic lithium fluoride tld chips (3mm3mm1 mm). The tlds (tld-100, harshaw, usa) were initially sorted into groups of equal sensitivity . This was accomplished by delivering a known dose (100 cgy) from 6 mv x - ray (siemens primus linac) and consequently measurement of the output light from each tld using a tld reader (harshaw model 3500 tld reader, usa). The annealing cycle of tlds consisted a heat cycle of 1 hour at 400c and then immediately 2 h at 100c . Calibration procedure was done by exposing tld to 6mv photons in the range of 0 - 250 centigray (cgy). In order to measure dose values, a calibration curve was plotted in which the tld reading in c is related to absorbed dose in cgy . Besides, the linear calibration equation which used for dose measurement was fitted on experimental data points (figure 2) [16 - 19]. Tps and direct measurement on the phantom an imaginary 4 cm putative tumor with 2 cm margin was considered in the right frontal lobe of the brain and phantom was ct planned . Three routine conventional techniques associated with frontal lobe brain tumors were considered as two lateral opposite - fields (technique 1), one lateral and an anterior field (technique 2), two lateral opposite - fields and an anterior field (technique 3). A new conventional technique with the benefits of conventional techniques and significantly reduced dose to chiasma was designed . This technique consists two 6 mv and 18 mv angled ap / pa fields with an oblique field with 18 mv photon beam . In angled ap / pa fields, the chiasma was brought out from radiation field with a couch rotation of 90 and gantry rotation of 10; plan of the new technique and chiasma are brought in figure 3 . The phantom was planned using above techniques (prowess panther treatment planning software, california, usa) with a total prescribed dose of 6000 cgy in 30 fractions . After contouring ptv and considered critical organs, dose distribution was obtained and absorbed dose to critical organs was extracted from the dose volume histograms (dvh). As the dose fall - off was severe in organs closer to ptv, the selected points on plan (roi) were about the same points where tlds were placed in the phantom . Tlds were inserted in considered places in the phantom (retina, optic nerve, chiasma, lens, submandibular glands, parotids and thyroid) and the phantom was irradiated with above techniques . The results of total absorbed dose to selected organs in selected points estimated from tps and tld in 30 fractions is presented in figure 4 and table 1 . In the assessed techniques, absorbed dose to all organs except lenses were at their tolerance dose levels and in the new technique, absorbed dose to chiasma is significantly reduced . Among all techniques, technique 2 (one lateral and an anterior field) was the best in terms of dose distribution, because of its localized irradiated volume at the side of brain . The differences between calculation and measurement in selected techniques are presented in table 2 as percentage difference . As it is obvious, the differences were as high as 70% in organs far enough from considered ptv such as thyroid and submandibular glands, although absorbed doses were not comparable with their tolerance dose . Also in the new technique, differences were in the range of three routine techniques . In clinical radiation therapy, usually tps is used to estimate dose to different organs; but the obtained values might be different from the actual values depending on the clinical circumstances . Our findings showed differences in the range of 1 - 5% in all techniques between tps and direct measurements for all organs except salivary glands and thyroid; although their distance from primary radiation field causes tld reading being small, but differs with tps (about 60%) which showed very smaller doses; this might be due to scattered radiation which is not well considered in the tps . It has been showed that an increase in parotid irradiated volume from 0% -40% to 90% -100% (carried out in patients who had received a dose of 35 - 45gy), results in decreased secretion from100% to 10% . Due to high probability of cataract induction in lens exposed to 400 cgy in three weeks to three months, and underestimation of doses obtained from plan and measurement at about 5% (~20 cgy), it is important during planning to consider these differences to reduce the incidence of cataract . As showed in table 1, in three routine techniques, chiasma receives high doses (1800 -3000 cgy). As previously has reported [13, 15], gensheimer et al . In a review on the outcomes of lacrimal gland adenoid cystic carcinoma treated with neutrons observed severe visual impairment which might be due to high dose to chiasma . In studying with sino - nasal undifferentiated carcinoma treated using three fields (weighted anterior field and two wedged laterals) found that mean dose to the optic chiasm was 54 gy which exceeded from tolerance dose level; these high doses are in accordance with our study . So, as showed in table 1, in the new technique we could reduce dose to chiasma as low as 596 cgy, and also save other organs better . It is notable that in presented technique, although sparing other organ at risks, the absorbed dose to chiasma was measured as 596 cgy which showed a considerable dose reduction . The present study sought estimates of the radiation doses received by organs at risk in the frontal lobe radiotherapy with direct measurement on the anthropomorphic phantom using tld dosimetry . In addition, a comparison between the estimated values from treatment planning system and direct measurement shows differences in the range of 1.1% to 70% between the estimated and measured values . The dose values of some organs despite the large difference between the two methods, was within their tolerance dose and so there will be no concern about elevated risk of radiation induced adverse effects . The present study sought estimates of the radiation doses received by organs at risk in the frontal lobe radiotherapy with direct measurement on the anthropomorphic phantom using tld dosimetry . In addition, a comparison between the estimated values from treatment planning system and direct measurement shows differences in the range of 1.1% to 70% between the estimated and measured values . The dose values of some organs despite the large difference between the two methods, was within their tolerance dose and so there will be no concern about elevated risk of radiation induced adverse effects.
