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The importance of nodal staging as a prognostic factor in oncology and as an accuracy indicator for resection in colorectal cancer has been demonstrated in many studies . The current nodal staging in the 7th american joint committee on cancer (ajcc) staging system is based on the number of metastatic lymph nodes, while the concept of lymph node ratio to compensate for the limitation of number of lymph nodes retrieved and to evaluate the adequacy of the resection has been introduced recently [2 - 4]. In addition, the distribution of lymph node metastasis and high ligation of the inferior mesenteric artery is a subject of intense debate [5 - 8]. After grinnell reported the concept of lymphatic spread of colorectal cancer, surgical treatment for rectal cancer has involved en bloc resection of the involved segment of rectum and the accompanying draining lymph nodes up to the level of the origin of primary blood supply . Lymphatic drainage of the rectum occurs in the downward, lateral, and upward directions . The upward spread is through lymphatic vessels along the superior rectal and inferior mesenteric artery to the central lymphatic drainage, whereas lymphatic spread in the lateral and downward directions does not occur through the inferior mesenteric nodes . Although lateral and downward spread are the possible routes for tumor metastasis, the upward spread is regarded as the main route of lymphatic metastasis . Based on these findings, hermanek and altendorf and hojo et al . Reported poor oncologic outcomes in patients with lymph node involvement along the trunks of the vessels compared to those in patients with lymph node involvement only at the branches of the vessels . Although there have been several studies that demonstrated the impact of the distribution of metastatic nodes, the current tnm staging system does not incorporate the location of involved lymph nodes as a prognostic indicator . This study is designed to evaluate the difference in oncologic impact according to the level of involved lymph nodes and to promote incorporation of the value of the level of nodal involvement in the current tnm staging system . A total of 577 consecutive patients, who underwent a low anterior resection for treatment of mid and upper rectal cancer at the department of surgery, chonnam university hospital, between 1995 and 2004, were identified from a prospectively maintained colorectal cancer database . After excluding the patients who were treated with palliative surgery, the stage of rectal cancer was determined by a pathologist using a surgical specimen according to the 6th edition of the ajcc tnm staging system . Follow - up was conducted every 3 months for 2 years after surgery, and then every 6 months for the next 3 years . At the time of follow - up, physical examination, including a patient interview and a digital rectal examination, was performed . The serum carcinoembryonic antigen level was assessed and a simple chest x - ray was taken at 2 - 3 month intervals . The recurrence pattern was classified based on the area where the recurrence was detected for the first time, and local recurrence was defined as tumors that recurred in the pelvic cavity and the anastomotic area . In general, all patients underwent standard total mesorectal excision and regional lymphadenectomy . After tumor removal, the surgeon identified and isolated the lymph nodes, and recorded both the number and distribution of the lymph nodes in the operating room . All regional lymph nodes were individually dissected from the adipose connective tissue of the specimen immediately by performing anatomical dissection along all the vessels . The location of metastatic lymph nodes was defined according to the location of the apical lymph nodes among the involved lymph nodes . We categorized the apical lymph nodes as d1 when they were located along the paracolic and/or pararectal artery (251), and as d2 when they were located along the superior rectal artery (252). D3 was defined when the apical lymph nodes were located at the root of the inferior mesenteric artery (253). An analysis of survival rate was performed using a kaplan - meier method with a log rank test, and a cox regression model was used for analysis of risk factors affecting the survival rate . A multivariate logistic regression test was performed for analysis of risk factors for recurrence . For the development of location - weighted prognostic scoring model, the lymph node status was included in the logistic regression test, while the other variables were excluded in the analysis . Using the coefficients of the estimated logistic regression model (table 1), we built a scoring model to predict a poor oncologic outcome (y) based on the 3 group categories: d1, d2, and d3 as follows: y = d10.630+d21.555+d32.543 - 1.094 . In the final model, one lymph node of the d1 category was scored as 1 (the coefficient 0.630 was re - calculated as 1), one lymph node of the d2 category was scored as 2.5 (the relatively calculated value as a ratio of the coefficient [d2]/the coefficient [d1]), and one lymph node of the d3 category was scored as 4 . The final scoring model was as follows: prognostic score (ps)=number of d11+number of d22.5+number of d34 . All patients were classified into 4 groups according to the quartile scores as follows: lymph node i (ln i) (ps<2), lymph node ii (ln ii) (2ps<3), lymph node iii (ln iii) (3ps<6), lymph node iv (ln iv) (ps6). A total of 577 consecutive patients, who underwent a low anterior resection for treatment of mid and upper rectal cancer at the department of surgery, chonnam university hospital, between 1995 and 2004, were identified from a prospectively maintained colorectal cancer database . After excluding the patients who were treated with palliative surgery, the stage of rectal cancer was determined by a pathologist using a surgical specimen according to the 6th edition of the ajcc tnm staging system . Follow - up was conducted every 3 months for 2 years after surgery, and then every 6 months for the next 3 years . At the time of follow - up, physical examination, including a patient interview and a digital rectal examination, was performed . The serum carcinoembryonic antigen level was assessed and a simple chest x - ray was taken at 2 - 3 month intervals . The recurrence pattern was classified based on the area where the recurrence was detected for the first time, and local recurrence was defined as tumors that recurred in the pelvic cavity and the anastomotic area . After tumor removal, the surgeon identified and isolated the lymph nodes, and recorded both the number and distribution of the lymph nodes in the operating room . All regional lymph nodes were individually dissected from the adipose connective tissue of the specimen immediately by performing anatomical dissection along all the vessels . The location of metastatic lymph nodes was defined according to the location of the apical lymph nodes among the involved lymph nodes . We categorized the apical lymph nodes as d1 when they were located along the paracolic and/or pararectal artery (251), and as d2 when they were located along the superior rectal artery (252). D3 was defined when the apical lymph nodes were located at the root of the inferior mesenteric artery (253). An analysis of survival rate was performed using a kaplan - meier method with a log rank test, and a cox regression model was used for analysis of risk factors affecting the survival rate . A multivariate logistic regression test was performed for analysis of risk factors for recurrence . For the development of location - weighted prognostic scoring model, the lymph node status was included in the logistic regression test, while the other variables were excluded in the analysis . Using the coefficients of the estimated logistic regression model (table 1), we built a scoring model to predict a poor oncologic outcome (y) based on the 3 group categories: d1, d2, and d3 as follows: y = d10.630+d21.555+d32.543 - 1.094 . In the final model, one lymph node of the d1 category was scored as 1 (the coefficient 0.630 was re - calculated as 1), one lymph node of the d2 category was scored as 2.5 (the relatively calculated value as a ratio of the coefficient [d2]/the coefficient [d1]), and one lymph node of the d3 category was scored as 4 . The final scoring model was as follows: prognostic score (ps)=number of d11+number of d22.5+number of d34 . All patients were classified into 4 groups according to the quartile scores as follows: lymph node i (ln i) (ps<2), lymph node ii (ln ii) (2ps<3), lymph node iii (ln iii) (3ps<6), lymph node iv (ln iv) (ps6). The median age of the study population was 60 years (range, 26 to 84 years) without gender predominance . Patients with positive proximal lymph nodes were significantly younger than elder patients (p=0.014). The median length of follow - up of overall patients was 49.5 months (range, 3 to 165 months). Among the 435 rectal cancer patients who underwent a curative low anterior resection, 160 patients had pathologically proven positive nodes . Of these 160 patients with positive nodes, lymph nodes in 108 patients were categorized as d1, those in 32 patients as d2, and those in 20 patients were categorized as d3 according to the protocol of this study . Tumors showed an infiltrative growth pattern in patients with proximal lymph node metastases (p=0.04). Of the 435 patients, 21 patients underwent preoperative chemoradiation therapy . In cases that received preoperative chemotherapy, there was a trend for a smaller number of harvested lymph nodes; however, it was not statistically significant . The distribution of metastatic lymph nodes was not significantly different between the group that underwent preoperative chemoradiation and the groups that did not undergo preoperative chemoradiation, although the patient volume was small (table 3). After a median 49.5 months of follow - up, the 5-year overall survival rate was 84.1% in patients without lymph node metastasis, 65.2% in patients with n1, and 45.0% in patients with n2 . In survival analysis according to our distribution - weighted nodal staging, the 5-year overall / disease - free survival rate was 72.2%/69.0% in patients with metastatic lymph nodes categorized as d1, 40.6%/35.7% in patients with metastatic lymph nodes categorized as d2, and 22.7%/0.0% in patients with metastatic lymph nodes categorized as d3 . 1 shows significantly different results for the overall and disease - free survival analyses according to the level of metastatic lymph nodes . According to our scoring model, 42 patients was classified into the ln i group and their median survival period was 77 months, 27 patients were classified into the ln ii group and their median survival period was 67 months, 52 patients were classified into the ln iii group and their median survival period was 52 months, and 39 patients were classified into the ln iv group and their median survival period was 26 months (p=0.011) (table 4). 2 shows a comparison of a location - weighted model with ajcc 7th edition nodal stage . Seventeen patients were classified as n2 according to our model; however, their stage was n1 according to the ajcc 7th edition, and the survival course of these patients was similar to that of patients in the n2 by both the staging systems . The median age of the study population was 60 years (range, 26 to 84 years) without gender predominance . Patients with positive proximal lymph nodes were significantly younger than elder patients (p=0.014). The median length of follow - up of overall patients was 49.5 months (range, 3 to 165 months). Among the 435 rectal cancer patients who underwent a curative low anterior resection, 160 patients had pathologically proven positive nodes . Of these 160 patients with positive nodes, lymph nodes in 108 patients were categorized as d1, those in 32 patients as d2, and those in 20 patients were categorized as d3 according to the protocol of this study . Tumors showed an infiltrative growth pattern in patients with proximal lymph node metastases (p=0.04). Of the 435 patients, 21 patients underwent preoperative chemoradiation therapy . In cases that received preoperative chemotherapy, there was a trend for a smaller number of harvested lymph nodes; however, it was not statistically significant . The distribution of metastatic lymph nodes was not significantly different between the group that underwent preoperative chemoradiation and the groups that did not undergo preoperative chemoradiation, although the patient volume was small (table 3). After a median 49.5 months of follow - up, the 5-year overall survival rate was 84.1% in patients without lymph node metastasis, 65.2% in patients with n1, and 45.0% in patients with n2 . In survival analysis according to our distribution - weighted nodal staging, the 5-year overall / disease - free survival rate was 72.2%/69.0% in patients with metastatic lymph nodes categorized as d1, 40.6%/35.7% in patients with metastatic lymph nodes categorized as d2, and 22.7%/0.0% in patients with metastatic lymph nodes categorized as d3 . 1 shows significantly different results for the overall and disease - free survival analyses according to the level of metastatic lymph nodes . According to our scoring model, 42 patients was classified into the ln i group and their median survival period was 77 months, 27 patients were classified into the ln ii group and their median survival period was 67 months, 52 patients were classified into the ln iii group and their median survival period was 52 months, and 39 patients were classified into the ln iv group and their median survival period was 26 months (p=0.011) (table 4). 2 shows a comparison of a location - weighted model with ajcc 7th edition nodal stage . Seventeen patients were classified as n2 according to our model; however, their stage was n1 according to the ajcc 7th edition, and the survival course of these patients was similar to that of patients in the n2 by both the staging systems . Our study showed that the distribution of involved lymph nodes may be an independent factor for the prediction of oncologic outcome in curatively treated patients with mid and upper rectal cancer . The distribution - weighted nodal staging model used in the present study showed that the modified nodal staging more precisely reflected the natural oncologic course in patients with rectal cancer . In the current ajcc nodal staging system, the prediction of oncologic outcome is based on the number of involved lymph nodes and the topographical distribution of lymph nodes is not taken into consideration . Although this study underscores the prognostic significance of proximal lymph node involvement, hermanek and altendorf reported that colorectal cancer patients with proximal lymph node involvement usually have at least four or more metastatic nodes . Lymph nodes along the inferior mesenteric artery are defined as regional lymph nodes in the recent edition of ajcc . However, kang et al . And kim et al . Reported the prognostic significance of lymph nodes along the inferior mesenteric artery (i m a) in their study . The presence of proximal lymph node metastasis not only has a prognostic impact, but it is also an independent prognostic factor . After comparing with some studies that reported the significance of the distribution of involved lymph nodes after categorizing them into proximal or distal lymph nodes, we could demonstrate a more precise grouping according to the lymphatic drainage from d1 to d3, and we could estimate the relative value of each groups . One lymph node of the d1 category was scored as 1, one lymph node of the d2 category was scored as 2.5, and one lymph node of the d3 category was scored as 4 . This significance value was used to calculate the statistical weight of the involved nodes, and the prognostic score was finally calculated . In this study, there may some concerns regarding the use of the location - weighted prognostic scoring model, such as small volume, and no consensus about lymph node harvesting; nevertheless, the significance of distribution of lymph nodes seems to differ according to their location . Location based nodal scoring model was essentially based on the extent of dissection and the level of the i m a ligation . Although several reports, including our study report, have demonstrated the significance of i m a lymph node metastasis as a prognostic factor, the oncologic benefit of complete removal of lymph nodes including those along the i m a is still under debate . Survival benefit of removal of i m a lymph nodes by high ligation has been observed in some studies, and it appeared to be due to the stage migration effect . Most studies found no significant difference in survival between high ligation of the i m a and low ligation of the i m a . Hence, the current evidence appears to demonstrate a preference for low ligation of the i m a because the oncologic benefit is insufficient, while high ligation of the i m a causes a significant reduction in perfusion and disruption of autonomic innervation . In our study, there were 20 patients (4.6%) with lymph node involvement along the i m a; this proportion of patients was not different from the reported range of proportion of patients with lymph node involvement along the i m a, between 0.3% and 8.6% . Nevertheless, our study suggested that the d2 category of lymph nodes had a significant impact on oncologic outcome; however, not as much as the d3 category of lymph nodes (the coefficient, 1.555 vs. 2.543; odds ratio, 1.878 vs. 4.735). Recently, there have been several studies that used the lymph node ratio, of the involved lymph nodes to the harvested lymph nodes, as an option to estimate the effect on the oncologic outcome in rectal cancer . Some studies have implicated that the neoadjuvant chemoradiation therapy used for treatment of mid and lower rectal cancer leads to limited lymph node harvest [3 - 6]. This study did not include patients with lower rectal cancer to avoid the effect of lateral pelvic node involvement and neoadjuvant chemotherapy; and according to this point of view, non - inclusion of these patients could be the major limitation of our study . Nevertheless, we tried to estimate the pure impact of the location of involved lymph nodes on oncologic outcome . This study confirms that the location of metastatic lymph nodes was significantly and independently associated with a poor oncologic outcome . The location - weighted prognostic scoring model may help to overcome the limitations associated with the use of number of involved nodes, lymph node ratio of involved nodes to harvested nodes, and high ligation of the i m a . A larger scale, multi - center study should be performed to confirm the impact of involved lymph nodes oncologic outcome in rectal cancer . The distribution of involved lymph nodes had a significant impact on prediction of the survival outcome . Our location - weighted prognostic scoring model, a prediction system that includes both the number and location of lymph node metastasis, may help to overcome the limitations of the current lymph node - related prognostic prediction system . A large scale study should be initiated and the significance of the distribution of metastatic lymph nodes may be included in the next tnm staging system.
Dengue is the most rapidly spreading mosquito - borne viral disease in the world and in the last 50 years, incidence has increased 30-fold . In pakistan (world health organization [who] eastern mediterranean region), the first confirmed outbreak of dengue hemorrhagic fever occurred in 1994 and since then dengue infections have been reported with increasing frequency and severity from large cities in pakistan . As the incidence of dengue is rising among adults more cases of dengue fever are being reported during pregnancy . Dengue infection in pregnancy increases the risk of hemorrhage for both the mother and the newborn, especially if the mode of delivery is operative . We present the successful management of hemorrhage and unanticipated complications of severe dengue in a young primigravida admitted to the intensive care unit (icu) after an emergency cesarean section . An 18-year - old primigravida with no known co - morbids presented to one of the secondary setups of a university hospital at 35 weeks of gestation, with a history of fever for 5 days and threatened preterm labor . Initial blood reports [table 1] were normal along with normal liver function tests and negative screening for hepatitis, typhoid, and malaria . A baby girl of 2.8 kg was delivered by emergency lower segment cesarean section as tocolysis failed to delay labor . Excessive bleeding per vaginum and hematuria was observed in the postanesthesia care unit, but she remained hemodynamically stable . Oxytocin infusion was continued, four units packed cells and four units fresh frozen plasma (ffp) were transfused, and she was referred to the tertiary setup in anticipation of need for admission to the surgical icu (sicu). On admission to the sicu, she was hypotensive (blood pressure of 104/64) and tachycardic (heart rate of 140/min) but fully conscious . Blood counts are shown in table 1, and dengue serology was reported as positive . Pharmacological measures (oxytocin infusion 40 iu in 500 ml normal saline at 100 ml / h and misoprostol 800 g) and balloon tamponade failed to control heavy per vaginal bleeding, and there was no structural or vascular injury, but the uterus was found to be atonic . B - lynch sutures were applied, and bilateral internal iliac artery ligation and vaginal tamponade was done to control the generalized ooze . In the icu, the vaginal bleeding continued, and she became hemodynamically unstable despite adequate volume replacement and transfusion of six units packed cells, 10 units of platelets, and 10 units of cryoprecipitate . Two doses of activated factor vii were given to avoid hysterectomy in this young primigravida . However, the bleeding continued, and supracervical hysterectomy with ovarian conservation was performed as a lifesaving procedure . She was shifted back to the icu for hemodynamic monitoring, vasopressor support, mechanical ventilation, and transfusion of blood products . During her 16-day icu stay, she received mechanical ventilation for 9 days and noninvasive ventilation for another 5 days along with deep venous thrombosis prophylaxis, stress ulcer prophylaxis, and enteral nutrition via a nasogastric tube . The postoperative course was further complicated by an ileus, wound infection leading to wound dehiscence, persistent thrombocytopenia [table 1] leading to hematuria, and ongoing fever treated with broad - spectrum antibiotics . A chest computed tomography after discharge from icu showed pulmonary embolism which was initially managed by heparin anticoagulation and later by warfarin . She also developed right sided leg weakness and pain which was diagnosed as acute right plexopathy on nerve conduction studies . She remained in the special care ward for another 6 days and was discharged home when she was afebrile, tolerated a regular diet, and mobilized with support . In addition, to the obstetrics and gynecology, she required follow - up by general surgery for wound management, chronic pain service for leg pain, and hematology for anticoagulation surveillance . Severe dengue should be considered if the patient is from an area of dengue risk presenting with fever of 27 days and evidence of plasma leakage, significant bleeding, and severe gi involvement, altered level of consciousness, or severe organ impairment . Diagnosis of dengue is difficult during pregnancy because the physiologic changes of pregnancy mask the pathognomonic features of severe dengue such as increased hematocrit, thrombocytopenia, and leucopenia . This patient had a normal white cell count, platelet count and hematocrit on presentation and thrombocytopenia was observed later [table 1]. Immunoglobulin m (igm) (elisa) is a widely used, simple and rapid method to detect dengue specific igm antibodies and should have been part of the initial workup of fever in this patient . Conservative obstetrical management is preferred in women with dengue infections, but the incidence of cesarean deliveries is between 44% (case reports) and 20.4% (case series). There are case reports of cesarean deliveries complicated by hemorrhage secondary to uterine atony and thrombocytopenia with variable outcome and the variable need for blood product transfusion . Our patient received a transfusion of 36 units packed cells (red cell concentrate), 44 units ffp, 49 units of platelets, nine single donor platelet mega units, and 20 units of cryoprecipitate during her hospital stay . She also received two doses of activated factor vii, as a last resort before hysterectomy was considered . The potential for thrombotic complications is known with the use of factor vii and this patient was diagnosed with pulmonary embolism after discharge from icu and may have resulted from activated factor vii use . Who recommends that all patients with severe dengue should be admitted to a hospital with access to intensive care facilities and blood transfusion . Icu admission may be required to manage thrombocytopenia (<20,000/mm), systemic inflammatory response or severe sepsis, severe bleeding, respiratory distress, hepatitis or jaundice, altered sensorium, shock, severe dehydration, or decreased urine output, and the associated mortality reported is 6.1% . Hemodynamic monitoring and support, massive transfusion of various blood products, need for mechanical ventilation, and management of unanticipated problems required an icu stay of 16 days in this patient . As the incidence of dengue is rising among adults more cases of dengue fever are being reported during pregnancy and dengue serology should be part of the workup of unexplained fever in pregnancy in the endemic areas . Surgical intervention in patients with suspected severe dengue should be undertaken cautiously, and early referral to healthcare centers where technical, transfusion and intensive care facilities are available may prove lifesaving.
A level of pain that completely incapacitates one individual may cause only a minor annoyance in another . Characterizing the genetic factors affecting susceptibility to chronic pain could lead to novel treatment or prevention strategies, which are desperately needed . A recent paper by costigan et al . The authors performed a multi - dimensional analysis using rat gene expression and human genetic association that identified a new genetic factor affecting susceptibility to chronic pain . This study was initiated by analysis of gene expression changes within rat dorsal root ganglia obtained after nerve damage was induced in three different models of chronic pain . The mrna for a potassium channel alpha subunit (kcns1) was constitutively expressed in sensory neurons, but was downregulated after three types of nerve injury in these models . Other studies have successfully identified a human genetic susceptibility factor on the basis of a candidate gene emerging from analysis of a rodent model . Costigan and colleagues therefore examined snp alleles within the human homolog (kcns1) in five independent cohorts (two with chronic low back pain, two after limb amputation and one after mastectomy) that have a high incidence of chronic pain . In four of the five cohorts studied, there was a significant association of the val allele at a nonsynonymous snp (rs734784, val / ile) with increased risk for chronic pain . For example, homozygous val / val individuals had a 2.4-fold increased relative risk of failing to achieve pain improvement 1 year after back surgery . The proportion of individuals without phantom limb pain after leg amputation was 45% in those without the val allele, but fell to 22% in val / val homozygotes . Among healthy volunteers that were subjected to multiple experimental pain stimuli, although experimental studies demonstrating an allelic effect were not presented, which is a substantial limitation of this study, these results make it likely that kcns1 alleles contribute to susceptibility to chronic pain . Although kcns1 by itself does not have potassium channel function when tested in heterologous expression systems, its expression has been shown to modulate the currents formed by other channels [3 - 5]. Voltage - gated potassium channels have important effects on neuronal function, including altering the resting membrane potential and the shape and frequency of the action potential . Similarly, gain - of - function mutations in the nav1.7 sodium channel cause syndromes associated with increased pain sensitivity . Chronic pain has a huge impact on our society, and its treatment is a major driver for the increasing cost of health care . An estimated 76 million people in the us have chronic pain, which costs the us public over $100 billion per year . Approximately 25% of chronic pain is neuropathic (caused by nerve damage), which is characterized by spontaneous pain that is burning or stabbing in nature . This can develop after limb amputation, mastectomy or lower back injury, or in individuals with a long history of diabetes . Although multiple kinds of treatment can be used, they often have limited efficacy, and chronic pain is often managed by administration of opioids (morphine and related synthetic compounds, including hydrocodone). The increased focus on pain management resulted in a six - fold increase in the per capita sales of prescription opioid medications in the us between 1997 and 2006 . Hydrocodone - acetaminophen is prescribed over 100 million times per year in the us, which is far more than any other medication, including lipid - lowering and blood - pressure - lowering agents . This has led to a dramatic increase in the incidence of opioid misuse, emergency room visits due to opioid analgesic poisoning, and fatal opioid overdose . Prescription opioids have now surpassed marijuana as the drug that is most commonly abused among the newly initiated . Because of the enormity of this public health problem, we desperately need new approaches for the prevention or treatment of chronic pain conditions . It is likely that genetic discoveries can lead to new approaches for chronic pain, because genetic discoveries have catalyzed new approaches for related clinical conditions . For example, haplotype - based computational genetic analysis has identified causative genetic factors affecting analgesic medication and inflammatory pain responses, and it has identified four genes affecting narcotic drug responses [11,14 - 16] in mice . The latter genetic discovery generated a new treatment strategy for preventing narcotic drug withdrawal symptoms, which was shown to be effective in humans . Moreover, the variable response to multiple classes of analgesic medications among inbred mouse strains was shown to be due to genetic variation within a genetic locus (kcnj9) that also encodes a potassium channel (girk3). Hopefully, characterizing the functional role of kcns1 in neuronal responses and pain perception, and the impact of the allelic differences, could lead to new approaches for treatment of chronic pain . Although the tractability of kcns1 (or other potassium channels) as a therapeutic target remains to be determined, small molecules targeting potassium channels have been produced (reviewed in), and one has attenuated neuropathic pain in animal models . Although the kcns1 val allele explains only a small percentage of the total variance (4.6 to 7.8%) in the chronic pain endpoints in the cohorts studied, the identification of an initial genetic factor for chronic pain is an important achievement for two reasons . First, it shows that genetic factors can be identified for this condition, and this study provides a template for subsequent studies . Second, it is likely that a greater percentage of the genetic susceptibility to chronic pain can be explained by combining this genetic factor with other subsequently identified factors . Moreover, pain is a subjective sensory symptom; it is difficult to measure; and there are substantial psychological and emotional components that contribute to the perception of pain . It has recently been found that there are different subtypes of chronic pain, which are distinct from the causative disease (diabetic neuropathy, radicular back pain and so on). It is likely that genetic factors determine whether a chronic pain syndrome will develop and the specific subtype that emerges after exposure to a triggering cause . Thus, the impact of a single genetic factor could be much larger if a specific pain subtype is examined relative to that measured in a large cohort of individuals with different types of chronic pain . Beyond finding new drug targets, identification of genetic factors affecting susceptibility to chronic pain syndromes could be the lever that drives advances in this difficult clinical area . A genetic risk factor could have a substantial impact on clinical practice because individuals at increased risk for developing a chronic pain syndrome could be identified before a surgical procedure or immediately after a traumatic incident . The genetic information could be used to develop proactive methods for prevention of chronic pain syndromes, enabling interventions to be targeted to the high - risk subset . Because of the great inter - individual variation in the response to painful stimuli and to analgesic drugs, knowledge of genetic risk factors could enable better stratification of chronic pain patients or aid in the selection of the appropriate therapy . Rather than focusing on the disease that is etiologically associated with the chronic pain, study is hopefully one of the first of many subsequent genetic studies that could lead to entirely new ways to approach chronic pain syndromes . Clearly, the clinical and research communities have a long way to go before genetically targeted therapies for chronic pain become a reality . However, kcns1 (or other channels) could perhaps become the next target for a new class of analgesics that are selectively used in the 20% of the population that are homozygous for the val alleles of kcns1 . The day might soon come when we no longer think of' chronic pain' as a single clinical entity, but have the tools to characterize the specific' channelopathy' that underlies the clinical presentation and targeted treatments for each subgroup . Gp is a professor and mz is the director of statistical research in the department of anesthesia at stanford university medical school.
Hepatitis c virus (hcv) is a parentally transmitted hepatotropic rna virus that causes chronic hepatitis, which may lead to cirrhosis and hepatocellular carcinoma (hcc). Evidence suggests that clearance and control of hcv infection during acute phase is dependent on vigorous and multispecific cd4 and cd8 t lymphocyte responses [2, 3]. On the contrary, the development and maintenance of chronic infection is linked to weak or absence of hcv - specific th1 response and to the presence of th2 cytokines (il-4 and il-10) [4, 5]. So, some studies have implicated il-10 in hcv pathogenesis [4, 6]. Regulatory t cell 1 (tr1) was first described in 1996 [7, 8] as a further subtype of cd4 t cells, without specific markers but with cytokine profile distinct from that of th1 or th2 cells . Indeed, they were shown as secreting high level of il-10, moderate levels of ifn- and il-5, quite undetectable il-4, and low amounts of transforming growth factor (tgf-). Recent studies have suggested that tr1 could be induced against bacterial, viral and parasite antigens in vivo and might prevent infection - induced immunopathology or prolong pathogen persistence by suppressing protective th1 response [9, 10] but has never been directly implicated in hepatitis c viral pathogenesis . The dysfunction of the immune response against hcv could be explained by immunosuppressive mechanisms supported by tr1 via their high production of il-10 . The recent identification of combined expression of cd4, cd18 (integrin 2), and cd49b (integrin 2) as specific markers for tr1 cells should facilitate their characterization in vivo . An increased frequency of tregs was recently described in the blood of patients with persistent hcv infection when compared with those who had cleared hcv [12, 13]. It has been proposed that tregs contribute to hcv persistence by suppressing hcv - specific t - cell responses [14, 15]. Some studies have shown a correlation between a reduced hcv - specific t - cell response and the secretion of il-10 and tgf- by liver - infiltrating tregs and that tregs may inhibit hcv - specific t - cell activity in a cell - cell contact manner [12, 13]. It has also been shown that treg levels are significantly enhanced in recurrent hepatitis c and that treg type 1 cell (tr1) levels are specifically higher in severe recurrent hepatitis c . Moreover, the opportunity of studying three histologically well - defined liver biopsies of an hcv genotype 1b infected patient followed from the chronic infection to the hcc allowed us to investigate the tr1 cells infiltration within these liver biospies . The first biopsy was performed during the chronic phase (date not specified in the file) and appeared normal without any liver injury, the second one was performed during the cirrhotic phase (march 1990), and the third one was performed during hcc (1998). Thus, we examined the intrahepatic t cell response in terms of cytokines, cytokine receptors, and adhesion molecules in a patient under ifn-/ribavirin treatment and who remained persistently infected> 18 years . The hcv infected patient was followed up in the hepatogastroenterology department of the necker university hospital . Information concerning the patient included age alat> three times the normal and negative anti - hav, hbv, and hev igm, and positive serology for hcv (third generation microparticle eia (abbott axsym, abbott park)) confirmed by riba strip immunoblot assay (chiron corporation, emeryville, ca). Genotyping of hcv was done using a multiplex pcr method with genotype - specific primers, and the concentration of hcv rna was determined by rt - pcr with the cobas amplicor hcv monitor test (roche diagnostics, branchburg, nj). The patient presented no human immunodeficiency virus (hiv) coinfection, immunosuppressive therapy, chronic liver disease due to hepatitis b virus, autoimmune hepatitis, or primary biliary cirrhosis and other causes of chronic liver disease . Three liver biopsies were performed for one patient who underwent several biopsies for a longitudinal study . The three biopsies were performed as part of the routine medical followup and were obtained by the standard menghini procedure (needle diameter: 1.6 mm) with a biopsy sample length of approximately 1 cm . Based on histologic fibrosis (f) and necroinflammatory (ni) scores, liver biopsies were divided into biopsies with small hepatitis and without liver lesion (f <or = 1/6; n <or = 1/18), biopsies with fibrosis grade and liver lesions corresponding to histologically proved cirrhosis (f = 6/6), and biopsies with hcc histologically proved by anatomopathologic expertise . The study was approved by the institut de biologie de lille (cnrs) institutional review boards and informed consent was obtained in writing from the donor . Extraction of total rna from frozen liver biopsy samples was performed using trizol reagent (gibco brl, invitrogen, scotland, uk) as described by the manufacturer . Homogenized samples were incubated for 5 minutes at room temperature and resuspended in 60 l of chloroform (prolabo, merck eurolab, france). Total rna was then immunoprecipitated with 100 l of isopropanol (acros organics, usa). The rna pellet was finally washed twice with 75% ethanol (carlo erba reagent, france) and dissolved in dnase, rnase - free distilled water (gibco brl, invitrogen, scotland, uk). Before reverse transcription, we performed dnase treatment using the kit message clean (genhunter corp ., france). Cdna was synthetized from total rna at a concentration of 100 ng/l using random hexamers and superscript reverse transcriptase (gibco brl, invitrogen, scotland, uk). The quantification of transcripts from liver samples was performed by real - time quantitative rt - pcr using the light cycler system (roche diagnostics, mannheim, germany). The pcr mixture contained the following: taq polymerase, 1x of light - cycler dna master sybr green i (roche diagnostics, meylan, france), 3 mm of mgcl2, 0.5 mol / l of each primer, and 1 l of cdna preparation (patient cdna samples) in a total volume of 20 l . Thirty - two samples were run in parallel by performing an initial denaturation at 95c for 8 minutes; the pcr reactions were cycled 35 to 40 times as follows: 15 seconds at 95c, 7 seconds at the appropriate annealing temperature (table 1), and 18 to 64 seconds at 72c according to the length of the target sequence, followed by annealing (40 s at 58c). The melting curve analysis was carried out immediately after amplification, following the manufacturer's instructions . Amplification of liver cdna was successfully repeated three times with cdna from the same extraction . All primers were designed for real - time pcr use and were purchased from mwg - biotech (germany) (cf . Samples are quantified using relative standard curves for each amplification reaction, and results were normalized to the internal controls -actin and g3pdh . After each light cycler run, agarose gel electrophoresis with tbe (tris - borate - edta) 1.5% agarose (sigma - aldrich, germany) gel, followed by dna staining with ethidium bromide, was performed to have an independent validation check of the presence of an amplicon . In order to control the length of the amplicon generated, a 100 bp dna ladder (gibco brl, invitrogen, scotland, uk) was used . As the lymphotropism of hepatitis c virus (hcv) has already been ascertained, and in the light of the fact that the immune defense system is an organized network composed of functionally immune cells, this study was carried out to verify the possible involvement of tr1 cells in the progression of hcv - related chronic hepatitis . In this study, we first analyzed the quantitative expression of the different intrahepatic cell populations by real - time pcr using light cycler system (figure 1), and we performed after each rt - pcr an agarose gel electrophoresis to have an independent validation check (figure 2). We observed a significant increase of cd19 expression in cirrhotic and hcc liver biopsies in comparison with the first biopsy without liver lesions . This observation confirmed data of the literature showing disturbances of b lymphocyte activation and function associated with hcv chronic infection [17, 18]. The cd8 but not cd8 gene expression was also increased in the cirrhotic and hcc biopsies . This observation was in correlation with recent data evidencing by a similar approach of real - time quantitative assays a significant increase and infiltration of cd8 during chronic hcv infection . But the major observation of our work was the significant increase of cd4 expression in the course of time and proportionally with the severity of the fibrosis . However, we did not observe a variation of ifn- or il-2 expression, suggesting that the cd4 marker detected in cirrhosis and hcc liver biopsies was probably not associated with the th1 protective phenotype . Due to the absence of il-4 expression, the cd4 cells with th2 phenotype were also excluded in accordance with the work of bergamini et al . . A further subtype of cd4 t cells, with immunosuppressive function and cytokine profiles distinct from either th1 or th2 t cells, termed regulatory t cells has been described . They include tr1 cells which secrete high level of il-10 and low to moderate levels of tgf-, th3 cells which primarily secrete tgf-, and cd4cd25 cells, which inhibit immune response through cell - cell contact (9). Although the level of il-10 and tgf- produced in vitro may not be significantly higher than that produced by classical th2 cells, what is important is that tr1 cells make these cytokines in the absence of significant levels of il-4 . We confirmed within cirrhotic and hcc biopsies a significant increase of expression of il-10, tgf-1 and their respective receptors il-10r, il-10r, and tgf-r2 whereas we did not detect an increase of il-4 . So, the production of il-10 and tgf- in the absence of il-4 argued in favor of the presence of tr1 in the cirrhotic and hcc biopsies . Although liver dendritic cells also secrete high levels of il-10, in the present study we could not detect cd11c expression, confirming a tr1 cells origin for il-10 production . In this sense, functional alteration of hcv - specific cd4 t cells or failure to develop a long - lasting t helper response was recently correlated to a loss of ifn- secretion and the presence of a significant antigen - specific il-10 and tgf-, which could contribute to chronic hepatitis c persistence . Moreover, tgf- plays a pivotal role in inducing fibrosis and has been proposed as its surrogate marker . Indeed, a very interesting study has shown that serum tgf-1 could be used to assess therapeutic outcome and short - term prognosis of hcv - related chronic hepatitis . Moreover, results obtained for tr1 specific markers cd49b and cd18 confirmed the tr1 phenotype and showed that tr1 cells increased during the progression of the pathology . Despite their low proliferative capacity, cloned tr1 express normal levels of t cell activation markers such as cd25 following tcr - mediated activation . They also expressed ccr5 and t1-st2, an il-1-r - like molecule, markers previously expressed preferentially on th1 and th2 cells, respectively . We clearly confirmed here within cirrhotic and hcc biopsies the expression of cd25, ccr5, and t1-st2 in relation to the presence of tr1 cells . In accordance with this tr1 phenotype, the expression of tgf--r2 was also observed . However, we cannot exclude the implication of other regulatory t cell subsets, insofar as we observed a significant increase of cd25, pselectin, and icam i expression, which were known as specific markers for cd4cd25 regulatory t cells . These observations were also conforted by other experiments we performed on liver biopsies of three well - defined cohorts of 45 hcv genotype 1b infected patients, including patients without liver lesions, patients with cirrhosis, and patients with histologically proved hcc that confirmed an increase of tr1 proportional to the aggravation of the pathology . A first implication of regulatory t cells during hcv infection was examined in peripheral blood of patients infected with hcv genotype 1b, showing a secretion of ifn- or il-10 but not il-4 by hcv core - specific cd4 t cell clones . This work demonstrated that helper type 1 and regulatory t cells were induced probably against the same epitopes on the core protein . Moreover, sugimoto et al . Have suggested for the first time that hcv persistence was associated with a reversible cd4-mediated suppression of hcv - specific cd8 t cells and with higher frequency of cd4cd25 regulatory t cells that could directly suppress hcv - specific type 1 cd8 t cells ex vivo . Indeed, it is possible that the induction of regulatory t cells during hcv infection may suppress antiviral th1 and consequently cd8 ctl responses in vivo, and this may explain the persistence of infection and prevalence of cirrhosis . Evidence of regulatory t cells installation in the liver of a chronically infected patient with cirrhosis and hepatocellular carcinoma (hcc) suggests a key role for these cells in the aggravation of the liver pathology and could potentially represent a predictive factor of liver damage aggravation . As previously described high serum il-10 levels may be related to a poor response to ifn treatment in patients with chronic hepatitis c. in this sense, it would have been interesting to investigate other markers such as spleen markers or beta2-microglobulin, although it has been described that neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy . In conclusion of our study, the increase of tr1 cells, which suppressed protective th1 responses via their high production of il-10, could explain the failure of the ifn treatment observed for this patient . In a more general way, the implication of tr1 in viral persistence and associated hepatic pathologies could have significant implications for our understanding of the role of t cells in immunity to infectious diseases and for the development of new therapies for the control of immune - mediated disorders.
