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The standard treatment of choice is anterior cervical discectomy and fusion (acdf). Although most patients wish to treat both the posterior neck pain and associated arm pain, some patients wish to be treated with less invasive methods, even though some tolerable symptoms may persist, due to their social / physical situations . Here all of the patients primarily complained of radiating arm pain, which was associated with a tolerable degree of neck pain . In korea, only physically healthy persons are allowed to serve in the military, and if a soldier was to undergo cervical spine surgery with implantation, the soldier discharged from the military against their will according to the military regulation . A pension after retirement is not enough to provide for a middle - aged father and his family . If they want to keep their job after undergoing surgery, they should be able to perform their military drills after the operation . Therefore, many doctors have agonized over how to treat middle - aged soldiers without forcing them to sacrifice their jobs and how to help them to maintain a level of physical fitness that is sufficient for soldiers . He had been suffering from radiating pain in the left c7 sensory dermatome (sd) for 2 yr . The pain intensity, measured by the visual analogue scale (vas), was 8/10 . He had been waiting for a promotion and endured the pain for 2 yr in hopes of getting the promotion . Physical examination revealed weakness in the right triceps (motor grade iv / v), and triceps jerk could not be elicited . Magnetic resonance imaging (mri) revealed disc protrusion in the right c6 - 7 foramen associated with disc degeneration at c5 - 6 (fig . Although he had already been promoted, he wanted to keep his job until he reached the retirement age of 54 yr . He has one son and one daughter, both of whom are in high school . He is a healthy man, and he did not want to be discharged from the army while in his socially and physically active stage of life . He complained of radiating pain at the left c7 sd (vas 7/10) and posterior neck pain (vas 2/10) that had lasted for 2 months . Physical examination revealed weakness in the left triceps (motor grade iv / v), and triceps jerk could not be elicited . He had been suffering from severe radiating arm pain at the left c7 sd (vas 10/10) for 2 weeks . Physical examination revealed weakness in the left triceps (motor grade iii / v), and triceps jerk could not be elicited . Mri showed disc rupture on the left posterior lateral side of the c6 - 7 disc space (fig . He wanted to treat the arm pain first, but he also wanted to keep his job as a soldier . All operations were performed after listening to the patients' needs and explaining the anticipated medical and social / physical outcomes . Mri was performed one week after the operations . During the follow - up period, motor grade was evaluated and flexion / extension films of the cervical spine were also checked . Each patient was situated in the prone position, and the neck was flexed without fixation . The entry point was 1 cm from the midline and just above the post - articulation . Gentle palpation of the inter - laminar space was made possible after the obturator (6.9 mm outer diameter) was introduced . The opened bevel of the working channel was directed toward the medial side in order to avoid accidentally compressing the spinal cord . Drilling was performed from the medial to lateral margin of the inter - laminar space, and the superior facet was drilled first . The ligamentum flavum was removed, and the lateral margin of the dura and exiting root was visualized . Decompression was confirmed by the lack of compressed lesion inferior and superior to the nerve root . All of the instruments used in the operations and the video system were manufactured by wolf (richard wolf gmbh, knittlingen, germany). All of the patients primarily complained of radiating arm pain, which was associated with a tolerable degree of neck pain . In korea, only physically healthy persons are allowed to serve in the military, and if a soldier was to undergo cervical spine surgery with implantation, the soldier discharged from the military against their will according to the military regulation . A pension after retirement is not enough to provide for a middle - aged father and his family . If they want to keep their job after undergoing surgery, they should be able to perform their military drills after the operation . Therefore, many doctors have agonized over how to treat middle - aged soldiers without forcing them to sacrifice their jobs and how to help them to maintain a level of physical fitness that is sufficient for soldiers . He had been suffering from radiating pain in the left c7 sensory dermatome (sd) for 2 yr . The pain intensity, measured by the visual analogue scale (vas), was 8/10 . He had been waiting for a promotion and endured the pain for 2 yr in hopes of getting the promotion . Physical examination revealed weakness in the right triceps (motor grade iv / v), and triceps jerk could not be elicited . Magnetic resonance imaging (mri) revealed disc protrusion in the right c6 - 7 foramen associated with disc degeneration at c5 - 6 (fig . Although he had already been promoted, he wanted to keep his job until he reached the retirement age of 54 yr . He has one son and one daughter, both of whom are in high school . He is a healthy man, and he did not want to be discharged from the army while in his socially and physically active stage of life . He complained of radiating pain at the left c7 sd (vas 7/10) and posterior neck pain (vas 2/10) that had lasted for 2 months . Physical examination revealed weakness in the left triceps (motor grade iv / v), and triceps jerk could not be elicited . He had been suffering from severe radiating arm pain at the left c7 sd (vas 10/10) for 2 weeks . Physical examination revealed weakness in the left triceps (motor grade iii / v), and triceps jerk could not be elicited . Mri showed disc rupture on the left posterior lateral side of the c6 - 7 disc space (fig . He wanted to treat the arm pain first, but he also wanted to keep his job as a soldier . All operations were performed after listening to the patients' needs and explaining the anticipated medical and social / physical outcomes . Mri was performed one week after the operations . During the follow - up period, motor grade was evaluated and flexion / extension films of the cervical spine were also checked . Each patient was situated in the prone position, and the neck was flexed without fixation . The entry point was 1 cm from the midline and just above the post - articulation . Gentle palpation of the inter - laminar space was made possible after the obturator (6.9 mm outer diameter) was introduced . The opened bevel of the working channel was directed toward the medial side in order to avoid accidentally compressing the spinal cord . Drilling was performed from the medial to lateral margin of the inter - laminar space, and the superior facet was drilled first . The ligamentum flavum was removed, and the lateral margin of the dura and exiting root was visualized . Decompression was confirmed by the lack of compressed lesion inferior and superior to the nerve root . All of the instruments used in the operations and the video system were manufactured by wolf (richard wolf gmbh, knittlingen, germany). Trimming of the protruded annulus was also performed after nucleus fragment removal in the first patient . After the operation, a computed tomography scan was obtained in order to determine the extent of the foraminotomy and to evaluate facet joint violation . In all patients, the patients were required to wear a neck brace for 7 days, and free neck motion was allowed from the 8th day after operation after the stitches were removed . The first and second patients showed significant improvement in radiculopathy within 1 day after the surgery, and both patients reported more than 90% improvement from the preoperative state, with slight improvements in neck discomfort (excellent outcome by macnab's criteria). The weakness in their triceps improved to motor grade v / v within 7 days after the operation . Dynamic film of the cervical spine taken one week after the operation showed no instability or change in disc height with preservation of motion (fig . They returned to their military base and were able to perform their military drills without difficulty within 10 days and 1 month of the surgery, respectively . The third patient complained of residual radicular pain (vas 4) 1 day after the operation . During the operation, a large disc fragment (10.8 cm) the direction of the herniated nucleus was posterior and lateral, and it was impossible to trim the annulus (capsule of ruptured nucleus) on the medial side because the spinal cord was not retractable . On follow - up mri, although the ruptured disc was removed, the thick residual of the annulus that formed a capsule around the herniated nucleus remained, and the nerve root was still compressed (fig . The patient was comfortable since the severe arm pain in his arm had disappeared, and further rehabilitation was continued one week after the operation . Dynamic film of the cervical spine taken one week after the operation showed no instability or change in disc height with preservation of motion . Trigger point injection relieved most of the myofascial pain around the shoulder and some of the arm pain . The weakness in his left triceps improved (motor grade v / v) within two weeks of the operation . His symptoms improved over time, and the severity of his arm pain was vas 1 (excellent outcome by macnab's criteria) 2 months after the operation . Follow - up mri performed two months after the operation showed slight shrinkage of the capsule (fig . He returned to his base two months after the surgery, and he is currently able to perform his military duties without difficulty . There are several treatment options for patients with cervical disc herniation when the pathology is located on the lateral or posterior lateral side and the primary symptom is radiculopathy . The first treatment option is traditional acdf, followed by cervical arthroplasty, anterior cervical endoscopic discectomy, anterior transcorporeal or transuncal anterior cervical discectomy, traditional posterior microforaminotomy with or without tubular retractor, and posterior endoscopic discectomy . However, this mode of treatment requires an anterior approach and fusion of the diseased segment, which leads to motion limitation and increased adjacent segment stress / degeneration (2 - 7). Some authors have reported that the rate of complications, such as recurrent laryngeal nerve injury or dysphasia, was higher than expected (8, 9). Implantation could be burdensome to physically and socially active people, such as soldiers because of the military regulation . Despite the development of improved operative procedures, postoperative scars on the anterior neck the artificial disc was invented to preserve the motion of the diseased segment and to reduce the amount of stress on the adjacent segment (3). However, biomechanical studies have shown that artificial discs are not a true substitute for original discs, as the range of motion on the implanted segment is greater than normal physiologic motion and the motion of the adjacent segment is mostly decreased (3). Moreover, the cost of this procedure is very high in comparison to other procedures . Until now, the artificial disc has not been a true solution for the replacement of human discs . As with acdf . The risk of approach - related complications and the cosmetic results could be similar to those of acdf . There are several other options for removing the pathology while preserving functional motion of the involved level . Anterior cervical endoscopic discectomy is a good choice for the treatment of lateral disc protrusion . This operation can be performed under local anesthesia and can be used to preserve the motion of the involved level (5 - 7). Excellent results were achieved in more than 90% of patients (5 - 7). Ahn and lee et al . Reported that further instability did not occur, even though the height of the disc was decreased (5 - 7). However, this procedure requires access through the disc anterior to the pathology with violation of the anterior structure, such as the annulus and nucleus . Radiological confirmation is necessary during the approach, and the operation cannot be performed if the c - arm does not show an involved level, such as the c6 - 7 or c7-t1 level (5 - 7). There is also a risk of major vessel complication or nerve injury with this approach (5 - 7, 10). Anterior transuncal microforaminotomy, developed by jho has a patient satisfaction rate higher than 95%, with functional preservation of the involved level (11, 12). However, the problems of disc space narrowing and anterior column violation still remain, even though there were no symptoms (2, 4 - 7). The transcorporeal approach was developed to solve the problem of uncovertebral joint violation (4, 13). Comparative study of the anterior transcorporeal approach and transuncal approach revealed that patients subjected to the anterior transuncal approach showed a significant decrease in disc height during the follow - up period in comparison to those subjected to the transcorporeal approach (13). Although different operations performed by different surgeons may have different results, the removal of an uncovertebral joint or the medial wall of the foramen exposing the vertebral artery might be a problem . Although the transcorporeal approach could resolve this problem, this procedure also has the risk of complication during the anterior approach and leaves a scar on the anterior side of the neck (4, 13). The procedure is similar to traditional foraminotomy, except that an endoscope is used instead of a microscope, with or without a tubular retractor (1, 2). Published their 2-yr results and reported a success rate of approximately 96% (2). A laterally localized herniated cervical soft disc without instability the complication rate was very low, and there was no operationinduced neck pain or instability (2). Contrary to the previous suggestion (4, 16), direct removal of the offending pathology is possible without violation of the nucleus or bone of the anterior column (2, 4, 15). Moreover, this procedure is possible, even at the c7-t1 level, with direct visualization during the approach (2, 15). The greatest difference between this procedure and conventional posterior foraminotomy is the lack of access - induced muscle injury (1, 2, 14 - 16). Muscle injury can also be reduced by microforaminotomy with the use of a tubular retractor system (1, 14, 16). There is hardly any difference between the operative skills necessary for this procedure and endoscopic discectomy, with the benefit of less traumatization of the paraspinal muscle (1, 2, 14, 16). The difference between the cosmetic result and accessinduced muscle pain associated with those two procedures may be regarded as minimal . However, the most distinguishing feature of endoscopy is its excellent magnification and illumination (2). Minimal manipulation of the root, facet joint and spinal cord are possible with the help of this feature of endoscopy (2, 15). However, although minimal, there is also the risk of disc narrowing, which occurs in 32% of patients without symptoms or instability (2). This procedure is not optimal for the removal of a centrally located pathology, hard disc or spur (2, 4, 15). A single method of operation would not be sufficient for all types of cervical disc pathology . A recent trend in disc surgery is' functional preservation' with a minimally invasive technique, and posterior endoscopic discectomy may be a good alternative choice for this reason (1, 2, 4 - 7, 11, 12, 14). The authors expect that stability will be maintained in the present cases because there was no violation of components in the anterior column and the extent of facetectomy was minimal (1, 15, 16). Further comparative study with a longer follow - up period in a larger patient group is warranted . In conclusion, the gold standard technique for the treatment of cervical nucleus herniation has been anterior cervical discectomy and fusion, until now . Even though the follow - up period in the present study was short, considering the physical / social activity levels and anticipated physical / social loads in some patients such as soldiers, posterior cervical endoscopic discectomy may be a promising alternative for selected cases.
Most industrialized countries are experiencing progressive aging of their populations.1 in italy, the percentage of people older than 65 years is currently equal to 21.7% and is projected to rise further, exceeding 33% by 2050.2 aging is associated with a progressive increase in the prevalence of frailty.3 frailty is a condition characterized by reduced resistance to stressful events, stemming from a functional decline of multiple physiological systems . This results in an alteration of the homeostatic mechanisms of the body and helps to increase the risk of adverse events, such as hospitalization, institutionalization and death.3 to date, there is no universally accepted definition of frailty . Several models have been proposed to identify and measure it,4,5 including some based on clinical criteria6 as proposed by fried et al3 and others based on laboratory tests.7,8 the progressive decline in the function of various organs and systems in the elderly contributes to a reduction in the homeostatic capacity of the body, leading to frailty . We argue that changes in the endocrine system can play a decisive role in this process . Physiologically, the endocrine system intervenes in regulating energy metabolism and stress response . In the elderly, the decline in endocrine function is particularly evident in the presence of stressful events.9 endocrine changes that occur with age involve all the glands and, although the relationship between frailty, declining age - related concentrations of androgens and growth hormone (gh) on the one hand10 and adrenal function on the other is well known, the role of thyroid hormones is still under debate . Thyroid abnormalities have been suggested as constituting a possible predisposing factor of frailty in elderly patients with chronic kidney disease (ckd) by abdel - rahman et al.11 however, in an exhaustive review, morley et al12 conclude that there is little evidence that thyroid hormone plays a role in the pathogenesis of frailty . Recently, fernndez - garrido et al13 assessed the clinical features of prefrail older individuals and emerging biomarkers and argued that the identification of biomarkers associated with prefrailty state would be very helpful . With advancing age, we observe a progressive reduction in serum triiodothyronine (t3) concentrations, while the levels of thyroxine (t4) and thyroid stimulating hormone (tsh) do not change significantly.9 these values are suggestive of nonthyroidal illness syndrome (ntis). This article evaluates the prevalence of ntis and describes the relationship between ntis and frailty . The data presented in this article are derived from an observational study conducted at the polyclinic tor vergata in rome; the study objective was to evaluate the main indicators of frailty and their relationship with changes in the endocrine system in patients aged 65 years or above, with or without low - energy fractures . Of these, 62 were enrolled among the patients hospitalized in the orthopedic department after a hip fracture and 50 control subjects were enrolled among outpatients evaluated at the department of medicine (clinical program on atherosclerosis). All investigations were carried out according to the declaration of helsinki, as modified in 2000, including obtaining written informed consent from all participants, and the study protocol was approved by the ethical committee of the polyclinic tor vergata . Inclusion criteria comprised an age of 65 years in both groups and current hip fracture in hospitalized patients . Each patient s sex and age were recorded, anthropometric parameters were measured (weight and height), and the body mass index (bmi) was calculated . All study participants underwent physical examination and blood sampling for laboratory assays (blood count, creatinine, glucose, albumin, serum cortisol, tsh, free t3 [ft3], free t4 [ft4], high - sensitivity c - reactive protein [hs - crp], interleukin-6 [il-6] and tumor necrosis factor- [tnf-]). The blood count and glucose concentration were assessed by routine laboratory tests (sysmex xe 2100; dasit), while creatinine and albumin were measured by homogeneous - phase chemiluminescent enzyme immunoassay (dimension vista 1500; siemens). Tsh (reference values: 0.354.5 iu / ml), ft3 (reference values: 2.34.2 pg / ml), ft4 (reference values: 0.81.75 ng / dl) and plasma cortisol were measured by chemiluminescence assay (advia centaur xp; siemens); hs - crp was measured using nephelometric method (dimension vista 1500). The levels of il-6 and tnf- were determined with enzyme immunoassay (drg diagnostics). Participating patients received a multidimensional geriatric evaluation comprising the following scales: activities of daily living (adl)14 and instrumental activities of daily living (iadl);15 mini mental state examination (mmse);16 geriatric depression scale (gds);17 and mini nutritional assessment (mna).18 comorbidities were assessed using the cumulative illness rating scale for geriatrics (cirs - g), from which we obtained the cirs severity (cirs - s), cirs comorbidity index (cirs - ci),19 and charlson comorbidity index (cci).20 in patients able to collaborate, muscle strength was measured through handgrip dynamometry, and the degree of frailty was subsequently calculated using the survey of health, ageing and retirement in europe frailty instrument (share - fi).6 patients enrolled in the study who were able to be moved without difficulty underwent dual - energy x - ray absorptiometry (dexa) for the evaluation of lumbar and femoral bone mineral density (bmd), lumbar and femoral t - score and z - score as well as the body composition . The kolmogorov student s t - test and analysis of variance (anova) were used to compare continuous variables . One - way anova and simple linear correlation were used to assess the relationship between continuous variables . Logistic regression was used to assess the relative influence of independent variables on ft3 and the frailty score . Analysis of covariance (ancova) was used to assess the weight of the fracture on the relationship between ft3 and the frailty score . An analysis of the receiver operating characteristic (roc) curve and the area under the curve (auc) was undertaken to assess the accuracy of ft3 in the distinction between frail and nonfrail subjects . Statistical analysis was performed using stat view 5 (sas institute, cary, nc, usa). Graphs were designed using graphpad prism 5 (graphpad software inc, san diego, ca, usa). Student s t - test and analysis of variance (anova) were used to compare continuous variables . One - way anova and simple linear correlation were used to assess the relationship between continuous variables . Logistic regression was used to assess the relative influence of independent variables on ft3 and the frailty score . Analysis of covariance (ancova) was used to assess the weight of the fracture on the relationship between ft3 and the frailty score . An analysis of the receiver operating characteristic (roc) curve and the area under the curve (auc) was undertaken to assess the accuracy of ft3 in the distinction between frail and nonfrail subjects . Values of p<0.05 were considered significant . Statistical analysis was performed using stat view 5 (sas institute, cary, nc, usa). Graphs were designed using graphpad prism 5 (graphpad software inc, san diego, ca, usa). The study population consisted of 112 patients, of both sexes, aged between 65 and 98 years, whose anthropometric characteristics and features related to body composition are reported in table 1 . The data relating to comorbidity, frailty and multidimensional assessment are reported in table 1 . Compared to subjects in the control group, patients with fractures had significantly lower average values of hemoglobin and albumin, as well as significantly greater average values of white blood cells (wbcs) and inflammatory markers (table 2). Patients with fractures had mean values of ft3 significantly lower than those observed in patients in the control group . The two groups did not differ in terms of mean values of tsh and ft4 (table 2). About 42.3% of the study participants had low levels of ft3: the prevalence of this condition was higher in the group with fractures compared with the control subjects (40.2% vs 2.1%, p<0.001). Unlike ft4, ft3 was correlated with nutritional status (mna), the degree of disability (adl and iadl), comorbidities (cirs - g) and muscle strength (handgrip), as shown in table 3 . A linear regression analysis (figure 1a and b) showed the absence of a statistically significant association between frailty score and ft4, as well as the presence of an inverse, statistically significant relationship between frailty score and ft3 (r=0.436; p<0.001). In figure 1b, it is evident how the two groups, namely, fractured and control, are clearly distinct from each other . An ancova showed that ft3 values, despite being strongly dependent on the fracture, remained closely associated with the degree of frailty of the subject (figure 1c). In the entire study population, ft3 was significantly lower in frail subjects compared to prefrail and not frail subjects (figure 2) (anova f=6.64; p<0.005). In a logistic regression model in which frailty was considered the dependent variable, the best predictor of the hormonal parameters of frailty was represented by ft3 (=4.358; p<0.05). Analysis of the roc curve (figure 3) showed that, in our study population, ft3 is an accurate parameter for identifying vulnerable subjects (auc = 0.8520; p<0.001). Taking ft3 values less than 2.3 pg / ml as cutoff, ft3 is able to distinguish frail subjects from the not frail ones, with a sensitivity of 73.91% (95% confidence interval [ci]: 51.60%89.77%) and a specificity equal to 74.19% (95% ci: 55.39%88.14%). Patients with hip fractures had a significant reduction in serum concentrations of ft3, but not of ft4 . It is well known that ntis is a clinical condition characterized by changes in the serum concentrations of thyroid hormones not caused by intrinsic abnormalities of thyroid function.21 often, markedly reduced ft3 is observed in elderly subjects suffering from acute diseases or undergoing surgery . However, a slight reduction of ft3 values can also be found in apparently healthy elderly subjects.22,23 the prevalence of ntis is high in the elderly, reaching 31.9% in elderly patients hospitalized for acute illness.24,25 the main alteration found in ntis is the reduction of serum concentrations of either t3 or ft3,26 generally in association with normal or reduced concentrations of t4 or ft4, normal levels of tsh and increased levels of reverse t3 (rt3).2628 from a pathogenic point of view, both the reduction of ft3 concentrations as well as the increase of rt3 concentrations appear to be the consequence of a reduction in the activity of type i deiodinase, an enzyme that physiologically promotes the conversion of t4 into t3 and the catabolism of rt3 into t2.21,23 in the presence of acute or chronic diseases, as well as after surgery, an inflammatory process is activated, characterized by an increase in plasma concentrations of cytokines, such as il-1, il-6 and tnf-, apart from cortisol.29 both the excess cortisol and cytokines appear to play an essential role in the pathogenesis of ntis . As reported in numerous studies in humans, high levels of glucocorticoids suppress the pituitary response to thyrotropin releasing hormone (trh), resulting in a reduction in serum concentrations of tsh and causing a modest decline in the levels of thyroid hormones.30 meanwhile, il-6 appears to have a direct inhibitory role on the activity of type i deiodinase,3133 preventing the conversion of t4 into t3 and the catabolism of rt3 . In our study, the reduction in serum concentrations of ft3 is a clear manifestation of stress associated with a fracture event . The effect of a fracture on ft3 concentrations, however, does not appear to be related only to the action of il-6, other proinflammatory cytokines and hormones responding to stress, such as cortisol, although their concentrations significantly increase in fracture patients compared with the levels in control subjects . Numerous preexisting factors, such as the fracture patient s nutritional status, sarcopenia, disability and comorbidities, which characterize the condition of frailty and influence its pathogenesis, are strongly correlated with the values of ft3 . Several studies have shown the existence of a significant relationship between frailty, disability and comorbidities3 and have established that, although frailty is a condition distinct from comorbidity and disability, it often overlaps with the latter two: when associated together, frailty and comorbidities can predict disability, disability can induce frailty and worsen comorbidity, and comorbidity may contribute to the development of frailty.34 these relationships, along with the direct inverse association between frailty and nutritional status, are well documented in several articles3537 and were confirmed in our study (data not shown). It is known that sarcopenia, defined as a syndrome characterized by a progressive loss of muscle mass and muscle strength,38 contributes to the development of frailty.39 a bone muscle cross talk is involved in maintaining bone and muscle integrity . In sarcopenic patients with hip fragility fractures, reduced expression of bone morphogenetic proteins (bmp2 - 4) has been demonstrated.40 our study also found that sarcopenia is inversely correlated with the frailty score (data not shown). Similarly to the results obtained for the variable frailty regardless of the event of fracture, in our study population, we observed lower values of ft3 in patients with a greater number and greater severity of comorbidity, as well as in those with a worse nutritional status and with greater disability . Therefore, on the one hand, a reduction in ft3 depends on the same factors that contribute to frailty, which, in turn, may be worsened by low values of ft3 or may simply coexist with them; on the other hand, low values of ft3 can contribute to the development of frailty . The hypothesis of a link between thyroid hormone abnormalities and frailty, which was recently suggested by abdel - rahman et al11 in patients with chronic kidney disease, is consistent with our data . A reduction in ft3 may be considered to be the consequence of multiple events, such as malnutrition as well as acute and chronic diseases . The latter also contribute to determining frailty and are independently present before the event of a fracture, which, in turn, is a further important cause of a decline in ft3 . Furthermore, as mortality is higher in frail subjects than in more robust subjects,3,41 several studies have shown that lower ft3 values are predictive of mortality.42 the diagnosis of ntis is not univocal: as the dosage of rt3 is not available in clinical practice, it is generally linked to overcoming the threshold corresponding to the lower limit of normal ft3 levels.43 both the increase in rt3 and a decline in ft3 are proportional to the severity of the syndrome and thus to the severity of the clinical condition . A decline in t3 is a progressive and continuous phenomenon that follows the increase in rt3, and it is therefore likely that the initial or milder forms of ntis are not diagnosed solely on the basis of ft3 values . We conclude that measuring ft3 can be a useful laboratory parameter in clinical assessment, which can play an important role in identifying vulnerable elderly subjects and in quantifying the condition of frailty . Furthermore, it deserves to be considered as an additional risk factor for mortality in elderly patients hospitalized for hip fracture . There are two main limitations to the study: 1) the small sample of participants; 2) a possible selection bias in that many patients with fractures were cognitively compromised and thus not in a position to give informed consent for their participation in the study . Nevertheless, this limitation should have had such an effect as to reduce the differences observed . There are two main limitations to the study: 1) the small sample of participants; 2) a possible selection bias in that many patients with fractures were cognitively compromised and thus not in a position to give informed consent for their participation in the study . Nevertheless, this limitation should have had such an effect as to reduce the differences observed.
Intriguingly, mtorc1 activity only regulates the expression of nedd41 but not of nedd42, the closest homolog of nedd41 in the mammalian genome, but the molecular mechanism underlying this isoform preference is unknown . Mtorc1 promotes global cap - dependent protein synthesis mainly by mediating the phosphorylation of eukaryotic initiation factor 4e binding protein 1 and 2 (4e - bp1 and 4e - bp2). 4e - bps bind to eukaryotic initiation factor 4e (elf4e) to prevent the formation of protein initiation complex and protein translation . Phosphorylation of 4e - bps leads to decreased affinity of 4e - bps to eif4e . This enables eif4e to be anchored to the 5-cap structure of mrnas, promotes the formation of protein initiation complex, and initiates protein synthesis . Of note, the 5 untranslated region (5'utr) of the nedd41 mrna (genbank accession number: nm_010890) contains a pyrimidine - rich sequence stretch that is related to the 5 terminal oligopyrimidine (5top) motif . This 5top motif is important for anchoring elf4e to the 5-cap structure of mrnas, to direct the assembly of the translation machinery, and to initiate protein synthesis . The pyrimidine - rich sequence in the 5utr of nedd41 mrna may play a similar role as the 5top motif in initiating the translation of nedd41 mrnas in an mtorc1 activity - dependent manner (fig . Further evidence supporting this model includes a study employing ribosome profiling to examine the translational efficiency of specific mrnas in the presence of an mtorc1 inhibitor, which showed that the translational efficiency of nedd41 mrna is reduced in non - neuronal cells upon mtorc1 inhibition . Figure 1.model of the regulation of nedd41 translation by mtorc1 . In response to growth factor signaling, . Elevated ptdinsp3 levels lead to the phosphorylation and activation of akt, which then activates mtorc1 . Phosphorylation of 4e - bps by mtorc1 reduces the binding of 4e - bps to eif4e, enabling eif4e to bind to the 5'utr of target mrnas and thus initiate protein synthesis . The 5'utr of nedd41 mrna (see sequence in the dialog box) contains a pyrimidine - rich sequence stretch (underlined) after the putative transcriptional start site (red), which is related to the 5' terminal oligopyrimidine (5'top) motif . This pyrimidine - rich sequence may play a key role in starting the translation of nedd41 mrnas in an mtorc1 activity - dependent manner . Nedd41 is a positive regulator of neurite growth and may have additional roles in regulating axon pathfinding, spine formation, and synaptic plasticity . In response to growth factor signaling, pi3k catalyzes the phosphorylation of ptdinsp2 to generate ptdinsp3 . Pten converts ptdinsp3 back to ptdinp2 and thus antagonizes the effect of pi3k . Elevated ptdinsp3 levels lead to the phosphorylation and activation of akt, which then activates mtorc1 . Phosphorylation of 4e - bps by mtorc1 reduces the binding of 4e - bps to eif4e, enabling eif4e to bind to the 5'utr of target mrnas and thus initiate protein synthesis . The 5'utr of nedd41 mrna (see sequence in the dialog box) contains a pyrimidine - rich sequence stretch (underlined) after the putative transcriptional start site (red), which is related to the 5' terminal oligopyrimidine (5'top) motif . This pyrimidine - rich sequence may play a key role in starting the translation of nedd41 mrnas in an mtorc1 activity - dependent manner . Nedd41 is a positive regulator of neurite growth and may have additional roles in regulating axon pathfinding, spine formation, and synaptic plasticity . Accumulating evidence indicates crucial roles of local protein synthesis in various aspects of normal neuronal development and function, including axon guidance, spine formation, and synaptic plasticity . All of these processes are highly dynamic and require spatially and temporally well - regulated and accurate changes in local protein levels in response to various stimuli . Locally regulated translation of mrnas provides an efficient way to spatially and temporally control local protein composition, which is of particular importance in distal neurites, where proteins synthesized at the soma may not be an economic source . The finding that nedd41 expression is regulated at the translational level is compatible with the notion that nedd41 mrnas are major targets of the local translational machinery in neurons . If so, it is very likely that nedd41 does not only regulate neurite growth regulation, as we reported previously, but may have important additional roles in axon guidance, spine formation, and synaptic plasticity . Indeed, drosophila melanogaster nedd4 regulates commisureless, which is an essential protein regulator of slit / robo signaling during axon pathfinding and midline crossing . Moreover, synaptic activation has been shown to cause a site - specific increase in nedd41 expression, which may mediate the ubiquitination and endocytosis of the ampa receptor subunit glua1 . In view of these findings, additional studies, ideally with higher spatiotemporal resolution, on how nedd41 expression is regulated in response to extracellular signals is very likely to provide further important insights into the physiological roles of nedd41 in neurons . This work has been supported by grants from the german research foundation (spp1365/ka3423/11; to h.k .) And the fritz thyssen foundation (to h.k . ).
Hypothermia defined as a core body temperature <35 can be a life - threatening emergency, especially in cold winter climates, but can be used therapeutically . Therapeutic hypothermia (th) has been used to improve neurological outcome in comatose patients after cardiac arrest.1)2) the exact mechanism for neuroprotection is unclear; potential mechanisms include attenuation of deleterious processes occurring after initial hypoxic tissue injury.3)4)5) generally, mild hypothermia is defined as a core body temperature between 32 and 34.2) this is thought to be appropriate temperature between neuroprotective effects and possible toxicities . The osborn wave (also known as the j wave) it is not pathognomonic of hypothermia, but usually occurs in patients with a core temperature <32 and the magnitude of the j point elevation correlates inversely with the body temperature.6) several studies have suggested an arrhythmogenic potential of the osborn wave,7)8)9)10) but there is no solid evidence that th - induced osborn wave itself is associated with increased mortality.11)12) here we report a case of recurrent ventricular fibrillation (vf) after appearance of osborn wave during mild th . A 56-year - old man with no underlying disease was admitted to the emergency department with sudden cardiac arrest . While having breakfast, he suddenly pounded his chest . He received cardiopulmonary resuscitation (cpr) from his son for about 20 minutes until paramedics arrived with a defibrillator . 1). Defibrillation with 200 j was delivered once followed by 5 cycles of cpr . Following restoration of cardiac rhythm, he was transferred to our hospital for further management . At our hospital, initial ecg (fig . 2a) showed sinus tachycardia with a heart rate of 114 beats per minute . On the first day of admission, echocardiography was done, which showed no regional wall motion abnormality of the left ventricle . The initial ph was 7.375 and the levels of electrolytes including sodium, potassium, calcium and magnesium were within the normal ranges . The target temperature was 33. soon after th was begun, osborn waves appeared in lead ii and v 4, v 5, and v 6 (fig . Two hours after th, vf occurred when the esophageal temperature was 34.9. defibrillation of 150 j was applied (fig . 2c). Not much later atrial fibrillation developed and the osborn waves were prominent in diffuse leads . Three more defibrillations were delivered to terminate vf and the height of osborn waves increased over time (fig . Two hours after a warmer was applied to the patient, ecg revealed less prominent osborn wave and vf did not recur . He was diagnosed with idiopathic vf and discharged from our hospital after implantable cardioverter defibrillator insertion (fig . It is generally accepted that th has the potential to improve the neurological outcome in comatose patients after cardiac arrest.1)2) following initial resuscitation, various mechanisms including ischemia, reperfusion injury, inflammatory response, and free radical production are involved in cerebral damage.1)3)5) th lowers the cerebral metabolic rate, blocks the apoptotic pathways, suppresses inflammatory responses, and attenuates production of free radicals.3)4)5) however, possible side effects, such as electrolyte imbalance, hemodynamic instability, infection, seizures, and arrhythmias, can rarely occur in patients undergoing th.1)2)5) electrocardiogram findings of hypothermia include osborn waves, atrial and ventricular dysrhythmias, and interval (pr, qrs, qt) prolongations.11)13)14) among these, the osborn wave is a positive deflection occurring at the qrs - st junction and may originate from a transmural voltage gradient during early repolarization due to a transient outward potassium current in the epicardium, but not in the endocardium.15) as the osborn wave can be seen in various conditions like acidosis, subarachnoid hemorrhage, and hypercalcemia, it is not pathognomonic of hypothermia . But, it has been reported that the height of j point elevation is related with the severity of hypothermia.6) several mechanisms have been suggested to explain the arrythmogenic potential of the osborn wave . The prominent action potential notch can lead to loss of the dome of action potential, which can heterogeneously cause a marked dispersion of repolarization . This eventually can cause extrasystolic activity by a mechanism called phase 2 reentry and vf.15)16) another proposed mechanism is triggered automaticity . Intracellular ca overload during hypothermia is a precursor for early or delayed after depolarization, which is associated with triggered activity.17) however, other reports have not associated the osborn wave with vf in hypothermic patients.18) therefore, the arrythmogenic potential of the osborn wave requires further study . Although various adverse events have been reported during th, th - induced arrhythmia seems not to be related with increased mortality.1)2) in one study of ecg changes in th, the osborn wave incidence during th was 30% and no association with unfavorable short - term outcome was evident.11) after rewarming, the osborn wave disappears in almost cases . So, on the basis of many studies, th is well tolerated and relatively safe . Hypothermia is also proarrhythmic and can cause lethal arrhythmias, especially at low temperature and in the presence of low serum ph.19) however, in our case, vf developed in normal serum ph, electrolyte levels, and not markedly low body temperature . Therefore, vf and osborn wave seem not always to be related to body temperature, and individual variations in response to hypothermia can be more important to determine the vulnerability to arrhythmias . Physicians need to monitor patient's vital signs including core temperature, ecg, and laboratory tests carefully during th.
The institutional review board approved our protocol, and all patients provided written informed consent . The study complies with the principles of the declaration of helsinki . During the period from september 2009 to february 2011, we enrolled 100 consecutive patients with drug - resistant af referred for catheter ablation at our institution . We excluded four patients from analysis because a three - dimensional image of the la prior to their first ablation was unavailable, leaving 96 patients in the cohort . Genotyping of snps rs10033464 (hgvs name: g.111720761t> g) and rs2200733 (hgvs name: g.111710169c> t) was performed with the massarray system of the sequenom genotyping platform as part of a single multiplex snp analysis (sequenom). Each sample was tested at least three times, and at least two concordant results were required . All individuals had either cardiac ct angiography (cta) or mr angiography (mra) to delineate end - diastolic left atrial dimensions . In patients who were undergoing their second or third ablation at the time of enrollment, we used cta or mra prior to the first ablation for lav measurements . Of all patients, 81 (84%) underwent cta and 15 (16%) underwent mra prior to their ablation . Cta was performed at the end of the expiratory phase after intravenous administration of 7080 ml of contrast (isovue 370) using a multi - detector ct scanner (toshiba s aquilion). Mra was performed after administration of 0.2 mmol / kg of gadopentetate dimeglumine (magnevist; bayer healthcare) with a 1.5-t magnetic resonance scanner (avanto; siemens) and a body phased - array coil . All scans were ecg - gated and were acquired at atrial end - diastole, just before opening of the mitral valve . Of all scans, 24 (25%) our detailed image acquisition protocols for cta and mri have previously been reported.9 a single experienced reader, who was blinded to patient genetic profile and demographic characteristics, performed image analysis . Lav was calculated using the prolate ellipsoid formula as previously described and validated.10 using the endocardial border, we measured three orthogonal dimensions of the la, including transverse (t), anterior posterior (ap), and longitudinal (lo) diameters . Transverse diameter of the la was defined as the distance between the midpoint of the right- and left - sided pulmonary veins in oblique axial images . The ap and lo diameters were measured at the midpoint of the transverse diameter in oblique axial and sagittal images (fig . These dimensions were then entered into the prolate ellipsoid formula: lav = [(t ap lo) (0.523)]. The association of lav with 4q25 genetic variants was examined using multivariable linear regression models, adjusting for age, ethnicity, gender, bmi, height type and duration of af, left ventricular ejection fraction, history of hypertension, antiarrhythmic drug use, and valve disease . Based upon univariate analyses of the association of lav with 4q25 genetic variants, a recessive model was assumed for polymorphisms . To estimate uncertainty associated with the sample size, we utilized a nonparametric bootstrap study to confirm the significance of the associations . This method involves resampling observations with replacement from the data 1000 times, therefore, estimating the sampling distribution, standard error, and confidence intervals without assumptions about the distribution of the underlying population . Thus, the method is useful in situations with small sample size or when the theoretical distribution of the statistic is complicated . We then executed the multivariable analysis, including those variables from model 3 that reached or approached statistical significance, and repeated the process for a total of 1000 times . These new samples were taken from the original data set using sampling with replacement, so it is not identical to the original sample.11,12 results are reported as significant if p 0.05, and with bonferroni correction for multiple comparisons, results should be considered significant at p 0.025 . The institutional review board approved our protocol, and all patients provided written informed consent . The study complies with the principles of the declaration of helsinki . During the period from september 2009 to february 2011, we enrolled 100 consecutive patients with drug - resistant af referred for catheter ablation at our institution . We excluded four patients from analysis because a three - dimensional image of the la prior to their first ablation was unavailable, leaving 96 patients in the cohort . Genotyping of snps rs10033464 (hgvs name: g.111720761t> g) and rs2200733 (hgvs name: g.111710169c> t) was performed with the massarray system of the sequenom genotyping platform as part of a single multiplex snp analysis (sequenom). Each sample was tested at least three times, and at least two concordant results were required . All individuals had either cardiac ct angiography (cta) or mr angiography (mra) to delineate end - diastolic left atrial dimensions . In patients who were undergoing their second or third ablation at the time of enrollment, we used cta or mra prior to the first ablation for lav measurements . Of all patients, 81 (84%) underwent cta and 15 (16%) underwent mra prior to their ablation . Cta was performed at the end of the expiratory phase after intravenous administration of 7080 ml of contrast (isovue 370) using a multi - detector ct scanner (toshiba s aquilion). Mra was performed after administration of 0.2 mmol / kg of gadopentetate dimeglumine (magnevist; bayer healthcare) with a 1.5-t magnetic resonance scanner (avanto; siemens) and a body phased - array coil . All scans were ecg - gated and were acquired at atrial end - diastole, just before opening of the mitral valve . Of all scans, 24 (25%) were obtained during af and the remaining scans were acquired during sinus rhythm . A single experienced reader, who was blinded to patient genetic profile and demographic characteristics, performed image analysis . Lav was calculated using the prolate ellipsoid formula as previously described and validated.10 using the endocardial border, we measured three orthogonal dimensions of the la, including transverse (t), anterior posterior (ap), and longitudinal (lo) diameters . Transverse diameter of the la was defined as the distance between the midpoint of the right- and left - sided pulmonary veins in oblique axial images . The ap and lo diameters were measured at the midpoint of the transverse diameter in oblique axial and sagittal images (fig . These dimensions were then entered into the prolate ellipsoid formula: lav = [(t ap lo) (0.523)]. The association of lav with 4q25 genetic variants was examined using multivariable linear regression models, adjusting for age, ethnicity, gender, bmi, height type and duration of af, left ventricular ejection fraction, history of hypertension, antiarrhythmic drug use, and valve disease . Based upon univariate analyses of the association of lav with 4q25 genetic variants, a recessive model was assumed for polymorphisms . To estimate uncertainty associated with the sample size, we utilized a nonparametric bootstrap study to confirm the significance of the associations . This method involves resampling observations with replacement from the data 1000 times, therefore, estimating the sampling distribution, standard error, and confidence intervals without assumptions about the distribution of the underlying population . Thus, the method is useful in situations with small sample size or when the theoretical distribution of the statistic is complicated . We then executed the multivariable analysis, including those variables from model 3 that reached or approached statistical significance, and repeated the process for a total of 1000 times . These new samples were taken from the original data set using sampling with replacement, so it is not identical to the original sample.11,12 results are reported as significant if p 0.05, and with bonferroni correction for multiple comparisons, results should be considered significant at p 0.025 . Of 96 patients in the cohort (mean age 60 9.9 years, 72% male), 89 (93%) were caucasians, 3 (3%) were african american and 4 (4%) were self - identified as other ethnicities . Af types were defined according to rhythm status prior to each patient s first ablation procedure . Overall, 55 (57%) patients had paroxysmal af, 36 (38%) had persistent af, and 5 (5%) had long - standing persistent af . The chads2 risk score was low (0 or 1) in 73 (76%) participants . Valve disease was present in six (6%) patients, including four with mild - to - moderate mitral regurgitation, one with severe mitral regurgitation, and one with mild aortic stenosis . There was a trend toward higher la volume in patients with valve disease (143.7 33.2 cm vs. 103.8 5.4 cm, p = 0.08). The minor allele frequencies at snps rs10033464 and rs2200733 were 0.14 and 0.25, respectively . The genotype frequencies for rs10033464 and rs2200733 were in hardy weinberg equilibrium (= 1.959, p = 0.162; = 0.474, p = 0.491, respectively). The r between rs1003464 and rs2200733 in our cohort was 0.003 . In 44 (46%) at least one risk allele at either rs10033464 or rs2200733 was detected in 12 (13%) or 31 (32%) patients, respectively . The remaining nine (9%) patients had at least one risk allele at both rs10033464 and rs2200733 . The baseline characteristics of all participants and of participant groups by genotype have been summarized in table 1 . The tt haplotype at rs10033464 was strongly associated with larger lav (table 2). In contrast, an association was not identified in those with the af risk allele at rs2200733 . In the unadjusted model, homozygosity at the t allele at rs10033464 was associated with 104.6 29.7 cm increase in lav (p = 0.001). To evaluate the effects of potential confounders, multivariable regression models with age, ethnicity, gender, af duration, type of af, bmi, height, left ventricular ejection fraction, history of valve disease, history of antiarrhythmic drug use, and history of hypertension were utilized . Model 1 controlled for demographic features such as age, ethnicity, and type of af . As illustrated in table 3, the magnitude of association for homozygosity at the t allele at rs10033464 with lav was unchanged at 104.4 31.6 cm, and statistical significance was maintained (p = 0.001). In the fully adjusted model 2, the magnitude of association of homozygosity at the t allele at rs10033464 with lav and statistical significance we further examined this finding by a nonparametric bootstrap method.11,12 this method confirmed that the magnitude of association of homozygosity for the t allele at rs10033464 with lav withstands resampling (91.9 38.2 cm, p = 0.016, table 4) and that the finding is unlikely to be incidental . Additionally, left ventricular ejection fraction (1.3 0.6 cm per 1% increase in ejection fraction, p = 0.026) and af duration 3 years (22.0 9.9 cm, p = 0.038) were associated with lav . The main finding of this study is that homozygosity for the t allele at snp rs10033464 is associated with larger lav in models adjusting for potential confounders of left atrial size . Importantly, the association of homozygosity for the t allele at snp rs10033464 with la volume was independent of the association of left ventricular ejection fraction and af duration with la volume . To the best of our knowledge, this is the first report of an association between a snp in close proximity to the pitx2 gene with left atrial structure in patients with af . We also observed that variation at both rs10033464 and rs2200733 was common in our cohort of patients with drug - refractory af referred for catheter ablation . In our cohort, this prevalence is higher than the prevalence of 35% reported in a population sample of european descent.2 left atrial dilatation can be the cause or the consequence of af . It has previously been shown that left atrial size is independently associated with af incidence.13 increased lav is also associated with decreased efficacy of catheter ablation for af . Each 10 ml increase in volume is associated with a 14% increased risk of af recurrence after ablation.14 however, the mechanisms that explain these associations are not yet well understood . Atrial dilatation and stretch may induce the activation of extracellular signal - related kinase erk1/erk2, leading to atrial fibrosis and thereby atrial arrhythmia.15 it appears, however, that atrial dilatation can increase af susceptibility and perpetuation independent of variations in atrial fibrosis or tissue refractoriness.16 conversely, it has also been shown that atrial dilatation may occur as a consequence of af in patients without prior left atrial dilatation.17 the non - coding snps at rs10033464 and rs2200733 have been associated with af, and are close to the pitx2 gene.2 the pitx2 gene product is a member of the pituitary homeobox family of transcription factors and appears to play an important role in embryologic morphogenesis.18 chinchilla and colleagues have demonstrated that the expression of pitx2c (the predominant cardiac isoform of pitx2) is decreased in left atrial biopsies of patients with af.19 additionally, using a murine model, the same group demonstrated that reduced pitx2 expression in atrial myocardium resulted in enlarged atrial chambers.19 this effect of reduced pitx2 expression on chamber size is likely mediated in part by selective upregulation of bmp10, a regulator of physiologic hypertrophy in the la.19 our findings support the findings of chinchilla and colleagues in the murine model . After adjusting for potential confounders, variation at rs10033464 was associated with increased lav in patients with drug - refractory af referred for catheter ablation . The study sample size is small and underpowered, and the findings may potentially be driven by false - positive, random findings . However, statistical analyses were driven by a specific hypothesis and limited to associations of polymorphisms at two specific sites with lav . The majority of our patients were caucasian; therefore, our results may not be generalizable to af patients with other ethnici - ties . Conversely, the inhomogeneity of ethnicity may also bias the results; however, the reported associations persisted after adjustment for ethnicity in our multivariable model . Our cohort included patients with paroxysmal, persistent, and long - standing persistent af; however, the associations persisted after adjustment for the type of af . The lav was measured using two modalities (cta or mra), which may introduce differential measurement error and/or variance . However, wen and colleagues reported that measurements of lav using these two modalities are very similar.20 in all patients with long - standing - persistent af and 13 (36%) patients with persistent af, images were obtained during af . In comparison, patients with paroxysmal af underwent imaging during sinus rhythm . In all cases, however, images were acquired just before the opening of the mitral valve, thus measuring maximum atrial volume . Previous reports have observed discrepant associations between the two snps and risk of af in different populations21 and validation samples.2 these discrepant associations likely reflect variations in linkage disequilibrium of the two snps across different population samples . We implemented a resampling simulation study that confirmed the significance of the association; however, our results lack external validation / control and will need to be confirmed in future studies . We did not investigate the mechanism for the association of polymorphism at rs10033464 with atrial volume . The study sample size is small and underpowered, and the findings may potentially be driven by false - positive, random findings . However, statistical analyses were driven by a specific hypothesis and limited to associations of polymorphisms at two specific sites with lav . The majority of our patients were caucasian; therefore, our results may not be generalizable to af patients with other ethnici - ties . Conversely, the inhomogeneity of ethnicity may also bias the results; however, the reported associations persisted after adjustment for ethnicity in our multivariable model . Our cohort included patients with paroxysmal, persistent, and long - standing persistent af; however, the associations persisted after adjustment for the type of af . The lav was measured using two modalities (cta or mra), which may introduce differential measurement error and/or variance . However, wen and colleagues reported that measurements of lav using these two modalities are very similar.20 in all patients with long - standing - persistent af and 13 (36%) patients with persistent af, images were obtained during af . In comparison, patients with paroxysmal af underwent imaging during sinus rhythm . In all cases, however, images were acquired just before the opening of the mitral valve, thus measuring maximum atrial volume . Previous reports have observed discrepant associations between the two snps and risk of af in different populations21 and validation samples.2 these discrepant associations likely reflect variations in linkage disequilibrium of the two snps across different population samples . We implemented a resampling simulation study that confirmed the significance of the association; however, our results lack external validation / control and will need to be confirmed in future studies . We did not investigate the mechanism for the association of polymorphism at rs10033464 with atrial volume . Polymorphism at rs10033464, but not rs2200733, near the pitx2 locus on chromosome 4q25 is associated with increased lav.
About 50% of cases of esophageal cancer occur in the middle thoracic esophagus which extends from 25 cm to 30 cm as defined in the most recent american joint committee on cancer report . The standard of care for these patients includes a multimodality approach involving surgery, chemotherapy, and radiotherapy in various combinations . For patients who are potential surgical candidates, commonly employed treatment strategy includes neoadjuvant chemoradiation followed by surgery, and adjuvant chemotherapy if indicated . For those patients with inoperable disease or at high - surgical risk, radical chemoradiation is the standard treatment . Multiple studies have demonstrated significantly better outcomes with multimodality approaches compared to surgery or radiotherapy alone, emphasizing the fact that completion of the planned treatment protocol plays an important role in determining the outcomes of the patients . Unfortunately, many patients suffer from disease - related malnutrition and cachexia even before the initiation of the treatment . Furthermore, the performance status of these patients often deteriorates during chemoradiation due to the associated acute toxicities, compromising the compliance of the patients to the treatment protocol, resulting in unexpected treatment breaks and shift of the treatment intent from cure to palliation . The purpose of the present audit is to determine the factors which influence the treatment compliance and might potentially predict unintended discontinuation of the planned treatment protocol in the resource constrained scenario of a developing country . The difference in treatment outcomes between the patients who completed their planned treatment and those who could not was analyzed as the secondary endpoint . All the patients registered in the department of radiotherapy and oncology of our institute between january 2008 and december 2012 with a diagnosis of locally advanced nonmetastatic middle thoracic esophageal cancer were included in this audit . Of the 1248 patients of esophageal cancer registered in the department, 91 patients (7.3%) of nonmetastatic middle thoracic esophageal cancer who planned for radical treatment apart from histopathological examination of the biopsied specimen, initial evaluation of these patients included endoscopy and barium study to assess the mucosal extent of the disease and contrast enhanced computed tomography (cect) scan of the chest to assess the extra - esophageal extent and nodal spread of the disease . Baseline evaluation of bone marrow, liver, and renal functions was carried out to assess the tolerance for chemotherapy . Potentially surgical candidates were planned for neoadjuvant chemoradiotherapy (group 1) and received 30 mg / m of cisplatin and 325 mg / m of 5-flurouracil (5-fu) for 4 days, followed by external radiation of 30 gy in ten fractions over 2 weeks as per the departmental protocol . Patients who were deemed inoperable per se were planned for definitive chemoradiotherapy (group 2) with external radiation of 60 gy in 30 fractions over 6 weeks, concurrently with 30 mg / m of cisplatin and 325 mg / m of 5-fu every week as per the departmental protocol . Inoperable patients who were not suitable candidates for concurrent chemotherapy because of deranged renal functions but otherwise with a good performance status were given radical radiotherapy (group 3) with external radiation of 60 gy in 30 fractions over 6 weeks as per the departmental protocol . All the patients in groups 1, 2, and 3 were planned on conventional simulator using open - posterior fields . The patients in groups 2 and 3 were planned using anteroposterior fields till a dose of 40 gy in twenty fractions, covering 5 cm of normal esophagus proximal and distal to the disease as determined by barium contrast, with a lateral margin of 3 cm on either side . The residual disease along with a proximal and distal margin of 2 cm and a circumferential margin of 2 cm is boosted using one anterior and two posterior oblique fields to a dose of 20 gy . All the patients were monitored once a week for acute treatment - related toxicity during the entire course of treatment . The monitoring included subjective and objective assessment of treatment - related toxicities using version 3 of common terminology criteria for adverse events . Weekly monitoring of complete blood counts and renal function tests was done for those undergoing concurrent chemotherapy . Any interruption in the planned treatment was documented, and the reason for the interruption was specified . The patients were followed up clinically at 2 monthly intervals, and response assessment was performed using barium study and endoscopy at the first follow - up (2 months posttreatment) and cect chest at the third follow - up (6 months posttreatment). The patients who could not complete all or a part of their initial plan of treatment were considered to be defaulters, and the factors determining the treatment compliance were evaluated for all the three groups separately . Various factors potentially influencing the compliance of the patients to the planned treatment protocol were evaluated in multivariate analyses using cox proportional hazard model . As the secondary endpoint, the loco - regional control and survival were calculated using kaplan - meier method and significance determined using the log - rank test . Spss 19, (ibm, armonk, ny, united states of america) was used for the analysis . Potentially surgical candidates were planned for neoadjuvant chemoradiotherapy (group 1) and received 30 mg / m of cisplatin and 325 mg / m of 5-flurouracil (5-fu) for 4 days, followed by external radiation of 30 gy in ten fractions over 2 weeks as per the departmental protocol . Patients who were deemed inoperable per se were planned for definitive chemoradiotherapy (group 2) with external radiation of 60 gy in 30 fractions over 6 weeks, concurrently with 30 mg / m of cisplatin and 325 mg / m of 5-fu every week as per the departmental protocol . Inoperable patients who were not suitable candidates for concurrent chemotherapy because of deranged renal functions but otherwise with a good performance status were given radical radiotherapy (group 3) with external radiation of 60 gy in 30 fractions over 6 weeks as per the departmental protocol . All the patients in groups 1, 2, and 3 were planned on conventional simulator using open - posterior fields . The patients in groups 2 and 3 were planned using anteroposterior fields till a dose of 40 gy in twenty fractions, covering 5 cm of normal esophagus proximal and distal to the disease as determined by barium contrast, with a lateral margin of 3 cm on either side . The residual disease along with a proximal and distal margin of 2 cm and a circumferential margin of 2 cm is boosted using one anterior and two posterior oblique fields to a dose of 20 gy . All the patients were monitored once a week for acute treatment - related toxicity during the entire course of treatment . The monitoring included subjective and objective assessment of treatment - related toxicities using version 3 of common terminology criteria for adverse events . Weekly monitoring of complete blood counts and renal function tests was done for those undergoing concurrent chemotherapy . Any interruption in the planned treatment was documented, and the reason for the interruption was specified . The patients were followed up clinically at 2 monthly intervals, and response assessment was performed using barium study and endoscopy at the first follow - up (2 months posttreatment) and cect chest at the third follow - up (6 months posttreatment). The patients who could not complete all or a part of their initial plan of treatment were considered to be defaulters, and the factors determining the treatment compliance were evaluated for all the three groups separately . Descriptive statistics was obtained for characterization of the treatment groups . Various factors potentially influencing the compliance of the patients to the planned treatment protocol were evaluated in multivariate analyses using cox proportional hazard model . As the secondary endpoint, the loco - regional control and survival were calculated using kaplan - meier method and significance determined using the log - rank test . Spss 19, (ibm, armonk, ny, united states of america) was used for the analysis . Patient characteristics table 2 shows the treatment details of the patients . Among the studied population about 40.7% of the patients could not complete the treatment completely, and the factors determining the treatment compliance were analyzed separately for each group . Factors influencing compliance to treatment completion among the 15 patients in group 1, nine patients could not tolerate the adjuvant chemotherapy though indicated . Postoperative weight loss, extreme loss of appetite, severe nausea, and poor hematological tolerance were the common causes of treatment interruption in this group . On multivariate analysis, loss of appetite postoperatively leading to nutritional inadequacy and excessive weight loss was the only factor which was significantly correlated to treatment interruption in the form of inability to take adjuvant treatment (p = 0.02). Among the 36 patients in group 2, 17 patients could not complete planned treatment . The only factors which significantly correlated with noncompliance to treatment completion in this group were loss of appetite and worsened nutritional intake (p = 0.05) and chemotherapy - induced myelosuppression (p = 0.05). Among the forty patients in group 3, 11 patients could not complete planned treatment . The factors which significantly correlated with noncompliance to treatment completion were radiation - induced acute mucositis (p = 0.02), loss of appetite and worsened nutritional intake (p = 0.000), and lost to follow - up (leading to noncompletion of planned radiotherapy) (p = 0.02). As the secondary endpoint, the patterns of failure, local control, and survival of each group were analyzed separately for the subsets who completed their intended treatment and the defaulters . The majority of the patients failed locally and the maximum failures were seen in group 3 [table 4]. Local control and survival rates are significantly higher in patients who completed their planned treatment compared to the defaulters [table 5a]. Local control and survival for patients who completed planned treatment and those who defaulted local control and survival for patients who completed planned treatment and those who defaulted within the treatment subset group table 5b shows that there are significantly higher local control and survival rates among the patients who completed their planned treatment in groups 2 and 3, compared to the defaulters . The patient in group 1 who failed at anastomotic site and all patients in group 2 who failed locally were further planned for surgery . When surgery was not feasible, palliative systemic taxol - based chemotherapy was planned . However, all the patients treated by radiation alone (group 3) were given supportive care only . About 40.7% of the patients could not complete the treatment completely, and the factors determining the treatment compliance were analyzed separately for each group . Factors influencing compliance to treatment completion among the 15 patients in group 1, nine patients could not tolerate the adjuvant chemotherapy though indicated . Postoperative weight loss, extreme loss of appetite, severe nausea, and poor hematological tolerance were the common causes of treatment interruption in this group . On multivariate analysis, loss of appetite postoperatively leading to nutritional inadequacy and excessive weight loss was the only factor which was significantly correlated to treatment interruption in the form of inability to take adjuvant treatment (p = 0.02). Among the 36 patients in group 2, the only factors which significantly correlated with noncompliance to treatment completion in this group were loss of appetite and worsened nutritional intake (p = 0.05) and chemotherapy - induced myelosuppression (p = 0.05). Among the forty patients in group 3 the factors which significantly correlated with noncompliance to treatment completion were radiation - induced acute mucositis (p = 0.02), loss of appetite and worsened nutritional intake (p = 0.000), and lost to follow - up (leading to noncompletion of planned radiotherapy) (p = 0.02). As the secondary endpoint, the patterns of failure, local control, and survival of each group were analyzed separately for the subsets who completed their intended treatment and the defaulters . The majority of the patients failed locally and the maximum failures were seen in group 3 [table 4]. The only patient in group 1 who failed treatment failed at anastomotic site . Local control and survival rates are significantly higher in patients who completed their planned treatment compared to the defaulters [table 5a]. Local control and survival for patients who completed planned treatment and those who defaulted local control and survival for patients who completed planned treatment and those who defaulted within the treatment subset group table 5b shows that there are significantly higher local control and survival rates among the patients who completed their planned treatment in groups 2 and 3, compared to the defaulters . The patient in group 1 who failed at anastomotic site and all patients in group 2 who failed locally were further planned for surgery . When surgery was not feasible, palliative systemic taxol - based chemotherapy was planned . However, all the patients treated by radiation alone (group 3) were given supportive care only . The benefit of multimodality therapy is clearly established for esophageal carcinomas, but its impact on toxicity is not well defined . The purpose of this study is to address the impact of multimodality therapy on treatment toxicity for patients of esophageal carcinoma treated at our institute and thereby identify the factors hindering the planned treatment completion . Taken individually, surgery, radiotherapy, and chemotherapy, each has its own toxicities and complications . When used in combination, not only can there be an additive effect of the adverse effects, but often, one modality can intensify the toxicities of the other . The incidence of toxic effects of radiotherapy can vary largely depending on dose, fractionation, treatment volumes, techniques, and whether administered concomitantly with chemotherapy . Still, the nature of toxic effects is consistent with the most common acute toxic effect being esophagitis . In patients of carcinoma esophagus, who often presents with dysphagia and poor nutritional status, the addition of esophagitis often results in dehydration and malnutrition requiring feeding tubes in half of the patients . Other common acute toxicities include gastrointestinal effects and fatigue . In the radiotherapy - alone arm of radiation therapy oncology group (rtog) 85 - 01, the most common acute toxicities were upper aerodigestive (18%). In our study also, acute radiation toxicity in the form of esophagitis and absolute dysphagia led to treatment interruption in three patients (7.5%). In four other patients (10%), loss of appetite during radiation led to excessive weight loss and overall deteriorated general condition, and thus further radiotherapy could not be delivered . Although acute esophagitis is most commonly ascribed as an adverse effect of radiotherapy, the addition of radiosensitizing chemotherapy does increase the incidence and severity of esophagitis . In rtog 85 - 01 trial, the addition of concurrent cisplatin/5-fu chemotherapy increased the incidence of severe upper aerodigestive toxicity from 18% with 64 gy of definitive radiotherapy to 33% with 50 gy of definitive chemoradiotherapy . The rates of severe (grade 3) esophagitis have ranged from 16% to 63% in studies using chemoradiotherapy . Other common gastrointestinal side effects are nausea, vomiting, and diarrhea . In clinical practice, patients receiving chemoradiotherapy, whether definitive or neoadjuvant, will almost universally experience some degree of esophagitis . In our study also, four patients (11.1%) on chemoradiation had acute esophagitis grade 3 and two patients (5.5%) had chemotherapy - induced severe nausea and vomiting because of which they could not complete their planned treatment . Hematologic toxicity is a well - expected toxicity observed with strategies using chemotherapy, the most common toxicity being neutropenia / granulocytopenia . Based on the trial comparing radiotherapy with chemoradiotherapy (rtog 85 - 01), the addition of chemotherapy to radiotherapy significantly increases acute hematologic toxicity from 3% to 48% . Myelosuppression leading to treatment interruption was seen in three patients (8.33%) in our study . The recent meta - analysis of neoadjuvant chemotherapy or chemoradiotherapy prior to surgery for esophageal carcinoma showed no significant association between the surgical complications and neoadjuvant interventions . Yet, the addition of chemoradiotherapy to surgery alone will induce both acute and late adverse effects that would not be seen with surgery alone . In the trials of multimodality therapy,, patients who could not complete the planned trimodality therapy can be categorized into two groups . The first group of defaulters (4 in number [26.7%]) experienced severe acute hematological and gastrointestinal toxicities after neoadjuvant chemoradiotherapy after which they could not proceed for surgery . Another group (five patients = 33.33%) completed neoadjuvant chemoradiotherapy and surgery but had excessive loss of weight postoperatively due to loss of appetite and inadequate diet and hence could not be delivered adjuvant chemotherapy . This aspect of treatment interruption can be seen commonly in resource constrained set up in developing countries where adequate postoperative nutritional counseling is lacking . In addition, fistula formation, disease progression during treatment, and lost to follow - up (leading to noncompletion of radiotherapy schedule) were the three factors which were unrelated to the toxicity of chemoradiotherapy but led to treatment interruption in nine of our patients . To achieve cure which is the main aim of treatment in all cancer patients, the treatment completion rates must be improved . Nutritional counseling should ideally be offered to all the patients before any cancer - directed therapy is initiated . Ours being a setup with heavy patient load, the nutritional counseling is done by oncologist only . We also have a palliative care section where patients with poor nutritional intake, are admitted, and are built up before starting radical treatment . However, due to limited resources in setup like ours, every patient cannot be provided such facilities . Therefore, for adequate diet counseling, a dietitian or nutritionist should be the part of the team management . European society for clinical nutrition and metabolism published their guidelines on enteral nutrition (en) for nonsurgical cancer patients in 2006 . En referred to oral nutritional supplements (onss) or tube feeding . According to the guidelines, en should be started if undernutrition already exists or if food intake is markedly reduced for more than 7 - 10 days . En is indicated preoperatively for 5 - 7 days in cancer patients undergoing major abdominal surgery . During radiotherapy of gastrointestinal regions, dietary counseling and ons treatment - related acute mucositis might lead to exaggeration of dysphagia in these patients, and it should be aggressively managed with enteral feeding through ryles tube (rt) or feeding jejunostomy (fj) or percutaneous gastrostomy (peg) depending on the institutional protocols . Esophageal stenting is another option to relieve malignant dysphagia before neoadjuvant or radical chemoradiation so as to maintain adequate nutrition during treatment . However, stenting is an expensive procedure compared to fj / peg and is associated with complications such as stent migration, tumor ingrowth, and overgrowth, esophagorespiratory fistula, for which stent replacements are required . Patients on chemotherapy, especially when combined with radiation, may require prophylactic treatment so as to prevent chemotherapy - induced nausea - vomiting and myelosuppression . For patients who receive concurrent chemoradiation, antiemetic therapy is dictated by the emetogenicity of chemotherapy unless the emetic risk of radiation therapy is higher . In our setup, patients who are given concurrent cisplatin are given a 5-hydroxytryptamine-3 (5-ht3) receptor antagonist for 5 days postchemotherapy . The recent update on the use of antiemetics by american society of clinical oncology (asco) however recommends that all patients who receive highly emetogenic chemotherapy regimens should be offered a three - drug combination of a neurokinin 1 receptor antagonist, 5-ht3 receptor antagonist, and dexamethasone . Preferential use of palonosetron is recommended for moderate emetic risk regimens, combined with dexamethasone . For low - risk agents, chemotherapy - induced myelosuppression is considered to be a significant negative prognostic factor for overall survival as it results in earlier discontinuation of the preoperative chemoradiation schedules, often requiring dose reduction of chemotherapeutic agents . Still according to the asco 2015 update of recommendations for the use of colony - stimulating factors (csfs) in cancer patients receiving chemotherapy, the use of csfs to prevent and treat chemotherapy - induced myelosuppression should be avoided in patients receiving concomitant chemoradiation, particularly involving the mediastinum . In the absence of chemotherapy, therapeutic use of csfs may be considered in patients receiving radiation therapy alone if prolonged delays secondary to neutropenia are expected . Psychosocial support is another aspect of treatment counseling which is often neglected in developing countries and at the centers with patient overload . It has been proven that patients with gastrointestinal cancer, who undergo surgery, benefit from a formal program of psychotherapeutic support during the inpatient hospital stay in terms of long - term survival . A well - trained psychotherapist should therefore be a part of the management protocols designed to treat patients . In resource - constrained centers, again, an oncologist can take over the role as is being done at our institute . Patients should be counseled both before and during the treatment by providing educational information regarding the need for adequate diet, disease prognosis, benefits of treatment, and a supportive relationship should be developed . Patients on radiation sometimes get relief from dysphagia in the middle of the course of radiotherapy schedule and stop treatment in between considering it to be the cure of disease . Some patients who develop radiotherapy- and chemotherapy - induced toxicities also leave the treatment in between . Adequate counseling should be done in such patients from the beginning of treatment for the need and impact of completing the entire treatment . Especially before discharge, the therapist should explore the patient's emotional and cognitive interpretation of the treatment and assist him in planning for future . Based on the results of this audit, to improve compliance rates in our patients, we have introduced following major changes . Before starting treatment, we nowadays refer all the patients with esophageal cancer to the dietitian for adequate diet counseling . An attempt is made to encourage all patients with more than or equal to grade 2 dysphagia for rt insertion or at least fj . All patients on concurrent chemotherapy with cisplatin are given a three - drug combination of neurokinin 1 receptor antagonist, 5-ht3 receptor antagonist, and dexamethasone if emesis is not controlled by 5-ht3 receptor antagonist alone . Patients of carcinoma esophagus by the nature of their disease are bound to have nutritional inadequacy either due to primary dysphagia or cancer - related loss of appetite . Radiation- and chemotherapy - induced toxicities further decrease the compliance of patients to treatment completion . Timely intervention by the management team consisting of an oncologist, dietitian, and psychotherapist can help overcome these factors and thereby improve the treatment completion rates.
Worldwide, open - angle glaucoma (oag) is estimated to affect almost 45 million adults over the age of 40 years, and the number is expected to reach 59 million by 2020.1 since the loss of vision associated with glaucoma is irreversible,2 early diagnosis and treatment are key to preserving visual function . Because intraocular pressure (iop) is a major risk factor in glaucoma and glaucoma suspects, all currently marketed treatments for glaucoma aim to lower iop . Accordingly, the administration of topical agents that reduce the production of aqueous humor and/or increase outflow is the mainstay of therapy.26 multidose formulations of topical antiglaucoma medications contain preservatives, of which the most commonly used is benzalkonium chloride (bak), a potent bactericidal and fungicidal agent.7,8 the majority of patients tolerate bak, but it has been associated with ocular sensitivity in some cases.912 patients who require multiple medications to reach / maintain target iop may be at higher risk of bak sensitivity, as are those with severe dry eye disease because of reduced dilution in the tear film.9 consequently, preservatives causing less irritation, as well as many single - dose, preservative - free (pf) formulations, have been developed . In clinical trials and clinical practice, pf formulations have demonstrated noninferior or equivalent efficacy to their respective preserved formulations, with potential for a reduced incidence of ocular adverse events.1322 bimatoprost / timolol (bim / tim) ophthalmic solution (ganfort; allergan plc, irvine, ca, usa) is a fixed - combination formulation of bimatoprost 0.03% (a synthetic prostamide) and timolol 0.5% (a nonselective -adrenergic receptor antagonist) preserved with bak.23 clinical trials have demonstrated that when dosed once daily, the fixed combination produces greater iop reduction than each of the active components dosed as monotherapy.24,25 accordingly, bim / tim is used in a number of countries worldwide to treat patients with primary oag (poag) or ocular hypertension (oht) who do not reach target iop with monotherapy . However, because of the increasing awareness of patient sensitivity to preservatives, bim / tim pf (allergan plc, irvine, ca, usa) was developed; this pf formulation is identical to the original bim / tim formulation, except for the removal of 50 ppm of bak . In a 12-week, multicenter, randomized, phase iii study of 561 patients with oag or oht, bim / tim pf demonstrated noninferiority and equivalence to bim / tim in terms of iop - lowering efficacy (without significant differences in safety / tolerability),26 which led to its marketing approval in the european union in september 2013.27 despite these findings, when the between - treatment difference in average eye iop was compared, a consistent trend toward lower iop favoring bim / tim pf over bim / tim was observed in seven of the nine time points measured.26 this observation suggested that bak removal might have enhanced the efficacy of bim / tim . Given that bak is a well - known permeability enhancer that has been reported to increase drug penetration into ocular tissues,2831 it was surprising that bim / tim pf produced a greater iop - lowering effect than the bim / tim formulation containing bak.26 a preliminary literature search exploring possible explanations for this clinical observation found data suggesting that timolol in preserved ophthalmic solutions may display an inverted, u - shaped dose response curve for iop lowering, with an optimal concentration between 0.25% and 0.5%.32 removal of bak from 0.5% timolol ophthalmic solution formulations could reduce timolol bioavailability enough to achieve a concentration that optimizes intraocular efficacy.32 this literature review aims to explain the findings of the phase iii study demonstrating enhanced efficacy of bim / tim pf over preserved bim / tim by exploring the relationship between the concentration of timolol in preserved ophthalmic solutions and its iop - lowering efficacy . An initial search of the literature for timolol dose response in humans was performed in scopus (elsevier, philadelphia, pa, usa). Articles that were published in english between 1960 and july 2014 were identified using the keywords timolol and (intraocular pressure or iop) and human (or derivatives) and (1% or 0.5% or 0.25%), and screened for relevance to iop lowering after ocular instillation of topical timolol 0.25%, 0.5%, or 1.0% . Similarly, a literature search spanning the years 1960 to july 2014 was conducted in pubmed (http://www.ncbi.nlm.nih.gov/pubmed) using the keywords timolol and (intraocular pressure or iop) and (1% or 0.5% or 0.25%), with filters set to display articles involving human patients in clinical trials published in english . Articles that reported direct comparisons of the effect of at least two concentrations of timolol on iop in adult patients were included . Studies in which patient subgroups received a higher concentration of timolol after responding poorly to an initial lower concentration were excluded . An initial search of the literature for timolol dose response in humans was performed in scopus (elsevier, philadelphia, pa, usa). Articles that were published in english between 1960 and july 2014 were identified using the keywords timolol and (intraocular pressure or iop) and human (or derivatives) and (1% or 0.5% or 0.25%), and screened for relevance to iop lowering after ocular instillation of topical timolol 0.25%, 0.5%, or 1.0% . Similarly, a literature search spanning the years 1960 to july 2014 was conducted in pubmed (http://www.ncbi.nlm.nih.gov/pubmed) using the keywords timolol and (intraocular pressure or iop) and (1% or 0.5% or 0.25%), with filters set to display articles involving human patients in clinical trials published in english . Articles that reported direct comparisons of the effect of at least two concentrations of timolol on iop in adult patients were included . Studies in which patient subgroups received a higher concentration of timolol after responding poorly to an initial lower concentration were excluded . Given its long history in the management of glaucoma, many reports of the iop - lowering effects of timolol have been published, but only a few provide data related to dose response . A comparable search in pubmed yielded 607 references, including ten relevant clinical trials . Accounting for overlap, the scopus and pubmed searches together found a total of 17 relevant studies (figure 1). All trials were randomized except one,33 and 14 were double - masked.3447 eight studies included parallel groups,34,37,41,43,44,4648 six involved dose escalation,33,35,38,42,45,49 and three had one or two crossover(s).36,39,40 five studies enrolled healthy volunteers,34,38,42,43,49 whereas the other 12 enrolled patients with oag or oht . Timolol is commonly prescribed as a 0.25% or 0.5% preservative - containing (usually bak) ophthalmic solution . However, the selected studies covered a broader range of concentrations: 0.008%, 0.025%, 0.0625%, 0.08%, 0.1%, 0.125%, 0.25%, 0.5%, 1.0%, and 1.5% (table 1). Nine studies measured iop after weeks to months of timolol administration once40,48 or twice33,3537,41,44,45 daily . The remaining eight studies assessed the short - term (ie, within 28 hours) iop - lowering effect following a single application.34,38,39,42,43,46,47,49 all studies that evaluated the efficacy of timolol over weeks or months found that the concentrations tested (ie, 0.1%, 0.25%, 0.5%, and 1%) provided statistically significant iop reductions compared with baseline and/or controls (untreated or placebo - treated eyes), regardless of the instillation schedule (ie, once versus twice daily).33,3537,40,41,44,45,48 however, results of short - term studies varied, depending on the study population . In patients with elevated iop, concentrations of 0.1%, 0.25%, 0.5%, 1.0%, and 1.5% provided statistically significant iop reductions compared with baseline and/or controls (untreated or placebo - treated eyes).34,38,43,46,47 even a concentration as low as 0.008% (administered as gel or solution) was effective in lowering iop in patients with oht and a baseline iop> 22 mmhg, compared with placebo controls.39 in contrast, studies of healthy volunteers with baseline iop of approximately 1314 mmhg reported no significant iop - lowering effect when treated with timolol 0.008% or 0.025%,42 or 0.0625%, 0.125%, or 0.25%.49 four studies compared the iop - lowering effect of timolol ophthalmic solution at concentrations of 0.5% or greater and found that timolol 0.5% was as effective as or more effective than higher concentrations . Three of these studies involved a single application of drug.38,46,47 in a parallel - group, single - dose study in 30 patients with oag, timolol 0.5% was as effective as or numerically more effective than timolol 1.5% at six of eight time points measured (figure 2a).46 in a similarly designed study in 20 patients with oag, timolol 0.5% was as effective as or numerically more effective than timolol 1.0% at the 4-, 12-, and 24-hour time points (figure 2b).47 also, in a dose escalation study in 30 healthy volunteers, katz et al38 found that iop reduction from baseline was numerically greater at 3, 5, and 7 hours after a single application of timolol 0.5% (25%, 26%, and 23%), compared with timolol 1.0% (23%, 19%, and 22%) or 1.5% (24%, 17%, and 17%), respectively . Timolol 0.5% was also numerically more effective than timolol 1.0% at 2 hours postinstillation (figure 2c). The fourth and most clinically relevant study involved administration of timolol 0.5% and 1.0% twice daily for 1 week in a dose escalation design,45 and found that patients with oht or oag had a numerically lower mean iop at four of six postinstillation time points during treatment with timolol 0.5% than during treatment with timolol 1.0% (figure 2d).45 the consistent findings suggest that on a concentration basis, timolol 0.5% is more effective than higher concentrations . Such an inverted u - shaped dose response relationship, in conjunction with the systemic cardiovascular risk associated with timolol as a nonselective -adrenoceptor antagonist,5056 explains why timolol is not prescribed at a dose strength higher than 0.5% . Of the 17 studies, 13 compared timolol 0.25% and 0.5%, currently the most commonly used concentrations . Among these, two studies showed that timolol 0.25% was either significantly or numerically more effective than timolol 0.5% in iop lowering . The first, a parallel - group study of timolol administered twice daily over 12 months, indicated that when statistically significant differences were found between doses, they always favored timolol 0.25% (figure 3a).41 the second, a double - masked, three - phase study of the effects of timolol 0.25% and 0.5% administered once daily for 10 days, reported that timolol 0.25% was numerically (but not statistically significantly) better than the 0.5% concentration in lowering iop over the majority of time points measured (figure 3b).40 three other studies provided data supporting a dose - dependent difference in iop - lowering favoring the 0.5% concentration . Results from a 1-week, dose escalation study of twice - daily administration indicated that iop lowering from baseline was numerically greater with timolol 0.5% (21% and 23%) than with timolol 0.25% (17% and 20%) at 1 and 6 hours postinstillation, respectively, but similar or greater with timolol 0.25% (21% and 24%) than with timolol 0.5% (21% and 15%) at 3 and 8 hours postinstillation, respectively.45 however, the iop - lowering effect appeared to be better sustained at 12 hours postinstillation (trough) with timolol 0.5% (19%) than timolol 0.25% (11%).45 similarly, two short - term studies with assessments that spanned 90 minutes43 and 28 hours47 postinstillation of timolol 0.25% or 0.5% found a dose - dependent, numerical difference in iop lowering at later time points that favored the 0.5% concentration (figure 4), although statistical analysis of the difference between concentrations was not provided . The remaining eight studies concluded that there was no difference in iop lowering between timolol 0.25% and 0.5% . In a double - masked, three - period crossover study of timolol administered twice daily for 4 weeks, the mean iop reduction at study end was 11.313.18 mmhg (34.67%) with timolol 0.25% versus 12.033.72 mmhg with timolol 0.5% (35.95%; p>0.5).36 findings were comparable when timolol 0.25% and 0.5% were dosed once daily for 8 weeks (figure 5a),48 or twice daily for 6 months (figure 5b).44 in a randomized, double - masked, dose escalation study, timolol 0.25% induced a 26% reduction in iop from baseline when administered twice daily for 1 week . An increase in concentration to timolol 0.5% only produced a modest, additional iop reduction of approximately 4%, but a statistical analysis of the between - concentration difference in iop lowering was not provided.35 in a similar dose escalation study of timolol 0.25% and 0.5% twice daily, increasing the concentration from 0.25% to 0.5% did not result in significant additional iop lowering from baseline; iop reduction reached 25% and 27% after 34 weeks of treatment with timolol 0.25% and 0.5%, respectively.33 a short - term study that assessed iop levels at 1, 2, 4, 6, 8, 12, and 24 hours postinstillation and included statistical analysis of the between - concentration difference in iop lowering from baseline also concluded that the effect of timolol 0.5% was not superior to that of timolol 0.25% (figure 5c).34 an area under the curve analysis of iop versus time (ie, 12 and 24 hours) yielded similar values for both concentrations: 43.66.2 and 74.911.0 for timolol 0.25% versus 38.19.4 and 69.116.4 for timolol 0.5%, respectively.34 importantly, the largest study identified in this systematic review compared the iop - lowering efficacy of timolol 0.25% and 0.5% administered twice daily as a maleate or hemihydrate solution over 8 weeks in 371 patients with poag or oht.37 the authors concluded that when either formulation was administered, the 0.25% and 0.5% dose strengths were equally effective from week 1 to week 12 (figure 5d); equivalence between the two concentrations at the final visit was established by statistical analysis . Taken together, the aforementioned results suggest that an inverted u - shaped dose response curve may exist for timolol in terms of iop lowering, and that the concentration that elicits maximum iop lowering likely lies between 0.25% and 0.5%, as illustrated in figure 6 . A possible explanation for the inverted u - shaped dose the association between repeated ocular timolol use and tachyphylaxis has long been described,5765 and although some patients exhibit relatively stable iop reductions for years in response to timolol ophthalmic solution, some demonstrate an upward iop drift after days or months, reflecting a partial or complete loss of response to timolol.59,62 timolol concentrations of 0.5% and higher may be associated with an increased occurrence of receptor upregulation,62,66,67 which leads to tachyphylaxis . In contrast, there has been no report of an association between bimatoprost and tachyphylaxis in the literature, and preclinical studies of bimatoprost in dogs and monkeys have revealed a flat dose response curve between 0.001% and 0.1%, suggesting that preservative removal is unlikely to affect the effective concentration of bimatoprost.68 given its long history in the management of glaucoma, many reports of the iop - lowering effects of timolol have been published, but only a few provide data related to dose response . A comparable search in pubmed yielded 607 references, including ten relevant clinical trials . Accounting for overlap, the scopus and pubmed searches together found a total of 17 relevant studies (figure 1). All trials were randomized except one,33 and 14 were double - masked.3447 eight studies included parallel groups,34,37,41,43,44,4648 six involved dose escalation,33,35,38,42,45,49 and three had one or two crossover(s).36,39,40 five studies enrolled healthy volunteers,34,38,42,43,49 whereas the other 12 enrolled patients with oag or oht . Timolol is commonly prescribed as a 0.25% or 0.5% preservative - containing (usually bak) ophthalmic solution . However, the selected studies covered a broader range of concentrations: 0.008%, 0.025%, 0.0625%, 0.08%, 0.1%, 0.125%, 0.25%, 0.5%, 1.0%, and 1.5% (table 1). Nine studies measured iop after weeks to months of timolol administration once40,48 or twice33,3537,41,44,45 daily . The remaining eight studies assessed the short - term (ie, within 28 hours) iop - lowering effect following a single application.34,38,39,42,43,46,47,49 all studies that evaluated the efficacy of timolol over weeks or months found that the concentrations tested (ie, 0.1%, 0.25%, 0.5%, and 1%) provided statistically significant iop reductions compared with baseline and/or controls (untreated or placebo - treated eyes), regardless of the instillation schedule (ie, once versus twice daily).33,3537,40,41,44,45,48 however, results of short - term studies varied, depending on the study population . In patients with elevated iop, concentrations of 0.1%, 0.25%, 0.5%, 1.0%, and 1.5% provided statistically significant iop reductions compared with baseline and/or controls (untreated or placebo - treated eyes).34,38,43,46,47 even a concentration as low as 0.008% (administered as gel or solution) was effective in lowering iop in patients with oht and a baseline iop> 22 mmhg, compared with placebo controls.39 in contrast, studies of healthy volunteers with baseline iop of approximately 1314 mmhg reported no significant iop - lowering effect when treated with timolol 0.008% or 0.025%,42 or 0.0625%, 0.125%, or 0.25%.49 four studies compared the iop - lowering effect of timolol ophthalmic solution at concentrations of 0.5% or greater and found that timolol 0.5% was as effective as or more effective than higher concentrations . Three of these studies involved a single application of drug.38,46,47 in a parallel - group, single - dose study in 30 patients with oag, timolol 0.5% was as effective as or numerically more effective than timolol 1.5% at six of eight time points measured (figure 2a).46 in a similarly designed study in 20 patients with oag, timolol 0.5% was as effective as or numerically more effective than timolol 1.0% at the 4-, 12-, and 24-hour time points (figure 2b).47 also, in a dose escalation study in 30 healthy volunteers, katz et al38 found that iop reduction from baseline was numerically greater at 3, 5, and 7 hours after a single application of timolol 0.5% (25%, 26%, and 23%), compared with timolol 1.0% (23%, 19%, and 22%) or 1.5% (24%, 17%, and 17%), respectively . Timolol 0.5% was also numerically more effective than timolol 1.0% at 2 hours postinstillation (figure 2c). The fourth and most clinically relevant study involved administration of timolol 0.5% and 1.0% twice daily for 1 week in a dose escalation design,45 and found that patients with oht or oag had a numerically lower mean iop at four of six postinstillation time points during treatment with timolol 0.5% than during treatment with timolol 1.0% (figure 2d).45 the consistent findings suggest that on a concentration basis, timolol 0.5% is more effective than higher concentrations . Such an inverted u - shaped dose response relationship, in conjunction with the systemic cardiovascular risk associated with timolol as a nonselective -adrenoceptor antagonist,5056 explains why timolol is not prescribed at a dose strength higher than 0.5% . Of the 17 studies, 13 compared timolol 0.25% and 0.5%, currently the most commonly used concentrations . Among these, two studies showed that timolol 0.25% was either significantly or numerically more effective than timolol 0.5% in iop lowering . The first, a parallel - group study of timolol administered twice daily over 12 months, indicated that when statistically significant differences were found between doses, they always favored timolol 0.25% (figure 3a).41 the second, a double - masked, three - phase study of the effects of timolol 0.25% and 0.5% administered once daily for 10 days, reported that timolol 0.25% was numerically (but not statistically significantly) better than the 0.5% concentration in lowering iop over the majority of time points measured (figure 3b).40 three other studies provided data supporting a dose - dependent difference in iop - lowering favoring the 0.5% concentration . Results from a 1-week, dose escalation study of twice - daily administration indicated that iop lowering from baseline was numerically greater with timolol 0.5% (21% and 23%) than with timolol 0.25% (17% and 20%) at 1 and 6 hours postinstillation, respectively, but similar or greater with timolol 0.25% (21% and 24%) than with timolol 0.5% (21% and 15%) at 3 and 8 hours postinstillation, respectively.45 however, the iop - lowering effect appeared to be better sustained at 12 hours postinstillation (trough) with timolol 0.5% (19%) than timolol 0.25% (11%).45 similarly, two short - term studies with assessments that spanned 90 minutes43 and 28 hours47 postinstillation of timolol 0.25% or 0.5% found a dose - dependent, numerical difference in iop lowering at later time points that favored the 0.5% concentration (figure 4), although statistical analysis of the difference between concentrations was not provided . The remaining eight studies concluded that there was no difference in iop lowering between timolol 0.25% and 0.5% . In a double - masked, three - period crossover study of timolol administered twice daily for 4 weeks, the mean iop reduction at study end was 11.313.18 mmhg (34.67%) with timolol 0.25% versus 12.033.72 mmhg with timolol 0.5% (35.95%; p>0.5).36 findings were comparable when timolol 0.25% and 0.5% were dosed once daily for 8 weeks (figure 5a),48 or twice daily for 6 months (figure 5b).44 in a randomized, double - masked, dose escalation study, timolol 0.25% induced a 26% reduction in iop from baseline when administered twice daily for 1 week . An increase in concentration to timolol 0.5% only produced a modest, additional iop reduction of approximately 4%, but a statistical analysis of the between - concentration difference in iop lowering was not provided.35 in a similar dose escalation study of timolol 0.25% and 0.5% twice daily, increasing the concentration from 0.25% to 0.5% did not result in significant additional iop lowering from baseline; iop reduction reached 25% and 27% after 34 weeks of treatment with timolol 0.25% and 0.5%, respectively.33 a short - term study that assessed iop levels at 1, 2, 4, 6, 8, 12, and 24 hours postinstillation and included statistical analysis of the between - concentration difference in iop lowering from baseline also concluded that the effect of timolol 0.5% was not superior to that of timolol 0.25% (figure 5c).34 an area under the curve analysis of iop versus time (ie, 12 and 24 hours) yielded similar values for both concentrations: 43.66.2 and 74.911.0 for timolol 0.25% versus 38.19.4 and 69.116.4 for timolol 0.5%, respectively.34 importantly, the largest study identified in this systematic review compared the iop - lowering efficacy of timolol 0.25% and 0.5% administered twice daily as a maleate or hemihydrate solution over 8 weeks in 371 patients with poag or oht.37 the authors concluded that when either formulation was administered, the 0.25% and 0.5% dose strengths were equally effective from week 1 to week 12 (figure 5d); equivalence between the two concentrations at the final visit was established by statistical analysis . Taken together, the aforementioned results suggest that an inverted u - shaped dose response curve may exist for timolol in terms of iop lowering, and that the concentration that elicits maximum iop lowering likely lies between 0.25% and 0.5%, as illustrated in figure 6 . A possible explanation for the inverted u - shaped dose response curve might involve receptor upregulation and tachyphylaxis . The association between repeated ocular timolol use and tachyphylaxis has long been described,5765 and although some patients exhibit relatively stable iop reductions for years in response to timolol ophthalmic solution, some demonstrate an upward iop drift after days or months, reflecting a partial or complete loss of response to timolol.59,62 timolol concentrations of 0.5% and higher may be associated with an increased occurrence of receptor upregulation,62,66,67 which leads to tachyphylaxis . In contrast, there has been no report of an association between bimatoprost and tachyphylaxis in the literature, and preclinical studies of bimatoprost in dogs and monkeys have revealed a flat dose response curve between 0.001% and 0.1%, suggesting that preservative removal is unlikely to affect the effective concentration of bimatoprost.68 the evidence gathered appears to support our hypothesis that the optimal iop - lowering concentration of timolol lies between 0.25% and 0.5% when administered as a bak - preserved formulation . The majority of studies found no significant differences in iop lowering between the 0.25% and 0.5% concentrations, and some studies showed statistically significantly increased efficacy with the 0.25% concentration . Therefore, if the removal of bak would result in lower ocular concentrations of timolol in patients treated with bim / tim pf (containing 0.5% timolol) than in those treated with preserved bim / tim (also containing 0.5% timolol), it is reasonable to believe that the reduced exposure to timolol 0.5% in the pf formulation at the target site may bring the effective concentration within the 0.25%0.5% range, thus maximizing the iop - lowering effect of timolol . This supposition, however, should be considered in light of the limitations of the available data: the variability of the study designs, the small sample sizes of some studies, and the lack of studies that compared the efficacy of various concentrations of timolol in preserved versus pf formulations . Early pharmacokinetic and pharmacodynamic studies showed that timolol is well absorbed through the cornea and rapidly distributes into ocular tissues following topical ocular administration; it can be measured in the human aqueous humor for up to 12 hours . Timolol significantly lowered iop in healthy volunteers and in patients with oag, and in dose ranging studies, the maximum effect appeared to occur with the 0.5% concentration.69 the current knowledge of the dose response relationship between iop lowering and timolol concentration in preserved topical ophthalmic solutions is highlighted in our systematic literature review . Studies that evaluated the efficacy of timolol over weeks or months in patients with oag or oht are likely more relevant to our hypothesis as they are indicative of the relationship between efficacy and steady - state concentrations of timolol in the eye (compared with studies assessing the iop - lowering effects within minutes or hours of treatment with a single dose). These studies concluded that timolol ophthalmic solutions at 0.1%, 0.25%, 0.5%, and/or 1% provide statistically significant iop reductions from baseline and/or compared with untreated or placebo - treated eyes, regardless of the instillation schedule . Nevertheless, all but two short - term studies (including one study in eleven patients that could not detect significant iop reduction at every time point with timolol 0.5%), reached the same conclusion, even for timolol concentrations 0.1% . Overall, these data suggest that on a concentration basis, timolol 0.5% is more effective at lowering iop than higher concentrations, and that the optimal iop - lowering concentration of timolol is likely between 0.25% and 0.5% . Furthermore, these findings support the clinical observation that removal of bak from the bim / tim formulation provided more optimal iop reduction.
Angioma serpiginosum is a benign vascular nevoid disorder with proliferation and ectatic dilatation of capillaries in the papillary dermis . Most cases are sporadic but familial cases suggesting an autosomal dominant inheritance have also been described . We report a case of late onset angioma serpiginosum localised to the breast, an unusual site . A 46-year - old female patient presented to the dermatology out - patient clinic with an asymptomatic, progressive red eruption on the right breast of 4 years duration . She did not provide a history of bleeding disorder, preceding trauma, or contact allergy prior to the eruption of the lesions . Clinical examination revealed multiple punctate macules in a serpiginous pattern against a bluish background, grouped at places, located on the lateral half of the right breast . A 3 mm, red, soft papule was noted overlying these lesions at their lower extent [figure 1]. Multiple, punctate macules in a serpiginious pattern against a bluish background on the right breast with a red smooth papule at the inferior aspect the differentials considered were angioma serpiginosum, unilateral nevoid telangiectasia, pigmented purpuric dermatoses and telangiectasia macularis eruptiva perstans . Epiluminescence microscopy with heine delta 20 dermatoscope (heine optotechnik, herrsching, germany) revealed multiple well demarcated oval to round red lagoons [figure 2]. Epiluminescence microscopy (20) revealing the well demarcated red lagoon appearance histopathological examination of the nonblanching punctate macules showed a normal to mildly orthohyperkeratotic epidermis with dilated thin walled capillaries in the papillary dermis . There was no evidence of extravasation of erythrocytes, inflammatory cell infiltrate or deposition of hemosiderin in the surrounding tissue [figure 3]. Periodic acid - schiff (pas) stain showed a thick cuff of amorphous acidophilic pas - positive diastase - resistant material surrounding the dilated vessels [figure 4]. The red papule overlying the punctate macules revealed a well delineated papillary dermal lesion composed of closely placed ectatic thin walled capillaries engorged with erythrocytes that was consistent with a diagnosis of cherry angioma [figure 5]. The clinical examination complemented by epiluminescence microscopy and she was advised ophthalmic examination, which was refused and no further treatment was sought . Dilated thin walled capillaries (arrow) in the superficial papillary dermis with an unremarkable deep papillary and reticular dermis . There is no extravasation of erythrocytes, inflammatory cell infiltrate or deposition of hemosiderin in the surrounding tissue (h and e, 200) cuff of periodic acid - schiff (pas) positive diastase - resistant material (arrow) surrounding the dilated vessels (pas, 200) well delineated papillary dermal lesion composed of closely placed ectatic thin walled capillaries engorged with erythrocytes (h and e, 100) first described by hutchinson in 1889 and named by radcliffe - crocker in 1893, angioma serpiginosum consists of multiple red, minute, nonblanchable and grouped macules, resembling purpura, in a serpiginous or gyrate pattern with a background of erythema or violaceous hue . . The erythematous or violaceous background hue may be due to dilatation of the subpapillary venous plexus . The eruption usually affects teenage females and in 90% cases has its onset before the age of 16 years . It commonly affects the lower extremities and buttocks and is often asymmetric . Any anatomic site with exceptions of the mucocutaneous junctions, palms and soles can be affected, though there has been a case report describing plantar involvement as well as reports of disseminated distribution . Majority of cases occur in females and are of childhood onset . In view of female preponderance and progression of lesions in pregnancy, the role of hormonal stimuli has been refuted by the absence of estrogen - progesterone receptor stimulation . Epiluminescence microscopy reveals demarcated red lagoon appearance due to the presence of increased and dilated vascular spaces in the papillary dermis and can help distinguish the condition from purpuric dermatoses on the basis of typical lagoon pattern . Histopathological findings include a normal epidermis, dilated thin walled capillaries in the papillary dermis without an extravasation of red blood cells or deposition of hemosiderin in the surrounding tissue . The capillary walls may be thickened due to deposition of pas positive material around blood vessels . The absence of dermal mast cell increase, hemosiderin deposition and red blood cell extravasation in our case ruled out the differential diagnoses that were considered . It was first described by campbell de morgan in 1872 . Generally developing after the third decade clinically, early lesions appear as flat, red macules that look like petechiae that later evolve into 1 - 5 mm red papules . These are known to be related to chemical exposures, with liver transplants, graft - versus - host disease, and secondary to cyclosporine treatment . In the absence of any known inciting factors, it is assumed that our patient had an incidental onset of solitary cherry angioma or as a result of chronological ageing . Histological examination of cherry angiomas shows numerous, newly formed capillaries in a lobular pattern in the papillary dermis . There have reports of two female patients with chest wall involvement with the onset at 7 and 9 years of age . Chest involvement has also been described as a part of disseminated lesions in three patients . Two of these patients were beyond 50 years of age at presentation with the onset of their lesions in early childhood . Angioma serpiginosum has been described to be associated with angiokeratoma of the vulva in contiguous areas . However, there has not been any report of cherry angioma in association with lesions of angioma serpiginosum or overlying the lesions . Our patient presented with a solitary cherry angioma superimposed on the angioma serpiginosum with the onset beyond 40 years of age and on the breast that is an unusual site for the latter . To the best of our knowledge, there has been no reported case of this entity with late onset lesions in the english language literature . Co - existence of cherry angioma is assumed to be a result of chronological ageing process or may be secondary to a common vascular origin of both these lesions.
Acute cytomegalovirus (cmv) infection is common in both immunocompromised and immunocompetent patients worldwide.12 the infection is often asymptomatic . Symptoms, when they occur, include fever, cervical lymphadenitis, and arthralgia.3 rarely, acute cmv infection may be complicated by pneumonia, colitis, myocarditis, pericarditis, or hemolytic anemia.4 vascular events caused by acute cmv infection, including thrombosis of the venous or arterial vascular system, are rarely reported in the english literature . We report a case of acute cmv colitis in an immunocompetent adult that was complicated by vascular thrombosis and pulmonary embolism . Further, we present a review of the literature regarding the association between vascular events and cmv infection in immunocompetent patients . A 78-year - old man was referred to our emergency department with a 3-day history of diarrhea and fever . He had no recent travel history, did not use steroids, and had not experienced recent trauma . He used medication for hypertension and diabetes mellitus, both of which were well controlled . On admission, his vital signs were: body temperature, 38.8; blood pressure, 145/85 mmhg; respiratory rate, 18/min; and pulse rate, 90/min . On physical examination, his pulse was regular and he had no cardiac murmurs, his chest was clear without signs of respiratory distress, and his abdomen was soft with hyperactive bowel sounds . Laboratory tests showed a white blood cell count of 8,270/mm (normal: 3,99010,500/mm), a platelet count of 199,000/mm (normal: 140,000450,000/mm), a prothrombin time of 14.10 seconds (control: 11.16 seconds), an inr of 1.21, an activated partial thromboplastin time of 26.0 seconds (control: 27.4 seconds), and crp level of 16.7 mg / dl (normal: <0.8 mg / dl). A coagulation profile, including protein c (80%, normal: 70%140%), protein s (75%, normal: 60%130%) and anti - thrombin iii (95%, normal: 75%125%), was normal . . Colonoscopy demonstrated multiple giant ulcers with skip lesions in the distal colon, sparing the rectum (fig . Multiple forceps - biopsy tissue samples, taken from the coloniculcers, were sent for pathological examination . However, 5 days post - colonoscopy, the patient experienced dyspnea and severe hypoxemia, necessitating emergent endotracheal intubation and ventilation . A chest radiograph obtained post - intubation revealed an engorged main pulmonary trunk with an abrupt cutoff of pulmonary vascularity in the distal portions bilaterally, indicative of the " westermark sign " (fig . Subsequently, a ct scan of the chest was performed that showed several filling defects within the pulmonary trunk and main pulmonary arteries (fig . Histological evaluation of the colonic biopsy specimens revealed cytomegalic cells with densely staining nuclei and intranuclear inclusion bodies in the epithelium (fig . These epithelial cells were positive for monoclonal anti - cmv antibody on immunohistochemical analysis, indicative of cmv infection (fig . The patient had no obvious risk factors for thrombosis, and had no family history of coagulopathy . Follow - up ct scan of the patient's chest showed complete resolution of the pulmonary emboli . Acute cmv infection in immunocompetent patients is common worldwide, with seroprevalence rates ranging from 40% to 100%, depending on country, socio - economic conditions, and age.1 colitis is the most common manifestation of severe cmv infection.2 patients with cmv colitis are generally asymptomatic; if symptomatic, they may present with diarrhea, fever, bleeding, abdominal pain, and perforation . 4 vascular events are a very rare manifestation of severe cmv infection in immunocompetent individuals.2 these vascular events include thrombosis of the portal, superior mesenteric, or splenic vein; cerebral venous thrombosis; deep vein thrombosis; and life - threatening pulmonary embolism . 567 vascular complications may be discovered only several weeks after the initial diagnosis of cmv infection . However, abgueguen et al . Proposed that venous thrombosis in immunocompetent patients with cmv infection may not be as rare as previously reported.8 although the pathogenesis of cmv - induced vascular events is unknown, several mechanisms have been postulated, including direct damage to endothelial cells, increased levels of hemostatic indicators, induction of smooth muscle proliferation, enhanced platelet - derived growth factor production, and the presence of antiphospholipid antibodies.91011 vascular thrombosis in immunocompetent patients is usually associated with predisposing factors such as heterozygous factor v leiden mutation, presence of antiphospholipid antibodies, heterozygous prothrombin gene mutation, protein c and/or protein s deficiency, or oral contraceptive use.12 in contrast, this patient had no family history of coagulopathy, was not bed - ridden, and had no other risk - factors for thrombosis . It is, therefore, likely that cmv infection was the precipitating factor for pulmonary embolism . In the diagnosis of vascular events caused by cmv infection, doppler ultrasound and ct scanning are the most useful diagnostic modalities.6 treatment of cmv - associated pulmonary embolism includes immediate administration of antiviral agents and anticoagulants . Advanced age, male gender, presence of immune - modulating comorbidities, and need for surgical intervention all negatively influence survival.13 although the cost - effectiveness of investigating for acute cmv infection in patients presenting with thrombosis has not yet been established, we believe that serum samples for cmv infection should be obtained and endoscopic examination should be considered in patients with idiopathic thrombosis . In conclusion, although pulmonary embolism caused by acute cmv colitis in immunocompetent patients is extremely rare, it should be considered in patients who present with severe respiratory symptoms . To the best of our knowledge, this is the first report of an immunocompetent patient with acute cmv colitis complicated by vascular thrombosis with pulmonary embolism in which no other obvious underlying predisposing factor for thrombosis was identified.
Insulinoma is a rare neuroendocrine tumor of the pancreas, accounting for 2% of all pancreatic tumors . Its occurrence is 1:100,000 and it is more common in women over 50 years of age . Insulinomas are usually solitary benign tumors, and less than 10% of them are malignant2,3). Malignant insulinomas are diagnosed when metastases occur, but distinguishing malignant insulinomas from benign insulinomas is difficult histologically . Focal nodular hyperplasia (fnh) represents a localized liver cell hyperplasia containing central and/or stellate fibrous scar4). Children account for only 7% of cases of this tumor, and fnh comprises only 2% of all pediatric liver tumors5,6). We report a case of an insulinoma in an 11-year - old child who subsequently developed multifocal nodular lesions of the liver after resections of the insulinoma . An 11-year - old girl was admitted for evaluation of recurrent seizures that had begun 5 months before admittance, especially in the morning, despite administration of oral antiepileptic medication . Although brain magnetic resonance imaging (mri) and electroencephalogram studies were all normal 2 months before admittance, she again had a seizure and was prescribed with oral levetiracetam to control her seizure attacks . Ten days prior to admittance, the patient had been brought to the emergency department due to an ongoing seizure . Her serum glucose level was 35 mg / dl and she was controlled seizure by intravenous infusion of 10% dextrose fluids . After discharge, she experienced 3 additional attacks of seizures that had been relieved by grape juice . Upon admittance, her height was 161.3 cm (> 97th percentile), and her weight was 47.6 kg (75th <percentile<90th) and her body mass index was 18.8 kg / m (50th <percentile<75th). All vital signs and the physical characteristics of the organs were all normal . Initial laboratory results, including a serum glucose level of 104 mg / dl, were unremarkable . A 72-hour fast test was initiated, and the patient demonstrated drowsiness, trembling, and tachycardia after less than 6 hours . Laboratory test results during the fast period showed hyperinsulinemic hypoglycemia without ketonuria: the serum insulin level was 50.1 u / ml (normal range, 2.0 - 25.0 u / ml), serum c - peptide level was 1.81 ng / ml (0.59 - 1.56 ng / ml), and plasma glucose level was 21 mg / dl (70 - 100 mg / dl). Subsequent glucagon stimulation test results revealed an increase in insulin level to 85.8 u / ml . The mri studies revealed a solid mass, 3 cm2.4 cm in size, with lobulated margins, in the pancreatic tail (fig . 1), with no metastasis, while abdominal ultrasonography (us) and computed tomography (ct) results were normal . The tumor was easily detected due to its superficial position at the end of the pancreatic tail, and no enlarged lymph nodes were detected around the pancreas or metastatic lesions in the abdominal cavity . The pathologic examination indicated that the resected tumor (3 cm3 cm2.5 cm in size) was multilobulated and not encapsulated, and no necrosis was evident . The tumor was of neuroendocrine origin, well differentiated, with an intermediate grade (mitoses 4/10 high - power fields) with angioinvasion . The ki-67 index, a cellular marker for proliferation which <3% means a low grade tumor, was 1% (fig ., fasting serum glucose levels were maintained between 110 to 140 mg / dl without any treatment . Follow - up mri studies were conducted 2 months after tumor removal, and revealed multiple hypointense nodular lesions in the hepatic segments s5, s6, and s8 (fig . Us - guided needle biopsy of the liver yielded a diagnosis of the lesions as fnh, with no necrosis or mitotic activity was noted . Since then the patient has been free of hypoglycemia for 2 years, and there was not the learning problem in her school . Recent mri studies have shown that the fnh lesions have decreased in size, without any evidence of metastasis . The classical diagnosis of insulinomas is based on whipple's triad: hypoglycemia of serum glucose lower than 50 mg / dl, neuroglycopenic symptoms (such as behavioral or personality changes and seizure), and prompt alleviation of symptoms after administering glucose . Malignant insulinoma is extremely rare in the pediatric population, with only a few cases reported in the english literature . Distinctions between benign and malignant insulinomas are based on metastasis or the invasion of the primary tumor into the lymph nodes, tissues, or other organs8). Insulinomas larger than 3 cm are also more likely be malignant, with local invasion or metastases to the liver9). Recently, janem et al.3) reported a pediatric case of malignant insulinoma in the context of a literature review of 9 preceding cases . Among the nine cases, five cases showed liver metastasis, two cases showed capsular invasion, and data for the remaining two cases were not available . Considering the criteria of malignant insulinoma, which require metastasis or local invasion of adjacent lymph nodes or tissues, these five cases with liver metastasis - together with the case reported by janem et al.3) involving metastasis to the liver, bone, and bone marrow - may be the only true reported malignant insulinomas to date . However, sata et al.10) and janez11) reported cases of malignant insulinomas initially diagnosed as benign, that recurred as liver metastases 8 and 15 years, respectively, after initial removal . Neither of these studies confirmed metastasis or invasion during the initial surgery or in subsequent histologic exams of the initial specimens . Thus, insulinomas may be considered as tentatively malignant because these rare tumors have unpredictable features and progression through the body . Neuroendocrine tumors have recently been identified as risk factors that are strongly associated with aggressive tumor behavior leading to malignancy12). Histopathologic grading of tumor cells based on mitosis may aid in prediction of future insulinoma outcomes, as the biologic behavior of low - grade tumors is often rather nonaggressive, whereas high - grade tumors are very aggressive12). Our histopathologic findings showed a well - differentiated, intermediate neuroendocrine tumor of the resected insulinoma and our operation field findings revealed no evidence of metastasis . Nevertheless, the possibility of malignancy was included due to the size of the tumor and its histological angioinvasion . The unusual multifocal hepatic lesions following tumor removal it has been infrequently documented in adults and children following chemotherapy, radiotherapy, and stem cell transplantation for different types of solid tumors13). The cause is unknown, although one hypothesis is that obstruction of hepatic vessels or abnormal vascularization could account for fnh, as suggested by the reported association with clinical and anatomic findings like hypoplasia or agenesis of the portal vein, vascular malformations, hemangioma and vascular dysplasia, budd - chiari syndrome, and hereditary hemorrhagic telangiectasia14). Although ct and mri findings of fnh may commonly include the presence of a central scar and typical enhancement patterns, biopsy is necessary in difficult cases with differential diagnosis of liver adenomas and carcinomas . Treatment recommendations regarding fnh are based on longitudinal follow - up of a small series of patients . Because fnh is a benign tumor with rare complications, most lesions are followed nonoperatively with serial us or mri, provided that a reliable diagnosis can be achieved using radiologic imaging3,15). Hepatic lesions can induce diagnostic problems when metastasis must be ruled out . In our case, hepatic nodular lesions were identified as fnh by needle biopsy, but the association of an insulinoma with the fnh after surgery remains unclear . To our knowledge, this is the first pediatric case report of an insulinoma in which diagnosis was confused due to newly discovered hepatic lesions after tumor resection . Close observation and follow - up imaging studies are required in patients who show malignant potential on histopathologic findings.
Cancer is the second leading killer in the usa, accounting for 25% of all deaths . Prostate cancer is the most common cancer in males, followed by lung and colorectal . For women however, it should be noted that lung cancer is the number one cancer killer for both men and women (1). An abundance of evidence suggests that lifestyle factors, including exposure to chemical carcinogens (smoking, etc . ), diet and inactivity play a major role in the development of these common cancers . In order to understand how lifestyle changes might reduce the risk for the most common cancers we must first understand how cancer develops . Unfortunately, many chemicals in the environment are capable of inducing free radical formation to damage dna in the body . Thus, at any given time most, if not all, of us might have a number of pre - neoplastic cells . If we are fortunate, the damaged dna sends a signal to genes such as p53 announcing the damage and increasing the protein products of these genes to stop the pre - neoplastic cell from dividing and hopefully repair the damaged dna . If the repair does not work, the cell should be directed to apoptosis or programmed cell death . Since cancer is so common in the usa, this might be the result of defects in the p53 gene or the fact that the gene is suppressed by certain factors . Defects in the p53 gene have been identified in about half of all cancers, but these defects are usually observed in end - stage cancer . For prostate cancer all early - stage cancer has an intact p53 gene that is suppressed from acting, as we will discuss later . If the p53 gene does not eliminate the defective pre - neoplastic cell, the cell is stimulated to divide (promotion stage) by certain co - carcinogens, leading to the formation of a tumor . Thus, to avoid cancer we should try to reduce the initiation stage by reducing exposure to many of the noxious chemical carcinogens present in our environment such as cigarette smoke, pesticides and many other commonly used household products . According to ames (2), the typical western diet also contains a variety of mutagens and carcinogens that may act through the generation of oxygen radicals and lead to the initiation of cancer as well as other degenerative diseases . A diet high in fat and/or refined sugar has been shown to induce oxidative stress (35). Consumption of red meat has been associated with cancers of the colon and rectum, breast and prostate . Whether it is the meat itself or the associated fat content is not known . Cooking meat at moderate to high temperatures forms carcinogenic heterocyclic amines, shown in animal studies to induce colon, breast and prostate cancer (6). Conversely, a diet high in whole grains, fruits and vegetables would contain large amounts of natural antioxidants that might play an important role in preventing free radical formation and cancer (2). This type of diet has been shown to reduce oxidative stress in the body (7). A diet high in whole grains, fruits and vegetables, especially if it includes an abundance of soy products would increase the intake of isoflavones, thought to reduce the risk of cancer (8). Genistein, the most abundant isoflavone in soy, has been shown in cell culture experiments to increase p21 and caspase 3 to cause cell cycle arrest and induce apoptosis of tumor cells (8). Consumption of soy might explain the large difference in many cancers between western and eastern cultures . Another factor that has been suggested to explain the difference is the consumption of green tea by eastern cultures . Green tea contains epigallocatechin gallate, also shown to be protective in cell culture and animal studies (8). Attention has also been focused on the value of omega-3 fatty acids found primarily in fish and certain nuts and seeds . These fatty acids block cox-2 expression and reduce inflammation that has been implicated in the early stages and/or progression of prostate, breast and colon cancers (911). Conversely, omega-6 fatty acids found in meat and many vegetable oils increases cox-2 expression . Recent studies have shown that non - steroidal anti - inflammatory drugs (nsaids) that block the cox pathway reduce the development of adenomas, precursors of colon cancer, and reduce the risk for prostate cancer (1214). Regular exercise has also been shown to increase the body's antioxidant mechanisms (15). In addition to preventing the initiation of cancer, diet and exercise may play an important role in reducing the promotion of cancer and inducing apoptosis by altering hormones such as insulin, testosterone and estrogen or growth factors such as insulin - like growth factor - i (igf - i). The link between smoking and lung cancer was established in the first surgeon general's report in 1964 . However, a number of epidemiological studies have reported that exercise lowers the risk for lung cancer as reviewed by lee (16). The large norwegian study of 81 516 men and women followed for 19 years reported a 25% reduction in lung cancer risk for men who walked or cycled for at least 4 h per week, after controlling for smoking habits and the number of cigarettes smoked (17). The mechanism by which exercise might reduce the risk for lung cancer has not been investigated but may be related to the ability of exercise to reduce serum insulin and subsequently igf - i . Igf - i has been reported to be a risk factor for lung cancer (18). A high consumption of fruit and vegetables has also been associated with a reduced risk for lung cancer (19). Prostate cancer is the most common male cancer in the usa, but has a very low incidence in asia . However, when asian men migrate to the usa and adopt the us lifestyle the incidence of prostate cancer approaches that of us men, suggesting the involvement of lifestyle factors as opposed to a genetic difference (20). In addition, as the developing countries with a low incidence of prostate cancer become more westernized, prostate cancer increases (21). The two lifestyle factors that have received the most attention are diet, especially the fat content, and being sedentary . The international data show a positive correlation between dietary fat and prostate cancer mortality with the lowest rates found in east asian men and the highest rates found in us and european men (22). Most of the prospective cohort studies within a given population, however, have failed to show a relationship between dietary fat or fatty food consumption and prostate cancer risk as discussed by moyad (23). The negative results from the cohort studies may be due to the fact that in any given population there is little variation in the dietary fat consumption, or may be due to the inability of questionnaires to accurately measure habitual fat consumption . Current evidence implicates omega-6 fatty acids in the promotion of cancers and omega-3 polyunsaturated fatty acids and omega-9 monounsaturated fatty acids as being protective (24). It may also be that in the international data not only do men with a low incidence of prostate cancer mortality consume much lower fat diets, they also tend to be more physically active . Epidemiological studies have reported that increased physical activity can reduce the risk for prostate, and other forms of cancer, in us and european men . In a recent review of the literature on physical activity and the risk for prostate cancer, thune and furberg (25) found that 14 of 28 epidemiological studies reported that increased occupational or leisure - time activity reduced the risk for prostate cancer by 1070% . In order to investigate possible mechanisms involved in the roles that diet and exercise play in the progression of prostate cancer, tymchuk et al . (26) developed a bioassay using serum to stimulate prostate cancer cells in culture . By placing men on a low - fat diet consisting primarily of grains, fruits and vegetables, and regular, supervised exercise they examined serum changes in vivo and their effect on prostate cancer cell growth in vitro . The subjects were participants or employees from the pritikin longevity center residential program . During their stay at the center, food was prepared and served buffet style to the participants and consisted of 1015% of calories from fat (polyunsaturated / saturated fatty acid ratio = 1.24), 1520% of calories from protein, and 6575% of calories from carbohydrates, primarily unrefined . Carbohydrates were in the form of high - fiber whole grains and other complex carbohydrates (5 servings / day), vegetables (4 servings / day), and fruits (3 servings / day). Protein was primarily derived from plant sources with non - fat dairy allowed for up to 2 servings / day . Fish or fowl was served in 3.5 ounce portions 1 day / week and in soups or casseroles 2 days / week . The diet contained <100 mg of cholesterol per day and alcohol, tobacco and caffeinated beverages were not allowed during the program . Prior to starting the exercise training, subjects underwent a graded treadmill stress test . Based on the results, the subjects were provided with an appropriate training heart rate value and given an individualized walking program . The exercise regimen consisted of daily walking at the training heart rate for 4560 min . In the initial study tymchuk et al . (26) found that just 11 days of the low - fat diet and exercise program reduced serum - stimulated lncap prostate cancer cell growth by 30% . Serum samples obtained from men (employees) who had adhered to the low - fat diet and exercise program for an average of 14 years showed an additional 15% reduction in lncap cell growth . Lncap is a well - established, androgen - dependent cell line, developed from a patient with prostate cancer . When the serum samples from the men following the low - fat diet and exercise program were used to stimulate pc-3 cells, an androgen - independent cell line typical of advanced prostate cancer, no reduction in cell growth was found (26). In a subsequent study ngo et al . (27) confirmed the diet and exercise reduction in lncap cell growth using another androgen - dependent, patient - derived cell line, lapc-4 . A control group of similar aged men without any intervention were studied over an 11-day period and no change in lncap cell growth was found . In an earlier study tymchuk et al . (28) had reported that men attending the pritikin longevity center low - fat diet and exercise program had significant reductions in serum insulin and significant elevations in sex hormone - binding globulin . The increase in shbg should lower the serum levels of free testosterone and estradiol, and might be a factor in the reduction in lncap cell growth observed following diet and exercise as this cell line is androgen dependent and has a mutated androgen receptor that binds both testosterone and estradiol . (26,29) confirmed the reduction in serum free testosterone in the pritikin participants and subsequently reported that insulin, testosterone and estradiol could individually stimulate the growth of lncap cells . However, when these hormones were added individually, or in combination, to the post diet and exercise serum they could only account for half of the reduction in lncap cell growth, indicating the involvement of other important factors . (30) developed a model similar to figure 2 and started to investigate igf-1 and its binding proteins . The igf axis consists of igf - i, igf - ii, six different binding proteins (igfbp 16) and two receptors, igf - ir and igf - iir . Igf - i is a peptide growth factor produced by the liver and other tissues, and is known to play a pivotal role in regulating cell growth, differentiation and apoptosis (programmed cell death) (18,31). It is also a potent mitogen for most tissues including the prostate (18,31). According to yarak et al . Igfbp-3 is the most abundant and binds 90% of the circulating igf - i but is not affected by different metabolic states and ngo et al . (33) found that the low - fat diet and exercise program decreased not only the fasting serum level of insulin, but also igf - i while increasing igfbp-1 . These changes in serum levels of igf - i and igfbp-1 are thought to be due to the changes in serum insulin and its impact on the liver . (34) reported that insulin stimulated the production of igf - i by hepatocytes . (27) added igf - i back to the diet and exercise serum, the reduction in lncap cell growth was completely eliminated . When igfbp-1 was added to the baseline serum, they observed a significant reduction in lncap cell growth . In addition to being a regulator of cell growth, igf - i has been reported to be a suppressor of apoptosis in several different studies (35). (27) studied apoptosis in their cell culture system using two different methods, annexin - v and tunel . Almost no apoptotic or necrotic cells were found in the fetal bovine serum (fbs) control or the pre diet and exercise samples . However, both the annexin - v and tunel staining methods showed a significant increase in apoptosis in the post diet and exercise - serum - stimulated samples . The fact that very little apoptosis was observed in the control serum - stimulated lncap cells may be the result of the high levels of igf - i and low levels of igfbp-1 in the control serum and may help to explain why prostate cancer is so prevalent in the usa . In an attempt to separate out the individual effects of diet versus exercise observed in their earlier studies with the androgen - dependent prostate cancer cell lines, barnard et al . (36) obtained serum samples from men who had been involved in the adult fitness program at the university of nevada, las vegas . The men were matched in age with a control group of men on no diet or exercise program, and for age and duration of participation to the long - term diet and exercise subjects previously studied . Volunteers were requested who had participated in the program for at least 10 years; the average was 14.7 years . The program was held 5 days per week for 1 h and consisted of warm - up and flexibility activities followed by 4550 min of continuous, strenuous exercise including calisthenics and swimming laps in the pool . Serum insulin and igf - i were lower in the exercise and diet + exercise groups compared to controls, but were not significantly different from each other . Igfbp-1 was higher in both the exercise and the diet + exercise groups compared to controls and was significantly higher in the diet + exercise group compared to the exercise group . When the serum was used to stimulate the lncap cells in culture, growth was reduced in both the exercise (65% fbs control) and diet + exercise (55% fbs control) groups compared to the control group where the growth was 99% of the fbs . The staining results from the two methods demonstrated that exercise as well as diet and exercise intervention increases apoptosis in the lncap cells . When the slides from the tunel staining were quantitatively analyzed, almost twice as much apoptosis was found in the diet and exercise samples compared to the exercise - only samples . The fact that apoptosis was higher in the diet + exercise subjects is in agreement with the significantly higher igfbp-1 levels compared to the exercise - only group . In an attempt to focus on the possible mechanisms involved in the reduction in lncap cell growth and increased apoptosis observed in the exercise - serum - stimulated samples, leung et al . Several studies have shown that igf-1 suppresses the action of p53 that is phosphorylated and stabilized when defects are found in dna . The increase in p53 protein normally activates other genes or factors to cause cell cycle arrest, dna repair or to induce apoptosis (34). The serum - stimulated cell cultures were lysed, centrifuged and the supernatant analyzed for p53 protein and was found to be significantly increased in the lysates from the exercise - serum - stimulated lncap cells compared to controls . These results also demonstrate that the lncap cells have an intact p53 pathway that is suppressed with serum from control subjects . According to gurumurthy et al . (35) an intact p53 pathway is characteristic of all early stage prostate cancer . In end - stage prostate cancer defects have been reported in the p53 gene . To further investigate the involvement of the p53 pathway in the exercise - serum - stimulated lncap cell growth reduction and induction of apoptosis, leung et al . Ln-56 is a lncap - derived cell line in which p53 was rendered non - functional by expression of a dominant negative fragment of p53, known as genetic suppressor element 56 . The results from the growth assay showed no significant difference between the control and exercise groups when the serum was used to stimulate the cells . The exercise - serum - stimulated growth in the ln-56 cells was 91% of the fbs control compared to 65% of fbs control for the lncap cells . When they examined apoptosis in the ln-56 cells, the exercise - serum - stimulated cells showed half the apoptosis observed with the control - serum - stimulated cells . This was opposite to the response observed in the lncap cells where apoptosis was greatly increased in the exercise - serum - stimulated cells . Collectively, the results from these experiments indicate that the reduction in serum igf - i and increase in igfbp-1 resulting from adopting a very - low - fat diet and/or regular exercise allows the prostate tumor cells to stabilize the p53 protein and activate downstream effectors to reduce cell growth and induce apoptosis . These data also provide a mechanism to explain the epidemiological data showing a reduction in the risk for prostate cancer in men who take part in regular exercise (25). If the observations of reduced cell growth and the induction of apoptosis reported for the cell culture studies with androgen - dependent prostate cancer cell lines also occur in the body, then a very - low - fat diet and exercise program might be of value in the treatment of prostate cancer patients, especially those with early - stage cancer . In order to investigate the possible effectiveness of a very - low - fat diet and exercise program on prostate cancer patients, ornish and colleagues (38,39) randomized a group of men on watchful waiting to control or to diet and exercise intervention . The patient all had biopsy - documented prostate cancer, psa ranging from 4 to 10 ng / ml, and a gleason sum of <7 prostate adenocarcinoma . The men in the diet and exercise intervention group were prescribed a vegan diet with 10% of calories from fat supplemented with soy, 3 g / day fish oil (omega-3 fatty acid) and 400 iu / day vitamin e. the exercise was to be aerobic (walking, jogging etc .) For 3060 min 6 days / week . Patients were also encouraged to practice stress management techniques including yoga, breathing, imagery etc . For 1 h / day . After 1 year, changes in serum psa were small but statistically significant, with the control group showing an increase and the diet and exercise group showing a drop . Also, at the end of 1 year, six of 43 in the control group had gone on for conventional treatment due to rising psa while none of the 41 in the diet and exercise group had treatment . There was very high adherence to the diet and exercise program and even some in the control group had made significant lifestyle changes . Serum samples from these patients were used in the lncap cell bioassay . Compared to baseline, cell growth was reduced by 9% in the control group and by 60% in the diet and exercise group . The growth rate in the baseline samples was not significantly different from what had previously been observed with serum from men without prostate cancer . Breast cancer is the most common female cancer in the usa and, like prostate cancer, is hormone - dependent in the early stages . The influence of lifestyle factors in breast cancer is supported by the large international variation and the migration studies (40,41). Even though breast cancer is initially an estrogen - dependent cancer, most breast cancer occurs in postmenopausal women . With the menopause estrogen levels drop but do not completely disappear as estrogen is produced via aromatase activity, primarily in fat cells . Thus, it is not surprising that obesity is a risk factor for postmenopausal breast cancer . Elevated serum estradiol has also been shown to be a risk factor for breast cancer in postmenopausal women (42). Like prostate cancer, the international data show a strong, positive correlation between per capita fat consumption and breast cancer incidence and mortality (40). Epidemiological studies in countries with a high incidence of breast cancer, such as the usa, have shown that regular exercise reduces the risk for breast cancer . In their review, thune and furberg (25) reported that occupational or leisure time physical activity reduced the risk for breast cancer in 26 of 41 studies by 30% in pre-, peri- and postmenopausal women . In 16 of 28 studies a graded dose heber et al . (43) studied postmenopausal women attending the pritikin center and reported that the 3-week, low - fat diet and exercise program lowered serum estradiol from 18.1 3.6 to 9.4 2.4 pg / ml . In a subsequent study (44) they reported serum changes in premenopausal women placed on the pritikin diet for 2 months at the ucla clinical research center . However, serum estradiol was reduced by 25% during the follicular phase and by 22% during the leuteal phase while serum estrone was reduced by 19 and 18%, respectively, during the two phases . Shbg, produced primarily in the liver, regulates the amount of free hormone available to interact with the hormone receptors . Insulin suppresses the production of shbg by the liver . In a more recent study (45) with postmenopausal women attending the pritikin 3-week diet and exercise program, serum insulin was reduced by 39% and shbg increased by 39% in women on hormone replacement therapy (hrt). In women not on hrt, insulin was reduced by 19% and shbg increased by 42% . The drop in insulin may be very important as insulin has been reported to be a risk factor for both estrogen positive and negative breast cancer (46). Barnard rs, liva m, ngo tm, varr bc and hong j (unpublished) recently studied changes in the igf axis in postmenopausal women attending the pritikin program . In 18 women on hrt, igf - i was reduced from 170 22 to 142 13 pg / ml and igfbp-1 was increased from 55 8 to 71 10 pg / ml while insulin was reduced from 14.5 2 to 9.1 1 iu / ml . These changes in the igf axis are important as igf - i has been reported to be a risk factor for breast cancer and it is now well established that there is interaction between the igf and estrogen receptor pathways in breast cancer (18,47). Cancer of the colon and/or rectum is the second leading cause of cancer deaths in the usa and is the third most common cancer in both men and women . Over the years there has been a lot of controversy regarding the involvement of lifestyle factors in intestinal cancer . This may be due, in part, to different site - specific etiologies of the cancer . Like the previous two cancers, colorectal cancer shows a large international variation that armstrong and doll (40) attributed to the variation in dietary fat consumption . Prior to this, burkitt (48) suggested that other dietary factors such as fiber and refined sugar played a more important role . He suggested that the removal of fiber by refining carbohydrates reduced stool bulk and increased transit time as well as adversely affecting intestional flora . Adenomas are precursor lesions of colorectal cancer and are informative endpoints for assessing cancer risk . (49) showed inverse associations between fiber intake and colorectal adenoma . On the other hand, studies in the usa, finland and sweden reported no protective effect of fiber (5052). One large cohort study in us men, the health professionals follow - up study, did find a significant inverse association of distal adenomas and soluble but not insoluble fiber intake (53). The exact reason for the inconsistency in the cohort studies is not known but may be due to the fact that the populations have been rather homogeneous with relatively low fiber intakes or may be due to the inability of food questionnaires to accurately predict habitual fiber intake . The recent european prospective investigation into cancer and nutrition (epic) study involved 519 978 individuals with fiber intakes that ranged from 13 g / day for the lowest quintile to 34 g / day for the highest quintile (54). Comparing the highest to the lowest quintile of fiber intake they found a relative risk for large bowel cancer of 0.75 . Even this intake of 34 g / day was lower that the 50 + g / day originally described by burkitt . After evaluating the scientific evidence, the european cancer prevention consensus panel concluded in 1998 that there was good evidence to support the protective effect of fiber against colon (and breast) cancer (55). Two recent papers have suggested that hyperinsulinemia resulting from consumption of high - fat, refined - sugar diets may be an important factor in the etiology of colorectal cancer (56,57). This might be related to our proposed model for prostate cancer as igf - i has also been found to be a risk factor for colon cancer (18). A high - fat diet (corn oil) has also been reported to increase colon mucosa igf - i receptors in rats (58). The involvement of hyperinsulinemia and elevated igf - i might also be related to the epidemiological studies showing a reduction in intestinal cancer with increased physical activity . According to friedenreich and orenstein (59), the most definitive evidence for an association between physical activity and cancer exists for colon cancer . Of 51 studies on colon or colorectal cancer, 43 demonstrated a reduction in risk in the most physically active men and women with an average reduction of 4050% and values as high as 70% for the most physically active . (60) recently examined the relationship between lifestyle factors and p53 mutations in colon tumors . They concluded that a western - style diet high in refined sugar and red meat was correlated with p53 mutations while no association was found with physical activity . This, to our knowledge, is the first study to show a relationship between diet and p53 mutations, whether or not a western - style diet can cause p53 mutations remains to be demonstrated . Reddy et al . (61) studied colon cancer risk factors in eight women attending the pritikin program by collecting stool samples over 2 days and diet records over 3 days before the start and at the end of the 3-week program . Dietary fat intake decreased from 34 to 7% of calories and fiber increased from 17 to 37 g / day during the residential program . Total stool bile acid was reduced from 9.7 2 to 4.5 1 mg / g dry weight . More important was the very significant reduction in the secondary bile acids, deoxycholic and lithocholic acid that suggests a change in the intestinal flora . The reduction in secondary bile acids should be important as they are thought to be the most carcinogenic bile acids (62). Intervention trials with bran, soluble fiber or vegetables have not reduced recurrence rates of adenomatous colorectal polyps (6365). Going back to burkitt's original work, not only did the african natives have a very low incidence of colorectal cancer with a high fiber intake, they also consumed a very - low - fat diet and were very physically active . Thus, it may be that total lifestyle modification is needed to reduce cancer risk, not simply modification of one aspect . In his presidential address to the american cancer society in 1966, joseph burchenal (66) suggested that the study of one form of cancer might provide guide - posts to the understanding of another related form of cancer . This suggestion seems to relate to hyperinsulinemia, the igf axis and their associations with several forms of cancer . However, the true unifying factor for most cancers seems to be lifestyle: smoking, a high - fat and refined - sugar diet along with a lack of physical activity . These lifestyle factors can easily be changed and may prevent most types of cancer . By adopting a diet consisting primarily of whole grains, fruits and vegetables with limited amounts of meat, primarily chicken or cold - water fish, and doing 4560 min of daily exercise, one could achieve significant changes in hormones and growth factors known to be associated with cancer as shown in figure 3 . Primary prevention trials to test this hypothesis are difficult to conduct due to the large number of subjects, the prolonged time required and the cost . However, secondary prevention trials like the ornish study (38,39) with prostate cancer or the colorectal adenoma recurrence studies may provide important scientific evidence for the value of these lifestyle changes . A model showing steps in the development of cancer . A model explaining the role of diet and a sedentary lifestyle in the development of prostate cancer [after barnard et al . A model explaining how a change in lifestyle to include a low - fat, high - fiber diet plus regular exercise might reduce the risk for cancer.
Microbial contamination and side effects of synthetic antioxidants are two important major concerns of food and pharmaceutical industries . Increasing propensity for replacing synthetic antioxidant by natural one on one side and development of microbial resistance to existing antibiotics from the other has encouraged researchers toward appraising medicinal plants for dual antioxidant and antimicrobial properties . Though, since immemorial time, medicinal plants have been used to treat and prevent various human ailments and they are considered as reservoir of bioactive compounds.1, 2 till date, biological properties and bioactive compounds of many medicinal plants are not studied . Extensive investigation of medicinal plants for biological activities and bioactive compounds is the crucial and the foremost step in development of effective alternative medications . In view of this, in the present study, bergenia ciliata sternb . (family saxifragaceae), a high value plant of the sikkim himalaya, has been investigated for antioxidant, antimicrobial activity and bioactive compounds . It is an important perennial medicinal herb that grows widely in the temperate himalaya between 1500 and 3000 m asl . The plant has been in use as folklore medicine since ancient times for dissolution of kidney and gall bladder stones.4, 5 in sikkim himalaya, bergenia rhizome is used to treat fractured bones, fresh cuts, wounds, diarrhea, pulmonary infections, vomiting, fever, cough and boils by locales.6, 7, 8 bergenia is also used for the treatment of heart disease, haemorrhoids, stomach disorders and ophthalmia . In addition, it is accredited with analgesic, antiviral, anti - inflammatory and antimalarial properties.9, 10 the plant's virtues, to a large extent, are attributed to its secondary metabolites such as bergenin, gallic acid and catechin which are therapeutic and account for its use in traditional medicine.11, 12 despite the widespread use of this plant in traditional medicine, the scientific literature with respect to its biological properties is scanty . To the best of our knowledge, so far, antioxidant, antimicrobial properties and the bioactive compounds associated with the leaf of bergenia plant, growing in the sikkim himalaya, have not been investigated . Therefore, the present study was conducted with the objectives to (1) obtain the most effective solvent for extracting the potent bioactive compounds, especially phenolics and flavonoids, (2) investigate different extracts for antioxidant and antimicrobial activities and, (3) quantify the amount of bergenin, catechin and gallic acid present in the leaf extracts of b. ciliata . Leaves of b. ciliata were collected during flowering season (march 2014) from the plants growing in the arboretum of g.b . Pant institute of himalayan environment and development (gbpihed), sikkim unit, gangtok, india (latitude 27 21 35.7n; longitude 88 37 24.4e), situated at the elevation of 2047 m asl . The leaves were washed thoroughly under running tap water and finally with distilled water and dried on blotting paper at room temperature to get consistent weight . 2 g powder was soaked separately in 10 ml of different solvents (ethyl acetate, methanol and hexane) for 24 h. the supernatant was transferred into a new tube and the residue was re - extracted twice with 10 ml solvent . Each extract was resuspended in the methanol to yield a 50 mg / ml stock solution . The yield of the extraction was calculated from {(w1/w2) 100}, where w1 is the weight of extract after evaporation of solvent and w2 is the dry weight of the plant sample . Solutions of each extract (100 l; 1 mg / ml) were taken individually in test tubes . To this solution, 2.5 ml of 10-fold diluted folin after 3 min, 2.0 ml of 7.5% na2co3 solution was added and the mixtures were incubated for 30 min . The absorbance of the reaction mixtures was measured at 760 nm by using a spectrophotometer (uv-1800, shimadzu, kyoto, japan). Gallic acid was used as a standard and tpc of bergenia extracts was expressed in milligram gallic acid equivalents (mg gae / g extract). Total flavonoid content was determined by the aluminum chloride calorimetric method, with some modifications . Then, the sample solution (2 ml) was mixed with 2 ml of 2% alcl3 . After 10 min of incubation at ambient temperature, the absorbance of the solution was measured at 435 nm by using a spectrophotometer (uv-1800, shimadzu, kyoto, japan). The flavonoid content was expressed as milligram quercetin equivalent (mg qe / g extract). The effect of extracts on 2,2-diphenyl-1-picrylhydrazyl (dpph) radical was determined using the method of liyana - pathiranan and shahidi . A solution of 0.135 mm dpph in methanol was prepared and 1.5 ml of this solution was mixed with 1.5 ml of extract in methanol . The reaction mixture was mixed thoroughly and left in the dark at room temperature for 30 min . The ability of sample to scavenge dpph radical was calculated by the following equation: dpph radical scavenging activity (%) = [(abs control abs sample)]/(abs control) 100where abs control is the absorbance of dpph radical + methanol; abs sample is the absorbance of dpph radical + extract / standard . Ic50 value is the concentration of extracts at which dpph radicals are scavenged by 50% . The lower ic50 value indicates higher radical scavenging capacity and vice versa . The 2,2-azino - bis(3-ethylbenzothiazoline-6-sulfonic acid) the abts radical cation solution was prepared by mixing equal quantities of abts (7 mm) and ammonium persulfate (2.45 mm) and allowing them to react for 1620 h at room temperature, in dark . The working solution was then prepared by diluting the previous solution with methanol until the absorbance at 734 nm was 0.706 0.02 . Plant extracts (1.5 ml) were allowed to react with equal volume of the abts working solution and the absorbance was taken at 734 nm after 7 min using the spectrophotometer . The abts scavenging capacity of the extracts was compared with that of bht and the percentage inhibition was calculated as: abts radical scavenging activity (%) = [(abs control abs sample)]/(abs control) 100where abs control is the absorbance of abts radical + methanol; abs sample is the absorbance of abts radical + extract / standard . Abts radical scavenging activity of extracts was determined by ic50 value as mentioned above in dpph assay . Total eight strains (4 bacteria, 2 actinomycetes and 2 fungi) were taken from microbial culture collection established in the microbiology laboratory of gbpihed institute, almora, india . Initially test organisms were grown on the respective media i.e. Bacteria and actinomycetes in tryptone yeast extract (ty) and fungus on potato dextrose broth (pd) in conical flask, at 25c for 24 h. antimicrobial activity was performed on ty and pd agar plates following disc diffusion assay at 25c, 2 days for bacteria and 6 days for actinomycetes and fungus . Minimum inhibitory concentration (mic) of extracts was determined following the protocol of clinical and laboratory standards institute (clsi).17, 18 all of the experiments were performed in triplicate, and results were expressed as diameters (mm) of inhibition . Detection and quantification of catechin, gallic acid and bergenin were carried out using shimadzu 20 ad, hplc system (shimadzu, japan) consisted of uv detector, a binary pump, a 10 l injection loop, and reverse phase c-18 column of dimensions 4.6 250 mm . The mobile phase used for catechin was 75% a (water + 0.1% trifluoroacetic acid) and 25% b (methanol) with a flow rate of 0.8 ml / min . The eluted samples were detected by the uv detector at 280 nm . For gallic acid, mobile phase used was 90% a (water + 1% acetic acid) and 10% b (acetonitrile) with a flow rate of 1.0 ml / min . For analysis of bergenin, elution was carried out with solvent 75% a (water + 0.1% trifluoroacetic acid) and 25% b (acetonitrile) as mobile phase having flow rate of 1.0 ml / min . Calibration curve was constructed by plotting the peak area (y) against concentration in g / ml of standard solutions (x). The standard equation obtained from the curve all the experiments were performed in triplicate and the experimental data were expressed as mean standard deviation (sd). One - way analysis of variance (anova) and duncan's multiple range tests were carried out to determine significant differences (p <0.05) between the means by spss (version 16.0). Results showed that leaf extraction yield of b. ciliata varied considerably as a function of solvent nature and ranged from 8.3 to 38.2% with a descending order of methanol> ethyl acetate> hexane (table 1). Extraction with methanol resulted in the highest amount of total extractable compounds whereas the extraction yield with hexane was small in comparison to other solvents . Higher extraction yield in methanol might be due to the fact that it easily penetrates the cellular membrane and extracts the intracellular ingredients from the plant material . Moreover, results indicated that the plant contains more of polar substances than the others . Earlier reports have also suggested that methanol give higher extraction yield than the other solvents such as acetone, diethyl ether, ethyl acetate and water.19, 20 the antioxidant activity of medicinal plants, fruits and vegetables has been reported to be positively correlated to their total phenolic contents due to their ability to scavenge free radicals.21, 22 it becomes, therefore, mandatory to estimate total phenolic compounds present in the bergenia extracts . In the present study, the content of extractable phenolic compounds was determined through a linear gallic acid standard curve (y = 2.271x + 0.18; r = 0.995). The highest content of total phenolic compounds was detected in the bergenia methanolic extract (473.4 mg gae / g extract) followed by ethyl acetate extract (249.7 mg gae / g extract) (p <0.05). In the hexane these results demonstrate clearly that the content of phenolic compounds is dependent on the polarity of the solvent used; higher the polarity of the solvent, higher the content of phenolic compounds . These results are in agreement with the previous study of singh et al . That reported methanol as an effective solvent for antioxidant extraction, phenolic compounds in particular . Flavonoids are common secondary metabolites present in plants which are responsible for many plant biological activities . The solvent efficiency on tfc, in ascending order was: hexane <methanol <ethyl acetate . The highest flavonoid content of 208.4 mg qe / g extract was observed in the extract of ethyl acetate followed by methanol extract (89.9 qe / g extract). These results are in agreement with the report of hajji et al . Which reported that flavonoid content in extract depend on solvent polarity . The dpph free radical scavenging activity in leaf extracts of b. ciliata is presented in fig . 1 . The methanolic leaf extract showed excellent free radical scavenging activity (ic50 = 53.5 g / ml). In other two leaf solvent extracts, the free radical scavenging activity in ethyl acetate extract (ic50 = 2593.3 g / ml) was superior to that of the hexane extract (ic50 = 3026.7 g / ml). Nevertheless, when compared to standard, the bht, all the tested bergenia leaf extracts showed significantly (p <0.05) lower dpph radical scavenging activity . These results are indicative of the influence of the solvent on the antioxidant activity of the biological extracts . It has been reported that in plant compounds with different polarity and structure are present that dissolve in specific solvents having similar polarity.24, 25 the difference in the dpph radical scavenging activity in different solvent extracts implies towards the preference of the solvents for extraction of different types and concentrations of bioactive compounds.24, 25 strong dpph radical scavenging activity of b. ciliata methanolic extract can be due to higher content of total phenolic compounds . The free radical scavenging activity of bergenia extracts was also determined using abts radical . Significant difference (p <0.05) was revealed between abts scavenging capacities of extracts measured as ic50 value (fig . 2). The highest abts radical scavenging activity was found in methanol extract with an ic50 value of 5.4 g / ml, whereas hexane extract exerted the lowest ability to scavenge abts radical with an ic50 value of 51 g / ml . Strong abts radical scavenging ability of b. ciliata methanol and ethyl acetate extracts can be attributed to the presence of flavonoids . Earlier, it has been reported that abts radical scavenging ability of bioactive compounds depends on its molecular weight, structure and presence of number of aromatic rings . The correlation coefficients determined between the antioxidant activities and the total phenolic and flavonoid contents in all the b. ciliata leaf extracts revealed the presence of significant high correlation (p <0.1). The correlation for dpph ic50 value vs tpc and dpph ic50 value vs tfc were 0.913 and 0.056, respectively . Results indicate clearly that the dpph activities of extracts are mainly due to the total phenolic compounds . Negative correlation indicated that with the increase in phenolic and flavonoid content, concentration of extract required to inhibit free radical decreases . The correlation for abts ic50 value vs tpc and abts ic50 value vs tfc were 0.911 and 0.782, respectively, imply that both the total phenol and flavonoids of b. ciliata are responsible for abts activities . These differences in correlation between phytochemicals and antioxidant assays could be attributed to the different mechanism of the radical antioxidant reaction . In accordance with this study, li et al . Have also reported high negative linear correlation between the phenolic contents and ic50 values in angelicae sinensis confirming that phenolics are likely to contribute to the antioxidant activity of the extracts . However, presence of other metabolites which have significantly contributed in antioxidant activity of b. ciliata extracts cannot be ruled out . The results of the disc diffusion assay (table 2) revealed that all the extracts tested were effective against the bacteria and actinomycetes studied, but showed no activity against fungi . Among the tested extracts, methanol extract was found to be the most effective which showed the highest effects against bacillus megaterium strain with inhibition zone of 9.8 mm, followed by nocardia tenerifensis and bacillus subtilis with inhibition zones of 9.7 and 8.8 mm, respectively . The ethyl acetate extract showed the similar pattern of microbial inhibition with highest inhibition zone of 7.5 mm for b. megaterium, followed by 6.2 and 5.5 mm inhibition zone for n. tenerifensis and b. subtilis, respectively . In contrary to methanol and ethyl acetate extract, the hexane extract had the greatest inhibitory effects against serratia marcescense (5.0 mm) and b. subtilis (4.7 mm). The minimum inhibitory concentration (mic) was determined in the methanolic extracts of b. ciliata due to its higher antimicrobial activity in disc diffusion assay against the tested organisms . The methanol extract gave the lowest minimal inhibitory concentration against b. subtilis with mic of 1250 g / ml (table 3). The highest antimicrobial activity of methanolic extract could be due to the high contents of phenolic compounds and flavonoids present in the extract . These results are in accordance with a previous study where similar observations were recorded in case of bergenia ligulata leaf extracts . B. ciliata is reported to contain a wide array of pharmaceutically important bioactive compounds.9, 29, 30 among all, three main compounds viz ., bergenin catechin and gallic acid are the most sought after compounds of the plant species . During the past years, efforts have been made for identification and quantification of the major bioactive compounds from bergenia spp.5, 28, 30, 31, 32 however, till date, b. ciliata plant of the sikkim himalaya has never been analyzed for secondary metabolites . To the best of our knowledge, this is the first report on this aspect . In the present study, different extracts of b. ciliata were analyzed by hplc for the identification and quantification of bergenin, catechin and gallic acid . Standards showed high linearity at tested concentrations with correlation coefficients (r) of 0.99, 0.96 and 0.99 for catechin, gallic acid and bergenin, respectively . The chromatographic peaks of the analytes were confirmed by comparing their retention time with those of the standards . From the standard equation obtained (table 4), the amount of bergenin, catechin and gallic acid calculated in different extracts is presented in table 5 . Bergenin was detected in all the extracts and amount of bergenin was found in the order: methanol> ethyl acetate> hexane . The methanol extract contained bergenin (2.43 0.08%) in amounts that were orders of magnitude higher than those in the ethyl acetate (1.63 0.17%) and hexane (0.0024 0.00%) extracts . Hplc analysis of catechin and gallic acid revealed that these compounds were present in methanol extracts only (table 5). In this study, the antioxidant and antimicrobial activities of b. ciliata plant growing in the sikkim himalaya are reported for the first time . The methanolic extracts of b. ciliata leaves, that were shown to contain the highest total phenolic and flavonoid compounds, exhibited high free radical scavenging activity against dpph and abts radicals . Further, hplc analysis of different extracts for catechin, gallic acid, and bergenin showed that methanolic extract contained significantly higher amount of these bioactive compounds.
Dyskeratosis congenita (dc) is a rare multisystem disorder first described in the early 20 century (1906); it is also known as zinsser - engman - cole syndrome.1 it is characterised by the classic triad of mococutaneous pigmentary disorder, nail dystrophy and oral leukoplakia seen in 89%, 80% and 45% of cases respectively.2 however abnormalities involving the digestive system (oesophageal stricture, liver cirrhosis), ocular (corneal ulcers, conjunctivitis, blepharitis, lacrimal duct stenosis with epiphoria), neurologic (mental retardation, delayed developmental milestones) have also been reported,3 and bone marrow failure occurs in about 75% of cases with x - linked form.4 bone marrow failure is the major cause of death, while other causes of mortality are pulmonary fibrosis and malignancy . There is scarcity of information on this disorder among nigerians to the best of our knowledge . Therefore, the case of a 9-year - old boy who presented with recurrent febrile illness, repeated blood transfusion with dystrophic nails and hyperpigmentation of the skin in whom a diagnosis of dc was made is reported . A 9-year - old boy presented with complaints of easy fatigability, recurrent nose and gum bleeding with recurrent blood transfusion of 2 months duration; he had four blood transfusions in the past 2 months . There was history of easy bruisability, but no history of prolonged bleeding following circumcision which he had at the age of 7 years . There was history of generalised darkening of the body, palms and soles with some whitish and dark patches on the tongue noticed around the age of 5 years . He was the 2 child in a monogamous non - consanguineous marriage setting of six children . The mother was a 30-year - old stay - at - home mother while the father was a 35-year - old self - employed trader . The patient had epistaxis from both nostrils; he was pale with hyperpigmentation of the skin of the neck, chest [figure 1], arms and palms of the hands [figure 2]; and soles of the feet with scattered areas of hypopigmentation . He had some white and black plaques [figure 3] on the tongue which were non - scrapable . There were dental caries, gum bleeds [figure 4], while the other mucosal surfaces of the oral cavity were normal . Both eyes had epiphoria [figure 5], but more on the right eye . The nails of both fingers and toes were cracked, ridged and atrophic [figures 6 and 7]. Hyperpigmentation of the skin of the neck, chest with areas of hypopigmentation hyperpigmentation of the palms white and black plaques on the tongue dental caries with gum bleeds epiphoria from both eyes dystrophic finger nails the full blood count showed haemoglobin of 56 g / l white blood cells count of 2.0 10/l (normal = 4 11 10/l), with an absolute neutrophil count of 1.0 10/l, platelet count of 23 10/l (normal = 150 - 450 10/l), clotting profile was normal and bone marrow biopsy revealed a hypocellular marrow [figure 8]; therefore, he had a pancytopenia . Based on the clinical and laboratory findings the diagnosis was dc . He was also referred for ophthalmologic evaluation which revealed lacrimal duct stenosis . Despite being on anabolic steroid for 12 weeks the bone marrow failure persisted and due to financial constraints he could not afford haematopoietic cell transplantation; he is currently on supportive care and is being followed up in the paediatric clinic . Dc occurs worldwide with very few cases reported so far.56 although x - linked, autosomal recessive and dominant modes of inheritance have been reported, our case had no family history of same disease and was a product of a non - consanguineous marriage, which makes a sporadic relationship most likely . Those with mild manifestations may have minimal physical features with normal bone marrow function, while those with severe manifestation may present with serve multisystemic disorders like revesz syndrome and hoyeraal hreidarsson syndrome7 presenting early in childhood . In classical dc, most of the somatic abnormalities are often absent at birth, but develops with increasing age; therefore, the diagnosis of dc is often delayed until adulthood, but our patient had all the major clinical features which made the diagnosis easy even in the absence of genetic analysis . Our patient had the classic triad of the disease, namely skin hyperpigmentation, nail dystrophy and oral leukoplakia which fulfilled the clinical diagnostic criteria . He also had a severe manifestation of the disease, with early onset of bone marrow failure . Bone marrow failure is often delayed until the second decade of life, but our case presented early - at the age of 9-years - involving all the three major blood cell lines (pancytopenia).5 dermatologic manifestation of dc can be reticular hyperpigmentation or hypopigmentation of the skin, especially affecting the neck and chest . The patient had hyperpigmentation of the skin of the neck, chest, arms and the palms and soles of the feet . White patches may affect the tongue, buccal mucosa or the palate, but the tongue is mostly affected . In our patient, the epiphoria was attributed to lacrimal stenosis, while the dental caries and repeated gum bleeds are due to deficiencies in the neutrophils and platelets respectively . The cause of dc has been linked to mutations in genes related to telomere maintenance (telomerase) and to date, eight genes have been implicated.89 mutations in a component of the shelterin complex are involved in autosomal dominant dc . The shelterin complex determines the structure of the telomeric terminus; it also controls the synthesis of telomeric dna . Therefore, without the shelterin complex telomeres are exposed to the dna damage - repair mechanisms, which may result in the ends of chromosome being incorrectly processed by the dna - repair pathways predisposing to fusion of chromosomal ends during replication.7 the fact that pancytopenia did not respond to oral androgen and the non - availability of facilities for haematopoietic stem cell transplant, which is the main stay of treatment,10 represent a big challenge in the management of this patient . Moreover, even if the patient had responded to steroids, a long - term anabolic steroid regimen may be complicated by side effects considering the age of this patient . The case of a 9-year - old boy, who had all the major clinical features - hypo-/hyperpigmentation of the skin, nail dystrophy, leukoplakia and early bone marrow failure- which before now had not been documented in nigeria, is reported.
Abo incompatible blood transfusions are rare, result in haemolytic transfusion reactions (htr), and they can be fatal in 2 to 6% of cases . It is enough to transfuse only 10 - 15 ml of abo incompatible blood to cause following symptoms of acute htr: fever, hypotension, disseminated intravascular coagulation (dic), complement - induced acute intravascular hemolysis and acute renal failure . In adults, thrombotic thrombocytopenic purpura (ttp) is idiopathic in approximately one - third of cases and in the remainder it is encountered in a variety of potentially triggering clinical situations (so - called secondary ttp), including pregnancy, malignancies, drugs, infection, auto - immune disorders and after haemopoietic stem cell transplantation . Secondary thrombotic ttp is a life - threatening condition and is characterized by an aggressive course, it requires prompt treatment at the outset . In case of delayed or inadequate treatment mortality rate may be close to 100% . To date, there is no report in the literature about secondary ttp caused by abo incompatible blood transfusion . We present a case of ttp developed after a htr in the setting of cardiac bypass surgery . A 64-year - old male patient developed nausea, vomiting and chills during red blood cell (rbc) suspension transfusion on the second day of coronary by - pass surgery . Caring physicians recognized an error in labeling of the rbc suspension . With a preliminary diagnosis of abo incompatible transfusion reaction, hemoglobin values dropped and lactate dehydrogenase (ldh) and serum creatinine levels started to increase proportionally . Urine color was darkened with a gradual development of oligo - anuria and acute kidney injury (aki). On the 7 day of transfusion, ttp was diagnosed according to following criteria: thrombocytopenia, microangiopathic anemia, increase in ldh level, and development of aki . After 11 and 15 sessions of hemodialysis and plasmapheresis, respectively, platelet, ldh, urea, creatinine levels and urine output improved . The patient showed a dramatic improvement, and plasmapheresis and hemodialysis were stopped subsequently . On the 41 day of admission he had bloody diarrhea and c. difficile toxin a was found to be positive in feces and vancomycin was administered for pseudo - membranous enterocolitis . On the 50 day, multiple infarctions were seen on cranial ct that was performed for the evaluation of confusion . Blood urea, creatinine and ldh levels increased, hemoglobin and platelet levels decreased again . The pertinent laboratory data and major interventions are chronologically illustrated in [table 1]. Every 1 of 14000 blood transfusions are erroneous, and among them 1 of 13000 are abo incompatible . The pathogenesis of htr is most likely related with activation of complement and hemostatic system, which leads to attachment of host antibodies to red blood cell antigens of incompatible donors blood . As a result, it can modify the intravascular hemolysis and initiate symptoms such as nausea, vomiting, chills, fever and dark appearance of urine, which were evident in our patient during and after transfusion . In addition, studies presented relation between abo groups, clearance of ultra large von willebrand factor multimers (ulvwf), deficiency of adamts13 and risk of ttp development . Adamts13 is a circulating zinc metalloprotease, responsible to cleave the ulvwf, thereby the multimers become progressively smaller due to cleavage by adamts13 . Deficiency of adamts13 leads to a shift of plasma ulvwf multimers to larger sizes, adhesion with platelets and its aggregation, leading to endothelial injury with activation of thrombosis cascade . Data from terrell et al . Demonstrated that blood group o is an independent risk factor for ttp associated with severe adamts13 deficiency . O abo blood group, but we could not documented the adamst13 deficiency due to unavailability of the lab technique at our institution at that time . The initiating mechanism of secondary ttp include drug toxicity, radiation and high - dose chemotherapy, angioinvasive fungal or viral infections, surgery and acute graft versus host disease . Ttp also appear to differ in their response to therapy . Primary or idiopathic form responds to plasmapheresis 80% of the cases whereas this rate is very low in secondary forms . The clinical course of our patient showed a severe relapse despite an initial complete biochemical and clinical response . Recently canadian apheresis group conducted a phase ii trial to test the beneficial effects of rituximab in idiopathic ttp . In this form of the disease, actually this study was planned after reporting of many cases in which rituximab was shown to be beneficial . And the authors concluded that autoimmune deficiency of adamts 13 may be due to yet unidentified autoantibodies and while plasmapheresis clears circulating autoantibodies, treatment modalities such as rituximab precludes further antibody production via blockage of b cells . Postoperative ttp have been described after a number of surgical operations, most common of which is open heart surgery . Ttp developed after five to nine days after surgical operation in most of the case reports . However, it has been proposed that extensive endothelial damage during surgery may lead to release of high - molecular weight von willebrand factor (hmw - vwf) multimers in substantial amounts sufficient to overwhelm the capacity of the vwf - cleaving enzyme . However, despite thousands of cabg operations worldwide at each year, only a handful of cases have been reported . This may in part be related to presence of confounding factors such as blood loss, hemodilution, severe infection, dic, and heparin induced thrombocytopenia commonly in the early course of cabg . Another explanation may be the rare patients who have genetically low levels of cleavage enzyme . Ttp secondary to abo incompatible blood transfusion has never been reported in the literature to date . We hypothesize a potential mechanism which may account for the development of ttp in this setting . However, ttp developed in our patient in the setting of open heart surgery . After the abo incompatible blood transfusion, dic usually develops if sufficient amount of incompatible blood is transfused . We know that dic is self - limited provided that underlying inciting disease is well controlled . However, thrombocytopenia in our patient developed 1 week after the offending event and lasted for weeks despite normal coagulation tests (inr and aptt), which is unusual in the setting of dic . Another supporting finding in favor of ttp was a favorable response to plasmapheresis performed with fresh frozen plasma . It is very difficult to discriminate whether transfusion of abo incompatible blood or open cardiac surgery or the combination of the two actually initiated the process . Perhaps, both factors worked hand - in - hand . However, one clinical observation supports our hypothesis of blood transfusion as the initiating factor . None of the ttp cases secondary to surgery had relapsed unless the patient underwent a second operation . However, this was not the case in our patient who developed a relapse after near complete improvement of laboratory and clinical parameters . Despite initial recovery, he developed a second bout which was complicated by multiple cerebral thrombi and subsequently died . Given the complex picture of early postoperative phase of cabg and the presence of potential confounders, many cases of postoperative ttp may have been missed . Prompt recognition is of utmost importance because of not only high mortality rate when left untreated but also to institute specific therapies availability of efficient therapeutic maneuvers to our knowledge, this is the first report of ttp secondary to abo incompatible blood transfusion.
Transurethral resection of bladder tumor (turbt) is well - established modality for treating non - muscle - invasive bladder cancer . Furthermore, it is essential for evaluating the pathological depth of bladder tumors in resected specimens . Despite improvements in endoscopic instruments and imaging systems, there are well - recognized complications associated with turbt, such as bladder perforation, irrigation fluid absorption, bleeding, infection, damage to the ureteric orifices, obturator nerve stimulation, and tumor cell implantation . Among these, bladder perforation is one of the most frequent complications of turbt and is sometimes associated with obturator reflex . Several researchers have reported that the risk of obturator nerve stimulation is from 10.6% to 11% . Several advances in instruments and techniques, including modifications of resectoscope design and snare resection, have been proposed to remove pedunculated bladder tumors . However, difficulties remain in safely collecting adequate specimens for flat tumors . We introduce an easy technique for superficial bladder cancer that uses a standard monopolar resectoscope loop . In this study, we demonstrate the easy resection technique for superficial bladder tumors that we have termed the " grasp and bite " turbt technique and compare it with conventional turbt . This was a retrospective study in the department of urology of chonnam national university hospital (gwangju, korea) between january 2012 and april 2013 . This study included a total of 29 men and 6 women who had bladder tumors with superficial lesions and who underwent turbt . Patients with a huge unresectable mass, an invasive tumor shown during preoperative imaging, or an irradiated bladder were excluded . Group 1 patients were treated by use of the conventional turbt technique, and group 2 patients underwent the grasp and bite technique . Conventional turbt was performed in 2012 and grasp and bite turbt was performed in 2013 . This study was approved by the institutional review board of chonnam national university hospital (irb no . Transurethral resection was performed with a monopolar resectoscope system (24-fr karl storz, tuttlingen, germany). For the grasp and bite technique, the operator initially defined the tumor morphology and safe margin . In small and flat lesions, the tumor and surrounding mucosal lesions were positioned between the resection loop and the end portion of the resectoscope sheath . We call this the " grasp " step (fig . 1; video clip, supplementary material). Using the grasping technique, the tumor lesions were lifted up . In the " bite " step, the surgeon maintained a tight hold and resected the tumor while grasping . A cystoscopic view and schematic illustrations 1 . For resection during the bite step, the movement of the electrode loop was straight backward in a linear direction . Resection was performed carefully so as not to cause bladder perforation without excessive bladder wall distention . The resection site was carefully inspected for bleeding and coagulation . In bulky or pedunculated tumors, after removal of the bulging mass, the base lesion and surrounding mucosa were resected carefully by using grasp and bite turbt as mentioned earlier . Specimens were measured by hematoxylin and eosin (h&e) staining and immunohistochemical smoothelin stain to determine the muscularis mucosae . Independent t - tests were used to compare the mean values of two independent parametric continuous variables . The mann - whitney test was used to compare the median values of two nonparametric continuous variables . This was a retrospective study in the department of urology of chonnam national university hospital (gwangju, korea) between january 2012 and april 2013 . This study included a total of 29 men and 6 women who had bladder tumors with superficial lesions and who underwent turbt . Patients with a huge unresectable mass, an invasive tumor shown during preoperative imaging, or an irradiated bladder were excluded . Group 1 patients were treated by use of the conventional turbt technique, and group 2 patients underwent the grasp and bite technique . Conventional turbt was performed in 2012 and grasp and bite turbt was performed in 2013 . This study was approved by the institutional review board of chonnam national university hospital (irb no . Transurethral resection was performed with a monopolar resectoscope system (24-fr karl storz, tuttlingen, germany). For the grasp and bite technique, the operator initially defined the tumor morphology and safe margin . In small and flat lesions, the tumor and surrounding mucosal lesions were positioned between the resection loop and the end portion of the resectoscope sheath . We call this the " grasp " step (fig . 1; video clip, supplementary material). Using the grasping technique, the surgeon maintained a tight hold and resected the tumor while grasping . A cystoscopic view and schematic illustrations are shown in fig . 1 . For resection during the bite step, the movement of the electrode loop was straight backward in a linear direction . Resection was performed carefully so as not to cause bladder perforation without excessive bladder wall distention . The resection site was carefully inspected for bleeding and coagulation . In bulky or pedunculated tumors, after removal of the bulging mass, the base lesion and surrounding mucosa were resected carefully by using grasp and bite turbt as mentioned earlier . Specimens were measured by hematoxylin and eosin (h&e) staining and immunohistochemical smoothelin stain to determine the muscularis mucosae . Independent t - tests were used to compare the mean values of two independent parametric continuous variables . The mann - whitney test was used to compare the median values of two nonparametric continuous variables . Statistical analyses were performed with ibm spss statistics ver . 20.0 (ibm co., armonk, ny, usa). From january to april 2012, 16 patients were enrolled who had undergone conventional turbt . From january to april 2013, the grasp and bite technique was applied to 19 patients with superficial bladder tumors . The two groups had comparable demographic and intraoperative data, including patient age, tumor size, patient gender, multiplicity, tumor morphology, and tumor location (table 1). The mean operative time was similar in the conventional and grasp and bite groups (35 minutes vs. 35 minutes, respectively). The mean duration of irrigation, duration of catheterization, and hospital stay were not significantly different between the groups . There were no cases of bladder perforation, excessive obturator reflex, or persistent hematuria in either group . 2). There was no significant difference in recurrence rates between the conventional and grasp and bite turbt groups during the 14-month follow - up (43.8% vs. 31.6%, p=0.458). The purpose of turbt is complete removal of all visible tumors for curative intent and acquisition of adequate tissue for pathologic evaluation . Also, tumor recurrence after turbt is relatively high . For non - muscle - invasive bladder cancer, the probability of recurrence after 1 year ranges from 15% to 70% . Many obstacles can arise during the procedure, such as bladder perforation, bleeding, obturator reflex, and damage to the ureteral orifices . To overcome these difficulties, many surgical techniques and innovative devices for example, endoscopic snare resection of a bladder tumor can be used to remove pedunculated tumors . Although the snare method offers the advantage of en block resection, smaller and sessile tumors are not suitable for this technique . New devices such as water jet hydrodissection and the thulium: yttrium aluminium garnet laser may be applicable for en block resection of superficial tumors . However, further studies and development of these techniques are necessary to determine efficacy and oncologic outcomes . Variable methods of tumor resection have been developed and used in association with tumor size and location . The side - to - side bidirectional lateral rotating handgrip and loop allows excellent precision and control over the loop under direct vision . Loop modification, the so - called " runner, " can push the bladder wall away during resection, thereby widening the view and decreasing the risk of perforation . We wanted a technique in which we could use a conventional resectoscope and loop without accessory equipment . When using a wire - loop resectoscope, the tumor must be resected thoroughly to obtain adequate muscle tissue . The movement of the loop is somewhat fast and deep to avoid charring of the surrounding mucosa and bladder perforation . Direct pressure to the bladder wall may allow deeper tissue to protrude into the bladder wall and then be easily removed by use of the resectoscope . Instead of the curvilinear movement of the resectoscope end according to the curved bladder surface, the easy linear motion can make a curved resection with grasping . Using our " grasp and bite " technique, beginners can easily perform turbt safely . Often, small and sessile tumors are missed and severely damaged during resection and cauterization . The grasp and bite technique, the protruding tumor can be removed f irst by the conventional method, and then the stalk and base lesion can be removed by the grasp and bite technique ., the grasp and bite technique could have difficulties in a posterior or posterolateral located tumor because of its moving mechanism . In our series, two posterior and four posterolateral tumors were successfully accessed by use of suprapubic pressure and nearly emptying the bladder . Similar to conventional turbt, it was also difficult to access posterior tumors with the grasp and bite method . In our consecutive cases, general anesthesia was preferred in the conventional and grasp and bite groups (81.3% vs 57.3%, p=0.138). Also, tumor locations did not differ significantly between the two groups (p=0.253). We postulated that the grasping method, which lifts the tissue away from the obturator nerve and tightly holds the grasped tissue, may eventually weaken the reflex . Considering this small population, however, thus, there could have been a selection bias . Also, the study population was small, and thus we were unable to perform statistical analysis of the oncologic outcome as recurrence - free survival . This was a preliminary study to develop a surgical technique performed by an experienced surgeon . Larger prospective studies with a long follow - up period will be necessary to show the oncologic advantage of this technique . To determine the feasibility of the technique for inexperienced surgeons, future study will address a teaching protocol comparing the conventional and grasp and bite methods . Investigation of tissue adequacy of proper muscle and effect of coagulation will be our next subject of study . Our study demonstrated that grasp and bite turbt is a safe and effective method for removing superficial bladder tumors . This technique has merit for flat tumors and can also be used for pedunculated and poorly accessible tumors . The grasp and bite method may be a shortcut for overcoming the difficulties of turbt . An accompanying video can be found in the' urology in motion' section of the journal homepage (www.kjurology.org). The supplementary data can also be accessed by scanning a qr code located on the fig . 1 of this article, or be available on youtube (http://youtu.be/2xjdgspzvy4).