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The matricellular proteins are a heterogeneous group of extracellular matrix (ecm) proteins that interacts with either other ecm proteins and with cell - surface receptors, growth factors and cytokines . The periostin, also called osteoblast - specific factor 2 (osf-2), is a non - structural matricellular protein that directly interacts with type i collagen and fibronectin regulating the biomechanical properties of connective tissues . It is an 811 amino acid, 93 kda secreted ecm protein and four main isoforms have been identified which are not uniformly but differentially expressed in various cell lines; a human periodontal ligament specific isoform has been recently characterized . Periostin is present in collagen - rich connective tissues like bone, skeletal muscle, tendons, joint ligaments, periodontal ligament, heart valves, adipose tissue, or skin . At present the functions of periostin in several biomedical areas like osteology, oncology, cardiovascular and respiratory systems, inflammatory diseases or dentistry, in both normal and pathological conditions, are rather well known . As a whole its main functions seem to be during the development and tissue repair, because the predominant role in mesenchymal remode ling . A series of recent studies have detected expression of periostin in developing and mature dental tissues, alveolar bone or periodontal ligament . Consistently, mice deficient in periostin display changes in ecm of dental tissues as well as defects in mineralized tissues . By contrast little information is available about the occurrence and distribution of periostin in the gingiva . It is known that gingival fibroblasts may be a source of periostin in response to some cytokines which are elevated in periodontitis, and decreased levels of periostin were found in gingival crevicular fluid proportionally with the progression and severity of periodontal disease . Moreover, periostin is involved in signaling pathways of drug - induced gingival over - growth . Since no data are available about the distribution of periostin in adult human gingival in normal conditions, and because its potential role in gingival pathologies, we used western blot and immunohistochemistry to analyze the occurrence and localization of periostin in these tissues . To establish whether periostin is cellular or extracellular we labeled in parallel keratinocytes and dermal fibroblasts samples of gingival tissues (sized 2x2x2 mm, approximately) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces, and were obtained from the instituto asturiano de odontologia . The age range was 16 to 32 years, and were males (n=8) and females (n=4). The tissue samples were washed with tap water followed with cold saline, then fixed in buffered 10% formalin for 24 h, and routinely processed for paraffin embedding . The pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . Moreover, fresh samples (n=6 from different subject) were quickly frozen, stored at -80c and used for western blot . This study was approved by the ethics committee of instituto asturiano de odontologia (oviedo, spain) and informed consent was obtained from each subject . Lysates prepared from gingival homogenates were processed as follows: representative samples of the free gingival, attached gingival and non - bone attached periodontal ligament were pooled and homogenized (1:2, w / v) in tris - hcl buffered saline (tbs, 0.1 m, ph 7.5) containing 1 m leupeptin, 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride . The homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8, 2% sds, 100 mm dithiothreitol, and 10% glycerol at 4c . After electrophoresis, proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk, and 0.1% tween-20 . The membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin (ls - bl10443, lifespan biosciences, inc ., after incubation, the membranes were washed with tbs ph 7.6 containing 20% tween-20, and incubated again for 1 h with goat anti - rabbit igg (diluted 1:100) at room temperature . Membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . Finally, the reaction was developed using a chemiluminescent reagent (ecl, amersham pharmacia biotech, buckinghamshire, uk) and exposed to hyperfilm . Relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva . Deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit (dako, copenhagen, denmark) following the manufacturer s recommendations . Moreover, anti - vimentin (clone 334, boehringer - mannheim, mannheim, germany), anti - pancytokeratin (clone pck-26, sigma - aldrich quimica, sl, madrid, spain) mouse monoclonal antibodies, and anti - collagen type i rabbit monoclonal antibody (erp778, abcam, cambridge, uk) were used to label dermal fibroblasts, epithelial cells and type i collagen fibrils, respectively . The sections were processed for simultaneous detection of periostin and vimentin, and periostin and pan - cytokeratin, as follows: the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs . The sections were then incubated overnight, at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies (both diluted 1:100 in the blocking solution); or anti - periostin and anti - pancytokeratin antibodies (diluted 1:200 and 1:100, respectively, in the blocking solution); or anti - vimentin and anti - type i collagen (both diluted 1:100 in the blocking solution). After rinsing with tbs, the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg (serotec, oxford, uk), diluted 1:1000 in tbs containing 5% mouse serum (serotec), then rinsed again, and incubated for another hour with cy3-conjugated donkey anti - mouse antibody (jackson - immunoresearch, baltimore, md, usa) diluted 1:50 in tbs . Both steps were performed at room temperature in a dark humid chamber . Finally, to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium (10 ng / ml). Triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope (photonic microscopy service, university of oviedo) coupled with a leica confocal software, ver . 2.5 (leica microsystems gmbh, heidelberg, germany) and the images captured were processed using the software image j version 1.43 g master biophotonics facility, mac master university ontario (www.macbiophotonics.ca). For control purposes representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies, or omitting the primary antibodies in the incubation . Under these conditions samples of gingival tissues (sized 2x2x2 mm, approximately) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces, and were obtained from the instituto asturiano de odontologia . The age range was 16 to 32 years, and were males (n=8) and females (n=4). The tissue samples were washed with tap water followed with cold saline, then fixed in buffered 10% formalin for 24 h, and routinely processed for paraffin embedding . The pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . Moreover, fresh samples (n=6 from different subject) were quickly frozen, stored at -80c and used for western blot . This study was approved by the ethics committee of instituto asturiano de odontologia (oviedo, spain) and informed consent was obtained from each subject . Lysates prepared from gingival homogenates were processed as follows: representative samples of the free gingival, attached gingival and non - bone attached periodontal ligament were pooled and homogenized (1:2, w / v) in tris - hcl buffered saline (tbs, 0.1 m, ph 7.5) containing 1 m leupeptin, 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride . The homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8, 2% sds, 100 mm dithiothreitol, and 10% glycerol at 4c . After electrophoresis, proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk, and 0.1% tween-20 . The membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin (ls - bl10443, lifespan biosciences, inc ., seattle, wa, usa), used diluted 1:200 . After incubation, the membranes were washed with tbs ph 7.6 containing 20% tween-20, and incubated again for 1 h with goat anti - rabbit igg (diluted 1:100) at room temperature . Membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . Finally, the reaction was developed using a chemiluminescent reagent (ecl, amersham pharmacia biotech, buckinghamshire, uk) and exposed to hyperfilm . Relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva . Deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit (dako, copenhagen, denmark) following the manufacturer s recommendations . The anti - periostin antibody was the same described above . Moreover, anti - vimentin (clone 334, boehringer - mannheim, mannheim, germany), anti - pancytokeratin (clone pck-26, sigma - aldrich quimica, sl, madrid, spain) mouse