San francisco, ca) is a monoclonal antibody against vascular endothelial growth factor (vegf) which was approved by the united states food and drug administration for the treatment of metastatic colorectal cancer.1 ophthalmologists have used intravitreal injection of avastin off - label, for a number of indications including age - related macular degeneration, diabetic macular edema, cystoid macular edema and retinal vascular accidents.2 acute endophthalmitis is a rare but devastating complication of intravitreal bevacizumab (ivb) injection . The incidence of endophthalmitis varies in the literature from 1:1000 to 1:5233 per injection.35 potentially, bilateral intravitreal injection of bevacizumab can lead to bilateral endophthalmitis . Our literature review during the research for this case report did not yield any reports of bilateral endophthalmitis following ivb injection . Here we describe the first two cases in peer review literature of bilateral endophthalmitis after ivb . A 76-year - old female was referred to our emergency clinic with complaints of bilateral ocular pain and decreased vision 1 day after receiving bilateral ivb injection for diabetic macular edema . On examination, her visual acuity was hand movement and light perception in right and left eyes, respectively . Significant anterior chamber reaction with a 0.7-mm hypopyon and severe vitritis were present in the left eye . Due a high index of suspicion for bilateral postoperative endophthalmitis, the patient underwent diagnostic and therapeutic vitreous tap and intravitreal injection of 2.25 mg/0.1 ml ceftazidime and 1 mg/0.1 ml vancomycin . Two days after antibiotic injection, visual acuity improved to 20/400 in both eyes and there was a significant improvement in the symptoms and signs of inflammation . A 52-year - old female with a history of bilateral ivb injection for diabetic macular edema, presented with bilateral ocular pain and photophobia 2 days after ivb injections . On examination, the patient underwent diagnostic and therapeutic vitreous tap and intravitreal injection of 2.25 mg/0.1 ml ceftazidime and 1 mg/0.1 ml vancomycin . Two days later despite intravitreal antibiotics therapy, the signs and symptoms progressed and pars plana vitrectomy and intravitreal antibiotic injection was performed in both eyes . During vitrectomy, five days after vitrectomy, the signs and symptoms of endophthalmitis resolved . However, there was no improvement in visual acuity due to the retinal necrosis . A 76-year - old female was referred to our emergency clinic with complaints of bilateral ocular pain and decreased vision 1 day after receiving bilateral ivb injection for diabetic macular edema . On examination, her visual acuity was hand movement and light perception in right and left eyes, respectively . Significant anterior chamber reaction with a 0.7-mm hypopyon and severe vitritis were present in the left eye . Due a high index of suspicion for bilateral postoperative endophthalmitis, the patient underwent diagnostic and therapeutic vitreous tap and intravitreal injection of 2.25 mg/0.1 ml ceftazidime and 1 mg/0.1 ml vancomycin . Two days after antibiotic injection, visual acuity improved to 20/400 in both eyes and there was a significant improvement in the symptoms and signs of inflammation . A 52-year - old female with a history of bilateral ivb injection for diabetic macular edema, presented with bilateral ocular pain and photophobia 2 days after ivb injections . On examination, the patient underwent diagnostic and therapeutic vitreous tap and intravitreal injection of 2.25 mg/0.1 ml ceftazidime and 1 mg/0.1 ml vancomycin . Two days later despite intravitreal antibiotics therapy, the signs and symptoms progressed and pars plana vitrectomy and intravitreal antibiotic injection was performed in both eyes . During vitrectomy, five days after vitrectomy, the signs and symptoms of endophthalmitis resolved . However, there was no improvement in visual acuity due to the retinal necrosis . Similar to our cases, most reports of endophthalmitis document decreased vision, ocular pain and redness, soon after ivb injection.36 recently, some standardization has been advocated to minimize the risk of postoperative endophthalmitis after ivb injection . These measures include preoperative cleansing of eyelids and conjunctiva with a 5% povidone - iodine solution, isolation of lids and lashes from the surgical field and treatment of high - risk patients with topical antibiotics . Sterile and infectious endophthalmitis after intravitreal injection presents with similar sign and symptoms such as a rapid decrease in visual acuity . Signs of infectious endophthalmitis include, inflammation, pain, fibrin, sudden and significant loss of vision within days of ivb . S. epidermidis is the most common pathogen isolated from the vitreous samples.78 the cause of infectious endophthalmitis after ivb remains contentious . Some studies have implicated the needle used during ivb as it contacts the ocular surface and inoculates the vitreous.8 others suggest pharmacological compounding during preparation of bevacizumab for ophthalmic use as the cause of infection.9 prophylactic measures are particularly important for bilateral procedures . In the current cases, we recommend performing ivb injection in diabetics or immunocompromised patients in separate sessions for each eye . Moreover, it is imperative to adhere to all prophylactic measures for each eye in all patients undergoing bilateral injection . We believe separate, surgical grade instruments (including speculum, drug vial and calipers) should be used for each eye in a bilateral procedure.
Mung bean (phaseolus radiatus l.) is a leguminous species grown in different parts of the world, primarily especially in asia including china, india, burma, and thailand . Mung bean commonly is a common source of protein in the asian diet or nutrient supplements . Mung bean has been reported to possess antioxidant, antidiabetic, anti - inflammatory, antitumor and antimelanocytes, and antiangiotensin i - converting enzyme activities [28]. Mung bean contains free phenolic acids, bound phenolic acids, total phenolic, and anthocyanin . Correlation analyses between bioactivities and phytochemicals demonstrated that antioxidant bioactivity may be mainly contributed to phenolic compounds, whereas anthocyanins play an important role in the antidiabetic bioactivities . Other reports showed that flavonoids including vitexin and isovitexin were the dominant components in mung bean [2, 9] and the content of vitexin was much higher than that of isovitexin in ethanol extracts . It has been reported that mung bean has a strong antioxidant activity and isovitexin and vitexin contribute to most of the 1,1-diphenyl-2-picrylhydrazyl, ferric - reducing antioxidant power or 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical scavenging ability . Various factors including geographical location, climate change, temperature, and illumination time have important effects on the types and contents of plant chemical components, which are related to their bioactivity, functionality, and applications . Light quality is one of the most important factors in the regulation of plant growth, morphogenesis, photosynthesis, metabolism, and gene expression [11, 12]. For the photobiological research, ultraviolet - visible spectrum between 200 nm and 800 nm wavelength plays an important role in changes of chemical compounds of the organisms by irradiating them, especially compounds with ultraviolet (uv) absorption property [13, 14]. Compared to the ordinary fluorescent light source, the light - emitting diode (led) light sources can provide a single wavelength of light quality with high photoelectric conversion efficiency, fixed wavelength, and low heat . Led light source is considered to be a new important light source in the field of plant physiology and plant cultivation . Previous studies indicated that the led light sources were used in the research of photomorphogenesis, chlorophyll synthesis, and photosynthesis . Recently the studies of this field attract more and more researchers focusing on the work [18, 19]. Many studies indicated that there were the practical problems of insufficient light intensity and limited spectral wavelength during the process of plant cultivation in the laboratory [2022]. It is necessary to find effective ways to replace or assist the ordinary fluorescent light source, to improve research method for the plant, and to promote the plant quality . In this study, we used the red leds and ultraviolet - b (uv - b) radiation as additional light sources for the process of plant cultivation in the laboratory to determine the role of the different qualities of light source on growth and photosynthetic characteristics of mung bean . Mung bean seeds were obtained from yangling breeding center of national bean engineering research center of china (shaanxi, china). The ordinary fluorescent light source (power 40 w) was purchased from philips inc . The light directly irradiated the seedling of mung bean from am 7:00 to pm 7:00 each day . The uv - b radiation was provided by filter qin brand (baoji lamp factroy, china) 30 w fluorescence sunlamps . They were filtered with 0.13 mm thick cellulose diacetate (transmission down to 290 nm) for uv - b radiation . The dose of uv - b irradiation was 0.861 kj / m per day . The lamps were suspended above the plant at the height of 40 cm perpendicular to the ground . Firstly, seeds were sterilized for 10 min by 0.1% hgcl2 and were grown in petri dish (diameter 18 cm) after being washed for 50 min by flowing water . Until seeds were germinated, they were transplanted in basin (diameter 25 cm) which was filled with the ratio of peat: vermiculite: perlite for 3: 1: 1 . One week after seed germination, the supplementary light treatments carried out seed germination . On the 20th day and 40th day of supplementary light treatments, organisms were sampled, respectively, for various analyses . The morphology including plant height, fresh weight, dry weight, root length, and stem diameter was measured . Mung bean seedlings were oven dried at 80c until constant weight and being weighed using electronic scale as biomass (g). The results of stomatal conductance, photosynthesis and water use efficiency were the mean values of the day . The method for the measure of the concentration of chl a and chl b was extracted by acetone and determined following the reported methods . Intact leaf samples of seedlings (fresh weight 0.5 g), which were at 5 - 6 leaves stage of development, were placed in a mortar and followed by the addition of silica of 0.2 g, caco3 of 0.2 g, and 15 ml 80% acetone . After thorough grinding, the samples were filtrated with two layers of filter paper by pump air and fixed to 25 ml with 80% acetone, and then the absorbance at 663 and 645 nm was determined, respectively . Chlorophyll concentration was calculated and expressed as mg / g fw . Fresh samples of 0.5 g were taken from the epicotyls and extracted in 10 ml acidified methanol (methanol - water - hydrochloric acid, 79: 20: 1, v / v) for uv - b absorbing compounds, according to the procedure of mirecki and teramura . Extract absorbance at 300 nm was measured with a spectrophotometer (uv-2100; shimadzu, columbia, md, usa) and the absorbance was arbitrarily used for analysis . The results were expressed as the means standard error (se) of triplicate . The data were subjected to one - way analysis of variance (anova) and the significance of difference between samples means was calculated by duncans' multiple range test and p values less than 0.05 were considered significant . It was observed that the values of the growth parameters of mung bean seedlings were irradiated for 20th day by red leds and uv - b was not significantly different compared with that of the ordinary fluorescent light (table 2). However, the red leds treatment caused a significant increase (p <0.05) of the values of plant height, fresh weight, dry weight, and root length compared with that of the ordinary fluorescent light for 40th day . With the uv - b radiation for 40th day, an obvious decrease (p <0.05) of plant height was observed, while it induced a marked increase (p <0.05) in fresh weight, dry weight, and stem diameter . The red leds treatment induced a significant increase (p <0.05) in the values of the photosynthetic characteristics for the two durations (table 3). However, a significant decrease (p <0.05) was observed in the values of the photosynthetic characteristics of the uv - b radiation for 40th day . The concentrations of chl a and chl b may affect the values of the photosynthetic characteristics to a certain extent . It was obvious that the red leds treatment induced statistically significant increases (p <0.05) not only in the values of the photosynthetic characteristics (table 3) but also in the concentrations of chl a and chl b (figure 1) for the two durations . Compared with the ordinary fluorescent light, the uv - b radiation did not cause significant differences . With the treatment of uv - b radiation, the concentrations of uv - absorbing compounds uv - b radiation - treated seedlings resulted in a notably increase in the concentrations of uv - absorbing compounds for the two durations (p <0.05). However, red leds did not cause significant difference in comparison to the ordinary fluorescent light . Leds are a promising irradiation source for plant growth in space for long life, minimal mass, volume, and being a solid state device . The red leds (wavelength 650 nm) were used as a supplementary light source for the greenhouse tomato in 1982, which was reported earlier by japan's mitsubishi corporation . During the process of laboratory cultivation, it has been reported that the ordinary fluorescent light lacked the ultraviolet part of the solar spectrum background, which was essential growth factor to play an important biological role [20, 26]. However, it was difficult to use a mixed - use led light sources during the process of plant cultivation completely . Therefore, we used red led and uv - b as supplementary light sources for the ordinary fluorescent light to study the role of these light sources in plant cultivation . Our results showed that these light sources were obviously increased for growth and photosynthetic characteristics of mung bean . The red led light source promoted the growth of the mung bean root (table 2), which was useful to absorb the nutrients and water of the soil . Chlorophyll concentrated in the chloroplast grana is the main pigment to capture the energy for photosynthesis in green plants . The results showed that the red led light source can significantly increase the concentrations of the chlorophyll (figure 1), which effectively promoted the photosynthesis and water use efficiency (table 3). On the contrary, uv - b radiation induced a notably decrease in the photosynthesis and water use efficiency . It was suggested that the uv - b treatment will reduce the stomatal opening degree of mung bean and then affect the gas exchange in photosynthesis . Uv absorption compounds in leaves are an important class of pigments including flavonoid, flavonol, cinnamon, and anthocyanin, which determine the color changes of many plants and are very sensitive to light . They play an important role in protective effect as a class of secondary metabolites [28, 29], which are related to antioxidant, antidiabetic, anti - inflammatory, antitumor and antimelanocytes and antiangiotensin i - converting enzyme activities [28]. The previous studies have reported that vitexin and isovitexin were major flavonoid in the ethanol extract of mung bean and vitexin content was much higher than isovitexin in ethanol extracts from mung bean sprout [3, 9]. However, another report showed that no significant difference in levels of vitexin and isovitexin was observed in mung bean sprout of the same cultivar tested in the experiments . Since uv absorption compounds have an absorption peak in uv - b radiation scope, it could be found that the uv - b treatment caused a significant increase in uv absorption compounds (figure 2). Compared with the ordinary fluorescent light, red leds did not induce significant differences in uv absorption compounds . Red led and uv - b as supplementary light sources have an important effect on plant growth and chemical components.
Sarcoidosis is a multisystemic granulomatous disease of unknown etiology characterized by the presence of non - caseating granuloma consisting of epithelioid cells . Heart, spleen, bone marrow and less often eye, skin and salivary glands are common extrapulmonary sites of disease manifestation . The liver has sarcoid nodules on occasions, however these nodules are generally multiple and diffuse . Granulomatous lesions in hepatic sarcoidosis are most often located at the catchment area of glisson's capsule . In these patients, portal hypertension is the predominant symptom . The diagnosis of hepatic sarcoidosis might be easy because patients with hepatic sarcoidosis have generally pulmonary lesions or hilar lymph node swelling at the same time . We present a case of hepatic sarcoidosis with a solitary giant nodule in the liver, but with radiological findings typical of hilar cholangiocarcinoma . A 51-year - old female was referred to our institution with abnormal laboratory data of liver function and the biliary system . The laboratory data on admission showed slight elevation of alkaline phosphatase (alp) and gamma - glutamyl transpeptidase (-gtp), but a normal level of tumor marker (table 1). Her physical condition upon admission was generally good; there was no anemia or jaundice, and the liver was not palpable . Abdominal ultrasonography showed a low echoic lesion of 37 mm diameter with an unclear margin at segment 4 . The intrahepatic bile ducts (ihbds) of the left lobe were dilated to 6 mm with obstruction by this tumor . A swelling round lymph node of 11 mm diameter was revealed in the hilar region of the liver . Abdominal dynamic computed tomography (ct) revealed a 4.0 cm tumor of low density at segment 4, which partially invaded segments 1, 5, and 8 ., the tumor was detected as a low - density lesion with an irregular shape (fig . The contrast enhancement of the tumor was slightly intense at the margin of the tumor during the equilibrium phase (fig . 1c) and became more intense during the delayed phase, but the margin became unclear (fig . Magnetic resonance cholangiopancreatography showed remarkable dilatations of the ihbds of segment 2 and 3 with an extreme stenosis of the left main hepatic duct . Endoscopic retrograde cholangiopancreatography also revealed conspicuous stenosis at the junction of the left hepatic duct and the common hepatic duct as well as stenosis and rigidity of the anterior and posterior branches of the right hepatic duct . Moreover, the junction of the hepatic duct draining the right caudate lobe had stenosis (fig . When deep cannulation was performed beyond the stenotic hepatic duct, dilated ihbds of segment 2, 3 and 4 were detected . 2b). Extended left lobectomy with caudate lobectomy and bile duct resection was performed with a diagnosis of hilar cholangiocarcinoma located predominantly at the left main hepatic duct . Some daughter nodules were found in the left lobe, which were considered to be intrahepatic metastasis . Pathological findings confirmed the presence of non - caseating granuloma with multinucleated giant cells . There were multiple nodules with severe fibrosis in the resected specimen . A lot of small non - caseating granulomas consisting of epithelioid cells were found in the dissected lymph nodes . The case reported here is important because of its morphologically distinct characteristics from radiological findings for hepatic sarcoidosis . Sarcoidosis is a systemic disease that primarily affects the lungs and lymphoid tissues of the body . In japan, sarcoidosis occurs mainly in the age group of 50- to 60-year - olds with a predilection for females . Hepatic involvement of sarcoidosis was described in 11.5% of 736 patients in the access study . Hepatic granulomas due to sarcoidosis were recognized most commonly as multiple and small nodules with less than 1.0 cm diameter . Liver involvement ranges from asymptomatic incidental granulomas to portal hypertension from granulomas in the portal triad, usually with relative preserved liver function . Angiotensin - converting enzyme (ace) level may be elevated in 70% of patients with sarcoidosis and is elevated in almost all patients with hepatic sarcoidosis . In general, in 2040% of patients with sarcoidosis, alp and -gtp levels are elevated, but in those with hepatic sarcoidosis the levels are usually much higher . Ultrasonography findings include parenchymal echogenicity, coarsening of the liver parenchyma with or without discrete nodules, and focal calcifications as well as contour irregularity . On ct, hepatic granulomas are visualized as multiple, discrete, low - attenuating, non - contrast - enhancing nodules of variable size . As they increase in size, they tend to become confluent and have to be differentiated from various infectious and neoplasmatic conditions . Magnetic resonance imaging also shows multiple diffuse, densely packed, uniform nodular foci with normal signal intensity with t1- and hypodense with t2-weighted sequence . On dynamic ct imaging, our case was compatible with hepatic sarcoidosis due to a poor enhancement effect on early arterial phase and a slight enhancement from the margin on delayed phase, even though this intrahepatic nodule was huge and solitary . In most patients with sarcoidosis, the course of hepatic involvement is asymptomatic . In a few patients, chronic intrahepatic cholestasis or portal hypertension portal hypertension could be due to obstruction of portal flow because of granulomas in the portal area, with fibrosis and hyalinization of the portal triad causing presinusoidal block . Another possible mechanism was advocated in which there might be arteriovenous shunts that increase portal blood flow . Asymptomatic patients with mild elevation in liver function enzymes should not be treated but should be followed with serial blood work . It has been reported that the majority of untreated patients who are clinically asymptomatic have spontaneous improvement in their liver function . Indeed, perry and vuitch reported that the mortality rate of patients with sarcoidosis was 1 of 28 at autopsy and 1 of 18 with portal hypertension . For patients with refractory liver dysfunction, chronic progressive cholestatic disease and portal hypertension that develop more severe liver disease, corticosteroids should be administered . However, corticosteroids cannot prevent disease progression, including development of portal hypertension in patients with early or established cirrhosis . For such cases, when medications are no longer efficacious, liver transplantation is the only option . There are other causes of hepatic granulomas, including bacterial infections such as tuberculosis, brucellosis, viral infections and parasite infections, primary liver disease such as primary biliary cirrhosis, autoimmune cholangiopathy and many other systemic illnesses . As mentioned above, there is a wide differential for granulomas in the liver . In western countries, a large portion is attributed to primary biliary cirrhosis (up to 55%) and sarcoidosis (1530%), but infective cases, like tuberculosis and brucellosis, should always be kept in mind during initial investigation [12, 13]. Evaluating hepatic granulomas the majority of cases in which granulomas appear only in the liver are caused by liver disease rather than multisystemic disease . In that case, the differential diagnosis may be frequently difficult compared to other tumor - forming diseases, such as neoplasm in the liver . The diagnostic approach to hepatic sarcoidosis must be made on the histological evidence of non - caseating granulomas in a liver biopsy . In this report, we describe a rare experience with hepatic sarcoidosis, which we presented with clinical and radiological findings characteristic of hilar cholangiocarcinoma . Our experience suggests that the diagnosis of patients with tumors in the liver with atypical radiological findings, who have a case history of sarcoidosis, need to be established by comprehensive and cautious consideration . There should be no hesitation to perform liver biopsy if there is difficulty in making a definitive diagnosis . Despite utilizing several detailed and extensive diagnostic modalities, we may occasionally encounter a patient with a solitary tumor in the liver for whom it is extremely difficult to make a differential diagnosis from the malignant neoplasm, much like our experience . In this case, a surgical treatment including liver resection should be seriously considered, because a patient with hepatic sarcoidosis occasionally had to undergo liver transplantation in the end after a short period of observation without any treatment due to the difficulty of diagnosis like our case.
Despite positive changes in professional definitions, such as removal of homosexuality from dsm in 1973, deviations from gender specific heterosexual development still constitute a reason for concern for most of the parents . Unique stressors associated with homosexuality such as stigma, discrimination and bully victimization may adversely affect mental health of young individuals having same - sex attraction . Review of relevant literature reveals that people having same - sex attraction are at higher risk of mental disorder, suicidal ideation, substance misuse, and deliberate self - harm than heterosexual people . Gay youths are more likely to engage in school refusal, runaway behavior and subsequent homelessness, prostitution and sexually transmitted diseases . While it is true that a great majority of people with same - sex attraction do not report any problem relating to their sexual behavior, there is also a significant amount of suffering as well . It is important to bear in mind that human history is loaded with accounts of severe persecution of individuals with same - sex attraction . It was not until the second half of the 20th century that most influential european countries decriminalized homosexual activity . Even today, in at least 76 countries, consensual same - sex relationships are considered as criminal offences punishable by imprisonment and even death, in at least five countries . Thus, the medical profession has a duty to alleviate this suffering in all legitimate ways possible . Besides fostering greater tolerance of homosexuality on behalf of society, clinicians are required to prevent negative health outcomes associated with it which may be better addressed by understanding the roots of homosexual behavior . There is a great deal of research that tries to identify biological factors influencing same - sex orientation . It is suggested that an individual s sexual orientation is determined by early biological events generated by genes and prenatal sex hormones, both of which hardwire brain morphology . In regard to heritable factors, despite several family, twin and molecular studies, much remains to be known about the genetics of human homosexuality . Although natural selection favors heterosexuality for transmission of genes from one generation to the next, persistence of homosexuality in a small but consistent percentage of the human population is of question . The prenatal androgen model, which has become a clich in the field, argues that homosexuality in men is due to under - exposure to prenatal androgens and in women, due to over - exposure . Attempts to support the model included hormone measurements, animal experimentations and studies of rare disorders (i.e., congenital adrenal hyperplasia and testicular feminization) all of which revealed inconsistent results . In the following surge of research in prenatal androgen model, indirect consequences of prenatal hormone impact, such as digit ratio, fingerprint patterns and oto - acoustic emissions have been studied, without reaching definitive conclusions . It is warranted that prenatal androgenization is essential for male gender identity development, but apparently not decisive . Thus, researchers and clinicians should think about what else may play a role in this puzzling area . The thyroid gland, located in front of the neck, produces two related hormones, thyroxine (t4) and triiodothyronine (t3). These hormones have a critical role in cell differentiation during development and help maintain thermogenic and metabolic homeostasis in the adults . Thyroid disorders are highly prevalent in the population and mostly affect women in childbearing age . In pregnancy, thyroid function is altered by 4 factors in pregnancy: i) the transient increase in human chorionic gonadotropin (hcg) during the first trimester, which cross - reacts with thyroid - stimulating hormone (tsh) receptor; ii) the estrogen - induced rise in thyroxine - binding globulin (tbg) which is the major transport protein for thyroid hormone; iii) increased autoimmunity; and iv) increased urinary iodide clearance, which can cause impaired thyroid hormone production in areas of low iodine supply . Consequently, the levels of both t3 and t4, the major hormones released by the thyroid, increase by ~50% and serum tsh levels decrease in the first trimester and increase in the second and third trimesters . Hypothyroidism may affect up to 3% of all pregnant women, while hyperthyroidism is less than 1% . Subclinical forms of thyroid disorders is also a growing concern as undiagnosed cases may result in serious consequences . It is a well - established fact that thyroid hormones are essential for both fetal and post - natal neurodevelopment and for the regulation of neuropsychological function in human beings . Either due to iodine deficiency or autoimmune thyroid dysfunction, reduction of circulating maternal thyroid hormones has been found to result in lower iq in infants and young children in several retrospective and prospective studies . During gestation, this is expected to include behavior relating to sexual attraction, however, there is no study focusing on this issue . In one related study by ellis and hellberg, fetal exposure to thyroid medications and diet pills another recent study mentioned autoimmune thyroid diseases among a group of autoimmune diseases that were found at increased rates in same - sex married danish individuals . In the present study, it was aimed to investigate the association between history of thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the offspring . This study was a retrospective chart review of patients evaluated by the author - clinician between 2005 to 2013 in 4 outpatient settings in istanbul; 2 of them attached to marmara university and the remaining ones as private clinics ., all diagnoses had been based on dsm - iv criteria . Considering the average age of the onset of same - sex attractions, the charts of the children and adolescents aged 8 to 17 years patients with mental retardation, autism and psychosis were excluded from the review . The excluded children with mental retardation charts with missing perinatal information were not included in the study . Although the study was a retrospective one, the approval of local ethics committee of marmara university medical school was obtained . Parents consent for treatment involved consent for the use of their data for audit and research purposes provided that their identity not to be disclosed . After the cases with overlapping conditions had been identified (n=12), a second - line consent for the use of their data in a study correlating complaints of the child with pregnancy history, was asked in telephone interviews and none of the mothers refused to participate . More detailed and updated data about the course of their thyroid dysfunction was collected in those interviews . A respectful approach to the subjects has been adopted because of the sensitivity of the topic . In accordance with current clinical practice parameters, the term same - sex attraction was reserved for the youngsters who display such behavior whereas the term childhood gender nonconformity was rather indicated for preadolescent cases who had behavior that fell outside gender norms . Patient records, kept as electronic office files were reviewed consecutively beginning from the earliest admission until the last one, at the end of october 2013 . Besides this, since all files are electronically searchable, all files were explored using relevant search terms in turkish and english, relating to pregnancy thyroid dysfunction in the mothers and same - sex attraction / gender nonconformity in the children . In order to prevent possible biases while searching for relevant cases, another clinician who had had no responsibility for any of the patients between 2005 to 2013 also reviewed the files . Based on this criteria, the statistical package for the social sciences version 15 was used for statistical analysis (spss 15, chicago, il, usa). Fisher s exact test and t - tests were conducted for the analyses of the data . The prevalence of same - sex attraction / gender nonconformity in the group of children having positive maternal history in pregnancy was compared with the total sample . We also used logistic regression analysis to assess variables predicting same - sex attraction / gender nonconformity . The level p<0.05 was taken as the cut - off value for statistical significance of results . The statistical package for the social sciences version 15 was used for statistical analysis (spss 15, chicago, il, usa). Fisher s exact test and t - tests were conducted for the analyses of the data . The prevalence of same - sex attraction / gender nonconformity in the group of children having positive maternal history in pregnancy was compared with the total sample . We also used logistic regression analysis to assess variables predicting same - sex attraction / gender nonconformity . The level p<0.05 was taken as the cut - off value for statistical significance of results . A total of 790 charts were identified as eligible for further search in this study . This group consisted 520 male children (65%) and 270 (35%) female children, with a mean age of 12.182.81 . The review of this archive revealed 39 mothers with thyroid dysfunction either in pregnancy, or currently . From this sample, twenty - four (61%) mothers reported current, 5 (12%) mothers reported only in pregnancy and 10 (25%) mothers reported both in pregnancy and current thyroid problem . In sum, 15 (38%) no statistically significant difference existed between the mean delivery age of mothers who had thyroid problem in pregnancy and those who did not . Any thyroid problem was found to affect 4.9% of the entire population (n=790), whereas the rate was 1.8% for pregnancy thyroid problem . The most common principal diagnoses given to the children of mothers in this group were attention deficit hyperactivity disorder (n=14; 35%) and depression (n=10; 25%). The mothers who had pregnancy thyroid dysfunction (n=15) reported their diagnoses as either hypothyroidism, goiter or hashimoto disease whereas three mothers could only recall the presence of a thyroid disorder . Among the mothers interviewed, only three reported medication use in pregnancy which were thyroid hormones . We have identified 16 children and adolescents in the total population who had behavior indicative of same - sex attraction / gender nonconformity . Of these children, 13 were males and 3 were females, having a mean age of 13.181.83 . Different diagnoses were given to these children, except for three, for whom no diagnosis was applicable . The percentage comparison of same - sex attraction / gender nonconformity in the focus group with other control groups are presented in figure 1 . For each group compared, there was a significant association between the history of thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the children born to these women (p<0.0001, by fisher s exact test). The association was also significant for each sex (p<0.0001, by fisher s exact test). In logistic regression analysis, no other factor has been found to influence same - sex attraction / gender nonconformity . Homophobic bullying, which targets children with same - sex attraction / gender nonconformity appeared to be a common problem (n=7; 58%). In table 1 no child was identified as having both mental retardation and perinatal history of thyroid dysfunction . In excluded cases, in this study, a significant association between thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the offspring was found ., this study is the only one that explored this association with a focused objective and found positive results . An initial point of note is that the prevalence of thyroid disorders in the study population, both currently and in pregnancy, is far less than the reported prevalence rates that may be expected . It is possible to hypothesize that only certain patients had received medical attention in pregnancy . Current case - finding procedures are estimated to fail to identify many pregnant women with subclinical and overt hypothyroidism . Also, a significant rate of children and adolescents with same - sex attraction may remain to be identified . First of all, same - sex interest may manifest later than the mean age we found in this population . Third, as can be seen in table i, most subjects were male children who assume a feminine role . Active or manly same - sex attraction was not a reason for referral . Similarly, behavioral manifestations of same - sex attraction in girls were lower than population estimates in this study . This finding of sex difference in referral rates is in line with previous reports of gender issues . Despite these difficulties, there existed sufficient cases to show the magnitude of underlying pathology . The assumption that same - sex interest is caused by child sexual abuse is no longer supported . Situational homosexuality, which occurs when heterosexual partner is inaccessible, like in prisons, constitutes only a small group . Therefore, it is likely that the index group in our study represents general characteristics of individuals with same - sex attraction in a population . As 3 children had not received any psychiatric diagnosis and the remaining children s disorders were either temporary or common ones seen in child psychiatry practice, this may call for further research about the generalizability of the present findings to general population . On the other hand, although the statistical association we found in this study is such strong, there were 3 cases who had no sex attraction / gender nonconformity despite maternal thyroid history dysfunction in pregnancy . Depending on the timing, duration, severity and type of maternal thyroid dysfunction, there may be a group of children who could survive the negative impact on behavior . As described in the methods section in accordance with current clinical practice parameters, although two different terms, gender nonconformity and same - sex attraction were used to identify the explored behavior, all cases have been merged in a single group as can be seen in table 1 . There is agreement that gender nonconformity often is a developmental precursor of homosexuality in males and it is also associated with negative health outcomes . The present study provides further evidence that both conditions are developmentally linked . On the other hand, in all youngsters in table 1, the extent of same - sex attraction was beyond the limits of sexual experimentation which is a feature of adolescent development . A particular point is that, no case had been diagnosed as having gender identity disorder, which is a different developmental pathway . However, gender identity disorder in the offspring should also be considered among the behavioral consequences of prenatal thyroid dysfunction and may be explored in further research . In contrast to what may be expected, no case was identified as having low iq because of early thyroid dysfunction, which is a well - established consequence . It is evident that the impact of early thyroid dysfunction on developing neurons produces more common but not less serious results which manifest later in life at a behavioral level . First of all, as different specialties are involved in this model, namely endocrinology, obstetrics, pediatrics, child and adult psychiatry, grasping the whole picture may be elusive . However, unlike other disciplines, child psychiatry has a unique advantage of being able to collect data for both parents and offspring . Secondly, the impact of thyroid dysfunction on sexual behavior unfolds after so many years, even after a couple of decades . Thirdly, because of understandable sexual privacy and secrecy in the context of strong moral, cultural and religious values, even very close relatives of an individual with same - sex attraction may be unaware of the condition . Fourth, a significant rate of pregnant women with thyroid dysfunction may go undiagnosed, or may have subclinical forms of thyroid dysfunction . All possible explanations of the findings will be based on thyroid dysfunction during pregnancy, as it is the most likely predictor of same - sex attraction in the majority of cases . Either present before pregnancy or developed afterwards, thyroid diseases may result in diminished supply of thyroid hormones which are crucial for optimal brain development . Developing brain areas, which will be responsible for determining heterosexual orientation remain undifferentiated without the influence of essential thyroid hormones . As the timing, duration, severity and type of insult during development determine the specific consequence, the characteristics of same - sex attraction (i.e., bisexual or entirely homosexual) in the offspring are expected to be shaped by these factors . Once the timely effects of specific hormones are missed, these functions may have been never recovered in a lifetime . Besides, these areas may also be overly sensitive to the availability of the thyroid hormones . We should also consider whether there is such a mechanism in the brain that in case thyroid hormones are low, less vital neurons are sacrificed to save more crucial ones to preserve optimal intelligence . Further to these explanations, we need to think about the possible role of autoimmunity in the generation of deviant sexual orientation, as hashimoto thyroiditis was quite common in the index group . It is known that, in hashimoto thyroiditis, tsh receptor blocking antibodies are capable of crossing the placenta and affecting the fetal thyroid . It is possible to hypothesize that besides lowering thyroid hormone levels, various antibodies may attack sensitive areas in the developing fetal brain and may produce permanent effects . Finally, there is also possibility that maternal thyroid dysfunction may impact prenatal androgen system and generate permanent consequences ., there are cases having same - sex attraction / gender nonconformity despite medication history addressing thyroid dysfunction during pregnancy . Although more detailed medication histories were not available, thyroid hormone use during pregnancy in the index cases will be discussed in two ways that i) medication use rate is low and ii) simple supply of thyroid medication may be ineffective in altering the pathogenesis if basic underlying factor is autoimmunity . At this point, i am going to refer to the study by ellis and hellberg, cited above . The most significant finding in that study was increased prenatal use of thyroid hormone and diet pills in the mothers of young females with homosexual / bisexual orientation . According to the authors, thyroid and dieting medications may trigger immune reactions that may result in homosexual / bisexual orientation in the female offspring . However, correction of hypothyroidism with synthetic levothyroxine is known to be safe, and in the present study there was no history of medication use in the majority of the cases . Thus, it appears that it is not the medication use that leads to same - sex attraction, but the basic thyroid dysfunction itself . Definitely, as there may be several cases who never come into medical attention, the subjects with medication use represent only a sub - group . I will also argue that a broad spectrum of thyroid disorders should be considered rather than only immune conditions . Finally, failure to find significant results in male subjects may stem from methodology: both sexes are affected in the present study as might be expected . Finally, a recent study that reported increased rates of certain autoimmune diseases in homosexual individuals but no increased overall risk can best be explained with the mechanism presented in our study . From a child psychiatry point of view, it is clearly visible that there is a new kid in the playground . What was found in the present study is obviously a challenge to pre - existing theories about homosexuality and there s sufficient ground to introduce a new model . The prenatal androgen model, which refers to rare disorders such as congenital adrenal hyperplasia and testicular feminization for explanation, is going to be affected by the decisive role of thyroid hormones in sexual orientation . It is not surprising to find significant results for homosexuality in genetic studies as prevalent thyroid disorders (i.e., hashimoto thyroiditis) have a very strong genetic background . In the search for possible factors for the persistence of homosexuality in human populations, thyroid disorders provide a reasonable explanation as they are prevalent and more common in women . Fraternal birth order effect, which refers to increasing odds of homosexuality in having older brothers, may simply reflect continuously decreasing capacity in thyroid disorders in subsequent pregnancies . In sum, the findings of this study and review of the preexisting views indicate the birth of prenatal thyroid theory to explain the underlying dynamics of same - sex attraction in human beings . There is a need to add relevant statements for the ethical position of this study and manuscript, as it is known that there exist views that oppose scientific study on the causes of same - sex attraction . We believe that clinicians as researchers do not have an option to suppress data, experience or insight gained in their practice . If there is a medical risk factor that may highly predict certain behaviors, clinical researchers cannot remain silent and cannot keep it hidden, which apparently constitutes an ethical violation . Individuals with same - sex attraction have the right to know the possible origins of their behavior which cannot be denied by making decisions on behalf of them . Apparently, in this age of information, this right extends to any individual who would like to be interested . People at childbearing age, for instance, may not be accepting homosexuality, because they do nt want offspring that is likely to be persecuted and susceptible to negative health sequelae . On the other hand, we are well aware that abuse of scientific knowledge is always a potential risk and we must be prepared to struggle against it at all times . First, the prenatal medical data relied on parental recall . However, as thyroid disorders are chronic in nature, the mothers may be pretty bright in reporting about this . Also, the parents and the children had not been questioned systematically about issues of sexual behavior . However, such limitations are inevitable in investigating an issue in which cause and effect are apart in time . Based on the insight gained from this work, is expected to focus on confirmation of present findings . Then, neurobiology of fetal impact and improvement of endocrinological care during pregnancy may be studied . Using current case - finding procedures, many pregnant women with subclinical and overt hypothyroidism may remain undetected . On the other hand, neither targeted case finding nor universal screening have been shown to lead to improved outcomes in population - based studies . In light of this study, screening for thyroid dysfunction in pregnancy should be rethought, which is controversial in current guidelines . Outcome criteria for intervention studies should be redefined considering same - sex attraction unfolding much later . Newer intervention strategies may be needed as same - sex attraction is a special kind of consequence . Psychological effects and therapeutic management of awareness of thyroid impact in affected individuals, either as mother or as offspring, may become a field of highest priority . A final note is that medical implications should always be developed with multi - disciplinary consensus . More convincing neurobiological evidence will be a positive step towards social acceptance and greater adaptation of young and adult people with same - sex orientation . Violence and prejudice against individuals with same - sex attraction will inevitably lose part of its ground . With further medical understanding and awareness, all medical specialties have to do their best to help all affected people in their potential problems . There is a real need to review all relevant cultural, legal, political and even historical aspects of the topic from the medical standpoint explained in this article . It is only with the rule of science and knowledge that certain misconceptions and misperceptions will be cleared up and humankind will benefit.