The occasional use of pesticides in large amounts may cause environmental pollution and therefore be a cause of concern . Residual amounts of organochlorine and organophosphate pesticides have been detected in soil, water reservoirs, vegetables, grains and other food products . Organophosphates induce toxicity in mammals through inhibition of acetylcholinesterase (ache) and subsequent activation of cholinergic receptors . Oxidative stress as a result of organophosphate exposure has been reported, and lipid peroxidation has been revealed to be one of the molecular mechanisms in organophosphate - induced toxicity [4, 5]. The use of natural remedies has been recently observed to be a good means of curbing oxidative stress by boosting the natural antioxidant defence system . In africa, medicinal plants are used traditionally to treat various ailments . Among the promising medicinal plants, ziziphus mucronata(zm) in the family ramnaceae various parts of the tree are already in use by common people as medicine and as food . The roots, bark and leaves are used in the treatment of arthritis, chest pains and boils . The sweet fruits of the plant are widely eaten in botswana, and they are dried, ground to powder and cooked as porridge . In botswana, a proposal has been made that the fruit might be useful in protecting the population from being affected by dimethoate toxicity . Hence, the present study has been aimed at establishing the antioxidant and hepatoprotective properties of zm fruit extracts . Other chemicals were of analytical grade and were bought from sigma - aldrich (st . The dried fruits were ground with a laboratory grinder to obtain 500 g of powder . The powder was soaked and extracted in hexane, 50% hexane / chloroform, chloroform, 50% chloroform / methanol, methanol and 70% methanol / water for 24 hr . Thirty male sprague dawley (sd) albino rats aged 20 - 21 weeks were kept in galvanized cages under a controlled temperature of 25 and a normal photoperiod (12-h dark, 12-h light). The animals were fed commercial food pellets (protein: 18%, fat: 6%, fiber: 6%, carbohydrates: 56%, calcium: 0.6% moisture: 10%) and water ad libitum . All procedures with the animals were performed in accordance with the protocols established by the university of botswana animal care committee (approval no: ub / irb/1084). The thirty adult male rats were divided into six groups of 5 rats each and treated as follows: group 1 - normal control (nc) received distilled water, group 2 - dimethoate control (dc) received 6 mg / kg.bw.day dimethoate dissolved in distilled water, group 3 - experimental group (e1) received dimethoate(6 mg / kg.bw) + zmfm (100 mg / kg.bw), group 4 - experimental group (e2) received dimethoate (6 mg / kg.bw) + zmfm (200 mg / kg.bw), group 5 - experimental group (e3) received dimethoate (6 mg / kg.bw) + zmfm (300 mg / kg.bw), group 6 - experimental control (eo) received zmfm (300 mg / kg.bw) only . The experiment was run for 90 days, and all extracts were administered orally with the help of a rubber tube and syringe . At the end of the experiment, rats were sacrificed after having been treated with mild ether anaesthesia . Blood was collected in heparinized tubes and centrifuged at a rate of 6000 rotations per minute, after which plasma samples were drawn and stored at -70 for further analysis . The rat liver tissue was removed and immediately fixed in 10% phosphate - buffered formalin for 24 hr . The tissue was dehydrated in an ethanol series (70%, 80%, 100%, and 100%), cleared in xylene and embedded in paraffin . Five--thick sections were taken by using a rotary microtone, and sections were affixed on clean glass slides by gentle heating . The sections were deparaffinised, stained with haematoxylin and eosin, and mounted with a cover slip by using neutral mounting media (dpx). The 2, 2-diphenyl-1-picryl hydrazyl radical (dpph) semiquantitative assay was performed on thin layer chromatography according to the method described by yeboah and majinda, and the spectrophotometric measurement of free - radical scavenging activities of the extracts by dpph was done using the method described by stoilova et al . . (abts) was determined as described by pellegrini et al ., and the total phenol content (tpc) was determined by using the method described by stoilova et al . . Activities of serum glutamate oxaloacetate transaminase (sgot), serum glutamate pyruvate transaminase (sgpt) and alkaline phosphatase (alp) and measurements of the levels of plasma low - density lipoproteins (ldl), highdensity lipoproteins (hdl), total protein (tp), triglyceride (tg) and cholinesterase activity were done using kits from aggape diagnostics, india . Thiobarbaturic acid reactive substances (tbars) were measured by using the method described by chaturvedi, and reduced glutathione (gsh) was measured by using the method described by ellman . Vitamin c was estimated according to the method described by omaye et al ., and superoxide dismutase (sod) was estimated according to flohe and otting, and the activity of catalase was assayed by using the method described by bisswanger . The total phenol content was estimated by using a linear regression on the standard curve of the standard (gallic acid). Various concentrations (0.1, 0.5,1.0, 5.0, and 10.0) of the fruit extracts from different solvents spotted on the tlc sheet showed high activity as indicated by the yellow color over the purple dpph background . Extracts from 100% methanol, 70% methanol / water, 70% methanol / chloroform,100%chloroform,50% hexane / chloroform and 100% hexane changed to yellow on the purple dpph background . The extent of yellow coloration was comparable to the gallic acid standard (fig 1). The percentage inhibition increased when going from low - polarity - solvent to a high - polarity - solvent extracts: namely, 100% methanol, 70% methanol/ chloroform, 100% chloroform, 50% hexane / chloroform, 100% hexane in that order . The standards, gallic acid, displayed 100% dpph inhibition (fig 2). As presented in table 1, the phenol content was found to increase from 30 mg gae / g for hexane extract to 100 mg gae / g for 50% methanol chloroform, 57 mg gae / g for chloroform, 48 mg gae / g for 50% hexane / chloroform and 250 mg gae / g for 70% methanol extract . * values are numbers or means sds . Effect of abts free radical scavenging activity of z. mucronata was assayed at various concentrations from 2.5, 5, 10, 25, 50 and 100 /. The method is based on the ability of antioxidant molecules to quench the long lived abts radical cation (abts). Significant abts scavenging activity was evident in 100% methanol extract 7.21, and a weak scavenging activity was observed in 100% hexane (table 2). Extracts were tested at 2.5, 5, 10, 25, 50 and 100 /. Reference standard (gallic acid) was tested at 1, 2, 4, 8 and 16 /. Daily administration of dimethoate resulted in elevated levels of tbars in the plasma of the dc group . A dosedependent reduction in the levels of plasma tbars was associated with the zmfm administration . Compared with the levels in nc rats, liver transaminases, serum glutamate oxaloacetate transaminase (sgot), serum glutamate pyruvate transaminase (sgpt) and alkaline phosphatase (alp) were increased significantly (p <0.001) in rats of the dc group . Significant dose - dependent declines in the levels of malonyldialdihyde and the activities of tranaminases were observed in plasma sgot (p <0.001), sgpt (p <0.001) and alp (p <administration of zmfm only resulted in no signs of toxicity as evidenced by the levels of tbars having no significant differences from those of the nc group . Sgot, sgpt and alp also had no significant differences when compared to the values for the nc group (table 3). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 ) compared with normal control (nc) and (p <0.05,p <0.001) compared with dimethoate control (dc). E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), e3 (300 mg / kg.bw + dm), and eo (300 mg / kg.bw). The results showed that the plasma levels of reduced gsh, vitamin c and e were reduced significantly (p <0.001) in dc rats compared to the levels in nc rats . When compared with nc rats, the levels were also reduced significantly (p <0.001) in experimental rats (e1-e3) in a dose - dependent manner . Although the activity of sod was low in the e3 group, it still differed significantly from levels in the dc group (p <0.05). The activities of catalase in the e3 group differed significantly from those in the dc group, but no difference was noted when comparison was made with the nc group . Administration of zmfm along with dimethoate helped the animals to ameliorate the levels of these non - enzymatic antioxidants (table 4). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 * ) compared with normal control (nc) and e1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). The results showed that the activities of sod and catalase were enhanced significantly (p <0.05) in dc rats as compared to nc rats, and significant decreases in the activities of sod and catalase were noticed in the experimental groups and were dose dependent . The highest significant reductions (p <0.05 for sod and p <0.001) for catalase were noticed in the e3 group when compared to the dc group . The sod and the catalase in the e3 group showed significant differences from the values for both the dc group and the nc group (p <0.05) (table 5). Values are expressed as means sems, n = 5 for each treatment (p <0.05,p <0.001 * ) compared with normal control (nc) and (p <0.05,p <0.001) compared with dimethoate control (dc). E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). Dimethoate administration was shown to significantly lower hdl levels (p <0.001) and total protein (p zmfm administration along with dimethoate successfully restored the hdl levels in the e2 and the e3 groups, and they differed significantly (p <0.05) from the dc group . On the other hand, dimethoate significantly increased the levels of low - density lipoprotein, cholesterol and triglyceride (p <0.001) as compared to the levels in the nc group . The ldl, cholesterol, triglyceride (p <0.001) and total protein (p <0.05) levels were found to decline significantly in the e3 group as compared to the dc group . Levels of total protein and triglyceride differed significantly from those in the nc group, as did the levels ldl and cholesterol (p <0.05). In the e2 group, a decline was noticed, and it showed a significant difference from the nc group . A similar response was noted for the e1group, but the changes were less significant than they were for the e2 group (table 6). Values are expressed as means sems, n = 5 for each treatment (p <0.05,p <0.001 * ) compared with normal control and (p <0.05,p e1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). The cholinesterase activity declined (p <0.005) in dimethoate - treated rats when compared to the normal control rats . There was a gradual and significant (p <0.005) increase in the cholinesterase activity during zmfm administration . The increment in cholinesterase activity occurred as a result of increasing zmfm dosage (table 7). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 * ) compared with normal control and (p <0.05,p <0.001) compared with dimethoate control . E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + zmfm administration revealed a preventive effect on the hepatocytes when compared to the dimethoate control (dc). The dc control group showed evident hepatic damage as shown by widening sinusoids, loss of both hepatocellular membrane and hepatocytes when compared to the normal control group . The preventive effect was shown by reduced necrosis in zmfm - fed rats when compared to dimethoate - fed rats (fig 3). (a) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver of the nc group . (b) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver of dc group . (c) light microphotograph of haematoxylin and eosin - stained and formalin - fixed livers of the e1 group . (d) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver in the e2 group . Most tissue was well preserved with no massive fatty changes, necrosis, and sinusoids not too wide, almost resembling the normal hepatocytes of the normal control . (e) light microphotograph of haematoxylin and eosin - stained and formalin - fixed livers of the e3 group . The hepatocytes are well preserved with no distinguishable damage to the cell membranes (x400). Various concentrations (0.1, 0.5,1.0, 5.0, and 10.0) of the fruit extracts from different solvents spotted on the tlc sheet showed high activity as indicated by the yellow color over the purple dpph background . Extracts from 100% methanol, 70% methanol / water, 70% methanol / chloroform,100%chloroform,50% hexane / chloroform and 100% hexane changed to yellow on the purple dpph background . The extent of yellow coloration was comparable to the gallic acid standard (fig 1). Zm fruit extracts showed high antioxidant activity . The increase in the antioxidant activity occurred in a concentration - dependent manner the percentage inhibition increased when going from low - polarity - solvent to a high - polarity - solvent extracts: namely, 100% methanol, 70% methanol/ chloroform, 100% chloroform, 50% hexane / chloroform, 100% hexane in that order . As presented in table 1, the phenol content was found to increase from 30 mg gae / g for hexane extract to 100 mg gae / g for 50% methanol chloroform, 57 mg gae / g for chloroform, 48 mg gae / g for 50% hexane / chloroform and 250 mg gae / g for 70% methanol extract . Effect of abts free radical scavenging activity of z. mucronata was assayed at various concentrations from 2.5, 5, 10, 25, 50 and 100 /. The method is based on the ability of antioxidant molecules to quench the long lived abts radical cation (abts). Significant abts scavenging activity was evident in 100% methanol extract 7.21, and a weak scavenging activity was observed in 100% hexane (table 2). Extracts were tested at 2.5, 5, 10, 25, 50 and 100 /. Reference standard (gallic acid) was tested at 1, 2, 4, 8 and 16 /. Daily administration of dimethoate resulted in elevated levels of tbars in the plasma of the dc group . A dosedependent reduction in the levels of plasma tbars was associated with the zmfm administration . Compared with the levels in nc rats, liver transaminases, serum glutamate oxaloacetate transaminase (sgot), serum glutamate pyruvate transaminase (sgpt) and alkaline phosphatase (alp) were increased significantly (p <0.001) in rats of the dc group . Significant dose - dependent declines in the levels of malonyldialdihyde and the activities of tranaminases were observed in plasma sgot (p <0.001), sgpt (p <0.001) and alp (p <administration of zmfm only resulted in no signs of toxicity as evidenced by the levels of tbars having no significant differences from those of the nc group . Sgot, sgpt and alp also had no significant differences when compared to the values for the nc group (table 3). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 * ) compared with normal control (nc) and (p <0.05,p <0.001) compared with dimethoate control (dc). E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), e3 (300 mg / kg.bw + dm), and eo (300 mg / kg.bw). The results showed that the plasma levels of reduced gsh, vitamin c and e were reduced significantly (p <0.001) in dc rats compared to the levels in nc rats . When compared with nc rats, the levels were also reduced significantly (p <0.001) in experimental rats (e1-e3) in a dose - dependent manner . Although the activity of sod was low in the e3 group, it still differed significantly from levels in the dc group (p <0.05). The activities of catalase in the e3 group differed significantly from those in the dc group, but no difference was noted when comparison was made with the nc group . Administration of zmfm along with dimethoate helped the animals to ameliorate the levels of these non - enzymatic antioxidants (table 4). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 * ) compared with normal control (nc) and (p <0.05,p <e1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). The results showed that the activities of sod and catalase were enhanced significantly (p <0.05) in dc rats as compared to nc rats, and significant decreases in the activities of sod and catalase were noticed in the experimental groups and were dose dependent . The highest significant reductions (p <0.05 for sod and p <0.001) for catalase were noticed in the e3 group when compared to the dc group . The sod and the catalase in the e3 group showed significant differences from the values for both the dc group and the nc group (p <0.05) (table 5). Values are expressed as means sems, n = 5 for each treatment (p <0.05,p <0.001 * ) compared with normal control (nc) and (p <0.05,p <0.001) compared with dimethoate control (dc). E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). Dimethoate administration was shown to significantly lower hdl levels (p <0.001) and total protein (p <0.001) in the dc group . Zmfm administration along with dimethoate successfully restored the hdl levels in the e2 and the e3 groups, and they differed significantly (p <0.05) from the dc group . On the other hand, dimethoate significantly increased the levels of low - density lipoprotein, cholesterol and triglyceride (p <0.001) as compared to the levels in the nc group . The ldl, cholesterol, triglyceride (p <0.001) and total protein (p <0.05) levels were found to decline significantly in the e3 group as compared to the dc group . Levels of total protein and triglyceride differed significantly from those in the nc group, as did the levels ldl and cholesterol (p <0.05). In the e2 group, a decline was noticed, and it showed a significant difference from the nc group . A similar response was noted for the e1group, but the changes were less significant than they were for the e2 group (table 6). Values are expressed as means sems, n = 5 for each treatment (p <0.05,p <0.001 * ) compared with normal control and (p <0.05,p <0.001) when compared with dimethoate control . E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + dm). The cholinesterase activity declined (p <0.005) in dimethoate - treated rats when compared to the normal control rats . There was a gradual and significant (p <0.005) increase in the cholinesterase activity during zmfm administration . The increment in cholinesterase activity occurred as a result of increasing zmfm dosage (table 7). Values are expressed as means sems, n = 5 for each treatment, (p <0.05,p <0.001 * *) compared with normal control and (p <0.05,p <0.001) compared with dimethoate control . E1 (100 mg / kg.bw + dm), e2 (200 mg/ kg.bw extract + dm), and e3 (300 mg / kg.bw + zmfm administration revealed a preventive effect on the hepatocytes when compared to the dimethoate control (dc). The dc control group showed evident hepatic damage as shown by widening sinusoids, loss of both hepatocellular membrane and hepatocytes when compared to the normal control group . The preventive effect was shown by reduced necrosis in zmfm - fed rats when compared to dimethoate - fed rats (fig 3). (a) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver of the nc group . (b) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver of dc group . (c) light microphotograph of haematoxylin and eosin - stained and formalin - fixed livers of the e1 group . (d) light microphotograph of haematoxylin and eosin - stained and formalin - fixed liver in the e2 group . Most tissue was well preserved with no massive fatty changes, necrosis, and sinusoids not too wide, almost resembling the normal hepatocytes of the normal control . (e) light microphotograph of haematoxylin and eosin - stained and formalin - fixed livers of the e3 group . The hepatocytes are well preserved with no distinguishable damage to the cell membranes (x400). Exposure to various organic compounds including a number of environmental pollutants and drugs can cause cellular damages through metabolic activation of those compounds by highly reactive substances such as reactive oxygen species (ros) [17, 18]. Dimethoate is one of the organophosphates frequently used in agriculture, and it has been reported to generate oxidative stress and impose toxicity . Organophosphates have been found to inhibit cholinesterase (ache) and subsequent activation of cholinergic receptors . The use of antioxidants like botanicals is an effective means to fight against the deleterious effects of oxidative stress . Botanicals are natural sources of antioxidants because of their richness in phenol, flavonoids and other anti - oxidants . In the present study, various extracts of zm were found to contain significant amounts of phenols . In the present study, the total phenol content was seen to increase when going from the less polar solvent extracts to the more polar solvent extracts .the highest concentration was present in the 70% methanol / water extract . Zm fruit extracts exhibited high antioxidant activity on the tlc / dpph antioxidant assay (fig 1). The tlc - dpph assay was confirmed by the dpph spectrophotometric assay(fig 2) and abts (table2) assays . The methanol fruit extract had the highest dpph scavenging activity (96 shown%) and exhibited 7.21/-inhibiting concentration (ic50) on abts other solvents showed lower antioxidant activities; hence, the highest antioxidant activity by the methanol extracts best represents the fruits antioxidant potential . It accumulates acetylcholine and prevents the smooth transmission of nerve functions, leading to convulsions and death . Dimethoate inhibits the activity of the enzyme cholinesterase that hydrolyses acytylcholine into acetyl and choline and, hence, prevents the return of cholinergic neurons to normal . The liver is the primary organ involved in xenobiotic metabolism and is a major target organ for chemicals and drugs . In the present study, oral administration of dimethoate to rats caused hepatic damage, as evidenced by the elevations in the hepatospecific enzyme activities, as well as by the severe alterations in different liver marker parameters . Tbars, sgot, sgpt and alp were significantly increased (p <0.05) in the dimethoate control (dc) group . Administration of zmfm (table3) significantly (p <0.005) lowered the levels of tbars, sgot, sgpt, and alp when coupled with dimethoate during its administration, thus demonstrating a dose - dependent preventive effect of the extract . The levels of gsh, vitamin c and vitamin e were observed to decline (p <0.05) in dimethoate - treated rats . Administration of the zmfm (table4) resulted in a gradual recovery of the non - enzymatic antioxidants in a dose - dependent manner . Glutathione is important in neutralizing the reactive intermediates and radical species generated during oxidative stress . The depletion of gsh may be a result of its being used up in quenching omethoate radicals . An oral feeding of the extract seems to scavenge free radicals or interfere with free radical formation in vivo . Administration of the extract is, thus, demonstrated to prevent gsh depletion, hence leading to normal gsh production . Vitamin e is thought to be an important chain - breaking antioxidant, is the major soluble antioxidant in all cellular membranes, and protects against lipid peroxidation . Zmfm may possibly play a role in scavenging the reactive oxygen species (ros). This thus allows the recovery of vitamin e which was used up in scavenging dimethoateinduced radicals . The observed significant (p <0.05) decline in the activity of sod in the experimental groups was possibly a result of the extracts that were administered to the rats . This decrease may be a possible indication that the extracts alleviate dimethoate effects on progenitor cells and possibly reduce the activation of compensatory mechanisms . This may possibly occur when the zm extract scavenges the free radicals produced as a result of dimethoate administration . The preventive effect may also occur through the blockage of the mechanisms leading to biotransformation of dimethoate to its destructive oxygen analogue (omethoate). The observed elevated catalase activity may be a result of an adaptive response to the generated free radicals . This directly indicates the failure of the total antioxidant defense system as shown by lipid peroxidation . The superoxide ion generated is, therefore, accounted for by the enhanced sod and is converted to h2 o2 or glutathione peroxidase . A decrease in the catalase activity suggests the possibility that zmfm scavenges the free radicals before they necessitate a natural adaptive response by the enzyme . The study, thus suggests the ability of zmfm to alleviate dimethoate - induced oxidative stress . Elevated low - density lipoprotein (ldl), cholesterol (cho) and triglyceride (trig) levels and decreased high - density lipoprotein (hdl) and total protein levels were observed in dimethoate - treated rats, when compared to normal control rats, after 90 days of treatment . Administration of zmfm extract resulted in significant (p <0.05) suppressions in ldl, cho and trig levels when compared to normal control rats . The extracts brought about significant (p <0.005) increases in the hdl and the total proteins levels when compared to the dimethoate control group . The increases in the total protein and hdl levels in the extract - treated rats possibly suggests the ability of the extracts to slow down dimethoate - damaging effects on the liver . Ldls are known to carry cholesterol to the peripheral tissues where it can be deposited and increase the risk of atherosclerotic attack and peripheral vascular disease . The orally - administered extract offers more benefits by increasing the level of hdls in rat plasma . Unlike the ldls, hdls hasten the removal of cholesterol from peripheral tissues to the liver for catabolism and excretion [1, 25]. The histological studies of the liver sections presented in fig 3provide supportive evidence for the protective effects of zmfm . The liver sections of normal control animals showed normal hepatic cells, each with preserved and prominent hepatocytes . The liver sections of dimethoateintoxicated rats showed loss of hepatocytes and loss of cellular boundaries and widened sinusoids; zmfm attenuated the degenerative effects of dimethoate toxicity . This was observed in the reduced intensity in liver - tissue degenerative changes . Here, less necrosis, limited loss of cell boundaries and reduced loss of intact hepatocytes were observed . In conclusion, zmfm extract could protect against oxidative stress by decreasing lipid peroxidation and enhancing the effects of enzymatic and non - enzymatic antioxidants.
Once considered diseases of affluence, the prevalence of non - communicable chronic health conditions, including overweight, has increased substantially in many low - and middle - income countries (13). As chronic conditions increase in these countries, it is imperative to examine how the social distribution of these conditions may be changing . Populations with low socioeconomic status (ses) within transitional countries, who have traditionally suffered disproportionately from communicable disease and undernutrition (4), may eventually come to experience the highest risk of these chronic conditions (5, 6). In the face of changing disease burdens, one hypothesized mechanism by which new health disparities may emerge is that higher ses populations adjust their preferences, choices and behaviors based on new medical knowledge, treatment or stigma (7), while lower ses populations face more constraints in making the same health - preserving adjustments (8, 9). This process of emerging and widening disparities under such conditions has been referred to as a social shaping of disease (7), and it can best be investigated under conditions of large changes in disease burden, new diseases, new treatments for diseases or new stigma associated with disease (7). For example, the epidemic increase in overweight among older adolescents in the us was associated with the emergence of a social disparity in overweight prevalence (10); see (8) (11) (12) for other examples). We use china as a case study to investigate the social shaping of disease (in this case, overweight) in a rapidly developing country (7). The dramatic increase in overweight prevalence in china over the past 20 years provides an excellent opportunity to investigate how the burden of overweight by socioeconomic status may have changed over time (13). China has experienced extremely rapid increases in economic development and national wealth over the last 20 years (14). With this economic growth and market restructuring have come major changes in food and physical activity environments and norms (1517). Using longitudinal panel data spanning 17 years, we test whether a social disparity in the relative odds of overweight has emerged since the late 80s in china among women or men . Data come from the china health and nutrition survey (chns), which is a panel study of nine chinese provinces that includes 7 observational periods between 1989 and 2006 . The chns was designed to be representative of the nine provinces, but not designed to be nationally representative . However, many of the general trends that have been reported among the chns sample are similar to those reported in other nationally representative surveys from china (18, 19). The retention rates are described in more detail in popkin et al (20), but briefly, the panel study design incorporated new households in each survey wave to replace those that were lost to follow - up . This is illustrated by the fact that while 68% of households included in the first wave (1989) were also included in the most recent wave (2006), the total sample size has increased slightly from 4,020 to 4467 households over the survey period (20). The study protocols were approved by the university of north carolina at chapel hill and the chinese center for disease control internal review boards . Our analyses are limited to non - pregnant women and men who were surveyed at least once and were younger than 50 years at their first included measurement and older than 18 years during at least one of the survey waves (measurements from age 18 and above are included for individuals who aged into our eligible sample). Of the 7789 eligible women, 7314 (94%) were included in the analyses; missing data were due to bmi (n=372), education (n=95), age (n=8). For men, of the 7141 eligible, 6492 (91%) were included in the analysis; missing data were due to bmi (n=345), education (n=27), age (n=10), and smoking (266). The average number of measurements per person is 3.2 for women and 2.9 for men . Bmi (weight (kg)/(height (m))) was used in its continuous form and also for overweight (bmi25) classification according to who guidelines (21). Action points for disease risk in asian populations, it is still recommended to use the standard cutpoints to enable international comparisons of overweight prevalence (22). We use education level to represent ses in our main analyses since, in general, education is believed to generally reflect social circumstance, particularly in lower income countries where the most disadvantaged groups may attend very little formal schooling (23). Additionally, attained education is typically correlated to some degree with earnings another indicator of socioeconomic status (23). Furthermore, there was a great deal of fluctuation into and out of income tertiles among families over the survey period, so we chose to focus on education as our indicator of ses, since it is more stable within individuals over the survey period . Highest level of education was ascertained for each individual at each survey wave and modeled as indicator variables according to the following categories: 1) less than primary school 2) primary school completed 3) secondary school completed 4) more than secondary school . Our key independent variable of interest is the ses - specific rate of bmi / overweight increase over time . We include calendar year to represent time and interact it with attained education level in the regression models in order to assess the education group - specific rate of increase in bmi / overweight risk over time . In the regression analyses, calendar time was recoded from 0 (1989) to 17 (2006) and was entered as an ordinal variable . Age, smoking, urbanicity, and birth cohort were hypothesized confounders of the relationship between socioeconomic status and bmi / overweight . Subject - specific mean age and mean age squared were entered as covariates; mean age was used rather than actual age at each time point so that the effect of age is the between - subject effect of age (24). This specification allows the coefficient on age to capture the effect of the being older or younger in a given year, and more importantly, allows coefficient on calendar time to capture the passage of time within and between subjects . Since smoking is quite common among men, but rare among women in this context (see table 1) and, since its inclusion results in the loss of observations from 1989, we included smoking as a covariate in the male analyses only . Birth cohort was categorized into 2 levels to keep adequate sample size yet distinguish between populations growing up during different periods in china . Following chen et al (25), we chose to divide the birth cohorts before or after 1956 to separate the early and late 50s since the great leap forward and the three year famine occurred during the late 50s . This results in an older birth cohort born in 19391955 and a younger cohort is born in 19561988 . We used a previously developed urbanicity scale (ranging from 0130) for the chns sample in its continuous, time - varying form in the analyses (26). Data come from the china health and nutrition survey (chns), which is a panel study of nine chinese provinces that includes 7 observational periods between 1989 and 2006 . The chns was designed to be representative of the nine provinces, but not designed to be nationally representative . However, many of the general trends that have been reported among the chns sample are similar to those reported in other nationally representative surveys from china (18, 19). The retention rates are described in more detail in popkin et al (20), but briefly, the panel study design incorporated new households in each survey wave to replace those that were lost to follow - up . This is illustrated by the fact that while 68% of households included in the first wave (1989) were also included in the most recent wave (2006), the total sample size has increased slightly from 4,020 to 4467 households over the survey period (20). The study protocols were approved by the university of north carolina at chapel hill and the chinese center for disease control internal review boards . Our analyses are limited to non - pregnant women and men who were surveyed at least once and were younger than 50 years at their first included measurement and older than 18 years during at least one of the survey waves (measurements from age 18 and above are included for individuals who aged into our eligible sample). Of the 7789 eligible women, 7314 (94%) were included in the analyses; missing data were due to bmi (n=372), education (n=95), age (n=8). For men, of the 7141 eligible, 6492 (91%) were included in the analysis; missing data were due to bmi (n=345), education (n=27), age (n=10), and smoking (266). The average number of measurements per person is 3.2 for women and 2.9 for men . Bmi (weight (kg)/(height (m))) was used in its continuous form and also for overweight (bmi25) classification according to who guidelines (21). Action points for disease risk in asian populations, it is still recommended to use the standard cutpoints to enable international comparisons of overweight prevalence (22). We use education level to represent ses in our main analyses since, in general, education is believed to generally reflect social circumstance, particularly in lower income countries where the most disadvantaged groups may attend very little formal schooling (23). Additionally, attained education is typically correlated to some degree with earnings another indicator of socioeconomic status (23). Furthermore, there was a great deal of fluctuation into and out of income tertiles among families over the survey period, so we chose to focus on education as our indicator of ses, since it is more stable within individuals over the survey period . Highest level of education was ascertained for each individual at each survey wave and modeled as indicator variables according to the following categories: 1) less than primary school 2) primary school completed 3) secondary school completed 4) more than secondary school . Our key independent variable of interest is the ses - specific rate of bmi / overweight increase over time . We include calendar year to represent time and interact it with attained education level in the regression models in order to assess the education group - specific rate of increase in bmi / overweight risk over time . In the regression analyses, calendar time was recoded from 0 (1989) to 17 (2006) and was entered as an ordinal variable . Age, smoking, urbanicity, and birth cohort were hypothesized confounders of the relationship between socioeconomic status and bmi / overweight . Subject - specific mean age and mean age squared were entered as covariates; mean age was used rather than actual age at each time point so that the effect of age is the between - subject effect of age (24). This specification allows the coefficient on age to capture the effect of the being older or younger in a given year, and more importantly, allows coefficient on calendar time to capture the passage of time within and between subjects . Current smoking habits were ascertained in each survey except 1989 . Since smoking is quite common among men, but rare among women in this context (see table 1) and, since its inclusion results in the loss of observations from 1989, we included smoking as a covariate in the male analyses only . Birth cohort was categorized into 2 levels to keep adequate sample size yet distinguish between populations growing up during different periods in china . Following chen et al (25), we chose to divide the birth cohorts before or after 1956 to separate the early and late 50s since the great leap forward and the three year famine occurred during the late 50s . This results in an older birth cohort born in 19391955 and a younger cohort is born in 19561988 . We used a previously developed urbanicity scale (ranging from 0130) for the chns sample in its continuous, time - varying form in the analyses (26). We used sex - stratified analyses due to the demonstrated variation in the association between ses and bmi by sex (27, 28). Additionally, we hypothesized the ses - specific growth rates for bmi might vary according to birth cohort and/or urbanicity (13, 26, 2932); we therefore tested these interactions . We calculated the mean bmi and proportion overweight for each education group in each survey wave for men and women separately to show unadjusted trends in bmi and overweight in the sample . To estimate the average bmi growth rate over time according to education group, we employ sex - stratified random - effects linear models of repeated bmi measurements . We used comparable random - effects logistic regression models to estimate the education - specific change in odds of overweight over the study time period . Random - effects models incorporate all available measurements from each subject, which maximized our analytic sample . For time - varying variables, the model employs a weighted average of the between and within cluster effects (24). In our models the cluster is each individual, inside which, repeated measurements are nested . The models included a random intercept for each participant to account for the correlation between bmi measurements within the same person over time . We used robust standard errors to account for the potential correlation of bmi between people of the same household and community and for heteroskedasticity of residuals at the lowest level (occasion) (24, 33). Our main variable of interests is the growth rate in bmi for each education group over time (represented by the interaction between education and survey year, as described above). Since we hypothesized that birth cohort and/or urbanicity might modify the effect of the education specific growth rates, we tested the significance of a three - way interaction between birth cohort and education and time as well urbanicity, education and time . In addition to the three - way interactions, we included all the lower order two - way interactions between the three variables and their main effects along with the covariates . Neither of the three - way interactions were significant, so we tested the remaining two - way interactions against the model with all two - way interactions included . After retaining the significant interactions, we test the assumption that the between and within subject effects are equivalent for the time - varying variables (time, time by education group, and urbanicity) by including the original variable and the person - specific mean of each variable (24). If the coefficient on the person - specific mean for any of these variables was significant it was retained in the model . We included a quadratic term for time if it was statistically significant to allow for curvilinearity . The final linear and logistic regression models for women and men differ slightly based on the significance of the various interaction terms in each model . For women, all of the interactions between birth cohort and education, urbanicity, age and age squared were retained due to statistical significance . The tests of statistical equivalence of the between and within effects for time and urbanicity led to the inclusion of both the main effect and the between effect for urbanicity and time . For males, the interaction between birth cohort and time was retained as was that of birth cohort and urbanicity, while the interactions between birth cohort and education, age and age squared were not included . Additionally, for males, we included smoking as a covariate and we included a quadratic term for time . We retained the person mean and occasion - specific deviation from the cluster mean for urbanicity only . Random - effects logistic regression models with the same specifications as linear models for female and male models were used to estimate the education - specific growth rate in the odds of overweight over the study time period . Model coefficients and post - estimation tests of confidence intervals were used to graph the predicted bmi and odds of overweight by education group . For sensitivity analyses, we added per capita household income adjusted for inflation as a covariate to the final linear and logistic models to see if the results changed substantially . This was a basic test that the association between ses, represented by education, was not entirely attributable to income . Additionally, we ran an unadjusted cox proportional hazards model to see whether the hazard rates for incident overweight were in the same direction and of same significance to the prevalence rates at the end of the survey . We chose to focus the main analyses on prevalence odds ratios and bmi trajectories over time since our research question specifically is concerned with tracking trends over time and since focusing on incidence substantially decreased the sample size, due to the elimination of all new prevalent cases at each wave in this panel study . Finally, we performed a post - hoc analysis to explore potential explanations for our main findings . We calculate the average minutes of physical activity spent engaged in heavy and light physical activity . Alpha was set at 0.05 for main effects and 0.10 for interactions . For linear models, model estimation was performed using xtreg with generalized least squares random - effects estimation and robust standard errors . For logistic models, we calculated the mean bmi and proportion overweight for each education group in each survey wave for men and women separately to show unadjusted trends in bmi and overweight in the sample . To estimate the average bmi growth rate over time according to education group, we employ sex - stratified random - effects linear models of repeated bmi measurements . We used comparable random - effects logistic regression models to estimate the education - specific change in odds of overweight over the study time period . Random - effects models incorporate all available measurements from each subject, which maximized our analytic sample . For time - varying variables, the model employs a weighted average of the between and within cluster effects (24). In our models the cluster is each individual, inside which, repeated measurements are nested . The models included a random intercept for each participant to account for the correlation between bmi measurements within the same person over time . We used robust standard errors to account for the potential correlation of bmi between people of the same household and community and for heteroskedasticity of residuals at the lowest level (occasion) (24, 33). Our main variable of interests is the growth rate in bmi for each education group over time (represented by the interaction between education and survey year, as described above). Since we hypothesized that birth cohort and/or urbanicity might modify the effect of the education specific growth rates, we tested the significance of a three - way interaction between birth cohort and education and time as well urbanicity, education and time . In addition to the three - way interactions, we included all the lower order two - way interactions between the three variables and their main effects along with the covariates . Neither of the three - way interactions were significant, so we tested the remaining two - way interactions against the model with all two - way interactions included . After retaining the significant interactions, we test the assumption that the between and within subject effects are equivalent for the time - varying variables (time, time by education group, and urbanicity) by including the original variable and the person - specific mean of each variable (24). If the coefficient on the person - specific mean for any of these variables was significant it was retained in the model . We included a quadratic term for time if it was statistically significant to allow for curvilinearity . The final linear and logistic regression models for women and men differ slightly based on the significance of the various interaction terms in each model . For women, all of the interactions between birth cohort and education, urbanicity, age and age squared were retained due to statistical significance . The tests of statistical equivalence of the between and within effects for time and urbanicity led to the inclusion of both the main effect and the between effect for urbanicity and time . For males, the interaction between birth cohort and time was retained as was that of birth cohort and urbanicity, while the interactions between birth cohort and education, age and age squared were not included . Additionally, for males, we included smoking as a covariate and we included a quadratic term for time . We retained the person mean and occasion - specific deviation from the cluster mean for urbanicity only . Random - effects logistic regression models with the same specifications as linear models for female and male models were used to estimate the education - specific growth rate in the odds of overweight over the study time period . Model coefficients and post - estimation tests of confidence intervals were used to graph the predicted bmi and odds of overweight by education group . For sensitivity analyses, we added per capita household income adjusted for inflation as a covariate to the final linear and logistic models to see if the results changed substantially . This was a basic test that the association between ses, represented by education, was not entirely attributable to income . Additionally, we ran an unadjusted cox proportional hazards model to see whether the hazard rates for incident overweight were in the same direction and of same significance to the prevalence rates at the end of the survey . We chose to focus the main analyses on prevalence odds ratios and bmi trajectories over time since our research question specifically is concerned with tracking trends over time and since focusing on incidence substantially decreased the sample size, due to the elimination of all new prevalent cases at each wave in this panel study . Finally, we performed a post - hoc analysis to explore potential explanations for our main findings . We calculate the average minutes of physical activity spent engaged in heavy and light physical activity . Alpha was set at 0.05 for main effects and 0.10 for interactions . For linear models, model estimation was performed using xtreg with generalized least squares random - effects estimation and robust standard errors . For logistic models, overweight more than doubled in women and more than tripled in men from baseline to final follow - up (table 1). Household income (adjusted for inflation) also increased over time, as did community - level urbanicity . Among women, unadjusted mean bmi levels increased for all education levels, but least so for those with the highest educational attainment (table 2). The mean bmi in 1989 for women in the two lowest education categories was slightly higher than that for women in the two highest categories . By 2006, the bmi for women in the three lowest education categories was approximately 2 units higher than that of women with the most education . Similarly, an estimated 12% of the least educated women are overweight in the first survey wave and this increased to 33% by the last survey wave (table 2). The increase in overweight is much lower for the most educated women, increasing from 9% in the first wave to 13% in the final wave . In the adjusted results from the longitudinal random - effects regression models, we estimated the education - specific bmi increases per year while controlling for confounders (table 3). Since the models included multiple interactions and were additionally complicated by the modeling of time, we used the model estimates to plot predicted bmi trajectories to aid in the interpretability of the results (figure 1). Similar to the crude results, the estimated growth rate for bmi was higher the least educated women compared to most educated . Specifically, the estimated growth rate for women with the lowest education (<primary school) was 0.11 bmi units per year (time: 0.11 (ci 0.10, 0.12) while women with highest education (> secondary school) had a lower rate of increase of 0.08 bmi units per year (yearhighest ed: 0.03 (ci 0.05, 0.005) (table 3). Additionally, the growth rate among those with the highest education was also lower than those with secondary and primary school education (figure 1). To translate these bmi gains into weight gains, we calculated the estimated weight gain for a female of average height (156 cm) with an initial bmi of 22 . At the bmi growth rate of the highest educated females this would amount to 0.19 kg / year or 3.31 kg over the 17-year period versus 0.27 kg / year or 4.54 kg at the growth rate of the least educated females . Figure 1 demonstrates that women in the older birth cohort have lower predicted bmis compared to the younger cohort throughout the survey period . However, in both cohorts the slower bmi growth rate for the highest education group results in a widening gap in predicted bmi between the most educated and least educated women over the survey period . At the beginning of the survey, the predicted bmi for the highest education compared to the lowest was not significantly different for the younger or older birth cohort (younger: secondary school 0.36 (ci 0.74, 0.02); older: 0.27secondary schoolxcohort (0.27, 0.82)). By 2006, this difference increased to almost one bmi unit lower for the young cohort by 2006 (0.88 (ci1.22, 0.53) and 0.61 bmi units lower for the older cohort (0.61 (ci 1.11, 0.11)) (figure 1). The covariates in the model of bmi (table 3) suggest that within each birth cohort, being older is associated with a higher bmi; this association is stronger for the older birth cohort, but decreases slightly with advancing age in this cohort . Increasing urbanicity over time is associated with increases in bmi for the younger cohort, but is not significant for the older birth cohort . The results examining the odds of overweight were generally similar to those found for bmi (table 4). Specifically, the highest education group experienced a significantly lower rate of increase (8% lower) in the odds of overweight compared to the lowest education group . At baseline, the odds of overweight between the highest and lowest education groups were not significantly different for the younger or the older cohorts . However, by 2006, the odds ratio for overweight for the highest versus the lowest education groups was or 0.22 (ci 0.11, 0.42) for the younger cohort and or 0.27 (ci 0.10, 0.72) for the older cohort (figure 2). Similar to the results from the linear regression, the inverse association between education and odds of obesity was of slightly larger magnitude for the younger cohort . The results for the covariates in the models of the bmi and odds of overweight from the linear and logistic regressions are generally similar in significance and direction, with the exception of that, the lowest education group in the older birth cohort has significantly lower bmis (table 3); however, this does not translate into significantly lower odds of overweight (table 4). Bmi increased for all education groups; however, for men, the highest education group had the largest increases in bmi (table 2). Specifically, the mean bmi for men in 1989 was similar across education category (21.221.8); however by 2006, the mean bmi for men with the most education had increased to 24.2 while that for men with the lowest education was 22.6 . The adjusted linear models also indicated that the estimated rate of increase in bmi was higher for the group with the highest education compared to those with the lowest (yearhighest ed 0.07 (ci 0.04, 0.10) (table 3). At baseline, predicted mean bmi for men with the highest education is not statistically different than that of men with the lowest education 0.07 (ci 0.45, 0.32). However, by 2006, compared to men with the lowest education, the predicted bmi for men with the highest education is more than one bmi unit higher (1.16 (ci 0.80, 1.52)) (figure 3). Figure 3 demonstrates that, for men, the older cohort has slightly higher initial bmi levels; however, the growth rate over time is faster for the younger cohort, who end up with higher bmis on average . Also for men, the trend is for generally increasing bmis, but the statistically significant quadratic term for time results in a curvilinear trend such that the slopes are less steep in more recent years . To translate these bmi gains into estimated kilograms we calculated the estimated weight gain for a male of average height (166 cm) with an initial bmi of 22 . At the bmi growth rate of the highest educated males this would amount to 9.83 kg vs 6.53 kg at the growth rate of the least educated males . The findings amongst the covariates are similar to those discussed for women; additionally, smoking is associated with a lower bmi (table 3). The trends in the odds of overweight for men are largely similar to those seen for bmi (table 4), with the exception that the education - specific growth rate for the most educated men was significantly higher for bmi, but does not reach statistical significance for odds of overweight . However, the odds ratio for overweight are still significantly higher for the most educated men (vs the least educated) in the final survey year or 3.2 (ci 1.85, 5.91) (figure 4) for men, the effect of time, but not that of education, varied by cohort, so the odds ratio of overweight comparing most educated to the least educated are the same for both the older and younger cohorts . Controlling for income in all analyses resulted in virtually no change to model estimates (results not shown). Among females, the coefficient for income was positive, but not statistically significant in either model . Among males, the coefficient on income was positive and statistically significant in the logistic models (or 1.01 for every 1000 yuan increase in real income; p=0.04). The hazard ratio of overweight was lower for the most educated versus the least educated (hr 0.72 (ci 0.54, 0.97) for women . In men, those with the most versus the least education had a higher hazard of overweight (hr 3.43 (ci 2.64, 4.46)) for men . These results are consistent in terms of direction and significance with our primary analyses of prevalence . Controlling for income in all analyses resulted in virtually no change to model estimates (results not shown). Among females, the coefficient for income was positive, but not statistically significant in either model . Among males, the coefficient on income was positive and statistically significant in the logistic models (or 1.01 for every 1000 yuan increase in real income; p=0.04). The hazard ratio of overweight was lower for the most educated versus the least educated (hr 0.72 (ci 0.54, 0.97) for women . In men, those with the most versus the least education had a higher hazard of overweight (hr 3.43 (ci 2.64, 4.46)) for men . These results are consistent in terms of direction and significance with our primary analyses of prevalence . Over the last 2 decades, chinese women experienced an emergence of a disparity in overweight by ses . Whereas odds of overweight did not differ significantly for women with low and high education in 1989, by 2006, a disparity in overweight risk was readily evident with higher overweight among women with lower education levels . For men, we observe the opposite, with the most educated men having higher levels of overweight by 2006 . Our findings among women provide empirical evidence that the burden of chronic, non - communicable conditions, such as overweight, might shift toward people with low socioeconomic status even in developing countries, and particularly for transitional countries undergoing rapid development . Our study adds to a small body of literature that traces the social distribution of various conditions over time to demonstrate emerging health disparities in the context of changing disease burden, medical technologies and/or stigma (1012, 35). These studies enhance a large body of literature that shows a robust cross - sectional inverse relationship between socioeconomic status and disease . Our findings among women are consistent with the findings of emergent disparities in overweight / obesity in which the lower ses groups now have higher rates of overweight in the us among adolescents (10) and among women in some regions of brazil (36). Our findings among men are also consistent with the trends among men in brazil (37). Our study improves upon these studies by following individuals longitudinally rather than using repeated cross - sectional data . In china, men and women with higher education likely have similar access to energy - dense foods, sedentary occupations and energy - saving modern conveniences (26, 38), but women with high education experienced a slowed growth rate in bmi while men with high education experienced an increased rate of growth bmi gains . Additionally, access to energy - dense foods, sedentary occupations, and labor - saving devices is likely more limited for the women with low education in comparison to the women with high education in this context (16, 38). The divergent patterns among high income men and women and high versus low ses women during a time in which the food and physical activity environments rapidly changed (13, 16, 39), could be consistent with a difference in response to a rapidly changing food environment by education group and sex . We briefly explore potential explanations that might account for the differences seen between men and women for the relationship of ses to bmi . First, women with high education might enact behaviors to limit bmi gains due to either health concerns / health knowledge or to a preference for a thinner body size . Preference for thinness due to stigma or health concerns may exist for women more so than for men, and, compared to lower educated women, highly educated women may have the better means to achieve thinner body sizes in what has become an increasingly obesogenic environment in modern china (13, 16, 39). The role of sex - specific stigma and desired body size has previously been speculated as a rationale for the difference in the associations between ses and body size between males and females (27, 40). Our study did not directly test this speculation; however, some evidence suggests that the desired body size among chinese women has been tall and thin since the 1970s (41) and that chinese girls currently on average expressed a desire for a thinner body, while boys were more likely to perceive themselves as underweight or normal weight (40, 42, 43). Differences in physical activity could be an explanation if the most highly educated men have substantially more sedentary jobs or get less total physical activity than the most highly educated women . However, despite early evidence from chns that men were more likely to transition out of farming into less physically demanding jobs while women remained in the more physically demanding farming jobs (44), more recent work has demonstrated that total physical activity for men and women in the chns has decreased more for women over time (45) and that men and women now have comparable levels of total activity on average . Additionally, examining the most recent data for our sample shows that for both men and women, compared to those with lower education, those with higher education report much lower levels of heavy activity and higher levels of light activity and that these levels are similar among men (heavy: 115 minutes / week; light 1797) and women (heavy: 61 minutes / week; light: 1867) of the same education level . Physical activity might explain the differences among men of high and low education, but it does not offer a convincing explanation for the male - female differences in the association between education and bmi or overweight . First, although this is a longitudinal study, not every participant was interviewed at every survey wave . However, due to the panel study design and to our use of random - effects models, we were able to retain a very high proportion of the participants in the analysis (94% of eligible women and 91% of eligible men) and we tested for differences on observed characteristics between those excluded and included . Second, education only addresses one aspect of ses; yet, we tested whether our results would remain the same if we added an additional control for income, finding virtually unchanged estimates . The doubling and tripling of overweight prevalence in women and men, respectively, in china has been accompanied by a shift in the social distribution of this overweight among women . Our work concurs with other predictions that the burden of chronic diseases, such as overweight, might shift toward the lowest social classes even in developing countries where higher ses individuals have historically had higher comparative risks for such diseases . The doubling and tripling of overweight prevalence in women and men, respectively, in china has been accompanied by a shift in the social distribution of this overweight among women . Our work concurs with other predictions that the burden of chronic diseases, such as overweight, might shift toward the lowest social classes even in developing countries where higher ses individuals have historically had higher comparative risks for such diseases.
The 62-year - old male patient in this case report had a smoking history of more than 30 pack - years and a medical history of hypertension and diabetes . He had had a percutaneous coronary intervention 6 years previously due to angina and had noticed a cold and numbing sensation in the extremities of both hands for the previous 3 years . Two years previously, the patient's fourth finger on the left hand had experienced blue discoloration, with extreme pain of greater than 90/100 mm on the visual analogue scale (vas), followed by ulceration . After a few tests at the orthopedic clinic, he had been referred to the pain clinic for conservative treatment one year previously . On the patient's upper extremity angiography, greater than 80% stenosis was observed in the first and third finger artery in the area of radial artery and palmer arch, and greater than 90% stenosis was also observed in distal ulnar artery . At the time of referral, the patient had been taking oxycodone 40 mg twice a day, as well as limaprost 5 g and gabapentin 300 mg 3 times day . Even with an increase in opiate dose and several chest sympathetic block and stellate ganglion blocks, the analgesic effect was temporal . The pain and ulcers on the fingers worsened, and a finger amputation was planned as arterial bypass surgery was not a valid method for this patient . Although a number of treatments were tried, the patient complained about extreme pain, 90/100 mm on vas, and there were severe gangrenous ulcers progressing on the fourth finger of the left hand and the index finger of the right hand . The patient was very much against the amputation even though he was under extreme pain and had been referred to the pain clinic several times . The blood pressure, heart rate, oxygen saturation, and electrocardiography were monitored, and, with the patient in the prone position, local anesthesia was performed at t2 - 3 intervertebral space . A 15-gauge tuohy needle was used for the paramedian approach with a c - arm fluoroscopic image . After ensuring the needle was positioned in the epidural space, the guidewire was inserted for easy insertion of the electrode, and the electrode was positioned in the posterior epidural space using the c - arm fluoroscopic image . The guidewire was removed, and the octrode electrode lead (advanced neuromodulation system inc, plano, texas, usa) was placed 2 mm left of the radiological center . The electrode was connected to the test stimulator, and the stimulation was profound in the areas with pain . After the test stimulation, the pain was reduced by 50% . During 1 week of test stimulation, the patient's pain was maintained to within 20 - 30/100 mm on vas, and the use of opiate analgesics was decreased by 50% . The genesis ipg (advanced neuromodulation system inc, plano, texas, usa) was buried under the lower left - side clavicle, and stimulation was well controlled with 3 - 4 + electrode combination, 4.0 v amplitude, 240 msec pulse width, and 34 hz frequency . After the spinal cord stimulation insertion, the ulcers on both hands began gradual recovery and were completely recovered after 6 months . The pain was maintained within 30/100 mm on vas, and the use of opioid analgesics was decreased more than 50% compared with the pre - procedure stage (fig . The cause of buerger's disease is unknown, but smoking is considered to be a major factor in the initiation and progression of the disease . The progression of the disease is relatively fast, with 2 - 5 years typically passing between the onset of the disease and loss of tissues . Therefore, the foundation of treatment is smoking cessation to stop the disease progression and prevent amputation of the lesions . Other than smoking cessation, systemic treatments such as thrombolytic agents, anticoagulants and prostaglandin agents can be used, as well as direct arterial bypass surgery, sympathectic block, and amputation [1 - 4]. A prostaglandin agent has been used in occlusive arterial disease due to its vasodilation and antiplatelet effects, but it has the disadvantage of 80 - 90% loss of efficacy through lung circulation when it is administered intravenously . Bypass surgery or percutaneous angioplasty tend to limit the treatment, as the saphenous vein for the graft can also accompany phlebitis, which often continues to progress even after the bypass surgery, and the lesions generally spread widely . Sympathectic nerve block is known to be effective for the ischemic ulcer but does not decrease the rate of the most severe limb amputations . According to a number of studies, spinal cord stimulation in patients with buerger's disease is not only a safe treatment method but also can prevent limb amputation, reduce pain, and improve blood flow and ulceration . Spinal cord stimulation in buerger's disease reduces ischemic pain and the rate of amputation by treating the ulceration . Hence, it is recommended for people who fail conservative or surgical treatment and for whom arterial bypass surgery is not feasible, as well as for people who complain about severe ischemic pain after failure of medication . Performed spinal cord stimulation in 29 patients with buerger's disease who did not respond to medications or arterial bypass surgery and followed up for up to 4 years . Most of the patients (limb salvage rate 93.1%) presented with a significant increase in microcirculation of the blood vessels . Lower limb amputation, one of the most serious problems in buerger's disease treatment, was performed in only 2 patients, a considerably lower rate than that seen in other studies that conducted sympathectic block or aterial bypass surgery . A number of other studies have suggested spinal cord stimulation in buerger's disease as a safe treatment method that can reduce the pain and improve blood flow and the treatment of ulcers . Therefore, it has been argued that spinal cord stimulation should be considered as an important alternative treatment choice . The reduction in the rate of limb amputation due to spinal cord stimulation is hugely beneficial in socioeconomic terms . The cross - sectional problem of the increase in treatment costs due to spinal cord stimulation can easily be offset by decreases in the length of hospital stay after the procedure, prevention of amputation, and the increase in quality of life due to pain reduction . Therefore, spinal cord stimulation should be considered in patients who do not find a sympathectic block to be effective in ischemic pain relief or for whom bypass surgery is not indicated . However, spinal cord stimulation is a high - cost treatment and only should be considered in patients with a high severity of disease, consistent symptoms, lack of success with medication, and no indication for arterial bypass surgery . In 1976, cook et al . Reported using electronic stimulation of spinal nerves and the dorsal root in vascular disease of limbs, and since then spinal cord stimulation has been successfully carried out in the treatment of atherosclerotic and vasospastic peripheral arterial disease and is mainly used in vascular diseases of lower limbs . There is no reported case of spinal cord stimulation in a patient with buerger's disease in korea . A case has been reported in which spinal cord stimulation was carried out in a 60-year - old male patient with raynaud's syndrome, and improvements in pain and ulceration were seen although the ulceration was not completely cured . Spinal cord stimulation improves microcirculation by inhibiting sympathetic vasoconstriction and stimulates the secretion of a number of inhibitory neurotransmitters, leading to a reduction in pain in patients with buerger's disease . Spinal cord stimulation reduces pain in buerger's disease by promoting the secretion of gamma - aminobutyric acid (gaba), serotonin, and substance p at the spinal nerve dorsal horn . In addition, it inhibits nociceptive neurotransmission, and intramedullary oxidative nitric oxide and gaba act as an important mediator for the induction of pain relief after spinal cord stimulation [11 - 13]. Spinal cord stimulation in peripheral obstructive arterial disease can treat chronic ischemic pain and ulcers and also prevent amputation . Unlike the pain reduction, the mechanism of action for the improvement in microcirculation is unknown, and the improvement in ulceration is considered to be the outcome of the reduction in vasoconstriction due to the inhibition of peripheral sympathetic nerves . The improvement in microcirculation after spinal cord stimulation in patients with peripheral obstructive arterial disease can be quantified by the ankle - brachial index, transcutaneous oxygen pressure, and regional perfusion index . It is simple and non - invasive and can effectively measure the degree of blood circulation of cutaneous tissue . In this case, the progression of ulcers was only observed through gross examination before and after the procedure, and objective measures were not carried out to determine the degree of microcirculation . Also, angiography was not carried out after spinal cord stimulation, and thus the change in peripheral vessels could not be confirmed . However, there was a reduction in redness on the patient's palms immediately after the spinal cord stimulation, and significant improvement was seen a week after the procedure . The ulceration on both hands seemed to improve slightly one week after the procedure, and the ulceration gradually improved to achieve complete recovery 6 months after the procedure . Although the patient fully recovered from the ulceration within 6 months of spinal cord stimulation, the pain still was present . However, the pain decreased by about 50% from 90/100 mm on vas immediately after the procedure and substantially improved after 6 months to 20 - 30/100 mm on vas . This pain reduction led to more than a 50% reduction in the use of opiate analgesics and the total amount of medication . After spinal cord stimulation, the patient completely recovered from ulcers on fingers of both hands and achieved a significant reduction in pain, although the cold sensation in his hands remained . Therefore, the authors suggest that spinal cord stimulation be considered in patients with buerger's disease who do not response to medications or sympathectic block and for whom arterial bypass surgery is not feasible . Spinal cord stimulation would be an excellent treatment choice in patients with buerger's disease when an amputation is planned due to the lack of successful response to various treatments.
Pari passu with the progress of science in medical education, simulation in medical education is also progressing in leaps and bounds . Something which was hardly ever talked about even a couple of years ago has now become the new buzz word in medical education circles, and now india is also getting on the bandwagon . Simulation has been variably defined as the imitation of the operation of a real - world process or system over time; an imitation of some real thing, state of affairs, or process for the practice of skills, problem solving, and judgment . The aim of medical simulation training is to imitate reality, or rather, to mimic reality to the closest extent possible so that the learner is in a state of suspended disbelief and he believes himself to be undergoing a real experience . Simulation has been used at its greatest effect in pilot training - indeed, it is mandatory for all pilots of commercial aircraft to undergo refresher courses on flight simulators, regardless of their real flying experience . Medical simulation has unfortunately not kept up with the development of simulation in engineering systems, flight training, etc . However, this situation is now rapidly changing . This change has been brought about by three main reasons: firstly, a big increase in student population in medical education, both at the undergraduate and post - graduate level . This unprecedented growth has occurred in the past two decades (mci vision 2015). Approximately 33,528 graduates pass out every year from these colleges (mci annual report 20092010). . There has also been legislation which limits the number of hours residents can now work, reducing further the opportunity for bedside learning . The american medical association's accreditation council for graduate medical education has limited the time that residents can be required to work to 80 h a week . Although this action was intended to improve patient safety, it also reduced the number of patient - contact hours needed to train residents . Secondly, there is a growing awareness of patients of their rights and reluctance to be practiced upon and the growing incidence of medical litigation leading to a reduction in the number of available patients . Finally, there has been a huge technological improvement in the quality and variety of simulators . Now, high fidelity computerised human patient simulators have been developed by various companies (e.g. Simman by laerdal, the meti family of human patient simulators by cae health care), which mimic very realistically the response of human beings to physiological insults, whether it be an acute cardiac event, or a trauma event; indeed, any disease process that affects the cardiovascular or respiratory system can be accurately modelled . Surgical simulators have been developed to practice laparoscopic surgery using a virtual reality environment (e.g. The lapvr laparoscopic simulator by cae health care), which even provide haptic feedback (a sense of tissue resistance)., many countries are now making certification on simulators mandatory before performing procedures on actual patients . Even in india, the mci 2015 vision document says that a mandatory and desirable comprehensive list of skills has been planned and would be recommended for bachelor of medicine and bachelor of surgery (mbbs) graduate . The educational technology section of the 2004 aem consensus conference for informatics and technology in emergency department health care concluded that there should be increasing use of web - based, virtual reality and human patient simulation in educating emergency medicine trainees . The authors postulate the philosophy of see one, simulate many, do one competently, and teach everyone . There are now many articles on how simulation can be used in medical training . A key challenge for surgical training is to provide conditions for effective learning without putting patients health at risk . Simulation presents an attractive solution, offering a safe, simulated clinical environment where the training agenda can be determined by the needs of the learner, not the patient . Evidence is now available to suggest that simulation training actually improves clinical performance . In a review of simulation articles, it was found that four (3%) journal articles provided evidence for the direct correlation of simulation validity with effective learning . Another study showed that the use of virtual reality surgical simulation to reach specific target criteria significantly improved the operating room (or) performance of residents during laparoscopic cholecystectomy . Simulation in wound management is still relatively in its infancy when compared to the use of simulation in other areas of medical education . It is however reasonable to believe that the use of simulation in wound management will result in improvement in wound care . Simulation in relation to wounds can be divided into use of simulation to study the effect of projectiles on tissue (wound modelling) and the use of simulation in the management of wounds . The purpose of this article is to outline the present status of simulation related to wounds, in terms of their creation and management . Moulage is the art of creating mock injuries to simulate wounds . Theatrical makeup and common everyday materials moulage is commonly used in mass casualty and disaster drills to add realism to the simulation . It is reasonable to believe, though not proven, that the use of moulage would create a more realistic environment to enhance learning in mass casualty and other trauma situations . As far back as in 1964, omissions and deficiencies in disaster ability were dramatically and conclusively revealed by use of what appeared to be a live disaster setting with smoke, fire, explosions; adverse weather and light conditions; realistically simulated casualties especially prepared not only to look but also to act the part . Moulage by author (drp) showing bleeding thigh wound and evisceration on metiman (cae health care) mannequin another area where simulation is used in wounds is to simulate the effect of injuring agents on the body so as to understand the various forces that are at work on the tissues during the causation of an injury . This would help in accurate wound assessment and anticipation of likely tissue damage if the causative agent is known . Work has mainly been done on cavitating injuries produced by high - velocity projectiles . Because gelatin and glycerin soap have approximately the same density as human tissue, the gunshot effects in them are comparable with those in human tissue . The experiments with these substitutes make it possible to understand what happens to the human body as the result of, for example, a penetrating gunshot . Some other authors have used transparent gel candles [15% kraton (polymers) in 85% white paraffin oil] for the same purpose . A synthetic skin skull brain model has been developed to simulate gunshot wounds to the head in such a realistic manner as to reconstruct the specific physical characteristics of a given trauma, including its progressive formation . This model uses a silicon cap containing synthetic fibres for artificial skin, a layered polyurethane sphere for the skull (containing the outer and inner tables as well as the diploe), a periosteum of latex and the brain itself is simulated with ordnance gelatin . High fidelity simulators are now available which can mimic the physiological effects of wounds on the body . The aim of management of a wounded individual in the emergency department is mainly to correct and prevent the life - threatening physiological derangements associated with the wound like airway obstruction, hypovolemia, respiratory distress, etc . As such these physiological derangements can be mimicked extremely realistically by these high fidelity systems, examples of which are the meti family of mannequins from cae health care or the simman family from laerdal . These simulators are used the world over to train emergency medical care providers, medical students, post - graduates and practicing physicians and surgeons in emergency trauma management [figure 2]. Military specific simulators (e.g. Caesar from cae healthcare) are now in advanced stages of development to train soldiers with non - medical backgrounds at the point of injury . John's ambulance team (malaysia) demonstration of trauma scenario on ecs (meti) simulator there are a few articles on the emergence of simulation in wound management, which deal essentially with teaching learners local wound management . This includes wound dressing techniques, performance of local surgery like escharotomy or wound debridement . Wound management simulators can be divided into basic part task trainers which use low - technology solutions that are cheap and high - technology virtual reality simulators which, of course, will be much more expensive . The low - technology solutions are discussed first, followed by the high - technology virtual reality solutions . Simulated wounds have been created on animal models like pig trotters to teach the technique of wound debridement . A simulated wound covered with thick slough can be created on a pig trotter by excising an area of skin and then applying a layer of hydrocolloid paste (e.g. Adapttm, hollister) on the denuded area . A layer of toothpaste may then be applied and covered with a piece of glad wrap to simulate the application of emla cream . A small curette is then used to debride the wound as in a human patient, after washing away the layer of toothpaste (= topical anaesthetic). Another study has created a simulated abscess in chicken breast by injecting mock purulent material under the chicken breast skin . The author felt that this new simulation model may be an effective tool to teach skin abscess management . Physicians who evaluated the simulated abscess found that it replicates the classic palpable fluctuance and ultrasound findings of an actual abscess, and it can be surgically incised and drained in a similar fashion . Yet another researcher has used an orange to simulate debridement - the student is required to remove the peel and the white fibres (representing slough) off the orange without puncturing the inner orange itself . Burn wounds offer a challenge to learners in terms of local wound care, and as a result this helps train burn care trainees in the management of such wounds, specifically to perform escharotomy . A major technological development has been that of a thigh wound virtual reality model by delp et al . They have used the data from the visible human project as the basis of reconstruction of the thigh anatomy, on which they superimposed wound models of bullet wounds gleaned from data available in the literature . Functional consequences of the injuries were also modelled, which provided both short - term consequences as well as long - term disability of the simulated patient . The user could interact with the virtual thigh model using virtual surgical instruments from a virtual tray to stop the bleeding, assess muscle bleeding and contractility, perform debridement, align bone, etc . The overall effect on the patient's physiology was also calculated and displayed in terms of vital signs readouts, which changed depending upon appropriate treatment, e.g. Control of bleeding, etc . The virtual reality medical center (vrmc) conceptualised and developed a unique injury simulator as an adjunct to current combat medic training . This initial technology, called injury creation science (ics), very realistically simulated a number of battlefield injuries such as amputations, eviscerations, blast injuries, punctures and burns . Since the initial prototypes, vrmc has developed this technology into wearable part - task trainers that simulate injuries as well as allow combat medics to practice actual medical procedures common to the battlefield . The procedures currently available or under development include treatment of pneumothorax, hemoperitonium and gunshot wounds to an artery . By integrating medical science with cutting - edge simulation and training technologies, realistic prosthetic tissue, wounds and part - task trainers one of the major issues with wounds is the risk of hospital - acquired wound infections, which are usually preventable by simple personal hygiene techniques . Unfortunately, many hospital workers are either unaware of them or unwilling to follow these steps . Training of all hospital staff to prevent cross infection is therefore high on the agenda for most hospital administrators, impinging as it does on patient safety . If the movement of bacteria that cause wound infection, and their removal from hands and other inanimate hospital objects could be seen, then it would be a very useful training tool and could markedly increase staff compliance with hygiene measures . This is exactly what was done in one study wherein ultraviolet polyethylene microspheres were able to simulate the spread of bacteria through direct and indirect contact on different surfaces and had the ability to be washed off under specific hand washing guidelines . The authors felt that due to the positive results, the use of microspheres as a simulator of bacterial presence and spread should be explored further in order to improve hand washing techniques and compliance . A simulation - based training system for surgical wound debridement has been developed and comprises a multimedia introduction, a surgical simulator (tutorial component) and an assessment component . The multimedia training component describes varieties of wound categories (e.g. Burns, lacerations, etc . ), methods of debridement (e.g. Sharp, mechanical, etc .) And equipment / materials used in the procedure . This module provides the pedagogical context for skills training and assessment, linking together all of the required elements for mastering the procedure . A software virtual agent is included and performs the role of an instructor by providing verbal guidance and assessment feedback . Designed to be comprehensive in order to reduce the need for a more senior instructor, the software addresses the patient's condition, initial description of the injury, and provides instruction on operation of the simulator . Specific instructions cover the removal of foreign objects with forceps, scrubbing with a brush, rinsing with saline solution and the maintenance of sterility . Trainees are instructed to remove debris with forceps, scrub with a brush and rinse with saline solution to maintain sterility . In conclusion, increasing sophistication in simulation technology has been also used in simulation of wounds, both in wound modelling and wound management . The available simulators include low - technology solutions and high - technology virtual reality simulators, all of which train learners in the art of wound management . However, this technology is yet to be validated in wound management to see if simulator training can result in better management of wounds in actual patients . Future developments would include research on application of simulator training to real patient care, and the development of better technology to make virtual reality simulators even more realistic by the improvement of design, graphics and haptic feedback.
Asthma is a chronic inflammatory disorder of the airways characterized by bronchial hyperresponsiveness, reversible airflow limitation, and respiratory symptoms (1, 2). Internationally, the prevalence of asthma has increased over the last 3 decades (3 - 5), and in the asia - pacific region asthma causes considerable morbidity, with 15% of teenagers troubled by exercise - induced symptoms during the past 12 months (6). Furthermore, asthma mortality rates in more affluent areas, such as hong kong and japan, are similar to those reported in western countries (6). The reported prevalence of asthma in korea ranges from 2 to 13% (9 - 18). However, its prevalence in the elderly has been reported to be very high, at 12.7% in those aged 65 or more (15). Indeed, with a population estimated at more than 48 million, and a life expectancy of 72.0 yr for males and 79.5 yr for females (19), korea faces an important public health challenge in terms of dealing with chronic diseases such as asthma . In addition to its increasing prevalence, the economic impact of asthma is also substantial and continues to grow . The total cost of the disease in the united states was estimated to be u$4.5 billion in the mid-1980s, whereas in the first half of the 1990s, this estimate had increased to between u$6.2 and u$10.7 billion (20 - 22). In korea, where health insurance is mandatory and the cost of medical care is only covered in part by health insurance, the importance of economic outcomes also continues to grow although little data is currently available . The purpose of this review is to present an overview of the current disease status with respect to the prevalence, mortality rate, socioeconomic burden, quality of life and the treatment patterns of asthma in korea, to identify barriers to improvements in asthma care and to provide recommendations for action at the national, organizational and individual levels . For this, all relevant english and korean language articles were retrieved using pubmed (www.ncbi.nlm.nih.gov/entrez/query.fcgi) and koreamed (www.koreamed.com) from november 2004 to january 2005 . Recently, several large - scale studies of different population in korea (9 - 17) have reported asthma prevalence ranging from 2 to 13% (table 1). These differences are probably ascribable to different case definitions, methodologies, and a tendency to survey children rather than adults due to the relative ease of implementing studies in a school environment . According to the latest summary issued by the global burden of asthma by the global initiative for asthma (gina) program, the prevalence of clinical asthma in korea is estimated to be 3.9% (23). The prevalence of asthma tended to be higher in children than in adults and was found to depend significantly on their place of residence, for example, it was found to be higher in seoul than in provincial cities (7). According to 1998 data (11), childhood asthma was less prevalent in korea than in other developed countries . However, among the elderly (aged 65 yr or older), its prevalence was found to be high at 12.7% in 2001 (15), which is about three times higher than among english or us elderly (24, 25). Although estimations of asthma prevalence in older age groups differ greatly between countries because of overlapping diagnoses, and poor patient perception of symptoms (24), this rate is unexpectedly high . In the elderly, asthma is an important problem because it is usually underdiagnosed and hence inadequately treated (24 - 26). Interestingly, korean adults and children appear to have a later average age of asthma onset than other asian populations (12) (fig . This is likely to be due to the underdiagnosis of early stage asthma by general physicians . Some investigators have reported that an underdiagnosis rate by general physicians was 21% (27). Another contributable factor may be that a high proportion of korean people, at least initially, turn to traditional medicine for medical help (28 - 30), which delays the diagnosis until they are referred to hospitals or emergency department with severe symptoms . Age, sex, affluence, genetic predisposition, climate, outdoor air quality and cigarette smoking have all been found to be associated with asthma (7, 12, 15, 31 - 33). Studies suggest a higher incidence of asthma in children than in adults (9 - 15, 17, 34), and male asthmatic patients were found to be more often hospitalized or treated at a tertiary medical centers than their female counterparts (34, 35). In a regression analysis of the asthma insights and reality in asia - pacific (airiap) data for the eight areas of asia - pacific countries including korea, it was found that a low household income is significantly associated with the likelihood of having moderate or severe persistent asthma symptoms (p=0.002 compared with a high income status) (12). It has been reported that the genetic variations of the chemokine (c - x - c motif) receptor 3 (cxcr3) (36), the signal transducer and activator of transcription 4 (stat4) (37), a disintegrin and metalloprotease 33 (adam33) (38), and tumor necrosis factor - alpha (tnf-) (39) are associated with the susceptibilities to the development of asthma and its intermediate phenotypes in the korean population . The il-4 cytokine gene cluster and/or the t cell receptor / gene complex are believed to be associated with the expression of bronchial responsiveness to methacholone in nuclear families (40), and a polymorphism in exon 7 of chromosome 11q13 was found to be significantly associated with histamine release from basophile to anti - ige stimuli in 80 randomly recruited asthmatic children (41). Spider mites (citrus red mite) (42, 43), the european red mite (panonychus ulmi), (44) and the two - spotted spider mite (tetranychus urtcae) (44), have been identified as important allergens in the development of work - related asthma and rhinitis symptoms and toluene diisocyanate has been most commonly implicated allergen in occupational asthma in korea (45). A number of studies have documented an association between air pollution and asthma - related morbidity in korean cities and have linked active smoking with asthma prevalence and severity (15, 28, 29, 46 - 48). The accurate determination of the asthma mortality in korea is difficult . Because large - scale medical databases tend to group asthma data along with those of other disease states, or to group it with less specific group diagnoses such as' chronic lower respiratory diseases' (49). According to the korea national statistical office data, the death rate from chronic lower respiratory diseases (including asthma) between 1992 and 2002 increased from 12.9 to 22.6 deaths per 100,000 of the population (50). In 1992, chronic lower respiratory diseases were ranked as the eighth leading cause of death in korea, and in 2002, this had risen to fifth place proceeded only by diabetes mellitus, heart disease, cerebrovascular disease, and cancer (50) (fig . 2). According to the latest gina estimates, there are 4.9 asthma related deaths per 100,000 asthmatics each year in korea (23). Globally, respiratory diseases are responsible for 6.3% of all death and asthma for 0.4% (51). These include cigarette smoking, chronic asthma severity, frequent hospitalization, the duration of recent asthma exacerbations, age and a female sex (28, 52, 53). At present, little data is available on the economic burden posed by asthma in korea . However, indirect estimates may be made based on the market shares of asthma medications in korea . According to the data from the pharmaceutical industry (54), from 2001 to 2004, the annual gross value of drugs sold in korea increased by nearly 50% (from u$3.48 to u$5.21 billion) and the proportion shared by drugs for asthma treatment in respiratory drug market increased by approximately 36% (from u$51.8 to u$70.6 million). Research in this area is underway, with a large - scale analysis being performed using data from health insurance review agency (hira), the 1998 national survey on health and nutrition in korea, and an ongoing patient survey . To adequately account for cultural and behavioral factors, a quality of life questionnaire for adult korean asthmatics (qlqaka) was developed (55), and in a multicenter study 5 using qlqaks, it was found that dyspnea (87%), difficulty in sputum discharge or throat clearing (87%), strenuous activity limitation (84%), and coughing (82.4%) were most frequently complained about by adult asthmatics (55). The airiap study revealed that approximately one - third of all korean asthmatic patients felt restricted in terms of their abilities to perform the routine activities of daily living such as, exercise, social activities, or sleeping (12). This survey also reported that 52% of asthmatic patients had experienced daytime symptoms during 4 weeks prior to completing the questionnaires, and that 41% of patients with asthma had been woken at night by their symptoms (12). Another study conducted to assess the quality of life of 189 patients showed that running, walking, and hurried movements appeared to be the most impaired daily activities (56). The characteristics that significantly impaired an individual's quality of life score were disease severity, level of asthma control and symptom attacks during the previous 3 months (all p<0.001) (56). Interestingly, a discrepancy was identified between perceived and actual asthma control in a substantial number of asthma patients . According to airiap data, about one - fifth of korean asthmatic patients with severe persistent symptoms considered that their asthma was adequately controlled, despite their current symptoms suggesting otherwise (12) (fig . This is due in part to the fact that some asthmatics have poor symptom perception (57 - 59) and partly because asthmatics in korea are not adequately educated about the practical and theoretical aspects of asthma management (60, 61). No national guidelines concerning best management practice of asthma have been issued in korea, although three academic associations have published separate guidelines (62 - 64). However, almost half of the physicians (43%) in 325 korean clinics surveyed by the hira either did not use or were unfamiliar with these guidelines (65). Therefore, few asthma patients used or were prescribed inhaled corticosteroid by their physicians to prevent symptoms (65) (fig . 4). The reasons for this reluctance to prescribing inhaled corticosteroid as cited by physicians in the hira survey were, in decreasing order; lack of patient education concerning inhaler use, high cost, patient refusal to use, and side effects (65). In addition, according to the airiap survey, 65% of patients with asthma in korea have never undergone a lung function test, and only 32% of patients reported that they had undergone a lung function test during the previous year (12). Moreover, although 6% of patients owned a peak flow meter for self - monitoring, only 20% of them used it daily, whereas another 20% used it on a symptomatic basis (12). Another survey found that patients who used a nebulizer at home were not properly instructed as to how to use it, and did not follow equipment maintenance procedures (66). However, paradoxically, most korean asthmatics were satisfied with their treatment, although many expressed a need for more information on the disease (12). This' disconnect' from the guidelines by physicians has had a major impact on the management of asthma patients . To reduce this gap, an' easy asthma management' study is currently underway in korea . The purpose of this study may provide a simple stereotyped algorithm using a computer based program to allow clinicians to easily diagnose asthma and implement its correct management . In december 2004, it was estimated that 11.9 million koreans had access to the world wide web (www) and the number of allocated ip addresses allocated in korea ranked eighth worldwide (67). Nowadays, computer - based educational programs available on the www are believed to be an effective alternative as they overcome the cost and time barriers that prevent implementation of education programs for asthmatics (68). However studies have shown that asthma educational material on the web sites is highly variable in quality and content, lacks detail of the core concepts essential for asthma education, and fails to meet patients needs (69, 70). This review showed that the prevalence of asthma in korea ranges from 2 to 13%, and that physicians and patients often underestimate its severity . Mortality from chronic lower respiratory diseases including asthma increased from 12.9 to 22.6 deaths per 100,000 of the population between 1992 and 2002 . Disease severity, level of control, and symptom state were all found to negatively impact the quality of life of asthmatics . Moreover, although international and korean asthma management guidelines are available, primary care physicians are largely unfamiliar with or fail to implement them.
Asthma in adults is a heterogeneous disease usually characterized by chronic airway inflammation with hyperresponsiveness of airways to various stimuli . The prevalence estimates of asthma, in general, and the prevalence of severe asthma, in particular, are well known and sufficiently illustrative . According to the global asthma report 2014,1 asthma affects 334 million people with projections of 400 million in 2025 . In addition, asthma still accounts for one of every 250 deaths worldwide, and almost all of these deaths are avoidable.2 in spain, prevalence rates of 5% and 10% have been estimated in adults and children, respectively, with more than three million people with asthma.35 in addition, there is a large geographical variability is asthma prevalence, with a trend toward stabilization following an increase in the prevalence of the disease observed in the past years.3,6 this complex disease affects patients of all ages . Although its diagnosis is usually easily established and most patients respond to therapy, approximately between 3% and 10% of adult asthma patients have disease that is difficult to control despite taking maximal doses of inhaled medications.7 patients with therapy - resistant or difficult - to - control asthma require a rigorous and systematic approach to their diagnosis and treatment . Despite rigorous, optimized follow - up treatment, 75% of severe asthma patients did not achieve adequate symptom control and presented with impaired quality of life.8 although much progress has been made in the understanding of difficult - to - control asthma, with consensus documents and clinical practice guidelines focused on severe uncontrolled asthma with proposals for a stepwise diagnostic procedure and phenotype - targeted treatment,9,10 improvements in clinical practice are still limited . In addition, involvement of pulmonology services in the specialized approach for difficult - to - control asthma is insufficient . In a survey carried out in spain, 47 (68.1%) of a total of 69 pulmonology services met criteria for an important level of health care activity in asthma, but only 29 (42%) had a monographic consultation for difficult - to - control asthma and 37 (53.6%) had implemented an education program . As for post - graduate education, only 31 (44.9%) provided their resident physicians with specific asthma training.11 in 2015, the asthma area of the spanish society of pneumology and thoracic surgery (separ) addressed the task of establishing the necessary requirements for the provision of official accreditation standards of the different levels of care for asthma units already existing in hospitals of the spanish national healthcare system . Accreditation levels included basic units, specialized units, or specialized units of high complexity, with or without the distinctive of excellence, according to the fulfillment of a series of criteria established by the society . The development and implementation of this nationwide strategic plan had several objectives, including improving the care and quality of life of asthma patients, particularly patients with severe difficult - to - control asthma in order to achieve a decrease and prevention of acute exacerbations, with a subsequent reduction in the number of visits to the emergency department, need of in - patient care, and inadequate use of asthma medications . All these actions have been complemented by the development and implementation of education and self - management strategies (where nursing plays a key and indispensable role) and follow - up of patients in monographic consultations for asthma and in the framework of multidisciplinary involvement of health care professionals . In a recent scientific meeting of health care professionals of the different asthma units, which underwent specialized accreditation, it was agreed that a perspective article should be drafted to achieve greater dissemination of the details regarding the characteristics, service portfolio, and resources available in these units . The present document written and approved by all attendees of this event and members of units provides information and describes the initial global experience of asthma units accredited in spain . It also aims to contribute to raising awareness among pulmonologists and other professionals about the problem of severe refractory asthma and the response offered by asthma units to improve patient care and to minimize the complications and burden of this complex pathology . In this respect, we believe that the spanish experience may be useful and potentially applicable to other settings, in particular with similar health care systems . However, workup details and assessment of outcomes were outside the scope of this report . Significant progress and advances in the understanding of asthma and in the care of asthma patients with an increase in the number of medications and development of new protocolized therapeutic strategies have been associated with a substantial reduction in asthma - specific mortality and hospitalization rates for asthma . However, despite all these improvements and the development of clinical practice guidelines with specific recommendations for the control of asthma, it is well known that in clinical practice, adequate asthma control is achieved in only one - third of patients.1214 uncontrolled severe asthma accounts for ~15% of all asthma patients but is associated with a higher morbidity and mortality as well as an important impact on health care costs and the consumption of resources.15 the asmacost study16 based on data of a prospective cohort of 627 patients throughout spain with asthma diagnosed according to the guidelines of the global initiative for asthma (gina)17 and the adapted spanish criteria (gua espaola de manejo del asma [gema])9 and followed up for 12 months revealed that the total societal cost for asthma was 1,726 per patient annually (1,533 to the national health service), with higher costs for patients older than 65 years and for those with a more severe disease (2,635 for severe asthma). Based on these findings, the total annual cost of asthma in spain was estimated to be 1,480 million euros . On the other hand, 70% of total costs were attributed to the poor control of the disease, which further reinforces the need to achieve clinical stability of the patients as it has been repeatedly emphasized in clinical practice guidelines.9,17 from another perspective, poor asthma control is a determining factor of the high indirect costs related to absenteeism and reduced productivity . In a naturalistic and observational study (tenor study) of 4,756 asthma patients recruited in 283 study sites from diverse geographical areas in the usa and followed up for 24 months, the mean annual costs for uncontrolled patients with difficult - to - treat asthma were more than double of those for controlled patients throughout the study.18 different studies carried out in other countries have obtained similar results.1921 moreover, chronic comorbidities contribute to the burden and costs of persistent asthma,22 and poor adherence to asthma medication regimens, including patients with difficult - to - control asthma, is a key problem contributing to poor disease control.23 adherence to asthma treatment in patients with uncontrolled difficult asthma is highly relevant, since treatments currently considered for step - up therapy (biologics, bronchial thermoplasty)9,17 are very expensive and, in many cases, are prescribed without adequate adherence to drug regimens in the lower steps.24 a further interesting aspect concerns to deficiencies in the development and implementation of effective education programs for asthma patients, promotion of health, and social support . It has been demonstrated that education in self - management of asthma with symptom or peak flow monitoring, combined with regular medical visits and written action plans, is effective in improving health outcomes in adults with asthma.25,26 in a 1-year cluster randomized controlled multicenter study with the participation of 230 adults with mild - to - moderate persistent uncontrolled asthma, an asthma educational program based on a repeated short intervention, given in four face - to - face sessions at 3-month intervals was effective in improving asthma symptom control, future risk, and quality of life.27 in this study, the education program included administration of a written personalized action plan and training on inhaler technique.27 other experiences reported similar results . In a controlled clinical trial in which a comprehensive asthma intervention program was evaluated in a population of medicaid - insured asthmatic children, a significant improvement in health outcomes (emergency department visits, hospitalizations) and asthma health care costs was observed in the intervention group in the year after enrollment.28 in a randomized patient selection study with crossover, a vigorous medical regimen and intensive educational program were able to decrease hospital use among a group of adult asthmatics who had previously required repeated readmissions for acute asthma exacerbations.29 in a large teaching hospital (glasgow royal infirmary) where asthma management was audited prospectively for 1 year, treatment of asthma patients in wards with a specialist interest in respiratory medicine was associated with a reduction in the rate of readmissions compared to patients admitted to general wards without this special interest (2% vs. 20%).30 given the complexity and multiple factors involved in the control of asthma, there is a need for establishing asthma units involved in the care of asthma patients, especially those patients with severe difficult - to - control disease . However, up to the present time, a few studies have demonstrated that assessment and management of patients in specific units for severe asthma are associated with substantial benefits in terms of health (asthma control, quality of life) and reduction in economic burden . In a study of 346 patients with severe asthma referred to specialist centers across the uk and followed up for a median of 286 days, significant reductions in health - care use (primary care or emergency department visits), hospital admissions, and steroid dose were observed, which were accompanied by significant improvements in quality of life and asthma control.31 in a crossover study carried out in spain, treatment of patients in asthma clinics was cost - effective and beneficial in asthma management in comparison with standard outpatient services.32 the national asthma program in finland, probably one of the most outstanding health care networks for asthma patients, has shown that integration of different health care levels involved in the management of asthma (pneumologists, primary care physicians, and pharmacists) improves control of the disease and reduces the morbidity of asthma.33 the separ has promoted the task of accrediting the levels of the different asthma units already existing in our country, with the following objectives: 1) to improve the level of care of asthma patients, ensuring a framework of quality of care; 2) to establish resources and facilitating their management; 3) to promote the development of training plans in asthma and to advance in the concept of accreditation of knowledge; 4) to favor collaboration with professionals from other clinical disciplines in a cooperative environment; and 5) to promote asthma research . Within the separ training framework, a manual on severe asthma and difficult - to - control asthma has been published, which includes a chapter on the provision and organization of a severe asthma unit.34 briefly, three levels of specialized asthma care have accredited based on available resources: specialized unit for highly complex asthma, specialized asthma unit, and basic asthma unit . Regardless of the level of accreditation obtained, the distinction of excellence could be granted when more requirements were met at each level . In order to obtain accreditation at each of the levels, there were indispensable requirements (irs) that had to be met; also, there were two other criteria, which included evaluable criteria (ec) and recommended criteria (rc). The certification of each level was achieved if at least 80% of the ec corresponding to each category were fulfilled . The quality of each level was excellent if the result of the formula (ec + rc) 100/(total number of ec + rc items at the level) was 80% . The integral care of the patient with severe asthma in a specialized multidisciplinary and high - quality setting presents many advantages, particularly related to the diagnosis of asthma, including identification of patients according to phenotypes, treatment of comorbidities, protocolized followup, optimization of the therapeutic arsenal, indication of specific treatments, and emphasis on health education for both patients and health care professionals . For instance, to have available a specialized nurse well trained in asthma education and use of inhaler devices will result in a better understanding of the disease, adherence to treatment, and control of the disease . In addition, there is a high prevalence of psycho - comorbidity in asthma, and acting at this level can improve the quality of life of the patients, control of anxiety, low consumption of resources, etc . In this respect, likewise, the possibility of implementing complementary programs contributes to better control of the disease . This approach is associated with cost savings as a result of a better and rational use of the resources . In addition, accredited asthma units could help patients with asthma through identifying misdiagnosed cases of asthma and providing more appropriate treatment / referral . Misdiagnosis of non - asthmatic conditions as uncontrolled asthma has been reported to be as high as 12%30%.35,36 the implementation of databases with relevant and updated clinical information is an essential tool for the independent analysis of the data of each unit or aggregated data from different units . This is effectively a proposal for the control and improvement of care provided to our patients, the results of which are pending to be collected and evaluated . We believe that diagnosis and treatment by highly specialized and experienced personnel in asthma will result in the achievement of a better control of disease . A recent study carried out in an asthma clinic of a university - affiliated hospital in lugo in 2012 showed that all cost variables except drugs and diagnostic tests were significantly reduced in comparison with standard outpatient services, giving an annual saving per patient of 338.32 the assessment and management of patients with severe difficult - to - control asthma in accredited asthma units aims to improve the quality of care and control of the disease . In addition, specialized asthma units can improve the cost - effectiveness of pharmaceutical expenditure, especially regarding the new and costly therapies . Health care professionals involved in the management of asthma should continue pursuing for unifying the quality of care of patients with asthma in a multidisciplinary collaborative setting.
There are similarities between children and adults suffering from heart failure (hf), such as the preferred pharmacological treatment (1), the use of pace - makers and heart transplants (2,3), the inability of the patient to reach the predicted heart rate for the patient's age during cardiopulmonary exercise testing (4,5), and the ergoespirometric response under similar clinical conditions (5). In adults, endothelial dysfunction is related to the development of diastolic dysfunction (6,7), chagas disease, left ventricular hypertrophy (8), ischemic cardiomyopathy, hf (8,9), obesity, type 1 diabetes, hyperlipidemia, arterial hypertension (10), peripheral arterial disease, chronic kidney disease (11) and atherosclerosis (12) because the dysfunction predisposes the vasculature to vasoconstriction, leukocyte adherence, platelet activation, and vascular inflammation (13). Nevertheless, there is a lack of data regarding endothelial function in children with cardiomyopathy . The severity of endothelial dysfunction is related to the cardiovascular risk (14), the severity of cardiovascular symptoms (15), and the inability to exercise (11) and represents a predictor for cardiac transplant and death (16). It is known that diseases, such as kawasaki's disease (8), hyperlipidemia (10), obesity, and type 1 diabetes, play important roles in systemic inflammation and endothelial dysfunction (17). These diseases may increase the likelihood of cardiovascular events (18) and may predispose children to the development of cardiomyopathy . Based on these considerations, we reviewed the published literature on endothelial function in pre - pubertal children to evaluate the endothelial function in pre - pubertal children with cardiomyopathy or children at risk of developing cardiomyopathy, and we conducted an analysis of the data from the relevant studies . This analysis was undertaken to help clarify the role of endothelial impairment in children at risk of suffering from cardiomyopathy . Endothelial function can be analyzed by noninvasive methods, including ultrasonography (us) (19) and peripheral artery tonometry (pat) (20). During a us examination, the baseline rest image of the subject's brachial artery is acquired, and a 5-min arterial occlusion is performed using cuff inflation to at least 50 mm hg suprasystolic pressure . The subsequent cuff deflation induces reactive hyperemia that results in an increase in flow or, more precisely, shear stress by dilating the brachial artery; this phenomenon is designated flow - mediated dilatation (fmd). After returning to the baseline, a second brachial artery image is recorded after the administration of nitroglycerine (ntg); this image corresponds to the contribution of the intima muscle relaxation to the dilation and is known as the endothelium - independent vasodilatation (19). In contrast to us, the pat evaluation is a method that does not require the administration of drugs, and it combines the assessment of the flow - mediated dilatation after the same 5-min arterial cuff occlusion, with the arterial pulse wave amplitude measurement taken using a pneumatic fingertip probe (20). A search of the pubmed, bireme, and scielo databases was conducted to perform a systematic review, according to the recommendations of prisma (21). The search was performed using the following keywords: endothelial, child, pediatrics, and infant . The results were limited to human studies published in english, spanish, and portuguese . The initial selection was based on the title and abstract, and those deemed relevant were retained for further analysis . The exclusion criteria included the following: analyses of the endothelial function in animals, cadavers, adolescents or adults; reviews; analyses of either the coronary or pulmonary arteries or the neurological or osteomuscular systems; if the patients had rheumatic, oncohematological, splenic or hepatic diseases; or if the studies were trials related to markers, genetics and interventions . The list of potential trials to include was verified by the authors of the present study . We note that there may be a risk of bias across the studies because not all of the trials published have been indexed in the selected databases . The following data were collected for analysis: the number of patients enrolled in the study; the patient's age; whether sexual maturity had been reached; any diseases involved; the protocol used for the endothelial function evaluation and the fmd, ntg, and pat values . Collected data in all tables were expressed following disease order, in order to better clarify data presentation and analysis . The values of endothelial function were expressed as the mean standard deviation (sd), as collected from the original trials . The data from healthy children were compared with the data for unhealthy children, and the differences were analyzed using the student t - test . A total of 559 articles were retrieved after the preliminary search, and 21 articles were considered potentially relevant based on the title and abstract . A careful analysis of these articles was performed, and ten trials were excluded for several reasons . Table 1 presents the general descriptions of the included studies and provides information regarding the participants, their ages, their diseases, the endothelial evaluation method used and whether there were differences between genders (table 1). All eleven of the selected studies provided data for healthy children for comparison, and, with the exception of one study (22), all of the studies compared healthy children to ill children . Four of the studies evaluated obese children (23 - 26), one evaluated children with obesity and type 1 diabetes (27), three studied children with type 1 diabetes (28 - 30), and two evaluated children with kawasaki's disease (31 - 32). These reviewed studies reported a mean age of 9.81.8 years old, and only four articles investigated the sexual maturation of the group of pre - pubertal children (23,25,26,29). Forty - five percent of all of the studies found no difference in the endothelial function between genders, and 55% of the reports did not provide these data (table 1). Most of the studies used ultrasound to determine the percent diameter changes for the fmd and ntg assessments (22 - 29,31,32). Only one study used reactive hyperemia peripheral artery tonometry (rh - pat) for the same purpose (30), showing that the endothelial function was impaired in diabetic children relative to that in healthy children (1.630.5 vs. 1.950.3 for diabetic children vs. healthy children) (30). The fmd response values from all of the other articles for obese and diabetic children were significantly lower than those for healthy children (5.91.29% for obese children, 4.50.7% for diabetic children, and 9.81.8% for healthy children; p<0.0008). The obese children had a 39% lower fmd response than the healthy children, and the diabetic children had a decrease in their fmd of 25% (table 2). The children with kawasaki's disease had 27% lower fmd responses than the healthy children (7.11.27% vs. 9.81.8%, respectively), but this difference was not statistically significant (p = 0.26). The fmd data for the obese children, diabetic children, and children with kawasaki's disease exhibited no statistically significant differences (p<0.38) (table 2). The ntg response (table 2) also showed no significant difference among the groups (22.77.1% for healthy children, 19.62.0% for obese children and 16.65.9% for diabetic children; p<0.19). Among the excluded studies, one contained no data on endothelial function (33), and most of the excluded studies did not list the data for pre - pubertal children, post - pubertal children (34 - 39) and adults (40,41) separately . Another study (42) published data for the same patients as used in a previously published study (32). Ultrasound is the most frequently used method to evaluate endothelial function because it has a low cost, is safe and is sufficiently reproducible . However, some limitations of this method are that the fmd response can be influenced by both temperature and age, and ultrasound requires drug administration and the recording of images for posterior analysis (19). Another technique for the assessment of endothelial function is the pat method, which has been proposed to be more practical and precise (20), and pat results are moderately significantly correlated with us results (43). The pat method combines the traditional flow - mediated dilatation measurement with pneumatic fingertip probes to measure the arterial pulse wave amplitude (20). One study (31) chose pat over us and confirmed that diabetic children have endothelial dysfunction, as do adults (44,45). In five studies (22,24,25,28,32), the gender of the individual did not correlate with the endothelial function, as has been found for adults (60) and adolescents (61). Pre - pubertal children show lower responses to beta - adrenergic receptors and have lower levels of circulating catecholamines (adrenaline and noradrenaline) than adolescents and adults (46). The obese children exhibit accelerated growth and, therefore, earlier puberty (47), which is evidenced by an advancement of menarche by 2.2 years (11.2 vs. 13.4 years, respectively) (48) because fat mass influences the levels of hormones (49,50). These data reinforce the need for the evaluation of sexual maturation in studies involving children . The extent of sexual maturation is widely accessed using the tanner - whitehouse scale, which is based on secondary sexual characteristics, such as breast development and menarche in girls, standards for penis development in boys, and pubic hair development in both sexes (51). This lack of sexual maturity assessment explains why those trials that combined information for children, adolescents (33 - 39), and adults (40,41) were excluded in the present study and explains the elevated fmd data from one study (24), which included a higher mean age of obese children of 124 years old . The evaluation of sexual maturity also explains why three of the included studies (23,25,26) that assessed the pubertal stage by clinical examination yielded similar fmd data . Diabetic and obese individuals have hyperinsulinemia, and both diabetes and obesity are related to the higher hormone (50) and inflammation levels (52) that contribute to increased arterial stiffness and vascular lesions, which are related to both endothelial function and structural arterial changes (53) and may explain the endothelial dysfunction results for these groups (23 - 30). In addition to some methodological disparities, all of the studies showed significantly impaired endothelial function (fmd response) in the obese and diabetic children despite the lack of a difference in the ntg response, a result that was reported for adults (54) when compared with healthy subjects (55). However, fmd impairment seems to be greater in adults than in children (44% in obese adults and 55% in diabetic adults); similarly, adults show no difference in ntg responses (54,56). The collected fmd values in children with kawasaki's disease are lower than those in healthy children (31,32), which can indicate a low, but persistent, level of inflammation (57), as evidenced by higher c - reactive protein (crp) levels (52,53,58). Only one study (32) included ntg data, and this lack of data limits the discussion of the information provided . The fmd values suggest that the major dysfunctions in these children occur as a result of the local nitric oxide (no) bioavailability in the endothelium (no production) because the shear stress induced by reactive hyperemia activates endothelial no production (59). The ntg response also suggests that the vascular smooth muscle cell function is preserved (19). Yet, one study showed significantly lower gt values in obese children than in healthy children (27), and two other studies did not publish values for the ntg response (22,24). Some reports (20,25,28) did not describe how long the subjects were at rest, according to corretti et al . (22),> 10 min is recommended, between the collections of the fmd and ntg response images, which might have contributed to the reported outcomes . It is also worth noting that three of the trials (24,27,29) allowed the arteries to return to the basal condition and that two of these studies (24,29) did not yield statistically significant data . In spite of the statistically significant differences in some of the endothelial function responses, the number of trials regarding this subject remains limited . Another limitation of the data search for pre - pubertal children is that the studies list the results for adults, teenagers, and children together, and do not divide the data by group according to the puberty state and age . The small number of studies in children may also be due to the use of the us method because drug administration is required . However, the new method of endothelial function evaluation, the pat method, might help increase the number of trials using this population . Although endothelial dysfunction has been identified in adults as a predictor of cardiac transplant and death, the clinical implications of endothelial dysfunction were not presented in the selected trials, nor were they the focus of the current study; however, this correlation should be further evaluated in children . Lastly, even though the number of pre - pubertal children with endothelial dysfunction is unknown, there is a great number of children who suffer from this condition . Thus, the barriers in the pediatric field must be broken, and more studies on this topic should be performed to understand this population better . In conclusion, endothelial function is an important clinical feature because it may indicate the severity of cardiovascular symptoms, prognosis, and mortality . Children at risk of developing cardiomyopathy exhibit endothelial dysfunction; however, the prevalence of endothelial dysfunction in cardiomyopathic children remains unknown because of the lack of data . We suggest that attention should be paid to the consequences of endothelial function in this group . A search of the pubmed, bireme, and scielo databases was conducted to perform a systematic review, according to the recommendations of prisma (21). The search was performed using the following keywords: endothelial, child, pediatrics, and infant . The results were limited to human studies published in english, spanish, and portuguese . The initial selection was based on the title and abstract, and those deemed relevant were retained for further analysis . The exclusion criteria included the following: analyses of the endothelial function in animals, cadavers, adolescents or adults; reviews; analyses of either the coronary or pulmonary arteries or the neurological or osteomuscular systems; if the patients had rheumatic, oncohematological, splenic or hepatic diseases; or if the studies were trials related to markers, genetics and interventions . The list of potential trials to include was verified by the authors of the present study . We note that there may be a risk of bias across the studies because not all of the trials published have been indexed in the selected databases . The following data were collected for analysis: the number of patients enrolled in the study; the patient's age; whether sexual maturity had been reached; any diseases involved; the protocol used for the endothelial function evaluation and the fmd, ntg, and pat values . Collected data in all tables were expressed following disease order, in order to better clarify data presentation and analysis . The values of endothelial function were expressed as the mean standard deviation (sd), as collected from the original trials . The data from healthy children were compared with the data for unhealthy children, and the differences were analyzed using the student t - test . A total of 559 articles were retrieved after the preliminary search, and 21 articles were considered potentially relevant based on the title and abstract . A careful analysis of these articles was performed, and ten trials were excluded for several reasons . Table 1 presents the general descriptions of the included studies and provides information regarding the participants, their ages, their diseases, the endothelial evaluation method used and whether there were differences between genders (table 1). All eleven of the selected studies provided data for healthy children for comparison, and, with the exception of one study (22), all of the studies compared healthy children to ill children . Four of the studies evaluated obese children (23 - 26), one evaluated children with obesity and type 1 diabetes (27), three studied children with type 1 diabetes (28 - 30), and two evaluated children with kawasaki's disease (31 - 32). These reviewed studies reported a mean age of 9.81.8 years old, and only four articles investigated the sexual maturation of the group of pre - pubertal children (23,25,26,29). Forty - five percent of all of the studies found no difference in the endothelial function between genders, and 55% of the reports did not provide these data (table 1). Most of the studies used ultrasound to determine the percent diameter changes for the fmd and ntg assessments (22 - 29,31,32). Only one study used reactive hyperemia peripheral artery tonometry (rh - pat) for the same purpose (30), showing that the endothelial function was impaired in diabetic children relative to that in healthy children (1.630.5 vs. 1.950.3 for diabetic children vs. healthy children) (30). The fmd response values from all of the other articles for obese and diabetic children were significantly lower than those for healthy children (5.91.29% for obese children, 4.50.7% for diabetic children, and 9.81.8% for healthy children; p<0.0008). The obese children had a 39% lower fmd response than the healthy children, and the diabetic children had a decrease in their fmd of 25% (table 2). The children with kawasaki's disease had 27% lower fmd responses than the healthy children (7.11.27% vs. 9.81.8%, respectively), but this difference was not statistically significant (p = 0.26). The fmd data for the obese children, diabetic children, and children with kawasaki's disease exhibited no statistically significant differences (p<0.38) (table 2). The ntg response (table 2) also showed no significant difference among the groups (22.77.1% for healthy children, 19.62.0% for obese children and 16.65.9% for diabetic children; p<0.19). Among the excluded studies, one contained no data on endothelial function (33), and most of the excluded studies did not list the data for pre - pubertal children, post - pubertal children (34 - 39) and adults (40,41) separately . Another study (42) published data for the same patients as used in a previously published study (32). Ultrasound is the most frequently used method to evaluate endothelial function because it has a low cost, is safe and is sufficiently reproducible . However, some limitations of this method are that the fmd response can be influenced by both temperature and age, and ultrasound requires drug administration and the recording of images for posterior analysis (19). Another technique for the assessment of endothelial function is the pat method, which has been proposed to be more practical and precise (20), and pat results are moderately significantly correlated with us results (43). The pat method combines the traditional flow - mediated dilatation measurement with pneumatic fingertip probes to measure the arterial pulse wave amplitude (20). One study (31) chose pat over us and confirmed that diabetic children have endothelial dysfunction, as do adults (44,45). In five studies (22,24,25,28,32), the gender of the individual did not correlate with the endothelial function, as has been found for adults (60) and adolescents (61). Pre - pubertal children show lower responses to beta - adrenergic receptors and have lower levels of circulating catecholamines (adrenaline and noradrenaline) than adolescents and adults (46). The obese children exhibit accelerated growth and, therefore, earlier puberty (47), which is evidenced by an advancement of menarche by 2.2 years (11.2 vs. 13.4 years, respectively) (48) because fat mass influences the levels of hormones (49,50). These data reinforce the need for the evaluation of sexual maturation in studies involving children . The extent of sexual maturation is widely accessed using the tanner - whitehouse scale, which is based on secondary sexual characteristics, such as breast development and menarche in girls, standards for penis development in boys, and pubic hair development in both sexes (51). This lack of sexual maturity assessment explains why those trials that combined information for children, adolescents (33 - 39), and adults (40,41) were excluded in the present study and explains the elevated fmd data from one study (24), which included a higher mean age of obese children of 124 years old . The evaluation of sexual maturity also explains why three of the included studies (23,25,26) that assessed the pubertal stage by clinical examination yielded similar fmd data . Diabetic and obese individuals have hyperinsulinemia, and both diabetes and obesity are related to the higher hormone (50) and inflammation levels (52) that contribute to increased arterial stiffness and vascular lesions, which are related to both endothelial function and structural arterial changes (53) and may explain the endothelial dysfunction results for these groups (23 - 30). In addition to some methodological disparities, all of the studies showed significantly impaired endothelial function (fmd response) in the obese and diabetic children despite the lack of a difference in the ntg response, a result that was reported for adults (54) when compared with healthy subjects (55). However, fmd impairment seems to be greater in adults than in children (44% in obese adults and 55% in diabetic adults); similarly, adults show no difference in ntg responses (54,56). The collected fmd values in children with kawasaki's disease are lower than those in healthy children (31,32), which can indicate a low, but persistent, level of inflammation (57), as evidenced by higher c - reactive protein (crp) levels (52,53,58). Only one study (32) included ntg data, and this lack of data limits the discussion of the information provided . The fmd values suggest that the major dysfunctions in these children occur as a result of the local nitric oxide (no) bioavailability in the endothelium (no production) because the shear stress induced by reactive hyperemia activates endothelial no production (59). The ntg response also suggests that the vascular smooth muscle cell function is preserved (19). Yet, one study showed significantly lower gt values in obese children than in healthy children (27), and two other studies did not publish values for the ntg response (22,24). Some reports (20,25,28) did not describe how long the subjects were at rest, according to corretti et al . (22),> 10 min is recommended, between the collections of the fmd and ntg response images, which might have contributed to the reported outcomes . It is also worth noting that three of the trials (24,27,29) allowed the arteries to return to the basal condition and that two of these studies (24,29) did not yield statistically significant data . In spite of the statistically significant differences in some of the endothelial function responses, the number of trials regarding this subject remains limited . Another limitation of the data search for pre - pubertal children is that the studies list the results for adults, teenagers, and children together, and do not divide the data by group according to the puberty state and age . The small number of studies in children may also be due to the use of the us method because drug administration is required . However, the new method of endothelial function evaluation, the pat method, might help increase the number of trials using this population . Although endothelial dysfunction has been identified in adults as a predictor of cardiac transplant and death, the clinical implications of endothelial dysfunction were not presented in the selected trials, nor were they the focus of the current study; however, this correlation should be further evaluated in children . Lastly, even though the number of pre - pubertal children with endothelial dysfunction is unknown, there is a great number of children who suffer from this condition . Thus, the barriers in the pediatric field must be broken, and more studies on this topic should be performed to understand this population better . In conclusion, endothelial function is an important clinical feature because it may indicate the severity of cardiovascular symptoms, prognosis, and mortality . Children at risk of developing cardiomyopathy exhibit endothelial dysfunction; however, the prevalence of endothelial dysfunction in cardiomyopathic children remains unknown because of the lack of data . We suggest that attention should be paid to the consequences of endothelial function in this group . Guilherme v guimares (cnpq #304733/2008 - 3) was supported by conselho nacional de pesquisa.
Acetone is the simplest ketone compound . In general, acetone is not considered harmful, and the world health organization has not classified acetone as carcinogenic . However, its prolonged inhalation cannot only cause irritation of the mucous membranes, headaches, confusion, and narcotic effects, but lead to coma as well [15]. For etiological reasons, acetonaemia is classified to be of endogenous and exogenous origin [6, 7]. Multiple toxicities and physiopathological conditions result in ketosis (acetonaemia particularly), including diabetes mellitus (dm), starvation coupled with physiologic stress, prolonged exercise, during pregnancy, and ethanol toxicity, other alcohol ingestions, drug toxicities, inborn errors of ketone metabolism, alcoholic ketoacidosis, delirium tremens, and hypothermia [69]. The main symptom of dm is a high blood glucose concentration depending on insulin deficiency . In this case the body cannot fully use glucose but could use fatty metabolism instead of glucose for energy . Dm, especially diabetes and autoimmune associated diseases, thyroid disease, and diseases that can accompany diabetes (hypertension, cardiovascular disease, cerebrovascular disease, and renal insufficiency) can cause pathological changes in most of the tissues, organs, and biological fluids depending on lipotoxicity and glucotoxicity . A lot of medical, chemical, and medicolegal investigations have been carried out with the determination of acetone in blood and other biological fluids [2, 6, 7, 10]. Over the decades, several methods have been used for its determination in biological samples . In the beginning, colorimetric methods were developed and used for the determination of acetone in plasma [1113]. These methods have common disadvantages such as the lack of specificity and detection limit . In recent decades, gas chromatographs equipped with flame ionization detectors or mass spectrometric detectors have been developed for determination of acetone concentrations in body fluids and in expired air [1418]. Enzymatic methods are more specific but more complex and have long assay times and gas chromatographic methods, although widely used, are applied with difficulty as routine tests . The determination of acetone in the blood is most important in clinical diagnostic laboratory studies . There are three ketone bodies, while the two main ketone bodies are acetoacetate (acac) and 3-b - hydroxybutyrate (3hb), the third ketone body; acetone (ac) is found minimum level . Ketone bodies are produced by the liver and used peripherally as an energy source when glucose is not readily available [9, 19]. Ketone bodies are three water - soluble compounds that are produced as by - products when fatty acids are broken down for energy in the liver and kidney . Ketone bodies are produced from acetyl - coa mainly in the mitochondrial matrix of hepatocytes when carbohydrates are so scarce that energy must be obtained from breaking down fatty acids [9, 20]. Acetone cannot be converted back to acetyl - coa, so it is excreted in the urine or exhaled . Recently, blood or urine testing kits have been used to test for the presence of acetone in clinical biochemistry laboratories . Acetone can also be quantified by sampling the human blood and testing by gas chromatography . Described a rapid and simple hplc procedure that can be used for the routine measurement of acetone in biological fluids, such as plasma and urine . According to fujii et al ., it is proposed that liquid chromatography with fluorescence (lc - fl) seems to be useful for the determination of acetone in the saliva . In this paper, we present a rapid and simple hplc technique using 2,4-dnph as a derivatizing reagent for quantitative determination and metabolomic research of acetone in biological fluid such as human blood . 2,4-dnph (sigma - aldrich, 97%), acetone (merck, 99.8%), acetonitrile (sigma - aldrich, 99.8%), and methanol (merck, 99.8%) were used in this study . For the chromatographic analysis, thermo scientific dionex ultimate 3000 hplc with a thermoacclaim - c18 (15 cm 4.6 mm 3 m) column and uv - vis dad were used . The deionized water was 18.2 mcm (millipore direct - q3 uv) and was used throughout the experiments . For the biochemical analyses, cobas 6000 (roche, germany) autoanalyzer was used for blood glucose levels . Qualitative analysis of total ketones in urine was evaluated by iris iricel 2000 analyzer (icem velocity). In the first stage of our study, following a 12-hour fasting venous blood samples were taken from patients admitted to hospital of the faculty of medicine, canakkale onsekiz mart university . The human blood and urine samples were directly collected into a tube (without a collection device) between 08:30 and 11:00 am . Clinical biochemistry laboratory blood and urine glucose tests were studied for routine biochemistry using a urine autoanalyzer . The patients were divided into high blood glucose and urine ketone positive subjects (group 1) and high blood glucose and urine ketone negative example subjects (group 2). The patients with hyperglycemia were 8 females and 7 males (age: 2187; n = 15), while 5 female and 2 male patients had positive urine ketones (age: 2168, n = 7) and 5 male and 3 female patients had negative urine ketones (age: 5587, n = 8). The blood glucose levels varied between 110 and 320 mg / dl in our patients (table 1). In the second stage of the study, to determine the probable positive acetone, its quantitative analysis was carried out in biological fluids using the hplc technique . The blood samples were immediately prepared for hplc analysis carried out within 8 hours after sample collection . Plasma specimens were deproteinized with acetonitrile (1: 1, v / v); 2,4-dnph is added to the supernatant (filtered blood samples) and treated with acetonitrile (2: 1, v / v) to prevent crystallization of the synthesized phenylhydrazone . An aliquot (20 microliters) of the reaction mixture was subjected to hplc at ambient temperature using thermoacclaim - c18 (15 cm 4.6 mm 3 m) column and uv - vis dad with (methanol / acetonitrile) water as eluent at a flowrate of 1 ml min and detection at 365 nm [2, 3]. The experimental procedures were conducted in accordance with the ethical standards of the helsinki declaration and approved by the canakkale onsekiz mart university human research ethics committee . Written informed consent was obtained from all the subjects . The 2,4-dinitrophenylhydrazone standards were prepared by mixing a and b solutions: (a): 0.40 g of 2,4-dnph dissolved in 2.00 ml of h2so4 + 3.00 ml of h2o + 10.0 ml ethanol; (b): 0.50 g or 1.00 ml of the acetone standard dissolved in 20.0 ml ethanol . After this mixing, a precipitate was formed in each case, isolated through filtration, and dried in vacuum [3, 24, 25]. Acetone was added into its 2,4-dnph derivatives by mixing 1.00 ml of a solution containing 200 mg/100 ml of 2,4-dnph with 1.0 ml of h3po4, and 4.00 ml of the human serum . After 2 h, a 40.0 l aliquot was withdrawn and analyzed by the hplc technique [3, 2427]. Chromatographic separation was achieved in a thermoacclaim - c18 (15 cm 4.6 mm 3 m) column at uv - vis dad detector (max 365 nm). The injection volume was 20.0 l and the detection was performed at 365 nm . The following gradient was used: (methanol / acetonitrile) (8: 2) water 60: 40 (v / v). Elution was achieved at retention time (tr) 12.10 and flow - rate of 1 ml min . Standard calibration curve was prepared with acetone (0.5, 2.5, 5.0, 10, and 20 mmol l) and 40 l 2,4-dnph in acetonitrile . Human samples were prepared by adding 40 l 2,4-dnph and 500 l acetonitrile to 200 l human serum . The calibration curve was constructed by plotting the peak area of the 2,4-dnph derivative of acetone (y) versus the concentration of acetone (x, mmol l) by linear regression (n = 4). The equation was found as y = 0.7361x + 0.0877 with a 0.9967, correlation coefficient (r). The method proposed was validated as described in ich guidelines in parameters of linearity, limit of detection and quantification, accuracy, and precision . The linearity of the method was determined at four concentration levels ranging from 0.5 to 20 mmol l. the calibration curves were constructed by plotting the peak area of the 2,4-dnph derivative of acetone (y) versus concentration of acetone (x). The slope, y - intercept, and correlation coefficient were calculated . To check the linearity, the lod was estimated using signal - to - noise ratio of 3: 1 or (3 s / m) and loq as 10: 1 or (10 s / m), at which accuracy and standard deviation were within 20% as per ich [28, 32, 34, 35]. Intraday accuracy and precision were performed for acetone at 5.0 mmol l in replicate (n = 3). Accuracy (expressed as recovery) and precision (expressed as relative standard deviation) should not deviate by 15% of the nominal concentration . As can be seen from figure 1, the retention time (tr) was obtained as 3.80 min . The most efficient separation of acetone as its 2,4-dnph was obtained with a thermoacclaim c18 column (15 cm 4.6 mm 3 m) at retention time (tr) 12.10 min and flowrate of 1 ml min using a (methanol / acetonitrile) water elution gradient . No elution problem for acetone as its 2,4-dinitrophenylhydrazone derivative was observed . Typical hplc chromatogram of 0.681 mmol l acetone as its 2,4-dnph derivative is given in figure 2 . The hplc chromatogram of 15 mmol l acetone in human blood in the first patient of group 1 is given in figure 3 . In our study, the patients were determined as high blood glucose and urine ketone positive subjects (group 1) and high blood glucose and urine ketone negative example subjects (group 2). The blood glucose levels, given in table 1, vary between 110 and 320 mg / dl in our patients, except for sample 7 . Many patients in our study have dm disease, except samples numbered 4, 5, 11, 13, and 15 (table 1). The calibration plot of peak area against concentration was obtained linear in the range 0.5 to 20 mmol l. the regression equation and correlation coefficient were obtained as y = 0.7361x + 0.0877 with a 0.9967, correlation coefficient (r). So this value is smaller than 2.2, and calibration curve is linear . For the human plasma samples, fcritical value for calibration curve at 3 degrees of freedom (p = 0.05) is 3.18 . So, this value is smaller than fcritical value, and obtained values are appropriate . The lod and loq were found as 0.041 and 0.136 mmol l, respectively . The accuracy for acetone in human plasma samples expressed as recovery was found as 98% . For interday assay, the accuracy for acetone in human plasma samples expressed as recovery was found as 96% . 2,4-dnph can be used to qualitatively detect the carbonyl functionality of a ketone such as acetone or aldehyde functional group . A positive test is signaled by a yellow, orange, or red precipitate known as a dinitrophenylhydrazone . If the carbonyl compound is aromatic, then the precipitate will be red; if aliphatic, then the precipitate will have a more yellow color . The reaction between 2,4-dnph and a ketone such as acetone is shown below:(1)rrco+c6h3no22nhnh2c6h3no22nhncrr+h2othis reaction can be described as a condensation reaction, with two molecules joining together with loss of water . It is also considered an addition - elimination reaction: nucleophilic addition of the -nh2 group to the c = o carbonyl group, followed by the removal of an h2o molecule . 2,4-dnph does not react with other carbonyl - containing functional groups such as carboxylic acids, amides, and esters . For carboxylic acids, amides, and esters, there is resonance associated stability as a lone pair of electrons interacts with the p - orbital of the carbonyl carbon resulting in increased delocalization in the molecule . Also with carboxylic acids there is the effect of the compound acting as a base, leaving the resulting carboxylate negatively charged and hence unable to be attacked by this nucleophile [36, 37]. In this study, an analytical method was applied for the quantitative determination of acetone in human blood . The determination was carried out using a uv - vis dad detector with hplc . In most of our patients' blood samples, higher levels of acetone have been measured in the patients who have high level of blood glucose and positive urine ketone (group 1). The hplc method based on the labeling of acetone with 2,4-dnph seems to offer a rapid, low cost, sensitive, selective, and reproducible methodology for quantification of the acetone level in clinical samples such as human blood . The hplc method described here overcomes many of the problems in the determination of acetone in biological fluids and the preanalytical errors . The volatile ketone is promptly stabilized by conversion into its dnph derivative and rapidly determined without recourse to a solvent extraction step . The method uses very inexpensive reagents . As can be stated by brega et al ., the proposed hplc method can therefore be used to great advantage over current gas chromatographic methods; it can be used in experiments requiring multiple samples and specific activity determination for the routine measurement of acetone in diabetic patients and in biological monitoring of exposed workers . We also presented acetone as a useful tool for the hplc - based metabolomics investigation of endogenous metabolism and quantitative clinical diagnostic analysis . In the medical and medicolegal practice however, the detection and early identification of acetone could be used as an initial indicator of detection of all these physiopathological conditions and the biological monitoring test and to determine further diagnostic management and timely treatment . Determination of acetone levels in blood may be a valid clinical approach in the symptomatic or / and nonsymptomatic cases in the literature for determining treatment strategy and controlling glycemic levels of patients . Consequently, the advanced studies which evaluate blood and urine ketone bodies level together should be performed in different physiopathological conditions including clinical and forensic toxicological studies.
In addition to being a crucial underlying circumstance in the primary cardiovascular risk factors (cvrfs), obesity is an independent risk factor for cardiovascular illness and mortality [1, 2]. Increased body mass index (bmi) alters the behavior of adipose tissue, which provides insulin resistance as well as resistance to type-2 diabetes, arterial hypertension, dyslipidemia, and proinflammatory and prothrombotic states, thereby favoring the onset of ischemic cardiopathy . An estimated 42.3% of coronary episodes in the spanish population may be attributable to excess weight after adjusting for age, sex, and other risk factors . It would therefore be logical to expect obesity to have a lethal effect on patients who have suffered a coronary event . However, some studies have reported better short- and medium - term prognoses in overweight coronary patients [5, 6]. This situation, in which obesity seems to protect patients with acute coronary syndrome (acs), has been called the obesity paradox and has been described in other facilities . Other authors have obtained conflicting results and question the existence of this protective effect [7, 8]. In light of this controversy, the goal of this study is to determine the relationship between bmi and intrahospital mortality in patients admitted consecutively for acs . All patients admitted consecutively between 2009 and 2010 for acs were included in the renaci database of the working group on ischemic heart disease and coronary care units of the spanish cardiology society . A total of 853 patients were initially admitted during the period studied with a discharge diagnosis of unstable angina or myocardial infarction . Therefore, the final sample consisted of 824 patients . The demographic characteristics and base anthropometry were recorded for all the patients studied . The patients were divided into three groups: normal (bmi <25 kg / m), overweight (bmi = 2530 kg / m), and obese (bmi> 30 we analyzed various clinical variables including the classic cvrfs (smoking, diabetes, hypertension, and dyslipidemia), cardiovascular background and treatment prior to the acute event, hemodynamic variables at admission, risk scores according to the timi and grace scales, electrocardiographic data, analytical parameters and noninvasive cardiologic examination parameters, treatment administered at admission, coronarography examination, and results, as well as percutaneous and surgical revascularization . All data were analyzed with the one - sample kolmogorov - smirnov test to evaluate the normality of their distribution . Continuous variables were expressed as the average and standard deviation and were compared using an unpaired student's t - test . The one - way anova test was used to compare more than two groups, and the bonferroni test was used to detect differences between groups . Percentages were compared using chi - squared statistics and fisher's exact test when any of the expected values in any of the cells were below 10 . Univariate and multivariate logistic regression analyses were used to determine the variables associated with hospital mortality . The average age of the sample was 65.84 + 0.4 years (ranging from 2595), and the sample was predominantly male (73.5%). Bmi had an inverse relation to mortality, that is, to say, higher bmis were associated with a reduced mortality rate . In the multivariate logistic regression analysis (table 1), bmi along with other clinical variables (age, diabetes mellitus, grace score, and cardiogenic shock) were the independent predictors of death in our sample . Table 2 shows the hospital admission data for the sample, and table 3 shows not only the distribution of subjects in the normal weight, overweight, and obese categories but also the main clinical variables (risk factors and relevant background factors). The incidence of risk factors is high in this population (95.8%). In the obese group, the prevalence of diabetes mellitus and chronic obstructive pulmonary disease is significantly higher than in the other two groups . A larger proportion of the obese group had previously undergone treatment with angiotensin ii receptor antagonists and diuretics although there were no other differences in prior treatments . At admission, overweight patients presented higher blood pressure levels, and lower killip classes and timi scores, as well as higher initial glycemia and cholesterol levels . A coronariography was conducted in 76% of the patients in our sample, and no differences were found between obese, overweight, or normal weight subjects . No differences were found among the three groups in the percentage of angioplasties conducted or in the medical treatment received during admission . The prevalence of complications such as angina pectoris, reinfarction, hemorrhage, and mechanical complications was similar across groups with the exception of stroke, which was lower in obese patients . Thirty - five patients died (4.2%) with no statistically significant differences among the different bmi groups . Most deaths occurred within 48 hours of having been admitted (19 patients), followed by 7 patients between day 2 and day 7, and a further 9 cases after day 7 . No statistical differences were found in the combined endpoint (mortality, reinfarction, bleeding, and cerebrovascular accident). Our study shows that bmi is an independent predictor of hospital mortality in that a higher bmi is associated with a lower mortality rate . This finding is correlated with a lower mortality rate in obese and overweight patients compared to patients in the normal range although this result is not statistically significant . Unlike other authors [5, 9], we did not find pronounced differences among these groups in terms of clinical data, hemodynamics, electrocardiographs, analyses, echocardiographs, angiographs, treatments administered, or resulting complications except that obese subjects have a greater incidence of diabetes, cardiovascular history, chronic obstructive pulmonary disease, and lower killip classes and timi scores . This data suggests that evolution would be less favorable and the mortality rate higher during hospital stays . Although bmi is an inverse predictor of mortality in our series, indicating that overweight patients with acs have better survival rates than normal weight subjects, when subjects are classified into bmi subgroups (normal weight, overweight, and obese), we were unable to demonstrate with statistical significance that patients classified as obese have lower hospital mortality although this tendency does exist . Among other reasons, this may be due to the sample size . Our data is consistent with other data published, among which are the results of the synergy, merlin - timi 36, and crusade studies, which also describe lower mortality rates in obese patients suffering from acs . In a meta - analysis of 40 studies with more than 250,000 patients, romero - corral et al . Observed that overweight patients with coronary disease have a lower risk of cardiovascular and total mortality than patients in the low and normal weight groups . However, this tendency disappears in the bmi 35 kg / m patient group (morbid obesity), which runs a greater risk of cardiovascular mortality . Analyzed the prognostic value of bmi in medium - term hospital mortality in a cohort of 1,063 consecutive patients with first infarction in 15 hospitals in spain and found no association between bmi and medium - term hospital mortality . Furthermore, in patients with acute st - elevated myocardial infarction, das et al . Described a less - favorable prognosis for very obese patients in a sample of 501,489 patients . Several hypotheses have been proposed to explain the inverse relationship between obesity and the prognosis of patients with ischemic cardiopathy . The higher mortality rate in the normal weight group may be due to a higher mortality rate in underweight patients . Some studies [1, 2] show that the optimal bmi for the general population is 22.525 kg / m in smokers, and that people in the lower range of normal bmi (18.522.5 kg / m) have a higher mortality rate than individuals in the upper range of overweight bmi (27.530 kg / m). So the high - risk, underweight group is subsumed within the group of patients that is often considered normal . In contrast, most studies have found that a higher mortality rate is associated with extreme obesity [8, 9, 11]. Another aspect that is often not given appropriate consideration is the role of pharmacological treatment, especially the adverse effects of multiple medicines used in acs (fibrinolytic agents, antiplatelet drugs, anticoagulants, inotropes, antiarrhythmic agents, diuretics, nitrates, beta blockers, etc . ), which could explain a higher mortality rate . Given the difficulty of anthropometric measurement in the first hours of cardiologic emergency treatment, errors in medicinal dosage may arise, primarily in those patients with lower body weight . Nevertheless, unlike other authors, in our series we found that bmi group had no effect on either treatments administered or hemorrhages . Since bmi cannot differentiate between muscle and fat mass, overweight and obese subjects with coronary disease may have more muscle mass . When bmi is very high and better reflects body adiposity, the obesity paradox disappears . The main approaches used to measure obesity are bmi, waist - hip ratio, and waist circumference . There is considerable disagreement on which of them is best [3, 1315]. Most authors recommend the simultaneous use of all of these parameters to better assess patient risk . Although the worst scenario is the obese individual with a high waist circumference, patients categorized as normal with a high waist circumference also face an elevated risk . Other authors suggest that obesity should be expressed as the percentage of body fat (> 25 for men and> 35 for women), but this is difficult to quantify clinically . Correctly identifying the different compartments of body fat, and specifically visceral fat, using more sophisticated techniques [16, 17] may help to clarify the role of obesity in acs patient mortality . Studies have also been conducted which measure the degree of activity or physical condition and classify the subgroups normal weight - sedentary, overweight active, obese active, overweight sedentary, and obese sedentary from least to greatest risk, with obese - sedentary being the group at greatest risk [18, 19]. Although the sample consisted of more than 800 patients, classifying the patients into three groups may prevent hospital mortality from being appropriately assessed because its incidence is relatively low . The clinical appraisal of obesity has many limitations, and an alternative might be the combined appraisal of other anthropometric measurements such as waist circumference, which was an unknown variable in our sample . Better anthropometric appraisal using data on abdominal obesity and an understanding of the physical condition of the patients so that they can be more accurately classified into groups could play a critical role in clarifying this paradox . The relationship between higher bmi and a greater incidence of coronary disease in the general population is well documented, and excess weight should clearly be avoided . However, once coronary heart disease arises, the association between bmi and the prognosis becomes more complex, even paradoxical according to some authors . So the controversy over the predictive value of bmi in patients with acs remains latent . Our study confirms that bmi is an independent predictor of hospital mortality; a higher bmi is associated with a lower mortality rate . Our overweight and obese patients had a higher incidence of diabetes, cardiovascular history, chronic obstructive pulmonary disease, and lower killip classes and timi scores . However, they did not show an increased mortality rate, which is apparently paradoxical.
Down syndrome (trisomy 21) is the most common and best known of all malformation syndromes . It is an easily recognized congenital, autosomal anomaly characterized by generalized growth deficiency and mental deficiency affecting 1 in 600 - 1 in 1000 live births . A compromised immune system is characteristic of these individuals, and it is this immunodeficiency that contributes to an increased susceptibility to infections . Various microorganisms are generally seen as normal commensals in the oral cavity, and these may become pathogenic under favorable conditions . Candida is one such endogenous pathogen, the prevalence of which has been reported to be 45 - 65% in healthy children and 30 - 45% in healthy adults . Opportunistic infections occur mainly when the host defenses are inadequate, candidiasis is the most common among them . Streptococci form a large portion of the resident oral micro flora . In the general population, half the isolates from the tongue and saliva are streptococci, but in down syndrome individual's oral streptococcal levels are relatively lower . This probably contributes to a disturbance in the normal microbial homeostasis and promotes the growth of other commensal organisms like candida . Here, we evaluated the candidial / streptococci carriage and the various species of candida / streptococci isolated from the down syndrome patients and correlated it with normal oral micro flora from health subjects . Fifty down syndrome individuals were selected for this study based on a karyotype of 47 xx, 21 + (female) and 47 xy, 21 + (male). A total of 50 normal children / adolescents were selected as a control group . To isolate candida and streptococci, swab / saliva was used to culture and smear preparation for gram stain and periodic acid schiff (pas) stain . Samples were taken from the oral cavity (dorsum of tongue) for each individual using a sterile cotton wool swab . Sabouraud's dextrose agar (sda) slope with antibiotic (gentamicin / actidione) was used for the isolation of candida . If colonies were white or cream to yellow, dull / shiny, pasty yeast like a smear was prepared from the colony, with a drop of saline . Smear was then air - dried, heat fixed, and stained with crystal violet (simple stain) to confirm the presence of yeast . The isolates were then subjected to the following tests for identification of various species of candida: germ tube testmorphology on corn meal agar for chlamydospore formation.carbohydrate fermentation testcarbohydrate assimilation testchromagar candida culture characteristics morphology on corn meal agar for chlamydospore formation . Carbohydrate fermentation test carbohydrate assimilation test chromagar candida culture characteristics smears taken from the oral cavity (tongue) were air - dried, heat fixed, and stained . Samples were taken from the oral cavity (dorsum of tongue) for each individual using a sterile cotton wool swab . Sabouraud's dextrose agar (sda) slope with antibiotic (gentamicin / actidione) was used for the isolation of candida . If colonies were white or cream to yellow, dull / shiny, pasty yeast like a smear was prepared from the colony, with a drop of saline . Smear was then air - dried, heat fixed, and stained with crystal violet (simple stain) to confirm the presence of yeast . The isolates were then subjected to the following tests for identification of various species of candida: germ tube testmorphology on corn meal agar for chlamydospore formation.carbohydrate fermentation testcarbohydrate assimilation testchromagar candida culture characteristics morphology on corn meal agar for chlamydospore formation . Smears taken from the oral cavity (tongue) were air - dried, heat fixed, and stained . The study included a total of 100 (50 down syndrome, 50 controls) subjects of both sexes . The study group of 50 down syndrome individuals comprised of 37 males and 13 females in the age range of 3 - 22 years and the mean age of 11.6 years . The control group of 50 normal individuals comprised of 34 males and 16 females in the age range of 3 - 18 years and a mean age of 9.5 years [tables 1 and 2]. Distribution of patients according to sex distribution of patients according to age of the 50 study group samples, which were cultured, 37 (74%) showed growth of candida colonies, whereas in the 50 control samples only 18 (36%) were positive for candida growth . In 4 sda culture slopes of the study group, more than one morphological type of colonies were observed [figure 1], and the colonies were considered as different isolates and subjected to further testing . A total of 42 isolates (1 isolate in 33 samples, 2 isolates in 3 samples, and 3 isolates in 1 sample) were obtained from the 37 culture positive samples, in the study group and 18 isolates were obtained from the 18 culture positive samples in the control group (1 isolate each in 18 samples) [table 3]. Sabouraud's dextrose agar slope showing more than one type of colony (white and cream) candidal growth pattern in the study group and control group smears taken from the oral cavity (tongue), for both the study group (50) and the control group (50) were stained with gram stain [figure 2] and pas stain [figure 3]. A tenfold increase in the positive staining for candida was observed in the study group when compared to the control group [table 4]. Gram - stained smear showing yeasts and pseudohyphae periodic acid schiff stained smear showing candidal hyphae and yeasts distribution of positive candidal staining in study group and control group of the 42 isolates from the 37 culture positive samples, 31 were germ tube positive, whereas all the 18 culture positive samples of the control group were positive for germ tube formation [figure 4]. Hence, 31 of the 42 isolates were candida albicans in the study group, and all 18 isolates of the control group were c. albicans . This was further confirmed by chlamydospore formation on corn meal agar [figure 5]. All the isolates that were germ tube positive produced chlamydospores on corn meal agar further confirming c. albicans . Of the 42 isolates and 37 culture positive samples, 31 isolates were c. albicans, 6 were candida tropicalis, and 5 were either candida parapsilosis / candida krusei / candida glabrata . Of the 18 control samples, which were culture positive, all 18 were c. albicans . Further confirmation and speciation was done by the carbohydrate assimilation test and supplemented with growth on chromagar candida . Germ tube formation by candida albicans chlamydospore formation by candida albicans this test was done with sugars: glucose, maltose, sucrose, lactose, galactose, cellulose, and trehalose [table 5]. Carbohydrate assimilation reactions for different candida species of the 37 samples that were culture positive in the study group, 31 (84%) samples had isolates of c. albicans, 6 (16%) samples had isolates of c. tropicalis and 5 (14%) had isolates of c. parapsilosis . 4 samples of the study group were colonized with more than one type of species . Of the 18 control samples that were culture positive all 18 isolates were c. albicans . Growth of all culture positive samples on chromagar candida was observed for the characteristic colony color [table 6]. Chromagar candida culture characteristics for different candida species in the chromagar candida, study sample showed 31 isolates of c. albicans, 6 of c. tropicalis, and 5 of c. parapsilosis [figure 6]. In the control samples, chromagar candida showing green colonies of candida albicans and cream colonies of candida parapsilosis saliva samples collected from both the study group (50%) and the control group (50) after inoculation on chocolate agar, and incubation at 37c for 24 h, the growth of alpha - hemolytic colonies were identified [figure 7]. The colonies were checked for optochin resistance to classify it as streptococcus viridans group [table 7]. Primary culture showing predominantly alpha hemolytic colonies growth pattern of streptococcus the growth of alpha hemolytic s. viridans colonies in the primary culture was classified as in table 8 . Distribution of viridans group of streptococci in control group and study group twenty - three out of 50 samples from study group had s. viridans colonies . In the 23 samples positive for streptococci 16 had many streptococcal colonies, and 7 had few streptococcal colonies in the primary culture . In the 32 samples, which were positive for streptococci, 29 had predominantly streptococcal colonies while 3 had few streptococcal colonies in the primary culture . The presence of candida with streptococci was compared in both the study group and the control group [table 9]. Comparison of candidal and streptococcal growth patterns in study group and control group candidal and streptococcal colonization was seen in 14 (28%) individuals in the study group and 6 (12%) individuals in the control group . Candida colonization alone in the absence of streptococcal colonization was seen in 23 (46%) individuals of the study group and 12 (24%) of the control group . Thus showing that candidal colonization occurred to a greater extent in the absence of streptococci (p <0.01, z = 2.96). Our results showed that in the study group (74%) candida colonization was comparatively higher when compared, with the control group (36%). Of the 37 culture - positive samples, 42 isolates were obtained, four samples had more than one isolate (one sample with three isolates, three samples with two isolates). The species isolated in the study group out of the 37 culture - positive samples were predominantly c. albicans (84%) followed by c. tropicalis (16%) and c. parapsilosis (14%). In the control group, only c. albicans were isolated . Thus, out of the total 50 down syndrome subjects, 31 (62%) showed colonization with c. albicans, 6 (12%) with c. tropicalis, and 5 (10%) with c. parapsilosis . In the comparative study of candida and streptococci, the oral findings of all the children and adolescents of the study group were recorded [figures 810]. 12-year - old male showing fissured tongue high arched palatal vault angular cheilitis with prognathic mandible distribution of oral findings in down syndrome subjects in the 50 down syndrome individuals, 17 (34%) of them showed an erythematous or a pseudomembranous lesion on the tongue (12 - erythematous, 5 - pseudomembranous), indicating possible oral candidiasis . This test was done with sugars: glucose, maltose, sucrose, lactose, galactose, cellulose, and trehalose [table 5]. Carbohydrate assimilation reactions for different candida species of the 37 samples that were culture positive in the study group, 31 (84%) samples had isolates of c. albicans, 6 (16%) samples had isolates of c. tropicalis and 5 (14%) had isolates of c. parapsilosis . 4 samples of the study group were colonized with more than one type of species . Of the 18 control samples that were culture positive all 18 isolates were c. albicans . Growth of all culture positive samples on chromagar candida was observed for the characteristic colony color [table 6]. Chromagar candida culture characteristics for different candida species in the chromagar candida, study sample showed 31 isolates of c. albicans, 6 of c. tropicalis, and 5 of c. parapsilosis [figure 6]. In the control samples, saliva samples collected from both the study group (50%) and the control group (50) after inoculation on chocolate agar, and incubation at 37c for 24 h, the growth of alpha - hemolytic colonies were identified [figure 7]. The colonies were checked for optochin resistance to classify it as streptococcus viridans group [table 7]. Primary culture showing predominantly alpha hemolytic colonies growth pattern of streptococcus the growth of alpha hemolytic s. viridans colonies in the primary culture was classified as in table 8 . Distribution of viridans group of streptococci in control group and study group twenty - three out of 50 samples from study group had s. viridans colonies . In the 23 samples positive for streptococci 16 had many streptococcal colonies, and 7 had few streptococcal colonies in the primary culture . In the 32 samples, which were positive for streptococci, 29 had predominantly streptococcal colonies while 3 had few streptococcal colonies in the primary culture . The presence of candida with streptococci was compared in both the study group and the control group [table 9]. Comparison of candidal and streptococcal growth patterns in study group and control group candidal and streptococcal colonization was seen in 14 (28%) individuals in the study group and 6 (12%) individuals in the control group . Candida colonization alone in the absence of streptococcal colonization was seen in 23 (46%) individuals of the study group and 12 (24%) of the control group . Thus showing that candidal colonization occurred to a greater extent in the absence of streptococci (p <0.01, z = 2.96). Our results showed that in the study group (74%) candida colonization was comparatively higher when compared, with the control group (36%). Of the 37 culture - positive samples, 42 isolates were obtained, four samples had more than one isolate (one sample with three isolates, three samples with two isolates). The species isolated in the study group out of the 37 culture - positive samples were predominantly c. albicans (84%) followed by c. tropicalis (16%) and c. parapsilosis (14%). In the control group, thus, out of the total 50 down syndrome subjects, 31 (62%) showed colonization with c. albicans, 6 (12%) with c. tropicalis, and 5 (10%) with c. parapsilosis . In the comparative study of candida and streptococci, the oral findings of all the children and adolescents of the study group were recorded [figures 810]. Table 10 shows in an ascending order the frequency of various findings . 12-year - old male showing fissured tongue high arched palatal vault angular cheilitis with prognathic mandible distribution of oral findings in down syndrome subjects in the 50 down syndrome individuals, 17 (34%) of them showed an erythematous or a pseudomembranous lesion on the tongue (12 - erythematous, 5 - pseudomembranous), indicating possible oral candidiasis . Down syndrome individuals are reported to exhibit an increased susceptibility to infections due to various factors such as increased activity of superoxide dismutase, increased activity of glutathione peroxidase, decreased levels of myeloperoxidase in leukocytes, defective neutrophil chemotaxis, abnormal serum immunoglobulin patterns and disturbed cell - mediated and humoral immunity . The present study determined the prevalence of oral candida carriage in down syndrome children and adolescents . Of the 37 individuals in the study group who exhibited candida colonization, only 14 showed colonization with streptococci while 23 remained negative and showed no streptococcal colonization . Thus, we concluded that the majority of the individuals showed candida colonization when there was no concomitant streptococcal colonization . The increased candida carriage in down syndrome individuals could probably be attributed to the relatively lower streptococcal levels . The increased susceptibility to candida infection may be a result of the disturbance in the microbial homeostasis . However, other factors such as a compromised immune system, poor oral hygiene, malnourishment and the like cannot be completely ruled out and are also expected to play a significant role . It may be also possible that the candida and streptococci may produce mutually inhibitive factors in the form of toxins or enzymes, thus producing this inverse relationship . The most common predisposing factor resulting in candidiasis is the presence of a suppressed immune status, thus this infection is seen in a higher frequency in down syndrome subjects, where a compromised immune status is the hallmark . In addition to this, a variety of local factors may also play a role, the most common being an altered intra oral milieu . As observed in this study, it may be assumed that the presence of microbial imbalance in the oral cavity and the occurrence of fissured tongue could be some major local factors contributing to the increased candida carriage in down syndrome individuals . The oral cavity is an environment heavily colonized by microorganisms, which are generally in harmony with each other . The down syndrome subjects run a greater risk of having opportunistic infections with a possibility of systemic spread, and the associated morbidity and mortality is also higher . Hence, to improve the quality - of - life of an individual with down syndrome, it becomes necessary to diagnose and treat these infections when they still remain localized.
This has been attributed to procedure time, cost, and the known risks, which include arterial puncture, pneumothorax, and infection . Numerous studies done in the last few years have paid attention to peripheral venous pressure (pvp) and more specifically its pressure waveform . Although controversy still exists concerning the role of peripheral veins and their contribution to the central volume in face of blood loss, many studies in the late 1990s and early 2000s have shown a consistent correlation between central venous pressure (cvp) and pvp . This implies that in emergency conditions and situations where anatomical sites are inaccessible for central venous catheterization as seen in burns patients, the estimation of cvp is possible via measurement of peripheral intravenous (iv) catheter . Hemodynamic monitoring has been shown to provide valuable additional information if burn resuscitation is not proceeding as planned or volume therapy guided by the typical vital signs is not attaining the desired effect . The goal of this study was to determine a reliable association between changes in cvp and pvp in patients with burns and to assess the long - term correlation in varied hemodynamic status during the first 10 h. after obtaining institutional ethical committee approval and informed consent from each patient, 30 consecutive patients admitted to our burns intensive care unit (bicu) from june to august 2014 were included in our study . The exclusion criteria were patients on the mechanical ventilator and untimely death during the study period . The sample size was decided after doing a pilot study for a month in our bicu and based on power of analysis . Central venous pressure access was obtained using a 7 french double - lumen, arrow international catheter placed via the left or right internal jugular or subclavian vein . Tip of central venous catheter was inserted at the junction of the superior vena cava and right atrium confirmed by chest x - ray . The peripheral measurement of cvp was obtained from a peripheral iv site (dorsum of the hand, forearm or antecubital region) using a standard iv catheter (18, 20 or 22 gauges). Cvp was measured from both the central venous catheter and the peripheral iv catheters using philips and spacelab monitors equipped with invasive blood pressure monitoring transducers, which were zeroed at the phlebostatic axis . Simultaneous measurements of cvp from central and peripheral venous catheters were made hourly for 10 consecutive hours . Age, weight, height, site of cvp and pvp and peripheral iv catheter size for each patient were recorded . The predictability of cvp by pvp was examined using linear regression analysis at a p 0.05 . Among the 30 patients in this study, there were 20 females and 10 males . The age range was from 18 to 65 years, and their weight ranged from 45 to 60 kg . The predictability of cvp by pvp was tested by applying the linear regression which is shown in figure 1 . This regression formula shows a reliable and significant association between cvp and pvp (p <0.001). The overall mean difference between cvp and pvp was 1.628 0.84 mmhg . The mean difference between cvp and pvp in each hour we used the bland - altman diagram for estimation of agreement between cvp and pvp during the 10 h period . This showed a perfect agreement (difference of 1.2 with an sd of + 1.96) as seen in figure 3 . The predictability of central venous pressure by measuring peripheral venous pressure tested by applying linear regression the hourly mean difference between central venous pressure and peripheral venous pressure agreement between central venous pressure and peripheral venous pressure during the 10 h period using bland the present study demonstrated reliable agreement between cvp and pvp over a 10 h period, suggesting that pvp monitoring can be used as a simple, cost - effective and less invasive substitute for cvp monitoring in patients admitted to the bicu . Previous studies comparing cvp measured from central and peripheral access have shown a consistent correlation, but most of these studies were done in surgical patients, and we could not find any similar study in burns patients despite extensive literature search . Cvp monitoring in the critically ill patients is usually performed by catheterization of either the subclavian or internal jugular veins . In burns patients, the site for catheterization of the central veins maybe inaccessible and in such patients monitoring the cvp via a peripheral vein is definitely an attractive option . The results of our study show that we can estimate cvp through simultaneous measurement of pvp and the difference between cvp and pvp measurements remain almost in a constant range over a period of time . Hence, evaluation of hemodynamic changes occurring with dehydration or volume overload can be made by measuring pvp ., showed that pvp measured from a peripheral iv catheter in infants and children with congenital heart disease was an accurate estimation of cvp and its changes had good concordance with cvp over a long period of time ., however, reported that pvp measurement in critically ill patients did not give an accurate estimate of absolute value of cvp . Tugrul et al ., also reported that pvp showed strong correlation with cvp and mean difference between pvp and cvp was 2 + 1.8 mmhg, but the upper limit of agreement of pvp - cvp (5.6 mmhg) indicated the difference between two pressures might reach a clinically unacceptable value . However, in our study, the overall mean difference between cvp and pvp was 1.628 0.84 mmhg and the upper limit of agreement correlated to the clinically acceptable limits and were comparable with data described in other studies ., found the mean difference between pvp and cvp to be 1.6 mmhg in all intraoperative patients and 2.2 mmhg in the postoperative period . Hoftman et al ., showed that pvp correlated with cvp even under adverse hemodynamic conditions in patients undergoing liver transplantation . Studies have also shown that neither the peripheral iv catheter size nor the site of catheter placement interfered with the agreement of pvp and cvp . In our study, we used three different sites in the upper limb and three sizes of the peripheral catheter but did not find any statistically significant variations in the agreement of pvp and cvp values . Although cvp waveforms characteristically showed a - waves, c - waves, and v - waves, pvp waveforms appeared as a more dampened sinusoidal pattern . We also noticed that the pvp tracing had more typical cvp waveforms when the peripheral catheter diameter was of a larger gauge and when the site was antecubital but since our subgroup sample size were small, we could not stratify this . Although by placing the peripheral iv catheter more distally and decreasing its diameter increased the pvp - cvp gradient, these failed to reach statistical significance in the study done by tugrul et al . Several studies have also demonstrated that the difference between cvp and pvp varies with the value of cvp . In nine patients undergoing orthotopic liver transplantation, hoftman et al ., reported a weaker correlation between pvp and cvp at lower cvp values . Another study by cave and harvey showed that when cvp increased, the difference between pvp and cvp tended to decrease . The number of patients was too small for subgroup analysis with regard to the percentage of burns and to the site and size of peripheral catheters affecting the pvp - cvp gradient . We also did not have a protocol for fluid management and its effect on our data . Hence, the trends in pvp may be useful as an alternative for hemodynamic monitoring in the bicu during emergencies, in situations where central venous site is inaccessible and also to avoid the complications of central venous catheterization in critically ill burns patients . Further studies are needed to determine the clinical usefulness of pvp as a trend monitor for evaluating the intravascular volume in this patient population.
Schizophrenia is a chronic disorder with serious physical, social, and economic consequences, with an impact on public health that has been unappreciated . Thus, it is a devastating illness that reflects in the productivity of individuals affected by it and requires permanent spending with hospitalizations, treatments, and rehabilitations.1,2 this chronic disorder is characterized by an onset in early adulthood, a lifelong course, debilitating symptoms, deterioration of functional ability, and lack of social acceptability, making it among the most disabling and economically catastrophic disorders.3 in clinical research, five symptom clusters have been described in persons with schizophrenia . They include negative, positive, excitement, cognitive, and depression and anxiety dimensions of schizophrenic psychopathology.4 positive symptoms are associated with hospital admission . They are identified as superimposed behaviors and principally involve delusions, hallucinations, and disorganized thinking . Delusions are the most common psychotic symptoms and occur in 65% of patients with schizophrenia.57 hallucinations and disorganized thinking are present in 50% of these patients . Auditory hallucinations are the most common sensory disturbance, but visual, tactile, olfactory, and gustatory hallucinations may also be present.8 negative symptoms represent an absence of a normal function . These symptoms comprise blunted affect, abulia, alogia, anhedonia, apathy, avolition, and asociality.79 excitement symptoms are often related to a manic - like syndrome in patients with schizophrenia . Particularly, they may be present in an acute phase of the disease and tend to respond well to pharmacological interventions . These symptoms are characterized by excitement, impulsivity, psychomotor activation, and uncooperativeness.7,10 cognitive domains are also affected by the disorder, and include deficits in attention, language, memory, executive function, processing speed, and social cognition . They are present in the premorbid phase of schizophrenic illness and persist throughout the long - term.7,11 depression and anxiety symptoms are present in a majority of schizophrenia patients at some point during the course of the illness . Depression may be part of the prodrome or the florid phase; it may follow an acute psychotic episode, or occur between psychotic exacerbations . Anxiety could be considered as a cormobity.7 treatment of schizophrenia requires long - term administration of antipsychotic drugs . Lack of efficacy, poor patient compliance, extrapyramidal symptoms (eps), weight gain, and sedation can interfere with long - term adherence to maintenance drug therapy.12 although atypical antipsychotic drugs have been associated with a lower risk of eps, which include abnormal involuntary movements like parkinsonism, dystonia, akathisia, and tardive diskinesia, these drugs pose a higher risk for the development of metabolic adverse events.1316 since the advent of chlorpromazine, there has been a great improvement in the treatment of people with schizophrenia . From that time, other antipsychotic agents were launched until the development of the second generation antipsychotics (sga). These new drugs (also known as atypical antipsychotics) are thought to be more efficacious in treating negative symptoms and also produce fewer side effects (eg, less eps) when compared to the first generation antipsychotics (fga).17 although patients report preference for the sga, we cannot forget the important side effects associated with their use (for instance, increased cardiometabolic risk). These drugs have different pharmacological profiles and side effects that may differ from one agent to another.18 in addition to promoting antipsychotic action without producing significant eps, other characteristics of the sga that define their atypicity include an absence of hyperprolactinemia and greater efficacy in positive (at least in the case of clozapine), negative, and cognitive symptoms . From a pharmacological perspective, the sga can be defined as antagonists of the serotonin - dopamine, d2-blockers with rapid dissociation and d2 or 5ht2 partial agonists.19 for some authors,2022 another class of antipsychotic agents for the pharmacological treatment of schizophrenia and other psychiatric disorders can be identified: the third generation antipsychotics (tga). Aripiprazole, an example of a tga, has functional antagonist activity under hyper - dopaminergic conditions, and functional agonist properties under hypodopaminergic conditions . Aripiprazole seems to produce d2-mediated functional effects that involve a broad range of classic pharmacological intrinsic activities . These variations in both intrinsic activity and potency have suggested that aripiprazole may be functionally selective at d2 receptors, and not just as a simple partial agonist.22 the effectiveness of current drugs for the treatment of schizophrenia may occur in only about 50% of patients.2325 poor symptom response is associated with premature treatment discontinuation, symptom exacerbation, relapse, and increased risk of hospitalization with resultant higher costs of treatment.2629 predictors of antipsychotic response should evaluate the disease state at baseline (before the initiation of treatment) and at early symptom changes after beginning treatment . Cognitive function deficits,30 poor premorbid functioning,31 earlier age of onset,32 duration of untreated psychosis,33,34 and male gender35 are baseline factors that have been found to be associated with poor treatment response . Patients may remain as nonresponders if early nonresponse is observed at the first 2 weeks of treatment . Correll et al36 reported that early nonresponse to treatment, as measured by a 20% reduction in the brief psychiatric rating scale (bprs) total score at week 1 predicted nonresponse at 4 weeks for 100% of patients . Other studies have suggested the hypothesis that early nonresponse to treatment within the first 2 weeks of treatment initiation is a good indicator of treatment refractoriness.37,38 double - blind, controlled studies have compared sga with fga, most often favoring sga insofar as greater symptom reduction (particularly for negative, cognitive, and affective symptoms) and better tolerability (notably regarding motor adverse effects) have been noted . In response to these positive studies, most international evidence - based guidelines advocate first - line use of sga.39 in the clinical antipsychotic trials of intervention (catie) study,24 the relative effectiveness of sga was compared with that of perphenazine in a double - blind fashion . A total of 1493 patients with schizophrenia were recruited at 57 us sites and were randomly assigned to receive olanzapine (7.5 mg to 30 mg per day), perphenazine (8 mg to 32 mg per day), quetiapine (200 mg to 800 mg per day), or risperidone (1.5 mg to 6.0 mg per day) for up to 18 months . Ziprasidone (40 mg to 160 mg per day) was included after its approval by the food and drug administration . The primary objective of the catie study was to discriminate between the differences in effectiveness of these five treatments . Before 18 months, 74% of patients discontinued the study medication (1061 of the 1432 patients who received at least one dose): 64% in the olanzapine group, 75% of those assigned to the perphenazine group, 82% of those randomized to the quetiapine group, 74% of those in the risperidone arm, and 79% of those who were assigned to the ziprasidone group . Time to the discontinuation of treatment for any cause was significantly longer in the olanzapine group than in the quetiapine (p <0.001) or risperidone (p = 0.002) group, but not in the perphenazine (p = 0.021) or ziprasidone (p = 0.028) group . Time to discontinuation because of intolerable side effects was similar among the groups, but the rates differed (p = 0.04); olanzapine was associated with more discontinuation for weight gain or metabolic effects, and perphenazine was associated with more discontinuation for extrapyramidal effects . In conclusion, the majority of patients in each group discontinued their assigned treatment due to inefficacy, intolerable side effects, or for other reasons . Olanzapine was the most effective drug in terms of the rates of discontinuation, and the efficacy of the conventional antipsychotic agent, perphenazine, appeared to be similar to that of quetiapine, risperidone, and ziprasidone . However, olanzapine was associated with greater weight gain and increases in measures of glucose and lipid metabolism . In the united kingdom, the cost utility of the latest antipsychotic drugs in schizophrenia study (cutlass)40 concluded that fga (largely sulpiride) and sga (olanzapine, risperidone, amisulpride, and quetiapine) did not differ in terms of overall efficacy as measured by the positive and negative syndrome scale (panss), extrapyramidal side effects, quality of life, and patient preference over a period of 52 weeks.40 leucht et al15 published a meta - analysis comparing nine sga and fga for overall efficacy; positive, negative, and depressive symptoms; relapse; quality of life; eps; sedation; and weight gain . Results showed that five sga (aripiprazole, quetiapine, sertindole, ziprasidone, and zotepine) were not significantly different from fga in their effects on overall, positive, and negative symptoms, whereas clozapine, amisulpride, olanzapine, and risperidone were more efficacious than fga . Olanzapine, risperidone, and sertindole proved to be significantly better than fga in terms of relapse prevention . With regard to quality of life, one issue regarding the efficacy of antipsychotic agents has been pointed out by recent publications . Data from randomized clinical trials (rct) of antipsychotics used in treating schizophrenia taken between 1991 and 2006, show that there is an increase in placebo response in more recent trials.41 some of the contributing factors include specific participant characteristics (eg, gender, treatment resistance and previous exposure, adherence to drug treatment, diagnostic precision, and illness severity), site characteristics (eg, academic or commercial, experience and training of site staff, recruitment pressures and procedures, recycling of subjects), trial design issues (eg, entry requirements, timing of assessments, double - blind placebo lead - ins, concomitant medications), and other issues.41 excessive placebo response in clinical trials includes expectancy bias (in patients and investigators), incentives, rater reliability, and data quality.42 similar issues were observed in antidepressant rct . The placebo response rate doubled between 1980 and 2000.43 the relatively modest effects of antidepressants (in comparison with placebo) in contemporary rct, as well as the lack of attention being paid to the contribution of placebo expectancy factors to individual outcomes, have often been underestimated . However, the modest benefits of antidepressants observed in grouped datasets may obscure large, specific, and very meaningful therapeutic effects for 10% to 20% of those treated with antidepressants.44 on the other hand, the placebo - controlled trial is the standard method used to verify the efficacy and safety of antipsychotic agents for the treatment of schizophrenia.45 currently, superiority over placebo is the only acceptable and reliable proof of efficacy of new antipsychotic medications.46,47 without the placebo - controlled design, one cannot show that improvements in symptoms do not represent nonspecific treatment effects or the natural course of the disease.45 also, in trials intended to show that a new drug is equivalent to established drugs, a variety of problems may reduce the power to detect clinically significant differences between the new drug and the active comparator.48,49 furthermore, without a placebo comparator, one cannot affirm whether the apparent equivalence of the standard and the new antipsychotic drug has occurred because the clinical population recruited for a study is nonresponsive to both drugs . In addition, if the experimental drug is ineffective and/or unsafe, the larger sample size required for noninferiority testing may expose more patients to harmful side effects or ineffective treatment compared with placebo - controlled designs.45 the placebo - controlled trial can protect research participants from the harmful effects of ineffective drugs.50 in terms of the clinical efficacy of antipsychotic agents, we are usually interested in treatment response and remission . In clinical trials, the efficacy of antipsychotics is measured through the use of scales like bprs51 or panss.52 among the available trials, there is no agreement in terms of when to consider a clinical response with an antipsychotic agent; different trials report that a response could be considered when reductions from the initial scores of 20%, 30%, 40%, 50%, and 60% are found.53 some studies using data from a large number of subjects,5458 rated simultaneously with the bprs / panss and the clinical global impression scale (cgi),59 concluded that a 25% reduction in the bprs / panss baseline scores is associated with a minimal improvement in the cgi score; moreover, a 50% reduction in the bprs / panss scores would correspond with much improved scores in the cgi . Remission is defined as a lack of significant symptoms . To obtain a uniformly accepted definition of remission, a group of experts60 proposed that a patient can be considered as being in remission if eight items on the panss52 or corresponding items on the bprs,51 scale for the assessment of positive symptoms,61 or on the scale for the assessment of negative symptoms56 are rated as being mildly present or better.62 other important outcome measures in schizophrenia trials include relapse and recovery . Relapse can be defined in different ways: exacerbation or recurrence of symptoms, change in functional status or change in treatment requirements, intensity of services, or locus of care.53 some studies define relapse when patients need to be hospitalized.6369 we have to remember that hospitalization due to social causes should not be considered as relapse . Also, some patients can be treated when presenting with worsening symptoms while avoiding hospitalization . Recovery is the goal of the treatment of patients with schizophrenia, and improvement of symptoms requires improvement of functional measures . Recovery is influenced by psychosocial treatments, family and community supports, supportive employment, and supportive education.53 there are many scales that measure clinical efficacy in schizophrenia trials . The following scales evaluate clinical symptomatology:53 cgi severity and improvement scale59bprs51panss52scale for the assessment of negative symptoms62calgary depression rating scale70 cgi severity and improvement scale59 scale for the assessment of negative symptoms62 calgary depression rating scale70 the importance of subjective measures has been repeatedly addressed in studies on the quality of life of patients with schizophrenia.71,72 improvement in quality of life and social functioning, with consequent reintegration into society, is clearly a major goal of treatment for schizophrenia.17,73 some studies have shown that the type of antipsychotic medication could influence treatment nonadherence . The use of atypical antipsychotics can improve adherence when compared with fga . In some studies, the use of atypical antipsychotics was seen to be associated with greater treatment adherence relative to conventional neuroleptics.7476 it has been suggested that better control of negative symptoms and fewer eps during treatment with sga are associated with better adherence to treatment.77 moreover, some studies suggest that patients receiving sga have better subjective responses to their current medication than those receiving conventional medications.7880 however, a prospective study reported that there was no difference between sga and fga in terms of adherence to treatment.81 patients subjective well - being is one important variable in the evaluation of the effects of antipsychotics and can be critical in determining compliance in schizophrenia.82 some studies have indicated that the severity of depressive symptoms can be associated with subjective well - being.8385 the evidence of the association between improvements in positive and negative symptoms is less consistent, suggesting that the identification and treatment of depressive symptoms in schizophrenic patients may affect perceptions of their own well - being.86,87 we can use self - report scales to measure patient outcomes, such as:53 drug attitudes inventory88subjective wellbeing under neuroleptic treatment scale (original / short form)8936-item short form health survey90euroqol91sheehan disability scale92 drug attitudes inventory88 subjective wellbeing under neuroleptic treatment scale (original / short form)89 36-item short form health survey90 sheehan disability scale92 other efficacy outcome scales will focus on cognitive impairment in schizophrenia: measurement and treatment to improve cognition in schizophrenia93 cgi of cognition in schizophrenia94 cogstate schizophrenia battery95 cogstate 12-minute battery96 schizophrenia cognition rating scale97 repeatable battery for assessment of neuropsychological status98 brief assessment of cognition in schizophrenia99 brief cognitive assessment tool for schizophrenia100 5-minute digit symbol coding task101 the effectiveness of a drug is characterized by its efficacy, tolerability, safety, function, and acceptability . The most common side effects are eps and sedation.102 sga produce fewer eps than fga; however, when doses of sga are increased, there is a possibility of emergence of eps . Clozapine and quetiapine have lower affinities for the dopamine receptors, resulting in a rare association with eps . In therapeutic dosages, clozapine only has a dopamine d2 receptor blockage of 40%.102 in doses of 450 and 750 mg / day, quetiapine has d2 receptor occupancies of 30% and 41%.103 also, these antipsychotic drugs have an antagonistic effect on the 5ht2 a receptor and a partial agonistic effect on the 5ht1 a receptor, which seems to aid in the production of low levels of eps, and gives these medications the character of atypicality.102 on the other hand, amisulpride has a high affinity for d2/d3 receptors, no significant binding to 5ht2 receptors, and also shows lower risk of eps . This is because amisulpride is a unique sga that selectively blocks d2-like receptors presynaptically in the frontal cortex, possibly enhancing dopaminergic transmission, as well as postsynaptically in the limbic areas, possibly reducing dopaminergic transmission in this region.104 in this case, an occupancy of 5ht2a receptors is not the cause of atypicality of the amisulpride, but it is likely that a fast dissociation from d2 receptors makes this drug more accommodating to physiological dopamine transmission, which permits an antipsychotic effect without eps.105 the monitoring of eps is important because these neurologic symptoms have been associated with poor compliance, reduced quality of life, increased suicide rates, and increased risks of developing tardive dyskinesia.102 since the beginning of the antipsychotic era, these drugs have been known to be associated with sudden death . Prolongation of the qt interval to more than 500 ms has been associated with an increased risk of developing the potential fatal cardiac arrhythmia, torsade de pointes . However, this mechanism may be responsible (to a minor degree) for the increased mortality rate seen in patients with schizophrenia . The antipsychotic - induced metabolic changes such as weight gain, increased risk of developing diabetes, and dyslipidemia are far more important . Clozapine, olanzapine, and quetiapine are the sga that cause the worst metabolic changes . Disturbances in glucose metabolism and diabetes mellitus can occur with the use of sga, and are associated with increases in insulin resistance and visceral adiposity with reduced responsiveness of pancreatic beta cells to glucose.106 for olanzapine and clozapine, a direct diabetogenic effect regardless of weight gain has been established . Olanzapine and clozapine seem to have the highest prevalence of ketoacidosis, but most cases are reversible when discontinuing the offending drug . Glycosylated hemoglobin (hba1c) is often elevated in these patients, and decreased levels of high - density lipoprotein and increased levels of triglycerides are associated with an increased risk of insulin resistance . Dyslipidemia increases the risk of cardiovascular disease due to arteriosclerosis, and it should be noted that sga affect both triglycerides and cholesterol levels . Results from the catie study suggest that clozapine, olanzapine, and quetiapine affect triglycerides and cholesterol levels the most.24 patients with antipsychotic - induced dyslipidemia might benefit from lifestyle interventions, or they may switch to a more metabolically tolerable antipsychotic drug . The metabolic side effects of sga were investigated in a meta - analysis of studies comparing the metabolic side effects of the following sga in a head - to - head comparison: amisulpride, aripiprazole, clozapine, olanzapine, quetiapine, risperidone, sertindole, ziprasidone, and zotepine.107 the authors found 48 studies with 105 treatment arms . Olanzapine was more likely to produce weight gain than risperidone, risperidone more than amisulpride, and sertindole more than risperidone . Olanzapine was more likely to produce an increase in cholesterol levels than aripiprazole, risperidone, and ziprasidone; no differences with amisulpride, clozapine, and quetiapine were found . Quetiapine produced a greater increase in cholesterol levels than risperidone and ziprasidone . Olanzapine produced a greater increase in glucose than amisulpride, aripiprazole, quetiapine, risperidone, and ziprasidone; no difference was found with clozapine . The clinician should take these side effects into consideration when choosing an atypical drug for each individual patient . Correll et al108 investigated patients with bipolar disorder and schizophrenia who were treated with sga . The authors suggested a shared susceptibility to antipsychotic - related metabolic deregulation not primarily related to psychiatric diagnosis or concomitant mood stabilizer treatment . Sga produce fewer eps than fga; however, when doses of sga are increased, there is a possibility of emergence of eps . Clozapine and quetiapine have lower affinities for the dopamine receptors, resulting in a rare association with eps . In therapeutic dosages, clozapine only has a dopamine d2 receptor blockage of 40%.102 in doses of 450 and 750 mg / day, quetiapine has d2 receptor occupancies of 30% and 41%.103 also, these antipsychotic drugs have an antagonistic effect on the 5ht2 a receptor and a partial agonistic effect on the 5ht1 a receptor, which seems to aid in the production of low levels of eps, and gives these medications the character of atypicality.102 on the other hand, amisulpride has a high affinity for d2/d3 receptors, no significant binding to 5ht2 receptors, and also shows lower risk of eps . This is because amisulpride is a unique sga that selectively blocks d2-like receptors presynaptically in the frontal cortex, possibly enhancing dopaminergic transmission, as well as postsynaptically in the limbic areas, possibly reducing dopaminergic transmission in this region.104 in this case, an occupancy of 5ht2a receptors is not the cause of atypicality of the amisulpride, but it is likely that a fast dissociation from d2 receptors makes this drug more accommodating to physiological dopamine transmission, which permits an antipsychotic effect without eps.105 the monitoring of eps is important because these neurologic symptoms have been associated with poor compliance, reduced quality of life, increased suicide rates, and increased risks of developing tardive dyskinesia.102 since the beginning of the antipsychotic era, these drugs have been known to be associated with sudden death . Prolongation of the qt interval to more than 500 ms has been associated with an increased risk of developing the potential fatal cardiac arrhythmia, torsade de pointes . However, this mechanism may be responsible (to a minor degree) for the increased mortality rate seen in patients with schizophrenia . The antipsychotic - induced metabolic changes such as weight gain, increased risk of developing diabetes, and dyslipidemia are far more important . Clozapine, olanzapine, and quetiapine are the sga that cause the worst metabolic changes . Disturbances in glucose metabolism and diabetes mellitus can occur with the use of sga, and are associated with increases in insulin resistance and visceral adiposity with reduced responsiveness of pancreatic beta cells to glucose.106 for olanzapine and clozapine, a direct diabetogenic effect regardless of weight gain has been established . Olanzapine and clozapine seem to have the highest prevalence of ketoacidosis, but most cases are reversible when discontinuing the offending drug . Glycosylated hemoglobin (hba1c) is often elevated in these patients, and decreased levels of high - density lipoprotein and increased levels of triglycerides are associated with an increased risk of insulin resistance . Dyslipidemia increases the risk of cardiovascular disease due to arteriosclerosis, and it should be noted that sga affect both triglycerides and cholesterol levels . Results from the catie study suggest that clozapine, olanzapine, and quetiapine affect triglycerides and cholesterol levels the most.24 patients with antipsychotic - induced dyslipidemia might benefit from lifestyle interventions, or they may switch to a more metabolically tolerable antipsychotic drug . The metabolic side effects of sga were investigated in a meta - analysis of studies comparing the metabolic side effects of the following sga in a head - to - head comparison: amisulpride, aripiprazole, clozapine, olanzapine, quetiapine, risperidone, sertindole, ziprasidone, and zotepine.107 the authors found 48 studies with 105 treatment arms . Olanzapine was more likely to produce weight gain than risperidone, risperidone more than amisulpride, and sertindole more than risperidone . Olanzapine was more likely to produce an increase in cholesterol levels than aripiprazole, risperidone, and ziprasidone; no differences with amisulpride, clozapine, and quetiapine were found . Olanzapine produced a greater increase in glucose than amisulpride, aripiprazole, quetiapine, risperidone, and ziprasidone; no difference was found with clozapine . The clinician should take these side effects into consideration when choosing an atypical drug for each individual patient . Correll et al108 investigated patients with bipolar disorder and schizophrenia who were treated with sga . The authors suggested a shared susceptibility to antipsychotic - related metabolic deregulation not primarily related to psychiatric diagnosis or concomitant mood stabilizer treatment . Progress in the treatment of psychosis has occurred in the last few decades, and more recently with the introduction of new antipsychotic agents . Some new challenges are now important points that should be taken into account by the clinician, such as the safety and tolerability of antipsychotics . Ultimately, choosing the best treatment for an individual patient can result in greater adherence to medication and more success in clinical outcomes.