monoclonal antibodies, and anti - collagen type i rabbit monoclonal antibody (erp778, abcam, cambridge, uk) were used to label dermal fibroblasts, epithelial cells and type i collagen fibrils, respectively . The sections were processed for simultaneous detection of periostin and vimentin, and periostin and pan - cytokeratin, as follows: the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs . The sections were then incubated overnight, at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies (both diluted 1:100 in the blocking solution); or anti - periostin and anti - pancytokeratin antibodies (diluted 1:200 and 1:100, respectively, in the blocking solution); or anti - vimentin and anti - type i collagen (both diluted 1:100 in the blocking solution). After rinsing with tbs, the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg (serotec, oxford, uk), diluted 1:1000 in tbs containing 5% mouse serum (serotec), then rinsed again, and incubated for another hour with cy3-conjugated donkey anti - mouse antibody (jackson - immunoresearch, baltimore, md, usa) diluted 1:50 in tbs . Finally, to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium (10 ng / ml). Triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope (photonic microscopy service, university of oviedo) coupled with a leica confocal software, ver . 2.5 (leica microsystems gmbh, heidelberg, germany) and the images captured were processed using the software image j version 1.43 g master biophotonics facility, mac master university ontario (www.macbiophotonics.ca). For control purposes representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies, or omitting the primary antibodies in the incubation . Under these conditions the expression of periostin at the protein level in the human gingiva was analyzed using western blot and immunohistochemistry . In gingival homogenates western blot associated to the anti - periostin antibody used throughout this study detected a single protein band with an estimated molecular weight of 94 kda (figure 1a) which is consistent with than expected for the human periostin . Imagej densitometric analysis demonstrated that the higher relative levels of periostin with respect to -actin corresponded to the non - bone attached periodontal ligament segment, followed by free gingiva and attached gingiva (figure 1b). To map the histological distribution of periostin in the human gingiva we used double immunofluorescence coupled with laser - confocal microscopy in three different sectors: the free gingiva, the attached gingiva, and the segment of connective tissue related to the junctional epithelium (which is especially rich in fibers of the periodontal ligament). The results were compared with the pattern of localization of cytokeratin and vimentin . As a rule periostin was never localized within the cells but always in the extracellular space . In the sections processed for the simultaneous demonstration of periostin plus cytokeratin (figure 1 c, d, g, h) it was observed that periostin - immunoreactivity is concentrated at the epithelium - connective tissue junction, presumably associated to ecm proteins of the basal membrane and was never found within the cytoplasm of the basal epithelial cells . On the other hand, the distribution of the immunoreactivity for periostin and type i collagen were similar at the epithelium - connective tissue junction (figure 1 e, f) although a faint widespread immunostaining for type i collagen was also detected in the connective tissue . Double immunofluorescence was also carried out for periostin and vimentin in order to determine the relation of periostin with the connective tissue fibroblasts . Independently of the gingival segment analyzed, free (figure 2 a - c) or attached gingival (figure 2 d - f), periostin immunoreactivity was dissociated from the vimentin positive fibroblast and was restricted to the epithelial - connective tissue junction . Regarding to the gingival segment containing the non - bone attached periodontal ligament, disposed under the junctional epithelium, the distribution of the periostin immunoreactivity was irregular, and never co - localized with the vimentin - positive fibroblasts (figure 2 g - i). Although some images occasionally might suggest co - localization of periositin witn vimentin or cytokeratin 2d cytofluorograms from the two detection channels of periostin (green) and cytokeratin or vimentin (red) demonstrated absence of co - localization (figure 2 j - l). As a summary, in human normal gingiva periostin is extracellular and is restricted to the epithelial - connective tissue junction, and among the fibroblasts forming the non - bone attached segments of the periodontal ligament . This study was designed to investigate the occurrence and distribution of the segregated matricellular protein periostin in the human gingiva . The localization of periostin in the dental tissues and the periodontal ligament in now rather well known, but no information is available about its distribution in the gingiva in spite of its potential role in the pathophysiology of some gingival disorders . The protein we have identified by western blot has a molecular weight of 94 kda . This is in good agreement with the expected molecular mass of this protein (93.3 kda), and was similar to that found in other human epithelial tissues like the cornea . The small differences between our results and others could be related to occurrence of tissue specific periostin isoforms, or post - translational processes of the protein . Based on image densitometry the relative level of periostin varies among different segments of the gingiva . The reason for these differences might be related to the density of cells producing periostin, and the functional significance of these findings if any remain to be elucidated . We have regularly observed that periostin in normal gingiva is restricted to the epithelium - connective tissue junction, and is also present around the fibers of the non - bone attached portion of the periodontal ligament . As far as we know this is the first study reporting the distribution of periostin in the human gingiva . In agreement with the identification of periostin as a matricellular protein we observed that it is localized extracellularly nevertheless, other studies on epithelial tissues reported the presence of periostin immunoreactivity in the basal keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin, or in the basal layer cells of the human corneal epithelium . According to those authors periostin only becomes extracellular during tissue remodeling of wound repair . Since in normal gingiva periostin is exclusively extracellular, its function may be distinct from that of skin, and the presence of intracellular periostin probably reflects non - secreted protein . The role of periostin in the interactions of the connective tissue and the epithelial cells has been extensively studied, however it remains still unknown in the gingiva . In the skin periostin plays pivotal functions in collagen fibrillogenesis, collagen cross - linking, and the formation of ecm meshwork via interactions with other ecm components . Whether or not it is the same for the gingiva remains to be investigated . In the skin periostin expression this is indicative of the physiologically protective functions of periostin, which promotes wound repair producing myofibroblast differentiation, keratinocytes proliferation and fibroblast proliferation and migration . The localization of periostin in the gingiva and the since it is an ecm secreted protein suggest it could signal in both epithelial cells . Matricellular proteins appear to be of importance in collagen assembly, and the expression of periostin in the epithelial - connective tissue junction suggest it may influence the biology of the basal membrane, including collagen fibrillogenesis (especially type i collagen which co - localizes) but probably its presence may be also related to cell migration, proliferation and adhesion of fibroblasts . On the other hand, the free and sub - junctional epithelium segments of the gingival contain fibers of the periodontal ligament subject to mechanical stress which in turn may activate latent tgf- 1 and to increase periostin mrna in periodontal ligament fibroblasts . Therefore, periostin may participate in the maintenance of the structure and reparative processes in adult human gingival as it occurs in the skin, although in these tissues have different localization, extracellular vs intracellular . As a summary, our study has demonstrated the localization of periostin in the ecm of the periodontal ligament and the cell - free zone of the dental pulp in adult human teeth.