Icter is the most prevalent disease in newborns and although most of the newborns affiliated with this seem healthy in other fields, there is always a fear for toxic complications of indirect hyperbilirobinemia on the central nervous system in these newborns . Icter happens when liver cannot pick enough amount bilirobin from the plasma, which may be due to high bilirobin production (generally because of increased hemoglobin degradation) and also the limitation of bilirobin clearance (reduced uptake, reduced conjugation and less excretion) by the liver . On the other hand, the increase in enterohepatic circulation, which is proven undeniably in newborns than the adult, may play a role in icter development together with the previously mentioned reasons . If the level of indirect bilirobin increases in the plasma to a level which denotes the probability of dangerous toxic complications, the basic intervention for it, i.e., phototherapy, must be carried out immediately . Nowadays phototherapy, although not defined as a standard, is not only method of decrease or avoidance of plasma indirect bilirobin, but also used widely in neonatal health care centers according to the devices available . Considering the effect mechanism of phototherapy in the treatment of, we should note that light, affecting the unconjugated bilirobin in the skin, subcutaneous and capillary reduces the level of indirect plasma bilirobin in three separate processes: one of these important processes is the creation of numeral photo isomers out of indirect bilirobin in a way that the indirect bilirobin, while normally exposing the 15z, 4z spatial formation, produces other spatial formations (4e, 15z; 4z, 15e; 4e, 15e) by the absorption of light especially in the range of 460 10 nm . These formations of photo isomers, known as e isomers, are bile soluble and enter the digestive system without the need for conjugationlumirubin, a structural isomer formed by the effect of light on unconjugated bilirobin, is bile and urine soluble and it can be excretion without the need for conjugationa new photo catabolism method is recently discovered for the unconjugated bilirobin which is followed by the products of unconjugated bilirobin oxidation (biliverdin, dipyrroles and monopyrroles) and is thought to be excreted in the liver or kidneys without any need for conjugation . One of these important processes is the creation of numeral photo isomers out of indirect bilirobin in a way that the indirect bilirobin, while normally exposing the 15z, 4z spatial formation, produces other spatial formations (4e, 15z; 4z, 15e; 4e, 15e) by the absorption of light especially in the range of 460 10 nm . These formations of photo isomers, known as e isomers, are bile soluble and enter the digestive system without the need for conjugation lumirubin, a structural isomer formed by the effect of light on unconjugated bilirobin, is bile and urine soluble and it can be excretion without the need for conjugation a new photo catabolism method is recently discovered for the unconjugated bilirobin which is followed by the products of unconjugated bilirobin oxidation (biliverdin, dipyrroles and monopyrroles) and is thought to be excreted in the liver or kidneys without any need for conjugation . Among all these routes, geometric photo isomerization has the most important role (80%) in reduction of the unconjugated bilirobin level through phototherapy . Considering the light spectral which can facilitate bilirobin photo catabolism, we can say that bilirobin widely absorbs the electromagnetic wavelengths in the range of 340 nm to 540 nm, which is identical to the range of spectral visible purple (less than 455 nm) to green (more than 492 nm); however, the absorption of photons is maximal in the range of blue color (420 nm to 480 nm) and specially for the wavelength of 458 nm (special blue) (for non - albumin bound bilirobin this wavelength is less [440 nm/460 nm] than the albumin bound bilirubin). The efficacy of phototherapy in the treatment of hyperbilirobinemia also depends on another characteristic than the quality of spectrum (wavelength), named irradiance . Irradiance in fact manages the packs of photons and as more photons are absorbed by the unit of surface, more energy is provided for photo catabolism of bilirobin . This effective factor in the dose of phototherapy is measured by a radiometer and the minimum irradiance acceptable for the phototherapy is about 6 - 12 w / cm / nm and if a light source can produce irradiance higher than 25 w / cm / nm in the surface unit of skin, it would be referred to as intensive phototherapy . We must also pay attention to the point that irradiance, other than the technical characteristics of the light from the source, relies on the distance of the light source from the skin . Light emitting sources which are used for phototherapy are categorized in the four following classifications based on the technology used in them and aim to produce a beam of light which has the highest care efficacy and the lowest risk of complications . Fluorescent lamps (fls); fls which have been used widely in the treatment of newborns icter during the last 40 years are categorized in two groups of long tubes and folded (compact) tubes and their most important advantage is that these devices are cheap; however, during their application it should be noted that theses lamps lose irradiance through time and they have a limited life span . Moreover, their irradiance alters according to the beam's color (blue, white, or green)halogen light emission sources or quarts halogen incandescent filament lamps (qhifl) or simply halogen bulbs; halogen lamps, which are designed based on emission of light from an incandescent string, provide a wide light output (white) based on the wavelength which covers the yellow to red bands with a high irradiance; however, due to the high level of heat emission we cannot place them close to the skin . These light sources, as opposed to fls, do not pose the limitation of losing irradiance through time and considering the white light emitted, their usage imposes less tension in the work atmospheremetal halide gas discharge lamps; this group of light sources, which are also referred to as exhaustive lamps, are designed on the basis of electric discharge in a gas environment, like fls and they produce a blue - white light but with high irradiancelight emitting diodes (led); blue led light sources are based on electric stimulation of a mineral (gallium nitride) and due to pure emission in the range of blue light, they produce less heat . Therefore, we can place these light sources very close to the skin, which in turn maximizes the absorbed irradiance at the surface of the skin . Fluorescent lamps (fls); fls which have been used widely in the treatment of newborns icter during the last 40 years are categorized in two groups of long tubes and folded (compact) tubes and their most important advantage is that these devices are cheap; however, during their application it should be noted that theses lamps lose irradiance through time and they have a limited life span . Moreover, their irradiance alters according to the beam's color (blue, white, or green) halogen light emission sources or quarts halogen incandescent filament lamps (qhifl) or simply halogen bulbs; halogen lamps, which are designed based on emission of light from an incandescent string, provide a wide light output (white) based on the wavelength which covers the yellow to red bands with a high irradiance; however, due to the high level of heat emission we cannot place them close to the skin . These light sources, as opposed to fls, do not pose the limitation of losing irradiance through time and considering the white light emitted, their usage imposes less tension in the work atmosphere metal halide gas discharge lamps; this group of light sources, which are also referred to as exhaustive lamps, are designed on the basis of electric discharge in a gas environment, like fls and they produce a blue - white light but with high irradiance light emitting diodes (led); blue led light sources are based on electric stimulation of a mineral (gallium nitride) and due to pure emission in the range of blue light, they produce less heat . Therefore, we can place these light sources very close to the skin, which in turn maximizes the absorbed irradiance at the surface of the skin . In fiber optic phototherapy systems, the light emission source can be one of the following sources, whereas the light is transferred to a contact pad on the newborn's skin surface through optical fibers . Considering the point that the technological characteristics implemented in these devices are comprehensively analyzed in vitro, it can be concluded that studies done on the relation of their performance in vivo compared with each other are very limited, hence, considering the development and evolution of these devices, making such comparisons through clinical trials seems increasingly necessary . This study is a randomized controlled trial done at shahid beheshti hospital, isfahan, iran from february 2012 to march 2013 . This study is approved by human ethical committee of isfahan medical university under reference number 391291 . The following formula was used to calculate the sample size: n = [(z1 /2) + (z1)] (s1 + s2)/d z1 /2 and = 0.05 1.96 z1 and = 0.2 0.84 the standard deviation (sd) in qhifl group will be equal to 0.6 (s1 = 0.6). The sd in fl group will be equal to 0.5 (s2 = 0.5). The study population included newborns with gestational age of 35 weeks or more who were hospitalized in the neonatal section during the 1 week after their birth due to icter . Inclusion criteria included: being healthy (no hypoxia, hypotonic, infectious, dehydration, or temperature instability disorder demonstrations observed) during medical examination, no foe to - maternal incompatibility of rhesus (rh) type, total serum bilirobin (tsb) level equal or higher than 11.7 mg / dl in week 35 - 37 of gestation, tsb level of 12.8 mg / dl at the gestational age of 38 weeks or more and last but not least, parents consent for the newborn to be included in the study . Exclusion criteria included: conjugated plasma bilirobin at the level of 1.3 mg / dl or more, tsb level equal or more than 15.4 mg / dl, severe hemolytic disease (anemia with hb <14 g / dl and/or hyperbilirobinemia before 48 h from birth), and phototherapy before the onset of the study . These newborns were entered in the study by a pediatric resident after careful examination and reviewing their conditions . In order to randomize the trial, newborns with even initial document numbers were put in qhifl and those with odd numbers were placed in fl group and sampling continued until the desired number of samples entered both groups . Fl group newborns were treated with 4 fls (tl/20w/52, philips holland) after they were put on the open crib . In qhifl group, the newborns were treated with halogen system phototherapy (bilispot, fanem sao paulo - brasil) after they were placed on the open crib . The light source was placed 45 - 50 cm from the newborn's skin in a way to illuminate a circle with the diameter of 18 cm . Irradiance level in both groups was measured by a radiometer (radiometer 2620, fanem sao paulo - brazil) at the beginning of the treatment and each 24 h after the start; the minimum acceptable irradiance was equal to 15 w / cm / nm . This measurement was done on the surface of face, xiphoid and the knees . Tsb assessment was done by capillary sampling, based on spectrophotometry on an 8 h basis for the first 24 h and after that each 12 h. phototherapy was discontinued if two successive tsb was categorized under the level of conventional phototherapy based on nomogram in american academy of pediatrics (aap) guidelines for phototherapy in hospitalized infants of 35 or more weeks gestation . Findings were recorded in a list designed for this purpose and they were analyzed by spss 20 software after entrance into computer database . The following formula was used to calculate the sample size: n = [(z1 /2) + (z1)] (s1 + s2)/d z1 /2 and = 0.05 1.96 z1 and = 0.2 0.84 the standard deviation (sd) in qhifl group will be equal to 0.6 (s1 = 0.6). The sd in fl group will be equal to 0.5 (s2 = 0.5). The study population included newborns with gestational age of 35 weeks or more who were hospitalized in the neonatal section during the 1 week after their birth due to icter . Inclusion criteria included: being healthy (no hypoxia, hypotonic, infectious, dehydration, or temperature instability disorder demonstrations observed) during medical examination, no foe to - maternal incompatibility of rhesus (rh) type, total serum bilirobin (tsb) level equal or higher than 11.7 mg / dl in week 35 - 37 of gestation, tsb level of 12.8 mg / dl at the gestational age of 38 weeks or more and last but not least, parents consent for the newborn to be included in the study . Exclusion criteria included: conjugated plasma bilirobin at the level of 1.3 mg / dl or more, tsb level equal or more than 15.4 mg / dl, severe hemolytic disease (anemia with hb <14 g / dl and/or hyperbilirobinemia before 48 h from birth), and phototherapy before the onset of the study . These newborns were entered in the study by a pediatric resident after careful examination and reviewing their conditions . In order to randomize the trial, newborns with even initial document numbers were put in qhifl and those with odd numbers were placed in fl group and sampling continued until the desired number of samples entered both groups . Fl group newborns were treated with 4 fls (tl/20w/52, philips holland) after they were put on the open crib . In qhifl group, the newborns were treated with halogen system phototherapy (bilispot, fanem sao paulo - brasil) after they were placed on the open crib . The light source was placed 45 - 50 cm from the newborn's skin in a way to illuminate a circle with the diameter of 18 cm . Irradiance level in both groups was measured by a radiometer (radiometer 2620, fanem sao paulo - brazil) at the beginning of the treatment and each 24 h after the start; the minimum acceptable irradiance was equal to 15 w / cm / nm . This measurement was done on the surface of face, xiphoid and the knees . Tsb assessment was done by capillary sampling, based on spectrophotometry on an 8 h basis for the first 24 h and after that each 12 h. phototherapy was discontinued if two successive tsb was categorized under the level of conventional phototherapy based on nomogram in american academy of pediatrics (aap) guidelines for phototherapy in hospitalized infants of 35 or more weeks gestation . Findings were recorded in a list designed for this purpose and they were analyzed by spss 20 software after entrance into computer database . A total of 25 newborns were placed in each of the groups of fl and qhifl . Demographic information of the study patients are given in table 1 . According to t - test, the average age at the time of hospitalization and average gestational age, direct and total bilirobin level showed no meaningful difference (p> 0.05). Moreover, according to chi - squared test and fisher's exact tests, gender distribution, blood type and rh also showed no meaningful difference (p> 0.05). Demographic and basic variables distribution in both groups as has shown in table 2, the mean and sd of total bilirobin level at the time of hospitalization (tsb at 0 h), 8 h after, 16 h after and 24 h after phototherapy and also at the time of discharge from hospital are given . T - test administration on the mentioned findings showed that the level of total bilirobin before and 8 h after the intervention showed no meaningful difference in both groups . However, the same level at 16 and 24 h after the phototherapy was lower meaningfully in fl group . The mean and the standard deviation of plasma tsb during the treatment (mg / dl) repeated variance analysis revealed that the change in total bilirobin during the intervention showed a meaningful difference (p <0.001). In figure 1, the change trend in the level of total serum bilirobin during the intervention in both groups the average hospitalization duration in fl group was 2 0/58 days and the same was 2/33 0/57 days in the qhifl group, which according to t - test, the mean of hospitalization duration in fl group was lower significantly (p = 0.047). In figure 2, the distribution of hospitalization duration is given for both groups . In a study done by sarin et al . For the comparison of phototherapy through special blue standard - length tube lights (stl) and special blue compact fluorescent lamp (cfl), icter affiliated newborns with gestational ages less than 34 weeks who were healthy from other aspects and not affiliated with rh iso - immunized and needed phototherapy according to aap guidelines, were included in the study . 50 newborns were included in each group (stl and cfl) and each 12 h their tsb level was assessed and if two successive assessments showed the level of tsb under the minimum level for phototherapy, this intervention would be discontinued . Findings of this study showed no meaningful difference between the two groups according to duration of treatment and the need for blood exchange . In a study done by kumar et al ., newborns with gestational age of 35 weeks or more who faced icter during their 1 week after birth were treated by phototherapy according to aap guidelines . They were placed in two groups of compact fluorescent tube (cft) and led . This study showed no significant difference according to treatment duration, unsuccessful phototherapy attempt, need for blood exchange and phototherapy complications . In a study by romagnoli et al ., done on newborns with gestational age of equal or less than 30 weeks affiliated with icter, who had plasma total bilirobin level of equal to 6 mg / dl, were treated with phototherapy in two groups through units of fluorescent and fiber optic systems with halogen light emitting sources (wallaby). The newborn's tsb level was assessed every 12 h while considering the phototherapy failure at the level of tsb = 14 mg / dl . There were 35 newborns in each group and this study revealed that the newborns treated with wallaby system meaningfully needed less duration of phototherapy intervention; moreover, the need for blood exchange was also lower in this group rather than the newborns in fluorescent group . In a study by seidman et al ., newborns with gestational age of equal or more than 35 weeks affiliated with icter, who were completely healthy in other aspects, were treated in two groups by halogen light systems (n = 57) and led systems (n-47) and their tsb level was assessed each 12 h. no meaningful difference was shown between the two groups according to their need for blood exchange . In a study by martins, newborns weighing 1000 g or more with icter were placed in two groups of 44 newborns each and they were treated with halogen and led phototherapy . Tsb samples were analyzed each 8 h. the mean tsb in led group at the beginning of the treatment was equal to 10.1 2.4 mg / dl, while the same was 10.9 2.0 mg / dl in the halogen group . This study showed that the speed of tsb decrease in led group was slower than the one in halogen group, hence making the duration of treatment less in halogen group . Considering the point that the general aim of this study is to compare the effect of light source in qhifl and fls on the treatment of jaundice for healthy newborns with gestational age of 35 weeks or more and considering the point that the demographic and basic variables showed no significant difference according to age at the time of hospitalization, gender, blood type, rh, age of pregnancy and the first tsb level, therefore these interfering variables are neutralized and the resulting findings are most probably related to the effect of the treatment on the newborns tsb level . Although in romagnoli study the speed of reduction of tbs level in the halogen group was meaningfully faster than fluorescent group and as a result the duration of treatment was meaningfully shorter in halogen group and this difference is against the findings of the current study, we should note that in romagnoli, the qhifl light source was directly connected with the skin of the newborn through optical fibers and a pad, whereas in the current study the qhifl light source is placed at the distance of 45 - 50 cm from the skin to avoid hyperthermia . Owing to the limited number of studies in this field, it is advised to use the qhifl sources for phototherapy with the hardware basis of optical fiber systems . According to the findings of this study, the technology used in qhifl cannot show the capabilities of the fls . However, more studies are needed to confirm the findings of this study are universal.
The demand for blood and blood products in nigeria is high due to road traffic accidents, surgical and obstetrical blood loss, and anemia from other causes . Despite several the goal of clinical blood transfusion is to provide qualitative, safe, and adequate blood to recipient.1 the challenges in achieving these goals are enormous and serious efforts are been made globally, by government, donor agencies, and voluntary organizations towards its actualization . The main sources of blood procurement are commercial (remunerated) blood donors, voluntary donors, and replacement donors . A commercial blood donor offers a unit of blood for a fee paid by a contracted hospital vendor . A replacement blood donor is a family member or relative of a patient, donating a unit of blood to be used for a specific patient, while a voluntary blood donor is a well - meaning member of the society who donates his or her blood without any inducement for use by a recipient not known to him or her . There is a general policy on blood screening before utilization in which a donor blood is screened for transfusion transmissible viral pathogens to ensure that only safe blood is transfused . It is a well - established fact that the safest source of blood is from a voluntary donor.24 in 1997, world health organization (who) set a goal of achieving 100% voluntary donation by 202056 but as at 2010 only 57 of 126 countries surveyed had established this as a standard.7 blood transfusion is a known risk factor for transmission of infectious diseases including viral infections such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv). The risk of transmission is highest with blood procured from commercial donors.2489 there is still a high dependence on remunerated donors especially in developing nations such as nigeria . Several nations have made frantic efforts and are making significant improvement towards achieving 100% voluntary donor blood procurement but not so much has been achieved in nigeria . Only 5% of voluntary donation has been achieved in some major donor centers in nigeria.7 the objective of this investigation is to evaluate donor blood procurement and the associated risk of transfusion transmissible viral infections in a tertiary health facility in south - south nigeria . Records of the blood transfusion unit of the health facility from 1 january 2009 to 31 december 2009 were reviewed in order to identify the methods of donor blood procurement and screening results for hiv, hbv and hcv . For every unit of blood, we studied the method of procurement (commercial, replacement or volunteer blood donation) and screening outcome . In this study, voluntary and replacement donors were grouped together due to the lack of proper differentiation in some of the records, the number of voluntary donors were very few and their blood were actually directed to specific recipients . Blood from commercial donors is usually obtained from outside of the hospital through several vendors . Collection by the vendor is usually not supervised by hospital personnel but adheres to routine guidelines for phlebotomy . The vendor brings the units of blood to the hospital in fulfillment of a contractual agreement . Blood from replacement donor or panel donor is collected at the blood transfusion unit of the hospital . Blood (450 ml) is usually collected into a bag containing 63 ml citrate phosphate dextrose with adenine (cpda) and stored at 2 - 8c . Prior to phlebotomy, the donor's blood group is determined and the packed cell volume (pcv) ascertained . Units of blood from commercial donors are inspected to determine good anticoagulation and exclude hemolysis . The strip has two horizontal lines labeled control and patient bars . At room temperature, and using automatic micropipette, 50 l of plasma or serum is added to the sample pad . The test strip is read within 15 - 60 min . A single red line at position c (control) on the strip indicates a valid control . A similar line is found in patient bar if positive for either hiv-1 or hiv-2 . The sample is negative for hiv if there is no colour change in the patient bar . Hepatitis b surface antigen screening is carried out using the clinotech's one - step hepatitis b antigen test strip . An already anticoagulated blood sample is collected into a clean tube and centrifuged at 3000 rpm for 10 min to obtain clear plasma at room temperature . A test strip is then inserted into the test plasma to a maximum indicated by the manufacturers for 10 s and laid face up on a flat non - absorbent surface . A test is positive if two transverse bands (t = test and c = control) are seen and negative when only the one band at control (c) is seen . Only units of donor blood found to have good collection and storage and free from these viral infections are offered for transfusion to patients . All infected commercial donor blood was discarded, while infected replacement donors are not bled . Statistical analysis was conducted using the statistical package for social sciences (spss) software . The unpaired student t - test was used to test the statistical significance between commercial and replacement donor status . The test of association and risk factor for viral transfusion transmissible infection calculated using the chi - square test and odd ratios . A total of 7,552 donors record were analyzed comprising 6,931(91.8%) commercial and 621(8.2%) replacement donors . 893 (11.8%) commercial donor blood and 23(0.3%) replacement donors test positive for viral transmissible infections [table 1]. The percentage of commercial donors tested positive was 12.9% comprising 7.2% for hiv only, 4.7% for hbv, 0.6% for hiv and hbv and 0.4% for hcv . While 3.8% of replacement donors tested positive for viral infection comprising of 2.6%, 1% and 0.2% for hiv, hbv and hcv respectively [table 2]. The chi square test was used to test the statistical significance of infection between commercial and replacement donors (=45.07, p<0.001, od=3.845). The hospital blood demand and utilization has been on the increase . In this 12-month transfusion record study, a related work in the same hospital by enosolease et al.8 recorded 11,021 over a 3 years period (january 2000 to december 2002). The hospital is still highly dependent on commercial donors as procurement from these donors accounted for 91.8% . A majority of the replacement donors are medical students and doctors who donate to their unit patients; some replacement donors are actually commercial donors recruited from commercial donor centers who disguise as patient relatives . The risk of viral transfusion transmissible infection is highest with blood procured from commercial donors . The findings are different from reports of earlier studies8 in the same centre but the prevalence of hiv in this study is comparable to that of the national hiv survey for 2010 which showed a rate of 7.5% in benin.10 this shows that the prevalence of hiv infection has increased over the years . Ejele et al.6 in a survey in niger delta nigeria reported lower prevalence rates, but the survey showed that commercial donors have the highest prevalence rates for viral transfusion transmissible infections . The unacceptably high rate of infections among commercial donors may be attributed to the fact that most of the donors may belong to the high risked group . It brings to fore the need to adhere to the strict practice of screening potential donors prior to donation . Furthermore, hospitals and donation centers should embrace and implement policies that will encourage voluntary blood donor scheme . The donor blood samples that tested positive were not subjected to further testing to validate the results . The donor blood samples that tested positive were not subjected to further testing to validate the results . Commercial blood donors are still the major source of blood to the hospital and they also have the highest prevalence of transfusion transmissible viral infections in this region thus constitute a major risk transmitting infections to potential recipients . Concerted efforts should be made towards encouraging voluntary blood donation, retention of donors through campaign and media enlightenment programmes so that reasonable progress can be made to achieve the who vision 2020 goal . Concerted efforts should be made towards encouraging voluntary blood donation, retention of donors through campaign and media enlightenment programmes so that reasonable progress can be made to achieve the who vision 2020 goal.