Iodinated contrast is frequently given to enhance the diagnostic utility of computed tomography (ct). The contrast material results in a large increase in body iodine stores, which is progressively cleared over several weeks to months . A typical ct study uses about 100 cc of intravenous contrast material, which translates to about 30 g of iodine . There is concern that this transient increase in iodine can compete with i and interfere with subsequent management, such as whole - body scans and treatment with radioactive iodine in thyroid cancer patients . In addition, many groups recommend iodine depletion diets to optimize the therapeutic response to radioactive iodine in these patients [2, 3]. Thus, avoidance of iodinated contrast material is particularly important as it can lessen the effectiveness of radioactive therapy [4, 5]. The duration of time required for body iodine stores to return to normal following iodinated contrast administration has not been well studied in thyroid cancer patients . Recommendations for avoidance of radioiodine administration after iodinated contrast vary from 4 weeks to 1 year, with most suggesting about 3 months . Iodinated contrast should be avoided if radioactive iodine therapy is planned within the subsequent few months . The european consensus guidelines also state that radioactive iodine administration should be postponed for two to three months after the event of iodine contamination . The normal thyroid has a considerable storage pool of organified iodine and since the body recirculates this iodine, thyroidectomised patients may possibly have a quicker elimination time than euthyroid individuals . If confirmed, then a briefer interval between iodinated contrast administration and subsequent radioiodine therapy could be considered and would expedite therapy . The objective of this study is to compare iodine clearance following iodinated contrast administration in thyroidectomised thyroid cancer patients and euthyroid individuals . A convenience study population was drawn from patients referred to the saint boniface general hospital diagnostic imaging department for iodinated contrast - enhanced ct studies . Thyroidectomised thyroid cancer patients were notified of the study by members of the cancercare manitoba thyroid cancer disease site group who identified eligible patients requiring contrast - enhanced ct for clinical reasons . Eligible thyroidectomised patients had previously undergone total or near - total thyroidectomy for thyroid cancer and were on a stable dose of suppressive l - thyroxine therapy . Control euthyroid subjects were recruited from routine diagnostic ct referrals coded to receive iodinated contrast . Eligible control individuals included those without documented thyroid disease and who were not taking thyroid hormone replacement . The university health research ethics board approved the study for the university of manitoba and signed and informed consent was obtained . Exclusion criteria included pregnancy, other major sources of iodine including any history of amiodarone use ever or iodinated contrast in the preceding 12 months, treatment with lithium, abnormal renal function (serum creatinine> 170 umol / l), inability or unwillingness to participate, and control subjects with known thyroid disease or those who were found to have abnormal thyroid function or anticipated survival of less than one year . Routine ct protocols used the same dye load (100 cc of omnipaque 350, ge healthcare) for all adult patients undergoing scanning of the neck, chest, abdomen, or pelvis . We recorded routine demographics and clinical characteristics including sex, age, height, weight, and any iodine - containing medications, vitamin supplements, or herbal products . Prior to the patient undergoing the iodinated contrast enhanced ct, a urine sample subjects were asked to collect a urine specimen every 2 weeks for the first 8 weeks and then monthly up to 6 months for 9 collections in total . Urinary iodine concentration is currently the most practical biomarker for determining the iodine nutritional status [8, 9]. Approximately 90% of all iodine ingested in the diet will be excreted in the urine . Urine samples were stored in a 60 c freezer until analysis at the end of the study . All samples were batch - run for assessment of urinary iodine in conjunction with creatinine at health canada . Urinary iodine concentrations were determined using ammonium persulfate digestion followed by colorimetric analysis based on the sandell - kolthoff reaction according to a modified microplate method . Urinary creatinine concentrations were determined by enzymatic idms - standardized two - point rate on an ortho - clinical diagnostics vitros 5, 1fs analyser . The urinary iodine to creatinine ratio (microg / g) was calculated as the measure of iodine clearance as used by the third national health and nutrition examination survey (nhanes iii) (19841994). A review concluded that spot urinary iodine concentration is a reliable measure of the iodine intake in the population when expressed as a function of urinary creatinine to correct for the influence of fluid intake . Demographic and clinical characteristics of the thyroidectomised thyroid cancer patients and of the euthyroid controls are described using medians, means, and standard deviations (sd) for continuous variables and frequencies and percentages for categorical variables . Group comparisons for continuous data were conducted with the student's t - test for specific time points . Models based upon generalised estimating equations (gee) were used to analyse combined data for all time points from 4 weeks onwards assuming an exchangeable correlation structure . Separate gee models were then constructed to look for an effect of time since contrast administration or a time group interaction . The dependent variable used for the primary analysis was the urinary iodine creatinine ratio . In a secondary analysis we enrolled 6 thyroidectomised thyroid cancer patients and 7 control subjects . In total 50 samples from the thyroidectomised thyroid cancer group there were no missing collections from the control group; there were 4 missing collections from the thyroidectomised thyroid cancer group . The mean urinary iodine creatinine ratio was 248.2 microg / g at baseline for the thyroidectomised thyroid cancer patients and 138.3 microg / g at baseline for the control patients . As anticipated, the two - week mean values were still elevated above baseline (thyroidectomised thyroid cancer 770.3 microg / g versus 670.3 microg / g control groups). Figure 1 shows the mean urinary iodine creatinine ratios for the two groups, demonstrating a return to baseline values 4 weeks following iodine contrast administration . Pairwise testing showed no significant group differences in urinary iodine creatinine ratios at baseline or any subsequent time point . When expressed as the change from baseline, only the 2-week sample showed significantly increased urinary iodine clearance (table 2). Gee was used to examine for differences in urinary iodine creatinine ratios between the two groups from week 4 onwards and no significant group effect was identified (p = 0.53). In a separate gee model we did not identify any significant time effect (p = 0.19) or time group interaction (p = 0.84). A gee model with change from baseline from week 4 onwards showed a statistically significant difference between the two groups, lower in the thyroidectomised thyroid cancer group (parameter 113.1 microg / g [95% confidence interval 12.8 to 213.3 microg / g], p = 0.027). Following exposure to a large iodine load, the plasma concentrations of free iodine remain elevated as the body's iodine stores are expanded in the interstitial fluids and in the thyroid colloid . Current literature suggests that, in euthyroid individuals, the body's iodine stores are increased for at least 3 months following iodinated contrast . Our study finds that the urinary iodine creatinine ratio returns to baseline by 4 weeks following iodinated contrast in both euthyroid and thyroidectomised thyroid cancer subjects . In addition, there was no significant difference between the thyroidectomised and euthyroid patients at 4 weeks, nor throughout the rest of the study period up to 6 months . In which urinary iodine levels returned to baseline at one month in thyroidectomised thyroid cancer patients after receiving iodinated contrast . A prospective study by nimmons et al . Showed that the median time for urinary iodine levels to normalize in euthyroid subjects was 43 days, with 75% of subjects returning to baseline by 60 days . Our study enrolled only 13 subjects in total and may have been underpowered to detect significant differences between the two groups . The small numbers of cases and controls are compensated for by the multiple time points assessed, which allowed for all urinary collections from 4 weeks onwards to contribute to the statistical analysis using gee . In addition, thyroid cancer patients need to be on a low iodine diet prior to radioiodine treatment . While this study demonstrates that urinary iodine creatinine ratio returns to baseline by 4 weeks in both thyroidectomised thyroid cancer and euthyroid subjects, it does not look at the effect of a low iodine diet on the urinary iodine creatinine ratio post iodinated contrast . We did not collect data on noncontrast sources of exogenous iodine (e.g., diet, vitamin supplements), but our analysis of the change in iodine excretion from baseline partially compensates for interindividual differences in dietary iodine intake . Increasingly, ct is being compared to ultrasound, magnetic resonance imaging, or positron emission tomography techniques . As more information is gained about the sensitivity, specificity, and accuracy of these techniques, ct may be used less frequently which would ameliorate the concern of iodinated contrast and therapeutic radioactive iodine . In conclusion, we found that 4 weeks may be sufficient for urinary iodine creatinine ratios to return to baseline value following iodinated contrast in both thyroidectomised thyroid cancer and euthyroid subjects . This suggests that clearance time may be more rapid than previously thought and that a shorter time interval between iodinated contrast and radioiodine therapy might be considered but requires confirmation in larger studies that include a broad case mix in terms of age and dietary iodine intake.
Cervical cancer (cc) is listed among the top five cancers that affect women globally . According to the international agency for research on cancer (iarc), in 2008 there were 530,000 new cases of cervical cancer worldwide, with 85% of the disease burden occurring in developing countries . Caribbean women are at an increased risk of dying from cc, which is the second most frequent cancer among women and also ranks as the third most frequently diagnosed cancer in both sexes . In the united states (us), cc has decreased in incidence and mortality since the mid 19th century, primarily because of screening . Even with the introduction and widespread use of the pap test, cc still ranks among the top ten cancers diagnosed in the us among minority populations, which includes blacks, american indians, and hispanics . Persistent high - risk (hr) human papillomavirus (hpv) infection, specifically hpv types 16 and 18, has been linked to the development of cc, anogenital cancers, and oropharyngeal cancers [4, 5]. In 2006, the fda approved the hpv vaccine gardasil (against hr hpv types 16 and 18 as well as low - risk types 6 and 11) for all females aged 9 through 26 . In 2009, the fda also approved the use of this vaccine in males aged 9 through 26 . Cervarix, which aims to protect against hr hpv types 16 and 18 only, was approved by the fda in the fall of 2009 for females aged 10 through 25 . The centers for disease control and prevention (cdc) advisory committee on immunization practices (acip) recommended routine hpv vaccination for adolescent boys and girls as young as 9 years and men and women as old as 26 . However, even with the availability of these vaccines, there is still a disparity in their utilization between races . Minorities are not receiving and/or completing the vaccine process at the same rate that their white counterparts are in the us . A study conducted among 363 african - american college females showed that only about 25% of young women report uptake of the hpv vaccine . Vaccine uptake was associated with significantly higher levels of hpv knowledge, lower perceived barriers to vaccination, and younger age . With respect to vaccine completion, niccolai et al . Reported that completing the vaccination series was associated with being white and having an annual household income> $75,000 . Reasons as to why disparity exists for hpv vaccine uptake and completion remain unclear in the literature . Some studies attribute racial disparity to income level differences, while others believe it is due to lack of knowledge [1012]. The poor response and completion rate of hpv vaccination amongst minorities still need to be fully assessed . In our earlier study of hpv knowledge amongst the general population, we reported that the general population was aware of hpv as well as the hpv vaccine; however, the benefits of the vaccine to the study population were not evident . When looking at knowledge according to race, people of african descent were less informed than whites (black 89% versus white 97%, p> 0.1). Cultural beliefs or perceptions and lifestyle are distinct between persons born in the us and those born in other countries [1416]. While there is a similar concern for high cc incidence among black women in the caribbean and the us, interventions that promote hpv vaccine uptake and completion may need to be tailored differently for each group . Prior to the development of needed interventions, information on knowledge and attitudes toward hpv and its vaccine is needed . Yet, no previous publication has assessed hpv knowledge and/or attitude towards hpv vaccination in the caribbean . Therefore we have expanded our previously published study to include an example of a caribbean nation (the bahamas) where cc incidence (17.6 per 100,000) is similar to the incidence of cc for the caribbean region (20.8 per 100,000). We sought to determine knowledge and attitudes of hpv and the vaccine in the bahamas and determine whether differences exist between different cultures of african descent . The original study population consisted of 202 volunteer participants from the university of pittsburgh and hampton university . The recruitment was expanded and involved two additional locations: the new york city (nyc) metropolitan area and the bahamas (bhm), west indies . Therefore, this study includes a diverse study population recruited from the general population between 2008 and 2010 from three urban cities in the north eastern us differing in size and demographics, (small (hampton, va, population 136,401) medium (pittsburgh, pa, population 307,484) and large (nyc, ny, population 8.245 million). Both hampton, va and pittsburgh, pa, comprise more of an african - american demographic compared to nyc where a higher proportion of the population consists of immigrant families, including the caribbean . We believe that this mix is diverse and may be more representative of the us population . Institutional review board approval was obtained from the university of pittsburgh, suny downstate medical center, and princess margaret hospital in the bahamas . For all study locations, study participants were recruited face - to - face or using posted flyers in various settings including hospitals, private and other clinics, and community locations (e.g., parks, restaurants, barber shops and laundromats, etc . ). The original english questionnaire was formally translated into haitian creole by the haitian embassy in the bahamas in order to accommodate creole - speaking participants . Those who were not able to read or write well had assistance in completing the questionnaire . A total of 793 surveys were administered and returned; of these, 231 were excluded from the present analysis because they were completed by people who identified themselves as some race other than black . The study population was then divided into two groups based on place of current residence us and west indies . The questionnaire used in this study consisted of demographic questions pertaining to age, race, sex, place of birth, religion, household income, level of education, health insurance status, parental status, and number of children . The study instrument included measures of hpv knowledge and the safety, efficacy, and impact of the hpv vaccine . A content assessment of the survey was conducted prior to the launch of the study . Descriptive statistics were generated for demographic and knowledge variables according to the place of residence for study participants . Fisher's exact test was used to determine statistical significant differences between proportions; differences in continuous variables were assessed using two - sample t - tests . Adjusted prevalence of correct answers to hpv and hpv vaccine knowledge questions was calculated and stratified by geographic location after adjusting for age, level of education, marital status, parental status, health insurance status, and income level . Ordered logistic regression was performed to assess differences in response to knowledge questions pertaining to hpv and hpv vaccination between the two groups, adjusting for demographic variables and the covariates previously mentioned . Statistical analysis was performed using stata version 10.1 software (stata lp, college station, tx, usa). A total of 555 black participants residing in the us and the bahamas (bhm) completed the survey (41% from the us and 59% from the bhm (table 1)). Between both populations, 4% of the subjects (n = 23) were immigrants and not born in their current country of residence . Twenty - five percent of the participants were male, and 75% were female; there was no difference in gender between the two geographic locations . In both locations, the majority of the participants were between the ages of 18 and 35, with a higher proportion of this age group in the us sample compared to the bhm (80.5% versus 49%, p <0.0001). Eighty - four percent of the us population was single compared to only 42% of the bhm population . Parenthood also demonstrated differences, where more bahamians tended to be parents (bhm: parent = 65.2% versus us: parent = 23.9%, p <0.0001). The correlation between parental status and marital status also differed between the us and bahamas . The majority of bahamian parents were married (45%), while the majority of us parents were single (41%). Us participants were more likely to have attended or completed college (us: some college = 55.3%, college graduate = 27.4% versus bhm: some college = 17.9%, college graduate = 22.0%, p <0.001). Income levels varied modestly between groups up to the us $50,000 mark; 40.7% of us respondents fell into this bracket compared to 11.7% of bahamian respondents reporting this as their income . General knowledge about hpv and the hpv vaccine differed between the two countries significantly (table 2). When asked if they ever heard of hpv, bahamian participants were less aware of the virus than participants in the us (us 89.5% versus bhm 61.5%, p <0.001). This difference in knowledge remained consistent for 6 out of the 10 questions related to hpv knowledge, while there was no difference in knowledge for four specific questions . Although the majority of respondents from both locations knew that only women could develop cervical cancer, lower proportions of respondents knew that hpv causes genital warts (28.2% of us and 33.2% of bahamians) and that genital warts are not caused by the same hpv types that cause cervical cancer (13.8% of americans and 7.5% of bahamians). For the questions related to hpv vaccination (table 3), the majority of both populations heard of the vaccine, however, the proportion was higher among american respondents (us 66.9% versus bhm 50.6%, p = 0.021). For two of the three questions assessing knowledge about the hpv vaccine (table 3), bahamian study participants were more likely to answer questions incorrectly when compared to american blacks . At the time the questionnaire was administered, the hpv vaccine had not yet been approved for males; when asked who was eligible to receive vaccination, approximately 51% of americans answered correctly versus only 35% of bahamian participants (p = 0.013). More than half of both populations knew that, in spite of vaccination, annual pap smears were still needed to screen for cervical cancer according to the recommendation at that time . Even so, a significantly lower proportion of bahamians answered this question correctly (us 75.3% versus bhm 51.5%, p <0.0001). There was no difference between the two groups when asked what age group was eligible to receive the vaccine; less than half of each group answered with the correct age group, 926 (us 47.6% versus bhm 40.4%, p = 0.264). For the participants that had previous knowledge of the hpv vaccine, the source of this information was similar between the two groups (figure 1). For the us, hpv vaccine awareness was first provided through an advertisement followed by a health care professional, the news, and school, although, in the bahamas, the primary mechanisms for promoting awareness were advertisement followed by a health care professional and the news with minimal contribution from school . Table 4 shows cumulatively the number of answers that respondents from each geographic location answered correctly . After adjusting for covariates (table 5), bahamian respondents were less likely to have higher numbers of correct knowledge answers when compared to americans (adjusted odds ratio [adj . Or] 0.47, 95% confidence interval [ci] 0.300.75). Older age, regardless of location, was also associated with answering fewer questions correctly (adj . Or 0.61, 95% ci 0.400.92). Having health insurance and higher income levels, regardless of location, was associated with answering higher numbers of questions correctly (adj . Or 1.76, 95% ci 1.072.92; adj . Or 1.21, 95% ci 1.081.35, resp . ). When asked whether or not the hpv vaccine should be given to both boys and girls, both us and bhm participants had mixed views; however, the majority of both groups agreed that both genders should receive the vaccine (figure 2). For both us and bahamian parents, the majority indicated that they were willing to vaccinate their daughters (us = 21/51, 41% and bahamas = 96/187, 51%). For the us parents unwilling to vaccinate their daughters, however, it was not possible to make comparisons between the two populations since 66% of the bahamian parents who were unwilling to vaccinate their daughters did not indicate the reason . For both groups, 90% or more respondents agreed that assurances were still needed for vaccine safety and efficacy . Overall, the majority of participants from both populations did not feel that administration of the vaccine would encourage risky sexual behavior and felt that discussing issues of sexuality before vaccination was necessary . When asked if an informed child should be able to request vaccination at sexual health clinics without parental consent, nearly 80% of bahamian respondents felt that children should not be able to receive the vaccine without parental consent compared to 57% of american respondents . A number of international studies have focused on knowledge and attitudes related to hpv and the hpv vaccine . However, a majority of these studies have been conducted in europe, asia, and africa [1723], with little work done in latin america and no studies conducted in the caribbean . This study is the first to report on overall hpv and hpv vaccine knowledge and perception in a sample of black volunteers containing a large number of subjects from the us and caribbean, and we have compared our findings by geographic residence . Our previous study showed a lack of knowledge among people of african descent in the us, which remained consistent with our current findings and other published literature [24, 25]. However, the present study indicates that overall knowledge was significantly lower in bahamian participants when compared to american blacks, even after adjusting for possible confounders . More than half of participants from each location were aware that a vaccine for hpv is available; however bahamian participants were less aware of this . Studies that focused on populations of african descent, outside of the us, report a similar lack of knowledge in regard to vaccine availability [21, 26], and educational attainment has been shown to be associated with familiarity of hpv and its vaccine [23, 25, 27]. In our study population, us participants had higher levels of education compared to bahamian participants, which could explain the lesser familiarity with hpv and its vaccine in the bahamas compared to the us . For persons who were aware of the hpv vaccine, there were slight differences between the us and bahamas in the type of resource from which they received this information . While advertisements were the major resource for obtaining information about hpv vaccines for both the us and bahamian populations, health professionals were also an important resource for the us population but not as much for the bahamas . Further investigations that help clarify the reasons why health professionals are not currently important resources for educating the bahamian population about hpv vaccines are warranted and might help provide insight for improving hpv vaccine awareness in this geographic region . Published literature suggests that in most cases, after parents were well informed about the risks and benefits of the vaccine, they were willing to vaccinate their children [11, 19, 28], and we have noted that in our study the majority of parents were willing to vaccinate their daughters . For the us, parents who were unwilling to vaccinate their daughters identified their reasons as safety concerns . However, in this study we were not able to compare the reasons for unwillingness of parents to vaccinate their children between the two countries, because of low response rates to those questions for the bahamian parents . It was not clear why the response rate for this question was low; however, it is possible that the participants may not have been comfortable with disclosing their reasons . In general, attitudes related to hpv vaccination were similar between the us and bhm but differed when the two populations were asked if a well - informed child should be able to request vaccination at sexual health clinics without parental consent . Us participants were more likely to agree with this statement (p <0.001). This difference may suggest that americans have a higher self - worth in regard to their health, which is not the case when it comes to children . Hughes reports that children considered themselves to be passive participants when determining their course of medical care . In our study population, the majority of parents from bhm were married in contrast to the parents from the us . In the west indies, the importance of family has been distinguished and deemed quite atypical to that of the western world . Although speculative, the rationale behind the majority of bahamian respondents disagreeing with children being able to receive the vaccine without parental consent might be explained by differences in overall family structure and child rearing practices, which have been shown to differ when compared to the us . Still, with the spread of american culture globally, family values have begun to change within the caribbean, which would explain why younger respondents from bhm tended to agree with the statement . Respondents who agreed with the statement about children being able to decide about their own vaccination tended to be younger people (<35 years), regardless of geographic location (p <0.001). Lack of knowledge in regard to hpv and the hpv vaccine is not a problem that is limited to peoples of african descent but extends to ethnicities within developing countries all over the world . Regardless of geographic location, whites have consistently displayed higher levels of knowledge concerning hpv and the vaccine when compared to other ethnic groups [13, 26]. Effective methods in disseminating necessary information to these populations with lower health education need to be assessed and put into action to minimize future cases of hpv - related cc . Given that the majority of study participants first heard about the hpv vaccine via advertisements or their health care providers, design of an intervention that combines the two may be effective in reaching the public . In cases where access to health care providers is limited, the use of community - based educators may prove to be more effective than gaining information from a physician . Moreover study findings suggest that interventions to increase the uptake of hpv vaccine in the caribbean must include parental involvement . In summary, the loss of productive lives and quality of life caused by morbidity and early death from mortality due to cervical cancer in the caribbean and latin american region is significant . Determining the best strategies to educate and increase hpv prevention practices in this population is crucial and needs to be treated with a sense of exigency in order to decrease the burden of cc in this region.
When biologists teach contemporary or historical discoveries in the life sciences, we often aim not only to impart the facts but also to explore how we know what we know focusing on the scientific process that led to the revelation of such new scientific information . Many scientist - educators would also like to include in their courses the examination of the future implications of such discoveries, encompassing both the future basic and applied scientific potential as well as the accompanying bioethical dilemmas . Both involve exploring the scientific reaches of new discoveries, but examining bioethical future concerns additionally requires some familiarity with ethical theory and ethical reasoning . In effect, looking forward biologically, we ask what can we do? While looking forward ethically we ask what should we do and why? To educate our students responsibly, and to introduce bioethics effectively, we should not simply allude to future bioethical challenges thereby raising awareness but should also help students to begin developing a framework for reasoning through those challenges to some resolution about what ought to be done ethically . Since its emergence as a discipline in the 1970s, bioethics has taken shape as an important academic discipline that intersects with the life sciences in myriad ways, and not only with genetic, biomedical, or biotechnological advances . The unesco universal declaration on bioethics and human rights (15) established in 2005 has identified 15 principles that range from human dignity, social responsibility, equality, and justice, to protecting the environment, the biosphere, biodiversity, and future generations . However, the integration of clearly reasoned bioethical thinking into undergraduate biology and microbiology curricula has not kept pace with this wide - ranging international awareness and is often limited to research or professional ethics . During this emergence of bioethics, the traditions of research and professional ethics have evolved with their respective mandates, and have been the default for integrating ethics into undergraduate or graduate courses in the life sciences . Research ethics prepares students to perform research ethically, in part by understanding the need to propose research with animals to institutional animal care and use committees (iacucs) or submit research proposed with humans to institutional review boards (irbs). Professional ethics teaches students the importance of abiding by professional codes of conduct such as the code of ethics for the american society for microbiology (http://www.asm.org/cclibraryfiles/filename/000000001596/asmcodeofethics05.pdf). Although research and professional ethics are necessary, they do not adequately address the range of bioethical questions that continue to emerge as the fields of biology and microbiology advance . These questions relate to scientific course content more directly than do policies, regulations, and codes . Indeed, there is some evidence that students appreciate the need for professional ethics but do not recognize the personal effects of morals and behavior (6), which is in itself a reason for folding ethics into biology courses . When women s studies began in the 1970s a favored pedagogical tactic advised instructors to add women and stirtake a traditional course and append a unit that would address women s issues . Though initially useful, the measure was eventually seen to marginalize the most crucial discussions about women (10). Since those early days, infusing women s issues into the general curriculum has proven an effective tactic, especially when viewed in relation to the original add women and stir approach, for infusion tactics require integration of material into the stream of the courses in question, discussing both together, contextualizing and enriching both standard and gender analyses . In this perspective, we heed that lesson and apply it to the teaching of bioethics in undergraduate science curricula: instead of adding ethics and stirring, that is, instead of inserting ethical issues at several points in each course necessarily very briefly, and without requiring much depth of thinking we recommend introducing one ethical topic allied to the professor s area of expertise or interest and taking the time to work this topic into the science material by means of ethical reasoning . Questions generated by such topics are many and varied . For instance, should we try to clone animals that are on the verge of going extinct even though that extinction is driven by habitat destruction? How do we balance the need to improve agricultural yields with stewardship of the environment? Is feeding fast food / junk food to children in school lunches ethical when we know it cannot only negatively affect their health but also potentially the health of subsequent generations? Should there be any limits placed on the genetic information commercially available to individuals? With advances in neuroscience, if pharmaceuticals that enhanced cognition were developed, who could / should take them? Should people, particularly those who are terminally ill, be able to choose the time and means of their own death? What responsibility do we have to maintain the increasingly large pool of embryos developed in vitro but which have not been chosen for implantation? Is it ethical to bypass the safety standards for developing safe drugs and vaccines when an epidemic such as ebola is rapidly expanding? This vertical infusion approach is also consistent with the reforms currently taking place in undergraduate biology education with more active, student - centered learning, greater interdisciplinary focus and problem - based learning, as detailed in the aaas report vision and change in undergraduate biology education: a call to action (1). In this report of a 2009 national conference designed to develop a blueprint for real change in biology education, the ability to understand the relationship between science and society is identified as a core competency in the practice of science with one example being evaluating ethical implications of biological research many excellent paths have been proposed to invite students of science disciplines to stretch their reasoning abilities toward the analysis of ethical issues . In 1990, when forming the national human genome research institute (nhgri) the national institutes of health (nih) included the ethical, legal, and social implications (elsi) of the human genome as a significant component of its funded research programs (8). Biology textbooks then began to incorporate elsi into their auxiliary information, enclosed in textboxes or simply as questions in the margins for those faculty and students who wished to explore these implications further . However, though most often these textbooks dutifully ensure that students get the science right, they do not ask students to reflect more deeply, reasoning with the aid of ethical principles, in order to reach a just resolution of the dilemmas presented . Another approach is the establishment of science - based bioethics courses within undergraduate biology curricula, as seen at columbia university in its crossroads in bioethics and bioethics for biomedical engineers courses . In this approach, the scientific issues, ethical challenges and ethical theories appropriate for resolving bioethical challenges are melded into one course (5). Columbia s integrated bioethics courses have the advantage of giving the students a chance to reason challenging dilemmas through to an ethical conclusion based on both the science and the ethical principles . One, entitled gynebioethics: reproduction and beyond, melded reproductive biology and bioethics, treating, for instance, the genetics of sex determination with the ethics of sex selection . Another course, the bioethics of food, explored the biological features of the western diet along with its consequent bioethical entanglements, such as, for example, the duty of a state to outlaw certain substances, such as trans fats, or genetically modified crops . The advantage of such courses is that the science content is more focused, so less competition exists for coverage of that content, leaving more time to actually explore and analyze bioethical issues in depth . However, not all biology programs can afford the faculty resources to develop such integrated approaches . Still another strategy for including bioethics in the undergraduate biology curriculum could be requiring life science majors to complete an allied bioethics course, as is often the practice for other foundational courses such as chemistry, physics, or math . Although we are not aware of many programs that do so (outside of pre - health studies, such as nursing) one example is the microbiology undergraduate program at rutgers (http://dbm.rutgers.edu/microbio.php) which requires a professional ethics course . We have mentioned the limits of such courses: they canvas professional codes and policies but not, as a rule, the types of ethical issues that scientists and citizens together may encounter as science advances, such as the possibility of three - parent embryos, the ownership of water rights as sources become scarce, the ethics of transgender fertility, the prospect of cloning beloved pets, or the question of what responsibilities biomedical research institutes have to patients whose tissues are harvested . Yet, even if expanded beyond professional ethical material, such stand - alone bioethics courses are most often taught by philosophers who are not necessarily prepared to integrate the science needed . In cases where this allied course requirement is the approach taken, it would be best to offer bioethics courses team - taught by a scientist and a philosopher to be able to incorporate effectively the science with the ethics . We have offered such team - taught bioethics courses and find students from many backgrounds biology, philosophy, anthropology, history, psychology, exercise science, and business fully engaged in the interfaces between science and ethics . Such courses are not required in our biology curriculum at transylvania university but remain very popular electives . Both of these kinds of stand - alone courses the science - based with constant bioethical integration and the team - taught courses with both philosopher and biologist have definite merits, but they do not address the concerns of biology professors who are committed to challenging their students directly, in the heat of a standard biology or microbiology course . Chamany and colleagues put it this way: as biology instructors we may choose to teach biology devoid of social context, believing that students can make these connections on their own . But students model their instructors behaviors, and follow their lead (4). Given the ever - increasing bioethical concerns attached to practical biological applications, it is not surprising to find many of us wanting to integrate bioethical questions into our own courses choosing to be the role models chamany and colleagues discuss . Confident voices of professionally - trained biologists will be needed more urgently as new biotechnologies and other bioscience discoveries emerge, especially in the span of our current students careers . If the question is, what can i do in my own courses to help stimulate mature and considered thinking about ethical aspects of what i teach? Biology professors should not apologize for being concerned primarily with scientific content and student ability to understand, apply, and integrate biology principles appropriately delivered to challenge student problem - solving and analytical thinking skills . When asked what impedes their treatment of ethical issues in their courses, most point to a lack of time (3). Whatever ethical content we examine, it should not interfere significantly with the science being taught but rather complement it and encourage students to see how important good science is to sound ethical reasoning and practice . Even though textbooks use elsi textboxes and thought - provoking questions to encourage discussion of bioethical issues, instructors may avoid doing so because these novel issues are thought to deflect student concentration at crucial stages of biology instruction . Even if the bioethical prompts are used to raise a number of ethical questions during a term (without going beyond merely showing that an ethical dimension exists), first, such bioethics instruction can be distracting and time - consuming; second, and just as important, since faculty may not want to spend much time on each insertion of bioethical thinking, students could easily get the impression that ethical issues are either too vague and indefinable to contemplate or too unimportant to squander cognitive resources on (11). Complaints like these, especially the latter, contributed to the diminishment of the add women and stir approach in women s studies (10). The horizontal approach seems most appropriate for specialized, integrated science - based bioethics courses discussed above, where one is not limited to brief analysis of each bioethical issue, rather than for introductory or advanced biology or microbiology courses in a standard curriculum . Vertical infusion would involve a single in - depth analysis of a relevant bioethical issue in each or several courses in a biology / microbiology program . Horizontal infusion would involve introduction of multiple bioethical issues in less depth across a single or a few courses in a biology / microbiology program . So what do we suggest for professors who responsibly commit to coverage and understanding of the science but also realize the importance of encouraging students to think seriously about ethical issues? We advise what we call a vertical approach to teaching the ethical dimension . Instead of trying to invest ethical import wherever it might fit, or, at several convenient points along the progress of the course, the vertical approach focuses on only one issue during the term and only for one occurrence but that issue is explored in more detail than would be possible if several had to be envisioned . Further, the topic in question could be selected in light of the professor s research interests, thus making preparation less onerous and more enticing for professor and students alike . If, for instance, one is studying the establishment of the human gut microbiome using 16s rdna analysis, and the course in question is an introduction to microbiology, then one could expand the normal flora section by discussing biobanking microbes from the human intestinal tract and the subsequent bioethical questions of consent, confidentiality, and privacy (see, for instance, the treatment of related ethical issues in ref . In this fashion, the question could be addressed more deeply, even to the point of encouraging students to think with the aid of ethical principles, such as the principlist ones now common in suggested bioethics curricula, both globally and nationally (5, 9, 12, 14). (note: we are not advocating for an exclusive focus on principlist ethical theory but suggest that it is a comfortable place to begin .) These basic ethical principles include autonomy / respect for persons, beneficence, non - maleficence, and justice . Their use became more common in 1979 after the release of the belmont report from the national commission for the protection of human subjects of biomedical and behavioral research (7) because they are acceptable principles in most cultural and religious traditions . Hence, instead of merely pointing out current policies guiding the just operation of biobanks (professional practice approach), one could ask why these practices are ethical, what principles justify them, and how these principles might radiate out to other similar biomedical practices . Undergraduate biology and microbiology professors have considerable content to cover, whether in an introductory or advanced course, as we have mentioned . We believe that the horizontal means of infusion of ethics (many examples in one course that are not covered in much depth) can distort the focus of the course, and diminish the importance and urgency of bioethical challenges . Conversely, a vertical approach, where each faculty member might develop a single ethical issue out of their own area of interest, would encourage deeper, more responsible thinking on the part of students given the parameters of a standard, single - term biology course . With the intense focus comes a relatively smaller portion of time one needs to allot to integrating ethical concerns . A lab period, or a couple of class periods, especially when devoted to and motivated by one s own passion, could suffice for deep engagement and productive conversation . Using the example of rights to a person s microbiome, a professor could explore how students have come to their conclusions about the issue, what ethical principle might lie behind that conclusion, and whether that ethical reasoning might itself be hiding a clash of principles . Imagine also if a number of faculty members in a department began vertically infusing a single ethical issue in each of their courses . Throughout a student s major pattern, they would have encountered several ethical issues and been encouraged to give them respectable consideration . On the best construal, each student would be integrating these issues from each course into a coherent style of thinking and critiquing . Any individual student would then have thought about the ethics of gmo foods, stem cells, antibiotic use, preservation of biodiversity, dna fingerprinting and biobanking, for example, finding similarities and seeking a consistent application of ethical principles . In such a program, students would grow to see their discipline in an even richer dimension and would not be hesitant to engage in the common social discourse about ethical issues related to scientific discoveries . With the vertical approach, instructors choose their own occasions for infusing bioethical questions, according to their own scientific strengths . How then to help students think clearly about ethical implications that attend those strengths? As we have mentioned, many excellent resources abound, attached to stand - alone courses and large programs, which might prove valuable, but by and large they are not targeted to aid undergraduate biology faculty who want to infuse a single issue . They are often too narrowly focused on medical ethics or human rights, or they target high school or professional school audiences . The nsf - sponsored national center for case study teaching in science (http://sciencecases.lib.buffalo.edu/cs/collection/) holds in its peer - reviewed collection more than 20 cases related to bioethics that are appropriate for introductory biology courses as well as upper - level ones . However, while well conceived, up to date, accurate with respect to science and technology, and framed with innovative pedagogical suggestions, they nonetheless do not offer help when it comes to actual reasoning about ethical principles . For biology professors using the vertical approach, we would, ideally, suggest that they consult a major classical formulation of principlism found in the principles of biomedical ethics by thomas beauchamp and james childress (2). Also, reliable but briefer introductions to those principles are widely available (for instance, thomas mccormick s at https://depts.washington.edu/bioethx/tools/princpl.html). The best way to learn on one s own how to infuse such bioethical principles into one s pedagogy is, as we have suggested, to begin where one is most comfortable, in one s own field, select an ethical issue, and work the principles into one s teaching with that single issue . The best assistance for busy faculty would be for asm through jmbe or microbelibrary to add a new area to their curriculum resources that focused on resources to support infusing ethics into biology and microbiology curricula . Ideally this would include a variety of resources on different ethical theories that could be applied in science courses, how they could be applied, and possibly additional case studies that demonstrate how to utilize various ethical theories to address bioethical issues . An online course designed by philosophers and biologists together or a summer team - taught workshop (possibly at the asm conference for undergraduate educators) on infusing bioethics into biology and microbiology courses would be most helpful . These would not be strictly human- or human rights - focused but would include the broad range of ethical issues appropriate for undergraduate biology and microbiology courses from responsible use of antibiotics, biotechnology, and neuroscience to agronomy, physiology, and climate change.
Real - time monitoring of the circadian gene expression was performed using rat-1 cells expressing a luciferase reporter under the control of the upstream region of bmal1 gene . Rat-1 cells were treated with 0.1 m dexamethasone for 2 h for the rhythm synchronization, and the bioluminescence signals from the cells were continually recorded at 37c under air with dish - type bioluminescence detector, kronos (atto, ab-2500) or lumicycle (actimetrics). The in vitro kinase assay was carried out at 30c in a reaction mixture (10 l) consisting of 1 mm dithiothreitol, 100 m [-p]atp, 10 ng 30k - camkii and 100 ng substrate protein . The substrate proteins, gst - sp, mbp - nt, gst or mbp (each 100 ng) were prepared as described previously . After incubation for 30 min, the reaction was stopped by the addition of 10 l of 2 sds sample buffer . Real - time monitoring of the circadian gene expression was performed using rat-1 cells expressing a luciferase reporter under the control of the upstream region of bmal1 gene . Rat-1 cells were treated with 0.1 m dexamethasone for 2 h for the rhythm synchronization, and the bioluminescence signals from the cells were continually recorded at 37c under air with dish - type bioluminescence detector, kronos (atto, ab-2500) or lumicycle (actimetrics). The in vitro kinase assay was carried out at 30c in a reaction mixture (10 l) consisting of 1 mm dithiothreitol, 100 m [-p]atp, 10 ng 30k - camkii and 100 ng substrate protein . The substrate proteins, gst - sp, mbp - nt, gst or mbp (each 100 ng) were prepared as described previously . After incubation for 30 min, the reaction was stopped by the addition of 10 l of 2 sds sample buffer . The animal experiments were conducted in accordance with the guidelines of the university of tokyo . The mice (c57bl/6 background, male, 10-week - old) were housed individually at 23c in cages with food and water available ad libitum . This work was supported in part by grants - in - aid for scientific research from jsps and mext, japan.
Discoveries of restriction enzymes by hamilton smith and daniel nathans, and reverse transcriptase by howard temin and david baltimore in the 1970s set the stage for an explosion in molecular and clinical genetics . Armed with a sufficient amount of the mrna product of a gene such as the beta globin gene, investigators could now produce a radioactive dna copy (cdna) of the mrna and use it as a valuable probe of gene expression . Cloned into an expression plasmid and transduced into an appropriate cell, the cdna would produce mature beta globin mrna, and cdnas could be manipulated at will in what became the era of recombinant dna technology . The new discoveries were greeted with enthusiasm by most biologists and clinical investigators but with mounting horror and suspicion by many members of the public and their elected officials, as well as some academics . Frankensteins were thought to be loose in biomedical laboratories; monsters would be created; plagues of vicious e. coli would be loosed on an innocent population; mad scientists would forever contaminate the food supply; the new genetics would lead to a resurgence of social darwinism . The specter of nazi medicine roiled some faculty meetings and communities in which the science was rapidly advancing . Rules and restrictions were demanded that would deliberately inhibit the work . To their credit, leaders of this new genetics revolution met in asilomar california, where they established laboratory standards intended to reassure themselves, their colleagues, and the general population that gene manipulation could be rendered safe and useful . While eager clinical investigators hoped to apply the new genetics in the treatment of inherited diseases, cooler heads recognized that the technology was not sufficiently powerful or predictable . The more cautious advised the national institutes of health to be very wary of human application because the biological' rules of the game' had not yet been established . That did not stop an american clinical entrepreneur from injecting beta globin cdna into the bone marrows of thalassemic patients in italy and israel . His reward was failure and opprobrium, and academic medicine encountered a congress increasingly determined to surround clinical research with an ever - growing net of regulation . How could a cdna be introduced efficiently into a rare, quiescent, mammalian cell, such as a bone marrow stem cell, remain potentially active, and be sufficiently expressed when that stem cell developed into a hematopoietic precursor and subsequently fully differentiated functional blood cell? Furthermore, could a defective gene be actually replaced in human cells in a targeted fashion by a normal counterpart and still maintain high transcription efficiency, or would such' plug and socket' technology be so inefficient that correction would be impossible? Instead, could cdnas such as a beta globin cdna be carried into the target cell chromosomes on the back of a virus, such as a retrovirus, and could the transduced sequence express its mature mrna regardless of its genomic location? The idea of a retrovirus as a gene - transfer agent was first seen as dangerously oncogenic, until 1983 and 1984 when mann, mulligan and baltimore devised cell lines that would produce replication - defective retroviruses that still exploited the capacity of the viruses to incorporate themselves efficiently in the dna of dividing cells . For the most part, the modified retroviral vectors infected human cells at comfortingly low multiplicities of infection . Shortly after this, williams and mulligan and dick and bernstein, and their colleagues, showed that murine bone marrow cells could be transduced with defective retroviruses carrying cdnas, and that mature nucleated blood cells would carry the foreign cdna for weeks, proving that the murine hematopoietic stem cell, despite its very low rate of division, could be so transduced . But the percentage of infected cells was very low and expression of the transferred gene was vestigial . The results suggested that successful gene transfer with cdnas borne on replication - defective retroviruses would require high recombinant viral titer, cell culture systems that would encourage stem cell division without differentiation, and a setting in which target cells would have a selective advantage following gene transfer . The entire field of gene therapy was energized by the findings of williams and dick and their colleagues, but the barriers to translation and clinical application were soon found to be almost insurmountable . The slow pace prompted orkin and motulsky to lower their expectations for immediate clinical application, and focus instead on solving the basic technical and biological problems . In an attempt to gain some clinical traction, blaese and his colleagues introduced retroviral cdna ex vivo into the mature t cells of patients with immunodeficiency due to mutations in adenosine deaminase . The treatment provided little if any clinical benefit, and the risk of malignancy was obviously high because the t cells were influenced to divide in culture in order to enhance transduction . Concerns about unwieldy and potentially unsafe clinical research protocols began to mount in the united states . Four levels of review, the recombinant dna advisory committee, individual institutional review boards, individual institutional biosafety committees, and the food and drug administration all established barriers that slowed the pace of gene therapy clinical research to a crawl . This necessary regulatory environment was onerous enough, but it became even more obstructive when investigators at the university of pennsylvania performed a study of gene replacement in a rare metabolic disorder using adenovirus as a vector in order to infect non - dividing liver cells . One young adult with the disease died after a high titer of virus was administered . An investigation revealed that the gene therapists had a financial stake in the company that produced the vector . That revelation initiated an even higher burden of regulation and added massively to a growing concern about conflict of interest in clinical research - a conflict that continues to roil academic waters to this day . Meanwhile, after four decades of development, the clinical application of allogeneic hematopoietic stem cells in transplantation for the treatment of congenital bone marrow diseases was moving ahead reasonably briskly (sans gigantic regulatory hurdles). This form of cell - based therapy, though initially applicable in only the 25 - 30% of patients with histocompatible donors, was associated with success in several patients with severe immunodeficiencies, congenital bone marrow failures of several types and even the inherited hemoglobin disorders . Adenovirus vectors were thought to be too immunogenic to be useful but they and their cousin, the tiny adeno - associated virus, were considered to be worthy of evaluation in the transduction of non - dividing cells such as liver cells or endothelial cells . Indeed, high and her co - workers have made quiet progress in the correction of canine hemophilia with adeno - associated virus . Most groups interested in hematopoietic targets or cancer vaccine protocols continue to focus on defective murine leukemia viruses or lentiviruses . The latter are thought to have a higher capacity than murine leukemia viruses to integrate within the dna of non - dividing cells . Both the science and the regulatory apparatus seemed to be daunting, and the funding was fragmented and difficult to obtain . But in 2002 cavazzana - calvo and his colleagues blew new life into the field when they made the startling announcement that they had successfully treated nine of ten patients with x - linked severe combined immunodeficiency (scid), utilizing a fairly standard murine leukemia viral vector that carried the common gamma chain of the interleukin-2 receptor into autologous bone marrow cells . Shortly thereafter, however, cynicism returned when the authors reported that several of the patients had developed t cell leukemia . Careful work by the investigators revealed that the long terminal repeat (ltr) of the vector may have a predilection for the lmo2 proto - oncogene on chromosome 11 . But even random integration of the ltr at the lmo2 site would favor selection of such cells . The lmo2 proto - oncogene is often activated by translocation (11:14) in t cell leukemia . Clearly, the gene - corrected immunocytes had a survival advantage, but the malignant t cells had an even greater survival advantage as well as a growth advantage . What was once a promising new start for gene therapy became an enormous set - back . Vector safety had always been a pressing issue - now it had become a yawning chasm: of 20 patients with x - linked scid treated by gene transfer in these two trials, 18 are currently alive and with good immune reconstitution, but five have experienced a serious side - effect . Of these five children, one died of therapy - related leukemia, and one died of complications of a subsequent stem cell transplant that was performed as a result of the failure of gene therapy . Have been the results of retroviral correction of oxidase deficiency in chronic granulomatous disease . In two well - described cases, correction of granulocyte oxidase deficiency has been achieved but at the cost of clonal proliferation of cells activated at the sites of the mds-1-evi1, prdm16 or setbp1 proto - oncogenes . Furthermore, other in vitro studies have demonstrated similar insertions by lentiviruses bearing beta globin genes . Finally, williams and his co - workers have temporarily discontinued their pioneering work on the correction of the deficiency of dna repair pathways in hematopoietic stem cells of patients with fanconi anemia, because they are concerned that current vectors that permit rescue of those cells may induce insertional mutagenesis and leukemia in the treated patients, and that focus should be on developing methods of expanding deficient hematopoietic stem cells in this disease . Despite these serious setbacks there have been some recent bright lights . In 2008, maguire and bainbridge and their colleagues reported progress on the treatment of leber's optic atrophy with an adeno - associated viral vector applied to the retina . And retrovirally transfected epidermal stem cells have been grown into keratinocytes to correct the lesions of epidermolysis bullosum . More follow - up is needed but the initial results hold promise for local applications of gene transfer . An encouraging report emerged at a recent annual meeting of the european society of gene and cell therapy . Lentivirus vectors have been used to transduce hematopoietic stem cells in x - linked adrenoleukodystrophy, a progressive demyelinating disease that causes severe debilitation by early teenage years . Long - term and stable gene modification has been observed in 20% of myeloid cells, well within the range to reverse phenotypes in some red cell and myeloid disorders . Finally, auiti and his colleagues have recently reported highly encouraging results in the treatment of adenosine deaminase deficiency . Thus, gene therapy of hematopoietic diseases with retroviruses lumbers in choppy straits twixt the scylla of insufficient gene transfer and the charybdis of leukemia, while the therapy of metabolic and coagulation disorders with adeno- and adeno - associated viruses is blunted by immune reponses to the vectors . To committed gene therapists, these are simply the challenges that they have always faced, while those who are engaged in stem cell transplantation, or in finding better halfway measures that support afflicted patients, work as best they can, all the while hoping that the holy grail of gene replacement will one day become a safe reality . The future could lie in the promising field of site - specific gene correction using modified nucleases, and the conversion of corrected somatic cells such as fibroblasts to functioning hematopoietic stem cells . Cdna: copy dna; ltr: long terminal repeat; scid: severe combined immunodeficiency . The authors are particularly grateful to our colleague david a williams, md, for his contributions to this field and his invaluable assistance during the development of this review.
Periodontal disease is a multifactorial disease, characterized by periodontal pocket formation, loss of clinical attachment, and alveolar bone resorption . However, dynamic interactions between bacterial factors and host response with genetic and environmental factors are considered as the primary cause for tissue destruction in periodontitis . In consideration to this bacterial - host response, treatment approach to periodontitis should be conventional mechanical debridement and the use of pharmacotherapeutic agents . Antibiotics can often be prescribed to the patients who are non - responsive to conventional mechanical therapy, for patients with acute periodontal infections associated with systemic manifestations, as a prophylactic agent in medically compromised patients and as an adjunct to mechanical therapy . Tetracyclines are broad - spectrum antibiotics which inhibit ribosomal protein synthesis and may also serve as a bacteria - reducing and host - modulating agent . This drug is mainly used for the treatment of acne, chronic respiratory diseases, and rheumatoid arthritis . Minocycline has many advantages over other tetracyclines, e.g., both anti - inflammatory as well as antibiotic properties, better absorption, increased antimicrobial activity, and negligible or no phototoxicity . Being lipid soluble, minocycline can easily penetrate into various body fluids, such as saliva and gingival crevicular fluid, and can act locally at the site of infection . A 28-year - old female patient reported with complaints of generalized swollen gums since last one year, preventing proper speech, articulation, and mastication causing inadequate lip apposition and poor aesthetics . Detailed case history disclosed that in last six years, she has been operated for the same problem four times; once full mouth open flap debridement and three times gingivectomy was performed but again causing the recurrence of gingival enlargement within 4 to 5 months . She did not give any history of drugs intake, fever, anorexia, weight loss, seizures, hearing loss, nor having any physical or mental disorder . Also, family and postnatal history was non - contributory . An intraoral examination revealed generalized diffused, nodular enlargement of gingiva, more on the right side . Gingiva was fiery red in color with soft and spongy consistency and bleeding on slightest of provocation . The teeth were covered with enlarged gingiva till the middle one third, with some teeth showing grade i mobility [figures 1a d]. Generalized gingival enlargement with inflammation frontal view generalized gingival enlargement with inflammation right lateral view generalized gingival enlargement with inflammation left lateral view generalized gingival enlargement with inflammation occlusal view panoramic radiograph revealed generalized bone loss [figure 2] and, teeth no . 11, 21, 45, and 46 were root canal treated with crowns on them . Panoramic radiograph showing generalized bone loss on the basis of detailed case analysis and clinical and histological findings, the case was diagnosed as refractory periodontitis with chronic inflammatory gingival enlargement . On the basis of clinical features and history of the patient, the diagnosis of refractory periodontitis with gingival hypertrophy was made, not responding to various periodontal surgical procedures . Thus, non - surgical periodontal therapy was chosen as the mode of therapy along with systemic administration of minocycline for which a written informed consent was signed from the patient . Minocycline 100 mg one a day was advised to start with for 1 week . At the end of 1 week on examination, patient showed dramatic improvement in gingival health, so the same medication was continued for another 1 week and on re - examination, gingival tissues showed overall reduction in gingival hypertrophy, inflammation, and pocket depth [figures 3a d]. Reduced gingival hypertrophy and inflammation on 2 week frontal view reduced gingival hypertrophy and inflammation on 2 week right lateral view reduced gingival hypertrophy and inflammation on 2 week left lateral view reduced gingival hypertrophy and inflammation on 2 week occlusal view for the better maintenance and to reduce plaque deposition, scaling and root planning was performed to remove local irritating and aggravating factors at the end of 2 weeks and medication was continued till 4 weeks . On recall visits at the end of 4 weeks, patient showed great improvement with disappearance of gingival enlargement completely with gingiva showing coral pink color, firm and resilient consistency and no bleeding on probing [figures 4a d]. Also, patient could maintain oral hygiene procedure routinely without any discomfort, pain, or bleeding . Patient was advised to stop medication after 4 weeks and was kept on regular follow - up for next six months with every month check - up . Healthy overall gingiva with better oral hygiene on 4 week frontal view healthy overall gingiva with better oral hygiene on 4 week right lateral view healthy overall gingiva with better oral hygiene on 4 week left lateral view healthy overall gingiva with better oral hygiene on 4 week occlusal view an intraoral examination revealed generalized diffused, nodular enlargement of gingiva, more on the right side . Gingiva was fiery red in color with soft and spongy consistency and bleeding on slightest of provocation . The teeth were covered with enlarged gingiva till the middle one third, with some teeth showing grade i mobility [figures 1a d]. Generalized gingival enlargement with inflammation frontal view generalized gingival enlargement with inflammation right lateral view generalized gingival enlargement with 11, 21, 45, and 46 were root canal treated with crowns on them . On the basis of detailed case analysis and clinical and histological findings, the case was diagnosed as refractory periodontitis with chronic inflammatory gingival enlargement . On the basis of clinical features and history of the patient, the diagnosis of refractory periodontitis with gingival hypertrophy was made, not responding to various periodontal surgical procedures . Thus, non - surgical periodontal therapy was chosen as the mode of therapy along with systemic administration of minocycline for which a written informed consent was signed from the patient . Minocycline 100 mg one a day was advised to start with for 1 week . At the end of 1 week on examination, patient showed dramatic improvement in gingival health, so the same medication was continued for another 1 week and on re - examination, gingival tissues showed overall reduction in gingival hypertrophy, inflammation, and pocket depth [figures 3a d]. Reduced gingival hypertrophy and inflammation on 2 week frontal view reduced gingival hypertrophy and inflammation on 2 week right lateral view reduced gingival hypertrophy and inflammation on 2 week left lateral view reduced gingival hypertrophy and inflammation on 2 week occlusal view for the better maintenance and to reduce plaque deposition, scaling and root planning was performed to remove local irritating and aggravating factors at the end of 2 weeks and medication was continued till 4 weeks . On recall visits at the end of 4 weeks, patient showed great improvement with disappearance of gingival enlargement completely with gingiva showing coral pink color, firm and resilient consistency and no bleeding on probing [figures 4a d]. Also, patient could maintain oral hygiene procedure routinely without any discomfort, pain, or bleeding . Patient was advised to stop medication after 4 weeks and was kept on regular follow - up for next six months with every month check - up . Healthy overall gingiva with better oral hygiene on 4 week frontal view healthy overall gingiva with better oral hygiene on 4 week right lateral view healthy overall gingiva with better oral hygiene on 4 week left lateral view healthy overall gingiva with better oral hygiene on 4 week occlusal view this case report presents a case of a female patient with severe gingival inflammation, bleeding gums even on the slightest provocation, and generalized deep periodontal pockets . The conventional periodontal therapy is the removal of bacterial biofilm and supra- and subgingival calculus, as much as possible . But, because of the lack of visibility to the subgingival areas, complete plaque and calculus removal is difficult to achieve . Waerhaug described that non - surgical scaling and root planing is not capable of removing subgingival deposits in sites with probing depths exceeding 5 mm . Therefore, the use of antimicrobial agents in conjunction with periodontal therapy was proposed . In the present case, also found reduction in gingival inflammation and probing pocket depth in the minocycline group and they suggested that additional use of systemic minocycline demonstrated improvement on mmp-8 and pge-2 . They also speculated that minocycline has a kind of host - modulating capacity when used systemically for 2 weeks . Migration of neutrophils with tetracycline to the infected site could potentially boost the concentration of tetracycline at local sites . Neutrophils tendency to penetrate site of infection in great numbers could result in enhanced tetracycline action . Systemic administration of antibiotics has certain advantages over topical administration such as penetration of the drug two multiple sites of disease activity and effect on extra dental sites, e.g., tongue and tonsillar areas . This enhances elimination of microorganisms from the entire mouth and decreases the risk of reinfection from the other sites . Disadvantages of systemic administration of antibiotics are the increased of drug reactions, increased selection of multiple antibiotic - resistant microorganisms, and chances of lower concentration of the drug in the gingival crevicular fluid . Even though recurrence cannot be predicted, psychological and functional benefits far outweigh the recurrence . Oral hygiene maintenance and the plaque accumulation have a very crucial effect on prognosis of any periodontal therapy . Long - term follow - up will be required to evaluate the predictability of the therapy carried out.
A 61-year - old woman was referred to department of thoracic and cardiovascular surgery with thrill on the right wrist . Two years earlier, she had visited the emergency department of smg - snu boramae hospital with a recently aggravated chest pain . She underwent emergency coronary angiography (cag) via the right femoral artery, and percutaneous coronary intervention for the proximal right coronary artery, which was successfully completed . One year after the intervention, she underwent diagnostic cag via the right radial artery, and was discharged one day after the procedure . About 11 months after the day of the diagnostic cag, she complained of palpable thrill on the right wrist at the outpatient clinic . She also presented dilated superficial veins on the right forearm, which was very close to the previous puncture site for the diagnostic coronary angiography (fig . 1). Suspecting radial arteriovenous fistula (avf) formation, local compressive dressing and elastic bands were applied on the right wrist, with the expectation of spontaneous closure, but there was no effect . A vascular ultrasound scan revealed a tract between the right radial artery and adjacent subcutaneous vein, which suggested radial arteriovenous fistula (fig . 2). Computed tomography (ct) angiography of the upper extremity was performed, and it showed early visualization of dilated deep and superficial veins of the right forearm in the arterial phase . In addition, the right cephalic, brachial and axillary veins were visualized by contrast media in the arterial phase (fig . The fistula tract between the radial artery and vein was ligated, and the arterial side fistula opening was closed primarily (fig . Transfemoral access is a widespread method for coronary diagnostic and interventional procedures, and it is rarely associated with access site vascular complications . The possible complications are major hematoma, avf, pseudoaneurysm, and arterial dissection, and surgical correction for these complications is very commonly recommended . Transradial access for coronary procedures had become an increasingly popular technique since it was first introduced by campeau in 1989 because it significantly reduces access site vascular complications compared to transfemoral access . In particular, avfs after transradial access are an extremely rare complication (0.08%) because of the absence of large veins near the radial artery . Reported that there were several factors predisposing patients to iatrogenic femoral avfs: age over 60, female gender, arterial hypertension, prolonged coumadin therapy, and high heparin dosage during the procedure . Most of the predisposing factors were associated with delayed healing of the access sites of the punctured arteries . In this case, there was atheromatous plaque in the fistulous portion of the right radial artery, which might have resulted in a poor healing process around the puncture site of the radial artery, and this, in turn, might have contributed to the formation of radial avf . The clinical presentation of iatrogenic avf is various: a pulsatile mass, thrill, pain, edema, and dilated and varicose veins can occur . However, radial avf is usually asymptomatic and manifests as a thrill or bruit over the wrist . The iatrogenic radial avfs usually have smaller shunt volumes than other large fistulae, such as hemodialysis fistula, so that significant hemodynamic change is not frequent . Several diagnostic tools, such as duplex ultrasonography, computed tomography, magnetic resonance imaging, and conventional angiography, can be used if there is clinical suspicion of iatrogenic avf . Duplex ultrasonography has become the routine screening test for patients with suspected avfs, but the use of a ct scan has gradually increased because ct is minimally invasive, rapid, and operator independent . In addition, 3-dimensional ct angiography shows the exact spatial relationship between the radial artery, superficial veins, and adjacent structures around the avf, which was the reason we performed additional ct angiography after the ultrasonographic diagnosis of the avf in this case . Three different therapeutic strategies have been reported so far for treating iatrogenic radial avf: conservative management, endovascular treatment, and surgical treatment . About one - third of iatrogenic avfs close spontaneously; for this reason, conservative management is usually considered to be the first line of treatment . Some have reported successful implantation of stents in radial avfs, and surgical repair also has frequently been recommended . In this case, we had applied local compression and elastic bands for a week, anticipating spontaneous closure of the radial avf . However, there was no effect; thus, we performed surgical repair of avf for the patient . We report an extremely rare case of delayed radial arteriovenous fistula formation, one year after cag, using transradial access, which was treated successfully with surgical repair.
A recent institute of medicine (iom) report on this subject acknowledges that the rates and impact of medication errors are huge but are poorly understood . The president of the institute of safe medication practices (ismp), michael cohen, in his testimony to a committee of the us congress estimated that the dollar cost of adverse drug events was about $200 billion across all settings . In ambulatory settings, medication errors and adverse drug events (ades) are one of the most important safety issues . A study based on the national ambulatory medical care survey (namcs) found that office - based physicians prescribed at least 1 inappropriate medication to nearly 8% of the elderly who received prescriptions . Another study of ambulatory elderly patients with polymorbidity and associated polypharmacy documented that 35% reported experiencing at least one ade within the previous year . Gurwitz and colleagues have estimated (by extrapolation) that medicare enrollees alone suffer approximately 500,000 preventable ades per year . A 2003 report of a multidisciplinary group (composed of the ahrq - supported medical group management association center for research, the centers for medicare and medicaid services, and the partnership for patient safety) draws attention to the fact that while safety risks are widespread in ambulatory settings, there has been insufficient attention paid to them . Some estimates suggest that ambulatory settings are at least equally important as inpatient settings, with up to 200,000 avoidable deaths annually in the united states of america (usa) alone [7, 8]. Lack of awareness of the type, the incidence, and consequences of errors in any setting is one of the most important barriers to reducing these errors and improving quality of care . The most commonly used method for estimating vulnerabilities in healthcare is to retrospectively collect and count errors through voluntary reporting systems (often referred to as these are fraught with difficulty due to underreporting (according to iom's 1999 report, only 5% of known errors are typically reported and then there are unknown errors) and abuse (e.g., reports filed and counterfiled as a means of retaliation against colleagues). Error reporting often does not promote understanding of the organizational structure and processes of care . Instead it tends to be associated with blame and shame, and frequently results in antagonism between team members undermining mutual respect, trust, and cooperation . The patient safety and quality improvement act (2005) was introduced in large part to stimulate increased error reporting through the creation of patient safety organizations (psos). Bates and colleagues have described difficulties involved in defining and quantifying errors; they report that even direct observational studies, which are highly labor intensive, often miss errors . An alternative approach that is prospective, rather than retrospective, and encourages involvement of all teammembers for identifying and prioritizing safety and quality problems invokes failure modes and effects analysis (fmea). This has been widely used in other high - risk industries and has been advocated by the iom as a means of analyzing a system to identify its weaknesses (failure modes), possible consequences of failure (effects), and to prioritize areas for improvement . We have adapted and tailored this methodology to allow for the levels of resources and expertise available in ambulatory settings, and developed an instrument that has been shown to be effective in a variety of these settings . The details of the rationale and processes behind this instrument termed safety enhancement and monitoring instrument that is patient centered have done, that a vital step toward creating the medical home is to close the physician staff divide so as to maintain communication and coordination . They draw attention to the fact that currently practice meetings are universally unpopular despite their indispensability . Our trm methodology, invoking the paradigm of complex adaptive systems, is designed to aid formation of central attractors in the form of self - empowered effective learning teams with a common vision to help their complex microsystems to adapt and thrive [1721]; thriving systems are endowed with simple rules, shared vision, and opportunities for team members to innovate . An outcome - oriented team has to be enabled to (a) own and identify vulnerabilities in their settings, (b) design and implement interventions tailored for its settings, (c) monitor the efficacy of these interventions, and (d) continue the never ending journey in pursuit of safe care, that is, continuing quality improvement . Interestingly, a recent study by quinn et al . Found that physicians from practices that were involved in evaluation of quality improvement activities had significantly less isolation, stress, and dissatisfaction . Objectivethe primary objective of this study was to evaluate the impact of the prospective team resource management methodology (trm), based on the semi - p instrument, (with and without the use of a practice enhancement associate (pea)) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades . The primary objective of this study was to evaluate the impact of the prospective team resource management methodology (trm), based on the semi - p instrument, (with and without the use of a practice enhancement associate (pea)) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades . The number of preventable ades in a vulnerable population aged 65 and above; the severity of these preventable ades . This was a cluster randomized trial in which 12 practices in the upstate new york practice - based research network were each randomized to one of 3 states (4 practices each): (1) team resource management intervention; (2) team resource management intervention with pea; (3) no intervention (comparison group). These can be repeated to make a cycle, as shown in figure 1, for continuous quality and safety improvement . This anonymous survey is an opportunity for all staff to freely express opinions about the care processes in their setting . This survey asks about each of the steps in the medication use process . To help orient staff to the overall process, the survey uses a diagram (figure 2) that shows who and what is involved in the processes and how they work (or are supposed to work) together . Each page of the survey is about a different area and consists of a list of errors or causes of error that can occur in that area . The lists are based on review of the literature and consultation with practicing physicians and nursing leaders . The survey asks staff to think about each of the errors in turn and, for each, to indicate their opinion about how often it occurs and, when it does happen, how severe the consequences usually are . The diagram of the testing process is included on each page, with red highlighting to show which part of the process is being asked about . If willing, they can mark their job category (provider, nursing, or administrative). Scores (called hazard scores obtained by multiplying frequency of each error with its respective severity of consequence) are calculated for each error in the survey, based on respondents' answers to the frequency and severity questions . The items are then listed in rank order (highest to lowest) and presented to staff in a graphical format in a group meeting for their discussion . This helps the team to form a common vision and consensus regarding which problems need to be addressed first . In further group meetings, staff discuss the prioritized problems and work together to design solutions, keeping in mind the resources available and the capabilities of their unique setting . Teams are formed to implement the solutions, with clear allocation of responsibilities and an agreed time schedule . She had completed a master's degree in nutrition but with no prior experience working with practices . She participated in all study - related team meetings at each practice and made herself available to each practice for up to half a day a week throughout the study period to support safety and quality improvement activities (not limited to study - related activities). The main contribution of the pea was to follow through on plans developed by the practice team, that is to support stages 3 and 4 in the trm cycle outlined above . Examples of pea activities include developing patient education materials, preparing draft protocols, and collecting resources for patient prescription assistance . The rate and severity of ades and preventable ades were measured by chart review for the two 12-month periods before and after the start of the intervention . Eligible patients were those aged 65 years and above, who had at least one visit for cardiovascular disease (icd-9-cm codes 390459) during the measurement period and at least one visit for any diagnosis in the prior year . If there were 200 or fewer eligible charts at a site, all were screened, otherwise a random sample of 200 was taken . The chart review was performed using a trigger tool method that involves 2 steps, namely a screening step and a review step [5, 2332]. The screening step involves identifying the presence of certain chart findings, known as triggers, that are known to be possible evidence of an adverse event . In this study, the triggers of interest are those that might represent an adverse drug event (ade). Examples include an elevated inr (often associated with adverse effects of warfarin) and abrupt discontinuation of a medication (that sometimes occurs because of an ade). In our study research assistants performed the screening step, using a previously published ade trigger tool that we adapted from the work of others [5, 25, 26]. In the second step, known as the review step, a physician and pharmacist reviewed the identified triggers to determine for each trigger: (1) whether an ade took place and if so, (2) whether the event was preventable, (3) the stage of the medication use process where the ade originated (prescribing, dispensing, administration, and monitoring), and (4) the effect on the patient (none / minimal, mild, moderate, or severe). Examples of preventable errors include missed allergy, wrong dosage, errors of dispensing, administration errors, and failure to order or complete laboratory monitoring . The physician and pharmacist worked together, reviewing, discussing, and recording their consensus opinion of each trigger . When they were unable to reach consensus a final determination was made by a third reviewer . If they identified any potential or actual harm that had not been previously recognized and addressed, they notified the primary care physician or site medical director . The study protocol was approved by the social and behavioral sciences institutional review board of the state university of new york at buffalo . Part way into the study, one of the comparison group practices withdrew from the study, citing concerns over the administrative burden of the chart reviews (the practice was undergoing administrative changes). Table 1 shows the characteristics of the 11 sites that completed the study . At baseline, of these, 1066 (54.1%) had triggers, yielding a total of 2898 triggers . This far exceeded our expectation and therefore posed a practical problem due to the effort (and therefore cost) associated with reviewing this number of triggers . Therefore, we elected to reduce the sample by randomly eliminating a proportion of patients at each site, sufficient to reduce the number to approximately 100 patients per site . This yielded a total of 1125 patients, of whom 598 patients had one or more triggers, all of which underwent review . A similar process was used to make the endpoint data manageable, yielding 1050 patients, of whom 564 had triggers that were reviewed . Table 2 summarizes the rates of preventable ades (normalized per 100 patient - years) for each arm of the study . For each arm, we compared the pade rate (per 100 patient - years) at the two time points (after versus before) by means of a paired t test with sites as the unit of analysis . As shown in table 2, in the intervention with pea group there was a statistically significant decrease in the overall rate of pades after the intervention compared to before (7.4 per 100 patient - years versus 12.6, p = 0.018) and in the rate of moderate or severe (combined) pades(1.6 versus 6.4, p = 0.035). In the comparison arm and the unaided intervention arms, analysis of variance (anova) with study arm and time as the factors and total pades as the outcome variable showed no significant interaction between arm and time . For the outcome of moderate or severe pades, the interaction term was significant (p = 0.023) supporting the notion that the intervention with pea practices had a greater reduction in moderate / severe pades over time than the comparison group . The trm approach, when aided by a pea, demonstrated a significant reduction in pades, especially those with the highest severity . The reduction in pades represents a significant improvement in patient safety through the collective efforts of practice staff guided by a structured trm process . It appears that the additional resource offered in the form of a pea was important, as the non - pea practices did not achieve the same statistically significant improvement that the pea practices did . While the mechanism of action of the pea is not clearly understood, it is reasonable to surmise that the extra human resource represented by the pea enabled practices to more effectively implement their planned interventions . The addition of a pea may have helped to minimize the incremental burden on teams that are already overburdened . While we did not formally evaluate this, it is the authors' observation that the humanistic self - empowerment approach used in this study helped to energize front - line workers to maintain and continually improve quality . Staff commented that seeing other people's perspectives (as reflected in semi - p results) helped to improve mutual understanding, leading to consensus . Further, we believe that by closing the physician staff divide, and encouraging closer communication and coordination between team members in addressing practice issues, the trm approach can achieve progress toward the creation of the medical home . Firstly, the outcome measure is based on a trigger tool methodology that has limited sensitivity . In fact, the sensitivity of the trigger tool is unknown because there is no gold standard against which to compare it . This means that the rates of pades reported should not be seen as estimates of true rates . Instead, they represent only a subset of pades, that is, those that are identifiable by the trigger tool . However there is no reason to expect that the sensitivity of the trigger tool would vary between study groups or over time so the validity of the study findings is not jeopardized . Another significant weakness is the small size of the study . Having only 3 - 4 practices in each arm limited the power . In addition, further exploration of the role of the pea is required to establish the most important active ingredients so that these can be deployed in an efficient way.
Brucellosis is a systemic infection that may affect any organ or system of the human body . Usually, a slight increase in the liver function tests and mild hepatosplenomegaly occur, and sometimes, acute hepatitis develops, but hepatic abscess is a rare manifestation of that disease 1, 2, 3 . A 33yearold woman was admitted to our university hospital with the complaints of temperature up to 40c, headache, nausea, and weakness for 2 weeks . She had a history of raising livestock and lived in rural area . On physical examination the laboratory data were as follows: total leukocyte count, 5600/mm (400010,000/mm); differential leukocyte count: neutrophils, 54%; lymphocytes, 36%; monocytes, 8%; basophils, 0.3%; eosinophils, 1.7%; platelets, 285,000/mm (normal range: 150,000450,000/mm); hemoglobin, 12 g / dl; hematocrit, 36.6%; serum alanine transferase (alt), 242 u / l (rr 035 iu / l); serum aspartate transferase (ast), 162 u / l (rr 032 iu / l); serum glutamyl transpeptidase (gtp), 60 u / l (rr 040 iu / l); alkaline phosphatase (alp), 95 iu / ml (rr 35114 iu / l); total bilirubin, 0.4 mg / dl (rr 01.2 mg / dl); sedimentation rate, 17 mm / h; and creactive protein, 11.3 mg / l (rr 05 mg viral hepatitis markers (the hbsag, antihbc igm, antihav igm, and antihcv tests) were negative . Abdominal ultrasonography showed multiple small echogenic foci are more prominent in the right lobe of the liver (fig . 1).with these findings, the patient was diagnosed as having hepatic microabscesses due to brucellosis . Doxycycline (2 100 mg / day p.o .) And rifampicin (1 600 mg / day p.o .) The patient received this treatment for 3 months . On the fiftyninth day of the treatment, the levels of serum alanine transferase level and serum aspartate transferase decreased to the reference range . Brucellosis is a zoonosis that has been virtually eliminated from most developed countries, but it is still endemic in many regions of the world including mediterranean areas, in parts of south and central america, and east and western africa . The disease is transmitted to man mainly after consumption of contaminated unpasteurized milk and dairy products and less often after direct contact with infected animals 1 . Hepatic involvement in brucellosis covers a wide spectrum, ranging from mild elevation of aminotransferases to hepatitis including granulomatous forms and to liver abscesses . Increases in aminotransferases are noted in onefourth to onethird of brucellosis cases and are more frequent in the acute stages . Hepatic involvement in brucellosis has been reported in the literature in around 23% of the cases . Although b. abortus tends to establish a granulomatous form of hepatitis, b. melitensis may cause both diffuse and granulomatous lesions in the liver 1, 2, 3, 4 . An abscess caused by brucella spp usually represents the chronic form of disease, but it can occur in acute or subacute brucellosis . Routine laboratory findings in brucellosis are not usually diagnostic that may include leukopenia, anemia, thrombocytopenia, pancytopenia, and mildtomoderate elevation of liver function tests 2, 5, 6 . Ultrasonography most commonly shows a single, hypoechoic lesion with 1 centrally located calcium deposits . Computed tomography findings most commonly depict a hypodense area, and often one or more saccular, loculated, heterogeneous mass, and one or more calcifications 5, 6 . The diagnosis of brucellosis can be established according to the isolation of brucella spp . In blood, bone marrow or any other body fluid or tissue sample, or the presence of a compatible clinical picture with the demonstration of specific antibodies at significant titers or seroconversion . Significant titers are considered to be a standard agglutination test (sat) result 1/160 or a coombs antibrucella or immunocapture agglutination test result 1/320 1, 6 . The best regimen for the treatment of localized lesions has not been clearly defined . The duration of treatment varies depending on the individual case and the response to treatment 1 . Small, multifocal abscesses which can be detected in the acute forms of the disease respond very well to medical treatment . Other types of abscesses with an indolent course have a much worse prognosis, which considered to be a true focal complications of the disease . However, no clear distinction is made of these groups 7 . In this case, the short duration of symptoms and the high titer of brucella agglutination test suggest that this case was acute . It was identified multiple millimetric foci were more prominent in the right lobe of the liver parenchyma to be significant in terms of microabscesses . Our patient was successfully treated with a combination of doxycycline and rifampicin for 3 months . The diagnosis of brucellosis in our case was confirmed with clinical findings, livestock farming history, positive serological tests, and complete response to medical treatment . In conclusion, brucellosis is a systemic infectious disease and it is still an important public health problem in endemic areas of the world including turkey that can cause serious complications and significant morbidity . Clinicians should be considered in the differential diagnosis of this unusual complication of brucellosis for those who live in or have visited endemic areas . Ie, rka, oo, pod, and ss: performed the analysis of case data . All authors: contributed toward data analysis, drafting and critically revising the manuscript, and agree to be accountable for all aspects of the manuscript.
Photonic forces [1, 2] are known and understood for more than 100 years . But the discovery that they can be employed to manipulate small objects like colloids, viruses, or quantum dots became possible only after the establishment of strong laser sources in the infrared . This enables one to exert and to control forces acting on a small object in the range between 10 to 100 pn with an extraordinary high resolution of 50 fn . By incorporating additional optical systems to determine the position of a microscopic particle in 3d - space with nanometer precision, the novel experimental tool of optical tweezers was invented this initiated a manifold of spectacular experiments in biophysics [4a j] and colloid research . It became, for instance, possible to measure the elastic properties of -phage dna on a single chain level, the rna polymerase was monitored for a single enzyme in action, or the work which has to be done to package bacteriophage dna into a single viral capsule was measured . In colloid physics, microscopic measurements of the pair interaction potential of two charge - stabilized colloids became possible or direct determination of the cross correlations between two particles in an external potential . Experiments with optical tweezers on the elastic properties of dna are mainly restricted to -phage dna which has a contour length of 16 m . In the presented article, novel experiments are reported in which the dna under study is systematically varied in its length by genetic engineering . This enables one to analyze in great detail the elastic properties of dna and to check how well it can be described by the worm - like chain model or competing approaches . 1) is based on an inverted microscope (axiovert s 100 tv, carl zeiss, jena, germany) which is designed for epi - illumination fluorescence microscopy . For the optical tweezers, a diode pumped nd: yag laser (1,064 nm, 1 w, lcs - dtl 322; laser 2000, wessling, germany) is used which is mounted to the microscope by the base port . The power and the profile of the laser a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light . The beam is expanded and coupled into the back aperture of the microscope objective (plan - neofluor 1001.30 oil, carl zeiss, jena, germany). After passing through the sample cell, the beam is recollimated by a condenser with high numerical aperture . A beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010 mm quadrant photodiode (s 5107, hamamatsu, herrsching am ammersee, germany) for the electrical position detection . Video imaging and the optical position detection is accomplished by a digital camera (kp f 120, hitachi, dsseldorf, germany). The optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators (controller: e-710, stage: p-517.3cd, pi gmbh, karlsruhe, germany). The sample cell consists of a closed chamber which can be flushed (inset fig . 1). A micropipette (self - made) with an inner diameter at the tip of 1 m is inserted into the chamber to hold one bead by capillary action . To determine the position of both beads and its separation image analysis is employed . By measuring and fitting the intensity profile of the two beads (fig . 2), its position relative to each other can be determined with an accuracy of 2 nm . Fig . 1scheme of the experimental set - up . In the enlargement, the sample chamber is shownfig . 2image analysis of two separate beads: measured (a, b) and calculated (c) intensity profile using the fit function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i{\left ({x, y} \right)}\; = \;i_{0} + {\sum\limits_{i = 1,2} {a_{i}}} {\left ({1 - a_{i} u_{i}} \right)}e^{{u^{3}_{i}}} $$\end{document} (3) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{i} \; = \;{\left ({\frac{{x - x_{i}}} {{r_{i}}}} \right)}^{2} \; + \;{\left ({\frac{{y - y_{i}}} {{r_{i}}}} \right)}^{2}$$\end{document} (4) where for each colloid (x i, y i) is the center position, r i the optical radius, a i the amplitude of the profile relative to the background image intensity . I 0 and a i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . In the enlargement, the sample chamber is shown image analysis of two separate beads: measured (a, b) and calculated (c) intensity profile using the fit function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i{\left ({x, y} \right)}\; = \;i_{0} + {\sum\limits_{i = 1,2} {a_{i}}} {\left ({1 - a_{i} u_{i}} \right)}e^{{u^{3}_{i}}} $$\end{document} (3) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{i} \; = \;{\left ({\frac{{x - x_{i}}} {{r_{i}}}} \right)}^{2} \; + \;{\left ({\frac{{y - y_{i}}} {{r_{i}}}} \right)}^{2}$$\end{document} (4) where for each colloid (x i, y i) is the center position, r i the optical radius, a i the amplitude of the profile relative to the background image intensity . I 0 and a i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where is the viscosity of the medium, r the radius of the bead, and its velocity relative to the surrounding solution . Varying the latter between 50 and 1,000 m / s enables one to calibrate the optical forces with an accuracy of 10% . As expected for the power dependence of the force constant of the optical trap, a linear relationship the whole experimental set - up is in a temperature controlled (1) room . Due to the fact that the focus of the microscope changes with 1 by about 500 nm, the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23 min . Afterwards, the z - positions have to be if necessary readjusted . 3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law streptavidin - modified polystyrene beads with a diameter of 2 m were purchased from polysciences europe (eppelheim, germany), oligonucleotides provided by metabion (martinsried, germany) and mwg (ebersberg, germany). Nucleic acid purification kits were supplied by qiagen (hilden, germany) and anti - dig antibodies were purchased from roche (penzberg, germany). All enzymes and standards were from mbi fermentas (vilnius, lithuania) and all other chemicals were delivered from sigma (deisenhofen, germany). The double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000 bp product, from pet28a+ in case of the 3,000 and 4,000 bp product, and from ptwin1 in case of the 6,000 bp product via standard polymerase chain reactions (pcr). The oligonucleotides which were used were primer a (5-bio - cca cct gac gtc taa gaa acc-3) and primer b (5-dig - ggc aac aac gtt gcg caa ac-3) for the 2,000 bp dsdna, primer c (5-dig - acg gcc tca acc tac tac tg-3) and primer d (5-bio - gcg ata agt cgt gtc tta cc-3) for the 3,000 bp dsdna, primer e (5-dig - cag ctt cct ttc ggg ctt tg-3) and primer f (5-bio - tga ttg ccc gac att atc gc-3) for the 4,000 bp dsdna, and primer g (5-dig - atg gag gcg gat aaa gtt gc-3) and primer h (5-bio - gttgaatcaccgcgtaatcg-3) for the 6,000 bp dsdna . Dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg (fig . The distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable (table 1). Fig . 4 a the dnas with the lengths 2,000, 3,000, 4,000, and 6,000 b result of the binding assay . In lane 1 and 3 60 fmol of 2,000 bp dna lane 2 shows the result of the binding of the 2,000 bp dna to the anti - dig antibody labeled beads . Lane 4 shows the negative control with carboxylated beads onlytable 1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000 bpa: 486 bp (24.2%)puc18c: 522 bp (26.0%)g: 532 bp (26.5%)t: 471 bp (23.4%)handle 3000 bd3,000 bpa: 665 bp (22.2%)pet28a+c: 858 bp (28.6%)g: 829 bp (27.6%)t: 648 bp (21.6%)handle 4000 bd4,000 bpa: 888 bp (22.2%)pet28a+c: 1,097 bp (27.4%)g: 1,114 bp (27.9%)t: 901 bp (22.5%)handle 6000 bd6,000 bpa: 1,474 bp (24.6%)ptwin1c: 1,528 bp (25.5%)g: 1,642 bp (27.4%)t: 1,356 bp (22.6%)lambda - dna48,502 bpa: 12,334 bp (25.4%)c:11,362 bp (23.4%)g: 12,820 bp (26.4%) t: 11,986 bp (24.7%) a the dnas with the lengths 2,000, 3,000, 4,000, and 6,000 bp were analyzed on a 1.0% agarose gel to check their correct size . B result of the binding assay . In lane 1 and 3 60 fmol of 2,000 bp dna lane 2 shows the result of the binding of the 2,000 bp dna to the anti - dig antibody labeled beads . Lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al ., for the production of antidigoxigenin (anti - dig) antibody covered polystyrene beads, carboxylated particles with a diameter of 2.0 m were used . The beads from a 50 l bead suspension (26.5 mg / ml) were washed twice with 200 l of carbonate buffer (100 mm sodium carbonate, 100 mm sodium bicarbonate, ph 9.6) and three times with 200 l buffer a (20 mm phosphate buffer, ph 4.5). In between beads were collected by centrifugation (5 min 13,000g). After resuspension of the final bead pellet in 50 l buffer a, the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50 l of a 2% edc solution (ethyl-3-(3-dimethylaminopropyl)-carbodiimide) in the same buffer . Particles were washed three times with buffer a. after resuspension in 80 l buffer b (200 mm borate buffer, ph 8.5), 10 l of anti - dig antibody solution (1 g ab/l) were added followed by overnight shaking at room temperature . To avert unspecific binding at remaining free reactive groups, the pellet was resuspended in 100 l bsa solution (10 mg bsa / ml in buffer b) and incubated under shaking for 30 min at room temperature . Finally, the particles were resuspended in 20 l pbs (136 mm nacl, 2.7 mm kcl, 15.6 mm na2hpo4, 17.6 mm kh2po4), and stored at 4c . To determine the coupling efficiency 5 l of particle suspension were mixed with 50200 ng of a 2,000 bp pcr - amplified dna fragment, carrying a digoxygenine and a biotine modification at each terminus, respectively, in a total volume of 10 l pbs . This mixture was then incubated for 15 min at room temperature . As a negative control, the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated (fig . To establish single dna strands between two colloids, the following procedure turned out to be effective and reliable (fig . 5): a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces . Afterwards, a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . If a molecular contact was established, a sudden decrease in the fluctuation amplitude of the bead could be observed . After a short incubation time of 1 min, the particles are pulled apart to ensure that a single ds - dna had bound only . A polystyrene (ps) bead modified with anti - dig antibodies is trapped with the optical tweezers (a) and fixed by a micropipette due to capillary forces (b). In the next step, a ps bead on which ds - dna is immobilized via biotin streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette (c). Binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap (d). By measuring the force extension dependence, it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids . A polystyrene (ps) bead modified with anti - dig antibodies is trapped with the optical tweezers (a) and fixed by a micropipette due to capillary forces (b). In the next step, a ps bead on which ds - dna is immobilized via biotin streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette (c). Binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap (d). By measuring the force extension dependence, it is ensured that just one ds - dna strand is fixed between the beads 1) is based on an inverted microscope (axiovert s 100 tv, carl zeiss, jena, germany) which is designed for epi - illumination fluorescence microscopy . For the optical tweezers, a diode pumped nd: yag laser (1,064 nm, 1 w, lcs - dtl 322; laser 2000, wessling, germany) is used which is mounted to the microscope by the base port . The power and the profile of the laser a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light . The beam is expanded and coupled into the back aperture of the microscope objective (plan - neofluor 1001.30 oil, carl zeiss, jena, germany). After passing through the sample cell, the beam is recollimated by a condenser with high numerical aperture . A beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010 mm quadrant photodiode (s 5107, hamamatsu, herrsching am ammersee, germany) for the electrical position detection . Video imaging and the optical position detection is accomplished by a digital camera (kp f 120, hitachi, dsseldorf, germany). The optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators (controller: e-710, stage: p-517.3cd, pi gmbh, karlsruhe, germany). The sample cell consists of a closed chamber which can be flushed (inset fig . 1). A micropipette (self - made) with an inner diameter at the tip of 1 m is inserted into the chamber to hold one bead by capillary action . To determine the position of both beads and its separation image analysis is employed . By measuring and fitting the intensity profile of the two beads (fig . 2), its position relative to each other can be determined with an accuracy of 2 nm . Fig . 1scheme of the experimental set - up . In the enlargement, the sample chamber is shownfig . 2image analysis of two separate beads: measured (a, b) and calculated (c) intensity profile using the fit function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i{\left ({x, y} \right)}\; = \;i_{0} + {\sum\limits_{i = 1,2} {a_{i}}} {\left ({1 - a_{i} u_{i}} \right)}e^{{u^{3}_{i}}} $$\end{document} (3) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{i} \; = \;{\left ({\frac{{x - x_{i}}} {{r_{i}}}} \right)}^{2} \; + \;{\left ({\frac{{y - y_{i}}} {{r_{i}}}} \right)}^{2}$$\end{document} (4) where for each colloid (x i, y i) is the center position, r i the optical radius, a i the amplitude of the profile relative to the background image intensity . I 0 and a i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . In the enlargement, the sample chamber is shown image analysis of two separate beads: measured (a, b) and calculated (c) intensity profile using the fit function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i{\left ({x, y} \right)}\; = \;i_{0} + {\sum\limits_{i = 1,2} {a_{i}}} {\left ({1 - a_{i} u_{i}} \right)}e^{{u^{3}_{i}}} $$\end{document} (3) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{i} \; = \;{\left ({\frac{{x - x_{i}}} {{r_{i}}}} \right)}^{2} \; + \;{\left ({\frac{{y - y_{i}}} {{r_{i}}}} \right)}^{2}$$\end{document} (4) where for each colloid (x i, y i) is the center position, r i the optical radius, a i the amplitude of the profile relative to the background image intensity . I 0 and a i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where is the viscosity of the medium, r the radius of the bead, and its velocity relative to the surrounding solution . Varying the latter between 50 and 1,000 m / s enables one to calibrate the optical forces with an accuracy of 10% . As expected for the power dependence of the force constant of the optical trap, a linear relationship the whole experimental set - up is in a temperature controlled (1) room . Due to the fact that the focus of the microscope changes with 1 by about 500 nm, the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23 min . Afterwards, the z - positions have to be if necessary readjusted . 3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law streptavidin - modified polystyrene beads with a diameter of 2 m were purchased from polysciences europe (eppelheim, germany), oligonucleotides provided by metabion (martinsried, germany) and mwg (ebersberg, germany). Nucleic acid purification kits were supplied by qiagen (hilden, germany) and anti - dig antibodies were purchased from roche (penzberg, germany). All enzymes and standards were from mbi fermentas (vilnius, lithuania) and all other chemicals were delivered from sigma (deisenhofen, germany). The double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000 bp product, from pet28a+ in case of the 3,000 and 4,000 bp product, and from ptwin1 in case of the 6,000 bp product via standard polymerase chain reactions (pcr). The oligonucleotides which were used were primer a (5-bio - cca cct gac gtc taa gaa acc-3) and primer b (5-dig - ggc aac aac gtt gcg caa ac-3) for the 2,000 bp dsdna, primer c (5-dig - acg gcc tca acc tac tac tg-3) and primer d (5-bio - gcg ata agt cgt gtc tta cc-3) for the 3,000 bp dsdna, primer e (5-dig - cag ctt cct ttc ggg ctt tg-3) and primer f (5-bio - tga ttg ccc gac att atc gc-3) for the 4,000 bp dsdna, and primer g (5-dig - atg gag gcg gat aaa gtt gc-3) and primer h (5-bio - gttgaatcaccgcgtaatcg-3) for the 6,000 bp dsdna . Dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg (fig . The distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable (table 1). 2,000, 3,000, 4,000, and 6,000 bp were analyzed on a 1.0% agarose gel to check their correct size . B result of the binding assay . In lane 1 and 3 60 fmol of 2,000 bp dna lane 2 shows the result of the binding of the 2,000 bp dna to the anti - dig antibody labeled beads . Lane 4 shows the negative control with carboxylated beads onlytable 1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000 bpa: 486 bp (24.2%)puc18c: 522 bp (26.0%)g: 532 bp (26.5%)t: 471 bp (23.4%)handle 3000 bd3,000 bpa: 665 bp (22.2%)pet28a+c: 858 bp (28.6%)g: 829 bp (27.6%)t: 648 bp (21.6%)handle 4000 bd4,000 bpa: 888 bp (22.2%)pet28a+c: 1,097 bp (27.4%)g: 1,114 bp (27.9%)t: 901 bp (22.5%)handle 6000 bd6,000 bpa: 1,474 bp (24.6%)ptwin1c: 1,528 bp (25.5%)g: 1,642 bp (27.4%)t: 1,356 bp (22.6%)lambda - dna48,502 bpa: 12,334 bp (25.4%)c:11,362 bp (23.4%)g: 12,820 bp (26.4%) t: 11,986 bp (24.7%) a the dnas with the lengths 2,000, 3,000, 4,000, and 6,000 bp were analyzed on a 1.0% agarose gel to check their correct size . B result of the binding assay . In lane 1 and 3 60 fmol of 2,000 bp dna lane 2 shows the result of the binding of the 2,000 bp dna to the anti - dig antibody labeled beads . Lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al . For the production of antidigoxigenin (anti - dig) antibody covered polystyrene beads, carboxylated particles with a diameter of 2.0 m were used . The beads from a 50 l bead suspension (26.5 mg / ml) were washed twice with 200 l of carbonate buffer (100 mm sodium carbonate, 100 mm sodium bicarbonate, ph 9.6) and three times with 200 l buffer a (20 mm phosphate buffer, ph 4.5). In between beads were collected by centrifugation (5 min 13,000g). After resuspension of the final bead pellet in 50 l buffer a, the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50 l of a 2% edc solution (ethyl-3-(3-dimethylaminopropyl)-carbodiimide) in the same buffer . Particles were washed three times with buffer a. after resuspension in 80 l buffer b (200 mm borate buffer, ph 8.5), 10 l of anti - dig antibody solution (1 g ab/l) were added followed by overnight shaking at room temperature . To avert unspecific binding at remaining free reactive groups, the pellet was resuspended in 100 l bsa solution (10 mg bsa / ml in buffer b) and incubated under shaking for 30 min at room temperature . Finally, the particles were resuspended in 20 l pbs (136 mm nacl, 2.7 mm kcl, 15.6 mm na2hpo4, 17.6 mm kh2po4), and stored at 4c . To determine the coupling efficiency 5 l of particle suspension were mixed with 50200 ng of a 2,000 bp pcr - amplified dna fragment, carrying a digoxygenine and a biotine modification at each terminus, respectively, in a total volume of 10 l pbs . This mixture was then incubated for 15 min at room temperature . As a negative control, the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated (fig . To establish single dna strands between two colloids, the following procedure turned out to be effective and reliable (fig . 5): a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces . Afterwards, a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . If a molecular contact was established, a sudden decrease in the fluctuation amplitude of the bead could be observed . After a short incubation time of 1 min, the particles are pulled apart to ensure that a single ds - dna had bound only . A polystyrene (ps) bead modified with anti - dig antibodies is trapped with the optical tweezers (a) and fixed by a micropipette due to capillary forces (b). In the next step, a ps bead on which ds - dna is immobilized via biotin streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette (c). Binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap (d). By measuring the force extension dependence, it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids . A polystyrene (ps) bead modified with anti - dig antibodies is trapped with the optical tweezers (a) and fixed by a micropipette due to capillary forces (b). In the next step, a ps bead on which ds - dna is immobilized via biotin streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette (c). Binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap (d). By measuring the force extension dependence, it is ensured that just one ds - dna strand is fixed between the beads the force extension dependence (fig . 6) of ds - dna of varying lengths between 2,000 to 6,000 bp can be described for forces 10 pn by the worm - like chain model suggesting for the force f stretching a chain: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f\; = \;{\left ({\frac{{k_{{\text{b}}} t}} {{l_{{\text{p}}}}}} \right)}\;{\left [{\frac{1} {{4{\left ({1 - \frac{x} {{l_{{\text{0}}}}}} \right)}^{2}}} \; - \;\frac{1} {4}\; + \;\frac{x} {{l_{0}}}} \right]}$$\end{document}where lp is the persistence length, l0 the contour length, x the extension, kb is boltzmann s constant, and t the absolute temperature . In principle, l0 is given by the number of base pairs assuming a length of 0.34 nm per pair . But due to the fact that the polystyrene colloid with antidigoxigenin antibodies had an uneven surface (with 100 nm deviations from the ideal spherical shape), the contour length was determined from the fits using eqs . 1 or 2 . Comparing these values (table 2) with the calculated lengths delivers agreement within the above mentioned absolute uncertainties of 100 nm . 6force - extension dependence for single ds - dna chains of different length as indicated . The solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach . The experimental uncertainly is 0.5 pntable 2contour lengths as calculated from the number of base pairs assuming 0.34 nm length per base pair and as determined from the fits using the wlc model or the winkler approachnumber of base pairstheoretical contour length l 0 (nm)experimentally determined contour length l 0 with wlc fit (nm)experimentally determined contour length l 0 with winkler fit (nm)2,0006806526543,0001,0209991,0044,0001,3601,3361,3416,0002,0401,9952,004the fits are restricted to forces 10 pn . The experiments are carried out in pbs - puffer with 137 mm nacl concentration . The uncertainty of fits as caused by the experimental accuracy of 0.5 pn in the force measurements and 2 nm in the determination of the position of the colloid in the optical trap force - extension dependence for single ds - dna chains of different length as indicated . The solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach . The experimental uncertainly is 0.5 pn contour lengths as calculated from the number of base pairs assuming 0.34 nm length per base pair and as determined from the fits using the wlc model or the winkler approach the fits are restricted to forces 10 pn . The experiments are carried out in pbs - puffer with 137 mm nacl concentration . The uncertainty of fits as caused by the experimental accuracy of 0.5 pn in the force measurements and 2 nm in the determination of the position of the colloid in the optical trap the data can be similarly well described by the model of a semiflexible chain of gaussian segments as suggested by winkler: 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f = \frac{{3k_{{\text{b}}} tx}} {{2l_{{\text{o}}} l_{{\text{p}}} {\left ({1 - \frac{{x^{2}}} {{l_{{\text{o}}} ^{2}}}} \right)}^{2}}} $$\end{document} the major difference between the approach by r. g. winkler and the worm - like chain (wlc)-model is based on the fact that in the former the magnitude of the tangent vector is not exactly one but only its average value . As a consequence, the contour length is not fixed . The persistence lengths which were calculated by applying these models are listed in table 3 . The persistence length ranges between 1620 nm (5 nm) for wlc and 2127 nm (4 nm) for the winkler fit . The measurements were carried out in pbs - buffer with a high ionic strength (137 mm nacl). Due to the high number of positively charged ions [na], the repulsion between the negatively charged groups of the phosphate backbone of the dna is reduced, which leads to an increase in flexibility and therefore to a decrease of the persistence length . Table 3persistence length as determined from the fits with the wlc model and with winkler s approachnumber of base pairsexperimentally determined persistence length l p with wlc fit (nm) (5)experimentally determined persistence length l p with winkler fit (nm) (4)2,00020273,00016214,00020276,0001926the fits are restricted to forces 10 pn . In all cases, the sample was kept in pbs buffer having 137 mm nacl concentration persistence length as determined from the fits with the wlc model and with winkler s approach the fits are restricted to forces 10 pn . In all cases, the sample was kept in pbs buffer having 137 mm nacl concentration from the wlc - fit for a 4,000-bp long dna in 10 mm nah2po4/na2hpo4 buffer ph 7 a persistence length of (table 4) lp=46 nm is obtained . The fit with the winkler model leads to a value of lp=65 nm (fig . These values are in good agreement with data published previously by wang et al . . As expected with increasing salt concentration a pronounced decrease in the persistence length is obtained (table 4). Fig . 7force - extension dependence for a single ds - dna chain of 4,000 bp . The solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach . The experimental uncertainly is 0.5 pntable 4nacl concentration dependence of the persistence length of dna with 4,000 bp as determined with the wlc model and the winkler s approachnacl concentration (mm)experimentally determined persistence length l p with wlc fit (nm) (5)experimentally determined persistence length l p with winkler fit (nm) (4)204665137 (pbs)29401502839the fits are restricted to forces 10 pn force - extension dependence for a single ds - dna chain of 4,000 bp . The solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach . The experimental uncertainly is 0.5 pn nacl concentration dependence of the persistence length of dna with 4,000 bp as determined with the wlc model and the winkler s approach the fits are restricted to forces 10 pn compared to wang et al ., the persistence length obtained in our experiments has a more pronounced dependence on the ionic strength of the surrounding media . We attribute this discrepancy to the different buffer systems used an effect well known and discussed in the current literature [16, 18, 19]. For the first time measurements of the force - extension dependence of single strands of ds - dna with systematically varying length are carried out . It is proven that the worm - like chain model, as well as the approach by r. g. winkler, describes the data well within experimental accuracy.
In pregnant women with mechanical heart valves, the frequency of valve thrombosis increases due to pregnancy - related hypercoagulability . Therefore, effective anticoagulation is critical in pregnant patients with mechanical heart valves but remains problematic because both oral anticoagulation and heparins have been associated with important fetal and maternal side effects (1). Coumarin derivatives are anticoagulants of choice for mechanical heart valves, but they cross the placenta and are associated with coumarin - induced fetal loss or embryopathy (1 - 3). Unfractionated heparin (ufh) provides an alternative therapy that avoids fetal side effects, however, the use of ufh is associated with increased maternal thromboembolic and bleeding complications (1, 4, 5). Low molecular weight heparin (lmwh) may be more advantageous than ufh (6), and appears a good alternative . However, little clinical information and no reliable data are available regarding its efficacy and safety . No consensus has been reached about optimal antithrombotic therapy in pregnant patients with a mechanical heart valve . Coumarins are contraindicated in pregnancy in north america due to fetal concerns, but european experts have recommended low dose coumarins (less than 5 mg daily) throughout pregnancy given a very low frequency of fetal anomalies (2, 3). Reports of lmwh use began to appear, and many physicians now use lmwh because of its good safety profile for both mother and baby (7 - 10). However, treatment failures have been reported (11, 12), and no lmwh has been licensed for use in pregnant patients with mechanical heart valves . Available published data regarding the efficacy and safety of lmwh in this clinical setting have been derived from small case series, and usually enoxaparin has been used . Here, we report our experience of nadroparin treatment, and its associated pregnancy outcomes and maternal complications . Between 1997 and 2005, 31 pregnancies were analyzed retrospectively in 25 women with mechanical heart valves . Basic characteristics and previous operative data are listed in table 1 . In 23 of these 31 pregnancies, nadroparin was used as an anticoagulant during the first trimester with given informed consent, concerning the risks and benefits of lmwh . Others were anticoagulated with coumarin derivatives (cmd) with or without aspirin due to unawareness of pregnancy until the first trimester, or because of refusal or poor compliance on self - injected lmwh . In the lmwh - treated group, when pregnancy was confirmed, coumarins were stopped and changed to subcutaneous nadroparin (7,500 u, twice daily), from 6 weeks to 12 weeks of gestation . Aspirin, at 100 mg / day, was also administrated throughout the pregnancy . At gestation week 38, women were scheduled for labor induction and changed to nadroparin to avoid the delivery of an anticoagulated fetus . After establishing labor, we carefully checked for hemorrhages and other complications, and babies were examined for congenital anomalies and weight . In the cmd - treated group, coumarins and aspirin were continued throughout the pregnancy and the target international normalized ratio (inr) was maintained between 2.5 to 3.5 . Coumarins were changed to nadroparin before 2 weeks prior to the expected delivery date to avoid fetal bleeding complications during delivery . Pregnancy outcomes, namely, numbers of healthy babies, fetal anomalies, fetal losses, and maternal complications, including thromboembolism or bleeding, were analyzed . To evaluate the efficacy and safety of lmwh, eight pregnancies, maintained using coumarins throughout pregnancy, were compared . Data were analyzed using spss for windows version 10.0 software and compared using the student's t - test, at a level of significance of p<0.05 . No maternal death or bleeding complication occurred in either the lmwh - treated group or the cmd - treated group . Frequencies of maternal thromboembolism were not different between the two groups (table 2). A maternal transient ischemic attack (tia) occurred in one case in each group, and both patients had previously undergone mitral valve replacement . Prosthetic mitral valve thrombosis occurred in three pregnancies, two in the lmwh group and one in the cmd group (table 3). The two of these three patients underwent redo surgery, and other patient was managed on thrombolytic therapy . Numbers of live born and healthy babies were higher in the lmwh group (table 4). In both groups, two babies had low birth weights of 2.1 kg and 2.4 kg, but were otherwise healthy . In the cmd group, one baby had hydrocephalus . However, the frequency of fetal loss including therapeutic abortion and stillbirth were significantly higher in the cmd group . Two fetal losses occurred in the lmwh group, both occurred in cases of maternal valve thrombosis . Four fetal losses occurred in the cmd group, and one of these involved maternal valve thrombosis . This study demonstrates that lmwh - based therapy is superior to coumarin therapy in pregnant women with a prosthetic heart valve, and that the use of nadroparin during the first trimester with 100 mg of aspirin throughout pregnancy could be a safe and effective protocol for thromboprophylaxis in these women . Recent recommendations, published in 2004 as part of the 7th american college of chest physicians (accp) consensus on antithrombotic therapy (12), included the following three regimens: 1) aggressive adjusted - dose lmwh throughout pregnancy; 2) adjusted - dose ufh, throughout pregnancy; or 3) either lmwh or ufh between 6 and 12 weeks and close - to - term only and the use of cmd at other times . Our protocol was similar to the third regimen, but we also administered aspirin (100 mg daily) throughout pregnancy to reduce coumarin dosages and the risk of thromboembolism . The overall frequencies of maternal thromboembolism, including valve thrombosis, were similar in both groups, but the frequencies of live and healthy baby births were higher in the lmwh group . These results demonstrate that lmwh - based therapy is a good alternative to coumarins, because it has similar anticoagulation effects with lower fetal side effects . Exposure to coumarins during the second part of the first trimester is associated with fetal loss, primarily due to spontaneous abortion or coumarin - induced embryopathy . The reported frequencies of coumarins - related embryopathy vary for debatable reasons (14, 15), a recent study suggested that coumarin risk is dose related and that adverse effects occur mainly in women taking> 5 mg daily ., the target inr was maintained with less than 5 mg of coumarins in all patients in the cmd group; however, a half of these lost their babies due to abortion or stillbirth . Our results represent only observational data, and the effect of dose - related embryopathy remains uncertain . Lmwh does not cross the placental barrier and offers potential advantages compared with ufh in terms of better safety profile with less thrombocytopenia, less bleeding, less osteoporosis with prolonged treatment, a more predictable and rapidly reached anticoagulant effect, and the possibility of self - administration of anticoagulant therapy without laboratory monitoring . However, treatment failures have been reported, and the use of lmwh for pregnant women with mechanical heart valves has become controversial due to small numbers of patients and a lack of accurate postmarketing data (16). A recent review of 81 pregnancies in 75 women treated with lmwh reported an 8.6% rate of valve thrombosis (17), and found that appropriate dose adjustments could reduce the frequency of thromboembolism . The 7th accp recommendations call for the use of lmwh at levels that achieve peak anti - factor xa values of around 1.0 u / ml (12). A recent prospective study with deltaparin reported that dosages based on body weight were inadequate to maintain a therapeutic level of lmwh in pregnancy (18). Our data demonstrate that valve thrombosis occurred in 2 patients treated with nadroparin; a prevalence of 8.7% . Unfortunately, we did not monitor anti - xa levels during nadroparin administration, and thus, we cannot conclude that valve thrombosis is associated with an inadequate nadroparin dose . Further studies, with sufficient statistical power, are required to clarify the clinical significance of anti - xa levels . In conclusion, despite the retrospective design of the present study, it might be worth to mention that lmwh appears a safe and effective substitute for any other anticoagulants in pregnant women with mechanical heart valves . We have experienced that pregnancy outcomes are acceptable with lmwh, but that its efficacy for preventing valve thrombosis remains uncertain . Further studies are needed in order to establish appropriate management protocols for pregnant women with mechanical heart valves.
The study population included patients over 18 years old who had an initial cabg or combined cabg and open chest aortic valve replacement (avr), from april 1, 1998 to october 31, 2011 in ontario . The date of first cardiac surgery was the index date, and eligible patients were followed for 1 year with respect to major outcomes, and 5 years for mortality . Preoperative data were included for 1 year prior to surgery, and outcomes for 1 year postoperatively . Patients for whom sex, age, height, weight were missing, and patients living outside of ontario or of unknown residence were excluded . Cardiac care network of ontario (ccn) data were used to identify baseline characteristics such as cardiac ejection fraction, number of grafts bypassed, prior myocardial infarction (mi), emergency or elective surgery, and other co - morbidities . Ccn data and the following datasets were combined from ices using deterministic linkage by unique ices key number identifiers: ontario health insurance plan, canadian institute of health information (cihi) discharge abstract database, national ambulatory care reporting system, same day surgery, and the registered persons database . Patients who had undergone either isolated cabg or combined cabg / avr were selected from the cihi discharge abstract database . Data for which other cardiac procedures had been performed during the same admission were excluded (eg, percutaneous coronary intervention or other valve procedures). Bmi was calculated as weight (kg)/height (m), and patients were divided into groups: underweight (bmi <20 kg / m), normal weight (bmi 20.0 to 24.9 kg / m), overweight (bmi 25.0 to 29.9 kg / m), obese (bmi 30.0 to 34.9 kg / m), and morbidly obese (bmi> 34.9 kg / m), closely based on world health organization (who) and health canada guidelines.1214 the following comorbidities were assessed for presence within 1 year prior to index date: diabetes, smoking history (current or ever smoked), peripheral vascular disease (pvd), chronic obstructive pulmonary disease (copd), dialysis within 1 year prior to surgery, cerebrovascular disease (cvd), congestive heart failure (chf, from cihi), hypertension, elective or emergent surgery, creatinine . Within 30 days prior to surgery, the following cardiac characteristics were captured: ejection fraction, prior mi, left main coronary disease, and previous cabg at date of surgery . At index date, the cardiac characteristics were number of grafts, off - pump surgery, and type of operation . The elixhauser index, a measurement score that includes 30 co - existing conditions, was used to account for baseline comorbidities that are predictive of long - term mortality.15,16 from index date to date of discharge, outcomes of interest were surgery type, total length of stay (los) in hospital, los excluding alternative level of care - los, surgery duration, reoperation, time in intensive care unit (icu), postoperative creatinine, blood transfusion, dialysis, stroke, mi, and death at discharge during hospital admission, and transfusion of blood products . Post - discharge outcomes collected included readmission rates within 60 days of discharge, dialysis, stroke, and mi within 60 days and 1 year of discharge, and mortality at 30 days, 1 and 5 years . Mean and standard deviation (sd) were used for continuous variables, and categorical variables were expressed as percentages . To detect differences between the bmi groups, anova was used, and mann - kendall trend test was used to test significant differences in bmi over time . Five - year mortality was compared using bivariate analyses, and cox multivariate regression analysis to investigate multiple confounders on the relationship between bmi and patient mortality, providing hazard ratios and 95% confidence intervals (ci). Age, gender, diabetes, smoking history, pvd, copd, dialysis, hypertension, serum creatinine, cvd, chf, elixhauser index, surgery type, los, reoperation, blood transfusion, stroke in hospital, mi in hospital, dialysis within 1 year, stroke within 1 year, mi within 1 year, ejection fraction, prior mi, elective or emergent surgery, left main coronary disease, previous cabg, and bmi were included in the model . Sas software (version 9.3, statistical analysis system institute) was used for statistical analyses, with p<0.05 considered significant . The study population included patients over 18 years old who had an initial cabg or combined cabg and open chest aortic valve replacement (avr), from april 1, 1998 to october 31, 2011 in ontario . The date of first cardiac surgery was the index date, and eligible patients were followed for 1 year with respect to major outcomes, and 5 years for mortality . Preoperative data were included for 1 year prior to surgery, and outcomes for 1 year postoperatively . Patients for whom sex, age, height, weight were missing, and patients living outside of ontario or of unknown residence were excluded . Cardiac care network of ontario (ccn) data were used to identify baseline characteristics such as cardiac ejection fraction, number of grafts bypassed, prior myocardial infarction (mi), emergency or elective surgery, and other co - morbidities . Ccn data and the following datasets were combined from ices using deterministic linkage by unique ices key number identifiers: ontario health insurance plan, canadian institute of health information (cihi) discharge abstract database, national ambulatory care reporting system, same day surgery, and the registered persons database . Patients who had undergone either isolated cabg or combined cabg / avr were selected from the cihi discharge abstract database . Data for which other cardiac procedures had been performed during the same admission were excluded (eg, percutaneous coronary intervention or other valve procedures). Bmi was calculated as weight (kg)/height (m), and patients were divided into groups: underweight (bmi <20 kg / m), normal weight (bmi 20.0 to 24.9 kg / m), overweight (bmi 25.0 to 29.9 kg / m), obese (bmi 30.0 to 34.9 kg / m), and morbidly obese (bmi> 34.9 kg / m), closely based on world health organization (who) and health canada guidelines.1214 the following comorbidities were assessed for presence within 1 year prior to index date: diabetes, smoking history (current or ever smoked), peripheral vascular disease (pvd), chronic obstructive pulmonary disease (copd), dialysis within 1 year prior to surgery, cerebrovascular disease (cvd), congestive heart failure (chf, from cihi), hypertension, elective or emergent surgery, creatinine . Within 30 days prior to surgery, the following cardiac characteristics were captured: ejection fraction, prior mi, left main coronary disease, and previous cabg at date of surgery . At index date, the cardiac characteristics were number of grafts, off - pump surgery, and type of operation . The elixhauser index, a measurement score that includes 30 co - existing conditions, was used to account for baseline comorbidities that are predictive of long - term mortality.15,16 from index date to date of discharge, outcomes of interest were surgery type, total length of stay (los) in hospital, los excluding alternative level of care - los, surgery duration, reoperation, time in intensive care unit (icu), postoperative creatinine, blood transfusion, dialysis, stroke, mi, and death at discharge during hospital admission, and transfusion of blood products . Post - discharge outcomes collected included readmission rates within 60 days of discharge, dialysis, stroke, and mi within 60 days and 1 year of discharge, and mortality at 30 days, 1 and 5 years . Mean and standard deviation (sd) were used for continuous variables, and categorical variables were expressed as percentages . To detect differences between the bmi groups, anova was used, and mann - kendall trend test was used to test significant differences in bmi over time . Five - year mortality was compared using bivariate analyses, and cox multivariate regression analysis to investigate multiple confounders on the relationship between bmi and patient mortality, providing hazard ratios and 95% confidence intervals (ci). Age, gender, diabetes, smoking history, pvd, copd, dialysis, hypertension, serum creatinine, cvd, chf, elixhauser index, surgery type, los, reoperation, blood transfusion, stroke in hospital, mi in hospital, dialysis within 1 year, stroke within 1 year, mi within 1 year, ejection fraction, prior mi, elective or emergent surgery, left main coronary disease, previous cabg, and bmi were included in the model . Sas software (version 9.3, statistical analysis system institute) was used for statistical analyses, with p<0.05 considered significant . Tables1 through 3 show preoperative characteristics and postoperative outcomes . Where specific values were not available for all study years, total sample size analyzed is indicated . Baseline characteristics anova indicates analysis of variance; chf, congestive heart failure; sd, standard deviation . Where data are missing, total perioperative characteristics occurring between surgery and discharge, unless otherwise indicated anova indicates analysis of variance; cabg, coronary artery bypass graft, sd, standard deviation . Where data are missing, total twenty - two percent of the patients were classified as normal weight (n=17 162), with 44% overweight (n=34 597), 23% obese (n=18 147), 10% morbidly obese (n=7628), and 2% underweight (n=1228). Underweight patients were the oldest (meansd: 6811), constituted the largest number of females (41%), had the highest rates of pvd, copd, dialysis, cvd, chf, left ventricular grades 3 or 4, and left main coronary disease, creatinine . The morbidly obese group was the youngest, and experienced the highest rates of diabetes and hypertension . Although more obese / morbidly obese patients had any smoking history, more underweight patients were current smokers at the time of surgery, compared with normal weight patients . There was no difference in mean serum creatinine, but a significant difference in creatinine concentration> the median number of bypass grafts for all bmi groups was 3; and 3.5% of patients had off - pump surgery, with the underweight group having the most at 5.6% . There was an overall statistical difference between bmi groups in all baseline characteristics, except for mean serum creatinine and prior mi . Table2 presents outcomes from index (surgery) date to date of discharge . The underweight group had the greatest hospital los, reoperation rates, icu hours, dialysis, mi, and blood (red cell, platelet) transfusion, and number of deaths . The difference between bmi groups in postoperative serum creatinine and stroke was non - significant . Table3 shows post - discharge outcomes with an overall statistical difference between bmi groups for all events, except stroke within 1 year of discharge . The underweight group experienced the highest rates of surgery readmission, mi, and mortality . The mean period of follow - up was 7.84 years, with 12 392 deaths . Overall, 30-day, 1- and 5-year survival rates were highest for the obese group (99.1% [95% ci, 98.9 to 99.2], 97.6% [95% ci, 97.3 to 97.8], and 90.0% [95% ci, 89.5 to 90.5]), respectively, and lowest for the underweight group (96.5% [95% ci, 95.4 to 97.7], 91.4% [95% ci, 89.6 to 93.2], and 75.9% [95% ci, 73.0 to 78.7]) (table4 and figure2). A reverse j - shaped relationship exists between bmi range and mortality (figure3), with their respective hazard ratios, using normal weight as the reference: underweight (1.35 [95% ci, 1.23 to 1.49]); overweight (0.94 [95% ci, 0.91 to 0.98]); obese (1.00 [95% ci, 0.96 to 1.04]); morbidly obese (1.12 [95% ci, 1.06 to 1.19]). Proportion of patients alive at 3 time intervals following date of surgery (95% confidence intervals) bmi indicates body mass index . The box in the boxplot displays the median (diamonds), mean (dashes), and encompasses the 25th and the 75th percentiles; lines include 1.5 interquartile range beyond the 25th and 75th percentiles; the outer circles represent outliers . There was no significant trend found over the years of the study (p=0.1005) using the mann - kendall trend test . Kaplan - meier curves indicating overall 5-year survival from date of surgery, by body mass index category . Hazard ratios for mortality with 95% confidence intervals by body mass index category, with reference to normal weight . Factors related to the risk of death using bivariate (table5) and multivariate (table6) analyses are presented . Clinical considerations dictated the choice between co - linear variables, which included age, sex, diabetes, smoking history, pvd, copd, dialysis, cvd, chf, elixhuaser index, surgery type, los, reoperation, blood transfusion, stroke in hospital, mi in hospital, dialysis within 1 year, stroke within 1 year, mi within 1 year . Bivariate cox analysis hazard ratios for mortality for each bmi category with respect to the normal weight group . For each comorbidity factor, overall hazard ratios are shown, followed by effect of bmi category adjusted for that factor . Cabg indicates coronary artery bypass graft; chf, congestive heart failure; copd, chronic obstructive pulmonary disease; mi, myocardial infarction; pvd, peripheral vascular disease . Multivariate cox analysis hazard ratios for factors with significant effect on mortality . Each line represents the effect of that factor on mortality, adjusting for all other factors in this model . Bmi indicates body mass index; cabg, coronary artery bypass graft . In the bivariate analysis, bmi significantly influenced survival when each of the above variables were factored into the model, with reference to the normal weight group for all variables, except pvd, cvd, mi in hospital for morbidly obese group . For example, adjusting for age (eg, underweight comprised the oldest bmi group), bmi independently influenced mortality (hazard ratio for underweight group 1.62, ci 1.48 to 1.79). Even when adjusting for co - morbidities (elixhauser index), bmi still affected mortality (hazard ratio for underweight group 1.39, ci 1.26 to 1.53). Similarly, considering the morbidly obese group was youngest, bmi still independently affected mortality . Using multivariate analysis, the reverse j - curve relationship between bmi and mortality was confirmed (figure3). Underweight patients had a significantly higher risk of death (hazard ratio, 1.35, 95% ci, 1.23 to 1.49) compared with patients with normal weight, as did morbidly obese patients (hazard ratio, 1.12, 95% ci, 1.06 to 1.19). There was a survival advantage in the overweight group, and no difference in the obese group, compared with normal weight . The elixhauser index was highly predictive of mortality, such that for each 1 point increase, there was a 22% increase in mortality rate . Age also impacted mortality, such that for every year of increase in age, the chance of death increased by 6%, taking bmi and all other factors relating to survival into account . However, bmi had an independent influence on mortality when adjusting for both age and risk score . Table1 lists baseline characteristics of the bmi groups . Twenty - two percent of the patients were classified as normal weight (n=17 162), with 44% overweight (n=34 597), 23% obese (n=18 147), 10% morbidly obese (n=7628), and 2% underweight (n=1228). Underweight patients were the oldest (meansd: 6811), constituted the largest number of females (41%), had the highest rates of pvd, copd, dialysis, cvd, chf, left ventricular grades 3 or 4, and left main coronary disease, creatinine . The morbidly obese group was the youngest, and experienced the highest rates of diabetes and hypertension . Although more obese / morbidly obese patients had any smoking history, more underweight patients were current smokers at the time of surgery, compared with normal weight patients . There was no difference in mean serum creatinine, but a significant difference in creatinine concentration> the median number of bypass grafts for all bmi groups was 3; and 3.5% of patients had off - pump surgery, with the underweight group having the most at 5.6% . There was an overall statistical difference between bmi groups in all baseline characteristics, except for mean serum creatinine and prior mi . The underweight group had the greatest hospital los, reoperation rates, icu hours, dialysis, mi, and blood (red cell, platelet) transfusion, and number of deaths . The difference between bmi groups in postoperative serum creatinine and stroke was non - significant . Table3 shows post - discharge outcomes with an overall statistical difference between bmi groups for all events, except stroke within 1 year of discharge . The underweight group experienced the highest rates of surgery readmission, mi, and mortality . The mean period of follow - up was 7.84 years, with 12 392 deaths . Overall, 30-day, 1- and 5-year survival rates were highest for the obese group (99.1% [95% ci, 98.9 to 99.2], 97.6% [95% ci, 97.3 to 97.8], and 90.0% [95% ci, 89.5 to 90.5]), respectively, and lowest for the underweight group (96.5% [95% ci, 95.4 to 97.7], 91.4% [95% ci, 89.6 to 93.2], and 75.9% [95% ci, 73.0 to 78.7]) (table4 and figure2). A reverse j - shaped relationship exists between bmi range and mortality (figure3), with their respective hazard ratios, using normal weight as the reference: underweight (1.35 [95% ci, 1.23 to 1.49]); overweight (0.94 [95% ci, 0.91 to 0.98]); obese (1.00 [95% ci, 0.96 to 1.04]); morbidly obese (1.12 [95% ci, 1.06 to 1.19]). Proportion of patients alive at 3 time intervals following date of surgery (95% confidence intervals) bmi indicates body mass index . The box in the boxplot displays the median (diamonds), mean (dashes), and encompasses the 25th and the 75th percentiles; lines include 1.5 interquartile range beyond the 25th and 75th percentiles; the outer circles represent outliers . There was no significant trend found over the years of the study (p=0.1005) using the mann - kendall trend test . Kaplan - meier curves indicating overall 5-year survival from date of surgery, by body mass index category . Hazard ratios for mortality with 95% confidence intervals by body mass index category, with reference to normal weight . Factors related to the risk of death using bivariate (table5) and multivariate (table6) analyses are presented . Clinical considerations dictated the choice between co - linear variables, which included age, sex, diabetes, smoking history, pvd, copd, dialysis, cvd, chf, elixhuaser index, surgery type, los, reoperation, blood transfusion, stroke in hospital, mi in hospital, dialysis within 1 year, stroke within 1 year, mi within 1 year . Bivariate cox analysis hazard ratios for mortality for each bmi category with respect to the normal weight group . For each comorbidity factor, overall hazard ratios are shown, followed by effect of bmi category adjusted for that factor . Cabg indicates coronary artery bypass graft; chf, congestive heart failure; copd, chronic obstructive pulmonary disease; mi, myocardial infarction; pvd, peripheral vascular disease . Each line represents the effect of that factor on mortality, adjusting for all other factors in this model . Bmi indicates body mass index; cabg, coronary artery bypass graft . In the bivariate analysis, bmi significantly influenced survival when each of the above variables were factored into the model, with reference to the normal weight group for all variables, except pvd, cvd, mi in hospital for morbidly obese group . For example, adjusting for age (eg, underweight comprised the oldest bmi group), bmi independently influenced mortality (hazard ratio for underweight group 1.62, ci 1.48 to 1.79). Even when adjusting for co - morbidities (elixhauser index), bmi still affected mortality (hazard ratio for underweight group 1.39, ci 1.26 to 1.53). Similarly, considering the morbidly obese group was youngest, bmi still independently affected mortality . Using multivariate analysis, the reverse j - curve relationship between bmi and mortality was confirmed (figure3). Underweight patients had a significantly higher risk of death (hazard ratio, 1.35, 95% ci, 1.23 to 1.49) compared with patients with normal weight, as did morbidly obese patients (hazard ratio, 1.12, 95% ci, 1.06 to 1.19). There was a survival advantage in the overweight group, and no difference in the obese group, compared with normal weight . The elixhauser index was highly predictive of mortality, such that for each 1 point increase, there was a 22% increase in mortality rate . Age also impacted mortality, such that for every year of increase in age, the chance of death increased by 6%, taking bmi and all other factors relating to survival into account . However, bmi had an independent influence on mortality when adjusting for both age and risk score . Obesity is a well - recognized risk factor for the development of cardiovascular disease, and patients with high bmi are highly represented among the population presenting for cardiac surgery (77% of the total in the current study). However, we have confirmed, using a cohort study with large administrative datasets and 5-year follow - up for survival analysis and mean follow - up of 7.8 years (4 years) that overweight and obese patients who undergo cardiac surgery have a survival advantage over underweight, normal weight, and morbidly obese patients . A reverse j - shaped relationship was found relating bmi with mortality, such that bmi independently predicted survival when multiple confounders were considered . At times of illness and stress, excess adiposity may confer an advantage.17 low bmi individuals may not have the reserve to tolerate the effects of weight loss as readily as obese individuals, potentially contributing towards high mortality.18,19 the improved survival of obese patients could be attributed to high metabolic reserves and body fat.20,21 this may be due to an increase in secretion of amino acids and dipokines, a decrease in the levels of b - type natriuretic peptide and in oxidative stress and inflammation.22 potential moderators of the relationship between obesity and mortality may include hormones and cytokines.20,21,23,24 it can be argued that the observed reverse j - shaped relationship in our study can be due to reverse causation, since the underweight group could have been malnourished or cachectic and were older, with a high prevalence of comorbidities.2527 in order to adjust for important confounders, obesity could be considered as a time - varying exposure to account for changes in weight status over the lifespan.28,29 however, no substantial change in mortality risk has been shown when excluding patients who had a history of cancer, or who died within the first 4 years of follow - up.3032 in the current study, bivariate and multivariate analyses demonstrated that effect modification due to age had an impact on results; however, when adjusting for age, there was still a significant relationship between bmi and mortality . Our findings may also be due to lead - time bias, since patients with high bmi tend to be treated sooner.23,33 this study demonstrated the lowest survival following cardiac surgery in the underweight group . As low bmi could represent a surrogate for serious underlying illness, we attributed a well - validated co - morbidity index to all patients . The elixhauser index was developed on administrative data from california, and is designed to be used with large data sets.15,16,34 the index includes 30 co - existing conditions using international statistical classification of disease codes icd-9 and icd-10 . Its performance has been shown in a recent systematic review to provide the best fit among various co - morbidity indices, especially when estimating mortality beyond 30 days.35 unsurprisingly, the elixhauser index was highest in the underweight and morbidly obese groups . When including the elixhauser index in our bivariate and multivariate models, low bmi remained the most significant independent predictor of mortality . Our results correspond with findings that have examined bmi and mortality, in that moderate obesity provides a protective effect in patients who undergo cardiac surgery20,21,23,3638 and the general population.1,39 however, limitations in past studies include a short follow - up of 3 years or less,33,36,38,40 a small sample, and limited numbers of deaths,23,4146 as well as not accounting for possible confounders, such as chronic illness and smoking.21,47 certain studies with relatively small sample sizes and short follow - up45,46 found no significant association between non - morbid obesity and mortality for cabg patients . Benedetto et al retrospectively studied 13 963 patients over 5 years and found that, using propensity score matching, obesity did not contribute towards operative mortality, but was related to a decrease in late survival in patients undergoing cabg.41 however, their analysis was not statistically significant because of the small number of morbidly obese patients (n=211). Other studies with follow - up longer than 3 years12,47 found similar patterns, with a reverse j- or u - shaped relationship between bmi and mortality . Gurm et al found a curvilinear relationship between bmi and 5-year mortality, with higher risk of death associated with bmi extremes in patients who had undergone cabg (n=1526).12 sarno et al investigated the impact of bmi on 7427 patients who had been treated by percutaneous coronary intervention (pci), assessing 5-year mortality.37 they found that the obesity paradox observed could have been due to a large proportion of elderly patients in the normal bmi group . However, as in our study, obese patients had a higher rate of risk factors compared with the normal bmi group . In contrast, morbidly obese patients were the youngest, constituted the largest group of smokers, and had the highest rates of diabetes . Also congruent with our findings, hastie et al examined 4880 patients who had undergone pci for cad over 5 years, and observed a u - shaped relationship between bmi and mortality, with the highest mortality risk for the underweight group.23 similarly, birkmeyer et al found that obesity was not associated with increased mortality (n=11 101 cabg patients) over 4 years.48 the main strengths of our study are the large sample, allowing for robust analyses among bmi categories (particularly underweight and morbidly obese patients), and the long follow - up (5 years). In addition, numerous potential confounding variables were adjusted for, revealing results that were attributable to bmi alone . In particular, although the baseline risk index differed between bmi groups, when this was factored into multivariate analysis, the difference in mortality attributed to bmi was independent of baseline risk index . Limitations include using retrospective data, determination of all - cause mortality only, lack of inclusion of ethnicity, and lack of data related to fitness or weight change before surgery . Additionally, a possible confounding relationship of socioeconomic status with bmi has yet to be studied in this population . The importance of our findings lies in risk assessment of patients presenting for cardiac surgery and the allocation of resources . Moderate obesity cannot be presumed to predict complications after surgery, whereas underweight and normal weight patients (aggregate bmi <25) may consume additional health care resources . The validity of bmi as an accurate adiposity measure has been questioned, given muscle mass decline with age,49,50 as it does not account for body composition or the location of adipose tissue.5153 nevertheless, several studies have shown that the obesity paradox exists with any measure of adiposity.24,51,5456 the definition of the bmi categories in the current study are closely based on who and health canada guidelines (with the exception of underweight being considered as bmi <18.5 in these guidelines).13,14 these definitions reflect relative levels of risk to health.14 following the results of this and other studies confirming an obesity paradox in various clinical situations, we suggest that traditional bmi categorization may need to be revisited . In summary, although obesity is a well - recognized risk factor for cardiovascular disease, overweight and moderately obese patients showed improved outcomes following cabg and cabg / avr with respect to patients with as outcomes were significantly worse for morbidly obese and underweight patients, focus should be placed on these populations in risk assessment, preparation, and resource allocation prior to cardiac surgery.
A 7month old baby girl was brought to our hospital with a 2 months history of an ill defined mass involving entire abdomen . Contrast enhanced computed tomography (cect) of the abdomen and pelvis revealed a small left kidney with a dilated renal pelvis that continued as a hugely dilated and tortuous ureter (figure 1). On a diethylene triamine pentaacetic acid (dtpa) micturating cystourethrogram (mcu) showed no reflux, with the bladder pushed laterally, probably due to the mass effect of the dilated ureter (figure 2). On cystoscopy, hemitrigone was noticed, with absent left ureteric orifice . A diagnosis of left ectopic ureter with congenital giant hydroureter and poorly functioning left kidney was made and open nephroureterectomy was done (figure 3). Ct scans with a small left kidney, an extremely dilated left ureter and a displaced bladder . This case has been presented to highlight the extent to which a ureter can dilate . In case of congenital giant hydroureter the ureter is dilated out of proportion to the kidney, which is usually small and atrophic . Very few such cases have been reported in literature [2, 3, 4] and surgeons should be aware of this entity as one of the causes of abdominal mass in an infant.
Pulmonary hypertension is an increasingly recognized complication of sickle - cell anaemia and a risk factor for early death [14]. Recurrent episodes of acute and subacute pulmonary crises reflecting in situ sickling within the lung has been postulated to be the primary event leading to pulmonary hypertension . Haemolysis may participate in its pathogenesis by limiting nitric oxide (no) bioavailability and producing vasculopathy [5, 6]. An initial study in howard university, usa, using echocardiographic assessment of tricuspid valve regurgitant jet velocity 2.5 m / sec as diagnostic criteria, demonstrated pulmonary hypertension in 32% of adult sickle - cell patients, and the prevalence appeared to increase with age of the patients . In patients between 40 and 49 years old, the prevalence was 40% and increased to 5560% by age 50 and above . Other studies have documented prevalence rates between 20 and 40% [4, 810]. Sickle - cell anaemia patients with pulmonary hypertension have a significantly increased mortality rate compared with patients without pulmonary hypertension . Sutton and colleagues reported 40% mortality rate in sickle - cell patients with pulmonary hypertension at 22 months after diagnosis (odd ratio 7.86; 95% confidence interval = 2.6323.4) compared with sickle - cell patients without pulmonary hypertension . Castro et al . In a study of 34 adult sickle - cell patients who underwent right heart catheterization for evaluation of pulmonary hypertension found increased pulmonary artery pressure in 20 (58.8%) on initial catheterization . During 2345 months of follow up, 11 of these 20 (55.0%) died compared with 3 of the 14 without pulmonary hypertension (21.0%). Every 10 mmhg increase in mean pulmonary artery pressure was associated with 1.7 increase in mortality (95% confidence interval = 1.12.7; cox proportional hazard model, p = 0.028). The study was aimed at comparing the clinical and electrocardiographic findings in sickle - cell anaemia patients with raised pulmonary artery pressure with those of patients without pulmonary hypertension . The study was carried out in the adult outpatient sickle - cell clinic and the cardiac centre of the university of nigeria teaching hospital (unth), enugu, nigeria . The study subjects were drawn from adult patients (age 18 years), attending the adult sickle - cell clinic of the hospital, who had haemoglobin genotype ss on haemoglobin electrophoresis, were in steady state, and consented to participate in the study . Steady state is defined as absence of any crisis in the preceding four weeks, absence of any symptoms, or signs attributable to acute illness . A total of sixty two sickle - cell anaemia patients, and sixty two age- and sex - matched normal controls were studied . Resting 12-lead electrocardiography was performed on all subjects using cardioline ar-600 model electrocardiography machine at a paper speed of 25 mm / s and standardized at 0.1 mv / mm . A single observer analyzed the electrocardiogram . Measurements of the heart rate, cardiac axis, pr- interval, qrs duration, and qtc interval were done in the standard fashion . Heart rate correction of the qt interval was performed using bazett's formula (qtc = qt/ rr). The dispersion of p - wave, qrs, and qtc intervals was measured manually under magnifying glass by the same observer and was taken as the difference between the maximum and minimum values of each parameter on standard 12-lead electrocardiogram . Left ventricular hypertrophy on electrocardiogram was based on sokolow and lyon voltage criteria, while right ventricular hypertrophy was based on the criteria described by allenstein and mori (dominant or tall r - waves or rs pattern in a vr, v1, and v2 with deep s - wave in i, avl, v5, and v6). Echocardiography was done using hewlett packard sonos 2500 echocardiography machine with 3.7 mhz transducer . Pulmonary artery flow acceleration time was obtained from a doppler signal of the pulmonary flow in the parasternal short - axis view at the aortic valve level and was described as the time from onset to peak flow velocity . The mean pulmonary artery pressure (mean pap) was calculated from the formula: mean pap (m m / h g) = 90 (0.62 a t), where at is the acceleration time of the pulmonary artery flow . Pulmonary hypertension was defined as calculated mean pulmonary artery pressure 30 mm / hg [4, 7]. Ethical clearance for the study was obtained from the ethical committee of the university of nigeria teaching hospital, enugu, nigeria . The declaration of helsinki's recommendations for guiding physicians in biomedical research involving human subjects were followed . Data were presented as means standard deviation for continuous variables and as proportions for categorical variables . Comparisons of continuous variables between groups were made with independent student's t - test . For discrete variables, distribution between groups was compared with chi - square test and fishers exact test as appropriate (where an expected cell is less than 5). Multivariate pearson's correlation coefficient was used to determine the relations between clinical, electrocardiographic data and mean pulmonary artery pressure . All statistical analyses were carried out using the statistical packages for social sciences (spss inc ., chicago, illinois) software version 11.0 and epi - info version 3.4 . Statistical tests with 2-tailed probability values less than 0.05 were considered statistically significant . The age, gender, and anthropometric parameters of the patients and controls are shown in tables 1 and 2 . Values for pulmonary artery pressure in the patients and controls are shown in table 3 . Elevated pulmonary artery pressures (pap) as defined by pap 30 mmhg was demonstrated in (26) 41.9% of patients with sickle - cell anaemia and in (2) 3.2% of the controls; = 26.571, df = 1, p <0.001 (figure 1). P - wave duration, qtc interval, and qtc dispersion were increased in patients with pulmonary hypertension (table 5). Significant correlation was found between mean pap and (1) frequency of crisis (spearman correlation = 0.320; p = 0.011), (2) body mass index (pearson's correlation = 0.297; p = 0.019), and (3) qtc interval (pearson's correlation 0.261; p = 0.040), (table 6). The prevalence of pulmonary hypertension in adult nigerian sickle - cell anaemia patients in this study was 41.9% . This value is slightly higher than previous report of 25% by aliyu et al . In northern nigeria . However, in that study, neither the mean pap nor the mean age of the subjects was stated . Previous studies have relied on tricuspid regurgitant jet velocity as an indirect estimate of pap . This has resulted in an underestimation of the prevalence of pulmonary hypertension in sickle - cell anaemia patients . Results of right heart catheterization for evaluation of pulmonary artery pressures in sickle - cell patients have shown prevalence rate of 58.8% . Autopsy studies suggest that up to 75% of sickle - cell anaemia patients have histological evidence of pulmonary arterial hypertension at the time of death . Mean pap derived from the echocardiographic estimation of pulmonary artery flow acceleration time in steady - state patients as was used in this study has been shown to correlate significantly with values from cardiac catheterization . Several studies have corroborated the role of vaso - occlusive crisis and acute chest syndrome in initiating nitric oxide depletion resulting in the pathological changes in pulmonary hypertension [6, 19]. This study identified a positive correlation between pulmonary artery pressure and frequency of vaso - occlusive crisis . Patients with pulmonary hypertension were found to have reduced body mass index when compared with patients without pulmonary hypertension . This difference could be due to increased disease severity in patients with pulmonary hypertension . Recurrent insitu vaso - occlusive crisis in the pulmonary vascular bed with resultant pulmonary hypertension and right ventricular hypertrophy has been demonstrated in sickle - cell disease patients . This is in keeping with the finding by this study of a significantly increased prevalence of right ventricular hypertrophy in patients with pulmonary hypertension . Right ventricular hypertrophy in these patients explains the significant positive correlation between pulmonary artery pressure and qtc interval . The significance of increased p - wave duration and the spatial dispersion of qtc - interval noted in this study in patients with pulmonary hypertension are unclear . Qtc dispersion is a measure of the disparity among qtc intervals in various electrocardiographic leads and reflects the variability of myocardial repolarization . This corroborates the finding in a study by akgul et al . In turkey which recorded higher qtc dispersion in sickle - cell patients with pulmonary hypertension . Pulmonary hypertension in adult sickle - cell anaemia patients is significantly associated with electrocardiographic evidence of right ventricular hypertrophy, increased p - wave duration, qtc interval, and qtc dispersion and correlates significantly with frequency of vaso - occlusive crisis and qtc interval . The observations by this study tend to suggest that these parameters could be useful for early detection and prevention of pulmonary hypertension in patients with sickle - cell anaemia.
The foley catheter has been a staple of medical care since its inception in 1935 by american urologist frederic foley (1). Though there have been modifications in the 80 years since its creation, the foley catheter has withstood the test of time and is considered an acceptable means for bladder drainage in the appropriate setting . One of the main complications of its use is catheter - associated urinary tract infection (cauti). The prevalence of cauti is high, as up to 40% of catheterized patients in the acute hospital care setting develop cauti (2). This, in conjunction with the fact that up to 25% of patients admitted to hospitals have a urinary catheter placed at some point during their stay highlights the healthcare burden generated by cauti (3). Although the cost of each cauti is estimated to be less than $600, the additive cost to hospitals for lack of medicare and medicaid reimbursement of a preventable complication is considerable (4, 5). For each day that a catheter is in place, 3 - 7% of patients may develop a cauti (6). By the 30th day of catheterization, the incidence of cauti or catheter - associated asymptomatic bacteriuria (caasb) is almost 100% (7). Failure to keep the urinary collection bag in a gravity dependent position is the most commonly violated catheter care - related recommendation, and can double the relative risk of developing cauti or caasb (8). One potentially modifiable factor to impact the incidence of cauti is inadequate drainage of the bladder . Stoller et al . Suggested that foley catheters do not consistently and constantly drain urine from the bladder, with 43% of patients found to have a residual urine volume> 50 ml (8). A previous study found that clinically asymp tomatic men with a post - void residual volume (pvr) of> 180 ml are at high risk for developing bacteriuria (9). Further, pvr> 150 ml in a non catheterized patient is associated with an increased risk of developing a urinary tract infection (10). Taking these findings together, it would seem logical that we can reduce the incidence of cauti by addressing the problem of inadequate bladder drainage by foley catheters . As such, the purpose of our study was to evaluate the efficacy of the foley catheter in bladder drainage . After obtaining approval from our institutional review board (approved protocol #3309371), we retrospectively identifed 167 consecutively catheterized patients via contrast enhanced and non - enhanced abdominal and pelvic ct studies from 7/1/2011 - 6/30/2012 . We assessed residual urine volume (ruv) by utilizing the 5 mm axial section demonstrating the greatest bladder cross sectional area . Using coronal multiplanar reformatted images to determine maximum bladder height, ruv was calculated by means of the formula for the volume (v) of a sphere in a cube with a given length (l), width (w), and height (h): v=(/6)(wl*h). Patients were stratified into three groups: ruv = 0 (n=68, 40.7%), ruv> 0 and <50 ml (n=95, 56.9%, mean=6.7 ml, standard deviation (st dev) = 7.9 ml), and ruv 50 ml (n=4, 2.4%, mean=394.7 ml, two of the four patients with ruv 50 ml were found to have improperly placed catheters by ct . The overall mean ruv was 13.2 ml with a st dev of 75.9 ml and margin of error of 11.6 ml for a 95% confidence interval . Thirty nine women (41%) had ruv = 0 ml, 54 (57%) had a ruv> 0 ml and <50 ml (mean=8.0 ml, st dev=8.3 ml), and 2 (2%) had ruv 50 ml, (mean=301.4 ml, st dev=172.6 ml). The overall mean ruv for women was 10.9 ml with a st dev of 50.0 ml . Twenty nine men (40.3%) had ruv=0 ml, 41 (57%) had a residual urine volume of> 0 ml and <50 ml, (mean=4.9 ml, st dev = 7.0 ml), and 2 (2.8%) had ruv> 50 ml (mean=488.1 ml, st dev=362.8 ml). The overall mean ruv for men was 16.2 ml, with a st dev of 100.2 ml . Contrary to the study performed by stoller et al ., we found that the vast majority of our patients (97.6%) had a ruv of <50 ml . Further, 40.7% of our patients were found to have no residual volume at all . This contrast may be attributed to differences in study design . In stoller's study, bladder sonography was performed at the bedside whereas in the present cohort, the patient was transported to the ct department . It is possible that during transport, manipulation and movement of the foley, as well as changes in abdominal pressure during transfer resulted in drainage in urine from the bladder . Since patients either move or are turned / moved frequently while in hospital beds, we feel that ct - derived measurement of bladder volume still represents a real world situation amongst catheterized inpatients . Further, sonographic measurement of the bladder is less precise when the bladder is collapsed (11, 12). In the present study, while only 2% of patients had a ruv of> 50 ml, 59% of patients a ruv of> 0 ml, indicating that for most patients the foley catheter does not completely drain the bladder . Though some of the smaller volumes may be considered negligible, 16% of our patients (n=26) had a ruv of> 10 ml . We found that half (2/4) of the patients with a ruv> 50 ml had an improperly placed catheter, emphasizing the importance of catheter placement technique and urine output monitoring . Hence, the vast majority of properly placed catheters appear to drain the bladder at least moderately well with an arbitrary cut point of 50 ml . The importance of finite ruv <50 ml in catheterized patients has yet to be determined . In summary, our retrospective study provides three principal findings: for the vast majority of patients the foley catheter adequately drained the bladder; there were no gender differences in the efficacy of the foley catheter in bladder drainage; improper placement of a foley catheter can lead to significant urinary retention.
Levetiracetam (lev) is used to treat generalized and partial seizures of various etiologies . Rhabdomyolysis postconvulsive seizure may limit kidney excretion of muscle breakdown products like creatine phosphokinase (cpk). In this case report, a 19-year - old right - handed male presented following a complex partial seizure with secondary generalization in the setting of stress and sleep deprivation . A second convulsive event lasting 2 min occurred in the emergency room . The patient was given 2 mg iv lorazepam, 2000 mg iv lev, and 1200 mg oxcarbazepine (oxc), and no further seizures occurred . Epilepsy etiology was due to a right frontal arteriovenous malformation (avm) located in the left leg primary motor cortex (fig . 1). The avm had previously been treated with gamma knife radiosurgery (20 gy at 50% isodose to encompass a volume of 1.2 ml). Outpatient anticonvulsant medications for this patient prior to the present hospitalization included oxc 600 mg in the morning and 900 mg in the afternoon or evening and lorazepam 0.5 mg prn for breakthrough seizures . Of note, this patient is an avid weight lifter and very muscular (fig . 2). Although an avid weight lifter, the patient denied using supplemental agents for muscle bulking such as anabolic steroids or protein powders . They occurred rarely in the daytime, and if they did, a warning of lightheadedness, dizziness, and a lost sense of balance would progress to bilateral muscle cramping, tonic this admission was complicated by a rapid rise in cpk and mild renal compromise, a new finding for the patient (fig . Maximum creatinine was 2.17 mg / dl and had normalized prior to cpk maximum . The patient's cpk levels were slow to resolve despite appropriate iv hydration . During the entire period of cpk elevation, the patient's urine remained clear / yellow; trace hematuria was noted only on day two . His day nine serum myoglobin was 228 ng / ml (normal range: 1092 ng / ml). An initial serum myoglobin level was not checked . With a marked drop of cpk on day 5 (cpk: 2736) however, blood work postdischarge showed cpk levels again in the 29,000 range, suggesting that the day five cpk measurement was a lab error . Levetiracetam was discontinued on day eight; day nine blood work showed a halving of cpk levels from 29,136 to 14,918 and a normal level of creatinine . Creatine phosphokinase levels continued to decline rapidly after lev discontinuation, with normalization occurring nearly a month later after resumption of a normal exercise regimen . Two convulsive seizures and subsequent lev therapy led to a dramatic increase in cpk in this muscular male . Although this could indicate a delayed rhabdomyolysis, the classic findings including marked elevations in myoglobin / myoglobinuria, decreased urine output, and muscle pain were not evident . Additionally, following muscular injury, rhabdomyolysis - related cpk elevations typically occur maximally by day three and decline by day five . The cpk elevation in this patient was both delayed and persisted beyond the typical time frame of a case of seizure - induced rhabdomyolysis, suggesting ongoing issues of muscle breakdown despite cessation of seizure activities . Creatine phosphokinase release occurs with insults to the sarcolemma, most notably failure of atp production or use . However, lev was the only new drug given, and its use even at a low maintenance dose and discontinuation closely mirror the trends in cpk levels in this patient . Although we suspect that lev may have contributed to cpk elevations in this heavily muscled patient, those suspicions remain unproven . A retrial of lev with cpk and renal function checks, a muscle biopsy, and a repeat renal ultrasound are not planned, given that the patient's clinical issues have resolved . This case revealed a marked and dangerous increase in cpk levels corresponding with modest lev use . Although this scenario could indicate a delayed rhabdomyolysis, the rapid decline in cpk with lev discontinuation suggests that, in this patient, lev may have had effects on kidney filtration and/or muscle breakdown.
Retinoblastoma (rb) is the most common intraocular malignant tumor in childhood, with an incidence of 1 in 15000 live births . It may affect one eye (unilateral rb) or both (bilateral rb) during the first five years of life . Although extensive epidemiologic studies have been done to study this tumor, the results have been more often misinterpreted at the expense of mutation theory which has prevailed until recently in spite of the outstanding evidence against it . In the present review the authors analyze the most relevant epidemiological issues concerning retinoblastoma, in the light of recent developments highlighting the role of aneuploidy and genetic instability in the pathogenesis of this eye cancer . The most important studies to investigate the pathogenesis of retinoblastoma began with a paper published by knudson in 1971, when the author, after investigating the age distribution and laterality of a cohort of 48 rb patients, concluded that the disease could be inherited and formulated the so - called two - hit theory in order to explain its pathogenesis . In reality, no clues about the inheritance of retinoblastoma could be deducted from such a small sample . In fact, in his first report on this matter, knudson referred to earlier, smaller series showing that, in retinoblastoma survivors with bilateral disease, the proportion of affected offspring closely approximated 50%, as in dominant (mendelian) inheritance . From an original, mathematical analysis of the above data, knudson inferred that retinoblastoma is caused by two sequential (two hit) mutational events . According to this hypothesis, in the dominantly inherited form of the disease, one mutation is inherited via the germinal cells and the second spontaneously occurs in somatic cells of the retina and other tissues of the body . On the contrary, in the nonhereditary form the different timing and cell type involved by the two mutations determines the different clinical phenotype, with all bilateral and a minority of the unilateral cases being classified as hereditary, and the remaining unilateral cases being included in the sporadic group (table 1). During the following forty years of epidemiological, clinical, genetic, and biological research in this field, with the discovery of the rb1 as the prototype tumor suppressor gene, the medical establishment agreed on the pathogenetic two - hit theory which was further expanded by knudson, in many other scientific articles and review papers [624]. This generated the widespread conviction that rb is caused by two mutational events leading to the loss or inactivation of both alleles of the rb1 gene, as still believed by some authors . As mentioned above, the original input into the possible genetic derivation of retinoblastoma was based on limited evidence showing an apparently dominant mendelian distribution of the disease in the offspring of bilaterally affected individuals, thus allowing knudson to conclude that bilateral rb is inherited through the germ cells . Minimum or no disagreement had been appeared on this account in the literature during the last four decades . In an attempt to elucidate this issue, we have performed an analysis of the distribution of the disease in the offspring of unilaterally affected rb survivors, referred to the department of ophthalmology we discovered that in a total of 16 children born to 12 unilaterally affected patients, 8 (50%) were healthy and 8 (50%) affected with rb (table 2). Using the reasoning of knudson, it would be easily concluded that the unilateral disease phenotype is inherited and not sporadic, and this would be in sharp contrast with the current knowledge according to which bilateral rb is always hereditary and unilateral rb is almost always sporadic . It was reported by knudson and confirmed by others that about 10% of all rb cases do have a positive family history . In other words, the family history of the index case offers at least one other affected member, either a parent or another close relative . In this case, it is assumed that the events leading to the inactivation of the rb1 gene run in the family, and therefore the first hit is transmitted through the germline, exactly what happens in bilateral rb, but with one difference; bilateral rbs, which after knudson are all to be considered hereditary, are also assumed to have inherited the first hit through a mutation in one of the parent's germ cells, but they are the only affected members in their families . We should therefore be reasoning that, since familial rbs share the same pathogenetic mechanism with bilateral (hereditary) rb, the vast majority of these cases should show the bilateral phenotype . To be more accurate, we could make a calculation of the percentage of familial rbs carrying the unilateral phenotype . As a matter of fact, table 1 shows that the unilateral phenotype accounts for about 1/3 of all hereditary cases (or about 30%), and since familial rb represents the 10% of all rbs, it comes out that rbs carrying the unilateral phenotype, within the familial group, should not be more than 3% (i.e., the 30% of the 10%). As a matter of fact, a meta - analysis of a cohort of 3584 patients (table 3), reported by us elsewhere [4, 5], reveals that on a total of 344 (9.5%) familial cases, 83 (24%) show the unilateral phenotype, instead of the predicted 3%, a rather unexplainable figure, in the light of the predictions made by the in 1986, potluri and coworkers observed that the association of rb with the constitutional chromosome 13q deletion syndrome and the finding of 13q deletions or monosomy 13 in rb cells in individuals with normal constitutional karyotypes seemed to suggest that chromosome 13q could contain a gene responsible for tumor development in retinoblastoma . Although the authors themselves acknowledged that other chromosome abnormalities, in addition to those involving chromosome 13, are evident in retinoblastoma (additional copies of 1q material in 44% of cases, isochromosome 6p, in 45% of cases, monosomy 16, in 18% of cases, marker 1p+, in 13% of cases, and homogeneously staining regions and double minutes, in 9% of cases), further investigations on this matter stressed the role of 13q deletions in the genesis of rb, thus reinforcing the belief that the loss or inactivation of the rb1 was the only responsible for rb to develop . But it is well known that retinoblastoma is only one among many different tumors associated with deletions of chromosome 13 . Cancers linked with these deletions include chronic lympocytic leukaemia (cll), chronic myeloproliferative disorders, multiple myeloma, hepatocellular carcinoma (hcc), nasopharyngeal carcinoma, benign and low - grade malignant lipomatous tumors, bladder cancer, malignant mesothelioma, and prostate cancer . The same pleiomorphism in the phenotypic expression of cancers associated with rb1 gene mutations is therefore evident in 13q deletion syndrome . But the most important consideration to be made about the association of 13q deletion syndrome and rb concerns the evident discrepancy existing between the expected and the real number of bilateral tumors among the patients affected by this genetic disorder . As a matter of fact, the 13q deletion syndrome must be confirmed by the cytogenetic analysis of peripheral blood lymphocytes, and it is due to a deletion of the long arm of chromosome 13 which involves, by definition, the rb1 gene locus . Since the constitutional deletion of the rb1 gene can only be present if transmitted through the germ cells of one parent, it follows that all patients affected by 13q deletion syndrome and retinoblastoma belong to the hereditary group of knudson's and must, therefore, express the bilateral disease phenotype . It happens, however, that this assumption does not fit the clinical reality . In table 4 a series of 13 cases of 13q deletion syndrome and retinoblastoma referred to us over the last four decades is reported . Of these patients, only 4 had the bilateral disease phenotype, while the remaining 9 were unilaterally affected (table 4). The mean age at diagnosis in this group, which is about 10 months, further reinforces the assumption that they must belong to the hereditary group of knudson's, but the unilateral disease phenotype is unexplainably high . In the light of the two hit theory, the data summarized above do not have any rationalization, and the only plausible conclusion is that the assumptions made in regard to the role of the rb1 gene in retinoblastoma are incorrect . Clinical epidemiology is a leading discipline in the understanding of disease pathogenesis and etiology, but, as any other scientific endeavor, it relies on the correct interpretation of the available data . The proper analysis of data, in turn, relies not only on the individual researcher's skill but also on social, economic, and political environment in which the data are analyzed . The presumed genetic origin of rb and its relationship with the rb1 gene represent a clear example of how an entire body of prominent researchers may fail to question a flawed pathogenetic hypothesis (i.e., the two - hit theory), for the sake of personal, academic, or other interests . It was not by chance that we had to approach many different scientific journals to have access to the medical community about the role of aneuploidy and genomic instability in the genesis of rb [4, 5, 39]. We are still optimistic, however, because our alternative pathogenetic explanation has finally appeared in recent ophthalmologic literature.
The etiology underlying temporomandibular joint osteoarthritis (tmj - oa) is not fully understood; however, some contributing factors are known, including internal derangement, macrotrauma, and parafunctional habits.1 - 3 in addition, functional overloading appears to be an important step in the cascade of events leading to osteoarthritis of the tmj,1,4,5 which can lead to physical disruption of cells, impaired cellular function, transient ischemia of certain cell populations and the production of neurogenic irritants.4 as a result, the joint tissues collapse . If the joint collapse occurs in both tmjs, condylar resorption causes morphologic collapse of the tmjs and a subsequent decrease in ramus height, which results in progressive mandibular retrusion with anterior open bite.4 this is called acquired open bite associated with tmj - oa . The malocclusion and retrognathic facial profile associated with acquired open bite can be improved by orthognathic surgery, including maxillomandibular advancement with counterclockwise rotation . However, patients with active tmj disease and concomitant or resultant maxillofacial skeletal discrepancies who are treated with orthognathic surgery alone often have poor outcomes and significant relapse.6 - 8 this implies that patients with presurgical tmj symptoms requiring mandibular advancement appear to have an increased risk of condylar resorption.9 in addition, orthognathic surgery requires surgical invasion, which carries risks and can result in significant postoperative discomfort.10 however, no significant alternative treatment for these patients has yet been reported . Therefore, we suggest orthodontic treatment for patients with skeletal mandibular retrusion and anterior open bite due to tmj - oa, including molar intrusion, which has a beneficial effect on both esthetic appearance and occlusion . As a result of the counterclockwise rotation of the mandible caused by molar intrusion, the condyle is repositioned, and a functional adaptation in circumoral musculature can be achieved . Here, we present an adult case of acquired open bite associated with tmj - oa treated using miniscrew anchorage . A 46-year 9-month - old woman presented with mandibular retrusion and circumoral musculature strain on lip closure (figure 1). She had a vertical and horizontal open bite and severe crowding of the upper anterior teeth . The only occlusal contacts were at the bilateral molars; however, significant abrasion was detected on the anterior teeth and premolars . Although the mandibular midline was shifted 2.4 mm to the left, the maxillary midline was nearly aligned with the facial midline . The molar relationship was angle class iii on the left side due to a missing lower left second premolar, and angle class i on the right side . During mandibular excursive movement, molar guidance with balancing side contacts was detected . In her history, she had been conscious of her retropositioned mandible and chin; but during the past several years, the retrognathia increased . Model analysis showed an arch length discrepancy of -5.8 mm in the upper arch and -3.5 mm in the lower arch . Panoramic radiography showed the absence of the upper and lower third molars, and that the lower left second premolar had been extracted (figure 2). The lower left second molar had little or no alveolar bone support and was a floating tooth . Lateral transcranial radiography of the tmj showed that both condyles were located anterior to the glenoid fossa, and no translations of the condyles were induced during mouth opening . Cephalometric analysis revealed a skeletal class ii malocclusion with a severe retropositioned mandible (figures 2 and 3). The mandibular plane and gonial angles were significantly larger than the japanese norms (frankfort - mandibular angle, 52.7; gonial angle, 134.1).11 the mandible exhibited a backward and downward rotation with a short ramus . Although the inclination of the maxillary incisors was within the normal range, the lower incisors were labially inclined . The patient had experienced frequent tmj pain during mastication and at maximum mouth opening for at least 5 years . Maximum mouth opening without pain was 28.0 mm, and tmj crepitus was detected on both sides . The diagnosis was skeletal open bite with a short mandibular ramus associated with tmj - oa . The treatment objectives were to correct the anterior open bite, establish an ideal interincisal relationship, and to achieve an acceptable occlusion with a good functional class i occlusion . This case was treated according to the following plan: extraction of both maxillary first premolars and the mandibular right second premolar.placement of multi - bracket appliances on both dentitions to align the teeth.placement of titanium miniscrews in both sides of the posterior maxilla to intrude the molars, since molar intrusion should lead to subsequent counterclockwise mandibular rotation.use of a transpalatal arch to prevent buccal tipping of the maxillary molars during intrusion . Placement of titanium miniscrews in both sides of the posterior maxilla to intrude the molars, since molar intrusion should lead to subsequent counterclockwise mandibular rotation . Use of a transpalatal arch to prevent buccal tipping of the maxillary molars during intrusion . Although mandibular advancement with orthognathic surgery is considered an effective treatment method, surgical treatment is not strongly recommended for patients with progressive mandibular retrusion associated with tmj - oa . In addition, surgical treatment requires prolonged hospitalization and higher medical costs, and is the most invasive option . We did not want to close the anterior open bite by extruding the anterior teeth, because the vertical relationship between the incisors and jaws was considered acceptable prior to orthodontic treatment, and tooth extrusion is generally considered an unstable movement . Therefore, intrusion of the maxillary molars and subsequent counterclockwise rotation of the mandible were considered good options to treat the anterior open bite in this patient . A transpalatal arch was placed between the maxillary first molars, and both the upper first and the lower right second premolars were extracted . After the extraction, standard edgewise appliances with 0.018 0.025-inch slots were placed on both dentitions, except for the upper incisors (figure 4a). After leveling and alignment, titanium miniscrews (dual top anchor; jeil medical co., seoul, korea) were placed at the buccal sites of the posterior maxilla, and molar intrusion and canine retraction were initiated using elastic chains from the anchor screws . At this time, the brackets were bonded onto the upper incisors . At 1 year, 0.016 0.022-inch co - cr alloy wires were placed on both arches (figure 4b). As a result of molar intrusion, her anterior open bite was nearly corrected without the aid of vertical intermaxillary elastics . After intrusion, the canine and molar relationships were changed to almost class i. at 1 year and 6 months, the space closing process and anterior retraction continued with the use of elastic chains from the miniscrews (figure 4c). After 2 years and 7 months of orthodontic treatment, an acceptable occlusion was achieved without any recurrence of tmj symptoms, and the multi - bracket appliances were removed . Immediately after removal, lingual bonded retainers were fixed on both dentitions, and a wraparound retainer was added to the upper arch . An acceptable occlusion was achieved, and overjet and overbite were increased to 2.2 mm and -0.7 mm, respectively . The canine and molar anteroposterior relationships were improved to class i on both sides (figure 5). Lateral transcranial radiography of the tmj showed that both condyles were still anterior to the glenoid fossa, and that condylar movements during mouth opening were still poor . Cephalometric analysis revealed approximately 1.5-mm maxillary molar intrusion and subsequent counterclockwise rotation of the mandible (figure 7). Reduction of the excessive overjet was due to lingual inclination of the upper incisors . Throughout the treatment period, maximum mouth opening without pain was 38.0 mm; however, tmj crepitus was still detected on both sides . Magnetic resonance imaging taken after treatment showed anterior disc displacement without reduction in both tmjs, and the condylar head was only black in color, which indicated cortical bone without cancellous bone and bone marrow, suggesting an avascular necrosis - like structure (figure 8). Two years after retention, the mandibular position was nearly unchanged (figure 7). The circumoral musculature strain upon lip closure disappeared, and an acceptable occlusion was maintained without recurrence of tmj symptoms (figure 9). The canine and molar relationships remained class i, and no relapse of the anterior open bite was found . Panoramic radiographs showed little or no change in condylar structure, with condylar resorption and deformity (figure 10). Lateral transcranial radiography of the tmj showed that the condylar movements during mouth opening were still poor . Although successful outcomes have been reported for orthognathic surgical management of maxillofacial skeletal discrepancies with signs and symptoms of tmj disease,12 the outcomes depend on the pretreatment tmj condition . Orthognathic surgeries in patients with active tmj disease and concomitant or resultant maxillofacial skeletal discrepancies often have poor results and significant relapse.6 - 8,13,14 this implies that patients with tmj symptoms have an increased risk of condylar resorption . Symptomatic and asymptomatic pre - existing tmj pathologies that can lead to unfavorable outcomes include the following; internal derangements, progressive condylar resorption, condylar hyperplasia, osteochondroma, and congenital deformities.14 since the tmjs are the foundation of orthognathic surgery, the resultant pathology of tmj conditions with gross erosive changes in the articulating components of the fossa and condyle resulting in vertical height loss offers a poor base for maxillofacial skeletal and functional reconstruction . Furthermore, the degenerative and osteolytic changes in the joint components due to these conditions make these tmj components highly susceptible to failure under the new functional loading resulting from orthognathic surgical repositioning of the maxillofacial skeleton . Maxillomandibular advancement with counterclockwise rotation of the occlusal plane is an established procedure for patients with healthy tmjs . However, patients who had presurgical tmj symptoms and underwent orthognathic surgery alone had a statistically significant rate of skeletal relapse related to condylar remodeling and resorption . In addition, orthognathic surgery requires surgical invasion, which carries risks and can cause postoperative discomfort . Therefore, orthognathic surgery is not recommended for patients with progressive mandibular retrusion associated with tmj - oa . In the present case, the patient had an anterior open bite with a retropositioned mandible caused by severe condylar resorption and deformity, indicative of tmj - oa . Morphologic collapse of the joint component by tmj - oa induces a decrease in ramus height, leading to clockwise rotation of the mandible and an anterior open bite . The results of finite element model analysis showed that an open bite can induce greater tmj stress than normal occlusion.15 furthermore, clockwise rotation of the mandible, which is a major character of skeletal anterior open bite, leads to a synergistic increase in tmj stress during clenching.15 this suggests that improvement of mandibular clockwise rotation, which results in the reduction of tmj overloading, may be indispensable for treatment of acquired open bite associated with tmj - oa . However, it is nearly impossible to provide skeletal improvement in patients with anterior open bites using traditional orthodontics . Several recent studies have demonstrated effective treatment of anterior open bite patients with class i or ii jaw relationships using temporary anchorage devices (tads), which has now been established as a new treatment strategy.10,16 - 19 absolute molar intrusion, which was previously impossible with traditional orthodontic mechanics, using tads results in counterclockwise rotation of the mandible, and the reduced overbite is increased without incisal elongation . Kuroda et al.10,17,18 reported that although the mandibular plane was rotated more than 5 by molar intrusion, the patients had no functional problems after treatment . In addition, the procedure is definitely less invasive than a le fort i osteotomy for maxillary impaction with a mandibular repositioning osteotomy, and provides superior morphologic improvement over orthognathic surgery.18 therefore, absolute anchorage with tads was used in the present case . Superimposition of the cephalometric tracings before and after treatment showed counterclockwise rotation of the mandible due to upper molar intrusion . Because of this mandibular rotation, the mandibular plane angle was decreased by 2.3. the mandibular condyles also exhibited rotational repositioning in the inferior direction . Consequently, due to increases in the anterior and superior areas, the joint space became nearly uniform . It would be reasonable to assume that these changes in condylar position, if accomplished with optimal occlusal support, may lead to biomechanical equilibrium in the tmj . When tmj - oa is progressing, the condyle deformity is more prominent with a shorter ramus height . Although tmj - oa often has an unpredictable course, optimal condylar position and stable occlusion can achieve biomechanical equilibrium in the tmj, and this may inhibit progression of tmj - oa, and occasionally lead to functional and adaptive remodeling of the condyles through resorption repair.20 in the current case, there was no recurrence of tmj symptoms during the orthodontic treatment . Although anterior disc displacement without reduction and condylar resorption and deformity persisted after treatment, all symptoms of tmj disease disappeared, and long - term stability of both the occlusal and symptomatic states was obtained . Growing evidence suggests that in tmj - oa, similar to oa in other joints, overloading may initiate a series of degenerative changes, such as condylar resorption, decreased mandibular ramus height, mandibular clockwise rotation, progressive mandibular retrusion, and an anterior open bite . To date, many treatment modalities for tmj - oa have been reported; however, the treatment outcomes depend on the preoperative tmj conditions . Therefore, understanding the pathogenesis of tmj - oa and its current clinical treatment is essential for developing a " good as new " treatment for tmj - oa, including the orthodontic approach . We showed here that orthodontic correction through molar intrusion using titanium miniscrews was effective for the management of an open bite and clockwise - rotated mandible associated with tmj - oa and jaw deformity.