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Incremental cost of pim use in the elderly is estimated to be $7.2 billion, pim use in ambulatory elderly patients has been found to be 50 per 1,000 person years . The elderly population is especially susceptible to use of pim as up to 94% take at least one pim . Pim is defined as use of a drug with risk of an adverse event that outweighs its clinical benefit, particularly when there is a safer or more effective alternative therapy for the same condition . Beers criteria to assess and reduce the risk of pim in the elderly by identifying pim has been developed . Utilization of this criteria has shown pims to be associated with a 25% increase in the risk of hospitalizations in elderly nursing home residents . In addition, the incidence of pim use has been reported to range from 22% to 66% during hospitalization, and 44% upon discharge . Geriatric population is very fragile and prone to medication side effects in view of changing physiological capacities of the body . The decline is much more rapid when co morbidities, especially diabetes and hypertension set in . There is a corresponding decline in other body systems, including: cardiac, respiratory, gastrointestinal, and neurovascular . This results in accentuated drug side effects and toxicities secondary to slowed pharmacokinetics and pharmacodynamics . This is the subset of population where more and more use of polypharmacy and pim use is observed . However, there are very few studies available evaluating the association of pims with hospital readmission . Mansur and colleagues evaluated several objectives in a cohort of hospitalized elderly patients, including the relationship of pims with readmission and mortality, 3 months post discharge . They found no significant difference in pim use among patients who were readmitted or died as compared to those who were not readmitted or survived . However, this trial had several limitations, including a small sample size of 212 patients . The present study was designed to see if polypharmacy and/or pim use was related to readmission rates to the hospital irrespective of the admission diagnosis . The objective of this study was to evaluate the association of pim use with hospital readmission within 30 days regardless of the admitting diagnosis . This nested case - control study included all patients readmitted within 30 days of discharge to a 250 bed community hospital located in scranton, pennsylvania usa . All patients 65 years of age or older admitted between january 2008 and december 2009 were eligible for study enrollment . Day 0 was defined as the day of discharge and day1 was defined as the day - after surgery . The present study included all the consecutive readmissions and no controls were used for comparison . The modified beer's criteria is the most widely followed drug list that is used to screen potentially inappropriate drug use in the geriatric population [table 1]. Modified beer criteria showing list of the potential associated with inappropriate usage of medicines this was a retrospective case record review of 414 patients who were hospitalized within 30 days of discharge from the hospital between january 2008 and december 2009 . There was no bias in the selection of patients on the basis of age, sex, co morbidities, height, weight, marital status, ethnicity, and socio - economic status . The institutional review board of hospital approved the study and waived the requirement for informed consent for the retrospective review of medical records . The goal of the study was to identify the patient characteristics associated with readmission to the hospital within 30 days of discharge from the hospital . Day 0 was defined as readmission on the same day before midnight and any readmission after midnight was defined as 1 day . Rest of the readmissions were defined as within 30 days of discharge but after day 1 . Demographic and laboratory data was retrieved from an electronic hospital database . After merging data from the different sources, automated and manual data verification was performed . Statistical analysis was carried out using a two - way anova on the data to see if polypharmacy and/or pim use was related to readmission within 30 days of discharge irrespective of admission diagnosis . This was a retrospective case record review of 414 patients who were hospitalized within 30 days of discharge from the hospital between january 2008 and december 2009 . There was no bias in the selection of patients on the basis of age, sex, co morbidities, height, weight, marital status, ethnicity, and socio - economic status . The institutional review board of hospital approved the study and waived the requirement for informed consent for the retrospective review of medical records . The goal of the study was to identify the patient characteristics associated with readmission to the hospital within 30 days of discharge from the hospital . Day 0 was defined as readmission on the same day before midnight and any readmission after midnight was defined as 1 day . Rest of the readmissions were defined as within 30 days of discharge but after day 1 . Demographic and laboratory data was retrieved from an electronic hospital database . After merging data from the different sources, automated and manual data verification was performed . Statistical analysis was carried out using a two - way anova on the data to see if polypharmacy and/or pim use was related to readmission within 30 days of discharge irrespective of admission diagnosis . A total of 312 patients were on polypharmacy (five or more medications at the time of admission) figure 2 . Gender profile of readmissions polypharmacy and its relationship to readmission to the hospital on the same day for readmission and readmission the immediate day after discharge was statistically significant . The impact of polypharmacy and pim combined on hospital readmission was statistically significant only when the readmission occurred on the same day or immediate next day . No polypharmacy and no inappropriate drugs were statistically significant as compared to polypharmacy and pim . At day 1 and day 0, data for all the readmissions was compiled and statistical analysis was carried out . The statistical comparison is expressed as significant (p <0.05) and highly significant (p <0.001) with regard to polypharmacy and pim . No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001)polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs: (p <0.05)poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001)no polypharmacy versus polypharmacy: (p <0.001). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001) polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs: (p <0.05) poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001) no polypharmacy versus polypharmacy: (p <0.001). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy versus polypharmacy: (p <0.01). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy versus polypharmacy: (p <0.01). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001)polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs: (p <0.05)poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001)no polypharmacy versus polypharmacy: (p <0.001). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001) polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs: (p <0.05) poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.001) no polypharmacy versus polypharmacy: (p <0.001). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05)no polypharmacy versus polypharmacy: (p <0.01). No polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs: (p <0.05) no polypharmacy versus polypharmacy: (p <0.01). There is a trend to pay for performance and hospitals are not being reimbursed for so - called never events . The list of the never events has been gradually increasing over the last couple of years . The expansion of never events and hospital acquired conditions by center of medicare and medicaid (cms) would have a substantial financial impact on tertiary care facilities . This is in sharp contrast to earlier trends when hospitals were reimbursed irrespective of the quality of the services offered . Costs have continued to rise at double - digit rates, and quality is far from optimal . In the 21 century, we see aging of the population; the fastest growing population being people over 85 years of age . Adverse drug events, which can be especially problematic in older adults, often can be prevented by detecting potential risk factors . Many primary care doctors possess a poor knowledge of pim and are unaware of prescribing guidelines such as the beers criteria . Also there is poor communication of drugs potential side effects profile between the physician and the patient . Hospital readmission within 30 days of discharge from the hospital is a multifaceted dragon which has to be dealt with a multipronged approach . A substantial amount of economic resources are spent on readmissions, especially in terms of finances . The most common cause of readmission by admitting diagnosis is congestive heart failure followed by psychosis [figure 4]. The usual approach is to blame it on the disease process without addressing the patients co - morbidities . At the hospital, where this study was conducted, this study was designed to quantify the use of inappropriate medications among older adult outpatients . Patients have a more difficult time trying to remember their drug regimen daily and can infringe on quality of life . Not to forget to mention the cost of the polypharmacy . Healthcare spending is also increased with more follow - up visits to doctor's offices . Healthcare spending can also involve the side effects caused by polypharmacy that can include acute drug reactions and readmissions . Tracking the number of patients who experience unplanned readmissions to a hospital after a previous hospital stay is another category of data used to judge the quality of hospital care . In usa, cms services began to look into the issue after a national push by the obama administration . In usa, the president's health - care reform has identified readmission rates as a key target for medical care savings . The hospital compare site allows patients to compare hospitals based on the how the rate of readmission for three treatments heart attack, heart failure, and pneumonia compare with the national average . The rates take into account how sick patients were before they were admitted to the hospital . The modified beer's criteria is the most widely followed drug list that is used to screen potentially inappropriate drug use in the geriatric population . For a complet e list of medications included in the beer's criteria although it remains a useful screening tool to screen for pim, its practical application in acute - care settings remains obscure . Critics of the beer's criteria opine that it is too big to be implemented in its entirety in the acute - care settings . In fact adrs could contribute to accelerated functional decline in elderly hospitalized patients independently of the use of beers listed pims . For example, pregabalin is very poorly tolerated by geriatric population and is not included in the beer's criteria . Furthermore, potentially any medication could be inappropriate in the geriatric population if it is not titrated by the physiological decline, especially renal decline . In this subset of population,, all anticholinergics, opiods, and benzodiazepams should raise a red flag, especially at the time of medication reconciliation . Consideration should be placed on benefit to risk ratio when a new medication is added . On each hospitalization there should be a diligent review of the drug profile and every effort should be made to minimize medications . Screening tool of older persons pim (stopp) have recently found favor as a tool for screening pim . In the hospital setting, proton pump inhibitors (ppi) are very commonly prescribed and are the most over prescribed medications . This not only compounds the cost of healthcare delivery but is detrimental to the patients medical management by increasing morbidity . Along with the over prescribed ppi's, a significant portion of the geriatric population is on clopidogrel because of coexistent coronary artery disease . Ppi added in conjunction with plavix has a 64% higher risk of re hospitalization for myocardial infraction mi or coronary stent placement than patients receiving plavix alone . There is also an increase in stent thrombosis in patients who are on both plavix and ppis . Healthcare professionals should be vigilant patient advocates when it comes to polypharmacy or at least be cautious of all the side effects . This is to drive home the point that pim use be at the back of the mind with a larger goal to minimize polypharmacy . Antidepressants are another class of medications, which is widely used in the geriatric population but does not figure into the beer's criteria . Considering the benefit to risk ratio the widespread use of antidepressants need to be minimized . If we look at the natural course of depression, 60% of the cases are self - resolving if we consider the side effects profile to antidepressants, there is an extensive list and of course like most other medication, side effects are more pronounced in the elderly . Most patients are on antidepressants forever because most physicians are hesitant to taper them off . A good discharge plan should include a meticulous medication reconciliation especially weighing the benefit to risk ratio . We would also recommend a mandated continued medical education on pim on renewal of state license . A good surgeon knows when not to operate and a good physician knows when not to give medicines . Polypharmacy is a big contributor to increasing healthcare cost and increasing morbidity as well as mortality in the elderly population . There should be focused attention on all patients whether in hospital, or in physician's follow - up clinic towards minimizing polypharmacy . If clinicians practice polypharmacy, they are bound to lead to use of pim . In our opinion, internship year should be geared towards orienting the physicians to focus on the geriatric aspect of medicine and improve medication use in elderly population . This is important because india is seeing rapid surge of elderly with infrastructure and health care system ill equipped to handle it . We would need a larger prospective multicenter randomized controlled intervention study to assess the effect of optimization strategies on the appropriateness of prescribing in elderly people.