Over many years, the interference of tracheostomy on swallowing has been studied; tracheostomy may increase the risk of aspiration, as it interferes directly in the pharyngeal phase of swallowing.1 the type of cervical incision, surgical technique applied, type of cannula used, and inflation of the cuff may, separately or together, contribute to a further fixing of the larynx, increasing aspiration . The loss of sensitivity of the larynx caused by lack of air traffic contributes to the reduction of coughing reflex, facilitating secretion and food aspiration.2 however, frequency of tracheostomy in traumatic brain injury (tbi) management when patients are receiving mechanical ventilation contrasts with the lack of evidence as to when tracheostomy should be removed.3 tracheostomy is indicated when there is upper airway obstruction, when the patient requires prolonged mechanical ventilation and/or has difficulty in weaning, when there are excessive tracheobronchial secretions, and when the patient needs continuous airway protection against aspiration.4 approximately 10% of critically ill patients undergo tracheostomy for respiratory support and facilitation of air passage into airways, providing better quality of life for patients and reducing hospital expenses.5 6 dysphagia presents a close relationship with tracheostomy, not only because this procedure is indicated in patients with swallowing problems and tracheal aspiration but because the tracheostomy itself may cause aspiration.1 although it is a common procedure, the presence of a tracheostomy tube is not devoid of complications . The cause of aspiration is unknown, but probably results from these multiple factors.7 five mechanisms are responsible for the aggravation of aspiration after tracheostomy: decreased laryngeal elevation, esophageal obstruction, cleaning of larynx with airflow, laryngeal desensitization, and uncoordinated laryngeal closure . This airflow deviation and the interruption of normal vocal function have great implications throughout respiratory, phonatory, and swallowing systems . In some cases, even post - tracheostomy scar, fixing the trachea to the skin, without any other disease can cause dysphagia.8 information is needed on the treatment of dysphagic and tracheostomized individuals, including information on intervention methods, which must be evaluated objectively . Patients were separated into two groups: the first group received a multidisciplinary approach and the other group was retrospectively assessed through medical charts, answering to a clinical protocol . The group that underwent a multidisciplinary treatment showed a significant decrease in mean length of cannulation time to 28 days, compared with those without the approach who had an average of 33 days.9 the early speech therapy intervention in a hospital aims a fast identification of dysphagia, which is an important factor for decreasing risks involving aspiration - related pneumonia and poor nutrition and preventing complications arising from the same clinics; this may reduce hospital stay, leading the patient to an early independence and an improvement of life quality.10 speech therapy within an interdisciplinary team is responsible for therapeutic management of dysphagic patient to optimize airway protection, aiming at reintroduction of oral intake in a fast and secure way, as well as helping with the decannulation process.11 12 following specific protocols in tracheostomy prescription is common, but there is no protocol description for the decannulation process . Most hospitals have an interdisciplinary team that is responsible for this process, and clinician, nurse, physiotherapist, and speech pathologist interaction reduces time of tracheostomy use, accelerates weaning, and increases safety for the patient while reducing risk of failure and complications.13 clinical speech therapy evaluation should occur prior to the actual condition before decannulation is attempted of the patient so that, under favorable conditions (high level of consciousness, adequate breathing pattern, minimum quantity of orotracheal secretions, good phonation, normal swallowing, and effective coughing), decannulation can be performed quickly and effectively, avoiding the impact of tracheostomy in the laryngotracheal region.14 recent research established some guidelines for speech pathologists prior to tracheal decannulation: the patient should be able to clean the oral tract; during cuff deflation, only minimum secretion from above the cuff should need to be suctioned; during tube occlusion, the patient must breath spontaneously and sufficiently through the upper airway with sufficient and stable oxygen saturation; the patient should have efficient spontaneous coughing with subsequent swallowing and must have improved vigilance.9 other researchers found the following criteria for tracheostomy decannulation: stable arterial gasometry, absence of respiratory distress, hemodynamic stability, absence of fever or active infection, paco2 <60 mm hg, absence of delirium or psychiatric disorder, normal endoscopic evaluation or revealing lesion occupying <30% of airway, adequate swallowing, and expectoration capability.4 because little is known about how health care professionals make a decannulation decision, a survey was conducted with professionals working in this field, in large centers around the world, to determine clinical factors they believe to be important in decannulation recommendation . Four determinant criteria for tracheostomy decannulation were found: level of consciousness, ability to tolerate tracheostomy tube capping, coughing effectiveness, and secretion quantity . Of mild importance were the following: patient comorbidities, etiology of respiratory failure, swallowing, and oxygen rates . These factors may drive the creation of specific decannulation protocols.15 even though there are specialist recommendations to guide management decisions for decannulation, there is no objective protocol to establish guideline criteria . Therefore, to aid in evaluating a tracheostomized patient's clinical state for possible decannulation and making speech therapy rehabilitation possible, the main objective of this research was to evaluate the applicability of a protocol tracheal decannulation . Twenty patients were assessed in this prospective study; the patients were between 21 and 85 years of age (average 33.55), and there were 4 women (20%) and 16 men (80%). All patients had been diagnosed by a neurologist as having tbi, and anatomical region of the lesion was known (table 1). Were adult patients over 18 years of age suffering from tbi, tracheostomized, alert and responsive at the time of evaluation, and with medical approval for a speech therapy evaluation . This research took place in the neurology wing of a general hospital in curitiba, brazil . Subjects signed a consent form with the knowledge of the objectives, procedures, and responsibilities and received answers to any questions regarding the survey . The subjects were evaluated following tracheal decannulation criteria, through a clinical assessment protocol developed by the authors: the speech therapy tracheal decannulation protocol (appendix i). Speech therapy clinical evaluation consisted of patient identification data analysis considering the variables age, gender, diagnostic, anatomical region of the lesion, and six criteria for tracheal decannulation as described: level of consciousness: measured by the glasgow coma scale, the level of consciousness was considered insufficient to protect the airway when the score was consistently less than 8 points.3 measurement was made by the medical team, and during speech therapy assessment, the patient's most recent score was noted . Respiration: tube material was noted (plastic or metal), as was whether the cuff was inflated, deflated, or partially inflated and if the patient kept the cuff deflated . Whether the patient maintained breathing pattern during tube tracheal secretion: secretions in the tracheostomy region were observed and amount, aspect, and color were noted . Amount was quantified as little, normal, or abundant, and thickness, consistency, and color (clear or yellowish) were noted . Orally responsive patients were checked for presence of wet (gurgly) voice . Patients not verbalizing were evaluated for reaction to pain, moaning, coughing, and/or frequent throat clearing . Swallowing: a clinical evaluation of swallowing was performed with occluded tracheostomy, offering food in various consistencies and noting the findings of swallowing phases . The functional oral intake scale (fois) graded the functional oral intake of patients at specific levels, with level 1 being nothing by mouth and level 7 being total oral diet with no restrictions.16 coughing: coughing was observed in food intake or in the event of tracheal aspiration, as was the presence of voluntary coughing and whether it was effective or ineffective, because coughing is a reflex mechanism for airway protection . Statistical analysis applied the fisher exact test, which was the basis for comparative process of tabulated data . All subjects in this study were diagnosed with a severe degree of tbi, characterized by anatomical region and hemisphere of the lesion; these variables were correlated with the decannulation indicator . Eleven (55%) patients had frontal lobe lesion, 1 (5%) had lesion in the temporal lobe, 3 (15%) in parietotemporal region, and 5 (25%) in the frontal - parietotemporal region . Of the total, in 8 (40%) patients the lesion was located in the right hemisphere, 6 (30%) in the left, and 6 (30%) bilaterally (table 1). We did not find any positive statistical correlation between sex and decannulation, which confirms other studies.17 patients underwent a clinical evaluation to assess their clinical conditions at the time of the procedure and to establish speech therapy criteria for tracheal decannulation . For criterion level of consciousness, patients were given a glasgow coma scale score; of the 20 patients, 6 (30%) had scores less than 8, which was considered insufficient for airway protection, and at the time of the evaluation 14 (70%) had scores greater than 8 . Of the 14, only 2 (14%) could not start the decannulation process, because one could not maintain cuff deflation and the other could not keep the breathing pattern with closed cannula . The correlation between level of consciousness by the glasgow coma scale and decannulation was significant (p = 0.0007). Of the 20 individuals who participated in the survey, 3 (15%) had a metal cannula and 17 (85%) with a plastic cannula with cuff at the time of assessment . Eight (47%) had inflated cuff, 1 (6%) was partially inflated, and 8 (47%) were deflated . Of those with plastic cannula cuff, 11 (65%) were eligible for decannulation, and the other 6 (35%) showed no conditions were ineligible for decannulation . Of the individuals with a metal cannula, 1 (33%) could be decannulated and the other 2 (67%) could not . Despite being relevant information for assessment, this study revealed that these data were not significant for the rate of decannulation (p = 0.3439). Of the 17 subjects using a cuff, 13 (76%) were able to keep it deflated and 4 (24%) were not . Two (15%) of those who were able to keep the cuff deflated were unable to be decannulated for other reasons; however, 11 (64.7%) were able to begin the decannulation process, proving this criterion to be significant (p = 0.0063). Patients were also observed with tracheostomy tube occluded; out of total population of the study, 12 (60%) maintained the breathing pattern after brief tube capping and 8 (40%) had alterations in the breathing pattern . Consequently, 12 (60%) patients were able to close the tube and 8 could not keep it occluded, which also confirmed a significant relationship between respiration and decannulation (p = 0.0000). Fifteen (75%) patients had secretions in the tracheal region and 5 (25%) did not . Of these 15, 8 (53%) had a small amount and could be decannulated, but those who had great amounts were not considered capable of decannulation . Of the patients who had secretions in the tracheal region, 1 (7%) had thick secretions and was decannulated . Four (20%) patients without secretion were able to close the cannula, and 1 (5%) patient, showing no secretion, was not able to close the cannula . Still, regarding the secretion criterion, it was possible to close the tracheostomy in 7 (47%) patients who had clear secretion . Of the total of 15 patients with tracheal secretions, 1 (7%) had yellowish secretion and could begin the process of decannulation . From these data, the amount, thickness, and color of the secretion in relation to decannulation indications were found to be significant (p <0.05 for every aspect). Thirteen patients (65%) were capable of verbalizing and with the exception of 1 (5%), all the others were able perform the training to close the tube . In the sample surveyed, 7 (35%) were considered to have no verbal responses, and none of these could be decannulated, which also confirmed a significant relationship between phonation and decannulation (p = 0.0001). Of the 20 total patients, 3 (15%) had wet voice and were to occlude the tracheostomy, and 9 (45%) did not have this vocal pattern and were also able to cap the tube . It should be noted that patients with absent phonation were not evaluated for wet voice because at the time of assessment they were not verbally responsive . Regarding swallowing, of 20 patients evaluated, 8 (40%) were graded as level 1 (nothing by mouth in fois) and could not have tracheostomy tube closed . Only 1 (5%) was graded at the same level, and the other 11 (55%) patients were classified at levels 2, 3, 4, and 5 and could occlude the tracheostomy tube . Swallowing was also assessed for signs of aspiration, considering cough reflex, dyspnea, wet voice, throat clearing, and/or discomfort at one or all offered consistencies . Of the 20 patients evaluated, 11 (55%) showed no suggestive signs of aspiration during intake and of these, 9 (82%) began training to close the tube and 2 (18%) were not considered capable . Of the total sample, 9 (45%) aspirated during the intake, and of them, 3 (33%) patients were able to cap the tracheostomy and 6 (67%) were not . These data showed that aspiration was significantly correlated to decannulation (p = 0.0399). Of the total of evaluated patients, 12 (60%) had coughing and could be decannulated, 2 (10%) had coughing but were not able to occlude the cannula, and 6 (30%) had no coughing, none of whom were able to be decannulated . The relationship between coughing and decannulation was significant (p = 0.0007). However, the effectiveness of coughing was not significant (p = 0.8571). This study evaluated 20 patients of both sexes, between 21 and 85 years of age, affected by tbi and tracheostomized with an open cannula . The decreased level of consciousness of the patient has been described as a related factor to oropharyngeal dysphagia and in the literature is associated with aspiration and pneumonia . This positive influence of the patient's awareness confirms the findings of other studies that a preserved level of consciousness decreases the risk of aspiration pneumonia and is considered so protective that it allows a better prognosis for improving communication and swallowing.18 19 deficient respiratory conditions have been reported as risk factors for aspiration and dysphagia, complicating the rehabilitation of patients with dysphagia . The more dependent on respiratory support, the less airway protection ability patients have, causing a greater risk to their clinical state and to speech therapy interventions with food.18 a recent study compared breathing through the tracheostomy cannula to breathing through the upper airway with capped cannula . The authors conclude that the work of breathing increases in 30% of patients due to higher ventilation requirements and therefore tracheal decannulation is extremely important.20 secretion should have acceptable volume and thickness (clear and fluid). Secretion aspect (thick or fluid) in the tracheal region or aspirated indicates the level of hydration and humidification . Patients with a large amount of secretion are more likely to have laryngotracheal penetration and aspiration.21 decannulation failures could be attributed to uncontrolled secretions and severe glottic stenosis.14 voice quality is the term used to denote a set of characteristics that identify the voice and aims to identify the presence or absence of wet voice after food intake by comparing the voice before and after swallowing . Changes in voice quality may suggest paresis or paralysis of the vocal fold, which in addition to altering vocal quality of the patient is an indication of impaired sphincter action of the larynx when swallowing, which could cause aspiration . A wet voice confers the possibility of presence of saliva, secretions, or food in the vocal cords and into the laryngeal vestibule . It is necessary to give prominence to the wet voice with spontaneous throat clearing or hoarse / breathy voice quality, in combination with other changes observed during the assessment, as these characteristics are often associated with increased risk of aspiration.22 23 the neurologic aspects often determine a condition for vocal change.24 in 2005 the fois was validated, which grades at specific levels the amount of oral intake; the grading may be applied throughout the therapy process to monitor patients' evolution, and so it was chosen to measure swallowing.16 tracheal aspiration occurs when the material being swallowed enters the airway below the true vocal folds . The material over the inflated cuff can drip down into trachea and lungs . When the cuff is deflated for swallowing tests, it is important to suction the area above the cuff before deflating it to remove all accumulated food and secretions.7 the identification of clinical signs of aspiration not only speeds up speech therapy but also enables airway assessment, eliminating any factor that can make weaning difficult or impossible (stenosis, granuloma, tracheomalacia, and significant dysphagia).25 26 the coughing reflex may be decreased due to both sensory (internal superior laryngeal nerve) and motor components (recurrent laryngeal nerve). The recurrent laryngeal nerve, in addition to laryngeal motricity, is responsible for cough as protection and cough in general, of a different etiology than aspiration . This distinction is very important as it allows understanding that the aspiration is likely to occur silently, without the normal response of cough and choking.18 the criteria described above were relevant to establish the beginning of decannulation process of tracheostomy . However, the data collected are based on the start of a further longitudinal research with an increase of the population assessed . This study used a protocol to establish six criteria for tracheal decannulation in patients affected by tbi: level of consciousness, respiration, tracheal secretion, phonation, swallowing, and coughing.
Papillon - lefevre syndrome belongs to a heterogeneous group of skin diseases that are characterized by hyperkeratosis of palms and soles . Papillon - lefevre syndrome is inherited as an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and early loss of primary and permanent teeth . Papillon - lefevre syndrome differs from other types of palmoplantar keratoderma in its severe periodontopathy and premature loss of primary and permanent dentition . Papillon - lefevre syndrome was first described in 1924 by papillon m m and lefevre p in two siblings . Fifty percent of the patients with papillon - lefevre syndrome appear to have been derived from consanguineous marriages . Hart et al ., showed that mutations of cathepsin c gene are responsible for papillon - lefevre syndrome and a region of chromosomes (11q14 - 21) is responsible for this . An 18-year - old boy reported to the department of periodontology and implant dentistry, bharati vidyapeeth university dental college and hospital, navi - mumbai for the treatment of generalized mobility of teeth and bleeding gums . Scaly skin lesions began to appear by third year of age, later fissures appeared on palms and soles . Intense itching was noted and skin lesions went on to involve fingers, knees and elbows . Patient showed bilateral hyperkeratosis of palms, soles, dorsal surface of fingers and knees [figures 1 and 2]. Intraoral examination showed that the patient had lost most of his permanent teeth and the remaining teeth showed grade ii mobility . Hyperkeratosis of palms and soles - case 1 hyperkeratosis of knees - case 1 intra - oral view - maxilla - case 1 intra - oral view - mandible - case 1 this patient too showed considerable hyperkeratosis of palms and soles [figure 6]. Orthopantomograph showed moderate horizontal bone loss and erupting permanent tooth buds [figures 79]. Hyperkeratosis of palms and soles - case 2 intra - oral view - maxilla - case 2 intra - oral view - mandible - case 2 complete blood count, serum calcium and alkaline phosphatase levels were done for both patients and were found to be within normal limits . On the basis of history, thorough scaling and root planing of all teeth was carried out along with administration of systemic antibiotics . Patient showed bilateral hyperkeratosis of palms, soles, dorsal surface of fingers and knees [figures 1 and 2]. Intraoral examination showed that the patient had lost most of his permanent teeth and the remaining teeth showed grade ii mobility . Hyperkeratosis of palms and soles - case 1 hyperkeratosis of knees - case 1 intra - oral view - maxilla - case 1 intra - oral view - mandible - case 1 this patient too showed considerable hyperkeratosis of palms and soles [figure 6]. Orthopantomograph showed moderate horizontal bone loss and erupting permanent tooth buds [figures 79]. Hyperkeratosis of palms and soles - case 2 intra - oral view - maxilla - case 2 intra - oral view - mandible - case 2 complete blood count, serum calcium and alkaline phosphatase levels were done for both patients and were found to be within normal limits . On the basis of history, patient showed bilateral hyperkeratosis of palms, soles, dorsal surface of fingers and knees [figures 1 and 2]. Intraoral examination showed that the patient had lost most of his permanent teeth and the remaining teeth showed grade ii mobility . Hyperkeratosis of palms and soles - case 1 hyperkeratosis of knees - case 1 intra - oral view - maxilla - case 1 intra - oral view - mandible - case 1 orthopantomograph showed moderate horizontal bone loss and erupting permanent tooth buds [figures 79]. Hyperkeratosis of palms and soles - case 2 intra - oral view - maxilla - case 2 intra - oral view - mandible - case 2 complete blood count, serum calcium and alkaline phosphatase levels were done for both patients and were found to be within normal limits . On the basis of history, thorough scaling and root planing of all teeth was carried out along with administration of systemic antibiotics . Papillon - lefevre syndrome is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and aggressive periodontitis . Skin lesions develop concurrently with oral lesions and may extend to dorsal surfaces of hands and feet . Another form of disease associated with palmoplantar keratosis and severe aggressive periodontitis has been named haim - munk syndrome . It differs from papillon - lefevre syndrome in symptoms such as arachnodactyly, acroosterolysis and onychogryphosis . In papillon - lefevre syndrome the permanent dentition starts to erupt at proper time, but around 8 - 9 years of age periodontal destruction is repeated in the same manner as in primary dentition . All permanent teeth are usually lost before 14 - 16 years of age . In subjects with papillon - lefevre syndrome the exact biochemical defect leading to periodontal disease is still unclear . In 1942, wannenmacher suggested that papillon - lefevre syndrome was due to a combination of ecto and mesodermal malformations . Suggested that a functional imbalance of collagenolytic activity in the periodontal ligament was responsible for periodontitis in papillon - lefevre syndrome . In 1984, vandyke suggested that neutrophils from an individual with papillon - lefevre syndrome exhibit decreased receptor affinity for chemotaxins such as formyl peptides . Papillon - lefevre syndrome is associated with myeloperoxide deficiency, low integrin expression, defective phagocytosis and chemotaxis . Hattab et al ., reported four cases of papillon - lefevre syndrome affecting two jordanian families with eight children . In all patients there was a relationship between increased severity of skin lesions and seasonal variations and intensified periodontal destruction . Lass et al . In three multiplex families, one ethiopian and two german, demonstrated linkage of papillon - lefevre syndrome with chromosome 11q13-q14 . Fischer et al ., conducted a primary genome wide search by homozygosity mapping in a large consanguineous family with four affected siblings . Toomes et al ., showed that in patients with papillon - lefevre syndrome gene for cathepsin c which lies in chromosome 11 had undergone mutation resulting in decreased cathepsin c production . Hart et al ., reported mutations in cathepsin c gene in patients with papillon - lefevre syndrome from five different countries . In conclusion, we have reported two cases of papillon - lefevre syndrome . Further studies in the field of microbiology and genetics are necessary to find the exact cause of periodontal destruction in such patients, so that best possible dental treatment can be rendered to them . In subjects with papillon - lefevre syndrome there is defective cathepsin c production . The gene for cathepsin c lies on chromosome 11 . The exact biochemical defect leading to periodontal disease is still unclear . In 1942, wannenmacher suggested that papillon - lefevre syndrome was due to a combination of ecto and mesodermal malformations . Suggested that a functional imbalance of collagenolytic activity in the periodontal ligament was responsible for periodontitis in papillon - lefevre syndrome . In 1984, vandyke suggested that neutrophils from an individual with papillon - lefevre syndrome exhibit decreased receptor affinity for chemotaxins such as formyl peptides . Papillon - lefevre syndrome is associated with myeloperoxide deficiency, low integrin expression, defective phagocytosis and chemotaxis . Hattab et al ., reported four cases of papillon - lefevre syndrome affecting two jordanian families with eight children . In all patients there was a relationship between increased severity of skin lesions and seasonal variations and intensified periodontal destruction . Lass et al . In three multiplex families, one ethiopian and two german, demonstrated linkage of papillon - lefevre syndrome with chromosome 11q13-q14 . Fischer et al ., conducted a primary genome wide search by homozygosity mapping in a large consanguineous family with four affected siblings . . Toomes et al ., showed that in patients with papillon - lefevre syndrome gene for cathepsin c which lies in chromosome 11 had undergone mutation resulting in decreased cathepsin c production . Hart et al ., reported mutations in cathepsin c gene in patients with papillon - lefevre syndrome from five different countries . In conclusion, further studies in the field of microbiology and genetics are necessary to find the exact cause of periodontal destruction in such patients, so that best possible dental treatment can be rendered to them.
Mitochondrial transmembrane potential apoptosis - inducing factor apoptotic protease - activating factor 1 bcl2-associated x protein b - cell cll / lymphoma 2 bh3 interacting domain death agonist dna - dependent protein kinase electron transport chain high temperature requirement protein a2 inhibitor of apoptosis inhibitor of caspase - activated dnase mitochondrial outer membrane permeabilization nicotinamide adenine dinucleotide nadh dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 nadh dehydrogenase (ubiquinone) fe - s protein 1/2 nadh dehydrogenase (ubiquinone) flavoprotein 1 nicotinamide adenine dinucleotide phosphate (nadph)-oxidase nuclear mitotic apparatus protein 1 12, 17 and 20 kda cleavage fragments of the maturation process of caspase 3 poly-(adp - ribose) polymerase reactive oxygen species human cytotoxic lymphocytes (ctls) eradicate pathogen - infected or transformed cells primarily through the cytotoxic granule pathway, which relies on perforin (pfn)- and granulysin - mediated delivery of the granzyme serine proteases into eukaryotic target cells or bacteria, respectively . Among the 5 human granzymes (a, b, h, k, and m) and 10 mouse homologues (a, b, c, g, e, f, g, k, m, and n), granzymes b and a (gb and ga) are the most abundantly expressed, and the best characterized . Like caspases, gb cleaves its substrates after aspartic acid residues to induce cell death . In fact, gb behaves as a hybrid between initiator and effector caspases like initiator caspases, gb activates the effector caspases 3 and 7 and the proapoptotic bh3-only protein bid; however, gb can also directly cleave important effector caspase substrates such as icad, parp-1, lamin b, numa, dna - pkc and tubulin for cell death induction . Mitochondria adjust both their morphology and function, acting as hubs that integrate cues for the appropriate cellular response . In response to activation of death receptors, dna damage, or er stress, activation of bh3-only proteins of the bcl2 family triggers the oligomerization of bax and bak leading to mitochondrial outer membrane permeabilization (momp). This critical step of many cell death pathways results in release of the apoptogenic factors cytochrome c (cyt c), endog, smac / diablo, htra2 and aif from the mitochondria into the cytosol . As a consequence, cytosolic cyt c triggers activation of apaf1 and caspase 9 apoptosome platform, and smac removes the repression of the inhibitor of apoptosis (iap) proteins ., momp was considered sufficient for apoptogenic factor release; however, recent findings suggest that the story is more complex . In fact, reactive oxygen species (ros) are necessary for cyt c release . Cyt c engages in both electrostatic and hydrophobic interactions with inner mitochondrial membrane cardiolipins that are disrupted by ros for optimal release through momp . Gb triggers optimal cyt c, endo g, and smac release in a ros - dependent manner (fig . It is likely that, similar to cyt c, ros - mediated untethering of endo g and smac is required for their release through the bid / bax / bak - mediated momp in the gb pathway . Ros are also necessary for gb - induced dna fragmentation since ros scavengers significantly reduce and delay oligonucleosomal dna fragmentation without affecting icad cleavage and activation of cad, the endonuclease involved in this process (fig . This result could be predicted by the compromised release of endog from mitochondria in the absence of ros . Furthermore, ros were found to be necessary for the nuclear shuttling of endog during cisplatin - induced death of head and neck squamous carcinoma cells, suggesting that ros may also be required for endog and cad nuclear translocation following gb and pfn treatment (fig . An additional contribution of ros in apoptosis progression was revealed by the work of christoph borner's group, who showed that during tnf apoptotic signaling, caspase 9 induced lysosomal membrane permeabilization (lmp) in a ros - dependent manner, leading to cathepsin release and further caspase activation and cell death . Overall, these findings are consistent with the inhibitory effect of ros scavengers on ctl - mediated cell death, implying that ros are active effectors of cell death . Gb can directly cleave parp1, lamin b, tubulin, numa, dna - pk, and icad to induce caspase - independent cell death . Gb also enters the mitochondria by an unknown mechanism and cleaves ndufs1, ndufs2, and ndufv1 from complex i to trigger ros production . Together with ros, momp allows the release of cyt c, endog, smac, htra2, and aif (the latter 2 are not represented here). Both cad and endog shuttle to the nucleus probably in a ros - dependent mechanism to induce oligonucleosomal dna fragmentation . Ros and caspase 9 cooperate to trigger lysosomal membrane permeabilization and cathepsin release, thus amplifying caspase 3 activation . Among other substrates, activated caspase 3 cleaves parp1, lamin b, tubulin, numa, dna - pk, and icad, which together contribute to cell death . Gb can directly cleave parp1, lamin b, tubulin, numa, dna - pk, and icad to induce caspase - independent cell death . Gb also enters the mitochondria by an unknown mechanism and cleaves ndufs1, ndufs2, and ndufv1 from complex i to trigger ros production . Together with ros, momp allows the release of cyt c, endog, smac, htra2, and aif (the latter 2 are not represented here). Both cad and endog shuttle to the nucleus probably in a ros - dependent mechanism to induce oligonucleosomal dna fragmentation . Ros and caspase 9 cooperate to trigger lysosomal membrane permeabilization and cathepsin release, thus amplifying caspase 3 activation . Among other substrates, activated caspase 3 cleaves parp1, lamin b, tubulin, numa, dna - pk, and icad, which together contribute to cell death . The production of ros during cell death has long been recognized; however, a molecular mechanism for their production has only recently begun to be unraveled . Although one report suggested an extra - mitochondrial origin of ros during apoptosis, douglas green's group first showed that staurosporine - mediated ros - dependent apoptosis requires caspase 3 cleavage of ndufs1, the 75-kda subunit of the mitochondrial electron transport chain (etc) complex i. later, judy lieberman's group reported that ga triggered a caspase- and momp - independent cell death that also required the disruption of complex i through the cleavage of ndufs3 to induce ros - dependent apoptosis . Interestingly, gb cleaves ndufs1, ndufs2, and ndufv1 subunits of this same complex to trigger ros - dependent apoptosis (fig . 1). Actually, gb also alters complex ii and complex iii function, disrupts the supercomplex organization of the respiratory chain, and triggers a loss of the mitochondria cristae junctions . In this pathway, electron flow is required for ros production since 0 cells that carry a non - functional respiratory chain are resistant to granzyme - mediated ros production and cell death . It is possible that cleavage of these complex i subunits exposes the electrons within the fe - s cluster to ambient oxygen for ros production . Additionally, the mitochondria targeted antioxidant mitoq completely abrogates gb - induced ros and cell death, further supporting a mitochondrial origin of ros in this context . Integration of these data indicates that 3 cell death pathways caspase 3, ga, and gb converge on complex i for ros production during apoptosis . I by ga and gb following human killer cell attack has been implicated in ros - dependent bacterial death, suggesting that cleavage of complex i subunits is an evolutionarily conserved mechanism of ros - dependent death . Our results indicate that ga and gb, which lack a mitochondrial targeting signal, still find their way into this double - membrane organelle to disrupt the respiratory chain and trigger an increase in ros . We have also found that granzyme mitochondrial entry is momp - independent but requires an intact membrane potential . Do ros act through non - specific systemic redox - modification of macromolecules or through a discrete set of targets? How many ros are needed? Which radical species are most effective for the various contributions of ros to cell death? Putting emphasis on redox signaling during apoptosis will undoubtedly open new windows of investigation and allow the characterization of useful biomarkers of cell death . Jerome thiery (gustave roussy cancer campus, villejuif, france) and dr michael walch (university of fribourg, switzerland) for proofreading this manuscript . This work was supported by ambizione grant snsf pz00p3_126710/1, erc starting grant erc-2010-stg_20091118 and subsides, from the carlos and elsie de reuter foundation and schmidheiny foundation.
Gingival health and appearance are the essential components of a charming smile . Although clinical gingival pigmentation does not indicate a medical problem, black gums may cause complaints regarding esthetic problems, particularly in patients with a high smile line . According to dummett and barens, the distribution of oral pigmentation in dark individuals is as follows: gingiva, 60%; hard palate, 61%; mucous membrane, 22%; and tongue, 15% . Gingival pigmentation occurs as a diffuse, deep - purplish discoloration or as irregularly - shaped brown and light brown patches . Melanin pigmentation can be treated by various methods that include chemical methods using phenol, alcohol, ascorbic acid, and surgical methods of depigmentation such as gingival abrasion technique, split - thickness epithelial excision, combination technique (gingival abrasion and split - thickness epithelial excision), free gingival grafting, and recent methodologies in gingival depigmentation lasers, cryosurgery, and radiosurgery . In recent years, cryosurgery, an effective method of tissue destruction by freezing, is gaining importance as a therapeutic technique . Cryosurgery leads to cell destruction and tissue death attributable to physical and chemical changes induced by freezing . The use of cryogen tetrafluoroethane (tfe) is easy, practical, and inexpensive as compared to lasers and other conventional methods such as scalpel or chemical methods . Kumar et al . In 2013 concluded by a case series that ultra - low temperature of tfe causes controlled cryonecrosis of gingival epithelium and effectively eliminates gingival pigmentation without any significant side effects and esthetically pleasing results . There are a number of case reports and case series conducted to evaluate the efficacy of tfe, but there is limited availability of higher level evidences . Hence, this randomized controlled split - mouth clinical study was carried out with an objective to compare the efficacy of gingival depigmentation by tfe cryosurgery and surgical scalpel technique . A randomized clinical split - mouth study was carried out in the department of periodontics, k m shah dental college and hospital, sumandeep vidyapeeth, after obtaining approval from the institutional ethics committee . A total of 25 patients with age range between 20 and 60 years were included in the study, and the sample size calculation was done by the following formula n = (z/2)./l, with 95% confidence interval and 20% relative precision . The patients who complained of hampered esthetics due to black gums and had healthy periodontium were included in this study . Exclusion criteria for the study were pregnancy, breastfeeding, systemic diseases which were associated or not associated with the gingival melanin pigmentation, malignancy, medications, uncontrolled diabetes, and the adverse reactions to cryosurgery . The relative contraindications such as cold intolerance and cold urticaria were considered thoroughly . A personal history regarding the smoking habit of the patients was recorded and also habits such as chewing tobacco in any form were also considered and patients with those were excluded from the study . Each patient signed a written informed consent prior to his / her participation in the study, and he or she was duly explained the procedure with its merits and demerits . Detailed extraoral / intraoral examinations which included the evaluations of the skin pigmentation to correlate with intraoral pigmentation, the perioral pigmented lesions, and the characteristics of the shape, color, surface, and borders of the gingival melanin pigmentation were recorded . Before undertaking any of the depigmentation procedures, all the patients underwent phase i therapy, following which oral hygiene instructions were given to them . Gingival pigmentation index (gpi) was recorded for all the 25 patients before the treatment and at 3, 9, and 12 months after the treatment . 0 - absence1 - spots of brown to black2 - brown to black patches but not diffuse3 - diffused brown to black pigmentation and attached . 1 - spots of brown to black 2 - brown to black patches but not diffuse 3 - diffused brown to black pigmentation and attached . Then, the patients were randomly allocated into control and test groups using coin toss method . Allotment was done by a co - investigator into test group (cryosurgery) and control group (scalpel technique). The primary investigator was blinded with respect to the site selection for the technique that was employed . Now, in each patient, tfe cryosurgery was carried out in three anterior tooth regions comprising central incisor, lateral incisor, and canine . (a) preoperative, (b) depigmentation by cryosurgery on the right side, (c) depigmentation using scalpel on the left side the test site before the tfe application was isolated and air - dried . Topical anesthesia with 15% tfe was sprayed on a cotton swab, which was immediately rolled gently over the pigmented area to include the papillae as well . A freezing zone was continuously maintained for 3040 s in each area by rolling the dampened swab continuously at the site . The patients were prescribed analgesics if there was any pain . Before scalpel technique, skin preparation was carried out by 15% povidone - iodine . Local infiltration with local anesthetic solution results were evaluated, and gpi was recorded at intervals of 3 and 6 months [figure 2]. The total area of the gingival pigmentation was traced and measured with image - analyzing photomatix software for accurate evaluation of the outcome achieved after the procedure . Postoperative photograph showing follow - up at (a) 30 days, (b) 90 days, and (c) 180 days the total number of participants included in this randomized split - mouth study was 25 . The chief complaint of the participants was black gums and they were concerned about their esthetic smile window, and consent was obtained before starting the procedure from each of them . Out of the 25 participants, 20 were males and 5 were females . The parameter used for evaluating gingival pigmentation was gpi, and the perception of pain was evaluated using vas . The gpi score was 2 at baseline for four control sites and three test sites and was of 3 for 21 control sites and 22 test sites [table 1]. Gingival pigmentation index at baseline similar gpi scores were noted in both the test and control groups . The statistical test used was pearson's chi - square test [table 2]. Gingival pigmentation index at 30 days the gpi scores showed no reoccurrence in both the groups at 90 days using pearson's chi - square test [table 3]. Gingival pigmentation index at 90 days gpi scores at 180 days were recorded, and the results denoted that only 8% of cases showed reoccurrence in tfe group when compared to scalpel group which showed 20% of reported cases with reoccurrence . However, it is interesting to note that, out of the 25 cases treated, 92% of cases in tfe group and 82% in scalpel group did not show any reoccurrence at 180 days (6 months). The statistical test used was pearson's chi - square test with p = 0.417 and this clearly showed that there was no statistical significant difference between the groups [table 4]. Gingival pigmentation index at 180 days meanstandard deviation for 0, 30, 90, and 180 days the mean gpi at baseline, 30, 90, and at 180 days clearly indicates that there is no reoccurrence at 30 and 90 days, and reoccurrence observed at 180 days showed no statistical significance between both the groups [figure 3]. Bar graph showing mean visual analog scale score for both scalpel and tetrafluoroethane groups comparing the perception of pain in between the two groups using the vas, participants preferred the test modality of tfe as compared to scalpel . Majority of the participants gave a vas score of 3 for scalpel whereas a score of 2 for tfe, indicating a preference for the newer treatment modality of using tfe . When mean scores for vas were calculated, the graphical outcome showed the same results [table 6 and figure 4]. Vas score at 180 days suggests minimal pain and discomfort in tfe group (p <0.001). Visual analog scale scores for perception of pain line graph showing mean gingival pigmentation index at baseline, 30 days, 90 days, and 180 days hence, with the results, we can conclude that in the present study the test group (tfe) has shown better results and outcome in all the parameters considered when compared with conventional surgical scalpel technique . We know that physiologic gingival melanin pigmentation is not a medical problem, but patients may complain of unesthetic black gums . People nowadays are getting more aware of the esthetics and that has given rise to the dawn of various treatment modalities for the management of gingival hyperpigmentation . In the present study, we have tried to compare the conventional scalpel surgery technique with the recently introduced cryogen tfe technique . The use of cryosurgery has been incorporated for the treatment of a wide range of lesions in the oral and maxillofacial region . James arnott (1851) was the pioneer to report the therapeutic use of extremely cold solutions . Arnott had noticed that cold temperatures have beneficial analgesic along with anti - inflammatory effects . By 1950s, simple methods, such as direct application to the gums with a cotton wool bud (open), and/or use of expensive probes (closed), have been tried with a fair bit of success in dentistry . With several animal and human studies, the safety and biocompatibility of tfe have been evaluated and concluded to be safe to use as a cryogen . Tfe was recently used in dentistry in the field of periodontics for depigmentation . In the present study, recurrence of pigmentation was observed in five cases in scalpel group and two cases in tfe group; the gpi score recorded was 1 in both the groups . Pigmentation may reappear in some cases as a result of the presence of active melanocytes in the basal cell layer of the epithelium, which may not have been removed completely . In general, the ultra - low temperature (818c for 10 s) formed by the cryosurgery method resulted in complete epithelial destruction . Complete elimination of the gingival epithelium along with the melanocytes could be achieved . However, in some cases, active melanocytes could have survived and hence could have become active again over time . Most of the past studies carried out on depigmentation modalities are case reports and case series with short- and long - term follow - up . The study done by kumar et al . In 2013 is a case series with ten patients, comparing gingival abrasive technique with tfe, which showed spots of melanin pigment in four patients with the use of gingival abrasion, and in tfe group, three cases showed very minimal spots of pigmentation at 30 days . However, at 90 days, seven cases were reported with recurrence in abrasive technique, but in tfe group, the results showed healthy pink gingiva and no more spots of recurrence . Gpi scores at 180 days showed reoccurrence in all the cases treated with gingival abrasion, but only one case showed spots of reoccurrence in cryosurgical group . Follow - up after 2 years in cryosurgical group showed no signs of any repigmentation and only one case was reported with gpi score of 1 . In a recently carried out study, singh et al . (2013) carried out a study comparing tfe and diode laser . The authors used a simple questionnaire to evaluate the perception of pain and found that the cryosurgical approach by tfe was more acceptable, caused less discomfort, and was less painful as compared to diode lasers . Another recent study carried out by kumar et al . In 2013 is a case series with 12 patients where they histologically assessed and correlated the effectiveness of the tfe for gingival depigmentation and they concluded that tfe can effectively destroy the gingival epithelium without any harm to the underlying connective tissue and clinically the results were more satisfactory with the color, healing, and longevity of the results . Thus, it can be concluded from the study that the cryosurgical treatment for depigmentation is more effective, easy to perform, and had better patient compliance when compared to scalpel technique . A randomized controlled trial evaluating the long - term results of cryosurgery, using efficient delivery system for tfe and evaluating histological events in the process of healing after treatment, should be planned to prove the external applicability of results of the present study . The present study evaluates the effectiveness and efficiency of the newly introduced cryogen tfe with conventional scalpel technique which is till now considered to be most widely used and accepted . The results of this study gave us the vision to think beyond these conventional techniques and also newer modalities such as lasers . The cryosurgical group (test) showed more convincing results in the perception of pain and discomfort in tfe group when evaluated with vas . Hence, the advantages of tfe not only stick to its storage, transport, ease of use, less technique sensitive, and cost - effectiveness but also the acceptance and compliance of the patient is greater when compared to scalpel technique . Hence, there is nothing wrong to establish the statement that cryosurgery with tfe has shown better and effective results in every aspect to conventional scalpel technique in the present study . And so, we should be prospectively looking forward to use the novel material (tfe) widely and wisely to treat gingival hyperpigmentation and correction of smile window of the patient.