Fisher used the expression crash migraines to describe acute, high - intensity headaches similar to those caused by ruptured saccular aneurysms, but with normal lumbar puncture and angiography . Day and raskin coined the term thunderclap headache to refer to a similar type of headache occurring in a patient who had three severe, acute episodes of headache within 1 week . The brain ct scan and the cerebrospinal fluid (csf) examination were normal in this patient, while cerebral angiography showed diffuse, multifocal, and segmental cerebral vasospasm with a saccular aneurysm at the origin of the right posterior cerebral artery . The authors concluded that unruptured intracranial aneurysms can present with thunderclap headache and that angiography is necessary in patients presenting headache episodes with the thunderclap profile thunderclap headache (tch) is a severe and headache of the explosive type that appears suddenly, like a clap of thunder, with peak intensity of the pain occurring at onset (usually within 30 s). The international classification of headache disorders, second edition (ichd - ii, 2004) categorizes headaches into primary and secondary forms based on the absence or presence of intracranial lesions . This classification has included tch in group iv other primary headaches (code 4.6) (table 1) and has proposed precise diagnostic criteria .table 1 4 . Other primary headaches 4.1 primary stabbing headache 4.2 primary cough headache 4.3 primary exertional headache 4.4 primary headache associated with sexual activity 4.4.1 preorgasmic headache 4.4.2 orgasmic headache 4.5 hypnic headache 4.6 primary thunderclap headache 4.7 hemicrania continua 4.8 new daily - persistent headache (ndph) the nosography of most headaches included in group iv is not immediate . Although some of them are simply identified according to their trigger it is mandatory to accurately rule out any possible organic cause (that is to exclude a possible secondary origin) when a patient with one of these provoked headaches is seen for the first time . Indeed a recent publication shows that 18 of 30 patients with sexual headaches with an abrupt and severe headache which was similar to tch in most cases had reversible cerebral vasoconstriction syndrome (rcvs) (code 6.7.3 in ichd - ii). Thunderclap headache is a rare type of headache with an incidence of 43 cases per 100,000 adults per year . Primary tch closely mimics secondary forms of tch and therefore appropriate instrumental investigations are absolutely mandatory to rule out possible organic causes . The close similarity between primary and secondary tch has led to the hypothesis that the primary form may, in some cases, actually be a wrong diagnosis resulting from the incapacity of instrumental diagnostics to identify the organic cause; for this reason, the alternative term thunderclap headache of undefined origin has also been proposed . Some authors showed that patients with recurrent tch can be divided in two groups based on the result of mra: those with diffuse vasospasm and those without any (visible) vasospasm . However, both groups had similar clinical features and both showed the same rate of ischaemic complications [67% of posterior reversible encephalopathy syndromes (pres) and of ischaemic stroke]. These authors therefore suggested that primary tch and rcvs may be part of the same spectrum . . Showed that 21% of 67 patients with a proven rcvs had an initial normal mra and they would have been classified as primary tch in the absence of a second mra repeated after a few days later or of a conventional angiogram . Furthermore, other authors showed that the arterial abnormalities of rcvs, as assessed by non invasive tools, increase several days after tch onset . Indeed mean flow velocities of middle cerebral artery (mca) measured by transcranial doppler are maximal at 1825 days after tch onset, and mra vasoconstriction scores are maximal 16 10 days after headache onset, close to headache resolution at 16 9 days . Therefore, it is possible that some of primary tchs are actually forms of rcvs whose arterial abnormalities went undetected by neuroimaging . The clinical and diagnostic overlapping is also reflected in the pathophysiological field where the proposed mechanisms for the so - called primary tch are vasospasm and autonomic dysfunction, which are also thought to underline rcvs . A certain degree of overlapping between tch and rcvs also exists as regards treatment because nimodipine shows beneficial effect both in the presence and in the absence of vasospasm . Rcvs is a vascular disorder that is frequently, but by no means always, benign . Stroke occurs in 515% of the prospective series [4, 9] and death has also been reported . Thus, the clinical spectrum of rcvs is large and it ranges from isolated recurrent and self - limiting tch to life - threatening forms . Severe forms of rcvs were previously misdiagnosed as benign forms of primary angiitis of the central nervous system (pacns), with good evolution and prognosis, and normalization of arterial irregularities after a short course of steroids . Calabrese et al . First proposed that these patients had benign angiitis of the central nervous system (bacns) and later on recognized that bacns was equivalent to rcvs . Although headache can recur within the first week after onset, it generally does not recur regularly over subsequent weeks or months . As regards the pathophysiology of primary tch the proximal portions of the large intracranial arteries are indeed innervated by neuropeptide y and noradrenaline - containing sympathetic afferents, which modulate vascular tone [13, 14]. It has been suggested that the head pain in tch may be due to acute vasoconstriction or alterations in vascular tone secondary to heightened sympathetic tone, and indeed there exist experimental and clinical data supporting a pivotal role of the sympathetic nervous system (e.g. Tch attacks associated with hypertension or preceded by events associated with elevated sympathetic tone, such as episodes of anger, sexual intercourse, and exertion) [15, 16]. Vasoconstriction per se does not seem to be the cause of pain, since in the cases of tch with rcvs it is a long - lasting phenomenon, which may persist for hours up or weeks, even when headache has already disappeared . It seems more likely that pain in tch results from a combination of vasoconstriction with systemic or local autonomic changes . In patients with sexual headache resembling tch associated with rcvs, additional factors that may contribute to pain induction are possibly represented by the psychological stress associated with sexual arousal and by the activation of systemic autonomic reflexes, which result in increased blood pressure and heart rate . As regards the pathophysiology of the arterial spasm, several factors encompassing mechanical stimulation, biochemical mediators, and neurogenic events vasoactive substances, i.e. Ergotamine, amphetamine, cocaine may trigger it . Alternatively, the formation of circulating metabolites (i.e. During eclampsia or pheochromocytoma) or the exposure to toxins (i.e. Angiographic contrast material) are all possible causes for vasospasm . It is essential to remember that a diagnosis of primary or secondary tch can be made only when exhaustive instrumental investigations have been performed . To manage a patient with tch correctly, the clinician must be aware of all the organic disorders that can act as a causative factors . These are, first of all, subarachnoid haemorrhage (sah), but also cerebral venous sinus thrombosis, carotid artery dissection, hypertensive encephalopathy, spontaneous retroclival haematoma, sentinel headache, ischaemic stroke, pituitary apoplexy, spontaneous intracranial hypotension, colloid cyst of the third ventricle, intracranial infection, pacns . It is also worth noting that a headache with clinical features of tch has been described in conditions not associated with a structural abnormality, such as bathing headache, primary cough, sexual and exertional headaches, and rcvs . Recently, some authors have proposed diagnostic criteria for tch attributed to idiopathic reversible cerebral vasoconstriction (tharcv) syndrome based on the clinical and radiological features . Table 2 shows the diagnostic work up and the main criteria for the differential diagnosis of primary tch versus other disorders that can cause / be associated with tchs [1288].table 2differential diagnosis of tchdiseaseneurological symptoms / signsprecipitating factorscommentssubarachnoid haemorrhage (sah) [1932]headachephysical exertionabout 70% of pts with sah present with headache aloneloss of consciousnesssexual intercoursefocal neurological symptomscerebral venous sinus thrombosis (cvst) [3342]headachepuerperium1530% of pts present with isolated headache that can worsen in the recumbent positionaltered consciousnessdehydrationfocal neurological symptoms / signscancercervical artery dissection (cad) [3, 4345]headachehead and neck injurygenerally headache is ipsilateral to the cadamaurosis fugaxhorner s syndromefocal neurological symptoms / signsacute hypertensive crisis (ahc) [4651]headachehypertensive crisisheadache occurs in about 20% of pts with ahcaltered mental statusseizuresfocal neurological symptoms / signsspontaneous retroclival hematoma (srh) [52, 53]headachenonesrh is very raremild nuchal rigidityoculomotor nerve palsyupper limb paresissentinel headache (sh) headachephysical exertionsh is present in 1040% of pts with sahfocal neurological symptoms / signs generally absentsexual intercourseischaemic stroke (is) [5558]headachenoneheadache is more common with large isfocal neurological symptoms / signspituitary apoplexy (pa) [22, 5860]headachepregnancypa commonly occurs in pts with no known pituitary tumour historyvisual disturbancesspontaneous intracranial hypotension (sih) [6164]orthostatic headachevalsalva manoeuvretch is present, at onset, in about 15% of pts with sihhearing disturbancesmild nuchal rigiditycolloid cysts of third ventricle [7375]headachenoneheadache can be relieved by recumbencyloss of consciousnessseizurescomareversible cerebral vasoconstriction syndrome (rcvs) [8, 6973]headachepostpartumrcvs is spontaneous in about 30% of casesfocal neurological symptoms / signssexual intercoursedrugs exposure (see bottom of table)prognosis is uncertain, but most pts do wellblood products (see bottom of table)head traumaneurosurgical proceduresbenign hot bath - related headache [7478]headachehot bathheadache disappears spontaneously after a period of 2 weeks to 3 monthsnormal neurological examinationhot showerprimary cough, headachecoughthese headache forms aresexual and exertional headaches [3, 79]normal neurological examinationphysical exertionan exclusion diagnosissexual activityprimary angiitis of the central nervous system (pacns) [72, 8083]headachenoneheadache is the most common presenting symptomseizuresbehavioural disturbancesfocal neurological symptoms / signsprimary thunderclap headache (tch) [3, 1215, 24, 26, 8489]headachenonetch is an exclusion diagnosis and has a relatively benign prognosisnormal neurological examinationdrugs exposure phenylpropanolamine, ergotamine tartrate, methergine, bromocryptine, lisuride, tricyclic antidepressants, selective serotonin reuptake inhibitors, sumatriptan, isometheptine, cocaine, ecstasy, amphetamine derivatives, marijuana, lysergic acid diethylamide, tacrolimus (fk-506), cyclophosphamideblood products erythropoietin, intravenous immune globulin, and red blood cell transfusions differential diagnosis of tch drugs exposure phenylpropanolamine, ergotamine tartrate, methergine, bromocryptine, lisuride, tricyclic antidepressants, selective serotonin reuptake inhibitors, sumatriptan, isometheptine, cocaine, ecstasy, amphetamine derivatives, marijuana, lysergic acid diethylamide, tacrolimus (fk-506), cyclophosphamide blood products erythropoietin, intravenous immune globulin, and red blood cell transfusions while defining the diagnosis, tch must always be managed as a medical emergency in order to avoid potentially catastrophic consequences that can occur with secondary tch . Non - contrast brain ct is the first examination in this assessment, to be performed within the first 12 h after the onset of the headache, preferably using third - generation ct scanners that have a specificity of 98% and a sensitivity close to 100% . The sensitivity of ct for the detection of sah declines with increasing time from haemorrhage onset, falling to 86% on day one, 76% after 2 days, and 58% after 5 days . Therefore, although the sensitivity of ct scan in detecting sah is very high in the early phase, if we consider also the possibility of human error, which, in the case of sah, may be fatal, we recommend to perform lumbar puncture even in the presence of a normal brain ct . Csf assessment is definitely mandatory in patients who come to clinical attention 12 h after tch onset and have normal or non - diagnostic brain ct scans . Blood and csf work up (routine cell counts, measurement of protein, glucose, opening pressure, and inspection for xanthochromia) should be performed . Because visual inspection for xanthochromia is associated with a high rate of false - negative interpretations, spectrophotometry should be performed when available . Spectrophotometry also helps to overcome the problem of false - positives; analysis for bilirubin by spectrophotometry has a sensitivity close to 100% when lumbar puncture is performed between 12 h and 2 weeks after sah . Magnetic resonance imaging (mri), which can detect many of the possible causes of secondary tch, should be performed in all tch patients with normal or non - diagnostic ct scans and csf analysis . In most cases, magnetic resonance studies should include cerebral mri, magnetic resonance angiography, magnetic resonance venography and, if necessary, mri of the cervical arteries using the fat saturation technique . Ct angiography can be used instead of magnetic resonance angiography for the diagnosis of an intracranial aneurysm . Evidence suggests that conventional cerebral angiography is not necessary in the assessment of patients with tch, normal neurological examinations, and normal ct and lumbar puncture . Some authors believe that conventional cerebral angiography should be avoided, since the contrast medium could enhance the vasospasm and this, in turn, would increase the chance of a stroke or even cause further deterioration of a critical neurological condition . However, since there is no adequate evidence to conclude that conventional angiography can result in worsening vasoconstriction, in highly selected cases, when the clinical suspicion of intracranial aneurysm remains high despite normal or non - diagnostic ct, lumbar puncture, and mri studies, conventional angiography has to be considered . The following indications have been derived from the information collected from a systematic analysis of the international literature . We conducted a literature search covering the period 19322010, employing available electronic databases (national library of medicine, national institute of health, embase) with the following medical search terms: thunderclap, cough, exertional, exercise, orgasmic, sex, or abrupt in association with unfortunately, the literature contains very few reports on the treatment of primary tch and those that are available are mostly represented by single case reports ., a woman with an apparent primary tch had frequent recurrences of the headache until she reached (by day 14) a therapeutic dosage of gabapentin 600 mg three times a day . The exact mechanism of action through which gabapentin decreases headache pain is not known, although multiple mechanisms might be involved . Gabapentin enhances gaba - mediated inhibition, inhibits gaba metabolism and modulates l - type calcium channels by binding to their 2 subunit . Iv nimodipine and magnesium were administered to a 63-year - old woman with tch and vasospasm; a posterior ischaemic infarct had occurred before infusion . The patient s headaches resolved within hours, and transcranial doppler sonography revealed normalized mean cerebral blood flow velocity, suggesting relief of the vasospasm . The mean cerebral blood flow velocity increased again when oral nifedipine, another calcium channel blocker, was used in place of nimodipine . Nimodipine infusion effectively stopped tch in another woman, aged 58 years, who had vasospasm and posterior ischaemic infarct . Described 11 patients with primary tch (nine without vasospasm and two with vasospasm) treated with nimodipine . In eight of the nine patients without vasospasm, tch stopped within 24 h of oral nimodipine administration; the other patient had one further attack 2 days later . The highest dosage of nimodipine was 30 mg every 4 h in four patients, 45 mg every 4 h in one, and 60 mg every 4 h in four . In the two patients with arterial vasospasm, oral nimodipine was only temporarily effective and tch recurred . When iv nimodipine was given instead, tch subsided in 6 h without recurrence . In one of the patients, the dose infused was 2 mg / h; in the other, it was lower (0.51 mg / h) because of the appearance of nimodipine - induced hypotension . The iv route was switched to oral when mr angiography or transcranial doppler sonography no longer showed evidence of vasospasm . The duration of iv nimodipine treatment was variable, ranging from 5 to 10 days . No patient reported a relapse of tch during a mean 6-month follow - up (range 119 months) after nimodipine discontinuation . These reports suggest that iv nimodipine is the drug of choice for primary tch with cerebral vasospasm, whereas oral nimodipine can be used in patients without vasospasm, although the optimal dose and time window remain to be determined . It is noteworthy that the patients with tch associated with vasospasm described in the above reports [8789], the correct diagnosis should be rcvs rather than tch, while only the nine without vasospasm described by lu et al . Can technically be classified as having primary tch, although with a certain degree of approximation since one cannot exclude a rcvs without visible vasoconstriction at the time of angiogram . Therefore, these nine subjects can be regarded either as probable rcvs or probable primary tch . Following this line of reasoning, if we consider that, in some cases, rvcs may be missed by mra because of timing issues, it seems wise to avoid tricyclic antidepressants (e.g. Amitriptyline) and propranolol because they may facilitate the development of vasoconstriction as suggested by valenca et al . . In analogy, vasoconstrictor medications, such as ergots and triptans, should be contraindicate during the treatment of headache of patients with potential cerebral vasoconstriction syndromes, at least in the acute phase and until ongoing or impending rvcs has been ruled out . All patients with tch profiles must be assessed urgently and thoroughly in order to consider and rule out all the possible organic causes . After the exclusion of all secondary causes, including rcvs, there are very few patients left with true primary tch . The data gathered in recent years suggest that, in order to avoid false primary tchs, it is appropriate to perform neuroimaging studies (brain mri angiography or ct angiography) 34 weeks after the onset of all cases of tch with negative instrumental findings during the acute phase in order to exclude the presence of delayed vasospasm [10, 11] and therefore identify the true primary tchs . Once the diagnosis has been defined with certainty, secondary forms of tch must be managed through treatment of the underlying brain disorder . For primary tch, as well as for forms associated with rcvs, the therapeutic options are restricted to nimodipine, intravenously or orally administered, although gabapentin was reported effective in one case of primary tch.
Anterior cruciate ligament (acl) reconstruction has significantly advanced over the last few decades; however, some literatures have shown that up to 20% of patients still have persistent instability on functional testing and degenerative arthritic changes after traditional single - bundle acl reconstruction.12 these outcomes have led to an attempt at anatomic double - bundle acl reconstruction . There has been a significant increase in double - bundle acl reconstruction . Although double - bundle acl reconstruction has theoretical benefits, such as more accurate reproduction of acl anatomy, it is a more technically demanding surgery . Therefore, double - bundle acl reconstruction may cause additional problems such as graft failure, graft impingement, femoral condyle fracture, and tunnel expansion.3 as the incidence of double - bundle acl reconstruction surgery has increased, the number of failures and need for revision reconstruction have also increased . Generally, when performing revision acl reconstruction, there are numerous problems that are anticipated to revision acl reconstruction such as a tunnel malposition, tunnel widening, preexisting hardware and injuries to concomitant structures in the knee.4 therefore, the determination of the cause of failure and preoperative plan is the first step in obtaining a successful result after revision acl reconstruction . The techniques available to treat the failed acl knee include: graft and hardware removal, dealing with bone loss and tunnel expansion, the need for a staged procedure or concomitant surgery, anatomic placement of the bone tunnels, graft selection and fixation and rehabilitation . This report describes a case of a one stage single - bundle revision acl reconstruction after a failed double - bundle acl reconstruction with vertical and widened tunnels . A 23-year - old female, who is a professional dancer, sustained an acute acl rupture during dancing in september 2009 . She underwent double - bundle acl reconstruction with a tibialis anterior allograft in december 2009 at another hospital . After 18 months of the primary surgery, she was fully able to return to dancing . She had a re - trauma on the same knee while performing a basic dance step 6 months after returning to performance . Magnetic resonance imaging (mri) performed elsewhere revealed a bucket handle tear of the medial meniscus but the acl graft was still attached but loose . The patient underwent arthroscopic medial meniscus repair with an inside - out technique; however, she was unable to return to dancing due to several episodes of instability after the arthroscopic meniscus repair . When the patients sought medical attention at our institute after 3 months, examinations revealed a 1 + lachman test, 3 + pivot shift test, and 3 mm kt-2000 (medmetric, san diego, ca, usa) side - to - side differences . X - ray and computed tomographic examinations showed tunnel widening in the femur and tibia (anteromedial [am] femoral tunnel: 15.8 mm, posterolateral [pl] femoral tunnel: 7.5 mm, am tibial tunnel: 13.1 mm, and pl tibial tunnel: 8.1 mm) [figure 1]. Under arthroscopic evaluation, the acl graft was lax with a probe and situated in a grossly vertically oriented position [figure 2a]. After the removal of all remnant grafts, the footprint of the previous femoral tunnels was revealed . A new femoral tunnel was made in the 2 oclock position between the previous two femoral tunnels via the previous am tibial tunnel [figure 2b]. An impacted morselized bone graft with a cancellous allograft to the previous femoral tunnels was performed, and a new 10 mm femoral tunnel was created . Since the previous am tibial tunnel was too wider than the width of autograft with semitendinosus and gracilis tendon, a hybrid substitute with semitendinosus tendon autograft and an achilles tendon allograft were used . A dual fixation was performed with endo - button cl (smith and nephew, andover, ma, usa) and rigid fix (mitek, norwood, ma, usa) to get the strong fixation on femoral condyle with severe bone defect [figure 2c]. The tibial fixation was performed with a spike staple (smith and nephew, andover, ma, usa) and reinforced with a postie after impacting the bone graft to the previously widened tibial tunnel [figure 3]. She underwent the delayed postoperative rehabilitation to prevent the complication of the large bone defect on the femoral condyle . 3 weeks of postoperative immobilization with a locked brace in full extension and another 3 weeks of partial weight bearing with crutches, were allowed, and followed by a gradual increase in the knee range of motion up to 90. running was allowed at 4 months, and plyometric functional exercise was performed 8 months postoperatively . Computed tomography (a) coronal view (b) sagittal view showing tunnel widening before revision anterior cruciate ligament reconstruction an arthroscopic view showing (a) previous double - bundle graft, stretched both anteromedial and posterolateral graft . (b) new femoral tunnel was aiming at the 2 oclock position between the previous two tunnels . (c) a revision single - bundle graft after a morselized bone graft with previous two femoral tunnels postoperative x - ray of knee joint anteroposterior view showing femoral tunnels of previous double - bundle anterior cruciate ligament (acl) reconstruction and revision single - bundle acl reconstruction . A new femoral tunnel was made at the 2 oclock position between the previous two femoral tunnels . Anteromedial (am): tunnel for am bundle of previous double - bundle acl reconstruction . Posterolateral (pl): tunnel for pl bundle of previous double - bundle acl reconstruction . Single - bundle (sb): tunnel for revisional single - bundle acl reconstruction at 24 months postoperatively, a clinical examination revealed a negative lachman and pivot shift test, a kt-2000 side to side difference of 0 mm and full restoration of the range of motion . There were improvements in the lysholm knee score from 79 to 91, and the international knee documentation committee subjective score improved from 73 to 96 . A followup mri after 10 months of revision showed sound bony healing with an intact revision graft [figure 4]. Coronal and sagittal magnetic resonance imaging (mri) at 10 months followup showing incorporated bone graft and completely filling the previous femoral tunnel defect and intact graft the most important aspect to this case report was an uncommon single - bundle revision surgery after primary double - bundle acl reconstruction and a case dealing with the large previous bone defect in one stage surgery . Double - bundle acl reconstruction first proposed in the 1980's.5 zantop et al.6 reported an anatomic double - bundle acl reconstruction technique with two femoral and two tibial tunnels; however, the definite position for a femoral tunnel is under debate . Whilst double - bundle technique may improve the bundle appearance of the acl, it increases the operating time in both the number of tunnels to be drilled, the placement of the tunnels and the operative complexity of passing and securing the two grafts . Double - bundle acl reconstruction with an inaccurate femoral tunnel position leads to many problems, such as persistent instability and serious femoral and tibial tunnel widening,7 as with our case . Having two tunnels within the femur may leave larger bony voids within the lateral femoral condyle to fill during revision surgery . Tunnel widening also makes revision surgery difficult, which prevents patients from fully returning to their previous sports activity . Therefore, orthopedic surgeons must consider their surgical technique during a double - bundle acl reconstruction because this surgery is more technically demanding than the traditional single - bundle acl technique . In the present case, the two femoral tunnels were located anterosuperior to the true anatomic acl footprint, and the am bundle was too vertical . A malpositioned femoral tunnel after double - bundle acl reconstruction results in an acl graft failure and a two - femoral tunnel bony defect results in severe bone stock deficiency for revision surgery . Therefore, orthopedic surgeons must consider revision surgery problems resulting from increasing the number of tunnels; additionally, a particularly delicate surgical technique is required prior to double - bundle acl reconstruction . Firstly, several choice of grafts were introduced, including bptb, hamstring, or quadriceps autograft; and those allograft.89 secondly, in technical terms, shino et al.10 reported revision acl reconstruction with a rectangular tunnel technique and taketomi et al.11 presented three - dimensional fluoroscopic navigation guidance for femoral tunnel creation in revision acl reconstruction . It was also reported than revision acl graft fixation is not different that different form primary acl graft fixation like aperture fixation (interference screw and cross pins) and extraarticular fixation (cortical fixation devices, femoral loops and tibial cortical fixation).4 lastly, many published reports about outcome of revision acl reconstruction reported that an analysis of the results of acl revision was difficult and that the outcomes of acl revision were various because of the multifactorial nature of acl failure, the varied revision techniques and the concomitant procedures.12 additionally, there are many methods for managing bony deficiencies and malpositioned tunnels during a revision acl reconstruction and large defects often require a two - stage procedure . The initial procedure consists of graft removal, tunnel curettage and bone grafting and the second stage is the revision acl reconstruction.13 in our case, the primary acl reconstruction failed, so the patient was unable to return to her professional occupation for 4 years . We have performed one stage revision acl reconstruction using an impacted morselized bone allograft14 and this technique provides good clinical outcomes . In other words, during one stage acl revision surgery, it is critical to correct the previously failed tunnels with an impacted morselized bone graft and a dual fixation could be beneficial . The limitations of this study are that this is a single case report and a case series is needed in the future . To conclude, one stage revision single - bundle acl reconstruction with impacted morselized bone graft is one of the techniques for managing a failure after primary double - bundle acl reconstruction with considerable bone defect.
Electronic health records (ehrs) are increasingly used by medical providers and offer a wide - reaching source of information on utilization of preventive services . Numerous measures used for quality assessment and public reporting are estimated based on ehr data . However, sources of error and misclassification can lead to over- or underestimation of true utilization rates . Ehr - derived measures of screening test use are subject to error due to misclassification of screening and diagnostic tests . The implications of this misclassification for ehr - based screening utilization estimates have not been well explored . We calculated the bias in estimates of screening test utilization associated with several published ehr - based algorithms for identifying screening colonoscopies and propose two simple methods to correct this bias . We apply these corrections to obtain adjusted estimates of screening colonoscopy utilization using ehr data from group health, an integrated health care system in washington state . The bias in screening colonoscopy utilization estimates ranged from an underestimation of 3 percentage points to an overestimation of 12 percentage points across classification methods . If the operating characteristics of the classification method are known or if a statistical model that returns predicted probabilities of screening indication is applied in the population of interest, this information can be used to obtain unbiased estimates through simple corrections to the utilization rates with little loss of precision . When applied to data on colonoscopies received at group health, we found that an unadjusted estimate was 4 percentage points higher than our adjusted estimate . Error in classification of tests as screening when using ehr data to study screening utilization should be accounted for in order to eliminate bias and prevent spurious findings . Electronic health records (ehr) data, including administrative data used for billing and clinical medical records collected as part of patient care, are potential sources of information on a wide range of quality measures . These data include information on a broad patient population and facilitate research and quality assessment that is representative of care in community medical practice . Ehr data will become increasingly available and valuable as adoption of electronic systems expands, a process that has been accelerated by incentives for health it provided under the affordable care act . Rates of breast, cervical, and colorectal cancer screening are examples of measures of preventive services utilization that are commonly estimated using ehr data . For instance, cancer screening measures estimated via ehr data are included among the national committee on quality assurance s healthcare effectiveness data and information set (hedis) measures,1 and have also been tied to reimbursement through incentives mandated by the affordable care act and the centers for medicare and medicaid services (cms) hospital outpatient quality reporting program.2 estimating these measures using ehr data is preferable to self - report of screening test utilization due to the possibility of bias in estimates based on self - report.3,4 use of ehr data for evaluating screening utilization is also far more efficient than manual chart review . Despite the potential value of ehr data for estimating screening test utilization, obtaining accurate estimates is challenging for several reasons . For instance, the national quality forum (nqf) colorectal cancer screening measure is an estimate of the proportion of individuals ages 5075 years who have been screened with colonoscopy within the past 10 years, flexible sigmoidoscopy within the last 5 years, or fecal occult blood test within the past year.5 an accurate assessment of this outcome requires reliable measures of the number of individuals in the screening - eligible population and the number of individuals receiving screening with each of the tests of interest . Identifying individuals in the screening - eligible population is challenging because individuals may have recently entered the health system population, making it difficult to determine if they have pre - existing diagnoses that should result in their exclusion from the eligible population . Similarly, identifying screened individuals is challenging because new enrollees may have been screened prior to enrollment in the health system . Additionally, there are no unique codes for screening as distinguished from diagnostic colonoscopy . Including diagnostic colonoscopies in estimates of screening utilization this may obscure important information about access to and utilization of cancer screening services and may produce biased or spurious findings regarding disparities in screening if there is differential misclassification across patient subgroups . The extent of the error in estimates of cancer screening utilization based on ehr data attributable to misclassification of screening indication depends on the operating characteristics of methods used to classify cancer screening tests . Several previous studies have estimated the operating characteristics of approaches to distinguishing screening and diagnostic tests using administrative ehr data.68 while these studies have focused on the use of administrative ehr data, similar considerations apply to studies using data from clinical encounters, if the classification approaches in question produce measures of screening indication that have errors . In the context of breast cancer screening, several algorithms have been developed for classifying mammograms as screening or diagnostics using diagnosis and procedure codes from claims data.6,9,10 a number of algorithms have also been developed for identifying screening colonoscopies based on ehr data with varying operating characteristics.7,1113 the sensitivity of these approaches ranges from 70 to 90 percent and specificity from 60 to 90 percent . A natural question arises: how might estimates of screening test utilization based on ehr data be influenced by misclassification of screening and diagnostic examinations? In this paper we explore the implications of using ehr data with imperfect information on screening indication to estimate rates of screening test utilization . We use numerical examples and simulations focusing on screening colonoscopy to demonstrate the magnitude of bias that can be induced by using an imperfect measure of screening indication . We then demonstrate via simulation studies how to correct this bias using information on the operating characteristics of the algorithm used to assign screening indication or a predicted probability that the test was performed for screening purposes . Finally, we demonstrate the application of unadjusted and adjusted measures of screening colonoscopy utilization using ehr data from group health, an integrated health plan and health care delivery system in washington state . To illustrate the bias arising from imperfect ascertainment of screening - test utilization we introduce the following notation . Let y represent the true classification of the test with y = 1 indicating a screening test and y = 0 indicating a diagnostic test . We assume that y is unobserved in ehr data but that a proxy is available, denoted y. for instance, in the case of colonoscopy, procedure codes do not distinguish colonoscopy performed for screening versus colonoscopy performed to evaluate signs or symptoms (i.e., a diagnostic exam). However, by applying existing algorithms to administrative data it is possible to obtain a predicted probability of screening indication that can then be dichotomized to yield an indicator that the examination was a screening test . In this example the operating characteristics of y are the sensitivity, se = p(y=1\|y=1), and specificity, sp= p(y=0\|y=0). In studying utilization of cancer screening tests, our objective is to estimate the proportion of the population receiving a screening test, p(y=1). If we use instead p(y=1) as a measure of screening utilization, the difference, p(y=1) we can express this bias as a function of the sensitivity and specificity of y, p(y=1) p(y=1) = se it can be easily seen that for a test with perfect operating characteristics (sensitivity and specificity equal to 1), p(y=1) will be unbiased . The relative impact of sensitivity and specificity on bias will depend on p(y = 1), which is unknown in practical applications . In table 1, we illustrate the bias in estimates of screening - colonoscopy utilization when using three different algorithms for identifying screening examinations . Previously, self - report data have been used to estimate screening - colonoscopy utilization.14,15 however, ehr data offer the opportunity to evaluate utilization in a broad population without the bias inherent in self - report . Motivated by estimates of lower endoscopy utilization,15 we assume that the true proportion of individuals who have been screened with colonoscopy in the past 10 years is 58.5 percent . In this setting, bias in screening - colonoscopy utilization ranges from underestimation by 5.7 percentage points to overestimation by 11.6 percentage points . In this section we present two methods for correcting the bias in estimates of screening utilization based on imperfect ehr - based algorithms for assigning screening indication . The first relies on the existence of validation studies that have established the operating characteristics of the classification algorithm in the population of interest . We can use the known sensitivity and specificity of the algorithm for classifying tests as screening to correct estimates through direct algebraic manipulation of the formula for bias, providing a simple approach for obtaining unbiased utilization estimates . Specifically, a corrected formula for screening - test utilization is given by (p(y=1)+sp1)/(se+sp1). Utilization is first estimated using algorithm - assigned screening indication, providing an estimate of p(y=1). This estimate and the estimated sensitivity and specificity of the algorithm are then substituted into the expression above to produce a bias - corrected estimate of p(y=1). The second approach to correcting estimates of screening utilization requires an algorithm that assigns individual - level probabilities of screening indication . Some methods for identifying screening tests return a predicted probability that a test was used for screening purposes . For instance, in the context of colorectal cancer screening, a lasso algorithm has been proposed that provides the probability that a given colonoscopy was a screening test.8 lasso is a statistical prediction model that combines variable selection and parameter estimation under a constraint on the size of the regression coefficients.16 assuming that these probabilities are well calibrated, summing the predicted probabilities of screening indication in the patient population of interest provides an unbiased estimate of the number of screening tests performed in the population . A well - calibrated probability implies that the proportion of tests with screening indication is equal to the predicted probability that a test has screening indication . For instance, among all tests with predicted probability of screening indication equal to 0.2, 20 percent will truly be screening tests if the prediction model is well calibrated . If the algorithm was developed in a different population, it may not be well calibrated and might over- or underestimate the probability of screening indication in a new population . Given a well - calibrated predicted probability, the utilization rate can be estimated without bias by taking the mean of the predicted probabilities in the population of interest . To demonstrate the bias and precision resulting from using uncorrected classifications of screening indication to estimate utilization as well as performance of the two proposed approaches to bias correction, we conducted a simulation study motivated by the context of screening with colonoscopy . In this study we assumed a population of size 10,000 and that 59 percent of this population were screened at least once over a 10-year period . We assumed that predicted probabilities of screening indication in this population arose from a beta(0.25, 0.17) distribution . This distribution implies that the cutpoint maximizing the average of sensitivity and specificity has a sensitivity of 0.89 and a specificity of 0.90 . These parameter values were selected to create a scenario similar to the adams algorithm for classifying screening colonos - copies.8 for each simulated individual, both the true classification of the test as screening or diagnostic and the algorithm - assigned predicted probability were known . We first computed an uncorrected utilization estimate by dichotomizing the predicted probability at the value maximizing the average of sensitivity and specificity and then computing the proportion of individuals classified as having received a screening examination . We then computed the two bias - corrected estimates, first by applying the correction factor presented above and then by computing the mean of the predicted probabilities . We repeated this process across 10,000 simulated populations and plotted the distribution of screening - test utilization estimates provided by each approach in figure 1 . In these simulated populations, the approach directly using the algorithm - assigned classification without adjustment underestimated screening utilization by 2.5 percentage points . Both of the bias - corrected approaches had a bias of less than 0.01 percentage points . The uncorrected method had a standard error of 0.5, the direct adjustment method of 0.6, and the mean probability method of 0.4 percentage points . To demonstrate the application of an adjusted utilization approach in a real data set derived from an ehr, we used data on receipt of colonoscopy from group health, an integrated health plan and health care delivery system in washington state . Because sensitivity and specificity in this population are unknown we used the mean probability method to obtain adjusted estimates . We constructed a sample consisting of members ages 50 years or older who were continuously enrolled in group health for at least 5 years between 2002 and 2012 with no prior diagnosis of colorectal cancer . In this population, we estimated the proportion of members receiving a screening colonoscopy at least once during a 5-year period . We used current procedural terminology (cpt) codes (45355, 4537845387, 45391, 45392), healthcare common procedure coding system (hcpcs) codes (g0105, g0121) and international classification of diseases ninth revision, clinical modification (icd-9-cm) codes (45.2145.24, 45.25, 45.42, 45.43) to identify each colonoscopy received by a member of the denominator population . We then applied the algorithm of adams8 to each colonoscopy to obtain a predicted probability that the colonoscopy was a screening examination . If an individual received more than one colonoscopy during this time frame we retained the colonoscopy with the highest probability of screening indication we first dichotomized the predicted probabilities at a threshold of 0.261, the threshold maximizing the average of sensitivity and specificity in a validation study,8 to obtain a binary indicator of receipt of screening colonoscopy . We then computed an unadjusted measure corresponding to the proportion of individuals with a screening colonoscopy based on this binary indicator . Finally, we computed an adjusted measure by taking the mean of the predicted probabilities . This approach is appropriate for accounting for misclassification in identification of screening tests when the sensitivity and specificity of the algorithm are unknown in the target population . In this population of 139,163 individuals, we compare this to an estimate of 7.8 percent based on averaging the predicted probabilities . Thus, the unadjusted approach appears to substantially overestimate screening - colonoscopy utilization in this population . Estimates of screening - test utilization based on algorithms for assigning screening indication with imperfect operating characteristics will be biased . This bias leads to under- or overestimation of the proportion of the population making use of a screening test . In the case of underestimation this could lead to unnecessary efforts to improve utilization, and in the case of overestimation this could lead to failure to address the needs of underserved populations . The magnitude and direction of the bias depend on the sensitivity and specificity of the ehr - based algorithm as well as the prevalence of screening utilization in the population of interest . We have demonstrated two simple approaches to correcting bias, which can easily be applied to ehr data, and implemented an adjusted approach using ehr data from group health . These methods require that either the operating characteristics of the algorithm are known based on a prior validation study or that the algorithm returns predicted probabilities of screening indication and is well calibrated in the population under study . We recommend that one of these two approaches be used to correct utilization estimates whenever possible . Use of these approaches is particularly well suited to ehr data because the depth of information available via data from clinical encounters facilitates validation studies for evaluation of the discrimination and calibration of the measure in a small sample of the target population . The measure and appropriate methods for correcting for misclassification can then be rapidly applied to a broad population using administrative ehr data . In this study, we focused on estimation of screening - test utilization when screening indication is imperfectly ascertained . However, there are a variety of other uses for ehr - derived data on cancer screening that rely on accurate ascertainment of test indications . For instance, estimates of screening - test effectiveness including the cancer stage at diagnosis and the mortality among screened and unscreened individuals may be of interest . Misclassification of screening and diagnostic tests will bias these measures of screening - test effectiveness . Several previous studies have discussed considerations for using algorithm - assigned outcomes and exposures in studies based on ehr data.17,18 these provide guidance on the relationship between algorithm operating characteristics and bias in various measures of effectiveness and can be used in conjunction with the current study to guide the choice of algorithm and analysis strategy when using data with known error in screening indication . Calibration and discrimination of the ehr - based measure are key to obtaining accurate estimates . If the measure is not well calibrated all three of the approaches discussed in this paper will return biased estimates . We recommend using a validation sample, in which both clinical and administrative data are available, to evaluate the calibration of any screening indication algorithm before it is applied broadly to ehr data . Additionally, an algorithm with poor discrimination (sensitivity and specificity) will lead to biased estimates and may inflate the standard errors of the estimates . The direct adjustment method, in particular, will have inflated standard errors if its denominator, se + sp 1, is close to 0 . This will occur in the case of a measure where the sum of sensitivity and specificity is close to 1, which would occur, for instance, if both cases and controls are correctly classified only about 50 percent of the time . If each site uses a different approach to classifying screening indication or if a common approach is used that has differing operating characteristics across study sites, then differential bias may lead to the spurious appearance of variability in screening utilization across sites . If patient characteristics differ across sites this could also lead to the appearance of associations between patient characteristics and utilization that are entirely attributable to bias . In the case of a multisite study the classification approach should be validated at each study site allowing for bias correction at the study site level . Similarly, if operating characteristics vary across patient subgroups this could result in apparent variation in utilization across subgroups where none truly exists or, conversely, might obscure true variation in utilization . To protect against this bias, validation studies within patient subgroups of interest must be conducted to provide estimates of operating characteristics within each population or to demonstrate that algorithm - assigned probabilities are well calibrated in each group . As use of electronic medical records expands, ehr data will become increasingly valuable as a source of information on screening test utilization . The considerations provided here allow for evaluation of the bias that will occur if algorithms for screening indication are used without correction for potential misclassification of screening tests as diagnostic tests . We have provided simple approaches to correct this bias that can be readily implemented and have demonstrated their potential for eliminating bias without inflating standard errors . The need to classify tests as screening or diagnostic is not unique to the setting of cancer screening using ehr data but exists across a broad range of prevention measures including use of tests for depression, high cholesterol, and osteoporosis . Moreover, the considerations described here do not apply solely to misclassification of diagnostic and screening tests but are relevant broadly to measures that rely on imperfect ascertainment of the service of interest . Error in ascertainment of utilization due to misclassification should be considered and appropriate adjustment methods such as those proposed in this paper should be applied to avoid bias.
Corynebacterium urealyticum, a gram - positive bacillus, has been identified as the most frequent causative pathogen . Urinary stasis is highly likely to contribute to stone formation, because large - scale spontaneous precipitation of crystals occurs when a critical concentration (e.g., supersaturation) is exceeded . Failed treatment of encrusted pyelitis in a transplanted kidney can cause graft failure or nephrectomy . To overcome urinary stasis with encrusted pyelitis in a transplanted kidney, we report surgical treatment by ileo - pelvic anastomosis without nephrectomy . In addition, in this case, augmentation cystoplasty was performed to strengthen the atrophied bladder following tuberculosis . A 51-year - old female patient received a kidney from her sister after a double nephrectomy owing to renal tuberculosis 12 years previously . At that time, the transplanted ureter was anastomosed to the ileum in the left lower abdomen with an ileal conduit on the opposite side . The patient experienced no problems for 12 years . At a routine checkup, she complained of abdominal pain, gross hematuria, and increased debris in the ileal conduit . Her serum creatinine was elevated to 1.7 mg / dl (normal, 0.6 to 1.1 mg / dl), and her transplanted kidney developed hydronephrosis with calcified debris observed by sonography (fig . Neither hydronephrosis nor calcified debris had been observed 6 months before in a routine outpatient checkup . Abdominopelvic computed tomography revealed hydronephrosis with encrusted pyelitis and ureteritis in the transplanted kidney (fig . A cystogram showed that the bladder was not refluxed but had a small capacity (50 ml). After 3 weeks, we operated to anastomose the transplanted renal pelvis to one end of the ileal conduit, performing augmentation cystoplasty with new ileum and to anastomosis the other end of the remnant ileum with the augmented bladder . Augmentation cystoplasty is a reconstructive technique for creating a compliant, large - capacity urinary storage unit to protect the upper urinary tract . The operation was performed via a midline incision . Because it was difficult to approach the major calices of the transplanted kidney in the left pelvic rim, we anastomosed one end of the ileal conduit to the pelvis of the transplanted kidney in an end - to - end fashion . The ileal conduit was lengthened by 30 cm to extend from the pelvis to the augmented bladder chimney . After exposure of the urinary bladder, a transverse wide v - shaped cystostomy incision was created, with the apex approaching the anterior bladder neck and the base extending posteriorly past the midcoronal plane of the bladder dome . A 20 cm segment of new ileum at least 15 cm proximal to the ileocecal junction was used for ileocystoplasty . For the small bowel, a v - shaped plate was created by a side - to - side anastomosis with 2 - 0 vicryl . A circumferential, continuous, full - thickness, single layer anastomosis of the bowel to the bladder was started posteriorly by using 2 - 0 braided absorbable sutures . The other end of the ileal conduit was anastomosed through small separate ileotomies (1.5 cm length) in the limb of the augmentation cystoplasty . A follow - up cystogram showed increased bladder capacity to approximately 300 ml with well - preserved end - to - end anastomosis of the ileo - pelvic portion with the other end of one chimney of augmentation cystoplasty with the elongated ileal conduit (figs . 3, 4). The most common causative agent in the formation of infection stones is a urease - positive urinary tract infection . Less invasive methods are preferable to preserve the connection between the renal pelvis and the upper ureter . Ileo - pelvic anastomosis must be adjusted for patients with infection stones in the upper urinary tract after renal transplantation . An alternative way to prevent these infection stones is to ensure a wider pathway through a ureter that has been narrowed in places by these stones . According to a report on the incidence of urolithiasis in cystectomy patients, 5 of 78 patients who had undergone diversion with an intestinal conduit developed urolithiasis, all in the upper tract . In contrast, 4 of 78 patients who had received a diversion had a renal stone at the time of presentation . Refluxing urine may contribute to an increased risk of stone formation after urinary diversion, whereas pouch stasis may contribute to stone formation in the diversion group . A case of failed treatment of encrustation in a kidney transplant recipient was reported and graft removal was performed after 6 months of failed management owing to intractable febrile urinary tract infections that became life threatening . They excised renal parenchyma from the caudal portion of the inferior calyx, which was then anastomosed to the ileostomy . In their case, bladder capacity was within the normal limit; thus, augmentation cystoplasty was not indicated . In conclusion, if urinary tract infection is controlled, we suggest that ileo - pelvic anastomosis combined with augmentation cystoplasty is an alternative for the treatment of encrusted pyelitis with atrophied bladder . In our patient, we performed ileo - pelvic anastomosis to prevent urinary stasis by elongating the ileal conduit . In addition, the augmentation cystoplasty technique is useful for increasing bladder capacity that has been diminished by renal tuberculosis (fig.
Laparoscopic surgery (ls) for both benign and neoplastic colonic disease has become a standard procedure worldwide [18], although its distribution is currently limited . Many authors reported adequacy and short - term benefits also for laparoscopic rectal procedures [1013]; nevertheless, large randomized studies and oncologic results are still lacking . In recent years, innovative endoscopic procedures such as single - port laparoscopic surgery (sils), natural orifices transluminal endoscopic surgery (notes), and needlescopic surgery (ns) have been introduced to further reduce surgical invasiveness and abdominal wall trauma . This goal has been achieved by reducing the number of ports (sils), avoiding transabdominal incisions (notes), or reducing port size (ns). This should possibly reduce postoperative pain and lower the incidence of wound infections and port site hernias, besides improving cosmetic results . Notes has been performed mainly on experimental models [17, 18], and its application in clinical environment is very limited [19, 20]. Several attempts with single - port technique have been made for various procedures, including appendectomy, cholecystectomy, splenectomy, inguinal hernia repair, and in paediatric, gynaecologic, and urologic surgery; few preliminary experiences are available also for colorectal surgery [2846]. Likewise, ns has been gradually introduced in the aforementioned surgical fields, with some preliminary results also in colorectal surgery [4755]. The main drawback of sils is the loss of triangulation of surgical instruments in the operative field, which despite recent development of curved instruments and flexible endoscopes enhances technical difficulty and requires a long learning curve . Needlescopic technique keeps port positioning unchanged compared to standard laparoscopic procedures and therefore has minimal impact on the surgeon . Since reports are limited in this field, we aim to review technical points such as instrumentation and its use in the different steps of the operation . In our practice, laparoscopic colorectal resections are currently performed with a 3- to 5-port (512 mm size) technique, intracorporeal anastomosis whenever possible, and specimen extraction through a suprapubic transverse incision . Laparoscopic instrumentation consists of 30 scope, atraumatic graspers, coagulating hook, bipolar grasper, clip applier, ultrasonic dissector (optional), suction device, retractor, needle holder, and linear stapler . Apart from the clip applier, the ultrasonic dissector, and the stapler, all instruments are available in 3 mm size (figure 1) still keeping a high standard of quality and performance . Only 3 mm laparoscopes, although providing a good vision, are still less performant than 5 mm hd scopes which may be preferable in advanced laparoscopic procedures . Since a minilaparotomy is always planned, open access with a hasson port may be performed at the suprapubic site allowing introduction of 1012 mm devices . Further trocars ranging from 3 to 5 mm size are placed after insufflation under direct vision . Port positioning for minilaparoscopic left colectomy is shown in figure 2 . After placement of the 12 mm hasson port at the site of the planned minilaparotomy, one 5 mm port is inserted through the umbilicus for the scope, and two 3 mm ports are placed in the right hypochondrium on the midclavicular line and in the right lower quadrant . Such position allows good triangulation in order to work between the left hypochondrium and the pelvis . An additional 3 mm port may be placed in the left lower quadrant for the surgeon to switch hands and improve triangulation during mobilization of the splenic flexure or dissection of the lower rectum . When in place, this port may be used by the assistant for additional grasping or to expose the operative field with a retractor when working in the pelvis . A standard medial to lateral approach clips for vascular ligation are inserted through the 12 mm suprapubic port (figure 3). Mobilization of the splenic flexure may be performed indifferently as a first step or before bowel section . Dissection is performed with the 3 mm coagulating hook; should the ultrasonic dissector be used, the 3 mm port in the right and/or left lower quadrant is to be replaced with a 5 mm port . Three mm instruments allow fine grasping of elements such as vessels and peritoneum, but care must be taken during lifting of the mesocolon as the small contact surface may result in the tearing of the vessels which need to be preserved; it is therefore advisable to interpone a sponge (inserted through the 12 mm port) between the grasper and the tissue to be handled . Similarly, since mesorectal integrity is of utmost importance during total mesorectal excision in rectal cancer surgery, grasping of the mesorectal fascia with small instruments is to be avoided, and a wad of gauze held by the grasper should be used to expose the holy plane (figure 4). If a stronger retraction is needed to achieve dissection of the lower rectum or in case of bulky tumours in obese patients, a 10 mm retractor may be introduced through the 12 mm suprapubic port . The same port is used to place the linear stapler and transect the rectum at any level down to the pelvic floor (figure 5). After specimen retrieval, the suprapubic minilaparotomy is closed leaving in place the 12 mm port which may be useful for extraction of the staple trocar, anterior retraction during confection of low colorectal anastomosis, and introduction of sutures if the peritoneum is to be closed . Alternatively, the suprapubic minilaparotomy may be performed as a first step of the operation and sealed temporarily with a device which allows air - tight placement of a 12 mm port . At the end of the procedure, the ports are removed under vision to check eventual bleeding, and the 12 mm port is extracted at last . The 12 mm hasson port is inserted above the pubis using the open technique, and two additional ports are placed under vision: one 5 mm port is placed in the left lower quadrant for the introduction of the scope and one 3 mm port in the left hypochondrium on the midclavicular line . Such position allows good triangulation when working in the right abdomen and on the middle transverse colon . The use of the ultrasonic dissector requires a 5 mm port in the left upper quadrant . An optional 3 mm port may be placed in the right hypochondrium to allow grasping and retraction by the assistant (figures 6 and 7). The clip applier and linear stapler are introduced through the 12 mm port . After completing the mobilization and the bowel transaction, a double enterotomy is performed in the distal ileum and transverse colon, and a stapled side - to - side isoperistaltic anastomosis is performed . Due to the direction of the linear stapler introduced through the suprapubic port, the visceral stumps must be correctly oriented using one or two traction sutures held by graspers . The anastomosis is completed with a running suture, and the ileal mesentery and transverse mesocolon are approximated . Five and 3 mm ports are retrieved under vision, and the specimen is extracted via a suprapubic incision . Laparoscopy has been widely proven to be a feasible, safe, and effective technique to perform colorectal resections [1, 2, 5661] leading to clinically relevant advantages in selected patients such as reduction of postoperative pain [1, 62] and complications, shortening hospital stay and improving recovery [1, 58, 63], wound healing [1, 64], and cosmesis [65, 66]. Moreover, minimally invasive surgery has facilitated the application of enhanced recovery programs in colorectal surgery [6769]. Long - term outcome of laparoscopic colonic resection for cancer is not different from what has been achieved by open surgery procedures . Therefore, some authors suggest that laparoscopy should be the preferred technique to perform colectomy in patients suitable for this approach . New trends have been developed in order to further reduce the impact of surgical procedure in patients undergoing colorectal resections . Three main directions have been undertaken in specialized centres: sils, which aims to the reduction of port number, notes, in which surgical instruments are inserted in hollow organs trough natural openings, and minilaparoscopic colorectal surgery, based on reduction of port size . Sils was first described by piskun and rajpal for cholecystectomy as early as 1999; this term currently identifies surgical procedures that provide the placement of one port having three or more working channels within the umbilicus . Surgeons who perform single - port colorectal surgery seem to agree that this technique, though should be suitable for the resection of colon cancer with respect to oncologic principles, is demanding because of the difficulties of exposure of the operative field and because of the risk of crowding while maneuvering laparoscopic instruments, although specially designed for this purpose . : this term currently identifies surgical procedures that provide the placement of flexible endoscopic systems through natural orifices (per - oral, transvaginal, transanal, transumbilical, or transvesical routes) entering the peritoneal cavity through an incision of hollow organs and approaching target organs to perform intra - abdominal procedures . Many procedures ranging in complexity from cholecystectomy to colorectal resections may be theoretically performed entirely endoscopically without the need for abdominal incisions [70, 71]. The advantages of such an approach include absence of incisional pain and wound complications (including infection and hernias), improved cosmetic results, and faster recovery . Although studies have shown the feasibility of an notes approach, significant constraints have been identified with the use of a flexible endoscopy platform, including a relative inability to apply off - axis forces, mechanical stability, inadequate triangulation, and limits in passing multiple instruments simultaneously into the peritoneal cavity . Concerns have also been expressed about the risk of postoperative leak and infections: with the intestinal closure systems currently adopted for notes access sites, it is doubtful that 100% safety can be achieved . At present, the need for improved technology remains a major limitation for sils and notes . The use of smaller ports to perform laparoscopic procedures is defined with different terms such as minilaparoscopy, microlaparoscopy, miniendoscopic or microendoscopic surgery, and microinvasive surgery . In general, ns is the term used to describe ls with instruments with an external diameter of 2 - 3 mm, as defined by gagner and garcia - ruiz . Santoro et al . Have defined miniendoscopic surgery as any procedure that uses endoscopic instruments and optics 5 mm in diameter or smaller . Needlescopic colorectal surgery is feasible, effective, and easy to perform since no specific training is required . Surgeons who experienced ns in the aforementioned surgical fields [4755] report several advantages over standard ls . In general, reduction of laparoscopic port size is associated with limited trauma on the abdominal wall . Smaller incisions result in decreased incisional pain and reduced risk of complications such as port - site bleeding, infection, and herniation . Moreover, minimal scarring allows better cosmetic results . On the other hand, narrow operative field, lower image quality due to lack of definition and reduced light transmission [16, 74], and blurred vision with the use of electrocautery achilles' heel of this technique and cause more stress for the surgeon especially when using 3 mm scopes . The use of modern 5 mm optics with high - definition cameras and powerful light sources is much more comfortable in performing advanced laparoscopic procedures, though a 3 mm optic inserted through an ancillary port may be useful if the 5 mm port is to be used for a larger instrument such as the clip applier . As for smaller instruments, they may show a weaker grasping capability and a lack of tensile strength due to increased flexibility, particularly in the presence of fibrosis or inflammation . Manipulation of tiny laparoscopic instruments may result in an increased risk of tissue damage during dissection [16, 74, 7679]. Apart from these precautions, moving from standard laparoscopic technique to needlescopic colorectal resections is not to be considered as approaching a new technique but simply an adaptation of a well - established practice and does not require a long learning curve . None of the steps of the operation has shown difficulties resulting from the use of miniaturized instruments . A good exposition of the surgical field has been always achieved during vessel ligation and viscera dissection, transection, and anastomosis . Building on the experience gained from needlescopic procedures such as cholecystectomy and appendectomy, we decided not to give up the greater definition provided by 5 mm scopes, since the 3 mm optics are still less performant for more advanced and complex procedures . The 3 mm grasper has been shown to provide good traction, also during gentle dissection . We used a simple trick to overcome its aforementioned limits: a wad of gauze held within the jaws of the instrument itself was used for lifting and retracting viscera in order to increase its strength and decrease the risk of injury of other organs . One aspect that has been reconsidered performing needlescopic colorectal surgery is the position of trocars: we thought it would be logical to incorporate the only 12 mm port that must necessarily be placed for the introduction of the stapler in the minilaparotomy which is generally a transverse suprapubic incision; we therefore started introducing the stapler from a suprapubic port not only for low rectal resection but also to transect the upper rectum and transverse colon . The use of the stapler from the suprapubic port did not result in substantial differences in bowel transection . Nevertheless, performing an intracorporeal side - to - side mechanical ileocolic anastomosis from the suprapubic port requires wider mobilization of the transverse colon in order to place it parallel to the stapler . Approximation and orientation of the ileal and colonic stumps is best achieved by pulling on two stitches placed at each end of the anastomosis, the proximal one being held by the 3 mm grasper in the right hypochondrium and the distal one passing through the 12 mm suprapubic port . The 3 mm grasper in the right hypochondrium is also useful during hand suturing of the enterotomies . Finally, attention must be paid when maneuvering 3 mm instruments, which must be done under direct vision throughout the operation . Our experience suggests that in well - trained hands and for properly selected patients, ports can be reduced in size safely without a negative impact on the surgeon's ability to perform laparoscopic colorectal resections . These findings should promote a larger prospective randomized comparison with conventional laparoscopy to determine whether this refinement of laparoscopic colorectal surgery confers concrete and incontrovertible benefits to the patients.
Gout is the most common form of inflammatory arthritis and presents with intermittent acute arthritis and chronic deformed joints with tophus formation . The prevalence of gout in the adult population was estimated at 3.23% (5.17% in men and 1.34% in women) in the united kingdom, 3.9% in the united states, and 3.8% in taiwan . The incidence and prevalence of gout has been increasing since the 1980s, and this may be related to the growing prevalence of obesity and metabolic syndrome, both of which are risk factors for venous thromboembolism (vte). Vte includes deep vein thrombosis (dvt) and pulmonary embolism (pe), both of which are common acute diseases that are potentially lethal . Pe is 1 of the most common causes of cardiovascular disease (cvd) mortality after myocardial infarction and cerebral stroke . The world reported incidence of vte was 1.43 per 10 person - years in norway and 1.22 per 10 person - years in quebec, canada . In addition to obesity and metabolic syndrome, as noted above, cigarette smoking, hypertension, and diabetes are other known risk factors for vte . On the other hand, although the reported incidence of vte in taiwan was much smaller, the annual incidence has increased gradually from 0.12 per 10 persons in 1998 to 0.23 per 10 persons in 2008 . Both the framingham study and the national health professional follow - up study reported a greater risk of coronary heart disease in men with gout than in men without gout (hazard ratio [hr] 1.60 and 1.55, respectively). The multiple risk factor intervention trial and a recent report in taiwan reported similar results . However, whether gout is an independent risk factor for vte is still unconfirmed . Dvt and pe have a common pathophysiology, namely inflammation, hyper - coagulation, endothelial injury, and hemostasis with thromboembolism, with other forms of cvd . We hypothesized that the modest but persistent inflammation in gout may promote atherosclerosis and thrombogenesis and thus the development of cvd and vte . This study aimed to examine the risk of vte, including both dvt and pe, in patients with gout . The national health insurance research database (nhird) was used for this population - based cohort study . Studies using this dataset have been published elsewhere . The accuracy and validity of diagnoses in the nhird have been reported . The subjects of interest were extracted from 1 million participants from all insured beneficiaries by a systemic sampling of the patient dataset (the longitudinal health insurance database 2000; lhid2000) in the nhird for the period of january 1, 1996 to december 31, 2000 in taiwan and were followed up until the year 2010 . This study was approved by the ethical review board of the china medical university in taiwan (dmr96-irb-241, dmr99-irb-074, and cmuh103-rec1 - 020). The international classification of diseases, 9th revision, clinical modification (icd-9-cm) code was used for coding the diseases of interest in the present study . To prevent medical fraud with overbilling for healthcare or inappropriate charges based on unconfirmed diagnoses, nhi sets strict rules on disease coding and regularly monitors coding and claims submitted for reimbursement by all participants of nhi beneficiaries . Nhird provides reliable information for the proposed studies to explore the risk of vte development in patients with gout . In the lhid2000, there were 58,614 enrollers with the new diagnosis of gout (icd-9-cm 274) identified during the study period (19982008), excluding the possible prevalent gout during 1996 to 1998 . Patients were excluded from the study if they were diagnosed with vte before they were diagnosed with gout (n = 633). The reference subjects were selected from people without gout and without vte history in the lhid2000 . The entry date of reference subjects was randomly assigned to match the entry date of a gout patient . For each identified gout patient, there were 2 reference subjects matched for age (within a 5-year age span), gender, and year of entry date (n = 115,961, only 1 reference subject was not satisfied for the matching criteria). The primary end point was the occurrence of vte during the study period (19982010) including occurrence of dvt (icd-9-cm 453.8) or pe (icd-9-cm 415.1) after excluding iatrogenic pe (icd-9-cm 415.11). All subjects were followed from the entry date until the date of vte diagnosis, withdrawal from the nhi program or the end of 2010 . The variables of relevance were age, gender, and the following comorbidities: hypertension (icd-9-cm 401405), diabetes mellitus (icd-9-cm 250), atrial fibrillation (icd-9-cm 427.31), stroke (icd-9-cm 430438), heart failure (icd-9-cm 428), fracture of lower limbs with / without operation (icd-9-cm 820, 821, 823, 81.51, 81.52, 81.53, and 81.54), all cancer (icd-9-cm 140208), and pregnancy (icd-9-cm procedure 7274 or icd-9-cm 640.x1676.x1, 640.x2676.x2, and 650659). The demographic factors and comorbidities were compared between the gout cohort and the reference cohort using a chi - squared test for categorical variables and a student t test for continuous variables . A kaplan meier survival curve was used to compare the incidence of dvt or pe between cohorts and was tested using a log - rank test . The gender-, age-, and comorbidity - specific incidence rates (per 10 person - years) of vte were compared between the case and reference cohorts . We used a poisson regression model to estimate the incidence rate ratio (irr) and corresponding 95% confidence interval (ci) of dvt and pe in gout patients relative to the reference subjects . The multivariate - adjusted hr and the corresponding 95% ci of dvt and pe in gout patients relative to the reference subjects were derived by the cox proportional hazard regression model with adjustment for age, gender, and comorbidities of atrial fibrillation, diabetes, hyperlipidemia, hypertension, stroke, heart failure, fracture of lower limbs with / without operation, cancer, and pregnancy . To address the concern of constant proportionality, we examined the proportional hazard model assumption using a test of scaled schoenfeld residuals . Results showed that there was no significant relation between schoenfeld residuals for gout and follow - up time in either the model evaluating dvt risk (p = 0.97) or the model evaluating pe risk (p = 0.53). We further compared the risk of vte development in patients with gout after stratification with respect to gender, age, and comorbidity . The age subgroups were 20 to 49, 50 to 64, and 65 years . Medication to treat gout, especially urate lowering agents, was considered to modify the risk of cvd in gout patients . The risk of vte in gout patients who did not use urate lowering agents was compared with the 1-to-2 matched reference subjects of nonusers (no gout, no urate lowering agent use) the national health insurance research database (nhird) was used for this population - based cohort study . Studies using this dataset have been published elsewhere . The accuracy and validity of diagnoses in the nhird have been reported . The subjects of interest were extracted from 1 million participants from all insured beneficiaries by a systemic sampling of the patient dataset (the longitudinal health insurance database 2000; lhid2000) in the nhird for the period of january 1, 1996 to december 31, 2000 in taiwan and were followed up until the year 2010 . This study was approved by the ethical review board of the china medical university in taiwan (dmr96-irb-241, dmr99-irb-074, and cmuh103-rec1 - 020). The international classification of diseases, 9th revision, clinical modification (icd-9-cm) code was used for coding the diseases of interest in the present study . To prevent medical fraud with overbilling for healthcare or inappropriate charges based on unconfirmed diagnoses, nhi sets strict rules on disease coding and regularly monitors coding and claims submitted for reimbursement by all participants of nhi beneficiaries . Nhird provides reliable information for the proposed studies to explore the risk of vte development in patients with gout . In the lhid2000, there were 58,614 enrollers with the new diagnosis of gout (icd-9-cm 274) identified during the study period (19982008), excluding the possible prevalent gout during 1996 to 1998 . Patients were excluded from the study if they were diagnosed with vte before they were diagnosed with gout (n = 633). The reference subjects were selected from people without gout and without vte history in the lhid2000 . The entry date of reference subjects was randomly assigned to match the entry date of a gout patient . For each identified gout patient, there were 2 reference subjects matched for age (within a 5-year age span), gender, and year of entry date (n = 115,961, only 1 reference subject was not satisfied for the matching criteria). The primary end point was the occurrence of vte during the study period (19982010) including occurrence of dvt (icd-9-cm 453.8) or pe (icd-9-cm 415.1) after excluding iatrogenic pe (icd-9-cm 415.11). All subjects were followed from the entry date until the date of vte diagnosis, withdrawal from the nhi program or the end of 2010 . The variables of relevance were age, gender, and the following comorbidities: hypertension (icd-9-cm 401405), diabetes mellitus (icd-9-cm 250), atrial fibrillation (icd-9-cm 427.31), stroke (icd-9-cm 430438), heart failure (icd-9-cm 428), fracture of lower limbs with / without operation (icd-9-cm 820, 821, 823, 81.51, 81.52, 81.53, and 81.54), all cancer (icd-9-cm 140208), and pregnancy (icd-9-cm procedure 7274 or icd-9-cm 640.x1676.x1, 640.x2676.x2, and 650659). All comorbidities were defined before the entry date . The demographic factors and comorbidities were compared between the gout cohort and the reference cohort using a chi - squared test for categorical variables and a student t test for continuous variables . A kaplan meier survival curve was used to compare the incidence of dvt or pe between cohorts and was tested using a log - rank test . The gender-, age-, and comorbidity - specific incidence rates (per 10 person - years) of vte were compared between the case and reference cohorts . We used a poisson regression model to estimate the incidence rate ratio (irr) and corresponding 95% confidence interval (ci) of dvt and pe in gout patients relative to the reference subjects . The multivariate - adjusted hr and the corresponding 95% ci of dvt and pe in gout patients relative to the reference subjects were derived by the cox proportional hazard regression model with adjustment for age, gender, and comorbidities of atrial fibrillation, diabetes, hyperlipidemia, hypertension, stroke, heart failure, fracture of lower limbs with / without operation, cancer, and pregnancy . To address the concern of constant proportionality, we examined the proportional hazard model assumption using a test of scaled schoenfeld residuals . Results showed that there was no significant relation between schoenfeld residuals for gout and follow - up time in either the model evaluating dvt risk (p = 0.97) or the model evaluating pe risk (p = 0.53). We further compared the risk of vte development in patients with gout after stratification with respect to gender, age, and comorbidity . The age subgroups were 20 to 49, 50 to 64, and 65 years . Medication to treat gout, especially urate lowering agents, was considered to modify the risk of cvd in gout patients . The risk of vte in gout patients who did not use urate lowering agents was compared with the 1-to-2 matched reference subjects of nonusers (no gout, no urate lowering agent use) in the lhid2000, we identified 57,981 gout patients as the gout cohort and 115,961 matched subjects without gout as the reference cohort (ratio of 1:2), for a total of 1,358,378 person - years of follow - up . The mean age of the gout cohort was 52.5 16.0 years . Among the 173,942 investigated subjects, the proportion of men was 2.8 times higher than that of women (73.9% vs 26.2%). The age of gout patients was split into subgroups: 15.7% of patients were ages 20 to 34 years, 30.2% were ages 35 to 49 years, 28.6% were ages 50 to 64 years, and 25.5% were ages 65 years . The prevalence of co - morbidities including hypertension, diabetes, hyperlipidemia, atrial fibrillation, stroke, heart failure, and fracture of lower limbs with / without operation was significantly higher in the gout cohort than in the reference cohort (table 1). Demographic characteristics and comorbidities in patients with gout and in reference subjects without gout although the reported incidence of vte in taiwan was small, we used the lhid2000 to estimate the standardized prevalence and incidence of vte with respect to the taiwanese population in 2010 as 6.39 (4.79 for dvt and 1.87 for pe) per 10 person - years and 1.69 (1.41 for dvt and 0.32 for pe) per 10 person - years, respectively . Figure 1 shows the cumulative incidences of the gout and reference cohorts for dvt and pe during 11 years of follow - up (all p 0.01, log - rank test). During the study period, 243 patients with gout (5.26 per 10 person - years) and 236 reference subjects (2.63 per 10 person - years) developed dvt, with a significant irr of 2.00 (95% ci = 1.932.07) and an adjusted hr of 1.66 (95% ci = 1.372.01; table 2). The effect of gout on the risk of dvt was similar for men and women . It is noteworthy that the greatest effect of gout on the risk of dvt was in the youngest subgroup and the risk steadily declined with increasing age (table 2). We further investigated if gout patients had a higher risk of pe . During the study period, 49 patients with gout (1.06 per 10 person - years) and 59 reference subjects (0.66 per 10 person - years) developed pe (adjusted hr = 1.53; 95% ci = 1.012.29; table 2). Cumulative incidence of dvt (a) and pe (b) in patients with gout and reference subjects without gout . Irr and hr of dvt and pe stratified by sex and age the impact of comorbidities on outcomes was examined by stratifying the gout and reference cohorts with respect to the status of comorbidities . The risk of dvt was significantly increased in gout patients with comorbidity (hr = 1.45, 95% ci = 1.171.79) and in gout patients without comorbidity (hr = 2.27, 95% ci = 1.563.31) relative to their respective reference subjects (table 2). However, the event number of pe was also too small to make significant estimates after stratification . To explore the joined effect of gout and comorbidity on dvt or pe (as both individual p values for interaction were> 0.05), we stratified the gout patients and reference subjects using a 2 2 stratification with respect to the status of comorbidity (table 3). Relative to the reference subjects without comorbidity, gout patients with and without comorbidity had significantly higher risk of dvt, and gout patients with comorbidity had a significantly higher risk of pe (table 3). Figure 2 demonstrates the joined effect of gout and each comorbidity on dvt (figure 2a) and pe (figure 2b). Relative to the reference subjects without comorbidity, the gout patients without comorbidity had a significantly higher risk of dvt, with hrs ranging from 1.87 to 2.32 across comorbidities (figure 2a, red lines). The hr of gout for dvt was significant for each comorbidity and was higher than the hr of the reference subjects with comorbidities for each comorbidity except cancer, because cancer is a major risk factor for dvt . Relative to the reference subjects without comorbidity, the gout patients with comorbidity had a significantly higher risk of dvt, with hrs ranging from 2.21 to 3.33 . Relative to the reference subjects without comorbidity, the gout patients without comorbidity had a significantly higher risk of pe, with hrs ranging from 1.58 to 1.75 across comorbidities except hypertension (figure 2b, red lines). Cox proportional hazards regression analysis for the combined effect of gout and comorbidity on the risk of dvt and pe the combined effect of gout and each comorbidity (atrial fibrillation, hypertension, diabetes mellitus, stroke, heart failure, fracture of lower limbs with / without operation, all cancer, and pregnancy) on the occurrence of dvt (a) and pe (b). The hazard ratio of vte in gout patients without comorbidity relative to the reference subjects without comorbidity is marked in red . Dvt = deep vein thrombosis, cva = cerebral stroke, pe = pulmonary embolism, vte = venous thromboembolism . Figure 3 demonstrates the risk of dvt and pe in the gout patients and reference subjects with respect to the number of comorbidities . The effect of gout on the risk of dvt and pe was comparable to that of any single comorbidity . However, the effect of gout combined with any other comorbidity on the risk of dvt and pe was higher than the effect of any 2 comorbidities combined . The relation between the number of comorbidities in patients with gout and reference subjects without gout and the risk of dvt (a) and pe (b) expressed as the incidence rate (per 10 person - years) (left) and adjusted hr (95% ci) (right). Ci = confidence interval, dvt = deep vein thrombosis, hr = hazard ratio, pe = pulmonary embolism . During the follow - up, there were 35,795 gout patients prescribed with urate lowering agents . These constituted 61.7% of total gout patients and can possibly modify the risk estimates of vte in gout patients . We compared the risk of vte between those 22,186 gout patients who did not use urate lowering agents and the matched reference subjects . The risks were markedly enhanced in the untreated gout patients with hrs of 2.16 (95% ci = 1.592.92) for dvt and 2.28 (95% ci = 1.174.46) for pe relative to the matched reference subjects . The present study of a large taiwanese database confirmed that gout increased the risk of vte by 50% to 60% after adjusting for age, sex, and comorbidities (metabolic syndrome of diabetes, hyperlipidemia, hypertension, and atrial fibrillation, stroke, heart failure, lower leg fracture with / without operation, cancer, and pregnancy). These estimates are comparable to those of the effect of gout on cvd, which emphasizes the importance of gout as a risk factor for dvt and pe . Besides, the risk of dvt was higher in gout patients without comorbidity than in gout patients with comorbidity, and the risk of vte in gout patients with any single comorbidity was markedly higher than that in reference subjects without gout but with any combination of 2 comorbidities . Urate lowering therapy in the united kingdom was only received in 37.63% of gout patients and 39.66% of whom were adherent to treatment . Obviously, the current management of gout is suboptimal, even though the risk factors and treatment of gout are best understood . In this study, more than 2 times increased risk of vte in gout patients who did not receive urate lowering agents relative to the reference subjects were demonstrated . This finding can imply that under - treatment of gout may have serious negative consequences for vte . Although prolonged immobilization or elevated body mass index is associated with the risk of vte, the relationship between gout and vte is less well evidenced . However, several lines of evidence can provide a possible biological explanation to support the increased vte risk in gout patients . The monosodium urate (msu) crystals that form during gout represent 1 of the damage - associated molecular patterns (damps) that trigger toll - like receptors, and contribute to the cyclic activation and resolution of chronic gouty inflammation . The persistent deposition of msu crystals with tophi formation in the inter - critical phase of gout may sequentially induce inflammatory mediators . Low - grade inflammation around asymptomatic tophus can be demonstrated by imaging studies including doppler ultrasonography, and the immune - histological method can also reveal proinflammatory cytokines around coronal zone of tophus . Nitric oxide production is thus reduced while reactive oxygen species is increased in blood vessels . Both aggravate vascular inflammation inhibit endothelial - cell growth and result in proliferation of vascular smooth muscle cells . Uric acid in the kidney damage can be similarly through activation of renin - angiotensin system, decreased nitric oxide production with microvascular rarefaction, afferent arteriolopathy, interstitial inflammation, and tubulointerstitial fibrosis . Multiple organs and systems taken together, these results indicate that the inflammation in gout may have potentiated arthrosclerosis and thrombogenesis to increase the likelihood of endothelial dysfunction and vte development . First, gout patients may have been misclassified in the population of lhid2000, because patients in the case cohort with a clinical diagnosis of gout defined by the physicians did not necessarily meet the classification criteria of american college of rheumatology . However, the utility and validity of using claims dataset of lhid2000 to identify gout has been assessed . In agreement with published reports, the gout patients in the present study were predominately male and were most often in the middle - age subgroups (age 3564 years). These characteristics support the validity of the current approach of identifying gout patients from the lhid2000 . Second, the lhid2000 does not include variables such as body mass index; inflammatory index; uric acid level; and synovial fluid analysis; the results of imaging studies and personal habits such as diet, cigarette smoking, and alcohol drinking; therefore, we were unable to include these variables as covariates in the current analysis . Although smoking and metabolic syndrome are reported as risk factors of vte, we were unable to assess the impact of smoking on the risk of vte in gout patients, and we can only assess the effect of respective component of metabolic syndrome except obesity on vte . However, patients with gout had a significantly higher prevalence of comorbidities than the reference cohort, and may have impacted the vte risk . Nevertheless, the independent risk of gout for vte was confirmed by adjusting for comorbidities and by sequential stratifications by comorbidity in both patients and reference subjects . The vte risk persisted in gout patients without comorbidities and was higher in younger patients, which strengthens our conclusion that gout is an independent risk factor for vte . Third, due to a difficulty in resolving the potential bias from indication of urate lowering agents, whether intervention in gout patients can reduce risk for dvt and/or pe were not demonstrated . However, a marked enhanced risk of vte in the untreated gout patients was shown relative to the matched reference subjects . Fourth, people can still argue that other residual confounding factors can exist; however, with the huge national insurance data and large sample size, these confounders may become nondifferential bias and all these evidences may strengthen the contention that gout can be an independent risk factor for vte . First, the nhird encompasses> 22 million individuals in taiwan who are enrolled in a national insurance program and therefore includes> 98% of the population . The bureau of nhi monitors and audits the insurance claims for reimbursement rigorously to prevent healthcare fraud, and thus the reliability of the diagnosis recorded for insurance claims is strengthened by this stringent nhi surveillance program . Second, the statistical power gained from the large sample size made subgroup analyses possible and allowed us to ascertain the impact of gout on vte risk . Although the increased risk of dvt in gout patients was significant across all age subgroups, the risk of vte in gout patients was highest in the youngest subgroup and declined with increasing age . The findings of increased risk in young people who often have a low prevalence of traditional risk factors and comorbidities support an independent risk of gout on vte . Third, the long observation period further increased the accuracy with which we could assess the effect of gout on future vte development . In conclusion, the present study extends research on the risk of gout for cvd development to confirm an independent effect of gout on vte . This message can further raise attention to the systemic effect of gout in addition to the intermittent arthritis attack and joint deformity, and also add to our current understanding of the association between gout and cvds.
A total of 3,614 consecutive patients underwent pet / ct scans between 14 march 2006 and 30 october 2008 at the nottingham pet / ct center (53.00n 1.20w). All scans were acquired using a siemens biograph 16 hirez lso pet / ct scanner . Temperature was not kept constant at the pet / ct center throughout the year, although there is a thermostat in the room and indoor temperature significantly exceeds outdoor temperature . Patients were indoors for at least 75 min to allow for 18f - fluorodeoxyglucose injection and uptake prior to imaging . The presence of bat activity is routinely included in the radiology report at our institution . The 154 patients with bat activity constituted the study group . Data were also obtained about age, sex, bmi, serum glucose, month scanned, and disease . Patients who underwent serial scans were identified, and variation of bat expression in these individuals was recorded . Bat activity was quantified by calculating the maximum standardized uptake value (suvmax) within a specified region of interest using a leonardo workstation in the area of maximal activity in these patients . Assessment of depots was made by a single observer experienced in pet / ct interpretation . The suvmax is dependent on a number of factors including time between injection and imaging, blood glucose, patient weight, and the equipment used (12). Although serial suvmax measurements are more reproducible in individuals, a semiquantitative method of analyzing the intensity of bat depots was also developed to give an approximate estimate of the amount of bat present in an individual . The five main depots (supraclavicular, mediastinal, axillary, paravertebral, and abdominal) were analyzed, and the amount of activity in each was assigned a score (0 = absent, 1 = low grade, 2 = moderate, 3 = high grade) that was then was summated for the five major depot sites to provide a total intensity score (015). All statistical analyses were performed using spss version 16.0 for windows (spss, chicago, il). A test was performed to ascertain whether there was a significant relationship between sex and total number of bat - positive scans and between season and total number of bat - positive scans . The suvmax is dependent on a number of factors including time between injection and imaging, blood glucose, patient weight, and the equipment used (12). Although serial suvmax measurements are more reproducible in individuals, a semiquantitative method of analyzing the intensity of bat depots was also developed to give an approximate estimate of the amount of bat present in an individual . The five main depots (supraclavicular, mediastinal, axillary, paravertebral, and abdominal) were analyzed, and the amount of activity in each was assigned a score (0 = absent, 1 = low grade, 2 = moderate, 3 = high grade) that was then was summated for the five major depot sites to provide a total intensity score (015). All statistical analyses were performed using spss version 16.0 for windows (spss, chicago, il). A test was performed to ascertain whether there was a significant relationship between sex and total number of bat - positive scans and between season and total number of bat - positive scans . The prevalence of bat activity in this cohort of patients was 4.6% (n = 167/3,614) with the location summarized in table 1 . Frequency of reporting of bat - positive anatomical depots with average age in years for each depot in male patients, 2.8% of scans demonstrated bat activity (n = 60/2,131) compared with 7.2% of scans in female patients (n = 107/1,483). The difference between male and female patients was highly significant (p = 0.00005, test). Irrespective of sex, for the 57 patients in whom bmi data were available, there was a trend for bat - positive individuals to have a low bmi, with a smaller proportion being obese . The proportion of patients with bat activity showed a very striking seasonal variation, being highest in winter and lowest in summer (fig . The month - to - month circannual variation is even more striking because it is inversely correlated with average monthly temperatures and positively related to night length in nottingham as recorded through the study period (fig . Additionally, the number of depots identified also varied with season, with more depots identified in winter than summer (fig . The difference in the monthly number of patients in which bat activity observed was therefore positively correlated with night length (r = 0.876; p <0.00001; pearson correlation) and negatively correlated with ambient temperature (r = 0.696; p <0.001). Results are expressed as either the number of individual depots recorded (a) or the percentage of all scans (b) together with the variation in ambient temperature (http://www.tutiempo.net/en/climate/united_kingdom/gb.html) (c) and photoperiod i.e., night length (http://www.timeanddate.com/worldclock/astronomy.html) (d) over the time period in which bat - positive scans were recorded . Significant effect of season assessed by either mann - whitney u test; * * p <0.01) or test * * * p <0.00001 . The number of patients with positive scans for each month is given and includes 16 patients who were scanned more than once . The seasonal variation in bat activity was also apparent in those individuals who were scanned in different months (fig . 2), with the mean suvmax value being 4 times higher in winter compared with summer (average suvmax 1.5 in july and 9.8 in january). Indeed each line represents an individual patient who underwent serial pet / ct scans throughout the study period, with the total number of patients being 16 . The age range of the bat - positive cohort was 1388 years (average age 51.8 years), and there was a trend for both the average number of depots identified and the semiquantitative bat score to decrease with increasing age (see fig . 3a and b), although the latter effect is primarily due to the decreasing number of depots . Results are expressed as either the mean total intensity (a) or the number of individual depots recorded (b). A total of 71 scans were analyzed because not all were available for this type of analysis . In the bat cohort, 11/167 (6.6%) of patients did not have a diagnosis of cancer, and all were investigated for solitary pulmonary nodules . Of those with cancer, 111/156 (71.2%) had active disease and 45/156 were in remission (28.9%). There was also no relationship between the plasma glucose and the occurrence of bat activity, with all patients being normoglycaemic (data not shown). The prevalence of bat activity in this cohort of patients was 4.6% (n = 167/3,614) with the location summarized in table 1 . Frequency of reporting of bat - positive anatomical depots with average age in years for each depot in male patients, 2.8% of scans demonstrated bat activity (n = 60/2,131) compared with 7.2% of scans in female patients (n = 107/1,483). The difference between male and female patients was highly significant (p = 0.00005, test). Irrespective of sex, for the 57 patients in whom bmi data were available, there was a trend for bat - positive individuals to have a low bmi, with a smaller proportion being obese . The proportion of patients with bat activity showed a very striking seasonal variation, being highest in winter and lowest in summer (fig . The month - to - month circannual variation is even more striking because it is inversely correlated with average monthly temperatures and positively related to night length in nottingham as recorded through the study period (fig . Additionally, the number of depots identified also varied with season, with more depots identified in winter than summer (fig . The difference in the monthly number of patients in which bat activity observed was therefore positively correlated with night length (r = 0.876; p <0.00001; pearson correlation) and negatively correlated with ambient temperature (r = 0.696; p <0.001). Seasonal variation in the occurrence of bat in adults . Results are expressed as either the number of individual depots recorded (a) or the percentage of all scans (b) together with the variation in ambient temperature (http://www.tutiempo.net/en/climate/united_kingdom/gb.html) (c) and photoperiod i.e., night length (http://www.timeanddate.com/worldclock/astronomy.html) (d) over the time period in which bat - positive scans were recorded . Summary showing the percentage occurrence of bat according to calendar month (e). Significant effect of season assessed by either mann - whitney u test; * * p <0.01) or test * * * p <0.00001 . The number of patients with positive scans for each month is given and includes 16 patients who were scanned more than once . The seasonal variation in bat activity was also apparent in those individuals who were scanned in different months (fig . 2), with the mean suvmax value being 4 times higher in winter compared with summer (average suvmax 1.5 in july and 9.8 in january). Indeed each line represents an individual patient who underwent serial pet / ct scans throughout the study period, with the total number of patients being 16 . The age range of the bat - positive cohort was 1388 years (average age 51.8 years), and there was a trend for both the average number of depots identified and the semiquantitative bat score to decrease with increasing age (see fig . 3a and b), although the latter effect is primarily due to the decreasing number of depots . Results are expressed as either the mean total intensity (a) or the number of individual depots recorded (b). A total of 71 scans were analyzed because not all were available for this type of analysis . In the bat cohort, 11/167 (6.6%) of patients did not have a diagnosis of cancer, and all were investigated for solitary pulmonary nodules . Of those with cancer, 111/156 (71.2%) had active disease and 45/156 were in remission (28.9%). There was also no relationship between the plasma glucose and the occurrence of bat activity, with all patients being normoglycaemic (data not shown). Obesity is a significant cause of morbidity and mortality, and there has been considerable recent interest in studying the physiology of bat in humans, given its protective role against obesity in animal experiments (6). A greater understanding of bat function could thus help to develop treatment strategies for obesity, especially because it appears that white adipocyte area is smaller in individuals possessing ucp-1 (13). One of the most striking findings of our study is the pronounced seasonal variation in bat expression seen in the entire cohort of patients, in individuals, and in the number of depots that are active . To our knowledge, this is the largest study of its kind documenting the distribution of active bat in multiple depots in adults and how bat varies with season . Two smaller studies have demonstrated increased bat activity in winter and/or early spring (14), although other studies have not confirmed this finding (15,16). We thus extend the findings from a small japanese cohort (17) in which the variation in temperature was much greater than in the u.k . Importantly, we raise the question whether it is the prevailing ambient temperature or photoperiod that may be the primary factor determining bat activity . Cold exposure is established to promote bat activity in both rodents (2) and humans (8). The extent to which it is the primary regulator of bat function remains uncertain because photoperiod can also determine bat activity irrespective of ambient temperature, although this effect is enhanced in the cold (18,19). Photoperiod is known to impact on prolactin release (20), which increases with day length (21). Prolactin administration also promotes the loss of ucp-1 (22), and prolactin secretion can be temperature sensitive (23). The prolactin receptor is essential for bat function in the newborn (24), in whom the direct stimulation of prolactin receptor promotes bat thermogenesis (25). It is thus possible that prolactin is an important factor determining the overall activity of bat we have observed with season . This would explain why bat that is inactive in the summer is then recruited in the winter for nonshivering thermogenesis, evidenced by increased activity within depots and a greater number of depots . Furthermore, it should be noted that patients undergoing scans are in a warm environment (indoors) while being prepared for their scans . The persistence of the strong variation in bat activity despite stable indoor temperatures could thus relate to the longer - term recruitment of bat depots (10). Although prolactin may be an important mediator, as discussed, the endocrine mechanisms controlling bat expression are likely to be complex and other hormones, for example the key player in mediating circannual rhythms in mammals melatonin, are likely to be involved (26). An understanding of the physiology of bat is likely to be important if recruitment of bat is to be considered as a strategy for weight loss in obese individuals . Global temperatures are rising in conjunction with increased use of artificial lighting and central heating (27). Our results demonstrate that seasonality and, thus, photoperiod and/or ambient temperature are pivotal determinants of bat activity in adult humans . In fact, the abundance of bat quadrupled between the summer and winter months in our study, thus emphasizing the importance of considering the time of the year in weight loss programs . The success or the failure of dietary or pharmacological interventions aimed at weight loss in obesity could depend on the capacity of bat to burn excess energy . This raises the question as to whether the greater quantity of bat we possess during the winter could be used to promote fat mobilization at this time of year . Our study also demonstrated significantly increased prevalence of bat in female patients, a finding that is consistent with other studies (9,14). Female rats have higher levels of ucp-1 in the interscapular bat depot compared with males when housed at the same temperature, suggesting that they may have a lower temperature threshold for cold - induced thermogenesis (28). Bat function is dependent on thyroid hormones (29), which may be under the control of estrogen in female rats . Bat also expresses estrogen receptors, which may further explain these differences (2). One caveat is that the study group mostly consists of patients undergoing treatment for cancer, although this is unlikely to affect the seasonal variation in bat activity . It has been suggested that bat activity may in part relate to increased expression of ucp-1 in cancer patients (30). In the present study, however, bat was shown to be present in a significant proportion of noncancer patients, as well as those that were in remission, indicating that the presence of bat is not exclusively due to cancer - related factors . In conclusion, our large retrospective study demonstrates the presence of a strong seasonal variation in bat activity (9) both in a cohort and in individual patients . Improved understanding of the influence of both photoperiod and ambient temperature on bat function is likely to lead to the development of novel treatments for obesity.
Asthma affects an estimated 8.9% of usa children under 17 years and continues to be one of the most common childhood chronic illnesses . Uncontrolled asthma is associated with more school days missed among children, more work days missed among caregivers, and poorer quality of life among both [2, 3]. A special case of poor asthma control, nighttime awakenings from asthma, have been linked to school absences, lower school performance, and parents' lost workdays . The national heart, lung, and blood institute (nhlbi) guidelines provide several recommendations for proper asthma management to minimize uncontrolled asthma . These guidelines include: use of pharmacologic therapy, patient education, reduce environmental triggers, and assess and monitor asthma control . The guidelines emphasize the importance of using a collaborative approach between providers, parents, and children to develop an appropriate asthma management plan for the child . However, recent studies have found that these guidelines are not being met, with less than half of families ever receiving any education about their child's asthma [7, 8]. In another study, most hospitalizations for asthma attacks were found to be preventable and had medications been taken regularly . Prior research indicates that asthma patients who report poor communication with their physicians are less adherent with inhaled steroids [10, 11]. Few studies have examined how physicians communicate about medications during medical visits using actual communication data and to our knowledge, no prior study has investigated how physicians communication about control medications during pediatric asthma visits [1215]. Findings from these prior adult studies suggest that communication about medications can be improved . In a sample of 40 veterans who were on continued or newly prescribed antidepressants, providers asked 6% of patients about adverse events and 15% of patients how well the antidepressants were working . Moreover, providers only gave 10% of patients' information about adverse events and 5% information on how well the medication works . Young et al . Used standardized patients (n = 131) and found that physicians provided information about side effects to 85% of patients but only gave information about how well the drug works to 38% of patients . To our knowledge, there are no studies that have examined how providers communicate about control medications during pediatric asthma visits . It is important to better understand how providers communicate about control medications during medical visits because the clinical practice guidelines of the national asthma education and prevention program of nhlbi encourage physicians to discuss medications with patients at every follow - up asthma visit . Therefore, the purpose of this study was to: (a) describe the extent to which providers discuss, educate, and ask children and their caregivers questions about control medications and (b) examine how child, caregiver, and provider characteristics are associated with provider communication about control medications during pediatric asthma visits . . Providers were recruited at five pediatric practices in nonurban areas of north carolina, and consent was obtained . Children and their caregivers of these participating providers were recruited . Children were eligible if they: (a) were ages 8 through 16 years, (b) were able to speak english, (c) could read the assent form, (d) had been seen at the clinic at least once before, (e) were present at the visit with an adult caregiver (parent or legal guardian) who could read and speak english and who was at least 18 years of age, and (f) had mild, moderate, or severe persistent asthma . Persistent asthma was defined as experiencing asthma - related daytime symptoms more than twice a week, asthma - related nighttime symptoms more than twice a month, or receiving one or more long - term control therapies for asthma [16, 17]. Clinic staff referred potentially eligible and interested patients to a research assistant who explained the study, obtained caregiver consent and child assent, and administered the eligibility screener . Providers and families were told that the study was examining communication during pediatric visits . All of the medical visit audio - tapes were transcribed verbatim, and a detailed coding tool was developed to assess provider communication behaviors . All of the transcripts were coded using the coding tool and more detail about the types of communication behaviors coded is provided below . The research assistants showed caregivers a list of asthma medications and asked them to indicate which one(s) the child was taking . Responses were dichotomized based on whether the caregiver reported that the child was taking a control medication versus not taking a control medication . Asthma severity was classified as mild versus moderate / severe by a research assistant based on recent symptoms and medication use reported by the caregivers when research assistants administered the eligibility screening instrument for the study [16, 17]. Our eligibility screening instrument utilized the primary asthma severity classification system that was being used when the study was designed and conducted [16, 17]. All child study information was then reviewed by a pediatric pulmonologist or a clinical pharmacist with expertise in asthma to verify the severity classification as mild or moderate / severe persistent asthma . The first method was medication use; any child receiving a single long - term control agent was considered to have mild persistent asthma . Any child receiving two or more long - term control agents was categorized as having moderate to severe persistent asthma . A long - term control medicine included inhaled corticosteroids, leukotriene modifiers, cromolyn, nedocromil, or a long - acting beta agonist as defined by the national heart lung and blood insititute's guidelines [16, 17]. Subjects who reported the occurrence of any one of eight symptoms as occurring two or more times per week or who reported awakening with asthma symptoms two or more times per month was classified as mild persistent . The eight daytime symptoms included: wheezing with a cold, wheezing without a cold, attack of wheezing that made it hard to breath or catch breath that lasted longer than a day or more, had a cough that would not go away, complained that chest felt tight or heavy, used rescue inhaler for symptoms, wheezed with exercise or running or playing hard, and coughed with exercise or running or playing hard . The nighttime symptoms asked about how often the child's sleep has been disturbed because of wheezing, coughing, chest tightness, or shortness of breath . Reports of daily symptom occurrence or awakening 5 times a month resulted in a classification as moderate or severe persistent . In situations where the two methods (medication use and symptom frequency) resulted in discordant classification, the more severe category was used . Child and caregiver age, caregiver education, and years the child had asthma were measured as continuous variables . Child race was recoded into four categories: white, african american, native american / american indian, or other (includes categories of: hispanic, asian american, other). However, for the bivariate analyses, child race was recoded into a dichotomous variable (white versus non - white). The child's insurance status was measured using the following categories: none, private insurance, medicaid, the state children's health insurance program (schip), and others . How well the child thinks the provider knows them as a person was measured with the following categories: hardly at all, slightly, moderately well, and very well . Reason for visit was measured as asthma - related versus other (e.g. Physical). Length of visit was measured in minutes, and whether the child was taking a control medication was measured as a dichotomous variable . The categories used in the coding tool for communication about asthma medications were adapted from the categories used in prior studies of provider - patient communication about medications [1215]. The transcripts were reviewed by two research assistants who met twice a month with the investigators to develop and refine the coding rules until themes were saturated . Using the coding tool for transcribed medical visits, coders recorded the following: was there any discussion of control medications, did the provider give any education about control medications, and how many questions did the provider ask the child and caregiver about control medications . The research assistants then coded whether discussion, education, and question - asking occurred in each of the following areas: adherence, fears / concerns, frequency / timing, generic / brand, how well it works, purpose, side effects, strength / dose, supply, and others . Two research assistants coded 20 of the same transcripts throughout the study period to assess intercoder reliability which was calculated using interrater correlations . Inter - rater reliability was 1.0 for whether control medications were discussed, 0.87 for the number of areas discussed, 0.91 for whether the provider educated about control medications, 0.80 for number of areas the provider educated about, and 0.95 for the number of questions the provider asked about control medications . All children were included in the analyses because even if children were not on a control medication, control medications could have been discussed during the visit as a possible treatment . First, we present descriptive statistics for the demographic, clinical, and provider communication variables . Second, we examine bivariate relationships between the demographic variables and provider communication variables using correlation coefficients, t - tests, or pearson chi - square statistics, as appropriate . Next, we used generalized estimating equations (gee) to examine how demographic and clinical characteristics of the child and caregiver were associated with: (a) whether the provider discusses control medications, (b) whether the provider educates about control medications, and (c) how many questions the provider asks about control medications . A poisson gee was used to examine provider question - asking because the variable was skewed . Forty - one providers agreed to participate in the study; two providers refused to participate for a participation rate of 95.3% . Three hundred and thirty - three of the 377 families (88%) that approached the research assistant to learn more about the study agreed to participate in the study . Two - hundred and ninety six patients of the 333 participating patients (89%) had useable audio - tape data, and these patients were seen by 35 of the 41 providers who agreed to participate in the study . Four of the 35 providers were nurse practitioners or physician assistants, and they saw seventeen of the participating children . Twenty - seven of the providers were white, two were american indian, three were african american, one was asian, and two classified their race as other . Providers ranged in age from 30 to 70 years (mean = 44.8 years, standard deviation = 9.4). Approximately 62% of the children were white, 30% were african american, and 10% were native american / american indian . In terms of the child's asthma providers discussed control medications during 87.4% of visits where children were taking control medications and during 63.8% of visits where children were not taking control medications . The average number of topic areas discussed was 2.92 (standard deviation = 2.0; range 0 to 8 areas). Table 2 shows the control medication areas that providers discussed most often during the medical visits: (a) frequency / timing of use (63%), (b) supply of medication (50%), (c) strength / dose of medication (48%), (d) adherence (47%), and (e) purpose of the controller medication (34%). Side effects were only discussed during approximately 11% of encounters and fears / concerns were only discussed during 4.4% of encounters . In the bivariate results, control mediations were significantly more likely to be discussed if a child was on a control medication (pearson chi - square = 16.1, p <0.001), if the child had moderate / severe persistent asthma (pearson chi - square = 12.8, p <0.001), and if the child was present for an asthma - related visit (pearson chi - square = 14.48, p <0.001). Providers were significantly more likely to discuss control medications if a child was on a control medication and during visits with children who had moderate to severe persistent asthma compared to children with mild persistent asthma . Providers were also more likely to discuss control medications when the reason for visit was asthma - related versus not asthma - asthma - related . The average number of areas educated about was 1.54 (standard deviation = 1.6; range 0 to 7 areas). As presented in table 2, providers educated about control medications most often in the following areas: (a) frequency / timing of use (37%), (b) strength / dose (32%), and (c) purpose (30%). Providers were significantly more likely to provide education about control medication side effects if a child was not on a control medication (pearson chi - square = 5.1, p = 0.02). In the bivariate results, providers were more likely to educate about control medications if the child had moderate / severe persistent asthma (pearson chi - square = 13.3, p <0.001), if the child was younger (t - test = 2.71, p = 0.007), and if the child was present for an asthma - related visit (pearson chi - square = 12.9, p <0.001). Table 3 demonstrates which child and caregiver characteristics were associated with whether providers educated families about control medications . Providers were significantly more likely to educate about control medications during visits with children who had moderate to severe persistent asthma, younger children, and during visits that were primarily asthma - related . The average number of questions asked was 2.68 (standard deviation = 3.1; range 0 to 16 questions). Providers were most likely to ask children and their caregivers questions about control medications in the following areas: (a) frequency / timing of use (38%), (b) adherence (37%), and (c) supply (29%). Providers only asked about side effects during 2% of encounters, fears / concerns during 1% of encounters, and how well the control medications were working during approximately 12% of encounters (table 2). In the bivariate results, providers were more likely to ask control medication questions if the child was on a control medication (t - test = 3.5, p = 0.001) and if the child was younger (pearson correlation coefficient = 0.12, p = 0.04). Table 4 presents the poisson gee results predicting the number of questions providers asked about control medications . Providers asked more questions about control medications if a child was currently treated with a control medication . Providers also were more likely to ask younger children more questions about control medications than older children . Providers discussed the frequency of use, supply of medication, and strength / dose of medication with families most often, but they only discussed the purpose of the control medication during about one third of all visits and how well the medication works during about a quarter of all visits . Providers rarely discussed side effects and fears / concerns about control medications . According to clinical practice guidelines of the national asthma education and prevention program of nhlbi the national clinical practice guidelines instruct providers to teach and reinforce the roles of control medications at every opportunity, yet providers in this study only educated children and their caregivers about control medications during about two - thirds of the visits . It is especially critical for families to understand the purpose of asthma control medications so that they understand the difference between control and rescue medications . Providers rarely educated about side effects and how well the medications work even though according to the practice guidelines of the national asthma education and prevention program of nhlbi, providers should ask about specific side effects from control medications during routine asthma visits . Our results provide evidence that discussion of asthma controller medication does not occur at every follow - up visit . Given recent evidence that poorly controlled asthma (56%) is common among children receiving asthma care from community pediatricians, this study points to provider discussion and education as a key area for improvement . If children and their caregivers better understand what to expect when taking the medications, they might be more adherent . Better adherence could lead to improved asthma control and fewer school days missed for children and reduced health care costs [2, 3]. Future work needs to assess the relationship between provider education about control medications and medication adherence and asthma control . Specifically, providers were more likely to educate younger children about control medications and they were more likely to ask younger children more questions about control medications . Perhaps this is because younger children are less likely to volunteer information about their use and experiences in using control medications . It is important to make sure that children of all ages understand how to use their control medications . Providers also were more likely to engage children with more severe asthma in discussions about controller medications . It is important for providers to ask about child adherence to control medications so they can work with families to improve adherence and asthma control . Providers asked few questions about side effects and how well the control medications were working, which is similar to findings of medication communication in adults with other medical conditions [12, 13]. If providers want to detect and prevent problems with asthma control medication use and adherence, they should consider asking at least one open - ended question about how the medications are working and a second question about any side effects or barriers to use (e.g., how are your asthma medications working? And what types of problems have you had with your medications?). The study's generalizability is limited in that it was conducted in five pediatric clinics in nonurban areas of north carolina . Another limitation is that clinic staff referred potentially eligible patients to the research assistant; thus, we do not know how many referred patients chose not to talk with the research assistant . However, we could not ask the clinic staff to track these numbers because of the busyness of the clinic and our promise not to interrupt clinic flow . Providers and caregivers knew they were being recorded and may have changed their communication, but they did not know the study hypotheses . It is also important to note that we did not assess the level of control in this study based on the current nhlbi guidelines because this study was initiated prior to the release of the new guidelines . Another limitation is that we did not include children ages 27 years and adolescents ages 17 - 18 years . Future research should examine provider discussion, education, and question - asking about control medications in these age groups . Despite its limitations, this study presents information on the extent to which providers discuss and educate children about control medications during pediatric medical visits.
Most dysentery cases in the tropics are caused by shigella, whereas dysentery in the developed countries is usually caused by salmonella . Death rates as high as 6.2% have been reported during epidemics of shigella dysenteriae type 1 . The provision of effective anti - microbial therapy is important especially for reducing the prevalence of shigella and other organisms causing dysentery in children . Decreasing the bacterial load excreted by a child with dysentery also reduces the probability of fecal oral transmission to close contacts, such as neighbours, friends or members of the child s household . Anti - microbial therapy is particularly important in developing countries, where prolonged diarrhoea episodes, including dysentery, can significantly decrease the growth and nutritional status in the affected children ., the world health organization (who) recommends that all episodes of diarrhoea with blood in the stool be treated with antibiotics . The who currently recommends treatment with ciprofloxacin (a quinolone) or one of the three second - line antibiotics, pivmecillinam, azithromycin and ceftriaxone (a third - generation cephalosporin). Here, we review the scientific evidence supporting the who - recommended antibiotics ciprofloxacin, ceftriaxone and pivmecillinam for the effective treatment of dysentery . We systematically reviewed all literature published between 1 january 1990 and 31 january 2009 to identify the studies describing the efficacy of ciprofloxacin, ceftriaxone and pivmecillinam for the treatment of dysentery in children aged 5 years . Methods paper), we searched pubmed, cochrane libraries and all who regional databases, including literature published in other languages . Shigella and salmonella. Studies were included if they reported the effect of the antibiotics on severe morbidity as observed by decreased blood in the stool or the effect of the antibiotics on shigella and/or salmonella bacteraemia, in the stool of paediatric dysentery cases . We abstracted data describing study identifiers and context, study design and limitations, intervention specifics and outcome effects, into a standardized abstraction form from any publications that met final inclusion and exclusion criteria (ref . Bacteriologic failure and bacteriologic relapse. Clinical failure was defined as an absence of marked improvement in, or worsening of, illness with the presence of bloody mucoid stools, more than a trace of blood in stool, abdominal pain, tenesmus and/or fever . Bacteriological failure was defined as failure to clear an enteropathogen isolated from an individual on admission to the study, by the end of the treatment period . Bacteriological relapse was defined as the reappearance of an enteropathogen in stool after that enteropathogen was cleared by treatment . Each study was assessed and graded according to the cherg adaptation of the grade technique . One- to two - point grade increases were allotted to studies with statistically significant strong levels of association (> 80% reduction). Any study with a very low final grade laird pooled relative risk and corresponding 95% confidence interval because there was heterogeneity in the study design . We also ran, but did not report, the mantel haenszel pooled relative risk and corresponding 95% ci . We summarized the evidence by outcome, including qualitative assessments of the study quality and quantitative measures, according to the standard guidelines for each outcome . We applied the cherg rules for evidence review to the collective diarrhoea morbidity outcomes to estimate the effects of ciprofloxacin, ceftriaxone and pivmecillinam on eliminating severe morbidity due to diarrhoea in children with dysentery . We identified 586 titles from searches conducted in all databases (figure 1). After screening titles and abstracts because very few studies reported data exclusively for children aged 5 years, we expanded our study population to include children aged up to 16 years . Eight papers were included in the final dataset with some papers contributing data for multiple antibiotics or more than one outcome measure (supplementary table 1). We found eight studies that reported on clinical failure (12 unique data points),, with most studies evaluating clinical failure status 3 days after treatment was initiated (range 36 days). Four studies reported on bacteriological failure (six unique data points),, and five reported on bacteriological relapse (seven unique data points),, (table 1). Figure 1synthesis of study identification to review effect of ciprofloxacin, ceftriaxone and pivmecillinam on diarrhoea treatment failure, bacteriological failure and bacteriological relapse table 1quality assessment of trials of antibiotics for the treatment of diarrhoeaquality assessmentsummary of findingsdirectnessno . Of eventsno . Of studies (ref)designlimitationsconsistency (based on the heterogeneity of the meta - analysis)generalizability to population of interestgeneralizability to intervention of interestinterventioncontrolpooled failure rate (95% ci)clinical failure: moderate outcome - specific quality eight, rctrct but only able to use treatment failure rates (0.5)0.55/12 data points from israel7/12 hospital based; 2/12 multicentre820.1% (0.2 to 0.5%)bacteriological failure: moderate outcome - specific quality four, rctrct but only able to use treatment failure rates (0.5)borderline heterogeneity based on the meta- analysis (p = 0.077) (0.5)3/6 data points from bangladeshgeneralizable90% (0.1 to 0.1%)bacteriological relapse: moderate outcome - specific quality five,,rctrct but only able to use treatment failure rates (0.5)consistent based on meta - analysis3/7 data points from israelgeneralizable70% (0.1 to 0.1%)random effects meta-analysis.rct, randomized controlled trial . Synthesis of study identification to review effect of ciprofloxacin, ceftriaxone and pivmecillinam on diarrhoea treatment failure, bacteriological failure and bacteriological relapse quality assessment of trials of antibiotics for the treatment of diarrhoea random effects meta - analysis . Rct, randomized controlled trial . In table 1, we report the quality assessment of trials by study outcome as well as results from corresponding meta - analyses . Based on 12 data points from eight studies, treatment with one of the three antibiotics resulted in a clinical failure rate of 0.1% (95% ci 0.2 to 0.5%). Based on six datasets abstracted from four studies evaluated in this review, the effect size of antibiotic therapy on a child s relative risk of bacteriological failure is 0% (0.1 to 0.1%). Seven datasets from five studies indicate that the effect size of antibiotic therapy on a child s relative risk of bacteriological relapse is 0.0% (0.1 to 0.1%). Assuming treatment failure rate to be an extremely conservative proxy for dysentery deaths not preventable with prompt antibiotic treatment, it can be estimated that treatment of dysentery with ciprofloxacin, ceftriaxone or pivmecillinam will reduce diarrhoea mortality attributable to dysentery by 99% (figure 2). Figure 2application of the cherg guidelines for the effect of antibiotics on dystenteric morbidity and mortality application of the cherg guidelines for the effect of antibiotics on dystenteric morbidity and mortality diarrhoeal disease, including dysentery, is a major cause of morbidity and mortality among children in developing countries . This systematic review of the literature summarizes the evidence supporting the use of the antibiotics recommended by who: ciprofloxacin, ceftriaxone and pivmecillinam . It also suggests that the bacteria isolated from a stool sample of a child with dysentery rarely relapses if the child has received full - course treatment with one of these antibiotics, and the disease - causing bacteria is sensitive to the antibiotic . Reducing a child s risk of bacteriological relapse is beneficial, because the likelihood of subsequent episodes of dysentery occurring in that child, and of transmission occuring to others, are reduced as a result . The studies contibuting data in this review were conducted in middle- and low - income countries increasing their generalizability to paediatric populations in countries with the highest diarrhoea mortality rates . Extrapolating clinical failure to mortality, our meta - analyses indicate that> 99% of dysentery deaths can be prevented with ciprofloxacin, ceftriaxone or pivmecillinam treatment . For application in the lives saved tool, it is essential to extrapolate severe morbidity to mortality, although this leap has many limitations . Children with functioning immune systems do not always progress to death as a result of dysentery . It is possible for some children to successfully fight the infection without antibiotics and make a full recovery . In addition, many children who present for medical care and are prescribed one antibiotic are put on a second - line treatment if the first choice fails, thus further reducing the treatment failure rate . Nearly, all studies were conducted in a clinic or hospital, where staff could monitor treatment . In a community or outpatient setting, the therapeutic effect of the antibiotics reviewed here may not be as great as our analyses indicate, because caregivers may not comply with the dosage and duration specifications of the treatment . Caregivers may also fail to manage the dehydration that often accompanies diarrhoea, thereby increasing a child s risk of death . The 99% reduction in diarrhoea mortality that we estimate is attributable to the treatment of dysentery with ciprofloxacin, ceftriaxone or pivmecillinam and assumes antibiotic susceptibility . The variability in the types of dysentery - causing organisms that occur worldwide and their sensitivity to the antibiotics recommended for treatment by the who may decrease the generalizability of the findings presented in this review . Because bacteria that cause dysentery can acquire resistance to antibiotics, drugs used for treatment should be selected based on resistance patterns prevalent in the community . Future research with regard to site - specific antibiotic resistance may provide additional data and help refine recommendations for national or local planning . There is strong evidence in favour of the continued use of the antibiotics recommended by who ciprofloxacin, ceftriaxone and pivmecillinam to reduce morbidity and mortality in children with dysentery . Us fund for unicef from the bill & melinda gates foundation (grant 43386 to promote evidence - based decision making in designing maternal, neonatal and child health interventions in low- and middle - income countries). National institutes of health (grant t32hd046405 for international maternal and child health).
Hypertrophic cardiomyopathy (hcm) is a common genetic disease that is characterized by a hypertrophied, nondilated, left ventricular (lv) cavity with normal or supernormal systolic function . However, a small number of hcm patients progress to lv remodeling and systolic dysfunction . Such a condition is referred to as end - stage hcm, which has attracted considerable interest because of its high risk of substantial cardiovascular mortality . The reported prevalence of end - stage hcm varies from 2.4% to 15.7% in different series . Despite recent progress in the differential diagnosis of dilated cardiomyopathy and underlying mechanisms, research focusing on end - stage hcm has been sparse, and the sample size of most studies was small . In addition, risk factors for cardiovascular mortality and the strategies for management of end - stage hcm remain obscure . Accordingly, the prognostic factors of end - stage hcm patients need to be clarified to target early management . Therefore, the purpose of this study was to evaluate the clinical characteristics, prognosis, and risk factors in chinese end - stage hcm patients . This retrospective study included 1844 consecutively enrolled hcm patients from april 2002 to november 2013 at fuwai hospital in beijing . Hcm was diagnosed as documentation of a hypertrophied or nondilated lv (maximum lv wall thickness [mlvwt] 15 mm in adults and the equivalent relative to body surface area in children), at some point during the patients clinical course, in the absence of another cardiac or systemic disease capable of producing a similar magnitude of hypertrophy by echocardiography or cardiac magnetic resonance imaging . End - stage hcm was defined by the detection of a lv ejection fraction (lvef) <50% on echocardiography during follow - up . Patients were excluded for the following reasons: a history of surgical or ablative septal reduction therapy; a history of coronary artery disease or documented coronary arterial narrowing (50% stenosis of at least one major artery by angiography). The magnitude of lv hypertrophy was assessed from two - dimensional images in accordance with the recommendation of the american society of echocardiography . Initial data regarding medical history, electrocardiograms, and echocardiograms at the diagnosis of end - stage hcm were collected . The follow - up data were obtained during serial clinical visits or by interview by telephone . All of the patients provided their informed consent to participate in this research, which was approved by the ethics committee of fuwai hospital . Cardiovascular death was defined as follows: (1) sudden cardiac death (scd), unexpected sudden collapse occurring within 1 h from the onset of symptoms in patients with a previously stable or uneventful clinical course; (2) heart failure - related death, occurring in the context of progressive cardiac decompensation 1 year before death and proceeded by signs and symptoms of heart failure or cardiogenic shock; (3) stroke - related death that occurred as a result of probable or proven embolic stroke; (4) heart transplantation, which was considered equivalent to heart failure - related death; and (5) aborted cardiac arrest or appropriate discharge of an implantable cardioverter - defibrillator (icd) for ventricular fibrillation that was regarded as surrogate scd . Conventional risk factors for sudden death, including a family history of sudden death, mlvwt 30 mm at the initial diagnosis of hcm, syncope, and nonsustained ventricular tachycardia at the diagnosis of end - stage hcm, were calculated for survival analysis . All of the data were expressed as mean standard deviation (sd) or frequency . Comparisons of characteristics between groups were made with the student's t - test, wilcoxon rank - sum test, chi - square test, or fisher's exact test as appropriate . The kaplan - meier method was used to calculate the rate of survival free from the primary survival curves among different patient groups . All reported p values were two - sided, and a p <0.05 was considered as statistically significant . This retrospective study included 1844 consecutively enrolled hcm patients from april 2002 to november 2013 at fuwai hospital in beijing . Hcm was diagnosed as documentation of a hypertrophied or nondilated lv (maximum lv wall thickness [mlvwt] 15 mm in adults and the equivalent relative to body surface area in children), at some point during the patients clinical course, in the absence of another cardiac or systemic disease capable of producing a similar magnitude of hypertrophy by echocardiography or cardiac magnetic resonance imaging . End - stage hcm was defined by the detection of a lv ejection fraction (lvef) <50% on echocardiography during follow - up . Patients were excluded for the following reasons: a history of surgical or ablative septal reduction therapy; a history of coronary artery disease or documented coronary arterial narrowing (50% stenosis of at least one major artery by angiography). The magnitude of lv hypertrophy was assessed from two - dimensional images in accordance with the recommendation of the american society of echocardiography . Initial data regarding medical history, electrocardiograms, and echocardiograms at the diagnosis of end - stage hcm were collected . The follow - up data were obtained during serial clinical visits or by interview by telephone . All of the patients provided their informed consent to participate in this research, which was approved by the ethics committee of fuwai hospital . Cardiovascular death was defined as follows: (1) sudden cardiac death (scd), unexpected sudden collapse occurring within 1 h from the onset of symptoms in patients with a previously stable or uneventful clinical course; (2) heart failure - related death, occurring in the context of progressive cardiac decompensation 1 year before death and proceeded by signs and symptoms of heart failure or cardiogenic shock; (3) stroke - related death that occurred as a result of probable or proven embolic stroke; (4) heart transplantation, which was considered equivalent to heart failure - related death; and (5) aborted cardiac arrest or appropriate discharge of an implantable cardioverter - defibrillator (icd) for ventricular fibrillation that was regarded as surrogate scd . Conventional risk factors for sudden death, including a family history of sudden death, mlvwt 30 mm at the initial diagnosis of hcm, syncope, and nonsustained ventricular tachycardia at the diagnosis of end - stage hcm, were calculated for survival analysis . All of the data were expressed as mean standard deviation (sd) or frequency . Comparisons of characteristics between groups were made with the student's t - test, wilcoxon rank - sum test, chi - square test, or fisher's exact test as appropriate . The kaplan - meier method was used to calculate the rate of survival free from the primary survival curves among different patient groups . Multivariate cox proportional hazards analysis was applied to evaluate the influence of possible predictors . All reported p values were two - sided, and a p <0.05 was considered as statistically significant . End - stage hcm patients were identified in 99 of 1844 (5.4%) patients [table 1], during a follow - up period of 12 9 years after the diagnosis of hcm . The mean age was 44 16 years old (range, 480 years) at initial diagnosis of hcm and 52 16 years old (range, 1482 years) at the diagnosis of end - stage hcm . Of them, 58 (59%) patients had a lv end - diastolic diameter (lvedd) 55 mm, whereas the remaining 41 (41%) patients had a lvedd <55 mm . Left atrial thrombus, and lv mural thrombus were observed in 5 (5%) and 14 (14%) patients, respectively . Patients with a q wave (n = 55) had a larger lvedd (59 13 mm vs. 53 9 mm, p = 0.018) and a lower lvef (42 7 mm vs. 38 7 mm, p = 0.007) compared with patients without a q wave (n = 44). Patients with a q wave had a higher frequency of severe symptomatic heart failure (new york heart association [nyha] class iii / iv, 67% vs. 43%, p = 0.016), sustained ventricular tachycardia / ventricular fibrillation (56% vs. 9%, p = 0.036), and use of amiodarone (55% vs. 44%, p = 0.045) than patients without a q wave . Baseline characteristics of patients at the diagnosis of end - stage hcm arb: angiotensin receptor blocker; hcm: hypertrophic cardiomyopathy; nyha: new york heart association; lbbb / rbbb: left / right bundle branch block; vt: ventricular tachycardia; vf: ventricular fibrillation; lad: left atrial diameter; lvedd: left ventricular end - diastolic diameter; mlvwt: maximum left ventricular wall thickness; ivs: intraventricular septal thickness; pw: posterior wall thickness; lvef: left ventricular ejection fraction; ash: asymmetric septal hypertrophic; con: concentric; ap: apical hypertrophic; lvo: left ventricular obstructive; mvo: midventricular obstructive; acei: angiotensin converting enzyme inhibitor; icd: implantable cardioverter - defibrillator; sd: standard deviation . During a follow - up period of 3.9 3.0 years, all - cause death occurred in 51 (52%) of 99 patients . Among the death events, heart failure - related death was the most common cause (n = 26, including an icds), followed by scd (n = 17), heart transplantation (n = 4), icds (n = 2), stroke - related death (n = 1), and noncardiovascular death (n = 1, lung cancer). In addition, 61 patients were nyha class iii / iv at the last evaluation and 39 patients developed refractory heart failure . Cardiac resynchronization therapy (crt) with or without a defibrillator was implanted in three and two patients, respectively . To evaluate the possible predictors of cardiovascular death for end - stage hcm patients, we divided patients into the cardiovascular death group and the survivor group [table 1]. Kaplan - meier analysis showed that a higher probability of cardiovascular death was observed in patients with nyha class iii / iv (p = 0.018), those with severe systolic dysfunction (lvef 35%) (p = 0.045), and those with the presence of left bundle branch block (lbbb) (p = 0.002) and an abnormal q wave (p = 0.001, figure 2). Kaplan - meier analyses of significant variables on the probability of cardiovascular death in patients with end - stage hypertrophic cardiomyopathy . (a) comparison of survival free of cardiovascular death with or without left bundle branch block; (b) abnormal q wave; (c) new york heart association functional (nyha) class iii / iv; (d) left ventricular ejection fraction 35% . Univariate cox regression analysis showed that the predictors of cardiovascular death included nyha class iii / iv (hazard ratio [hr]: 2.08; 95% confidence interval [ci]: 1.123.87; p = 0.02), and the presence of lbbb (hr: 2.36; 95% ci: 1.324.22; p = 0.004) and a q wave (hr: 2.68; 95% ci: 1.425.04; p = 0.002). In the multivariate model, after adjusting for confounding factors, nyha class iii / iv at the diagnosis of end - stage hcm (hr: 1.99; 95% ci: 1.053.80; p = 0.036), the presence of a q wave (hr: 2.21; 95% ci: 1.164.23; p = 0.016), and lbbb (hr: 2.80; 95% ci: 1.475.31; p = 0.002) were identified as independent predictors of cardiovascular death . Similar results were obtained for sudden death, all - cause death, and heart failure - related death [table 2]. Results of univariate and multivariate cox proportional - hazards analyses of the relation between baseline clinical variables and outcome nyha: new york heart association; lvef: left ventricular ejection fraction; lbbb: left bundle branch block; hr: hazard ration; ci: confidence interval . End - stage hcm patients were identified in 99 of 1844 (5.4%) patients [table 1], during a follow - up period of 12 9 years after the diagnosis of hcm . The mean age was 44 16 years old (range, 480 years) at initial diagnosis of hcm and 52 16 years old (range, 1482 years) at the diagnosis of end - stage hcm . Of them, 58 (59%) patients had a lv end - diastolic diameter (lvedd) 55 mm, whereas the remaining 41 (41%) patients had a lvedd <55 mm . Left atrial thrombus, and lv mural thrombus were observed in 5 (5%) and 14 (14%) patients, respectively . Patients with a q wave (n = 55) had a larger lvedd (59 13 mm vs. 53 9 mm, p = 0.018) and a lower lvef (42 7 mm vs. 38 7 mm, p = 0.007) compared with patients without a q wave (n = 44). Patients with a q wave had a higher frequency of severe symptomatic heart failure (new york heart association [nyha] class iii / iv, 67% vs. 43%, p = 0.016), sustained ventricular tachycardia / ventricular fibrillation (56% vs. 9%, p = 0.036), and use of amiodarone (55% vs. 44%, p = 0.045) than patients without a q wave . Baseline characteristics of patients at the diagnosis of end - stage hcm arb: angiotensin receptor blocker; hcm: hypertrophic cardiomyopathy; nyha: new york heart association; lbbb / rbbb: left / right bundle branch block; vt: ventricular tachycardia; vf: ventricular fibrillation; lad: left atrial diameter; lvedd: left ventricular end - diastolic diameter; mlvwt: maximum left ventricular wall thickness; ivs: intraventricular septal thickness; pw: posterior wall thickness; lvef: left ventricular ejection fraction; ash: asymmetric septal hypertrophic; con: concentric; ap: apical hypertrophic; lvo: left ventricular obstructive; mvo: midventricular obstructive; acei: angiotensin converting enzyme inhibitor; icd: implantable cardioverter - defibrillator; sd: standard deviation . During a follow - up period of 3.9 3.0 years, all - cause death occurred in 51 (52%) of 99 patients . Among the death events, heart failure - related death was the most common cause (n = 26, including an icds), followed by scd (n = 17), heart transplantation (n = 4), icds (n = 2), stroke - related death (n = 1), and noncardiovascular death (n = 1, lung cancer). In addition, 61 patients were nyha class iii / iv at the last evaluation and 39 patients developed refractory heart failure . Embolic stroke was observed in 20 patients and peripheral artery thrombus in two patients . Cardiac resynchronization therapy (crt) with or without a defibrillator was implanted in three and two patients, respectively . To evaluate the possible predictors of cardiovascular death for end - stage hcm patients, we divided patients into the cardiovascular death group and the survivor group [table 1]. Kaplan - meier analysis showed that a higher probability of cardiovascular death was observed in patients with nyha class iii / iv (p = 0.018), those with severe systolic dysfunction (lvef 35%) (p = 0.045), and those with the presence of left bundle branch block (lbbb) (p = 0.002) and an abnormal q wave (p = 0.001, figure 2). Kaplan - meier analyses of significant variables on the probability of cardiovascular death in patients with end - stage hypertrophic cardiomyopathy . (a) comparison of survival free of cardiovascular death with or without left bundle branch block; (b) abnormal q wave; (c) new york heart association functional (nyha) class iii / iv; (d) left ventricular ejection fraction 35% . Univariate cox regression analysis showed that the predictors of cardiovascular death included nyha class iii / iv (hazard ratio [hr]: 2.08; 95% confidence interval [ci]: 1.123.87; p = 0.02), and the presence of lbbb (hr: 2.36; 95% ci: 1.324.22; p = 0.004) and a q wave (hr: 2.68; 95% ci: 1.425.04; p = 0.002). In the multivariate model, after adjusting for confounding factors, nyha class iii / iv at the diagnosis of end - stage hcm (hr: 1.99; 95% ci: 1.053.80; p = 0.036), the presence of a q wave (hr: 2.21; 95% ci: 1.164.23; p = 0.016), and lbbb (hr: 2.80; 95% ci: 1.475.31; p = 0.002) were identified as independent predictors of cardiovascular death . Similar results were obtained for sudden death, all - cause death, and heart failure - related death [table 2]. Results of univariate and multivariate cox proportional - hazards analyses of the relation between baseline clinical variables and outcome nyha: new york heart association; lvef: left ventricular ejection fraction; lbbb: left bundle branch block; hr: hazard ration; ci: confidence interval . The present study showed that the morphological features of end - stage hcm appeared to be more diverse than previously thought . The phase of end - stage hcm showed a varied prognosis, but overall, proved to be largely unfavorable . In addition, nyha functional class at entry and the presence of lbbb and a q wave in electrocardiography were independent predictors of cardiovascular mortality in end - stage hcm . In accordance with our result, the reported incidence of end - stage hcm is relatively uniform, ranging from 0.5% to 1.5% of patients with hcm per year . Also, restrictive - hypokinetic morphological end - stage hcm was comparable with the dilated - hypokinetic subtype . In addition, we found that midventricular obstructive hcm was a main subtype of evolution into the end - stage phase, secondary to asymmetric septal hypertrophy . As previously shown, midventricular obstructive hcm is disposed to develop systolic dysfunction, especially with apical aneurysms . In addition, lv mural thrombus was present in 14% of patients without ischemic or dilated cardiomyopathy . This finding suggested that the dyskinetic or akinetic walls provide a substrate for ventricular mural thrombus formation in end - stage hcm . Our results also suggested that a simple electrocardiogram could be a reliable prognostic predictor of end - stage hcm . The presence of abnormal q waves was also closely related to ventricular enlargement and systolic dysfunction in hcm patients, in accordance with our results . In addition, our study indicated that an abnormal q wave was a risk factor for mortality in end - stage hcm . To date, there are two underlying mechanisms of abnormal q waves in hcm: loss of electrical forces due to transmural myocardial fibrosis, and an altered direction of the resultant initial qrs vector due to increased electrical forces of disproportionate hypertrophy . However, a relationship between the location and severity of lv hypertrophy and the presence of abnormal q waves is controversial . Recently, an increasing amount of studies have identified late gadolinium enhancement (equal to myocardial fibrosis) by cmr as a risk factor for sudden death and development of the end - stage phase in patients with hcm . In addition, papavassiliu et al . Found that the segmental and transmural extent of late gadolinium enhancement rather than the mere presence of myocardial late gadolinium enhancement is the underlying mechanism of abnormal q waves in hcm . Therefore, abnormal q waves are associated with an unfavorable prognosis in end - stage hcm, probably by extensive myocardial fibrosis ., we observed that the presence of lbbb was associated with a poorer prognosis in multivariate analysis, supporting the prognostic importance of lbbb in hcm patients from a national study in japan . Lbbb is defined as a marker of unfavorable prognosis in chronic heart failure, mainly due to contractive asynchrony . In hcm, inter- and intra - ventricular asynchrony additionally, lbbb is associated with more marked lv dilation, depressed lvef, and mitral valve regurgitation in patients with heart failure . Therefore, we hypothesized that lbbb is a new marker for lv systolic dysfunction with a poor prognosis in end - stage hcm . Recently, biventricular pacing was reported to improve heart failure symptoms and reverse remodeling in end - stage hcm patients with lbbb in case reports and a series report . In view of these findings, crt is considered for hcm patients with refractory symptoms, lvef <50%, and lbbb in the newly enacted european society of cardiology guideline (class iib, level c). In addition, our study showed that the risk of mortality of mild systolic dysfunction was equal to that of severe systolic dysfunction by echocardiography, which strengthened the importance of early crt management, even in mild systolic dysfunction . Therefore, the presence of lbbb in an electrocardiogram is an indicator of routine crt implantation in end - stage hcm for an early management of refractory heart failure . Previous studies have indicated direct correlations of conventional risk factors (left ventricular wall thickness and syncope) with cardiovascular death in end - stage hcm . However our results supported recent findings that these risk factors might not necessarily be predictors for sudden death or cardiovascular death and that they should not be considered as isolated risk factors for cardiovascular mortality in patients with hcm . As proposed by olivotto et al ., mlvwt may be a risk factor for sudden death only in patients diagnosed with hcm at a young age . A high proportion of included pediatric patients (39%) can explain the difference between a previous study and our study . In addition, the different definitions of endpoint and length of follow - up could change the composition of mortality . Patients in end - stage hcm are at higher risk of atrial fibrillation (54%) than in general hcm (2030%). Lv hypertrophy aggravated the incidence of left atrial thrombus in atrial tachyarrhythmia and additional frequent mural thrombus complicates embolic events, even in sinus rhythm . Atrial fibrillation is still a strong risk factor for embolic stroke in hcm . Because of left atrial enlargement is a marker of susceptibility for atrial fibrillation, we strongly recommended anticoagulants for each patient with end - stage hcm . In addition, the icd was not effectively implanted as required for primary prevention in most patients . This indicated that strong interventions will be effective for improving the prognosis of end - stage hcm . These interventions include health education, financial support, intensive surveillance, icd or crt implantation, and heart transplantation . First, our study was performed in a referral hospital, and selective bias was inevitable . Second, genetic analysis was not undertaken in this cohort despite the increasing value of genetic testing in hcm . Third, late enhancement analysis by cardiac magnetic resonance imaging was not available in our study, and its prognostic significance was overlooked . Finally, the follow - up time was relatively short . Therefore, more detailed data on a larger multicenter scale are encouraged to evaluate the detailed risk factors related to end - stage hcm . In conclusion, in end - stage hcm, atrial fibrillation, mural thrombus, and thromboembolic events are fairly frequent incidents . Heart failure - related death and sudden death are the major outcomes in end - stage hcm . Patients with lbbb and an abnormal q wave have a high probability of cardiovascular death and need early targeted management for mild systolic dysfunction.
Oral drug delivery systems are essential to optimize both the residence time of the system within the gastrointestinal tract and the release rate of the drug from the system . Various attempts have been made to prolong the residence time of the dosage forms within the stomach . Rapid gastrointestinal transit could result in incomplete drug release from the drug delivery system to absorption window leading to diminished efficacy of the administered dose . Prolonged gastric retention is important in achieving control over the gastric residence time because it helps to maintain the controlled release system in the stomach for a longer time in an expected manner . Floating systems (hydrodynamically controlled systems) are low - density systems which means they are less dense than gastric fluid . These systems have sufficient buoyancy to float over the gastric contents and remain buoyant in the stomach for a prolonged period of time without disturbing the gastric emptying rate . While the formulation is floating on the gastric contents, drug is released slowly from it at a desired rate [3, 4]. For oral sustained release, multiple unit dosage forms (i.e., microballoons) are more beneficial than single unit dosage forms . Because single unit dosage forms have the disadvantage of a release all or nothing emptying process and multiple unit dosage forms have advantages of disperse widely and release drug uniformly along the gastrointestinal tract, which results in more reproducible drug absorption, less dose dumping, and reduced risk of local irritation than the use of single unit dosage form [57]. These microballoons are characteristically free flowing powders consisting of proteins or synthetic polymers, ideally having a size less than 200 m . Pentoxifylline (ptx), trisubstituted xanthine derivative (3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1h - purine-2,6-dione), is a hemorheologic agent used for the treatment of peripheral arterial disease and intermittent claudication . The apparent plasma half - life of the drug and its metabolite is 2 - 3 hours . On the basis of using ptx as drug of choice in chronic occlusive aterial diseases, it is of a wise candidate drug to be formulated in sustained release oral dosage form . Thus, the aim of the present research work was to design, develope, and characterization of pentoxifylline loaded floating microballoons . Mumbai, india . Ethyl cellulose, hydroxypropyl methylcellulose (hpmc k4 m), and tween 80 were purchased from central drug house (cdh), new delhi, india . All other solvents and chemicals were of analytical grade . Pentoxifylline, hpmc k4 m, and ethyl cellulose were dissolved in a mixture of ethanol and dichloromethane at room temperature . These were poured into 250 ml of water containing 0.01% tween 80 maintained at a temperature of 3040c and consequently stirred at ranging agitation speed to allow the volatile solvent to evaporate . The formulated microballoons were filtered, washed with distilled water, and dried at 40c . The size of microballoons of each formulation was determined using a microscope fitted with an ocular micrometer, and stage micrometer and average particle size was determined . The surface morphology of microballoons was examined by scanning electron microscopy (jeol jsm-1600, tokyo, japan) operated at 15 kv on samples gold sputtered at 10 ma, under argon at low pressure . The weight of microballoons was divided by the total weight of all the nonvolatile components that were used for the preparation of the microballoons and multiplied by 100 gives the% yield of microballoons as follows: (1)% yield=(weight of microballoons collected) (weight of all nonvolatile components used for the preparation)1100 . Percentage loading efficiency . To determine loading efficiency, microballoons were taken, thoroughly triturated, and suspended in a minimal amount of alcohol . The estimation of drug was carried out using uv spectrophotometer (uv - vis double beam spectrophotometer 2201, systronics) at 272 nm max . The percentage loading efficiency was calculated as follows: (2)loading efficiency (%) = amount of drug actually presenttheoretical drug load expected100 . The buoyancy test of the microballoons was carried out using usp ii (paddle type) dissolution apparatus (ds 8000, labindia). Dissolution test solution simulated gastric fluid (sgf) containing tween 80 (0.02% v / v) was used as a dispersion medium to simulate gastric fluid . The microballoons were spread over the surface of the sgf, ph 1.2 (900 ml, 37 0.5c), which was agitated by a paddle rotated at 100 rpm for 12 h. after agitation for a previously determined interval, the microballoons that were floating and the ones that settled to the bottom of the flask were recovered separately . After drying, the fraction of the microballoons was weighed . The% buoyancy of the microballoons was calculated by the following formula: (3)% buoyancy = weight of floating microballoons after dryingweight of floating+settled microballoons after drying 100 . In vitro drug release studies . The dissolution test was performed using 0.1 n hcl (ph 1.2) as dissolution fluid (900 ml) maintained at 37 0.5c at 100 rpm . The samples (5 ml) of the solution were withdrawn from the dissolution apparatus for 12 h, and the samples were replaced with fresh dissolution medium each time to maintain the sink condition . Withdrawn samples were analyzed using uv - vis double beam spectrophotometer at 272 nm against suitably constructed calibration curve . All measurements were carried out in triplicate, and average values were plotted . Statistical analysis . Two - way analysis of variance (anova) was applied to check significant differences in drug release from different formulations . The kinetic models used were zero order (cumulative percentage of drug release versus time), first order (log cumulative percentage of drug remaining versus time), the higuchi model (cumulative percentage of drug release versus square root of time), and korsmeyer - peppas (log cumulative percent drug release versus log of time). Regression (r) values were calculated for the linear curves obtained by regression analysis . Preparation of pentoxifylline loaded floating microballoons was done by using hpmc k4 m and ethyl cellulose as sustained release polymers by the solvent evaporation technique . Ethanol and dichloromethane were used as solvents to keep both polymers and drug in solution . The solution was poured with the help of syringe into 250 ml water containing 0.01% tween 80 maintained at a temperature of 3040c and subsequently stirred at ranging agitation speed to allow the volatile solvent to evaporate . The formulations f1, f2, and f3 were formulated by varying the concentration of ethyl cellulose, and formulations f4, f5, and f6 formulated by varying the concentration of hpmc k4 m . Microballoons with higher concentration of ethyl cellulose gave much retarded drug release than higher concentration of hpmc k4 m . From the result of this study, the average particle size of microballoons were found to be 74.63 1.04, 85.18 3.12, and 104.0 2.87 for f1, f2, and f3 formulations and 82.96 2.13, 99.32 1.45, and 110.4 2.94 for f4, f5, and f6 formulations, respectively . This is due to the increase in viscosity of the solution and the decrease in stirring efficiency . Also with increasing polymer concentration, therefore, a shorter time was provided for the breakup of droplets, and larger microballoons were formed . The scanning electron microphotograph showed that the developed floating microballoons were spherical with porous surface which facilitate diffusion of drug as shown in figure 1 . Production yields were found to be 75.76 1.54, 78.13 1.21, 80.89 2.24, 76.79 1.38, 74.66 2.61, and 72.57 1.85 for f1, f2, f3, f4, f5, and f6 formulations, respectively, as shown in table 2 . The percentage loading efficiencies were found to be 75.5 1.82, 76.36 1.27, 77.85 0.61, 76.22 0.82, 77.29 0.12, and 77.66 1.35% for f1, f2, f3, f4, f5, and f6 formulations, respectively . The buoyancy percentage for all batches was almost above 70%, which was studied for 12 h. the highest percentage was obtained with formulation f6 . Average buoyancies in percentage were found to be in the range of 72.43 0.21% to 78.19 0.63% for f1 to f6 formulations . In general, with the increase in the amount of polymers, there was an increase in the buoyancy percentage . The increase in the buoyancy percentage may be attributed to air and gel - forming polymer hpmc k4 m which caused swelling because of increased amount of the polymers present . The in vitro drug release of formulations f1, f2, f3, f4, f5, and f6 was found to be 96.81 0.16, 88.84 0.46, 82.21 1.29, 93.13 1.48, 90.16 0.98, and 87.09 1.73 in 12 h, respectively . Results indicate that proportion of polymers in formulation was the key factor governing the release of drug from microballoons . As the concentration of polymer increased, there was an increase in diffusional path length . Formulations comprised of ethyl cellulose in higher proportion exhibited much retarded drug release as compared to formulations comprised of hpmc k4 m in higher proportion . The release profile of pentoxifylline from microballoons for all formulations was shown in figure 2 . The release profile of pentoxifylline from microballoons containing varying concentrations of ethyl cellulose and hpmc two - way analysis of variance (anova) was applied to check significant differences in drug release from different formulations containing different concentrations of hpmc k4 m and ethyl cellulose . On increasing the amount of polymers, a significant decrease (p <0.05) was obtained in the cumulative drug release . The kinetics and mechanism of drug release were determined using zero order, and first order, higuchi's model, and further analysis was performed using korsmeyer - peppas equation . All formulations were found to be following higuchi's model as the plot showed high linearity (r = 0.985 to 0.991) as shown in table 3 . This equation indicates that the cumulative amount of drug release is proportional to the square root of time for diffusional release of drug from the formulation . N values from the power law equation (korsmeyer - peppas equation) for drug release profiles were between 0.776 and 0.842, suggesting that drug release mechanism from formulations followed the non - fickian (anomalous) transport mechanism, which may indicate that diffusion was predominant mechanism of drug release . The release kinetic data obtained from different plots of models for all formulations are given in table 3 . Floating microballoons of pentoxifylline were prepared by the solvent evaporation technique using different concentrations of polymers like hpmc k4 m and ethyl cellulose (ec) dispersed in ethyl alcohol and dichloromethane as a solvent system . Prepared floating microballoons showed significant floating ability, good buoyancy, and sustained drug release . In vitro drug release of microballoons was influenced by polymers concentration . From the percentage loading efficiency and in vitro drug release studies, it was observed that f3 formulation exhibits greater drug loading efficiency and sustained release behavior . On fixing the in vitro drug release data of optimized formulation to various kinetic models, it was found that it exhibits the higuchi order of kinetics followed by zero order and first order . The formulation undergoes anomalous (non - fickian) diffusion, which indicates that the drug release rate was controlled by swelling, erosion, and diffusion from microballoons . Thus, pentoxifylline loaded floating microballoons can prove to be potential pharmaceutical dosage form for prolonging the gastric retention time of dosage form.
Hemimandibular hypertrophy along with its variants was first described by obwegeser and makek as a developmental deformity of unknown etiology affecting the mandible unilaterally . Various studies have assumed lowered genetic control over the formation and development of bilateral structures of the face or environmental inuences and accidents during development to be the cause . A 34-year - old male patient reported to the out - patient department with a complaint of asymmetric appearance of his face . He had been involved in a road accident 15 years ago that had caused severe trauma to his chin . There were no other contributory findings in his dental, medical, and family history . On clinical examination, marked facial asymmetry was observed in the lower region of the face with deviation of the chin to the left . The mandibular body and ramus appeared longer and wider on the right side as compared to the left side [figure 1a]. Posterio - anterior view of the skull demonstrated elongation of the condyle on the right side . Height of the ramus and body of the mandible were also larger along with transverse canting of the occlusal plane on the right side [figure 1b]. Three - dimensional computed tomography (ct) image showed elongation and enlargement of condyle, ramus, and body of the mandible on the right side [figure 1c]. Scintigram demonstrated hot spots indicative of increased uptake of radioisotope in the condylar region on the right side [figure 1d]. Thus on the basis of clinical and radiographic features, diagnosis of hemimandibular hypertrophy - elongation hybrid variant of the right side was confirmed . Patient was referred to the oral and maxillofacial surgery department, where orthognathic surgery was done and both function and esthetics of the patient's facial structure were restored [figure 1e]. 34-year - old male patient presenting with facial asymmetry due to hemimandibular hypertrophy hybrid variant . (b) x - ray posterio - anterior view of skull shows mandibular enlargement and transverse canting of occlusal plane on the right side (white arrow). (c) three - dimensional computed tomography shows right sided hemimandibular hypertrophy along with bilateral crossbite . (d) scintigram demonstrates hot spot (arrow) in the condylar region, indicative of increased activity in the mandible.on the right side . An 18-year - old female patient reported to the out - patient department with a complaint of gradually developing asymmetry of her face . She first noticed asymmetry of her face after a trauma she suffered about 5 years earlier . Since then the condition had gradually deteriorated and attained the present status . No information relevant to the condition was elicited from past dental, medical, and family history . On extra - oral examination, marked facial asymmetry was observed in the lower face region with deviation of chin toward the left side . On intra - oral examination, mandibular central incisor midline was found not to coincide with facial midline . Panoramic radiograph revealed elongation of condyle, condylar neck, ramus and body of mandible on the right side . Horizontal dimension of the mandibular body was also larger as compared to that on the left side . The distance between the mandibular roots and the inferior alveolar nerve on the right side was more than that on the left side [figure 2c]. Further, ct demonstrated elongation and medial rotation of right lower border of the mandible [figure 2d and e]. On the basis of all these findings, case 2 . 18-year - old girl with asymmetry of the face due to hemimandibularr hypertrophy hybrid.variant . (a) photograph of the face shows enlargement of mandible on the right side . (c) panoramic radiograph demonstrates elongation (arrow) and increased distance between the mandibular molar roots and inferior alveolar nerve canal on the right side (double - headed arrow). (d) three - dimensional computed tomography (ct) shows elongation and enlargement of the mandible on the right side (arrow). (e) ct axial view reveals asymmetry due to increased growth of the mandible on the right side (arrow). A 34-year - old male patient reported to the out - patient department with a complaint of asymmetric appearance of his face . He had been involved in a road accident 15 years ago that had caused severe trauma to his chin . There were no other contributory findings in his dental, medical, and family history . On clinical examination, marked facial asymmetry was observed in the lower region of the face with deviation of the chin to the left . The mandibular body and ramus appeared longer and wider on the right side as compared to the left side [figure 1a]. Posterio - anterior view of the skull demonstrated elongation of the condyle on the right side . Height of the ramus and body of the mandible were also larger along with transverse canting of the occlusal plane on the right side [figure 1b]. Three - dimensional computed tomography (ct) image showed elongation and enlargement of condyle, ramus, and body of the mandible on the right side [figure 1c]. Scintigram demonstrated hot spots indicative of increased uptake of radioisotope in the condylar region on the right side [figure 1d]. Thus on the basis of clinical and radiographic features, diagnosis of hemimandibular hypertrophy - elongation hybrid variant of the right side was confirmed . Patient was referred to the oral and maxillofacial surgery department, where orthognathic surgery was done and both function and esthetics of the patient's facial structure were restored [figure 1e]. 34-year - old male patient presenting with facial asymmetry due to hemimandibular hypertrophy hybrid variant . (b) x - ray posterio - anterior view of skull shows mandibular enlargement and transverse canting of occlusal plane on the right side (white arrow). (c) three - dimensional computed tomography shows right sided hemimandibular hypertrophy along with bilateral crossbite . (d) scintigram demonstrates hot spot (arrow) in the condylar region, indicative of increased activity in the mandible.on the right side . An 18-year - old female patient reported to the out - patient department with a complaint of gradually developing asymmetry of her face . She first noticed asymmetry of her face after a trauma she suffered about 5 years earlier . Since then the condition had gradually deteriorated and attained the present status . No information relevant to the condition was elicited from past dental, medical, and family history . On extra - oral examination, marked facial asymmetry was observed in the lower face region with deviation of chin toward the left side . On intra - oral examination, mandibular central incisor midline was found not to coincide with facial midline . Panoramic radiograph revealed elongation of condyle, condylar neck, ramus and body of mandible on the right side . Horizontal dimension of the mandibular body was also larger as compared to that on the left side . The distance between the mandibular roots and the inferior alveolar nerve on the right side was more than that on the left side [figure 2c]. Further, ct demonstrated elongation and medial rotation of right lower border of the mandible [figure 2d and e]. On the basis of all these findings, (a) photograph of the face shows enlargement of mandible on the right side . (c) panoramic radiograph demonstrates elongation (arrow) and increased distance between the mandibular molar roots and inferior alveolar nerve canal on the right side (double - headed arrow). (d) three - dimensional computed tomography (ct) shows elongation and enlargement of the mandible on the right side (arrow). (e) ct axial view reveals asymmetry due to increased growth of the mandible on the right side (arrow). Obwegeser and makek classified condylar hyperplasia into two distinct types namely hemimandibular hypertrophy and hemimandibular elongation . Later, certain cases were seen with overlapping features and were categorized as a hybrid variant that showed a combination of hemimandibular hypertrophy and hemimandibular elongation . Hemimandibular elongation presents with a marked lateral displacement of the chin to the unaffected side and depressed commissure of lips on the affected side . Due to the asymmetric shift of underlying skeletal component to one side, occlusion gets de - arranged and clinically the mandibular dental centerline does not coincide with the midfacial line . Crossbite is observed on the unaffected side . Since there is minimal or almost no discrepancy in the vertical component to the abnormal mandibular growth, open bites or transverse canting of the maxillary occlusal plane are not evident . Radiographically, there is elongation of the body of the mandible on the affected side . Differences between hemimandibular hypertrophy and hemimandibular elongation hemimandibular hypertrophy is a three - dimensional developmental enlargement of one side of the mandible including the condyle, condylar neck, ramus and body along with medial rotation . The anomaly terminates exactly at the symphysis of the affected side and for this reason it is called hemimandibular hypertrophy . It is distinct from hemimandibular elongation as the latter shows increase only in the horizontal component . The etiology of hemimandibular hypertrophy is still under discussion . In the literature, genetic factors, circulatory problems, hormonal disturbances, traumatic lesions and arthrosis there is an increase in the height of the affected side, giving the face a rotated appearance . If the anomaly occurs before adolescent growth spurt then the maxilla follows the downward growth of the mandible and the teeth of the affected side are at a lower level, resulting in tilting of the occlusal plane in the transverse dimension . If the maxilla is unable to follow the mandibular growth, an open bite becomes evident on the affected side . Panoramic radiograph shows that both the ascending ramus and condyle are elongated vertically along with thickening of the condylar neck . The inferior border is bowed downward to a lower level compared to the unaffected side . Due to elongation of the mandibular body, the distance between the molar roots and the mandibular canal is increased . The thickness of the proliferative zone increases, the fibro - cartilaginous zone becomes hypertrophic, endochondreal bone formation occurs while the articular zone remains remarkably intact . It can be used to determine the side that is affected, to demarcate between abnormal condylar growth and generalized mandibular growth, and finally to assess whether hyperplasia is still active or has become stable . Hybrid forms are the combination of unilateral hemimandibular hypertrophy and hemimandibular elongation and lead to marked facial asymmetry . Such cases present with both vertical elongation and lateral displacement, causing large degree of facial asymmetry (seen in case 2). Due to overlapping features, there are certain conditions such as hemifacial hypertrophy, hemifacial atrophy of opposite side, hyperactive condyle, monostotic fibrous dysplasia and segmental odontomaxillary dysplasia that need to be considered in differential diagnosis . In cases of hemifacial hypertrophy, entire half of the face is involved and the affected side shows enlarged teeth with rapid eruption of dentition . Other features include premature loss of primary teeth along with enlarged tongue and alveolar bone . Secondly, it is often associated with mental deficiency, skin abnormalities, compensatory scoliosis, wilms tumor and beckwith - weidemann syndrome . Monostotic fibrous dysplasia and segmental odontomaxillary dysplasia can be differentiated due to their unique radiographic features . . Acquired mandibular asymmetries involve pain, symptomatic changes, alterations in facial appearance and function with time . Other features include temporomandibular joint (tmj) crepitation, limited mandibular movements, severe crossbite and irregular condyle anatomy . Whereas, developmental changes do not involve pain, symptoms usually remain unchanged over time, no functional changes take place in the tmj, there may be limited protrusion without limiting mandibular rotation movements, a pronounced dental compensation in the asymmetric mandible may be present and the condyle remains pronounced and smooth, even in the presence of volumetric changes . Mandibular hypertrophy can be seen in proteus syndrome that is characterized by striking facial abnormalities, one of two subforms show unilateral condylar overgrowth causing progressive craniofacial asymmetry . Unilateral overgrowth of mandible can be clearly seen in female patients with klinefelter's syndrome (47, xxy). Finally, there are craniofacial malformations that do not affect the jaws directly, but lead to indirect, presumably compensatory, alterations of otherwise relatively normal condylar growth . For example, in cases of craniosynostoses such as crouzon, pfeiffer, and apert syndromes, unusual transverse mandibular growth may be regarded as an attempt at adapting to the impaired expansion of the cranial vault . Treatment for hemimandibular hypertrophy varies according to the age of the patient . In a growing child, prime goal is to obtain a proper functional occlusion, which is achieved by occlusal adjustments and maxillary expansion . However, in an adult patient apart from orthodontic procedures, an additional muscle memory deprogramming with diagnostic splints is indicated to set the centric relation position . Patients suffering from hemimandibular hypertrophy often seek seclusion due to abnormal shape of the face and thus early diagnosis with computed tomography can aide in identifying the underlying malformations.
A 65-year - old male patient presented to us with diminution of vision of one - day duration in the right eye . The best - corrected - visual - acuity (bcva) in the right eye was 10/200 . Fundus of the eye showed an smh along with a large pigmentary epithelial detachment (ped) [fig . Leakage of the dye on indocyanine green angiography (icg) and variable reflectivity on optical coherence tomography (oct) suggested a subfoveal cnvm [fig . 2]. Red free image of right eye showing submacular hemorrhage (blue arrow) along with pigmentary epithelial detachment (red arrow) optical coherence tomography showing variable reflectivity of the retinal pigmentary epithelium (blue arrow) suggestive of choroidal neovascular membrane with subretinal blood and large pigmentary epithelial detachment (red arrow) a 62-year - old male presented with sudden diminution of vision in the right eye since one week . Oct showed variable reflectivity of the retinal pigment epithelium (rpe) along with an increase in the retinal thickness suggestive of subfoveal cnvm with macular edema . A 35-year - old female presented to us with diminution of vision in the left eye since seven days . Patient had undergone refractive surgery for myopia 10 years back . Left eye fundus showed a subretinal hemorrhage and oct showed hyper - reflectivity in the rpe suggestive of cnvm . A 65-year - old female came to us with complaint of diminution of vision in the left eye since one week . . Left eye fundus showed smh along with chorio - retinal atrophy at the edge . Ffa showed blocked fluorescence in the area of hemorrhage with late hyperfluorescence of the chorioretinal atrophy . All patients were given an intravitreal injection of 1.25 mg or 0.05 ml bevacizumab (one vial of bevacizumab contains 100 mg in 4 ml) along with pneumatic displacement of the smh by 0.4 - 0.5 ml of sf6 [micro sf, micromed s.r.l, italy]. Intravitreal gas was injected using 30 g needles (pricon, iscon surgicals ltd ., a 65-year - old male patient presented to us with diminution of vision of one - day duration in the right eye . The best - corrected - visual - acuity (bcva) in the right eye was 10/200 . Fundus of the eye showed an smh along with a large pigmentary epithelial detachment (ped) [fig . Leakage of the dye on indocyanine green angiography (icg) and variable reflectivity on optical coherence tomography (oct) suggested a subfoveal cnvm [fig . 2]. Red free image of right eye showing submacular hemorrhage (blue arrow) along with pigmentary epithelial detachment (red arrow) optical coherence tomography showing variable reflectivity of the retinal pigmentary epithelium (blue arrow) suggestive of choroidal neovascular membrane with subretinal blood and large pigmentary epithelial detachment (red arrow) a 62-year - old male presented with sudden diminution of vision in the right eye since one week . Oct showed variable reflectivity of the retinal pigment epithelium (rpe) along with an increase in the retinal thickness suggestive of subfoveal cnvm with macular edema . A 35-year - old female presented to us with diminution of vision in the left eye since seven days . Patient had undergone refractive surgery for myopia 10 years back . Left eye fundus showed a subretinal hemorrhage and oct showed hyper - reflectivity in the rpe suggestive of cnvm . A 65-year - old female came to us with complaint of diminution of vision in the left eye since one week . . Left eye fundus showed smh along with chorio - retinal atrophy at the edge . Ffa showed blocked fluorescence in the area of hemorrhage with late hyperfluorescence of the chorioretinal atrophy . All patients were given an intravitreal injection of 1.25 mg or 0.05 ml bevacizumab (one vial of bevacizumab contains 100 mg in 4 ml) along with pneumatic displacement of the smh by 0.4 - 0.5 ml of sf6 [micro sf, micromed s.r.l, italy]. Intravitreal gas was injected using 30 g needles (pricon, iscon surgicals ltd ., treatment, if delayed, leads to poor recovery due to the delay in diagnosis and treatment of the primary pathology as well as due to the harmful effect of persistent subretinal blood on photoreceptors . Treatment options have included cnvm and hemorrhage removal with forceps, pneumatic displacement with/ without intravitreal tissue plasminogen activator (tpa), injection of subretinal tpa with displacement by perfluorocarbon liquid, injection of subretinal tpa followed by hemorrhage evacuation, vitrectomy with intravitreal/ subretinal injection of tpa with an intraocular gas bubble to help displace the hemorrhage inferiorly . Previously, pneumatic displacement was the treatment of choice in patients of smh in cnvm, at our center . Subsequent management of the underlying pathology used to be done in a second sitting . With the widespread use of intravitreal bevacizumab in treatment of wet amd, we decided to combine the two approaches for a faster visual recovery . Intravitreal injection of sf6 displaces the hemorrhage from the macular area, allowing for increase in visual acuity as well as better visualization of the primary pathology . India being a developing country with limited availability and high cost of tpa, bevacizumab seemed a good alternative . This resulted in a better definitive delineation of the primary pathology on subsequent ffa and oct [figs . 3 and 4]. Red free image of the right eye showing clearing of submacular hemorrhage after intra - vitreal bevacizumab and sf6 injection (red arrow) optical coherence tomography of the same patient showing normal reflectivity of the retinal pigmentary epithelium after the procedure with normal foveal contour (red arrow) gopalakrishan et al ., have reported a series of 20 cases of smh who were treated with pneumatic displacement out of which five had amd . In these patients in contrast, all the four cases in our series had good recovery of visual acuity . This is an observation with very few patients but seems a promising technique in patients of amd with smh . Since earlier studies have had poor results with the other techniques available, more patients need to undergo this treatment for a better analysis of outcome.
Infantile systemic hyalinosis (ish) is a rare and fatal genetic disorder with mutations in capillary morphogenesis gene-2 cmg2 / human anthrax toxin receptor gene-2 antxr2 resulting in spindle cell proliferation, altered collagen metabolism with extensive deposition of amorphous eosinophilic pas positive hyaline material in the connective tissues of various organs . The common presenting features would be progressive stiffness of multiple joints, skin lesions, multiple episodes of protracted infections, prolonged diarrhoea and failure to thrive . Ish is a rapidly progressive painful disorder of infancy with a very short life expectancy . 3 months old identical twins born to a 5th degree consanguinous couple were brought with complaints of excessive cry, deformity of all four limbs, recurrent episodes of respiratory tract infections and diarrhoea since birth . Evaluation of both the probands revealed facial dysmorphism with perinasal nodules, gingival hypertrophy, fixed deformities of multiple joints bilaterally, umbilical hernia, fleshy perianal nodules and pigmented patches over knuckles and ankle . A clinical diagnosis of ish was suspected and confirmed by detection of homozygous c.277_278insattattt (or p.l93yfs14) in exon 3 of the antxr2 gene . The probands were managed symptomatically and parents were counselled regarding prenatal diagnosis in future pregnancies . Ihs is commonly misdiagnosed as arthrogryposis multiplex congenita and is often mismanaged with manipulation of the stiff joints and invasive surgical procedures . Prenatal diagnosis by chorionic villus biopsy is possible once causative mutation in a family is identified . Invasive surgical interventions for histopathological analysis can be avoided as clinical features are most often classical and genetic analysis is confirmatory . We report this case of identical twins with features of ish in view of its rarity as timely clinical suspicion can avoid painful and invasive procedures for diagnosis and management . Infantile systemic hyalinosis (ish) is a rare autosomal recessive genetic disorder caused by biallelic mutations in the antxr2 gene . It is exemplified by progressive deposition of amorphous / hyaline material in various connective tissues usually affecting subcutaneous regions on the scalp, ears, neck, face, hands, feet, and manifests as pearly papules or fleshy nodules with severe and painful restriction of joint movement, gingival hypertrophy and systemic involvement of varying severity . Ish is a rapidly progressive painful disorder of infancy with a very short life expectancy . It is allelic to a condition previously called juvenile hyaline fibromatosis, which is now known to be a later onset, relatively milder phenotype with less frequent systemic involvement and improved survival . The largest series reported till date was of 19 cases and most of them were offsprings of consanguinous couples . We report a case of identical twins with features of ish, a rare entity to cite in the literature . 1) born to a consanguinous couple (fig 2: pedigree) were brought with complaints of excessive cry, deformity of all four limbs, recurrent episodes of upper respiratory tract infections and diarrhoea since birth . Any attempt of manipulation by parents lead to excessive cry, without any relief from stiffness . Both the children were examined by several doctors and were referred to us with a query of arthrogyrposis multiplex congenita . Clinical photograph of identical twins it was noted that the identical twins were delivered by caesarean section at full term with birth weight of 2.3 and 2.1 kg respectively . Both of them cried at birth and had stiff hips and knees . On examination, both the probands had facial dysmorphism, fixed flexion deformity of hips and knees, umbilical hernia and pigmented patches over knuckles and ankle . They were lost to follow up for one year subsequently . At the age of 15 months, the children were brought back with increased severity of earlier symptoms along with additional features like macrocephaly with wide open fontanelle, thickened skin and subcutaneous tissue around multiple joints with fixed deformity of hips, knees and ankles, deviation of neck to one side, extension deformity of elbow in one child and flexion deformity in the other . Nodular lesions around the nostrils, hypertrophied gums and fleshy nodular excrescence around the anus were noted (fig 3: a - f). 4). Genetic analysis of antxr2 gene revealed that the babies were homozygous for insertion mutation c.277_278insattattt (or p.l93yfs14) in exon 3 confirming the diagnosis of ish (fig . All the invasive procedures described in the literature for diagnosis and management of ish were thus avoided . Parents were counselled regarding the need for prenatal diagnosis by chorionic villus sampling in future pregnancies . A & b: clinical photograph showing deviation of neck to right, umbilical hernia, flexion deformity of both hips and knees and extension attitude of both the elbows . D: photograph of ankle with foot depicting hyper pigmented patches, swollen ankle and foot . E: photograph showing swollen hand with clawing of fingers and hyper pigmented patches in the creases . F: photograph of perineum depicting perianal fleshy excrescence / nodule anteroposterior and lateral radiographs of both lower limbs show decreased bone density . No other bony abnormality is noticed a. sequence chromatogram of proband showing homozygous insertion mutation c.277_278insattattt in exon 3 b. sequence chromatogram of control exon 3 ishikawa and hori introduced the term systemic hyaline fibromatosis in 1964 and detailed description of ish was given by landing in 1986 . Al from saudi arabia reported the largest series (19 cases) of ish in the literature . Ish is an autosomal recessive disorder with identified mutations in von willebrand factor a (vwa) like domain of capillary morphogenesis gene-2 (cmg2)/ human anthrax toxin receptor gene-2 (antxr2) gene located on chromosome 4q21.21 . Mutations in this gene lead to excessive proliferation of spindle cells, abnormal glycosaminoglycans and collagen synthesis with extensive deposition of amorphous eosinophilic hyaline material in the connective tissues of various organs like skin, cardiac and skeletal muscles, gut, thyroid and adrenal glands . Two allelic disorders namely ish and juvenile hyaline fibromatosis (jhf) have been described with the mutation in cmg2/antxr2 gene . A detailed comparison of ish and jhf along with the present case is tabulated for ready reference (table: 1). The children with ish present with deformities of multiple joints, excessive cry and difficulty in handling, coarse facies, inflexible and thickened skin with papulo - nodular lesions, gingival hyperplasia with difficulties in feeding, persistent diarrhoea, recurrent respiratory infections, failure to thrive and succumb early before 3 years of age . Jhf is presently believed to be a milder phenotype of ish and not a distinct clinical entity . Differential diagnosis of arthrogyrposis multiplex congenita, infantile stiff skin syndromes, torg winchester syndrome, farbers syndrome, nomid (neonatal onset multisystemic inflammatory disease) and amyloidois are usually considered in a child presenting with features of ihs / jhf . Histopathological evidence of abundant homogenous amorphous eosinophilic material with sparse cellularity in the dermis was the corner stone for diagnosis of ish in most of the cases described in the literature . As the clinical features of ish are diagnostic in most cases, molecular analysis of the antxr2 gene can be the first and stand alone investigation for the confirmation of diagnosis . The management of children with ish is primarily symptomatic and there is no effective treatment . Genetic counselling plays an essential role in preventing recurrences by providing information about recurrence risk and option of prenatal diagnosis to the family . Ish is a rapidly progressive, debilitating and fatal genetic disorder of infancy with abnormal glycosaminoglycan and collagen synthesis and amorphous hyaline matrix accumulation in various organs owing to the deletion / nonsense mutation in cmg-2/antxr2 gene . Prenatal diagnosis by chorionic villus biopsy is possible once causative mutation in a family is identified . Invasive surgical interventions for histopathological analysis can be avoided as clinical features are most often classical and genetic analysis is confirmatory . Being a very rare entity, infantile systemic hyalinosis is commonly misdiagnosed and mismanaged as arthrogryposis multiplex congenita in the initial stages . High index of suspicion, clinical knowledge of ish and confirmation by genetic analysis are desirable to avoid misdiagnosis and mismanagement.