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Chronic obstructive pulmonary disease (copd) is a major cause of chronic morbidity and mortality throughout the world . The prevalence and burden of copd are projected to increase in the coming decades due to continued exposure to copd risk factors and the changing age structure of the world s population, with copd likely to become the third leading cause of death by 2020.1 morbidity and mortality among patients with copd are in large part related to acute exacerbations (aecopd), which impair respiratory, physical, social, and emotional functioning both acutely and longitudinally.24 the management of aecopd is empirical and includes oral corticosteroids, often combined with antibiotics, although the need to prescribe these antibiotics is still not convincingly demonstrated.5 most placebo - controlled antibiotics trials that have been performed have important limitations and are difficult to compare because different definitions of copd and aecopd and different endpoints have been used.6,7 further, these trials were conducted several decades ago, before systemic steroids were widely introduced for the treatment of aecopd.811 in a more recent placebo - controlled study by llor et al, treatment with amoxicillin / clavulanic acid did show a beneficial effect; however, only 17% of these patients received systemic steroids.12 in a study by daniels et al, in which the add - on effect of antibiotics was investigated, no difference in clinical outcome after 30 days was observed.13 theoretically, to be maximally effective, the antibiotic concentration at sites of infection should exceed the minimum inhibitory concentration at which 90% (mic90) of the growth of potential copd pathogens such as haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis is inhibited . Levels of antimicrobial agents in sputum, where as a representation of the site of infection many potential pathogenic microorganisms are located, may be a more relevant predictor of efficacy of antibiotics in treatment of an aecopd than concentrations in serum.1416 an antibiotic widely used in the treatment of aecopd is amoxicillin / clavulanic acid . In a former study by brusse - keizer et al, in which 33 copd patients were treated with amoxicillin / clavulanic acid for aecopd, sputum amoxicillin concentrations proved to be an important determinant of clinical outcome . Patients with sputum amoxicillin concentrations <mic90 were hospitalized for 4 days longer than patients with an amoxicillin concentration in sputum mic90 (7 versus 11 days). Moreover, 67% of patients in this study had a sputum amoxicillin concentration <mic90.17 the sputum amoxicillin concentration may differ markedly from the concentrations in serum due to various factors, such as the diffusion of amoxicillin into the airways,18 which could be both a host - related as well as a drug - related factor . A well - known drug - related factor associated with amoxicillin, there are several potential pathogens (eg, h. influenza and m. catarrhalis) that produce these beta - lactamase enzymes.19 the use of beta - lactamase inhibitors, such as clavulanic acid, allows inactivation of certain beta - lactamases.20 an excessive beta - lactamase activity in sputum of copd patients compared with the dosage of clavulanic acid could possibly be an explanation for the earlier observed low numbers of copd patients in which an amoxicillin concentration above the mic90 was reached . We therefore conducted a study to investigate whether there was any difference in beta - lactamase activity between copd patients with a sputum amoxicillin concentration <mic90 and those with a concentration mic90 . This study was part of the cohort of mortality and inflammation in copd (comic) study, a single - center prospective cohort study on the immune status of copd patients as a determinant of survival . From december 2005 until april 2010, 795 patients were included in the cohort, with a follow - up period of 3 years . To be eligible for the cohort, patients had to meet the following criteria: a clinical diagnosis of copd, as defined by the global initiative for chronic obstructive lung disease criteria;21 current or former smoker; age 40 years or over; no medical condition compromising survival within the follow - up period or serious psychiatric morbidity; absence of any other active lung disease (eg, sarcodosis); no maintenance therapy with antibiotics; and ability to speak dutch . In this exploratory study, we had no data available on which we could base our power calculations . We therefore included all patients from the comic study when they were hospitalized for an aecopd that was treated with amoxicillin / clavulanic acid between november 2009 and march 2010 . An aecopd was defined as an acute event characterized by a worsening of the patient s respiratory symptoms that is beyond normal day - to - day variations and leads to a change in medication.21,22 patients with pneumonia were not excluded, and pneumonia was defined as an acute respiratory infection combined with an infiltrate of the lungs, which was visible on an x - ray of the chest . The infiltrate had to be non - pre - existent, nor reasonably caused by another cause than pneumonia . The medical ethics committee of medisch specrtum twente, enschede, the netherlands approved the comic study and the amendment for the current study, and all patients provided written informed consent for both the comic study itself and the amendment . All patients received usual care, which included oral corticosteroids and amoxicillin / clavulanic acid started on the day of admission . Amoxicillin / clavulanic acid was administered according to regular care, which was either orally (500/125 mg three or four times a day) or intravenously (1,000/200 mg four times a day). Some patients received oral and intravenous amoxicillin / clavulanic acid sequentially . On the first day of admission, c - reactive protein was measured in blood using the nyocard crp single test (clindia diagnostics, leusden, the netherlands). On the third day of treatment with amoxicillin sample collection was performed on the third day of treatment because, theoretically, steady - state amoxicillin concentrations in both serum and sputum should be reached by then.23 serum samples were stored at 80c . Sputum samples were stored at 80c after a culture was performed . Before measuring amoxicillin concentrations and beta - lactamase activity in sputum, sputum samples were thawed and homogenized using a magna lyser (360 seconds at 6,500 rpm; roche diagnostics, indianapolis, in, usa). After homogenization, vials were centrifuged at 10,000 g for 5 minutes to separate cell debris . Amoxicillin concentrations in serum and homogenized sputum samples were determined using a high - performance liquid chromatography / tandem mass spectrometry method . For amoxicillin, an a priori cut - off level of 2 mg / l was defined as an adequate concentration in both serum and sputum . The mic90 