One hundred twenty - one thoroughbred mares (222 years old) in hokkaido, japan housed under natural conditions were used for measuring the levels of circulating estradiol . For determining the correlation between the results for whole blood and serum, five pregnant mares, 819 years of age, were used . Whole blood samples were collected weekly from the jugular vein into commercially supplied plastic tubes containing heparin sodium as an anticoagulant during day 233 of pregnancy and the day of delivery . For serum sample collection, whole blood was drawn from the jugular vein of the same mares into plain blood collection tubes . The concentrations of estradiol in all samples were measured by pathfast . For the experiments determining the correlation between the results of pathfast and time - resolved fia, two pregnant mares, 4 and 15 years old, were used . Serum samples were collected monthly from the jugular vein into commercially supplied plastic tubes at the day of copulation and the day of delivery . The concentrations of estradiol in all samples were measured by pathfast and fia . For evaluating the pattern of circulating estradiol during gestation in mares, fifty - nine pregnant mares, ranging from 422 years of age, were used . Serum samples were collected during gestation from the jugular vein into commercially supplied plastic tubes . All procedures were carried out in accordance with the guidelines established by the tokyo university of agriculture and technology for the care and use of horses in research . Pathfast: the concentrations of estradiol in whole blood and serum samples were determined using the pathfast analyzer with the pathfast reagent kit for estradiol as described previously . In brief, estradiol measurement by pathfast was performed using a single reagent cartridge containing 100 l of whole blood and serum samples without extraction . In the competitive assays, estradiol in the samples was inhibited from forming an immunocomplex by an alkaline phosphatase - conjugated antigen . Following immunoreaction for 5 min, separation of bound and free hormones was performed using the magtration technology . After addition of the chemiluminescent substrate to the immune complex, the amount of chemiluminescence was measured . The intra -assay coefficients of variance were 7.012.4% for whole blood, and 6.312.9% for serum samples . Fia: serum estradiol concentrations were determined by a time - resolved fluoroimmunoassay using dissociation - enhanced fia systems (perkinelmer, waltham, ma, usa) as described previously [19, 20]. The intra- and inter - assay coefficients of variation were 5.0% and 5.1%, respectively . Pearson s r was calculated to determine the correlation between variables using graphpad prism software version 5 for windows (graph pad software, san diego, ca, usa). One hundred twenty - one thoroughbred mares (222 years old) in hokkaido, japan housed under natural conditions were used for measuring the levels of circulating estradiol . For determining the correlation between the results for whole blood and serum, five pregnant mares, 819 years of age, were used . Whole blood samples were collected weekly from the jugular vein into commercially supplied plastic tubes containing heparin sodium as an anticoagulant during day 233 of pregnancy and the day of delivery . For serum sample collection, whole blood was drawn from the jugular vein of the same mares into plain blood collection tubes . The concentrations of estradiol in all samples were measured by pathfast . For the experiments determining the correlation between the results of pathfast and time - resolved fia, two pregnant mares, 4 and 15 years old, were used . Serum samples were collected monthly from the jugular vein into commercially supplied plastic tubes at the day of copulation and the day of delivery . The concentrations of estradiol in all samples were measured by pathfast and fia . For evaluating the pattern of circulating estradiol during gestation in mares, fifty - nine pregnant mares, ranging from 422 years of age, were used . Serum samples were collected during gestation from the jugular vein into commercially supplied plastic tubes . All procedures were carried out in accordance with the guidelines established by the tokyo university of agriculture and technology for the care and use of horses in research . Pathfast: the concentrations of estradiol in whole blood and serum samples were determined using the pathfast analyzer with the pathfast reagent kit for estradiol as described previously . In brief, estradiol measurement by pathfast was performed using a single reagent cartridge containing 100 l of whole blood and serum samples without extraction . In the competitive assays, estradiol in the samples was inhibited from forming an immunocomplex by an alkaline phosphatase - conjugated antigen . Following immunoreaction for 5 min, separation of bound and free hormones was performed using the magtration technology . After addition of the chemiluminescent substrate to the immune complex, the amount of chemiluminescence was measured . The assay range of pathfast was 202000 pg / ml . The intra -assay coefficients of variance were 7.012.4% for whole blood, and 6.312.9% for serum samples . Fia: serum estradiol concentrations were determined by a time - resolved fluoroimmunoassay using dissociation - enhanced fia systems (perkinelmer, waltham, ma, usa) as described previously [19, 20]. The intra- and inter - assay coefficients of variation were 5.0% and 5.1%, respectively . Pearson s r was calculated to determine the correlation between variables using graphpad prism software version 5 for windows (graph pad software, san diego, ca, usa).
United states healthcare costs continue to escalate with no immediate relief in sight . In 2005, healthcare spending totaled us$2.0 trillion16% of the gross domestic product (gdp) (poisal et al 2007). Further, healthcare spending is projected to account for 18.4% of gdp by 2013, when more than one out of every four dollars of personal consumption will be spent on healthcare (heffler et al 2004). The aging of americans is a central component fueling cost increases . In the year 2000, 12.4% of americans were over age 65, but this will increase to 19.6% in 2030 (goulding et al 2003). Chronic diseases, especially prevalent in the senior population, generate a large proportion of medicare spending . In 19951999 (the most recent data), only 5% of medicare beneficiaries, presumably many with chronic diseases, accounted for almost half (47%) of total spending . A much larger segment of the population (40%), those in relatively good health, accounted for only 1% of the total (liberman et al 2003). Preventing or postponing the onset of chronic disease among seniors has become a crucial public health issue, as has the potential to compress the period of morbidity prior to death (vita et al 1998). It has been estimated that approximately 70% of the total burden of disease (as measured in terms of premature deaths and potential years of life lost) can be traced back to illnesses that are preventable (department of health and human services 1991). Particularly noteworthy is research by mcginnis and foege (1993) and mokdad and colleagues (2004) who concluded that about half of all deaths in the us are caused by modifiable risk factors . Although death is inevitable, it is now becoming clear that successful health promotion programs can improve health, prevent much disability, delay mortality, and thereby substantially improve the quality of seniors lives (aldana 2001; chernoff 2001). The growing awareness of the value of prevention and health promotion is evident at the executive branch of government . The president launched the healthierus initiative, promoting healthier lifestyles and a reduction of risk factors such as inactivity, obesity, and smoking (healthierus 2003). The rationale for this initiative, especially in relation to health promotion efforts directed at seniors, is described on the white house website: poor health should not be a foregone consequence of aging . Improvements in diet and physical activity can greatly improve the quality of life at any age . Regular physical activity also helps older americans maintain joint strength and mobility and substantially delays the onset of functional limitations and loss of independence . Improvements in diet and physical activity can greatly improve the quality of life at any age . Regular physical activity also helps older americans maintain joint strength and mobility and substantially delays the onset of functional limitations and loss of independence . The rapidly rising cost of healthcare, an aging population, and the high prevalence of chronic disease among the elderly generate a sense of urgency for finding innovative solutions to this country s healthcare crisis, including health promotion and disease prevention initiatives . In this paper, we highlight several key studies that underscore the value of introducing health promotion and risk reduction programs directed at seniors . Furthermore, we present evidence suggesting that health promotion and risk reduction programs may reduce unnecessary healthcare utilization and related expenditures for the medicare program, yielding a favorable return - on - investment (roi) for such interventions . We finish our discussion with a description of an innovative demonstration project that centers for medicare and medicaid services (cms) will be introducing in 2007 to test whether health promotion programs can improve the health of seniors and also achieve a positive cost - benefit ratio . A growing literature presents convincing evidence that seniors who reduce their modifiable health risks can forestall disability, reduce their utilization of health services, and ultimately save medicare money (hickey et al 1997; king et al 1998; vita et al 1998; fries 2002; omenn 2003). Lubitz and colleagues (2003) presented evidence that elderly persons in better health at age 70 live about 2.7 years longer than those in poor health, and their cumulative medicare healthcare expenditures are no greater than those who die earlier; in fact, they may be slightly lower . The macarthur study on aging, consisting of dozens of studies conducted over a decade, helped researchers identify components of successful aging (seeman et al 2001). For example, quitting smoking and initiating other lifestyle changes even later in life have produced substantial health benefits (hickey et al 1997; hermanson et al 1988). Importantly, several studies confirm the value of physical activity for improving health among the elderly (king 2001). Physical activity can extend life, help prevent heart disease and colon cancer, (christmas and andersen 2001; penedo et al 2004) mitigate the effects of chronic diseases such as arthritis or diabetes (christmas and andersen 2001; bean et al 2004), improve coordination and flexibility to help avoid falls (christmas and andersen 2001; bean et al 2004), and alleviate depression among older adults (christmas and andersen 2001). Adults who practice even simple physical activity can improve their health status and use fewer health and social services (aldana 2001). To emphasize the importance of physical activity, a comprehensive review by himes (2002) identified these two overriding determinants of health status for seniors: social isolation as a strongly negative influence and regular physical activity as a strongly positive factor . In the private sector, evidence suggests that multicomponent health promotion and risk reduction programs can permanently change lifestyle habits and reduce population health risks for nonseniors (heaney and goetzel 1997). A report by rand (2001) concluded that successful risk reduction programs directed at working and senior populations share certain characteristics that make them effective in changing life - long health habits . Successful programs are anchored in behavior change theory, employ tailored and personalized interventions, are sufficiently intensive, and are complemented by adequate social supports . Such programs appear to work effectively even if they are not delivered face - to - face, but instead provided by telephone, internet, and mail . In addition, health promotion programs that engage participants in self - care activities and increase their involvement in healthcare decision - making can achieve long - term behavior change and risk reductions (heaney and goetzel 1997). Although much of this private sector research has been conducted with those under age 65, the underlying principles and methods employed by these programs possess sufficient flexibility to be tailored to the unique needs of diverse populations, suggesting that risk reduction programs targeting seniors have a high potential to achieve lasting behavior changes and potentially lower utilization of healthcare services (rand 2001). Other research in the private sector is pointing in the direction that evidence - based health promotion and risk reduction programs can save money . Several literature reviews that weighed the evidence from experimental and quasi - experimental research studies suggest that programs grounded in behavior change theory and that utilize tailored communications and individualized counseling for high - risk individuals produce a positive roi (goetzel et al 1999; aldana 2001; us department of health and human services 2003). Much of the roi research has emerged from employer - sponsored health promotion programs directed at active employees (goetzel et al 1999; pelletier 2001). In a review of 32 health promotion program evaluations, aldana (2001) found 28 studies that reported medical cost savings . Of the seven studies that calculated cost - benefit ratios, financial returns averaged $3.48 for every dollar expended . Studies often cited with the strongest research designs and large numbers of subjects included those performed at johnson and johnson (bly et al 1986; breslow et al 1994), citibank (ozminkowski et al 1999), dupont (bertera 1990), the bank of america (leigh et al 1992; fries et al 1993), tenneco (baun et al 1986), duke university (knight et al 1994), the california public employee retirees system (calpers) (fries et al 1994), procter and gamble (goetzel, jacobsen, et al 1998), and chevron corporation (goetzel, dunn, et al 1998). Even accounting for certain inconsistencies in design and results, most produced positive cost outcomes . The most recent review on this topic conducted by chapman (2003), summarizing the results from 42 qualifying financial impact studies conducted over the past two decades, concluded that worksite programs achieve a 25%30% reduction in medical and absenteeism costs in an average period of about 3.6 years (goetzel, jacobsen, et al 1998). Similarly, the rand report (2001) concluded that health promotion and risk reduction programs using health risk assessments (hras) and ongoing tailored interventions have the potential to be cost - beneficial . Five financial analyses are especially pertinent to cost - benefit calculations for medicare enrollees engaged in health promotion programs: four (fries et al 1993; fries et al 1994; fries and mcshane 1998; ozminkowski et al 2006) focused on risk reduction programs for seniors (at the bank of america, calpers, and at general motors corporation), and one for working - age individuals at citibank (ozminkowski et al 1999). Employing available medical claims data to estimate changes in medical expenditures, that roi estimate was similar to one obtained in a randomized trial using the same intervention program in a different population (fries et al 1993). Using methods that imputed changes in medical expenditures from changes in utilization, the evaluations of bank of america and calpers programs for seniors reported rois of approximately $5.00 to $1.00 . The general motors study was not an roi study, because it contained no program cost information . However, it did demonstrate that health promotion programs offered to seniors, particularly those that are based upon administering a health risk appraisal, can save $101 to $648 per person per year (at the us dollar value for 2006), depending upon who participates and how many programs they use . Admittedly, the methodological rigor of evaluations performed at many corporate health promotion programs has not been consistent . In fact, randomized trials are hard to find in the literature, largely because they are not well accepted in a business environment . However, methodological shortcomings in earlier analyses have diminished significantly over the past two decades . The most recent evaluations using sophisticated econometric methods that control for selection bias do assess impact over several years (with some extending for three to five years and one, performed at johnson & johnson, lasting nine years) (ozminkowski et al 2002). These advances should inform future study designs of similar health promotion interventions directed at seniors . In short, a focus on prevention and health promotion offers a most promising approach to the urgent challenges that the medicare program faces today and into the future . Research in the private sector presents compelling evidence to warrant large scale federally funded demonstrations that test the prospect for well - designed health promotion and risk reduction efforts to pay for themselves through lower healthcare expenditures . Although some corporate studies have recently begun to focus on productivity impacts from these programs (eg, reduced absenteeism, shortened disability periods, decreased presenteeism), much of the private sector research has emphasized healthcare cost savings . This is the same focus of policy makers and legislators with oversight responsibility for medicare . There are still several unanswered questions related to the application of health promotion and risk reduction programs in an elderly population . For example, will living a healthy life truly reduce seniors illnesses and incidence of disability prior to their demise or will they simply live longer and their illnesses and disability be delayed? Thus, will a longer life span cost medicare more because seniors are alive longer and may still experience significant end - of - life illness and costs? These are testable hypotheses . While successful health promotion interventions could extend the period of healthy life and result in costs being incurred to the medicare program over a longer period of time, the alternatives are not appealing . Researchers at rand recently estimated the costs of the future obese elderly, and found that medicare will spend 35% more caring for an obese 70-year - old person over his or her lifetime than for a normal weight person, at a cost of $36,000 (lakdawalla et al 2005). To explore these very complex issues, in 2007, medicare will initiate a three and a half year research project entitled senior risk reduction demonstration (srrd), to test the health and economic impacts of providing health promotion services, modeled after programs provided to those under age 65 . The srrd will provide insights on how to deliver such programs practically, an important step in advancing the medicare program from one focused on the diseases of older people to one focused on improving their health and quality of life . Cms will offer risk reduction services to noninstitutionalized medicare beneficiaries between the ages of 67 and 74, to be delivered by private sector vendors . Five vendors will be given contact information for a random sample of beneficiaries from across the us, as well as from communities that have exemplary information and referral / assistance (i&r / a) programs for seniors.voluntary participation . Incentives will be offered to induce high participation and retention rates, but participation will be at the discretion of the beneficiary.focus on self - care . The srrd will emphasize health improvement and risk reduction to complement the clinical aspects of medical services that are offered by other healthcare professionals . The program will contain elements of chronic disease self - management that take the form of patient education, advice, and counseling but will be primarily targeted at seniors who are well and do not need disease management services.tailoring to beneficiaries . Vendor programs will be tailored to the needs, concerns, and learning styles of seniors . The goal is to develop personalized materials and instruments, followed by interventions tailored to the risks presented by the participants.central coordination . Programs will deliver interventions via the mail, internet, and/or telephone counseling and coaching.referral to local community resources . Vendors will be required to refer beneficiaries to national or local community resources . For beneficiaries residing in preselected exemplary i&r / a communities, vendors will be required to make referrals to the local i&r / a organizations.multiple behavior change modules . Programs will address several modifiable health risk categories simultaneously in the assessment, triage, and follow - up phases of the intervention . Risk factors addressed in the srrd include the following: 1) physical inactivity / lack of exercise; 2) poor nutrition; 3) smoking / tobacco use; 4) excessive alcohol consumption; 5) high blood pressure; 6) high blood glucose; 7) high total cholesterol; 8) being overweight / obese; 9) inappropriate use of clinical preventive services; 10) depression; 11) high stress; 12) lack of general well - being; 13) burden of providing care giving; 14) social isolation; 15) lack of motor vehicle / home safety; 16) falls (preventable accidents); and 17) polypharmacy / medication issues . Target population . Cms will offer risk reduction services to noninstitutionalized medicare beneficiaries between the ages of 67 and 74, to be delivered by private sector vendors . Five vendors will be given contact information for a random sample of beneficiaries from across the us, as well as from communities that have exemplary information and referral / assistance (i&r / a) programs for seniors . Incentives will be offered to induce high participation and retention rates, but participation will be at the discretion of the beneficiary . Focus on self - care . The srrd will emphasize health improvement and risk reduction to complement the clinical aspects of medical services that are offered by other healthcare professionals . The program will contain elements of chronic disease self - management that take the form of patient education, advice, and counseling but will be primarily targeted at seniors who are well and do not need disease management services . Tailoring to beneficiaries . Vendor programs will be tailored to the needs, concerns, and learning styles of seniors . The goal is to develop personalized materials and instruments, followed by interventions tailored to the risks presented by the participants . Programs will deliver interventions via the mail, internet, and/or telephone counseling and coaching . Vendors will be required to refer beneficiaries to national or local community resources . For beneficiaries residing in preselected exemplary i&r / a communities, vendors will be required to make referrals to the local i&r / a organizations . Programs will address several modifiable health risk categories simultaneously in the assessment, triage, and follow - up phases of the intervention . Risk factors addressed in the srrd include the following: 1) physical inactivity / lack of exercise; 2) poor nutrition; 3) smoking / tobacco use; 4) excessive alcohol consumption; 5) high blood pressure; 6) high blood glucose; 7) high total cholesterol; 8) being overweight / obese; 9) inappropriate use of clinical preventive services; 10) depression; 11) high stress; 12) lack of general well - being; 13) burden of providing care giving; 14) social isolation; 15) lack of motor vehicle / home safety; 16) falls (preventable accidents); and 17) polypharmacy / medication issues . The srrd will be judged on its ability to achieve high enrollment, participation, and retention rates; improve beneficiaries health, health risks, and functionality; and achieve a positive roi for the medicare program . Readers interested in learning more about the srrd are invited to visit cms website (cms 2006). This administration s interest in prevention, combined with the impending insolvency of the medicare trust fund, make it timely for cms to explore and test evidence - based approaches to risk reduction and health promotion that may not only help beneficiaries to take better care of themselves, but that are likely to generate a positive roi . The srrd will provide insights on how to deliver such programs practically, an important step in advancing the medicare program from one focused on the diseases of older people to one focused on improving their health and quality of life . In short, a focus on prevention and health promotion offers a most promising approach to the urgent challenges that the medicare program faces today and into the future.
In recent years, it has been revealed that astrocytes perform a significantly wider range of functions than previously appreciated . Interest in astrocyte function has increased dramatically because of their newly discovered roles in synapse formation, maturation, efficacy, and plasticity . Today, astrocytes are recognized as multifunctional cells with well - defined essential neuron supporting functions . Mounting evidence suggests that these versatile cells participate in a multitude of diverse processes in the central nervous system (cns). These roles include regulating blood flow, providing much needed energy to neurons, and supplying the building blocks of neurotransmitters that fuel synapse activity . The addition of their role in synaptic function to the known repertoire of astrocyte activities over the past decade has enhanced our conception of their seminal importance in normal functioning of the adult brain . More comprehensive reviews highlighting astrocyte function include jacobs et al ., wang and bordey, and kimelberg . In the developing nervous system, the assembly of synaptic circuits is a complex and dynamic process, requiring the coordinated exchange of signals between pre- and postsynaptic neurons and neighbouring glia . The formation, maintenance, and modulation of synaptic connections are required for normal cns function and ongoing plasticity . In the diseased nervous system, however, the structural and functional integrity of synaptic connections is often modified or lost, resulting in profound cognitive and behavioral deficits . Yet until recently, no exact roles had been identified for astrocytes in the pathogenesis of specific cns diseases . While some aspects of the mechanisms underlying the formation, maintenance, and plasticity of cns synapses in the developing and diseased nervous system have been elucidated, many more remain enigmatic . As our knowledge about astrocytic function in normal physiology has expanded, exploration into their likely role in disease pathology has followed . In the case of fragile x syndrome (fxs), a compelling case can be made for the abnormal dysfunction of astrocytes . Fxs is the most common form of inherited mental impairment, and it typically results from the transcriptional silencing of the fmr1 (fragile x mental retardation 1) gene and loss of the encoded protein, fmrp (fragile x mental retardation protein). Fxs symptoms include neurodevelopmental delay, anxiety, hyperactivity, and autistic - like behavior . Fmrp was once thought to be expressed solely in neurons; however, it was later shown to have specific roles in astrocytes . Pacey and doering found that fmrp is expressed in early development in cells of the glial lineage both in vitro and in vivo . Although few studies focus specifically on the role of astrocytes, recent work provides important examples of how a better understanding of astrocyte biology during development can enhance our knowledge about human disease . In this paper, we discuss the landmark findings and recent advances in our understanding of astrocytes and their featured roles in regulating synapse formation, maturation, and synaptic transmission . Further, we assess how astrocytes contribute to the extensive plasticity that occurs during development, highlighting the dynamic morphology of astrocyte processes and their involvement in synaptic development . Lastly, we explore the means by which perturbations in astrocyte function may contribute to neurological diseases, such as fxs, in the context of synaptic defects . We propose here that, by investigating the precise roles of astrocytes during neurological disease, we are likely to achieve a broader understanding of how the brain works, in addition to new insights into disease prognosis, diagnosis, and treatment . They were so named due to their stars in the night sky appearance obtained from golgi stained samples . In the late nineteenth century and the early twentieth century, camillo golgi and santiago ramn y cajal noticed that, although different astrocytes share a stellate feature, their morphology is extremely diverse, perhaps as diverse as neurons . Since cajal's time, modern scientists have confirmed the morphological diversity of astrocytes in vitro and in vivo [10, 11]. Astrocytes are divided into two main classes distinguished on the basis of their morphology and primary location [12, 13]. Their processes, which are long, thick, and highly ramified, are closely associated with synapses as well as blood vessels . In the hippocampus, protoplasmic astrocytes ensheath more than half of the synapses, most of which are excitatory . The other subtype is composed of fibrous astrocytes found mainly in the white matter of the brain, where their processes pass between nerve fibers . In contrast to protoplasmic astrocytes, fibrous astrocyte processes are long, cylindrical, smooth, and branch infrequently . From the cell soma radiate primary branches that gradually divide into finer and finer processes to generate a dense network of delicate terminal processes, which associate very closely with synapses . A number of immunological markers have been used over the years to characterize astrocyte morphology . Until recently, our understanding has been predominantly based on classical immunostaining with the widely used astrocyte marker gfap (glial fibrillary acidic protein, an intermediate filament protein), which grossly underestimates the complexity of astrocytes and their interactions with neurons and other cells . Gfap only reveals the structure of primary branches, which represent a meager ~15% of the total volume of the astrocyte . Other markers include aldh1ll (aldehyde dehydrogenase 1 family, member l1), glt-1 (glial glutamate transporter 1), and glast (glutamate - aspartate transporter). To date, no marker has been identified that is expressed exclusively in mature astrocytes . Moreover, no pan - astrocytic marker has been identified with which to determine the fraction of astrocytes that are gfap+, although recent studies on aldh1l1 seem promising . Recent physiological and gene expression profiling studies indicate that astrocytes, like neurons, are a diverse cell population with distinct properties in different brain regions and at different periods of development . They function as neural stem cells and guide axon projections; they promote synapse formation and maintain neuronal survival [20, 21]. Subsets of astrocytes, or astrocyte - like cells, in the adult subventricular zone (svz) and in the subgranular zone (sgz) of the dentate gyrus of the hippocampus act as neural stem cells, whereas most astrocytes in other parts of the adult brain do not normally proliferate . Heterogeneity of astrocytes, however, is not exclusive across brain regions, as it can also exist within the same areas of the brain . The number and size of astrocytes in the brain also vary between species relative to species brain size and cognitive ability . For example, the human brain contains several more populations of astrocytes than the rodent brain, and human astrocytes are threefold larger than their rodent counterparts . Therefore, these classifications may not be adequate to appreciate the full extent of astrocyte diversity . Astrocytes have unique cytoarchitectural and phenotypic features that ideally position them to sense their surroundings and respond in dynamic ways to changes in their microenvironment . Astrocytes are, therefore, well suited to share synaptic function with neurons as they extend numerous processes, forming highly organized anatomical domains with little overlap between adjacent cells . The territory of a single astrocyte is estimated to contact between 300 to 600 dendrites and upwards of 10 synapses [16, 26]. This extensive synaptic interaction not only ensures that astrocytes are able to fulfill their metabolic support roles but also positions astrocytes to directly influence the structure and function of the synapse . While some astrocyte processes (which express a wide range of receptors and ion channels) closely ensheath synapses, others are in close contact with intraparenchymal blood vessels via specialized processes called endfeet . In line with this, the formation of synaptic contacts is paramount for the proper development and function of the cns . Although most neurons are produced during embryonic stages, the major waves of synaptogenesis follow and depend on astrocyte production . Given their proximity to synapses, astrocytes can directly promote and regulate these processes through both secreted and contact - mediated signals . The traditional assumption that neurons are intrinsically able to form synapses led early studies on synaptic development to focus on neuronal signals and surface molecules . Remarkably, neurons cultured with media conditioned by astrocytes control the number and effectiveness of synapses [2830], indicating that soluble factors secreted from astrocytes play an important role in synapse formation . These include matricellular proteins, such as thrombospondins (tsps-14), sparc, sparc - like 1 (hevin), and tenascin c, which are all expressed by astrocytes in the cns of rodents . A possible role for glial involvement in cns synaptogenesis was first elucidated by a series of studies on rat retinal ganglion cells (rgcs). Cholesterol complexed to apolipoprotein e (apoe) released by astrocytes increases the number of glutamatergic synapses in rgc cultures . When cholesterol is applied directly to cultured rgcs, the frequency of spontaneous synaptic events increases . The researchers further demonstrated that cholesterol acts to increase the quantal content of synaptic vesicles and the overall efficacy of vesicle release . This is in concert with other findings that cholesterol is an essential component of synaptic vesicle production whose presence serves as a limiting factor in vesicle formation . The rgc culture technique has also been used to identify other key synaptogenic - secreted factors including thrombospondins 1 and 2 (tsp-1 and tsp-2), members of oligomeric extracellular proteins . Christopherson and colleagues identified tsps as the signals coming from astrocytes that can induce an increase in synapse number . When directly applied to rgc cultures, immunodepletion of tsps from astrocyte - conditioned medium (acm) decreased its synaptogenic effect down to control levels indicating that tsps are the key synaptogenic component of acm . The expression of tsp-1/2, which is elevated in the developing brain when the majority of synapses are formed (during postnatal week 1), ceases in the mature adult brain (by postnatal week 3). This suggests that astrocytes downregulate pathways that strongly promote synapse formation when the synaptogenic period of neurons is reduced, and other tsp genes may be functioning to stabilize synaptic structures . Besides tsps 1 and 2, other tsps (tsps-35) are detected in mammals . In contrast to other tsp - s, tsp-4 expression is only detected in mature astrocytes after p17 . This suggests that tsp-4 could represent the adult isoform of tsp in the cns and is important for the control of synaptogenesis and enhanced plasticity in the adult brain . Recently, gabapentin receptor 2-1 has been identified as the tsp receptor responsible for mediating excitatory cns synaptogenesis . Despite the critical role of tsps in promoting synaptogenesis, additional signals are likely required for synapse maturation, as tsp - induced synapses are ultrastructurally normal, but postsynaptically silent, underscoring the complexity of astrocyte contribution to synapse formation . A more recent study has identified two closely homologous glypicans, glypican-4 and glypican-6, as astrocyte - secreted proteins that are sufficient to increase ampa glutamate receptor levels on synapses, thus inducing postsynaptic function . Additionally, tenascin - c (tn - c), another extracellular matrix glycoprotein, seems to play a role in synaptogenesis and synaptic function [38, 39]. Tn - c is highly expressed by astrocytes during early stages of development, while its expression ceases in the adult cns, with the exception of specific cell populations, particularly those in close proximity to areas of active neurogenesis, such as the hippocampus, subventricular zone borders, and the rostral migratory stream . Following stimulation of synaptic activity, tn - c was found upregulated in the hippocampus within a few hours . In tn - c knockout mice, stimulation of schaffer collaterals resulted in a reduction of long - term potentiation (ltp) at ca1 synapses, whereas ca1 long - term depression (ltd) was completely abolished [42, 43]. These expression patterns reveal important roles for tn - c in the remodeling of the cns, both during development and in adulthood . In fact, recent studies reveal astrocyte contributions to inhibitory synapse formation and function in cultured hippocampal neurons . While astrocyte - expressed extracellular matrix protein hevin has been found to induce the formation of synapses in cultured rgcs [44, 45], its homolog, sparc, which is also secreted by astrocytes, antagonizes the synaptogenic function of hevin, thereby acting as a negative regulator of synapse formation . Sparc expression is typically high in early development, where it then becomes downregulated in certain parts of the brain by the time of synaptogenesis . Alternatively, hevin expression increases with development in agreement with synapse formation and is also present in adulthood, most likely functioning in the maintenance of existing synapses . Unlike tsp-1 and tsp-2, the expression of which is decreased during maturation, hevin and sparc mrna levels remain high even in the adult . Taken together, the secretion of both positive and negative regulators of synapse formation allows astrocytes to regulate the timing and location of synapse formation with greater precision . Moreover, a recent study has provided evidence that astrocytes play a role in the elimination of redundant synapses during development . In the developing postnatal brain and retina, immature astrocytes seem to be a source of a signal that triggers the expression of complement component c1q in developing neurons . C1q's best - known role in the innate immune system is to opsonize or c1q localizes to synapses that are thus tagged for elimination through the activation of the complement cascade and deposition of c3b, an opsonin derived from the proteolytic activation of the complement component c3 . Mice deficient in c1q or the downstream complement cascade protein c3 exhibit large sustained defects in cns synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons . Also, c1q - deficient mice show enhanced neocortical excitatory synaptic connectivity and epileptiform activity . Together, these findings implicate a role for astrocytes during the critical period when neural circuits are formed . While astrocyte - secreted factors induce the formation and function of synapses, other evidence proposes further regulatory roles for astrocytes through contact - mediated mechanisms . An elegant study by hama et al . Local contact with astrocytes via integrin receptors facilitated excitatory synaptogenesis through the activation of protein kinase c (pkc) in individual dissociated hippocampal neurons . The researchers observed that pkc activation, while initially focal, subsequently spread throughout the entire neuron . Thus, propagation of pkc signaling could signify an underlying mechanism for global neuronal maturation following local astrocyte adhesion . Astrocyte processes, which are highly mobile, contribute to the stabilization of new synapses during synaptogenesis . Astrocytes may induce local structural and functional modifications of dendritic segments or individual synapses through a contact - mediated mechanism involving bidirectional ephrin / epha signaling [4951]. Membrane - bound ligands on astrocytes, such as ephrin - a3, have been shown to upregulate spine morphology in the hippocampus, suggesting local activation of epha receptors on spines by astrocytic ephrin - a3 . Dendritic spines are small protrusions visible on dendrites of neurons that serve as postsynaptic sites for excitatory input [5254]. Live imaging of organotypical hippocampal slice preparations showed that astrocytes rapidly extend and retract fine processes to engage and disengage from postsynaptic dendritic spines . Studies with two - photon microscopy that tracks the dynamics of astrocyte processes and the fate of dendritic protrusions also revealed contributions of astrocyte contact . Dendritic protrusions with astrocyte contacts had a longer lifetime and were morphologically more mature . Thus, dendritic protrusive activity and transient contacts with astrocytes act to stabilize newborn synapses and promote subsequent spine maturation . Spine dynamics are largely controlled through changes in cytoskeletal proteins . Expressing a dominant negative mutant rac1, a gtpase that mediates actin motility, reduces astrocyte process motility and provides evidence that cytoskeletal rearrangements underlie motility, similar to mechanisms of spine extension and retraction [56, 57]. Liu et al . Showed that local contact between neurons and astrocytes significantly increased the amplitude and density of gabaa currents in developing hippocampal neurons . This contact - dependent increase in gabaergic synaptic activity relied on ca signaling in astrocytes . In addition, astrocytes were shown to regulate cl gradient in cultured spinal cord neurons and convert gabaergic synapses from excitatory to inhibitory . This finding is particularly exciting given the importance of local gabaergic inhibitory circuits in both activity - dependent wiring of developing neural circuits and the consolidation of critical period plasticity [60, 61]. Overall, these studies reveal that contact - mediated signaling between astrocytes and neurons is important for the structure and maintenance of synaptic connections and suggests a model in which physical and molecular interactions between neurons and astrocytes provide instructive cues that control synapse formation, morphology, and plasticity . As our understanding of the extent of their influence at the synapse unfolds, it is much more apparent that astrocytes are well poised to modulate multiple aspects of synaptic plasticity than was previously imagined . A turning point in our understanding of astrocytes was elicited by the recognition of their active communicative properties [6264]. Astrocytes, which are bidirectional, can communicate and exchange information with both pre- and postsynaptic elements . Communication is primarily controlled by the change in ca concentrations, causing excitability within the astrocyte [6466]. Astrocytes use their ability to respond to neurotransmitters and secrete neuromodulators to actively regulate a number of processes involving synaptic plasticity [6769]. In addition to secreting factors that influence and modulate synapse formation, astrocytes are known to release factors that can directly affect synaptic transmission . Briefly, of the gliotransmitters released by astrocytes, the most well characterized and extensively reviewed are glutamate [71, 72], adenosine triphosphate (atp), and d - serine [74, 75]. Glutamate serves as the principal excitatory