Traumatic optic neuropathy (ton) is an impact injury to the optic nerve, usually secondary to blunt head trauma, resulting in partial or complete loss of vision . A meta - analysis reported that both steroid administration and surgical decompression improved the prognosis of patients with ton . After consideration of a lateral or medial orbitotomy, as well as transcranial approaches to the orbital apex and optic nerve canal, an endonasal transethmoidal - sphenoidal approach represents an alternative strategy for the treatment of ton . The endoscopic anterior - to - posterior technique offers many advantages, including decreased morbidity, preservation of olfaction, rapid recovery time, more acceptable cosmetic results without external scars and no risk of injury to developing teeth in children . However, despite its safety, effectiveness, and minimal invasiveness, the long - term effects of the iatrogenic trauma (removal of uncinate process and anterior ethmoidal sinus) resulting from the complete ethmoidectomy procedure used to gain full access to the optic nerve canal is unknown, and sequelae such as nasal synechia and sinusitis should not be ignored . Anatomically, the optic canal is just superolateral to the sphenoid sinus as a part of the lesser sphenoid wing . The canal is wide with thin walls proximally toward the optic chiasm, and narrow with thick walls distally at the optic tubercle . The optic tubercle is the thick bulge of the medial aspect of the bone surrounding the optic foramen . Depending on the degree of pneumatisation or the presence of superspheno - ethmoid (onodi) cellulae, the optic tubercle can be visualized within the sphenoid sinus or at the junction of the sphenoid and posterior ethmoid sinuses . In the case of high sphenoidal pneumatisation, the use of an angled endoscope allows clear visualisation of the lateral sphenoidal wall and the orbit apex . This raises the possibility that optic nerve decompression can be performed through a single trans - sphenoidal approach rather than using complete ethmoidectomy . Herein, we report five cases of ton in which a modified surgical procedure for endoscopic optic nerve decompression was performed, using a 45 endoscope . We include discussion of the operative events, along with the apparent advantages and limitations . Five patients with ton, all of whom were unresponsive to high - dose methylprednisolone (500 mg / day) for more than 7 days, were enrolled to undergo modified endoscopic optic nerve decompression . Only patients with high sphenoidal pneumatisation and without onodi cellulae identified by preoperative fine - cut computed tomography (ct) scan [figure 1] were included . This study was approved by the ethical committee of our hospital, and a written informed consent was obtained from each subject . Demographics of five ton patients performed modified optic nerve decompression representative preoperative computed tomograpgy (ct) scan of a traumatic optic neuropathy (ton) patient (a) coronal view; (b) horizontal view the surgical procedure was modified from what we have previously described . Briefly, patients were prepared in the routine manner for endoscopic sinus surgery under general anesthesia . Cotton pledgets soaked in epinephrine solution were placed in the nasal cavity to ensure vasoconstriction . In contrast to the standard endoscopic ethmoidectomy procedure, we located the ostium of the sphenoid sinus by removing the superior turbinate [figure 2a]. When the sphenoid sinus was opened and the ostium was enlarged to the lateral wall, the optic nerve canal was clearly identified using a 0 or 45 endoscope [figure 2b]. In two cases, the posterior ethmoids were slightly opened retrograde from the sphenoid to improve the exposure of the optic nerve canal . Representative endoscopic view of optic nerve decompression using a modified trans - sphenoidal approach (a) sphenoidal ostium; (b) optic nerve canal; (c) optic nerve; (d) intact middle meatus so, sphenoidal ostium; st, superior turbinate; onc, optic nerve canal; on, optic nerve; mt, middle turbinate the orbital apex was then dissected to expose the lateral wall of the sphenoid sinus . Under endoscopic observation, a long - hand microdrill with a diamond burr was used to remove the medial wall of the bony optic canal . To avoid thermal injury to the optic nerve, the optic nerve canal was drilled 180 medially from the optic tubercle to near the optic chiasm until the bony canal became very thin . A special instrument was then used to elevate the thin bony canal from its position over the optic nerve . Extreme care was taken to not exert pressure on the optic nerve with the elevator [figure 2c]. We modified the routine procedure for endoscopic optic nerve decompression as follows: we used a endonasal trans - sphenoidal approach instead of a endonasal transethmoidal - sphenoidal approach to keep the anterior ethmoidal sinus and middle meatus intact [figure 2d]we drilled off the distal bony wall of the optic nerve canal using a 0 endoscopewe drilled off the proximal bony wall of the orbital apex using a 45 angled endoscopewe maintained the integrity of optic nerve without incision of the optic sheath . We used a endonasal trans - sphenoidal approach instead of a endonasal transethmoidal - sphenoidal approach to keep the anterior ethmoidal sinus and middle meatus intact [figure 2d] we drilled off the distal bony wall of the optic nerve canal using a 0 endoscope we drilled off the proximal bony wall of the orbital apex using a 45 angled endoscope we maintained the integrity of optic nerve without incision of the optic sheath . All patients received regular follow - up care including visual acuity and field chart testing 10 days postoperatively . A patient's vision was considered to have improved if they showed any of the following: an improvement from no light perception to light perception or better; an improvement from light perception to hand motion or better; an improvement from hand motion to finger counting or better . Five patients with ton, all of whom were unresponsive to high - dose methylprednisolone (500 mg / day) for more than 7 days, were enrolled to undergo modified endoscopic optic nerve decompression . Only patients with high sphenoidal pneumatisation and without onodi cellulae identified by preoperative fine - cut computed tomography (ct) scan [figure 1] were included . This study was approved by the ethical committee of our hospital, and a written informed consent was obtained from each subject . Demographics of five ton patients performed modified optic nerve decompression representative preoperative computed tomograpgy (ct) scan of a traumatic optic neuropathy (ton) patient (a) coronal view; (b) horizontal view briefly, patients were prepared in the routine manner for endoscopic sinus surgery under general anesthesia . Cotton pledgets soaked in epinephrine solution were placed in the nasal cavity to ensure vasoconstriction . In contrast to the standard endoscopic ethmoidectomy procedure, we located the ostium of the sphenoid sinus by removing the superior turbinate [figure 2a]. When the sphenoid sinus was opened and the ostium was enlarged to the lateral wall, the optic nerve canal was clearly identified using a 0 or 45 endoscope [figure 2b]. In two cases, the posterior ethmoids were slightly opened retrograde from the sphenoid to improve the exposure of the optic nerve canal . Representative endoscopic view of optic nerve decompression using a modified trans - sphenoidal approach (a) sphenoidal ostium; (b) optic nerve canal; (c) optic nerve; (d) intact middle meatus so, sphenoidal ostium; st, superior turbinate; onc, optic nerve canal; on, optic nerve; mt, middle turbinate the orbital apex was then dissected to expose the lateral wall of the sphenoid sinus . Under endoscopic observation, a long - hand microdrill with a diamond burr was used to remove the medial wall of the bony optic canal . To avoid thermal injury to the optic nerve, the optic nerve canal was drilled 180 medially from the optic tubercle to near the optic chiasm until the bony canal became very thin . A special instrument was then used to elevate the thin bony canal from its position over the optic nerve . Extreme care was taken to not exert pressure on the optic nerve with the elevator [figure 2c]. We modified the routine procedure for endoscopic optic nerve decompression as follows: we used a endonasal trans - sphenoidal approach instead of a endonasal transethmoidal - sphenoidal approach to keep the anterior ethmoidal sinus and middle meatus intact [figure 2d]we drilled off the distal bony wall of the optic nerve canal using a 0 endoscopewe drilled off the proximal bony wall of the orbital apex using a 45 angled endoscopewe maintained the integrity of optic nerve without incision of the optic sheath . We used a endonasal trans - sphenoidal approach instead of a endonasal transethmoidal - sphenoidal approach to keep the anterior ethmoidal sinus and middle meatus intact [figure 2d] we drilled off the distal bony wall of the optic nerve canal using a 0 endoscope we drilled off the proximal bony wall of the orbital apex using a 45 angled endoscope we maintained the integrity of optic nerve without incision of the optic sheath . All patients received regular follow - up care including visual acuity and field chart testing 10 days postoperatively . A patient's vision was considered to have improved if they showed any of the following: an improvement from no light perception to light perception or better; an improvement from light perception to hand motion or better; an improvement from hand motion to finger counting or better . The cause of ton included motorcycle accidents (4/5) and a fall (1/5). After performing a direct sphenoidotomy through the natural ostium of the sphenoid sinus rather than a complete ethmo - sphnoidectomy, we found that the endonasal trans - sphenoidal approach provided adequate access to the optic nerve canal and the apex using a 45 angled endoscope . Successful decompression of the canal optic nerve was performed trans - sphenoidally in all five patients using an angled endoscope . No surgical complications occurred, and none of the patients suffered from anterior ethmoidal sinus or skull base damage . In this study, we successfully developed a modified endoscopic optic nerve decompression procedure for five patients with ton with severe sphenoidal pneumatisation and without onodi cellulae . Our results suggest that modified endoscopic optic nerve decompression is a safe, effective, and minimally invasive strategy for specially selected ton patients . Ton is an important cause of severe visual impairment following blunt or penetrating head trauma . Treatment options include steroids, surgical decompression, or both, with no consensus on the optimal protocol . The use of high - dose steroids after optic nerve injury became more common during the 1980s, following the rationale that steroids may reduce post - traumatic edema, contusion necrosis, and vasospasm, and thus aid functional recovery . Previous studies have reported improvement in between 44% and 82% of patients following steroid therapy . However, in other studies, 57% of untreated patients showed improvement in visual acuity, indicating spontaneous remission . The indications for endoscopic optic nerve decompression are also cause for debate . It has been suggested that such surgery should only be performed following failure of high - dose steroid therapy . Some authors suggested that endoscopic optic nerve decompression be performed if vision fails to improve after 72 hours of methylprednisone therapy; or if progressive visual loss during steroid therapy is observed . Additionally, complete blindness with ct evidence of optic nerve compression was also put forth as an indication for surgery . Despite the lack of evidence showing a clear benefit for either steroids or surgery or combined therapy, patients with ton have a strong desire for endoscopic optic nerve decompression for vision salvage . In this study, we sought to develop a novel, less invasive surgical approach rather than to address the clinical efficacy of and indications for endoscopic optic nerve decompression . Currently, a traditional transethmoidal - sphenoidal approach is generally performed to gain wide access to the optic nerve canal and optic apex by means of a complete endoscopic ethmoidectomy and sphenoidotomy . However, the complete endoscopic ethmoidectomy includes removal of the uncinate process and anterior ethmoidal sinus, and the long - term effects of this trauma are unknown . Additionally, common postoperative sequelae such as nasal synechia and sinusitis should not be ignored . To avoid iatrogenic trauma as much as possible, we developed a modified surgical procedure that leaves the uncinate process, ethmoidal bulla, and middle meatus intact . In this study, we modified the endonasal transethmoidal - sphenoidal approach by using an endonasal trans - sphenoidal approach to avoid removing the uncinate process and anterior ethmoidal sinus . In cases of high sphenoidal pneumatisation, we were able to drill off the distal bony wall of the optic nerve canal using a 0 endoscope, and drill off the proximal bony wall of the orbital apex using a 45 angled endoscope . In addition, the modified endonasal trans - sphenoidal approach may be especially appropriate for patients with lamina papyracea bony fragments, in whom the orbital fat herniating into the ethmoidal sinus typically blocks access to the sphenoidal sinus and orbit apex . We successfully conducted trans - sphenoidal decompression of the canal optic nerve in all five patients . The decision to perform nerve sheath incision during optic nerve decompression is also under debate . Based on our unpublished data, we found no additional optic vision improvement following incision of the optic sheath . Thus, we made a second modification by maintaining the optic nerve intact without incision of the optic sheath . Consequently, we showed that incision of the optic sheath is not a prerequisite for successful optic nerve decompression . First, although the uncinate process and anterior ethmoidal sinus were kept intact, the posterior ethmoids were slightly opened retrograde from the sphenoid to improve the exposure of the optic nerve canal in two of five cases; thus, the minimal trauma of this approach cannot be over - interpretated for some patients . It is also worthwhile to note that all five patients had high sphenoidal pneumatisation without onodi cellulae (identified by a preoperative fine - cut ct scan), as the optic nerve canal may not be accessible in the case of onodi cellulae . Additionally, when the ostium of sphenoidal sinus is enlarged to the lateral wall, the optic nerve canal need to be identified using a 45 endoscope to gain appropriate access to the lateral sphenoidal wall and orbit apex, which may increase the technical difficulty of the surgery . Our study demonstrates that a modified trans - sphenoidal optic nerve decompression is a feasible, safe, effective, and minimally invasive approach for ton patients with high sphenoidal pneumatisation and without supersphenoid - ethmoid cellulae . In cases of high sphenoidal pneumatisation, the use of an angled endoscope allows for easy access to the optic nerve canal and the orbit apex using a trans - sphenoidal approach while keeping the uncinate process and anterior ethmoidal sinus intact.
Klippel - trenaunay syndrome (kts) is characterized by the triad of port wine stain, venous and lymphatic malformation, and soft tissue hypertrophy of the affected extremity . Depending on the type of vessel involved and its flow characteristics kts is classified as a slow flow complex combined capillary venous or capillary venous lymphatic malformation . The localized variants are solitary angiokeratoma, fordyce's angiokeratoma, angiokeratoma of mibelli and angiokeratoma circumscriptum naeviforme (acn). The systemic form, angiokeratoma corporis diffusum, is usually associated with an inborn error of metabolism . Among all the types, acn is the rarer and the only congenital variant of angiokeratoma . The lesions of acn are bluish red, well defined and are classically seen on the lower extremity in a unilateral distribution . A 4-year - old male child presented with complaints of a linear eruption on his right leg since birth . His parents gave history of pain and several episodes of bleeding from the lesion after trauma ., there were multiple hyperkeratotic discrete and closely aggregated papules and plaques of varying size on an erythematous base arranged linearly along the lateral aspect of the right thigh extending up to the knee joint [figures 1 and 2]. Right lower limb arteriovenous (av) doppler revealed a hypoplastic right lower limb deep venous system with compensatory dilatation of the superficial venous system . The superficial femoral vein, popliteal vein, anterior tibial and posterior tibial vein on right side were hypoplastic along with thickening of subcutaneous tissue and associated dilatation and malformation of the superficial venous system [figures 5 and 6]. Histological examination revealed numerous dilated thin walled, congested capillaries mainly in the papillary dermis and very few in reticular dermis . Overlying epidermis showed compact hyperkeratosis, irregular acanthosis with elongated rete ridges consistent with diagnosis of angiokeratoma [figures 7 and 8]. On the basis of clinical, histological and radiological findings dilated superficial venous system verrucous plaque with soft tissue hypertrophy prominent superficial veins on dorsum of foot plain radiograph showing soft tissue hypertrophy magnetic resonance imaging angiography showing dilated superficial veins on the right side dilated superficial venous system hyperkeratotic epidermis with congested capillaries in papillary dermis with normal deep dermis and subcutaneous tissue hyperkeratotic epidermis with dilated congested capillaries in papillary dermis in 1900, two french physicians maurice klippel and paul trenaunay described two patients with hemangiomatous lesions of the skin with associated bone and soft tissue hypertrophy and coined the term naevus variqueux osteohypertrophique . In 1907, parkes weber reported the association of kts with av fistula and called it hemangiectatic hypertrophy . In 1965, lindenauer proposed that the syndrome originally described by klippel and trenaunay without av malformation be considered as a specific entity the kts and the one associated with av fistula be designated as parkes weber syndrome . In a study of 252 patients with kts at the mayo clinic, 63% had all three features of kts . Port wine stains were found in 98% of patients, venous malformations in 72% and limb hypertrophy in 67% patients . The port wine stain is apparent at birth and usually involves the affected limb, often stopping at the midline with a sharp linear border . The nevus may involve the whole of one side of the body and may sometimes be present on the contralateral limb . Varicose veins may be obvious at birth, but frequently become evident after walking starts . The venous abnormalities of the deep venous system that occur in kts include aneurysmal dilatation, duplication, aplasia, and hypoplasia . Patients with at least two of the three cardinal features have been classified as having an incomplete form of kts . It is the only congenital variant and is more common in women and is usually unassociated with systemic disease . The histopathological finding is same irrespective of the type of angiokeratoma and consist of numerous thin walled congested capillaries mainly in the papillary dermis underlying an epidermis that shows variable degree of acanthosis with elongation of rete ridges and hyperkeratosis . Unusual variants of acn described in literature are along the lines of blaschko, and a systematized band like pattern . Verrucous hemangioma clinically resembles angiokeratoma but they can be differentiated histopathologically as the former involves the dermis and the subcutaneous fat and the latter involves only the dermis . Our case showed typical clinical features of acn, which was confirmed by histopathologic examination . Associated soft tissue hypertrophy and deep venous malformation on mri are classical features of kts . There are very few case reports of association of kts with acn in world literature . Schimpf and wehberg in their study have reported three cases of acn on the lower limb associated with soft tissue hypertrophy . Bone hypertrophy was present in two cases, varicose veins in one case and only one case had associated port wine stain on the back . The case reported by odeh had acn of the right leg associated with soft tissue hypertrophy and bone hypoplasia of the right half of pelvis . They preferred the term hemangiectatic hypertrophy as there was no associated bony and venous abnormalities of kts . To the best of our knowledge, this is the first case report of this rare association from india.
Transgenic cre and r26r reporter mouse lines used in this study are listed in table 1 . Experimental animals were generated by crossing tg(ins2-cre) (termed rip - cre) (11), tg(ins2-cre) (termed rip - cre) (10), tg(ins2-creesr1) (termed rip - cre / ert) (12), tg(pdx1-cre) (termed pdx1-cre) (13), tg(ipf1-cre) (termed pdx1-cre) (14), tg(pdx1-cre / ert) (termed pdx1-cre / ert) (15), or tg(ins1-cre / ert) (termed mip - cre / ert) (tamarina et al ., unpublished data) transgenic lines with a reporter strain expressing either lacz gt(rosa)26sor (termed r26r) (17,19) or enhanced yfp gt(rosa)26sor (termed r26r) (18). Both r26r reporter strains on c57bl/6 background were obtained from jackson laboratories (bar harbor, me). Complete details of the sources for all mouse strains used in this study are listed in supplementary table 1 (available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0624/dc1). For timed pregnancies, noon on the day of the vaginal plug was considered embryonic day 0.5 (e0.5). All animal studies were approved by the institutional animal care and use committees at the relevant institutions . Mouse transgenic cre and r26r reporter lines used in this study primary antibodies included guinea pig anti - porcine insulin igg (1:500; dako, carpinteria, ca), guinea pig anti - insulin antibody (1:1,000; millipore, billerica, ma), rabbit anti-gal igg (1:5,000; mp biomedicals, solon, oh), goat anti-gal igg (1:1,000; biogenesis ltd, poole, uk), rabbit anti - stat3 phosphorylation (pstat3) igg (1:1,000; cell signaling technologies, beverly, ma), rabbit anti - orexin igg (1:2,000; calbiochem, emd biosciences / merck, darmstadt, germany), and rabbit anti - cre antibody (1:1,000, cat . Fluorescent - labeled secondary antibodies were purchased from jackson immunoresearch (west grove, pa) and invitrogen (carlsbad, ca). Recombinant mouse leptin was obtained from the national hormone and peptide program (los angeles, ca). Over a 5-day period, mice were injected subcutaneously or intraperitoneally with 3 doses of 18 mg tamoxifen (sigma, t5648) freshly dissolved in corn oil (sigma, c8267) at 10 mg / ml, 20 mg / ml, or corn oil vehicle . The subcutaneous injection site was sealed with a drop of vetbond tissue adhesive (3 m). Following tamoxifen administration, the mice were housed individually for 510 days before being analyzed for cre - recombinase mediated activity . -gal activity was detected by 5-bromo-4-chloro-3-indolyl--d - galactopyranoside (x - gal) staining as described previously (20) with slight modifications . Briefly, pancreata and brains were dissected in ice - cold 10 mmol / l pbs and fixed in freshly prepared 12% paraformaldehyde for either 24 h at room temperature or overnight at 4c . Brains (2-mm slices) and pancreata were permeabilized for 5 h in 2 mmol / l mgcl2, 0.01% sodium deoxycholate, 0.02% np-40, 10 mmol / l pbs, and then stained overnight in the dark in 2 mmol / l mgcl2, 5 mmol / l potassium ferricyanide, 5 mmol / l potassium ferrocyanide, 1 mg / ml x - gal, 0.01% sodium deoxycholate, 0.02% np-40, 10 mmol / l pbs ph 7.4 at ambient temperature or 37c . Tissues were washed in pbs, postfixed in 4% paraformaldehyde for 1 h, washed in pbs, and placed into 70% ethanol prior to whole mount imaging . For yfp detection, embryos were dissected at e15.5 and imaged in whole mount . Mice were injected intraperitoneally with leptin (5 mg / kg) or vehicle (pbs) and then rested for 2 h prior to perfusion . Immunodetection of pancreatic cre expression was performed in 5-m paraffin sections prepared from paraformaldehyde - fixed pancreata of rip - cre, rip - cre / ert, pdx1-cre / ert, and mip - cre / ert mice . Transgenic lines expressing cre / ert received the third dose of tamoxifen on the day prior to being killed . After antigen retrieval, sections were incubated with primary antibodies to cre and insulin (millipore). For immunodetection of -gal expression in the brain, mice were perfusion - fixed, and brains were removed and postfixed overnight as described previously (21). Following cryoprotection, brains were sectioned into 30-m coronal slices, collected in four consecutive series, and stored at 20c . Sections were then incubated with primary antibodies to pstat3, -gal (biogenesis), or orexin overnight at 4c . Immunolabeling was visualized with appropriate fluorescent - labeled secondary antibodies . One - way anova analysis was used to compare the percent of -cells that express cre in the islets of the different transgenic lines . Islets (22) and hypothalamus were isolated from adult tg(pdx1-cre) (13) mice and their controls . Total cellular rna was isolated using the rnaqueous small scale phenol - free total rna isolation kit (ambion, austin, tx), and trace contaminating dna was removed with the turbo dna - free kit (ambion). High - quality rna had a 28s to18s ratio from 1.2 to 2.0 and an rna integrity number from 8.2 to 8.9 . Single - stranded cdna was generated by reverse transcription from 180-ng total rna using the superscript iii first strand synthesis kit (invitrogen). Cdna (40 ng / reaction) was analyzed by quantitative rt - pcr using the abi prism 7900 sequence detection system and power sybr green master mix (applied biosystems, foster city, ca). Samples were analyzed in duplicates, and relative cdna levels were determined by comparing cycle threshold values of cre cdna to hypoxanthine phosphoribosyl transferase (hprt) cdna . Samples with cycle threshold values greater than 40 were considered to have undetectable amounts of template . Primer sequences were the following; cre (5tgcaacgagtgatgaggttc3 and 5gcaaacggacagaagcattt3), hprt (5tacgaggagtcctgttgatgttgc3 and 5gggacgcagcaactgacatttcta3), and pdx1 (5ctgagggacaaagatgcaga3 and 5ttctaattcagggcgttgtg3). One - way anova with newman - keuls multiple comparison tests were used to compare outcomes in mice of different genotypes . Transgenic cre and r26r reporter mouse lines used in this study are listed in table 1 . Experimental animals were generated by crossing tg(ins2-cre) (termed rip - cre) (11), tg(ins2-cre) (termed rip - cre) (10), tg(ins2-creesr1) (termed rip - cre / ert) (12), tg(pdx1-cre) (termed pdx1-cre) (13), tg(ipf1-cre) (termed pdx1-cre) (14), tg(pdx1-cre / ert) (termed pdx1-cre / ert) (15), or tg(ins1-cre / ert) (termed mip - cre / ert) (tamarina et al ., unpublished data) transgenic lines with a reporter strain expressing either lacz gt(rosa)26sor (termed r26r) (17,19) or enhanced yfp gt(rosa)26sor (termed r26r) (18). Both r26r reporter strains on c57bl/6 background were obtained from jackson laboratories (bar harbor, me). Complete details of the sources for all mouse strains used in this study are listed in supplementary table 1 (available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0624/dc1). For timed pregnancies, noon on the day of the vaginal plug was considered embryonic day 0.5 (e0.5). All animal studies were approved by the institutional animal care and use committees at the relevant institutions . Primary antibodies included guinea pig anti - porcine insulin igg (1:500; dako, carpinteria, ca), guinea pig anti - insulin antibody (1:1,000; millipore, billerica, ma), rabbit anti-gal igg (1:5,000; mp biomedicals, solon, oh), goat anti-gal igg (1:1,000; biogenesis ltd, poole, uk), rabbit anti - stat3 phosphorylation (pstat3) igg (1:1,000; cell signaling technologies, beverly, ma), rabbit anti - orexin igg (1:2,000; calbiochem, emd biosciences / merck, darmstadt, germany), and rabbit anti - cre antibody (1:1,000, cat . Fluorescent - labeled secondary antibodies were purchased from jackson immunoresearch (west grove, pa) and invitrogen (carlsbad, ca). Recombinant mouse leptin was obtained from the national hormone and peptide program (los angeles, ca). Over a 5-day period, mice were injected subcutaneously or intraperitoneally with 3 doses of 18 mg tamoxifen (sigma, t5648) freshly dissolved in corn oil (sigma, c8267) at 10 mg / ml, 20 mg / ml, or corn oil vehicle . The subcutaneous injection site was sealed with a drop of vetbond tissue adhesive (3 m). Following tamoxifen administration, the mice were housed individually for 510 days before being analyzed for cre - recombinase mediated activity . -gal activity was detected by 5-bromo-4-chloro-3-indolyl--d - galactopyranoside (x - gal) staining as described previously (20) with slight modifications . Briefly, pancreata and brains were dissected in ice - cold 10 mmol / l pbs and fixed in freshly prepared 12% paraformaldehyde for either 24 h at room temperature or overnight at 4c . Brains (2-mm slices) and pancreata were permeabilized for 5 h in 2 mmol / l mgcl2, 0.01% sodium deoxycholate, 0.02% np-40, 10 mmol / l pbs, and then stained overnight in the dark in 2 mmol / l mgcl2, 5 mmol / l potassium ferricyanide, 5 mmol / l potassium ferrocyanide, 1 mg / ml x - gal, 0.01% sodium deoxycholate, 0.02% np-40, 10 mmol / l pbs ph 7.4 at ambient temperature or 37c . Tissues were washed in pbs, postfixed in 4% paraformaldehyde for 1 h, washed in pbs, and placed into 70% ethanol prior to whole mount imaging . For yfp detection, embryos were dissected at e15.5 and imaged in whole mount . Mice were injected intraperitoneally with leptin (5 mg / kg) or vehicle (pbs) and then rested for 2 h prior to perfusion . Immunodetection of pancreatic cre expression was performed in 5-m paraffin sections prepared from paraformaldehyde - fixed pancreata of rip - cre, rip - cre / ert, pdx1-cre / ert, and mip - cre / ert mice . Transgenic lines expressing cre / ert received the third dose of tamoxifen on the day prior to being killed . After antigen retrieval, sections were incubated with primary antibodies to cre and insulin (millipore). For immunodetection of -gal expression in the brain, mice were perfusion - fixed, and brains were removed and postfixed overnight as described previously (21). Following cryoprotection, brains were sectioned into 30-m coronal slices, collected in four consecutive series, and stored at 20c . Sections were then incubated with primary antibodies to pstat3, -gal (biogenesis), or orexin overnight at 4c . Immunolabeling was visualized with appropriate fluorescent - labeled secondary antibodies . One - way anova analysis was used to compare the percent of -cells that express cre in the islets of the different transgenic lines . Islets (22) and hypothalamus were isolated from adult tg(pdx1-cre) (13) mice and their controls . Total cellular rna was isolated using the rnaqueous small scale phenol - free total rna isolation kit (ambion, austin, tx), and trace contaminating dna was removed with the turbo dna - free kit (ambion). High - quality rna had a 28s to18s ratio from 1.2 to 2.0 and an rna integrity number from 8.2 to 8.9 . Single - stranded cdna was generated by reverse transcription from 180-ng total rna using the superscript iii first strand synthesis kit (invitrogen). Cdna (40 ng / reaction) was analyzed by quantitative rt - pcr using the abi prism 7900 sequence detection system and power sybr green master mix (applied biosystems, foster city, ca). Samples were analyzed in duplicates, and relative cdna levels were determined by comparing cycle threshold values of cre cdna to hypoxanthine phosphoribosyl transferase (hprt) cdna . Samples with cycle threshold values greater than 40 were considered to have undetectable amounts of template . Primer sequences were the following; cre (5tgcaacgagtgatgaggttc3 and 5gcaaacggacagaagcattt3), hprt (5tacgaggagtcctgttgatgttgc3 and 5gggacgcagcaactgacatttcta3), and pdx1 (5ctgagggacaaagatgcaga3 and 5ttctaattcagggcgttgtg3). One - way anova with newman - keuls multiple comparison tests were used to compare outcomes in mice of different genotypes . Using the r26r reporter line, the rip - cre line (11) was previously shown to have robust cre - mediated recombination within the -cells and the ventral brain during development (9). To investigate whether cre - mediated recombination occurred within the brain of other transgenic cre lines using the rat ins2 or pdx1 promoter (table 1), these mouse strains were crossed with the r26r reporter strain and analyzed for -gal activity in whole mount brain slices (figs . 1 and 2). No x - gal staining was detected in the brain or pancreas from control r26r littermates indicating that -gal is not expressed in the absence of cre activity (fig . Widespread x - gal staining was detected in most brain areas with robust expression in the mid - brain and ventral regions, which was consistent with previous reports (9) (fig . X - gal staining was less widespread and had a more punctate pattern without any obvious regionalization (fig . The brains of rip - cre / ert;r26r mice with cre activity induced by three 2-mg doses of tamoxifen revealed a diffuse intermediate pattern of x - gal staining that was more extensive than in rip - cre;r26r mice but less than in rip - cre;r26r mice (fig . All three transgenic lines, rip - cre, rip - cre, and rip - cre / ert, showed a high level of recombination in pancreatic islets (fig . Rip - cre transgenic lines display cre - mediated recombination in multiple regions of the brain . Adult brains were sliced into four or five coronal sections and subjected to whole mount x - gal staining . 14 . A: sagittal and coronal views of mouse brain (adapted from allen mouse brain atlas, http://www.brain-map.org/) (29). A dashed vertical line marks coronal sectioning plane spanning the hypothalamic region of the brain . B e: images of coronal brain slices located on the left side of the sectioning plane in the sagittal view in a. b: r26r littermate control mice (n = 17) lacked x - gal staining in the brain . The cortex (ctx) and hypothalamus (hy) are labeled and correspond to regions marked on the coronal view in a. c: rip - cre;r26r mice (n = 8) showed x - gal staining throughout the brain with high signal intensity in the mid - brain and ventral regions . D: rip - cre;r26r mice (n = 14) showed weaker, punctate x - gal staining throughout the brain without obvious regionalization . E: rip - cre / ert;r26r mice (n = 4) injected intraperitoneally with three 2-mg doses of tamoxifen over a 5-day period displayed strong, punctate x - gal staining throughout the brain with expression pattern more restricted than in rip - cre; r26r mice . Brains from littermate controls injected with corn oil vehicle were negative for x - gal staining (data not shown). F i: whole - mount x - gal staining of pancreas from r26r in f, rip - cre; r26r in h, rip - cre;r26r in g, and rip - cre / ert;r26r mice in i. (a high - quality digital representation of this figure is available in the online issue .) Pdx1-cre transgenic lines show localized cre - mediated recombination within specific regions of the brain including the hypothalamus . B d: images of coronal brain slices located on the left side of sectioning plane in the sagittal view in a. the schematics of the mouse brain are from the allen mouse brain atlas (http://www.brain-map.org/) (29). B: x - gal staining in pdx1-cre;r26r brain (n = 7) was localized to the brain stem and hypothalamus . C: x - gal positive cells in pdx1-cre;r26r brain (n = 4) were localized to hypothalamic region . D: adult pdx1-cre / ert;r26r mice (n = 4) were injected subcutaneously with three 8-mg doses of tamoxifen (right panel) and analyzed for lacz expression . X - gal staining had a broader punctate pattern with high - intensity signal localized to the hypothalamic region . Brains from littermate controls injected with corn oil vehicle (n = 2) were negative for x - gal staining (data not shown). E: brains from adult pdx1 mice (n = 4) were negative for x - gal staining . I: whole - mount x - gal staining of pancreas from pdx1-cre;r26r in f, pdx1-cre;r26r in g, pdx1-cre / ert;r26r mice in h, and pdx1 mice in i. j and k: brains from pdx1-cre;r26r embryos at e15.5 (n = 6) in j were analyzed for lacz expression . X - gal staining indicated expression of pdx1-cre transgene in the brain stem and ventral region of the brain that gives rise to the hypothalamus (arrows). Pancreas in k had expected x - gal staining . Brain and pancreas from r26r (n = 5) and r26r (n = 6) e15.5 controls were negative for x - gal staining and yfp fluorescence, respectively (supplementary fig . L and m: brains from e15.5 pdx1 embryos (n = 7) in l were negative for x - gal staining, while pancreas showed expected x - gal positivity in m. in e15.5 pdx1 embryos (n = 10), both brain and pancreas were x - gal negative (supplemental fig . 9). Ctx, cortex; d, duodenum; dp, dorsal pancreas; hy, hypothalamus; iiiv, third ventricle; ph, posterior hypothalamic region; sp, spleen; st, stomach; vp, ventral pancreas . (a high - quality digital representation of this figure is available in the online issue .) Cre - mediated recombination within the brain of pdx1-cre transgenic lines has not been examined, but ectopic recombination was reported in the pharyngeal region of pdx1-cre;r26r embryos (23). Unlike the widespread recombination in brains from rip - cre transgenic lines, x - gal staining in pdx1-cre;r26r brains (fig . Analysis of cre mrna by quantitative rt - pcr in the pdx1-cre line confirmed expression in the hypothalamus with levels of hypothalamic expression 12.6-fold lower than in islets (supplemental fig . 5). Injection of a single dose of tamoxifen into pregnant females (2 mg/40 g body weight) at e16.5 did not result in recombination in the brains of pdx1-cre / ert;r26r embryos dissected at e20.5 (15). In adult pdx1-cre / ert;r26r mice injected with three 1-mg doses of tamoxifen, recombination was detected mainly in the hypothalamus (supplementary fig . However, three 8-mg doses of tamoxifen induced much broader recombination throughout the brain, suggesting that the extent of recombination in the adult brain is dependent upon the tamoxifen dose (fig . These data suggest that the pdx1-cre / ert transgene is expressed in the adult brain but not in the e16.5 brain, although it is possible that higher tamoxifen levels may be needed to induce cre - mediated recombination within the embryonic brain . To further examine the timing of cre expression in the brains of the pdx1-cre lines expressing constitutively active cre, we studied the pdx1-cre transgenic line crossed into either r26r or r26r reporter mice and analyzed embryos at e15.5 (fig . Both reporter strains demonstrated cre activity in the brain stem and ventral region of the developing brain that gives rise to the hypothalamus, indicating that functional cre protein is expressed in the ventral region of the pdx1-cre brain prior to e15.5 . To determine whether cre activity in the hypothalamus of the three different pdx1-cre transgenes was due to previously unrecognized endogenous pdx1 expression, a mouse line with a lacz reporter cassette in the pdx1 locus both adult and embryonic (e15.5) pdx1 (fig . 2e and l and supplementary fig . Furthermore, expression of the endogenous pdx1 gene was undetectable in the hypothalamus by real - time rt - pcr (data not shown) indicating that pdx1-cre, pdx1-cre, and pdx1-cre / ert transgenes are ectopically expressed in the brain . Detection of cre - mediated recombination in the hypothalamus of pdx1-cre;r26r mice (fig . 2b, supplementary fig . 5, and supplementary fig . 10) raised the possibility that cre protein may be expressed in neurons involved in the regulation of energy and glucose homeostasis . To determine the extent of cre - mediated recombination within these specific neuronal populations, -gal positive cells in brain sections from leptin - treated pdx1-cre;r26r mice were co - localized with orexin and leptin - induced pstat3, respectively . In the lateral hypothalamus, -gal protein was expressed in a complex pattern that partially overlapped with both the orexin - expressing and leprb - expressing neuronal populations (fig . 3), although significant populations of -gal positive cells did not overlap with the neuronal cell population in either the lateral hypothalamus or in other hypothalamic regions including the arcuate nucleus . Nonetheless, these data clearly illustrate that the pdx1-cre line induces cre - mediated recombination in subpopulations of hypothalamic neurons involved in energy expenditure and glucose metabolism . Pdx1-cre;r26r mice display a complex pattern of cre - mediated recombination that partially overlaps with orexin - positive and leptin - responsive neuronal populations . Adult pdx1-cre;r26r mice were treated with leptin (5 mg / kg, intraperitoneally, 2 h), perfusion - fixed and brains isolated for immunohistochemical detection of pstat3, orexin, and -gal positive . Localization of -gal signal in brain sections of adult pdx1-cre;r26r mice is available in supplementary fig . A c: pstat3 (green) and -gal (red) immunoreactivity do not co - localize efficiently in the arcuate nucleus (arc). D i: co - localization of pstat3 (green) and -gal (red) immunoreactivity in a subpopulation of neurons in the lateral hypothalamus (lha). Despite extensive -gal labeling within the preoptic area, there was essentially no co - localization with leptin - responsive neurons (data not shown). J o: co - localization of orexin (green) and -gal (red) immunoreactivity within neurons in the lha . (a high - quality digital representation of this figure is available in the online issue .) A new transgenic line, mip - cre / ert, which employs an 8.5-kb fragment of the mouse ins1 promoter has been recently developed to express the tamoxifen - inducible cre / ert in -cells (tamarina et al ., unpublished data) (table 1). Following three doses of 2-mg tamoxifen, strong -gal activity was detected in the pancreatic islets of adult mip - cre / ert;r26r mice but not in their r26r littermates (fig ., no -gal activity was detected in any region of the brain from mip - cre / ert;r26r mice (fig . When the tamoxifen dose was increased to three doses of 8-mg, -gal activity was not detected within the brain (data not shown). Cre expression efficiency in the -cells, as determined by immunohistochemistry, was similar in mip - cre / ert (89.0 8.0%), rip - cre (85.4 5.5%), rip - cre / ert (88.9 5.8%), and pdx1-cre / ert (92.1 6.6%) mice (supplemental fig . Brains from adult mip - cre / ert; r26r (n = 5) and their r26r littermates (n = 5) (24) were sliced into five coronal sections and subjected to whole - mount x - gal staining the schematic of the mouse brain is from the allen mouse brain atlas (http://www.brain-map.org/) (29). Brain slices examined are shown in capital letters (a, b, c, etc . ). Vertical dashed lines mark coronal sectioning plane designated as face in lowercase letters (a, b, c, etc . ). C: sagittal brain sections from mip - cre / ert; r26r (top panel) and r26r littermates (bottom panel). D: whole - mount x - gal staining of pancreas from mip - cre / ert; r26r (top panel) and r26r littermates (bottom panel). Brains from mip - cre / ert; r26r mice and controls in b and c were negative for x - gal staining, while mip - cre / ert; r26r pancreas showed robust x - gal labeling in the islets in d. ctx, cortex; hy, hypothalamus . (a high - quality digital representation of this figure is available in the online issue .) The studies examining the in vivo role of genes associated with biological processes in the pancreas and -cells have relied largely upon fragments of the rat ins2 gene promoter or the pdx1 gene promoter (3,813,16). Although insulin secretion from -cells plays an important role in glucose control, many other tissues including the brain are intimately involved in the regulation of glucose metabolism . Previous studies have demonstrated that the 668 bp rat ins2 promoter fragment drives cre - recombinase expression within the central nervous system of the mouse transgenic line, rip - cre [tg(ins2-cre)] (9,24,25). In this study, we examined whether cre - mediated recombination occurred in the brain of six mouse transgenic lines that have been extensively used to express cre specifically within the pancreas or islet -cells (table 1). Analysis of cre - mediated recombination using the r26r reporter strain demonstrated that all six transgenic lines expressed cre recombinase to varying extents within the brain, raising the possibility that alterations of gene expression in the brain may complicate the analysis and that the observed phenotype may not be solely due to changes in the -cell . This possibility was highlighted by a recent study that used the rip - cre line to selectively delete the stat3 gene in the -cells (24). Stat3-deficient mice displayed increased food intake, obesity, and leptin resistance; physiological effects that the authors attributed to stat3 deficiency in the brain leading to impaired leptin signaling . There are no previous studies reporting cre - mediated recombination in the brain with pdx1-cre lines . Our findings indicate that the pdx1-cre line causes recombination in a subset of hypothalamic neurons involved in energy and nutrient homeostasis . The similarity of -gal expression patterns in the hypothalamus with the other two pdx1-cre transgenic lines suggest that cre - mediated recombination in these lines may also affect similar neuronal subpopulations . The lack of -gal activity in the brains of pdx1 mice indicates that the cre expression in the brain of the pdx1-cre, pdx1-cre, and pdx1-cre / ert mice is not a reflection of endogenous pdx1 gene expression . A likely explanation for this spurious expression is the removal of these pdx1 gene promoter fragments from their endogenous gene context . Furthermore, the similar recombination pattern generated with pdx1-cre lines makes it unlikely that this brain expression is a result of neighboring sequences at the sites of integration, which are almost certainly different for each line . While a lacz knock - in reporter to analyze endogenous ins2 expression is currently not available, a recent study demonstrated expression of the ins2 gene but not the ins1 gene in the mouse hypothalamus (26). Thus, in contrast to the infidelity of cre transgene expression found with the pdx1 promoter, cre expression observed using the ins2 promoter may reflect, in part, endogenous promoter activity . It is not known whether the ectopic expression of the insulin or pdx-1 transgenes occurs during the embryonic period, adult periods, or in both periods . First, we did not examine all currently available insulin, pdx-1, or other gene promoters used to direct cre expression to the pancreas or -cell . Thus, it is essential that investigators examine brain expression in any cre line thought to be pancreas- or -cell specific . Second, our results should not be interpreted to indicate specific expression (or lack of expression) in any brain region or nuclei as we did not perform detailed mapping of brain regions following lacz staining . We did note regions with strong x - gal staining, but this should not be taken as evidence that other areas do not express cre, and it is possible that a more isolated or diffuse expression in other brain regions may also lead to cre activity . More detailed work is needed to identify which brain regions are positive or negative for cre activity . Third, whether cre expression leads to excision of a floxed dna fragment is an incompletely understood process that depends on both cre expression and the floxed allele . We mostly used a single reporter line, and we do not know if the results would differ with other lines that express other reporters such as alkaline phosphatase . In fact, we predict that some reporter lines will not show the same cre activity we observed given that a range of sensitivity of floxed alleles to cre - mediated recombination is likely (with the r26r line being more cre - sensitive) (27). This possibility further complicates interpretation of studies using cre to inactivate a gene of interest . Thus, we urge caution in extrapolating that the lack of cre - mediated recombination with a certain reporter gene predicts a lack of cre - mediated recombination of a gene of interest in the brain . Based on the current study, it is clear that the r26r floxed allele is susceptible to cre - mediated recombination in several brain regions, including orexin - positive and leptin - responsive neuronal populations . If lineage tracing is the goal, is it preferable to use a sensitive or insensitive reporter? If gene inactivation is the goal of cre - mediated recombination, then whether other tissues or cells endogenously express the gene of interest becomes a critical factor . If the gene of interest is expressed in places other than the pancreas or -cell (especially in the brain where a large number of genes are known to be expressed in both tissues), then the current finding of cre - mediated recombination in brain regions involved in glucose homeostasis, appetite, weight, and energy expenditure make attributions of the phenotype to the -cell more difficult . The above analysis clearly illustrates that new transgenic lines are needed to ensure fidelity of conditional cre expression in islet -cells . In transgenic tg(ins1-egfp) mice (28), an 8.5-kb fragment of the mouse ins1 gene promoter was successfully used to express enhanced green fluorescent protein (egfp) specifically within islet -cells in the absence of egfp expression in other tissues . This same ins1 promoter fragment was used in the mip - cre / ert transgenic line to direct cre / ert gene expression in -cells (tamarina et al ., unpublished data). The improved fidelity of cre expression observed in the mip - cre / ert line is likely due, in part, to the additional regulatory elements within the larger promoter fragment employed and because the mouse ins1 gene is not expressed in the hypothalamus (26). Thus, the mip - cre / ert mice appear to represent a transgenic line to express cre efficiently and specifically in islet -cells . In conclusion, this study reveals that the current transgenic lines utilizing ins2 and pdx1 promoter fragments target cre expression not only to the islet -cells, but also to the brain . While not invalidating the use of these lines, our data indicate that studies conducted using these cre transgenic mice should be interpreted carefully to assess whether manipulation of the target gene within the brain could contribute to the observed phenotype . The lack of cre - mediated recombination in the brain of mip - cre / ert;r26r mice suggests that the newly developed mip - cre / ert line is currently the only available -cell specific cre line . As with all newly developed cre transgenic lines, caution must also be exhibited when using this line until its potential as a -cell specific cre line has been validated through further experimental analysis.
Musicians often experience musculoskeletal pain as a result of lengthy and long - term instrument performance . More students enrolled in music schools note upper limb pain compared with students enrolled in ordinary schools1 . It has also been reported that the incidence of upper limb pain is higher in those who have longer practice times . Therefore, it can be said that upper limb pain in musicians is a symptom of overuse, with lengthy and long - term instrument performance being contributing factors . Musculoskeletal pain in musicians is common not only in the upper limbs but also in the lumbar and cervical regions, as well as in the shoulder girdle2 . For example, it is conceivable that when playing a heavy instrument such as the tuba, a large load is applied to the trunk to maintain the performance posture . Furthermore, as instruments in a marching band are played while marching, a large load is probably also applied to the lower limbs . However, the performance postures of various types of instruments have not been sufficiently investigated . Thus, the present study clarified the characteristics of the standing performance posture for the trumpet and the marching euphonium and investigated the effect of the performance postures of these instruments on the lower limb musculoskeletal system . The subjects were 10 female university students in japan . Their (mean sd) age was 20.5 1.3 years, their height was 159.6 6.2 cm, and their weight was 55.1 7.2 kg . Because the subjects were not professional trumpet or marching euphonium performers, they all received sufficient instruction from the instructor regarding the trumpet and marching euphonium performance posture prior to measurements . The purpose and contents of the study were sufficiently explained to the subjects, whose consent to participate in the study was obtained . Additionally, approval was obtained from the shijonawate gakuen university ethics committee (approval number 23 - 2). The subjects adopted a closed - leg resting standing position, a trumpet performance posture, and a marching euphonium performance posture . All instrumental performance postures were adopted in the standing, and the subjects focused directly ahead on a mark positioned at eye level . Subjects maintained performance posture without actually playing the instrument . The angle and muscle activity of the trunk and lower limbs were measured while the postures were maintained . The measurement was performed in the sequence of the resting standing position, the trumpet performance posture, and the marching euphonium performance posture . The trunk and lower limb angles were measured using a three - dimensional motion analysis device (cms - hs, zebris medical gmbh, isny, germany). Markers were attached to the lateral malleolus, lateral epicondyle of the femur, greater trochanter, and acromion on the left side of the subjects . The sampling frequency was 100 hz, and the measurement time was 10 s. measurements were performed as soon as the subjects could maintain a stable performance posture . The collected data were processed by image analysis software (win - date, zebris medical gmbh), and the angles of knee flexion, hip flexion, and anterior tilt of the trunk were calculated . The angle of knee flexion was considered as the angle formed between the line linking the lateral malleolus and the lateral epicondyle of the femur and the line linking the lateral epicondyle of the femur and the greater trochanter . The angle of hip flexion was considered as the angle formed between the line linking the lateral epicondyle of the femur and the greater trochanter and a vertical line . The anterior tilt angle of the trunk was considered as the angle formed between the line linking the acromion and the greater trochanter and a vertical line . A surface myograph (myosystem 1200, noraxon inc ., the muscle activities measured were those of the cervical paraspinal muscles, upper fibers of the trapezius, lumbar paraspinal muscles, gluteus maximus, rectus femoris, biceps femoris, and lateral head of the gastrocnemius on the left side . After treating the skin sufficiently to create a skin resistance of no greater than 10, electrodes (blue sensor p-00-s, ambu a / s, ballerup, denmark) were attached parallel to the muscle fibers at the center of each muscle . Spacing between the electrodes was 25 mm, the sampling frequency was 1,000 hz, and the sampling time was 10 s. measurements were performed as soon as subjects could maintain a stable performance posture . The collected data were subjected to full - wave rectification using electromyogram analysis software (myoresearch, noraxon inc . ), and the average amplitude of the central three seconds was determined . The average amplitude of each muscle was normalized as 100% according to the average amplitude of the 3-s maximum voluntary contraction . Statistical analysis entailed a one - way analysis of variance and multiple comparison using tukey's method . Mean sd . A: significant difference between the standing position and the trumpet performance posture (p<0.01). B: significant difference between the standing position and the marching euphonium performance posture (p<0.01). C: significant difference between the trumpet performance posture and the marching euphonium performance posture (p<0.05) mean sd . A: significant difference between the standing position and the marching euphonium performance posture (p<0.01). B: significant difference between the standing position and the marching euphonium performance posture (p<0.05). C: significant difference between the trumpet performance posture and the marching euphonium performance posture (p<0.01) the anterior tilt angle of the trunk decreased significantly in the trumpet and marching euphonium performance postures compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture (table 1). The muscle activity of the cervical paraspinal muscles, upper fibers of the trapezius, and lumbar paraspinal muscles increased significantly in the marching euphonium performance posture compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture (table 2). However, there were no significant differences in other measurement items between the postures . The anterior tilt angle of the trunk can be rephrased as an increase in the posterior tilt angle of the trunk . This is likely a means for maintaining the combined center of gravity of the instrument and the body in the most stable position possible . An increase in the posterior tilt angle of the trunk increases the lumbar lordosis angle . This increase results in tension being applied to the anterior tissues of the lumbar spine, such as the anterior longitudinal ligament, and compressive force being applied to the zygapophyseal joints4 . Christie et al . Reported that an increase in the lumbar lordosis angle while standing is associated with chronic low back pain5 . Consequently, maintaining the performance posture for the trumpet and the marching euphonium for a long period can contribute to low back pain . No significant difference was found in the muscle activities of the lumbar paraspinal muscles between the resting standing position and the trumpet performance posture, but a significant increase was noted in the marching euphonium performance posture compared with the resting standing position . The lumbar paraspinal muscles are antigravity muscles which are contracted continuously while standing, thus easily incurring mechanical stress . It is conceivable that, since the muscle activity of the lumbar paraspinal muscles increases during the marching euphonium performance posture, holding the performance posture for a long time increases the load on the lumbar paraspinal muscles and is therefore a factor leading to lumbar fatigue and low back pain . The muscle activity of the upper fibers of the trapezius increases in line with instrument weight in the trumpet to marching euphonium postures . When holding these instruments, the shoulder joint flexes and abducts, while the scapula rotates up and elevates . The muscle activity of the upper fibers of the trapezius is likely to increase to maintain such a posture . We also found that muscle activity of the cervical paraspinal muscles increased significantly in the euphonium performance posture compared with the resting standing position . Since the posterior tilt angle of the trunk increases in the marching euphonium performance posture, it is necessary to tuck in the chin to face forward chin - in posture and is considered a posture in which the muscle activity in the cervical area including the cervical paraspinal muscles increases6 . Sustained contraction of these muscles is a likely factor of muscle fatigue . Furthermore, obstruction of blood flow by sustained muscle contraction will result in myalgia7 . Therefore, holding the trumpet and marching euphonium performance postures for a long time would be factors of myalgia . The present findings suggest that the trumpet and marching euphonium performance postures increase the load on the cervical and trunk musculoskeletal system . Moreover, the load on the musculoskeletal system increases in line with increased instrument weight . However, the trumpet and marching euphonium performance postures do not affect the musculoskeletal system of the lower limbs . Therefore, we consider it important to evaluate the cervical and trunk musculoskeletal systems and to conduct conditioning coaching appropriate for trumpet and marching euphonium performers . Because these instruments are supported by the left hand and played with the right hand, the trunk and lower limb musculoskeletal system on the left side, that is, the supporting side, was measured in the present study . We plan to measure the trunk and lower limb musculoskeletal system on the right side to elucidate the effect of performance postures on the whole body . In addition, it is possible that the trunk and lower limb musculoskeletal system is affected differently by the technique used to hold the instrument with the left hand during trumpet and the marching euphonium performance . Because the angles and muscle activities of the upper arm were not measured in the present study, we plan to perform these measurements in a future study to elucidate the influence of different instrument holding techniques on the trunk and lower limb musculoskeletal systems.
Protein glycosylation is a phenomenon shared by all domains of life . Over 70% of the eukaryotic proteome although it is too early to predict the full extent of prokaryotic glycosylation, it is clear from the diversity of prokaryotic glycoproteins discovered in recent years that glycosylation in these organisms is the norm rather than the exception . A great deal of progress has been made in understanding prokaryotic glycosylation since the seminal review of szymanski and wren in 2005 which focused on the discovery, five years earlier, of a general n - glycosylation system in campylobacter jejuni . The best understood prokaryotic glycoproteins are s - layers, pilins, and flagellins plus a selection of cell surface and secreted proteins which are known to be involved in adhesion and/or biofilm formation . Notably, novel general o - glycosylation systems have recently been uncovered in both pathogenic and symbiotic bacteria . In this paper, our primary aim is to articulate commonalities and differences in eukaryotic and prokaryotic glycosylation rather than provide full coverage of specific areas . There are many excellent specialist reviews referred to throughout our paper which the reader should consult for in depth coverage of particular topics . Until recently it was widely believed that n - glycosylation of proteins is a eukaryotic phenomenon . Nevertheless, it was as long ago as 1976 that mescher and strominger reported that the s - layer protein from an archaeal prokaryote, halobacterium salinarum, contained glycans covalently linked to asparagine residues . Over the ensuing three decades sporadic evidence emerged suggesting that n - glycosylation was likely to be common in the s - layers of archaea . Then, in the early years of the 21st century, groundbreaking research on the bacterial pathogen campylobacter jejuni, showed that this prokaryote has a general n - glycosylation system [3, 4]. It soon became clear that all three domains of life (eukarya, bacteria, and archaea) perform n - glycosylation in a similar manner . Thus, all engage in stepwise assembly of sugars in the cytoplasm, donated by soluble nucleotide - activated sugars, to form an oligosaccharide precursor attached via pyrophosphate (all domains) or phosphate (archaea) to a lipid carrier (the so - called lipid - linked oligosaccharide or llo). After assembly of the oligosaccharide, the llo is flipped from the cytoplasm to face the lumen of the endoplasmic reticulum (er), or the periplasmic face of the inner membrane, in eukaryotes and gram - negative bacteria, respectively (figures 1(b) and 1(c)). Thus far n - glycosylation has not been observed in gram - positive bacteria . In the case of archaea, which do not have a compartment equivalent to the er or periplasm, flipping across the cytoplasmic membrane will position the llo on the exterior surface of the cell where the subsequent transfer to proteins is believed to occur (figure 1(a)). In all three cases, the oligosaccharide is subsequently transferred en bloc from the lipid carrier onto the acceptor protein in a step catalysed by the ubiquitous oligosaccharyltransferase enzyme (n - ost). Shared and unique aspects of the three hallmark events of n - glycosylation (cytoplasmic assembly of the llo, flipping across the er / periplasmic / cytoplasmic membranes, and en bloc transfer of the oligosaccharide to the protein acceptor) within the three domains are examined in more detail in the next sections . Interestingly these hallmark processes are mirrored in the biosynthesis of bacterial lipo - oligosaccharides (los) and lipo - polysaccharides (lps) (compare figures 1 and 2). In eukarya and archaea, the lipid constituent of the llos is dolichol, which is a polymer of isoprene units (ch3c(ch3)=ch ch2) numbering about 12 in archaea, 14 in yeast, and up to 19 in mammals . Bacteria also have a polyisoprene as their llo lipid but, instead of dolichol, they use undecaprenol (11 isoprene units) which has one more double bond than the same length dolichol . This double bond is located between carbons 2 and 3 with respect to the alcohol group (see figure 3). The absence of this specific double bond will confer greater rotational mobility to the oligosaccharidic chain in the dolichol llos, compared with the undecaprenol llos, which might facilitate chain extension after flipping . The llo biosynthetic pathway has been exhaustively characterized in eukaryotes and is very well understood [7, 8]. Thus the cytoplasmic llo carries a unique heptasaccharide (man5glcnac2; figure 1(b)) which is further elaborated in all higher eukaryotes, after flipping to the lumen of the er, by the stepwise addition of 4 additional mannoses plus 3 glucoses, donated by dolichol - phosphate - linked sugars, to form glc3man9glcnac2-p - p - dol (figure 1(b)). The glucoses play a pivotal role in lectin - mediated quality control of glycoprotein folding in the er and are removed by glucosidases during the folding process . In protozoa, however, there is some divergence from the conserved 14 sugar llo [9, 10]. It has been discovered that these primitive eukaryotes are characterized by llos that lack glucose and some are further deficient in the four er - derived mannoses . This lack of llo processing in the protist's er is reminiscent of periplasmic events in bacteria which do not appear to involve the addition of further sugars to their translocated llos (see below). Although the n - biosynthetic pathways of the three domains have much in common, the archaeal and bacterial llo processes differ from eukaryotes in two key respects . Firstly, there is no evidence for oligosaccharide sequences being conserved amongst the archaea and bacteria, in contrast to the conserved glc3man9glcnac2 sequence of all higher eukaryotes . Indeed, as shown in figure 4, a great diversity of glycans are known to be transferred by n - osts to bacterial and archaeal proteins . Despite this diversity, there is some commonality with respect to the type of linking sugar utilized in the three domains . Secondly, the bacterial and archaeal llos do not appear to be further elaborated after flipping . However, it should be borne in mind that knowledge of bacterial and archaeal n - linked glycosylation is only just emerging, and very few biosynthetic pathways have been investigated thus far . Therefore, it remains an open question as to whether llos can be extended by stepwise addition of extra sugars in the periplasmic and cell surface compartments . Although there exists a very substantial body of evidence, assembled over more than three decades, demonstrating unequivocally that in eukaryotes the llo precursor is assembled in the cytoplasm and then flipped across the er membrane to the lumen, remarkably no er flippase has yet been biochemically identified . Hence comparisons of flippase structures and mechanisms between the three domains are not yet possible . Fortunately, genetic tools have enabled considerable progress to be made in uncovering likely candidates for flippases . Thus, genetic experiments in yeast have provided very good evidence that the rft1 protein is involved in transfer of the man5glcnac2-llo across the er membrane . In accordance with the conclusion that they play a role in translocation, rft1 proteins are conserved in eukaryotic organisms, although it is still not clear whether they are actually the elusive flippases . The elucidation of archaeal n - biosynthetic pathways is still in its infancy (see calo et al . For an in - depth review of recent discoveries) and in contrast, bacterial flippases are quite well understood, not the least because llo translocation is integral to lps biosynthetic pathways which have been intensively studied for many years . It is known, for example, that the product of the wzx gene, a non - abc - type transporter, mediates transport of undecaprenol - linked o - antigen subunits across the plasma membrane in lps biosynthesis . With respect to the bacterial n - glycosylation pathway, which has been rigorously studied in the paradigm organism, c. jejuni, aebi, and coworkers have shown that pglk (previously called wlab), which is an abc - type transporter, is responsible for flipping the llo . Interestingly, these workers found that pglk has a relaxed substrate specificity exemplified by its ability to complement a wzx deficiency in o - antigen biosynthesis in e. coli . Notably, all bacterial n - glycans identified to date have seven or fewer sugar residues, with many archaeal structures being of a similar size (figure 4). As described earlier, the eukaryal cytoplasmic llo contains seven sugars (see figure 1(b)). These observations suggest that a maximum of seven sugars might be optimal for the flipping mechanism, though it has also been suggested that the flipping process might be affected by monosaccharide composition at the reducing end of the glycan . In this context, it could be significant that the large archaeal n - linked polysaccharide shown in figure 4 is composed of tandem repeats of a short oligosaccharide . This type of structure is reminiscent of bacterial lps and could therefore be assembled from short llo precursors, after flipping across the cytoplasmic membrane, in a similar way to wzx / wzy - dependent o - antigen polymerization in the periplasm of bacteria . Alternatively, it possible that this n - linked polysaccharide might be flipped across the membrane in an atp - binding cassette (abc) transporter - dependent manor . The transfer of oligosaccharides from the llos to asparagine acceptors in n - linked glycoproteins is catalysed by homologous oligosaccharyltransferase enzymes (n - osts) in the three domains of life . In eukaryotes and archaea; n - osts are ubiquitous . Consequently, n - linked glycoproteins are found in abundance throughout both domains . On the other hand, bacteria have probably not evolved n - osts of their own (see below) and n - glycosylation is restricted to a limited number of species . This is the pglb gene of campylobacter jejuni which was found to be highly homologous to the catalytic subunit (called stt3) of eukaryotic n - osts . A similar degree of homology was found in archaeal n - ost genes (which are called aglb) when their identity was confirmed a few years later [16, 17]. When the general n - glycosylation system was first discovered in c. jejuni, it was thought to be unique, and it was postulated that this organism might have acquired the pglb gene by lateral gene transfer from either the archaeal or the eukaryal domains . It is now considered most likely that pglb originated from archaea rather than eukarya (brendan wren, london school of hygiene and tropical medicine, personal communication). This conclusion is based on knowledge emerging from searches of bacterial genomes for pglb orthologues . Thus far, bacterial n - ost candidates have been found exclusively in a subset of species belonging to the phylogenetic grouping known as the epsilon subdivision of the proteobacteria, which include campylobacter, helicobacter, and wolinella genera . Amongst these, n - glycosylation has been rigorously confirmed by mass spectrometry for c. jejuni, w. succinogenes, and h. pullorum (figure 4) [1, 18, 19]. Note, however, that although h. pullorum has the machinery for n - glycosylation, the pglb gene is absent in related mammalian pathogens such as h. pylori and h. hepaticus . It may be significant that in primordial deep sea vents, which are the homes for many archaea, the majority of bacteria are epsilon proteobacteria . So it is tempting to speculate that these extreme environments have provided the conditions for n - ost gene transfer between the prokaryotic domains (brendan wren, personal communication). We now overview current understanding of the biochemistry of n - osts across the three domains of life . N - osts in archaea, bacteria, and primitive eukaryotes (protozoa) are comprised of a single subunit (the catalytic subunit) which is the product of the aforementioned aglb, pglb, and stt3 genes, respectively . In contrast, all n - osts of higher eukaryotes are multi - subunit complexes in which the catalytic subunit (stt3) is accompanied by a total of seven additional proteins whose roles remain poorly understood [14, 2022]. Suggested functions of these accessory proteins include regulating substrate specificity, possibly by expanding the range of acceptor sequences, and assisting in protein translocation and/or folding . Why primitive eukaryotes do not require a multiprotein complex remains enigmatic, but even more enigmatic is the observation that a single stt3 from leishmania major can substitute for the whole n - ost complex in yeast [23, 24]. For example, l. major expresses four stt3 paralogs, whilst trypanosoma brucei has three . Yeast, however, has only a single stt3 gene (called stt3p), whilst vertebrates, insects, and plants have two, encoding for stt3a and stt3b, respectively . It has been shown, via sirna knockdown experiments in mammalian cells, that stt3a glycosylates cotranslationally, whilst stt3b, which is normally coexpressed, acts posttranslationally, although the protein must not be folded . Also, stt3b is required for efficient glycosylation adjacent to the n - terminal signal sequence . Thus the two isoforms appear to function in concert to ensure maximal efficiency of n - glycosylation . Information is only just beginning to emerge concerning the number of n - ost genes in prokaryotes . Campylobacter has only a single pglb gene but h. pullorum has two unrelated genes, denoted pglb1 and pglb2, the first of which has been proven to mediate glycosylation . Bioinformatic searches for aglb genes in archaea have suggested multiple candidates in individual organisms but confirmation of expression and activity has not yet been determined experimentally . N - osts from all domains have been found to exhibit quite relaxed specificity with respect to the oligosaccharide donor . Thus each is capable of transferring short glycans from biosynthetic llo intermediates in addition to the full length glycans of the mature llos . Eukaryotic and bacterial n - osts transfer glycans whose reducing sugar carries at least one acetamido (nac) group . Thus the eukaryotic n - glycan has a chitobiose core (glcnacb1 - 4glcnac) and the linking sugar in characterized bacterial n - glycans is either 2,4-diacetamido-2,4,6-trideoxyglucopyranose (bacillosamine; campylobacter and wolinella) or hexnac (h. pullorum). Interestingly, the pglb protein of c. jejuni is capable of transferring a variety of o - antigen oligosaccharides onto protein acceptors in engineered e. coli cells, provided their reducing sugar has an nac moiety . This discovery has important implications for the development of o - antigen containing neoglycoprotein vaccines . Archaea appear to have a greater diversity of linking sugars, including glc as well as glcnac and galnac [5, 17]. Interestingly, a sulfolobus archaeal species, which is very close phylogenetically to primitive eukaryotes, has mannose rich chitobiose - linked n - glycans reminiscent of the eukaryotic core sequence . When comparing mechanisms of prokaryotic and eukaryotic n - glycosylation, it is important to remember that the folding status of their proteins is very different at the time of glycosylation . Thus, eukaryotic oligosaccharides are transferred to nascent proteins before they are folded, whilst in prokaryotes the proteins are presumably fully folded, having already been transported from the cytoplasm, where translation occurs, into the periplasm or onto the surface, where glycosylation takes place . In all three domains, the asparagine acceptor must normally be located in a consensus sequence (asn - x - ser / thr or, rarely, asn - x - cys, where x cannot be proline); however, not all consensus sequences are glycosylated . Sequence motifs contributing to specificity of site occupancy are not yet fully understood, but it is already clear that bacterial glycosylation is much more restricted than eukaryotic glycosylation . For example, consensus sites in c. jejuni require an upstream glu or asp residue in the extended consensus sequence d / eznxs / t, where neither z nor x can be proline . High throughput glycoproteomic efforts are beginning to provide comprehensive site - occupancy data in eukaryotic systems . It is hoped that these and similar experiments will facilitate the development of algorithms that will be capable of accurately predicting which consensus sequences in eukaryotic proteomes are likely to be occupied . In contrast to eukaryotes, very few prokaryotic glycoproteins have had their glycosylation sites determined . Based on a limited body of data, some predictions have been made for sequences favouring archaeal glycosylation but emerging data from studies of sulfolobus s - layers suggest that these rules probably will not be universally applicable (see and unpublished work from our laboratory). Bearing in mind that glycosylation in prokaryotes occurs posttranslationally, it is conceivable that general rules for site occupancy may not prevail in these organisms, because of the unique nature of individual proteins with respect to accessible consensus sequences . This could be especially relevant in s - layer glycosylation, because these proteins self - assemble into crystalline monolayers and all their consensus sequences are therefore likely to be in exposed locations [30, 31]. In concluding this section on n - osts, we draw attention to the fact that crystal structures are now available for the c - terminal domains, that include the wwdyg motif implicated in the catalytic mechanism, of both an archaeal aglb and the pglb of c. jejuni, although not so far for the eukaryotic stt3, despite many valiant efforts . Mechanistic and evolutionary understanding provided by the crystal structures has been reviewed very recently so will not be covered here . All eukaryotic glycoproteins are subjected to extensive remodelling in the golgi apparatus after they exit the er, resulting in heterogeneous mixtures of glycoforms exhibiting a great variety of peripheral structures, many of which are rich in functionally important sugars such as fucose and sialic acid [7, 8]. Prokaryotes have no counterpart to the golgi apparatus, and there is no evidence so far that they remodel their n - linked glycoproteins . About seven years ago a study of the hmw1 adhesin of h. influenzae uncovered a potentially novel n - glycosylation pathway occurring in the cytoplasm of this bacterium . This intriguing discovery has now been confirmed by rigorous structure analyses which, remarkably, have identified 31 glycosylated asn residues within the hmw1 protein [33, 34]. All sites carry either hex or hex - hex, where hex can be gal or glc, and all but one of the glycosylation sites has the normal n - glycosylation consensus sequence (asn - x - ser / thr, see section 2). Interestingly it transfers glucose to all glycosylated asparagines but only transfers galactose to a subset of these sites . Moreover, the same enzyme appears to be responsible for the hex - hex glycoforms as well as those carrying a single glc or gal . Homology analysis suggests that a variety of other bacteria possess hmw1c - like proteins, so it is likely that this type of cytoplasmic n - glycosylation will be found elsewhere . Whether similar glycosylation occurs in archaea is not known . As shown in figure 4, glucose has been observed as a linking sugar in some archaeal n - glycans, but it is likely that the n - ost pathway is employed in their biosynthesis . The presence of a glc - asn moiety was reported in eukaryotic laminin in 1994 but this observation has not been independently confirmed . During the past five years, intensive research on neisseria and pseudomonas pilin glycosylation has uncovered a general o - glycosylation pathway that, remarkably, has all the hallmarks of n - linked glycosylation (figure 5). Moreover, this general pathway does not appear to be restricted to a few pilin proteins in a handful of pathogens . Thus, very interesting data are emerging from research on bacteroides species that suggests that these bacteria are capable of glycosylating a great number of proteins in this way . So far oligosaccharyltransferase - mediated o - glycosylation has only been found in gram - negative bacteria, which is perhaps not surprising, bearing in mind that it mirrors lps biosynthesis . Much of our knowledge of bacterial o - linked glycosylation pathways has been elucidated from studies in neisseria species . O - linked glycosylation was first characterised in neisseria meningitidis, where the pilin protein was shown to be modified by a trisaccharide, with a similar glycan being found on n. gonorrhoeae . Subsequent bioinformatics and directed mutagenesis led to the identification of an o - ost, called pgll, in n gonorrhoeae [38, 39]. Pgll o - osts belong to a family of bacterial osts responsible for o - linked glycosylation of type iv pilins . This family appears to be widespread amongst pathogenic bacteria, including some strains of pseudomonas aeruginosa, where it is called pilo [40, 41]. Moreover, it has recently been demonstrated that neisseria are able to decorate a diverse set of proteins via the o - ost pathway [42, 43]. Research using neisseria and pseudomonas glycosylation systems in engineered e. coli cells has demonstrated that the biosynthesis of the o - linked glycan has a number of similarities to its n - linked counterpart (compare figures 1 and 5). The o - linked glycosylation pathway involves llos, and the glycans are transferred en bloc by the o - osts from the llos carrier onto the protein . The translocation of the llo substrate into the periplasm is required for activity and it has been shown that pglf, a protein with homology to o - antigen flippase, is required for pilin glycosylation which is thought to occur in an analogous manner to the wzy - dependent addition of o - antigen to the core - lps . In a similar fashion to the n - osts in archaea, bacteria, and lower eukaryotes, the o - ost's catalytic subunit is sufficient for glycosylation . As with o - glycosylation in eukaryotes, there appears to be no consensus sequence for defining sites of o - glycan attachment . Interestingly, the neisseria o - osts display a pronounced substrate promiscuity when compared to n - osts, as demonstrated by their ability to transfer virtually any glycan from an llo carrier onto pilin in engineered e. coli cells . For example, it was shown that the neisseria pgll could transfer peptidoglycan subunits onto pilin, highlighting the potential for exploitation of such pathways for biotechnological purposes . As such it appears that the substrate specificity of the o - osts is found in the lipid carrier, a hypothesis nicely demonstrated using an in vitro glycosylation system that utilised purified neisseria pgll, pilin, and the lipid farnesyl pyrophosphate carrying a synthetic pentasaccharide that was successfully transferred onto the pilin protein . Bacteroides comprise one of the most abundant genera of commensals in the human colon . Exciting recent research suggests that these bacteria are not only capable of o - glycosylating many of their proteins but, unusually, they exploit a host - like pathway to add fucose (apparently acquired from their host glycans and/or from plant polysaccharides present in the gut) onto their glycoproteins and polysaccharides . A combination of cell biology and molecular biology experiments has provided convincing evidence for the existence of a general o - glycosylation system in these symbiotic bacteria which has all the hallmarks of the pilin o - ost - mediated pathogen pathway described earlier . Notably, o - glycosylation appears to be central to the physiology of b. fragilis as well as its ability to colonise its ecological niche . Although the structures of the b. fragilis o - glycans remain to be defined, many elements of the biosynthetic pathway are beginning to be unraveled . Thus five glycosyltransferases, plus an unrelated fucosyltransferase, have been proposed to be involved in assembly of the llo on the cytoplasmic face of the inner membrane . Translocation to the periplasm is thought to be mediated by the o - antigen flippase (wzx, see figure 2). However, there is no candidate gene as yet for the putative o - ost . Interestingly, very recently it has been reported that fucosylated o - glycans are present on the fimbriae of porphyromonas gingivalis . Like b. fragilis, it is conceivable, therefore, that p. gingivalis glycosylates its proteins via a similar pathway to b. fragilis . Monosaccharide compositional analysis has shown that the p. gingivalis glycans are likely to be complex (fuc, xyl, man, gal, glc, galnac and glcnac have all been detected) but so far no sequences are available for this glycoprotein . All eukaryotic o - glycosylation is processive that is, it is a stepwise process which begins with the attachment of the linking monosaccharide to the acceptor serine or threonine . Many eukaryotic o - glycans are of the mucin - type which are linked via galnac, but other classes exist which are attached to proteins via a variety of sugars including fuc, man, glc, gal, ara, xyl, and glcnac . Most eukaryotic o - biosynthetic events take place in the golgi, although some classes of o - glycans, for example, o - man linked glycans (see later), are initiated in the er . An enormous variety of sequences can be attached to these linking sugars . Thus there is a great diversity of o - glycosylation in the eukaryotic domain [6, 7]. The only substantive study was about twenty years ago when the s - layers of halobacterium salinarum and haloferax volcanii were shown to carry several o - linked disaccharides of sequence glc1 - 2gal . In contrast, there is a large body of evidence pointing to a rich diversity of o - glycans in the bacterial domain . The most complex structures have been found on bacterial s - layers which have been investigated in many species of bacteria over the last thirty years . Their structures and biosynthesis have been comprehensively reviewed on several occasions [5, 31, 49, 50] and the reader is referred to these articles for further information . Below we discuss emerging understanding of other families of bacterial cell surface and secreted o - linked glycoproteins, many of which, in contrast to the s - layers, have structural and/or functional counterparts in the eukaryotic domain . Mucins are high molecular weight eukaryotic glycoproteins, produced in abundance by epithelial and goblet cells, whose polypeptide chains are coded by the muc genes . Mucins are characterized by the presence of tandem repeats of serine / threonine / proline - rich sequences which are extensively o - glycosylated . Mucins readily form gels and are a key component of most gel - like secretions in eukaryotes where they have functions ranging from lubrication to serving as receptors for microbes . In recent years, it has become evident that many bacterial biofilms contain glycoproteins whose compositions indicate that they are mucin - like molecules [5153]. The best characterized are the serine - rich repeat (srr) glycoproteins belonging to the fap1 family, which are conserved in streptococci, staphylococci, and lactobacilli, and are required for bacterial biofilm formation and pathogenesis . The polypeptides of srr family members are comprised of a long signal peptide followed in turn by a short serine - rich domain, an acidic or basic region, a long serine - rich domain and a c - terminal anchoring motif . The srr sequences are reminiscent of the eukaryotic mucins in that the majority of the polypeptide is comprised of tandem repeats of short motifs which have related sequences (see figure 6 for comparison of a portion of an srr domain compared with part of the human muc-1 sequence). The serine - rich domains have the key hallmark of the mucins, namely, variable repeated sequences rich in potential o - glycosylation sites (see figure 6), but in contrast to the mammalian mucins, proline is absent from the srr domains . Thus their proline residues are important for ensuring that exposed, accessible sites are available for glycosylation . Currently little is known about the process of glycan attachment to the bacterial srrs, other than the fact that attachment of the linking sugar appears to occur very rapidly in the cytoplasm, before transport to the cell surface via the accessory sec transporter . It has been proposed that the glycosylation mechanism is a two - step process, with the second step requiring several accessory secretion components and thus is probably coupled with secretion . Recent electron microscopy structural studies have indicated that the serine dipeptide repeat domains have a super - helical extended structure with exposed serine side - chains, which are expected to be readily accessible to o - glycosylation . The glycan content of the srrs has been explored in five species, streptococcus parasanguinis, s gordonii, s. agalactiae, s. pneumoniae, and staphylococcal aureus . This has been largely done using lectins such as wheat germ agglutinin (wga) which recognize terminal glcnac, and by sugar compositional analyses . Lectin blotting, supplemented by sugar composition data, has indicated that the linking sugar in the srrs is probably glcnac . Interestingly two glycosyltransferases, called gtf1 and gtf2, are required for this initial glycosylation step by s. parasanguinis . Gtf1 and gtf2 homologs from s. pneumoniae also form an enzyme complex that catalyzes the transfer of glcnac to serine - rich sites of psrp . A similar requirement for two glycosyltransferases adding a single sugar occurs in the o - mannosyl glycans of higher eukaryotes (see later) whose biosynthesis is initiated by a heterodimer enzyme complex composed of protein o - mannosyltransferases (pomt) 1 and 2 . It should be noted that although o - linked glcnac is found on eukaryotic cytoplasmic and nuclear proteins, it is probably not analogous to srr o - glycosylation, because eukaryotic o - linked glcnac residues are not further extended . Moreover, this ubiquitous eukaryotic glycosylation is unusual because it is dynamic and involves cross - talk with phosphorylation . A glucosyltransferase has recently been identified in s. parasanguinis that transfers glucose to the glcnac - modified fap1 . Although the structure of the product of glycosylation remains to be determined, it is tempting to speculate that these bacterial proteins might carry glycans whose core sequences are glucosyl analogues of the core type 1 sequence, gal1 - 3galnac, that is ubiquitous in mammalian mucins . Sugars additional to glc and glcnac, including galnac and rha, have been observed at low levels in sugar analyses of the fap1 glycoproteins . It remains to be established whether the putative glc - glcnac moieties are further elongated or whether other glycans account for the compositional data . The title of a recent review protein o - mannosylation: conserved from bacteria to humans o - mannosyl glycans are abundant in yeast and fungi, whilst in mammals they occur on a restricted number of proteins, such as -dystroglycan where their impairment is a cause of congenital muscular dystrophy [63, 64]. Yeast and fungi express short mannosyl oligomers, with galactose being present on terminal sites in some species . In contrast, mammalian o - mannosyl glycans carry sialylated and fucosylated n - acetyllactosamine sequences similar to those found in mucins . Eukaryotic o - mannosylation is initiated in the er by the concerted action of two protein o - mannosyltransferases (pomt1 and 2) which employ dol - p - man as the mannose donor . Chain extension subsequently takes place in the golgi . O - mannosyl glycans analogous to those found in yeast and fungi have been found on glycoproteins from members of the actinomycetes class of gram - positive bacteria which include the mycobacteria and streptomyces genera [62, 65]. The first to be characterised were the surface glycoproteins of mycobacterium tuberculosis [66, 67]. Subsequently m. bovis, corynebacterium glutamicum, and streptomyces coelicolor were shown to be similarly glycosylated . All contain o - glycans whose sequences are restricted to short stretches of mannose (usually three residues or less). Like in eukaryotes, the mannosyl donor is a polyprenol phosphate, and their protein o - mannosyltransferases (pomts) are membrane associated . The activities of the products of candidate pomt genes in m. tuberculosis, c. glutamicum, and s. coelicolor have been genetically and biochemically confirmed . The fact that bioinformatic screening has uncovered a plethora of pomt homologs in other species, indicates that o - mannosylation constitutes a general o - glycosylation pathway in actinomycetes . Steps equivalent to the eukaryotic golgi processes of o - mannose extension have not been determined thus far in bacteria . There are, nevertheless, candidate mannosyltransferases, for example, those involved in the biosynthesis of the mannan core of cell wall lipomannan / lipoarabinomannan in mycobacteria . It has been suggested that o - mannose extension in mycobacteria is coupled with sec - dependent secretion in a manner akin to that proposed for the serine - rich proteins in streptococci and staphylococci described earlier . Aida - i, tiba, and ag43 are three autotransporter proteins in pathogenic e. coli which are associated with virulence phenotypes, such as the formation of biofilms and aggregates . All three are extensively glycosylated with o - linked heptose on their so - called passenger domains [7174]. Glycosylation occurs in the cytoplasm and the heptoses are derived from adp - glycero - manno - heptopyranose which is recruited from the lps biosynthetic pathway . The passenger domains are secreted to the extracellular environment where their glycosylation appears to enhance bacterial attachment to human cells . The heptoses are attached at multiple sites in ser / thr rich domains (figure 7) that are reminiscent of eukaryotic mucin sequences (see earlier) although they lack the hallmark tandem repeats of the latter . Flagellin o - linked glycosylation has been widely reported in a number of bacteria, where it appears to be restricted, with the archaeal counterparts being n - linked . Current knowledge of the o - linked sugars involved in flagellin glycosylation has been covered in recent reviews [75, 76]. Probably the best studied flagellin glycans are found to be glycosylated with a family of sialic acid - like monosaccharides, based around the sugars pseudaminic acid and legionaminic acid, with a diversity being generated by variation in their decorating appendages . These sugars have been found in several species including campylobacter jejuni, helicobacter pylori, and aeromonas caviae . In c. jejuni, the o - linked glycosylation gene cluster has been identified and the function of a number of gene products involved in the pseudaminic acid biosynthetic pathway has been elucidated . It appears to have evolved to share some of the same biosynthetic machinery as the n - linked glycosylation pathway, allowing the organism to maintain a compact genome and avoid redundancy . Flagellin glycosylation in p aeruginosa has also been shown to share biosynthetic machinery, in this case with the o - antigen pathway . The most complex flagellin o - glycans identified thus far have been found in hypervirulent strains of clostridium difficile [75, 78]. The c. difficile flagellins carry hexnac - linked oligosaccharides up to at least five sugars in length . In contrast to the en bloc transfer in the n - linked pathway, it is apparent that flagellin o - linked glycosylation is likely to proceed in a sequential fashion . Given that a single sugar residue is often added to sites of attachment, specific glycosyltransferases are thought to be involved in the glycosylation process, but our present understanding of the glycosyltransferases involved in the glycosylation process remains limited . The current proposed model for o - linked flagellin glycosylation occurs at the cytoplasmic face of the inner membrane in the vicinity of the type iii secretion complex . Nucleotide activated sugars are utilised by specific glycosyltransferases in the glycosylation machinery and are added to exposed serine and threonine residues . The glycosylated flagellin monomers are then secreted to the tip of the growing flagellin filament . However, it is well known that certain sequence motifs are preferred in mucin type o - glycosylation . Indeed the netoglyc open access tool, which can be very helpful for predicting possible sites of eukaryotic mucin o - glycosylation, was developed using knowledge of preferred sequence motifs . The rapid progress that is being made in defining o - glycosylation sites in diverse prokaryotic glycoproteins, coupled with the fact that some researchers are beginning to employ the netoglyc tool to guide them in the choice of targets for mutation in searches for prokaryotic glycosylation [67, 80], have made it timely to assess the applicability of the netoglyc tool to prokaryotic glycoprotein research . In a preliminary unpublished study, we have ascertained netoglyc predictions for selected members of each of the families of prokaryotic o - glycoproteins described in the previous sections, for which there is published experimental data on site occupancy . The outputs send the very clear message that netoglyc does not, in fact, correctly predict o - glycosylation in most families of prokaryotic glycoproteins . Thus we found that no sites were correctly predicted in the pilins, flagellins, serine - rich proteins, the autotransporters, or the bf2494 glycoprotein of b. fragilis . Experimental data suggests that the latter has an essential three - residue glycosylation site motif (d)(s / t)(a / i / l / v), which is not typical of eukaryotes, so it is therefore not surprising that netoglyc appears to be not suitable for predicting this type of o - glycosylation . In conclusion, netoglyc is likely to only be useful for predicting o - glycosylation in prokaryotic glycoproteins like mycobacterial lipoproteins whose pro / ser / thr - rich glycosylation domains have sequence characteristics which they share with the mammalian mucins . Prediction in other families will require the development of new algorithms which take account of their specific glycosylation domain characteristics . Although much remains to be uncovered concerning protein glycosylation in prokaryotes, several themes are emerging from the discoveries of the past decade . Firstly, like in eukaryotes, n - glycosylation is largely restricted to asn - x - ser / thr consensus sequences, even when the canonical oligosaccharyltransferase pathway is not involved . Secondly o - glycosylation is far more abundant in bacteria than in archaea whilst the reverse is true for n - glycosylation . Thirdly, oligosaccharyltransferase - mediated o - glycosylation is likely to be widespread in gram - negative bacteria . In contrast this type of o - glycosylation has not thus far been proven to occur experimentally in gram - positive bacteria or archaea . Finally cytoplasmic o - glycosylation appears to be both more common and more diverse in gram - positive compared with gram - negative bacteria, possibly because of the existence of the alternative periplasmic o - ost pathway of the latter.
All reactions were carried out under a nitrogen atmosphere in oven- or flame - dried glassware . All catalysts and reagents were obtained from commercial sources and were used without further purification with the exception of bf3oet2, which was purified by distillation from cah2 . N - allyl-4-methylaniline, 2-allyl-4-methylaniline, 2-allylaniline, 2-bromocyclohex-1-ene-1-carbaldehyde, and 2-(but-3-en-2-yl)aniline were prepared according to published procedures . Structural and stereochemical assignments were made on the basis of 2-d cosy, hsqc, and noesy experiments . Ratios of diastereomers were determined by h nmr analysis . High resolution esi mass spectra were acquired using an instrument equipped with at tof mass analyzer, and high resolution ei mass spectra were acquired using an instrument equipped with a magnetic sector mass analyzer . Yields refer to isolated yields of compounds estimated to be 95% pure as determined by h nmr analysis unless otherwise noted . The yields reported in the supporting information describe the result of a single experiment, whereas the yields reported in scheme 2 and table 1 are average yields of two or more experiments . Thus, the yields reported in the supporting information may differ from those shown in scheme 2 and table 1 . A thick - walled glass pressure tube equipped with a stir bar was sealed with a septum . The pressure tube was flame - dried and cooled under a vacuum and then purged with nitrogen . The flask was charged with n - allyl-4-methoxyaniline (2.5 g, 15.2 mmol) and xylenes (30.5 ml). The solution was cooled to 0 c, bf3oet2 was added dropwise, and the resulting solution was then allowed to warm to room temperature . The septum was removed, the pressure tube was replaced with a teflon screw cap, the tube was immersed in a 170 c oil bath, and the reaction mixture was allowed to stir for 2 h. the mixture was then cooled to room temperature, and 1 m naoh (15 ml) was added . The resulting mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was extracted with dichloromethane (3 15 ml). The combined organic layers were dried over anhydrous na2so4, filtered, and then concentrated in vacuo . The crude product was purified via flash chromatography on silica gel using 5% ethyl acetate: hexanes as the eluent to afford 870 mg (35%) of the title compound as an orange oil: h nmr (700 mhz, cdcl3) 6.666.62 (m, 3 h), 5.975.91 (m, 1 h), 5.135.08 (m, 2 h), 3.74 (s, 3 h), 3.40 (s, br, 2 h), 3.28 (d, j = 5.6 hz, 2 h); c nmr (175 mhz, cdcl3) 152.9, 138.3, 135.7, 125.7, 116.9, 116.2, 116.0, 112.7, 55.7, 36.6; ir (film) 3357, 1500 cm; ms (esi+) 164.1071 (164.1070 calcd for c10h13no, m + h). A flame - dried flask equipped a stir bar was cooled under a vacuum and then purged with nitrogen . This flask was charged with 2-iodonitrobenzene (3.6 g, 14.5 mmol) and thf (58 ml). The resulting solution was cooled to 40 c in a mecn / co2(s) bath, and then a solution of phmgbr (16 ml, 16.00 mmol, 1 m in thf) was added dropwise . The resulting mixture was stirred at 40 c for 5 min, and then a solution of cucn2licl (29 ml, 29 mmol, 1 m in thf) was added dropwise . The reaction mixture was stirred at 40 c for 10 min, and then 2-methylallyl bromide (2.4 g, 17.5 mmol) was added dropwise . The mixture was stirred at 40 c for 1 h, and then the reaction was quenched with nh4cl (50 ml) and transferred to a separatory funnel . Water (50 ml) was added, the layers were separated, and the aqueous layer was extracted with etoac (2 50 ml). The organic layers were then combined, washed with brine, dried over anhydrous na2so4, filtered, and concentrated in vacuo . The crude product was purified via flash chromatography on silica gel using 5% ethyl acetate: hexanes as the eluent to yield slightly impure 2-(2-methylallyl)nitrobenzene (1.8 g, 70% yield), which was carried on to the next step without further purification . A flame - dried flask equipped with a stir bar was cooled under a vacuum, purged with nitrogen, and charged with 2-(2-methylallyl)nitrobenzene (1.8 g, 10 mmol) and etoh (67 ml). The resulting mixture was stirred at rt until the 2-(2-methylallyl)nitrobenzene was completely dissolved, then powdered zinc (9.9 g, 151 mmol) and acoh (8.6 ml, 151 mmol) were added . The resulting mixture solution was stirred at rt for 1 h and then was filtered through celite, and the celite was washed with et2o . The crude product was purified via flash chromatography on silica gel using 25% ethyl acetate: hexanes as the eluent to afford 1.16 g (54%) of the title compound as a yellow oil that contained ca . Data are for the major product: h nmr (400 mhz, cdcl3) 7.097.02 (m, 2 h), 6.74 (t, j = 7.6 hz, 1 h), 6.67 (d, j = 7.6 hz, 1 h), 4.87 (s, 1 h), 4.74 (s, 1 h), 3.68 (s, br, 2 h), 3.28 (s, 2 h), 1.73 (s, 3 h); c nmr (100 mhz, cdcl3) 145.2, 143.6, 131.0, 127.5, 123.9, 118.7, 115.9, 111.6, 41.1, 22.3; ir (film) 3394, 1480 cm; ms (esi+) 148.1125 (148.1121 calcd for c10h13n, m + h). A flame - dried flask equipped with a stir bar and a reflux condenser the flask was charged with the appropriate 2-allylaniline derivative (1.25 equiv), the appropriate 2-bromobenzaldehyde derivative (1.0 equiv), 4 molecular sieves (1 g / mmol), and toluene (0.2 m). The resulting solution was heated to reflux with stirring overnight . The mixture was then cooled to rt and filtered, and the solids were washed with etoac . The combined organic solutions were then concentrated in vacuo, and the resulting crude imine was immediately used without further purification . A flame - dried flask with a stir bar was cooled under a vacuum and then purged with nitrogen . The flask was charged with the crude imine (1 equiv) and et2o (0.5 m) and was cooled to 0 c . A solution of lialh4 (2 equiv, 4 m in et2o) was added dropwise, and the resulting solution was allowed to warm to rt and stir overnight . The mixture was diluted with et2o, and then water (0.1 ml / mmol of lah) was carefully added dropwise to the solution followed by dropwise addition of 1 m naoh (0.1 ml / mmol of lah). Additional water was then added dropwise until a white solid precipitated and clung to the walls of the flask . The solution was filtered, the flask was washed with et2o, and the resulting solution was also filtered . The combined organic solutions were then concentrated in vacuo, and the crude product was then purified via flash chromatography on silica gel using 25% ethyl acetate: hexanes as the eluent . The coupling of 2-allylaniline (894 mg, 6.72 mmol) with 2-bromobenzaldehyde (994 mg, 5.37 mmol) was accomplished according to general procedure a. this procedure afforded 1.01 g (62%) of the title compound as a white solid: mp 4244 c; h nmr (400 mhz, cdcl3) 7.62 (d, j = 8 hz, 1 h), 7.39 (d, j = 7.6 hz, 1 h), 7.267.22 (m, 1 h), 7.147.06 (m, 3 h), 6.71 (t, j = 7.2 hz, 1 h), 6.53 (d, j = 8 hz, 1 h), 6.035.93 (m, 1 h), 5.165.09 (m, 2 h), 4.43 (s, 2 h), 4.27 (s, br, 1 h), 3.35 (d, j = 6 hz, 2 h); c nmr (175 mhz, cdcl3) 145.7, 138.2, 136.0, 132.8, 129.9, 129.0, 128.6, 127.7, 127.5, 123.6, 123.2, 117.6, 116.4, 110.9, 48.2, 36.6; ir (film) 3440, 1512 cm; ms (esi+) 302.0544 (302.0539 calcd for c16h16brn, m + h). The coupling of 2-allyl-4-methoxyaniline (1.22 g, 7.47 mmol) with 2-bromobenzaldehyde (1.10 g, 5.97 mmol) was conducted according to general procedure a. this procedure afforded 1.09 g (55%) of the title compound as an orange oil: h nmr (400 mhz, cdcl3) 7.56 (d, j = 8 hz, 1 h), 7.34 (d, j = 7.2 hz, 1 h), 7.267.23 (m, 1 h), 7.12 (t, j = 8 hz, 1 h), 6.726.63 (m, 2 h), 6.49 (d, j = 8.8 hz, 1 h), 6.025.92 (m, 1 h), 5.155.09 (m, 2 h), 4.38 (s, 2 h), 3.97 (s, br, 1 h), 3.74 (s, 3 h), 3.33 (d, j = 6 hz, 2 h); c nmr (175 mhz, cdcl3) 152.1, 139.8, 138.5, 135.7, 132.7, 129.1, 128.5, 127.5, 125.7, 123.3, 116.6, 116.6, 112.2, 112.1, 55.7, 49.0, 36.5; ir (film) 3428, 1508 cm; ms (esi+) 332.0645 (332.0645 calcd for c17h18brno, m + h). The coupling of 2-allyl-4-methylaniline (2.20 g, 15.0 mmol) with 2-bromobenzaldehyde (2.21 g, 12.0 mmol) was conducted according to general procedure a. this procedure afforded 2.07 g (55%) of the title compound as an orange oil: h nmr (400 mhz, cdcl3) 7.56 (d, j = 8 hz, 1 h), 7.34 (d, j = 7.6 hz, 1 h), 7.257.21 (m, 1 h), 7.11 (t, j = 7.2 hz, 1 h), 6.916.89 (m, 2 h), 6.44 (d, j = 8.8 hz, 1 h), 6.035.93 (m, 1 h), 5.145.09 (m, 2 h), 4.40 (s, 2 h), 4.14 (s, br, 1 h), 3.32 (d, j = 6.4 hz, 2 h), 2.23 (s, 3 h); c nmr (175 mhz, cdcl3) 143.4, 138.4, 136.1, 132.7, 130.7, 129.0, 128.5, 128.0, 127.5, 126.7, 123.8, 123.3, 116.3, 111.1, 48.5, 36.6, 20.4; ir (film) 3437, 1513 cm; ms (esi+) 316.0695 (316.0695 calcd for c17h18brn, m + h). The coupling of 2-allylaniline (832 mg, 6.25 mmol) with 2-bromocyclohex-1-ene-1-carbaldehyde (945 mg, 5.00 mmol) was conducted according to general procedure a. this procedure afforded 989 mg (65%) of the title compound as a yellow oil that contained ca . Data are for the major product: h nmr (400 mhz, cdcl3) 7.15 (t, j = 8 hz, 1 h), 7.04 (d, j = 6.8 hz, 1 h), 6.70 (t, j = 8 hz, 1 h), 6.64 (d, j = 8 hz, 1 h), 6.005.90 (m, 1 h), 5.145.08 (m, 2 h), 4.123.91 (m, 3h), 3.29 (d, j = 6.4 hz, 2 h), 2.602.50 (m, 2 h), 2.182.09 (m, 2 h), 1.711.60 (m, 4 h); c nmr (175 mhz, cdcl3) 146.2, 136.1, 133.9, 129.8, 127.8, 123.6, 121.3, 117.3, 116.3, 110.8, 49.4, 36.8, 36.6, 29.3, 24.7, 22.4; ir (film) 3438, 1508 cm; ms (esi+) 306.0852 (306.0852 calcd for c16h20brn, m + h). The coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 2-bromo-5-fluorobenzaldehyde (2.02 g, 10.0 mmol) was conducted according to general procedure a. this procedure afforded 2.28 g (72%) of the title compound as a pale yellow solid: mp 4647 c; h nmr (700 mhz, cdcl3) 7.49 (dd, j = 4.9, 8.4 hz, 1 h), 7.107.06 (m, 3 h), 6.83 (t, j = 7.7 hz, 1 h), 6.72 (t, j = 7 hz, 1 h), 6.43 (d, j = 7.7 hz, 1 h), 6.025.96 (m, 1 h), 5.175.12 (m, 2 h), 4.38 (s, 2 h), 4.30 (s, br, 1 h), 3.37 (d, j = 4.9, 2 h); c nmr (175 mhz, cdcl3) 162.4 (d, j = 245.3 hz), 145.3, 140.8 (d, j = 6.8 hz), 136.0, 133.9 (d, j = 7.3 hz), 130.1, 127.8, 123.7, 117.9, 116.8 (d, j = 2.6 hz), 116.5, 115.9 (d, j = 23.8 hz), 115.6 (d, j = 22.9 hz), 110.8, 48.1, 36.6; ir (film) 3440, 1463 cm; ms (esi+) 320.0450 (320.0445 calcd for c16h15brfn, m + h). The coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 6-bromo-1,3-benzodioxole-5-carboxaldehyde (2.29 g, 10.0 mmol) was conducted according to general procedure a. this procedure afforded 2.51 g (73%) of the title compound as a white solid: mp 5355 c; h nmr (400 mhz, cdcl3) 7.137.06 (m, 2 h), 7.02 (s, 1 h), 6.85 (s, 1 h), 6.71 (t, j = 7.6 hz, 1 h), 6.51 (d, j = 8.4 hz, 1 h), 6.035.94 (m, 3 h), 5.165.09 (m, 2 h), 4.31 (d, j = 5.6 hz, 2 h), 4.22 (s, br, 1 h), 3.34 (d, j = 6 hz, 2 h); c nmr (175 mhz, cdcl3) 147.6, 147.4, 145.6, 136.0, 131.6, 129.9, 127.7, 123.7, 117.6, 116.4, 113.3, 112.8, 110.9, 109.0, 101.6, 48.1, 36.5; ir (film) 3439, 1478 cm; ms (esi+) 346.0440 (346.0437 calcd for c17h16brno2, m + h). The coupling of 2-allylaniline (1.7 g, 12.5 mmol) with 1-bromo-2-naphthaldehyde (2.35 g, 10.00 mmol) was conducted according to general procedure a except after addition of lialh4 the reaction mixture was heated to reflux overnight . This procedure afforded 2.77 g (79%) of the title compound as an orange solid: mp 9597 c; h nmr (400 mhz, cdcl3) 8.34 (d, j = 8.4 hz, 1 h), 7.80 (d, j = 8 hz, 1 h), 7.74 (d, j = 8.8 hz, 1 h), 7.60 (t, j = 7.2 hz, 1 h), 7.527.48 (m, 2 h), 7.107.06 (m, 2 h), 6.71 (t, j = 7.2 hz, 1 h), 6.55 (d, j = 8.8 hz, 1 h), 6.055.95 (m, 1 h), 5.165.11 (m, 2 h), 4.66 (s, 2 h), 4.39 (s, br, 1 h), 3.37 (d, j = 6.0 hz, 2 h); c nmr (175 mhz, cdcl3) 145.8, 136.6, 136.0, 133.9, 132.4, 130.0, 128.1, 127.8, 127.8, 127.4, 126.9, 126.3, 126.1, 123.7, 123.0, 117.6, 116.5, 111.0, 49.2, 36.6; ir (film) 3442, 1541 cm; ms (esi+) 352.0700 (352.0695 calcd for c20h18brn, m + h). The coupling of 2-(2-methylallyl)aniline (1.16 g, 7.84 mmol) with 2-bromobenzaldehyde (1.16 g, 6.28 mmol) was conducted according to general procedure a. this procedure afforded 767 mg (39%) of the title compound as a white solid: mp 4346 c; h nmr (400 mhz, cdcl3) 7.56 (d, j = 8 hz, 1 h), 7.32 (d, j = 6 hz, 1 h), 7.257.21 (m, 1 h), 7.147.04 (m, 3 h), 6.70 (t, j = 7.2 hz, 1 h), 6.51 (d, j = 8 hz, 1 h), 4.88 (s, 1 h), 4.76 (s, 1 h), 4.424.39 (m, 3 h), 3.33 (s, 2 h), 1.73 (s, 3 h); c nmr (175 mhz, cdcl3) 146.1, 143.7, 138.3, 132.7, 130.8, 128.8, 128.5, 127.7, 127.5, 123.5, 123.2, 117.4, 112.0, 110.9, 48.1, 41.4, 22.3; ir (film) 3430, 1510 cm; ms (esi+) 316.0692 (316.0695 calcd for c17h18brn, m + h). The coupling of 2-(but-3-en-2-yl)aniline (298 mg, 2.02 mmol) with 2-bromobenzaldehyde (300 mg, 1.62 mmol) was conducted according to general procedure a. this reaction afforded 451 mg (88%) of the title compound as a yellow oil that contained ca . Data are for the major product: h nmr (400 mhz, cdcl3) 7.57 (d, j = 8 hz, 1 h), 7.34 (d, j = 7.6 hz, 1 h), 7.247.22 (m, 1 h), 7.157.07 (m, 3 h), 6.75 (t, j = 8 hz, 1 h), 6.54 (d, j = 8 hz, 1 h), 6.035.94 (m, 1 h), 5.125.07 (m, 2 h), 4.424.37 (m, 3 h), 3.52 (quint, j = 6.4 hz, 1 h), 1.43 (d, j = 7.2 hz, 3 h); c nmr (175 mhz, cdcl3) 145.2, 142.3, 138.3, 132.8, 129.0, 128.6, 128.5, 127.5, 127.4, 126.9, 123.3, 117.7, 114.2, 111.1, 48.3, 38.1, 18.9; ir (film) 3434, 1506 cm; ms (esi+) 316.0693 (316.0695 calcd for c17h18brn, m + h). The coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 2-bromoacetophenone (2.00 g, 10.0 mmol) was conducted according to general procedure a except after addition of lialh4 the reaction mixture was heated to reflux overnight . This procedure afforded 1.75 g (55%) of the title compound as a yellow oil that contained ca . 6% of an unknown impurity: h nmr (400 mhz, cdcl3) 7.54 (d, j = 8 hz, 1 h), 7.37 (d, j = 7.6 hz, 1 h), 7.19 (t, j = 7.2 hz, 1 h), 7.087.02 (m, 2 h), 6.97 (t, j = 8 hz, 1 h), 6.63 (t, j = 7.6 hz, 1 h), 6.18 (d, j = 8 hz, 1 h), 6.075.97 (m, 1 h), 5.235.18 (m, 2 h), 4.84 (quint, j = 6.4 hz, 1 h), 4.25 (s, br, 1 h), 3.40 (d, j = 6 hz, 2 h), 1.48 (d, j = 6.4 hz, 3 h); c nmr (175 mhz, cdcl3) 144.7, 143.6, 136.3, 132.9, 129.7, 128.3, 128.0, 127.6, 126.7, 122.9, 122.6, 117.2, 116.3, 111.6, 52.4, 36.9, 23.1; ir (film) 3746, 1506 cm; ms (esi+) 316.0692 (316.0695 calcd for c17h18brn, m + h). A flame - dried schlenk tube equipped with a stir bar was cooled under a vacuum and then purged with nitrogen . The tube was charged with naobu (96 mg, 1 mmol), pd2(dba)3 (9 mg, 0.01 mmol), and pcy3hbf4 (15 mg, 0.040 mmol). The substrate (0.5 mmol) was dissolved in toluene (5 ml) and added to the flask . The resulting solution was heated 105 c with stirring for 18 h, at which time the substrate was judged to be completely consumed by gc, tlc, or h nmr analysis of an aliquot removed from the reaction mixture . The mixture was then cooled to rt, and saturated aqueous nh4cl (10 ml) was added . The mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was extracted with etoac (3 15 ml). The combined organic layers were then dried over anhydrous na2so4, filtered, and concentrated in vacuo . The crude product was then purified via flash chromatography on silica gel using 2% ethyl acetate: hexanes as the eluent to afford a product that still contained small amounts of impurities . The product was then dissolved in 30% et2o / hexanes (5 ml, 0.1 m) and 2 m hcl in et2o (1.5 ml, 3 equiv) was added dropwise . The resulting solution was stirred at rt for 1 min, and a white solid (the product hcl salt) precipitated from the solution . The salt was then dissolved in dicholoromethane (10 ml) and transferred to a separatory funnel . A solution of 4 m naoh was added, and the mixture was shaken vigorously for 1 min . The layers were then separated, and the aqueous layer was extracted with dichloromethane (3 15 ml). The combined organic layers were dried over anhydrous na2so4, filtered, and concentrated in vacuo to afford the desired product . The cyclization of 3a (151 mg, 0.500 mmol) was conducted according to general procedure b to afford 94 mg (85%) of the title compound as a yellow solid: mp 125127 c; h nmr (400 mhz, cdcl3) 7.207.10 (m, 6 h), 6.72 (t, j = 7.2 hz, 1 h), 6.55 (d, j = 8 hz, 1 h), 4.58 (d, j = 15.6 hz, 1 h), 4.02 (d, j = 15.2 hz, 1 h), 3.583.50 (m, 1 h), 3.15 (dd, j = 7.6, 15.2 hz, 1 h), 3.053.00 (m, 2 h), 2.75 (dd, j = 11.2, 15.0 hz, 1 h); c nmr (175 mhz, cdcl3) 151.8, 134.7, 133.5, 129.5, 129.3, 127.5, 126.9, 126.3, 126.1, 124.4, 118.5, 107.0, 61.6, 48.5, 35.7, 35.6; ir (film) 2778, 1456 cm; ms (ei) 221.1201 (221.1204 calcd for c16h15n, m). The cyclization of 3b (166 mg, 0.500 mmol) was conducted according to general procedure b except only 1 equiv of 2 m hcl in et2o was used . This procedure afforded 86 mg (68%) of the title compound as a yellow solid: mp 134 c (dec); h nmr (700 mhz, cdcl3) 7.197.14 (m, 4 h), 6.80 (s, 1 h), 6.68 (d, j = 7 hz, 1 h), 6.50 (d, j = 7 hz, 1 h), 4.54 (d, j = 14.7 hz, 1 h), 3.93 (d, j = 14.7 hz, 1 h), 3.74 (s, 3 h), 3.453.40 (m, 1 h), 3.11 (dd, j = 7.7, 14.7 hz, 1 h), 3.053.02 (m, 2 h), 2.73 (dd . J = 11.9, 14.7 hz, 1 h); c nmr (175 mhz, cdcl3) 153.4, 146.2, 134.6, 133.6, 131.2, 129.3, 126.9, 126.3, 126.0, 112.2, 111.6, 107.3, 62.6, 56.0, 49.6, 36.0, 35.4; ir (film) 2781, 1486 cm; ms (ei) 251.1314 (251.1310 calcd for c17h17no, was conducted according to general procedure b to afford 98 mg (81%) of the title compound as a brown solid: mp 6870 c; h nmr (400 mhz, cdcl3) 7.217.15 (m, 4 h), 6.976.92 (m, 2 h), 6.49 (d, j = 8 hz, 1 h), 4.58 (d, j = 15.2 hz, 1 h), 3.98 (d, j = 15.2 hz, 1 h), 3.523.44 (m, 1 h), 3.11 (dd, j = 14.8, 7.6 hz, 1 h), 3.043.02 (d, j = 6.8 hz, 2 h), 2.74 (dd, j = 14.8, 11.2 hz, 1 h), 2.27 (s, 3 h); c nmr (175 mhz, cdcl3) 149.8, 134.8, 133.7, 129.9, 129.3, 128.0, 127.6, 127.0, 126.4, 126.1, 125.4, 107.0, 62.2, 49.2, 35.7, 35.6, 20.9; ir (film) 2918, 1491 cm; ms (ei) 235.1358 (235.1361 calcd for c17h17n, m). The cyclization of 3d (153 mg, 0.500 mmol) was conducted according to general procedure b. in order to remove a small amount of unreacted starting material from the product, the crude product was dissolved in dichloromethane (2.5 ml), and dmap (6.1 mg, 0.050 mmol) and et3n (104 l, 0.750 mmol) were added to the solution . Acetic anhydride (61 l, 0.650 mmol) was added, and the solution was allowed to stir at rt for 3 h and was then concentrated in vacuo . The crude product was purified via flash chromatography to afford 62 mg (55%) of the title compound as a brown solid: mp 8890 c; h nmr (700 mhz, cdcl3) 7.097.06 (m, 2 h), 6.67 (t, j = 7.0 hz, 1 h), 6.45 (d, j = 7.7 hz, 1 h), 3.68 (d, j = 15.4 hz, 1 h), 3.343.29 (m, 1 h), 3.18 (d, j = 15.4 hz, 1 h), 3.03 (dd, j = 7.0, 14.7 hz, 1 h), 2.61 (t, j = 12.6 hz, 1 h), 2.21 (t, j = 14.7 hz, 1 h), 2.12 (d, j = 16.1 hz, 1 h), 2.021.91 (m, 4 h), 1.771.71 (m, 2 h), 1.571.52 (m, 2 h); c nmr (175 mhz, cdcl3) 151.9, 129.6, 127.5, 127.3, 125.7, 124.2, 118.1, 106.8, 61.5, 49.6, 36.5, 35.8, 30.2, 27.7, 22.9, 22.8; ir (film) 2916, 1607 cm; ms (ei) 225.1518 (225.1518 calcd for c16h19n, m). The cyclization of 3e (161 mg, 0.505 mmol) was conducted according to general procedure b to afford 105 mg (87%) of the title compound as a yellow solid: mp 9293 c; h nmr (400 mhz, cdcl3) 7.137.07 (m, 3 h), 6.896.85 (m, 2 h), 6.73 (t, j = 7.2 hz, 1 h), 6.54 (d, j = 8 hz, 1 h), 4.54 (d, j = 15.6 hz, 1 h), 3.98 (d, j = 15.6 hz, 1 h), 3.553.47 (m, 1 h), 3.15 (dd, j = 4.6, 14.8 hz, 1 h), 3.022.90 (m, 2 h), 2.74 (dd, j = 11.2, 14.8 hz, 1 h); c nmr (175 mhz, cdcl3) 161.3 (d, j = 242.6 hz), 151.6, 135.4 (d, j = 7 hz), 130.7 (d, j = 7.5 hz), 130.3 (d, j = 2.6 hz), 129.5, 127.6, 124.5, 118.8, 113.6 (d, j = 21 hz), 113.3 (d, j = 21.7 hz), 107.1, 61.8, 48.6 (d, j = 1.9 hz), 35.6, 34.9; ir (film) 2789, 1480 cm; ms (ei) 239.1116 (239.1110 calcd for c16h14fn, m). The cyclization of 3f (173 mg, 0.500 mmol) was conducted according to general procedure b to afford 95 mg (72%) of the title compound as a white solid: mp 9092 c; h nmr (400 mhz, cdcl3) 7.127.09 (m, 2 h), 6.72 (t, j = 7.2 hz, 1 h), 6.60 (d, j = 6.8 hz, 2 h), 6.53 (d, j = 8 hz, 1 h), 5.88 (s, 2 h), 4.46 (d, j = 14.8 hz, 1 h), 3.92 (d, j = 14.8 hz, 1 h), 3.523.43 (m, 1 h), 3.13 (dd, j = 7.6, 15.0 hz, 1 h), 2.90 (d, j = 7.2 hz, 2 h), 2.74 (dd, j = 10.8, 15.0 hz, 1 h); c nmr (175 mhz, cdcl3) 151.6, 146.1, 146.1, 129.4, 127.6, 127.4, 126.2, 124.3, 118.5, 108.8, 106.9, 106.5, 100.8, 61.5, 48.5, 35.5, 35.5; ir (film) 2891, 1484 cm; ms (ei) 265.1103 (265.1103 calcd for c17h15no2, m). The cyclization of 3 g (173 mg, 0.491 mmol) was conducted according to general procedure b to afford 106 mg (80%) of the title compound as a red solid: mp 9597 c; h nmr (400 mhz, cdcl3) 7.94 (d, j = 8.4 hz, 1 h), 7.79 (d, j = 8 hz, 1 h), 7.66 (d, j = 8.4 hz, 1 h), 7.50 (t, j = 7.2 hz, 1 h), 7.44 (t, j = 6.8 hz, 1 h), 7.22 (d, j = 8.4 hz, 1 h), 7.167.12 (m, 2 h), 6.75 (t, j = 7.2 hz, 1 h), 6.60 (d, j = 8 hz, 1 h), 4.64 (d, j = 15.6 hz, 1 h), 4.12 (d, j = 15.6 hz, 1 h), 3.603.51 (m, 2 h), 3.22 (dd, j = 7.2, 14.8 hz, 1 h), 3.09 (t, j = 12.8 hz, 1 h), 2.85 (dd, j = 11.6, 14.2 hz, 1 h); c nmr (175 mhz, cdcl3) 151.9, 132.5, 132.4, 130.9, 129.9, 129.8, 128.6, 127.6, 126.6, 126.3, 125.4, 125.4, 124.6, 122.9, 118.8, 107.3, 61.8, 49.3, 36.1, 32.0; ir (film) 2924, 1482 cm; ms (ei) 271.1351 (271.1361 calcd for c20h17n, m). The cyclization of 3h (163 mg, 0.517 mmol) was conducted according to general procedure b to afford 97 mg (80%) of the title compound as a yellow solid: mp 4750 c; h nmr (700 mhz, cdcl3) 7.197.14 (m, 3 h), 7.06 (m, 3 h), 6.63 (t, j = 7.0 hz, 1 h), 6.41 (d, j = 7.7 hz, 1 h), 4.50 (d, j = 16.1 hz, 1 h), 4.25 (d, j = 16.1 hz, 1 h), 3.10 (d, j = 15.4 hz, 1 h), 2.94 (s, 2 h), 2.70 (d, j = 15.4 hz, 1 h), 1.15 (s, 3 h); c nmr (175 mhz, cdcl3) 150.5, 134.6, 132.9, 129.8, 128.3, 127.6, 126.5, 126.4, 126.0, 124.8, 117.2, 106.2, 62.3, 43.7, 43.7, 40.0, 20.9; ir (film) 2924, 1482 cm; ms (ei) 235.1364 (235.1361 calcd for c17h17n, the cyclization of 3i (64 mg, 0.202 mmol) was conducted according to general procedure b to afford 36 mg (75%) of the title compound as a yellow oil . This product was judged to have been formed with 18:1 dr by h nmr analysis . Data are for the major isomer: h nmr (700 mhz, cd3od) 7.197.15 (m, 4 h), 7.097.06 (m, 2 h), 6.76 (t, j = 7.7 hz, 1 h), 6.62 (d, j = 7.7 hz, 1 h), 4.58 (d, j = 15.4 hz, 1 h), 3.83 (d, j = 14.7 hz, 1 h), 3.06 (d, j = 14.7 hz, 1 h), 2.922.84 (m, 3 h), 1.37 (d, j = 6.3 hz, 3 h); c nmr (175 mhz, cd3od) 152.7, 136.1, 135.6, 134.6, 130.3, 128.4, 127.9, 127.4, 127.1, 123.5, 120.3, 108.7, 71.5, 50.3, 43.3, 35.0, 16.1; ir (film) 3676, 1506 cm; ms (ei) 235.1363 (235.1361 calcd for c17h17n, m). The cyclization of 3j (165 mg, 0.500 mmol) was conducted according to the general procedure b to afford 64 mg (52%) of the title compound as a yellow oil . 2:1:1 mixture of the two possible diastereomers and a side product tentatively assigned as 6-methyl-11-methylene-5,6,11,12-tetrahydrodibenzo[b, f]azocine, which results from competing intramolecular heck reaction . Data are for the mixture: h nmr (700 mhz, cdcl3) 7.267.24 (m, 1 h), 7.227.20 (m, 1.5 h), 7.197.16 (m, 2.5 h), 7.167.14 (m, 2.5 h), 7.127.07 (m, 4.5 h), 7.02 (d, j = 7.7 hz, 1 h), 6.97 (t, j = 10.5 hz, 1 h), 6.686.63 (m, 2 h), 6.60 (d, j = 7.7 hz, 0.5 h), 6.48 (d, j = 8.4 hz, 1 h), 6.45 (d, j = 7.7 hz, 0.5 hz), 6.29 (ddd, j = 3.5, 10.5, 17.3 hz, 0.5 h), 5.235.13 (m, 1 h), 4.844.78 (m, 1.5 h), 4.534.50 (m, 1 h), 3.973.92 (m, 1 h), 3.763.60 (m, 1 h), 3.23 (dd, j = 8.4, 14.7 hz, 1 h), 3.062.92 (m, 4 h), 2.772.72 (m, 1.5 h), 1.55 (d, j = 6.3 hz, 1.5 h), 1.53 (d, j = 7.0 hz, 1.5 h), 1.36 (d, j = 7.0 hz, 3 h); c nmr (175 mhz, cdcl3) 151.3, 149.8, 146.0, 143.5, 140.9, 140.4, 139.8, 139.2, 134.5, 134.0, 132.6, 130.2, 129.6, 129.3, 129.0, 128.8, 127.7, 127.7, 127.5, 127.4, 127.4, 127.2, 127.1, 127.0, 126.5, 126.3, 126.0, 124.5, 124.2, 123.5, 122.3, 117.9, 117.8, 117.5, 117.3, 115.3, 106.9, 106.3, 62.7, 56.4, 54.6, 53.9, 50.6, 48.0, 36.3, 36.1, 35.8, 35.3, 24.9, 19.2, 17.2; ir (film) 3362, 1617 cm; ms (esi+) 236.1442 (236.1434 calcd for c17h17n, m + h).
One of the shortcomings of the available treatments for major depressive disorder (mdd) is the time delay between commencing the treatment and achieving an antidepressant response . Most of the approved drugs for such treatment need at least 2 to 4 weeks for the onset of their effects, and sometimes the peak of drug efficacy is achieved after 6 - 8 weeks (1). The fast effectiveness and the short period between the onset of action and the relief of symptoms are the major advantages of electroconvulsive therapy (ect) compared with common antidepressant drugs; however, similar to drugs, the time delay between the start of ect and its effect is still a weak point of this treatment modality (2, 3). Despite the relative superiority of ect to drugs in the treatment of mdd, the patients suffering caused by the symptoms and the possibility of harmful behaviors in the period between the start of treatment and the response to it still remains one of the major challenges in the treatment of depression . Ketamine is a non - competitive inhibitor of the n - methyl - d - aspartic acid (nmda) receptors . It is sometimes used for induction of anesthesia in ect because it neither raises the convulsive threshold nor impairs cognition (4). Furthermore, there are some evidences showing the elevated mood in patients receiving ketamine (5 - 7). Hence it seems that the induction of anesthesia by ketamine, which has mood enhancing effects, during ect, may expedite the process of mdd recovery . Recent studies suggest the probability of the synergic effects of ect and ketamine in accelerating the recovery of patients, but controversial results make performing more research in this field mandatory (8). Some reports show that ketamine during ect can have neuroprotective effects and reduce its cognitive side effects to some extent (7, 9), while other studies report effects to the contrary . Additionally, according to the results of some other investigations, ketamine itself may cause cognitive impairments (10). It, therefore, seems that more studies are needed to illuminate the effect of ketamine on patients cognitive conditions after ect . The present report evaluates the role of ketamine in accelerating antidepressant effects and its cognitive effect after ect . This double - blind randomized clinical trial study recruited mdd patients who received ect in qods hospital, sanandaj, west of iran, between july 2010 and june 2012 . The inclusion criteria were comprised of age between 18 and 65 years, normal iq, suffering from mdd according to the diagnostic and statistical manual of mental disorders, 4th edition, text revision (dsm - iv - tr) criteria, and having a score of 20 or higher in the hamilton rating scale for depression (hrsd). The hrsd scores, vital signs, and duration of reorientation were collected by an author who was blind to group assignment . The patients were also blind to the received medication and only the anesthesiologist responsible for the induction of anesthesia knew which patient received which drug . The exclusion criteria consisted of substance or drug dependence for up to at least 3 months before the study, any contraindication for receiving ect, cognitive impairment, epilepsy, receiving ect in the previous 3 months, and anesthesiology class> ii according to the american society of anesthesiologists (asa) physical status classification system . This study was approved by the ethics committee of kurdistan university of medical sciences and was registered in the iranian registry of clinical trials (irct.ir#irct138811022935n2). As the study was done under close observation in the hospital, no changes were made in the type and doses of the already prescribed drugs . Considering = 0.05, = 0.1, = 3.5, and d = 2.5, the sample size was calculated as 22 patients in each intervention and control group . The patients in the intervention group received ketamine with propofol and those in the control group received normal saline and propofol . The hrsd scores were obtained one day before ect onset, the day after the third session, the day after the last session, and finally 2 weeks after the last session . The minimum score needed to participate in the study was considered 20 and response to treatment was considered as at least a 50% reduction in the score after the last session . At time 0, the intervention group received 0.3 mg / kg of ketamine hydrochloride (manufactured by rotexmedica, germany) intravenously (iv) which was diluted with 5 ml of saline; and in the control group, ketamine was replaced with 5 ml of normal saline . Thirty seconds later, after the loss of eyelid reflex, anesthesia was induced with 1 mg / kg of propofol 1% iv (manufactured by fresenius kabi, austria gmbh). Muscle relaxation was achieved by the administration of 0.5 mg / kg of succinylcholine iv . Before succinylcholine administration, a manometer cuff was fastened to one of the legs and blown up to 30 ml higher than systolic pressure and was retained until the end of seizure . Propofol complementary doses were administered if needed . Then, in all the patients, lung ventilation with 100% oxygen was performed by mask . The anesthesiologist was the only person aware of the administered drugs . After complete relaxation, electric stimulation was performed using an ect apparatus (iec 601 - 1 type bf class 1, iran) with bifrontotemporal electrode placement . For the first session, the patients received 30% to 50% of maximum output stimulus . The blood pressure and heart rate of the patients were also evaluated 5 minutes after the motor seizures were ended . To assess the patients cognitive performance recovery time, 10 questions relating to orientation were asked at 5-minute intervals, after terminating the motor seizures (box 1). The minimum cognitive performance recovery time was considered when the patient was able to answer all the 10 questions correctly . Similar assessments have been performed to measure the patients cognitive performance recovery time in previous studies (7, 11). Repeated measurement was used to compare the trend of the hrsd, blood pressure, heart rate, seizure duration, and cognitive performance recovery time at different time points . The within - subject contrasts test was employed to assess the interaction effects between group and time . The average of the p values after adjusting the degree of freedom through the greenhouse - geisser and huynh - feldt was reported for some of the variables for which sphericity assumption was rejected . In the control group, one patient was excluded because of consent withdrawal for ect and one more patient was excluded because of early discharge from the hospital . Finally, of the 42 patients whose data were analyzed at the end of the study, 22 patients were in the intervention group (27.3% men and 72.7% women) and 20 patients (35% men and 65% women) were in the control group (figure 1). The two groups had no significant difference in sex distribution (p= 0.74, = 0.29, df = 1), education level (p = 0.72, = 1.5, df = 3), and mean age (p = 0.82, t = 0.23, df = 40) (table 1). There was no significant difference in the motor seizure duration between the two groups (p = 0.395, df = 5, f = 1.04) (table 2). The mean cognitive performance recovery time in the intervention group was lower than control group (p = 0.042, df = 5, f = 2.36) (figure 2 and table 2). There were no significant differences between the groups in terms of the rate and speed of the recovery process (p = 0.82, df = 3, f = 0.31) (figure 3, table 2). T1, one day before ect onset; t2, a day after the third session; t3, a day after the last session; t4, two weeks after the last session . The results of this study did not show any significant difference in cardiac side effects between the patients in both groups before and after ect (table 2). Other plausible side effects of ketamine such as postictal agitation, hallucination, or derealization were not seen in the participants of either group . In this study, we assessed the efficacy of ketamine on the rate of recovery from mdd by adding it to one of the routine methods of anesthesia induction in ect . We could not find significant differences in the symptom recovery, seizure duration, and blood pressure alterations between the two groups . However, the cognitive performance recovery time in the ketamine group was better than in the control group . In the current study, the evaluation of cognitive performance recovery time in both groups revealed that the patients who received ketamine were able to answer the cognitive ability questions faster than the control group . In mc daniel and colleagues study, the ketamine - administered patients for ect showed less memory impairment than the etomidate - administered patients.the authors suggested the antagonistic effect of ketamine on the nmda receptors as a protective agent against the side effects of ect on the cognitive abilities of patients (9). Krystal and colleagues also reported a reduction in cognitive disabilities caused by ect after the ketamine usage (7). In contrast, in a report by loo and colleagues, administering ketamine with thiopentone did not affect the neuropsychological side effects of ect in the treated patients . The ketamine dose was 0.5 mg / kg in that study, and the patients underwent ultrabrief pulse - width right unilateral ect (8). As unipolar ect has normally fewer side effects, one of the probable reasons for the lack of efficacy of ketamine on cognition in their study could be the different ect modality . In addition, evidence has also shown that the ketamine effect on glutamate transportation via the nmda receptors is dose - dependent and is shaped like an inverted u (14 - 16), which can be a possible explanation for the variations in different reports . In our study, although in 5 out of 6 sessions of ect, the motor seizure in the ketamine - administered patients was more than that in the control patients, the difference was not significant . In both groups, our finding is in line with that reported by kayhan and colleagues, who stated that their groups had no difference in motor seizure duration, but the quality of seizure was better in the ketamine group . In that study, the ketamine dose was 0.5 mg / kg and the authors also succeeded in recording the seizure time by electroencephalography (eeg) (13). On the other hand, krystal and colleagues showed that increasing the ketamine dose (0.7 to 2.8 mg / kg) could promote the seizure duration significantly compared with the administration of methohexitone only (7). It seems that the difference in results stems from the ketamine dose or mixing it with other drugs and the methods of seizure recording . As we could not record the seizures using eeg, we can make no claims regarding the quality of seizure . The present study did not show any significant differences in terms of the cardiac side effects of ketamine between the two groups . Although ketamine may cause cardiac side effects due to the systemic release of catecholamines (17), the small dose of ketamine used in this study may be the reason for the absence of cardiac side effects . It is deserving of note that some studies have shown that the combination of ketamine and propofol may provide better hemodynamic stability (18, 19). In our study, the decreasing trend of the scores in the hrsd during the treatment for both groups showed the efficacy of both anesthesia induction methods . However, there were no significant differences in the rate and quality of recovery between the two groups (p = 0.92). In machado - vieira and colleagues study, which used ketamine as an iv single dose, the patients showed recovery in the first 24 hours (20). The rapid antidepressant effects of ketamine were also shown in a report by pheilps and colleagues, who studied 26 treatment - resistant patients with major depression (24). Similar effects have been reported about using ketamine on patients with major depression and chronic pain syndrome (22), treatment - resistant patients with depression at risk of suicide (23), and also during ect (24). In wang s study, the patients who received ketamine or a ketamine - propofol combination during ect experienced less depression on days 2, 3, and 7 after ect . It is important to note that the patients in that study received only one ect session and underwent further ect if necessary (12). In a study done by goforth and colleagues, the depression symptoms of a patient were relieved 8 hours after administering 100 mg of ketamine and one ect session (24). Loo showed that adding ketamine led to better treatment efficacy in the first week but this effect had no continuity (8). It is noteworthy that there are limited studies on the combined effect of ketamine and ect and that they were retrospective (25) or case reports (26) or limited to one ect session (12). Moreover, a few other similar studies did not assess the continuity of the effect (21, 22). Consequently, it seems that although our study could not show the effect of ketamine on the mdd recovery in a 2-week follow - up period, the fact that there are conflicting reports on the effects of ketamine means that such effects on depression cannot be ruled out and that they may be caused by the ketamine dose, the onset of administering the drug before ect, and the time of evaluating the changes in depression intensity . First and foremost among the limitations of the present study is that we could not record the seizure duration by eeg . Another salient drawback to this study is its small sample size due to the unavailability of patients for follow - up in iran, which may have affected the results . Additionally, ect discontinuation because of patient noncompliance and incomplete hrsd questionnaires because of low literacy are also the weak points of our study . Another shortcoming is the small ketamine dose in our study by comparison with other similar studies . Indeed, achieving more reliable results requires the use of higher doses of ketamine . Finally, some degree of misinterpretation by our patients in answering the hrsd questionnaire may be a possible source of bias in this study.
Traumatic gallbladder rupture occurs rarely and the incidence is reported to amount to approximately 2% of all blunt abdominal injuries . The main reason for the low incidence is that the gallbladder is protected by the surrounding organs, including the liver, intestines, omentum, and ribs . Preoperative diagnosis of gallbladder rupture is generally difficult because no specific symptoms are observed and other concomitant organ injuries are common . Gallbladder rupture is usually diagnosed at laparotomy because emergency operation is necessary due to other organ injuries . However, even in the isolated gallbladder rupture which does not need an emergency operation, prompt diagnosis of gallbladder rupture is quite challenging due to the lack of specific symptoms . It is very difficult to detect a defect in the gallbladder wall even when using imaging such as ultrasonography, computed tomography (ct), and magnetic resonance imaging (mri). However, such images can detect the accumulation of intraperitoneal fluid including hemorrhage and bile . If accumulated fluids contain bile, endoscopic cholangiography is useful not only to diagnose the gallbladder injury but also to determine the therapeutic strategy . We present the details of a patient who underwent laparoscopic cholecystectomy after an exact preoperative diagnosis of traumatic gallbladder rupture . A 43-year - old man fell off a tractor and was subsequently run over by the tractor . On admission to our emergency department, his vital signs were stable and he was alert . Complete blood count revealed a white blood cell count of 21,800/mm and a hemoglobin level of 14.0 g / dl . Enhanced abdominal ct suggested a mild liver laceration in hepatic segment 5 and a small amount of hemorrhagic ascites around the liver (fig . The tentative diagnosis was liver injury with edward's classification grade i, and conservative treatment was initiated . The day after admission, the right upper quadrant pain and ascites increased and a muscular defense sign appeared . Percutaneous drainage of the ascites was performed, and the aspirated fluid was bloody and almost pure bile . Although his abdominal pain improved after continuous drainage, the amount of biliary discharge reached 800 ml / day . Mri and ultrasonography revealed little about the source of the bile leakage; therefore, on the 10th day after abdominal blunt injury, endoscopic retrograde cholangiography was performed, and a diagnosis of gallbladder rupture was made (fig . 2). Laparoscopic cholecystectomy was subsequently performed and revealed biliary ascites accumulated in the upper right abdomen with the gallbladder covered by edematous greater omentum . There were no injuries in other abdominal organs and no obvious injury in segment 5 of the liver . After isolating the gallbladder, a perforation was seen in the gallbladder body, as suggested by endoscopic retrograde cholangiography (fig . The perforation in the gallbladder wall was sutured closed, and then standard cholecystectomy was performed . Blunt traumatic gallbladder injury is rare, and the most common causes are accidents associated with motor vehicles, falls, blows, kicks, or industrial work . Blunt gallbladder injury is classified into three types: contusion, avulsion, and laceration (rupture or perforation), and lacerations are the most common after blunt injury . The gallbladder injury in our case was categorized as a laceration with an exact preoperative diagnosis . Ct scan is the most useful examination when diagnosing abdominal injuries, and avulsion gallbladder injuries can easily be diagnosed by ct because the gallbladder is torn from the liver and hemorrhage is obvious . However, no specific signs are revealed on ct for laceration gallbladder injuries . Despite the substantial leakage of bile into the peritoneal cavity in our case, we could not determine whether it was caused by a gallbladder injury or liver laceration, and further examinations and treatments were required because of the persistent high - volume bile leakage . As magnetic resonance cholangiopancreatography revealed little about the origin of the bile leakage, endoscopic retrograde cholangiopancreatography was planned . Because gallbladder rupture is often associated with multiple organ injuries leading to peritonitis, laparotomy is recommended as soon as possible after the diagnosis . However, ruptured gallbladder alone has no associated hemodynamic disorders after successful abdominal drainage; therefore, emergent laparotomy is not required, and patients are good candidates for laparoscopic surgery . Also, the final diagnosis of gallbladder rupture can be confirmed laparoscopically and simultaneous minimally invasive treatment can then be performed . In conclusion, management of gallbladder injuries varies with the presence of other concomitant organ injuries and the degree and type of gallbladder injury . In cases with gallbladder rupture simply diagnosed by direct cholangiography,
An estimated 207 million cases of human schistosomiasis have been reported worldwide and about 90% of these live in sub - saharan africa, with nigeria having the highest prevalence . Schistosoma infections cause significant morbidity and mortality with peak prevalence and intensity of infection occurring between the ages 10 and 20 years and subsequent decline by age 65 years . Chronic human circulatory system infection by schistosoma haematobium is reported to affect the urinary bladder and is a possible risk factor in the aetiology of cancers of the bladder and the urinary tract system . S. haematobium infection has been linked with the development of squamous cell carcinoma of the bladder [4, 5]. S. haematobium associated bladder damage has been closely linked to the immune reaction elicited against the parasite egg deposited in the bladder which eventually induces chronic inflammation related granulomatous injury . Schistosomiasis and bladder cancer share common symptoms such as haematuria, dysuria, and pain with micturition . This may prevent early diagnosis of bladder cancer and the resultant severe bladder damage particularly in people living in s. haematobium endemic areas . In nigeria, most studies have focused on the epidemiology of s. haematobium infection [3, 7, 8] particularly in school - age children, with limited information about the morbidity resulting from urinary schistosomiasis in adults . This study was therefore aimed at determining the prevalence of schistosomiasis and associated bladder pathology in adults living in eggua, yewa, north local government area, ogun state, nigeria . The study was carried out in eggua, a rural agrarian community, between august 2012 and may 2013 . It is one of the wards that make up yewa north local government area as previously described . It consists of settlements at sagbon, imoto, tata, agbon - ojodu, and igan alade . Two major rivers (yewa and iju) flowing through the area serve as the main water source, resulting in high water contact by the inhabitants . These rivers are used for religious, domestic, and entertainment activities which enhance the transmission of schistosomiasis . Participants aged 30 to more than 60 years old from the community were enrolled for the study . Children were excluded from the study in line with the objective of the study to determine the effect of chronic urinary schistosomiasis on adult members of the community . Informed consent was obtained from each participant under a protocol approved by the local government and local health officials . Ethical approval for the study was also obtained from the ogun state ministry of health . Blood (5 ml) and urine specimens were collected from each study participant . The urine samples were collected between 10:00 and 14:00 hours to ensure maximum egg yield . The urine samples (10 ml) were processed for microscopic examination and egg count [3, 10]. The eggs were quantified by counting under the microscope and classified as light infection if there were 50 (149) eggs/10 ml urine and heavy infection if there were> 50 eggs/10 ml urine . A blind ultrasound examination was carried out on each participant approximately 1 h after drinking potable water (0.11.5 litre depending on the age of the participant) to distend the bladder . The classification of bladder pathology or damage was based on the definition of the who [11, 12] and shiff et al . ; the abnormalities assessed included abnormal bladder shape, bladder wall irregularities, bladder masses, presence of polyps, calcification, and presence of hydronephrosis in the kidneys . Bladder lesions were considered severely abnormal when four of the above conditions or three conditions as well as hydronephrosis were present in a single individual . Lesions were considered moderate if fewer conditions were seen and negative when no specific lesions were observed . A structured, pretested questionnaire was used to obtain information about participants' habits regarding smoking and alcohol consumption, which are determinants of bladder cancer . Statistical analysis of data obtained was done using spss version 20.0 (p <0.05). A total of 257 (79 males and 178 females) participants aged 3090 years were screened for s. haematobium infection and associated bladder pathologies . The overall prevalence of s. haematobium in the sampled population was 25.68% (66/257), 21 (31.8%) in males and 45 (68.2%) in females . The highest prevalence of infection was observed in participants over 60 years old (table 1). The majority (56/66) (84.8%) of those positive for s. haematobium had a light intensity of infection with the egg mean intensity of 16.7 eggs/10 ml urine . The yewa river was the main source of water for most (49/62) (79.0%) of the participants infected with s. haematobium (table 4). Bladder pathologies were observed in 33.9% (87/257) of the sample population and included abnormal bladder wall thickness (39/66) (59%), abnormal bladder shape (10/66) (15.2%), bladder wall irregularities (15.2%), bladder masses (1.5%), and bladder calcification (1.5%) (table 2). Bladder wall thickness, the most common abnormality, was recorded in 46/79 (58.2%) males and 90/178 (50.6%) females (table 3). Among the participants, 56 (84.8%) with bladder pathologies also had an existing schistosomiasis infection, 48 (87.3%) of which were light intensity and 8 (72.7%) of which were heavy intensity: = 267.5, p = 0.001 (table 5). Thus, there was an association between urinary tract pathology and the intensity of s. haematobium infection (= 375.4, p = 0.001, table 2). Among the participants with light and heavy intensity of s. haematobium infections, bladder wall thickness was the most common bladder structural pathology identified in 33/56 (58.9%) and 6 (60.0%) participants with light and heavy s. haematobium infections, respectively (table 5). Abnormal bladder shape and bladder wall irregularity were seen in 8/56 (14.3%) and 2 (20%) participants with light and heavy infections, respectively (figures 13). Hydronephrosis was present in only one participant with light infection, while calcification was identified in only one participant with heavy infection . Mild bladder pathology was more common than severe bladder pathology in this study and was found in 48 of the participants (table 5). There was a higher incidence of bladder pathologies among female participants (table 3); bladder mass and hydronephrosis were also seen only in female participants . There was no significant relationship between cigarette smoking and bladder pathology in the study (table 6). Among participants with bladder pathology, 29 (33.3%) admitted consuming alcohol while 58 (66.7%) said that they had never consumed alcohol (table 7). The overall prevalence rate (25.98%) of adults with s. haematobium infection recorded in this study was slightly higher than 20.8% and 20.0% reported in yewa north local government, ogun state, and owan east local government, respectively, in nigeria [3, 13]. Most (81.3%) of the participants depended solely on the s. haematobium contaminated river water, which could account for the higher s. haematobium prevalence; and little or no schistosomiasis control (drug) intervention targeted to adults has been recorded in this area . The higher frequency of light intensity s. haematobium infection observed in this study could be explained by some level of acquired protected immunity by adults in that community due to chronic exposure to schistosomiasis . . Found that the proportion of egg - positive individuals falls progressively with age and is a feature in populations with lifelong exposure to the parasite . Therefore, chronicity of infections in older people will more likely be difficult to ascertain using egg count method . The higher frequency of mild bladder pathology observed in this study was also similar to another study which observed a higher incidence of mild bladder than severe bladder pathology . This result could be explained by the low number of participants who smoked cigarettes and consumed alcohol; these conditions may serve as promoting factors either in progression of bladder pathology to cancer or in making the bladder pathology more severe (table 4). In addition, this lifestyle could buttress the possibility of s. haematobium being the principal cause of the reported bladder structural pathology in the study population . The close relationship between the intensity of s. haematobium infection and the presence of bladder abnormalities was similar to previous reports [3, 1416]. The presence of hydronephrosis in participants with light infection is however at variance with the report of nmorsi et al . Although hydrocalycosis (a condition mostly mistaken for hydronephrosis) was observed in some patients with heavy infection, indicating the likely contribution of this infection to kidney pathology . Females (64.7%) had more structural bladder pathology compared to males (35.3%). This may be due to higher water contact by females and also to the higher number of female study participants than an indication of a female predilection to bladder pathology . However, since hydronephrosis and bladder mass or bladder calculi were found together in a female participant, female predilection to bladder pathology may not completely be ruled out . The structural changes to the bladder recorded in this study were in consonance with observations in west madagascar and nigeria [3, 15] where bladder irregularities and bladder wall thickness were identified as the most common pathologies in individuals infected with s. haematobium . In conclusion, there is evidence that s. haematobium infections may be associated with bladder pathology, on ultrasound examination . Individuals with bladder pathologies could have heavy or light intensity of schistosomiasis infection or have no existing infection at all . However, a long term exposure to schistosomiasis is necessary for the development of bladder cancer . Further research on the determinants and progress of the bladder pathologies seen in this study population is needed.
Therapeutic high intensity focused ultrasound (hifu) or focused ultrasound (fus) is a noninvasive medical treatment that allows the deposition of energy inside the human body . The energy levels carried in the ultrasound beam are several orders of magnitude greater than those of a standard diagnostic ultrasound beam . In the case of focused ultrasound, the high energy levels carried in a hifu beam can therefore be magnified further and delivered with precision to a small volume, while sparing surrounding tissues . Fus energy can be deposited in small areas providing a substantial advantage for drug targeting . The volume of energy deposition following a single hifu exposure is small and will vary according to transducer characteristics but is typically cigar shaped with dimensions in the order of 13 mm (transverse) 815 mm (along beam axis). Hifu transducers deliver ultrasound with intensities in the range of 10010,000 w / cm to the focal region, with peak compression pressures of up to 30 mpa peak and rarefaction pressures up to 10 mpa . The ultrasound wave propagates through tissues, causing alternating cycles of increased and reduced pressure (compression and rarefaction, resp . ). In the case of tissue ablation during hifu treatments, the temperature at the focus can rise rapidly (up to 80c) which can cause cell damage . Ultrasound affects the molecular structure of the tissues during the alternating cycles of compression and rarefaction . During rarefaction, gas can be drawn out of the solution to form bubbles, which can collapse rapidly . In this case injury is induced through a combination of mechanical stresses and thermal effects at a microscopic level . When ultrasound is applied in biological systems it can induce local tissue heating, cavitation, and radiation force, which can be used to initiate local (focal) drug delivery, increase permeation through membranes, and enhance diffusivity of drugs, respectively, only at the site of sonication therefore allowing control of local drug release . The ability of fus to induce thermal or mechanical effects at very defined (focal) locations in living tissue has been first described in 1942, when lynn et al . They used a sonication system in combination with x - rays to determine the target location relative to skull and to focus the ultrasound beam through a craniotomy into deep brain for effective functional neurosurgery . Later on, in the 1980s the first fda - approved fus system, sonocare cst-100, was developed to treat ocular disorders such as glaucoma and many patients were successfully treated with this system . More recently substantial technological developments have led to new fus equipment for a number of different applications . Current research and development aims to explore transducer technology and array design to achieve faster delivery of focal sonications, to improve transducer accessibility (smaller devices) or fit them to certain parts of the body such as a helmet of arrays for brain focal treatment . These devices can operate under image guidance to provide real - time monitoring of the treatment . Guidance and monitoring of acoustic therapy controls the treatment region and minimizes damage to adjacent structures . Monitoring using real - time imaging, such as with sonography (diagnostic ultrasound), ensures that the targeting of the fus beam is maintained on the correct area throughout the procedure . Mri and sonography are the two imaging modalities currently being used for guidance and monitoring fus therapy . Mri has the advantage of providing temperature data during fus treatment . However, mri guidance is expensive, labor - intensive, and of lower spatial resolution in some cases . Sonographic (ultrasound) guidance provides the benefit of imaging using the same form of energy that is being used for therapy . Therefore, if the target cannot be well visualised with sonography, then it is unlikely that fus therapy will be effective . Insightec manufactures the exablate2000 which uses mri for extracorporeal treatment of uterine fibroids (fda - approved) with significant success, and extensive current research focuses on investigating its application in other parts of the body [7, 8]. Exablate technologies are used for prostate cancer or bone metastasis (exablate 2100 conformal bone system); these applications are currently under development by insightec . The ablatherm hifu / us consists of a transrectal probe for prostate treatment and has ce mark approval . The sonablate 500, an ultrasound guided system uses a transrectal probe to carry out prostate cancer focal ablation . The sonalleve hifu / mr is an mr compatible device developed to examine a series of applications as fibroids and other body sites ., tirat carmel, israel) with each element driven separately, providing individual correction of skull distortion as well as electronic steering . The device has been used for the treatment of neuropathic pain essential tremor and there is also evidence of possible application for brain tumours [12, 13]. Essential tremor noninterventional functional neurosurgery treatment has shown an immense potential of transcranial mrgfus application to induce lesions focally and treat patients nonsurgically . Ultrasound propagates as mechanical vibrations that induce molecules within the medium to oscillate around their positions in the direction of the wave propagation . The rate of energy flow through a unit area, normal to the direction of the wave propagation, is called acoustic intensity . At 1 mhz the ultrasound wave is attenuated about 50% while it propagates through 7 cm of tissue . The attenuated energy is transformed into temperature elevation in the tissue [15, 16]. Ultrasound is transmitted from one soft tissue layer to another . Usually in soft tissues a small amount of wave is reflected back except at the soft tissue - bone interface where approximately one - third of the incident energy is reflected back . In addition, the amplitude attenuation coefficient of ultrasound is about 1020 times higher in bone than in soft tissues . Each particle moves small distances from its rest position but the vibrational energy is propagated as a wave traveling from particle to particle through the material . Ultrasound is attenuated as it travels through a tissue due to beam divergence, absorption, and deflection of the acoustic energy . The energy required for a sound wave to travel through a tissue must overcome the internal friction intrinsic to any material . As a sound wave travels through tissue, it continually loses a proportion of its energy to the tissue (attenuation). Divergence of the sound beam spreads the acoustic energy over a larger beam area and reduces the intensity along the beam axis . The greatest cause of attenuation in the body is absorption, in which energy is transferred from the sound beam to the tissue and ultimately is degraded to heat . The amount of absorption depends on the frequency of the ultrasound beam . Whenever a sound beam encounters a boundary between two materials, the direction of the reflected wave, or the echo, depends on the orientation of the boundary surface to the sound wave . The major physical effects of ultrasound are heat, mechanical effects, cavitation, and chemical effects . Acoustic impedance is a measure of the resistance that a material offers to the passage of an ultrasound wave and is expressed in units of rayls (kg / m / sec). Acoustic impedance of water is 1.5 10 mrayls whereas that of bone is 8 10 mrayls . The greater the difference in acoustic impedance between two materials, the stronger the echo (reflected wave) arising from their interface . When the rate of heat generation is higher than the rate of heat dissipation in the body, the body temperature will rise significantly . Mechanical effects, such as the breaking of bonds, can occur if the amplitude of the ultrasound wave is significantly large . Cavitation occurs when an ultrasound beam of sufficient intensity travels through a liquid in which gas bubbles have been generated . The alternating high- and low - pressure periods of the ultrasound wave forces the bubbles to contract and expand . The amplitude of the bubble oscillation increases with increasing ultrasonic intensity . During the bubble contraction, the internal pressure can increase and the temperature can reach 10,000c . A sonic explosion can occur, releasing a large amount of energy, although for very short (m) distances . Chemical effects, such as the acceleration of chemical reactions, can occur due to an increase in the temperature and pressure . When ultrasound beams are focused a focal diameter of 1 mm can be achieved at 1.5 mhz . If the ultrasound beam is transmitted from an applicator 23 cm in diameter, the ultrasound intensity at the millimeter - sized focal spot can be several hundred times higher than in the overlying tissues . Typical diagnostic ultrasound transducers deliver ultrasound with time - averaged intensities of approximately 0.1100 mw / cm or compression and rarefaction pressures of 0.0010.003 mpa, depending on the mode of imaging . In contrast, hifu transducers deliver ultrasound with intensities in the range of 10010,000 w / cm to the focal region, with peak compression pressures of up to 30 mpa and peak rarefaction pressures up to 10 mpa . The ultrasound exposure drops off rapidly across the area within the sonication path and therefore focusing provides a method to overcome attenuation losses and to concentrate energy deep in the body while avoiding the surrounding tissues . Focusing is dramatically improved with the use of transducer arrays that are driven with signals having the necessary phase difference to obtain a common focal point . The main advantage of these phased arrays is that the focal spot can be controlled . In addition, the electronically focussed beam allows multiple focal points to be induced simultaneously or fast electronic scanning of the focal spot which increases the size of the focal region . The combination of high - intensity focused ultrasound together with high - resolution mr guidance has created a system that can produce tissue destruction deep within solid organs without any invasion . Accurate targeting and detailed accurate thermal mapping are provided by mri . In recent years imaging has been combined with fus to provide real - time manipulation of drug guidance within the targeted area . Ultrasound and magnetic resonance (mr) imaging are widely used clinical imaging modalities that can be combined with fus for image guided fus treatments . In the area of drug delivery ultrasound microbubbles or nanocarriers providing contrast enhancement can be used . When using nanocarriers sensitive to mechanical forces (the oscillating ultrasound pressure waves) and/or sensitive to temperature, the content of the nanocarriers can be released locally . Thermosensitive liposomes have been suggested for local drug release in combination with local hyperthermia more than 25 years ago . Real - time imaging methods, such as magnetic resonance, optical and ultrasound imaging, have led to novel insights and methods for ultrasound triggered drug delivery . Image guidance of ultrasound can be used for: (1) target identification and characterization; (2) spatiotemporal guidance of actions to release or activate the drugs and/or permeabilize membranes; (3) evaluation of biodistribution, pharmacokinetics and pharmacodynamics; and (4) physiological read - outs to evaluate the therapeutic efficacy . The enhanced permeability and retention effect has served as a basic rationale for using liposomes and other nanoparticles to treat solid tumors . However, it has been recently noticed that the enhanced permeation and retention effect does not guarantee a uniform delivery . This heterogeneous distribution of therapeutics is a result of physiological barriers presented by the abnormal tumor vasculature and interstitial matrix . In a recent review by jain and stylianopoulos first, the abnormal structure of tumor vessels results in heterogeneous tumor perfusion and extravasation, and a hostile tumor microenvironment that supports drug resistance and tumor progression . Second, in highly fibrotic tumors, the extracellular matrix blocks penetration of large nanoparticles leaving them concentrated in perivascular region . To overcome these barriers the authors suggest normalization of the vascular network and the extracellular matrix as well as development of nanoparticles that release therapeutic agents in response to the tumor microenvironment or an external stimulus (such as heat light and hifu). However, development of thermosensitive liposomal carriers for cancer was only introduced as recently as 1999 when needham's group evaluated phase transition enhanced liposomal permeability . In vivo data using cancer models were presented one year later when the authors described a new lipid formulation containing doxorubicin optimized for mild hyperthermic temperatures (39c to 40c) that are readily achievable in the clinic leading to very rapid release times of the drugs . This new liposome, in combination with mild hyperthermia, was found to be significantly more effective than free drug or current liposome formulations at reducing tumour growth in a human squamous cell carcinoma xenograft . These low temperature - sensitive liposomes (ltsl) were further developed in dogs having canine tumours to show a superior efficiency [27, 28]. A formulation based on these thermosensitive liposomes took the brand name thermodox and was further developed by celsion corporation . Thermodox liposomes can be triggered to release their payload by any heat - based treatment such as radiofrequency thermal ablation (rfa), microwave hyperthermia, and high intensity focused ultrasound (hifu). Results from a phase i study that used thermodox was recently published . In a phase i study researchers used escalating dose of thermodox with radiofrequency (rf) ablation and concluded that thermodox can be safely administered at 50 mg / m in combination with rf ablation . Currently thermodox in combination with rf ablation is being tested in a large phase i study to treat hepatocellular carcinoma . The concept of using liposomes and hifu was introduced recently, in 2006 when frenkel et al . Used liposomal doxorubicin (doxil) in combination with pulsed high - intensity focused ultrasound (hifu) exposures in a murine breast cancer tumor model . Doxil is a stable liposomal preparation that has no response to increased temperature and was developed to minimise doxorubicin's cardiotoxicity, by encapsulating doxorubicin within stealth liposomes . Although doxil achieves long circulation of doxorubicin with minimum cardiotoxicity it does not rapidly release the drug within the tumour . Pulsed - hifu exposures were not found to enhance the therapeutic delivery of doxorubicin and did not induce tumour regression . However, a fluorescent dextran showed blood vessels to be dilated as a result of the exposures . Experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumours . Although this study did not achieve or prove a therapeutic advantage of the use of hifu with temperature stable liposomes it showed clearly that pulsed hifu induces a substantial increase of permeation of macromolecules and nanoparticles in tumours . Presented the first study on thermosensitive liposomes (low temperature sensitive liposomes (ltsl)) and hifu . The authors investigated pulsed - high intensity focused ultrasound as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in murine adenocarcinoma tumours . In vitro treatments simulating the pulsed - hifu thermal dose (42c for 2 min) triggered release of 50% of doxorubicin from the thermosensitive liposomes; however, no detectable release from the nontemperature sensitive liposomes (similar to doxil) was observed . Similarly, in vivo experiments showed that pulsed - hifu exposures combined with the ltsl resulted in more rapid delivery of doxorubicin as well as significantly higher concentration within the tumour when compared with ltsls alone or nonthermosensitive liposomes, with or without exposures . In a later study the same team developed mr imageable thermosensitive liposomes (iltsl), with the objective to characterise drug release in phantoms and in vivo . An mri contrast agent (prohance gd - hp - do3a) and doxorubicin were loaded and drug release was quantified by spectroscopic and fluorescence techniques, respectively . Release with hifu under mr guidance was examined in tissue - mimicking phantoms containing iltsl and in a vx2 rabbit tumour model . Release of doxorubicin and prohance from iltsl was minimal at 37c but fast when heated to 41.3c . Relaxivity of iltsl increased significantly from 1.95 0.05 to 4.01 0.1 mms when liposomes were heated above the phase transition temperature indicating the release of prohance from liposomes and its exposure to the aqueous surroundings . Importantly, the signal increase corresponded spatially and temporally to mr - hifu - heated locations in phantoms . In vivo, iltsl injection and after each 10-min heating, with greatest increase in the heated tumour region . The authors concluded that mr - hifu combined with iltsl may enable real - time monitoring and spatial control of drug release from liposomes . In a follow - up study the authors investigated the effect of iltsl in rabbits bearing vx2 tumours . In that study image - guided noninvasive hyperthermia was applied for a total of 30 min, completed within 1 h after ltsl infusion and quantified doxorubicin release in tumours with hplc and fluorescence microscopy . Sonication of vx2 tumours resulted in accurate and spatially homogenous temperature control in the target region . Ltsl+mr - hifu resulted in significantly higher tumour doxorubicin concentrations (3.4-fold greater compared ltsl resp . ). The authors observed that free doxorubicin and ltsl treatments appeared to deliver more drug in the tumour periphery as compared to the tumour core indicating that hifu induced hyperthermia and ltsl increases doxorubicin's permeability as doxorubicin was found in both the tumour periphery and core . The group further developed a heating algorithm using the same rabbit tumour model proving that the use of the binary feedback algorithm results in accurate and homogenous heating within the targeted area . A computational model that simulated the tissue heating with hifu treatment and the resulting hyperthermia that leads to drug release was developed by haemmerich . In this model a spatiotemporal multicompartmental pharmacokinetic model simulated the drug release in the blood vessels and its transport into the interstitium as well as cell uptake . Two heating schedules were simulated each lasting 30 min, the first corresponding to hyperthermia, (ht; 43c) and the second corresponding to hyperthermia followed by a high temperature (50c) for 20s pulse, (ht+). Using the computational model (validated in rabbit vx2 tumours) the study indicates the importance of simulations in the application of drug delivery mechanisms to tumours . In addition to the progress in the understanding of the physical mechanism of drug delivery from well validated thermosensitive liposomes carrying doxorubicin, researchers further investigated the chemical composition of such liposomes in response to hifu induced hyperthermia . De smet et al . Two temperature - sensitive systems composed of the following lipids dppc: mppc: dppe - peg2000 (low temperature - sensitive liposomes; ltsl) and dppc: hspc: cholesterol: dppe - peg2000 (traditional temperature - sensitive liposomes; ttsl) were investigated for their stability and release profile at 37c and 42c in phantoms using mri 1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc), 1-palmitoyl - sn - glycero-3-phosphocholine (mppc), 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n[methoxy(polyethyleneglycol)-2000] (dppe - peg2000), hydrogenated - l--phosphatidylcholine (hspc). The ltsl system showed a higher leakage of doxorubicin at 37c, but a faster release of doxorubicin at 42c compared to the ttsl system indicating that lipid composition plays an important role on stability and release profile . The authors further investigated the more stable traditional temperature sensitive liposomes carrying doxorubicin and prohance in vivo in rats bearing 9l gliosarcoma tumours . A clinical mri - hifu system was applied in a proof - of - concept study to induce local hyperthermia for 30 min . The local temperature - triggered release of prohance was monitored with interleaved t1 mapping of the tumour . A good correlation between the r1 (change in longitudinal relaxation rate r1 = (1/t1)) and the intratumour doxorubicin and gadolinium concentration was found, implying that the in vivo release of doxorubicin from the thermosensitive liposomes can be probed in situ with the longitudinal relaxation time of the coreleased mri contrast agent (dose painting). Temperature sensitive liposomes release their encapsulated drugs at the melting phase transition temperature (tm) of the lipid bilayer . At this tm the lipid membrane changes its structure as it transfers from a gel to the liquid crystalline phase . When the liposomal membranes are in the gel phase they show less permeability to molecules and water compared to the liquid crystalline phase . The liposomes' transition to the liquid crystalline phase can be achieved with the incorporation of a lyso - phospholipid such as mspc (r = c17h35). . A potential disadvantage of mspc containing liposomal formulations is their rapid doxorubicin leakage at 37c . Prepared temperature sensitive liposomes using nonionic surfactants brij which are peg - ylated lysolipids resembling the chemical structures of mspc and dspe - peg(2000). Results indicated that the optimal acyl chain length of the surfactant was between c(16) and c(18) with a saturated carbon chain and a peg repeating unit ranging between 10 and 100 with a molecule weight above 600 da . In the panel of surfactants tested, brij78 was optimal and could be incorporated into the liposomes by the thin film hydration or the postinsertion method with an optimal range of 1 to 8 mol% . The authors continued with in vivo experiments in mice bearing mammary carcinoma cells emt-6, investigating gddtpa (diethylene triamine pentaacetic acid) release with relaxometry . The authors observed a good correlation between relaxation enhancement in the heated tumour and the inhibition of tumour growth at day 21 after treatment . Kono et al . Investigated the effect of poly [2-ethoxy(ethoxyethyl)vinyl ether] chains (having a lower critical solution temperatures) and polyamidoamine g3 dendron - based lipids having gd chelate residues into pegylated liposomes . These designed liposomes exhibited excellent ability to shorten the longitudinal proton relaxation time . When administered intravenously into tumour - bearing mice, accumulated liposomes in tumours increased with time, reaching a constant level 8 h after administration by following t1-weighted mri signal intensity in tumours . Liposome size affected the liposome accumulation efficiency in tumours: liposomes of about 100 nm diameter were accumulated more efficiently than those with about 50 nm diameter . Tumour growth was strongly suppressed when liposomes loaded with doxorubicin were administered intravenously into tumour - bearing mice and the tumour was heated mildly at 44c for 10 min at 8 h after administration . In our group we have investigated the potential of an mri labelled phospholipid / lysolipid containing liposome to accumulate in tumours and release the drug under conditions of mild hyperthermia induced by fus . We label the liposome nanoparticles with a lipid that consists of a dota [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid] headgroup (figure 1) [44, 45]. Introducing the imaging lipid in the lipid bilayer provides a better and clearer monitoring of liposomal particle kinetics and a better knowledge of the time required for maximum nanoparticles accumulation in tumours (monitored by mri). Although most research studies have focused mainly in thermoresponsive liposomes and fus activation of drug release, there is limited work on the use of polymers (thermoresponsive or not) and their application in fus triggered drug delivery . The effect of ultrasound on drug release from polymers was studied in 1989 by kost et al . And indeed the authors found that ultrasound can increase the polymer degradation rate leading to 20 times higher release rate . Interestingly the authors observed that the release rate increased in proportion to the intensity of ultrasound proposing that cavitation appeared to play a significant role . Triggered drug delivery using an external physical force provides the required control of drug deposition in certain tissues avoiding exposure of healthy tissues to high (toxic) concentrations . The trigger induced delivery should be acute and the effect induced on nontargeted tissues nondamaging and reversible . Hyperthermia induced by a means like ultrasound can be exploited as an external trigger in drug delivery [3, 47]. Mild hyperthermia can be induced by pulsed fus that can reduce extreme tissue heating by allowing the tissue to cool down between us exposures . The increase in temperature can be 35c (hyperthermia) despite the high energy deposited in the tissue . Studies with canine soft tissue sarcoma and human tumour clinical studies have also demonstrated that hyperthermia improves tumour oxygenation and enhances response of such tumours to radiotherapy or chemoradiotherapy . The increased blood flow and vascular permeability caused by temperatures such as 42c may also improve the delivery of chemotherapy drugs, immunotherapeutic agents and genes to tumour cells . Fus exposures in pulsed mode lower the rates of energy deposition and generate primarily mechanical effects for enhancing tissue permeability to improve local drug delivery . These pulsed exposures can be modified for low - level hyperthermia as an enhancement of drug delivery that would lead to better drug deposition and better therapeutic effect . Mild hyperthermia of 42c can improve the degree of nanocarrier extravasation as shown by kong et al . . The reason that this leads to increased extravasation maybe due to downregulation of ve - cadherin that contributes to vascular integrity as it was shown in huvec endothelial cells . It is clear that hyperthermia can provide a boost to extravasation and drug deposition in tumours . This should provide an adjuvant effect when nanocarriers are used and accumulate in tumours due to enhanced permeation and retention effect . It would be interesting to investigate the effect of hyperthermia on tumour / tissue drug clearance . Acoustic cavitation can be defined as the growth, oscillation, and collapse of gas containing bubbles under the influence of the varying pressure field of sound waves in a fluid and can have an effect on the permeability of a biological tissue [5355]. The noninertial (stable) cavitation occurs when bubbles persist for a number of acoustic cycles . In this case the bubble's radius increases and decreases (expands and contracts) according to the applied us frequency . Inertial (transient cavitation) occurs when bubbles grow faster expanding 2- or 3-fold their resonant size, oscillate unstably, and collapse in a single compression half cycle . It has been considered that the primary mechanism to affect the structure of intact cells is inertial cavitation that can induce irreversible damage as well as increase cell membrane permeability [56, 57]. An important application of hifu and microbubbles lies in the area of altering the permeability of the blood brain barrier (bbb). In a study in 2002, hifu induced reversible, nondestructive, bbb disruption in a targeted area and this opening reversed after 72 h. the authors showed with microscopy that hifu either entirely preserved brain architecture while opening the bbb, or generated tissue damage in a small volume within the region of bbb opening . Further electron microscopy suggested that hifu disrupted the bbb by opening capillary endothelial cell tight junctions, a mechanism that was not observed in other methods used to open bbb . The effect of fus on tight junctions' integrity was later confirmed in a study investigating rat brain microvessels after this bbb disruption . The authors used immunoelectron microscopy to identify tight junctional proteins such as occludin, claudin-1, claudin-5, and submembranous zo-1 after sonication . Monitoring the leakage of horseradish peroxidase (mw 40 kda) the authors observed that the bbb disruption appears to last up to 4 h after sonication . In a later study the role of caveolin in the mechanism of fus - bbb enhanced permeation was suggested . In a study investigating caveolae density it was found that caveolae and caveolin-1 were primarily localized in the brain microvascular endothelial cells of all the animals tested (rats) regardless of treatment, and that caveolin-1 expression was the highest in the rats treated with both fus and microbubbles . The authors concluded that caveolin-1-mediated transcellular transport pathway may cooperate with other transport pathways (e.g., tight junctional disruption) to induce opening of the bbb . The bbb opening was measured by an mri contrast agent evaluating the local enhancement in the brain . The authors found that low ultrasound powers and pressure amplitudes were found to cause focal enhancement of bbb permeability . Trypan blue injected before animals were sacrificed indicated blue spots in the areas of the sonicated locations . The authors concluded that hifu disruption of bbb could be used enhancing drug delivery to the brain . Tested the safety of this method by searching for ischemia and apoptosis in areas with bbb disruption induced by pulsed ultrasound in the presence of gas bubbles and by looking for posttreatment effects up to one month after sonication . Pulsed ultrasound exposures (sonications) were performed in the brains of rabbits under monitoring by mri . Whole brain histologic examination was performed using staining for ischemic neurons and tunel staining for apoptosis . Tiny regions of extravasated red blood cells scattered around the sonicated locations, indicated capillaries . Despite these vasculature effects, only a few cells in some of the sonicated areas showed evidence of apoptosis or ischemia . The authors found that ultrasound - induced bbb disruption is possible without inducing substantial vascular damage that would result in ischemic or apoptotic death to neurons . The method could find application in the delivery of large therapeutic molecules that do not normally permeate the bbb . Herceptin (trastuzumab), a humanized anti - human epidermal growth factor receptor 2 (her2/c - erbb2) monoclonal antibody, was delivered locally and noninvasively into the mouse central nervous system through the blood - brain barrier under image guidance by using an mri - guided focused ultrasound . The amount of herceptin delivered to the target tissue was correlated with the extent of the mri - monitored barrier opening, making it possible to estimate indirectly the amount of herceptin delivered . It was further shown that dopamine d(4) receptor - targeting antibody could also be delivered using the same technique in the brain [65, 66]. Delivery of small molecules can also be enhanced with the use of hifu cavitation disruption of the bbb . Demonstrated relatively high concentrations of doxorubicin in the brain with minimal healthy tissue damage effects . Mri signal enhancement in the sonicated region correlated strongly with tissue doxorubicin concentration, suggesting that contrast - enhanced mri could perhaps indicate drug penetration during image - guided interventions . Konofagou and coworkers assessed the spatial permeability of the bbb - opened region using dynamic contrast - enhanced mri (dce - mri) in mice . The authors processed dce - mr images using the general kinetic model and the reference region model . Permeability maps were generated and the ktrans (the transfer rate constant from the intravascular system to the extracellular extravascular space) values were calculated for a predefined volume of interest in the sonicated and the control area for each mouse . The results demonstrated that ktrans in the bbb - opened region was at least two orders of magnitude higher when compared to the contralateral (control) side . There are several parameters to affect the level of bbb enhanced permeability and the endothelial tight junctions disruption; the pulse sequence comprising short bursts, the spacing between bursts or the rate of infusion of the microbubbles, and the size of microbubbles were found to affect the effect on bbb disruption [69, 70]. The brain - derived neurotrophic factor (bdnf) was delivered to the left hippocampus in mice through the noninvasively disrupted blood - brain barrier (bbb) using focused ultrasound . The bdnf bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the ptrkb receptor and activated pakt, pmapk, and pcreb in the hippocampal neurons . It was shown that bdnf delivered this way induced signalling effects in a highly localized region in the brain . However it is the area of targeting brain tumours that have attracted most interest in the fus disrupted bbb . Mei and colleagues investigated the effects of targeted and reversible disruption of the blood - brain barrier by mri - guided focused ultrasound and delivery of methotrexate to the rabbit brain . The authors recorded that the methotrexate concentration in the sonicated group was notably higher than that in both the control group (intravenous administration) and the internal carotid artery administered group . They observed a greater than 10-fold increase in the drug level compared to internal carotid administration without fus . Liu et al . Investigated the delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea (bcnu) to glioblastomas in rats with induced tumours with the help of fus . The authors found that fus significantly enhanced the penetration of bcnu through the bbb in normal and tumour - implanted brains without causing bleeding . Surprisingly, treatment of tumour - implanted rats with focused ultrasound alone had no beneficial effect on tumour progression . However, treatment with focused ultrasound before bcnu administration controlled tumour progression and improved animal survival relative to untreated controls . Liu and colleagues recently assessed fus - mediated delivery of an iron oxide magnetic nanoparticle (mnps) conjugated to an antineoplastic agent, epirubicin . They used mnps because of the favourable mr imaging characteristics, which could facilitate imaging . They demonstrated a substantial accumulation of mnps, as well as epirubicin, up to 15 times the therapeutic range in the brain when delivered with fus . They further showed decreased tumour progression in animals with brain tumours that received mnp with epirubicin via fus . Receptors targeting liposomal nanocarriers have been combined with mrgfus to treat brain tumours . In a recently presented study it was shown that pulsed hifu and human atherosclerotic plaque - specific peptide-1- (ap-1-) conjugated liposomes containing doxorubicin (ap-1 lipo - dox) acted synergistically in an experimental brain tumour model . Prior to each sonication, ap-1 lipo - dox or unconjugated lipo - dox were administered intravenously, and the concentration in the brain was quantified . Drug injection with sonication increased the tumour - to - normal brain doxorubicin ratio of the target tumours by about twofold compared with the control tumours . Moreover, the tumour - to - normal brain ratio was the highest after the injection of ap-1 lipo - dox with sonication . The results of this study indicate that combining targeting strategies can substantially enhance delivery of chemotherapy in the brain . In a separate study the authors investigated the pharmacokinetics of i - labeled ap1-lipo - dox using microspect . The authors confirmed that sonication increased liposomal doxorubicin concentrations in tumour areas (murine glioblastoma) and that molecular targeting acts synergistically with fus . Targeted gene transfer into central nervous system was investigated using mri - guided focused ultrasound - induced blood - brain barrier disruption . The results of this study showed that mri - guided fus achieved plasmid dna transfer across the opened bbb furthermore plasmid ware internalized into the neurons presenting heterogeneous distribution and numerous transparent vesicles were observed in the cytoplasm of the neurons in the sonicated region, suggesting vesicle - mediated endocytosis . Bdnf (and bdnf - egfp) expressions were markedly enhanced by the combination of ultrasound and pbdnf - egfp - loaded microbubbles about 20-fold than that of the control group . The method by using mri - guided fus to induce the local bbb disruption could accomplish effective targeted exogenous gene transfer in the cns . In this the investigators conjugated plasmid onto the surface of microbubbles and they coated these carriers using polymers in a layer by layer technique . An exciting application is the delivery of therapeutic stem cells to the brain using fus to potentially treat neurodegenerative diseases, traumatic brain injury, and stroke . Mri guidance was used to target the ultrasound beam thereby delivering iron - labeled, green fluorescent protein (gfp) expressing neural stem cells specifically to the striatum and the hippocampus of the rat brain . Immunohistochemical analysis confirmed the presence of gfp - positive cells in the targeted brain regions suggesting that mrigfus may be an effective alternative to invasive intracranial surgery for stem cell transplantation . Although a very efficient approach, the use of microbubbles to enhance drug permeation through tissues, it may require significant safety consideration . In a key study in 2005 prentice et al . Presented clearly in a well - designed experimental setup that there are important interactions between individual cells and violently cavitating microbubbles leading to large pores in the cell membrane (sonoporation). These effects on cell membrane will need to be thoroughly investigated at microscopical and molecular level to design efficient and safe fus regimes . During the last few years the main dosage forms tested in mrgfus drug delivery strategy are the thermosensitive liposomes and the lipid based microbubbles that can be conjugated with drugs or other liposomes on their surface [78, 81]. Rapoport discussed recently the potential of using micelles and fus for enhanced tissue permeation . Micelles are nanosized carriers able to carry hydrophobic drugs; their combination with fus could substantially enhance their delivery in tissues . Kostarelos and colleagues suggested the incorporation of thermosensitive peptides onto liposome bilayers to enhance thermoresponsiveness, and the group of lammers designed polymer - based microbubbles for ultrasound drug release . It is clear that already established delivery systems such as different structurally nanocarriers have not been investigated in combination with image guided fus . It would be interesting to see the effect of fus on the enhanced permeability of micelles, polymers (dendrimers cyclodextrins), or metal nanoparticles (gold - iron) to tissues . Polymers or proteins that respond to small change of temperature could form suitable image guided fus triggered platforms . The effects of fus in biological tissues with or without carriers will require a more thorough investigation to understand the short- and long - term effects of ultrasound in the body and the complex environments such as tumours, blood vessels, and bone . The mechanism of fus induced hyperthermia and/or the fus tissue permeability increase is not well understood at cellular and molecular levels . There is limited knowledge on the effects of fus on genomic dna and if certain proteins are overexpressed after fus treatment . In addition to the above, the frequency of fus drug delivery treatments (or dosing) and the long - term effects in the body will have to be investigated in preclinical studies in order to design a fus drug treatment regime . An imaging modality will have to be used for accurate image guided fus therapy . In the case of mri clinically approved contrast enhancing agents will have to be added to the delivery system to monitor carriers' distribution in the treatment area as well as efficient and rapid release . Considering the approval in clinical applications, such treatments will require the control of several factors such as drug and drug carrier, mri contrast enhancing agents, and mrgfus parameters, and this could mean several regulatory hurdles . However, the fact that most of the components (fus, liposomes) have been tested in clinical trials is encouraging for such approach to move forward . Most of the current strategies to increase tumour specificity of nanocarriers include the use of tumour biomarkers for either targeting (receptors) or for triggered release (internal stimuli; ph proteases) and/or the use of external stimuli such as light and ultrasound . Biomarkers and internal stimuli may vary in different tumours indicating that such nanocarriers for cancer treatments should be individualised . External stimuli can be used independent the tumours characteristics and therefore guarantee a more uniform effect . It also shows the significant advantages of being noninvasive as well as controlled and focused . Overall mrgfus drug delivery is a novel and valuable tool to increase drug targeting and tissue specific drug delivery . It is expected that future studies will prove the clinical efficacy of mrgfus drug delivery applications.