used in this study is derived from local susceptibility tests from the regional laboratory of public health and is comparable with data published by the european committee on susceptibility testing.24 beta - lactamase activity was measured directly in homogenized sputum samples by measuring the turnover rate of nitrocefin (calbiochem, san diego, ca, usa). Nitrocefin is a beta - lactam with chromogenic properties; it changes color from yellow to red under the influence of beta - lactamase activity . Nitrocefin turnover was measured with spectrophotometry at =490 nm in a mixture of 50 l of homogenized sputum and 50 l of a 0.025% nitrocefin solution . Beta - lactamase activity was quantified by comparing the measured activity with the activity of a beta - lactamase positive lysate from a h. influenzae culture . Beta - lactamase activity was calculated as a percentage of the activity of the h. influenzae lysate . Continuous variables are expressed as the mean (standard deviation [sd]) or as the median (interquartile range [iqr]). Categorical variables are displayed as numbers (percentages). The crude relationship between beta - lactamase activity and sputum amoxicillin concentrations (<mic90 or mic90) whitney u test . To identify confounders in this relationship, first the association of a priori selected possible confounders with beta - lactamase activity was analyzed by pearson correlation tests, independent samples t - tests, and analysis of variance for normally distributed (continuous / dichotomous / categorical) variables . For non - normally distributed variables, this was performed with, respectively, spearman correlation tests, mann variables associated with beta - lactamase activity with a significance of p<0.15 were tested for an association with sputum amoxicillin concentration (<mic90 or mic90). For categorical variables, these associations were tested by chi - square tests or by fisher s exact tests, and for continuous variables by independent samples t - tests or mann whitney u tests . Variables that were also associated with sputum amoxicillin concentration with a significance of p<0.15 were considered as potential confounders in the relationship between beta - lactamase activity and sputum amoxicillin concentration and were entered in a multivariate logistic regression model . Subsequently, variables with the highest p - values were eliminated step by step, until the fit of the model decreased significantly, based on the 2 log likelihood . The statistical analyses were performed using statistical package for the social sciences version 15.0 software (spss inc ., chicago, il, usa). This study was part of the cohort of mortality and inflammation in copd (comic) study, a single - center prospective cohort study on the immune status of copd patients as a determinant of survival . From december 2005 until april 2010, 795 patients were included in the cohort, with a follow - up period of 3 years . To be eligible for the cohort, patients had to meet the following criteria: a clinical diagnosis of copd, as defined by the global initiative for chronic obstructive lung disease criteria;21 current or former smoker; age 40 years or over; no medical condition compromising survival within the follow - up period or serious psychiatric morbidity; absence of any other active lung disease (eg, sarcodosis); no maintenance therapy with antibiotics; and ability to speak dutch . In this exploratory study, we had no data available on which we could base our power calculations . We therefore included all patients from the comic study when they were hospitalized for an aecopd that was treated with amoxicillin / clavulanic acid between november 2009 and march 2010 . An aecopd was defined as an acute event characterized by a worsening of the patient s respiratory symptoms that is beyond normal day - to - day variations and leads to a change in medication.21,22 patients with pneumonia were not excluded, and pneumonia was defined as an acute respiratory infection combined with an infiltrate of the lungs, which was visible on an x - ray of the chest . The infiltrate had to be non - pre - existent, nor reasonably caused by another cause than pneumonia . The medical ethics committee of medisch specrtum twente, enschede, the netherlands approved the comic study and the amendment for the current study, and all patients provided written informed consent for both the comic study itself and the amendment . All patients received usual care, which included oral corticosteroids and amoxicillin / clavulanic acid started on the day of admission . Amoxicillin / clavulanic acid was administered according to regular care, which was either orally (500/125 mg three or four times a day) or intravenously (1,000/200 mg four times a day). On the first day of admission, c - reactive protein was measured in blood using the nyocard crp single test (clindia diagnostics, leusden, the netherlands). On the third day of treatment with amoxicillin sample collection was performed on the third day of treatment because, theoretically, steady - state amoxicillin concentrations in both serum and sputum should be reached by then.23 serum samples were stored at 80c . Sputum samples were stored at 80c after a culture was performed . Before measuring amoxicillin concentrations and beta - lactamase activity in sputum, sputum samples were thawed and homogenized using a magna lyser (360 seconds at 6,500 rpm; roche diagnostics, indianapolis, in, usa). After homogenization, vials were centrifuged at 10,000 g for 5 minutes to separate cell debris . Amoxicillin concentrations in serum and homogenized sputum samples were determined using a high - performance liquid chromatography / tandem mass spectrometry method . For amoxicillin, an a priori cut - off level of 2 mg / l was defined as an adequate concentration in both serum and sputum . The mic90 used in this study is derived from local susceptibility tests from the regional laboratory of public health and is comparable with data published by the european committee on susceptibility testing.24 beta - lactamase activity was measured directly in homogenized sputum samples by measuring the turnover rate of nitrocefin (calbiochem, san diego, ca, usa). Nitrocefin is a beta - lactam with chromogenic properties; it changes color from yellow to red under the influence of beta - lactamase activity . Nitrocefin turnover was measured with spectrophotometry at =490 nm in a mixture of 50 l of homogenized sputum and 50 l of a 0.025% nitrocefin solution . Beta - lactamase activity was quantified by comparing the measured activity with the activity of a beta - lactamase positive lysate from a h. influenzae culture . Beta - lactamase activity was calculated as a percentage of the activity of the h. influenzae lysate . Continuous variables are expressed as the mean (standard deviation [sd]) or as the median (interquartile range [iqr]). Categorical variables are displayed as numbers (percentages). The crude relationship between beta - lactamase activity and sputum amoxicillin concentrations (<mic90 or mic90) first the association of a priori selected possible confounders with beta - lactamase activity was analyzed by pearson correlation tests, independent samples t - tests, and analysis of variance for normally distributed (continuous / dichotomous / categorical) variables . For non - normally distributed variables, this was performed with, respectively, spearman correlation tests, mann variables associated with beta - lactamase activity with a significance of p<0.15 were tested for an association with sputum amoxicillin