neurotransmitter in most regions of the cns, and its release from astrocytes has been shown to modulate synaptic transmission . Glutamate released from neurons activates metabotropic glutamate receptors on astrocytes, leading to an increase in astrocyte ca concentrations and a subsequent astrocytic release of glutamate . D - serine, perhaps the most interesting, is an important neurotransmitter that serves as a coagonist with glutamate, promoting nmda (n - methyl - d - aspartate) receptor activity at synapses in the hypothalamus . Moreover, astrocytes release atp to communicate with each other and other glia by activating purine receptors localized on neighbouring cells . These findings have led to the establishment of a new concept in synaptic physiology, the tripartite synapse, in which astrocytes exchange information with neuronal synaptic elements [6, 67, 77]. Consequently, astrocytes are an integral part of the synapse, being involved not only in passive homeostatic control of adequate conditions for synaptic function, but also actively in synaptic function . With an evident role of astrocytes in normal neural function at all cellular and molecular levels, it is not surprising that astrocytes contribute in some capacity to almost all pathological conditions of the nervous system [7984]. For most disorders, it remains unclear whether astroglial changes are causative of the disease or if they merely represent an accompanying phenomenon . Accordingly, astrocyte - dysregulated function has been fundamentally linked with the progressive pathology of ischemic stroke, epilepsy, and to a number of neurodegenerative disorders including, but not limited to, amyotrophic lateral sclerosis, huntington's disease, and parkinson's disease . Further involvement of astrocytes has also been implicated in the development of neurodevelopmental disorders such as rett syndrome (rtt), down syndrome (ds), fragile x (fxs), and autism . Among these conditions, fxs has emerged as the prototypical disorder in which to study how altered signaling may lead to synaptic defects and dysfunctional neural circuitry underlying pathology . Both dysregulated astrocyte signaling and abnormal synaptic function stand as prominent contributing factors to the learning disability phenotype expressed in fxs . Fragile x syndrome (fxs) is the most common form of inherited intellectual disability . It affects approximately 1 in 4,000 males and 1 in 6,000 females and is characterized by cognitive impairments, attention deficits, and autistic - like behaviors . Fxs is caused by an expanded cgg trinucleotide repeat in the 5 untranslated region of the fmr1 gene leading to gene silencing and the consequent loss of fmrp expression [87, 88]. To understand the etiology of the synaptic phenotypes that accompany fxs, it is first important to discuss the purported function of fmrp . Fmrp acts as a regulator for the transport and local translation of specific synaptic mrnas in response to neural stimulation . Fmrp is found in growth cones, immature axons, and mature dendrites, as well as dendritic spines . The loss of fmrp results in the aberrant expression of its mrna targets, which in turn leads to functional deficits that characterize fxs . The reason that fmrp has been implicated in synaptic plasticity is on the basis of dendritic spine abnormalities and exaggerated long - term depression (ltd) displayed by fmr1 mutant mice . This finding led to the mglur theory of fmrp, whereby synaptic signaling of metabotropic glutamate receptor 5 (mglur5) leads to the localized translation of fmr1 mrna . As such, the newly synthesized fmrp acts as a translational repressor of specific target mrnas, resulting in the downregulation of mglur5 activity through a negative feedback loop (figure 1). Several exceptional reviews on the genetic and clinical features of fxs or molecular functions of fmrp include bear, huber et al . Current knowledge surrounding the pathophysiology of fxs has been greatly advanced by the development of animal models . These transgenic animals do not carry the trinucleotide expansion but do have functional deletions of fmrp . The first model developed was the fmr1 knockout (ko) mouse, which recapitulates behavioural and cognitive deficits reminiscent of the human condition . Drosophila and zebrafish models also exist and have contributed to our understanding of the conserved roles of fmrp in neural development [9799]. Although they are not perfect models of the human disease, they have helped to reveal the cellular and molecular mechanisms underlying fxs, and they have immensely enhanced glial - neuronal research . During development, selective elimination or pruning of inappropriate synaptic connections occurs for the proper formation and establishment of neural circuitry . Current models regarding the neurobiological changes that underlie fragile x have largely focused around the synapse . This is based in part on the structural synaptic changes and alterations in synaptic function, which are observed in human patients and fxs animal models . Spines develop around the time of synaptogenesis and are dynamic structures that continue to undergo remodeling over time . Developmental changes in the shape of dendritic protrusions reflect the progressive replacement of thin, elongated, and highly motile filopodia, characteristic of immature neurons, with more stable spines that acquire a mature morphology . Spine morphogenesis is fundamental to the development of neuronal networks and the regulation of synaptic plasticity . Some of the first neuroanatomical findings associated with mental impairment were alterations in dendritic spine structure . The first such evidence of altered synapse structure in fxs came from analysis of postmortem cortical tissue, which exhibited an increased number of dendritic spines relative to control individuals . This data revealed that excitatory synapse number was increased in fxs patients and further provided a potential mechanism for the increased rates of epilepsy in fxs . It was additionally noted that a large proportion of the spines of fxs patients appeared abnormally long, thin, and tortuous, a phenotype reminiscent of the immature spine precursors (filopodia), and indicative of alterations in synapse development and/or function . At this point, it was not clear if the excess filopodia - like spines in fxs represented functional synapses or immature synapse precursors . Much of the evidence for a role for fmrp in synaptic and neurite pruning is derived from the drosophila melanogaster model of fxs (dfxr). During development, fmrp has been shown to control the pruning of immature dendrites in developing neurons . In support of a pruning function for dfxr, most neurons of dfxr null flies exhibit an overgrowth and elaboration of axons and dendrites into the peripheral and cns [98, 103106]. Parallel to human studies, work with the fmr1 ko mouse has largely confirmed the spine phenotype observed in fxs patients . Numerous studies agree that fmr1 mutant brains display an increase in long, thin, immature dendritic spines [102, 107] mirroring human neuroanatomical abnormalities . It is important to note that many of these defects in spines and in synaptic / circuit plasticity occur during critical periods of development in the first postnatal weeks, coinciding with the maximal expression of fmrp . However, the existence and/or magnitude of the spine alterations in the fmr1 ko mouse varies according to brain region, developmental age, and genetic background, indicating the complex and multifactorial regulation of spines . In a study by cruz - martin et al ., spines of l2/3 layered pyramidal neurons were imaged at various developmental stages, and it was revealed that fmr1 ko mice demonstrated a delay in the stabilization of dendritic spines, due to high turnover during the second postnatal week . This happens to correspond to the time when fmrp protein expression is highest in the cortex . In the absence of fmrp, hippocampal neurons have fewer spines that colocalize with synaptic markers, which suggests a loss of functional spines this provides compelling evidence that fxs might be caused by a failure in the transition from filopodia (earliest dendritic protrusions) to mature spines, consequently resulting in an increase of immature synapses . The failure of spines to stabilize during the critical period in the barrel cortex strongly suggests that fmr1 ko mice could have problems in maintaining the proper balance between stable and dynamic connections that is necessary to establish mature synapses . Since dendritic spines are the primary sites of excitatory synapses and information exchange in the cns, perturbations in their structure and function can result in synaptic and circuit alterations leading to disrupted brain function and pathology . While it has been recognized that astrocytes play multiple critical roles in the regulation of normal cns function, the possibility that astrocyte - specific dysfunction might cause diseases that manifest as pathologies of neurons is a relatively recent idea . Previously, it was thought that fmrp expression in the brain was exclusively confined to neurons . Our laboratory initially identified fmrp in the astrocyte lineage in the fxs mouse . When studying stem and progenitor cells from the brains of wild - type (wt) and knockout (ko) fxs mice, approximately half of the cells in culture coexpressed fmrp and gfap . Parallel immunocytochemical studies in vivo also showed the coexpression of fmrp and gfap in the embryonic and adult developing hippocampus . With the identification of fmrp in astrocytes and knowledge of their role in synaptogenesis, our laboratory was prompted with further experiments to explore neuronal development and synapse formation in fxs . Utilizing a coculture design adapted by jacobs and doering, hippocampal neurons (e17) and cortical astrocytes (p0 - 1) were independently isolated to explore four different combinations of neuronal - astrocyte cultures (wt neurons + wt astrocytes, wt neurons + fmr1 ko astrocytes, fmr1 ko neurons + wt astrocytes, fmr1 ko neurons + fmr1 ko astrocytes). The cells were grown for 7, 14, or 21 days in vitro and then processed for immunocytochemistry to analyze morphological and synaptic profiles . These experiments are novel and exciting as they are the first to establish a potential role for astrocytes in the altered neurobiology of fxs . The first group of experiments focused on neurons in each of the four combinations to elucidate the effects of fmrp on dendritic morphology and excitatory synapse expression . The neurons were studied with antibodies directed against the neuronal (dendritic) marker, map-2, the presynaptic protein synaptophysin, and excitatory postsynaptic protein, psd-95, respectively . Through sholl analyses, morphological assessments were performed on neurons under parameters of dendritic branching and the area of the cell body . Synaptic protein distribution was determined by the quantification of synaptic puncta (spots of intense staining). Wt neurons grown on fmr1 ko astrocytes exhibited significantly altered dendritic arbor morphologies, with a shift toward a more compact and highly branched dendritic tree . Specifically, wt neurons grown on fmr1 ko astrocytes resulted in a decrease in the length of the longest primary dendrite and area covered by dendritic arbor, and an overall increase in branch number and density in comparison to their wt counterparts . These neurons also displayed a significant reduction in the number of pre- and postsynaptic protein aggregates . However, when the fmr1 ko neurons were cultured with wt astrocytes, the alterations in dendritic morphology and synaptic protein expression were remarkably prevented . In fact, their morphological characteristics and synaptic protein expression approached the appearance of normal neurons grown with wt astrocytes . These experiments were the first to suggest that astrocytes contribute to the abnormal dendritic morphology and the dysregulated synapse development seen in fxs . In the next phase of this research, we wanted to determine if these altered characteristics represented a developmental delay imparted by the fmr1 ko astrocytes . Focusing on wt neurons grown in the presence of wt or fmr1 ko astrocytes, we evaluated the dendritic arbor morphology and synaptic protein expression at 7, 14, and 21 days in culture . Our results revealed that wt neurons grown with fmr1 ko astrocytes displayed significantly altered morphological and synaptic protein profiles at 7 days (when compared to the wt condition). Strikingly, by 21 days in culture, these differences were no longer significantly different from normal . In light of these findings, it appears that astrocytes in the fxs mouse may contribute to the altered characteristics of neurons seen in fxs in a developmentally regulated manner . Thus, these results suggest that timing is crucial in brain development . Despite these outcomes, it is noteworthy that conclusions about synapse maturity cannot be drawn . It is possible that the increase in synapses observed in the neurons grown on fmr1 ko astrocytes reflects an increased number of immature synapses . Given that the dendritic spine is the site for the majority of excitatory synapses, this finding would be in agreement with numerous studies that identified neurons in fxs with an abnormally high number of immature dendritic spines . As a note, the methods used in the current study did not permit the assessment of alterations in dendritic spine morphology . Understanding the role of astrocytes in human neurological diseases requires a comprehensive picture of how astrocytes develop and what roles they play in development . Given these findings, it is highly plausible that fxs astrocytes lack functional fmrp, specifically at a time during development when astrocyte support of neuron growth and synapse formation is vital, and that this lack of fmrp could contribute to the abnormal neuron phenotype seen in fxs . However, it is uncertain whether the alterations in astrocytes are due to a lack of fmrp or if they are abnormal because they develop and function in a diseased microenvironment . Also, if the absence of fmrp in astrocytes is the primary source of dysfunction, how are these effects translated to neurons? For instance, is astrocyte - neuron signaling disrupted due to a lack of astrocyte fmrp? How, where, and when do these signals act? Is the abnormal astrocyte - neuron communication mitigated by a membrane associated or a soluble factor? Could it be a combination of both direct and indirect contact? These questions, among many others, about the fxs astrocyte are now important targets for fxs research . The answers will allow us to gain a full understanding of the underlying neurobiology that contributes to the morphological phenotype seen in fxs and in the potential of a future treatment for individuals with fxs . Recent evidence indicates that the interface between astrocytes and neurons is necessary for normal synapse development, including synaptic pruning . Dendritic spines, which are highly dynamic during development, become more stable in the adult brain; thus, a correlation exists between age - dependent spine dynamics and the plasticity of the brain . This decrease in spine motility in the mature brain could be attributed to the close association of astrocytes with synapses, with astrocytes providing both physical constraints that inhibit spine movement as well as molecular interactions that stabilize spines . Importantly, epha4r (expressed on dendritic spines) interacts downstream with members of the rho / ras pathways, suggesting that ephar / ephrin - a interactions may underlie aspects of actin - driven astrocyte motility observed during synapse formation [27, 115]. Therefore, this raises the possibility that in vivo defects in dendritic spine development are at least partly related to neuron - glia interactions during development . Astrocyte involvement has also been fundamentally implicated in neurodevelopmental disorders such as rtt and ds . A common finding in many of these studies is that astrocyte dysfunction has profound non - cell - autonomous effects on surrounding neurons . Rtt, which is an x - linked neurodevelopmental disorder, is caused by the loss of the transcriptional repressor methyl - cpg - binding protein (mecp2). A study by ballas et al . Showed that wild - type hippocampal neurons cocultured with cortical astrocytes or conditioned medium from mecp2-deficient mice had abnormally stunted dendrites, suggesting that mecp2-deficient astrocytes may dominantly affect normal neuronal development . Furthermore, in ds patients, cognitive deficits have been associated with structural changes in the architecture and alterations in dendritic spine number . Garcia et al . Found that ds astrocytes are directly involved in the development of spine malformations and reduced synaptic density . These researchers also indicated that the astrocyte - secreted protein tsp-1 possesses a potent modulatory effect on spine number and morphology . Taken together, these studies serve to identify astrocyte dysfunction as a significant factor of spine and synaptic pathology . Future experiments will focus on the assessment of dendritic spines in fxs and the role of direct / indirect neuronal - astrocyte cell contact in the altered developmental sequences that we observed in our tissue culture paradigm . It is highly conceivable that the absence of astrocyte fmrp would directly affect spine morphology or dynamics via dysregulated protein synthesis, and a defect in the maturation of dendritic spines could explain deficits in the intellectual ability seen in individuals with fxs . Armed with novel experimental techniques, powerful imaging tools, and a better understanding of astroglia, neuroscientists are uncovering a new view of the synapse . Neuroscientists are now in a better position to explore and uncover the long - standing mysteries of astrocytes and gain new insights into the cellular and molecular underpinning of the nervous system . The recent findings discussed in this paper place astrocytes in an important position to actively exchange signals with neurons and other glial cells to coordinate synaptic networks . While studies help to distinguish the effects of astrocyte contact from secreted factors on neuronal form / function, it is unlikely that they are separate in vivo . Also, given the central role of the synapse in neuronal communication and plasticity, it comes as no surprise that dysregulation of the synapse accounts for many, if not most, of pathological and developmental disorders in the brain . Thus, the involvement of astrocytes and how they interface with neuronal circuitry should be taken into consideration when interpreting future studies in the pathophysiology of fxs and/or other related neurological diseases . A unifying theme from these recent findings is that astrocytes can promote the development and plasticity of synaptic circuits . Much of the current literature surrounding fxs focuses on synaptic control of protein synthesis because it appears to be proximal to the biology of fmrp and the pathogenesis of the disease in multiple animal models . In addition to targeting synaptic protein synthesis, other therapeutic approaches show promise, for example, in changing the balance of excitation to inhibition by enhancing gaba signaling . Whether different approaches will converge on the same pathophysiological processes or whether they will target distinct aspects of the disease remains to be determined . As we continue to expand our understanding, insights into how these mechanisms may be perturbed in fxs and other diseases states may pave the way for promising future therapeutic interventions and treatments . Potential modes of pharmacological therapy should indeed concentrate on the astrocyte as a gatekeeper of neuronal health and function.
The evaluation of vibration sensation informs the clinician about the integrity of mechanoreceptors in the skin, rapidly conducting large - diameter afferent fibers in the peripheral nerves and the dorsal column - medial lemniscus pathways in the central nervous system [1, 2]. Therefore, diminished vibration sensation is an important finding in the diagnosis of disorders affecting the dorsal column - medial lemniscal system and may also be an early sign of peripheral neuropathies . It is known in everyday practice that patients, due to the physical properties of this sensory modality, can perceive vibration through layers of clothes . This recently resulted in a debate during a case presentation in our clinic in which one of the authors tried to perform the examination on a clothed subject for the sake of speed . The present study was designed under the inspiration of this dispute with the hope of providing some new insights into the characteristics of vibration sensation . Fifty healthy volunteers (h group; median age was 37 years, 22 male) and 19 patients with diabetic polyneuropathy (pnp group; median age was 57 years, 5 male) were included in the study (table 1). The h group mainly consisted of volunteer relatives of patients, healthcare professionals, and medical students stratified into 4 age groups, as follows: ages 1825 (n = 14), 2640 (n = 13), 4155 (n = 13), and 55 + (n = 10). Their mean height was 168.5 9.1 cm (range 153 to 183) and their mean weight was 72.2 14.6 kg (range 49 to 110). A brief neurological examination including superficial sensation, muscle strength, and tendon reflexes was made and found to be normal in all the subjects in this group . Those who had any neurological and systemic disease or who were using medications which could affect the peripheral or central nervous systems were excluded from the study . The pnp group consisted of patients with known type 2 diabetes mellitus and the clinical diagnosis of diabetic polyneuropathy visiting the diabetic outpatient clinic for their regular controls (median follow - up duration was 14 years; range 126). Vibration sensation was measured with the common psychophysical technique, whereby the duration of vibration sensation is dependent on each subject's judgment . The subjects lay supine in a comfortable quiet room with an ambient temperature of 2025c during the measurements . A 128 hz standard tuning fork was used to assess the vibration sensation throughout the study (riester tuning fork w / clamps, aluminum, no . 5162, c 128 hz, california, usa). The socks used in this study all had the same thickness and tissue characteristics (the same model from a certain manufacturer; cotton, 70 denier linear mass density) which had been purchased in bulk and were disposed of after a single use . Four sites were selected for measurements: dorsum of the interphalangeal joint of the great toe (gt) and the vertex of the medial malleolus (mm) on both sides . The tuning fork was applied vertically to these points with gentle pressure after the prongs were maximally activated (by tapping the prongs against the examiner's hypothenar eminence) for each measurement (figure 1). Two investigators took the measurements; the first one applied the tuning fork while the second investigator measured with a stopwatch the duration of vibration sensation from the activation time of the tuning fork until the subject's verbal indication that the sensation was over . Those who were found to have no vibration sensation at all on any of the measurement sites (usually at gt) were excluded from the study at this point . In the measurement phase, 2 measurements without and 2 measurements with socks were performed on each site in the following sequence, allowing a five - minute break inserted so as not to exhaust the subjects and to help them maintain their concentration: without, with, interval, with, without . Sixteen measurements in total were taken for each subject, requiring about 20 minutes for the entire procedure . The averages of each pair of measurements with and without socks were used as the statistical input . The procedure was approved by the istanbul faculty of medicine ethics committee (2012/1244) and all participants provided informed consent . All the statistical analyses were performed using the statistical package for the social sciences (spss), version 17.0, for windows (spss, chicago, il, usa).samples were tested for their distribution with kolmogorov - smirnov test.correlations between the measurements in the same conditions (first and second measurements on the same side without socks and with socks) and between the measurements on both sides were tested with pearson's r.after showing a high correlation between the items in, the results of the two measurements for each condition and the results elicited on the right and left sides were averaged to constitute the main inputs for the statistical analyses . The difference between the mean durations of vibration sensation without and with socks in both groups was analyzed using paired samples t-test.statistically significant results elicited in were analyzed for the effect size, using cohen's d.in the h group, correlations of vibration sensation with age, height, and weight were assessed with pearson's r.independent samples t - tests were performed to evaluate the difference between the vibration sensations measured at the gt and mm.differences between the results elicited in the pnp group and those found in an age - matched subgroup of h (13 females and 9 males, aged 40 years (median 53, min 40, max 68)) were analyzed with independent samples t - tests . Correlations between the measurements in the same conditions (first and second measurements on the same side without socks and with socks) and between the measurements on both sides were tested with pearson's r. after showing a high correlation between the items in, the results of the two measurements for each condition and the results elicited on the right and left sides were averaged to constitute the main inputs for the statistical analyses . The difference between the mean durations of vibration sensation without and with socks in both groups was analyzed using paired samples t - test . Statistically significant results elicited in were analyzed for the effect size, using cohen's d. in the h group, correlations of vibration sensation with age, height, and weight were assessed with pearson's r. independent samples t - tests were performed to evaluate the difference between the vibration sensations measured at the gt and mm . Differences between the results elicited in the pnp group and those found in an age - matched subgroup of h (13 females and 9 males, aged 40 years (median 53, min 40, max 68)) were analyzed with independent samples t - tests . The mean durations of vibration sensation and the differences between the results elicited without and with socks are given in table 1 . The effect of wearing socks was found to be insignificant except that found on the mm in the h group, which was seen to have a small effect size upon further analysis with cohen's d <0.2 (d = 0.17). Vibration sensation was found to decline with age in the h group for two measurement points and both conditions . Pearson's r in measurements without socks were 0.502 (p <0.001) and 0.432 (p = 0.001), and with socks were 0.467 (p = 0.002) and 0.416 (p = 0.003), respectively (figure 2). The duration of vibration sensation was shorter at the mm as compared to gt in the h group (the mean difference was 4.3 s, p <0.001). A smaller difference in the same direction was also observed in the pnp group (1.5 s mean difference, p = 0.012) (figure 2). The vibration sensation of only one of the 50 participants in the h group, a 68-year - old male, measured less at the gt than at the mm, whereas 3 patients in the pnp group were found to have shorter vibration durations at the gt . As compared to the age - matched subjects from the group h, the duration of vibration sensation in the pnp group had a significantly shorter vibration duration at the gt but not at the mm measurement points (p <0.01 and p = 0.12, resp . ). The present study reveals that the mean durations of vibration sensation measured without and with socks are nearly the same; there is practically no difference between them in normal subjects and patients with diabetic polyneuropathy . Although a statistically significant reduction with socks was found in the measurements at the mm in group h, its effect size was small and the mean difference was less than one second . Since clinicians generally express the duration of vibration sensation by whole seconds, without using fractions, the size of the difference found at the mm does not seem important enough to affect the results of clinical examination . As far as we know, this is the first study on humans to investigate the perception of vibration over clothing . Other examples of clinical studies which investigated the effect of clothing during physical examination do exist, including the auscultation of lung sounds through thin clothing and measurement of blood pressure through sleeves using the auscultatory method . These studies all resemble each other in that the focus of interest is sounds and vibrations and the spectrums in question are also similar . Kraman showed that lung sound perceived by stethoscope was somewhat deteriorated by one or two layers of indoor clothing but this effect can be reduced by force on the stethoscope head . Liebl et al . Did not found statistically significant difference between sleeved and nonsleeved arm during manual auscultatory sphygmomanometric and automatic oscillometric blood pressure measurement . An everyday experience was also found to be congruent with these results, that of a vibrating cell phone . It was observed that one is easily able to perceive the vibrations of a cell phone in her / his pocket through a significant amount of clothing . With this observation in mind, we measured the frequencies of incoming call vibrations of several models of cell phones and found that the range was 116 to 250 hz . It is not surprising that this range includes the optimum frequency range for pacinian corpuscles, namely, between 60 and 400 hz . All these data suggest that the interference from garments on vibration energy is little . With regard to the vibration of a cell phone in vibration sensing, the use of a pager device in its vibration mode was reported to be used for clinical vibration testing in an era when cellular phones were not so common . The tuning fork is the most readily available instrument in daily clinical practice for measuring vibration sensation and is sufficiently reliable when compared to other techniques of measurement [7, 8]. Several studies report the use of the rydel - seiffer graduated 64 hz tuning fork because of its semiquantitative properties [79]. Although one was available at the installation phase of the present study, it was discarded because the semiquantitative scale of this device seemed to provide insufficient time resolution for our requirements . The other reason for discarding this tuning fork was that its 64 hz was thought to be in the preferential frequency range of meissner's corpuscles rather than the pacinian afferents . The physiology of pacinian corpuscles may play a critical role in the sensation of vibration through clothing, due to their subcutaneous localization and wide field of perception [1, 10]. The shorter vibration durations at the mm than at the gt are an unexpected finding for clinicians familiar with axonal polyneuropathies, due to the distal to proximal progression of degeneration . It has been concluded that substantial tissue damping affects the tuning fork while taking measurements at the mm . This is probably caused by the solid mass of the tibia which is substantially larger than that of the toe bones beneath the gt measurement points . We think that the shorter vibration durations at the mm can be attributed to this phenomenon, which has been analyzed in detail in the article by goldberg and lindblom . Mean vibration duration at the gt was higher than that at the mm even in the pnp group, although the difference was less . This may be due to the fact that this study involved only mild to moderate cases of diabetic pnp and excluded the more advanced cases in whom vibration sensation was measured as totally absent distally . It might be expected that, with more advanced disease, the gt measurement would have been able to exceed the mm in shortening of vibration sensation duration due to the distal axonopathy process . In conclusion, this study shows that no practically significant difference arises from wearing socks when measuring vibration sensation with a tuning fork; any interference thus caused is negligibly small . This is true for the healthy population over a wide age range and also for patients within the clinical context of a diabetic polyneuropathy . However, this study does not aim to recommend measuring vibration sensation over socks . In the patients with chronic polyneuropathy, careful examination of the feet is crucial for the screening of ulcers and infections and leaving the socks in place during examination is strongly discouraged . Besides, the socks would still have to be removed in order to perform the other modalities of neurological examination . Bearing these in mind, we hope that the results of the present study will be considered as a clinical clue indicating one of the remarkable properties of vibration sensation.
Chronic arsenicosis is a major health and occupational problem in rural parts of west bengal such as in the parts of the gangetic plain of india . Chronic arsenicosis occurs due to accidental ingestion of repeated amounts of small doses by those working with metal or by taking food or drink in which there are traces of arsenic . Sources of arsenic are ash, soil, water (20 - 100 m depth of subsoil water), some sea fish, and prawn . Chronic arsenic exposure may affect almost all systems of the body; but, major external cuteneous manifestations include palmar - plantar hyperkeratosis and rain drop pigmentation with arsenical punctate keratosis . Papillon - lefvre syndrome is a rare disease characterized by skin lesions caused by palmar - plantar hyperkeratosis and severe periodontal destruction involving both the primary and permanent dentitions . The sharply demarcated, erythematous keratotic plaques involve the entire surface of the palms and soles, sometimes extending onto the dorsal surface of the hands and feet . There may be associated hyperhidrosis of the palms and soles, which may cause a foul smell . The second major feature of papillon - lefvre syndrome, severe periodontitis, starts at the age of 3 or 4 years . The development and eruption of the deciduous teeth proceeds normally, but their eruption is associated with severe gingival inflammation in the absence of any local etiologic factor . Until date, palmoplantar hyperkeratosis is a major manifestation in both chronic arsenicosis and papillon - lefvre syndrome . We report herein a rare case of chronic arsenicosis in a patient from rural bengal, whose all features mimic papillon - lefvre syndrome . It is probably the first case from india having papillon - lefevre syndrome - like presentation in chronic arsenicosis . A 20-year - old unmarried, young girl from the remote village of south bengal, who had slightly poor physical but normal mental development, presented with occasional high rise of temperature, frequent dental caries, and fall - out of teeth from time to time since the age of 34 years along with persistent thickening, flaking, and scaling of the skin of her palms and soles for past 14 years . She also complained of poor formation of saliva (xerostomia), poor formation of tear (xerophthalmia), recurrent conjunctivitis, alopecia, frequent pharyngitis, otitis, rhinitis, and pyogenic skin lesions since childhood . The past dental history revealed that her deciduous teeth had erupted normally, but exfoliated gradually by the age of 46 years . Similarly, her permanent teeth too were lost prematurely after having erupted normally . At the age of 6 years, her parents had noticed the presence of rough skin on the plantar surface of her feet, with subsequent involvement of the palmar surface of the hands by the age of 910 years . She also informed that many of her neighbors of her locality had similar palmar - plantar hyperkeratosis without any other complaints she presented, particularly dental problems . On examination, there were symmetrical, well - demarcated, keratotic, and confluent plaques affecting the skin of the palms and soles [figures 1 and 2]. She also had chronic eczematous dermatitis in elbows and knees, few areas of hypo / hyper - pigmentation of anterior and posterior part of the chest . Her hair was sparse, fine, and lusterless, brittle along with alopecia; she also had decreased body hair and sparse or absent eyebrows and eyelashes . On intraoral examination, it was found that patients central incisors, mandibular lateral incisors, maxillary first premolars, and all permanent first molars were missing [figure 3]. All permanent teeth that were present exhibited marked mobility and were malformed, rudimentary, and conical or pegged teeth . Serological tests like anti - nuclear antibody, anti - cytoplasmic neutrophilic antibody, hiv - i and ii, and vdrl were negative . Orthopantomonograph (opg) of the patient showed severe alveolar bone loss in relation to the existing permanent teeth up to the level of apical third of roots, giving the teeth a floating in air appearance . At this point, we made a clinical provisional diagnosis of papillon - lefevre syndrome, with a possible differential diagnosis of chronic arsenicosis . All the clinical features were in favor of papillon - lefevre syndrome, except for those that were similar palmo - plantar hyperkeratotic lesions of her parents and of few neighbors . Histopathology of skin lesions was not done as her parents declined a biopsy . To rule out chronic arsenicosis, we decided to send her hair and nail for estimation of arsenic level by neutron activation analysis at our own costs as she was from a poor family . She and her parents and affected neighbors were advised to change the source of drinking water . She was initiated with a standard dose of penicillamine as a chelating agent and was referred to a dermatologist for treatment of palmo - planter hyperkeratosis . Symmetrical, well - demarcated, keratotic, and confl uent plaques affecting the skin of the palms symmetrical, well - demarcated, keratotic, and confluent plaques affecting the skin of the soles intraoral examination of patient showing missing central incisors, mandibular lateral incisors, maxillary first premolars, and all permanent first molars papillon - lefvre syndrome, first described by two french physicians papillon and lefvere in 1924, is an extremely rare genodermatosis inherited as an autosomal recessive trait, affecting children between the ages 14 years . The disorder is characterized by diffuse palmo - plantar hyperkeratosis and premature loss of both deciduous and permanent teeth . The palmo - plantar hyperkeratosis typically has its onset between the age of 1 and 4 years . The sharply demarcated hyperkeratotic plaques may occur focally, but usually involve the entire surface of the palms and soles, sometimes extending onto the dorsal surfaces of the hands and feet . Often, there is associated hyperhidrosis of the palms and soles resulting in a foul - smelling odor . The second major manifestation of papillon - lefvre syndrome is severe periodontitis, which starts at the age of 3 or 4 years . The development and eruption of the deciduous teeth proceed normally, but their eruption is associated with gingival inflammation and subsequent rapid destruction of the periodontium . However, with the eruption of the permanent dentition, the process of gingivitis and periodontitis is usually repeated and there is subsequent premature exfoliation of the permanent teeth, although the third molars are sometimes spared . Nail changes are apparent in advanced cases, as in this case, and manifested by transverse grooving and fissuring . In addition to the skin and oral findings, patients may have immune suppression and an increased susceptibility to bacteria, associated with recurrent pyogenic infections of the skin . Papillon - lefvre syndrome is caused by loss - of - function mutations affecting both the alleles of the cathepsin - c gene, located on chromosome 11q14.1-q14.3 . The skin manifestations of papillon - lefvre syndrome are treated with emollients, with salicylic acid and urea added to enhance the effect . Oral retinoids including acitretin, etretinate, and isotretinoin are the mainstay of treatment of both the keratoderma and the periodontitis associated with papillon - lefvre syndrome . Treatment is more beneficial if it is started during the eruption and maintained during the development of permanent teeth . Frequent periodontal cleaning, oral hygiene instruction, and antibiotic therapy can, at best, only delay the shedding of teeth . Chronic arsenicosis is a major health and occupational problem in parts of rural west bengal, as in the parts of the gangetic plain of india . High levels of arsenic in the subsoil water has been reported from all over the world, and globally many areas with as high a content have been identified by the who . These include areas surrounded by the great ganga basin in india, where 25% of the wells have been found to such a high content, as reported by who . Its compounds include arsenic trioxide (as2o3), arsenic pentaoxide (as2o5), arsine (ash3), copper arsenite (cuas), arsenic trisulfide, and arsenic bisulfide . Chronic arsenicosis occurs due to accidental ingestion of repeated amounts of small doses by those working with metal or by taking food or drink in which there are traces of arsenic . Sources of arsenic are ash, soil, water 20 - 100 m depth of subsoil water, some sea fish, and prawn . Arsenic is mainly absorbed from the gastrointestinal (gi) tract, but gaseous arsenic is absorbed by the lung . Arsenic concentrates mainly in the liver, kidney, skin, lung, bone, and muscles . Non - specific symptoms like easy fatigability, malaise, lack of interest and concentration, pain in joint, constipation and loose motion, loss of weight, vomiting, and sensory and motor polyneuritis are some systemic manifestation . Chronic arsenic exposure may affect almost all systems of the body, but major external cuteneous manifestations include palmar - plantar hyperkeratosis and rain drop pigmentation with arsenical punctate keratosis . Chronic exposure may result in its accumulation in the hair, nail, and skin . Lung carcinoma, lung fibrosis, bowens disease of sole, and squamous cell carcinoma of skin have also been reported in some cases . Blood levels are not a good indicator of exposure level due to its short half - life . Chronic arsenicosis is diagnosed by neutron activation analysis of hair or nail sample, where more than 75 g% in hair and/or more than 100 g% in nail confirm diagnosis . The source of poisoning should be identified and further exposure should be avoided . According to who guidelines, levels of arsenic in drinking water should not exceed 10 g / l . In areas where the drinking water supply contains unsafe levels of arsenic, the immediate concern is finding a safe source of drinking water . There are three main options for this: (1) finding a new source, (2) increasing the depth of tubewell to more than 100 m, and (3) removing arsenic from the contaminated source . In our present case, all the clinical features of the patient were in favor of papillon - lefevre syndrome, except for those similar to palmo - plantar hyperkeratotic lesions of her parents and of few neighbors who were in favor of chronic arsenicosis . However, in our case, genetic testing could not be performed to identify the gene mutation of cathepsin - c because of the low economic status of the parents, but the dermatological, periodontal, and radiological features strongly suggested the diagnosis of papillon - lefevre syndrome . Therefore, we concluded from this rare case that (1) this may be a rare papillon - lefevre syndrome - like presentation of chronic arsenicosis or (2) chronic arsenic exposure (transplacental and her life - time exposure) may cause mutation of cathepsin - c gene expressed as papillon - lefevre syndrome.