The online version of this article (doi:10.1007/s00125 - 010 - 1903 - 9) contains supplementary material, which is available to authorised users . Possible environmental diabetogenic factors are dietary products and bacterial / viral infections [1, 2]. These diabetogenic triggers can induce an immune cascade, eventually leading to the autoimmune process that is typical of type 1 diabetes [1, 2]. We and others have shown that diet and gut microbiota are critical for autoimmune diabetes pathogenesis in the diabetes - prone (dp) biobreeding (bb) rat model of type 1 diabetes [1, 38]. When fed a hydrolysed casein (hc) diet, only 50% of dp - bb rats develop autoimmune diabetes [1, 36]. These studies also showed that besides lack of diabetogenic antigens in the food, skewing the (mucosal) immune response towards a less pathogenic th2 phenotype and the induction of islet neogenesis are important mechanisms in the prevention of type 1 diabetes by the hc diet [1, 36]. There is growing evidence that a third factor, increased intestinal permeability, underlies the pathogenesis and/or perpetuation of at least some autoimmune disorders such as crohn s disease and coeliac disease [9, 10]. Intriguingly, type 1 diabetes patients and animal models of type 1 diabetes show an impaired intestinal barrier function and signs of intestinal inflammation that precede the onset of type 1 diabetes [1121]. In type 1 diabetes patients and dp - bb rats increased serum zonulin levels were found [13, 14]. Zonulin, the human analogue of zot from vibrio cholerae, is an intestinal physiological modulator of tight junctions . By binding to its receptor, this protein activates signalling pathways that cause opening of the tight junctions and therewith increased intestinal permeability . Blocking the zonulin receptor by a specific antagonist led to reduced intestinal permeability in vitro and in vivo [14, 22, 23], and prevented the increase of intestinal permeability caused by bacterial agents or gliadin [23, 24]. Interestingly, zonulin antagonists restored the intestinal barrier function and subsequently delayed or prevented the development of autoimmune diabetes in dp - bb rats . Food components and gut - derived bacteria can affect intestinal barrier integrity [12, 23, 24], but the role of the intestinal barrier in preventing autoimmune diabetes by hc diet has not been thoroughly investigated . It was shown that a hc diet improves the intestinal integrity of dp - bb rats [7, 16], but these studies did not investigate the effect of the hc diet on tight junctions, the zonulin system, gut micro - biota and the relation with type 1 diabetes development . We therefore investigated whether, besides the above - mentioned mechanisms, restoration of the impaired intestinal barrier function (with emphasis on tight junction proteins) also contributes to the prevention of autoimmune diabetes by a hc diet in dp - bb rats . Animals rats were derived from the worcester dp - bb and diabetes - resistant (dr) bb strain, but were maintained and bred at our institutional central animal facility under specific - pathogen - free and viral - antibody - free conditions . The animals received humane care in compliance with the principles of laboratory animal care (nih publication no . 85 - 23; revised 1985) and the dutch law on experimental animal care . The university ethical board for animal studies approved all animal experiments reported in this study . Dietary intervention protocol two dietary intervention experiments were performed.in the first, experiment 1, dp - bb rats were given the hc diet (n = 18) or a standard diet (n = 16) from weaning onwards and monitored for the development of autoimmune diabetes . At 65 days of age, just before dp - bb rats start to develop autoimmune diabetes, a lactulose after subjection to the la / ma test, the rats were monitored until 140 days of age . Animals were weighed three times per week . In case of weight loss or suspicion of diabetes, blood glucose was measured in tail vein blood using a glucose sensor (reflolux s; boehringer, mannheim, germany). When blood glucose exceeded 15 mmol / l (non - fasting), rats were considered diabetic and killed.in the second intervention, experiment 2, another group of dp - bb rats was fed the hc diet (n = 37) or the standard diet (n = 33) from weaning onwards . At fixed time points (25, 35, 45, 55 and 65 days of age) rats were killed . Ileum tissue was collected to investigate the effect of the hc diet over time on the intestinal barrier function by measuring mrna expression of tight junction proteins and the ileal transepithelial electrical resistance (teer). In addition, ileal mrna expression of the cytokines ifn- (also known as ifng), tnf- (also known as tnf), il-10 (also known as il10) and tgf - (also known as tgfb1) was measured . Dr - bb rats of 21 to 50 days of age (n = 5) or 51 to 70 days of age (n = 25) were used as control rats . Diet the standard plant - based diet used was a standard laboratory rodent diet (rmh - b diet; arie blok, woerden, the netherlands). The hc diet (td99482; harlan - teklad custom research, madison, wi, usa) is a modification of the ain-93 g diet and contained 200 g / kg hc (as source of amino acids), 3 g / kg l - cysteine, 509.8 g / kg corn starch, 120 g / kg sucrose, 70 g / kg soy - bean oil, 50 g / kg cellulose, 35 g / kg mineral mix, 10 g / kg vitamin mix, 2 g / kg choline bitartrate and 0.20 g / kg butylated hydroxyanisole antioxidant . La / ma test for measuring intestinal permeability in vivo the la / ma test is a non - invasive technique to measure intestinal permeability in vivo . The sugar alcohol mannitol, which is a small molecule, permeates the intestinal mucosa via a transcellular pathway through the water - filled pores of the cell membrane, whereas the larger disaccharide molecule lactulose uses a paracellular route through the junctional complexes between adjacent enterocytes . An increased uptake of lactulose (associated with a high lactulose: mannitol ratio) indicates a damaged barrier . Therefore, a reduction of the intestinal permeability will lead to a lower lactulose uptake and a decreased lactulose: mannitol ratio in the urine . This test allowed us to investigate whether the rats with high intestinal permeability in the prediabetic phase would indeed develop autoimmune diabetes.a la / ma assay as described by meddings et al . Briefly, a stock solution was made containing 4 g mannitol and 6 g lactulose per 100 ml distilled water . Enough solution was made to give each rat 2 ml of the probe.rats were placed in stainless steel metabolism cages with wire bottoms to separate faeces from urine . Rats were denied access to water for 3 h, at which point they were allowed free access to water for the remainder of the experiment . Urine was collected for a total of 24 h, after which rats were returned to their normal cages . Urine volumes were measured and the urine composition was analysed by hplc as described previously .final data were reported as a ratio of fractional excretions (lactulose: mannitol). Fractional excretion is defined as the fraction of the gavaged dose recovered in the urine sample . Snapwell assay for measuring teer an indicator of intestinal permeability, teer was measured on ileum samples in vitro by snapwell assay as described by watts et al . A small sample (length ~50 mm) was taken from the ileum . During the time between killing of the animal and mounting in the snapwells (corning, schiphol - rijk, the netherlands), the samples were kept in dmem (with 4.5 g / l glucose) (gibco, breda, the netherlands) at 4c . Before mounting, the intestinal pieces were cleaned of faeces by gently pulling the intestine over a 1 ml pipette and then cut open longitudinally . The intestine was cleaned further by gently moving it, with the mucosal layer facing downwards, in a petri - dish with approximately 1 ml medium . A piece was cut out, placed on a filter with the mucosal layer facing upwards, sandwiched between two disks and put in the snapwell insert . This insert was placed in a pre - warmed six - well plate containing 2 ml dmem in each well . On top of the insert 450 l dmem was added . The tissue was mounted within 15 min after excision and the plates were incubated at 37c . The teer measurements were done using a millipore millicell - ers meter (millipore, amsterdam, the netherlands) at 30, 60, 90, 120 and 180 min after excision . The teer shown in this paper was measured at 60 min after tissue excision . Quantitative pcr of tight junction proteins and cytokines rna was isolated from ileal tissue and expression of myo9b, cldn1, cldn2 and ocln mrna (encoding the tight - junction - related proteins myosin ixb, claudin-1, claudin-2 and occludin) and ifn-, il-10, tgf - and tnf- mrna (encoding cytokines) was analysed . To isolate rna from the intestine, frozen tissue (1 cm, stored at 80c) was homogenised in 1 ml of tri reagent (sigma - aldrich, zwijndrecht, the netherlands) and mrna was isolated using the tri reagent mrna isolation method . The concentration of the isolated mrna was determined using a nanodrop (nd-1000; isogen, maarsen, the netherlands). Isolated mrna (5 g) was converted to cdna using a kit (superscript ii reverse transcriptase kit; invitrogen life technologies, breda, the netherlands). To measure differences in expression of genes for tight junction - related proteins, transcript levels of myo9b, cldn1, cldn2, ocln and the housekeeping gene hypoxanthine phosphoribosyl - transferase (hprt) were quantified using real - time pcr (for primer sequences see electronic supplementary material [esm] table 1). Real - time pcr analysis was performed using iq sybr green supermix (bio - rad laboratories, veenendaal, the netherlands) according to the manufacturer s instructions; detection was by icycler iq real - time pcr detection system (bio - rad), using the following programme: 3 min 95c, 40 cycles of 30 s at 95c and of 30 s at 60c, and of 10 s at 58c, then 80 times an increase in temperature of 0.5c to create a melting curve . Results were expressed as ratio of target gene, hprt, according to a mathematical method described by pfaffl et al . . Zonulin elisa serum zonulin levels were analysed by the um center for celiac research at the university of maryland medical center . Statistical analysis difference in survival was calculated by the logrank test for kaplan meier survival curves . Differences in teer and expression of tight junction proteins and cytokine levels were calculated by kruskal wallis test followed by the mann correlations were tested for significance using the spearman correlation method . A p value of <0.05 was considered significant . Relation between the level of prediabetic gut permeability and autoimmune diabetes development, and effects of the hc diet on intestinal barrier function in dp - bb rats as previously reported by our group and others [1, 37], the hc diet prevented and delayed the onset of autoimmune diabetes in dp - bb rats (fig . 1hc diet, intestinal permeability and autoimmune diabetes development in dp - bb rats . A kaplan meier survival curve showing the development of autoimmune diabetes in dp - bb rats fed a hc diet (white circles) or a standard diet (black circles). B urinary lactulose: mannitol ratio established at 65 days of age in dp - bb rats on the indicated diet . Standard diet, n = 12 (three urinary samples not analysed owing to sampling problems; one rat excluded because it developed diabetes during la / ma test); hc diet, n = 18 . * p <0.05 c, d correlation between lactulose: mannitol ratio at 65 days of age and the age of diabetes onset in dp - bb rats fed the hc diet (white circles, dotted lines) or the standard diet (black circles, continuous lines). The data in c were analysed including the rats who did not develop type 1 diabetes before 140 days of age (marked by the oval). Dotted line: hc diet - fed dp - bb rats, r = 0.4463, p = ns; continuous line: standard diet - fed dp - bb rats, r = 0.6819, p = 0.0146 . Data in d were analysed excluding the rats who did not develop type 1 diabetes before 140 days of age . Dotted line: hc diet - fed dp - bb rats, r = 0.7122, p = 0.0209; continuous line: standard diet - fed dp - bb rats, r = 0.6041, p = 0.0490as seen in fig . 1b, our results show that hc diet fed rats had a reduced urinary lactulose: mannitol ratio (i.e. Less leaky gut). Interestingly, in both groups, there was a significant correlation between the day of autoimmune diabetes onset and the prediabetic urinary lactulose: mannitol ratio at 65 days of age (fig . 1c, d). In the rats on a standard diet this correlation was present when all the rats are included (diabetic and non - diabetic rats) (fig . 1d). In rats fed a hc diet, the correlation was only sizable when the diabetic rats were analysed . These results indicate a relation between prediabetic intestinal barrier function and the moment of diabetes outcome . Hc diet, intestinal permeability and autoimmune diabetes development in dp - bb rats . A kaplan meier survival curve showing the development of autoimmune diabetes in dp - bb rats fed a hc diet (white circles) or a standard diet (black circles). B urinary lactulose: mannitol ratio established at 65 days of age in dp - bb rats on the indicated diet . Standard diet, n = 12 (three urinary samples not analysed owing to sampling problems; one rat excluded because it developed diabetes during la / ma test); hc diet, n = 18 . * p <0.05 c, d correlation between lactulose: mannitol ratio at 65 days of age and the age of diabetes onset in dp - bb rats fed the hc diet (white circles, dotted lines) or the standard diet (black circles, continuous lines). The data in c were analysed including the rats who did not develop type 1 diabetes before 140 days of age (marked by the oval). Dotted line: hc diet - fed dp - bb rats, r = 0.4463, p = ns; continuous line: standard diet - fed dp - bb rats, r = 0.6819, p = 0.0146 . Data in d were analysed excluding the rats who did not develop type 1 diabetes before 140 days of age . Dotted line: hc diet - fed dp - bb rats, r = 0.7122, p = 0.0209; continuous line: standard diet - fed dp - bb rats, r = 0.6041, p = 0.0490 the hc diet increases ileal teer in dp - bb rats the ileal teer of dp - bb rats fed the hc diet was measured ex vivo at 65 days of age using the microsnapwell assay described previously . As control groups, dr - bb rats and dp - bb rats fed the standard diet were used . Dp - bb rats on the standard diet had a lower teer (i.e. Increased intestinal permeability) than dr - bb rats on the same diet (fig . Dp - bb rats that were fed the hc diet showed an increased teer as compared with dp - bb rats on the standard diet (fig . 2), confirming and extending the la / ma data as shown in fig . 1 . Fig . 2dp - bb rats have reduced ileal teer as compared with dr - bb rats or dp - bb rats fed the hc diet . Rats were killed at 65 days of age and their ileal teer measured as described . Dr - bb rats fed standard rodent diet, n = 17; dp - bb rats on the standard diet, n = 10; dp - bb rats on the hc diet, n = 21 . The data are presented as a scatter dot plot with the mean indicated by a horizontal line . * p <0.05 dp - bb rats have reduced ileal teer as compared with dr - bb rats or dp - bb rats fed the hc diet . Rats were killed at 65 days of age and their ileal teer measured as described . Dr - bb rats fed standard rodent diet, n = 17; dp - bb rats on the standard diet, n = 10; dp - bb rats on the hc diet, n = 21 . The data are presented as a scatter dot plot with the mean indicated by a horizontal line . * p <0.05 impaired ileal mrna expression of tight junction proteins in dp - bb rats is restored by a hc diet tight junctions in gut epithelia are pivotal for maintenance of intestinal barrier function [2729]. Therefore, we investigated the effect of the hc diet on mrna expression of myo9b, cldn1, cldn2 and ocln, which encode proteins necessary for tight junction functionality.no differences were observed regarding ileal ocln mrna expression (fig . Myob expression in the ileum of dp - bb rats was increased as compared with that in dr - bb rats (fig . Strikingly, feeding the hc diet to dp - bb rats led to a significant decrease in myo9b expression in the ileum of dp - bb rats . Interestingly, the longer the dp - bb rats were fed the hc diet, the lower the expression of myo9b was . 3ileal mrna expression of ocln (a), myo9b (b), cldn1 (c) and cldn2 (d) in dp - bb rats fed the standard diet (black bars), dp - bb rats on hc diet (white bars) and dr - bb rats fed the standard diet (hatched bars). A no difference in ileal ocln mrna expression between dp - bb rats fed the standard diet and those on the hc diet . B increased ileal myo9b mrna expression in 51- to 70-day - old dp - bb rats fed the standard diet, as compared with other groups . C reduced ileal cldn1 mrna expression in dp - bb rats fed the standard diet as compared with dp - bb rats on the hc diet and dr - bb rats on the standard diet . D at 51 to 70 days of age dp - bb rats had increased ileal cldn2 mrna expression compared with dr - bb rats . Group sizes: dp - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8); dr - bb rats 2150 days of age (n = 5), 5170 days of age (n = 8). * p <0.05; * * p <0.01; p <0.1 . 3c, cldn1 expression in the ileum of dp - bb rats declined over time and at> 50 days of age was lower than in dr - bb rats . Feeding the hc diet to dp - bb rats resulted in an increase of cldn1 expression in the ileum of the dp - bb rats . Above 50 days of age, cldn2 expression in the ileum of dp - bb rats 3d). Feeding dp - bb rats the hc diet reduced the expression of cldn2 to levels observed in dr - bb rats on the standard diet (fig . Ileal mrna expression of ocln (a), myo9b (b), cldn1 (c) and cldn2 (d) in dp - bb rats fed the standard diet (black bars), dp - bb rats on hc diet (white bars) and dr - bb rats fed the standard diet (hatched bars). A no difference in ileal ocln mrna expression between dp - bb rats fed the standard diet and those on the hc diet . B increased ileal myo9b mrna expression in 51- to 70-day - old dp - bb rats fed the standard diet, as compared with other groups . C reduced ileal cldn1 mrna expression in dp - bb rats fed the standard diet as compared with dp - bb rats on the hc diet and dr - bb rats on the standard diet . D at 51 to 70 days of age dp - bb rats had increased ileal cldn2 mrna expression compared with dr - bb rats . Group sizes: dp - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8); dr - bb rats 2150 days of age (n = 5), 5170 days of age (n = 8). * p <0.05; * * p <0.01; p <0.1 . Nd, not determined increased ileal mrna expression of tgf - and il-10 in dp - bb rats fed a hc diet the expression of tgf - and il-10 in the ileum of hc - fed dp - bb rats was increased as compared with that in dp - bb rats on the standard diet (fig . 4). Expression in the ileum of hc - fed rats was comparable to expression levels found in the dr - bb rats . No differences were observed regarding the expression of ifn-, tnf- and forkhead box p3 (data not shown). Fig . 4a increased ileal tgf - mrna expression in dp - bb rats on the hc diet (white bars) as compared with dp - bb rats on the standard diet (black bars). At 51 to 70 days of age dp - bb rats on the standard diet had reduced ileal tgf - mrna expression as compared with dr - bb rats on the standard diet (hatched bars). B between 21 and 50 days of age, dp - bb rats fed the hc diet had an increased ileal il-10 mrna expression as compared with age - matched dp - bb rats on the standard diet . Group sizes: dp - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8); dr - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8). Nd, not determined a increased ileal tgf - mrna expression in dp - bb rats on the hc diet (white bars) as compared with dp - bb rats on the standard diet (black bars). At 51 to 70 days of age dp - bb rats on the standard diet had reduced ileal tgf - mrna expression as compared with dr - bb rats on the standard diet (hatched bars). B between 21 and 50 days of age, dp - bb rats fed the hc diet had an increased ileal il-10 mrna expression as compared with age - matched dp - bb rats on the standard diet . Group sizes: dp - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8); dr - bb rats 2150 days of age (n = 8), 5170 days of age nd, not determined expression of ileal cldn1 and tgf - mrna in diabetic and non - diabetic dp - bb rats the diabetic rats were killed at diabetes onset at the age of 70 to 100 days . The non - diabetic rats were killed at 140 days of age . Of the rats fed a hc diet, ten diabetic and five non - diabetic rats were analysed . Of the rats on a standard diet, ten diabetic rats and one non - diabetic rat were analysed . Only the expression of cldn1 and tgf - was analysed, because the difference in expression of these variables was the most pronounced around 60 days of age.in both the control rat groups and the hc diet - fed dp - bb rats, the diabetic and non - diabetic rats displayed lower expression of cldn1 and tgf - than rats younger than 50 days of age . No differences were observed between diabetic and non - diabetic rats (esm fig . 1). Reduced serum zonulin levels in dp - bb rats fed a hc diet serum zonulin levels were measured in serum samples, which had been cross - sectionally obtained in previous experiments, of prediabetic dp - bb rats fed the standard diet or the hc diet, and killed between 50 and 70 days of age . As shown in fig . 5, dp - bb rats receiving the hc diet had reduced serum zonulin levels as compared with rats on the standard diet . Because zonulin levels strongly correlate with intestinal permeability [12, 14, 15], this result indicates that the hc diet improves intestinal barrier function in dp - bb rats via downregulation of zonulin . Zonulin levels were measured by elisa in serum of prediabetic dp - bb rats between 50 and 70 days of age . Dp - bb rats on the standard diet, n = 11; dp - bb rats on the hc diet, n = 13 . The data are presented as scatter dot plot with the mean indicated by a horizontal line . * p <0.05 dp - bb rats fed the hc diet have reduced serum zonulin levels . Zonulin levels were measured by elisa in serum of prediabetic dp - bb rats between 50 and 70 days of age . Dp - bb rats on the standard diet, n = 11; dp - bb rats on the hc diet, n = 13 . The data are presented as scatter dot plot with the mean indicated by a horizontal line . * here we provide evidence that a hc diet restores the impaired intestinal barrier function in prediabetic dp - bb rats as reflected by increased ileal teer, reduced serum zonulin levels and a reduced urinary lactulose: mannitol ratio . Rats with a low prediabetic intestinal permeability developed autoimmune diabetes later or were protected against the disease . Myosin ixb is an adaptor protein involved in linking the cytoskeleton with tight junctions, whereas claudins and occludin are involved in the architecture of tight junctions itself [2729]. High levels of myosin ixb and claudin-2, and low levels of claudin-1 and occludin will result in the opening of tight junctions and a subsequent increase in intestinal permeability [2729]. Therefore, our results for tight junction proteins may explain why, at 65 days of age, the intestinal barrier function of dp - bb rats on the standard diet is reduced compared with that of dr - bb rats . Interestingly, watts et al . Demonstrated that intestinal permeability of dp - bb rats increases from the age of 50 days, which correlates with the mrna expression pattern of tight junction proteins shown in the current report . Besides tight junctions, mucins are also important for maintaining intestinal barrier function . It was shown by courtois et al . That a hc diet increased intestinal mucin levels and subsequently improved intestinal barrier function in bb rats as demonstrated by reduced uptake of fitc - labelled dextran across the gut epithelium . We show that in dp - bb rats, as opposed to dr - bb rats, the expression of cldn1 strongly declined over time, leading to very low levels after the age of 50 days . The mrna expression levels of myo9b, ocln and cldn2 showed no significant changes over time, but myo9b and cldn2 mrna expression levels were increased as compared with dr - bb rats . In dr - bb and dp - bb rats of 34 and 41 days of age, neu et al . Found reduced ileal claudin-1 protein levels as compared with wistar rats, but observed no differences between dp - bb and dr - bb rats . Interestingly, like neu et al ., we also found no difference in cldn1 mrna expression between dp - bb and dr - bb rats below the age of 50 days . However, we did observe a difference between these two rat groups above the age of 50 days, a difference caused by the decline of cldn1 mrna expression in dp - bb rats . Interestingly, in dp - bb rats, the hc diet seemed to normalise tight junction mrna expression levels towards levels observed in dr - bb rats . Therefore, the observed patterns of decreased myo9b and cldn2 expression and increased cldn1 expression in dp - bb rats on a hc diet fits well with the hypothesis that the hc diet has a beneficial effect on gut permeability through modification of tight junction functionality . A proinflammatory intestinal cytokine environment affects tight junction functionality and subsequently increases intestinal permeability [12, 29]. Here, the hc diet increased the anti - inflammatory cytokines il-10 and tgf -, whereas tnf- and ifn- levels were not affected (data not shown). The hc diet therefore creates an anti - inflammatory cytokine milieu in the small intestine, which might lead to improved intestinal barrier function . In rats fed the standard diet and in dp - bb rats on a hc diet, the animals that did not develop diabetes had no higher ileal cldn1 and tgf - expression than the diabetic rats . This result was not expected, but might be explained by ageing . As shown in figs 3 and 4, and in esm fig . 1, cldn1 and tgf - expression declined with age in the dp - bb rats, but not in the dr - bb rats . The non - diabetic dp - bb rats were on average 50 days older than the diabetic rats . Therefore, the comparable mrna expression levels of cldn1 and tgf - in diabetic and non - diabetic rats might have been due to ageing . Unfortunately, we did not kill non - diabetic dp - bb rats between 70 and 100 days of age . Previous research by our group and others has shown that the hc diet affects autoimmune diabetes pathogenesis mainly between 30 and 70 days of age [1, 3]. Dp - bb rats that were fed a cereal - based diet until 60 to 70 days and then switched to the hc diet were not protected against autoimmune diabetes development, suggesting a crucial window [1, 3]. Therefore, prevention of exposure to diabetogenic triggers in this window can affect diabetes outcome . The results presented here show that the decline of intestinal barrier function with age in dp - bb rats is delayed, but not completely prevented by the hc diet . However, this delay might be enough to prevent exposure to diabetogenic triggers in the diabetes - susceptible window and subsequently prevent or delay autoimmune diabetes onset . Food components and intestinal bacteria can affect the integrity of the intestinal barrier [8, 12, 2224]. Recently, mojibian et al . Demonstrated that ~50% of established type 1 diabetes patients have t cell responses against wheat polypeptides . These results indicate that wheat might be a major dietary antigen capable of inducing type 1 diabetes . An important question raised by the mojibian study is why in ~50% of type 1 diabetes patients a wheat polypeptide - specific response is found . Interestingly, studies by our group and others have shown that also ~50% of type 1 diabetes patients have intestinal barrier defects [13, 15]. Although mojibian et al . Did not investigate whether the wheat - specific t cell responses correlated with impaired intestinal barrier function, it is reasonable to hypothesise that impaired intestinal barrier function leads to an increased passage of intestinal diabetogenic antigens (e.g. Wheat peptides, bacterial agents) that induce the autoimmune cascade typical of type 1 diabetes [11, 12, 31]. To prove this hypothesis, further investigation of the relationship between intestinal barrier function and immune responses against intestinal antigens and beta cells in type 1 diabetes patients will be required . In summary, the results presented here show that in dp - bb rats the level of prediabetic gut permeability is associated with autoimmune diabetes development later in life and that improvement of this intestinal barrier function might contribute to the prevention of autoimmune diabetes by dietary intervention with the hc diet . Changes to the local intestinal cytokine profile, direct effects on tight junctions and reduced zonulin production might be important mechanisms for this effect . Taken together, these results suggest that a hc - based diet probably prevents diabetes development in the dp - bb rat by: (1) improving intestinal barrier function thereby contributing to no or less induction of the autoimmune cascade responsible for autoimmune diabetes development [1, 7, 15]; (2) skewing the autoreactive t cells to a less pathogenic phenotype [1, 3, 6]; and (3) the induction of islet neogenesis . Modification of intestinal permeability either directly by tight junction modulators or indirectly via environmental factors like bioactive peptides, prebiotics or probiotics could be a promising approach to the development of a whole new field of therapeutic strategies to prevent or treat autoimmune diabetes . Below is the link to the electronic supplementary material . Primer sequences (pdf 52.7 kb) a prediabetic (below the age of 70 days) dp - bb rats on standard diet (black bars) showed reduced ileal cldn1 mrna expression as compared with hc diet - fed dp - bb rats (white bars) and dr - bb rats on standard diet (hatched bars). No differences in claudin-1 expression between diabetic or non - diabetic rats fed either the standard diet or hc diet were observed . B prediabetic (below the age of 70 days) dp - bb rats fed the standard diet showed a reduced ileal tgf - mrna expression as compared with dp - bb rats on hc diet and dr - bb rats on standard diet . No differences in tgf - expression between diabetic or non - diabetic rats fed either the standard diet or hc diet were seen . Group sizes: dp - bb rats 2150 days of age (n = 8), 5170 days of age (n = 8); dr - bb rats 21 to 50 days of age (n = 5), 5170 days of age (n = 8). Diabetic dp - bb rats on standard diet, n = 10; non - diabetic dp - bb rats on standard diet, n = 1; diabetic dp - bb rats on hc diet, n = 10; non - diabetic dp - bb rats on hc diet, n = 5 . * p <0.05; * * p <0.01 . D (x - axes), diabetic rats 73 to 100 days of age; nd, non - diabetic rats 140 days of age (pdf 20 kb)
A 33-year - old male (height 172.3 cm and weight 61.5 kg) visited our pain clinic due to neck pain radiating to both shoulders, which started 3 years ago . He complained of tingling sensations of the bilateral hand along with shoulder pain, and difficulty in neck flexion . Upon physical examination, the results of the jackson compression test and the spurling's test did not appear positive . On the cervical mri, c5/6 and c6/7 intervertebral disc protrusions were found and chronic right c6 radiculopathy was found on the electromyography . This patient had already received an epidural steroid injection and decompressive neuroplasty using a racz's catheter at a local hospital . Therefore, we decided to perform cervical nucleoplasty at the c5/6 and c6/7 intervertebral discs . On the pre - procedural laboratory tests, there were no abnormalities in the cbc, esr, and blood chemistry test (table 1). Nucleoplasty was performed on the c5/6 and c6/7 intervertebral discs without any perioperative events . Under a supine position, skin preparation was performed with betadine soap and 2% chlorhexidine-70% isoprophyl alcohol solution, and a sterile surgical drape was applied . After the internal carotid was laterally displaced, a 19 gauge 3 inch introducer needle was introduced till the anterolateral annulus fibrosus . After the needle was advanced deeper, the position of the needle was adjusted to reach the target site of the herniated disc under a c - arm guide . When the needle reached the proper target site, the stylet of the introducer needle was withdrawn, and the perc dc spine wand (arthrocare co., sunnyvale, ca, usa) was replaced and fastened to the needle hub . Next, the perc dc spine wand was connected to the arthrocare system 2,000. after confirming that there was no cervical root stimulation, coblation was then carried out by rotating the flange 180 for 20 seconds . After this, the wand was retracted 1 - 2 mm under c - arm guidance and the same procedure was repeated 3 times . Stay, the body temperature was 36.7 - 36.9 and there were no signs or symptoms of acute complications . An additional amount of intravenous cefazolin was administered three times a day and a routine prophylaxis for 2 more days during the hospital stay . Additional oral cefradine was prescribed 1,000 mg / day for 1 week after the patient's discharge . After 8 days, the patient revisited our outpatient clinic for routine postoperative checkup, but there were no improvements in the patient's symptoms . In addition, there were no symptoms or signs of infection including fever or chills, and the laboratory findings, such as the cbc and esr, were in the normal range . After 2 months, during his second postoperative visit to our clinic, the patient complained of right shoulder pain and worsening neck pain during flexion . He also complained of feelings of weakness in both arms, but his motor functions were intact when a physical examination was performed . In addition, new physical signs developed; the jackson compression test and spurling's test were positive, with radiating pain in both shoulders . There was tenderness on the right cervical facet joint, but no tenderness was observed on the left side . The body temperature was normal, and there was no sensation of chills . In order to evaluate the focus of the pain, a cervical mri was performed, and the findings were compatible with spondylodiscitis at the c6/7 intervertebral disc and the vertebral body (fig . The patient was referred to the neurosurgery department immediately, and intravenous antibiotics therapy was planned . However, with the patient's will, he was transferred to another hospital for long - term antibiotics therapy . Early diagnosis and treatment of spondylodiscitis is important for the prevention of catastrophic sequelae and huge additional expenses . Therefore, it is essential to understand the natural course of postoperative spondylodiscitis . Usually, short - term relief of the symptoms comes first after surgery, and then back pain recurs within 6 weeks . According to bavinzski et al . In addition, fever was a typical symptom associated with this infection . In our case, neck pain was aggravated and new positive signs in the physical exam appeared 8 weeks after the procedure . Moreover, our patient was afebrile, which can be explained by the lack of elevation of an inflammatory marker . The esr and crp are known as the earliest and most sensitive screening tools for the infection . Meyer et al . Suggested the sensitivity and specificity of crp as 100% and 95.8%, respectively, when predicting postoperative infections . In addition, mustard et al . Conducted a study on 108 patients, and a positive crp response was defined as meeting two criteria: on days 3 and 4, the crp level is> 80% of day 2 (positive diagnosis by day 4); and after day 4, the crp rises on 2 consecutive days with a level greater than 15 mg / l for each day (positive diagnosis by day 6). The above criteria had a sensitivity of 63%, a specificity of 82%, a positive predictive value of 68% and a negative predictive value of 78% . Recommended that an esr of more than 50 mm / hr for more than 2 weeks after surgery be used as an early diagnostic marker for spondylodiscitis, indicating a need to perform more tests . However, during the early postoperative period, the crp and esr may rise as a consequence of tissue damage caused by the surgery itself . Kwon et al . Found that the crp and white blood cell (wbc) rose starting on the first day of surgery and decreased after the third postoperative day, and the esr elevation lasted up to 11 days after spine surgery . To screen postoperative infections with laboratory tests, adequate follow up periods and tests should be considered, with further evaluations being needed to confirm spondylodiscitis . Based on the previous studies, crp should be checked 4 days after the surgery and compared with a baseline value (preoperative value), while the esr should be checked 12 days after the surgery . There has been a reported case of spondylodiscitis following cervical nucleoplasty: in theat case, the complication was detected 3 weeks after the procedure . In our case, outpatient follow - ups were performed 9 days and 8 weeks after the operation . During the first visit, in our case, neither the infection nor the procedure itself increased the esr and crp . Further research is needed to clarify the changes of the inflammatory marker after nucleoplasty . At the second visit, the crp was checked and was identified to be within the normal range . During the 8-week postoperative follow - up, we could not find any abnormalities in the laboratory tests even when the mri showed apparent infection . Bavinzski et al . Reported a series of 13 patients with postoperative disc space infection . All patients suffered from severe local lumbar pain with muscle cramps and radiating pain to the hips, abdomen, legs, scrotum, groin, or perineum . Many patients were unable to stand, with the pain exacerbated by motions of the spine segments . In a recent review article, the typical course of spondylodiscitis is the return of low back pain approximately six weeks later . Similar to the previous studies, our patient also complained about pain being aggravated by motion and local tenderness around the infection site about 8 weeks after the spinal procedure . Therefore, clinicians should be careful if patients complain of aggravated pain about 6 weeks post - operation, while additional studies are required to diagnose the infection . If there is no neurologic or structural instability, it is recommended that antimicrobial therapy should be withheld until positive identification of the organism is achieved unless back pain is accompanied by systemic symptoms . However, in our case, the infection was identified at the site of nucleoplasty 2 months later, and the patient's symptoms were aggravated . Therefore, the infection was considered to be a procedure - related problem and empirical antibiotics therapy was initiated . Surgical intervention is recommended in cases of impending pathologic fractures, functionally significant neurologic deficits, paravertebral or epidural abscess formations, persistent septicemia despite antibiotic treatment, intractable pain, and unacceptable saggital or coronal plane deformity . We postulate that the pathogen may be sequestrated from the blood stream completely, therefore, systemic inflammatory changes were not triggered, or the immunity of this patient (33 year - old man) was strong enough to overcome the dispersion of the pathogen . For detecting postoperative discitis, mri is the imaging method of choice, with its high sensitivity and specificity . Mri is considered to be the most sensitive and specific method, 93% and 97%, as reported . Furthermore, mri is the gold standard for diagnosing spondylodiscitis . In conclusion, therefore, an mri should be taken when there is clinical suspicion of infection in order to not miss the complications after interventional procedures, even if the laboratory findings were normal.
Traditionally, barium examinations have been the mainstay of radiological investigations of the small and large intestines . However, the standard barium follow - through (sbft) examination has high false - negative and false - positive rates for the detection of abnormalities this is because only 2535% of the bowel length can be adequately assessed on a standard examination due to overlapping loops, poor coating, and other factors . Several new techniques have therefore evolved that provide better diagnostic capabilities and higher accuracy rates. [24] in this article, the newer techniques and modalities being employed for imaging of the bowel are detailed, with an overview of their strengths and weaknesses . Innovations in intestinal imaging have been driven by the demand for detailed clinical information and the limitations of standard methods of small - bowel examination . Alternative techniques such as the pill camera that allow high - definition color views of the bowel have appeared . At an average frequency of two frames per second, a total of> 50,000 frames are collected in 78 h as the pill camera travels through the bowel . Despite the high - resolution images provided by this technique, it has become increasingly apparent that not all mucosal abnormalities seen on wireless capsule endoscopy are clinically relevant . Mucosal erosions may be seen in as many as 14% of asymptomatic healthy subjects, with the rate of such abnormalities being more than double this in users of nonsteroidal anti - inflammatory drug (nsaid) agents . Furthermore, the pill camera does not provide any information about mural and extramural abnormalities . Contraindications to the pill camera include bowel obstruction, history of bowel strictures or fistulae . The most accurate conventional radiologic method in the diagnosis of small - bowel obstruction, inflammatory bowel disease (ibd), gastrointestinal bleeding, and neoplasms is double - contrast enteroclysis . However, sbft has remained the most commonly performed method for the investigation of small - bowel diseases because of its ease of performance and the relative technical complexity of enteroclysis as well as the discomfort to patients during intubation . The sbft has a low diagnostic yield and low negative predictive value . In a recent study, 8% of patients with known crohn disease encountered capsule retention despite a previous normal sbft . Non - intubation examinations do not fully distend the bowel, and therefore partial strictures, obstructions, and small polyps or masses can be easily missed . The advantage of enteroclysis examinations is that there is optimal bowel distension, which allows detailed evaluation of all segments [figure 1]. Methylcellulose bowel examination shows linear filling defects (webs) (arrows) in the distal ileum secondary to the use of nonsteroidal anti - inflammatory drugs (nsaids) enteroclysis examination with barium methylcellulose is the most commonly performed enteroclysis method in many countries . However, the disadvantage with this technique is that methylcellulose has a washout effect on superficial mucosal features . Subtle surface abnormalities get effaced as more methylcellulose is infused to achieve a good double - contrast effect and bowel distension . Therefore, this method should be reserved for detection of obstruction or mass lesions or it should be used in combination with ct scan or mri exams. [247] fluoroscopic evaluation with positive enteral contrast can help distinguish fixed (stenotic) segments of small - bowel narrowing from spasm caused by active inflammation in patients who have crohn disease and can also differentiate mild stenosis from normal peristaltic contractions . On the other hand, double - contrast enteroclysis with air / co2 and barium is by far the best method for showing mucosal details of the small bowel. [578] a scientific study comparing double - contrast air enteroclysis with histopathology showed good correlation between the two for the visualization of aphthae and small ulcers [figure 2]. Magnified view from an air barium double contrast enteroclysis examination shows early linear mucosal breaks and ulcers (arrow) in a patient with proven crohn disease therefore, if capsule endoscopy is unavailable or contraindicated, double - contrast air enteroclysis should be the preferred modality for the detection of early ulcerations, irregularities, and erosions of the small bowel . This technique should also be employed if other imaging tests have been negative and a suspicion of small - bowel pathology still persists . This technique can be performed on its own or it can be combined with ct scan or mri . The pill camera remains the most sensitive method for the evaluation of gastrointestinal tract bleeding. [89] the most important factor for obtaining good intestinal imaging is optimal distension of the bowel with enteral contrast . Enteral contrast can be positive, neutral, or negative, depending on its density (hu). Positive enteral contrast agents range from a 415% contrast solution to a dilute 0.36% barium solution . The author prefers an 810% iodine concentration of water - soluble contrast because this density allows diagnostic fluoroscopic observations as well as diagnostic radiographs . Neutral contrast agents can be methylcellulose, mannitol, polyethyl glycol, or other bulk fibers and have a density similar to water (0 hu). Negative contrast is mainly used in the imaging of the colon [ct colonography (ctc)], where co2 or air is insufflated per rectum . Enteral contrast can be administered via the intubation method (ct enteroclysis / mri enteroclysis) or the enterographic method [figure 3]. For enterographic examination, the patient ingests a large volume of contrast over a set period of time prior to imaging . The 1300 ml is divided into two aliquots: the first lot is ingested with a prokinetic agent (metoclopromide) over 25 min and the second lot is consumed over the next 25 min . Just prior to the scan, 200 ml of contrast the main advantage of the enterographic examination is that it is more acceptable to patients than enteroclysis as intubation may be an unpleasant experience for patients . The disadvantage of enterograpic examination is that it may not provide uniform or adequate bowel distension as compared to enteroclysis . Axial ct enteroclysis examination demonstrates a segment of kinked bowel (arrowhead) and several adhesive bands across other segments (arrows). This patient had undergone several negative ct examinations previously water is also routinely used as a neutral enteral contrast agent at many centers; however, its use in dedicated small - bowel studies should be discontinued . Water has been found to provide inadequate distension (at various volumes and ingestion times) of the small bowel in many studies. [1011] this is because water undergoes rapid reabsorption in the bowel and fails to adequately distend the distal small - bowel segments . Therefore, additives (such as contrast, mannitol or other agents) need to be mixed with water to provide an iso - osmolar solution that does not get reabsorbed and remains in the lumen to provide distension . The use of water as a contrast agent may lead to false - negative examinations . Furthermore, collapsed loops are known to demonstrate hyperenhancement on post - contrast studies and this may lead to false - positive diagnoses . Therefore, the author strongly discourages the use of water as an enteral agent in dedicated studies of the small bowel . The only instance where water may be used as an enteral agent is in the enteroclysis technique where the operator exclusively controls distension of the bowel and rate of infusion . The advantage of current - generation multidetector ct (mdct) scanners is increased anatomical coverage with thinner sections, which provide high - quality mutiplanar (mpr) images and fewer motion artifacts. [1213] positive - contrast ct enteroclysis (cte) or enterography (cteg) may be used mainly for the diagnostic workup of small - bowel obstruction or in suspected cases of small intestinal tumors or metastases . One of the drawbacks of positive contrast is the production of streak artifacts on mpr reconstruction, particularly on older ct scanners that cannot produce isotropic voxels (slice thickness> 2 mm). The advantages of neutral contrast are that it does not obscure the mucosal lining and does not produce streak artifacts . Therefore, neutral - contrast cte or cteg is indicated for the workup of patients with inflammatory bowel bleed, gastrointestinal bleeding, and suspected bowel pathologies . Several studies have shown that ctc has high sensitivity and specificity (> 95%) for the detection of polyps and cancers [figure 4a]. Datasets of the distended colon are obtained, which can then by manipulated by software to provide virtual colonoscopy images . Screenshot (a) from a ct colonographic examination shows a polyp in the colon . Top two axial images show the location of the polyp within a marker box, whereas the bottom two are virtual 3d images in the supine and prone positions which detail polyp size and distance from the rectum . Mucosal views (b) show a virtual dissection view of the colon with a small polyp (arrow) in the left image . On the right, translucency rendering shows this lesion to be homogenously dense (red), implying this is retained fecal matter mri is an emerging technique for comprehensive and functional bowel imaging . The absence of ionizing radiation and high - contrast resolution are the major advantages of mri over ct scan . The nonionizing aspect of mri makes it particularly suited for use in patients with ibd who may need repeated imaging . The inherent high - contrast resolution can provide high diagnostic confidence and also facilitate the detection of discriminating features of intestinal diseases . T1w and t2w imaging sequences for the imaging of the bowel have been reported in earlier studies. [101718] however, in recent years, advances with ultra - fast sequences based on steady - state precession have revolutionized bowel imaging . These sequences can be performed within a single breath - hold, and when combined with bowel paralysis (using antiperistaltic agents, e.g., buscopan), the technique allows rapid imaging of the small bowel . These sequences are called true fast imaging with steady - state precession (true - fisp), balanced fast field - echo (ffe), or fast imaging employing steady - state acquisition (fiesta). These sequences are relatively insensitive to motion artifacts and provide high contrast between the bowel wall, lumen, and the mesentery . The problem with these sequences is the presence of a black boundary artifact along the bowel wall, which may mask small lesions or abnormalities . The addition of fat suppression may help in reducing the effects of the black boundary artifact [figure 5]. Fast sequences also obviate the need for long breath - holds, and thus reduce motion or respiratory artifacts . A patient with crohn disease . Mri enterography examination shows good opacification of the small and large bowel with thickening of the inflamed cecal wall (arrow) t2w fast sequences based on rapid acquisition and relaxation, such as half - fourier single - shot turbo spin - echo (haste) or single - shot fast spin - echo (ssfse) sequences, are also used and can provide high contrast between the lumen and the bowel wall, producing images akin to a conventional barium study . Fat saturation can be used to increase contrast resolution and also for better assessment of bowel enhancement . It is possible that with this technique, high - resolution three - dimensional datasets comprising isotropic voxels of the entire abdomen could be produced within a single breath - hold . Three - dimensional datasets allow high - quality mpr reconstructions, which can increase diagnostic confidence . Usg also allows observation of normal or abnormal bowel peristalsis, fixity, and compressibility . Some studies have employed bowel distension using oral contrast (hydrosonography) although its use remains limited . The current - generation high - frequency probes provide greater spatial resolution than ct scan or mri [figure 6]. Contrast - enhanced usg can provide detailed information about vascularity and inflammation of the bowel . High - resolution usg image shows an inflamed bowel segment, with marked enhancement of the mucosal vessels (arrowheads) and engorgement of the penetrating blood vessels (arrow) the most important factor for obtaining good intestinal imaging is optimal distension of the bowel with enteral contrast . Enteral contrast can be positive, neutral, or negative, depending on its density (hu). Positive enteral contrast agents range from a 415% contrast solution to a dilute 0.36% barium solution . The author prefers an 810% iodine concentration of water - soluble contrast because this density allows diagnostic fluoroscopic observations as well as diagnostic radiographs . Neutral contrast agents can be methylcellulose, mannitol, polyethyl glycol, or other bulk fibers and have a density similar to water (0 hu). Negative contrast is mainly used in the imaging of the colon [ct colonography (ctc)], where co2 or air is insufflated per rectum . Enteral contrast can be administered via the intubation method (ct enteroclysis / mri enteroclysis) or the enterographic method [figure 3]. For enterographic examination, the patient ingests a large volume of contrast over a set period of time prior to imaging . The 1300 ml is divided into two aliquots: the first lot is ingested with a prokinetic agent (metoclopromide) over 25 min and the second lot is consumed over the next 25 min . Just prior to the scan, 200 ml of contrast the main advantage of the enterographic examination is that it is more acceptable to patients than enteroclysis as intubation may be an unpleasant experience for patients . The disadvantage of enterograpic examination is that it may not provide uniform or adequate bowel distension as compared to enteroclysis . Axial ct enteroclysis examination demonstrates a segment of kinked bowel (arrowhead) and several adhesive bands across other segments (arrows). This patient had undergone several negative ct examinations previously water is also routinely used as a neutral enteral contrast agent at many centers; however, its use in dedicated small - bowel studies should be discontinued . Water has been found to provide inadequate distension (at various volumes and ingestion times) of the small bowel in many studies. [1011] this is because water undergoes rapid reabsorption in the bowel and fails to adequately distend the distal small - bowel segments . Therefore, additives (such as contrast, mannitol or other agents) need to be mixed with water to provide an iso - osmolar solution that does not get reabsorbed and remains in the lumen to provide distension . The use of water as a contrast agent may lead to false - negative examinations . Furthermore, collapsed loops are known to demonstrate hyperenhancement on post - contrast studies and this may lead to false - positive diagnoses . Therefore, the author strongly discourages the use of water as an enteral agent in dedicated studies of the small bowel . The only instance where water may be used as an enteral agent is in the enteroclysis technique where the operator exclusively controls distension of the bowel and rate of infusion . The advantage of current - generation multidetector ct (mdct) scanners is increased anatomical coverage with thinner sections, which provide high - quality mutiplanar (mpr) images and fewer motion artifacts. [1213] positive - contrast ct enteroclysis (cte) or enterography (cteg) may be used mainly for the diagnostic workup of small - bowel obstruction or in suspected cases of small intestinal tumors or metastases . One of the drawbacks of positive contrast is the production of streak artifacts on mpr reconstruction, particularly on older ct scanners that cannot produce isotropic voxels (slice thickness> 2 mm). The advantages of neutral contrast are that it does not obscure the mucosal lining and does not produce streak artifacts . Therefore, neutral - contrast cte or cteg is indicated for the workup of patients with inflammatory bowel bleed, gastrointestinal bleeding, and suspected bowel pathologies . Several studies have shown that ctc has high sensitivity and specificity (> 95%) for the detection of polyps and cancers [figure 4a]. Datasets of the distended colon are obtained, which can then by manipulated by software to provide virtual colonoscopy images . Screenshot (a) from a ct colonographic examination shows a polyp in the colon . Top two axial images show the location of the polyp within a marker box, whereas the bottom two are virtual 3d images in the supine and prone positions which detail polyp size and distance from the rectum . Mucosal views (b) show a virtual dissection view of the colon with a small polyp (arrow) in the left image . On the right, translucency rendering shows this lesion to be homogenously dense (red), implying this is retained fecal matter the absence of ionizing radiation and high - contrast resolution are the major advantages of mri over ct scan . The nonionizing aspect of mri makes it particularly suited for use in patients with ibd who may need repeated imaging . The inherent high - contrast resolution can provide high diagnostic confidence and also facilitate the detection of discriminating features of intestinal diseases . T1w and t2w imaging sequences for the imaging of the bowel have been reported in earlier studies. [101718] however, in recent years, advances with ultra - fast sequences based on steady - state precession have revolutionized bowel imaging . These sequences can be performed within a single breath - hold, and when combined with bowel paralysis (using antiperistaltic agents, e.g., buscopan), the technique allows rapid imaging of the small bowel . These sequences are called true fast imaging with steady - state precession (true - fisp), balanced fast field - echo (ffe), or fast imaging employing steady - state acquisition (fiesta). These sequences are relatively insensitive to motion artifacts and provide high contrast between the bowel wall, lumen, and the mesentery . The problem with these sequences is the presence of a black boundary artifact along the bowel wall, which may mask small lesions or abnormalities . The addition of fat suppression may help in reducing the effects of the black boundary artifact [figure 5]. Fast sequences also obviate the need for long breath - holds, and thus reduce motion or respiratory artifacts . A patient with crohn disease . Mri enterography examination shows good opacification of the small and large bowel with thickening of the inflamed cecal wall (arrow) t2w fast sequences based on rapid acquisition and relaxation, such as half - fourier single - shot turbo spin - echo (haste) or single - shot fast spin - echo (ssfse) sequences, are also used and can provide high contrast between the lumen and the bowel wall, producing images akin to a conventional barium study . Fat saturation can be used to increase contrast resolution and also for better assessment of bowel enhancement . It is possible that with this technique, high - resolution three - dimensional datasets comprising isotropic voxels of the entire abdomen could be produced within a single breath - hold . Three - dimensional datasets allow high - quality mpr reconstructions, which can increase diagnostic confidence . The main advantages of usg are its nonionizing character and general availability . The dynamic, real - time qualities of usg provide high temporal resolution . Usg also allows observation of normal or abnormal bowel peristalsis, fixity, and compressibility . Some studies have employed bowel distension using oral contrast (hydrosonography) although its use remains limited . The current - generation high - frequency probes provide greater spatial resolution than ct scan or mri [figure 6]. Contrast - enhanced usg can provide detailed information about vascularity and inflammation of the bowel . High - resolution usg image shows an inflamed bowel segment, with marked enhancement of the mucosal vessels (arrowheads) and engorgement of the penetrating blood vessels (arrow) mri fluoroscopy: changes in bowel kinetics can be evaluated on mri fluoroscopy to demonstrate either an obstructive element or abnormalities in peristalsis . Mri fluoroscopy can provide functional information regarding bowel motility and may help distinguish between fibrotic strictures and functional bowel spasm . Pet / ct: positron emission tomography (pet)/ct depends on the uptake of tracer by abnormal or highly metabolic tissue . Typically, cancers or metastases show up as hot spots due to their higher metabolic rate as compared to the surrounding tissues . The role of pet / ct in intestinal imaging is mainly related to cancer imaging and the detection of distant metastases . Pet - ct also has a role is the detection of recurrent cancers and in quantifying malignancy in suspicious nodes [figure 7]. [2324] pet / ct image shows a recurrent rectal tumor as a hot spot (arrow) just anterior to the presacral fascia diffusion - weighted mri imaging: diffusion - weighted mri signal is derived from the motion of water molecules within cells or extracellular spaces . Highly cellular tumors have restricted water diffusion and show up as areas retaining high signal intensity on high b value (8001000 s / mm) images . By performing diffusion imaging using different b values, it is possible to calculate the apparent diffusion coefficient (adc, measured in m / s). Areas of restricted diffusion show low adc values and adc values are inversely correlated with tumor cellularity . Therefore, reduction in adc has been shown to have good correlation with response to cytotoxic therapy . Currently, diffusion imaging is mainly used for detection of abnormal cancerous tissue and involved lymph nodes . Studies have also been conducted on the detection of active inflammation in ibd patients using diffusion imaging [figure 8]. Diffusion - weighted mri image shows high signal in the cancer tissue (arrow) and involved lymph node in the mesorectum (short arrow) perfusion imaging: perfusion ct is a technique that integrates anatomic detail with assessment of vascular physiology . Analysis of tumor enhancement, tumor blood flow, blood volume, mean transit time, and permeability surface area product are possible . Perfusion ct is a reflection of angiogenesis in tumors, and therefore its main use is in the assessment of colorectal tumors and their response to treatment.
Methylmercury is efficiently accumulated through estuarine food webs, such that concentrations in fish are controlled by initial ch3hg assimilation at the base of the food web . Sources and pathways of ch3hg and hg accumulation in lower trophic levels are therefore fundamental to understanding hg cycling in estuaries and controlling hg levels in top predator fish . Estuarine sediments are a sink for hg and also a dominant site for methylation of hg to the more bioavailable species, ch3hg . Benthic organisms, which dwell at the sediment water interface, are exposed to hg from both sediments and the water column and are also a food source to larger invertebrates and fish . As such, they present an important link between hg bioaccumulation in benthic and pelagic food webs, yet dominant sources and pathways of hg uptake in benthic fauna are not well understood . Benthic species are exposed to dissolved metals from porewater and overlying water and also take up metals from dietary sources, which can originate in both the sediment and water column . Identifying the uptake and trophic transfer between sediment and water food sources and primary consumers is important to linking water and sediment quality data to bioaccumulation . An important benthic species along the east coast of north america is the estuarine amphipod, leptocheirus plumulosus, which is abundant over a wide range of sediment types and salinity and temperature conditions.l . Plumulosus is also used as a test organism to assess both acute and chronic toxicity of estuarine sediments . L. plumulosus are facultative feeders; they live in u - shaped burrows through which they filter water and ingest suspended particles and also roam the surface of the sediment and deposit feed, thus deriving their nutrition and exposure to contaminants from both the water column and sediments . Microcosm and modeling studies have shown dietary ingestion of organic matter, rather than uptake from the dissolved phase, to be the dominant process for ch3hg and hg accumulation in l. plumulosus(6,18) over a range of assimilation efficiencies (ae) and ingestion rates (ir), the relative contribution of food sources to body burden was modeled to be> 80% for ch3hg and 1060% for hg, although accumulation was determined in water with no appreciable dissolved organic carbon (doc), and uptake of both species from natural water is inversely correlated to doc concentrations . Ingestion pathways are ignored in toxicity testing, which assumes metal exposure is predominantly from sediment porewater . Biokinetic models of metal accumulation currently lack estimates of uptake from deposit vs suspension feeding . Feeding behavior in l. plumulosus is a critical piece of information to understanding hg accumulation and trophic transfer . In this study, we used enriched isotopic ch3hg and hg tracers in microcosm experiments to directly determine hg uptake pathways in l. plumulosus . Two experiments were conducted to study feeding pathways of ch3hg (experiment 1) and hg (experiment 2). This approach had several advantages: (1) using multiple enriched isotopes of hg allowed us to simultaneously decipher uptake from different pathways . (2) we could expose amphipods to ch3hg and hg at environmentally relevant levels . (3) unlike radiometric tracer studies, this technique enabled hg species transformation to be traced throughout the experiment . Because ae of ch3hg is much higher than that of hg, methylation of hg will increase availability to the biota . Methylation occurs in anoxic zones in sediment and has also been observed in algal cultures . If hg speciation is not monitored during incubation in uptake experiments, bioaccumulation of hg species may be misinterpreted . Enriched stable isotopes of hg species have been used as a powerful tool to study hg cycling and uptake into freshwater foodwebs . In this study, we applied the technique to identify trophic transfer of hg species from live phytoplankton in water and sediment compartments in estuarine benthic infauna . All solutions were made up using ultrapure water (> 18 m cm) produced by a purelabpluswater purifier (us filter, ma, usa). Enriched stable isotopes of hg (as hgo, hgo, and hgo) single isotope spikes of hg and hg were prepared by dissolution of hgo in hcl, followed by dilution to 400 mg / l in 5% hcl (fisher optima grade, pittsburgh, pa, usa). Solutions of ch3hgcl and ch3hgcl were synthesized by conversion of hgo to hgcl2 followed by reaction with methylcobalamin . Stock solutions were diluted to 50 mg / l of ch3hg and 5 mg / l of ch3hg in 0.5% acetic acid and 0.2% hcl (fisher optima). Isotopic abundances of natural hg and the enriched isotope spikes are given in the supporting information . The marine phytoplankton species, i. galbana, was used as the food source for the ch3hg and hg exposures of l. plumulosus . Cells of i. galbana were grown in a 19 l carboy of 20 ppt artificial seawater (instant ocean prepared in ultrapure water) to a density of 1.3 10 cells per milliliter and then reduced by centrifugation to 800 ml . The concentrated culture was then divided into four 200 ml solutions, and each algal solution was spiked with one isotopically enriched hg species . In experiment 1 (ch3hg uptake), the suspensions were tagged with 2.5 g ch3hg or ch3hg and in experiment 2 (hg uptake), with 34 g of hg or hg . The isotopically labeled cultures were allowed to equilibrate for 24 h and then spun down into pellets and the supernatant discarded . Aliquots (1 ml) of each cell suspension were removed and then centrifuged, and the resulting algal pellets were freeze - dried and weighed . The pellet and supernatant solutions (both n = 4) were analyzed for ch3hg and thg, and the fraction of algal - bound hg was determined (94 1% for ch3hg, and 95 2% for ch3hg; 98.1 0.1% for hg and hg). Concentrations of hg spikes in the algal slurries are included in tables 1 and 2 . Leptocheirus plumulosus was purchased from aquatic research organisms (hampton, nh) and cultured according to epa method 1994 . Similarly sized (> 1 mm) amphipods were retrieved from culturing tanks prior to the experiment and depurated for 4 h in salt water . Microcosm experiments were conducted in 100 ml (5 cm diameter, 10 cm height) glass jars (qorpak, bridgeville, pa, usa). The organic carbon content was determined to be 3.2 0.1% by loi (dry sediment was heated at 550 c for 4 h). For each experiment, 16 identical microcosms were assembled . In each microcosm, 1 ml of algal suspension containing enriched isotope ch3hg (experiment 1) or hg (experiment 2) was mixed with 16 g of wet sediment, and the other enriched isotope - tagged algae was mixed with 60 ml of 20 ppt artificial seawater (table 2; henceforth isotopically enriched hg species are denoted by a subscript (hgsed or hgwater) indicating the compartment to which the spiked algae was added). The sediment was added to a jar (1 cm depth), along with 5 amphipods, and overlying water was added . Microcosms were kept in an environmental chamber (20 c, 10:14 (l / d) h photoperiod), covered loosely with plastic to minimize evaporation, and slowly agitated on an orbital shaker (100 rpm) to maintain aeration and suspension of algae . A water sample was taken from four microcosms immediately after assembly (t = 0); then four microcosms were dismantled for analysis after 12, 24, 36, and 48 h. this two - day time series was chosen to monitor relative accumulation of hg from different sources in amphipods over time, and to minimize the effects of algal death and decay on bioavailability of ch3hg and hg associated with longer incubation periods . Changes in hg concentration and speciation were monitored in whole and filtered water and sediment throughout the time series . A second set of microcosms was assembled under the same conditions but using algae suspensions not spiked with hg species, to monitor dissolved oxygen (do), ph, and oxidation reduction potential (eh) throughout the experiment . Results are reported in the supporting information (si). To dismantle microcosms after each time point, a 15 ml aliquot was filtered through a 0.45 m poresize 25 mm syringe tip filter (fisherbrand), and a second 15 ml sample was collected unfiltered . Both filtered and whole water samples were acidified to 0.5% hcl (fisher optima). Amphipods from each microcosm were removed from the sediment by pasteur pipet and thoroughly rinsed in two sequential water baths and placed in a preweighed 15 ml glass vial (i - chem). Sediments were removed from the microcosm jars and placed in 60 ml pfa vials (sarstedt, nmbrecht, germany). Amphipod and sediment samples were freeze - dried; all concentrations are reported on a dry weight basis . All sample containers were double bagged and stored refrigerated in the dark until analysis . Freeze - dried amphipod samples were weighed and extracted in glass vials . To each vial, 1.5 ml 4 m hno3 was weighed, and samples were heated overnight at 60 c . A 50 l aliquot of each extract was transferred to a 40 ml amber glass vial with teflon - lined septa (brooks rand laboratories, seattle, usa). Samples (8 ml) of filtered and whole overlying water were weighed into 40 ml vials . Portions (0.5 g) of freeze - dried, homogenized sediment samples were leached with kbr / h2so4/cuso4, extracted into ch2cl2, then back extraction into 10 ml water; then 2 ml of the extract was weighed into 40 ml glass vials . Samples were buffered and derivatized, then analyzed using a merx - m methylmercury analysis system (brooks rand laboratories, seattle, usa) coupled with a 7700x agilent icp - ms (agilent technologies, santa clara, usa); the details of this technique are given in taylor et al . Isotope deconvolution was used to separate hg species derived from three sources: the ambient hg in the sample (determined from the hg signal) and hg inputs from two enriched hg - tagged algal spikes added to the water and sediment compartments . External calibration was performed using six standards (0.025 to 25 ng / l), and a secondary source calibration check was analyzed every 10 samples . Method detection limits (mdl) were 0.05 ng / g (sediment), 5 ng / g (amphipods), and 0.13 ng / l water . Recoveries for ch3hg were 85 13% (n = 2) in bcr-580 estuarine sediment (irmm, geel, belgium) and 106 10% (n = 3) in nist 2976 mussel; spike recoveries in water were 99 5% (n = 5). Quantification of total hg from biological tissues was determined from 4 m hno3 extracts . Aliquots (0.5 ml) of amphipod extracts were diluted 10 times by weight and analyzed directly by icp - ms (agilent 7700x), with a mdl of 20 ng / g . Portions of sediment samples (0.25 g) were weighed into 60 ml pfa tubes (sarstedt) and 5 ml of 45% hno3: 5% hcl v / v was added to each tube . Samples were digested in an open vessel microwave (mars xpress, cem corps, matthews, nc, usa) at 95 c for 1 h. digested samples were diluted to 50 ml with ultrapure (18 m) water, and weighed, then analyzed by icp - ms, with a mdl of 0.1 ng / g . Signal intensities were deconvoluted (si) prior to conversion to concentration by external calibration . Recovery of standard reference materials nist 2711a (n = 2) and nist 2976 (n = 3) were 80 1% and 115% 11%, respectively . Filtered and whole overlying water and supernatant samples from algal suspensions were analyzed by cold vapor - icp - ms, using an automated merx t purge and trap system (brooks rand) coupled with icp - ms based on epa method 1631 . Samples (8 ml overlying water or 0.4 ml supernatant) were weighed into 40 ml clear glass vials with teflon - lined septa (brooks rand) and diluted to 25 ml with 1% hcl . Samples were digested overnight in capped vials, by the addition of 0.1 ml freshly prepared brcl (0.16 g kbr was dissolved in 15 ml hcl, then 0.38 g kbro3 added and stirred for 1 h). To each sample, 0.1 ml sncl2 and 0.1 ml hydroxylamine (brooks rand) were added, then vials were recapped for analysis . Bioaccumulation factors (baf) were calculated for each uptake pathway to normalize body burden to different exposure concentrations . Body burdens of isotopically enriched hg from 36 and 48 h time points were divided by sediment concentrations in each microcosm; e.g., in experiment 1, baf for ch3hgsed was calculated as the concentration of ch3hgsed (ng / g) in amphipods relative to its concentration in sediment . Metal accumulation from ingestion has been described by the model in eq 1(40,41)1where the body burden of a metal species taken up from food, css, is calculated from the metal concentration in food (cf), the assimilation efficiency of the metal species (ae, unitless), the ingestion rate, ir (mg food / g body wt / d), the metal efflux rate constant following uptake from food, kef (1/d), and the growth rate constant, g (1/d). The equation was rearranged to calculate ir . Reported values of ae (ch3hg = 80%, hg = 6%), kef (ch3hg = 0.052 d, hg= 0.089 d), and g (0.07 d) for l. plumulosus were applied, and concentrations of isotopic tracers in algae cf and amphipods css were used to distinguish ingestion from benthic and pelagic sources . Nonparametric analyses were chosen because distribution could not be ascertained due to small sample sizes . Significant trends in spike concentrations in sediment, water, and amphipods with time were assessed by kruskal wallis rank tests; specific differences in concentration between time points were determined using the mann whitney rank sum statistic . Using the two enriched isotopes, ch3hg concentrations from spiked algae added simultaneously to the water column and sediment were tracked in the sediment, water column, and in amphipods over time (table 1). For sediment, differences in concentrations of both enriched isotope hg species were not significant between time points (kruskal wallis: d.f . = 3, p> 0.05), so data for all time points were pooled for each experiment . Only 0.2% of the recovered ch3hgwater was suspended in the water column after 12 h, whereas concentrations of ch3hgwater and ch3hgsed in the homogenized sediment samples were similar . Variation in ch3hg tracer levels occurred in the water column between time points (table 1). Levels of ch3hgwater in the initial whole water sample (t = 0) were elevated and variable between microcosms . Concentrations of ch3hgwater decreased from 0 to 24 h and then reached a steady state between 24 and 48 h (mann whitney u, p> 0.05). In the filtered water fraction, low levels of the ch3hgsed tracer were also present in the water compartment . Enriched isotope ch3hg tracers were present in amphipods after 12 h (figure 1), and concentrations of both tracers in amphipods did not vary significantly across time series (kruskal wallis, d.f . = 3, p> 0.05), although variance between replicates at each time point was large (1646%). Body burdens of isotopically enriched ch3hg tracers in l. plumulosus over time (experiment 1). The two enriched isotopes of hg were tracked over time in microcosms (table 2). As with experiment 1, both tracers were present in the bulk sediment, including 99.9% hg - tagged algae added to the water column . Concentrations of hg tracers in sediment did not vary with time (kruskal wallis d.f.= 3, p> 0.05), whereas tracer concentrations in the whole water fraction were variable . Whole water samples were elevated in hgwater at t = 0 (mann whitney u, z = 2.3, p = 0.02) immediately following the addition of spiked algae to the surface water but reached an apparent steady state from 24 to 48 h (p> 0.05). Concentrations of hgwater in the filtered water were also highly variable at t = 0 but did not change significantly from 0 to 48 h (kruskal wallis, d.f . Methylation of the hg spikes was evident during incubation of the i. galbana suspensions (table 2; 0.70.8% ch3hg). The% ch3hg of tracers in sediment increased slightly relative to the starting algal suspension (mann whitney u,% ch3hgsed: z = 2.5, p = 0.01;% ch3hgwater: z = 2.0, p = 0.04). The% ch3hg in the hgsed tracer, mixed with the sediment, was slightly higher than in the hgwater tracer initially added to the water column (mann whitney u, z = 2.2 p = 0.03), although variability in% ch3hg in tracers among microcosms was high (rsd = 5863%). In the whole and filtered water samples,% ch3hgsed was higher than in the algal suspensions (table 2) prior to addition to the sediment (mann whitney u, z = 2.5, p = 0.01). In the overlying water,% ch3hgsed was not significantly different than in the sediment (p> 0.05). There was no difference between% ch3hgwater in the water column and in the algal suspensions or sediments (p> 0.05). Neutral ph (8) and oxic conditions (do> 6 mg / l) in the sediment and water column were maintained over the time course under these experimental conditions (si). Body burdens of isotopic tracers in amphipods, as thg, ch3hg, and% ch3hg are shown throughout the time series in figure 2 . Ambient thg in amphipods (from hg) was 15 ng / g with 39 12% ch3hg (all time points; n = 16). At t = 12 h, body burdens of hgwater were higher than in later time points, even when one high value (1812 ng / g) was removed (mann whitney u, z = 2.3, p = 0.02). Conversely, increases in hgsed body burden were significant with time (figure 2a). (a) body burdens of isotopically enriched hg tracers, as thg concentrations . (b) ch3hg concentrations and% ch3hg in l. plumulosus over time (experiment 2). Letters (a, b, c for hgsed; or y, z for hgwater) signify statistical differences between time points by mann amphipods accumulated methylated forms of both tracers, comprising up to 18% of thg (figure 2b and c). Uptake of ch3hgwater was evident, but there was no relationship between accumulation and time (kruskal wallis, d.f . = 3, p> 0.05), whereas ch3hgsed body burden was significantly related with time . The trend was also significant when normalized to total hgsed (as% ch3hgsed). Because 99.8% ch3hgwater and 99.9% hgwater added to the water column were recovered in the bulk sediment, bafs for both enriched isotopes were calculated as body burdens normalized to sediment concentrations . The baf for ch3hgwater was 682 442 (unitless), whereas for ch3hgsed, the baf was 30 19, such that uptake of ch3hg from organic material of pelagic origin was 23 higher than from algae mixed with the sediment . For experiment 2, bafs for hg were 0.9 0.2 for hgsed and 5.8 2.0 for hgwater, and bioaccumulation from settling algae was only 6 higher than for sediment sources . For the ch3hg experiment, ir was determined to be 65 mg / g / day from ingestion of algae added to the water column and 3.1 mg / g / day for ingestion of algae mixed in with the sediment . For the hg experiment, ir was 30 mg / g / day for algae originating in the water column and 5.2 mg / g / day for sediment feeding . The ir is a measure of the milligrams of food consumed (per body wt / day) and should not vary with metal species, although its calculation does depend on the accuracy of the applied constants (ae, kef, g) as well as the experimentally derived food concentration and body burden . Microcosm studies using enriched stable isotopes showed trophic transfer from sedimenting algae was a major source of hg to benthic - dwelling amphipods . This suggests that newly deposited phytoplankton is an important and potentially overlooked source of hg to benthic infauna in ecosystem studies and toxicity testing . Enriched stable isotopes are a powerful tool for studying accumulation of hg in aquatic food webs; we applied this technique to a microcosm study to compare trophic transfer of hg by two ingestion pathways . Microcosms were scaled down from recommended conditions for culturing l. plumulosus, to minimize the quantity of enriched isotopes needed, but maintaining recommended amphipod densities (<1.5 cm), sediment depth (1 cm), and water conditions (20 ppt, 20 c, do> 4.4 mg concentrations of ch3hg and thg in sediments and whole surface waters in microcosms were within ranges found in new england and estuaries worldwide, although dissolved water concentrations were slightly higher . Body burdens of hg from tracers were up to 6 higher than those found in new england estuaries (chen, unpublished: 17105 ng / g). In estuaries, sediment characteristics and food quality have been shown to affect ae and kef in amphipods, as well as survival and reproduction, but specific effects on amphipod feeding behavior have not been reported . Because amphipods feed selectively, behavior may change with habitat and food availability . Accumulation of hg species from sediment mixed with algae was shown to be higher than from sediment alone, suggesting the abundance of high quality food spiked with hg in this experiment may lead to higher accumulation in amphipods than in a natural environment . Homogenized sediment and standardized conditions used in this experiment allow for direct comparison of metal accumulation from different feeding modes under optimal conditions . Algae added to the water column was rapidly deposited to the sediment surface, such that> 99.8% of ch3hg and hg were recovered from the bulk sediment within 12 h. concentrations of hg species added to the water column varied over time, due to settling of algae added to the water, and diffusion / suspension of tracers added to the sediment . Variability between samples was high at initial time points due to the capture of large suspended particles during sampling . While sediment concentrations were similar for both tracers added to the microcosm, spiked algae originating in the water column was deposited to the sediment surface, whereas the spikes originating in the sediment were thoroughly mixed in the bulk sediment . Tracking speciation throughout the experiment proved important as methylation of hg spikes was observed . In radiometric methods, hg species cannot be distinguished, and species transformation alters the bioavailability of hg, which could lead to inaccuracies in uptake modeling . Methylation of tracers was observed in spiked algal cultures (0.70.8%) and has previously been observed in cultures of periphyton . Further methylation was also evident during sediment incubation (23% ch3hg), despite maintaining oxic conditions throughout the experiment . This may also occur during toxicity testing, where sediment incubation conditions are similar to those used here, altering the bioavailability of hg to test organisms relative to natural environments . Because the experiment focused on uptake, a short exposure duration was used to minimize artifacts from algal decay, water quality changes, and amphipod death / reproduction . The body burden of ch3hg reaches an apparent steady state after 12 h in this experiment, although variability at each time point was high, possibly due to feeding behavior, and to variable metal distribution in the algal food source . Lawrence and mason reported an apparent steady state in amphipods sampled at three and six days, but calculated much longer exposures (50 day) are needed to achieve equilibrium . The apparent steady state in body burden may be due to the long time intervals (12 h) used in this study, relative to reported gut passage times of 35228 min for l. plumulosus fed i. galbana . Longer exposure to hg sources may lead to higher accumulation if a true steady state was not reached in this experiment, but body burdens represent relative accumulations from different feeding pathways . Because tracers were ingested simultaneously and prepared from the same algal source, ae and kef will be the same for both ingestion pathways . Amphipods in this study were not depurated, although for ch3hg, this is likely to have little effect on the determined body burden due to its high ae where rate of loss from excretion is negligible relative to growth dilution . Variability in body burden of hg tracers was high, likely due to spatial variability of tracers in both food sources . The body burden of hgsed increased significantly between time points, but hgwater did not . The pattern may be explained by preferential uptake of hgwater from settling algae during the first time interval (t = 0 to 12 h), then increased consumption of hgsed as amphipods stir up sediment while they burrow . This may be explained by the higher ae of ch3hg (80%) than hg (6%), causing hg to be excreted while ch3hg is mostly retained . This difference in assimilation should be consistent for both isotopic tracers, however, but was not significant for ch3hgwater . Uptake of hg tracers initially added to the water column and deposited on the sediment surface was significantly higher than for tracers mixed in the sediment, suggesting ingestion from the sediment surface is the major feeding mode in amphipods . While bafs are useful to normalize body burden to different exposure concentrations, they can be difficult to evaluate between different food sources and habitats . In this study, because the same algal food source was added to both compartments, bafs were used to compare relative uptake from different sources . For both spikes, bafs were determined using concentrations in amphipods relative to the sediment; this assumes that ingestion from the sediment surface or the bulk sediment were distinct uptake pathways and ignores accumulation from the water column . Conversely, if both sediment sources of ch3hg were assumed to have the same baf, then bafch3201hg+ (for bulk sediment) could be applied to the concentration of ch3hgwater in the sediment compartment, to predict an amphipod body burden of 45 ng / g ch3hgwater . To accumulate another 625 ng / g of ch3hgwater from the fraction of algae suspended in the water, a 5 mg amphipod would have to ingest 3.9 ng of ch3hgwater (assuming ae = 80%). Whole water ch3hgwater concentrations were 2.7 to 16.5 ng / l (12 to 48 h), meaning that only 0.15 to 1 ng of ch3hgwater was available for uptake from the entire in 60 ml water compartments . Similar calculations suggest amphipods would need to acquire 40 ng of hgwater from suspended algae to achieve determined body burdens, whereas only 1.54 ng of hgwater were present in the microcosms . Only a small portion of hg spikes was present in the dissolved water fraction . Lawrence and mason found an inverse effect of doc - binding on hg uptake and derived a laboratory - based relationship between doc concentration (mg / l) and water bioaccumulation factor (wbaf): log wbafch3hg = 1.740.173[doc], where wbafch3hg is the concentration of ch3hg in amphipods (ng / g wet wt) divided by the dissolved concentration of ch3hg in the water column (ng / l). Using this equation, wbafch3200hg was estimated to be 7.5 for a doc concentration of 5 mg / l measured in this study . For a dissolved ch3hg concentration of 2 ng / l (experiment 1; time points 12 to 48 h), amphipod concentrations are calculated to increase 100 ng / g (dry wt, assuming 15% dry / wet weight ratio), which is less than 15% of the determined ch3hgbody burden . Wbafhg2 + is reportedly 10 times lower than wbafch3hg, under the same conditions . Ng / l (experiment 2; time points 12 to 48 h), amphipod concentrations are calculated to increase 80 ng / g (dry wt), which is also 15% of the determined body burden . Uptake from both dissolved and suspended algae sources was therefore considered minor in this study, and while newly deposited algae may be considered part of the sediment, this study suggests accumulation was much higher from this new surface layer than from the bulk sediment . As expected, bafs for ch3hg were significantly higher than for hg . In phytoplankton, ch3hg accumulates predominantly in the cytoplasm of algal cells, whereas hg adheres to the cell membrane; the fraction of metal in the cytoplasm is attributed with higher ae during trophic transfer . Comparing the bioaccumulation of the two hg species, bafch3hg was relatively higher than bafhg for tracers originating in the water column than for sediment sources . Uptake of ch3hg into the cytoplasm of phytoplankton only occurs in live cells, whereas both hg species are bound to the cell membrane in dead cells, causing lower assimilation of ch3hg during trophic transfer . Accumulation of ch3hg in some invertebrates (copepods) was shown to decrease with decay of the marine algae, thalassiosira weissflogii; however, ch3hg assimilation in l. plumulosis was not affected, which is thought to be due to this species being well adapted to detrital feeding . Release of trace metals from decaying algae decreases exponentially with time and varies between elements . In this study, death and decay may have occurred more rapidly in the phytoplantkon mixed in the sediment, which would explain this relative difference, but the microcosm incubation time was relatively short (48 h), and algal decay has been shown not to affect ch3hg assimilation in amphipods . Although amphipods were rinsed, residual particle binding may lead to adsorption of hgsed to the amphipod exoskeleton elevating apparent body burdens from the sediment . Given that hg is strongly particle reactive, adsorption of hgsed to the exoskeleton may have been higher than for ch3hgsed, causing the relatively higher baf from sediment sources . To advance biokinetic modeling studies, ir for both feeding pathways of l. plumulosus were calculated . Values of ir for surface feeding determined here are in reasonable agreement with values for suspension feeding determined from five different metal species using radiometric techniques (50 to 151 mg / g / day). In this study and in williams et al ., ir was calculated using values of ae determined by radiometric techniques, which may have led to overestimation of ae for hg if methylation of hg spikes occurred during incubation . This would lead to underestimation of ir, which was lower for hg than for ch3hg in both studies . No other studies report ir from deposit feeding in amphipods, although shlekat et al . Reported a single measurement of 3 g / g / day, which is much higher than was observed here . This may reflect variation in feeding behaviors and effects of food substrate on test organisms . Phytoplankton rapidly accumulate ch3hg, but rapid algal growth has been shown to dilute hg in algal food sources and therefore decrease uptake into higher trophic levels . Deposition of phytoplankton blooms is a major source of organic carbon flux to sediment surfaces, and this study suggests deposited algae are a source of hg species to primary benthic infaunal consumers . In estuaries, increases in growth and reproduction of l. plumulosis have been correlated to chl a production associated with sedimenting algae . In a study of c - labeled tracers, however, phytodeposition was found to be a less prominent food source than microphytobenthos production for benthic fauna, including amphipods . However, the study also demonstrated that pelagic organic carbon sources were consumed during pulses which mimicked deposition of spring phytoplankton blooms . . Turbulence and resuspension of sediment can increase transfer of sediment ch3hg into organisms, and sedimenting algal blooms have also been shown to increase ch3hg production in surface sediments . Previously, bioavailability of hg species in estuaries has been inversely correlated with sediment organic matter . This study suggests hg in suspended and freshly deposited algal cells are an important uptake source, and that newly deposited algal particles should be distinguished from bulk sediment organic carbon as compartments controlling hg accumulation . In field collection, bulk sediments are typically collected from the top 4 to 15 cm . Field collection of amphipods is reported within the top 57 cm of the sediment, but imaging of amphipod burrows in sediment incubations show the highest burrow volume within the top 1 cm of sediment . Our findings suggest that amphipods feed primarily from the sediment surface, suggesting much smaller sampling depths (<1 cm) are appropriate for assessing hg sources to benthic infauna . L. plumulosus are routinely used in both acute and chronic toxicity tests for estuarine sediments . In acute (10 d) tests, sediments are the only food source, whereas during 28 day chronic toxicity tests, l. plumulosus are fed by an external uncontaminated food source 23 times per week . If in natural systems, algae originating in the water column are a preferred food source, these tests do not accurately assess exposure to contamination, as both tests overlook the influence of contamination originating in the water column, and acute toxicity tests ignore ingestion pathways completely . Bioaccumulation varies with feeding behavior, suggesting uptake pathways in test organisms need careful investigation to relate to sediment and water quality criteria.
Spinal epidural abscess (sea) is a relatively rare disease but frequently, emergent surgery is needed2,9,12,18). Their incidence is on the rise because of the various factors such as the increase of the elderly population, intravenous drug use, epidural steroid injection for pain control, epidural anesthesia and development of imaging methods such as computed tomography (ct) and magnetic resonance imaging (mri). With the development of diagnostic radiological evaluations such as ct or mri, rapid and accurate diagnosis has been available, leading to the significant improvement of mortality and morbidity . However, neurological complete recovery rate and mortality were reported to be still 16% and 41 - 47%, respectively despite appropriate treatments4,15). The aim of this study is to describe the clinical characteristics of 35 patients with sea and to disclose the risk factors, treatments and neurologic outcomes . This study was conducted on 35 patients who had been diagnosed with sea at the department of neurosurgery in our hospital for 24 years from april 1987 to april 2011 . We excluded osteomyelitis or discitis without true epidural collection and sea due to tuberculous spondylitis . We retrospectively reviewed the following data from the medical records including age, gender, clinical manifestation, laboratory data, imaging finding such as ct and mri and neurological outcome data at the time of discharge . The patients who underwent either emergent operation within 24 hours or delayed surgery after 24 hours, or conservative management were dealt case by case . Emergency surgeries within 24 hours were conducted when patients had neurological deficit or neurological deterioration and 16 patients received antibiotics treatment alone without surgery . Neurological outcome and clinical outcome were classified by comparing the mrc scale at the time of diagnosis and at the time of discharge . To elucidate improved to worsened outcomes of patients by treatment methods on admission to our department, there were 22 males (62.9%) and 13 females (37.1%). The presented symptoms were neck pain or back pain in 12 cases (34.3%), radiating pain in 22 cases (62.9%), and neurologic deficits such as motor weakness and sensory change in 28 cases (80%). Fever upon hospitalization was shown in 10 cases (28.6%), and the mean number of white blood cell (wbc) was shown to be 9,8863,776 cells / ml . The mean c - reactive protein (crp) was shown to be 4.944.05 mg / l, and the increased crp was shown in 19 cases (54.3%). Non - spinal infections were the most common cause of sea . Among them, paravertebral abscess was the utmost cause, and skin and soft tissue abscess was followed by it . In addition, risk factors were found in spinal procedure such as epidural injection for pain control, acupuncture and previous spine surgery after spinal trauma . Risk factors such as diabetes mellitus, liver disease, and cancer were also found . The location of epidural abscess collection was in the cervical spine in 4 patients, thoracic in 8, thoracolumbar in 3, lumbar in 19, and lumbosacral in 1 patient . The extents of spinal epidural abscesses ranged from 1 to 6 segments, and the mean was 2.7 segments . The spinal epidural collection was dorsal segment to the thecal sac in 4 cases, ventral segment to the thecal sac in 24 cases, and circumference to the thecal sac in 7 cases (table 2). Gadolinium enhanced mri was performed in 34 cases and ct was conducted in 1 case when mri was unavailable . Mri showed iso or low signal intensity in t1 weighted image (t1wi), whereas it exhibited high signal intensity in t2 weighted image (t2wi). After gadolinium administration, rim enhancement was found in 22 cases, whereas heterogeneous enhancement was found in 12 cases (fig . The initial mrc scale of patients was 1/5 in 5 patients, 2/5 in 1 patient, 3/5 in 3 patients, 4/5 in 18 patients, and 5/5 in 8 patients . And the post treatment mrc scale was 1/5 in 1 patient, 2/5 in 4 patients, 3/5 in 6 patients, 4/5 in 6 patients, and 5/5 in 18 patients (table 3). Once sea was diagnosed in the patients, an immediate treatment was initiated to all sea patients . Laminectomy, decompressive debridement, abscess drainage, or spinal fusion was performed in the patients with minor or severe neurologic deficit, and spinal fusion was performed in the patients with instability or spinal cord compression in the imaging study even if there were no neurologic deficit . Laminectomy was performed in 15 cases and laminectomy with spinal fusion was performed in 4 cases . Antibiotics alone therapy was performed in patients with no neurologic deficit and minimal spinal cord compression in the imaging study . Empirical antibiotics covering staphylococcus aureus was initially used and was changed with sensitive antibiotics according to the culture of abscess . Causative pathogens were identified in 13 cases (37.1%) in the abscess culture, of which staphylococcus aureus was found in 7 cases (53.8%). Streptococcus species was found in 2cases (15.3%), klebsiella, and burholderia cepacia were found in one case (7.6%) each . A positive result in the blood culture was shown in 8 cases (22.9%), and staphylococcus aureus was most frequently found . For the period of antibiotics use, an intravenous antibiotic was used for 4 - 8 weeks while monitoring wbc count, erythrocyte sedimentation rate (esr) and crp, and an oral antibiotic was additionally used in some patients for 2 - 3 weeks more . 14 cases (40%) showed improvement of mrc scale and 13 cases (37.1%) were unchanged . Nevertheless, mrc scale was worsened in 8 cases (22.9%). Of the 14 cases showing improvement, 10 patients underwent the emergent surgery within 24 hours and 3 patients had the delayed surgery over 24 hours and 1 patient received conservative treatment with antibiotics alone . Among 13 patients who showed no change in mrc scale, 1 patient underwent the emergency surgery, 2 patients experienced the delayed surgery and 10 patients underwent conservative treatment with antibiotics alone . Of the 8 patients who showed worsened neurologic deficit, no patient received emergency surgical treatment, 3 patients underwent delayed surgical treatment and 5 patients experienced antibiotic treatment (table 4). Between operative and conservative management, operation, regardless of emergency or delay, was more effective than conservative management to improve neurological deficit measured by mrc scale (table 5). Initially, patients with decreased mrc scale and rapidly progressing neurological deficit underwent emergency surgery within 24 hours . Patients with intact mrc scale and minor neurological deficit got delayed surgery over 24 hours or conservative management with antibiotics (table 6). Among 19 patients those who underwent emergent operation within 24 hours showed better prognoses than those who received delayed operation after 24 hours . 13 patients who had positive culture revealed more improvement of mrc scale than the patients with negative culture (p=0.009) (table 8). Sea mainly occurs in patients aged from 30 to 70 years old and a very rare disease with a prevalence of 1 - 2 persons/10,000 patients before 1990s . However, it has been recently increasing with a prevalence of 10 - 12 persons/10,000 patients due to increased elderly population, intravenous drug abuse, patient controlled analgesia, and spinal anesthesia for pain treatment as well as decreased immune response caused by various reasons1,10,22). In our series, a specific co - morbid condition with spinal epidural abscess was non - spinal infection in 19 cases (55%), spinal procedure in 6 cases (17%), diabetes mellitus in 1 case (3%), spinal trauma in 4 cases (11%), liver disease in 3 cases (8%), cancer in 1 case (3%) and no risk in 1 case (3%). Three main mechanisms of sea are as follows: first, results from hematogenous dissemination of distant infectious foci such as urinary tract infection, pneumonia, and laryngitis, account for the highest proportion . Second, infections are directly transmitted from adjacent structures such as vertebral osteomyelitis, spondylodiscitis, and perispinal mass abscess . Third, infections are transmitted via the hands such as spinal surgery, epidural injection therapy, and spinal anesthesia7,21). Staphylococcus aureus has been known to be the most common causative pathogen accounting for 42 - 84%, and streptococcal strains and gram - negative bacteria are followed by staphylococcus aureus4). In this study, staphylococcus aureus was shown to be the most frequently found causative pathogen . However, due to the early use of antibiotics prior to bacteria culture and insufficient tissue for bacteria culture, the unidentified causative pathogen and the negative result in the blood culture has been known to account for 24.4% and 50%, respectively6,8,19). In this study, the successful bacteria culture in the abscess and in the blood was shown in 13 cases (37.1%) and in 8 cases (22.9%), respectively . Sea most frequently occurs in the thoracic region, and then in the lumbar region, whereas it has a lowest prevalence in the cervical region . The higher prevalence of sea in the thoracic region is attributable to larger epidural fat tissue and space . The lower prevalence of sea in the cervical region is attributable to the limited epidural fat tissue and space7,11). In this study, sea was shown to occur in the lumbar regions in 19 cases, almost half of all the presented cases (54.2%). Mri with gadolinium enhance is the most commonly used radiologic diagnostic method in these days . Ct has an advantage of identifying small bone destruction or paraspinal abscess, but has disadvantages of difficulties in identifying the small amount of abscess and accurately assessing the spreading level of abscess into the adjacent area due to low contrast among the tissues11). In addition, the distribution of epidural abscess can be accurately identified via mri10,13,20). Typical findings include iso or low signal intensity in t1wi and heterogeneous high signal intensity in t2wi3). As mri has a disadvantage of a difficulty in assessing the change of the concurrent adjacent bone structure, simple x - ray or ct is conducted with mri to accurately assess the state of the lesions14). Treatment of sea is comprised of immediate decompression and surgical drainage, and appropriate administration of antibiotics before and after the surgery3,10,14). Through immediate decompression and surgical drainage, the neurologic deficit can be repaired as well as pathological diagnosis, and the identification of causative pathogens can be achieved15,20). Operation strategy should be determined according to abscess location, concurrent osteomyelitis, and vertebral body destruction . Laminectomy is the most commonly used in the case of abscess located in the posterior spine without lesions in the vertebral body . In addition, in the cases of compression rate of the vertebral body more than 50%, defected posterior spinal structure and the kyphotic angle of more than 20, internal fixation with autologous bone graft is required16,17). Because most infections are caused by staphylococcus aureus, empirical antibiotics covering staphylococcus aureus was initially used and was changed with sensitive antibiotics according to the sensitivity . The 4-week period of antibiotics use is generally recommended in the case of abscess without pyogenic spondylitis . The 8-week period of antibiotics use is recommended in the case of abscess with pyogenic spondylitis . Furthermore, an oral antibiotics is additionally used for extra 2 - 3 weeks while monitoring esr and crp4,8). In the past, a mortality of 100% was reported in the case of no surgical treatment, and a mortality of 32% was reported in the case of surgical treatment, which showed high morbidity and mortality11). At present, a neurologically complete recovery rate of 41 - 47% and a mortality of 16% are still reported even in the case of appropriate treatments4,15). In general, there were no statistical differences between groups in age, gender, location of abscess, number of involved vertebral levels or risk factors . Complete recovery can be expected in the cases of no paraplegia before operation or paraparesis period of 36 hours or less . However, neurological recovery cannot be achieved in the case of paraplegia of more than 48 hours as neurological paraplegia causes not only mechanical decompression, but also circulatory disturbance due to thrombosis caused by poor inflammatory response, thereby causing spinal neurologic deficit5,11,20). Our study suggests that neurological improvement in spinal epidural abscess is more easily achieved via decompressive laminectomy or abscess drainage than conservative treatment after neurological deterioration . Poor prognoses are expected in the cases of intraspinal high signal intensity in t1wi, thrombocytopenia, increased esr, increased crp or pancytopenia in hematologic test, pure abscess with no granulation tissue, and antibiotics treatment alone without surgical treatment4,6,8). In particular, prognoses are poorer in the case of thoracic region than in the cases of cervical or lumbar region . This is likely to be attributable to rapid neurologic deficit due to narrow spinal subarachnoid space and poor circulation14). In this study, better recovery of neurologic deficit was shown in the patients with surgical treatment than in the patients with antibiotics treatment alone . Patients who had decreased mrc scale at diagnosis, had more incidence to receive surgery than who had intact mrc scale . We had decompressive surgery and abscess drainage to patients with decreased mrc scale at diagnosis if condition of patient was not excessively bad for operation . In the patients with surgery, better prognoses were shown in the patients with emergent surgery within 24 hours than in the patients with delayed operation after 24 hours . Each patient had its own reasons for treatment and as our study is retrospective, we could not compare with the other treatments . Thus, it is insufficient to conclude early surgery is better than the other treatments and it is the limitation of our study . However, good outcomes can be expected if decompression and surgical drainage are conducted immediately after diagnosing sea . And the improvement of neurological symptoms can be expected through emergency surgery particularly in the patients with neurological deficit with decreased mrc scale . Sea is a rare disorder that can cause neurological deterioration or death if it is not recognized or treated early . Therefore, sea should be considered in patients with back or radiating pain, motor weakness or sensory change . And if sea is diagnosed, although the patients have minor neurological deficit or no motor weakness, early surgical removal of sea should be considered . We can expect desirable outcomes through immediate surgical drainage and decompression when sea is diagnosed . In particular, satisfactory outcomes can be achieved from emergent surgery within 24 hours from diagnosis of sea even if neurological deficit is observed at the time of diagnosis.
We used data from the 2002 china national nutrition and health survey (cnnhs), which is a nationally representative cross - sectional study on nutrition and chronic diseases . As has been described in detail previously (8), a stratified, multistage probability cluster sampling design was used in this survey . Subjects were categorized into five exposure cohorts according to the subject s birthday and corresponding exposure period (8). To minimize misclassification of the famine exposure periods, subjects who were born between 1 october 1958 and 30 september 1959 or between 1 october 1961 and 30 september 1962 were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions . Adults who were born between 1 october 1962 and 30 september 1964 were classified as nonexposed cohort . Subjects who were born between 1 october 1959 and 30 september 1961 were classified as fetal - exposed cohort whose mother was exposed to famine during the whole gestation period . Subjects who were born between 1 october 1952 and 30 september 1958 were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . As previously described, excess death rate of each province was used to determine the severity of the famine (8,9). The excess death rate was calculated as the percent change in mortality rate from the mean level in 19561958 to the highest value during the period of 19591961 . The excess death rate of 100% was used as the threshold: regions that had equal or higher rate than this cutoff were categorized as severely affected famine areas, and otherwise as less severely affected famine areas . Accordingly, all five birth cohorts were divided into severely affected and less severely affected famine areas . The protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety, chinese center for disease control and prevention . Signed consent forms were obtained from all participants . Fasting plasma glucose (fpg) concentration was measured using glucose oxidize enzymatic method within 3 h of plasma preparation (8). Plasma triglyceride and hdl cholesterol were measured enzymatically with a hitachi 7060, 7180 auto - analyzer (hitachi, tokyo, japan). Subjects seated blood pressures were measured on the right arm after 5 min of rest to the nearest 2 mmhg according to 1999 world health organization / international society of hypertension guidelines on hypertension (15). The waist circumference was measured to the nearest 0.1 cm at the midpoint between the bottom of the rib cage and the top of the iliac crest at the end of exhalation . The atp iii criteria (16) were used for the definition of metabolic syndrome, as three or more of the following variables and cutoff points; 1) fasting triglyceride: 1.69 mmol / l (150 mg / dl); 2) hdl cholesterol: men <1.04 mmol / l (40 mg / dl), women <1.29 mmol / l (50 mg/ dl); 3) fasting glucose: 5.5 mmol / l (100 mg / dl) (17); 4) waist circumference: men> 102 cm, women> 88 cm; 5) systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg . Dietary patterns and bmi measured in 2002 were used as measures of the nutritional environment in adulthood . A validated semiquantitative food frequency questionnaire (ffq) the method for assessing dietary patterns has been described in detail elsewhere (18). Briefly, two major dietary patterns, the traditional dietary pattern and western dietary pattern (8), were derived through cluster analysis of food consumption data collected by the ffq . We classified subjects as overweight if bmi 24 kg / m, or normal otherwise, using criteria recommended for chinese adults . Risks of metabolic syndrome among fetal- and childhood famine - exposed subjects, compared with nonexposed subjects, were examined with the method of maximum likelihood by using a survey logistic regression model . Interactions between famine exposure cohort (fetal or childhood exposed vs. nonexposed) and area (severely affected vs. less severely affected) were tested by adding a multiplicative factor in the survey logistic regression model . Analyses were adjusted for sex, economic status, family history of diabetes and hypertension, educational level, current smoking, alcohol use, and physical activity level, all assessed in 2002 . To explore whether the associations between fetal exposure to severe famine and metabolic syndrome were modified by improved nutritional environment in later life, we subsequently stratified the analyses by dietary patterns and bmi in adulthood . We conducted two sensitivity analyses: one applying lower cutoff points of waist circumference (men 90 cm and women 80 cm) (19) to the definition of the metabolic syndrome based on the asian criterion for abdominal obesity and the other excluding abdominal obesity from the definition of the metabolic syndrome (three or four of: elevated fasting triglyceride levels, lower hdl cholesterol levels, elevated fasting glucose levels, and high blood pressure). We used data from the 2002 china national nutrition and health survey (cnnhs), which is a nationally representative cross - sectional study on nutrition and chronic diseases . As has been described in detail previously (8), a stratified, multistage probability cluster sampling design was used in this survey . Subjects were categorized into five exposure cohorts according to the subject s birthday and corresponding exposure period (8). To minimize misclassification of the famine exposure periods, subjects who were born between 1 october 1958 and 30 september 1959 or between 1 october 1961 and 30 september 1962 were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions . Adults who were born between 1 october 1962 and 30 september 1964 were classified as nonexposed cohort . Subjects who were born between 1 october 1959 and 30 september 1961 were classified as fetal - exposed cohort whose mother was exposed to famine during the whole gestation period . Subjects who were born between 1 october 1952 and 30 september 1958 were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . As previously described, excess death rate of each province was used to determine the severity of the famine (8,9). The excess death rate was calculated as the percent change in mortality rate from the mean level in 19561958 to the highest value during the period of 19591961 . The excess death rate of 100% was used as the threshold: regions that had equal or higher rate than this cutoff were categorized as severely affected famine areas, and otherwise as less severely affected famine areas . Accordingly, all five birth cohorts were divided into severely affected and less severely affected famine areas . The protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety, chinese center for disease control and prevention . Signed consent forms were obtained from all participants . Fasting plasma glucose (fpg) concentration was measured using glucose oxidize enzymatic method within 3 h of plasma preparation (8). Plasma triglyceride and hdl cholesterol were measured enzymatically with a hitachi 7060, 7180 auto - analyzer (hitachi, tokyo, japan). Subjects seated blood pressures were measured on the right arm after 5 min of rest to the nearest 2 mmhg according to 1999 world health organization / international society of hypertension guidelines on hypertension (15). The waist circumference was measured to the nearest 0.1 cm at the midpoint between the bottom of the rib cage and the top of the iliac crest at the end of exhalation . The atp iii criteria (16) were used for the definition of metabolic syndrome, as three or more of the following variables and cutoff points; 1) fasting triglyceride: 1.69 mmol / l (150 mg / dl); 2) hdl cholesterol: men <1.04 mmol / l (40 mg / dl), women <1.29 mmol / l (50 mg/ dl); 3) fasting glucose: 5.5 mmol / l (100 mg / dl) (17); 4) waist circumference: men> 102 cm, women> 88 cm; 5) systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg . Dietary patterns and bmi measured in 2002 were used as measures of the nutritional environment in adulthood . A validated semiquantitative food frequency questionnaire (ffq) the method for assessing dietary patterns has been described in detail elsewhere (18). Briefly, two major dietary patterns, the traditional dietary pattern and western dietary pattern (8), were derived through cluster analysis of food consumption data collected by the ffq . We classified subjects as overweight if bmi 24 kg / m, or normal otherwise, using criteria recommended for chinese adults . Risks of metabolic syndrome among fetal- and childhood famine - exposed subjects, compared with nonexposed subjects, were examined with the method of maximum likelihood by using a survey logistic regression model . Interactions between famine exposure cohort (fetal or childhood exposed vs. nonexposed) and area (severely affected vs. less severely affected) were tested by adding a multiplicative factor in the survey logistic regression model . Analyses were adjusted for sex, economic status, family history of diabetes and hypertension, educational level, current smoking, alcohol use, and physical activity level, all assessed in 2002 . To explore whether the associations between fetal exposure to severe famine and metabolic syndrome were modified by improved nutritional environment in later life, we subsequently stratified the analyses by dietary patterns and bmi in adulthood . We conducted two sensitivity analyses: one applying lower cutoff points of waist circumference (men 90 cm and women 80 cm) (19) to the definition of the metabolic syndrome based on the asian criterion for abdominal obesity and the other excluding abdominal obesity from the definition of the metabolic syndrome (three or four of: elevated fasting triglyceride levels, lower hdl cholesterol levels, elevated fasting glucose levels, and high blood pressure). The prevalences of metabolic syndrome among adults in non-, fetal-, and early childhood, midchildhood-, and late childhood exposed cohorts were 5.7, 7.7, 8.4, 7.6, and 8.9%, respectively, in 2002 . Table 1 shows that in severely affected famine areas, adults who were exposed to the famine during fetal life had a higher risk of metabolic syndrome, as compared with nonexposed subjects (odds ratio [or] 3.13 [95% ci 1.247.89, p = 0.016]). No difference was observed in less severely affected famine areas (p for interaction between famine cohort and area = 0.016). Similar associations were observed among adults who were exposed to the famine during early childhood . As compared with the nonexposed subjects, the risks of metabolic syndrome among adults who were exposed to the famine during their early childhood were 2.85 (95% ci 1.196.83, p = 0.019) and 0.91 (95% ci 0.551.51, p = 0.72) in severely and less severely affected areas, respectively, with a significant interaction between famine cohort and area (p for interaction = 0.026) (table 1). Prevalence and ors of metabolic syndrome among different birth cohorts stratified by severity of chinese famine area* * metabolic syndrome was defined using criteria of atp iii as at least three of the following: 1) fasting triglycerides 1.69 mmol / l (150 mg / dl), 2) hdl <1.04 mmol / l (40 mg / dl) for men and <1.29 mmol / l (50 mg / dl) for women, 3) waist circumference> 102 cm for men and> 88 cm for women, 4) fasting glucose 5.5 mmol / l (100 mg / dl), and 5) blood pressure 130/85 mmhg . Ors were adjusted for sex, family income, family history of diabetes and hypertension, educational level, current smoking, alcohol use, physical activity level, and bmi . Results for the analyses stratified by adult dietary patterns and bmi for the associations of early famine exposure with metabolic syndrome are shown in fig . 1 . The fetal famine - exposed cohort with a western diet in later life had a particularly high prevalence of the metabolic syndrome (34.6%), whereas the prevalence among the fetal famine - exposed cohort with a traditional diet in later life was only 4.2% (fig . No significant difference of metabolic syndrome was found in less severely affected area (fig . Higher prevalences of metabolic syndrome were observed among overweight adults who were exposed to the chinese famine during their fetal life (22.6%) and early childhood (22.5%) as compared with nonexposed subjects (10.2%) (fig . The prevalences of metabolic syndrome were low among adults who were exposed to famine during early childhood (1.6%) and fetal period (2.3%) or were not exposed to famine (0.2%) (fig . Overweight adults had significantly higher risk of metabolic syndrome regardless of the severity and age period of the famine exposure (fig . In sensitivity analysis, applying lower cut points for waist circumference (men 90 cm and women 80 cm) to the definition of the metabolic syndrome (supplementary fig . 1) or excluding abdominal obesity from the definition of the metabolic syndrome (supplementary fig . Prevalence of metabolic syndrome among birth cohorts according to early life famine exposure and later life dietary patterns (1) and bmi (2) in severely (a) and less severely (b) affected famine areas . We found that exposure to chinese famine during fetal life and early childhood was associated with a higher risk of metabolic syndrome in later life . These associations were stronger in subjects who had a western dietary pattern and subjects who were overweight in adulthood . (12) and gluckman and hanson (13,20) suggested that environmental factors during fetal life and infancy play a role in the origin of the metabolic syndrome and its components . A mismatch between fetal and postnatal environments might exacerbate this effect . To support this hypothesis, the dutch famine study found that prenatal exposure to famine was associated with higher bmi, higher waist circumference, lower glucose tolerance, higher risk of coronary heart disease, more atherogenic lipid profile, and increased levels of plasma fibrinogen in later life (21). Men who had experienced the siege of leningrad (19411944) during early life had increased mortality from ischemic heart disease and stroke in their adulthood (22). Early life exposure to chinese famine (19591961) was also associated with the increased risk of overweight (9,10), hyperglycemia (8), and hypertension (3) in adult life . The current study provides further evidence that both fetal and infant exposure to severe famine increased the clustering of the metabolic risk factors that predispose a person to type 2 diabetes and cardiovascular disease . The association between fetal exposure to famine and higher risk of metabolic syndrome was exacerbated by a nutritionally rich environment in later life represented by western dietary pattern and overweight . This dietary pattern was characterized by high intake of meat, eggs, dairy, sugary beverages, and edible oils and a low intake of vegetables (18). Our results provide evidence to support the hypothesis that the mismatch between the early life starve environment and later life rich environment magnified the risk of metabolic diseases in later life . A potential limitation of this study is resident migration across different regions in china, which might lead to misclassification of famine exposure . However, the mobility of the population in rural china is relatively low because relocation of permanent residents needed to be approved by authorities . According to the 2000 china national population census, only 2.68% of the rural population lived in provinces other than their birthplaces (23). Therefore, we do not expect that intraprovince migration led to serious misclassification in famine exposure . Another limitation of our study is the lack of birth weight data, which precludes us from examining the interactions between birth weight and adult overweight or dietary patterns on risk of metabolic syndrome . Despite these limitations, the current study provided a unique opportunity to test the hypothesis of the joint association between early life famine exposure and later life environment with the risk of metabolic syndrome in adulthood . When compared with other famine studies, the chinese famine lasted much longer and affected more people . In conclusion, we found that exposure to severe famine in fetal life and infant period was associated with higher risk of metabolic syndrome in adulthood . A nutritionally rich environment, represented by a western dietary pattern and overweight, further strengthened the association . Our study indicates that both the early life environment and later life overnutrition are critical for the cardiometabolic health in adult life.
With an annual death toll of more than one million people, the tropical disease malaria is considered one of the most significant infectious diseases worldwide . Malaria is caused by protozoan parasites of the genus plasmodium and transmitted by blood - feeding anopheline mosquitoes . During their life cycle, plasmodia alternate between the human host and the insect vector, and thus the transmission stages of the parasite had to develop mechanisms for rapid adaptation to the new environment in order to coexist with the respective host . Like most apicomplexan parasites, plasmodia further switch between tissue - specific multiplication cycles and a phase of sexual reproduction, which mediates the transition from the human to the mosquito and thus plays a crucial part in the spread of the disease . The malaria sexual phase begins with the differentiation of gametocytes in human erythrocytes, followed by their uptake during the blood meal of the mosquito and the formation of gametes within the insect midgut . The transformation of the fertilized zygote into the infective ookinete subsequently marks the end of the malaria sexual phase (reviewed in). Historically, scant research has been devoted to the malaria sexual stages, namely, gametocytes, gametes, and zygotes, since they neither contribute to the clinical picture of patients nor do they play a role for vector control . However, within the last two decades the dramatic increase of drug resistance in malaria parasites has forced researchers to broaden their consideration of tactics to combat the disease, including transmission blocking strategies aimed at the sexual stages . Such transmission blocking strategies, on the level of either drugs or vaccines, are designed to disrupt parasite reproduction and further development in the mosquito midgut, thus breaking the life cycle of the parasite . Research on the malaria transmission stages, however, was formerly hampered by cost- and time - consuming cultivation, as well as by the technically challenging infections of mosquitoes with parasites . This was particularly true for work on p. falciparum, the causative agent of malaria tropica . In recent years knowledge on the malaria sexual phase has benefited from a dramatic resurgence provided by proteomic, microarray, and annotation projects that arose out of the genome sequence projects for multiple malaria species (e.g., [27]). As a result, a number of new sexual stage antigens have been identified, and progress has been made in the identification and functional characterization of enzymes and regulatory proteins that are involved in gametocyte differentiation and fertilization (reviewed in). Nowadays, three main questions regarding the malaria sexual phase are in the focus of interest . (1) which are the mechanisms that cause a subset of erythrocytic parasites to enter the sexual stage pathway and to differentiate to gametocytes? . (2) how do gametocytes become activated within the mosquito midgut and how do they transform into gametes? . (3) in which way do the sexual stages interact with factors of the mosquito midgut? This review addresses the role of gametocytes during malaria transmission and particularly discusses the recent findings on gametocyte activation following entry of the mosquito midgut, as well as their egress from the host erythrocyte and transformation into gametes . Additional aspects of gametocytogenesis, sexual stage proteins, and malaria transmission can be found in other recent reviews [1, 811]. The gametocytes are the only stages within the life cycle of malaria parasites that are able to mediate the transition from the human host to the insect host . The development of asexual blood stage parasites to intraerythrocytic gametocytes, which is referred to as gametocytogenesis, starts approximately 715 days after the appearance of parasites in the human blood . It is not known to which degree gametocytes develop stochastically, with a small proportion of committed parasites leaving the asexual cycle and entering the sexual pathway, versus gametocytogenesis as a response to complex environmental signals during infection . Gametocytogenesis was previously shown to be influenced by different kinds of stress, including parasite density, anemia, host immune response or drug treatment (e.g., [1220]; reviewed in [8, 10]). Up to date, little is known about parasite genes that regulate gametocytogenesis, but it was observed that reduced levels of gametocytes in parasite cultures are often associated with the loss of genetic information following subtelomeric deletion in the right arm of chromosome 9 . While in most plasmodium species the sexual stages mature within less than two days, a time period of about 10 days is required for gametocyte development in the human malaria pathogen p. falciparum . Gametocyte maturation can be classified into five morphological stages (stages i v), and mature p. falciparum gametocytes show an eponymous falcipare form . In these stages, the host erythrocyte has conformed to the crescent shape of the parasite and is reduced to a small cytoplasmic hem . The intraerythrocytic gametocyte lies within the parasitophorous vacuole (pv) and is shielded from the erythrocyte cytoplasm by the pv membrane (pvm), which is located adjacent to the parasite plasma membrane (ppm) (figure 1(a)). Underneath the ppm is a typical gametocyte feature, the pellicular complex, which consists of a subpellicular membrane (spm) vacuole subtended by an array of longitudinally oriented microtubules . These structures probably give the gametocyte stability, and the electron - dense spm disappears during gametogenesis (figures 1(a), 1(b), 1(c), and 1(d)). Besides morphological changes, maturation of gametocytes also includes alterations on the molecular level in order to prepare the parasites for the rapid adaptation to the mosquito midgut . For instance, a large amount of mrna is transcribed and stored in the cytoplasm of female gametocytes, as shown for the transcripts of the sexual stage surface proteins pbs25 and pbs28 in the rodent malaria model p. berghei, which will be transcribed only in the mosquito vector (see below). Gender specificity becomes established in the schizont committed to gametocytogenesis, and the gender ratio is typically female - biased with one male for about five female gametocytes, depending on the respective parasite clone . This difference might be explained by the fact that one male gametocyte forms approximately eight microgametes, thus establishing a roughly 1: 1 ratio of micro- and macrogametes in the mosquito midgut, thereby leading to most efficient fertilization in a monoclonal infection [8, 28]. Recently, reece et al . Demonstrated in the mouse model that parasites were able to adjust the sex ratio according to parasite density and the number of parasite clones coinfecting the mammalian host . For instance, a less female - biased sex ratio would increase the probability of a successful fertilization of females of the respective clone, when competing with others [2932]. In contrast, in vitro studies on p. falciparum did not show an adjustment of sex ratio to gametocyte density . However, an impact of the sex ratio on the infection rate, depending on gametocyte density, was observed . The fact that single, haploid asexually replicating malaria parasites are able to develop into gametocytes of both sexes in the absence of sex chromosomes indicates that gametocyte gender determination is governed by differential gene expression . Gametocyte stages i to iv were reported to sequester in the bone marrow and spleen, while terminally differentiated stage v gametocytes are then released in the peripheral blood system [35, 36] and only become infectious to mosquitoes after a further two or three days of circulation [37, 38]. Bloodstream gametocytes might not be distributed homogeneously, as evidenced by a significant aggregation pattern observed in midgut smears of p. falciparum - fed mosquitoes, and it is an intriguing hypothesis that parasites increase the likelihood of fertilization in the mosquito midgut by promoting uptake in preformed complexes of gametocytes . While feeding on an infected human, the female mosquito takes up malaria gametocytes together with the blood meal . By entering the midgut, the parasites receive environmental signals, which indicate the switch from warm - blooded host to insect vector and which initiate the development of gametes . Such signals include a drop of temperature by approximately 5c, and the presence of the mosquito - derived molecule xanthurenic acid (xa), a byproduct of eye pigment synthesis [41, 42]. An additional signal reported to induce gametocyte activation is an increase of ph from 7.2 to about 8 [40, 43], but such a ph shift was later discussed to be an artificial inductor of exflagellation . While xa appears to initiate a number of signaling events in the parasite (see below), the quest for a receptor that binds xa was hitherto unsuccessful . Gametocyte activation is routinely measured by the formation of exflagellation centers, although a time period of almost 15 minutes lies between these two events . This is due to the fact that exflagellation can easily be observed under the light microscope and quantified by counting of exflagellation centers . Exflagellation is the process when the activated male microgametocyte forms motile flagellar microgametes, which detach from the residual body by binding to erythrocytes (see below). Two previous studies showed that induction of exflagellation involves a fast increase in intracellular calcium and cgmp [45, 46]. An initial benchmark in elucidating sexual stage signaling was the identification of two guanylyl cyclases (gc and gc) as integral membrane proteins in p. falciparum, which are activated by addition of xa (figure 2). Noteworthy, the subsequent disruption of the gc ortholog in p. berghei resulted in normal exflagellation, but motility - impaired ookinetes, indicating that the role of gc is not essential for gametocyte activation . The increase of cgmp triggers the activation of a cgmp - dependent protein kinase, pkg . Activation of pkg leads to rounding up of the gametocyte, a process that appears to be independent from calcium increase . Gametocyte exflagellation further involves the presence of the second messengers diacylglycerol and inositol triphosphate (ip3), hydrolysis products of phospholipase c activity . The latter eventually mediates the release of intracellular calcium from the endoplasmic reticulum (er) (figure 2). It is not yet known how the signaling pathway involving ip3 and calcium release and the pathway involving cgmp and pkg activation are linked together, and whether pkg has an additional effect on calcium release from the er (figure 2). Current data suggest that at least three effector pathways exist (discussed in): (1) a pkg - dependent, calcium - independent pathway that mediates rounding up of the activated gametocytes, (2) a calcium - dependent pathway that initiates microgamete formation, and (3) a calcium - dependent pathway that regulates emergence of activated gametocytes of both genders . Following uptake by the mosquito, both male and female gametocytes round up and then escape from the enveloping erythrocytes within about 10 minutes postactivation (figures 1(b), 1(c), and 1(d)). In this period the microgametocyte replicates its genome three times in order to produce eight motile microgametes (reviewed in [1, 9]). Egress of the activated gametocyte from the host erythrocyte has been linked to the presence of osmiophilic bodies, gametocyte - specific secretory organelles that were first identified by electron microscopy due to their electron - dense features (figure 1(b)) [24, 52]. They appear first in stage iv gametocytes and are particularly present in the female sexual stages . The osmiophilic bodies migrate to the ppm during activation and disappear within a few minutes post - activation, coevally with the rupture of the pvm (figures 1(b), 1(c), and 1(d)) (g. pradel, unpublished observations). Osmiophilic bodies contain a gametocyte - specific and highly hydrophilic protein, pfg377, which is considered a marker for these organelles [53, 54]. Gene - disruption studies showed that female p. falciparum gametocytes lacking this protein reveal a reduced number of osmiophilic bodies and fail to egress from the host erythrocyte, pointing to a pivotal role of this protein in gametocyte emergence . Another protein, which was only recently identified, mdv-1/peg3, has also been implicated with gametocyte egress . In p. falciparum, expression was reported to be initiated in stage i gametocytes in association with all membranous structures of the pvm and to persist until gametocyte maturation [5658]. First gene disruption studies on p. falciparum described a reduced formation of particularly male gametocytes . Two subsequent studies, however, indicated a role of the protein post - activation of gametocytes.lal et al . Reported the presence of mdv-1/peg3 in p. berghei gametocytes of both sexes and a subsequent focal localization at the anterior pole of the developing ookinete . Studies on parasites in which the respective gene was knocked out resulted in reduced ookinete formation . A study by ponzi et al ., on the other hand, showed that p. berghei mdv-1/peg3 was associated with the gametocyte osmiophilic bodies . Gametocytes lacking this protein failed to egress from the host erythrocyte, thus resulting in reduced fertilization and ookinete formation . Therefore suggested that mdv-1/peg3 plays a major role in disrupting the pvm and the erythrocyte membrane (em). Independent from the life - cycle stage, host cell egress of malaria parasites involves rupture of two membranes, pvm and em . The time line of rupture, however, was recently object to several brisk discussions . Particularly two models are currently under investigation, the inside - out model, in which the pvm ruptures prior to the em, and the outside - in model, in which the em is degraded first [62, 63] (reviewed in). The timeline of parasite egress was hitherto mainly investigated in the asexual blood and liver stages, and no data are available for the egress of gametocytes . In the above suggested that mdv-1/peg3 is involved in pvm destabilization and that em rupture depends on the absence of the pvm . In accord with this hypothesis, new studies from our laboratory indicated that the pvm disappears within a few minutes after gametocyte activation, and that the rupture of the em follows several minutes later (g. pradel, unpublished observations), thus supporting the inside - out model of egress . The coming - out of malaria parasites from the host cell requires protease activity . A number of new studies engaged with the identification of proteases that mediate emergence of asexual blood stage merozoites . Data point to the involvement of the cytoskeleton - degrading malaria proteases falcipain-2 and plasmepsin ii . Particularly sera (serine - rich antigen) proteins, which were identified in the pv of blood stage schizonts (e.g., [65, 66]), are supposed to mediate pvm rupture . It was shown for sera-5 of p. falciparum that the protease is proteolytically activated by the serine - like subtilisin protease pfsub1 . While no detailed studies were yet performed on malaria gametocytes, it is worth mentioning that transcripts of select representatives of the above mentioned protease families are expressed in these stages, including falcipain-1, plasmepsin vi, sera-6, sera-7, and pfsub3 [68, 69]. A popular strategy in investigating protease activity during rupture is the treatment of parasites with protease type - specific inhibitors . Again, these studies were mostly performed on blood stage parasites, particularly using cysteine protease inhibitors like e64 . Treatment with this inhibitor, however, resulted to date in contradictory results, and it was reported that the inhibitor blocked either degradation of the pvm [62, 63] or rupture of the em . A similar egress study using protease inhibitors was recently performed on activated p. berghei gametocytes and showed that exflagellation can be blocked by the cysteine / serine protease inhibitors tpck and tlck . Treatment of activated gametocytes with tpck, tlck, pmsf, or two novel falcipain - targeting cysteine protease inhibitors during activation reduced the formation of microgametes . Furthermore, the aspartic protease inhibitor epnp appeared to interfere with rounding up of gametocytes . The exact modes of action for these proteases during gametocyte egress from its host cell remain to be investigated . Exflagellation is the process in which the newly formed microgametes adhere to neighboring erythrocytes, thus forming rosettes called exflagellation centers, and then detach from the residual body of the activated male microgametocyte . The exflagellating microgamete adheres to sialic acids and glycophorin a of the erythrocyte surface and this binding is probably mediated by pfs230, an abundantly expressed adhesion protein that is associated with the gamete surface . Interestingly, pfs230 is proteolytically processed during gametocyte activation, and this processing can be inhibited by the metalloprotease inhibitor 1,10-phenanthroline [75, 76]. The same inhibitor blocks exflagellation by leaving the microgamete amotile, and it is tempting to speculate that processing of pfs230 increases the adhesive properties of this protein, which are needed for the binding of the exflagellating microgamete to erythrocytes . This might be explained by the fact that sexual stage proteins can easily be disrupted because of their nonessentiality for parasite proliferation, and thus a functional characterization can be obtained by phenotype analysis of parasites, in which the respective genes have been knocked out . Genome annotation has revealed an extensive catalog of parasite - encoded kinases, the malaria kinome, with at least 86 hypothetical kinases identified in p. falciparum [77, 78]. An initial elegant study showed that the calcium - dependent protein kinase pbcdpk4 is involved in sexual stage signaling and regulation (figure 2). The kinase becomes activated by calcium increase following xa activation, resulting in genome replication in microgametocytes . In p. falciparum, pfcdpk4 was reported to be gametocyte - specific and activated by phospholipase c . In a subsequent step, the mitogen - activated protein kinase pbmap-2 controls formation of male gametes at the stage of cytokinesis [8183]. Downstream of these events, the protein kinases pbnek-2 and pbnek-4 trigger genome replication to the tetraploid level in the zygote stage [81, 84, 85]. Furthermore, pbcdpk3 is required for ookinete motility and engagement with the mosquito midgut epithelium [86, 87]. When highlighting these novel signaling pathways during gametogenesis, it has to be taken under consideration, however, that in some cases the results obtained for p. berghei and p. falciparum might differ . For example, a recent reverse genetics approach on the p. falciparum ortholog pfmap-2 pointed to an essential function of this kinase for the parasite asexual blood cycle, contradictory to the abovementioned results on pbmap-2 . This indicates that insights gained by studying the rodent malaria model p. berghei cannot be as easily applied to human malaria pathogens as has so far been assumed . Ingestion by the blood - feeding mosquito triggers molecular changes in the sexual stages of p. falciparum, with approximately 20% of stage - specific genes being activated during sexual stage development and parasite transmission [3, 5, 6]. This molecular switchover adjusts the gametocytes to the invertebrate host and on one hand initiates reproduction but on the other hand prepares the emerging gametes for the hostile environment of the mosquito midgut . Gametocyte development and gamete formation are particularly accompanied by the coordinated expression of numerous surface - associated proteins, including the egf domain - containing proteins pfs25 and pfs28, the cysteine motif - rich proteins pfs230 and pfs48/45, as well as the multiadhesion domain pfccp proteins . These proteins and their potential as transmission blocking targets were discussed previously and will therefore not be focus of this review . Noteworthy is that the majority of these surface proteins have adhesive properties and can be divided in two classes . One class of sexual stage proteins, including pfs230, pfs48/45, and the six pfccp proteins, is expressed within the pv of the developing gametocyte and subsequently present on the gamete surface, but expression of these proteins usually ceases during fertilization (figure 3). The expression of the second class of surface proteins starts at the time point of fertilization, as was shown for pfs25 and pfs28, and expression often persists until the ookinete has formed . The reason for this sudden onset of protein expression during fertilization is the translational repression of messenger rna encoding for these proteins . This was interalia shown for the repression of pbs25 and pbs28 by the p. berghei rna helicase dozi (development of zygote inhibited) as part of a ribonucleoprotein complex . The factor, however, is only translated in the ookinete stage, where it then activates a set of genes encoding for adhesion proteins important for midgut invasion . The reason for such a high number of adhesive proteins in the malaria parasite sexual stages remains elusive, but a new study from our laboratory might provide a first step towards answering this question . We showed that the six pfccp proteins, which are characterized by a high number of adhesion modules, assemble to form multiprotein complexes during their expression in the pv, and these complexes are subsequently present on the surface of the newly emerged macrogametes . Preliminary data point to an additional involvement of other surface - associated adhesion proteins in these complexes, like the transmembrane protein pfs48/45, which might link the complex to the gamete surface (s. scholz, a. kuehn, n. simon, and g. pradel, unpublished observations). We hypothesize that these protein complexes cover the macrogamete in the form of a sticky coat and that they are involved in important adhesive processes during malaria transmission to the mosquito . The complexes might play a role in promoting contact between the emerging gametes within the blood meal or in protecting the gametes from the aggressive environment of the mosquito midgut . Noteworthy, the gametes and zygotes are the only stages within the parasite's life cycle that, for more than one day, have to persevere outside a host cell . Here they are exposed to factors of the blood meal, including midgut bacteria and digestive enzymes, as well as components of the human immune system . This exposure results in an approximate 300-fold loss of parasite abundance during transmission to the mosquito, and the malaria transmission stages are therefore considered bottleneck stages of the parasite's life cycle . During exflagellation, the microgamete detaches from the residual body and is freely motile, moving via sinusoidal or helical waves . It is not known whether the microgamete meets the macrogamete by coincidence, whether it actively scans the blood meal, or whether it migrates along a gradient of an attractant that is released by the macrogamete . Interestingly, we recently identified filamentous protrusions of the p. falciparum gamete surface, which form immediately upon activation and which appear to establish long - distance contacts between parasites in the mosquito midgut (g. pradel, unpublished observations). These filaments fit the typical characteristics of socalled nanotubes, novel organelles that were recently described for a number of animal cells (reviewed in [95, 96]). It has been proposed that nanotube - like filaments can be formed by almost all cells serving as a medium for exploring the extracellular environment and therefore are likely to represent ancient features of unicellular eukaryotes . Nanotubes were reported to have a function in communication between cells, including calcium signaling and organelle transfer [95, 96]. We therefore hypothesize that the nanotubes of malaria gametes might be tools to facilitate association within the midgut in order to increase the chance of parasite mating . Once the microgamete adheres to a macrogamete, fertilization begins by fusion of the plasma membranes . Two recent studies on p. berghei described the identification of the microgamete protein gcs1 (generative cell specific 1), also termed hap2, which enables gamete fusion, and disruption of the respective gene results in male sterility and blocked fertilization [97, 98]. Gcs1/hap2 is a conserved protein of algae and plants, where it is involved in pollen tube guidance and seed formation [99, 100], and was also identified in protozoan parasites [97, 98, 101]. Importantly, gcs1/hap2 does not mediate the initial binding between the two mating partners, which appears to involve other adhesion proteins . Cell fusion is followed by nuclear fusion, and over the next 3 hours, meiosis occurs and the zygote becomes tetraploid . During the following 24 hours, the zygote transforms into the infective ookinete stage, thus marking the end of the malaria sexual phase . The ookinete is motile and possesses an apical complex which enables it to disrupt and traverse the midgut epithelium, before settling down between epithelium and basal lamina . Parasite tetraploidy persists throughout the ookinete stage until sporozoite budding in the oocyst restores the haploid state . Despite intense work on the sexual stages of malaria parasites, they represent the least understood stages of the parasite's life cycle . Gametocyte differentiation and gametogenesis have mostly been studied in the human malaria pathogen p. falciparum, and for these sexual stages, a variety of proteins have been identified and characterized . On the other hand, the implication of malaria sexual stage proteins for malaria transmission was preferentially investigated in the murine p. berghei model, which is more easily accessible for genetic manipulations and transmission studies . Up to date it is challenging to combine information gained by both systems to receive the big picture on the malaria sexual phase . We expect that in the near future research on the sexual stages of malaria parasites will be dominated by two major tasks: (i) the big hunt for the gene, which enables the blood stage parasite to enter the sexual pathway and (ii) the analysis of the molecular mechanisms and signaling events of sexual stage parasites during fertilization in the mosquito midgut.
Protein solvation determines to a large extent the physicochemical behavior of proteins, and it is therefore an essential part of their exerted function . In particular, complex formation, which is involved in almost all protein functions, depends on the detailed balance between solvent solute and solute solute forces . Too strong solvent solute forces would impair protein function, while too strong solute solute forces would lead to unspecific protein aggregation . Protein folds are generally stable under physiological conditions, but changes in the environment can induce unfolding and denaturation . Urea is among the most frequently used denaturants in studies of protein folding and stability . The effect of cosolvents on proteins has been studied experimentally and theoretically (for reviews, see refs (13)), but to date the molecular mechanisms and particularly the driving forces of urea - induced protein denaturation are not yet fully understood . Several theoretical studies using molecular dynamics (md) on single amino acids, peptides, and proteins have been performed to elucidate the atomic details of the interactions between urea and amino acids, peptides, or proteins . Most simulation studies focused on the energetic components governing the interaction between the proteic solute and the water urea solvent mixture . Different interaction mechanisms have been suggested, debating whether urea - induced denaturation is driven by polar interactions or by hydrophobic interactions and if these are induced by direct or indirect interactions with the solute atoms, but in the meantime there is sufficient experimental evidence to exclude a urea - induced structural change of water . A recent experimental study expands thermodynamic data to the spectrum of atoms occurring in proteins with the conclusion that urea interacts favorably, compared to water, with most atom types . A general consensus attributes a dominant role to van der waals interactions between urea and protein atoms over pure electrostatic contributions . The hydration shell model explained the solvation terms of small aliphatic hydrocarbons in urea water mixtures . An essential term of this model is an enthalpic contribution arising from the van der waals forces between the solute and the cosolvent urea . However, the model neglected the cavity formation, an integral element of solvent transfer models . Urea does not reduce the free energy of cavity formation; on the contrary, the strong interaction between urea and water increases the surface tension . Therefore, the replacement of water from the solvation shell around the hydrophobic solute seems not to contribute to the transfer free energy . Also, the hydrogen bond reorganization in the first solvation shell has only a compensatory energetic effect; i.e., enthalpic interaction of water with the protein surface is replaced by a gain of entropy in the bulk solvent . An alternative thermodynamic approach is the solvent exchange model that considers the solute surface as a collection of small interaction sites . This model is closer in spirit to molecular simulations, because (i) system trajectories provide direct information about the urea protein interactions and (ii) the model allows for the existence of heterogeneous sites; i.e., the heterogeneity of the protein surface may be accounted for by the formalism . A strong interest in protein unfolding and the principles underlying conformational transitions comes from observations that denatured cellular proteins tend to aggregate, a phenomenon that is often associated with a diseased cellular state . A prominent example of a potentially fatal condition caused by protein misfolding and aggregation is the prion disease that is associated with the formation of aggregates of the prion protein (prp). While the native structure of the globular c - terminus of the cellular prion protein (prp) is mostly helical, its amyloid fibrillar aggregates contain -rich conformers (prp). The details of the conformational transition as well as the structure of the fibrillar aggregates are still elusive . In a quest to determine a minimal set of structural elements that retain the aggregation propensity of the prion protein, the 36-residue fragment h2h3, comprising the helices h2 and h3, has been designed . As demonstrated by our previous studies, h2h3 shows fibrillation, gpi anchoring, and insoluble pk - resistant aggregate formation analogous to prp . We evaluate here the forces acting at the initial stage of urea - induced denaturation on three prp constructs, a stable structure and two h2h3 intermediates isolated from misfolding simulations of h2h3 in water . These two intermediates of h2h3 represent an -rich state h2h3 as an analogue of prp and a -rich state h2h3 as an analogue of prp . These two conformers have been previously extensively characterized by md simulations, and their role in the assembly of misfolded states has been evaluated . Besides being very different in their secondary structure content, they differ remarkably in their solvent exposure, with the h2h3 state exposing considerably more hydrophobic surface . H2h3 and h2h3 are therefore ideal systems to study the solvation effects . The solvent forces on h2h3 and h2h3 in pure water and in 4 m aqueous urea solution were analyzed . We have previously introduced shannon entropy maps and analyzed the hydration properties of water at the surface of prp, identifying particular loci with different entropies . By studying the effect of urea on folded and intermediate structures, we (i) isolate the urea effect on defined conformers from the unfolding process itself and by restraining the protein structure (ii) observe the competition of urea and water for protein interaction in the absence of protein dynamics . It is clear that urea denaturation is induced spontaneously, because protein surface atoms interact stronger with urea than with water . To our knowledge, no study has yet been directed to the role of solvent forces in the initiation of urea - induced unfolding . Here, the preference of the proteic solute to interact with either urea or water was computed on the basis of atomic force distributions . Shannon entropies of the solvent were computed to illustrate the replacement of water from the solvation shell . We compare our results of atomic solvation forces to thermodynamic data and observe good correlations, as well as general trends in hydrophobicity scales extracted from molecular dynamics simulations and based on energetical considerations . Three starting structures were used in this study: -rich h2h3 (h2h3), -rich h2h3 (h2h3), and the crystal structure of the c - terminal globular domain of prp (pdb: 1uw3). H2h3 is a truncated form of the prion protein comprising mainly helices h2 and h3 (residues 183218). H2h3 has been shown previously to undergo a conformational transition from an -rich (h2h3) to a -rich (h2h3) conformation . These two conformations were used here to compare forces on the same sequence in two different folds . The c - terminal globular domain of prp is used as a native reference protein for the analyses . The urea model of smith et al . Was used in its implementation as molecular building block' urea' in this force field . The temperature was set to 300 k and controlled by weak coupling to a temperature bath with a coupling constant t = 0.1 ps . The non - bonded pair list was updated every time step for pairs within 0.8 nm and every fifth time step for the range 0.81.4 nm . Twin - range cutoff radii of 0.8/1.4 nm were used to compute non - bonded interactions . Long - range electrostatic interactions were approximated by a reaction - field force, using a dielectric constant of 54 . Simulations were kept at 0.061020 kj mol nm (1 atm) with a coupling time of p = 0.5 ps and an isothermal compressibility of 5.575 10 (kj mol nm). Initial protein structures h2h3, h2h3, and 1uw3 were energy minimized using 100 steps of steepest descent . Energy minimized protein conformations were solvated in a periodic box of 5.2 edge length . The minimum solute systems were electrostatically neutralized by replacing water molecules with sodium ions to compensate the net charge (h2h3, 1; 1uw3, 2) of the protein at a neutral ph value . The neutralized systems were energy minimized using 100 steps of steepest descent, while the protein was harmonically positionally restrained using a force constant of 2.5 10 kj mol nm . The systems were run for 5 10 steps (1000 ps) of md while keeping the solute positionally constrained . Configurations, energies and forces were saved at intervals of 250 steps (0.5 ps), yielding 2000 conformations per trajectory . The final urea concentrations were curea(h2h3) = 3.8 m, curea(h2h3) = 4.5 m, and curea(1uw3) = 4.2 m. forces between the following groups of atoms were recorded: protein, ion, water, and urea . Two sets of simulations were performed, (i) in pure water (ii) and in a urea / water mixture . Since water equilibration around solutes occurs on the time scale of 1020 ps, explicit solvent simulations of 1000 ps are sufficiently long for the pure water simulations to sample representative force distributions . The urea / water systems equilibrated after about 5 ns, as shown by the radial distribution functions of urea and water in supporting information figure s1 . Simulations of these systems were run for 10 ns, and the last 3 ns were used for the analysis . Given that we estimate the equilibration to occur at approximately 5 ns, we considered 10 ns as sufficiently long to obtain accurate equilibrium solvent properties . The first 300 ps of the trajectories were excluded from analysis to remove potential equilibration effects . The contact coefficient is the contact ratio, the fraction of protein urea contacts, normalized by the mole ratio of urea:1where n is the number of contacts, m is the number of molecules, p: u are protein urea interactions, and p: s are protein(co)solvent (urea and water) interactions . Normalized force differences f were computed for protein atoms with at least one contact to urea via2where f denotes the mean force over a specified interval of the trajectory . Rescaled forces were computed for protein atoms with at least one contact to urea via3 statistical values were computed over the last 700 ps of the trajectories using the statistics functions of the gnu scientific library and plotted using the r - project software . The ability of prp to form fibrils is well recognized, but only recently has the neurotoxic activity been correlated with the formation of soluble oligomeric species . We have shown that oligomers from the h2h3 subdomain have very similar physicochemical charateristics to those of the entire ovine prp protein . The formation of these oligomeric species is accompanied by an increase in -sheet (h2h3) content . We have studied in detail this conformational transition in water by molecular dynamics simulations and have characterized the process of interconversion from an all -helical to a -rich conformer in water . After only 90 ns, a -sheet seed forms close to the disulfide bridge (nucleating at residues v183n184); later the elongation of this sheet resulted in the refolding of the entire structure to what we call a double--hairpin (first hairpin, v183t191/i208m216; second hairpin, v192t196/q199q203) (figure 1a, b). This conformer was very stable in solution and has been repetitively observed in simulations of oligomer - forming mutants . We therefore suspect that this is one of the -rich species in solution leading to oligomer formation . Transition from (a) the -rich h2h3 to (b) the -rich h2h3 conformation . (c) c - terminal globular domain of ovine prp (1uw3). The model systems illustrated in figure 1 were selected, because we assume that they are among the species present in the oligomerization pathway . The comparison of the organization of the urea mixture around these structures can reveal the tendency of denaturants to favor the species that is more prone to forming oligomers . By computing the solvent accessible surface area (sasa) calculated by pops and divided into hydrophilic and hydrophobic contributions (figure 2), one observes the exposure of backbone atoms in the transition from the -rich to the -rich conformer observed in pure water solution (hydrophobic sasa in yellow, hydrophilic sasa in red). This is intriguing for the study of solvation forces, because one of the postulated effects of urea - induced denaturation is strong interactions with backbone atoms that become exposed . The only region in which the -rich conformer exposes more hydrophobic surface is observed in the stretch 198204, which corresponds to the loop connecting the two helices . This loop becomes quite buried in the -rich conformer, forming a minimal core between the two aforementioned hairpins . Solvent - accessible surface areas (in) of the prion protein conformers h2h3 (blue) and h2h3 (red). The bars are divided into hydrophobic (light colors) and hydrophilic (dark colors) contributions . In denaturant - induced unfolding experiments, the number of denaturant molecules bound to the protein can be derived from the depression of the transition temperature, and thermodynamic parameters can broken down into increments per contact . In computational studies, this type of information is directly accessible if one defines a contact distance threshold (here 3.5) between protein atoms and (co)solvent atoms . The relative preference of protein surface atoms to bind to either the cosolvent (urea) or the solvent (water) can be expressed by the contact coefficient, which is the fraction of contacts to a given cosolvent normalized by the mole fraction of the cosolvent in the mixture . A contact coefficient above 1 indicates a favored contact to the cosolvent and below 1, a disfavored contact . In figure 3, the normalized force difference between the protein hydrophobic c atoms show the strongest preference for urea, and their force difference is positive; i.e., protein urea forces are stronger than protein water forces for hydrophobic atoms . The polar amide atoms n and o have a preference for urea contacts, but the interaction forces with urea are on average lower than those with water . Polar oh and charged o and n atoms show the highest preference and force differences for water . The normalized force differences between protein urea and protein water interactions in figure 3 follow a hydrophobicity scale . Urea and protein water interactions per atom type as a function of the contact coefficient . The linear fit through all data points (solid line) has a regression coefficient of r = 0.6, exclusion of the outlier solvent forces on individual protein atoms are cumulative; i.e., the recorded forces originate from interactions with one to several (co)solvent atoms and molecules . The effect of denaturants is concentration dependent, because the number of interactions and therefore the cumulative solvation forces increase with the mole ratio . The concentration effect can be compensated computationally by a scaling of the solvation forces with the contact ratio (eq 3). This creates a useful theoretical scenario, in which solvent and cosolvent form an equal number of contacts with the protein, and therefore the forces are represented on a symmetric basis . A detailed picture of this scenario is obtained from a plot of the force distributions . The quantile quantile (q q) plots in figure 4 provide a graphical summary of the differences between the force distributions . Q plots, while increased frequencies in one distribution bend the curve toward that dimension . The axes show the scaled forces fc of protein urea versus protein it is apparent that almost all atom types favor urea interactions compared to water interactions, with the exception of hydroxyl o and partially carboxylate o and amide n establish particularly strong forces with urea, while forces on amide o are moderate . The increased backbone exposure of h2h3 compared to h2h3 is notable in the relatively large forces on the backbone atoms amide n and amide o. the surface interaction potentials (in units 10 m) determined by guinn et al . However, the potentials aromatic c 8.9 and amide o 8.7 were not reproduced by our simulations, possibly because the exposure of these atoms was significantly higher in the model compounds of the thermodynamic study than in the protein folds used here . The mean forces (not scaled), contacts, and contact ratios are tabulated in table 1 . Interaction forces normalized by the contact ratio between h2h3 and urea (fcp: u) compared to h2h3 and water (fcp: w). Shown are the force distributions of selected types of atoms as q q plot . (a) h2h3, (b) h2h3, and (c) 1uw3 . Data points above the diagonal line indicate a preferred interaction with urea, below a preferred interaction with water . Fc: mean force per contact standard deviation in units kj mol nm . Entropic solvation effects are as important as the enthalpic force contributions discussed in the previous section . As urea replaces water in the first solvation shell, water gains entropy, but the reorganization of hydrogen bonds is an endothermic process . Therefore, the water replacement has been refuted as a driving force (gibbs energy change) of denaturation . The solvent entropy in simulated systems can be approximated by the shannon entropy h of the solvent . The atom occupancy within cells of a virtual grid were recorded along the simulation and transformed into an entropy hper grid cell (for details, see the methods). The orange distributions show the values of grid cells close to the protein surface (00.25 distance); the blue distributions show the values of the second solvation shell (0.50.75 distance). The differences between the orange and blue distributions illustrate that the h values are a sensitive measure of local atom fluctuations, although the resolution of the grid cells is limited (here 2.0) to provide a sufficiently diverse cell occupancy, because each additional atom contributes to the shannon term . Water mixtures (hugc) and pure water (hwgc) are given as q q plots in the right column of figure 5, the color code representing the same distance ranges as in the left column . The entropy distributions of the bulk are about the same for protein urea mixtures and pure water, with the exception of the lower entropy range of the h2h3 system . H2h3 shows a reduction, compared to the pure water system, of the number of water molecules in the urea / water mixture in the entropy range of 0.40.7, which indicates a loss of low entropy water molecules in the solvation shell of the protein . H2h3 exposes the largest fraction of hydrophobic surface area among the three prion conformers, and this entropic effect is most likely due to the replacement of water by urea at the protein surface . This effect is not detectable in the urea / water mixture systems of the helical proteins h2h3 and 1uw3 . However, all urea / water systems show an increase, compared to the pure water system, in the number of water molecules in the entropy range 0.71.2, which is caused by the presence of urea in the solvation shell and the concurring reduction of solvent mobility . Shannon entropy distributions of the solvent around prion conformers h2h3 (a, b), h2h3 (c, d), and 1uw3 (e, f). Distributions are divided into grid cells close to (00.25, orange) and far from (0.50.75, blue) the protein surface . Right: q q plots of the shannon entropy distributions of the solvent environment of water (hwgc) and urea (hcgc) atoms . Urea does not replace water molecules that are tightly bound to the protein surface and therefore create a low entropy environment . Urea replaces water molecules at protein surface locations where the bound solvent water fluctuates . The water urea shannon entropy maps are shown as isosurfaces in figure 6 for both h2h3 and h2h3 . These maps highlight the structural and dynamical properties of the solvent mixture around the molecules . High entropy solvents close to the protein surface are visible as locations where the contour map touches the protein . These are sites that are prone to (water) desolvation by exchange with cosolvents like urea that form stronger interactions with these sites . For the h2h3 -rich conformer, the loop between the two helices shows a high entropy environment, and urea molecules interact closely with the backbone . For both conformers, one can observe clustering of urea molecules at exposed amide n atoms (blue space - filling spheres), in agreement with observations in the atomic force analysis, where amide n atoms gave rise to the largest scaled forces in water urea mixtures . Shannon entropy of the solvent surrounding (a) h2h3 and (b) h2h3 illustrated as isosurface in wire mesh rendering . Solvation forces are a sensitive measure to gauge the solution properties of protein atoms in urea water mixtures . The individual preferences to interact with urea or water follow a hydrophobicity scale in a similar trend as force field energies of amino acid analogues . The comparison of the scaled forces showed largely agreement with data of surface interaction potentials . Particularly strong force differences were observed for carboxylate o and amide n atoms . The direct comparison of urea and water forces on the protein was performed on rescaled forces to compensate for the dependence of the solvation forces on the urea concentration . Solvent species appear in two forms: occupying a site and as a solution component . This has implications for the relative weights of the contributions to the solvation force of the (co)solvent components . In the direct comparison of the force distributions of the protein urea and protein water interactions (figure 4), the forces were scaled by the inverse of the contact ratio, but not additionally by the mole fraction as in the contact coefficient . Therefore the interacting solvent components were viewed as occupying a site, which reflects the fact that the relative concentrations of urea and water are different close to the protein surface compared to in solution, as has been shown by radial distribution functions here and in previous studies . The urea molecules close to the protein surface remained largely outside the first water solvation shell, except at locations where the solvent entropy is relatively high . It was noted earlier for the 1uw3 molecule in pure water that surface sites surrounded by high entropy water molecules are likely to be structural defects or interaction sites, because of the low energetic cost of desolvation . One of these sites, the loop between the two helices, is also an apparent interaction site in the h2h3 -rich conformer: it is surrounded by high entropy solvent, and urea molecules bind to the backbone amide n atoms . The use of other cosolvents like fluorinated derivatives in the stabilization of folded and unfolded species of prpc has been explored in experimental and theoretical approaches . Generally, a stabilization of helical structure is observed in these solvents . Hexafluoro-2-propanol has been shown to affect the ultrastructure of prp amyloid and to decrease the -sheet content as well as prion infectivity . In contrast, 1,1,1-trifluoro-2-propanol does not inactivate prion infectivity but alters the morphology of the rods and abolishes congo red binding . Protein simulations in urea water mixtures provide a rich source of information about solvation effects that can be used to detect potential structural defects and interaction sites . The solvent interaction forces of individual atom types could be converted to solvation parameters and embedded in an implicit solvation model for urea water mixtures . Another, more challenging, perspective is the combination of the observed surface site interactions in md simulations with a thermodynamic site model.
Melting point determinations were performed by the open capillary method and are reported uncorrected . H and c nmr chemical shifts () are reported in parts per million (ppm) relative to tms, and coupling constants j are reported to the nearest 0.1 hz . C, ch, ch2, or ch3c signals are assigned from dept-90 and 135 spectra . In a number of cases, carbon atoms attached to boron gave very broad peaks in c nmr spectra, and these could not always be distinguished . Hydrogen atoms attached to these carbons were not always observed in h nmr spectra . Low- and high - resolution mass spectra were recorded on a time - of - fight mass spectrometer using electron impact (ei). Ir spectra were recorded on a ft - ir spectrometer as a thin film (liquid samples) or applied as a solution in chloroform, and the chloroform was allowed to evaporate (solid samples). Gc determinations were performed using a gas chromatograph fitted with a zb-5 column (30 m, 0.32 mm inner diameter, 1.0 m film thickness). The carrier gas was he at 69.3 kpa, and a split injection mode was used . The oven temperature was increased from 70 to 260 c at 6 c min and then held for 4 min . Authentic samples of products were used to determine response factors relative to tetradecane, a known amount of which was added to reaction mixtures to allow quantification of product yields . A mixture of propiolic acid (8.79 ml, 0.143 mol) and aq hbr (48%, 40 ml) was heated at 95 c for 2.5 h, then left to cool . The crude product was filtered, washed with cold water (3 25 ml), and dried in air to give (e)-3-bromoacrylic acid as colorless needles (13.49 g, 63%); mp 120121.5 c (lit . A two - necked round - bottomed flask equipped with a magnetic stirrer bar, septum, and septum - capped dropping funnel was assembled hot and flushed with n2 . (e)-3-bromoacrylic acid (8.00 g, 53 mmol) was dissolved in dry diethyl ether (40 ml) and transferred via syringe to the dropping funnel . Powdered lialh4 (4.02 g, 106 mmol) was transferred quickly to the reaction flask, and dry diethyl ether (40 ml) was added . The reaction mixture was cooled in an ice bath, and the solution of 3-bromoacrylic acid was added dropwise via the dropping funnel with vigorous stirring . The mixture was stirred for 2 h, then water (2.5 ml) was added dropwise, followed by 15% naoh (5.25 ml) and more water (7.4 ml). The resulting salts were washed with diethyl ether (3 50 ml), and the combined extracts were dried over sodium sulfate and concentrated under reduced pressure to give fairly pure (e)-3-bromoprop-2-en-1-ol as a yellow liquid (4.16 g, 57%). A 50 ml round - bottomed flask equipped with a magnetic stirrer bar was charged with (e)-3-bromoprop-2-en-1-ol (3.52 g, 25.7 mmol) and cooled using an ice bath . Hexachloroacetone (7.79 ml, 51.4 mmol) was added and the solution stirred for 5 min . Powdered triphenylphosphine (6.74 g, 25.7 mmol) was added portionwise over a period of 25 min . The mixture was stirred for 2 h at 0 c, then fractionally distilled under reduced pressure to give 90% pure (e)-1-bromo-3-chloroprop-1-ene, which was fractionally distilled a second time to give pure (e)-1-bromo-3-chloroprop-1-ene (5) as a colorless liquid (0.52 g, 13%): h (500 mhz, cdcl3) 6.48 (1h, dt, j = 13.5, 1.1 hz), 6.34 (1h, dt, j = 13.5, 7.1 hz), 4.02 (2h, dd, j = 7.1, 1.1 hz); c (125 mhz, cdcl3) 133.1 (ch), 110.8 (ch), 43.6 (ch2). Boronate esters 3 where r = bn and 4-meobn were purchased from sigma - aldrich and tci, respectively, and used without further purification . Boronate esters 3 where r = bu, i - pr, and cyclohexyl were prepared from the reaction of their corresponding boronic acids with pinacol in pentane in 73, 36, and 32% yields, respectively . Boronate ester 3 where r = tert - bu was prepared by the literature method from pinacolborane and tert - bumgcl in 40% yield . Boronate ester 3 where r = thexyl was prepared by the method previously described from the monohydroboration of 2,3-dimethyl-2-butene and subsequent reaction with pinacol, while boronate esters 11 and 12 were also prepared as described previously . The procedure for preparation of 13 (r = c - hex, r = phch = ch) is typical . Characterization details of the other boronate esters 13 can be found in our previous publication . Dicyclohexylborane was prepared by the dropwise addition of cyclohexene (2.12 ml, 20.9 mmol) to borane dimethyl sulfide complex (10 m, 1.00 ml, 10.0 mmol). The mixture was stirred at room temperature for 2 h, dry thf (10 ml) was added, then the mixture was kept at 0 c during the dropwise addition of phenylacetylene (1.15 ml, 10.5 mmol). The mixture was stirred for 1 h at 0 c and 1 h at room temperature then recooled to 0 c . Dichloromethyl methyl ether (1.36 ml, 15.0 mmol) was added dropwise, followed by a freshly prepared solution of lithium triethylcarboxide (dropwise addition of n - buli (1.6 m in hexanes, 9.38 ml, 15.0 mmol) to a solution of 3-ethyl-3-pentanol (2.12 ml, 15.0 mmol) in thf (10 ml)) dropwise via a cannula over a 20 min period . The ice bath was removed and the mixture left to stir for a further 1 h. 2,2-dimethyl-1,3-propanediol (1.57 g, 15.0 mmol) in dry thf (5 ml) was added and the reaction mixture left to stir overnight at room temperature . The crude product following workup was separated by column chromatography on silica (petroleum ether) to give pure (e)-2-(1,1-dicyclohexyl-3-phenylallyl)-5,5-dimethyl-1,3,2-dioxaborinane (1.19 g, 20%) as cubic crystals: mp 188189 c; h (400 mhz, cdcl3) 7.40 (2h, d, j = 7.2 hz), 7.29 (2h, app t, j = 7.6 hz), 7.16 (1h, t, j = 7.3 hz), 6.36 (1h, d, j = 16.7 hz), 6.25 (1h, d, j = 16.7 hz), 3.65 (4h, s), 0.911.82 (22h, m), 1.01 (6h, s); c (125 mhz, cdcl3) (quat c next to boron not seen) 139.4 (quat c), 135.9 (ch), 129.0 (ch), 128.4 (ch), 126.2 (ch), 126.1 (ch), 71.9 (ch2), 41.1 (ch), 31.4 (quat c), 30.5 (ch2), 29.2 (ch2), 27.7 (ch2), 27.4 (ch2), 27.3 (ch2), 22.6 (ch3); b{h} (96.2 mhz, cdcl3) 29.3; hr - ei ms m / z (%) calcd for c26h39bo2 394.3043, found 394.3049 (m, 39%). A mixture of the appropriate boronic ester 3, 11, 12, or 13 (1 equiv), 1-bromo-3-chloroprop-1-ene (5, 1.1 equiv), and dry thf in a dry 50 ml round - bottomed flask equipped with a magnetic stirrer bar and septum was cooled to 78 c, and t - buli in hexanes (1.1 equiv) was added dropwise with vigorous stirring, and the mixture was stirred for an additional 30 min . The cooling bath was removed and the reaction mixture left to warm to 20 c over 3 h. an accurately weighed amount of tetradecane (0.2 ml) was added as a standard to enable gc determination of the amount of desired product formed following workup . The reaction mixture was cooled to 0 c, then oxidized by dropwise addition of excess naoh (0.6 g in 5 ml of h2o), followed by hydrogen peroxide (30% by weight, 3 ml). Once the initial exothermic reaction subsided, the cooling bath was removed and the mixture left to stir overnight at room temperature . Diethyl ether (10 ml) was added, the aqueous layer saturated with sodium chloride, and a small aliquot taken from the organic phase for gc analysis . The extract was washed with water (10 ml) and brine (10 ml), then dried over mgso4 to give a crude product containing the desired 3-alkylprop-1-en-3-ol 9 or 14, which was estimated by gc . In some cases (see below), mixtures were separated by column chromatography on silica (prewashed with 3% triethylamine in petroleum ether, petroleum ether / ethyl acetate 95:5 through 85:15) to provide the pure products . From 3 (r = thexyl) (0.564 g, 2.66 mmol), 5 (0.416 g, 2.68 mmol), thf (15 ml), and t - buli in hexanes (1.9 m, 1.41 ml, 2.68 mmol) (note that the excess of 5 and organolithium reagent was smaller in this example); colorless oil (0.105 g, 28%); h (400 mhz, cdcl3) 5.94 (1h, ddd, j = 17.2, 10.5, 6.7 hz), 5.23 (1h, d, j = 17.2 hz), 5.18 (1h, d, j = 10.5 hz), 4.01 (1h, d, j = 6.7 hz, choh), 1.73 (1h, app sept, j = 7.0 hz), 1.47 (1h, br s), 0.87 (3h, d, j = 6.9 hz), 0.850.82 (6h, m), 0.73 (3h, s); c (125 mhz, cdcl3) 138.5 (ch), 116.5 (ch2), 78.1 (ch), 39.6 (quat c), 32.9 (ch), 19.0 (ch3), 18.6 (ch3), 17.5 (ch3), 17.4 (ch3); hrms (ei) m / z calcd for c9h18o 124.1252, found 124.1252 (m h2o). From 13 (r = r = et) (0.335 g, 1.58 mmol), 5 (0.274 g, 1.76 mmol), dry thf (10 ml), and t - buli in hexanes (1.6 m, 1.09 ml, 1.74 mmol); colorless liquid (0.042 g, 17%); h (500 mhz, cdcl3) 6.02 (1h, ddd, j = 17.2, 10.5, 6.6 hz), 5.24 (1h, app dt, j = 17.2, 1.6 hz), 5.16 (1h, app dt, j = 10.5, 1.6 hz), 4.003.96 (1h, m), 1.411.28 (6h, m), 0.84 (9h, t, j = 7.6 hz); c (125 mhz, cdcl3) 138.8 (ch), 115.9 (ch), 78.4 (ch), 41.7 (quat c), 26.0 (ch2), 8.5 (ch3); hrms (ei) m / z calcd for c10h20o 156.1514, found 156.1509 (m). From 3 (r = bn) (0.280 g, 1.28 mmol), 5 (0.226 g, 1.45 mmol), dry thf (10 ml), and t - buli in hexanes (1.5 m, 0.97 ml, 1.46 mmol); colorless oil (0.097 g, 52%); h (400 mhz, cdcl3) 7.24 (2h, app t, j = 6.9 hz), 7.197.15 (3h, m), 5.86 (1h, ddd, j = 17.2, 10.5, 5.8 hz), 5.18 (1h, app dt, j = 17.2, 1.4 hz), 5.06 (1h, app dt, j = 10.5, 1.3 hz), 4.314.24 (1h, m), 2.82 (1h, dd, j = 13.6, 5.1 hz), 2.72 (1h, dd, j = 13.6, 8.0 hz), 1.64 (1h, br s); c (125 mhz, cdcl3) 140.2 (ch), 137.8 (quat c), 129.7 (ch), 128.6 (ch), 126.7 (ch), 115.1 (ch2), 73.8 (ch), 43.9 (ch2); lrms (ei) m / z 148 (m, 4%). From 4-methoxybenzylboronic acid pinacol ester (0.292 g, 1.18 mmol), 5 (0.210 g, 1.35 mmol), dry thf (10 ml), and t - buli in hexanes (1.5 m, 0.84 ml, 1.26 mmol); colorless oil (0.185 g, 52%); h (400 mhz, cdcl3) 7.15 (2h, d, j = 8.7 hz), 6.86 (2h, d, j = 8.7 hz), 5.93 (1h, ddd, j = 17.2, 10.5, 5.8 hz), 5.24 (1h, app dt, j = 17.2, 1.4 hz), 5.13 (1h, app dt, j = 10.5, 1.4 hz), 4.344.27 (1h, m), 3.79 (3h, s), 2.83 (1h, dd, j = 13.7, 5.1 hz), 2.73 (1h, dd, j = 13.7, 7.9 hz), 1.65 (1h, br s); c (125 mhz, cdcl3) 158.4 (quat c), 140.3 (ch), 130.5 (ch), 129.7 (quat c), 114.8 (ch2), 114.0 (ch), 73.7 (ch), 55.3 (ch3), 42.9 (ch2); lrms (ei) m / z 178 (m, 45%). An oven - dried 50 ml round - bottomed flask equipped with a magnetic stirrer bar and septum was flushed with n2 . Vinylmagnesium bromide solution in thf (0.7 m, 1.2 equiv) was added, and the mixture was cooled to 0 c . The appropriate aldehyde (1 equiv) was added dropwise, and the mixture was stirred for 15 min . Distilled water (10 ml) was added and the product extracted with diethyl ether (3 20 ml). The combined organic extracts were washed with water (2 10 ml), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the product . From vinylmgbr (0.7 m, 20 ml, 14 mmol) and valeraldehyde (1.24 ml, 11.7 mmol); light yellow liquid (0.73 g, 55%); h (400 mhz, cdcl3) 5.86 (1h, ddd, j = 17.2, 10.5, 6.3 hz), 5.21 (1h, app dt, j = 17.2, 1.4 hz), 5.09 (1h, app dt, j = 10.5, 1.4 hz), 4.08 (1h, app q, j = 6.7 hz), 1.77 (1h, br s), 1.601.44 (2h, m), 1.411.23 (4h, m), 0.89 (3h, app t, j = 7.2 hz); c (125 mhz, cdcl3) 141.5 (ch), 114.6 (ch2), 73.4 (ch), 36.9 (ch2), 27.6 (ch2), 22.8 (ch2), 14.1 (ch3). From vinylmgbr (0.7 m, 30 ml, 21 mmol) and isobutyraldehyde (1.60 ml, 17.5 mmol); colorless liquid (1.49 g, 85%); h (500 mhz, cdcl3) 5.86 (1h, ddd, j = 17.2, 10.5, 6.4 hz), 5.22 (1h, app dt, j = 17.2, 1.5 hz), 5.15 (1h, app dt, j = 10.5, 1.5 hz), 3.86 (1h, app t, j = 6.1 hz), 1.781.67 (1h, m), 1.58 (1h, br s), 0.93 (3h, d, j = 6.8 hz), 0.90 (3h, d, j = 6.8 hz); c (125 mhz, cdcl3) 139.7 (ch), 115.7 (ch2), 78.4 (ch), 33.8 (ch), 18.3 (ch3), 17.9 (ch3). From vinylmgbr (0.7 m, 10 ml, 7 mmol) and cyclohexanecarboxaldehyde (0.71 ml, 5.9 mmol); colorless liquid (0.50 g, 61%); h (500 mhz, cdcl3) 5.86 (1h, ddd, j = 17.2, 10.4, 6.6 hz), 5.20 (1h, app dt, j = 17.2, 1.6 hz), 5.14 (1h, app dt, j = 10.4, 1.6 hz), 3.84 (1h, app t, j = 6.4 hz), 1.881.81 (1h, m), 1.791.70 (2h, m), 1.701.62 (2h, m), 1.55 (1h, br s), 1.440.93 (6h, m); c (125 mhz, cdcl3) 140.0 (ch), 115.6 (ch2), 77.9 (ch), 43.7 (ch), 28.9 (ch2), 28.5 (ch2), 26.7 (ch2), 26.3 (ch2), 26.2 (ch2). Borane dimethyl sulfide (10 m, 1.94 ml, 19.4 mmol) was added to a 50 ml round - bottomed flask equipped with a stirrer bar and under n2 . The flask was cooled in an ice bath, and a solution of pinacol (2.29 g 19.4 mmol) in dry dichloromethane (10 ml) was added dropwise over a period of 10 min . The reaction mixture was stirred for an hour at 0 c and a further hour at room temperature before being transferred dropwise via cannula to a 100 ml round - bottomed flask equipped with a stirrer bar and a suspension of wilkinson s catalyst (0.16 g, 0.17 mmol, 1 mol%) in dry dichloromethane (5 ml). The reaction mixture was stirred for 5 min, then tert - butyl but-3-enoate (2.50 g, 17.6 mmol) was added dropwise over a period of 10 min . Hexane (20 ml) was added, the mixture was filtered, and the filtrate was concentrated to give the crude product (4.30 g, 91%), which consisted of boronic esters 15 and 16 (85:15 by relative integrations by h nmr). This was separated by column chromatography on silica (98:2 petroleum ether / ethyl acetate, followed by 95:5, 90:10, and finally 85:15) to give boronate ester 15 as a colorless oil (1.80 g, 50%): h (400 mhz, cdcl3) 2.21 (2h, t, j = 7.6 hz), 1.69 (2h, app pent, j = 7.7 hz), 1.43 (9h, s), 1.23 (12h, s), 0.79 (2h, t, j = 7.8 hz); c (125 mhz, cdcl3) 173.1 (quat c), 83.1 (quat c), 80.0 (quat c), 38.0 (ch2), 28.3 (ch3), 24.9 (ch3), 19.9 (ch2), 10.7 (br, ch2b); b{h} (96.2 mhz, cdcl3) 32.8; hr - ei ms m / z (%) calcd for c10h18bo3 197.1349, found 197.1353 (m oc(ch3)3, 41%). Tert - butyl-5-hydroxyhept-6-enoate (17, 96% yield by gc) was prepared under the standard conditions (see section on reactions of alkylboronic esters with 5 and tert - buli; general procedure) from boronate ester 15 (0.631 g, 2.34 mmol), 5 (0.423 g, 2.72 mmol), dry thf (10 ml), and t - buli in hexanes (1.5 m, 1.81 ml, 2.72 mmol), except that after oxidation of the organoboron compound with alkaline hydrogen peroxide the mixture was made acidic (ph 3) by addition of hcl, prior to saturation with sodium chloride and extraction . The crude product was separated by column chromatography on silica gel (90:10 petroleum ether / ethyl acetate, followed by 85:15) to give 17 as a colorless oil (0.183 g, 39%): h (400 mhz, cdcl3) 5.84 (1h, ddd, j = 17.2, 10.4, 6.2 hz), 5.21 (1h, dt, j = 17.2, 1.4 hz), 5.09 (1h, dt, j = 10.4, 1.4 hz), 4.09 (1h, app q, j = 6.6 hz), 2.24 (2h, app t, j = 7.5 hz), 2.01 (1h, br), 1.721.57 (2h, m), 1.591.48 (2h, m), 1.42 (12h, s); c (125 mhz, cdcl3) 173.1 (quat c), 140.9 (ch), 114.8 (ch2), 80.3 (quat c), 72.7 (ch), 36.3 (ch2), 35.2 (ch2), 28.1 (ch3), 20.8 (ch2); hr - ei ms m / z (%) calcd for c7h11o2 127.0759, found 127.0759 (m tert - butyl 5-hydroxyhept-6-enoate (17, 0.069 g, 0.345 mmol), dry benzene (10 ml), and p - toluenesulfonic acid monohydrate (0.018 g, 0.093 mmol) were added to a 25 ml round - bottomed flask equipped with a stirrer bar and a condenser . The reaction mixture was heated to reflux (bath temperature = 100 c) for 4 h. the reaction mixture was concentrated under reduced pressure, hexane (20 ml) was added, and the mixture was filtered . The filtrate was concentrated under reduced pressure to give lactone 18 as a light yellow oil (0.044 g, 100%): h (500 mhz, cdcl3) 5.86 (1h, ddd, j = 17.2, 10.6, 5.5 hz), 5.34 (1h, dt, j = 17.2, 1.4 hz), 5.23 (1h, dt, j = 10.6, 1.4 hz), 4.844.79 (1h, m), 2.622.55 (1h, m), 2.522.44 (1h, m), 2.021.81 (3h, m), 1.701.61 (1h, m); c (125 mhz, cdcl3) 171.2 (quat c), 136.1 (ch), 117.0 (ch2), 80.4 (ch), 29.7 (ch2), 28.0 (ch2), 18.2 (ch2); hr - ei ms m / z (%) calcd for c7h10o2 126.0681, found 126.0678 (m, 20%).