concentration (<mic90 or mic90). For categorical variables, these associations were tested by chi - square tests or by fisher s exact tests, and for continuous variables by independent samples t - tests or mann whitney u tests . Variables that were also associated with sputum amoxicillin concentration with a significance of p<0.15 were considered as potential confounders in the relationship between beta - lactamase activity and sputum amoxicillin concentration and were entered in a multivariate logistic regression model . Subsequently, variables with the highest p - values were eliminated step by step, until the fit of the model decreased significantly, based on the 2 log likelihood . The statistical analyses were performed using statistical package for the social sciences version 15.0 software (spss inc ., chicago, il, usa). Between november 2009 and march 2010, 147 patients were screened for eligibility (figure 1). The univariate analysis showed no difference in beta - lactamase activity between patients with a sputum amoxicillin concentration <mic90 and patients with a concentration mic90 with, respectively, a median beta - lactamase activity of 0.35 (iqr 0.260.59) and 0.32 (iqr 0.180.62; p=0.79). Also when individual data of beta - lactamase activity and sputum amoxicillin concentrations were plotted in a scatter diagram (figure 2) no correlation could be observed (r=0.06, p=0.80). In 18 of 23 sputum samples (78%), amoxicillin concentrations were below the mic90 . In six of those samples (26%), seven of 23 serum samples (30%) had an amoxicillin concentration below the mic90 . . Only route of administration, median daily clavulanic acid dose, and c - reactive protein concentration at admission were univariately associated with beta - lactamase activity (p<0.15; table 2) and were tested for an association with sputum amoxicillin concentration . All three were also significantly associated with sputum amoxicillin concentration <mic90 or mic90 (table 3). Multivariate logistic regression analysis showed no significant relationship between beta - lactamase activity and sputum amoxicillin concentrations <mic90 versus mic90 (odds ratio 0.53 for a 0.1% increase in beta - lactamase activity; 95% confidence interval 0.231.2; p=0.13). The likelihood of reaching a sputum concentration mic90 was 80 times greater when amoxicillin / clavulanic acid was administered intravenously versus via the oral route (odds ratio 80.6; 95% confidence interval 1.64100; p=0.03). No difference in beta - lactamase activity was found between patients with an amoxicillin concentration in sputum <mic90 and patients with a concentration mic90 when treated with amoxicillin / clavulanic acid . Although we found that 78% of the patients had a low sputum concentration of amoxicillin (<mic90), beta - lactamase activity does not seem to be the reason for this observation . Beta - lactamase activity could not explain the low numbers of patients with an adequate amoxicillin concentration in sputum, so it seems that there should be other host - related or drug - related factors that influence the penetration across the blood - bronchus and alveolar - capillary barriers . The most important host - related factor is the integrity of the anatomical barriers which may be damaged by inflammation and mechanical injury . In the presence of inflammation, the distribution of amoxicillin may be altered because of increased membrane permeability.25,26 as observed in this and our previous study a high level of c - reactive protein, a marker of systemic inflammation, was related to higher amoxicillin levels.17 c - reactive protein levels could therefore possibly be used as a marker to determine copd patients in whom adequate concentrations could be reached . However, this does not solve the problem of low concentrations in the majority of patients; this is a worrying feature, especially because our previous study showed that this is also associated with significantly worse clinical outcomes.17 this warrants further investigation into more optimal treatment in these patients . Since beta - lactam antibiotics such as amoxicillin do not cross membranes readily,27 it might be interesting to look at other antibiotics for the treatment of aecopd that have a better penetration in sputum.28 also, the possibility of individually tailored amoxicillin dosing could be investigated, since it has been shown that increased systemic dosing of amoxicillin is associated with increased levels in sputum . We observed that intravenous administration was associated with a higher probability of reaching a sputum amoxicillin concentration mic90 and led to higher serum concentrations (data not shown), but still 50% of patients had a concentration <mic90 . Further, due to the wide confidence interval that was observed with this probability and since we did not observe any differences in the numbers of patients who reached the mic90 between intravenous and oral administration in our earlier study,17 we cannot recommend a preference for use of either intravenous or oral administration to reach adequate amoxicillin sputum levels . Aerosolized antibiotics have been proven to deliver high concentrations of antibiotics into the airways with low systemic bioavailability . Stockley et al treated patients with bronchiectasis with nebulized amoxicillin and observed significant reductions in sputum purulence and volume after failure with the same drug given orally.29 it would therefore be interesting to administer amoxicillin / clavulanic acid via inhalation to reach the mic90, and perhaps to improve the efficacy of antibiotic treatment, as seen in cystic fibrosis.29,30 the observed concentration of amoxicillin in serum is in the range of concentrations found in earlier published studies, which showed concentrations between 3.87 and 45.2 mg / l . Observed concentrations of amoxicillin in sputum ranged between 0.23 and 4.4 mg / l.3136 however, these concentrations were measured in non - copd patients and in healthy subjects . Our study could have been influenced by selection bias, since we only included patients who were able to produce a sufficient amount of sputum on day 3 . There might have been a difference in beta - lactamase activity and/or amoxicillin concentration between patients who were able to produce sputum and those who were not . Patients who were able to produce sputum may have had other characteristics in terms of presence / absence of bacteria or cause of exacerbation (eg, viral / bacterial infection). To overcome this issue, induction of sputum with hypertonic saline and the effects on possible dilution of amoxicillin concentration sputum induction has proven to be safe even during exacerbations in copd patients.37 since the majority of patients had an amoxicillin concentration <mic90, the beta - lactamaseactivity of these patients could be compared to the beta - lactamaseactivity of only a small group of patiets with a concentration mic90 . Although there seems to be no difference in median beta - lactamase activity, this study cannot definitely conclude that no relationship exists, and further studies are still necessary . We observed no relationship between beta - lactamase activity and sputum amoxicillin concentration (<mic90 or mic90) in patients treated with amoxicillin / clavulanic acid for an acute exacerbation in copd . Further, we repeatedly observed that the majority of patients had low sputum concentrations of amoxicillin (<mic90), suggesting that current treatment with antibiotics should be optimized.
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