Alzheimer's disease (ad) is a prominent neurodegenerative disorder characterized by chronically progressive global cognitive decline . Mild cognitive impairment (mci) has been recognized as intermediate between normal elderly cognition and dementia . A proportion of individuals with mci may progress to ad and have typical biological changes of this kind of dementia . To date, the underlying mechanisms involved in the degeneration of cerebral neurons and synapses in ad remain an enigma, but the theory about the involvement of inflammatory processes and immune dysregulation in their pathogenesis has been widely demonstrated . Neuropathological and neuroradiological studies have demonstrated that inflammatory changes in ad brains are a relatively early pathogenic event that precedes the process of neuropil destruction [3, 4]. In addition, studies have demonstrated that the brains and cfs of ad and mci contained various proinflammatory substances, such as cytokines, acute phase proteins, and complement proteins [57], which have a fundamental role in inducing cognitive decline, memory loss, and dementia . It has been proposed that the autoimmune mechanism may be a trigger for ad, which was confirmed by the presence of autoantibodies and the apparently good outcome after immunotherapy as seen both in the animal model and in a few patients tested . Circulating autoantibodies against a are several times higher in individuals with ad, and antibody titers correlate with cognitive dysfunction . Active immunization and the use of monoclonal antibodies for passive vaccination have provided encouraging data in transgenic mouse models of ad . Osteopontin (opn), also called early t cell - activation gene 1, is a negatively charged acidic hydrophilic protein that is produced by various cell types and participates in diverse physiological and pathological processes, including bone mineralization, oxidative stress, remyelination, wound healing, inflammation, and immunity [1113]. The expression of opn was elevated in the brains of rats with experimental autoimmune encephalomyelitis (eae) but not in brains of rats protected from eae, and severity of eae was significantly reduced in opn deficient mice . In concordance with those findings in animal models, opn transcripts were frequently detected and were exclusive to the multiple sclerosis mrna population, but not found in control brain mrna . In addition, the expression levels of opn in plasma and tissues are also elevated in other several inflammatory or autoimmune disorders, such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and lymphoproliferation disease [1619]. It has been well studied that interactions between opn and its receptors (including v3, 51 and cd44) mediated survival, migration, and adhesion in many types of cells [20, 21]. As a proinflammatory mediator, opn plays a role in the progression of chronic inflammatory and autoimmune diseases through various mechanisms, including involving in generation of th1 and th17 cells that are pathogenic t cells for various inflammatory diseases [2224], inhibiting apoptosis of autoreactive immune cells and recruitment of leukocytes to sites of inflammation [21, 25]. Recently, the important role of opn in ad has been investigated both in humans and animals model . In ad brains, there was a significant 41% increase in the expression of opn in pyramidal neurons compared with age - matched control brain, and there was a significant positive correlation between opn staining intensity and amyloid - beta load . By means of proteomic analysis of csf samples, simonsen and colleagues identified a phosphorylated c - terminal fragment of opn that was increased in patients with mci progressing to ad as compared to patients who remain stable over time and healthy controls . Demonstrated that opn levels are increased in the csf of ad subjects as compared to controls and its levels are more markedly raised in the early stages of the disease and correlate with cognitive decline . In addition, upregulated opn expression has also been demonstrated in app / ps1 ki mice, an animal model of ad with severe pathological alterations . Collectively, these findings strongly suggest the involvement of opn in the development of ad . To further clarify the role of opn in the progression of cognitive decline, we longitudinally assessed the opn expression changes in the plasma and csf in the same individuals . On the other hand, we transversely analyzed opn levels in patients with mci, newly diagnosed ad, and chronical ad, comparing the results with those obtained in the groups of healthy control and other noninflammatory neurologic diseases (ond). Thirty - five patients affected by ad and thirty - one patients with a diagnosis of mci were selected for the study . Table 1 lists the demographic data of the subjects, including gender, age, and the mini - mental state examination score (mmse), which is a general measure of cognitive performance . The clinical diagnosis of ad was performed according to nincdsadrda work group criteria and dsm iv - r [32, 33]. The mean age of ad patients (16 males and 19 females) was 78.2 years (age range 5880 years). All patients underwent complete medical and neurological evaluation, laboratory analysis, ct scan, or mri to exclude reversible causes of dementia . Standard laboratory tests performed at the time of diagnosis included complete blood count, serum electrolytes, serum glucose, blood urea nitrogen, b12, folate, thyroid function tests, and serology for syphilis . Neuropsychological evaluation and psychometric assessment was performed with a neuropsychological battery including mmse, digit span forward and backward, logical memory and paired associated words tests, token test, supra span corsi block tapping test, verbal fluency tasks, raven colored matrices, the rey complex figure, clinical dementia rating scale (cdr), and the hachinski ischemic scale . Thirty - two patients were late and three early ad were onsets; all cases were sporadic . Ad patients were divided into two subgroups according to disease duration: 17 newly diagnosed ad patients (adn, disease duration 2 years) and 18 chronical ad patients (adc, disease duration> 2 years). The diagnosis of mci was based on the following unanimously adopted criteria: (1) reported cognitive decline; (2) impaired cognitive function; (3) essentially normal functional activities; and (4) exclusion of dementia [35, 36]. The mean age of mci patients (17 males and 14 females) was 74.2 years (age range 5782). All of these patients received neurological examination, laboratory test, and brain mri to exclude intracranial mass, infarcts, moderate to severe nonspecific white matter disease, and reversible causes of cognitive impairment . All patients had a follow - up visit every 6 months, and the monitoring period was 3 years . Based upon subsequent diagnosis status at follow - up evaluations, mci participants can be divided into two subgroups: 13 mci patients who have converted to ad (mci converters, mcic) and 18 mci patients who have not converted to ad (mci nonconverters, mcinc). Twenty patients with ond (11 females and 9 males, age 5979 years, mean age 76.4 13.1 years) were also enrolled in the study . These patients with the following conditions: 2 strokes, 5 transient ischemic attacks, 4 chronic intractable headache, 3 status epilepticus, 3 normal pressure hydrocephalus, and 3 peripheral neuropathies . Plasma samples were also obtained from 24 healthy elderly subjects (hc), age and sex matched with the patient (11 males and 10 females, age 5781 years, mean age 74.7 14.5 years). These individuals were either unrelated healthy spouses of ad and mci patients or healthy volunteers, and they had no family history of dementia or evidence of acute or chronic diseases at the time of enrollment . The cognitive status of ond and hc was assessed by administration of mmse (score for inclusion as normal control subjects 28). All formal neurocognitive test scores for these participants were within 1.5 standard deviations of normative data in published studies or manuals . Patients with an inflammatory or infectious disease, with a history of immunological or malignant disease, medication of immunologically relevant drugs, abnormal white blood cell count, and abnormal csf findings, were not included into the study . All study procedures were approved by the harbin medical university, china, institutional review board, and all participants or their representatives gave informed consent . All of blood and csf samples were obtained at the initial visit . In those mci converters, the second blood and csf samples were available when they received a diagnosis of ad during followup . After lumbar puncture, csf samples (2030 ml) were obtained and collected in polypropylene tubes . The samples were centrifuged at 2,000 g at 4c for 10 minutes to eliminate cells and other insoluble material and were then immediately frozen and stored at 80c pending biochemical analyses, without being thawed or refrozen . Cell count was performed on the csf samples and no sample contained more than 500 erythrocytes/l . The opn protein content in serum and csf was measured using a commercial elisa according to the manufacturer's instructions (assay designs, inc . Briefly, serum and csf samples were diluted, respectively, 1: 20 and 1: 50 into assay buffer provided by the manufacturer and were incubated at 37c for 1 h in microtiter plates precoated with a polyclonal n - terminal capture anti - opn antibody (assay designs). Then, the plates were washed and wells were incubated at 4c for 30 min with a horseradish peroxidase labeled opn - specific monoclonal antibody (assay designs). After washing, the wells were incubated with tetramethylbenzidine - h2o2 solution for 30 min . The color reaction was stopped by adding a solution containing 1 n sulfuric acid . Optical densities were measured at 450 nm with reference wavelength set at 590 nm . The opn concentrations were calculated using a standard curve of recombinant human opn provided by the manufacturer . The lower detection limit of the kit was 3.33 ng / ml . Baseline and follow - up csf and plasma samples from a patient were measured on the same plate . Assays were repeated when the difference between the two probes of one sample was more than 10% . Normally distributed data sets were analysed by student's t - test, paired t - test, analysis of variance (anova), and linear regression and correlation analysis (using primer for biostatistics). There was no difference in sex distribution in mci, ad, and ond patients and the healthy control subjects . Plasma opn concentrations in healthy controls (51.4 9.8 ng / ml) and ond (53.3 10.3 ng / ml) did not differ significantly and did not correlate with age and sex . Plasma opn concentrations in mcinc (52.3 11.7 ng / ml), mcic (53.7 11.1 ng / ml), adn (74.4 13.7 ng / ml), adc (54.8 10.1 ng / ml), ond patients, and healthy controls differed significantly (figure 1(a); p <0.001). Adn patients had higher plasma concentrations of opn than the healthy controls (p <0.005), whereas plasma concentrations of opn in the mcinc, mcic, and adc patients did not differ significantly from plasma opn concentrations in ond or healthy controls (figure 1(a)). We next questioned whether the plasma opn concentrations would vary within the same individual in relation to disease status . In 13 mcic individuals tested longitudinally, the plasma opn concentrations were significantly elevated when they received a diagnosis of ad during followup (figure 1(b)). The csf represents the fluid compartment that is closest to reflect the inflammatory situation in the degenerative processes of the nervous system, so we then sought to compare the concentrations of opn in csf from patients with mci, ad, and ond . Csf opn concentrations differed significantly in mcinc (128.6 17.7 ng / ml), mcic (173.2 20.6 ng / ml), adn (226.5 21.2 ng / ml), adc (165.6 20.4 ng / ml), and ond patients (134.5 19.3 ng / ml) (figure 2(a)). Patients with mcic, adn, and adc (p <0.001) had significantly higher opn concentrations in the csf than the neurological controls . The concentrations of opn in csf from patients with adn was significantly higher than that from patients with adc (p <0.001). We also determined whether the csf opn concentrations would vary within the same individual in relation to disease status . In 13 mcic individuals tested longitudinally, the csf opn concentrations were significantly elevated when they received a diagnosis of ad during followup (figure 2(b)). When comparing paired csf and blood samples from patients with mci, ad, and ond, the concentrations of opn in the csf were significantly higher than plasma concentrations in all patients (figure 3). First, we determined whether there was a correlation between the levels of opn in the csf and the degree of cognitive decline in ad patients . A strongly positive correlation between the csf opn concentrations and the mmse score (r = 0.53, p <0.001) was observed (figure 4(a)). Secondly, we explored whether there was a correlation between the levels of opn in the csf and disease duration of ad patients . Our result showed that the csf opn concentration was correlated inversely with the disease duration (r = 0.51, p <0.001) (figure 4(b)). No other clinical parameters, such as gender, age, a42, tau, and p tau levels, had any significant correlation with the levels of opn in the csf of ad patients . Although a trend was noted toward the positive correlation between the plasma opn concentrations and the mmse score in the adn patients, it was not statistically significant . By contrast, no clear correlation was found between the csf opn concentrations of mcic and both the degree of cognitive decline and disease duration . In this study, the crosswise comparison demonstrated that csf opn concentrations were significantly increased in ad and mci converters compared to ond, and opn protein levels both in the csf and plasma of newly diagnosed ad patients were higher than that of chronical patients . Furthermore, in mci converters individuals tested longitudinally, both plasma and csf opn concentrations were significantly elevated when they received a diagnosis of ad during followup . Finally, opn csf levels displayed direct correlation with the mmse score and inverse correlation with disease duration . Our data have shown that the plasma concentrations of opn were significantly increased in the group of newly diagnosed ad than the other groups, and there was a trend toward the positive correlation between the plasma opn concentrations and the mmse score, although it was not statistically significant . One previous study on serum opn concentrations in ad patients gave a different result from ours . Found that there were no significant differences in serum opn concentrations comparing ad to controls . The results of the present study suggest that the differences between the results of the previous study may, at least partly, be explained by that we stratified patients according to their disease duration . Studies demonstrated that high levels of plasma opn were well correlated with the activities of various disease conditions: plasma opn concentrations were significantly higher in systemic lupus erythematosus patients and increase in opn concentration correlated positively and significantly with sledai score in all patients; serum opn concentrations of patients with idiopathic retroperitoneal fibrosis were elevated compared to healthy controls and correlated with the transverse diameter of the periaortic cuff as determined by imaging studies . In addition, elevated plasma opn levels have been also shown to play an important role in inflammatory and degenerative processes of the central nervous system: opn plasma levels were elevated in secondary progressive ms compared to relapsing - remitting ms patients in remission and healthy controls, supporting a role for opn in the chronic disease activity; opn serum levels were elevated in parkinson's disease and higher serum levels were associated with more severe motor symptoms . Prospective epidemiological studies show that elevated plasma levels of acute phase reactants can be considered as a risk factor for ad . Therefore, we speculate that elevated opn plasma levels in the initial stages of ad may contribute to the progression of cognitive decline, although the exact role of opn and its underlying mechanism as a key proinflammatory cytokine in ad is not understood . Th17 cells have been shown to be a pathogenic effector cell for development of various inflammatory and autoimmune diseases . Shinohara and colleagues found that opn plays a critical positive role in the differentiation of th17 cells by repressing il-27 secretion in mouse dendritic cells . A recent report showed that th17 t cells were increased in ad patients, which favors the speculation that elevated opn plasma levels in ad may be associated with the differentiation of th-17 cells . Accumulating evidence suggests that inflammation mainly occurs in pathologically vulnerable regions of the ad brain (such as the entorhinal, temporoparietal, and cingulate cortex), with increased expression of acute phase proteins and proinflammatory mediators . Therefore, we further examined the concentrations of opn in the csf, which directly contact with brain and can accurately reflect the ongoing inflammatory process in the central nervous system . Our results showed that the csf concentrations of opn were significantly increased in patients with mci progressing to ad than that in stable mci . Prediction of conversion from mci to ad is of major interest in ad research, which would allow for the appropriate application of disease - modifying treatments at a point where clinical manifestations are limited . Presently, there are few clinical or imaging markers for the early identification of mci which progresses to ad and mci which does not progress . Recently, simonsen and colleagues found that a phosphorylated c - terminal fragment of opn was increased in the csf of patients with mci progressing to ad as compared to patients who remain stable over time and healthy controls and proposed opn as a biomarker to predict the progression of mci to overt ad . The findings of these two studies showed that not only the intact forms of opn but also the cleaved form of opn was increased in the csf of patients with mci progressing to ad . More importantly, our results showed that the csf opn concentrations of mcic were significantly elevated when they received a diagnosis of ad during followup . Our findings are in agreement with another study, which showed that ad patients displayed about a two - fold increased opn levels in the csf compared to age - matched controls and it was particularly striking in the early stages of the disease . To identify protein changes during the presymptomatic phase of ad, ringman and colleagues performed proteomic analyses of csf from persons with or at risk for inheriting familial ad using high - resolution liquid chromatography - mass spectrometry . Their results showed that opn was elevated in the csf of familial ad mutation carriers compared to related noncarriers, which suggest changes of opn in the csf occurring a decade before clinical dementia . In addition, increased serum and csf opn levels were also detected in the lewy dementia, and the genotypic variation of snp-66 was associated with the occurrence of the lewy body dementia . Studies showed that opn seemed to act as a double - edged and might exert two opposite functions in the progression of neurodegenerative diseases . On one hand, opn functions as a neuroprotectant by upregulating myelination and remyelination . On the other hand, opn had a disease - accelerating role by triggering neuronal toxicity and death . In the current study, though we could not directly conclude that the increased levels of opn within the csf stimulate the degeneration of cerebral neurons and synapses in ad, we conjecture that opn may favor ad development because mcic patients had high levels of csf opn and the concentrations of opn in the csf and plasma were further increased when they received a diagnosis of ad . Their results demonstrated that opn expression was increased in the pyramidal neurons of the ca1 region of the hippocampus of ad patients and opn staining intensity positively correlated with both amyloid - beta load and age . Increased opn expression may exacerbate the abnormal immune response presented in the ad brain by enhancing the survival of activated t cells, which were detected in the brain tissues of ad patients . In conclusion, the bell - shaped curve of csf opn expression in disease progression of cognitive decline has extended the evidence for a role of opn in ad pathogenesis . It will be important to study larger cohorts of individuals with longer durations of followup, and from different centers, to further evaluate whether higher baseline level of csf opn was associated with a more marked decline of mmse over followup.
Renal angiomyolipoma (aml) is an infrequent tumor that, in most cases, follows a benign course and has clearly defined radiological and histological characteristics . We present a case of giant renal aml, which is the heaviest ever reported, encountered in a tertiary care hospital in india . A 49-year - old lady, a native of afghanistan, presented with a 3-year history of steadily increasing abdominal girth and bloating sensation in the abdomen . She underwent investigation in another institute and was found to have a huge abdominal mass . Review of contrast enhanced computed tomography (cect) scans of abdomen revealed a large fat containing tumor measuring 40 cm 24 cm 10 cm (<20 hounsfield units), replacing the left kidney and displacing the rest of the abdominal contents toward the other side of the midline [figure 1]. There was absence of enhancement, inhomogeneity, necrosis, or calcification ruling out the possibility of malignancy . A provisional diagnosis of benign renal neoplasm, possibly aml causing pressure effects was made . Hence, cect scan of the head was done, which excluded any lesion suggestive of tuberous sclerosis (ts) [figure 2] and decision of to perform nephrectomy was taken . 49-year - old lady with complaints of bloating sensation in the abdomen and a steadily increasing abdominal girth, which was subsequently diagnosed as angiomyolipoma of left kidney . Contrast enhanced computed tomography scan axial section film of abdomen reveals a lipomatous neoplasm 40 cm 24 cm 10 cm in dimension (hu <20 hu) replacing the entire left kidney, while displacing the rest of the abdominal contents (arrows) toward the other side of the midline . 49-year - old lady with complaints of bloating sensation in the abdomen and a steadily increasing abdominal girth, which was subsequently diagnosed as angiomyolipoma of left kidney . Contrast enhanced computed tomography scan axial section films of the head reveals no lesion suggestive of tuberous sclerosis . Under intubated general anaesthesia, an abdominal midline incision was made extending from xiphisternum to just above pubic symphysis . Simple nephrectomy was performed with intact tumor retrieval and minimal blood loss, without necessitating any blood transfusion . The recovered surgical specimen measured 39 cm 25 cm 9 cm and weighed 7500 g with compressed renal hilar vessels and ureter [figure 3]. Grossly, it was well- circumscribed and with a glistening yellow (fatty) appearance . Histopathological examination of the specimen revealed majority of the cells were mature adipocytes associated with thick - walled blood vessels and a few epithelioid stromal cells . There was a pattern with typical fat and perivascular epithelioid cells arranged around a blood vessel, suggesting it to be aml [figure 4]. Immuno - histochemical staining of the tissue by beta - hydroxy beta - methylbutyric acid-45 (hmb-45) (a melanosome - associated protein) indicated the presence of epithelioid component [figure 5]. 49-year - old lady with complaints of bloating sensation in the abdomen and a steadily increasing abdominal girth which was subsequently diagnosed as angiomyolipoma (aml) of left kidney . Excised specimen shows a giant aml 39 cm 25 cm 9 cm in dimension and weighing 7500 g, replacing the whole kidney . Renal hilar vessels and ureter are compressed, and emerging out of the mass . 49-year - old lady with complaints of bloating sensation in the abdomen and a steadily increasing abdominal girth, which was subsequently diagnosed as angiomyolipoma of left kidney . Histopathology slide of the excised mass shows admixture of tortuous thick - walled blood vessels (arrows), sheets of mature adipose tissue (f) and bundles of smooth muscle fibres (m) (stain used hematoxylin and eosin, 100). 49-year - old lady with complaints of bloating sensation in the abdomen and a steadily increasing abdominal girth, which was subsequently diagnosed as angiomyolipoma of left kidney . Photomicrograph (x40) of immunohistochemical stained specimen shows mature adipocytes (arrow heads) and strong staining of the epithelioid cells with the beta - hydroxy beta - methylbutyric acid-45 (arrows). The patient recovery was uneventful and she was discharged from the hospital on the 5 post - operative day . At 6 months post - operative follow - up aml is a benign hamartous lesion consisting of varying amounts of mature adipose tissue, smooth muscle, and thick - walled vessels . Renal aml are benign tumors known to occur sporadically and in association with genetic syndromes like ts and lymphangioleiomyomatosis . However, in our patient there were no clinical or radiological signs suggestive of any systemic syndrome . According to vitaly et al ., an estimated 20 - 30% of amls are found in patients with ts syndrome, which is an autosomal dominant disorder characterized by mental retardation, epilepsy, and adenoma sebaceum . Mean age at presentation is 30 years, and there is a 2:1 female - to - male predominance . However, 70 - 80% of patients with aml who do not have ts, there is a more pronounced female predominance and most patients present later in life, during the fifth or sixth decade, as was in the present case . Although, renal aml in patients with ts can be associated with significant morbidity, mostly related to complications from bleeding, sporadic cases do occur mostly as incidental lesions . The detection of sporadic cases has increased due to greater use of abdominal imaging for the evaluation of a wide variety of non - specific complaints . Occasionally, the increase in abdominal swelling or epigastric fullness may be the presenting feature, as in our case . Aml is the only benign renal tumor that is confidently diagnosed on cross - sectional imaging . The presence of fat (confirmed on non - enhanced thin - cut computed tomography by a value of 20 [hu] or less) seen within a renal lesion on imaging is considered the diagnostic hallmark . Findings of more than 20 pixels with attenuation less than 20 hu and of more than 5 pixels with attenuation less than 30 hu have been shown to have a positive predictive value of 100% . Histopathologically, aml consists of mature adipocytes, thick - walled blood vessels, and epithelioid stromal cells in various proportions . Usually it displays as a pattern of typical fat and perivascular epithelioid cells arranged around a blood vessel . Positive immunoreactivity for hmb-45, a monoclonal antibody raised against a melanoma - associated antigen, is characteristic for aml and can be used to differentiate this tumor from sarcoma and other tumors . The indication for surgical treatment is usually a symptomatic aml or an incidental aml of size larger than 4 cm . In cases of bilateral lesions, as in ts, nephron - sparing surgery (by either selective embolization or open or laparoscopic / robotic partial nephrectomy) must be performed . In our case, the tumor was so large that there was no option but to excise the whole lesion in totality . On review of cases in the literature, we find our case represents the heaviest aml (7500 g) ever reported, both in ts and sporadic categories . This giant lesion surpasses the aml mass weighing 6300 g in a case associated with ts reported by rossell barbar et al ., and the lesion (weighing 3500 g each) in the cases reported by tsutsumi et al ., and katz and poster . The only other aml, which has dimensions larger than the present one (39 cm 25 cm 9 cm) has been reported by katz and poster, where the mass measured 45 cm 20 cm 15 cm and weighed 3500 g (weight less than 7500 g measured in the present case), and was associated with ts . Thus the present case is the largest sporadic renal aml in size and heaviest renal aml in both syndromic and sporadic categories ever reported in the literature . Aml can be identified when on ct image more than 20 pixels show attenuation less than 20 hu and if more than 5 pixels show attenuation less than 30 hu . Histologically, aml lesions show a pattern of typical fat and perivascular epithelioid cells arranged around a blood vessel . Positive immuno - histochemical staining by hmb-45 (a melanosome - associated protein) for epithelioid component confirms the lesion as aml . We present a case where the patient presented with a huge abdominal mass in the left kidney diagnosed as aml and was managed by left nephrectomy . The giant tumor is the heaviest aml ever reported in the literature . At 6 months post - operative follow - up
The eye is an immune privileged site where the introduction of antigens does not elicit an inflammatory immune response . The first line of protection is the blood - ocular barrier, which impedes harmful pathogens from entering the eye via the peripheral bloodstream . A regional immune system provides a second multilayered defense in case the blood - ocular barrier is breached . The anterior chamber is bathed in aqueous humor fluid, which is strongly immunosuppressive and profoundly inhibits t cell activation . In addition, low expression of major histocompatibility complex (mhc) class ii molecules limits antigen presentation to immune cells reducing the chances of an immune response . Stromal cells of the iris and ciliary body have the ability to convert effector t cells to regulatory t cells and expression of death - inducing molecules results in apoptosis of immune cells keeping the immune response in check . Furthermore, aqueous humor outflow regulation, which is important in glaucoma, may be impacted by the innate immune system . The glaucomas are a group of optic neuropathies with a characteristic pattern of damage to the optic nerve that leads to loss of peripheral vision (see figure 1). A leading cause of global irreversible blindness, glaucoma will impact 111.8 million individuals by 2040 . The number of affected individuals is likely to be much higher than the reported number because the disease is usually asymptomatic up until major neural damage has occurred [7, 8]. It is clear that it is a heterogeneous group of disorders with multiple causative factors including gene mutations, environmental factors, and certain medications . Moreover, there are several risk factors that predispose to the disease including advanced age, elevated intraocular pressure, race, and family history [912]. While lowering intraocular pressure often reduces the rate of vision loss, many patients continue to go blind despite apparently successful pressure control [10, 11, 13]. Since there is no single causative factor, there are potentially many disease mechanisms behind glaucoma . Some lead to higher intraocular pressures, while others affect how the optic nerve withstands either pressure fluctuations or sustained elevation of intraocular pressure . Thus, glaucoma research has focused on the front of the eye, where intraocular pressure is regulated by a tissue called the trabecular meshwork, the back of the eye, where studies have focused on retinal ganglion cell physiology and optic nerve damage, and distal changes in retinal ganglion cell axons in terminal projection sites such as the superior colliculus and the lateral geniculate [1418]. Diagnostic criteria for glaucoma have been revamped significantly over the past 40 years with increased emphasis on characteristic changes in the optic disc and retinal nerve fiber layer and less reliance on elevated intraocular pressure [9, 19]. Normal tension glaucoma, that is, patients with statistically normal intraocular pressures, makes up 30 to 40% of glaucoma cases [13, 20]. Even for these patients, the only effective treatment for glaucoma continues to be reduction of intraocular pressure levels by either pharmaceutical or surgical means . Physiological intraocular pressures are established by maintaining a balance between production and drainage of aqueous humor in the anterior chamber . Aqueous humor is continuously produced by the ciliary processes and it bathes tissues in the anterior chamber before exiting out to schlemm's canal via a filter - like tissue called the trabecular meshwork (see figure 2). Building resistance to aqueous humor outflow in the trabecular meshwork produces a tunable system by which this filter can increase or decrease outflow when needed . Dysfunction of this conventional outflow pathway leads to impaired drainage and elevated intraocular pressure, as is seen in poag patients . Increased intraocular pressure places excessive mechanical stresses on the lamina cribrosa of the optic nerve in the posterior segment of the eye and loss of retinal ganglion cells ensues . Since these cells are responsible for transmitting visual signals to the brain, irreversible blindness occurs . Inflammatory responses may contribute to the glaucomatous process as shown by studies in humans and rodent models [2326]. In the anterior segment, certain inflammatory cytokines have altered expression levels in the aqueous humor of glaucomatous eyes compared to age - matched normal eyes . These include interleukin-6 (il-6), transforming growth factor beta-1 (tgf1), tgf2, il-6, il-8, il-10, il-12, -serum amyloid a, interferon- (ifn), and cxl9 [2733]. The source of these cytokines in the aqueous humor of glaucoma patients is not clear . However, acute elevation of intraocular pressure, such as in primary angle - closure glaucoma, damages the blood - aqueous - barrier (bab), which can lead to leakage of the cytokines into the aqueous humor . Inflammation - related changes also occur in the posterior segment . In a mouse model of laser - induced ocular hypertension, there was upregulated expression of mhc - ii and glial fibrillary acidic protein (gfap) in the microglia of contralateral eyes . The authors suggest that microglial activation in the nontreated eye could be related to an immune response [35, 36]. In humans, however, a systemic autoimmune response is less convincing because the contralateral eye in patients with unilateral glaucoma does not appear to exhibit glaucomatous degenerative changes . However, proinflammatory cytokines such as tumor necrosis factor- (tnf) and its receptor are upregulated in glaucomatous human optic nerve [3840]. Moreover, use of bupropion, which suppresses tnf production, significantly lowered the risk of developing poag in humans in a large retrospective study, while anti - tnf medication (etanercept) was found to be neuroprotective in a rodent model of glaucoma . Thus, both in glaucoma patients and in rodent models of glaucoma, there is accumulating evidence for a potential role of the immune system in contributing to deleterious changes in anterior and posterior ocular tissues . Our recent identification of a mutation in interleukin-20 receptor - b (il-20rb) supports this contention . Our group mapped a gene in a large poag oregon family to chromosome 3, the glc1c locus . Eighty - six family members, ranging in age from 8 to 91 years old, had extensive ophthalmic examinations . Thirteen family members were diagnosed with poag and twelve of these had elevated intraocular pressure (22 to 49 mm hg). An additional nine individuals, who do not have poag at this time, had elevated intraocular pressures . After refining the region to 4 cm, we sequenced all 49 genes in the glc1c locus and identified one nonsynonymous mutation: a t104 m change in il-20rb (rs367923973). This is an extremely rare variant with a reported frequency of 0.02% in dbsnp (http://www.ncbi.nlm.nih.gov/snp). This mutation, t104 m, lies in il-20rb's active binding site for the cytokines, il-19, il-20, and il-24, which are all members of the il-20 subfamily of interleukins (see below). Substitution of t104 with a methionine would replace the hydroxyl group that forms a hydrogen bond with s111 in il-20 with a sulfate group, thus disrupting the bond between the cytokine and its receptors . Based on these findings, this il-20rb mutation is highly likely to impact the il-20 signaling pathway, which may contribute to the pathogenesis of glaucoma in this family . The il-20 subfamily of cytokines and receptors are members of the larger il-10 family, which are grouped together based on their utilization of common receptor subunits, similarities in their target - cell profiles, and biological functions . This subfamily consists of the cytokines, il-19, il-20, il-22, il-24, and il-26, as well as the receptors, il-20ra, il-20rb, il-10rb, and il-22ra1 . Il-19 exclusively signals through the il-20ra / il-20rb heterodimer, while il-20 and il-24 can use both the il-20ra / il-20rb heterodimer and the il-22ra1/il-20rb receptor . It should be noted that il-20ra and il-20rb are also known as il-20r1 and il-20r2, respectively . The il-20 subfamily participates both in amplifying inflammatory responses particularly during autoimmune and chronic inflammation and alternatively in anti - inflammatory responses, such as tissue protection and regeneration [47, 48]. Thus, understanding how regulation of this subfamily is occurring is paramount in devising new treatment strategies for patients in this large poag family . Both il-20ra and il-20rb are expressed in normal human trabecular meshwork cell lysates and are upregulated in response to cytokine treatment . Il-20rb mrna is also expressed in moderately high levels in both the retinal ganglion cell layer and the optic nerve head in rats (dr . Elaine johnson, personal communication). Il-20, il-24, il-20ra, and il-20 rb are expressed in the retina and optic nerve head in the dba/2j mouse model of glaucoma (see later). However, il-19 is not expressed in the retina or optic nerve head of dba/2j mice . Binding of il-20 to its receptor activates the janus kinase- (jak-) signal transducer and activator of transcription (stat) pathway (jak - stat) [51, 52]. Elevation of intraocular pressure has also been shown to activate the jak - stat pathway, which is involved in retinal ganglion cell survival . Activation of stat3 can be both pro- and anti - inflammatory even within the same cell type, but how the desired response is elicited remains a question . For example, il-6 and il-10 both activate stat3, but they generate different cellular responses with il-6 generating a proinflammatory response and il-10 producing an anti - inflammatory one . These differences appear to correlate with the level of stat3 over time, with il-6 producing a transient activation while il-10 generates a sustained level of stat3 activation . The stat3-induced protein, suppressor of cytokine signaling-3 (socs3), may be involved because it mediates signaling dynamics due to its ability to inhibit signals from the il-6 receptor, but not the il-10 receptor . Several studies have linked il-20 signaling to mmp levels and activity . In breast cancer cells, il-20 appears to act synergistically with il-1 in these cells: higher levels of tnf, il-1, il-6, il-8, mmp-3, and monocyte chemoattractant protein-1 (mcp1) were found when disc cells were treated with both cytokines compared to exposure to il-20 or il-1 alone . Induction of mmp activity decreases the resistance to aqueous humor outflow through the trabecular meshwork which, in turn, lowers intraocular pressure . To investigate whether normal downstream signaling pathways were active in mutant cells, we asked if il-20 treatment induces stat3 phosphorylation and mmp activity in normal dermal fibroblasts and in poag patient fibroblasts with the il-20rb mutation . Fibroblasts from glaucoma patients with the t104 m il-20rb mutation had higher basal levels of phosphorylated stat3 compared to wild - type fibroblasts (see figure 3). Stimulation of wild - type fibroblasts by il-19, il-20, or il-24 cytokines led to a significant increase in phosphorylation of stat3 after 15 minutes, but this was not found in glaucomatous fibroblasts with the t104 m il-20rb mutation . Using a quenched fluorescent peptide assay that produces fluorescent signal when cleaved by a mmp, we showed that il-20 increased mmp activity in wild - type human fibroblasts, but not in fibroblasts with the t104 m il-20rb mutation (see figure 4). The differential response of mutant and wild - type cells to cytokine treatment suggested that poag cells with the t104 m mutation are unable to launch an appropriate cell signaling response upon stimulation by the il-20 family of cytokines . Collectively, these observations suggest that, in normal cells, il-20 and related cytokines bind to il-20 receptors on the cell surface (figure 2(c)). This would in turn activate stat3, which would translocate to the nucleus to modify transcription of inflammatory - related genes . The anti - inflammatory response would include upregulation of il-20 receptors by trabecular meshwork cells and activation of mmps leading to remodeling of the extracellular matrix . This would lead, ultimately, to greater outflow of aqueous humor . In glaucoma patients with the il-20rb mutation (figure 2(d)), the il-20 family of cytokines would not bind efficiently to the il-20ra / rb receptor so stat3 would not be activated and translocated to the nucleus . Therefore, the anti - inflammatory response would not be adequate and sustained expression of proinflammatory genes would remain . Subsequently, many secondary downstream effects may contribute to the elevated intraocular pressure observed in glaucoma patients harboring the il-20rb mutation and, ultimately, to glaucomatous damage . The dba/2j mouse model of glaucoma is an inbred mouse strain that progressively develops glaucoma - like abnormalities with aging . In the anterior chamber, the mice develop a form of pigment dispersion syndrome with the primary action being an inflammatory response resulting in elevation of intraocular pressure . The dba/2j mouse has two distinct phenotypes: iris pigment dispersion, which may be involved in immune dysfunction in dba/2j eyes, and iris stromal atrophy . These phenotypes are caused by mutations in the gpnmb and tyrp1 genes, respectively, [66, 67]. Gpnmb is expressed in some types of dendritic cells [68, 69], which are potent professional antigen presenting cells, whereas tyrp1 is an antigen that is involved in inflammatory eye disease . As discussed above, however, the aqueous humor of dba/2j mice lacks immunosuppressive properties and the capacity to support anterior chamber associated immune deviation . Iris pigment is shed into the aqueous humor, where it enters the outflow pathways and eventually causes blockage of the drainage channels . Dba/2j mice between 6 and 7 months of age begin to lose retinal ganglion cells . By 1012 months, significant retinal ganglion cell loss has occurred in the majority of dba/2j mice [72, 73]. However, if the mice are exposed to a high dose of -irradiation, the dba/2j mice are protected from developing glaucomatous damage, although they still have high intraocular pressures [73, 74]. Both retinal and optic nerve morphology appear normal in the irradiated mice, whereas untreated dba/2j mice show optic nerve atrophy, as well as clear loss of retinal ganglion cell axons . A unique finding in the dba/2j mice is a high level of activated microglia in the inner central retina and optic nerve region at 3 months, which occurs well before the loss of retinal ganglion cells . Il-19 is the mostly highly upregulated gene in activated microglia and, in addition, the il-20ra and il-20rb receptors are expressed . Il-19 has anti - inflammatory activity in microglia via stat3 activation, which leads to an anti - inflammatory response . Irradiation reduces the number of proliferating microglia in the optic nerve head along with a reduction in the levels of microglia activation in the central retina, optic nerve head, and laminar region in the dba/2j mouse [74, 77]. Minocycline, a neuroprotective tetracycline derivative that suppresses chronic neuroinflammation and microglial activation, also has a protective effect on retinal ganglion cell viability in the dba/2j mouse . In dba/2j mice with moderate axon damage, il-24 expression is significantly increased in the retina, but no significant differences were seen for either il-20 or il-20rb . Conversely, il-20ra levels are significantly reduced in optic nerves from eyes with severe axon damage in dba/2j mice compared to dba/2j - gpnmb . / il-20rb signaling is impaired, which could ultimately lead to altered extracellular matrix remodeling by mmps via inappropriate stat3 activation as described above . Changes in extracellular matrix composition and organization in the glaucomatous optic nerve head have been well documented and include deposition of extracellular matrix materials in areas formerly occupied by axons . The extracellular matrix changes likely contribute to the altered biomechanical properties of the glaucomatous optic nerve head and increase the vulnerability of the remaining axons to cell death [80, 81]. In conclusion, several lines of evidence provide compelling evidence for a role of the il-20 family of cytokines in glaucoma . First, we have identified a mutation in il-20rb at a residue that is critical for binding of the receptor to the il-20 family of cytokines in a large poag pedigree . Second, il-20 stimulation of the mutant il-20rb leads to abnormal stat3 activation and mmp activity in glaucoma fibroblasts . Third, il-20 family members and their receptors have altered expression levels in the retina and optic nerve head of the dba/2j mouse, which develops glaucoma - like symptoms with aging . While the il-20rb mutation most likely is not a causative factor in the majority of glaucoma cases, the identification of this mutation has revealed that defective il-20 signaling may lead to glaucoma in this large poag pedigree . Future studies will focus on the role of the il-20 subfamily members in both aqueous outflow regulation in normal trabecular meshwork and their anti - inflammatory role in the posterior tissues of the eye . This may lead to the development of novel therapeutic treatments to maintain tissue homeostasis and prevent glaucomatous vision loss in this large oregon poag family.