Survival rates continue to improve with the advent of more refined treatments and better supportive care . Today over 80% of children with cancer are alive for at least five years, and the majority of these are cured . As a consequence, many sufferers of childhood cancer are now living into adulthood and having families of their own . It has been estimated that about 1 in 900 adults aged 18 to 44 years is a cancer survivor . Among this group, those with an inherited susceptibility to cancer will transmit their genetic fault to a proportion of their children . They are also at risk of developing a second cancer during their adult life . While the causes of the majority of childhood cancers are largely unknown, there are a number of clinical syndromes for which the evidence for an excess cancer risk in children is most persuasive . These include li - fraumeni syndrome, neurofibromatosis type i, inherited retinoblastoma mutations, familial wilms tumor, and some disorders of dna repair . These syndromes collectively account for less than 10% of all childhood cancers, however . Beyond this, it is not clear if cancer in childhood increases the risk of cancer in relatives . A number of recent studies have described an association between childhood cancer and cancer in first - degree relatives [57], particularly siblings [5, 7] and mothers . In this study, we describe the distribution of cancers in australian children and estimate the risks of cancer in their relatives . It was established from the clinical services at the royal children's hospital (rch) and the monash medical centre (mmc), between which over 95% of children under 10 years of age and 83% of children aged 1015 years with cancer within the state of victoria, australia, are treated . Children were eligible for inclusion if they had a diagnosis of any cancer before age 15 and when initial treatment was given at one of the participating centres between october 1998 and october 2002 . The human research ethics committee at each participating institution reviewed and approved the protocol before enrolment commenced . A total of 486 (422 at rch and 64 at mmc) incident cases of childhood cancer were diagnosed during the study period . Of these, 21 families were found to be ineligible (seven non - english speaking parents, seven where the proband was aged over 15 years, six where the proband did not have their initial treatment at either service, and one where the proband was adopted). Of the remaining 465 families, 21 were not contactable, 53 refused to participate, 6 were lost to followup, and 6 later withdrew consent . This left 379 (82%) families participating (340 at rch and 39 at mmc), from which we interviewed 351 (92.6%) fathers and 371 (97.9%) mothers . In 343 (90.5%) families, both the mother and father were interviewed, while in 28 (7.4%) families only the mother was interviewed and in 8 (2.1%) families only the father was interviewed . At the time of enrolment, both parents of the affected child were asked a baseline questionnaire that included information on lifestyle and environmental risk factors and a detailed family history of cancer . Where half siblings were identified, the appropriate, biologically relevant family history was obtained . The family history of cancer questionnaire asked about all of the affected child's siblings, parents, aunts, uncles, and grandparents (i.e., all first - degree and second - degree relatives). Parents were asked to identify each first - degree and second - degree relative, any known cancer diagnoses with the age (in years) at diagnosis, and type of malignancy, together with their current age or age at the time of death . The information collected was reviewed and classified by the authors with expertise in cancer (jah; es). The world health organization's international classification of diseases for oncology, 3rd edition (id - o-3) scheme, was used for classification of site of malignancy . Nonmelanoma skin cancers, nonmalignant tumours, and in situ cancerswere not included in the analyses . To address the potential contribution of known familial cancer syndromes, we examined the medical records of the 26 affected children who had a first - degree relative with cancer . In particular, we sought clinical evidence for li - fraumeni syndrome, neurofibromatosis type 1, familial retinoblastoma, and familial wilm's tumour syndrome . Person - years at risk for the cohort of relatives were calculated as the time to date of diagnosis of cancer, date of death, or date of interview completion, whichever occurred earliest . Australian population - based cancer incidence data, specific for gender, age, and year of birth, were obtained from the australian institute of health and welfare . Standardised incidence ratios were used to compare the number of observed cancers in relatives with the number expected in the australian population . Robust estimates for confidence intervals were calculated to account for potential clustering within a family . All statistical tests were two - sided and a p value <0.05 was considered statistically significant . The distribution of childhood cancers diagnosed in the cohort of 379 children is summarized in table 1 . A family history for cancer was identified in 4,736 (1,337 first - degree and 3,399 second - degree) relatives with a total of 211,394 person - years of followup . Fourteen of the 379 (3.7%) families reported a positive history for childhood cancer in any relative, with none having more than one case identified . Twenty - six children with cancer (6.9%) had a first - degree relative (parent or sibling) with a history of cancer . The results for the family history of cancer in all childhood cancer patients are summarized in table 2 . There was a higher than expected, though not statistically significant, incidence of childhood cancer among first - degree relatives (sir 1.43; 95% ci 0.545.08). There was also a higher than expected, though not statistically significant, incidence of cancer among first - degree relatives (sir 1.45; 95% ci 0.932.1). There was a statistically significant increase in cancer among female first - degree relatives (sir 1.82; 95% ci 1.263.39). The increased family cancer history in first - degree females was largely attributable to an effect in mothers (sir 1.78; 95% ci 1.273.33) but was also observed in sisters (sir 2.15; n = 1). Elevated cancer incidence rates were also observed in aunts (sir 1.48; 95% ci 1.112.00). The gender - specific association was reflected in higher than expected incidence rates of breast cancer in mothers (sir 1.92; 95% ci 0.726.83), aunts (sir 1.64; 95% ci 0.982.94) and to a lesser extent grandmothers (sir 1.04; 95% ci 0.781.40). Although numbers were small, an increased cancer incidence in relatives appeared to be most apparent in the children with sarcomas (sir 2.58; 95% ci 0.998.52), embryonal tumors (sir 2.12; 95% ci 0.719.34), and brain tumors (sir 1.52; 95% ci 0.703.92). Of the 26 families with a history of cancer within a first - degree relative, only one met the criteria for a clinically recognizable familial cancer syndrome (li fraumeni syndrome). The increased rates of cancer in female first - degree relatives (sir 1.68; 95% ci 1.032.93), including mothers (sir 1.78; 95% ci 1.093.09) and aunts (sir 1.49; 95% ci 1.122.02), remained when this child and family were removed from the analyses . Our small, population - based study suggests an increased risk of childhood cancer and some adult cancers among relatives of childhood cancer patients that are not accounted for by clinically identifiable familial cancer syndromes . These findings were seen in both first- and second - degree relatives . Although some of our findings did not reach statistical significance, they are largely consistent with previously published unselected childhood cancer cohorts [57, 12] and add to a growing body of evidence that unidentified genetic risk exists in some families . In addition to the small size of the study, other limitations include the use of family history in isolation and the absence of some parental input to family history of cancer . While we were not able to cross - reference reported history of cancer with cancer registry data, a similar study in sweden confirmed underreporting of cancer in relatives by history . Furthermore, in the small minority of cases where both parents were not available to provide the relevant family history of cancer, underreporting of the family history of cancer is also likely . Allowing for these two study biases towards the null hypothesis is therefore likely to strengthen the positive findings we have reported . This underreporting may also explain why a large number of apparent protective effects occurred in second - degree relatives, where direct, personal access to a family history of cancer is not always possible . Overall, first - degree relatives (siblings and parents) had an increased incidence of childhood tumors . This is consistent with the higher occurrence of childhood cancers among siblings demonstrated in a study of 51,000 children who developed cancer under the age of 15 in the uk, even after those with a genetic etiology were excluded . It was also reported in a cohort of 13,703 childhood cancer survivors in north america (sir 1.5; 95% ci 1.351.7). The apparent association of childhood cancer with adult cancers in female relatives is fascinating and supports a recently published population - based study from utah, usa, where a higher risk of adult cancer was restricted to mothers and siblings (sir 1.31; 95% ci 1.111.56) but was not observed in fathers . It is interesting to note that the effect was greatest for children younger than 5 years at diagnosis (sir 1.48; 95% ci 1.131.95), suggesting a strong ante- or perinatal effect . Factors which may be related to maternal effects on childhood cancer risk include maternal age of pregnancies, altered in utero exposures, and birth weight . Our finding of an increased prevalence of breast cancer in female relatives also supports recently published swedish data . A number of related studies have also observed higher rates of breast cancer in mothers and sisters [1416]. The potential link between breast cancer and childhood cancer is of great interest, given past reports of increased rates of childhood cancer in families carrying a brca1 or brca2 mutation [17, 18]. Our study was not designed nor powered to examine the relationship between specific childhood cancer types and family history of cancer, so it must be acknowledged that potential associations may have gone undetected . Certainly, others have postulated a link between family history of cancer and the more common subtypes, childhood leukaemia, childhood lymphomas, and childhood brain tumours . The epidemiology of childhood cancer described here is the starting point for further explorations into identifying previously unrecognized genetic predisposition to childhood cancer . We are currently undertaking whole genome sequencing of these children with cancer and their affected first - degree relatives' germline dna in order to identify the role of previously described and novel genetic mutations to account for our findings.
Women of childbearing age appear to be particularly susceptible to the exacerbation of existing mental illness and the development of new mental illness [15]. Indeed, it has been estimated that over 500,000 pregnancies annually are complicated by psychiatric illness that either precedes pregnancy or arises during pregnancy . Untreated psychiatric disease during pregnancy is associated with increased risks for the mother (including self - harm / suicide, self - neglect, and reduced compliance with prenatal and postnatal care) and risks for the child (including impaired fetal development, infanticide, and impaired mother - child bonding) (reviewed in [7, 8]). Historically, antipsychotics have been extensively and effectively used for the treatment of schizophrenia and bipolar disorder, and more recently they are becoming part of the treatment of depression [912]. Conventional antipsychotics (also known as typical or first generation antipsychotics) which were more commonly used to treat these conditions caused a significant decrease in fertility . However, the newer atypical antipsychotics do not have this side - effect . As a consequence, the number of women taking antipsychotics, who are becoming pregnant, is on the rise . Indeed, appointments at a motherisk program clinic related to the use of antipsychotic medications increased 170% between 1989 and 2001; a rise which was for the most part attributable to an increased use of second - generation or atypical antipsychotics . The vast majority of women who use antipsychotics during pregnancy do so because of ongoing illness . In fact, only in cases of where the first schizophrenic episodes are reported in pregnancy or there is a risk of puerperal psychosis would the exposure of antipsychotic exposure be restricted to the pregnancy period . Functionally, typical antipsychotics exhibit high affinities for d2 receptors [15, 17] in the mesolimbic, mesocortical, and nigrostriatal dopamine pathways . This, rather nonspecific targeting of dopaminergic pathways, can result in a range of undesirable motor disorders . Atypical antipsychotics, however, are more selective for the d2 receptors in mesolimbic pathway as compared to those in the mesocortical and nigrostriatal pathways . In addition, they also target the serotonin receptor subtype 2a (5ht2a) which may help to reduce the negative side - effects associated with typical antipsychotics [19, 20]. It is important to note that the proper correlation between the ratio of the clinical dosage and d2 receptor affinity necessary to treat the symptoms of conditions such as schizophrenia has not been completely established . It is well understood that the suitability of antipsychotic medications during pregnancy is a balance between the risks of adverse obstetrical and neonatal outcomes and the risks associated with untreated or inadequately treated psychiatric illness . The most common atypical antipsychotics administered during pregnancy are olanzapine, clozapine, risperidone, quetiapine, and aripiprazole . Complicating the matter further is that almost 4057% of women taking atypical antipsychotics during are prescribed a combination of these drugs (polytherapy) [2325]. In general, current practice guidelines discourage changing medications during pregnancy as this may leave the patient on nontherapeutic doses during a period of time; a situation which is not in the best interests of the mother . Therefore, the usual standard of care dictates that dosages be increased or polytherapy be implemented . Although general clinical practice guidelines have been established (acog, 2008), there remains significant uncertainty regarding effects of these drugs on the fetus . To date, the teratogenic effects of antipsychotic exposure have received significant attention; however, the effects of these medications on long - term health outcomes of the offspring have not been well studied . In adults, one of the major side - effects of antipsychotic use is the dysregulation of body weight homeostasis (reviewed in [26, 27]). Similarly, maternal use of antipsychotics has been reported to result in aberrant fetal growth . Indeed maternal antipsychotic use has been reported to result in an increased incidence of both low and high birth weight relative to the general population [23, 24, 28, 29]. Since being either too small or too large at birth is a risk factor for the development of metabolic syndrome in postnatal life [3032], children exposed to antipsychotic medications in utero may be at increased risk of developing obesity in postnatal life . However, there are no human studies which have tested this hypothesis and the mechanism(s) by which atypical antipsychotics may affect fetal growth have yet to be elucidated . It is very difficult to decipher, based on the available literature, why atypical antipsychotics can cause both small for gestational age (sga) and large gestational age (lga) fetuses . For example, mckenna et al . Report an increased risk of sga in women taking the atypical antipsychotics olanzapine, risperidone, quetiapine, and clozapine . Report an increased risk of lga for women taking the atypical antipsychotics amisulpride, clozapine, olanzapine, quetiapine, and risperidone . Since the sample sizes in these studies are small for women taking each of the individual drugs, the risks associated with each drug cannot be accurately reported . Furthermore, the paucity of data evaluating the action of antipsychotics in pregnancy using animal models makes the discussion of mechanisms for these drugs difficult . It is well accepted that atypical antipsychotic use can alter body weight homeostasis in humans and nonpregnant rodent models, resulting in significant drug - induced weight gain and visceral fat accumulation [26, 27, 33], an effect which is more pronounced in females . The weight gain varies among the atypical antipsychotics, ranging from 2 to in excess of 25 kg [27, 35], and can affect up to 60% of patients after 312 months of use . Furthermore, there is now considerable evidence from animal experiments and clinical studies that the use of atypical antipsychotics is a major risk factor for impaired glucose homeostasis and type 2 diabetes in the nonpregnant population [26, 27, 3640]. However, the effects of atypical antipsychotics on gestational weight gain, postpartum weight retention, and gestational diabetes have not been systematically addressed; although, one study has reported that the use of antipsychotics during pregnancy significantly (or 1.78, 95% ci 1.043.01) increases the risk of developing gestational diabetes . In addition to metabolic consequences of antipsychotic medications, there are a number of other risk factors which may explain the increased incidence of diabetes in mentally ill patients including hereditary factors, poverty and poor access to good nutrition, physical inactivity, and antipsychotic medication use (reviewed in holt et al ., 2004). These changes in glucose homeostasis may explain, in part, the increased incidence of large for gestational age (lga) babies born to women taking atypical antipsychotics during pregnancy as lga is regarded as the most common fetal complication when women have gdm or preexisting diabetes [4346]. The mechanisms by which atypical antipsychotics can cause maternal obesity and dysglycemia are varied, and the relative contribution of each pathway to an aberrant metabolic state is not completely understood . Atypical antipsychotic - induced weight gain and fat accumulation may be the result of altered food intake (i.e., hyperphagia and decreased satiety) [4750], direct effects of the drug on adipocytes to alter lipogenesis and lipolysis in favour of lipid accumulation [5153], or effects on peripheral tissues to induce insulin resistance [51, 54, 55]. This insulin resistance in combination with increased gluconeogenesis [51, 54, 56] and impaired insulin secretion from the pancreatic beta cell [55, 57, 58] may in turn be responsible for the observed increase in type 2 diabetes with the use of atypical antipsychotics . However, there is a great deal of heterogeneity amongst the responses evoked by different antipsychotics . Indeed, while some evoke insulin release, others such as risperidone, ziprasidone, and quetiapine do not . The paucity of data using individual antipsychotics makes it difficult to conclude how these drugs induce such heterogeneous responses . Regardless of the mechanism, aberrant maternal glucose control and maternal obesity can have significant implications for the long - term health of both the mother and her child . There is compelling evidence that obesity in women, whether as a result of prepregnancy obesity, excessive gestational weight gain, and/or postpartum weight retention, is associated with an increased risk for many pregnancy - related health complications such as gestational diabetes and hypertensive disorders of pregnancy [60, 61]. In addition, maternal obesity also considerably increases the risk of fetal complications such as spontaneous abortion, stillbirth, congenital anomalies, neonatal death, and altered fetal growth . Indeed, in humans, obesity during pregnancy is associated with a significantly increased risk of macrosomia (commonly defined as birth weight 4500 g (8 lb 13 oz to 9 lb 15 oz)) as well as an increased risk of delivering a low birth weight baby [43, 60, 6266]. Similarly, animal studies have also shown that maternal overweight and obesity results in altered fetal growth with reports of both increased and decreased birth weight [30, 6769]. Therefore, based on the well - documented relationships between maternal obesity / diabetes and altered fetal growth, it is plausible that the altered fetal growth in women taking atypical antipsychotics may be a reflection of antipsychotic - induced changes in maternal metabolic or nutritional status . In addition to the impact of maternal metabolic status on the pregnancy, atypical antipsychotics may also be transferred across the placenta and directly impact fetal growth . While there is limited evidence quantifying the extent to which these drugs are transferred across the placenta, the work of schenker et al . Suggests that somewhere in the range of 514% of a labeled olanzapine can be transferred from maternal to fetal system in 4 h. however, the effects of atypical antipsychotics on fetal growth may be also mediated via altered placental development and/or function (figure 1). Prospective and retrospective analyses of maternal antipsychotic use and fetal outcomes have provided evidence that the use of atypical antipsychotics during pregnancy can result in dysregulated fetal growth . The results from these studies are conflicting; with some studies reporting that maternal use of atypical antipsychotics results in an increased incidence of high birth weight babies relative to the normal population and some studies reporting an increased incidence of low birth weight babies [23, 24, 28, 29]. Such observations suggest the actions of atypical antipsychotics on fetal growth may be described as a u shaped growth curve . Understanding the mechanisms driving such complex interrelationships may prove to be difficult, but the most likely place to initiate this search may be placental development and function . Mechanistically, it is known that atypical antipsychotics can act via dopamine d2 or serotonin 5-ht2a receptors . In addition to their well documented, but rather heterogeneous effects on various regions of the brain, the atypical antipsychotics also affect a number of the other tissues . Recent work from viau and colleagues reports the identification of the 5-ht2a serotonin receptor in human trophoblasts, a lineage of cells which are of central importance to placental development and function . In other cell types, signaling via the 5-ht2a receptor has been shown to affect cellular differentiation, proliferation, and migration, all of which are central to the function of placental trophoblast cells in the establishment of proper placentation [75, 76]. Such a relationship becomes more relevant because serotonin is synthesized de novo in human trophoblast cells where it likely serves an important endocrine, paracrine, and autocrine role in regulating placental function . Indeed, treatment of cultured trophoblasts, bewo and jeg-3 cells, with serotonin results in increased aromatase activity; an effect which may result in altered estrogen biosynthesis . Control of estrogen biosynthesis is important not only for successful implantation of the blastocyst but also for the regulation of leptin expression (review in). Furthermore, the levels of leptin in both maternal and fetal plasma have been associated with the regulation of fetal growth [81, 82]. Taken together, this evidence raises the possibility that atypical antipsychotics may affect fetal growth through the regulation of estrogen biosynthesis and leptin expression . Dopamine receptors, another target of atypical antipsychotics, are also known to be present in the human placenta, in trophoblast cells . Two subtypes, d1 and d2 receptors, have been localized to the spongiotrophoblasts and giant cells of the junctional zone of rat placenta at gestational days 1216 (term 2123 days). These cell lineages play important roles in the establishment, development, and maintenance of pregnancy in rodents [85, 86]. Like the serotonin receptor, d2 receptors have also been linked to important hormone regulatory processes such as the inhibition of basal and hormone stimulated release of human placental lactogen (hpl) from trophoblast cells . Furthermore, the observation that the pattern of d2 receptor expression varies during the course of a normal pregnancy argues for an important role for dopamine signaling during placental / fetal development [87, 88]. While the function of these receptors have not been directly related to altered fetal growth, their presence in the placenta suggests that drugs which target these receptors may have potential regulatory consequences for placental function and fetal development . While compounds such as clozapine also have a relatively high affinity for the dopamine d4 receptor subtype, which has been detected in the placenta, the functional role(s) of these receptors in placenta there is currently a paucity of data supporting the direct action of atypical antipsychotics on placental function . However, it is biologically plausible that this class of drugs might impact placental function given the presence of the putative receptors for these drugs on trophoblast cells as discussed above . Moreover, activation of dopamine receptors by other drugs, namely, bromocriptine, has been shown to alter the expression of pit-1; a pituitary - specific transcription factor which is synthesized in the rat placenta and is involved in the regulation of rat placental lactogen (rpl) gene expression, a hormone known to impact fetal development . Alternatively, atypical antipsychotics may affect placental development and/or function via alterations in oxidative stress in this tissue . Oxidative stress is a term used to describe the imbalance between the production of reactive oxygen species and the ability of the cell to limit their damage . There is some evidence that olanzapine and quetiapine may act as antioxidants in cultured peripheral neurons by decreasing oxidative stress associated with an increased accumulation of -amyloid protein [92, 93] and can even work to reverse the effects of compounds known to increase oxidative stress . However, the majority of evidence suggests that atypical antipsychotics are associated with increased oxidative stress [95, 96]. These, apparently, divergent effects of atypical antipsychotics may once again reflect the paucity of data in this area of research . Systematic investigation of these drugs on both neuronal and uterine systems will be important understanding the effects of these drugs on cellular stress response pathways . Clozapine, olanzapine, and aripiprazole have been found to differentially evoke oxidative stress in different regions of the brain . For example, chronic treatment (28 days) of rats with clozapine increased oxidative damage in the hippocampus . Olanzapine and aripiprazole actually decreased oxidative damage in the prefrontal cortex, despite the observation that the aripiprazole increased mitochondrial superoxide formation, a radical species known to cause both mitochondrial and cytosolic oxidative damage [98, 99], in the same tissue . Atypical antipsychotics have also been linked to increased superoxide formation and apoptosis in nave neutrophils . Furthermore, clozapine treatment of isolated lymphoblasts increased oxidative damage of a limited group of proteins . The increased damage of proteins associated with cellular metabolism suggests that the oxidative stress may arise from the mitochondria; known to be a primary producer of cellular superoxide . Production of free radicals from mitochondrial sources tends to preferentially damage mitochondrial proteins, the majority of which are associated with energy metabolism . Evidence that mitochondria are a target of atypical antipsychotics arises from the observation that the cerebral cortex and hippocampus of rats exposed to clozapine exhibited altered mrna expression for 14 mitochondrial proteins . Six of these proteins comprise various subunits of the electron transport chain, including complex i as well as complex v (atp synthase). This is particularly pertinent to oxidative stress since inhibition of the mitochondrial respiratory chain inhibition is known to be associated with increased oxidative stress [101, 103]. While typical and atypical antipsychotics have both been shown to inhibit mitochondrial complex i activity [104, 105], the inhibitory effects of atypical antipsychotics generally require higher concentrations . There are only minimal inhibitory effects of antipsychotics reported on complex ii of the mitochondria [104, 106, 107]. In addition, activation of the 5-ht2a receptor has also been associated with mitochondrial biogenesis . Taken together, it is feasible to propose that some of the actions of the atypical antipsychotics may occur via mitochondrially generated oxidative stress in the placenta especially since the placenta is characterized by particularly high levels of mitochondria . Increased oxidative stress along with poor mitochondrial function in the placenta has been linked to growth restriction and premature fetal demise . Mechanistically, oxidative stress may impact the ability of trophoblasts to transport nutrients between maternal and fetal circulation or carry out their endocrine functions . However, the exact linkage between placental oxidative stress and altered fetal growth and development remains undiscovered (figure 2). Oxidative stress and endoplasmic reticulum (er) stress have been linked and may jointly influence a number of cellular processes [19, 113, 114]. However, there has been very little work done to determine whether antipsychotics impact cellular er stress . Research by kurosawa et al . Suggests that the atypical antipsychotic olanzapine alleviates chemically induced er stress in cultured neurons . In addition to their effects on neuronal function, these drugs also impact reproductive functions such as fertility and may also effect placental function . The mechanisms by which these drugs directly, as well as indirectly (via modulation of metabolic homeostasis), influence fetal programming need to be more thoroughly investigated . There is currently considerable uncertainty regarding prescribing practices for pregnant women with severe and persistent psychiatric disorders . The physician and the mother have to balance the risks of untreated psychiatric illness, during pregnancy, against the potential fetal toxicity of atypical antipsychotic medications . Atypical antipsychotics are suspected to be teratogenic, although there are no randomized controlled studies for obvious ethical reasons . The adverse metabolic side - effects of these medications compound the risks associated with teratogenicity . Given that the number of women exposed to atypical antipsychotic drugs during pregnancy is increasing, it is important to more clearly delineate the risks associated with the administration of these drugs . There is limited information regarding their impact of these compounds on placental development and/or function . The use of appropriate animal models may be crucial in understanding the effects of these drugs on fetal growth and development which have profound consequences for the long - term health of the offspring.
The prognosis of the newly diagnosed breast cancer patient depends on a number of factors, among the most important of which is the extent of spread of disease to the axillary lymph nodes [1, 2]. Because treatment is influenced by the presence and number of axillary lymph nodes involved, evaluation of the axillary nodes has been performed in every patient that could tolerate it after a diagnosis of invasive carcinoma . In the past, a complete surgical dissection of the axilla was performed, resulting in significant morbidity, including a 30% incidence of lymphedema . The development of sentinel node mapping resulted in a notable reduction in morbidity; however, if a sentinel node was positive, often not discovered until final pathologic processing done postoperatively, complete axillary dissection would be performed at a later date to assess the total number of lymph nodes involved, thus requiring a second surgical procedure and anesthesia [57]. A preoperative diagnosis of axillary metastasis by ultrasound guided node biopsy would streamline patient care and reduce operating room time and expense by allowing definitive breast surgery and complete axillary dissection in the same operative setting, eliminating an unnecessary sentinel node procedure . While the need for preoperative node sampling in patients with nonpalpable nodes and t1 and t2 cancers has been challenged by the acosog z 11 study, there are subgroups of women for whom initial ultrasound guided node sampling is desirable, including those planning mastectomy or neoadjuvant chemotherapy . Using current high frequency transducers, studies have been performed attempting to identify malignant lymph nodes by their morphology but there is overlap both in the appearance of benign and malignant lymphadenopathy and in the appearance of normal and abnormal lymph nodes, with benign causes of axillary adenopathy being relatively common [911]. Therefore, tissue diagnosis of axillary node status remains important when it will affect patient management . Tissue sampling of suspicious axillary lymph nodes with ultrasound guidance has been performed for many years with a fine needle to obtain aspirates for cytologic evaluation . Reports in the literature have shown that fna is useful for evaluation of metastatic disease, with sensitivities ranging from 44 to 100%, the variability being likely due at least in part to patient selection [1218]. Studies comparing fna to large needle core biopsy of breast masses have shown core biopsy to be more accurate and easier to interpret than fna, leading breast care specialists to wonder if that is the case for sampling of axillary lymph nodes . While it is reasonable to expect the use of a core biopsy device to obtain a larger and architecturally intact piece of tissue to increase diagnostic accuracy as compared to obtaining an aspirate of cells with a fine needle, if the only question to be answered in sampling the axillary node is whether or not metastatic carcinoma cells are present along with lymphocytes, a larger architecturally intact piece of tissue may not be required . Although core biopsy is more invasive than fna due to the larger needle size, reports have shown that core biopsy of axillary nodes is a safe and effective procedure [20, 21]. Retrospective comparisons of fna and core accuracy in the axilla have been performed [22, 23], but to our knowledge, only one other prospective study has been published directly comparing fine needle aspiration to core biopsy of an axillary lymph node for the diagnosis of metastatic breast cancer . The purpose of this study was to determine if there is a difference between ultrasound guided core biopsy and fna in their ability to detect metastatic disease in the axillary lymph nodes of patients with a current diagnosis of ipsilateral breast cancer . From december 2008 through december 2010, women with suspected or recently diagnosed breast carcinoma and at least one lymph node in the ipsilateral axilla judged to be abnormal in sonographic appearance were approached for participation in this irb - approved, hipiaa - compliant prospective study . 105 women gave written informed consent to undergo ultrasound guided fna, immediately followed by core biopsy of the same node, followed by clip placement . Patients unable to consent due to language or comprehension difficulties and patients deemed emotionally too fragile to discuss the subject of metastatic disease required in the consent form were excluded . Other patients excluded themselves, not wishing to undergo a second needle procedure for research purposes . One patient was excluded due to difficulty accessing the node with a core biopsy due to its location . The outcome of percutaneous node sampling was correlated with surgical pathology from sentinel node excision or axillary dissection . Prior to node sampling, the cortical thickness, presence or absence of a hilus, presence or absence of cortical flow, node shape, and number of nodes thought to be abnormal were recorded . A phillips iu22 ultrasound unit (phillips healthcare, andover, ma) with 12 or 17 mhz transducers was used for imaging . Criteria used in determining appropriateness for node sampling were loss of the normal hilus, abnormal shape including focal bulging of the cortex, presence of cortical flow, and cortical thickening . A strict threshold for cortical thickness was not used; a node with a cortex between 2 and 3 mm was considered suspicious if the other nodes had cortices less than 2 mm . The patients were asked to rate their pain during each procedure on a scale of 1 to 10 and were informed of which procedure was being performed . The degree of bleeding (minimal or moderate) or hematoma formation, if any, was documented . Of the 105 patients, 7 patients' percutaneous breast biopsies were unexpectedly negative for malignancy (their node biopsies were all negative also). Of the remaining 98 with breast malignancy, 3 patients with both fna and core negative nodes were excluded due to lack of histopathologic axillary surgical correlation . We included 4 patients who did not have axillary surgery but were assumed to be true positives as they were both core and fna positive . Tumor size ranged from 6 mm to 10.7 cm, with 12 patients having tumor size greater than 5 cm . Cortical thickness of the sampled node ranged from 2 mm to over 6 mm . Table 1 shows features of the study population . To mimic the actual range of clinical practice, variability in sampling devices fna was performed using a 21 or 25 g, 2-inch needle, with one (90 cases), 2 (11 cases), or 3 (4 cases) needle entries of multiple needle excursions through the cortex of the node . The number of excursions was not recorded, but radiologists preferring more than one entry typically made fewer excursions per entry, estimated at 10 versus 20 to 30 . The aspirated material was placed both on slides in 95% alcohol and in buffered formalin for cell block preparation; a pathologist was not present at the time of the procedure to evaluate the adequacy of the samples . Core biopsy was performed immediately following the fna with either a 14 g (86 cases), 12 g (16 cases), or 18 g (3 cases) biopsy device . The 12 g device used was celero (hologic, bedford, ma). The 14 g devices were bard monopty, bard maxcore, finesse (bard, tempe, az), and achieve (cardinal health, dublin, oh), and the 18 g devices were achieve . One to four cores were obtained (one core in 23 cases, 2 cores in 54 cases, 3 cores in 20 cases, and 4 cores in 8 cases). With 14 g devices, only one core was obtained in 11 of 86 cases . With 12 g devices, one core only was obtained in 12 of 16 cases . With 18 g devices, 2, 3, or 4 the procedures were performed by 12 different academic radiologists experienced (range 321 years, with over half performed by those with over 16 years of experience) in breast imaging and biopsy (figure 1). However, for a given patient, the same radiologist performed both the fna and the core biopsy . The cytologic material obtained was evaluated by one of three pathologists experienced in breast pathology and cytology, without the knowledge of the core biopsy result . The fna result was categorized as negative if reported as containing suspicious cells but not actually stating that metastatic cancer cells were present . Suboptimal specimens were categorized as negative for both core and fna, because the course of action in our institution in most cases would be to proceed with sentinel node biopsy rather than to repeat the percutaneous biopsy . The sensitivities of the fna and core biopsy procedures were compared using mcnemar's exact test for correlated proportions . The trends in sensitivities with changing numbers of passes, entries, or needle sizes were assessed with the exact cochran - armitage test . Patients' subjective perception of pain levels was compared using the exact wilcoxon sign test . Of the 95 patients in the study cohort, 70 patients (74%) had metastatic adenopathy . This group included 5 patients that were both core and fna positive at percutaneous biopsy but were node negative after chemotherapy, 2 discordant (fna negative, core positive or fna positive, core negative) cases with complete pathologic response to chemotherapy resulting in negative nodes at surgery, and 4 core and fna positive patients without axillary surgical correlation . Fna was positive in 55/70 (78.6%) and core was positive in 61/70 (87.1%) (p = 0.18 (95% ci 0.0320.166)). 65 of the 70 (92.9%) patients had a positive axillary node by tissue sampling: 51 by both fna and core, 4 only by fna, and 10 only by core biopsy . Thus, in this group of 70, there were 14 cases (20%) where fna and core results were discordant (k = 0.3 (95% ci 0.03 to 0.58)). The sensitivity for single pass core biopsy was 78.6% (11/14) and for multipass cores was 89.3% (50/56), which was not a statistically significant difference (p = 0.37). The sensitivity for the 12 g device was 89.9% (8/9), for 14 g devices was 86.7% (52/60), and for 18 g devices was 1/1; the differences in sensitivity using the 12 g vacuum assisted device versus other devices together were not statistically significant (p> 0.99). The sensitivity for 21 g single entry fna was 76.1% (35/46) not different from 25 g single entry fna at 78.6% (11/14) (p = 0.85). The sensitivity for single entry fna was 76.7% (46/60) not statistically significantly different from multientry fna at 90% (9/10) (p> 0.41) (table 2). The sensitivities of fna and core biopsy were compared in table 2 for numbers of suspicious nodes, for node hilus, cortex, and shape, and also for tumor size . Fna sensitivity was inferior to core sensitivity (p = 0.04) when the node hilus was visible but improved with hilar absence . Both fna and core sensitivities improved with cortical thickness increasing beyond 2 mm, and when 3 or more abnormal appearing nodes were noted . Of the 10 fna negative / core positive patients, all but one had positive nodes at surgery; that patient had a complete pathologic response to chemotherapy and 20 negative nodes . One cytology specimen was reported as less than optimal, and 2 mentioned suspicious cells but were not diagnostic for malignancy . Of the 4 fna positive / core negative cases, each performed by a different radiologist, all of the core specimens were reported as suboptimal, 2 with scant lymphoid tissue, and two with absent lymphoid tissue . In 2 of the 4 (one scant and one absent lymphoid tissue), only one core was obtained . Three of the 4 had positive nodes at surgery, and one patient had negative nodes but had a complete pathologic response to chemotherapy with neoadjuvant related changes in the axilla . Of the 5 core and fna negative patients with positive nodes at surgery, the clip was noted to be in a negative node in 2 cases indicating that the wrong node was chosen for sampling . The presence of a clip or evidence of biopsy was commented on in 59% (54/91) axillary surgical reports . There was no difference in bleeding between the 2 procedures, which was minimal for all but one case that was moderate for both fna and core . The mean pain score for fna was 2.0 and for core was 2.4 while the range was from 1 to 8 for fna and from 1 to 10 for core . Reported pain levels were similar during fna and core in 63 patients (60%), greater with core in 31 patients (29.5%), and greater with fna in 11 patients (10.5%). The higher pain level was reported significantly more frequently for core than for fna (p <0.01). Our results show that although core biopsy had greater sensitivity than fna in detecting metastasis, it did not approach statistical significance, probably primarily due to the small number of patients . These results are in agreement with the meta - analysis by houssami et al . Who reported a sensitivity for fna (24 studies) of 72.2% and sensitivity for core biopsy (4 studies) of 83.3%, which were not statistically significantly different . The only other prospective comparison study reported a significantly greater sensitivity for core biopsy (88.2%) than for fna (72.5%) but had a small sample size of 51 patients undergoing percutaneous node biopsy with axillary metastasis . Our study included several experienced radiologists and allowed a variety of sampling devices to simulate actual clinical practice . While axillary node fna is technically easy to perform for one experienced in image - guided procedures, the radiologist must obtain an aspirate that is both sufficient in the amount of material and at the same time not overly bloody, to enable an optimal interpretation . It is not clear why there were fewer false negative results when multiple fna entries were performed, as the total number of needle excursions likely did not differ greatly . Perhaps the chance of obtaining a better sample was increased by using different entry sites or obtaining less blood mixed with cells from the node . The number of slides used, actual number of excursions, and length of procedure were not recorded, which could have affected the results . In some institutions, a pathologist is present when cytologic samples are obtained and can request additional sampling if the specimen is deemed suboptimal; the presence of a pathologist at the time of sampling could have improved the yield from fna . In our institution, immunostains may be used to aid in interpretation when fna alone is performed . Our pathologists have extensive experience in cytopathology but in this study there were no immunostains used in the cytologic evaluation; because the pathologists knew that additional tissue would be examined by core biopsy, a factor that may have decreased the sensitivity of fna . As demonstrated by the core negative / fna positive cases, care must be taken to be certain that the core specimen is being taken from the node; the node may be more difficult to visualize due to its depth and is frequently very mobile, making the core biopsy procedure quite challenging . Obtaining more than one core sample should insure a greater chance of obtaining an adequate specimen, as shown by the trend (albeit statistically not significant) for increased sensitivity with a greater number of core passes . Obtaining one core with a 14 g device was the least sensitive technique in this study and would not be recommended . If it is not certain that adequate cores were obtained, the radiologist should perform fna of the node or take additional cores . In either case, samples should be taken from the node's cortex, where the metastatic cells would lodge, avoiding the hilus where the vascular supply to the node is located . Both fna and core biopsy (excluding the insufficient cores of ill - defined nodes) were least sensitive when the node appearance was least abnormal . This can be due to difficulty in choosing the appropriate node for sampling or due to smaller metastatic deposits in the sampled node . In 4 of the 5 cases that were both core and fna negative, the nodes had a normal shape, visible hilus, and cortical thickness of 2.1 to 4 mm . In our study core biopsy had no more morbidity than fna, even with the largest gauge device . Use of a biopsy device with a nonthrow option should diminish the chance of vascular injury . However, patients whose suspect node was immediately adjacent to a vessel or very deep and difficult to access were not asked to participate in the study and hence were not subjected to core biopsy . Despite the statistically significant difference we observed in the number of patients reporting pain being greater during core than fna, the majority of patients tolerated the pain equally well during both procedures, and we do not believe this should be a factor in deciding which procedure to perform . Limitations of our study included its small size, in particular, the small size of subgroups of needle types and number of samples obtained . Although there may have been some selection bias due to excluding patients with nodes not suited to core biopsy, the aim of the study was to compare the two methods when both were possible . In all cases, the core biopsy was performed after the fna, with additional lidocaine, which may have minimized the pain associated with core biopsy . Fna was always performed first because of concern that core biopsy might cause sufficient bleeding to have to abort the second sampling procedure, but bleeding was not a significant problem . A large fraction of patients underwent neoadjuvant chemotherapy, which was not predicted at the time of initiation of the study . This could have rendered some patients node negative that were initially node positive, but there were only 7 that were node negative after chemotherapy and node negative by both core and fna . If fna and core were both falsely negative, there would be a similar reduction in sensitivity for each method . However, the 5 patients which were both fna and core negative and with positive nodes at surgery did not have neoadjuvant chemotherapy . Table 2 shows the sensitivities of core biopsy and fna in patients chosen to receive chemotherapy to be better than in those going directly to surgery, which is likely a reflection of the fact that the neoadjuvant group had more abnormal appearing nodes with thicker cortices . Our study began before the acosog z0011 trial that reported in 2010 no statistically significant differences in local or regional recurrence after median follow - up of 6.3 years between those randomized to sentinel node dissection alone versus completion axillary dissection in patients with clinically negative axillae and t1 or t2 invasive breast cancers treated with lumpectomy and radiation and 1 or 2 positive sentinel nodes . As a result of this trial, surgeons indicated that they would perform sentinel node biopsy even after a positive percutaneous node biopsy to determine if only one or two nodes were positive rather than perform axillary dissection in patients with tumors less than 5 cm . Consequently some surgeons have requested that radiologists not biopsy suspicious nodes in these patients . However, the z0011 patient population had a low tumor burden with median tumor sizes of 1.6 and 1.8 cm, and a high percentage of micrometastases and solitary positive nodes, suggesting that their outcome may be different than those with positive percutaneous node biopsies . Although the z0011 trial results have called into question the value of preoperative tissue sampling of axillary nodes in a selected population, the preoperative detection of metastatic adenopathy at the time of breast cancer diagnosis will continue to be helpful in management of patients with a larger tumor burden and allow many women to have axillary dissection at the time of definitive breast surgery, sparing them an unnecessary sentinel node procedure . For patients who will undergo neoadjuvant chemotherapy, the z0011 results do not apply; percutaneous axillary node sampling will aid in proper staging prior to treatment . The decision to perform core biopsy versus fna should be based on the pathologist's experience in interpreting cytology and the accessibility of the lymph node . Fine needle aspiration is a good alternative to core biopsy when a smaller needle is desired due to node location or other patient related factors.
Although liver resections have been associated with high mortality and morbidity rates, recent advances in anesthetic and surgical management have significantly reduced the operative risk . The techniques of vascular control during hepatectomy are highly demanding and should be performed under special anesthetic considerations . Hepatic vascular control methods can be categorized as those involving occlusion of liver inflow and those involving occlusion of both liver inflow and outflow . Hepatic pedicle occlusion: continuous pringle maneuver (cpm),intermittent pringle maneuver (ipm). Selective inflow occlusion . Hepatic pedicle occlusion: continuous pringle maneuver (cpm),intermittent pringle maneuver (ipm). Continuous pringle maneuver (cpm), intermittent pringle maneuver (ipm). Inflow and outflow vascular exclusion total hepatic vascular exclusion (thve), inflow occlusion with extraparenchymal control of the major hepatic veins: with selective hepatic vascular exclusion (shve). Total hepatic vascular exclusion (thve), inflow occlusion with extraparenchymal control of the major hepatic veins: with selective hepatic vascular exclusion (shve). When performing these techniques, the conduct of anesthesia should take into account hemodynamic management, risks of vascular air embolism, ischemia reperfusion liver injury, intraoperative blood loss, and the need for transfusion, factors which usually complicate hepatic vascular control methods . Special attention should also be paid to the preoperative assessment and induction of anesthesia, as patients undergoing liver resection usually have a compromised health status . Careful selection of the anesthetic drugs can minimize the effects of hepatic blood flow decrease induced by the surgical technique adopted . Our objective was to identify the anesthetic considerations in techniques of hepatic vascular control methods . All prospective randomized studies were thoroughly evaluated and presented, as they are the most important source of information on the outcomes of surgical and anesthetic manipulations . Few case reports and smaller studies are mentioned, given the fact that they highlight special anesthetic aspects . Healthy patients undergo a routine preoperative assessment including a full blood count and a standard biochemical and coagulation test . Preexisting hepatic impairment is a risk factor, even for nonhepatic surgery, with higher blood transfusion requirements, a longer hospital stay, a higher number of complications, and increased mortality rates of 16.3% in cirrhotic patients as compared to 3.5% in controls . Estimating the health status of patients presenting for hepatectomy is quite challenging: coagulopathy, volume and electrolyte disturbances, viral infections (hep c), hepatorenal [24] and hepatopulmonary syndrome, portopulmonary hypertension, and low cardiovascular reserve capacity can occur in patients with chronic liver disease . The identification of patients at risk to develop postoperative hepatic or renal failure is important and, ideally, involves many related disciplines such as surgery, anesthesia, and intensive care . Although vascular occlusion techniques have minimized hepatic bleeding, the risk for postoperative liver and/or renal failure remains high for patients of advanced age and those with steatosis and cirrhosis, on preoperative chemotherapy and with small remnant liver volumes . Have developed and validated a prediction score for postoperative acute liver failure following liver resection based on the preoperative parameters of cardiovascular disease, chronic liver failure, diabetes, and alt levels, which seems to be an easily applicable and attractive tool in clinical practice . Hepatic ischemia and reperfusion on subsequent liver dysfunction is associated with unexpected responses to surgical stress [79] and poor prognosis . Patients with end - stage liver disease have a characteristic hemodynamic profile: increased cardiac output with blunted response to painful stimuli, splanchnic vasodilatation and central hypovolemia . As a result, silent moderate - to - severe coronary artery disease cannot be easily recognized . Currently, there are no specific guidelines for the identification of coronary artery disease in patients with advanced liver disease [11, 12]. Preoperative invasive assessment of preexisting cardiovascular dysfunction is indicated only for high risk patients, provided that any coagulopathy is corrected . In the noninvasive assessment of coronary artery disease in patients with cirrhosis, beta blockade discontinuation in order to permit adequate cardiac function assessment may be hazardous in patients with advanced liver disease . Beta blockers reduce portal hypertension, decrease cardiac workload, and their use seems to be beneficial to both the liver and the heart in the setting of hepatectomy . In general, the preoperative assessment needs to be adapted to the individual patient to minimize the perioperative liver insults of hepatic vascular control . Cis - atracurium is the nondepolarizing muscle relaxant of choice in patients with liver disease as it is hydrolyzed by hoffman elimination . Atracurium can provide stable neuromuscular blockade, as its requirements remained unchanged during exclusion of the liver from the circulation . An intravenous hypnotic is used for induction and a halogenated volatile agent in air - oxygen mixture is used for maintenance . Hepatic vascular control techniques depress cardiovascular function in addition to the depression caused by general anesthesia . Careful selection of the volatile agent is required . Most commonly used volatile anesthetics for maintenance are isoflurane and sevoflurane . Isoflurane has mild cardiodepressive effects but maintains hepatic oxygen supply, due to vasodilatation in the hepatic artery and portal vein . Both isoflurane and sevoflurane upregulate heme - oxygenase-1, release iron and carbon monoxide, and thus decrease portal vascular resistance in rats . In humans, sevoflurane decreases portal vein blood flow but increases hepatic artery blood flow ., in a randomized controlled trial on patients undergoing liver surgery, showed that ischemic preconditioning with sevoflurane before inflow occlusion limited postoperative liver injury, even in patients with steatosis . Although various inhalational anesthetics are used in liver surgery, no optimal anesthetic technique has been established for the maintenance of anesthesia . Additionally, desflurane undergoes only minor biodegradation (it is metabolized at a ratio of 0.02%) and in fact it may cause less hepatocellular damage due to its reduced metabolism . Ko et al ., comparing the effects of desflurane and sevoflurane on hepatic and renal functions after right hepatectomy in living donors reported better postoperative hepatic and renal function tests with desflurane as compared to sevoflurane at equivalent doses of 1 mac without, however, being able to validate the clinical importance of their study . Arslan et al . Comparing the effects of anesthesia with desflurane and enflurane on liver function, showed that during anesthesia with desflurane, liver function was well preserved; glutathione - s - transferase and aspartate aminotransferase levels were significantly lower in the desflurane group . On the other hand, laviolle et al . Suggested that propofol has an early protective effect against hepatic injury compared with desflurane after partial hepatectomy under inflow occlusion . However, few studies on the effects of general anesthesia during hepatectomies under vascular control techniques are available in patients with significant comorbidities . Portal triad clamping increases systematic vascular resistance by up to 40% and reduces cardiac output by 10% . . However, the systemic circulation in patients with cirrhosis is hyperdynamic and dysfunctional, with increased heart rate and cardiac output, decreased systemic vascular resistance, and low or normal arterial blood pressure . Thus, maintaining adequate organ perfusion may be difficult to achieve and preoperative optimization of the patient is required . The anesthetic management is dictated by the surgical approach and the patient's health status . Invasive monitoring provided by a central venous line or pulmonary catheterization is reserved for patients with poor cardiovascular status or when prolonged vascular occlusions are performed . A low cvp (between 2 and 5 mmhg), while aiming at euvolemia, reduces blood loss during liver surgery and improves survival [30, 31]. A low cvp can be achieved by limitation of intravenous fluids administration pre- and intraoperatively . Maintenance fluids and crystalloids to stabilize blood pressure> 90 mmhg and ensure diuresis of at least 0.5 ml / kg / h can be used safely with minor hemodynamic disturbance . If fluid restriction is ineffective to keep a low cvp, vasoactive agents are used . Nitroglycerin reduces cvp to the desired level during the resection phase or when excessive oozing is observed from the resected surface [13, 16]. Cpm with a cvp of 5 mmhg or less is associated with minor blood loss and a shorter hospital stay . If, however, it is applied under a low cvp during transection, blood loss and hemodynamic changes are minimal [3437]. In an experimental animal study, sivelestat, a neutrophil elastase inhibitor, reduced hepatic injury and stabilized hemodynamics after ischemia - reperfusion following ipm . The advantages of a low cvp must be weighed against inadequate perfusion of the vital organs and loss of volemic reserve in case of bleeding and/or air embolism . Support that in low cvp anesthesia during liver resection, the incidence of perioperative renal failure does not increase significantly . (1) total hepatic vascular exclusion (thve)in thve, rapid hemodynamic changes (table 1) are frequent due to surgical events such as caval clamping, sudden blood loss, and hepatic reperfusion . Cross - clamping of the inferior vena cava and portal vein result in a 4060% reduction of venous return and cardiac output, with a compensatory 80% increase in systemic vascular resistance and a 50% increase in heart rate . Although systemic vascular resistance and heart rate increase, the cardiac index is reduced by half, secondary to a preload reduction . Unclamping is followed by an increase in cardiac index and a significant reduction in systemic vascular resistance .the anesthetist should take prompt steps to manage the preload reduction and the sudden decrease in cardiac output evoked by the inferior vena cava and portal vein clamping . Intraoperative monitoring includes ecg, pulse oximetry, etco2 tension, invasive blood pressure monitoring through an arterial line, and cvp monitoring through a large bore central venous line . In addition, the presence of a pulmonary artery catheter allows the tailored administration of vasopressors in case of massive hemorrhage due to vena cava injury . The vigileo, an uncalibrated arterial pulse contour cardiac output monitoring system, has been proved to be unreliable in cirrhotic patients with hyperdynamic circulation undergoing major liver surgery .before colloids, beyond correcting volume deficits, improve splanchnic circulation, displace fluid into the blood compartment, and reduce bowel edema . Blood pressure and circulatory support vasopressin or norepinephrine are administered if volume loading is inadequate to maintain blood pressure following clamping of the vena cava .there is no standard approach to the use of vasoactive agents in thve . Vasoactive agents should be used carefully, as they improve cardiac output at the expense of microcirculatory blood flow . During vascular isolation of the liver in eight pigs, norepinephrine infusion (0.7 g / kg / min) decreased hepatic vascular capacitance by activation . In a recent study in septic patients, krejci et al . Showed that norepinephrine increased systemic blood flow but reduced microcirculatory blood flow on liver's surface.vasopressin on the other hand, is known to rapidly restore blood pressure during septic shock . However, in an experimental study, vasopressin proved to be inferior to norepinephrine in terms of improving hepatosplanchnic blood flow . The response to both norepinephrine and vasopressin is blunted in patients with cirrhosis [44, 45]. Renal autoregulation ceases below a renal perfusion pressure of 70 to 75 mmhg, below which, flow becomes pressure dependent . Perioperative fluid shifts, intravascular hypovolemia, and sympathetic activation during thve result in a reduction of renal blood flow . Low dose dopamine have been used with the aim of preventing intraoperative renal injury without evidence of substantial benefit . Fenoldopam had beneficial effects on postoperative creatinine levels and creatinine clearance of critically ill patients . Recently, terlipressin along with volume expansion have been shown to improve renal function, without, however, improving survival .hemodynamic intolerance to thve or ischemia under thve exceeding 30 or 60 minutes, require venovenous bypass [50, 51]. Thve should be limited to selected cases, as hemodynamic intolerance has been observed in 1020% of patients, as well as increased morbidity and hospital stays (table 2). In thve, rapid hemodynamic changes (table 1) are frequent due to surgical events such as caval clamping, sudden blood loss, and hepatic reperfusion . Cross - clamping of the inferior vena cava and portal vein result in a 4060% reduction of venous return and cardiac output, with a compensatory 80% increase in systemic vascular resistance and a 50% increase in heart rate . Although systemic vascular resistance and heart rate increase, the cardiac index is reduced by half, secondary to a preload reduction . Unclamping is followed by an increase in cardiac index and a significant reduction in systemic vascular resistance . The anesthetist should take prompt steps to manage the preload reduction and the sudden decrease in cardiac output evoked by the inferior vena cava and portal vein clamping . Intraoperative monitoring includes ecg, pulse oximetry, etco2 tension, invasive blood pressure monitoring through an arterial line, and cvp monitoring through a large bore central venous line . In addition, the presence of a pulmonary artery catheter allows the tailored administration of vasopressors in case of massive hemorrhage due to vena cava injury . The vigileo, an uncalibrated arterial pulse contour cardiac output monitoring system, has been proved to be unreliable in cirrhotic patients with hyperdynamic circulation undergoing major liver surgery . Before thve, colloids can be administered to prevent the abrupt decrease in cardiac output . Colloids, beyond correcting volume deficits, improve splanchnic circulation, displace fluid into the blood compartment, and reduce bowel edema . Blood pressure and circulatory support vasopressin or norepinephrine are administered if volume loading is inadequate to maintain blood pressure following clamping of the vena cava . Vasoactive agents should be used carefully, as they improve cardiac output at the expense of microcirculatory blood flow . During vascular isolation of the liver in eight pigs, norepinephrine infusion (0.7 g / kg / min) decreased hepatic vascular capacitance by activation . In a recent study in septic patients, krejci et al . Showed that norepinephrine increased systemic blood flow but reduced microcirculatory blood flow on liver's surface . Vasopressin on the other hand, is known to rapidly restore blood pressure during septic shock . However, in an experimental study, vasopressin proved to be inferior to norepinephrine in terms of improving hepatosplanchnic blood flow . The response to both norepinephrine and vasopressin is blunted in patients with cirrhosis [44, 45]. Renal autoregulation ceases below a renal perfusion pressure of 70 to 75 mmhg, below which, flow becomes pressure dependent . Perioperative fluid shifts, intravascular hypovolemia, and sympathetic activation during thve result in a reduction of renal blood flow . Low dose dopamine have been used with the aim of preventing intraoperative renal injury without evidence of substantial benefit . Fenoldopam had beneficial effects on postoperative creatinine levels and creatinine clearance of critically ill patients . Recently, terlipressin along with volume expansion have been shown to improve renal function, without, however, improving survival . Hemodynamic intolerance to thve or ischemia under thve exceeding 30 or 60 minutes, require venovenous bypass [50, 51]. Thve should be limited to selected cases, as hemodynamic intolerance has been observed in 1020% of patients, as well as increased morbidity and hospital stays (table 2). (2) selective hepatic vascular exclusion (shve)shve is a flexible technique that can be applied in a continuous or intermittent manner . Should accidental tears of major hepatic veins occur, rapid conversion to thve must be undertaken . The literature suggests that many institutions favor shve as one of the standard methods of vascular control because it provides a bloodless surgical field and it is tolerated by most patients . No special anesthetic considerations regarding the hemodynamic management of shve are referred, as this method diminishes blood pressure and heart rate fluctuations during liver resection (table 1).in a cohort study among 246 patients, hemodynamic tolerance to shve was excellent with only a slight increase in systemic and pulmonary resistance during clamping . No deaths were reported and the mean hospital stay was 9.6 days.shve is the method of choice in cases when cvp cannot be lowered (i.e., right heart failure, poor cardiovascular status) [5356]. In a retrospective study on 102 patients, shve was shown to be unaffected by cvp levels and the authors concluded that it should be used whenever cvp remains high despite adequate anesthetic management . Although the performance of shve requires significant surgical expertise, it is tolerated by most patients and has a hemodynamic profile similar to that of cpm [53, 54]. Furthermore, it controls backflow bleeding of the hepatic veins . In a large clinical study, shve proved to be more effective than cpm in controlling intraoperative bleeding, preventing blood loss, and reducing postoperative complications and mortality rates (table 2). Combined shve and perioperative fluid restriction has also been suggested as a liver and renal protective procedure in partial hepatectomy . . Demonstrated that active preoperative dehydration of the patient, low cvp anesthesia and shve resulted in minimal blood loss, low morbidity, and zero mortality in patients undergoing partial liver resection . In conclusion, shve which is not associated with cardiorespiratory and hemodynamic alterations is well tolerated by the majority of patients and requires shorter hospitalization times . Shve is a flexible technique that can be applied in a continuous or intermittent manner . Should accidental tears of major hepatic veins occur, rapid conversion to thve must be undertaken . The literature suggests that many institutions favor shve as one of the standard methods of vascular control because it provides a bloodless surgical field and it is tolerated by most patients . No special anesthetic considerations regarding the hemodynamic management of shve are referred, as this method diminishes blood pressure and heart rate fluctuations during liver resection (table 1). In a cohort study among 246 patients, hemodynamic tolerance to shve was excellent with only a slight increase in systemic and pulmonary resistance during clamping . Shve is the method of choice in cases when cvp cannot be lowered (i.e., right heart failure, poor cardiovascular status) [5356]. In a retrospective study on 102 patients, shve was shown to be unaffected by cvp levels and the authors concluded that it should be used whenever cvp remains high despite adequate anesthetic management . Although the performance of shve requires significant surgical expertise, it is tolerated by most patients and has a hemodynamic profile similar to that of cpm [53, 54]. Furthermore, it controls backflow bleeding of the hepatic veins . In a large clinical study, shve proved to be more effective than cpm in controlling intraoperative bleeding, preventing blood loss, and reducing postoperative complications and mortality rates (table 2). Combined shve and perioperative fluid restriction has also been suggested as a liver and renal protective procedure in partial hepatectomy . . Demonstrated that active preoperative dehydration of the patient, low cvp anesthesia and shve resulted in minimal blood loss, low morbidity, and zero mortality in patients undergoing partial liver resection . In conclusion, shve which is not associated with cardiorespiratory and hemodynamic alterations is well tolerated by the majority of patients and requires shorter hospitalization times . Although the relative risk of air embolism in hepatic surgery is low (<5%), factors predisposing to vascular air embolism during liver resections include: (a) surgical technique, (b) size and place of the tumor, (c) blood loss, and (d) low cvp anesthesia . Clinical signs of vascular air embolism during anesthesia with respiratory monitoring are: a decrease in end - tidal carbon dioxide and decreases in both arterial oxygen saturation (sao2) and tension (po2), along with hypercapnia . From the cardiovascular system monitoring, tachyarrhythmias, electromechanical dissociation, major hemodynamic manifestations such as sudden hypotension may occur before hypoxemia becomes present . When performing techniques of inflow vascular occlusion (cpm, ipm, selective inflow occlusion), air embolism may be observed during parenchymal transection under low cvp anesthesia or during reperfusion, due to mobilization of air bubbles trapped in opened veins . Resection of large tumors situated in the right lobe, close to the inferior vena cava or the cavohepatic junction, put the patient at risk of venous air embolism . Recent clinical trials assessing the efficacy of shve and pringle maneuver in preventing vascular air embolism showed that embolism occurred in three out of 2100 patients or in one out of 29 patients of the pringle group, following massive blood loss during tumor resection . Air embolism did not occur in any case of the shve group [6264]. Massive bleeding (> 5000 ml) and subsequent air embolism can even result in intraoperative death in patients undergoing major liver resections . The morbidity and mortality of air embolism depend on the volume and rate of air accumulation . From case reports of accidental intravascular delivery of air, the adult lethal volume has been described as between 200 and 300 ml or 35 ml / kg [67, 68]. Low cvp further enhances the negative pressure gradient at the surgical field compared to the right atrium and increases the possibility of air embolism . Currently, the most sensitive monitoring devices for vascular air embolism are transesophageal echocardiography and precordial doppler ultrasonography, detecting as little as 0.02 ml / kg and 0.05 ml / kg of air, respectively [6971]. The consequences of air embolism can be minimized by placing the patient in a 15 degree trendelenburg position [7274]. Furthermore, moulton et al . In a small study among ten patients, showed that patient positioning alone during liver surgery does not affect the risk of venous air embolism . Thus, the beneficial effects of low cvp in liver resections must be carefully weighed against adequate hydration and volume status optimization . Additionally, cirrhotic patients undergoing hepatectomy have pulmonary abnormalities including intrapulmonary shunting, pulmonary vascular dilatation, and arteriovenous communications . In these patients, air can pass into the systemic circulation (paradoxical air embolism), even if cardiac abnormalities (patent foramen ovale) are not present, evoking fatal consequences . Recent literature suggests that shve prevents vascular air embolism and provides operative tolerance . However, recognizing the risk for vascular air embolism and planning the appropriate level of monitoring and treatment is the key to patient safety . Liver resections may result in significant blood loss and subsequent transfusion of rbc (red blood cells) in about 25%30% of patients . The two main sources of bleeding during a liver resection are (a) the inflow system (hepatic artery and portal vein) and (b) the outflow system (backflow bleeding from the hepatic veins). Bleeding may also occur during liver mobilization, hepatic transection, and dissection of biliary structures . Blood loss has been linked to morbidity and mortality since 1989, whereas rbc transfusions are associated with multiple disadvantages, risks, and side effects . Furthermore, operative blood loss independently predicts recurrence and survival after resection of hepatocellular carcinomas . Operative mortality in patients refusing blood transfusions was 7.1% for patients with hemoglobin levels> 10 g / dl and 61.5% for patients with hemoglobin levels <6 g / dl [79, 80]. The refinement of inflow and outflow occlusive techniques as well as the appropriate anesthetic management has reduced intraoperative bleeding and the need for blood transfusions . Study of the literature reveals the following results regarding bleeding with different vascular occlusion techniques: pringle maneuver has been shown to be effective in reducing blood loss during parenchyma transection . Portal triad clamping is associated with less bleeding compared with no clamping . In procedures of liver ischemia time belghiti et al ., in a prospective study of ipm versus cpm, found no difference in total blood loss or the volume of blood transfused between the two groups, despite higher blood loss during parenchyma transection ., in two prospective studies of ipm versus no use of vascular control at all, showed lower total blood loss and fewer transfusions in the ipm group [8385]. Hemihepatic vascular clamping was shown superior to ipm and to no application of vascular control, with reduced both blood loss and transfusion requirements . Shve provides a bloodless surgical field similar to thve, but is better tolerated by patients . Many authors favor shve as one of the standard methods of vascular control, as it substantially prevents massive blood loss and diminishes transfusion needs . From an anesthetic standpoint, a low cvp level plays an important role in reducing intraoperative blood loss and transfusion rates [30, 57, 87]. Maintaining a cvp <5 mmhg by volume restriction and intravenous infusion of nitroglycerine and a systolic blood pressure above 90 mmhg by intravenous infusion of dopamine (46 g / kg) the anesthetist should also provide normothermic conditions to the patient undergoing liver resection, because hypothermia reduces blood coagulation, especially platelet function, and increases intraoperative blood loss . Of the pharmacological methods, desmopressin, although used in treating hemophilia, was not effective in reducing blood loss and transfusion needs in patients undergoing liver resection . In a randomized clinical trial, the use of recombinant factor viia in major liver resections failed to reduce the number of units transfused . A significant reduction in blood transfusion needs in liver resections has been shown with the use of aprotinin . Aprotinin was found to reduce intraoperative blood loss by 25% and transfusion requirements by 50% . Used half dose aprotinin (10 kiu followed by 2.5 10 kiu / hour infusion) during hepatic transplantation in patients who have a significant coagulopathy or portal hypertension and in those who had previous abdominal surgery . However, lentschner et al . Cautioned against the routine use of aprotinin due to the incidence of life threatening allergic reactions, thrombotic potential, and renal failure . Currently, there is no scientific support for the routine use of aprotinin in patients undergoing partial hepatectomy, whereas its efficacy in liver transplantation is well established . Tranexamic acid has also been shown to reduce blood requirements in liver resection surgery but safety concerns have been raised and require further investigation [92, 93]. In the future, two artificial oxygen carriers (hemoglobin solutions and perfluorocarbons) may become essential in reducing the need for allogeneic rbc transfusions [9496]. Artificial oxygen carriers improve o2 delivery and tissue oxygenation as well as the function of organs with marginal o2 supply . More studies examining their efficacy in ischemic liver during hepatectomy need to be performed . Undoubtedly, the improvement of vascular control techniques during hepatectomy has permitted an aggressive approach for liver resections with low mortality rates (4%). In addition, anesthesia orientated towards an almost transfusion free setting has also improved mortality and morbidity following liver surgery . To this direction, a transfusion risk score, including variables of: (a) preoperative hemoglobin concentrations below 12.5 g / dl, (b) largest tumor more than 4 cm, (c) need for exposure of the vena cava, (d) need for an associate procedure, and (e) cirrhosis, accurately predicted the likelihood of blood transfusions in liver resections . Recently, cescon et al ., in a retrospective review assessing the outcome of 1500 consecutive patients who underwent hepatic resection, estimated overall mortality and morbidity at 3% and 22.5%, respectively . Their multivariate analysis revealed that blood transfusions, primary liver tumors, and additional procedures were associated with an increased risk of postoperative complications, whereas blood transfusions, cirrhosis, biliary malignancies, and extended hepatectomy were associated with an increased risk of postoperative mortality ., evaluating the long - term outcomes of liver resection for hepatocellular carcinoma, estimated that 86.9% of the patients did not require perioperative blood transfusion and that pringle maneuver and rbc transfusions are independent prognostic factors influencing survival . Blood transfusions are well known to carry the risk of transmitted infections, acute or delayed reactions and wrong blood incidents . In liver resections, blood transfusions there is strong evidence that blood transfusions have an impact on tumor recurrence for patients with early stages of hepatocellular carcinoma . However, no such effect could be demonstrated for patients undergoing partial liver resection for late stages of hepatocellular carcinoma, colorectal metastasis, or cholangiocarcinoma . Transfusion evoked immunosuppression is also responsible for trali (transfusion - related acute lung injury). Dyspnea, hypotension, fever, and bilateral noncardiogenic pulmonary edema, present within 6 h of transfusion and complicate the postoperative outcome of patients following major liver surgery . Patients with chronic liver disease have the greatest risk of developing trali, in comparison to other populations [101, 102]. Although all blood products can lead to this life - threatening situation, plasma - containing products were responsible for the majority of cases in patients undergoing liver transplantation . Recent studies suggest that trali fatalities followed plasma transfusion components were linked to multiparous female donors with leukocyte antibodies [103, 104]. Therefore, the establishment of new strategies in blood donation excluding multiparous women as donors, as potential carriers of trali - inducing antibodies, is expected to eliminate this entity . In conclusion, given the influence of blood loss and transfusions on the surgical outcome, techniques of liver vascular control and anesthetic management should be adjusted to the individual patient . The tumor location, the underlying liver disease and the patient's cardiovascular status should therefore be taken into account, in order to minimize blood loss and transfusion requirements . Ischemia / reperfusion (i / r) injury is a serious complication of liver surgery, especially after extended hepatectomies . It causes a local and systemic inflammation response and its clinical manifestations may vary from transient arrhythmias to multiorgan dysfunction and death . Reperfusion injury is mediated via reactive oxygen species which damage cellular membranes, stimulate leukocyte activation and endothelial adhesion, and activate the complement . Hepatic i / r injury affects patient recovery after major surgery and bears a risk of poor postoperative outcome . In liver surgery, ischemic preconditioning (ip) and intermittent clamping are the only established methods to provide protection against tissue damage due to ischemia during inflow occlusion [98, 108]. Ip is defined as a process in which a short period of ischemia, separated by intermittent reperfusion, renders an organ more tolerant to subsequent episodes of ischemia [107, 109]. It was initially described for a canine heart by murry et al . In 1986 . As far as the liver is concerned, the beneficial effect of ip was first demonstrated in a rodent model by lloris - carsi et al . . It leads to improvement of hepatic microcirculation, reduction in tissue apoptosis, and improvement of survival . Experimental data suggest that generation of adenosine, activation of adenosine a2 receptors with subsequent generation of no and release of no cause vasodilation and prevent the increase in endothelins, thus protecting the liver from reperfusion injury . Ip stimulates adenosine receptors on kupffer cells in nonischemic lobes to produce oxygen radicals, leading to the promotion of liver regeneration after partial hepatectomy . In a clinical study of 61 patients undergoing liver surgery performed by heizmann et al ., the absence of preconditioning was found to be an independent risk factor for postoperative complications . It has been stated that ip might also be less beneficial during extended liver resections, due to hyperperfusion - induced derangement in hepatic microcirculation . Similarly, the effect of preconditioning was lost in patients undergoing tissue loss above 50% . In small liver remnants of about 30%, it may in fact have detrimental effects . This is because the small remaining tissue suffers from shear stress - associated microvascular injury . Ischemic preconditioning seems to attenuate the apoptotic response of hepatic cells in major hepatectomies performed under shve . On the other hand, azoulay et al . Found that ip failed to protect human liver against ir injury after major hepatectomy under continuous vascular occlusion with preservation of caval flow . Ip and salvialonic acid - b have been shown to possess synergistically protective effects in rats, mediated through reduction of postischemic oxidative stress, higher atp levels and reduction in hepatocellular apoptosis . The preconditioning effect fades away when the ischemic time is prolonged . In this case, intermittent vascular occlusion, although more complex surgically, seems to be the method of choice . Van wagensveld et al . Demonstrated that prolonged intermittent vascular inflow occlusion in pig liver surgery caused less microcirculation impairment and hepatocellular necrosis compared with continuous occlusion and recommend it when a prolonged period of vascular inflow occlusion is expected . It has been found that when ischemia persists for more than 40 minutes, intermittent vascular occlusion offers better protection of liver cells, demonstrated by lower ast values, lower apoptotic activity and reduced capsase-3 activation . In several animal models, pharmacological preconditioning with a volatile anesthetic has been proven to provide protection against ischemic injury . Beck - schimmer et al . Evaluated the effects of sevoflurane preconditioning before liver ischemia and concluded that this particular volatile anesthetic limited the postoperative increase of serum transaminase levels by 261 u / l for the alt and by 239 u / l for the ast . The sevoflurane group had less major complications (such as sepsis, bilioma, bleeding, and infection) than the control (propofol) group . The protective effects were more pronounced in patients with liver steatosis . However, according to wang et al ., propofol also seems to have the ability to protect human hepatic l02 cells from h2o2-induced apoptosis . Intraportal administration of l - arginine, a precursor of no, has been recently studied in pigs and appears to reduce cell damage during the early phase of reperfusion, by downregulating capsase-3 activty and by preserving mitochondrial structure . Clinically, it resulted in a reduction of ast and an increase in bile production . In another animal study, simvastatin (5 mg / kg) protected the rat liver from i / r injury by regulating the inflammatory response and by improving microvascular flow . Prostaglandins have also been found to have protective effects on i / r - injured livers by inhibiting the generation of reactive oxygen species, preventing leucocyte migration, improving hepatic insulin and lipid metabolism and regulating the production of inflammatory cytokines . Finally, ramalho et al . Reported that angiotensin ii type i receptor (at1r) antagonist increased regeneration in nonsteatotic livers, while in the presence of steatosis both at1r and at2r antagonists increased liver regeneration . Intolerance to thve is not unusual and this method should be reserved for patients in need for extensive reconstruction of the inferior vena cava . Inflow occlusion techniques, although simple and effective, require specific anesthetic manipulations to reduce liver injury and prevent backflow bleeding . Every method of hepatic vascular control applied under a carefully selected anesthetic plan can improve the outcome of patients undergoing hepatectomy . Anesthetic vigilance along with thorough knowledge of the surgical manipulations promotes team - based health care in the operative room.
The ageing process is characterised by progressive loss in cellular homeostasis and a decline in physiological function (balcombe and sinclair, 2001), resulting in an overall decline in fecundity and increased risk of mortality over time . While ageing was historically believed to be an inevitable and intractable process (for discussion see (kirkwood and holliday, 1979; kirkwood, 2005, 2008; munch et al ., 2008)), we now know that ageing can be modulated through various environmental, genetic and pharmacological interventions such as dietary restriction (dr) (kenyon, 2005; mair and dillin, 2008; rikke et al ., 2010; swindell, 2012; gems and partridge, 2013). Dr is defined as the reduction in overall energy intake, or in specific components of the diet, relative to that consumed normally by individuals with free ad libitum (al) access to food . Dr has been conclusively shown to extend lifespan in a wide range of taxonomically diverse organisms (mair and dillin, 2008; selman, 2014). In addition, studies have demonstrated that particular genetic pathways modulate ageing through the observation that deletion of single key genes can extend lifespan and healthspan in model organisms . In particular, reduced signalling via the insulin / insulin like growth factor-1 (igf1) signalling (iis) pathway or the target of rapamycin (tor) pathway shows highly conserved effects on lifespan across wide evolutionary distances (kenyon, 2005; piper et al ., 2008; broughton and partridge, 2009; indeed, differential expression of several iis and tor genes is associated with longevity in humans (deelen et al ., 2013; more recently pharmacological interventions, including rapamycin and metformin, have been shown to extend lifespan in model organisms . This has led to renewed hope in identifying realistic, efficacious and safe pharmacological interventions that will increase the period of our life free from age - related diseases (selman, 2014). Excitingly, evidence exists that some molecular processes affected by these different interventions may overlap, thereby suggesting some level of commonality (selman et al ., 2009; yan et al ., 2012), although other studies suggest that specific interventions may function through distinct mechanisms (masternak et al ., 2004; bhattacharya et al ., 2012; fok et al ., furthermore, recent research has suggested that the effectiveness of each intervention on ageing is highly sensitive to the effects of genetic background . Therefore, the use of such interventions in genetically diverse human beings becomes questionable unless we fully understand the precise role that genetic background plays . In this review, we will discuss the evidence that genetic background plays a role in lifespan extension in response to dietary, genetic and pharmacological interventions in mice . The first studies on lifespan extension in animals in response to dr were published almost 100 years ago (osborne et al ., 1917; mccay et al ., 1935). Consequently, dr is the most reproducible and widely used intervention to modulate lifespan (masoro, 1995; speakman and mitchell, 2011; selman, 2014). In addition, dr has been shown to induce significant health benefits, with mouse studies showing dr protects animals against a range of age - related and non - age - related pathologies, including various cancers, glaucoma, glucose intolerance and sarcopaenia (weindruch and walford, 1982; sheldon et al ., 1995a, 1995b; mckiernan et al ., 2004; hempenstall et al ., furthermore, dr decreases pathogenesis and increases survival in a range of mouse models of disease including parkinson's disease (duan and mattson, 1999), alzheimer's disease (halagappa et al ., 2007), viral myocarditis (kanda et al ., 2007) and pancreatic cancer (lanza - jacoby et al ., 2013). The positive effects of dr on health also extend to rhesus monkeys, where it has been shown that the incidence of cancer, cardiovascular disease, type - ii diabetes mellitus and brain atrophy with advancing age are reduced relative to al controls (colman et al ., 2009, 2014; similarly, dr has been shown to induce significant beneficial effects on metabolism (weiss et al ., 2006) and cardiac function (stein et al ., 2012) in humans, and lowers several risk factors associated with coronary heart disease (fontana et al ., 2007). Despite the large number of studies that have used dr to extend lifespan we still do not understand the precise mechanism(s) through which dr acts to modulate lifespan . In addition, studies using an increasing number of different model systems have implicated that the effects of dr on lifespan extension are not universal and question whether dr is indeed a public modulator of longevity . Several studies in organisms ranging from yeast to non - human primates have reported no effect of dr on lifespan (harrison and archer, 1987; kirk, 2001; carey et al ., 2002;, 2010; rikke et al ., 2010; mattison et al ., 2012; another caveat has recently emerged from research on mice which indicates that specific genotypes can affect the extent and direction of the lifespan response to dr . As an example, the effect of dr on lifespan in dba/2 mice has ranged from lifespan extension to lifespan shortening (fernandes et al ., 1976; turturro et al . Clear differences in key variables including terms of husbandry conditions, extent of restriction, diet, gender and age of dr initiation will undoubtedly vary across different studies which may help explain the differences in the findings reported . However, it is also well established that dba/2 mice show distinct differences in a range of metabolic parameters (e.g., insulin sensitivity, glucose tolerance, metabolic rate) under both al and dr feeding when compared to strains such as c57bl/6 mice (funkat et al ., 2004; goren et al ., 2004; berglund et al ., 2008; hempenstall et al ., it is also clear that extended lifespan, when observed, in dba/2 mice in response to dr is more moderate than the extension reported in c57bl/6 mice (turturro et al ., 1999). Recently, lifespan was assayed in heterogeneous ilsxiss recombinant inbred (ri) mice derived from eight distinct mouse strains (liao et al ., 2010; in two separate studies undertaken by the universities of texas and colorado, clear lifespan differences existed between distinct ilsxiss lines under 40% dr . The earlier study examined 39 female lines and 41 male lines under dr and reported that only 21% of female and 5% of male lines showed a significant lifespan extension under dr (liao et al ., 2010). In this study, a higher number of lines (26% and 27% for males and females, respectively) showed a significant shortening of lifespan under dr . Similarly, the second study examined 42 female lines again and reported a significant difference in terms of the response to dr across different lines, with 21% of females showing lifespan extension and 19% showing significant truncation of lifespan under dr (rikke et al ., 2010). Similarly, the differential effect of dr on lifespan reported in rhesus monkeys by the wisconsin national primate centre (colman et al ., 2009, 2014) and the national institute of aging (mattison et al ., 2012) may be partly explained by inter - study differences in geographical origin and genetic background of the experimental animals (mattison et al ., 2012; partridge, 2012; currently, we do not understand how genetic background may impact on how dr acts to extend life . In ilsxiss mice at least it may be that the optimal dr regime differs between lines and consequently the 40% dr regime employed (liao et al ., 2010; rikke et al ., 2010) was too extreme to maximise lifespan in all lines (swindell, 2012; selman, 2014). Furthermore, it is not known if specific lines are more prone to particular pathologies under both al and dr conditions . However, comparative - type approaches by taking advantage of the differential responses to dr across different mouse strains or ri lines may be a powerful approach to understand precisely how dr acts to slow ageing (selman, 2014). Consequently, a larger subset of more genetically diverse rodent models should be studied under dr, along with the greater use of non - model organisms to increase our understanding of how dr acts . Significant research efforts over the last couple of decades, initially using invertebrate model organisms, have demonstrated that decreased iis and tor signalling extends lifespan in a highly conserved fashion across model organisms (kenyon, 2005; piper et al ., 2008; gems and partridge, 2013). In addition, in mice a large number of studies have also shown that specific disruptions in somatotrophic function also extends lifespan significantly relative to wild type controls (bartke, 2011). Furthermore, it is evident that long - lived genetically mutant mice tend to display a much greater proportion of their life free from age - related pathologies (selman and withers, 2011). As genetic interventions modulating lifespan is a more recent finding compared to studies on dr, it is unsurprising that few studies, so far, have investigated how genetic background can impact on the life - extending effects of specific mutations . The most widely used genetically modified mice in ageing research are those which harbour mutations that result in growth hormone (gh) deficiency or gh resistance (for review see bartke, 2011; bartke et al ., 2013); ames mice (prop1), snell mice (pit1) including growth hormone receptor knockout (ghrko) and the little mouse (ghrhr). In addition to altered somatotrophic function, these animals appear to have secondary suppression of iis (bartke, 2011; bartke et al ., 2013). These models show reproducible lifespan extension across different studies and appear to be protected against a range of age - related pathologies (brown - borg et al ., 1996; flurkey et al ., 2001; kinney et al ., 2001;, 2002; coschigano et al ., 2003; ikeno et al ., the impact of genetic background on lifespan has been studied in snell mice, where it has been shown that the lifespan extension in these animals is consistent across different genetic backgrounds (flurkey et al ., 2001; flurkey et al ., however, it should be noted that the additive effects on lifespan with dr reported on a mixed genetic background (bartke et al ., 2001) were lost with a greater penetrance of the c57bl/6j background (garcia et al ., 2008). In addition, ames mice backcrossed on to a c57bl/6 or 129s1/svlmj background led to a significant increase in perinatal mortality (nasonkin et al ., 2004). In 2003, two separate studies were the first to demonstrate that reduced iis extended lifespan was also conserved in mammals . These studies reported that the loss of the insulin receptor specifically in white adipose tissue (bluher et al ., 2003) or haploinsufficiency in the gene encoding the insulin like growth factor 1-receptor (igf1r) (holzenberger et al ., 2003 (2003) showed that female igf1r mice maintained on the 129sv background were 33% longer lived than wild type mice, although the effect in male igf1r mice was non - significant (16%). In addition, they showed that female but not male mice were resistant to the effects of the oxidant stressor paraquat . This study has since been criticised due to the short lifespan of the control animals and the small sample size used (liang et al ., 2003). Lifespan and end - of - life pathology were examined under housing conditions optimised to maximise the lifespan of the control mice (bokov et al ., 2011). In addition, the igf1r mice were re - derived on the c57bl/6 background for at least 10 generations before lifespan was assayed . This second study reported a more modest increase in female lifespan (5%; significant using the log - rank test) relative to controls and a slight, but non - significant, reduction in male lifespan . While genetic background and the targeting event used may help explain some of the differences reported between the two studies, the authors in the second study (bokov et al ., 2011) tend to discount this as significant overlap in terms of the metabolic and oxidative stress resistant phenotype which was seen between each study . (2011) also examined lifespan in igf1r female mice on a f1 hybrid c57bl/6 129sv background and showed no effect of haploinsufficiency on lifespan . Consequently, the authors of the second study (bokov et al ., 2011) proposed that sub - optimal housing conditions may have led to increased stress exposure of all mice in the earlier study (holzenberger et al ., 2003). This was hypothesized to lead to a survival advantage of the stress resistant igf1r females over wild type females, which was not enjoyed by igf1r males . While evidence suggests dr may not have the ubiquitous effect on lifespan as originally proposed, animals under dr tend to retain a longer period of life free from ill health, including the onset and impact of age - related pathologies such as type-2 diabetes, cardiovascular disease and cancer . Consequently, much current research aims to identify drugs which mimic the effects of dr, i.e., extend vitality in old age . Several compounds have been identified, including rapamycin, metformin and resveratrol, which appear to have significant potential in the development of efficacious and safe dr mimetics (selman, 2014). However, the precise mechanism through which these interventions act is still unclear and research from murine studies has demonstrated that genotype appears to be important in how particular dr mimetics impact on an individual's phenotype . The tor pathway appears to be a highly conserved lifespan determinant (kapahi et al . 2010; lamming et al ., 2012; wu et al ., 2013), which plays a key role in growth and metabolism (bjedov and partridge, 2011) by responding to a range of stimuli including various growth factors, nutrients and energy status (fig . 1). Tor kinase, the central component of the tor pathway, forms two functionally different complexes: tor complex 1 (torc1), which plays a central role in regulating cellular processes associated with growth and differentiation, and torc2 which has a regulatory role in the insulin signalling cascade (lamming and sabatini, 2011; lamming et al ., 2012, 2013; selman and partridge, 2012). Rapamycin, a macrolide compound, has been shown to be a highly potent inhibitor of mtorc1, and more recently also of mtorc2 (lamming and sabatini, 2011; lamming et al ., 2012, 2013a, 2013b; selman and partridge, 2012). In model organisms,, 2009; anisimov et al ., 2010b; bjedov et al ., 2010; neff et al ., 2013) and in mice can attenuate some, but not all, ageing - related phenotypes (wilkinson et al ., 2012; however, mouse studies using rapamycin to modulate lifespan have shown that its effects on metabolism appear to be highly sensitive to genetic background . As an example, rapamycin treatment induces overt insulin resistance in c57bl/6 mice (lamming et al ., 2012) but insulin resistance was not reported in young or old het3 mice (lamming et al ., 2013a). In addition, rapamycin appears to have temporal effects on metabolism in mice, leading to hyperinsulinaemia, insulin resistance and glucose tolerance after 2 weeks of rapamycin treatment but hypoinsulinaemia and insulin sensitivity after 20 weeks of treatment (fang et al ., 2013). Recent findings have shown that rapamycin induces sex - dependent effects on lifespan (miller et al ., 2014) and that there appears to be less overlap with dr, in terms of metabolism and transcription, than was originally believed (fok et al ., 2014; miller et al ., 2014). Similarly, the effect of the biguanide metformin on lifespan and health in mice appears to be, at least partly, dependent on genotype, but also dependent on sex and age at which metformin treatment is initiated (anisimov et al . It is clear that the dramatic rise in the proportion of elderly individuals making up our population is going to have significant ramifications . Ageing is associated with a decrease in the quality of life linked to an increase in the risk of developing a range of pathologies, including various dementias, type-2 diabetes, osteoporosis, many cancers and cardiovascular disease . Consequently understanding the fundamental processes that drive ageing and increase the susceptibility to develop disease is undoubtedly one of the greatest current challenges in biomedical research . To add to this challenge, it is becoming increasingly prevalent in the literature that genetic heterogeneity is likely to play a critical role in the response to interventions which modulate lifespan and healthspan . Another potential confounding issue is the focussed use of model organisms in such studies, for example, the small number of mouse strains used in ageing research has been highlighted as a potential limitation to the identification of longevity - associated genes (yuan et al ., 2013). Future research efforts should perhaps complement model organism studies with those examining ageing in non - model organisms (selman et al ., 2012), for example, employing comparative - type approaches to study ageing in fast - ageing and slow - ageing species (austad, 2010a, 2010b). Additionally, we should also take greater advantage of the inherent differences in longevity and pathology at death seen across different mouse strains (storer, 1966) to further understand how specific interventions, including dr, genetic inactivation of iis / mtor and rapamycin treatment, act to modulate lifespan and health in the background of increasing genetic heterogeneity.
We report the case of a 19-year - old female football spectator who suffered a duodenal rupture as a result of being hit in the abdomen by a football . A 19-year - old girl presented to the emergency department at 1 am after having a football kicked from a distance of 510 m into her abdomen 7 h previously whilst watching a game . Her observations were stable; however, on examination, her abdomen was diffusely tender and there was evidence of generalized peritonism . She had a white cell count of 18.8 10/l, but the rest of her blood tests, including amylase, was normal . A computed tomographic (ct) scan of her abdomen and pelvis was arranged, which showed abnormal areas of low attenuation and multiple pockets of air in the right flank, surrounding the right kidney and in the right sub - hepatic space, consistent with a perforation of either the ascending colon or the duodenum (fig . 1). A laparotomy was performed and a 0.5 cm perforation was seen in the antero - lateral border of the duodenum at the junction of the first and second parts of the duodenum following kocherization . Figure 1:ct of abdomen showing free air in right para - renal space (white arrow). Ct of abdomen showing free air in right para - renal space (white arrow). The duodenum is a c-shaped organ primarily situated in the retroperitoneum and is anatomically divided into four sections (d1d4). It is vulnerable to damage by shearing or compression forces, as d1 and d4 are relatively mobile in comparison with d2 and d3, which are fixed . Commonly injuries occur at the junction between d3 and d4 and at the junction between d1 and d2, as seen in this case [1, 2]. The retroperitoneal nature of the duodenum may also result in delays in the diagnosis of duodenal rupture as patients may not present with frank peritonism initially . It is therefore important to consider both mechanism of injury and also other clinical signs such as tachycardia and raised white cell count as delays in diagnosis and subsequent management have been shown to adversely affect morbidity and mortality [18]. However, in this case, due to the positioning of the perforation, duodenal contents entered the peritoneal cavity directly from the point of rupture, thus causing peritoneal irritation . Duodenal injuries secondary to blunt trauma can range in severity from an intramural haematoma to a complete transection and devascularization of the duodenum, and are graded 15 by the american association for surgery of trauma [2, 5, 6]. Ct scanning is a useful adjunct to in the diagnosis of duodenal rupture, and can aid in the differentiation between full thickness rupture that requires surgical intervention and a haematoma which can be managed conservatively . The finding of fluid in the right anterior para - renal space on a ct scan may be seen in the presence of a duodenal haematoma, whereas if air is seen in this area duodenal rupture is likely to have occurred [14, 6, 9]. In this case, ct scanning was useful in delineating the possible area where a perforation may be found, especially as the patient presented with generalized abdominal pain . There are multiple ways to repair a duodenal rupture and these are obviously dependant on the severity of the injury . The literature suggests that kocherization (mobilization) of the duodenum should be performed to allow full examination of the duodenum to rule out multiple perforations [1, 2, 4, 7, 9]. Duodenal perforations are uncommon secondary to blunt trauma, comparative to penetrating trauma . A retrospective study analysing a trauma database of 103,864 patients showed that only 0.2% (208 patients) had a duodenal injury related to blunt trauma and of these only 30 patients had full thickness duodenal rupture . The most common mechanism of injury was involvement in a road traffic accident [1, 3]. Duodenal rupture as a result of a sporting injury is very rare and there are no reports of duodenal injury from the specific mechanism described in this report [8, 10]. This case demonstrates that even if the mechanism of injury seems trivial, a high level of suspicion should be maintained in the presence of suggestive clinical findings . It also shows the benefit of ct scanning in the diagnosis and management of duodenal rupture.
Adenomyosis is a common gynecological condition that is characterized by ingrowths of the endometrial cells into the myometrium . Adenomyosis and leiomyoma are benign conditions that are often responsible for uterine enlargement, menorrhagia, anemia, and infertility . Adenomyosis or internal endometriosis may occur as a result of increased overgrowth of the endometrium with invasion of the underlying myometrium or the displacement of the endometrium during pregnancy, delivery, endometrial curettage, cesarean section, myomectomy, or metroplasty . Because of its similarities to leiomyoma, it can be difficult in some cases to accurately diagnose adenomyosis by ultrasound . Most gynecologists will consider conservative medical treatment more often for adenomyosis only, while surgical options will be chosen frequently for leiomyoma or combined adenomyosis and leiomyoma, especially in large uteri . The three most common methods of clinical diagnosis of adenomyosis are magnetic resonance imaging (mri), transabdominal ultrasonography (tas), and transvaginal ultrasonography (tvs). There is limited diagnostic capacity with tas, while tvs is a more feasible option . Moreover, tvs is also much more cost effective than mri and is generally more readily available in the office to most practicing gynecologists . The aim of this retrospective study was to determine the accuracy, sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of tvs in the diagnosis of adenomyosis and leiomyoma confirmed by postsurgical histopathological findings . The importance of an accurate noninvasive diagnostic method like ultrasound for adenomyosis, leiomyoma, or combined is an ongoing need in the gynecological community . This was an institutional review board (irb) approved retrospective consecutive case series study . From november 2006 to may 2012, patients underwent surgery for the treatment of adenomyosis, leiomyoma, or combined adenomyosis and leiomyoma . Diagnostic criteria of uterine adenomyosis include two of the five sonographic features on tvs: (1) no distinction of the endometrial - myometrial junction; (2) asymmetry of the anterior and posterior myometrium; (3) subendometrial myometrial striations; (4) myometrial cysts and fibrosis; and (5) heterogeneous myometrial echotexture . Diagnostic criteria of uterine leiomyoma include two of the five sonographic features on tvs: (1) clear demarcation of the tumor margin; (2) whorly appearance of the tumor content; (3) the presence of blood vessels (by color doppler) surrounding the tumor; (4) irregularities of the uterine surface (subserous and intramural tumors); and (5) irregularities of the endometrial surface (submucous tumors type 1 and 2). Symptomatic patients diagnosed with adenomyosis and leiomyoma via tvs underwent myomectomy with excision of the surrounding myometrium which presumably contained adenomyosis . Following surgery, a histopathological examination was performed by the hospital pathologists . Student's t - tests were used for parametric continuous variables; and the chi - square or fisher's exact test, where suitable, was used for categorical variables . Sensitivity, specificity, npv, ppv, positive and negative likelihood ratios, and accuracy were determined for ultrasound findings as they corresponded to the final histopathological diagnosis . From november 2006 to may 2012, 163 patients underwent surgery for the treatment of adenomyosis, leiomyoma, or combined adenomyosis and leiomyoma . One hundred and twenty - three patients were diagnosed with adenomyosis, and 134 patients were diagnosed with leiomyoma . Twenty - nine patients were diagnosed with adenomyosis only, 40 patients were diagnosed with leiomyoma only, and 94 patients were diagnosed with combined adenomyosis and leiomyoma, as illustrated in figure 1 . Patient diagnosis groups one hundred and thirty patients diagnosed with adenomyosis or combined adenomyosis and leiomyoma via tvs underwent hysterectomy . The mean age was 43.7 years with a standard deviation (sd) of 6.7 years and median of 43 years . There was no significant difference in the mean age, weight, height, gravidity, and parity of the patient diagnosis groups . Patient characteristics for 163 females who underwent hysterectomy or myomectomy between 2006 and 2012 of the 123 patients (75.46%) who were positively diagnosed with adenomyosis, 93 of the patients (75.61% ppv) diagnoses were confirmed by the histopathological findings . Histopathological reports found 23 (57.50% npv) confirmed negative diagnoses and 17 (42.50%) positive findings . The sensitivity of tvs in the diagnosis of adenomyosis was 84.55% (95% ci 76.4 - 90.7, p <0.0001) and the specificity was 43.40% (95% ci 29.8 - 57.7, p = 0.41). Table 2 shows the sensitivities, specificities, ppvs, npvs, and accuracy for each of the initial tvs diagnoses . This could be explained by the difficulty to diagnose adenomyosis in patients with other intrauterine abnormalities or conditions . Patients positively diagnosed with adenomyosis via tvs are 1.49 (95% ci 1.16 - 1.92, p <0.002) times more likely to have the condition . Conversely, patients negatively diagnosed with adenomyosis via tvs are 2.81 (95% ci 1.65 - 4.79, p = 0.0002) times less likely to have the condition . Number of patients, positive and negative predictive values, sensitivity, specificity, and accuracy of transvaginal ultrasound for initial diagnosis with histopathological correlation (n=107) one hundred and thirty - four patients (82.21%) were diagnosed with leiomyoma and 29 patients (17.79%) were negative for leiomyoma upon the initial tvs . Of the 134 patients diagnosed with leiomyoma, 133 (99.25% ppv) had a confirmed diagnosis represented in their postsurgical pathology report and one patient's (0.75%) pathology report did not confirm the diagnosis . Of the 29 patients whose tvs diagnosis was negative for leiomyoma, 24 (82.76%) patients histopathological findings were also negative, and five patients (17.24%) had a positive histopathological diagnosis . The corresponding sensitivity and specificity of tvs as a diagnostic test for leiomyoma was 96.38% (95% ci 91.75 - 98.81) and 96.00% (95% ci 79.65 - 99.90), respectively . Patients with a positive diagnosis of leiomyoma via tvs are 24.09 times (95% ci 35.30 - 164.45, p = 0.001) more likely to have leiomyoma . Patients with a negative diagnosis of leiomyoma via tvs are 26.53 times (95% ci 11.16 - 62.89, p <0.0001) less likely to have leiomyoma . For the statistical calculations of adenomyosis and leiomyoma as coexisting conditions, analyses were performed excluding the patients who were diagnosed with a singular condition . These patients were classified as negative solely for the purpose of calculations regarding the combined condition . Of the 94 patients (57.67%) that were positively diagnosed with combined adenomyosis and leiomyoma, 70 (74.47% ppv) had histopathological confirmation of both conditions and 24 patients (25.53%) did not have evidence of both conditions in their post - surgical pathology report . Sixty - nine (42.33%) patients had a negative tvs diagnosis for combined adenomyosis and leiomyoma, and 49 (71.01% npv) had histopathological confirmation of the negative diagnosis . However, 20 patients (28.99%) had a positive histopathological diagnosis for both adenomyosis and leiomyoma . The sensitivity and specificity of tvs in the diagnosis of combined adenomyosis and leiomyoma was 77.78 (95% ci 67.79 - 85.87) and 67.12% (95% ci 55.13 - 77.67), respectively . Patients who obtained a positive tvs diagnosis are 2.37 times (95% ci 1.67 - 3.34, p <0.0001) more likely to be diagnosed with combined adenomyosis and leiomyoma . Alternatively, patients who are negatively diagnosed with adenomyosis and leiomyoma via tvs are 3.02 times (95% ci 1.99 - 4.59, p <0.0001) less likely to be diagnosed with combined adenomyosis and leiomyoma . Adenomyosis is a gynecological disorder that is characterized by the overgrowth of the endometrium into the underlying myometrium . The difficulty in diagnosing adenomyosis clinically is due to the lack of strong positive pathognomonic signs and/or clinical findings . The explanation for this wide range of values, as described by azziz, is the result of differences in the histological criteria for the diagnosis of adenomyosis, the care of which the pathologic specimens are handled, and the number of blocks of sampling specimens taken . Various measures of accuracy were calculated for the diagnosis of adenomyosis, leiomyoma, and combined adenomyosis and leiomyoma . Table 3 shows a comparison of sensitivity, specificity, ppv, and npv of this study with several previous studies that investigated the diagnosis of adenomyosis . It is important to note that the mean number of patients included in the 13 previous studies in table 3 is 76.8 patients; however, this study utilized 163 patients in the analyses . Along with differences in inclusion criteria, ultrasound equipment, and/or the differences in the criteria to diagnose adenomyosis, the larger sample size could contribute to the difference in results . The sensitivity and specificity of tvs for the diagnosis of adenomyosis in this study was 84.55 and 43.40%, respectively . The associated p value for the specificity was not significant, suggesting tvs as a diagnostic tool is sensitive, but not specific in the diagnosis of adenomyosis . The sensitivity in this study is similar to those previously reported in table 3, but the specificity is the lowest of those reported . This could be due to the difficulty in diagnosing adenomyosis in the presence of other uterine abnormalities and conditions, especially uterine leiomyomata that may distort the uterus . Patients that were diagnosed with multiple intrauterine conditions were not excluded from this study, since the principle inclusion criteria was patients who underwent hysterectomy or myomectomy and a preoperative tvs . Sensitivity, specificity, positive and negative predictive values of tvs for the diagnosis of adenomyosis from previous series compared with this series the sensitivity of tvs for the diagnosis of leiomyoma was 96.38% and the specificity was 96.00%, which is similar to that of a previous study . The sensitivity for the diagnosis of combined adenomyosis and leiomyoma was 77.78%, and the specificity was 67.12% . It was accurate, sensitive, and specific in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma . Tvs was both accurate and sensitive in the diagnosis of adenomyosis, but not specific . Moreover, tvs is cost effective and readily available in the office to the majority of practicing gynecologists . This study demonstrated that tvs is a valuable noninvasive method that should be utilized in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma.
The human gastrointestinal mucosa constitutes the largest mucosal surface area in the human body interfacing the external environment . A network of complementary regulatory interactions between different types of immune and nonimmune cells maintains mucosal homeostasis in the gut . These regulatory interactions occur in the midst of a complex mixture of proteins, known as extracellular matrix (ecm) or stroma [1, 2], which together with soluble mediators such as cytokines and growth factors from mesenchymal cells, immune cells, and epithelial cells regulate cell activation and differentiation . Mesenchymal cells are actively involved in the inflammatory process in the gut and can perpetuate chronic gut inflammatory conditions like inflammatory bowel disease (ibd) [35]. Transforming growth factor- (tgf-) is one of the potential soluble mediators in homeostatic mechanisms in the gut that downregulates effector t - cell responses in the mucosa by reducing proliferation and interferon- (ifn-) production [4, 6]. However, tgf- together with il-6 and il-1 from the inflamed mucosa induces proinflammatory th17 cells, suggesting an innate regulatory function of the gut mucosal microenvironment [4, 7, 8]. Research is ongoing to elucidate the role of soluble factors and ecm in immunopathological mechanisms in ibd, but no such studies have so far been performed on microscopic colitis (mc) where the inflammation is subtler . Mc comprises two entities, collagenous colitis (cc) and lymphocytic colitis (lc). Both conditions are characterized by chronic nonbloody, watery diarrhoea, often associated with abdominal pain and weight loss [911]. The colonic mucosa is macroscopically normal or almost normal and the diagnosis relies on microscopic assessment of mucosal biopsies . Cc is presented with increased densities of lymphocytes and a thickened subepithelial collagen band (10 m) adjacent to the basal membrane . The pathophysiological data of cc are still limited, but it is postulated to be at least partially caused by disturbed immune responses to various luminal antigen(s), such as drugs, gluten, or infectious agents, in predisposed individuals . Nonsteroidal anti - inflammatory drugs (nsaids), proton pump inhibitors, aspirin, and selective serotonin reuptake inhibitors have been associated with cc . In the majority of patients, however, no precipitating factor is found . Dysregulated myofibroblast function has also been implicated for collagen deposition in cc patients [10, 13]. Recently, we reported on increased local activation of both cd4 and cd8 t cells in the lamina propria and epithelium of cc patients, demonstrated as increased expression of cd45ro and the proliferation marker ki67, using flow cytometric analysis of freshly isolated lymphocytes from colonic biopsies . In addition, mucosal transcript levels of ifn-, il-12, il-1, il-6, il-17a, il-21, il-22, and il-23 are enhanced in the inflamed mucosa of cc patients compared to normal mucosa, together with elevated protein levels of il-6, il-21, and tnf . Although the above findings suggest that the mucosal microenvironment is involved in cc immunopathology, the interplay between these factors and the proinflammatory activity of local mucosal t cells in cc patients has not been elucidated . We therefore investigated the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells using a novel in vitro model system with the aim of mimicking the in vivo exposure of newly recruited peripheral blood t cells to the soluble factors in the colonic milieu of inflamed cc and normal mucosa . Cc diagnosis was confirmed by clinical symptoms: 3 loose stools / day and/or abdominal pain and a macroscopically normal colonic mucosa with characteristic histopathological findings: increased numbers of lymphocytes in the epithelium and lamina propria with deposition of a 10 m thick subepithelial collagen layer . Patients with enteric infection, ischemic colitis, colonic cancer, or a previous history of crohn's disease or ulcerative colitis were excluded . We investigated colonic biopsies from 7 cc patients (female; n = 6) and 20 noninflamed controls (female; n = 11) without diarrhoeal symptoms, recruited among patients undergoing colonoscopy for examination of gastrointestinal bleeding or of abnormal radiological findings . Twelve biopsies from the hepatic flexure from each individual were obtained using standard biopsy forceps, placed in phosphate buffered saline (pbs), and processed within 1 hour . The colonoscopies were performed at the division of gastroenterology, rebro university hospital, sweden, between november 2012 and november 2013 . Peripheral blood from healthy donors (n = 6) was collected in heparin tubes for cd4 lymphocyte isolation as described below . The study was approved by the regional ethical committee of rebro - uppsala county, sweden (i d no . We investigated the influence of two different preparations of the mucosa: one where the epithelium and intraepithelial cells were removed enzymatically, the denuded biopsies (dnb), and one where collagenase was used to digest the lamina propria after the removal of the epithelium . The dnb and the isolated mononuclear cells were cultured overnight, and the latter were termed lamina propria mononuclear cells (lpmcs). The cell populations are intact in the dnb fraction, whereas the lamina propria mononuclear cells (lpmcs) fraction is composed of a free leukocyte population as well as tissue, collagen, and cell debris . Twelve biopsies were thoroughly washed with pbs and incubated with prewarmed hank's balanced salt solution (hbss) (sigma aldrich, st . Louis, mo, usa) containing 1 mm edta, 20 mm hepes, and 5% heat inactivated fetal bovine serum (fbs) at 37c, with constant stirring 4 times (15 min), to remove the epithelial layer . Six denuded biopsies were kept in serum - free rpmi-1640 containing 20 mm hepes, 100 g / ml streptomycin, 10 g / ml gentamycin, and 100 u / ml penicillin (hereafter referred to as culture medium) on ice until further use, and the remaining six biopsies were further digested with collagenase type viii and dnase i type iv (sigma aldrich) for 11.5 hrs to digest the collagen . Isolated lamina propria mononuclear cells were washed twice in pbs and resuspended in culture medium . Dnbs and lpmcs were cultured in culture medium overnight at 37c under 5% co2 - 95% air, to generate conditioned medium (cm). Cm from the dnb and lpmc fractions from cc patients and noninflamed controls, respectively, were pooled . Endotoxin levels were quantified using the pierce lal chromogenic endotoxin quantitation kit (pierce, rockford, il, usa) according to the manufacturer's protocol . The maximum endotoxin level for dnb - cm and lpmc - cm was 4 eu / ml and 4.2 eu / ml, respectively . Total protein concentrations were determined using the bio - rad dc protein assay kit (bio - rad, hercules, ca, usa). Dnb - cm was used at the total protein concentrations of 250 and 62.5 g / ml, whereas lpmc - cm was used at the total protein concentrations 125 and 62.5 g / ml, the highest possible concentrations of the respective conditioned media . Cm from the intestinal mucosa of noninflamed controls is referred to as ctrl - dnb - cm and ctrl - lpmc - cm, whereas cm derived from cc patients is referred to as cc - dnb - cm and cc - lpmc - cm . Cd4 peripheral blood lymphocytes were isolated from healthy donors using a human cd4 t - cell enrichment cocktail kit (stemcell technologies, grenoble, france) according to the manufacturer's protocol . The purity of cd4 t cells was 9095% as determined by flow cytometric analysis (epics altra, beckman coulter, fullerton, ca, usa). Purified cd4 pbls were cultured in 96-well flat bottom assay plates (sarstedt, newton, nc, usa) precoated with 50 l of 5 g / ml anti - cd3 (ucht1, bd biosciences, san diego, ca, usa) for 2 hrs at 37c, followed by two washes with pbs . 1 10 cd4 t cells were added to the washed wells together with 1 g / ml soluble anti - cd28 (bd biosciences) and were incubated in culture medium with the addition of 2 mm l - glutamine and 5% ab serum, with or without dnb - cm or lpmc - cm from noninflamed controls or cc patients in a total volume of 200 l . As controls, ctrl / cc - dnb - cm and ctrl / cc - lpmc - cm were cultured without cd4 t cells, and cd4 t cells alone were incubated without anti - cd3/anti - cd28 . The assay was performed in duplicate wells for cytokine analysis and triplicate wells for the proliferation assay . The cells were cultured at 37c under 5% co2 - 95% air for three days; thereafter, supernatants were harvested and stored at 80c until determination of cytokine content, and t - cell proliferation was measured using the celltiter 96 aqueous one solution cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium, promega, madison, wi, usa). Forty microliters of aqueous one solution reagent was added into each well and incubated at 37c for 4 hrs in a humidified, 5% co2 incubator followed by recording of the absorbance at 490 nm . The pooled conditioned medium from inflamed cc mucosa and controls as well as supernatants from peripheral cd4 t cells were analyzed for il-1, il-4, il-6, il-10, il-17a, ifn-, and tnf using the xmap technology developed by luminex (austin, tx, usa), using two milliplex map kits (cat . Number hcyp2mag-62k), according to the manufacturer's instructions (millipore, ma, usa). The assays were performed in duplicate and the levels of different cytokines were expressed as pg / ml, according to a standard curve with known amounts of each analyte (millipore). Tgf- levels were determined by elisa, according to the manufacturer's instructions (bd biosciences, san diego, ca, usa). Cytokine amounts were calculated as follows: cytokine amounts released by peripheral t cells incubated with cm minus cytokine amounts in cm alone . For each blood donor, the cytokine amounts released by cd4 t cells incubated with cm from cc patients were compared with the cytokine amounts released by cd4 t cells incubated with cm from controls . As the data obtained were not normally distributed, wilcoxon's signed rank nonparametrical test was used for statistical comparison between groups . To mimic in vitro the exposure of peripheral t lymphocytes that have newly arrived into the colonic mucosa and to determine whether the local intestinal milieu affects t - cell activation and differentiation, we investigated t - cell cytokine production by -cd3 plus -cd28 stimulated cd4 peripheral blood t cells in the presence of soluble factors from the intestinal mucosa . There was significantly increased production of the proinflammatory cytokines ifn-, il-17a, il-6, and il-1 in the presence of cm generated by culture of denuded biopsies (dnb - cm) from the colon of collagenous colitis patients, compared to dnb - cm from noninflamed controls (figure 1). This was evident with the lower protein concentration of cm tested for il-17a, il-6, and il-1 and with the higher concentration of cm tested for ifn- production . We also noted a significantly increased production of the anti - inflammatory cytokines il-4 and il-10 in the presence of dnb - cm from cc patients compared to noninflamed controls, with the lower protein concentration of cm (figure 1). In contrast, no significant differences were noted for tgf- production by peripheral cd4 t cells in the presence of dnb - cm from cc patients compared to noninflamed controls (data not shown). In general, lpmc - cm had less impact on peripheral cd4 t - cell activation and differentiation . A trend towards increased production of il-17a and il-10 (both p = 0.06) was noted in the presence of lpmc - cm from cc patients compared to noninflamed controls in the lower protein concentration (figure 2). We next investigated the ability of soluble factors from the colonic mucosa to inhibit t - cell proliferation, as this has previously been demonstrated in vitro [4, 16, 17]. A tendency towards reduced proliferation inhibition of peripheral cd4 t cells was noted in the presence of cm from culture of denuded biopsies (dnb - cm) (p = 0.06) from inflamed cc patients compared to noninflamed controls (figure 3). This was evident with both total protein concentrations of dnb - cm tested for peripheral t - cell proliferation . In contrast, no differences in proliferation inhibition were observed in the presence of lpmc - cm from noninflamed controls compared to cc patients (figure 3). As t - cell differentiation and function are regulated by different cytokines, we next analysed eight cytokines in pooled conditioned medium from dnb and lpmc fractions derived from inflamed cc mucosa compared to controls . We found more than twofold and eightfold increased levels of il-6 and il-1, respectively, in dnb - cm from cc patients compared to controls, whereas no alterations were found in the levels of ifn-, il-17a, tnf, il-4, and il-10 (table 1) or tgf- (data not shown). Similar trends of increased levels of il-6 and il-1 were noted in lpmc - cm from cc patients compared to controls, though it was investigated in pooled cm from only two cc patients (data not shown). We here report on a novel in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and differentiation . We found that despite the subtle inflammation in the mucosa of collagenous colitis patients, not visible by the naked eye upon colonoscopy, soluble factors in the mucosa are sufficient to significantly enhance the production of ifn-, il-17a, il-6, il-1, il-4, and il-10 by peripheral cd4 t cells exposed to them in vitro . This is the first study to investigate the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells, where this novel system reflects the impact of in vivo exposure of newly recruited peripheral blood t cells to soluble factors in the colonic milieu of inflamed mucosa from cc patients compared to normal mucosa . Cm from denuded biopsies from inflamed cc mucosa induced increased production of both pro- and anti - inflammatory cytokines by peripheral t cells . As microscopic colitis is subtler compared to ulcerative colitis and crohn's disease, this may suggest that the colonic microenvironment in cc promotes production of anti - inflammatory cytokines to counterbalance inflammatory responses . A study on the effects of stroma conditioned medium from crohn's patients mucosa on cytokine production by t cells demonstrated increased ifn- and il-17 production but provided no data on the effect of anti - inflammatory cytokine production . No differences were noted in tgf- production by peripheral cd4 t cells in the presence of cm from cc patients compared to controls . Whereas tgf- in the normal mucosa likely suppresses t - cell function, the significantly increased amounts of il-6 and il-1 in the inflamed cc mucosa instead likely promote differentiation of proinflammatory th17 cells [7, 18] producing large amounts of il-17a . The colonic milieu from cc patients might also promote differentiation of peripheral cd4 t cells into il-17/ifn- double producing th17/th1 cells that have been suggested to mediate gut inflammatory processes [1921], corroborating our findings of enhanced levels of both il-17a and ifn-. We found a trend towards reduced inhibition of t - cell proliferation by soluble factors from denuded biopsies from the colonic mucosa of cc patients compared to controls . Older studies have demonstrated that the mucosal microenvironment reduces the proliferative responses of lamina propria lymphocytes to antigen receptor stimulation [17, 22] but they are still active in their helper and cytolytic functions [22, 23]. The present study together with the study by huff et al . Indicates that these effects are at least partly imprinted in the t lymphocytes by the local milieu, rather than an intrinsic characteristic . In contrast to cm from denuded biopsies, lamina propria mononuclear cell- (lpmc-) cm from cc patients did not affect t - cell proliferation compared to lpmc - cm from controls . These different effects of the two types of cm are unclear and further experiments need to be performed to elucidate the differences in the composition of the cms . Despite our observed enhanced production of il-10 by peripheral t cells in the presence of cm from cc patients, known to inhibit both t - cell proliferation and cytokine production, we found neither reduced proliferation of peripheral t cells nor production of proinflammatory cytokines . This indicates that other immunoregulatory molecules drive synthesis of these proinflammatory cytokines in collagenous colitis . The production of cytokines did not increase with higher total protein concentrations in the cm . One explanation could be the presence of inhibiting and/or toxic factors in the cm limiting the t - cell responses . In addition, various molecules have different optimal concentrations for their function and high concentrations can limit their activity . To further elucidate this and explain the differences observed on cd4 t - cell differentiation we want to investigate a larger panel of cytokines and compare the protein profile between cm from cc patients and that from noninflamed controls and between dnb and lpmc fractions by proteomics . In conclusion, we have set up an in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and cytokine production . Despite the subtle inflammation in cc, our data demonstrate significant alterations in cytokine production by peripheral cd4 t cells in the presence of mucosa - derived soluble factors from cc patients compared to controls . One of our future goals is to test this in vitro model on differentiation of cd8 t cells, as we have previously reported on their increased numbers in the colonic mucosa of cc patients . We also want to evaluate its use in evaluating the effect of drugs, including those in present use, on the colonic mucosal milieu and the lymphocytes there within, thereby facilitating the decision on optimum molecules as well as doses required for suppression of t - cell inflammatory responses.
Technological advances in three - dimensional (3d) imaging such as cone - beam computed tomography (cbct) appear to offer significant advantages in both quality and quantity of data representing true anatomy, . The increased use of computed tomography in dentistry has also spurred the improvement of existing software designed primarily for dental use, as well as the development of new software . However, it is necessary to select protocols and to assess the reliability and accuracy of landmark identification in 3d images . Development of 3d imaging into a practical and usable cephalometric method was hindered for many years by technologic limitations, high costs, and high radiation doses . Advances in cbct technology make its use feasible for specific indications, such as impacted teeth, craniofacial anomalies, airway and temporomandibular joint disorders . In these cases, cbct images could be used for routine cephalometric purposes beyond the diagnostic purposes for which they were primarily taken . This has been extensively done for cephalometric measurements, but to a much lesser extent for maxillofacial computed tomography imaging . Whether cbct should be used routinely in all orthodontic patients the voxel is the smallest unit of ct images and its size has an influence on image spatial resolution . By decreasing the voxel size the image spatial resolution increases as there is a decrease in partial volume averaging . On the other hand, by decreasing the voxel size, the scan time increases, as does the probability of patient movement . This is especially of concern in the case of younger individuals, who constitute the overwhelming majority of orthodontic patients . This issue of increased radiation dose to children, and specifically for orthodontic purposes, has caused widespread concern and has been the source of much discussion even in the lay literature . In order to verify the clinical reliability of measurements on multiplanar sections and 3d reconstructions derived from cbct, it is also necessary to evaluate the measurements using anatomic landmarks without the use of metallic markers . The most recent studies involving cbct scans have shown reproducibility and accuracy of cephalometric measurements performed on lateral cephalograms reformatted from cbct images or in 3d - cbct images . However, most of the studies used radiopaque markers and these might have an influence on the accuracy and reproducibility of the measurements . The presence of metallic markers eliminates errors from landmark identification because metallic landmarks can be easily identified and located with a high degree of accuracy and precision . The aim of this study was to verify the influence of voxel size on the accuracy and reproducibility of linear measurements of the mandible performed without metallic markers on 3d cbct images, comparing multiplanar sections and 3d rendering images . Approval for this study was received from the ethics committee at the university of so paulo, bauru, brazil . Ten dry adult human mandibles, selected from the collection of dry skulls of the department of anatomy from the bauru school of dentistry were scanned . To fit the inclusion criteria, the mandibles had to be well preserved with no large metallic restorations on the teeth, in order to avoid scattering and artifacts . The sample size of each group was calculated based on an alpha significance level of 0.05 and beta of 0.2 to achieve 80% of power . To provide soft - tissue equivalent attenuation, the mandibles were positioned with the mandibular plane parallel and the midsagittal plane perpendicular to the floor, in accordance with the manufacturer s instructions for performing the scans in the clinical setting . The cbct images were acquired using the i - cat classic 3d dental imaging system (i - cat classic, imaging sciences international, hatfield, pa, usa). Each mandible was scanned twice: once with a 0.20 voxel resolution (36.12 mas, 40 sec, fov of 8 cm, 120 kv) and once with a 0.40 voxel resolution (18.45 mas, 20 sec, fov of 8 cm, 120 kv). Cbct data were exported in dicom (digital imaging and communication in medicine) multi - file format and imported into dolphin11.5 software (dolphin imaging & management solutions, patterson technology, chatsworth, ca, usa). A computer a505-s6975 (satellite, toshiba, tokyo, japan) with a dedicated 512-mb video card on a 17-in crystalbrite lcd flat - panel color screen with a maximum resolution of 1440x900 pixels measurements were made separately on the multiplanar sections (2d) and on 3d rendering images display window of dolphin 3d . The multiplanar window displays the sagittal, coronal and axial slices, as well as the 3d rendered image . It was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . Linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks (figure 1). For multiplanar groups, each landmark (figure 2) was identified and marked on the three orthogonal slices simultaneously (axial, sagittal and coronal). The corresponding linear measurements (figure 3) were determined electronically with the measurement tool on the sectional images . For performing the linear measurements which landmarks were not located in the same sectional image, as c mf, the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . As the volume rendering was reoriented, the positions of the landmarks were verified in the axial, sagittal and coronal slices and, if necessary, relocated . Figure 1frontal (left) and sagittal (right) views of mandible showing the points figure 2points . Reference in the anatomical view and in the cone - beam computed tomography (cbct) images figure 3definitions of mandibular linear measurements for 3d rendering images, the landmarks were identified directly on the surface for 3d images . The measurements were performed twice by 2 blinded independent examiners (tmff and ja). The physical measurements of the dry mandibles were obtained with a high - precision digital caliper (mitutoyo, mitutoyo sul americana, suzano, sp, brazil), identical to those used in similar studies . In this initial study, we selected landmarks that are relatively easy to identify linear measurements were identified on two occasions, one month apart, by one operator (mlp). The mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . The measurements were grouped into five groups: physical measurements (g1); measurements performed on multiplanar slices with 0.20 mm voxel size (g2); measurements performed on multiplanar slices with 0.40 mm voxel size (g3); measurements performed on 3d images with voxel size of 0.20 mm (g4) and measurements performed on 3d images with voxel size of 0.40 mm (g5). Intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . Analysis of variance for repeated measures (anova) and dunnett s test were used to compare the groups on cbct images and the physical measurements . All tests were performed with statistica software 6.0 (statistica for windows; statsoft, tulsa, ok, usa) and spss version 10 (spss, spss inc . Measurements were made separately on the multiplanar sections (2d) and on 3d rendering images display window of dolphin 3d . The multiplanar window displays the sagittal, coronal and axial slices, as well as the 3d rendered image . It was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . Linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks (figure 1). For multiplanar groups, each landmark (figure 2) was identified and marked on the three orthogonal slices simultaneously (axial, sagittal and coronal). The corresponding linear measurements (figure 3) were determined electronically with the measurement tool on the sectional images . For performing the linear measurements which landmarks were not located in the same sectional image, as c mf, the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . As the volume rendering was reoriented, the positions of the landmarks were verified in the axial, sagittal and coronal slices and, if necessary, relocated . Figure 1frontal (left) and sagittal (right) views of mandible showing the points figure 2points . Reference in the anatomical view and in the cone - beam computed tomography (cbct) images figure 3definitions of mandibular linear measurements for 3d rendering images, the landmarks were identified directly on the surface for 3d images . The measurements were performed twice by 2 blinded independent examiners (tmff and ja). The physical measurements of the dry mandibles were obtained with a high - precision digital caliper (mitutoyo, mitutoyo sul americana, suzano, sp, brazil), identical to those used in similar studies . In this initial study, we selected landmarks that are relatively easy to identify . Linear measurements were identified on two occasions, one month apart, by one operator (mlp). The mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . The measurements were grouped into five groups: physical measurements (g1); measurements performed on multiplanar slices with 0.20 mm voxel size (g2); measurements performed on multiplanar slices with 0.40 mm voxel size (g3); measurements performed on 3d images with voxel size of 0.20 mm (g4) and measurements performed on 3d images with voxel size of 0.40 mm (g5). Intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . Analysis of variance for repeated measures (anova) and dunnett s test were used to compare the groups on cbct images and the physical measurements . All tests were performed with statistica software 6.0 (statistica for windows; statsoft, tulsa, ok, usa) and spss version 10 (spss, spss inc ., chicago, il, usa). The measurements for investigator 1 showed excellent reliability with intraclass correlation coefficients (icc) ranging from 0.93 to 1 (icc average g2:0.97; g3:0.97; g4:0.98 and g5:0.98). Additionally, the mean absolute differences between the first and second measurements were less than 0.40 mm (mean average difference g2:0.11 mm and g3:0.02 mm) for multiplanar images and less than 0.64 mm (mean average difference g4:0.14 mm and g5:0.03 mm) for 3d model images . For investigator 2, the results also showed excellent reliability, with icc coefficients ranging from 0.88 to 0.99 (icc average - g2:0.96; g3:0.95; g4:0.97 and g5:0.97). Table 1intraobserver and interobserver variation for each group and analysis methods [intraclass correlation coefficient (icc) and bland - altman] multiplanar images3d model imagesvariable g2g3g4g5 0.2 voxel (n=10)0.4 voxel (n=10)0.2 voxel (n=10)0.4 voxel (n=10)intraobserver obser veradsd95% ciiccadsd95% ciiccadsd95% ciiccadsd95% ciiccc c 10.010.36 - 0.69; 0.711 - 0.010.15 - 0.31; 0.2910.120.67 - 1.20; 1.440.990.180.77 - 1.34; 1.700.98 2 - 0.410.75 - 1.89; 1.070.98 - 0.680.75 - 2.16; 0.800.970.170.87 - 1.54; 1.880.980.460.73 - 0.98; 1.900.98mf mf 1 - 0.130.46 - 1.03; 0.770.99 - 0.190.49 - 1.14; 0.760.980.170.72 - 1.25; 1.590.96 - 0.210.49 - 1.17; 0.750.98 2 - 0.301.18 - 2.16; 2.010.93 - 0.080.68 - 1.41; 1.250.97 - 0.210.82 - 1.82; 1.400.96 - 0.031.33 - 2.63; 2.570.92c mf 1 - 0.041.09 - 2.18; 2.100.970.310.67 - 0.99; 1.610.980.640.78 - 0.90; 2.180.980.260.81 - 1.32; 1.840.98 2 - 0.581.21 - 2.95; 1.790.95 - 0.920.97 - 2.81; 0.970.960.480.92 - 1.33; 2.290.970.420.75 - 1.04; 1.880.98c mf 10.190.84 - 1.46; 1.840.990.240.90 - 1.53; 2.010.970.390.67 - 0.93; 1.710.980.100.70 - 1.28; 1.480.99 2 - 0.660.81 - 2.24; 0.920.97 - 0.811.27 - 3.30; 1.680.940.100.73 - 1.34; 1.540.990.630.85 - 1.03; 2.290.97c mf 10.401.22 - 2.0; 2.800.950.051.40 - 2.68; 2.780.930.041.00 - 1.93; 2.010.960.261.06 - 1.83; 2.350.95 2 - 1.050.90 - 2.82; 0.720.91 - 0.291.01 - 2.27; 1.690.95 - 0.090.61 - 1.28; 1.100.990.410.68 - 0.92; 1.740.97c mf 10.121.40 - 2.62; 2.860.94 - 0.041.13 - 2.27; 2.190.950.090.90 - 1.67; 1.850.970.090.73 - 1.35; 1.530.98 2 - 0.650.88 - 2.36; 1.060.96 - 0.690.72 - 2.10; 0.720.96 - 0.020.99 - 1.96; 1.920.970.061.01 - 1.93; 2.050.97mco lco (right)1 - 0.030.39 - 0.80; 0.740.98 - 0.070.40 - 0.84; 0.700.980.140.28 - 0.42; 0.700.99 - 0.030.45 - 0.91; 0.850.98 20.260.47 - 0.66; 1.180.970.180.71 - 1.22; 1.580.950.770.54 - 0.29; 1.830.900.680.55 - 0.40; 1.760.92mco lco (left)10.390.44 - 0.47; 1.250.94 - 0.380.53 - 1.42; 0.660.930.010.35 - 0.67; 0.690.98 - 0.050.53 - 1.10; 1.000.95 2 - 0.050.53 - 1.08; 0.990.950.300.81 - 1.29; 1.890.88 - 0.080.43 - 0.93; 0.770.980.140.33 - 0.50; 0.780.98lco lco 10.300.38 - 0.34; 0.940.99 - 0.220.50 - 1.22; 0.780.99 - 0.030.31 - 0.65; 0.591 - 0.150.70 - 1.52; 1.220.99 2 - 0.300.70 - 1.09; 1.690.980.150.67 - 1.15; 1.450.990.770.53 - 0.28; 1.820.970.780.53 - 0.27; 1.830.97mco mco 1 - 0.070.35 - 0.76; 0.620.990.080.54 - 0.98; 1.140.98 - 0.220.35 - 0.91; 0.470.99 - 0.120.50 - 1.11; 0.870.99 2 - 0.330.51 - 1.34; 0.680.98 - 0.620.35 - 1.31; 0.070.97 - 0.080.35 - 0.78; 0.620.99 - 0.051.29 - 0.62; 0.520.99total10.11 0.970.02 0.970.14 0.980.03 0.98 2 - 0.41 0.96 - 0.35 0.950.18 0.970.35 0.97interobserverc c 1 x 2 - 0.540.98 - 2.46; 1.380.960.000.49 - 0.95; 0.960.99 - 0.110.88 - 1.84; 1.630.98 - 0.150.56 - 1.25; 0.950.99mf mf 1 x 21.000.61 - 0.20; 2.190.910.940.54 - 0.12; 1.990.923.591.490.66; 6.510.263.251.450.38; 6.090.45c mf 1 x 2 - 1.201.18 - 3.51; 1.150.93 - 1.491.09 - 3.63; 0.640.90 - 2.420.80 - 3.99; -0.840.84 - 2.560.66 - 3.86; -1.260.82c mf 1 x 20.631.05 - 1.45; 2.700.970.060.80 - 1.51; 1.640.980.071.20 - 2.30; 2.430.96 - 0.340.57 - 1.46; 0.790.99c mf 1 x 2 - 1.091.34 - 3.72; 1.530.89 - 1.070.94 - 2.91; 0.790.92 - 2.421.38 - 5.13; 0.300.70 - 2.471.06 - 4.56; -0.370.69c mf 1 x 20.400.60 - 0.78; 1.590.98 - 0.790.81 - 0.79; 2.380.950.070.68 - 1.28; 1.410.980.100.58 - 1.05; 1.240.99mco lco (right)1 x 2 - 1.770.54 - 2.83; -0.710.69 - 1.840.50 - 2.83; -0.860.67 - 0.180.84 - 1.83; 1.460.92 - 0.490.46 - 1.40; 0.410.95mco lco (left)1 x 2 - 1.430.54 - 2.49; -0.360.63 - 1.390.63 - 2.63; -0.140.63 - 1.130.84 - 2.79; 0.520.72 - 0.730.56 - 1.85; 0.380.86lco lco 1 x 2 - 1.420.55 - 2.50; -0.340.92 - 1.450.45 - 2.34; -0.550.93 - 0.091.16 - 2.35; 2.190.96 - 0.150.93 - 1.96; 1.670.97mco mco 1 x 21.990.461.08; 2.890.762.120.431.27; 2.960.761.340.610.14; 2.530.861.170.480.21; 2.130.90total -0.34 0.86 - 0.49 0.87 - 0.13 0.82 - 0.24 0.86ad: average difference; sd = standard deviation; ci: confidence interval; icc: intraclass correlation coefficient ad: average difference; sd = standard deviation; ci: confidence interval; icc: intraclass correlation coefficient cbct multiplanar reconstruction measurements showed good to high interobserver reliability with icc ranging from 0.63 to 0.99 (mean average g2=0.86 and g3=0.87) with the absolute differences between the first and second measurements ranging for 0 to 2.12 mm (mean average difference g2:-0.34 mm and g3:-0.49 mm, table 1). For 3d reconstruction measurements, independently of the voxel size (g4 or g5), most of the variables presented good to high interobserver reliability, with the exception of inter - mental foramen width (mf mf) that had fair reliability on 0.2 mm voxel images (g4=0.26) and moderate reliability for 0.4 voxel size (g5=0.45). The mean values and standard deviations of the physical measurements and of the cbct measurements for all groups are presented in table 2 . Two variables (mf mf and c mf) at groups g2 and g3 were significantly smaller than physical measurements (g1). For 3d rendering images (g4 and g5), only two variables out of 10 were not significantly smaller than the physical measurements . No significant difference was found between the voxel size both for 3d rendering and multiplanar sections . Table 2mean (mm) and standard deviation (sd) of linear measurements for dry mandibles compared to cone - beam computed tomography (cbct) images with different voxel sizes [analysis of variance (anova) for repeated measures and dunnett s tests] physical measurementmultiplanar images3d model images variableg1g2g3g4g5p 0.2 voxel0.4 voxel0.2 voxel0.4 voxel (n=10)(n=10)(n=10)(n=10)(n=10) meansdmeansdmdmeansdmdmeansdmdmeansdmd c c 101.033.98101.154.150.11100.974.01 - 0,06101.124.060,09101.184.260.140.697mf mf 49.17 3.0347.91 2.98 - 1.2647.91 2.81 - 1,2647.16 2.64 - 2,0147.76 3.01 - 1.420.000c mf 76.43 4.7176.50 4.420.0776.24 4.17 - 0,1875.59 4.60 - 0,8475.51 4.49 - 0.920.000c mf 104.41 4.63103.82 4.79 - 0.58103.56 4.26 - 0,85102.76 4.38 - 1,64103.10 4.47 - 1.310.000c mf 75.16 3.9975.66 3.640.5075.69 3.580,5374.48 3.46 - 0,6874.25 3.30 - 0.910.000c mf 102.65 3.92102.44 4.02 - 0.21102.33 3.76 - 0,32101.29 3.90 - 1,36101.38 3.81 - 1.270.000mco lco 21.42 2.4321.68 2.280.2621.69 2.280,2619.61 2.08 - 1,8120.07 2.19 - 1.350.000mco lco 21.37 2.0521.28 1.67 - 0.0921.02 1.68 - 0,3419.31 1.82 - 2,0619.14 1.74 - 2.230.000lco lco 124.25 4.05124.00 4.03 - 0.25123.78 4.10 - 0,47122.73 4.21 - 1,53122.74 4.04 - 1.510.000mco mco 83.873.0984.072.860.2084.132.970,2684.072.810,283.762.98 - 0.110.251sd, standard deviation; md, mean difference * statistically significantdifferent letters represent statistically significant differences (dunnett s test) sd, standard deviation; md, mean difference statistically significant different letters represent statistically significant differences (dunnett s test) the availability of this technology is undoubtedly expanding the use and application of 3d imaging in the field of orthodontics . However, while cbct provides many advantages, patient radiation dose is still higher than conventional cephalometric and panoramic digital imaging modalities . This study was performed to evaluate the reliability and accuracy of linear measurements between common landmarks of the mandible taken from cbct multiplanar sections and 3d volumetric renderings with different voxel sizes . In addition, larger voxel sizes are consistent with the alara principle, keeping the dose as low as reasonably achievable for the intended purpose of the scan . Most of the previous studies performed to validate cbct for 3d cephalometrics measured linear distances between anatomical landmarks in dry skulls in combination with radiopaque markers . Cephalometric analyses are subject to the influence of the examiner, landmark identification and type of image analyzed (2d sections or 3d image). The major question of this study was if the difficulty in identifying the landmarks in 3d images can influence the reliability and accuracy of the measurements, and for this reason, metallic markers were not used . This method is close to the real clinical procedures and is more clinically relevant than making measurements between well - defined metallic landmarks . There was excellent intraobserver reliability for both examiners with the mean icc for all groups above 0.95 and mean average differences less than 0.41 mm, independently of the protocol analyzed (table 1). These differences are not clinically significant for orthodontic / surgical diagnosis and treatment planning, and acceptable for skeletal measurements . Additionally, good to high interobserver precision was identified for all groups, with exception of measurement mf the main explanations for the good reliability found in this study are the use of landmarks which are easily identifiable, the calibration between observers and the high spatial definition of cbct images . The differences between multiplanar sections and 3d volume rendering images were probably because landmarks on multiplanar images were identified on the orthogonal slices simultaneously instead of being identified directly on the surface of 3d reformatted images . Our study also evaluated the accuracy of measurements performed in cbct images (table 2). Two variables at g2 and g3 and 8 variables at g4 and g5 were significantly smaller than physical measurements (g1). (2009), who showed a tendency for the cbct measurements to slightly underestimate the gold standard . The consistency of the differences we found suggests a systematic error in the 3d model measurement method . One possible explanation is that this error might have been introduced by the measurement software, mainly building line . Although few software systems currently have a 3d cephalometric module, most of them have not been tested or validated . The results of the validation measurements for a specific machine or software cannot be extrapolated to the entire cbct machine and software . Most of them are designed differently and software updates are constant and frequent . In addition, the difference ranged from 0.06 to 2.23 mm . In agreement with previous reports, the average difference in our study is below clinical significance . According to grauer, et al . (2009), generating measurements in 3d volumetric images rather than simultaneously in sets of 2d multiplanar images introduces error because of the difficulty involved in locating landmarks in 3d space and the inaccuracies of the user entered threshold used for the construction of 3d virtual surface models . Rendered for this reason, greater caution is suggested for linear measurements performed on 3d models . The spatial resolution of the image has an influence in the accuracy of the measurements . Most studies on cbct with a large field of view used 0.4 mm voxel size, whereas others with a small field of view used a 0.2 mm or smaller voxel sizes . None of the measurements showed significant difference between the voxel sizes, independently of the method of measurement (table 2). (2010), who have suggested that 0.4 mm voxel resolution is adequate for performing measurements of craniofacial structures . While small voxel sizes increase the image resolution, they also may increase the image noise . The benefits of a shorter scanning time, with its reduced likelihood of patient movement and especially the lower exposure dose compensates for the poorer resolution . Besides, large measurements are less influenced by small differences in spatial resolution . On the other hand, the diagnostic ability of cbct images for evaluating small structures appears to be influenced by voxel size . The two most common voxel sizes used in orthodontics 0.3 and 0.4 mm provide lower spatial resolution than smaller voxel sizes . Orthodontic scans with resolutions of 0.3 and 0.4 voxels are ideal for general treatment planning but should be used with caution if the goal is to assess small variations in bone thickness . (2009) investigated the usefulness of cbct for identifying artificial external root resorption in images with voxel sizes of 0.4, 0.3, and 0.2 mm . They concluded that, even though the results were the same for the different voxel sizes, diagnosis was easier at smaller voxel sizes of 0.3 and 0.2 mm . Although the benefits of a shorter scanning time satisfy the as low as reasonably achievable principle, the risks of misdiagnosis and treatment complications must also be weighed . Consequently, a scanning protocol with a 0.40 mm voxel size might not be suitable for every patient . Voxel size may vary according to the size of the structures to be analyzed as well as to the level of detail desired . In this study, we measured distances that are in excess of the resolutions 0.2 and 0.4 mm of the scan, so that voxel size would not very likely influence the measurements . Unlike in conventional cephalometrics, where all the landmarks are identified in one image, namely, the lateral cephalogram, with cbct each landmark must be identified in three different images (axial, coronal and sagittal), making the process of performing the measurements in cbct images more time - consuming for the orthodontist . Further, cbct demands a higher radiation dose than traditional cephalometric images . For these reasons, its use should be limited to specific indications, for example, patients with impacted teeth, or those with facial asymmetries or craniofacial anomalies where cbct is better able to quantify the differences between the right and the left side of craniofacial structures . Evidence - based will tell whether future advances, especially in terms of dose reduction, will make cbct appropriate for routine use in all orthodontic patients . Linear measurements obtained on multiplanar 2d cbct images with 0.2 and 0.4 voxel sizes using i - cat scanner and dolphin software are reliable and accurate for clinical diagnosis and treatment planning . Caution should be taken in linear measurements on 3d rendering images, because the measurements were reliable, but not accurate . Reducing the voxel size from 0.4 to 0.2 mm does not influence the accuracy and reliability of measurements of large craniofacial structures on cbct-3d images.
Patients with schizophrenia present major impairment in social functioning, independent living and work status, compared to healthy subjects [1, 2]. Besides the well - established positive and negative symptoms, recently it has been largely recognized that a generalized cognitive deficit is at the core of schizophrenia, and it significantly affects patient's social functioning [2 - 5]. Cognitive impairment in schizophrenia ranges from sensory and perceptual dysfunctions to higher order cognitive dysfunctions like working and episodic memory, attention, problem solving, processing speed [6 - 8]. The course of cognitive impairment in schizophrenia is characterized by deterioration close to and during the first psychotic episode and a relative stability afterwards . Nevertheless, patients with schizophrenia tend to present lower cognitive performance than the general population even in the premorbid phase [9, 10]. Antipsychotic medications have shown modest positive effects on different cognitive domains, without a preferential effect over a specific function [11 - 14]. Better treatment approaches to cognition could be achieved with a greater understanding on the pathogenesis of cognitive impairment in schizophrenia, which remains poorly understood . Evidence from studies with other neuropsychiatric disorders indicates that immuno - inflammatory processes may play a central role in cognitive deficits . For instance, it has been shown that inflammation may be an important neuropathological mechanism underlying cognitive decline and dementia in elderly population . Executive dysfunction was associated to inflammatory parameters in bipolar patients, with inhibitory control being positively correlated to tnf- levels . Levels of inflammatory markers have also been linked to cognitive function in major depressive disorder . For instance, high il-6 levels were associated to low performance in immediate and delayed verbal recall tests in recurrent depressed women . In schizophrenia, there is considerable evidence of changes in the immune system . Previous studies have shown an imbalance between type-1 and type-2 immune responses with a predominant type-2 response . However, a recent meta - analysis showed dominant pro - inflammatory changes in schizophrenia, but not type-2 or type-1 predominant immune response . After controlling for antipsychotic use as a confounding factor, only il-1ra and il-6 levels were elevated in schizophrenia . Another meta - analysis, that considered clinical status and antipsychotic effects, did not find an increase in type-2 cytokines either . Instead, the authors suggested that some cytokines are state markers of acute exacerbations (il-1, il-6 and tgf-), while others may be trait markers (il-12, ifn-, tnf-, sil-2r). Th1 derived cytokines il-12 and ifn- were elevated only in acute relapses and first episode psychotic patients, as well as macrophage derived cytokines il-6, tnf- and il-1 . Prenatal and perinatal infections could disrupt fetal neurodevelopmental processes, leading to brain long - lasting changes and increasing the risk of psychotic disturbances in early adulthood . Prenatal and perinatal infections could also act on the priming of immune activation in this early period of life . In this scenario, altered levels of il-1 and il-6 could influence the release of hormones by the hypothalamic - pituitary - adrenal axis, contributing to changes in monoamine neurotransmission . Therefore, infection and inflammation may trigger pathological mechanisms, resulting in proneness to psychosis and, possibly, cognitive dysfunction . Given the relevance of the cognitive impairment and the immune changes in schizophrenia as well as the putative role of the immune system in cognitive dysfunction, it turns relevant to investigate the association between cognitive and immune variables in schizophrenia . Therefore, the aim of the present study was to systematically review all the papers published concerning schizophrenia, cognition and immunity . We focused on studies that assessed cognitive and immune variables directly on the patients . By excluding studies that used other populations (e.g. Alzheimer, bipolar disorder) or basic research we tried to reduce complexity, homogenize results and to emphasize on studies with direct clinical implications nevertheless, articles not included in our selection, but whose content is relevant to our results, were included in the discussion . A search was conducted in the medline database, up to april 2013, comprising studies written in english, with the following terms in the abstract and/or title: schizophrenia or psychosis or psychotic and inflamm * or immun * or cytokine or il- * or tnf- * or kynureni * or kyna, and cognit * or attention or memory or executive function . Only original papers were included . Seventy five papers were identified using the selected terms . After a critical analysis of the abstracts, the papers excluded were either too basic, concerning chemical or biological in vitro research or animal studies with murine models with little contribution for clinical practice; or too broad, revising immunological aspects in other neuro - psychiatric disorders or concerning different symptomatological domains . Papers that only inferred that immune changes could affect cognition in schizophrenia, without actual tests, were also excluded to homogenize the sample and focus on clinical and practical aspects . Papers that assessed cognition and immune and/or inflammatory markers in patients with schizophrenia are displayed on table 1 . Clinical trials that added immuno - modulatory drugs to antipsychotic regimen are shown in table 2 . Four studies addressed associations between immune / inflammatory markers and cognitive functions in schizophrenia or first - episode psychosis . Elevated serum c - reactive protein (crp) levels were associated with the severity of cognitive impairment, but not with positive or negative symptoms . Patients with crp levels above 5 mg/l (high level group) had a lower score on the repeatable battery for the assessment of neuropsychological status (rbans), which is based on scales for immediate and delayed memory, visuo - constructional, language and attention . A recent study of the same group showed that the effects of elevated cpr levels and herpes simplex virus type 1 (hsv-1) seropositivity on cognitive impairment as assessed by rbans in patients with schizophrenia were additive and statistically independent . The largest cognitive impairment was found in the group with both factors, i.e. High levels of crp and hsv-1 antibodies, reinforcing the hypothesis that inflammation and infection may play a role in cognition . Another study correlated levels of either the chemokine monocyte chemoattractant protein 1 (mcp-1/ccl-2) or oxidative stress markers (nitrites and glutathione) with performance on cognitive tasks in a group of first - episode psychosis patients . Mcp-1 levels were negatively correlated to learning and memory performance, while oxidative stress markers were associated with poorer executive functioning . A recent chinese study found a positive association between interleukin 18 (il-18) levels and rbans visuospatial and constructional indexes in first episode and drug nave psychotic patients . Three studies yielded data about the effect of anti - inflammatory or immunomodulatory drugs as add - on therapy to the antipsychotic regimen on cognition of patients with schizophrenia . Using celecoxib (cyclooxygenase type 2 (cox-2) inhibitor) 400mg / day or placebo as add - on to risperidone, mller et al . Patients using celecoxib had a greater reduction of total score in the positive and negative symptoms scale (panss) when compared to the placebo group . Specific effects on cognition were evidenced after re - evaluation of data that showed that the effect on the panss cognition factor (items difficulty in abstract thinking and conceptual disorganization) was the most pronounced . The effect seems to be stronger in cases with more recent onset and shorter disease duration . Another trial added aspirin (1000 mg) to antipsychotic treatment in a group of 70 patients with schizophrenia for a three month period . It was argued that aspirin has a cardioprotective profile and that its unselective inhibition of cox-1 and cox-2 enzymes would allow a wider range of action . Statistically significant effects on the total score and positive subscale of panss were observed . Interferon- and the effect was larger in patients with a lower type-1/type-2 cytokine balance (defined by the median interferon-/ interleukin-4 ratio), suggesting that the reduction of positive symptoms was larger in patients with an immune profile tending to the type-2 response . Although aspirin seemed to improve psychiatric symptoms, cognition was not affected . A double - blind, randomized, placebo - controlled study with minocycline, an antibiotic which also exerts immuno - modulatory activity, as augmentation to antipsychotics found that it may alleviate negative symptoms and improve cognitive functioning in early - phase schizophrenia . Patients using minocycline showed improvement in executive functions (working memory, cognitive shifting and planning) in comparison to the placebo group measured by the cambridge neurospychological automated battery (cantab). Although the research linking cognition and inflammation in schizophrenia is still scarce, initial results are promising . Mcp-1 and il-18 levels, as well as crp levels and hsv-1 seropositivity, were linked to cognition in schizophrenia . Interventional studies that added immunomodulatory drugs to antipsychotic regimen also yielded interesting results, opening new possibilities to improve cognition and other psychiatric symptoms in schizophrenia . Elevated high sensitivity crp may be also a marker of memory and visuospatial impairment in the elderly, being associated with dementia . Crp is known to be elevated in those with risk factors common to stroke and dementia, such as diabetes, obesity and smoking . Recent studies suggested that assessment of low grade inflammation by high sensitivity crp can be related to cerebral microstructural disintegration, affecting frontal lobe pathways and leading to executive dysfunction . Frontal dysfunction is a common feature in schizophrenia and these findings may help explain how inflammation may undermine cognitive functions . Crp assessment could also help identify groups of patients with schizophrenia that would benefit from immunomodulatory drugs, improving cognitive function or even psychiatric symptoms . As well as inflammatory markers such as crp, it appears that immune activators such as viruses could be related to cognition in schizophrenia . The concept of early - life programming of adult disease postulates that specific environmental factors acting during sensitive prenatal or early postnatal developmental periods can induce persistent changes in physiological, emotional and behavioral functions throughout life . These factors could prime not only the immune system and its responses in adult life, but cause neurodevelopmental changes and increase proneness to psychosis . Prenatal maternal immune activation, inflammation, viral infections, as well as psychological stress and malnutrition appear to affect offspring development, increasing the risk of psychotic disorders later on, impairing sensorimotor gating, information processing, cognition, social function and leading to subcortical hyperdopaminergia [22, 36]. Prenatal influenza infections are associated with altered neuronal migration in cortical and hippocampal neurons, which are essential for broad functioning of many cognitive domains . Different kinds of viruses are known to alter the monoaminergic balance within the brain after experimental infection, particularly serotonin and noradrenaline - mediated systems . Hsv-1 lifelong cycles in the brain may cause neuronal damage and dysfunction and may also be associated with longitudinal gray matter loss in the posterior cingulate gyrus and decline in executive functioning among subjects with schizophrenia . Significant association between the neurocognitive summary score, verbal memory, vigilance and processing speed; and antibodies to hsv-1 was described in the catie trial (clinical antipsychotic trial of intervention effectiveness). These findings may help explain the association of hsv-1 and cognition in schizophrenia described in the results by dickerson [25, 26] and also possible underlying pathological changes . We could also hypothesize that microglial activation may occur in early neurodevelopment in these cases and be linked to first episode psychosis as well, converging previous neuro - structural abnormalities and monoaminergic imbalance to behavioral and cognitive impairment . Mcp-1 is a chemokine that recruits monocytes, dendritic and t cells to the inflammation site . It was hypothesized that in psychotic patients the systemic oxidative - inflammatory status may influence cognitive performance through inflammatory mediators in cerebral vasculature or increase in blood - brain permeability . Mcp-1 has already been linked with cognitive deficits associated with hiv dementia and alzheimer s disease [41, 42]. Therefore, inflammatory processes initiated by mcp-1, like microglia activation and leucocyte migration, appear to be associated to cognition in schizophrenia as shown by the negative association between mcp-1 levels and learning and memory (p=0.009). This is in line with the microglia hypothesis of schizophrenia, which postulates that pro - inflammatory cytokines and free radicals produced by an activated microglia may decrease neurogenesis, result in white matter abnormalities and favor neurodegeneration, contributing to the neuropathology of the disorder . Animal experiments have shown that il-6 can increase dopaminergic neurotransmission in the hippocampus, and il-2 also increases serotonin and noradrenaline - mediated neurotransmission . Although no differences were found between il-18 levels in first episode patients with schizophrenia and healthy controls, there was a positive association between il-18 levels and rbans visuospatial / constructional cognitive index . These findings seem paradoxal since there are studies associating il-18 levels to worse cognitive function in alzheimer s disease and multiple sclerosis . Due to the association between viral infections and schizophrenia, it is proposed that il-18 release after viral infection would lead to microglial activation with interferon gamma (ifn-) release in the brain parenchyma and viral clearance . Another explanation is that the visuospatial index may be more resilient to be impaired by il-18 than other cognitive functions measured by rbans . It is still uncertain how each immune marker affects neurodevelopment, and therefore also influences cognition . Damage control, for example, following viral infection in pre or perinatal period, avoiding or minimizing structural abnormalities associated to schizophrenia and cognitive impairment . Most clinical trials that used anti - inflammatory or immunomodulatory drugs in addition to antipsychotics in order to verify cognitive effects yielded positive results . It is an antibiotic with anti - inflammatory, anti - oxidative and anti - apoptotic properties . Its neuroprotective properties may arise from astrocytic and microglial caspase 1 inhibition, as well as nitric oxide synthase inhibition, which also affects the glutamatergic system . The dopaminergic system may also be affected, since minocycline attenuates dopamine elevation following nmda agonist administration . Preclinical inflammation models also showed impact on inflammatory markers, such as tnf-, il-1, pge2 and cox-2 [32, 46, 47]. Pro - inflammatory cytokines and free radicals produced by microglia can contribute to neuron degeneration . There have been reports of inhibitory effects of typical and atypical antipsychotics in inflammatory and oxidative stress mediated by the microglia; factors which have been recently associated with reduced neurogenesis and white matter abnormalities . Since patients were described as having early - phase schizophrenia, authors suggest that intervention with immunomodulatory drugs may display better results in initial stages of the disease and improve clinical course . Celecoxib also seems to improve cognition, although the observed improvement derived from panss cognitive factors and not neurocognitive tests per se . Celecoxib is a selective anti - inflammatory drug that inhibits cox-2, but not cox-1 . Contrasting with its isoform cox-1, cox-2 is induced by stimulation in most tissues, but it is constitutively expressed in the central nervous system structures such as frontal cortex, amygdala and hippocampus which are critically involved in cognitive functioning [38, 48]. Cox-2 activity can be stimulated by tissue damage and also by glutamate excitation, showing its high sensitivity to neuronal stress . Cytokines such as il-2, il-6 and il-10 activate cox-2, and result in inflammatory response also in the central nervous system . Hence, cox-2 inhibition seems to balance type 1/ type 2 immune responses by inhibition of pge2 and stimulation of type 1 immune response, and also by inhibition of kyna (kynurenic acid) production . Kyna is an antagonist at the glycine site of the n - methyl - d - aspartic acid receptor (nmdar) and at the 7 nicotinic acetylcholine receptor (7nachr), both of which implicated in the cognitive impairment of schizophrenia [51, 52]. Increased immune function, primarily in the blood, would raise most kynurenine metabolites and in result, elevate also kyna levels in the brain . Since the kynurenine pathway of tryptophan metabolism is induced by immunological activation and stress, it can mediate the effects of environmental factors in cognition and behavior . Therefore, it may be a promising metabolic pathway for developing new pharmacological interventions to treat and prevent cognitive dysfunction in schizophrenia and other neuro - psychiatric disorders . The beneficial effects of cox-2 inhibition may also be due to alteration in glutamate neurotransmission, with inhibition of the nmda receptors and activation of kainate receptors . Mller et al . Also proposed that cox-2 inhibitors may have a role in learning and memory by affecting long - term potentiation and long - term depression, as well as attenuating cholinergic dysfunction . Therefore, many mechanisms could explain the positive effects of cox-2 inhibitors on cognition in schizophrenia . As argued by mller and schwarz when evaluating the effect of anti - inflammatory drugs on cognition in schizophrenia therapeutic studies have shown beneficial effects of anti - inflammatory drugs mostly in early stages, with little effect in late stages . Mller et al . Stated that patients with a shorter duration of disease improved more in the aforementioned clinical trial . These findings could reflect that an early anti - inflammatory therapy could prevent neuronal damage and structural changes caused by chronic inflammation in schizophrenic patients over many years of disease evolution . Aspirin (acetylsalicylic acid) is a widely used anti - inflammatory agent that inhibits inflammatory cyclo - oxygenase pathways and suppresses prostaglandins and tromboxane . . Found that the group of patients with lower th1/th2 balance benefited more from the use of aspirin than the others . This finding is in line with other studies and the hypothesis of an imbalance between pro and anti - inflammatory forces . Cognition was not affected, but authors argued that it was a stable domain over time in these patients, and that a longer time could be necessary to observe changes . Laan et al . Also hypothesized that aspirin may act by antagonizing nmda dysfunction and that it exerts its effects by interacting with antipsychotics . As only a few studies that addressed simultaneously cognition and immunological changes in schizophrenia are available to date, the results must be interpreted cautiously . The exclusion of research or animal models may have narrowed the discussion, excluding other hypothetical mechanisms linking immune changes and cognition in schizophrenia that have not been transposed into clinical studies yet . Regarding immunomodulatory drugs, many parameters are still undefined, such as duration, dose and time of initiation of a possible immunomodulatory drug . Moreover, it appears that positive effects are more evident in the initial stages of schizophrenia . One of the clinical trials revised did not use standard neuropsychological assessment battery in schizophrenia (panss cognition factor). It is advised a careful selection of the neuropsychological battery used to obtain more reliable results in future trials, in order to compare drug efficacy and cognitive improvement . Schizophrenia is a heterogeneous syndrome and immune changes may be another neuropathogenic mechanism among many already described . Cognition is one of the main compromised domains in schizophrenia and accounts for a great part of the patient s functioning and quality of life . Clinical trials have shown improvement on cognitive parameters and other symptoms with the addition of immunomodulatory drugs . It seems very likely, though, that inflammation and immune dysfunction affect neurodevelopment, increase risk of psychotic illnesses and continue to affect cognitive processes throughout life . Possible underlying mechanisms include monoaminergic imbalance, microglial activation, structural abnormalities in white matter, frontal lobe, hippocampus; as well as changes in the kynurenine pathway of the tryptophan metabolism affecting glutamatergic and cholinergic neurotransmission . Future clinical trials using either anti - inflammatory drugs, nicotinic or glutamatergic agents or manipulation of brain kyna levels should address cognition more directly . More basic and clinical studies are needed in order to further enlighten the mechanisms underlying cognitive deficits and pursue new cognitive - enhancing drugs to the treatment of the disorder.
Anorexia is an eating disorder that generally affects adolescents, triggering severe relational imbalance to the family . The emotional functioning of families with an anorexic adolescent plays a pivotal role in the evolution of the disorder and in the therapies addressed not only to the adolescents but to the family as well.1 nevertheless, still little is known about emotional functioning in this kind of family . Studies conducted on the general population have found a link between family pathological functioning and alexithymia, in particular between family emotional dysfunctions and difficulties identifying feelings.2 despite the significance of this link, literature has focused greatly on individual alexithymia, producing only scarce and conflicting studies on alexithymia in families of anorexic patients.35 evidence, however, is now highlighting the importance of family dynamics in the development of the anorexic disorder,68 especially in younger patients.1,9 literature seems to confirm the link between use of body symptoms and difficulties in managing and expressing emotions at the symbolic verbal level . In fact, patients with eating disorders, anorexia in particular, reveal significantly higher alexithymic traits and difficulties in regulating affect.1012 in first diagnosed anorexic adolescents, emotional dysregulation is associated with lower levels of diazepam - binding inhibitor, a peptide involved in mood eating behavior regulation,13 and directly correlated to the severity of autonomic dysregulation.12 difficulties in identifying feelings have proven to be an important prognostic factor in the evolution of anorexia.14 it must, however, be noted that research on the topic, conducted on adolescent samples,1519 continues to be scarce, although this pathology typically arises in adolescence . The emotional deficits of anorexic patients may be connected with their parents emotional functioning.9 studies on adult patients have yielded contradictory results . In dahlmans3 first study, the mothers of anorexic patients resulted more alexithymic than controls, but this has not always been confirmed in subsequent studies.4 furthermore, even in studies reporting higher levels of alexithymia in parents of an anorexic daughter,5 this alexithymic state seems mostly linked to considerable emotional distress, often linked to traumatic experiences.4,5 in general, studies conducted using self - report questionnaires (eg, the toronto alexithymia scale [tas-20]) show that the high levels of alexithymia reported by patients are often correlated to depression and negative affectivity.14,2023 the high levels of alexithymia measured by self - report are not always confirmed by performance - based measures devised to assess the actual ability to attribute and express emotions.24,25 this would trigger doubts on the validity and reliability of alexithymia self - administered measuring systems . Self - report instruments are inevitably influenced by the subject s will and ability to refer to what he or she feels, thus by the very capacity an alexithymic is not supposed to have.2628 a more reliable assessment of such a complex construct as alexithymia would generally entail multimethod measuring29,30 that combines a categorial (diagnostic) approach as well as a dimensional (trait intensity) approach, attained using both self - administered and interviewer - administered instruments . An interesting study by berthoz et al31 conducted on eating disorders (ed) patients has paved the way in this direction, comparing various alexithymia measuring instruments, among which we can find the widespread tas-20 self - report and another, lesser known, report (observer alexithymia scale dhaviland32). Considering the interesting results of the aforementioned study, the prime aim of this research was to use a multimethod measurement to assess alexithymia in anorexic adolescent patients and in their parents . For such a purpose two different instruments were chosen, a self - administered questionnaire and a structured clinical interview, tas-2033,34 and the toronto structured interview for alexithymia (tsia).35 both instruments developed by the toronto - based group are theoretically similar, thus enabling a comparison of their sensitivity and ability to detect the presence and intensity of alexithymic components in families of anorexic patients . The second aim of the study was to broaden the limited knowledge on alexithymia in adolescent anorexic patients and in their parents, considering them not only as isolated individuals but as members of an interconnected triad, a restricted family nucleus that has specific ways of expressing and managing feelings . We considered each triad as a restricted family nucleus in order to compare the alexithymia of the daughter, the mother, and the father and to determine the mean alexithymia score for each family nucleus . The sample included 46 subjects: 16 anorexic adolescent patients (aged 1317 years) and their 30 parents . The subjects were consecutive attendees at the child and adolescent neuropsychiatric university unit of the san gerardo hospital, university of milan - bicocca, monza, italy, from march to june 2013 . We considered each triad (daughter, mother, father) as a restricted family nucleus . Due to ethical problems, siblings, where present, were excluded from this study, avoiding their medicalization . The study was carried out according to the standards for good ethical practice of san gerardo hospital . Selection was based on the following inclusion criteria: restrictive diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm iv - tr) diagnosis of anorexia nervosa, female, aged from 13 to 17 years, and parents consent to participate in the study . Exclusion criteria instead included another major diagnosis (autism, schizophrenia, mental retardation) and undergoing psychotherapy for an extended period (more than 3 months). The anorexic adolescent patients mean age was 15.81 years, ranging from 13 to17 years, and their mean current body mass index was 16 (standard deviation = 2), resulting in being quite representative of the population usually attending the child and adolescent neuropsychiatric university unit and entering clinical research programs.12,13,19,36,37 among the 32 parents, one of the fathers had passed away and one of the mothers could not be traced at the time of the research . The mothers mean age was 45.8 years, ranging from 36 to 52 years, and fathers mean age was 49.13 years, ranging from 38 to 61 years . This instrument provides the total score and also measures three major components of alexithymia: difficulty identifying feelings (f1), difficulty describing feeling to others (f2), and externally oriented thinking (f3). There are 20 items, each evaluated using a five - point likert scale, starting with 1 (strongly disagree) to 5 (strongly agree). The total score can vary from a minimum of 20 to a maximum of 100 . The italian translation of the tas-20 has demonstrated factorial validity, internal consistency, and high test it comprises a total of 24 items subdivided into four subscales (each with six items) designed to evaluate four core dimensions of alexithymia: difficulty identifying feelings (dif), difficulty describing feelings (ddf), externally oriented thinking (eot), and imaginal processes (imp). Each question is scored on a three - point likert scale ranging from 0 to 2 . Higher scores indicate a higher degree of alexithymia, and total scores range from 0 to 48 . Its design stems from the need to compensate the weaknesses of the tas-20 (table 1): self - assessment and exclusion of a measure of poor imaginal processes . The presence of an external interviewer guarantees, contrarily to what happens with the self - report questionnaire, that the answer be investigated and clarified whenever the interviewee shows scarce consciousness of his / her problems . The tsia used along with the tas-20 allows a multimethod assessment based on different measuring instruments, self and other report.30,39,40 in the validation study of the toronto group,35 as well as in the validation of the italian version,41 the tsia obtained a good interrater, test retest, and internal reliability and concurrent validity with the tas-20 . The hierarchic factor structure is made up of four subscales grouped into two higher order factors: conscious emotional awareness (subscale 1 and 2) and operational thinking (subscale 3 and 4). A neuropsychiatrist with special interest and clinical experience in anorexia evaluated all patients, collecting a comprehensive family and medical history . Patients, diagnosed using the dsm - iv - tr criteria for restricting type anorexia nervosa, also completed the eating disorder inventory (edi-3).42 the diagnosis of comorbid disorders was supported by the kiddie - sads semistructured interview.43 patients were asked to fill out the tas-20 questionnaire and undergo the tsia structured interview . The answers to the tsia were collected by two specifically trained interviewers, both unaware of the scores obtained in the tas-20 questionnaire . Interviews were then scored by two judges, one being an expert in using the instrument . In rare cases of disagreement in attributing the score, the study relied on the use of rasch analysis.44 the andrich rating scale model45 was applied to estimate the measurements obtained from likert type ratings provided by participants for each of the two scale items . Model program46 was used to estimate the level of alexithymia expressed by each person on the rating scale . Since the study s main aim is to compare the item responses4751 provided by participants, rasch analysis was applied to the data because of its psychometric features, in particular the invariance of comparisons property . As is generally known, when data fit the rasch model properly and when comparing the responses of two people, such comparison is independent from stimuli or items that determine the responses . Rasch analysis was applied separately for the two scale total scores and for each of the common factors of the scales (factor 1, dif; factor 2, ddf; factor 3, eot). Furthermore, to compare the total scores and scale factors between the two scales, the estimates of each person were standardized . Subsequently, an alexithymia mean standard score was calculated for each family nucleus . To evaluate the difference between alexithymic levels of family members, a contrast standard value was calculated for each couple in the family, more specifically daughter versus mother, daughter versus father, and mother versus father . To summarize the data related to the 16 families, three categories, representing different levels of alexithymia, were created for the families and separately for daughters, mothers, and fathers: category 1 (low) included standard scores below 1.64 (p<0.05), thus respondents with low alexithymia levels; category 2 (middle) ranges between 1.64 and 1.64, thus with middle alexithymia levels; and category 3 (high) above 1.64 (p<0.05), thus high alexithymia levels . Considering the contrasts between family members, denominating a and b the members of a couple, category 1 (low) included couples with statistically different levels of alexithymia, where a is less alexithymic than b (below 1.64, p<0.05); category 2 (middle) included couples with statistically nonsignificantly different levels of alexithymia (between 1.64 and 1.64, p>0.05); and category 3 (high) contained couples with statistically different levels of alexithymia, where a is more alexithymic than b (above 1.64, p<0.05). Due to the low number of respondents, the frequencies in the categories were converted into percentages.52 the estimated standard scores were categorized instead of calculating mean scores, based on the hypothesis that a comparison between the two scales at low and high levels of distributions should be particularly suited for the purpose of this study . A series of chi - square analyses were elaborated to analyze the differential functioning between the two scales in detecting alexithymia in families, family members, and couple contrasts . Chi - square analyses were performed separately for the common factors and the two scale total scores and for the two extreme categories, low and high . The two extreme categories were considered and the middle one was excluded to highlight statistically significant results . The sample included 46 subjects: 16 anorexic adolescent patients (aged 1317 years) and their 30 parents . The subjects were consecutive attendees at the child and adolescent neuropsychiatric university unit of the san gerardo hospital, university of milan - bicocca, monza, italy, from march to june 2013 . We considered each triad (daughter, mother, father) as a restricted family nucleus . Due to ethical problems, siblings, where present, were excluded from this study, avoiding their medicalization . The study was carried out according to the standards for good ethical practice of san gerardo hospital . Selection was based on the following inclusion criteria: restrictive diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm iv - tr) diagnosis of anorexia nervosa, female, aged from 13 to 17 years, and parents consent to participate in the study . Exclusion criteria instead included another major diagnosis (autism, schizophrenia, mental retardation) and undergoing psychotherapy for an extended period (more than 3 months). The anorexic adolescent patients mean age was 15.81 years, ranging from 13 to17 years, and their mean current body mass index was 16 (standard deviation = 2), resulting in being quite representative of the population usually attending the child and adolescent neuropsychiatric university unit and entering clinical research programs.12,13,19,36,37 among the 32 parents, one of the fathers had passed away and one of the mothers could not be traced at the time of the research . The mothers mean age was 45.8 years, ranging from 36 to 52 years, and fathers mean age was 49.13 years, ranging from 38 to 61 years . This instrument provides the total score and also measures three major components of alexithymia: difficulty identifying feelings (f1), difficulty describing feeling to others (f2), and externally oriented thinking (f3). There are 20 items, each evaluated using a five - point likert scale, starting with 1 (strongly disagree) to 5 (strongly agree). The total score can vary from a minimum of 20 to a maximum of 100 . The italian translation of the tas-20 has demonstrated factorial validity, internal consistency, and high test it comprises a total of 24 items subdivided into four subscales (each with six items) designed to evaluate four core dimensions of alexithymia: difficulty identifying feelings (dif), difficulty describing feelings (ddf), externally oriented thinking (eot), and imaginal processes (imp). Each question is scored on a three - point likert scale ranging from 0 to 2 . Higher scores indicate a higher degree of alexithymia, and total scores range from 0 to 48 . Its design stems from the need to compensate the weaknesses of the tas-20 (table 1): self - assessment and exclusion of a measure of poor imaginal processes . The presence of an external interviewer guarantees, contrarily to what happens with the self - report questionnaire, that the answer be investigated and clarified whenever the interviewee shows scarce consciousness of his / her problems . The tsia used along with the tas-20 allows a multimethod assessment based on different measuring instruments, self and other report.30,39,40 in the validation study of the toronto group,35 as well as in the validation of the italian version,41 the tsia obtained a good interrater, test retest, and internal reliability and concurrent validity with the tas-20 . The hierarchic factor structure is made up of four subscales grouped into two higher order factors: conscious emotional awareness (subscale 1 and 2) and operational thinking (subscale 3 and 4). This instrument provides the total score and also measures three major components of alexithymia: difficulty identifying feelings (f1), difficulty describing feeling to others (f2), and externally oriented thinking (f3). There are 20 items, each evaluated using a five - point likert scale, starting with 1 (strongly disagree) to 5 (strongly agree). The total score can vary from a minimum of 20 to a maximum of 100 . The italian translation of the tas-20 has demonstrated factorial validity, internal consistency, and high test it comprises a total of 24 items subdivided into four subscales (each with six items) designed to evaluate four core dimensions of alexithymia: difficulty identifying feelings (dif), difficulty describing feelings (ddf), externally oriented thinking (eot), and imaginal processes (imp). Each question is scored on a three - point likert scale ranging from 0 to 2 . Higher scores indicate a higher degree of alexithymia, and total scores range from 0 to 48 . Its design stems from the need to compensate the weaknesses of the tas-20 (table 1): self - assessment and exclusion of a measure of poor imaginal processes . The presence of an external interviewer guarantees, contrarily to what happens with the self - report questionnaire, that the answer be investigated and clarified whenever the interviewee shows scarce consciousness of his / her problems . The tsia used along with the tas-20 allows a multimethod assessment based on different measuring instruments, self and other report.30,39,40 in the validation study of the toronto group,35 as well as in the validation of the italian version,41 the tsia obtained a good interrater, test retest, and internal reliability and concurrent validity with the tas-20 . The hierarchic factor structure is made up of four subscales grouped into two higher order factors: conscious emotional awareness (subscale 1 and 2) and operational thinking (subscale 3 and 4). A neuropsychiatrist with special interest and clinical experience in anorexia evaluated all patients, collecting a comprehensive family and medical history . Patients, diagnosed using the dsm - iv - tr criteria for restricting type anorexia nervosa, also completed the eating disorder inventory (edi-3).42 the diagnosis of comorbid disorders was supported by the kiddie - sads semistructured interview.43 patients were asked to fill out the tas-20 questionnaire and undergo the tsia structured interview . The answers to the tsia were collected by two specifically trained interviewers, both unaware of the scores obtained in the tas-20 questionnaire . Interviews were then scored by two judges, one being an expert in using the instrument . In rare cases of disagreement in attributing the score, the study relied on the use of rasch analysis.44 the andrich rating scale model45 was applied to estimate the measurements obtained from likert type ratings provided by participants for each of the two scale items . Model program46 was used to estimate the level of alexithymia expressed by each person on the rating scale . Since the study s main aim is to compare the item responses4751 provided by participants, rasch analysis was applied to the data because of its psychometric features, in particular the invariance of comparisons property . As is generally known, when data fit the rasch model properly and when comparing the responses of two people, such comparison is independent from stimuli or items that determine the responses . Rasch analysis was applied separately for the two scale total scores and for each of the common factors of the scales (factor 1, dif; factor 2, ddf; factor 3, eot). Furthermore, to compare the total scores and scale factors between the two scales, the estimates of each person were standardized . Subsequently, an alexithymia mean standard score was calculated for each family nucleus . To evaluate the difference between alexithymic levels of family members, a contrast standard value was calculated for each couple in the family, more specifically daughter versus mother, daughter versus father, and mother versus father . To summarize the data related to the 16 families, three categories, representing different levels of alexithymia, were created for the families and separately for daughters, mothers, and fathers: category 1 (low) included standard scores below 1.64 (p<0.05), thus respondents with low alexithymia levels; category 2 (middle) ranges between 1.64 and 1.64, thus with middle alexithymia levels; and category 3 (high) above 1.64 (p<0.05), thus high alexithymia levels . A and b the members of a couple, category 1 (low) included couples with statistically different levels of alexithymia, where a is less alexithymic than b (below 1.64, p<0.05); category 2 (middle) included couples with statistically nonsignificantly different levels of alexithymia (between 1.64 and 1.64, p>0.05); and category 3 (high) contained couples with statistically different levels of alexithymia, where a is more alexithymic than b (above 1.64, p<0.05). Due to the low number of respondents, the frequencies in the categories were converted into percentages.52 the estimated standard scores were categorized instead of calculating mean scores, based on the hypothesis that a comparison between the two scales at low and high levels of distributions should be particularly suited for the purpose of this study . A series of chi - square analyses were elaborated to analyze the differential functioning between the two scales in detecting alexithymia in families, family members, and couple contrasts . Chi - square analyses were performed separately for the common factors and the two scale total scores and for the two extreme categories, low and high . The two extreme categories were considered and the middle one was excluded to highlight statistically significant results . The alexithymia rasch estimate standard score for each family member, the family mean standard score, the alexithymia contrast standard score between family members in relation to the tsia and the tas-20 total scores, and their common factors are presented in table 2 . The data were obtained for each of the 16 families that participated in the study . For illustrative purposes, only the data related to a single family are introduced herein . In the selected family, the tsia detects significantly high alexithymic scores at the total score level for both daughter and father, whereas the mother does not show a significant score . The daughter shows a significantly high score, in particular in relation to factor 1, whereas the mother shows a significantly low level of alexithymia . Concerning factor 2, mother and father have significantly high levels of alexithymia, whereas in factor 3 only the father has a significantly high score . The tas-20 provides a very different functioning both at the individual and at the family levels . All members of the family, as regards the total score and, in particular, factors 1 and 2, show significantly low levels of alexithymia . Only for factor 3, although the alexithymia is low, is the result not significant . It is worth noting that even the distance between levels of alexithymia of family members differs based on the two scales . Considering the tsia, the daughter proves to have notably greater difficulties in identifying feelings (factor 1) compared with the mother or the father . This is not the case in the tas-20, where all the family members scored similarly low . The tsia factor 2 instead shows that the daughter is better able to describe her own feelings compared with her father . The opposite occurs with the tas-20, where the father considers himself less alexithymic than his daughter . As stated, to summarize the data of the 16 families, three categories representing different levels of alexithymia were created for the families and separately for daughters, mothers, and fathers . Furthermore, to evaluate the contrasts between alexithymic levels of couples in the family, the same categories were constructed for daughter versus mother, daughter versus father, and mother versus father . The results are illustrated graphically, where the two scales are compared in relation to the three common factors and total score . In figure 1, the distribution of frequency percentages is associated with the low and high categories for family alexithymia mean standard score; similarly, in figure 2, the distribution of frequency percentages with the family members; and, in figure 3, the distribution of frequency percentages relates to the contrasts between family members for the categories low and high . Successively, chi - square analyses were performed to test the statistical significance of the differential functioning between the two scales in relation to the factors and to the total scores . The data related to the 16 families in figure 1 indicate that, for all the factors and for the total score, the tas-20 is characterized by a significantly (p<0.001) larger number of cases (range 18.5%75%) than the tsia (range 0.0%2.5%) associated with the low category, which contains the families with average low levels of alexithymia, whereas the tsia is specified by a significantly (p<0.001) higher number of cases (range 6.25%25%) (except for factor 1) associated with the high category, which includes the families with average high levels of alexithymia . Particularly evident is the differential function of the two scales in relation to factor 1 and to the total score at the low category level and to factor 3 at the high category level . The data from our sample in figure 2 indicate that, for each family member, for all factors and total scores, the tas-20 shows a significantly (p<0.001) larger number of cases (range 20%100%) than the tsia (0.0%3.3%) associated with the low category, whereas the tsia in general shows a significantly (p<0.001) higher number of cases (6.7%53.3%) than the tas-20 (range 0.0%8.75%) associated with the high category . Considering the low category, a particularly salient difference is between the two scales with regard to factor 1, factor 3, and the total score for the daughter, factor 3 and the total score for the mother, and factor 1 for the father . In the high category, the two scales show different functioning, especially in relation to factor 3 for the daughter, factor 2 for the mother, and factor 3 for the father . In figure 3, the distribution of frequency percentages is concerned with the contrasts between members in the family for the low and high categories . The low category contains the cases where the first member in the couple has a significantly lower level of alexithymia than the second member in the couple . On the contrary, the high category includes the cases where the first member in the couple has a significantly higher level of alexithymia than the second one . Once again, the tsia scale can serve to better illustrate the relations among and between family members compared with the tas-20, both at the low and the high level . The alexithymia rasch estimate standard score for each family member, the family mean standard score, the alexithymia contrast standard score between family members in relation to the tsia and the tas-20 total scores, and their common factors are presented in table 2 . The data were obtained for each of the 16 families that participated in the study . For illustrative purposes, only the data related to a single family are introduced herein . In the selected family, the tsia detects significantly high alexithymic scores at the total score level for both daughter and father, whereas the mother does not show a significant score . The daughter shows a significantly high score, in particular in relation to factor 1, whereas the mother shows a significantly low level of alexithymia . Concerning factor 2, mother and father have significantly high levels of alexithymia, whereas in factor 3 only the father has a significantly high score . The tas-20 provides a very different functioning both at the individual and at the family levels . All members of the family, as regards the total score and, in particular, factors 1 and 2, show significantly low levels of alexithymia . Only for factor 3, although the alexithymia is low, is the result not significant . It is worth noting that even the distance between levels of alexithymia of family members differs based on the two scales . Considering the tsia, the daughter proves to have notably greater difficulties in identifying feelings (factor 1) compared with the mother or the father . This is not the case in the tas-20, where all the family members scored similarly low . The tsia factor 2 instead shows that the daughter is better able to describe her own feelings compared with her father . The opposite occurs with the tas-20, where the father considers himself less alexithymic than his daughter . As stated, to summarize the data of the 16 families, three categories representing different levels of alexithymia were created for the families and separately for daughters, mothers, and fathers . Furthermore, to evaluate the contrasts between alexithymic levels of couples in the family, the same categories were constructed for daughter versus mother, daughter versus father, and mother versus father . The results are illustrated graphically, where the two scales are compared in relation to the three common factors and total score . In figure 1, the distribution of frequency percentages is associated with the low and high categories for family alexithymia mean standard score; similarly, in figure 2, the distribution of frequency percentages with the family members; and, in figure 3, the distribution of frequency percentages relates to the contrasts between family members for the categories low and high . Successively, chi - square analyses were performed to test the statistical significance of the differential functioning between the two scales in relation to the factors and to the total scores . The data related to the 16 families in figure 1 indicate that, for all the factors and for the total score, the tas-20 is characterized by a significantly (p<0.001) larger number of cases (range 18.5%75%) than the tsia (range 0.0%2.5%) associated with the low category, which contains the families with average low levels of alexithymia, whereas the tsia is specified by a significantly (p<0.001) higher number of cases (range 6.25%25%) (except for factor 1) associated with the high category, which includes the families with average high levels of alexithymia . Particularly evident is the differential function of the two scales in relation to factor 1 and to the total score at the low category level and to factor 3 at the high category level . The data from our sample in figure 2 indicate that, for each family member, for all factors and total scores, the tas-20 shows a significantly (p<0.001) larger number of cases (range 20%100%) than the tsia (0.0%3.3%) associated with the low category, whereas the tsia in general shows a significantly (p<0.001) higher number of cases (6.7%53.3%) than the tas-20 (range 0.0%8.75%) associated with the high category . Considering the low category, a particularly salient difference is between the two scales with regard to factor 1, factor 3, and the total score for the daughter, factor 3 and the total score for the mother, and factor 1 for the father . In the high category, the two scales show different functioning, especially in relation to factor 3 for the daughter, factor 2 for the mother, and factor 3 for the father . In figure 3, the distribution of frequency percentages is concerned with the contrasts between members in the family for the low and high categories . The low category contains the cases where the first member in the couple has a significantly lower level of alexithymia than the second member in the couple . On the contrary, the high category includes the cases where the first member in the couple has a significantly higher level of alexithymia than the second one . Once again, the tsia scale can serve to better illustrate the relations among and between family members compared with the tas-20, both at the low and the high level . The prime aim of this study is to examine how the two measuring instruments, the self - report and the clinical interview, developed by the toronto - based group differ in their ability to detect the presence and intensity of alexithymia within the anorexic patients families . Despite its widespread use and unquestionably easy application in research domains, the tas-20 questionnaire has nonetheless revealed some important limits in terms of accuracy and clinical validity, especially when examining psychopathological conditions.27,53 families with anorexic adolescents can prove to be a particularly difficult context that calls for more in - depth clinical evaluation of alexithymia rather than the faster, more economical self - report evaluation . Differently from other studies in literature, latent trait analysis adopted in this research allowed us to examine each subject s alexithymia at a latent level, independently from the content of the items and from other intervening variables, due to the administering set.54 latent trait analysis therefore allows the patients and parents alexithymic levels to be compared as detected by the two instruments . Significant discordance was found between the two measures, both in the total as well as in the components of alexithymia, common to both scales (f1-dif, f2-ddf, f3-eot). The clinical instrument detected a greater level of alexithymia compared with the self - report . In particular, in our adult parent sample, alexithymia was higher when based on the structured interview administered by clinicians rather than on the answers the subjects provided themselves, the sole exception being the first factor for the mothers . In such case, the lower scores detected by the two instruments were not significantly different, because mothers generally tend to be aware of their own ability to identify feelings (f1), which is correctly reported in the self - administered test too . In the young anorexic girls, the tsia interview, compared with the tas-20, detected a greater level of alexithymia in the total score and in externally oriented thinking (f3). The results on the difficulties in identifying (f1) and expressing personal feelings (f2) are not as clear . Tas-20 detects a greater number of lower scores compared with tsia, perhaps due to the girls lack of awareness or attempt to deny difficulties,55 whereas the clinical interview and the self - report have a comparable tendency to score higher in f1 and f2 . This is in line with literature, as studies conducted using the tas-20 self - report alone have succeeded in detecting the significant difficulties anorexic people have in recognizing and verbalizing their emotions (f1, f2).4,10,14,22 the clinical interview in tandem with the self - report questionnaire adopted in our study, as the authors of the two instruments suggest,35,40 have yielded substantially different and clinically relevant information on our sample, especially on adults . In fact, a sense of deep crisis and distress for the daughter s condition can lead parents to adopt denial and massive defensive attitudes toward self - administered questionnaires . Otherwise, parents may be so anxious and under such stress that they are unable to answer the questionnaire appropriately, despite their willingness to do so.5 some studies have reported high levels of emotional distress, negative affectivity, depression, and anxiety in the parents of anorexic patients, probably linked to the high levels of alexithymia revealed by self - reports.4,5,9 we can therefore assume that the interview administered by trained clinicians might be more sensitive to the actual level of alexithymia, as it excludes or attenuates the confounding factors generated by parents negative affective state, defense mechanisms, varying levels of awareness, and capabilities to reflect on feelings.35,36 the study results on the overall level of family alexithymia are worthy of notice . Compared with the tas-20, the tsia interview has identified a much higher level of family alexithymia for all of the factors . In the total score, the mean family alexithymia index was very high in some families (13%) only when using the tsia measurement, while no families had low alexithymic scores . No family had high alexithymic scores, with as much as 75% of the families scoring very low, based on the answers provided by the subjects . This marked difference between the two measures of family alexithymia is attributable especially to the scores that fathers obtained, which are lower for all of the tas-20 factors, thus when they were the ones to answer the questionnaire rather than when evaluated by the clinician . This marked difference may highlight the fathers inability to detect their emotional difficulties, perhaps linked to a defense style founded on external oriented thinking and concrete factual problem solving.5 considering the role of paternal rather than maternal alexithymia is somewhat innovative, compared with the approaches in current studies on adult patients and their parents.4,5 conceivably, this particular way of functioning of fathers may, in part, be a previous salient characteristic of their personality . Within the couple, concrete thinking and cutting off emotions could lead these men to have a collusive relationship with more emotional reactive women . The complex dynamics created in this kind of couple may subsequently include young anorexic daughters . Nonetheless, a pivotal point to bear in mind is that the adolescent daughter s life - threatening condition of being is a traumatic factor that often leads such men, who may perhaps already be inclined, to adopt massive defense mechanisms and to freeze their emotions.5 this type of paternal response to the daughter s pathology can, in turn, negatively affect the evolution, triggering a vicious circle . Currently, plenty of documented proof illustrates how a lack of communication and emotional communication perceived by the patients in the families,6,9,56 and a greater extent of coldness and hostility,7,9 even on the father s part,7,57 can play a negative role in eating disorders . Recent evidence,9 in fact, has pointed to the important role of fathers in anorexic adolescents . If paternal warmth and participation have a positive effect on the outcome of the daughter s anorexia, on the contrary, the paternal alexithymia studied herein can imply lesser involvement and therefore negatively impact the quality of family relations and thus the girl s outcome.9 we therefore wish to underscore that, under a therapeutic perspective, the father s role and the reciprocal emotional communication among family members need to be tackled . Encouraging the participation and support of fathers, who generally tend to slip away, wishing to remain excluded emotionally and concretely, can prove crucial when trying to modify intrafamily dynamics, which appear to be the key for successful family therapy in adolescent patients with anorexia.1 family relationships are well worth investigating from such a perspective . In the case of alexithymia even at this level the tsia and the tas-20 differ, especially when considering, within the couples, the father s alexithymia, which turns out to be lower in the self - report and higher in the interview . In considering total alexithymia, this difference is even more striking from the use of the clinical interview, which minimizes the paternal negation tendency . Total alexithymia, when measured with the tsia, revealed a wide gap between the two partners in as much as 71% of the couples, with husbands being more alexithymic than wives . The level of alexithymia between the two partners is therefore comparable only in a very limited number of couples where there is a sort of alexithymic accordance, whereas the opposite case, that of a more alexithymic wife, was not detected in any couple . Many daughters also have a lower level of alexithymia compared with their father s when measured with the tsia clinical interview . The exact contrary is true with the tas-20 self - report, as fathers are more inclined toward negation, whereas, in both self - report and interview, most cases resulted in more alexithymic daughters compared with their mother, whose alexithymia is generally not high . It may be a significant clinical index that could influence family relationships.58 subsequent studies could explore the value of this index as a discriminant factor in relation to the kind of family functioning, the potential therapeutic alliance with the families, and perhaps the daughter s outcome . In fact, literature considers family relationships to be more relevant than the parents individual personality and psychopathological traits in predicting the anorexic daughter s alexithymia.4 in addition, unhealthy family functioning and affective responsiveness have proved to be connected with the specific risk factors that may trigger anorexia.8 likewise, after the onset of the disorder in the daughter, individual psychological traits of family members and the way they express emotions continue to be interrelated to the clinical severity of anorexia.9,56,59 one of the major innovations in our study is the use of the tsia . To date, no other study has explored the alexithymia of adolescent anorexic patients and their parents using self - report in tandem with a clinical interview . Results suggest that a clinical interview may be a more sensitive instrument in detecting alexithymia . Compared with the tas-20, the clinical interview may detect different, and perhaps finer, elements and thus be better at capturing the gap among the different family members alexithymia . This would justify carrying out the validation process of the tsia for the italian adolescent population . Aside from the aforementioned lack of validation for italian adolescents, the study s other weaknesses include not having a control group and requiring an expanded adolescent parent sample . It is important to note that the rasch modeling approach applied in this study, due to the model s property invariance of comparisons, should substantially overcome the limitation of lacking a comparator group.54 future research should thus be extended to include other families within the sample and, whenever possible, the patient s siblings within each family . Presently, this was not possible due to ethical problems and to the family s reluctance to adhere . Future studies could also integrate an evaluation of other psychological aspects (ie, attachment,51 depression19,20) studied not only at the individual level but also within the family system . The value and the relevance of the alexithymic index at the family level could thus be assessed . In the future, the authors expect to carry out a prolonged observation of the patients evolution to gain better understanding of whether the variables studied are prognostically significant . Results seem to indicate that the clinical interview may be a more sensitive instrument compared with the self - report, allowing data integration on the functioning of adolescent anorexic patients and their parents . The tsia interview can therefore be an instrument worth validating for the adolescent age group . Moreover, the study of a couple s functioning in terms of alexithymia has spotlighted a significant alexithymic gap within families, particularly within parental couples, where fathers are noticeably more alexithymic than mothers . Clinical questions have arisen on how useful it may be to give greater weight to family functioning rather than to individual personality traits in predicting the possibility of establishing a therapeutic alliance and thus the outcome of the anorexic adolescent.
During the course of sepsis pattern recognition receptors (prrs) of the innate immune system such as toll - like receptors (tlrs) have been shown to play a decisive role in the disease process . Toll - like receptors bind pathogen associated molecular patterns (pamps) originating from non - self - sources as well as damage associated molecular patterns (damps) deriving from the host organism itself . In the human organism eleven tlrs are expressed, among those tlr4 the receptor for endotoxins has been described most extensively . However, in the last few years another tlr has entered the focus of scientific interest: the receptor for viral and bacterial dna tlr9 . Bacterial dna is characterized by unmethylated cytosine - phosphate - guanine oligodesoxynucleotide (cpg motifs), which are much less prevalent in mammalian dna . This receptor has been found to be of pivotal importance during polymicrobial sepsis [3, 4]. The reason for its relevance may lie in the common appearance of cpg motifs in all bacterial and viral dna . Therefore, tlr9 can be regarded as a central receptor for inflammatory response in any kind of sepsis . Importantly, the presence of impaired cardiovascular function in sepsis is associated with significantly increased mortality [5, 6]. Hence, any successful intervention should aim to improve sepsis - dependent suppression of cardiovascular function . In vitro tlr9 signaling induced by the application of the synthetic stimulatory oligonucleotide 1668-thioate suppressed cardiac contractility via increased inos expression, which could be antagonized by s - methylisothiourea (smt). In addition, myocytes with tlr9-deficiency (tlr9-d) proved to be insensitive to stimulation with a synthetic substitute for bacterial dna . Thus, pharmacological antagonism of tlr9 might protect the cardiovascular system from the deleterious effects of bacterial dna . Dilatation of blood vessels exposed to polymicrobial stimuli can be prevented by the synthetic odn (h154-thioate) inhibiting tlr9 . However, the in vivo effect of tlr9 antagonism on the heart has not yet been investigated . Therefore, we wondered whether application of a synthetic oligodesoxynucleotide (1668-thioate) would be sufficient to depress cardiac function in vivo . With respect to the importance of effective inhibition of tlr signaling in sepsis, the aim of the present study was to test different tlr9 inhibitors (h154-thioate, irs954-thioate, and chloroquine) for their effectiveness in protecting the cardiovascular system during systemic inflammation . In order to distinguish tlr9 signaling from other inflammatory pathways the specific tlr9 ligand 1668-thioate was chosen as stimulus . Ten - to - twelve - week - old male c57bl/6 mice were purchased from charles river, sulzfeld / germany . The body weight varied from 20 to 25 g. tlr9-deficient mice (tlr9-d; backcrossed on c57bl/6 background) were kindly provided by professor shizuo akira (department of host defense, research institute for microbial diseases, osaka university, japan). All animal studies were approved by the state office for nature, environment and consumer affairs (landesamt fr natur, umwelt und verbraucherschutz) of north - rhine westphalia (recklinghausen, germany) and conformed to the guidelines for animal experimentation of the national institute of health (nih publication no . Buprenorphine was administered (0.1 mg / kg subcutaneously). In a pilot study cultured macrophages of the line raw 264.7 were stimulated with heat - inactivated feces of c57bl/6 mice for 24 h (concentration 10 bacteria / ml). Thereafter, tnf- protein was measured by elisa in the supernatant of the cell culture . Stimulation led to a 30-fold increase of tnf- protein compared with control (figures 1(a)1(c)). This increase was used to test the suppressive effect of the tlr9 inhibitors h154-thioate, irs954-thioate, and chloroquine [810]. H154-thioate was applied simultaneously with the polymicrobial stimulus in three different concentrations (50 mg / l, 25 mg / l, and 0.5 mg / l). All applied concentrations of h154 were able to reduce the tnf- protein significantly in a concentration - dependent manner (figure 1(a)). Comparable experiments were performed with irs954-thioate and chloroquine (figures 1(b) and 1(c)). Chloroquine was applied in four different concentrations (2.5, 10, 50, and 100 mg / l); the lowest effective concentration was 10 mg / l . In order to ensure efficaciousness of the antagonists a single dose of 8 mg / kg bw of h154- and irs954-thioate and 10 mg / kg of chloroquine was applied i.v . To the animals . All animals were initially treated with d - galactosaminen (d - galn; 1 g / kg bw, roth, karlsruhe, germany). D - galactosaminen was applied to slow down the hepatic degradation of cpg - odns [11, 12]. In control experiments d - galn alone did not induce an inflammatory response . In order to induce a systemic inflammation thirty minutes later all mice were injected i.p . With 1668-thioate (5-tccatgacgttcctgatgct; tibmolbiol, berlin, germany; 2 nmol / g bw = 12.1 mg / kg bw) in a total volume of 400 l pbs or with the same amount of pbs . Another 30 minutes later the mice received intravenous (i.v) treatment with one of four agents: (1) a control odn (1612-thioate; 5-gctagatgttagcgt-3; tibmolbiol, berlin, germany; 2 nmol / g bw = 9.3 mg / kg bw), (2) with h154-thioate (5-cctcaagcttgagggg-3; tibmolbiol, berlin, germany; 8 mg / kg bw), (3) with irs954-thioate (5-tgctcctggaggggttgt-3; tibmolbiol, berlin, germany; 8 mg / kg bw), (4) with chloroquine (10 mg / kg bw; bayer vital gmbh, leverkusen, germany). The mrna expression levels of tnf-, il-6, and il-1 were determined using taqman real - time quantitative pcr (rt - qpcr, applied biosystems, darmstadt, germany). Upon excision of the hearts total rna was isolated (trizol, applied biosystems) and first - strand cdna was synthesized using the high - capacity cdna transcription kit (applied biosystems) with random hexameric primers according to the manufacturer's protocol . Rt - qpcr was performed and analyzed with cdna (diluted 1: 10) on an abi prism 7900 sequence detection system and sds2.2 software (applied biosystems). Target gene expression was normalized to an internal control (glyceraldehyde-3-phosphate dehydrogenase, gapdh). Relative rt - pcr was performed using taqman gene expression master mix (part 4369016; applied biosystems) with the following primers: gapdh (mm99999915_g1), tnf- (mm00443258_m1), il-1 (mm99999061_g1), and il-6 (mm01210732_g1). All murine primers were measured using fam tamra chemistry and the relative standard curve method . At the end of rt - qpcr cycle six hours after stimulation with 1668-thioate hemodynamic parameters which included left ventricular systolic pressure (lvsp), stroke volume (sv), left ventricular end - diastolic pressure (lvedp), cardiac output (co), and contractility indices (dp / dtmax and dp / dtmin) were recorded using a pressure - volume catheter according to the manufacturer's manual (millar instruments, houston tx). Additionally, body temperature was monitored in representative mice using a rectal probe (figure 2(a)). For detailed descriptions significance testing included one - way anova followed by newman - keuls post hoc analysis . Clinical appearance as well as body temperature was investigated in wt and tlr9-d mice up to 18 h after 1668-thioate stimulation . In addition, survival was monitored in all groups of stimulated wt mice . After only 2 h wt mice started to display sepsis - like symptoms such as ruffled fur, food refusal, and limited ability to respond to external stimuli . In the following neither the specific tlr9 antagonist irs954 nor the unspecific tlr9 inhibitor chloroquine improved the clinical appearance of wt mice stimulated with 1668-thioate in vivo . However, the application of h154-thioate improved clinical behavioral patterns obviously: none of the mice in this group showed external signs of systemic inflammation . In accordance with this tlr9-d mice also did not exhibit any clinical symptoms . Six hours after 1668-thioate stimulation body temperature in both wt and tlr9-d mice significantly decreased; however, body temperature in tlr9-d mice remained above that of wt mice (figure 2(a)). Interestingly, wt mice cotreated with h154-thioate presented nearly the same temperature as tlr9-d mice (figure 2(a)). These findings concur with the observation that all wt animals treated with 1668-thioate were dead shortly after 6 hours . H154-thioate proved to be the most effective substance improving survival by 40% (figure 2(b)). Furthermore, all tlr9-d animals survived longer than 18 hours after 1668-thioate application (data not included in figure 2(b)). Two hours and six hours after 1668-thioate stimulation mediators of inflammation (tnf-, il-1, il-6) were monitored in cardiac tissue (figures 3(a)3(f)). In wt animals, 1668-thioate challenge resulted in a significant upregulation of all three proinflammatory cytokines at both time points with maximal expression at 2 h (figures 3(a), 3(c), and 3(e)). The additional application of the control oligonucleotide 1612-thioate led to a further increase in il-1 and il-6 mrna expression at 2 h (figures 3(c) and 3(e)). Four hours later, all three cytokines had decreased by at least 40% (figures 3(b), 3(d), and 3(f)). The application of h154-thioate diminished the 1668-thioate - dependent cytokine increase of all three mediators 2 h after stimulation . After 6 h this suppression was no longer detectable in neither tnf- nor il-1 (figures 3(b) and 3(d)). Neither irs954-thioate nor chloroquine suppressed cardiac tnf- or il-1 mrna expression at any time point (figures 3(a)3(d)). However, there was a significant downregulation of cardiac il-6 mrna expression in the 1668-thioate + irs954-thioate group 2 h after stimulation (figure 3(e)). Chloroquine application induced a biphasic response of il-6, enhancing its expression at 2 h and diminishing it at 6 h (figures 3(e) and 3(f)). In cardiac tissue of tlr9-d animals, application of 1668-thioate did not influence the mrna expression of the investigated mediators . In clinical symptoms and survival as well as in cytokine parameters of cardiovascular performance monitored with a pressure - volume catheter in wt as well as in tlr9-d mice 6 h after stimulation with 1668-thioate are given in figures 4(a)4(f). There was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . In agreement with cytokine mrna expression, additional application of h154-thioate significantly improved lv function heart rate (hr; data not shown), left ventricular systolic pressure (lvsp), stroke volume (sv), cardiac output (co), end - diastolic volume (edv), velocity of pressure increase (dp / dtmax), and velocity of pressure decrease (dp / dtmin) were improved by h154-thioate in comparison to 1668-thioate + pbs (figures 4(a)4(f)). Deficiency for tlr9 entirely prevented any deterioration of cardiac function (figures 4(a)4(f)). This study aimed to extend the in vitro finding that tlr9 stimulation decreases cardiomyocyte contractility to the in vivo setting . To our knowledge the present results are first to formally demonstrate that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation . The second attempt of the present investigation was to test possible tlr9 inhibitors (h154-thioate, irs954-thioate, and chloroquine) for their cardioprotective properties . The three tested inhibitors exhibited differential potencies; that is, h154-thioate proved to be the most effective substance . 1668-thioate has been applied in various experimental settings for the induction of a tlr9-dependent systemic inflammation as well as organ dysfunction [7, 11, 14, 16, 17]. The effect of tlr9-dependent systemic inflammation as well as organ dysfunction seems to be dose dependent as a low concentration (0.25 nmol / g) can serve as a mild stimulus for cardiac preconditioning, whereas higher concentrations in the range from 0.5 to 1 nmol / g induce sepsis - like inflammation [7, 17]. In order to warrant clear changes in cardiac function the formerly applied dose of 1 nmol / g was doubled to 2 nmol / g bw in the current study . Furthermore, the relatively high dose of 1668-thioate applied here seemed to be meaningful with respect to testing antagonizing strategies . Treatment either with a control odn (1612-thioate) or with one of the three inhibitors . Application of the antagonists was to avoid a possible direct interaction between stimulating and inhibitory substances in the peritoneal cavity . Specific tlr9 stimulation led to a time - dependent upregulation of cardiac tnf-, il-1, and il-6 mrna expression with a peak 2 h after stimulation and a reduced level 4 h later . This time course as well as the tlr9-dependent cytokine induction confirms earlier findings of our group . An interesting new aspect is that costimulation with 1612-thioate increased the expression of all three pro - inflammatory mediators at 2 h, reaching significance in the case of il-1 and il-6 mrna . However, prestimulation with 1668-thioate may sensitize the organism to succeeding stimuli in a way that originally inert substances (1612-thioate) are able to act as second hits, thereby further increasing the inflammatory response . A possible explanation for the enhanced mediator response may lie in the weak immune stimulatory properties of the charged phosphorothioate backbone of 1612-thioate . H154-thioate was the sole antagonist, which significantly depressed the upregulation of the three tested cytokines 2 h after stimulation with 1668-thioate . Chloroquine failed to develop any protective influence at this time point, but even enhanced the expression of il-6 (figures 3(a), 3(c), and 3(e)). Four hours later, the expression of the three cytokines had fallen to at least 60%, which was still significantly above the pbs and tlr9-d controls . Also in the groups with additional application of antagonists the absolute levels of cytokine expression had fallen . Significant antagonistic effects appeared only for the combinations of il-6 and h154-thioate as well as il-6 and chloroquine (figures 3(b), 3(d), and 3(f)). Ligands applied from the extracellular space have to be internalized and transported to the endosomal compartment . Chloroquine has been shown to disrupt this vesicle trafficking and acidification, which can explain the inhibition of the innate immune response . These data were mainly derived from in vitro experiments . However, there are reports that chloroquine is also effective in vivo [8, 20]. In a model of polymicrobial sepsis, chloroquine improved survival and reduced renal injury as well as systemic inflammation . With respect to survival these findings appear to contradict ours . However, there are differences between the model in this study and that of yasuda et al . . We applied a specific tlr9 stimulus in a concentration high enough to induce a drastic systemic inflammation causing death of 100% of the animals shortly after 6 h, whereas yasuda et al . Applied cecal ligation and puncture (clp) with a mortality of 50% after more than 48 h; that is, the clp - dependent inflammation was less severe than the one in the present study . In addition to the inhibition of vesicle trafficking, it has been proposed that chloroquine decreases tlr9 protein . Thus, tlr9 signaling itself may be a major target for the protective actions of chloroquine . This downregulation of tlr9 protein was detected 18 h after application of chloroquine; this mechanism is possibly too slow to develop a beneficial influence in our model . In another attempt, hong et al . Demonstrated that chloroquine at a dose of 30 mg / kg bw could protect mice from lethal challenge by 1668-thioate, whereas chloroquine at a dose of 25 mg / kg bw could decrease serum tnf- and il-6 in rats injected with sublethal doses of cpg - odn . In our hands, preliminary tests of a high chloroquine dose of 30 mg / kg bw caused a 100% mortality immediately after i.v . We therefore stayed with a dose of 10 mg / kg bw as explained in material and methods section . This divergence in chloroquine sensitivity may be attributed to differences in mouse strains (balb / c versus c57bl/6n mice). Taken together, in our model of cpg - induced sepsis the protective abilities of chloroquine could not be confirmed . Synthetic oligonucleotides containing inhibitory properties such as h154-thioate and irs954-thioate are frequently used in experimental studies to suppress tlr signaling [21, 22]. Pisetsky's group described synthetic oligonucleotides containing poly - g sequences, which block bacterial dna - induced activation [23, 24]. Furthermore, it has been suggested that suppressive odns interfere with the phosphorylation of signal transducer and activator of transcriptions 1 and 4 (stat1 and stat4, resp . ), thereby blocking inflammation mediated by stat - associated signaling cascades . This interaction may be highly specific because suppressive odns do not bind to other molecules in the nfb and mapk regulatory cascade, and control odns do not bind to stat1 or stat4 [25, 26]. Furthermore, in vitro studies have demonstrated that inhibitory odns preferentially bind to the c - terminal of tlr9, competing for cpg - odn binding . With respect to the structural details of inhibitory odns, ashman et al . Demonstrated in vitro that a specific motif (ccx(not - c)(not - c)xxggg (x = any base)) provides the sequences required to block tlr9 in human b cells and hek cells transfected with human tlr9 . Extending the sequence by four to five bases at the 5 end enhanced activity, even more so when a phosphorothioate backbone replaced the native phosphodiester backbone (for further details, see [25, 26]). In addition to the molecular composition of an inhibitory odn also the targeted cell type has been shown to influence the inhibitory potency of the respective odn; that is, some inhibitory sequences that function well in macrophages have been shown to be inefficient in b cells . Taken together, the workings of molecular blocking mechanisms of inhibiting odn have not yet been unraveled; differences in the potency of h154-thioate and irs954-thioate up to now cannot be attributed to their molecular structure . Are generated in a highly controlled environment, which is not the case in living organisms . Therefore, transfer from in vitro to in vivo cannot be assumed to be self - evident . Thus, the presented cardiodepressive effect of 1668-thioate stimulation in vivo enhances our knowledge of the understanding of tlr9 stimulation . This is further supported by the total insensitivity of tlr9-d mice to this stimulation . To our knowledge, this study is the first demonstration that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation . In our experimental setting h154-thioate there was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . In agreement with cytokine mrna expression, additional application of h154-thioate significantly improved lv function when compared to mice treated with 1668-thioate + pbs . Specifically, heart rate, left ventricular systolic pressure, stroke volume, cardiac output, end - diastolic volume, velocity of pressure increase, and velocity of pressure decrease were improved by h154-thioate in comparison to 1668-thioate + pbs (figures 4(a)4(f)). Deficiency for tlr9 entirely prevented any deterioration of cardiac function (figures 4(a)4(f)). Our finding that h154-thioate protected the heart against tlr9-stimulation complements the earlier observation that h154-thioate prevented tlr9-dependent vascular relaxation . The importance of tlr9 signaling in polymicrobial infection has already been shown by others and by our group [3, 4]. A possible reason for this may lie in the ability of tlr9 to bind the dna of gram - positive as well as gram - negative bacteria and thus to react to a broad range of stimuli [3, 30, 31]. This may have contributed to the disappointing results of the application of eritoran (e5564), a tlr4 antagonist, evaluated in the access (a controlled comparison of eritoran and placebo in patients with severe sepsis) trial, a global, randomized, double - blind, placebo - controlled phase iii study . Taken together, here we show that systemic tlr9 stimulation is able to depress cardiac function in vivo and that a pharmacological intervention is possible and may be a promising strategy for human clinical trials in future.
For centuries, the human brain remained impervious to direct observation and experimentation (penfield, 1958), but with the advent of magnetic resonance imaging (mri) this situation changed very quickly . The technique supported the fastest and most significant progress toward understanding the functional architecture of the human brain; after three decades of mri, thousands of individuals of all ages, conditions, and denominations have been scanned, leading to the emergence of comprehensive models of brain structure and function . Functional imaging studies recently broadened their focus from measuring local phenomena associated with specific perceptual and cognitive tasks, to identifying long - range networks involved in the orchestration of neural activity (sporns et al ., 2005). These networks are revealed statistically using resting - state functional mri (r - fmri) based on the time course of slow fluctuations in the bold signal when the subject is not engaged in any particular task and when the brain is at rest whether r - fmri - derived models depend on hard - wired anatomical connectivity is an open question, so this technique is often combined with diffusion tensor imaging (dti) to show major fiber tracts in the deep white matter (pierpaoli et al ., 1996; bandettini, 2009). These non - invasive neuroimaging modalities are the most widely utilized methodologies for mapping brain circuitry at the system level, and thus also the primary tools of the human connectome project (hcp; akil et al ., 2011). The problem with images of the brain acquired in vivo with mri and dti is that they are limited in both resolution and contrast . The data needs be averaged across many subjects who represent broad demographic or clinical groups in order to extract significant measures . In fact, even the quantification of biomarkers that are crucial clinically for the individual patient is contingent on population - based atlases that only contain very rudimentary anatomical information . In practice, it is not possible with mri to localize the exact borders of neighboring neuroanatomical structures and identify the underlying histological features on which radiologic images depend . The latter can only be determined postmortem . Given the undeniable importance of validating mri, why have neuroanatomical and histological studies have only played a small, tangential role in human brain mapping and why does the hcp, despite the large investment by the national institute for health (nih), not formally include a parallel histological program? The constraints are not conceptual; everyone agrees it would be very useful to corroborate non - invasive measures with baseline information on the actual properties of brain tissue . However, dti - based models of fiber tract orientation in the human brain are currently validated only on the basis of classic atlases (dejerine, 1895; flechsig, 1901; ludwig and klinger, 1956; yakovlev and lecours, 1967). These contain anachronistically zealous, yet subjectively construed illustrations and were derived from the dissection of a few undocumented brain specimens, so the level of comparison is severely limited . The challenges in creating a comprehensive histological model of the human brain that matches the scope and usability of mri population - based templates, including the proposed connectome, are primarily logistical . To begin with, any study that aims at examining cellular - level features in a brain depends on the expiration of the subject, and consequently neuroanatomical studies have been confined to those few cases where autopsies were prescribed because of clinical or legal concerns or to individuals who enrolled in tissue donation programs . Because in the first scenario the rate of autopsy has universally been decreasing (kretzschmar, 2009), the enhancement of brain donation programs (via incentives, outreach, and education) represents the only realistic future opportunity of supporting a large - scale, neuro - histological mapping effort . Assuming that the necessity of enrolling a large enough number of brain donors has been addressed, noting that the benefits of such donor program cannot be expected to roll out in the short - term, what pre - mortem information should characterize the specimen and how should the brain be processed in order to create images that can complement and validate mri studies? Because the main goal is to bridge the gap between non - invasive neuroimaging and histology it would be useful to have access to radiologic images acquired in vivo for each case that comes to autopsy . When comparisons are made between images from different modalities in the context of the same subject all the information necessary to localize and quantify microscopic features underlying the patterns revealed in mri images is available . Furthermore, the correspondence between images acquired in vivo and those from histopathology is enormously improved if the latter cover the whole brain, just like mri . These methodological prerogatives are the basis for a novel neuroimaging resource that will be used to cross - reference different modalities and establish fundamental properties of brain structure at multiple scales . Her neuroimaging profile was created using a palette of multiple modalities that provided correlated images of the brain at different levels of resolution . The study that is described in this communication was approved by the university of california's institutional review board . H.t . Was a woman of caucasian descent born in rochester, ny in 1920 . A smoker for 50 years, her only major medical concern was hypertension and she took medication to lower her blood pressure . She died of heart failure . During the last year of her life she experienced a slight decline in memory and cognitive proficiency (it should be noted that these phenomena were observed by her closest relatives; h.t . Never recognized or admitted to having any impairment). The examination of the clinical scans by a trained neuroradiologist did not report major abnormalities in the brain of h.t . With the exception of several small lacunar infarcts . H.t . Died of cardiac arrest at the age of 88 . Because she wore a pacemaker at the time of her enrollment in our study (thus precluding her from undergoing mri scanning while she was living), multiple scans of the brain in situ were acquired postmortem . Images of the brain were acquired on an hdx twinspeed excite 1.5 t scanner (milwaukee, wi .) Using an eight - channel phased - array head coil . Scan parameters were selected to allow robust reconstruction of the brain's cortical surface using freesurfer software and automated segmentation of cortex and subcortical structures (fischl et al ., 2002, 2004; dti imaging consisted in the collection of 51 diffusion gradient directions (figure 2a). (a) t1-weighted scan of the brain of h.t . Acquired in situ 3d ir - spgr, matrix size: 256 256, voxel size of 0.9375 0.9375, slice thickness = 1.2 mm . The volume underwent the automated labeling of subcortical structures and cortical gyri that is part of the morphometry pipeline included in the software freesurfer (fischl et al ., 2002, 2004). (b) high - resolution t1-weighted scan of the formalin - fixed specimen imaged ex situ . The brain was scanned in a dedicated chamber filled with phosphate buffer (matrix size: 512 512, pixel size: 0.5/0.5, slice thickness: 1.2 mm). Multiple acquisitions were repeated in order to increase the signal - to - noise ratio . (c) coronal blockface image from the same brain at the level of the posterior hippocampus . (d) corresponding histological slice stained for myelinated fibers [protocol modified from gallyas (1979); annese et al . Comparison between (a) fiber orientation maps derived from dti and (b) the underlying myelo - architecture in the region of intersection of the fibers belonging to the corpus callosum (cc) which travel along the coronal plane (parallel to the plane of the figure) and the superior corona radiata (scr). In this example, the region of intersection between cc fibers and the scr is an area that shows significant crossing over of fibers of small caliber (blue box). (c) one dti voxel - wide selection from silver - stained tissue slice (magnification = 20). (d) the density and orientation of single white matter fibers is quantified using a template matching algorithm [bartsch et al . The insert in panel d shows the discrete orientations of the templates used to classify fiber bundles . Dti acquisition parameters: image matrix size = 96 96, b0 = 1000, 51 directions, fov = 24 cm, 47 axial slices, slice thickness = 2.5 mm . Fiber track definition derived from the atlas of oishi and colleagues (2011); bcc: body of the corpus callosum; cgc: cingulum; slf: superior longitudinal fasciculus . Scale bars: panel b = 5 mm; panel c = 100 microns . After the scans of the brain in situ were completed the brain was extracted at autopsy and suspended by the basilar artery in fixative solution (4% paraformaldehyde phosphate - buffered solution) at 4c . After six weeks of fixation in formaldehyde, a second series of scans were performed ex situ (figure 1b). The protocol lasted approximately 36 h, using the same sequences that were run in the previous session but with multiple acquisitions (432 nex) in order to improve the signal - to - noise ratio in the fixed tissue and increase resolution . Fixation was protracted for 10 weeks after the second mri session and the tissue was subsequently cryo - protected by immersion in 30% sucrose . Following the removal of superficial blood vessels and pial membranes, the whole specimen was embedded in a block of 10% gelatin and frozen in a bath of chilled isopentane (40c). The gelatin - brain block was attached to a custom - engineered freezing stage and sectioned on a large motorized sliding microtome (leica microsystems inc ., bannockburn, il) during an uninterrupted cutting procedure . Digital images of the cut surface were acquired before each tissue slice was collected using a digital, single - lens reflex camera (nikon d700; nikon inc ., melville, ny) that was mounted directly in line with the microtome stage . Image acquisition during the sectioning procedure produced an unabridged series of tomographic anatomical images through the brain (figure 1c). These data contain excellent tissue contrast, ideal for anatomical delineations and the stack is the basis for the correct alignment and 3-d reconstruction of corresponding tissue slices . Two adjacent, regularly spaced series of giant histological slices were stained using thionin (nissl staining) and a sensitive silver impregnation technique based on the original gallyas (1979) protocol for myelinated fibers (annese et al ., 2004; figure 1d). Tissue slices were mounted on large glass slides (5 7 inches) and the tissue was imaged on custom - engineered large - format microscope scanners . The core digitizing unit is composed of a computer - controlled microscope, a linear - encoded motorized stage, a line scanner camera, and storage server (annese et al . The images representing the entire giant tissue slice are typically composed of 40,000 image tiles that are acquired systematically at 20 magnification (resolution: 0.4 m / pixel). Slices that allow for fast exploratory viewing and the topographic localization of microscopic features (mikula et al ., 2007; the images are very large (dimensions in pixels are: 334,500 266,200; file size on disk 250400 gb) nevertheless, as jpg2000 compressed pyramidal files (size reduced to 15 gb), the images can be examined through increasing magnification levels while a fixed number of pixels spanning progressively smaller fields of view are shown . This technology, combined with the possibility of surveying slices at multiple locations in the 3d model of the brain, is the key to the proper comparison of histological and neuroimaging data sets . The anatomical and dti data were aligned using tools for automated affine registration included in amira (developer package; visage imaging inc, san diego, ca). A binary mask based on color segmentation (annese et al ., 2006) was created to separate brain tissue from the surrounding gelatin matrix in the tomographic images acquired during the sectioning procedure . These were reconstructed into a volume and this dataset was registered to the average b0 volume acquired during the dti protocol . Because the anatomical volume is composed of images that correspond to unique histological sections, it is possible to identify which sections cross any region of interest in the dti data . The direct, landmark - based comparison of two equivalent coronal images at the level of the posterior hippocampus reveals the histological complexity underlying tensor - based maps of fiber orientation (figures 2a, b). This approach permits quantitative correlations between diffusion - based measures of fiber integrity and directionality (derived from fractional anisotropy values) with formal descriptions of axonal architecture . The latter can be produced from high - resolution histological images (figure 2c) using automated (hence potentially large - scale) image analysis routines (figure 2d). Quantification at the histological level produces actual values representing main fiber directionality, axonal diameter, and cross - over (bartsch et al ., 2011). It should be noted that myelination is not the only histological feature that affects mri signal; gliosis, and perivascular neuropathogenetic phenomena produce visible effects in mri images of the deep white matter . In order to decipher the relationship between non - invasive imaging and the underlying histology, multiple sequences with different scanning parameters must be correlated with the co - localization of different histopathologic markers of structure and disease . Using specific antibody staining, it is possible to localize neuropathological phenomena that produce visible mri abnormalities . Each subject - specific data set should be analyzed as a registered stack of anatomical layers that contain overlapping maps of biomarkers . In the context of our methodological pipeline, mri and dti are also stains in the sense that they show properties of the tissue that are complementary to the features detected by histochemical or immuno - cytological methods . A better understanding of local white matter architecture at the microscopic level can help defining dti acquisition and modeling parameters; these may need to be specific to different regions of the white - matter if the goal is to generate accurate whole - brain models of connectivity . In general, the approach demonstrates the possibility of extending animal neuroimaging studies and resources that combine non - invasive and histological mapping (johnson et al ., 2010) to the human brain . The power of our approach lies in the fact that the resolution of the images of h.t . 's brain created with different modalities spans from several millimeters (2.5 mm / voxel in our 3-d dti data) to a few hundred nanometers (0.3 m / pixel in the 2-d histological images). The mri (and dti) of the brain acquired in situ, containing gray matter (gm), white matter (wm), and cerebrospinal fluid (csf) and representing the original geometry is necessary because these scans link the microscopic maps created from h.t . 's brain to the very large base of non - invasive structural and functional neuroimaging data that will be collected by the hcp . Furthermore, the neuroimaging sequences that were used to acquire images of the brain of h.t . Can be matched to the standard protocols employed by the hcp and other ongoing neuroimaging projects to make direct correlations and inform the development of analysis tools . This way, the growing sample of brain specimens that will be fully characterized at the microscopic level will also be accessible as anatomical reference to large - scale neuroimaging databases . This cross - sectional data will serve as a base line reference for the interpretation of low - resolution images . One could additionally envisage embedding mri data - sets, like that belonging to the brain of h.t ., into the processing pipelines of the hcp and other purely non - invasive studies . The output of these pipelines could be evaluated based on corresponding histological images, providing an efficient means to algorithm optimization . As noted in the previous section, mapping local microscopic features within a 3d model of the whole brain can lead to the formulation of signature models that classify neuroanatomical features in specific compartments of the brain . Histological models may help predict individual anatomy (and pathology) from the macroscopic markers that are visible and measurable in non - invasive low - resolution images . In other words, local micro - architectural patterns that can be localized consistently in a large number of subjects may eventually be used to resolve ambiguities in the interpretation of low - resolution data (leergaard et al ., 2010). The systematic characterization and classification of local micro - architectural patterns as exemplified in figure 2 will be extremely useful for the calibration of the next generation of fiber tracking software and interpretation of the results from diffusion imaging . Our methodology may not be universally feasible for routine implementation . Aside from equipment and operational costs, the computational requirements for managing massive histological data - sets are considerable . However, depreciation of digital storage will occur sufficiently rapidly to justify the timely implementation of the protocols described above . Many web - based products and initiatives, like google maps and the hubble telescope, leverage on remote access and interoperability with digital images in the gigapixel range . The digitization at 20 magnification of whole histological sections of the human brain actually produces images in the terapixel range; these can nonetheless be shared on the web with the support of appropriate hardware and bandwidth . To demonstrate this functionality, the brain observatory developed the infrastructure for a web - based atlas of the human brain that can deliver terapixel histological images representing whole brain sections at cellular resolution; these are associated with other views of the brain created in 2d and 3d using other neuroimaging modalities (annese et al . The development of robust quantitative methods for registration and analysis combined with the availability of adequate computational resources for handling very large data - sets will make comparisons between neuroimaging data and the underlying microscopic features increasingly effective . Furthermore, as the number of single - subject neuroimaging studies like the one involving h.t, where we combined in vivo and postmortem modalities and for whom we were able to compute maps that span multiple spatial scales, increases the predictive power of the digital neuroanatomical catalog will become stronger, representing a useful resource for data mining, cross - modality validations, and algorithm development . The example provided in this article should support the notion of multi - modal, single - subject paradigm as a complement to mri data obtained in the context of large - scale neuroimaging initiatives such as the hcp (marcus et al ., 2011). Histological images only contain a final, freeze - frame picture of the brain's microanatomy; whereas we know that the architecture of neural circuits, from the system level to local dendritic properties, changes with maturation, behavior, and disease . Secondly, histology should not be considered unquestioningly as the gold standard for neuroimaging . Postmortem methods produce well - known artifacts and admittedly it is extremely difficult to produce consistent results on a large scale; nevertheless, the implementation of industrial and carefully controlled protocols for tissue processing can greatly increase the quality and reproducibility of microscopic data . Common standards are needed for histological processing, as well as for the acquisition, storage, and sharing of digital histological files . The importance of incorporating maps of microscopic anatomy into the hcp and other neuroimaging studies ought to justify these efforts and promote substantial investments that will provide solutions to the logistic and technical challenges that still hinder postmortem studies of the human brain (crick and jones, 1993). Given that modern clinical strategies for prevention and treatment of neurological disease depend largely on knowledge acquired via non - invasive mr - based tools and thus from the indirect observation of developmental and neuropathogenetic phenomena, it is crucial at this point in time to support a new phase of validation studies based on rigorously prepared histological material . These studies ought to be commensurate both in scale and funding to the ambitious neuroimaging initiatives like the hcp . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A sustained research activity has been devoted to lanthanide complexes, because of their successful application as diagnostic tools in biomedical analysis as mri contrast agents [1, 2]. They have been used even as effective catalysts for the hydrolytic cleavage of phosphate ester bonds . 4(4h)-quinazolines have been tested successfully against cancer and hiv virus [5, 6]. The synthetic analogues of quinazolines have been found to exhibit antimalarial, anti - inflammatory, and anticancer activity . Apart from the above, 2,3-disubstituted quinazoline-4-(3h)-ones have been reported for their wide variety of pharmacological activity [12, 13]. The title ligand hmpbaq possesses multiple coordinating sites such as phenolic oxygen, carbonyl oxygen, nitrogen of the benzoyl amino group, and nitrogen of the quinazoline ring . We have observed its tridentating behavior with transition metal ions . In continuation of our work on quinazolines, here we report the conformational changes in the ligand on coordination with lanthanides and the relative antimicrobial activity . Hydrazine hydrate, methyl 2-aminobenzoate, and o - vanillin were procured from rankem, merck, and himedia, respectively . The lanthanide nitrates were obtained by heating lanthanide oxides (99.9%) (indian rare earths limited, mumbai, india) with dilute nitric acid (50%) and evaporating the excessive acid . Synthesesthe ligand hmpbaq was synthesized as in our earlier report (see scheme 1). A solution of ln(no3)3 (1 mmol) and hmpbaq 0.838 gm (2 mmol) was refluxed in ethanol (25 ml) for 2 - 3 hours . The ph of the solution was then raised to 6.5 by the addition of sodium acetate and refluxed further for an hour . The precipitate obtained after concentrating the solution was filtered off, washed with water, and dried in air (yield: 90%, mp> 245 c). The ligand hmpbaq was synthesized as in our earlier report (see scheme 1). A solution of ln(no3)3 (1 mmol) and hmpbaq 0.838 gm (2 mmol) was refluxed in ethanol (25 ml) for 2 - 3 hours . The ph of the solution was then raised to 6.5 by the addition of sodium acetate and refluxed further for an hour . The precipitate obtained after concentrating the solution was filtered off, washed with water, and dried in air (yield: 90%, mp> 245 c). Elemental analysis was performed on a carlo erba strumentazione (milan, italy) chn analyzer . Ir spectra were obtained on a nicolet 170 sx ft - ir spectrometer using kbr pellets, in the range 4004000 cm . H- and c nmr spectra were monitored on a jeol - amx-400 nmr spectrometer, using dmso - d6 as the solvent . Tg / dta were recorded on a perkinelmer (mass, usa) (pyris diamond) analyzer in n2 atmosphere at a heating rate of 10c . Mass spectra of the ligand and complex were recorded on a thermofinnigan 1020-automated gcms and jeol sx 102/da-6000 mass spectrometer / data system using argon and xenon (6 kv, 10 ma) as the fab gas, respectively . Epr spectra of the gd(iii) and tb(iii) complexes were monitored on a varian e-4x band spectrometer . The metal contents were determined by complexometric titrations with edta using xylene orange as an indicator . The antibacterial activity of the ligand, metal salts, and the corresponding complexes were assayed simultaneously against pseudomonas aeruginosa (pa), bacillus cirroflagellosus (bc), by cup - plate method . Nutrient broth was prepared by dissolving peptone (0.5%), yeast extract (0.15%), beef extract (0.15%), sodium chloride (0.36%), and monopotassium phosphate (0.13%) in distilled water (100 ml). The ph of the solution was adjusted to 7.2 by adding sodium hydroxide solution (4%) and the resulting solution was autoclaved for 20 minutes at 15 psi . One day prior to the experiment, the cultures of pseudomonas aeruginosa and bacillus cirroflagellosus were inoculated in nutrient broth (inoculation medium) and incubated overnight at 37c . Nutrient agar medium was prepared by dissolving peptone (1%), yeast extract (0.6%), beef extract (0.5%), and sodium chloride (0.5%) in distilled water . The ph of the solution was adjusted to 7.2 by adding 4% aqueous sodium hydroxide solution . Agar (2.4%) was then added and the whole solution was autoclaved for 20 minutes at 15 psi . Each test sample (1 mg) was dissolved in dmso (1 ml), and 0.1 ml of this solution (10 g) was used for testing . Inoculation medium containing 24-hours grown culture was added aseptically to the nutrient medium and mixed thoroughly to get the uniform distribution . This solution was poured (25 ml in each dish) into petri dishes and then allowed to attain room temperature . Thereafter, punching the set of agar with a sterile cork borer and scooping out the punched part made the cups the test samples and the standard were tested at a concentration of 10 g . The plates were allowed to stand for an hour in order to facilitate the diffusion of the drug solution . The antifungal activity of the ligand and the corresponding metal complexes were tested against the pathogenic fungi aspergillus niger (an) and penicillium notatum (pn) by cup - plate method . Nutrient agar medium was prepared by the same method as explained under evaluation of antibacterial activity . One and half day prior to the experiment, the fungal cultures of aspergillus niger and penicillium notatum prepared in the inoculation medium were incubated at 37c for 36 hours . The fungal medium was prepared by dissolving peptone (0.5%), sodium chloride (0.36%), monopotassium phosphate (0.13%), and glucose (2%) in distilled water (100 ml). The ph of the solution was adjusted to 7.2 by adding sodium hydroxide solution (4%) and the resulting solution was autoclaved for 20 minutes at 15 psi . One and half day, grown cultures were added aseptically to this medium and mixed thoroughly to get uniform distribution . The solutions of the test samples and standard were evaluated for antifungal activity by cup - plate method at a concentration of 10 g . The zone of inhibition was measured in millimeter for the particular test sample with each organism at 48 hours interval . The analytical data (see table 1) indicate that the complexes have 1: 2 (metal: ligand) stoichiometry and can be represented by the general formula [la(mpbaq)2(h2o)2]no3 . They are soluble in dmso and dmf, sparingly soluble in ethanol and methanol, but insoluble in benzene, ether, and chloroform . The molar conductance data (see table 1) of the complexes in dmso at 10 m is in the range of 44.0049.00 ohmcmmol, which indicates 1: 1 electrolytic nature of the complexes . The diagnostic ir frequencies of the ligand hmpbaq and its complexes are compiled in table 2 . In the spectrum of the hmpbaq, the strong band observed at 1653 cm is assigned to (c = o) of the quinazoline ring . The appearance of this band at a lower wave number is due to the existence of strong intermolecular hydrogen bonding between the oxygen of c = o and the hydrogen of the 2-[2-hydroxy-3-methoxy phenyl) group, as observed in its crystal structure . The two nonequivalent phenolic oh groups are observed at 34602940 cm as a strong unresolved broad band . A medium intensity band at 3280 cm is assigned to (nh) of the quinazoline ring . In the spectra of all the complexes, the (c = o) has shifted to lower wave numbers indicating the involvement of carbonyl oxygen in coordination . The band due to (c = n) has shifted to lower energy (16161572 cm), indicating coordination through benzyl amine nitrogen . The band due to phenolic oh groups was not observed in the spectra of the complexes, it might have been obscured by a broad band due to water . However, the coordination through only one or both of the phenolic oxygen / s was further confirmed by h nmr spectral studies . Strong band at 1381 cm in the spectra of all the complexes indicates the presence of ionic nitrate . The presence of coordinated water molecules is suggested by the appearance of characteristic rocking frequency at 825 cm . H nmr spectra of the hmpbaq and its la(iii) complex were recorded in dmso - d6 and the data along with the assignments are displayed in table 3 . The h nmr spectrum of hmpbaq exhibits two sharp peaks at 11.31 and 9.42 ppm, corresponding to o(2)h (d2o exchangeable) and o(4)h (d2o exchangeable), respectively . Two singlets and one doublet at 8.43, 7.43, and 7.81 ppm are due to c(21)h, n(3)h (d2o exchangeable) and c(13)h, respectively . N(3)h and c(13)h, being cis to one another, coupled together and this coupling results in the splitting of c(13)h signal . Four triplets [c(17)h, c(16)h, c(10)h, and c(4)h] and six doublets [c(18)h, c(15)h, c(11)h, c(9)h, c(5)h, and c(3)h] have merged to give a multiplet at 7.316.6 ppm corresponding to ten aromatic protons . The resonance due to o(3)c(22)h3 and o(5)c(23)h3 appeared at 3.85 ppm . In the spectrum of la(iii) complex, the resonance due to o(4)h has appeared at the same position as in the ligand (9.40 ppm) indicating the noninvolvement of o(4)h in the coordination . The singlet at 11.31 ppm in the ligand spectrum ascribed to o(2)h group is not observed in la(iii) complex . The other signals [c(21)h, n(3)h, c(13)h, c(17)h, c(16)h, c(10)h, c(4)h, c(18)h, c(15)h, c(11)h, c(9)h, c(5)h, c(3)h, o(3)c(22)h3, and o(5)c(23)h3] in la(iii) complex did not show any considerable shift . The effective magnetic moments (see table 1) of all the complexes indicate that they are paramagnetic in nature except la(iii) and y(iii) which are diamagnetic . The values obtained are similar to the van vleck and frank and hund's values except in case of sm(iii) and eu(iii) where slightly higher values were obtained . This is due to low j - j separation, which leads to thermal population of higher energy levels . The values obtained are similar to those of typical lanthanide ions and indicate the noninvolvement of 4f electrons in bonding due to their very effective shielding by the 5s 5p octet . The energy level of the lowest excited state is very high with no contribution from orbital angular momentum and the anisotropic effect . Value of 1.99 (at room temperature) and 2.04 (at liquid nitrogen temperature) compared to the free - ion value of tetracynoethylene (2.00277) with broad resonance lines was obtained . The g values being almost the same and similar line widths indicate that the line widths are independent of temperature . Further, the complete absence of zero - field hyperfine splitting and the presence of broad bands indicate that the gd(iii) ion is located in a rather disordered environment caused by strain . These strains d - strain for the zero field splitting distribution) arise due to random hydrogen bonds between water molecules and the complex leading to distortions, which lead to broad resonance epr lines [19, 20]. The electronic spectral data of the two representative complexes are given in table 4 . The free ligand shows an intense band at 32786 cmand two weak bands at 28328 and 38610 cm of which the first two are assigned to the n * and the latter to the * transitions, respectively . The electronic spectra of the complexes are dominated by ligand bands, with a slight shift to higher or lower energy levels . This slight shift was attributed to the effects of crystal field upon the interelectronic repulsion between the 4f electrons . The bonding parameter (b), sinha's covalence parameter (), nephelauxetic parameter (), and angular covalence () have been calculated according to literature procedure [22, 23]. Values being less than unity and positive value of b and indicate metal - ligand covalent bonding . According to karraker, the shape of the hypersensitive transition reflects the environment of the metal ion . On comparison of the spectra with that of known compounds the tg / dta study of la(iii) and pr(iii) complexes was carried out as the representative . In case of la(iii) complex, the initial weight loss of 11.24% (cal . The next weight loss in the temperature range of 250350c is due to the loss of one ionic nitrate molecule (obs . A further weight loss in the range of 350800c corresponds to two ligand molecules (obs . 69.00%; cal . 69.89%). Finally, the most stable oxide was formed, on further heating up to 1000c . The percentage of metal obtained is in confirmation with the analytical values for the metal content . In case of pr(iii), the thermogram exhibits a loss of 3.00% (cal . A loss of 10.00% (cal . 10.35%) between 240260c is due to the removal of one ionic nitrate molecule . A further weight loss of 69.00% (cal . The mass spectrum of hmpbaq shows the formation of molecular ion peak at m / z = 419 and corresponds to the total molecular weight of the ligand . The fab mass spectra of [sm(mpbaq)2(h2o)2]no3 and [eu(mpbaq)2(h2o)2]no3 show the molecular ion peaks at m / z = 1084 and 1086, respectively, supporting the composition of the complexes . The results of the antibacterial and antifungal studies are given in table 5 . The ligand hmpbaq was less active against pn, pa, and bc except against an where it has shown moderate activity . All the complexes, except la(iii), pr(iii), and nd(iii) complexes, show moderate activity against bc . It was observed that, compared to the ligand and metal salts, the complexes exhibited enhanced antibacterial activity, which is due to the synergistic effect that increases the lipophilicity of the complexes . Chelation decreases the polarity of the metal ion, which further leads to the enhancement of complex's lipophilicity . Since the microorganism cell is surrounded by a lipid membrane which favors the passage of lipid soluble materials, increased lipophilicities allows the penetration of complex into and through the membrane and deactivates the active enzyme sites of the microorganisms based on the above data, structure i is proposed for the lanthanide(iii) complexes having the general formula [ln(mpbaq)2(h2o)2]no3, where ln = la(iii), pr(iii), nd(iii), sm(iii), eu(iii), gd(iii), tb(iii), dy(iii), and y(iii). In the present complexes, due to rotation about the n n bond, the ligand changes its conformation to facilitate the coordination in onn fashion as in the case of cd(ii) complex as reported earlier . The antibacterial activity of the ligand is enhanced on complexation, whereas no distinct change is observed in case of antifungal activity.
Bisphosphonates have an important role in the prophylaxis and treatment of postmenopausal and corticosteroid osteoporosis . This case report presents a patient who presented to the ent department with sinus symptoms from oroantral fistulae following bisphosphonate use requiring surgery . This case report aims to raise awareness among ent surgeons to these patients on bisphosphonates that could present to them with sinus disease from oroantral fistulae . A 66 year old lady presented with facial pain, chronic purulent discharge into the mouth and signs of an oroantral communication . Her medical history included type 2 diabetes, rheumatoid arthritis on prednisolone, hypertension and chronic pain syndrome . She had been on oral alendronic acid (one of the bisphosphonates) weekly for at least 9 months ., there was purulent material discharging from a sinus in the upper left fifth area in the oral cavity with a tender left maxilla . Ct scans (fig . 1 & 2) showed opacity and thickening of the left maxillary antrum with dehiscence of the medio - inferior and anterior walls of the sinus and a maxillo - oral fistula . There was destruction of bone in the floor of the left maxillary sinus consistent with an area of osteonecrosis secondary to bisphosphonate . Coronal ct scan of the head showing osseo - sclerotic changes / oroantral fistula floor of left maxillary sinus axial ct scan of the head showing osseo - sclerotic changes and defect in left maxillary sinus she underwent exploration of her left maxilla with debridement of large necrotic sequestrum from the left maxillary alveolus . There was a large fistula into the maxillary sinus which was repaired with an advancement of buccal flap . Bisphosphonates like alendronic acid, disodium etidronate, and risedronate are effective for preventing postmenopausal and corticosteroid induced osteoporosis . They are also useful in the treatment of paget's disease, hypercalcaemia of malignancy and in bony metastases . Osteonecrosis of the jaw (onj) has been increasingly reported following intravenous bisphosphonate use and rarely in those taking them orally . The vast majority of onj reported in the english language literature were seen following administration of intravenous pamidronate and zoledronate . Of the only 28 reported cases following oral bisphosphonate use, onj frequently affects the mandible followed by the maxilla and in a number of cases, is preceded by a tooth extraction . Patients can present with non - healing ulcers of the mandible or maxilla and can progress to pan - sinusitis with presentation to otolaryngologists (2). In a series of 15 patients with onj, some had background breast cancer, osteoporosis, multiple myeloma and prostate cancer (3). Osteonecrosis of the jaw has also been shown in association with cancer chemotherapy in 10 patients who received bisphosphonates as treatment for their malignant bone disease (4). The most common radiologic finding in bisphosphonate - associated osteonecrosis has been shown to be osseous sclerosis (3). The spectrum varied from thickening of the lamina dura and alveolar crest (subtle) to attenuated osteopetrosis - like sclerosis . Management will include the use of antibiotics, chlorhexidine mouthrinses and in cases of oroantral fistula, sequestrectomy, and surgical debridement . The aim of surgery is to try and remove all evidence of necrotic bone and to obtain mucosal coverage . There is an on - going 2 year audit by oral and maxillofacial surgeons in the uk in conjunction with the faculty of general dental practice, documenting the emerging trend of oroantral fistulae from bisphosphonate use (bronj) (5). This case report highlights the need for awareness among ent surgeons to these patients on bisphosphonates who could present to them with sinus disease . With the millions of patients receiving various oral bisphosphonates for osteopenia and osteoporosis, health care practitioners should be aware of the potential for the onset of osteonecrosis and familiar with its management (1).
A balanced diet during childhood and adolescence is crucial not only for the well - being and growth of the child, but also for the establishment of sound dietary habits that will persist in later life . For example, adequate intake of energy and macronutrients has an essential role in the overall physical growth of the adolescent . On the other hand, vitamins and minerals have specific individual and synergistic roles in supporting metabolic function (b vitamins), bone mineralization (calcium), hemoglobin production (iron), and growth (zinc). Micronutrient deficiencies (mnds) such as vitamin a deficiency (vad), iron deficiency anemia (ida), and iodine deficiency disorders (idds) have been major nutritional problems in developing countries, adversely affecting adolescents health and performance, and thereby becoming major impediments to economic development . In india, these mnds continue to be of public health significance . Nearly half of the world's micronutrient - deficient population is found in india . Moreover, factors that sought to reduce the macro- and micronutrients intake of adolescents could be unequal intrafamilial distribution of food and adverse and harmful dietary practices including dieting, specific food taboos, and dietary restrictions . Also, low lean body mass is associated with many concurrent and future adverse health outcomes . Thus, achievement of optimum growth during this period should be of utmost importance for maintaining good health thereafter . Early detection of the morbidities through regular survey helps in prompt treatment and prevention of serious complications . Therefore, keeping the magnitude of micronutrient malnutrition among the adolescents in view, the present study was carried out to assess the dietary pattern and nutritional deficiency disorders among the school - going adolescents . The specific objectives of the above study were the following: to evaluate the dietary intake and nutritional status of the adolescentsto assess the prevalence of nutritional deficiencies among the adolescentsto determine whether the parents education and socioeconomic status are associated with the nutritional status of the adolescents or otherwise . To evaluate the dietary intake and nutritional status of the adolescents to assess the prevalence of nutritional deficiencies among the adolescents to determine whether the parents education and socioeconomic status are associated with the nutritional status of the adolescents or otherwise . This was a cross - sectional study conducted in schools and colleges in the urban areas of jhansi district in uttar pradesh among adolescents (age group of 10 - 19 years). A predesigned and pretested 3-day food frequency intake questionnaire, along with examination schedule for recording information, was used by adopting the face - to - face interview method . The data obtained were collated and statistically analyzed by simple proportions and using the tests of significance (chi - square test). To calculate the optimum sample size required, the current prevalence of nutritional deficiency that is common in both boys and girls was taken . The relevant literature suggested that 62% of school - going children, both boys and girls, are found to be deficient in vitamin a. for the above purpose, the formula 4pq / l was used, where p = 62, q = 38, and l = allowable error in p (12%), i.e., 7.4 . Thus, the sample size was worked out to be 172 . Considering the design effect to be 2, the required sample size was 172 2 = 344 . To be on the safe side and for analytical convenience, it was increased to 400 adolescents (200 boys and 200 girls). Out of all the educational institutions in the urban areas of jhansi, subject to permission from the principal of each institution, each adolescent was then individually contacted . After explaining our objectives to them and obtaining the written informed consent from the parents of each adolescent, they were interviewed in person . Dietary intake in terms of calorie, protein, fat, vitamin a, thereafter, the averages were compared with the corresponding recommended dietary allowances (rdas) recommended by the indian council of medical research (icmr). The socioeconomic class and the level of education of the parents ethical clearance was sought from the ethics committee of maharani laxmibai medical college, jhansi, uttar pradesh, india . Nutritional status of the adolescents was assessed through body mass index (bmi), according to the world health organization (who) asian criteria . Of the 400 participants, majority (53.5%) were malnourished, i.e. 38.6% were underweight, 10.1% were overweight, and 4.75% were obese . It was further seen that in comparison to boys (31.5%), more girls (46%) were underweight . This may be due to particular cultural practices where the well - being of the boys are preferred to that of girls . Late adolescents (51%) were found to be more in the normal weight category than early adolescents (44%). However, the early adolescents (42%) were found to be more susceptible to underweight problems compared to the late adolescents (37%). It has also been observed that a significant percentage of adolescents from nuclear families belong to the normal weight category in contrast to joint / extended families . With better education of the parents and higher socioeconomic status of the families, majority of the adolescents were able to retain their weight within the normal category, and the association was found to be highly significant [table 1]. Nutritional status of adolescents and its association with sociodemographic determinants (n=400) majority of the participants consumed chapatti (94.5%) and rice (81.2%) as daily food . Most of them did not consume green leafy vegetables, milk products, fruits, and salad . Of the 400 participants, only 39.75% preferred to eat meat and fish [table 2]. More than 50% of the participants had calorie consumption less than 80% of the rda . Furthermore, it was also found that the consumption of macro- and micronutrients were below the par level of rda [figure 1]. It was further seen that the average calorie intake was 60.85% of the estimated rda . For protein and fat, it was 71.46% and 52.10% of the estimated rda, respectively . Vitamin a intake was only 29.30% of the estimated rda . For calcium, iron, and vitamin c, the average intakes were 56.75%, 73.88%, and 45.00% of the estimated rda, respectively, among the participants [table 3]. Dietary consumption pattern (n=400) nutrients intake in percentage to rda (n = 400) average nutrient intake (n=400) despite the fact that several national nutrition programs are in operation for the benefit of adolescents, the prevalence of nutritional deficiencies among adolescents continues to be a public health concern . In the present study, selected school - going adolescents in the age group of 10 - 19 years were interviewed and examined . A significant difference between the nutritional status and gender (<0.05) moreover, the early adolescents are more vulnerable to malnutrition than the late adolescents . The study conducted by dambhar et al . Had comparatively similar findings with the present study, namely, that 48.3% of the adolescents were normal and 51.7% were undernourished . Reported that 32.3% of urban school adolescents were in the normal range while 65.3% were either overweight or obese . The extent of undernutrition is lower in the present study than that reported in one indian study (53%) by kurz et al . And in two other kenyan studies (61%) and (57%). The present study shows that dietary deficiency of both macro- and micronutrients are alarming in the study area . For vitamin a and vitamin c, these facts are so glaring that for these nutrients, urgent intervention is required . Dietary deficiencies in adolescents have also been observed by chaturvedi et al . And eglesadi et al ., where they found the deficiency of vitamin a to be glaring in their respective study areas . A study published in british nutrition foundation (bnf)9 section has also reported that a large proportion of adolescents have low intakes of some vitamins and minerals (particularly vitamin a, riboflavin, iron, calcium, and magnesium), with more girls aged 11 - 18 years having low intakes compared to boys of a similar age another study by akkamahadevi et al . Also reported lower intake of energy and blood - forming micronutrients in adolescent girls . In the present study, it was also found that the type of family, parents education, and socioeconomic status of the adolescents play a pivotal role in their nutritional status . It was also seen that higher the socioeconomic status, better the nutritional status of the adolescents [table 4]. Documented findings in developing countries like india and ghana report a positive association between socioeconomic status and bmi . Importance of parental education in raising the nutritional status of the children is well - known . Mothers education has profound effect on preschool children, whereas fathers education seems to have a greater importance in the care of adolescents . Choudhury et al ., in their study, found that when the nutritional status of adolescents was examined against the highest education in the family, it was evident that with the increasing level of education decline in undernutrition was noticed among the subjects . In comparison to joint families, nuclear families were more able to maintain the normal nutrition status of the adolescents . Relation of bmi with family characteristics of adolescents (n=400) the strength of the study is that it is able to show the poor nutritional status among the urban adolescents . It also reveals that problem of malnutrition is multifaceted and has links with various socioeconomic and demographic factors . This study had a limitation, in that a 3-day food intake questionnaire was used instead of a 7- or 21-day one . The reason behind using a shorter version of the questionnaire was to exclude recall biases . This study also lacks biochemical assessment of the various nutrients that would have been able to give more accurate findings . In the light of the findings, there is need and scope to design and implement adolescent nutrition education programs and establishment of operation research models . Finally, gender differences in food habits should be taken into account, as school - based interventions aiming to improve health behaviors have been shown to influence boys and girls differently . Future researches should emphasize the need for more systematic evaluation of dietary habits, including studies of consumer behavior and the role of education on food intake . Moreover, future researches should evaluate which factors contribute to social inequalities in food habits, as a basis for future differentiation of interventions among the adolescents groups . Finally, we emphasize the need for future studies of trends in the consumption habits to evaluate how adolescent food habits continue to develop . The health and nutritional status among the adolescents was found to be low, more in girls than in boys . Such practices may be due to differences in the food allocation at the family level and because of individual likes and dislikes . These deficiencies lead to a decrease in the growth spurt, for both physical and mental health . A periodical and regular health check - up with concerted efforts toward their nutrition, along with focused health education will improve the health and nutritional status of these school - going adolescents . There is a need for the sensitization of adolescents and their parents through health and nutrition education (hne), information education, and communication (iec), and appropriate behavioral change communication (bcc) activities . Socioeconomic status of any family depends primarily on the per capita monthly income, which in turn is related to the family size; therefore, there is a need to encourage people to adapt small family norms to combat with nutrition deficiency . A periodical and regular health check - up with concerted efforts toward their nutrition, along with focused health education will improve the health and nutritional status of these school - going adolescents . There is a need for the sensitization of adolescents and their parents through health and nutrition education (hne), information education, and communication (iec), and appropriate behavioral change communication (bcc) activities . Socioeconomic status of any family depends primarily on the per capita monthly income, which in turn is related to the family size; therefore, there is a need to encourage people to adapt small family norms to combat with nutrition deficiency.
The successful pathogen, toxoplasma gondii, is able to infect virtually all warm - blooded animals including birds and mammals in worldwide, obligating to the zoonotic toxoplasmosis (1). Human beings acquired the disease mainly through ingestion of tissue cysts or oocysts of t. gondii, containing in food or water (2). T. gondii infection in healthy adults rarely causes clinical symptoms, but can lead to be fatal in immunocompromised individuals (24). In wildlife, many animals also were infected by t. gondii . Some studies reported high infection rate of t. gondii in zoo animals and wild birds . For example, murata in early 1989 just showed 29.28% (53/181) and 16.94% (61/179) of t. gondii igg in mammals and wild birds, respectively, by serological survey (5). Zhang et al . Also reported there had 25% (4/16) of t. gondii antibody in primates, 69.4% (25/36) in carnivores, 27.6% (8/29) in herbivores and 11.11% (4/36) in birds in china (6). There was 36.17% (51/141) in felids and 10.81% (4/37) in prosimians (7). Other recent reports also showed the high infection rate such as 36.1% (73/202) of wild birds captured from the wild environment (8) and 12.46% (39 /313) of house sparrows in china (9). In recent years, different sources of t. gondii strains were identified using multilocus polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) method, which was established for genotyping by su and other scholars (1014). The distribution of genetic diversity of t. gondii in wildlife animals is of great importance to understand the transmission of this parasite in the environment . Some studies have identified several genetic characterizations of t. gondii isolates from cats, bats, sheep, and birds in china (1518). However, little information concerning t. gondii isolates from zoo animals and pet birds in southeast china . The objectives of the present study were to determine the prevalence and genetically characterize t. gondii in wild animals from fuzhou zoo and pet birds farms in fujian province, southeastern china . A total of 185 animals were randomly collected from fujian province during 2012 and 2014 . The 45 heart tissue samples from were collected from zoo animals, and 140 heart tissue samples were collected from pet birds . Genomic dna was extracted from heart tissue samples of wild animals and pet birds using tianamp genomic dna kit (tiangen, beijing, china). In brief, 30 mg of heart tissue were treated with sodium dodecyl sulphate / proteinase k at 56 c for overnight digestion in a thermostat water bath . Dna samples were prepared according to the previous study (19) and eluted into 60 ul with elution buffer . The positive genomic dna of t. gondii rh (type i) and pru (type ii) strains were kindly provided by fujian normal university and lanzhou veterinary research institute, respectively . At first, all the dna samples from tissues of wild animals and pet birds were identified using semi - nested pcr method based on t. gondii b1 gene as described previously (20). In brief, the first round were amplified at 35 cycles with 93 c for 10 s, 57 c for 10 s and 72 c for 30 s. then the nested pcr was beginning at 93 c 10 s for denaturation, following by annealing at 62.5 c for 10 s and the 72 c 10 s for extension (21). The pcr amplification was performed by veriti 96-well thermal cycler (abi), and the products were separated by 1.5% agarose gel, visualized under ultraviolet light . The positive samples were then genotyped using multilocus pcr - rflp method as previously described by amplifications of 11 markers, including sag1, 5 and 3-sag2, alternative sag2, sag3, btub, gra6, c22 - 8, c29 - 2, l358, pk1, apico, and cs3 ((13, (15, (22). The reaction volume (25 l) contained 10 mm tris - hcl, 50 mm kcl, 1.5 mm mgcl2, 0.3 mm each primer, 0 . 25 mm each dntp, 1.25 u golden dna polymerase (tiangen, beijing, china), and 2 l t. gondii dna . All samples were incubated at 95 c for 5 min, then 35 cycles of pcr at 94 c for 30 s, 55 c for 30 s, 72 c for 40 s and 72 c for 7 min . The nested pcr was carried out with an annealing temperature at 60 c for 60 s for all the markers except apico, which was amplified at 55 c . The previous studies ((9, (13, (14, (15) are used to compare with the results of the genetic characterization of the chinese t. gondii in our present work . A total of 185 animals were randomly collected from fujian province during 2012 and 2014 . The 45 heart tissue samples from were collected from zoo animals, and 140 heart tissue samples were collected from pet birds . Genomic dna was extracted from heart tissue samples of wild animals and pet birds using tianamp genomic dna kit (tiangen, beijing, china). In brief, 30 mg of heart tissue were treated with sodium dodecyl sulphate / proteinase k at 56 c for overnight digestion in a thermostat water bath . Dna samples were prepared according to the previous study (19) and eluted into 60 ul with elution buffer . The positive genomic dna of t. gondii rh (type i) and pru (type ii) strains were kindly provided by fujian normal university and lanzhou veterinary research institute, respectively . At first, all the dna samples from tissues of wild animals and pet birds were identified using semi - nested pcr method based on t. gondii b1 gene as described previously (20). In brief, the first round were amplified at 35 cycles with 93 c for 10 s, 57 c for 10 s and 72 c for 30 s. then the nested pcr was beginning at 93 c 10 s for denaturation, following by annealing at 62.5 c for 10 s and the 72 c 10 s for extension (21). The pcr amplification was performed by veriti 96-well thermal cycler (abi), and the products were separated by 1.5% agarose gel, visualized under ultraviolet light . The positive samples were then genotyped using multilocus pcr - rflp method as previously described by amplifications of 11 markers, including sag1, 5 and 3-sag2, alternative sag2, sag3, btub, gra6, c22 - 8, c29 - 2, l358, pk1, apico, and cs3 ((13, (15, (22). The reaction volume (25 l) contained 10 mm tris - hcl, 50 mm kcl, 1.5 mm mgcl2, 0.3 mm each primer, 0 . 25 mm each dntp, 1.25 u golden dna polymerase (tiangen, beijing, china), and 2 l t. gondii dna . All samples were incubated at 95 c for 5 min, then 35 cycles of pcr at 94 c for 30 s, 55 c for 30 s, 72 c for 40 s and 72 c for 7 min . The nested pcr was carried out with an annealing temperature at 60 c for 60 s for all the markers except apico, which was amplified at 55 c . The previous studies ((9, (13, (14, (15) are used to compare with the results of the genetic characterization of the chinese t. gondii in our present work . Of the total 185 samples, 10 samples were detected to be positive for the t. gondii b1 gene, including 7 zoo animals (1 black - capped, 1 wild red dog, 1 lemur, 1 zebra, 1 red panda, 1 peacock, 1 red - crowned crane, 15.56%, 7/45) and 3 pet birds (1 budgerigar, 1 black - billed magpie, 1 zebra finch, 2.14%, 3/140,) (table 1). The positive rate of t. gondii dna in zoo animals was significantly higher than that of pet birds in fujian, china (p<0.05 . Further genotyped the positive samples using multi - locus pcr - rflp method showed that 3 dis - tinct genotypes (toxodb genotype #9, #2 and #10) of t. gondii from black - capped (cebus apella), peacock (peafowl) and budgerigar (melopsittacus undulatus) respectively were identified (table 2). By using sequence - specific primers, the relative number of copies of a particular dna sequence can be determined . There are many applications of this technique in the field of diagnosis (2325). T. gondii can be detected by semi - nested polymerase chain reaction (pcr) using b1 genes in pig, cat, and other wild animals (2629). In the present study, we identified the t. gondii infection in black - capped, wild red dog, lemur, zebra, red panda, peacock, red - crowned crane, budgerigar, black - billed magpie, zebra finch in china, which indicated these animals could be served as a potential source of infection for other animals and even humans . Pcr - rflp method is frequently used to identify differences between homologous dna sequences (13,15), which have been successfully used for genotyping of t. gondii from cat, chicken, human, cougar, sheep, birds, sparrow, pig and other animals (2, 9, 13, 1518, 26). Further genotyped the positive samples using multi - locus pcr - rflp method showed that 3 distinct genotypes (toxodb genotype #9, #2 and #10) of t. gondii from black - capped (cebus apella), peacock (peafowl) and budgerigar (melopsittacus undulatus) respectively were identified (table 2). Zoo animals and pet birds have formed a huge industry in order to prevent rare wild animals from extinction and to boost the economy . Fujian province has a great advantage in convenient transportation because of its southeast coastal location in china, so the exchange of wild animals and pet birds becomes more ordinary . The results in this present study will enhance our understanding of the epidemiology and prevention of t. gondii in zoo wild animals and pet birds . One sample isolated from budgerigar belonged to toxodb genotype #10, supporting the previous study that toxodb genotype #10 had been isolated from 3 sparrows in fuzhou (18). This result indicated that toxodb genotype #9 was predominant not only in southern, southwestern and central parts of china but also in southeast part of china (15, 17, 3032). In addition, our finding support that toxodb genotype #9 is at cs3 locus . Our study is also the first report of genetic typing of t. gondii from zoo wild animals and pet birds in china . The high genetic diversity of t. gondii genotypes in fujian may cause by the high number of cats in city and other feline in mountainous areas . The positive rate of t. gondii dna was high in zoo animals (7/45, 15.56%) but very low in birds (3/140, 2.14%) in fujian, china . Further genotyping analysis confirmed that the presence of t. gondii in zoo wildlife and pet birds were toxodb genotype #10, genotype #9 and genotype #2, which suggested the integrated measures should be taken to prevent t. gondii infection in humans or other animals.
According to world health organisation (who) data every twelfth women, or 8% of women are affected by breast cancer . Fortunately, the mortality rate hangs behind in comparison to the number of affected thanks to the risk prevention, promotive activities of the health sector, government sector and individual activities (1, 2). Along with todays known facts, in 85 - 90% of cases ethiology of breast cancer remains unknown, even though it is considered that the leading role has the environmental factor and that they are important for 75% cases of breast cancer genesis (3, 4, 5). Breast cancer incidence has the growing trend with the increase of the maternal age, it doubles every ten years till menopause, and then it grows really slowly . It is extremely rare with women younger than twenty, but with women at the age of eighty 200 - 300/100 000 gets affected by breast cancer (1). Solely 0,8% of breast cancer evolves with women younger than 30 years old, and aproximately 6.5% occurs with women between 30 and 40 years old (1, 2, 3, 4). Recent study in usa and china showed that obesity in combination with insufficient physical activity increases the risk for breast cancer evolution (5, 6). It is considered that insufficient consumption of calories intake is a cause of weight gaining and that is direct consequence for the impact on the growth hormone and estrogenes hormone that is tightly connected to breast cancer incidence increase (7). Obesity, by itself, is a breast cancer risk factor because it leads to the concentration increase of the endogenous estrogene (8). In the uk, 8% of cases are connected to the overweight (bmi 26 - 30) and obesity (bmi>30) (9, 10, 11, 12, 13). Breast cancer incidence is higher with women who are of a better socioeconomical status and with higher education . Women who live in urban surroundings are more likely to get affected by breast cancer than the ones who live in rural areas (12, 13, 14, 15). The aim of the study is to examine the factors of the quality of life as a risk for increasing breast cancer with women in central, industrial zenica doboj canton in bih, a developing country . A case control study conducted by polls has its purpose to research the connections between the individual and quality of life factors as breast factor risks with women who are treating at family medicine centers in ze - do canton . This research encompassed two hundred women, among which, one hundred women diagnosed with breast cancer according to clinical features (experimental group) and one hundred examinees who are not diagnosed with breast cancer or any other malignant diseases, but who take up preventive ultrasonography examination with general practicioner with aim of prevention, early detection and screening breast cancer (chosen practicioner did ultrasonic breast examination along with clinical examination). Examinees from the experimental group were diagnosed with breast cancer in the period from january the 1st 2003 till december 31st 2007 . The diagnosis was confirmed based on the clinical examination, ultrasonographic breast examination, mamography and pathohistologically confirmed biopsy diagnosis or during the operative treatement ex tempora. Selection of the examinees depended on the assent of the examinee in order to participate into the study, and as well on the breast cancer patient s general condition which had depended on the severity of the clinical features . Patients from the experimental group in the terminal phase of the disease along with patients with benign breast tumors and as well clinicly unsecured breast cancer diagnosis were not encompassed with the sampling . All examinees were stratified according to: the age, occupation / working place e.g. (housewives, retired persons, teachers, health workers, administration workers, workers in manufacturing and last but not least examinees employed in the service industry; body mass index bmi, employment / secure existence; wealth status and place of living (city, xountryside, or near the industrial facilities). The research has been conducted on survey method, and the instrument of the research was a questionnarie specially made for this research: the questionarrie about breast factor risks came into existence based on the experience of evidence based medicine . Prior to research accession, examinees were put through a short education and they were provided with necesssary information about the aims and the purpose of the research . Filling in the questionnarie met the conditions of ethical anonimity . The questionnarie holds a group of questions about individual and demographic data (age, education, occupation, employment, place of living, wealth status, satisfaction with the wealth status); in addition, place of living is categorized to urban, rural areas or somewhere near the industrial facilities; the wealth status is categorized from one (the best of all) to seven (the worst of all); the perception of satisfaction with wealth status is categorized from one (very much satisfied) to five (unsatisfied); and in distinction to previous data, breast cancer history within a family is researched separately . The following group of questions is about menarche and the reproduction that deals with giving birth (whether there was a childbirth, and if yes how many and at which age); if there was a miscarriage and if positive how many; whether the examinee had breast fed, if positive how many children and for how long; did the ecaminee use contraception, and for how long; did she use hormon therapy and for how long; was there a menopause and when it had happened; did she use hormone replacement therapy? The following question deal with breast cancer types (cystical formation, atipical hyperplasia, carcinoma in situ, previous forms of carcinoma etc . ). The following set of questions deals with the way of living, about the exposition to stress (was the examinee exposed to stress, for how long and how often); physical activity (is she active, and how much); smoking habits (is she smoking? Do any family members smoke? Is the examinee surrounded by smokers and if yes, how many hours? ), also questions on alcohol consumption (does she drink alcohol, how much and for how long?) (16, 17, 18, 19, 20). When it comes to statistic analysis, standard methods of descriptive statistics were used (central tendency measures and dispersion measures). In favour of testing differences of statistical significance, among the samples (x2 test, man - whitney z - test). For linear correlation analysis a tukey test is used (anova). But for multivariate correlation analysis we used anova (logistic regression analysis). The odds for significant differences (odds ration- or; statistical significant or> 1.0) were on 95% confindential interval (95% confidence interval). Selection of the examinees depended on the assent of the examinee in order to participate into the study, and as well on the breast cancer patient s general condition which had depended on the severity of the clinical features . Patients from the experimental group in the terminal phase of the disease along with patients with benign breast tumors and as well clinicly unsecured breast cancer diagnosis were not encompassed with the sampling . All examinees were stratified according to: the age, occupation / working place e.g. (housewives, retired persons, teachers, health workers, administration workers, workers in manufacturing and last but not least examinees employed in the service industry; body mass index bmi, employment / secure existence; wealth status and place of living (city, xountryside, or near the industrial facilities). The research has been conducted on survey method, and the instrument of the research was a questionnarie specially made for this research: the questionarrie about breast factor risks came into existence based on the experience of evidence based medicine . Prior to research accession, examinees were put through a short education and they were provided with necesssary information about the aims and the purpose of the research . Filling in the questionnarie met the conditions of ethical anonimity . The questionnarie holds a group of questions about individual and demographic data (age, education, occupation, employment, place of living, wealth status, satisfaction with the wealth status); in addition, place of living is categorized to urban, rural areas or somewhere near the industrial facilities; the wealth status is categorized from one (the best of all) to seven (the worst of all); the perception of satisfaction with wealth status is categorized from one (very much satisfied) to five (unsatisfied); and in distinction to previous data, breast cancer history within a family is researched separately . The following group of questions is about menarche and the reproduction that deals with giving birth (whether there was a childbirth, and if yes how many and at which age); if there was a miscarriage and if positive how many; whether the examinee had breast fed, if positive how many children and for how long; did the ecaminee use contraception, and for how long; did she use hormon therapy and for how long; was there a menopause and when it had happened; did she use hormone replacement therapy? The following question deal with breast cancer types (cystical formation, atipical hyperplasia, carcinoma in situ, previous forms of carcinoma etc . ). The following set of questions deals with the way of living, about the exposition to stress (was the examinee exposed to stress, for how long and how often); physical activity (is she active, and how much); smoking habits (is she smoking? Do any family members smoke? Is the examinee surrounded by smokers and if yes, how many hours? ), also questions on alcohol consumption (does she drink alcohol, how much and for how long?) (16, 17, 18, 19, 20). When it comes to statistic analysis, standard methods of descriptive statistics were used (central tendency measures and dispersion measures). In favour of testing differences of statistical significance, among the samples (x2 test, man - whitney z - test). For linear correlation analysis a tukey test is used (anova). The odds for significant differences (odds ration- or; statistical significant or> 1.0) were on 95% confindential interval (95% confidence interval). Namely in both groups, the experimental one with 83% breast cancer and 79% in the control group without cancer . In comparison to control subject poorer wealth status have the examinees diagnosed with breast cancer . We have discovered significantly lower representation of secure personal existence through employement and statisticaly significant difference of the examinees of the experimental group for (13.15, p= 0.004) category . Body mass index reveals unhealthy aproach to personal health improvement and unhealthy diet for 71% of examinees with breast cancer . It shows that 63% of the examinees are overweight or obese (= 1.95, p=0.749). When we analyse physical activity as a factor of healthy way of living, we can say that 70% of patients in both groups does not practice it . The genesis od menopause came earlier with patients diagnosed with breast cancer than with control subjects (=10.87, p=0.012; table 1). Menopause posed earlier with examinees diagnosed with breast cancer (=10.87, p=0.012; table 1). According to our samples, housewives, 52% of them, most oftenly get affected by breast cancer, retired women are second ones with 24%, and when it comes to employed women, the ones from service industry are third (table 2). Linear regression analysis at which breast cancer was dependent variable, and way of living factors were independent variables, it came to our knowledge that there were some differences for breast cancer . Satisticaly significant relative risk for breast cancer genesis is increasing age, over 45 (experimental gropup) (or= 1.2586, 95%ci, 0.6919- 2.289). Statisticaly significant relative risk for breast cancer origin is bad wealth status (or=1.1449) and displeasure with mentioned situation (or=1.1664; table 3). When we analyse family predisposition to breast cancer we see there is significant relative risk if a sister was affected by breast cancer (or=1.5247), if grandmother was affected by the breast cancer the risk grows lower (or= 2.211), and if a distant relative had breast cancer the relative risk doubles comparing to the case with grandmother (or= 4.422; table 3). Statistical significant risk factors for genesis of breast cancer from the area of jatrogen radiation was exposure to x - ray radiation untill three years old (or=1.290). It is important to point out that the exposure to ct diagnosis two or more times doubles the risk or= 2.022; table 3). Examinees who did not gave birth to a child have the growing risk of breast cancer (or=1.394), while the usage of hormon replacement therapy (or= 1.826) and time of the menopause (or=1.394) as well represent important breast cancer risk factors . Statisticaly significant is the time of the menarche (or= 2.651) and previously cistino changed breasts (or= 1.165; table 3). The lenght to the cigarette smoke exposition represents important risk factor with examinees who smoke (or= 1.531), the exposure to smoke by family members (or= 1.260), the exposure to smoke at a wirking place with examinees who are non - smokers (or= 1.220). Not practicing the physical activity is significant predictor of the breast cancer genesis and it has been discovered by multiple regression analysis (exp (b)=0.067 95% ci 0.009 - 0.504; p=0.009, the result is not shown). Zenica- doboj canton is located in the central part of bosnia and herzegovina, with an area of 3343.3 km2 with the population of 400 602 inhabitants (population density of 119.8 inhabitants / km 2). A cantonal is a unit of the federation of bosnia and herzegovina (fb & h). The breast cancer accounted for 74.5% of all registered malignant diseases of women (26.1% index structure) and a major cause of women mortality . What is the older female person has a higher risk of this malignancy (18, 19, 21, 22, 23). Thus increasing age two patients were aged 26 - 35 years (2% of 100 women with breast cancer). Women older than 45 years of age to have about 1.3 times more likely risk for develop breast cancer . In developed countries, often the risk is shifted by age category in women of advanced age> 60 or> 65 years . In the united states in the period from 2000 to the breast cancer was diagnosed in women younger than 20 years, 1.9% of them were aged 20 - 34 years, 10.6% aged 35 - 44 years, 22.2% aged 45 - 54 years, 22.9% aged 55 - 64 years, 20.2% aged 65 - 74 years, 16.7% aged 75 - 84 years and 5.4% aged over 85 godina (19). Similarly indications are for other countries . In the uk, according to the national cancer center (23), in 1996 risk of developing breast cancer at age 25 years is 1 in 15 000 women; at 30 age years 1 in 1900 women; under age 40 years 1 in 200 women; at age 50 years 1 in 50 women, from 50 to 60 years 1 in 23 women; at age 70 years 1 in 15 women; at age 80 years 1 in 11 women; from 80 to 85 years 1 in 10 women (10, 18). Our results reveal a higher incidence of breast cancer in younger age groups compared to developed countries (at age to 35 years, 2 in 100 women) and prevalence of diseases in the shift to younger age groups (18, 19). This is confirmed by research conducted by saric (2009) in b&h, in sarajevo canton . The median age of cancer was 58 (22).with increasing age continuously increases length of exposure to carcinogens and aggravating factors? It should be noted that research in our country suggest that breast cancer occurs in younger age groups than in developed countries . Specifically, patients with cancer are significantly poorer compared to the control group (31% vs. 17%, p <0.001, table 1). Subjects with cancer had a significantly uncertain existence in comparison to control subjects (only 15% of them employed). According to published studies women with higher level of socio- economic status had higher risk of breast cancer (12, 13, 26, 27). Our study results are contradictory and in fact poverty is a relative risk or worsening factor in the development of this serious disease, but is not a predictor for breast cancer . Studies worldwide showed that women in the higher risk of developing the disease compared to women living in rural areas (17, 19). There were no significantly difference in the occurrence of diseases among women living in villages and cities . If the grandmother had cancer increases the relative risk for nearly twice times more compared to the subjects without family predisposition (or = 2.2), but risk is 4 times higher if the breast cancer had a distant cousin (or = 4.4). It is known that obesity and physical inactivity is a poor combination of increased risk of developing breast cancer (10, 26). Routine physical activity is associated with increased incidence of breast cancer (10, 12, 28). Women who do not have the habit of routine physical activity have a predictor of developing breast cancer (28, 29), as is the case in our patients (p=0.009). The previous experience in developed countries can be explained by factors of breast cancer associated with exposure to estrogens during reproductive time or change the concentration of this hormone in obese people, alcohol and persons with reduced physical activity (4, 5, 6, 7, 8, 9, 21). Women who have undergone the menopause have a lower risk of breast cancer than pre - menopausal women of the same age and childbearing pattern . Risk increases by almost 3% for each year later at menopause (natural or induced by surgery), so that a women who has the menopause at 55 rather than 45, has approximately 30% higher risk (30, 31, 32, 33, 34). However, we found that menopause among patients with breast carcinoma come much earlier related to control subjects . Did wars distress, and along with poor financial status and unemployment may be risk factors for breast cancer in the territory of bosnia and herzegovina remains to be explored! We found numerous contradictory study results related to results from other authors, for example our patients with breast cancer have: increasing breast cancer risks among younger age groups (opposite to thesis that with increasing age increases breast cancer risk); low level of income (opposite to thesis that in develop country patients with breast cancer have usually high level of income); early menopause (opposite to thesis that later menopause is predictor for breast cancer); negative growth trend for breast cancer in the conditions of clearance to close relatives; our patients with breast cancer have mainly overweight or obesity which is not significantly breast cancer risk . Patients are be educated on medical treatment side effect prevention (diagnostic ct or hormonal substitute treatment risks), elimination of workplace predictors of breast cancer (as rotating night shifts), or healthy food intake and protection with continued physical activity (29, 30, 31, 32, 33, 34).
Conversion from laparoscopic to open surgery is common in colorectal surgery with an incidence of 10% to 23% . Many studies have examined risk factors for conversion in laparoscopic colorectal surgery in an effort to identify high - risk patients . Some of the reported risk factors include high body mass index, americans society of anesthesiology score> 2, and the surgeon's operative experience . There is limited data regarding outcomes of patients who had conversion from laparoscopic to open colorectal surgery . Also, the best surgical approach (laparoscopic vs. open) for high - risk patients is unclear . There is controversy regarding the outcomes of patients who required conversion during laparoscopic colorectal operations . Some previously published data demonstrates poor outcomes with up to 50% morbidity rate in patients requiring conversion . Longer operative time for colorectal laparoscopic surgery and a high rate of conversion to open surgery in high - risk patients has raised concerns about benefits of laparoscopic surgery in this subset of patients . Also, higher local recurrence and reduced cancer - free survival have been reported in colorectal cancer patients who had conversion from laparoscopic to open procedures . However, some studies report only a small difference in outcomes of patients who underwent conversion and justify implementation of laparoscopic surgery into daily practice . Deciding on surgical technique in high - risk patients while considering most previous studies had limited numbers of patients; therefore, a large nationwide study analyzing the contemporary rate of conversion and the impact of conversion on outcomes of colorectal patients is needed . Using a large database, we aim to report contemporary rates of conversion and outcomes of converted procedures compared with planned open and successfully completed laparoscopic operations . This study was performed using the nis (national inpatient sample) database from january 1, 2009, to december 31, 2012 . The nis database is the largest publically available all - payer inpatient care database in the united states and contains data on> 7 million hospital stays each year . Approval for use of the nis data in this study was obtained from the human research protection of the university of california, irvine medical center and the nis . We analyzed discharge data on patients who had undergone colorectal resections for the diagnoses of benign or malignant colorectal tumors, diverticular diseases, crohn disease, and ulcerative colitis using the procedural and diagnosis codes as specified by the international classification of diseases, ninth revision, clinical modifications (icd-9-cm). To identify patients who underwent colorectal resection the icd-9 procedure codes of 17.31 to 17.39, 45.71 to 45.76, 45.81 patients' diagnoses were defined based on the following icd-9 codes: malignant neoplasm of colon and rectum (153.0153.9, 154.0, 154.1, 230.3, and 230.4), benign neoplasm of colon and rectum (211.3, 211.4), diverticulosis (562.10 and 562.12), diverticulitis (562.11 and 562.13), crohn disease (555.0, 555.9), and ulcerative colitis (556.0556.9). Conversion of laparoscopic surgery to open surgery was defined as the icd-9 diagnostic code of v64.41 . Patients were excluded if they were younger than 18 years old or did not undergo colon or rectal resection . Preoperative factors analyzed, as conveyed in table 1, included patient characteristics (age, sex, and race) and 12 comorbid conditions including chronic pulmonary disease, chronic renal failure, fluid and electrolyte disorders, obesity (body mass index 30 kg / m), hypertension, diabetes mellitus, metastatic cancer, liver disease, coagulopathy, alcohol abuse, history of previous abdominal surgery, and weight loss> 10% in the preceding 6 months . Other factors analyzed included pathologic conditions (crohn disease, ulcerative colitis, colorectal cancer, diverticulosis or diverticulitis, and benign colorectal tumor), procedure type (cecectomy, right colectomy, left colectomy, transverse colectomy, sigmoidectomy, and proctectomy), surgical techniques (laparoscopic, open, and converted procedures), and postsurgical complications (intra - abdominal infection, acute renal failure, acute respiratory failure, deep vein thrombosis, urinary tract infection, pulmonary embolism, wound infection, wound disruption, bowel obstruction, postoperative prolonged ileus, and hospitalization for> 7 days from admission date). Patients were divided into 3 groups: successful laparoscopic surgery, open surgery, and conversion from laparoscopic to open surgery . Risk - adjusted analysis was performed to compare postoperative complications of the 3 groups of patients . Female sex, age <70 years, and benign colorectal tumor were used as reference data points for comparison in line with the literature . Demographics of patients who have undergone colon and rectal surgery in the united states, nis 20092012 values are n (%) unless otherwise indicated . Multivariate analysis using logistic regression was used to compare outcomes of the 3 groups of surgical techniques (open, laparoscopic, and laparoscopic procedures that required conversion). P values <.05 were considered statistically significant . For each outcome, the adjusted odds ratio (aor) with a 95% confidence interval (95% ci) was calculated and reported to estimate the relative risk associated with each surgical technique . Adjustments were made for age, sex, race, chronic pulmonary disease, chronic renal failure, history of abdominal surgery, obesity, hypertension, diabetes mellitus, metastatic cancer, liver disease, coagulopathy, alcohol abuse, weight loss, type of admission, type of the procedure, type of surgical technique, and pathology type . Multivariate analysis using logistic regression was used to compare outcomes of the 3 groups of surgical techniques (open, laparoscopic, and laparoscopic procedures that required conversion). P values <.05 were considered statistically significant . For each outcome, the adjusted odds ratio (aor) with a 95% confidence interval (95% ci) was calculated and reported to estimate the relative risk associated with each surgical technique . Adjustments were made for age, sex, race, chronic pulmonary disease, chronic renal failure, history of abdominal surgery, obesity, hypertension, diabetes mellitus, metastatic cancer, liver disease, coagulopathy, alcohol abuse, weight loss, type of admission, type of the procedure, type of surgical technique, and pathology type . The most prevalent comorbidities included hypertension (52.5%) and diabetes mellitus (18.5%). The most common laparoscopic colorectal procedure was right colectomy (41.2% of all laparoscopic procedures) followed by sigmoidectomy (40.3%). Among patients who underwent planned laparoscopic colorectal resection, 48,265 patients (14.3%) had conversion to open surgery . The mean length of total hospital stay was 9 days in patients with conversion, while patients who had successfully laparoscopic surgery had mean hospitalization of 6 days . The mean risk - adjusted difference in hospitalization length was statistically significant (mean difference = 2 days; 95% ci, 1.601.70; p <.01). The mortality rate in patients who underwent laparoscopic colorectal resection with and without conversion was 1.4% and 0.6%, respectively, while the adjusted risk of mortality in patients who underwent conversion was higher than in patients without conversion (aor, 1.61; 95% ci, 1.451.78; p <.01). Also, the risk - adjusted mortality of patients who underwent planned open surgery was higher than for converted patients (3.9% vs. 1.4%; aor, 1.70; 95% ci, 1.571.85; p <.01). The mean hospital charge was $77 186 in patients with conversion, while patients who underwent successfully completed laparoscopic surgery had a mean hospital charge of $56 032 . The mean difference in hospital charge was statistically significant (mean difference = $12 215; 95% ci, $11 648-$12 782; p <.01). The risk - adjusted analysis for postsurgical complications associated with conversion of laparoscopic surgery to open surgery is reported in table 2 . Specific postsurgical complications linked with conversion of laparoscopic surgery include intra - abdominal abscesses (aor, 2.64; p <.01), and postoperative wound infection (aor, 2.38; p <.01). Hemorrhagic complications and wound infection in converted procedures were more than in both laparoscopic and planned open procedures (table 2). Risk - adjusted analysis for postoperative complications of open and converted procedures compared with laparoscopic procedures values are n (%) unless otherwise indicated . The risk of conversion of laparoscopic surgery to open surgery is reported by perioperative factors and type of colorectal resection in tables 3 and 4 . Factors such as history of previous abdominal surgery (aor, 3.50; p <.01) and presence of metastatic cancer (aor, 1.73; p <.01) have strong associations with conversion . Also, compared with right colectomy procedure, the highest risk of conversion exists in proctectomy procedure (aor, 2.81; p <.01) followed by transverse colectomy (aor, 1.88; p <.01). Risk - adjusted analysis of factors associated with conversion of laparoscopic colorectal surgery to open surgery abbreviations as in table 2 . Risk - adjusted analysis of conversion of laparoscopic colorectal surgery to open by procedure type abbreviations as in table 2 . Table 5 estimates the increased risk of conversion associated with the presence of multiple risk factors in colorectal surgery . For example, patients with history of previous abdominal surgery who were admitted emergently with one of the comorbidities of liver disease, obesity, or metastatic cancer had at least 7 times increased risk of conversion . Multivariate risk estimating of conversion in colon and rectal surgery patients (increased risk calculated compared with female patients admitted nonemergently without any comorbidity) finally, table 6 describes the associations between preoperative variables and mortality of patients who underwent converted procedures . Conversion of laparoscopic to open colorectal surgery is associated with increased mortality and morbidity rates, as well as an increase in length of hospitalization . However, the outcomes of patients who required conversion are still better than those of patients who underwent planned open colorectal operations . This study reinforces implementation of laparoscopic surgery into daily practice in colorectal surgery . Also, considering the prognosis of patients who underwent converted laparoscopic procedures compared with patients who underwent open colorectal procedures, laparoscopic surgery in high - risk patients (even accepting higher chance for conversion) may still have benefits . Our results show that laparoscopic colorectal resectional surgery has a contemporary rate of 43.5% in the united states . This is in line with the previous report of 41.6% rate of laparoscopic colorectal surgery by the surgical care and outcomes assessment program collaborative in 2010 . Reported the use of laparoscopic procedures in colorectal surgery increased from 23.3% in 2005 to 41.6% in 2010 . The conversion rate of laparoscopic surgery has been previously reported between 5% and 29% in colorectal surgery.the wide variance of conversion rate is related to the patient selection, surgeon's experience, and procedure - related factors that affect the need for conversion in different studies . However, the conversion rate in colorectal surgery remains higher than for most other abdominal laparoscopic procedures such as cholecystectomy (4.9%)or splenectomy (5%). Our results show proctectomy and right colectomy have the highest and the lowest rates of conversion, respectively . High conversion rates of proctectomy in colorectal surgery have been previously reported . Also, patients suffering from crohn disease have the highest conversion rate for any pathologic condition . The incidence of conversion in patients suffering from crohn disease in this study was 23.2% . Also, risk adjustment shows that crohn disease increases the risk of conversion> 2 times than do benign colorectal tumors . This is in line with the previous report by schmidt et al of significant increase in conversion rate of laparoscopic colectomy for patients suffering from crohn disease . Converted laparoscopic procedures have higher mortality and overall morbidity than do successfully completed laparoscopic procedures . We reinforce previous reports of an increased risk of wound infection, prolonged hospitalization, and postoperative ileus with conversion compared with successful laparoscopic surgery . Other postoperative complications we found that have associations with conversion included intra - abdominal abscess, acute renal failure, respiratory failure, myocardial infarction, pulmonary embolism, hemorrhagic complications, pneumonia, wound disruption, postoperative bowel obstruction, and urinary tract infection . Our result reinforces the importance of controlling the correctable risk factors for conversion including surgeon - related factors (learning curve, experience, technical ability) by adequate training . Converted laparoscopic procedures, compared with planned open colorectal procedures, have lower rates of mortality and overall morbidity . Although converted laparoscopic procedures are performed with open surgical techniques and outcomes of such patients should not be better than planned open procedures, our results show mortality and postoperative complications were lower in converted procedures versus open procedures . This can be partly be explained with the difference in case selection between laparoscopic and open surgery, and partly by the progress made (eg, mobilization) prior to conversion . Further studies are indicated to compare outcomes of converted colorectal operations with planned open operations in 2 complete homogeneous groups of patients . After risk adjustment with a multivariate analysis, wound infection, postoperative ileus, and postoperative hemorrhagic complications in patients who underwent converted procedures are higher than both open and completed laparoscopic procedures . Further studies are indicated to confirm the significant increase of these 3 complications in patients undergoing converted procedures . Our study, which represents the largest to date on this topic, shows that 13 preoperative factors have associations with conversion of laparoscopic surgery to open surgery . Also, we confirm the previous reports of older age, male sex, and obesity as risk factors of conversion of laparoscopic surgery . Surprisingly, male sex has been cited as a predictive factor of conversion multiple times . It can be best explained by the differences in pelvic anatomy between male and female sexes and the higher amount of visceral obesity . Patients suffering from metastatic cancer and liver disease have increased risk of conversion of laparoscopic surgery . Our results show metastatic cancer and liver disease increase the risk of conversion of operation 73% and 27%, respectively . The higher rate of conversion in patients with advanced cancer has been reported previously . Emergently admitted patients with a history of previous abdominal surgery and comorbidities including liver disease, obesity, or metastatic cancer have at least a 7 times higher risk of conversion than do patients admitted nonemergently without any comorbidities . Because comorbidities of liver disease, obesity, and metastatic cancer are not acutely correctable risk factors, open planned surgery for such high - risk patients with multiple risk factors may have benefits . Although none of the identified risk factors is a contraindication to laparoscopic surgery, using this information may help surgeons in estimating the risk of conversion and expected outcomes for high - risk patients . Finally, age> 70 years and coagulopathy have strong associations with mortality of patients who underwent converted procedures . This study is a retrospective study of an immense database and is subject to selection bias and coding errors . Also, a wide variety of hospital settings and surgeons' expertise can affect the study results . The 3 groups of patients compared in the study were not 3 homogeneous groups of patients and their demographic data, comorbidities, and disease stage varied . Due to the restrictions of the database, some of the potentially important factors, such as the reason for the conversion and surgeon - related factors (eg, surgeon's experience), were not included in this study . Despite these limitations, this study is one of the most comprehensive and largest studies investigating outcomes of conversion of laparoscopic colorectal surgery to open surgery . This study is a retrospective study of an immense database and is subject to selection bias and coding errors . Also, a wide variety of hospital settings and surgeons' expertise can affect the study results . The 3 groups of patients compared in the study were not 3 homogeneous groups of patients and their demographic data, comorbidities, and disease stage varied . Due to the restrictions of the database, some of the potentially important factors, such as the reason for the conversion and surgeon - related factors (eg, surgeon's experience), were not included in this study . Despite these limitations, this study is one of the most comprehensive and largest studies investigating outcomes of conversion of laparoscopic colorectal surgery to open surgery . Although patients with converted procedures have higher rates of morbidity and compared with successfully completed laparoscopic procedures, morbidity and mortality of converted patients are still lower than those of planned open colorectal procedures . Wound infection, postoperative ileus, and postoperative hemorrhagic complications in patients who underwent converted procedures are higher than for both open and successfully completed laparoscopic procedures . In colorectal resectional operations, crohn disease patients have the highest risk of conversion of any pathology . A history of previous abdominal surgery is the most important predictor of conversion in colorectal surgery . Among procedures, proctectomy has the highest risk of conversion . The most important mortality predictor of patients who underwent converted operations are age> 70 years and coagulopathy . Improved control of coagulation disorders may decrease postoperative mortality of such patients.
Vigabatrin, an irreversible inhibitor of gamma aminobutyric acid (gaba) transaminase (gaba - t) was specifically designed to be a suicide substrate for this enzyme and thereby augment tissue gaba concentrations for the treatment of epilepsy . It was first licensed as an antiepileptic agent in the uk and the republic of ireland in 1989 . By the late 1990s, it had been accepted into mainstream clinical practice in the care of adult and pediatric patients in over 40 countries.1 in the us, vigabatrin has experienced a stuttering regulatory course . In 1998, the food and drug administration (fda) notified the manufacturer, hoechst marion roussel, that vigabatrin was not approvable based on the data submitted up until that time, in the light of the then emerging concerns about vigabatrin . These concerns included reports of severe, persistent visual field defects noted in association with the use of vigabatrin.2,3 interestingly, the development of vigabatrin in the us had been delayed for several years due to earlier concerns about white matter toxicity (intramyelinic edema) encountered in experimental animals,4 then resumed when such toxicity could not be demonstrated in humans.1 after years of having being shelved and after renewed efforts by a different company (ovation pharmaceuticals, now lundbeck), vigabatrin received fda approval in 2009 for the treatment of infantile spasms, as well as for refractory complex partial seizures . The indication for infantile spasms can be considered quite remarkable because vigabatrin is the first fda - approved treatment for this very difficult to treat disorder . Adrenocorticotropic hormone (acth), the legacy treatment for infantile spasms, has never been formally approved for that use in the us and is now entering regulatory hearings at the us fda . As described here, infantile spasms represents a rare and distinctive disorder of multiple etiologies and presents many challenges to the conduct of an adequately powered controlled study . Infantile spasms represent a devastating childhood epileptic syndrome, and has been described as one of the catastrophic epilepsies of infancy.5 what makes infantile spasms such a form of devastating epilepsy is the combination of extremely difficult to control seizures and its association with mental retardation . This clinical presentation, accompanied by a distinctive high - voltage chaotic electroencephalogram (eeg) pattern called hypsarrhythmia and developmental delay, is called west syndrome . Hypsarrhythmia is the characteristic interictal eeg pattern, consisting of a triad of features, including high amplitude and disorganized or chaotic background and multifocal independent spike discharges . Infantile spasms are slightly more common in males.6 this syndrome represents the response of the brain to a severe insult during a specific developmental window, and can result from hypoxic - ischemic encephalopathy, infections, focal cortical dysplasia, intraventricular hemorrhage, tuberous sclerosis, down syndrome, trauma, aicardi syndrome, neonatal hypoglycemia, ohtahara syndrome, and pyridoxine deficiency . Advances in genetic testing have shown that mutations within the aristaless - related homeobox gene on chromosome xp22.13 is associated with an x - linked infantile spasms syndrome.7 the cyclin - dependent kinase - like or serine - threonine kinase 9 gene located on the xp22.3 chromosome is also a cause of x - linked infantile spasms . Mutations in the lissencephaly-1 and the doublecortin genes are related to classical lissencephaly, double cortex syndrome, and infantile spasms.8 epileptic spasms have a variety of clinical and electrographic forms . The typical clinical spasms is a flexor spasms that is a rapid flexion of the neck, trunk, and extremities lasting less than two seconds, which is frequently followed by a sustained tonic phase for up to 10 seconds.9 on occasions, spasms can be extensor, with a rapid extension of the neck and arching of the back, or a mixture of both flexion and extension postures . The intensity of spasms can vary, even within a cluster, from brief eye rolling or subtle head nodding to a violent shock - like event . On occasion electrographically, there are a variety of eeg patterns which can accompany the spasms, but the most common is a generalized medium to high amplitude slow wave, followed by an electrodecrement and attenuation.6 children with symptomatic infantile spasms can have focal findings both clinically and electrographically . The availability of vigabatrin in much of europe, and the finding that it was often efficacious in the treatment of infantile spasms, resulted in its adoption as the preferred agent, because of its relative ease of use . In europe and many other countries, vigabatrin is considered the initial drug of choice . The european expert opinion statement from 2007 recommended vigabatrin as the treatment of choice for patients with tuberous sclerosis and other symptomatic etiologies.10 in the us, where vigabatrin was not fda - approved until very recently, acth has been considered the initial drug of choice, even though it awaits regulatory approval at this time . Many additional agents and treatments have been tried as second and third options, including valproic acid, pyridoxine, zonisamide, topiramate, levetiracetam, intravenous immunoglobulin, and the ketogenic diet, each with a handful of uncontrolled open - label studies or retrospective case series . Resective surgery for some symptomatic cases (in particular focal cortical dysplasia) has been successful . In 2004 the practice parameter: medical treatment of infantile spasms was released by the american academy of neurology and the child neurology society . Vigabatrin is possibly effective for the short - term treatment of infantile spasms (level c, class iii and iv evidence), 2 . Vigabatrin is also possibly effective for the short - term treatment of infantile spasms in the majority of children with tuberous sclerosis (level c, class iii and iv evidence), and 3 . Serious concerns about retinal toxicity in adults suggest that serial ophthalmologic screening is required in patients on vigabatrin . However, data are insufficient to make recommendations regarding the frequency or type of screening that would be of value in reducing the prevalence of this complication in children (level u, class iv studies).11 vigabatrin was finally approved by the fda on august 21, 2009 with indications for refractory complex partial seizures in adults and infantile spasms in children aged one month to two years . All health care providers using vigabatrin will need to enroll in the share (support, help and resources for epilepsy) program and complete the risk evaluation and mitigation strategy (rems) course . This was specifically designed to reduce the risk of vision loss, and includes baseline and regular vision monitoring, frequent assessments of effectiveness, and education . It is suggested to discontinue the medication if patients fail to show substantial improvement in seizures within three months . Vigabatrin is now the only medication in the us with an fda approval for infantile spasms.12 it was designed specifically to achieve irreversible inhibition of gaba - t,13 which is present in neurons and glia and causes oxidative deamination of gaba to succinic semialdehyde . Unlike several other inhibitors of gaba - t which also display some activity against the enzyme responsible for the synthesis of gaba (glutamic acid decarboxylase) vigabatrin the obvious structural similarity is responsible for the ability of vigabatrin to interact with gaba - t . The presence of the vinyl moiety in the structure of vigabatrin renders it a mechanism - based suicide substrate which is covalently (and irreversibly) bound to the enzyme . Thus, even a single dose produces a lasting, dose - dependent inhibition of brain gaba - t in experimental animals, and elevated gaba levels are seen for at least 24 hours.13 after a dose of 1500 mg / kg of vigabatrin in mice, gaba levels increased to a maximum of 650% of control in four hours,15 levels of the excitatory amino acids, glutamate and aspartate, reached a nadir of approximately 80% of control values in about the same duration . Noninvasive measurement of brain gaba levels in humans by the application of 1 magnetic resonance spectroscopy has permitted the demonstration of 2.5- to 3-fold elevations in gaba levels in the brains of epileptic patients treated with standard doses of vigabatrin.16,17 our present understanding of gaba receptor activation is based on its action at postsynaptic gaba(a) receptors (gabaar), which have a specific subunit composition, and differently assembled extrasynaptic receptors . Gabaar exhibit low affinity for gaba (and thus require the high levels of gaba reached by synaptic release) and rapid inactivation (such that the receptor is ready for a new signal again). The extrasynaptic receptors, on the other hand, have a stoichiometry that substitutes a 4 subunit (for the 1 or 2 in the synaptic receptors mediating phasic inhibition); these receptors are highly sensitive to low ambient gaba concentrations and the inhibition produced is lasting, and is called tonic inhibition . They lack a -subunit that confers benzodiazepine sensitivity to the synaptic receptors, and are assembled instead with a -subunit . Neurosteroids, related to progesterone and pregnenolone derivatives, activate extrasynaptic gaba receptors mediating tonic inhibition . This is also the postulated mechanism for ganaxalone, a synthetic neurosteroid that has shown some activity against infantile spasms.18 the action of vigabatrin likely augments both phasic and tonic inhibition by enhancing gaba concentration in different pools . Benzodiazepines like nitrazepam have been used to treat infantile spasms in the past . In a randomized multicenter study, nitrazepam was found to be comparable with acth in efficacy and better in tolerability.19 the impact on tonic inhibition can be viewed as having some similarity to one of the potential actions of acth, which can stimulate the adrenal formation of deoxycorticosterone and its tetrahydro metabolite, which is a strong agonist at extrasynaptic receptors . This potential convergence in the mechanistic basis of seemingly unrelated compounds used to treat infantile spasms is illustrated in figure 2 . In the developing brain, there may also be an important effect of ambient gaba on presynaptic gabaar to increase the strength of gabaergic synaptic transmission . Low concentrations of gaba or zolpidem, an agonist at the benzodiazepine binding site, increased the frequency of miniature gabaergic synaptic currents,20 representing a positive feedback loop . This effect was seen only in rat pups up to postnatal day 14, and has been demonstrated in the cerebellum . The presence of these presynaptic gabaar was confirmed by immunocytochemistry.20 the role of such receptors as targets for modifying spasms remains to be confirmed . The efficacy and safety of vigabatrin for use in infants has been evaluated in numerous studies (see table 1). However, some were open - label monotherapy trials or randomized comparative trials versus hydrocortisone and acth . We describe selected studies that contributed significantly to the development of vigabatrin for infantile spasms . An early large european retrospective analysis involving 250 infants evaluated vigabatrin as initial therapy for infantile spasms.21 of the initial 250 enrolled, 192 were determined to have classic spasms, based on semiology, age of onset, and eeg criteria . The mean dose of vigabatrin used was 105 mg / kg . The efficacy in this survey is among the highest reported . Cessation of spasms occurred in 69% of patients with cryptogenic spasms and 60% of patients with symptomatic spasms, excluding tuberous sclerosis . Among the responders, 82% registered their response within one week after initiation of treatment . A dramatic response rate of 96% was achieved in treating infantile spasms attributed to tuberous sclerosis . This report, like many others, found a low rate of adverse effects (14%), mainly somnolence . Importantly, only two of the original 250 patients discontinued therapy because of adverse effects . Appleton et al22 reported on a randomized, placebo - controlled, blinded study of treatment of infantile spasms using vigabatrin employing a distinctive design . In their study (n = 40, all meeting eeg criteria), the randomized, placebo - controlled phase was held for five days, after which all patients were eligible to have vigabatrin on an open - label basis . The average percent reduction in spasms in the vigabatrin - treated group was 78% compared with 26% in the placebo - treated group (p = 0.020). This study did not enroll any patients with tuberous sclerosis, a subset whose spasms have emerged to be uniquely responsive to treatment with vigabatrin . At the end of the 24-week trial period, 15 of the original 40 patients were spasm - free on monotherapy with vigabatrin, while four more were seizure - free on vigabatrin in combination with other medications . Separation between two groups was not significant because both groups had been on vigabatrin except for the first five days of the 24-week period . The study did establish that initial therapy of vigabatrin was effective, safe, and well tolerated . The first prospective, randomized, open - label trial comparing vigabatrin and acth as first - line monotherapy for infantile spasms was published in 1997.23 in a crossover study, 42 patients with untreated infantile spasms were randomized to vigabatrin 100150 mg / kg / day or depot acth at 10 iu / day . Patients who did not respond to initial therapy by three weeks were crossed over to the other treatment arm . After the first stage of treatment, 48% of those treated with vigabatrin and 74% treated with acth became seizure - free (p = 0.12). During the second stage, 92% of patients who failed vigabatrin became seizure - free on acth, and 40% of patients who failed acth became seizure - free on vigabatrin (p = 0.052). Overall, 46% treated with vigabatrin and 81% treated with acth became seizure - free (p = 0.007). More patients in the acth - treated group showed normalization of the eeg, and improvements in eeg occurred earlier . Side effects (somnolence, hypotonia, irritability) were more prevalent with acth (37%) while the 13% adverse event rate for vigabatrin was similar to that reported by aicardi et al.21 this study also confirmed that all but one of 11 responders to vigabatrin did so within eight days of therapy, and 100 mg / kg / day seemed the optimal dose . Furthermore, this study demonstrated that vigabatrin was more effective as initial therapy in children with brain malformations or tuberous sclerosis, while acth was superior in children who developed infantile spasms after hypoxic - ischemic insults . It is important to note that the dose of acth used in this study is substantially less than what has emerged as the standard in the us (150 iu / m / day). Important clinical trial data for the fda s deliberations on the approval of vigabatrin come from the multicenter, randomized, single - masked (as to dose) study by elterman et al.24,25 initial results were published in 2001 in peerreviewed form24 and the results including a larger population were presented in abstract form at the 2005 meeting of the american epilepsy society.25 this multicenter, randomized, single - blinded study evaluated 220 subjects who had not been treated with acth, prednisone, or valproic acid . They were randomized into two groups, ie, low - dose (1836 mg / kg / day) or high - dose (100148 mg / kg / day). A strict definition of treatment response was used (ie, within the first 14 days of therapy, free of spasms for at least seven consecutive days, and absence of spasms or hypsarrhythmia on an eight - hour eeg). Those who did not respond to the low - dose treatment in 14 days entered an open - label phase during which the dose of vigabatrin could be freely titrated according to response and/or tolerability . Only 18% were considered treatment responders by the strict definition, but 26% were considered responders if the 14-day window was extended . Eighty - eight percent of the responders remained spasms - free during the entire study . The response rate of the high - dose group (26%) was significantly higher than that of the low - dose group (11%). In addition, in concordance with other studies, patients with tuberous sclerosis (58%) had higher response rates than symptomatic (20%) or cryptogenic (37%) patients . In a separate abstract at the same meeting, the same group26 reported their safety data for vigabatrin . Overall, vigabatrin was well tolerated, with only 5.4% of patients having adverse effects classified as related to vigabatrin that caused a discontinuation of the drug . There are important differences in this study, which confirmed the safety recorded by earlier studies, but achieved a lower rate of efficacy . The inclusion criteria permitted patients up to 24 months of age, and duration of spasms for up to three months, which most likely contributed to the observed lower rate of response . Further, those who responded beyond the first two weeks are not counted in the comparison between groups . Thus, the response rate of 58% among patients with tuberous sclerosis and infantile spasms compares quite well with the initial observations of chiron et al27 who studied patients during the first year of life and reported an 86% response rate for infants with tuberous sclerosis . The united kingdom infantile spasms study compared the effect of vigabatrin with that of hormone therapy (prednisolone or tetracosactide depot) on developmental and epilepsy outcomes when the children were evaluated at 1214 months of age.28 absence of spasms was similar in the final clinical assessment, ie, hormone 41/55 (75%) versus vigabatrin 39/51 (76%), as were the developmental outcomes determined by the vineland adaptive behavior scale (vabs). The vabs scores were hormone 786 (sd 168) versus vigabatrin 775 (sd 127). Among patients considered to be cryptogenic, hormone therapy resulted in a higher developmental outcome (hormone 88.2 [17.3] versus 78.9 [14.3]). This study excluded children with a diagnosis of, or at high risk of, tuberous sclerosis, and thus eliminated that group that is considered to represent the strongest beneficiaries of receiving therapy with vigabatrin . Chiron et al reported on a prospective trial comparing vigabatrin with hydrocortisone as initial treatment for infantile spasms attributable to tuberous sclerosis . This randomized, crossover study utilized vigabatrin at 150 mg / kg / day or hydro - cortisone 15 mg / kg / day as initial treatment . All the patients who were first treated with vigabatrin (11/11) were spasms - free compared with 5/11 hydrocortisone infants (p <0.01). All the nonresponders to hydrocortisone became spasms - free after being crossed over to vigabatrin . Mean time to disappearance of infantile spasms was 3.5 days on vigabatrin versus 13 days on hydrocortisone (p <0.01). This study was responsible for establishing the special place of vigabatrin in the treatment of infantile spasms in patients with tuberous sclerosis . It is worth pointing out that such a high response rate is seldom seen in even so - called benign epileptic syndromes of childhood . Retinal toxicity and white matter changes on magnetic resonance imaging (mri) are the effects that merit separate discussion . However, the common side effects encountered commonly in the various studies include drowsiness / somnolence, nystagmus, hyperexcitability / hyperkinesia, insomnia, fever, memory impairment, depression, and confusion . Less frequently encountered are axial hypertonia, hypotonia, agitation / irritability, asthenia, laryngitis, myoclonus, diarrhea, weight gain, and vomiting . Rare cases of psychotic reactions, mild anemia, and decreased liver function tests (aspartate aminotransferase and alanine aminotransferase) have also been reported . Overall, in comparison with many other antiepileptic drugs, vigabatrin is well tolerated.1,21,22,29 one of the most discussed and concerning adverse effects of therapy with vigabatrin is retinal toxicity leading to visual field constriction . Reports of visual field deficits have appeared in the literature since 1997.30 in various studies, this effect has been documented in up to 40% of treated patients . Pale disc, mild optic nerve pallor, retinal artery narrowing, and problems with visual acuity, color discrimination, and contrast sensitivity have been reported . First, the increase of gaba in the retina is likely to have a role.3 second, there may be a relationship between retinal damage, vigabatrin, and exposure to light . In the rat retina, administration of vigabatrin produced a decrease in gaba - t activity to undetectable levels and a five - fold increase in gaba.31,32 in a study of chronic vigabatrin exposure in rats, vigabatrin for 45 days produced a decrease in visual tests, including photopic erg, flicker response, and oscillatory potentials.33 the authors described disorganization of the outer retina, as well as damage to the inner and outer cone segments, most prominent in the central areas with a decrease in numbers in up to 20% of photoreceptors . The toxicity of vigabatrin to retinal cells is likely affected by development . In the rabbit retina, the fraction of amacrine cells capable of taking up and accumulating vigabatrin was very small at postnatal day 1 and increased to near - adult levels by 10 days postnatum.34 it is still not entirely clear if the toxicity is related to the cumulative dose or if retinal involvement improves after discontinuation of the drug.35 there is a positive trend in one retrospective review of higher doses and longer treatment periods correlating with visual field constriction, abnormal erg, and abnormal visual - evoked potentials (veps),36 although these findings have not been consistent . Somewhat encouragingly for short - term treatment of infantile spasms with vigabatrin, a study of children observed that the most severe damage was not observed in the youngest patient.37 more recently, a finnish study of 16 children with a history of treatment of infantile spasms with vigabatrin tested the subjects with kinetic perimetry between six and 12 years and encountered only mild visual field loss in one patient and normal fields in 15.38 nevertheless, careful monitoring for discernible signs of retinal injury should be stressed, and has resulted in the risk evaluation and mitigation strategy (rems) described below . As part of rems, vision testing is required at baseline, at least every three months during therapy, and at least once within three to six months after discontinuation . While erg and veps are recommended, these studies are often difficult to accomplish, given the age of the patient population with infantile spasms (http://www.lundbeckshare.com/pg510_info_physicians.aspx).12 thus, periodic ophthalmologic examinations (at initiation of therapy, and subsequently, every three months while on treatment, and three to six months after discontinuation of therapy), while mandated, may or may not include erg and vep testing . It is also suggested that, because of the risk of permanent vision loss, vigabatrin should be withdrawn from a pediatric patient treated for infantile spasms (one month to two years of age) who fails to show substantial clinical benefit within 24 weeks of treatment initiation, or sooner if treatment failure becomes obvious . A recommended screening algorithm based on evidence - based review of visual function testing in patients treated with vigabatrin has been published recently.39 evaluation that includes perimetry is suggested only for children over the age of nine years . A critical role for the amino acid taurine in mediating vigabatrin - associated retinal ganglion cell toxicity has been demonstrated in rats and mice.40,41 animals treated with vigabatrin had 67% lower levels of taurine than controls . The hypothesis that the addition of taurine - rich foods or direct supplements may prevent or slow down the development of the retinal cell lesions has been proposed by the authors . However, excessive limiting of visual stimuli in very young patients may impact adversely on neural plasticity leading to ocular cortical organization and contribute to risk for amblyopia . The observation of intramyelinic edema and vacuolation of myelin of the brain in rats has been alluded to.4 during the period following the initial observation, human studies in adults using mri and veps reassured investigators that these findings in animals may have very limited significance for humans . Transient mri abnormalities unrelated to underlying cause have been seen in children and adults treated with vigabatrin . Areas of abnormality included the basal ganglia, thalamus, anterior commissure, corpus callosum, and brain - stem . 42 wheless et al43 reviewed 332 mris from 205 infants with infantile spasms and rereviewed mris from 668 children and adults with complex partial seizures . Abnormalities were defined as any hyperintensity on t2-weighted or fluid - attenuated inversion - recovery sequences that were diffusion restriction negative and not explained by specific pathology . Infants treated with vigabatrin were statistically more likely to have mri abnormalities than those not treated with vigabatrin . Another series involving infants (22 subjects, 34 mris) confirmed transient white matter signal changes.44 an example of such lesions from one of our patients is shown in figure 3 . It should be noted that the rems program does not address monitoring for white matter lesions by mri for infants treated with vigabatrin . Chiron et al reported on a prospective trial comparing vigabatrin with hydrocortisone as initial treatment for infantile spasms attributable to tuberous sclerosis . This randomized, crossover study utilized vigabatrin at 150 mg / kg / day or hydro - cortisone 15 mg / kg / day as initial treatment . All the patients who were first treated with vigabatrin (11/11) were spasms - free compared with 5/11 hydrocortisone infants (p <0.01). All the nonresponders to hydrocortisone became spasms - free after being crossed over to vigabatrin . Mean time to disappearance of infantile spasms was 3.5 days on vigabatrin versus 13 days on hydrocortisone (p <0.01). This study was responsible for establishing the special place of vigabatrin in the treatment of infantile spasms in patients with tuberous sclerosis . It is worth pointing out that such a high response rate is seldom seen in even so - called benign epileptic syndromes of childhood . Retinal toxicity and white matter changes on magnetic resonance imaging (mri) are the effects that merit separate discussion . However, the common side effects encountered commonly in the various studies include drowsiness / somnolence, nystagmus, hyperexcitability / hyperkinesia, insomnia, fever, memory impairment, depression, and confusion . Less frequently encountered are axial hypertonia, hypotonia, agitation / irritability, asthenia, laryngitis, myoclonus, diarrhea, weight gain, and vomiting . Rare cases of psychotic reactions, mild anemia, and decreased liver function tests (aspartate aminotransferase and alanine aminotransferase) have also been reported . One of the most discussed and concerning adverse effects of therapy with vigabatrin is retinal toxicity leading to visual field constriction . Reports of visual field deficits have appeared in the literature since 1997.30 in various studies, this effect has been documented in up to 40% of treated patients . In addition to visual field loss, abnormal funduscopic examination, including pale disc, mild optic nerve pallor, retinal artery narrowing, and problems with visual acuity, color discrimination, and contrast sensitivity have been reported . First, the increase of gaba in the retina is likely to have a role.3 second, there may be a relationship between retinal damage, vigabatrin, and exposure to light . In the rat retina, administration of vigabatrin produced a decrease in gaba - t activity to undetectable levels and a five - fold increase in gaba.31,32 in a study of chronic vigabatrin exposure in rats, vigabatrin for 45 days produced a decrease in visual tests, including photopic erg, flicker response, and oscillatory potentials.33 the authors described disorganization of the outer retina, as well as damage to the inner and outer cone segments, most prominent in the central areas with a decrease in numbers in up to 20% of photoreceptors . The toxicity of vigabatrin to retinal cells is likely affected by development . In the rabbit retina, the fraction of amacrine cells capable of taking up and accumulating vigabatrin was very small at postnatal day 1 and increased to near - adult levels by 10 days postnatum.34 it is still not entirely clear if the toxicity is related to the cumulative dose or if retinal involvement improves after discontinuation of the drug.35 there is a positive trend in one retrospective review of higher doses and longer treatment periods correlating with visual field constriction, abnormal erg, and abnormal visual - evoked potentials (veps),36 although these findings have not been consistent . Somewhat encouragingly for short - term treatment of infantile spasms with vigabatrin, a study of children observed that the most severe damage was not observed in the youngest patient.37 more recently, a finnish study of 16 children with a history of treatment of infantile spasms with vigabatrin tested the subjects with kinetic perimetry between six and 12 years and encountered only mild visual field loss in one patient and normal fields in 15.38 nevertheless, careful monitoring for discernible signs of retinal injury should be stressed, and has resulted in the risk evaluation and mitigation strategy (rems) described below . As part of rems, vision testing is required at baseline, at least every three months during therapy, and at least once within three to six months after discontinuation . While erg and veps are recommended, these studies are often difficult to accomplish, given the age of the patient population with infantile spasms (http://www.lundbeckshare.com/pg510_info_physicians.aspx).12 thus, periodic ophthalmologic examinations (at initiation of therapy, and subsequently, every three months while on treatment, and three to six months after discontinuation of therapy), while mandated, may or may not include erg and vep testing . It is also suggested that, because of the risk of permanent vision loss, vigabatrin should be withdrawn from a pediatric patient treated for infantile spasms (one month to two years of age) who fails to show substantial clinical benefit within 24 weeks of treatment initiation, or sooner if treatment failure becomes obvious . A recommended screening algorithm based on evidence - based review of visual function testing in patients treated with vigabatrin has been published recently.39 evaluation that includes perimetry is suggested only for children over the age of nine years . A critical role for the amino acid taurine in mediating vigabatrin - associated retinal ganglion cell toxicity has been demonstrated in rats and mice.40,41 animals treated with vigabatrin had 67% lower levels of taurine than controls . The hypothesis that the addition of taurine - rich foods or direct supplements may prevent or slow down the development of the retinal cell lesions has been proposed by the authors . In addition, this study demonstrated that limiting light exposure might minimize cellular injury . However, excessive limiting of visual stimuli in very young patients may impact adversely on neural plasticity leading to ocular cortical organization and contribute to risk for amblyopia . The observation of intramyelinic edema and vacuolation of myelin of the brain in rats has been alluded to.4 during the period following the initial observation, human studies in adults using mri and veps reassured investigators that these findings in animals may have very limited significance for humans . Transient mri abnormalities unrelated to underlying cause have been seen in children and adults treated with vigabatrin . Areas of abnormality included the basal ganglia, thalamus, anterior commissure, corpus callosum, and brain - stem . 42 wheless et al43 reviewed 332 mris from 205 infants with infantile spasms and rereviewed mris from 668 children and adults with complex partial seizures . Abnormalities were defined as any hyperintensity on t2-weighted or fluid - attenuated inversion - recovery sequences that were diffusion restriction negative and not explained by specific pathology . Infants treated with vigabatrin were statistically more likely to have mri abnormalities than those not treated with vigabatrin . Another series involving infants (22 subjects, 34 mris) confirmed transient white matter signal changes.44 an example of such lesions from one of our patients is shown in figure 3 . It should be noted that the rems program does not address monitoring for white matter lesions by mri for infants treated with vigabatrin . Vigabatrin, the only presently approved therapy for infantile spasms in the us, has shown efficacy and tolerability, as well as relative safety (especially compared with the available options, including nontreatment),45 representing a meaningful advance in the treatment of this catastrophic childhood epileptic encephalopathy . Availability of vigabatrin for infantile spasms is especially meaningful to those patients in whom it is a manifestation of tuberous sclerosis . Guidelines have been developed to monitor for visual system toxicity, and typical use will be limited to a few months, enhancing the benefit to risk ratio.
Noncoding regions are the major part of eukaryote genomes, and most of them are believed to evolve neutrally (kimura 1983). Under this assumption, we expect that the frequency of a particular short oligonucleotide, or dna word, of 10 bp or shorter should be primarily determined through accumulation of neutral mutations, and the total set of frequencies of all dna words of certain length should follow some simple statistical rules . Oligonucleotide frequencies of one genome can provide a useful mechanism of genome comparison (karlin 2005), including phylogeny reconstruction (takahashi et al . Most frequently, such comparisons are based on a dinucleotide composition model (karlin and mrazek 1997; gentles and karlin 2001) or on self - organizing maps (abe et al . 2003). We created a series of statistical models predicting the frequencies of word of up to 4 nt in a genome . We retrieved all available complete eukaryote and prokaryote genomes, constructed such models for them, and compared the actual word frequencies with those predicted by the models to determine the discrepancy . Here, we present a database, called genome composition database (gcd), which shows how accurately each genome can be approximated by a model . The unique point of this database is that it allows to compare compositional complexity of genomes and to analyze over- or underrepresentation of particular oligonucleotides . 2007), ensembl (http://uswest.ensembl.org/; flicek et al . 2012), university of california santa cruz (http://genome.ucsc.edu/; fujita et al . 2011), flybase (http://flybase.org/; mcquilton et al . 2012), and wormbase (http://www.wormbase.org/; harris 2010). Genome sequences of a total of 1,228 species (101 eukaryotes, 1,043 eubacteria, and 84 archaea, as of june 2010) were used to construct the database . For every genome, we created a series of five composition models: uniform (composition of a, c, g, and t are set to be all 25%), mononucleotide, dinucleotide, trinucleotide, and tetranucleotide . Each composition model is based on the total size and word frequencies of an actual genome . We use both dna strands to perform the word counting, so the number of g bases is always same with number of c, same for a and t, and each dna word has the same frequency with its reversed complementary counterpart . Among the 16 dinucleotides, there are 12 that differ from their reversed complementary dinucleotide and 4 that are identical to their reversed complementary one (cg, gc, at, and ta). Therefore, the first group of dinucleotides can be described with six frequencies (12/2) and the second with four . Subtracting one, and adding the genome size, we obtain ten parameters for the dinucleotide model . In case of trinucleotide frequencies, none of the trinucleotides are identical to their reversed complementary counterpart, so the model has 4/2 = 32 parameters . In tetranucleotide case, there are 16 tetranucleotides that are identical to their reversed complementary counterparts, so the tetranucleotide model has (4 16)/2 + 16 = 136 parameters . For a genome g of total length m and a dna word w, a composition model can be used to compute p(w), which is the probability of observing w at any particular position in the genome . For example, the uniform composition model gives(1)where l is the length of w. the mononucleotide composition model predicts(2)where wi is the ith nucleotide of w, f(x) is the observed frequency of x in the genome sequence, and c(x) is the complementary sequence to x. using the same principle, p(w) from dinucleotide, trinucleotide, and tetranucleotide composition models can be computed . The model expectation of the frequency of word w in both strands of the modeled genome is then given as follows:(3) then, we can define the deviation of the observed frequency from the expected frequency:(4) because each of the composition models assumes independence of different genome positions from each other, e(w) follows the binomial distribution, and its variance can be computed as follows:(5) the standard deviation of e(w) is its square root . We then can define the relative abundance of w, under this particular model, as follows:(6) this r(w) is 0 for dna words, occurring in the genome with exactly the same frequency, as predicted by the composition model . R(w) is positive when the actual frequency is larger than expected by the model . In such cases, we describe that w is overrepresented in the genome, according to this model . When the actual frequency is smaller than expected by the model, r(w) is negative, and w is underrepresented . Now we can summarize the overall magnitude of over- or underrepresentation of all dna words of length l in the genome (using a particular composition model of choice) as follows:(7)where w is the set of all dna words of length l and because r is the standard deviation of a sample of all r(w) for a particular word length l, the unit of r is the same with that of r(w), which is e(w) (standard deviation of the word frequency, predicted by the model). For each w, re(w) gives the relative number of occurrences of w, which would make w averagely rare or abundant . R is computed for a particular genome, composition model, and l and summarizes the ability of the composition model to predict the frequencies of words of length l in the genome . Large r implies that many w's have large absolute values of r(w), which means that their actual frequencies are far from those expected by the model . Thus, a large value of r signifies that the model's ability to describe the actual genome is poor . A good composition model has small value of r, with r being 0 for the perfect model . An example of such perfect model is the l - bp composition model used to predict the frequencies of words of the same length l bp or shorter . For instance, the dinucleotide composition model has the exact information about dinucleotide frequencies, so it gives perfect predictions for 1-bp or 2-bp word frequencies, resulting in r value of 0 . For the longer words, r is typically much larger than 0 for nonrandom sequences . On the other hand, when a random sequence is modeled using any composition model, the actual variances of the word frequencies are the same with the variances predicted by the model; therefore, r is close to 1 in this case (approaching 1 as the sequence becomes longer). This is also the case for semirandom sequences, where the deviation from uniform randomness is at most as complex (controlled by at most as many parameters) as the model used to analyze the sequence . For example, a semirandom gc - biased sequence can be accurately modeled by the nucleotide composition model, or any more complex model, but not by the uniform composition model . The r values obtained with the uniform composition model for such sequence are much larger than 1, whereas other models still produce r close to 1 . Figure 1 illustrates this by showing the example histograms of relative abundances for all words of length 8 in the human genome, using five different models . The strange bimodal - looking shape of the uniform model histogram results from the extreme depletion of cpg dinucleotide in mammalian (including human) genomes . Any 8-bp word containing cpg will appear as strongly underrepresented when comparing the actual frequencies with those predicted by the uniform model . So, all such words contribute to the left peak on the histogram, whereas words without cpg form the other peak, in agreement with the model . Histograms of relative abundances of all oligonucleotides of 8 bp in human genome, according to the five composition models . The words placed to the left of the line are underrepresented and to the right overrepresented . We computed r for all five composition models for available complete genomes, both eukaryotes and prokaryotes . We then extracted unusually rare and unusually abundant words, which we define as those having \|r(w)\|> r. these dna words, together with the corresponding statistics, are available for viewing and downloading at the gcd online . R value comparison for selected species note.this table compares the r values of e. coli, yeast, plant, fruit fly, fish, lizard, and mouse, respectively, for each of the five models we used, based on words of 8 bp . Next, we analyzed the spacing patterns of individual dna words in complete genomes . Looking at all occurrences of a particular dna word in the genome, we can extract the distances between the genomic locations of every two neighboring occurrences and use this set of distances as a spacing data set for this particular word . Sample parameters (mean, standard deviation, skewness, and kurtosis) are computed for such data set . The mean distance approximately equals to the genome size divided by total number of occurrences, so it correlates with the reciprocal of the word frequency . Skewness shows whether extremely unusual spacing values for this word tend to be large or small . Kurtosis shows if the word tends to form clusters and the density of those clusters relative to the distance between them . Taking a particular parameter for all words of length l furthermore, selecting only subset of dna words with parameters falling into particular ranges, we can extract interesting dna words . In order to verify the models and better understand the parameters, we constructed a range or semirandom sequences using a random sequence generator (kryukov k, unpublished data). Each semirandom sequence was based on particular real genome used as template (e.g., the human genome): it had the same size with the template genome, and it imitated n - bp composition of the template genome, with n ranging from 1 to 4 . We used genomes of five species as templates: human, anolis carolinensis (lizard), xenopus tropicalis (frog), oryzias latipes (fish), and drosophila melanogaster (fruit fly). Figure 2 shows the comparison of r values for 101 eukaryote genomes used in this study, as well as representative prokaryote genomes, computed for 5 bp oligonucleotides . Such r values represent how well different composition models can predict 5-bp composition of the genome . Supplementary figure 1 (supplementary material online) shows comparison of all prokaryote genomes included in this study . R values of five composition models are displayed as differently colored areas . As can be seen mammals are compositionally more complex than nonmammal vertebrates, land vertebrates are more complex than fishes, and fishes are more complex than most invertebrates and plants, which are still more complex than fungi and protists . Compositional genome complexity of prokaryotes, represented by r values, is comparable with that of fungi . Comparison of r values based on oligonucleotides of 5 bp and all five composition models . (a) eukaryote genomes (all available in public databases by october 2010). Figure 3 shows the average r values for different groups of organisms, with standard deviation . Under all five composition models, statistically significant difference is observed between the r values of mammals and nonmammal vertebrates (mann whitney p <0.001, see supplementary table 1, supplementary material online for test results). Interestingly, r values of invertebrates are close to those of plants and significantly higher than those of fungi, protists, or prokaryotes (archaea and eubacteria). In terms of r values, average r values for different groups of organisms, with standard deviations, using five different composition models . Tables 2 and 3 show the partial lists of under- and overrepresented words of 10 bp in human genome, using tetranucleotide composition model . The complete lists of under- and overrepresented words, for every of the included genomes, for each of the five composition models, and for dna words of up to 10 bp for eukaryotes and 8 bp for prokaryotes, are available at the gcd online . Both the actual and the expected frequency are given for both dna strands combined, so each word's frequency is identical with that of its reversed complementary counterpart (given in parentheses). Underrepresented oligonucleotides of 10 bp, example from human genome note.showing ten most underrepresented oligonucleotides, according to the tetranucleotide composition model . Both the actual and the expected frequency are given for both dna strands combined, so each word's frequency is identical with that of its reversed complementary counterpart (given in parentheses). Overrepresented oligonucleotides of 10 bp, example from human genome note.showing ten most overrepresented oligonucleotides, according to the tetranucleotide composition model . Other than the reporting the general compositional complexity, the gcd can be used to compute the distances between the composition vectors of various complete genomes and submitted sequences (similar to the method taken by takahashi et al . We used this tool to analyze three classes of human sequences: random sample from the human genome, conserved sequences of unknown function, and conserved functionally important sequences . Although sequences from these three classes are all found in the human genome, they have different nature and evolutionary history, allowing interesting comparison . The uce data set (human mouse rat ultraconserved elements, 481 sequence, 126 kbp in total, bejerano et al . Human microrna (mirna) seed sequences (1,100 sequences from mirbase, 7.7 kbp in total, kozomara and griffiths - jones 2011) were used as functionally important conserved sequences . Figure 4a shows the average euclidean distances between the composition vectors obtained from randomly sampled human sequence and composition vectors of complete vertebrate genomes . Each sample was chosen to have the same number of sequences and average sequence length with the uce data set: 481 sequences, 262 bp each . Di-, tri-, and tetranucleotide composition vectors are used for comparison . As expected, primate genomes are the closest to human sample, and more diverged species show progressively larger distances, with some fluctuations . Euclidean distances between composition vectors (oligonucleotide frequencies) of sample data sets and complete vertebrate genomes for three composition models (dinucleotide, trinucleotide, and tetranucleotide). One thousand samples were used, where each sample consisted of 481 sequences of 262 bp each (for a total size of each sample same with the uce data set), taken from the random locations in the complete human genome . (b) the composition of the uce data set is compared with that of complete vertebrate genomes . (c) the composition of human mirna seed sequences is compared with that of complete vertebrate genomes . The compositional distances between the uce and the complete vertebrate genomes appear to be relatively uniform among vertebrates and much larger than those for the random human sample . Interestingly, these sequences appear to be compositionally close to lizard, fish, and frog . Figure 4c shows the compositional distances between human mirna sequence data set and complete vertebrate genomes . Platypus and the fishes are compositionally the closest to this data set . To further investigate the differences between these three data sets, we computed the average distances by combining the genomes into four groups (fig . 5). 5a), while much more uniformity can be seen for uce and mirna seed data sets (fig . 5b and c). Average compositional distance (euclidean distances between the composition vectors) between sample data sets and complete genomes grouped into four groups . Panels (a, b, and c) correspond to panels (a, b, and c) of figure 4 . Figure 6 shows the plots for the pairs of spacing parameters, taken for 8 bp oligonucleotides for six species human, lizard, fish, fruit fly, yeast, and escherichia coli . Although the interpretation is difficult, more structure can be seen in the plots of more complex organisms . Plots of the spacing distribution parameters for six species, based on oligonucleotides of 8 bp . Different columns show plots for different pairs of parameters, from left to right: mean spacing (x axis) versus standard deviation (y axis), mean (x) versus skewness (y), mean (x) versus log(kurtosis) (y), standard deviation (x) versus skewness (y), standard deviation (x) versus log(kurtosis) (y), and skewness (x) versus log(kurtosis) (y). Each dot in the plot represents a particular 8 bp dna word, so 48 words constitute the data set in each case . Figure 7 shows spacing plots for four random genomes (generated using human genome as a template), the complete actual human genome and the repeat - masked version of the human genome . Repeat - masked is included because complexity is often associated with repetitive sequences . In case of the those elements correspond to the groups of dna words containing different number of gc . With gc contents being the only parameter for constructing the sequence, dna words with the same number of gc will have exactly same compositional properties, blurred only by randomness of the sequence . In case of hs random 2 similar grouping happens, this time depending on number of cpg each particular word may contain . Going into more complex semirandom sequence, the discreteness becomes less clear, and the plots are getting closer to that for the real human genome . Still significant difference remains between the plots of semirandom and real sequences and very little difference between the plots of repeat - masked and the complete human genome . Plot of the spacing distribution parameters for semirandom sequences, compared with the real human genome, as well as repeat - masked one . Based on oligonucleotides of 10 bp . Semirandom genomes 1, 2, 3, and 4 are constructed using 1-, 2-, 3-, and 4-bp composition of the actual human genome . The gcd provides a convenient measure of relative complexity of various genomes from statistical point of view . A genome is compositionally simple if its composition can be accurately described by a simple model . A set of r values for various word length and models can tell us how complex a particular genome is? As figure 2 shows, r values become smaller with the increase of model complexity as expected, a more complex model can describe genome composition more accurately, which results in smaller discrepancy . We observe that, generally speaking, r values are related to the general complexity of the organism . Remarkably, even tetranucleotide compositional models are unable to give good predictions of 5-bp composition in case of complex genomes, particularly for mammals and land vertebrates . Figure 3 confirms that compositional complexity of a genome is in good correlation with general complexity of the organism . Compositional discrepancy r computed with different composition models seems to be useful as a measure of compositional complexity of the genome . The extremely rare and extremely abundant sequences, as shown in tables 2 and 3, suggest the possible mechanisms of creating compositional complexity . The most underrepresented 10 bp dna words (using tetranucleotide composition model) seem to be found on the boundary of mononucleotide repeats, particularly poly - a to poly - t boundary (words 1, 2, 4, 6, 7, 8, 9, 10 in table 2) also poly - a to poly - c (words 3 and 5 in table 2). This means that such boundary is much less common, than suggested by the 4-bp composition . Among the top overrepresented words, there are poly - a (word 5 in table 3), dinucleotide repeats (words 1 and 2 in table 3), as well as fragments of sequence gcctgtaatcccagc (words 3, 4, 6, 7, 8, 9 in table 3), which has about 800,000 occurrences in the human genome compared with the expected number of about 7,00010,000 . This sequence being unusual is already reported by valle (1993); however, no explanation for the cause was given . Figure 4 shows the compositional distances between three sequence data sets (human sample, uce, and mirna seeds) and vertebrate genomes . Although in all three cases, the sequences are contained in the human genome, the compositional distances of those sequences to various genomes show very different pictures . The random sample behaves as expected the compositional distance is increasing with the increase of divergence from human . However, uce and mirna seed data sets show more or less uniform compositional distances from various vertebrate genomes . This suggests that those sequences became conserved before the emergence of mammals . In case of mirna seed sequences, the composition distances to all vertebrate genomes are more or less uniform, suggesting those sequences were fixed much earlier than the emergence of vertebrates . Composition of the uce and mirna seed sequences is frozen and represents the composition of the ancestoral genome, at the time where the fixation occurred . The compositional distance from the current day vertebrates is larger for mirna seed data set because the mirna fixation occurred much earlier, so larger compositional distance exists between the ancestoral genome and current day genomes . Thus, this allows us to discuss the composition of premammal vertebrate genome (in case of uce data set) and early animal genome (in case of mirna seeds). Oligonucleotide spacing patterns, summarized as sample parameters and displayed as scatterplots (figs . 6 and 7), provide a further interesting view into the compositional complexity . It is apparent that the human genome is very different from the semirandom sequences that imitate only some compositional properties of the actual genome . However, the spacing scatterplots for the repeat - masked human genome looks similar to those of the complete genome and different from those based on the semirandom sequences . It remains to be seen whether the apparent complexity results from the isochore structure of the mammalian genomes (bernardi et al . 1985), the online gcd provides the means of comparing the compositional complexity of various complete genome and extracting unusual dna words . The composition parameters computed using five models, as well as histograms, are available . Also spacing patterns, summarized as parameter histograms and 2d scatterplots, in addition that database features a facility for submitting a sequence data set and performing composition analysis and comparison with various complete genomes . Compositional models that we used in this study only utilize the word frequencies as parameters . The natural next challenge is to design an integrated composition model, which would be based on both frequencies and spacing patterns . Such model would better approximate the genome and thus would allow focusing more closely on the real source of complexity . Supplementary figure 1 and table 1 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).
Nowadays cardiac surgeons must face the problem of severely impaired left ventricle function almost on a daily basis . Heart transplantation, due to the limited donor pool, can be offered only to a small number of patients suffering from cardiac failure [1, 2]. Furthermore, the idea of long - term mechanical support as a destination therapy is still evolving . Hence, efforts to optimize the results of routine cardiac surgery have to be undertaken . In a properly selected group of patients coronary surgery alone or combined with mitral surgery can improve both quality of life and long - term survival . With borderline ventricular function prior to surgery this may result in failed weaning from cardiopulmonary bypass . In isolated coronary surgery beating heart strategy, either on or off pump seems to be a safer option . Its use however is hardly possible when mitral valve repair or replacement needs to be done . Intra - aortic balloon pump (iabp) is a gold standard here, but some patients may require more sophisticated mechanical assistance [3, 4]. The idea of short - term, perioperative mechanical support, preferably with a low anticoagulation regime, looks very convenient in this respect . Impella microaxial pumps (abiomed europe gmbh, aachen, germany) seem to meet these criteria . They use the idea of pulling the blood from the left ventricle into the aorta, which is accomplished by means of a rotating impeller positioned in the aorta with the tip of the device and inlet area positioned in the left ventricle . This results in reduction of both left ventricle end diastolic volume (lvedv) and left ventricle end diastolic pressure (lvedp). The systemic pressure and flow are increased . The less stretched left ventricle, with better subendocardial perfusion, has more time to recover . On the other hand, the impella pump can be applied either by cardiologists (impella 2.5/impella cp) in a cath lab setting or by cardiac surgeons (impella 5.0/impella ld) in an operating theatre with either peripheral vascular or central direct access . Offering flow up to 5 l / min, the anticoagulation management requires only heparin infusion to keep activated clotting time (act) not shorter than 160 s. thanks to the automated controller with an intuitive, user friendly interface, operating the device can be easily mastered by doctors and nursing staff . A 64-year - old woman suffering from ischemic dilated cardiomyopathy associated with mitral insufficiency was referred for combined treatment consisting of resynchronisation therapy followed by simultaneous coronary and mitral surgery . Initial echocardiography revealed impaired global contractility (ejection fraction [ef] 10 - 15%), dilated left ventricle (left ventricle end diastolic diameter [lvedd] 7.8 cm), significant ventricular asynchrony (50 ms) and moderate mitral regurgitation (vena contracta [vc] 6 mm) due to both restriction of the posterior leaflet and dilation of the mitral annulus (5.5 cm). There were no significant abnormalities regarding the right ventricle, pulmonary valve and tricuspid valve . Clinically the patient presented with end stage cardiac failure including resting dyspnea (nyha iv). The patient underwent uneventful implantation of an icd crd (maximo ii), which resulted in improved exercise tolerance . This was consistent with post - procedure echocardiography showing no significant ventricular asynchrony (4 ms), slightly improved contractility (ef 20%) and unchanged moderate mitral regurgitation (vc 6 mm). Therefore the patient was referred for combined cardiac surgery including mitral repair and coronary grafting . Because of poor lv function and the complexity of the procedure, it was decided to apply short - term mechanical support during and after surgery . The impella ld microaxial pump was chosen due to the simplicity of implantation and the low anticoagulation regime . Also, the impella ld does not require a hybrid theater with fluoroscopic guidance . Antegrade cold blood cardioplegic solution was administered through the aortic root . The posterior descending artery (pda) and left anterior descending artery (lad) were grafted with the long saphenous vein and left internal thoracic artery respectively . The left atrium was entered in a routine manner after dissecting sondergaard's plane . On inspection the echocardiographic findings were confirmed . The only surgical issue was severe atherosclerosis of the ascending aorta affecting both anastomosis of the vein graft and implantation of the impella device . Before the implantation a 10 mm dacron graft was anastomosed to the ascending aorta using a side biting clamp (fig . The impella catheter was inserted through the graft and then, under transesophageal echocardiographic (tee) guidance, was forwarded through the ascending aorta and the aortic valve into the left ventricle (figs . 2 and 3). Immediately after confirming the position the impeller rotation was initialized . Meticulous attention was paid to maintain proper left ventricle volume preloading in order to avoid pump malfunction . After securing hemostasis the chest was closed, letting the driveline out above the suprasternal notch and through the upper end of the sternotomy wound . The patient was transferred to intensive treatment unit (itu) in a stable condition . There she was kept sedated and ventilated electively . Therapeutic activated clotting time (act) the position of the pump was checked daily and in case of any suspicion of malfunction . Adequate flow was maintained most of the time, with one episode of stopping of the machine, which was easily managed with fluid infusion . The inotropic support was reduced gradually without affecting hemodynamic stability . On the 2 postoperative day the further postoperative course was mainly uneventful, although prolonged mechanical ventilation was required (8 days). The first follow - up visit two months after discharge confirmed good exercise tolerance and satisfactory echocardiographic findings (trace mitral regurgitation, lvedd 7.6 cm, ef 25%). Impella device inserted through the tubing graft anastomosed to the ascending aorta impella device in the ascending aorta, approaching the aortic valve impella device finally positioned in the left ventricle doppler scan showing the flow from the left atrium to the left ventricle (blue) and within the device from the left ventricle to the aorta (red) patients with impaired left ventricle function require a particular surgical strategy . Obviously, it is crucial to perform a routine procedure such as coronary grafting or valve surgery with meticulous surgical technique and good timing . Therefore, these patients should be operated on by senior surgeons . However, even in experienced hands, such cases may prove quite tricky . Sometimes, even perfect coronary anastomoses and the shortest ischemic time cannot prevent failed weaning from cardiopulmonary bypass . The possibility of perioperative mechanical support, exceeding the intra - aortic balloon pump, is quite tempting . One option is extracorporeal membrane oxygenation (ecmo), which can support the circulation for up to a few weeks . This requires, however, both arterial and venous cannulation, an aggressive anticoagulation regime and specially trained perfusionists and nursing staff . Extracorporeal membrane oxygenation is also associated with a long list of potential complications including infection, bleeding, thromboembolic events, hemolysis and finally multiorgan failure (mof). There is no doubt that ecmo is a well - established and recognized life - saving procedure; however, the question may be raised whether some patients could benefit from an easier technology used in a planned manner ., there is quite a wide range of indications for their use, two in cardiology: as a rescue treatment in cardiogenic shock caused mainly by acute coronary syndrome and, electively, as a support in high - risk percutaneous interventions including pci and vt ablation [7, 8]. Because of endovascular insertion and therefore limited device size (12 fr) the generated flow cannot be higher than 2.5 l / min, which usually is enough in the above situations . Impella pumps may be applied in post - cardiotomy shock when an intra - aortic balloon is not enough to succeed in weaning from cardiopulmonary bypass . Cardiac surgeons may also use them more electively as a perioperative support in patients with significantly impaired left ventricular function . The surgical insertion can be performed either peripherally (impella 5.0) or directly through the ascending aorta (impella ld). In peripheral access the femoral or axillary artery is exposed, then the device is inserted using a tubing graft and forwarded to the left ventricle under fluoroscopic guidance . The hybrid theatre setting is required, but on the other hand it is possible to perform less invasive, thoracoscopic surgery . In direct access, after sternotomy, the implantation is quite easy under tee guidance . Whatever the access, the surgical technique allows one to insert a bigger device (21 fr), which is able to generate flow up to 5 l / min (!). That means that after the procedure, which is slightly more complex than insertion of an iabp, and having a device which is almost as easy to operate as an iabp, we achieve an effect that is very close to the benefit of left ventricular assist device (lvad). The aforementioned problem of left ventricle venting is solved by definition, with impella being an intraventricular device . It should not stay in for longer than 5 days, which is more than enough to rethink and rearrange a new strategy . (impella, ecmo, lvad, recovery or transplant). When it is applied electively, as in our case, the chances are very high that only the first stage will be necessary . This being the case, the impella pump can be deservedly regarded as a short bridge to recovery.

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