Kidney diseases are common in hiv - infected patients.17 the most common one is hiv - associated nephropathy (hivan), as well as drug toxicities of various kinds . Interstitial nephritis is a possible complication of hiv infection.8,9 it is usually caused by drugs such as indinavir, foscarnet, abacavir, and co - trimoxazole; mycobacterial infections; infections by other viruses; or dysimmune syndromes such as immune reconstitution inflammatory syndrome and diffuse infiltrative lymphocytosis syndrome (dils; table 1).10,11 interstitial nephritis might occur as a direct consequence of hiv infection, but cases demonstrating this through exclusion of other etiologies are rare . Herein, we present a case of interstitial nephritis that was likely caused directly by hiv infection and not by other etiologies . A 34-year - old african man was referred to our hospital because of microscopic hematuria identified at an annual health checkup at his workplace . The patient had no significant past medical history and was not taking any medications . The patient gave written informed consent to be included in this case report . Upon initial routine workup, the patient s serum creatinine level was 0.86 mg / dl, with blood urea nitrogen of 10.1 mg / dl . Urinalysis showed red blood cell (rbc) 3 + and urinary sediment showed dysmorphic rbcs (> 100/high power field) with rbc casts and absence of white blood cells . Urine 2-microglobulin was 913 g / l, urine n - acetyl - beta - d - glucosaminidase was 14.9 the patient was subsequently diagnosed with pulmonary tuberculosis and was treated with a standard regimen including four drugs for 2 months, followed by isoniazid and rifampin for 4 months . Because of persistent hematuria, the patient was hospitalized to undergo renal biopsy . The histopathological analysis revealed focal interstitial infiltration of lymphocytes and plasma cells in the renal cortex as well as in the corticomedullary junction, accompanied by mild tubulitis without microcysts (figure 1). No tubular necrosis was observed, with erythrocytic casts and flattened tubular epithelium (figure 2). Analysis of glomeruli showed no evidence of podocyte hypertrophy, glomerular collapse, or endocapillary hypercellularity (figure 2). Neelsen staining of the biopsied specimens was negative, and there were no pathological findings suggestive of tuberculosis . Two weeks after the initiation of treatment for tuberculosis, antiretroviral therapy (art), including lamivudine, abacavir, and dolutegravir, was started . Eight months after the initiation of art, urinary levels of 2-microglobulin and n - acetyl - beta - d - glucosaminidase normalized and microscopic hematuria resolved completely . The onset and diagnosis of interstitial nephritis occurred prior to initiation of art or any other medications, eliminating the possibility of it being drug induced . Inflammatory disorders such as immune reconstitution inflammatory syndrome and dils could not have been the cause of interstitial nephritis in the present case because art was started only after the onset of renal disease . Moreover, other infections were unlikely to be causes of interstitial nephritis, as there were no signs of infectious diseases other than hiv and tuberculosis . Hivan is the most common cause of renal dysfunction in patients with hiv, and only up to 10% of renal dysfunction is caused by interstitial nephritis.4,5 hivan is characterized by proteinuria, with histopathological changes such as focal and segmental glomerulosclerosis or collapsing or noncollapsing nephropathy . However, diagnostic confirmation by kidney biopsy is often important, particularly when the typical proteinuria is not observed, and other diagnoses such as those related to diabetes or hypertension can be confirmed frequently by kidney biopsy.12,13 hivan frequently accompanies interstitial inflammation of the kidney,1417 but the lack of pathognomonic findings in glomeruli in the present case made hivan unlikely . The lack of other glomerular changes such as podocyte hypertrophy and hyperplasia also contributed to excluding the diagnosis of hivan . The pathogenesis of hiv - associated renal diseases, including hivan, has been thoroughly investigated, and the majority of current knowledge was gained from studies using animal models . Hiv-1 can infect renal epithelial cells through infected cd4 + lymphocytes, and viral proteins such as nef and vpr may have a synergistic role in inducing podocyte dysfunction . This also leads to renal tubular epithelial cell apoptosis and tubulointerstitial inflammation, which results in one of unique histopathological changes of hivan.15,18,19 through survey of the literature, we were unable to find studies on the pathogenesis of interstitial nephritis associated with hiv . Whether similar pathophysiological observations related to tubulointerstitial inflammation observed in patients with hivan applies to our case remains unknown . The optimal therapy for hiv - associated interstitial nephritis is unknown,4 but art is likely to be effective, as shown in the present case . An early study suggested that corticosteroids may be beneficial for improving the inflammatory lesions of hivan,20 but this treatment regimen is not commonly prescribed in the era of art . Additionally, we cannot confirm that the application of corticosteroids would be beneficial for hiv - associated pure interstitial nephritis . The exact incidence and morbidity of interstitial nephritis caused by hiv physicians should be aware of the possibility, and kidney biopsies should be performed for differential diagnosis of interstitial nephritis . We identified interstitial nephritis without glomerular lesions in a treatment - naive hiv - infected patient, which was considered to be a complication of hiv infection . To our knowledge, the occurrence of interstitial nephritis in hiv - infected patients without other etiologies is rare, given the lack of similar reports . The findings presented herein are rare and should be further investigated to understand the potential role of interstitial nephritis on the prognosis of hiv infection.
Tiered approaches are the basis of environmental risk assessment schemes that support the registration of pesticides (e.g. Campbell et al . 2008). In this context, a tier is defined as a complete exposure or affects assessment resulting in an appropriate predicted environmental concentration (pec) or regulatory acceptable concentration (rac). The concept of tiered approaches is to start with a simple conservative assessment and to do additional more complex work if necessary, but all tiers within the same scheme need to address the same specific protection goal (efsa 2010; nienstedt et al . This approach implies a cost - effective procedure both for industry and regulatory agencies . In the aquatic effect assessment tier-1 normally is based on results of laboratory toxicity tests with a limited number of standard test species and the application of an appropriate assessment factor (af). Subsequent higher tiers may include results of laboratory toxicity test with additional test species (allowing, e.g. The species sensitivity distribution (ssd) approach), aquatic micro-/mesocosm tests (model ecosystem approach), and the tiered system as a whole needs to be: (1) appropriately protective, (2) internally consistent, (3) cost - effective and (4) address the problem with a higher degree of realism and complexity when going from lower to higher tiers . In pesticide risk assessment under regulation (ec) no . 1107/2009 (ec 2009), the basic data requirement for the tier-1 effect assessment are strictly defined (ec 2011). The current tier-1 basic data requirement, however, are under discussion (see below), since the new regulation 1107/2009 not only aims an appropriate protection of crops against harmful organisms but also a higher level of protection of the environment and non - target organisms than under the former plant protection product directive (91/414/eec) (ec 1991).fig . 1schematic presentation of the tiered approach within the acute effect assessment for pesticides showing the refinement of the process through the acquisition of additional data and the possibility to use results of micro-/mesocosms (model ecosystem approach) to calibrate the lower tiers . Racsw; ac the regulatory acceptable concentration for surface water within the context of the acute effect assessment scheme, pecsw; max the maximum concentration predicted for surface water, ssd species sensitivity distribution approach (redrafted after solomon et al 2008 and efsa 2010) schematic presentation of the tiered approach within the acute effect assessment for pesticides showing the refinement of the process through the acquisition of additional data and the possibility to use results of micro-/mesocosms (model ecosystem approach) to calibrate the lower tiers . Racsw; ac the regulatory acceptable concentration for surface water within the context of the acute effect assessment scheme, pecsw; max the maximum concentration predicted for surface water, ssd species sensitivity distribution approach (redrafted after solomon et al 2008 and efsa 2010) a logical consequence of the principles of the tiered approach in the environmental risk assessment for pesticides is that results of an appropriate higher tier effect assessment may act as a reference to calibrate the lower tier effect assessment, because the assessment endpoint derived from a higher tier is closer to the actual objectives of the adopted protection goal (fig . 1). In the aquatic effects assessment for pesticides, a micro-/mesocosm test may provide the appropriate higher tier effect endpoint when invertebrates or primary producers are at risk (brock et al ., we therefore use threshold concentrations for treatment - related effects as observed in micro-/mesocosm studies treated with insecticides to calibrate the tier-1 acute effect assessment procedure . The main objective of this paper is to evaluate the tier-1 effect assessment procedure for insecticides based on the current and the proposed new, but not yet adopted, eu dossier data requirement for acute toxicity to aquatic invertebrates . The risk assessment procedure on basis of the current data requirement is described in the eu guidance document on aquatic ecotoxicology (ec 2002) and in commission regulation no 544 (ec 2011). According to these documents, the acute insecticide risk to invertebrates is assessed by comparing the predicted peak concentration (pecmax) for edge - of - field surface water with the daphnia 48-h ec50/100 value . In addition, according to ec (2002), an effect assessment on basis of the 28-day water - spiked chironomus test in the presence of sediment (oecd 2004; streloke and kpp 1995) should be provided if the test substance concerns an insecticide with a specific toxic mode - of - action (e.g. Neonicotinoid insecticide and insect growth regulator (igr); daphnia 48-h ec50> 1, the exposure concentration of the test compound is not maintained in the water compartment, and the effect endpoint (i.e. Noec or ec10) is expressed in terms of the initial (nominal) exposure concentration in water . In the risk assessment on basis of this test the derived noec / ec10 is divided by an af of 10, and the concentration thus obtained is compared with the pecmax . The guidance document on aquatic ecotoxicology (ec 2002) also states that for the acute effect assessment of insecticides like neonicotinoids and igrs, a water - only acute chironomus test may be appropriate, but that an official guideline for such a test needs to be developed . For pesticide risk assessment under the new plant protection product regulation (ec 2009), commission regulation (eu) proposals circulate to revise the basic data requirement for the first - tier aquatic effect assessment . An important change in the proposed update is the inclusion of an acute test for a second aquatic arthropod species (besides daphnia) as basic data requirement for insecticides and substances with insecticidal activity . The mysid shrimp (americamysis bahia) and/or larvae of the insect chironomus spp . Are mentioned as candidate . Since a. bahia is a saltwater crustacean, the insect chironomus is considered a more relevant freshwater test species and an official oecd guideline for an acute aquatic tests with chironomus has recently been published (oecd 2011; also see weltje et al . Consequently, when in the near future the proposed new data requirement will be implemented, the acute toxicity to aquatic invertebrates will, in the first instance, be assessed on basis of (1) the acute laboratory 48-h ec50 for daphnia (preferably daphnia magna) and (2) the acute 48-h ec50 for a. bahia and/or c. riparius (or another chironomus species mentioned in oecd (2011) such as chironomus dilutus or chironomus yoshimitsui). In the first - tier effect assessment an af of 100 the highest predicted peak concentration (pecmax) in edge - of - field surface water should not exceed the tier-1 regulatory acceptable concentration (tier-1 rac) thus obtained . This risk assessment procedure is believed to sufficiently protect non - target invertebrates that dwell in edge - of - field surface waters from short - term exposures to insecticides . In the current paper, we intend to evaluate the implication of the proposed new aquatic data requirement (inclusion of acute tests with a. bahia and/or chironomus spp .) For the tier-1 acute effect assessment for insecticides . In addition, we also explore whether the macro - crustacean gammarus pulex is potentially a suitable standard test species for the risk assessment of insecticides since this species has been widely used in toxicity testing and is often used as focal species for developing ecotoxicological models (e.g. Galic et al . Single - species acute toxicity data and micro-/mesocosm data were collected from existing open access toxicity data bases such as ecotox (www.epa.gov/ecotox/), footprint (www.eu-footprint.org/ppdb.html), open grey literature including eu draft assessment reports or dars (http://dar.efsa.europa.eu/dar-web/provision), rivm reports (www.rivm.nl/bibliotheek/index-en.html), summary reports of eu member states (e.g. Www.ctgb.nl) and scientific papers in the open literature (see table 1). In addition, confidential data from industry was used that was provided to alterra and used in the paper of maltby et al . Insecticides were allocated to one of the following categories: organophosphates, carbamates, pyrethroids, insect growth regulators, neonicotinoids, biopesticides and other types of insecticides (table 1).to respect the confidentiality of the data provided by industry, we made the different insecticides anonymous in the graphs but allocated them to one of the insecticide categories listed above . Selected endpoints were the median effect concentrations for immobility or mortality observed in toxicity tests (ec50). The test duration selected was 4896 h. geometric means were calculated when more than one toxicity value was reported for the same endpoint of a species.table 1insecticides used for the evaluation and related scientific papers in the open literature that were consulted in addition to the open access toxicity data bases mentioned in the materials and methods sectiongroupcompoundopen literature referencesorganophosphatesazinphos - methylvan wijngaarden et al . (2005)parathion - ethylvan wijngaarden et al . (2005a) and maltby et al . (2005), and de knecht and van herwijnen (2008)esfenvaleratebeketov (2004), beketov and liess (2008a), lozano et al . (2005)gamma - cyhalothrinvan wijngaarden et al . (2009) and giddings et al . (2011), beketov and liess (2008a), posthuma - doodeman (2008), and stoughton et al . (2005)flubendiamidespiromesifen insecticides used for the evaluation and related scientific papers in the open literature that were consulted in addition to the open access toxicity data bases mentioned in the materials and methods each study was classified into one of two exposure categories, namely (1) a single pulse exposure regime or (2) a repeated exposure regime . In addition, responses observed for the most sensitive endpoint of a study were ascribed to one of five effect classes (sensu brock et al . 2006; de jong et al . 2008). For each compound and exposure regime the noececo (= threshold concentration for treatment - related effects) was derived from test concentrations at which no statistical and ecological significant effects (effect class 1) or slight / transient effects on individual samplings only (effect class 2) were observed for the most sensitive population and/or community endpoint . When possible, for each compound a separate noececo data point for a single and a repeated treatment regime was derived . Only effect class 1 values were available, then this value was used as the noececo . In case only effect class 2 values were available, then this value was divided by two ((effect class 2 concentration)/2) to estimate the noececo . When both an effect class 1 and an effect class 2 value were available then the geometric mean of the class 1 and class 2 values was used as the noececo . In case more effect class 1 values were available for a compound (e.g. From different micro-/mesocosm studies), then the highest of these values was used . In the same situation for effect class 2 values, the lowest class 2 value was chosen . Ecosystem threshold levels (noececo) were then compared with arthropod first - tier regulatory acceptable concentrations (= tier-1 racs) based on acute toxicity data for d. magna, a. bahia, chironomus spp . And these tier-1 racs were obtained by dividing the acute toxicity values by an af of 100 (i.e. (4896 h e(l)c50)/100). In addition, the noec / ec10 values of the 28-day water - spiked c. riparius test was used to derive a tier-1 rac by dividing it by an af of 10 . Note that in the aquatic risk assessment for insecticides these tier-1 racs always are compared with the pecmax . Noececo values for each insecticide were plotted against the rac based on: the acute toxicity (ec50) for d. magna and an af of 100 (= dm/100);the noec / ec10 of the water spiked 28-day c. riparius test and an af of 10 (= 28dcr/10);the lowest toxicity value from the acute ec50/100 for d. magna and the 28d - noec/10 for c. riparius (= dm/100 and 28dcr/10);the acute toxicity for a. bahia and an af of 100 (ab/100);the acute toxicity for an oecd - chironomus sp . (i.e. C. riparius, c. dilutus (= chironomus tentans), c. yoshimitsui (oecd 2011)) and an af of 100 . When data were available for more than one species, the most sensitive was selected (= chir/100);the acute toxicity (ec50) for g. pulex and an af of 100 (gp/100);the lowest acute toxicity value (ec50) from the d. magna and a. bahia tests and an af of 100 (= (dm & ab)/100);the lowest acute toxicity value (ec50) from the d. magna and oecd - chironomus tests and an af of 100 (= (dm & chir)/100);the lowest acute toxicity value (ec50) from the d. magna, a. bahia or oecd chironomus tests and an af of 100 (= (dm & ab & chir)/100). The acute toxicity (ec50) for d. magna and an af of 100 (= dm/100); the noec / ec10 of the water spiked 28-day c. riparius test and an af of 10 (= 28dcr/10); the lowest toxicity value from the acute ec50/100 for d. magna and the 28d - noec/10 for c. riparius (= dm/100 and 28dcr/10); the acute toxicity for a. bahia and an af of 100 (ab/100); the acute toxicity for an oecd - chironomus sp . (i.e. C. riparius, c. dilutus (= chironomus tentans), c. yoshimitsui (oecd 2011)) and an af of 100 . When data were available for more than one species, the most sensitive was selected (= chir/100); the acute toxicity (ec50) for g. pulex and an af of 100 (gp/100); the lowest acute toxicity value (ec50) from the d. magna and a. bahia tests and an af of 100 (= (dm & ab)/100); the lowest acute toxicity value (ec50) from the d. magna and oecd - chironomus tests and an af of 100 (= (dm & chir)/100); the lowest acute toxicity value (ec50) from the d. magna, a. bahia or oecd chironomus tests and an af of 100 (= (dm & ab & chir)/100). These racs were compared with the 1:1 tier-1 rac / noececo ratio . Compounds falling below the 1:1 line indicate that tier-1 rac values derived from single - species toxicity tests are protective of ecological effects towards arthropod communities in semi - field studies characterised by a single or repeated pulsed treatment regime . Single - species acute toxicity data and noececo values could be compared for 31 insecticides which were categorized in one of seven groups (table 1). The tier-1 rac based exclusively on the acute ec50 values for d. magna and the application of an af of 100 was generally protective for organophosphates, carbamates and seven of the eleven pyrethroid cases (fig . In contrast, similar racs were not protective for any of the neonicotinoids evaluated since their daphnia ec50/100 values were considerably higher (a factor of 28 to 17,020) than the noececo, irrespective of exposure regime . Also three of the six igr cases showed a rac that was more than a factor of 5 to 10 greater that the line representing the 1:1 ratio . One biopesticide was a factor of 2 above this line as well as two compounds of the other insecticides category by a factor of 17 to 1,718 (fig . 2plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs exclusively based on the acute toxicity data for d. magna (dm/100); b racs exclusively based on 28d noec / ec10 for c. riparius (28dcr/10); c racs based on the lowest toxicity value of the combination acute ec50/100 for d. magna and the 28d- noec/10 for c. riparius (dm/100 and 28dcr/10). Op organophosphates, pyr pyrethroids, igr benzylurea / insect growth regulators, neonic neonicotinoids, biopest biopesticides, carb carbamates, other other types of insecticides, single single insecticide application in micro-/mesocosm study, multiple repeated insecticide application in micro-/mesocosm study plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs exclusively based on the acute toxicity data for d. magna (dm/100); b racs exclusively based on 28d noec / ec10 for c. riparius (28dcr/10); c racs based on the lowest toxicity value of the combination acute ec50/100 for d. magna and the 28d- noec/10 for c. riparius (dm/100 and 28dcr/10). Op organophosphates, pyr pyrethroids, igr benzylurea / insect growth regulators, neonic neonicotinoids, biopest biopesticides, carb carbamates, other other types of insecticides, single single insecticide application in micro-/mesocosm study, multiple repeated insecticide application in micro-/mesocosm study racs exclusively based on long - term toxicity data (28d noec) for c. riparius (water spiked test in the presence of sediment) and the application of an af of 10 also appeared not to be sufficiently protective for eight of the twenty - three insecticide cases evaluated (fig . 2b), although the deviations from the 1:1 line were less extreme (a factor of 2 to 25) than observed for the rac based on acute toxicity of daphnia . These eight insecticide cases comprised four pyrethroids and one organophosphate, igr, neonicotinoid and other insecticides each . When tier-1 racs were derived on the basis of the lowest value from the 28d noec/10 for c. riparius and 48 h ec50/100 for d. magna (fig . 2c) then the protection improved compared with the tier-1 racs based on the separate species (fig . 2c the tier-1 rac values for four of the 23 insecticide cases were a factor of 2 to 25 higher than their noececo values . Tier-1 racs exclusively based on the acute toxicity for a. bahia (ec50/100) resulted in three exceedences of the noececo . The a. bahia ec50/100 value for an igr was a factor of 75 higher than the corresponding noececo while that was a factor of 6 to 7 for two neonicotinoids (fig . 3plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs exclusively based on the acute toxicity for a. bahia (ab/100); b racs exclusively based on the acute toxicity for chironomus (oecd species; chir/100); c racs exclusively based on acute toxicity data for g. pulex (gp/100). For explanation of symbols, see fig . 2 plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs exclusively based on the acute toxicity for a. bahia (ab/100); b racs exclusively based on the acute toxicity for chironomus (oecd species; chir/100); c racs exclusively based on acute toxicity data for g. pulex (gp/100). For explanation of symbols, see fig . 2 tier-1 racs based on acute toxicity measured using the acute oecd chironomus (ec50/100) test exceeded the noececo by a factor 19 to 185 for three igr cases and a factor of 3 for an organophosphate (fig . All nicotenoid cases are below the 1:1 line indicating that for neonicotenoids the acute chironomus test is the best second tier-1 test species, but less so for igrs . Tier-1 racs based on the acute toxicity for g. pulex (ec50/100) resulted in exceedences of the noececo for two neonicotinoids (by a factor of 2 and 90) one biopesticide (by a factor of 5) and one igr (by a factor of 350) (fig . Tier-1 racs (acute ec50/100) based on the most sensitive acute toxicity data for d. magna and a. bahia, showed a similar pattern of noececo exceedences (fig . 4a) than when the effect assessment is based on acute toxicity of a. bahia alone (fig . 3a), illustrating that in general the acute ec50 value for a. bahia is lower than that for d. magna.fig . 4plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs based on the lowest acute toxicity value for the combination d. magna and a. bahia ((dm & ab)/100); b racs based on the lowest acute toxicity value for the combination d. magna and chironomus (oecd species; (dm & chir)/100); c racs exclusively based on the lowest acute toxicity data for the combination d. magna, a. bahia and/or chironomus ((dm & ab & chir)/100). For explanation of symbols, see fig . 2 plot of the acute tier-1 racs for insecticides against noececo values derived from aquatic micro-/mesocosm tests . A racs based on the lowest acute toxicity value for the combination d. magna and a. bahia ((dm & ab)/100); b racs based on the lowest acute toxicity value for the combination d. magna and chironomus (oecd species; (dm & chir)/100); c racs exclusively based on the lowest acute toxicity data for the combination d. magna, a. bahia and/or chironomus ((dm & ab & chir)/100). For explanation of symbols, see fig . 2 tier-1 racs (acute ec50/100) based on the most sensitive acute toxicity data for d. magna and an oecd - chironomus generally appeared to be protective for the insecticides investigated, except for the igr fenoxycarb (fig . An overall high level of protection was achieved when deriving tier-1 racs (acute ec50/100) based on the most sensitive available acute toxicity value for d. magna, a. bahia and oecd - chironomus, but again the tier-1 rac of one igr (fenoxycarb) considerably exceeded its noececo (fig . Whether the tier-1 effect assessment procedure on basis of the old data requirements (as presented in fig . 2) is protective or not seems to depend on the specific toxic mode - of - action of the insecticide evaluated . In the case of organophosphates, carbamates and pyrethroids most tier-1 racs based on acute toxicity data for d. magna, or the combination of these data with the 28-d noec / ec10 data for c. riparius, are below the noececo . However, the tier-1 racs for some other types of insecticides are above the noececo, hence, not protective, particularly when these tier-1 racs are exclusively based on acute toxicity data for d. magna . Our evaluation clearly demonstrates that for insecticides with very specific modes - of - action, such as neonicotinoids and insect growth regulators, the concentration on basis of the ec50 of daphnia and the application of an af of 100 may not always protect sensitive invertebrates in micro-/mesocosm studies . Although by additionally using results of the 28-d water spiked c. riparius test (and application of the af of 10) the tier-1acute effect assessment considerably improves for most of the insecticides evaluated, this combination is still not fully protective for 4 out of 23 cases (for which both the 48-h ec50 data of daphnia and the 28-day noec / ec10 data of chironomus were available) (fig . 2c). The tier-1 effect assessment procedure on basis of the new data requirements (acute ec50 values for d. magna and a. bahia and/or chironomus) appears to be protective for the vast majority of insecticides evaluated in micro-/mesocosms (fig . 4). For the combination d. magna and a. bahia in 3 out of 22 cases the tier-1 rac was not protective (fig . 4a) while that was 1 out of 26 cases for the combination d. magna and chironomus (fig . 3b) and 1 out of 30 cases for the combination d. magna and either a. bahia or chironomus (fig . The one case for which the tier-1 rac based on the proposed new data requirements was clearly not protective for effects observed in micro-/mesocosms concerned the igr insecticide fenoxycarb . For this compound the tier-1 rac exceeded its noececo by a factor of 76 to 164 (fig . 4a c). The position of fenoxycarb in the plots has to do with the exceptionally broad range of effect class 2 concentrations (0.0963.2 g / l) reported in the cosm study available (in smit and vonk 2008). Moreover, the effects of fenoxicarb treatment observed in the cosms only involved single - event reductions in abundance of the cladoceran bosmina longirostris . Clear long - term effects were reported at the next higher concentration of 11 g / l (smit and vonk 2008). In addition, an experimental stream study, focussing on two mayfly populations, demonstrated a noec of 5 g / l (licht et al . 2004). This might suggest that the lowest effect class 2 concentration that we used to derive the noececo (by dividing this effect class 2 concentration by 2) for fenoxicarb was overly conservative . The crustacean g. pulex is increasingly used as test species to underpin the aquatic effect assessment of pesticides, the advantages being that it is easy to handle in the laboratory and large enough to measure body burdens, and consequently, it is frequently used to study effects of time - variable exposures by means of toxicokinetic - toxicodynamic models (ashauer et al . Magna, tier-1 racs based on the acute toxicity of g. pulex are generally protective for neurotoxins (organophosphates, carbamates and pyrethroids), however, for more recently developed chemistries (e.g. Neonicotinoids and igrs) g. pulex may not be a representative sensitive species (fig . Our evaluation demonstrates that the tier-1, acute aquatic effect assessment on basis of laboratory ec50 values for daphnia, a. bahia and/or chironomus (eu commission regulation proposal for new data requirements) overall is protective for ecological effects due to pulsed insecticide exposures in the (semi-)field, in contrast to a tier-1 effect assessment on basis of daphnia alone . A lesson learned is that the tier-1 effect assessment procedure needs to be critically evaluated / calibrated each time new insecticides with a novel toxic mode - of - action are placed on the market, e.g. By requiring not only the mandatory tier-1 data but also an appropriate micro-/mesocosm study if read - across information is not available . For neonicotinoids, and to a lesser extend igrs, the key issue seems to be that crustaceans, and d. magna in particular, may be substantially less sensitive than aquatic insects (see e.g. Beketov and liess 2008b). It can be argued that, based on the toxic mode - of - action of the active ingredient evaluated, tier-1 insecticide studies always should include acute toxicity data for the most sensitive taxonomic group, which may be insects (e.g. The 48-h water only test with chironomus). A comparison of the racs for igrs based on the 48-h acute chironomus test (fig . 3b) and the 28-day water - spiked c. riparius test in the presence of sediment (fig . 2b), however, reveals that a test duration of 48 h may be too short to fully express the acute effects of igrs . Also in mesocosm experiments latency of effects following short - term exposure to igrs has been demonstrated (e.g. Brock et al . 2009). A direct consequence of the possible new data requirements is that the tier-1 effect assessment procedure becomes more conservative for those types of insecticides (e.g. Organophosphates, carbamates, pyrethroids) that are already sufficiently covered by the tier-1 rac on basis of daphnia . 4c, that compares the noececo values with the acute tier-1 racs on basis of the new data requirements, the organophosphates and pyrethroids are on average a factor of 158 and 59 below the 1:1 line . A possible solution to overcome unnecessary higher - tier testing is to calculate the tier-1 rac for insecticides by applying an af of 100 to the geometric mean ec50 value (see efsa 2005) of the available aquatic arthropod taxa in the core data set, if (1) sufficient read - across information for related compounds is available to underpin this approach and, (2) for the insecticide under evaluation the acute ec50 values for d. magna en chironomus differ less than an order of magnitude . Alternatively, the default approach may be to apply the geomean approach separately for crustaceans and insects and to select the lowest value . In a future paper we will calibrate the different options for the geomean approach on basis of acute ec50 values for standard and additional arthropod test species and insecticides by comparing the racs thus obtained with results of micro-/mesocosm tests.
Several guidelines have been published regarding the treatment and prevention of deep vein thrombosis (dvt)1, 2 . The guidelines underscore the fact that comprehensive measures for prevention of dvt are necessary3 . Guidelines for the diagnosis, treatment and prevention of pulmonary thromboembolism and deep vein thrombosis (jcs 2009)2 state that elastic stockings are effective for the prevention of dvt . The mechanisms by which wearing elastic stockings prevents dvt are prevention of blood stasis by increasing the blood flow volume and decrease of the caliber of venous blood vessels by compression of the lower limbs4 . However, these effects are still under debate5, 6 . Nara et al . Reported that there is little effect when elastic stockings are used as a single application7, but scurr et al . Indicated that a combination of elastic stockings and intermittent pneumatic compression was effective for prevention of dvt8 therefore, examination of the effects of combining elastic stockings with something else on prevention of dvt may be of clinical significance . Previously reported that ankle positions affect the blood velocity in the common femoral vein during ankle pumping exercises9, which was recommended for the prevention of dvt . The aim of this study was to identify how to effectively use elastic stockings to change the blood flow velocity in the common femoral vein under different ankle positions . The study subjects were 10 males without a history of cardiovascular diseases and no contraindications for exercise testing and training according to the guidelines of the american heart association10 . One experimenter (y.f .) Measured the blood flow velocity in all of these experiments . Time - averaged maximum flow velocity (tamv) in the left common femoral vein was measured using a pulse doppler method with a diagnostic ultrasound system (acuson p300, siemens healthcare, erlangen, germany). Tamv is the averaged blood flow velocity per unit time in the left common femoral vein . The subject s heart rate, systolic blood pressure (sbp), and diastolic blood pressure (dbp) were monitored (suntech tango+, suntech medical, usa) continuously during measurement of tamv . In this study, stocking were used, and the right size for each subject according to the instruction manual was selected . Subjects were asked to assume three different positions: supine (supine position), supine with the legs up after the examination bed was raised to an 18-degree angle (leg - up position), and supine with the head up after the examination bed was raised to a 30-degree angle (head - up position) (fig . 1.experimental protocol). At the beginning of each condition, the subjects had a 3-min rest period to acclimatize themselves to each position . After the 3 min of rest, the tamv in the left common femoral vein was measured for 20 seconds . In total, the subjects were asked to undergo testing under six conditions, that is, each of the 3 positions with and without stockings . Experimental protocol two - way anova for repeated measures was used to compare the tamv values of blood flow velocity . All analyses were performed using spss statistics ver . 17.0 (spss inc ., chicago, il, usa), and statistical significance was accepted at an alpha level of 0.05 . This study was approved by the human ethics review of tokyo university of technology (approval number; e14hs-026). Under the without stockings condition, the tamv in the head - up position (16.6 6.1 cm / sec) was significantly lower than those in the supine (30.1 17.8 cm / sec) and leg - up positions (28.9 17.5 cm / sec) (supine vs. head up, p<0.05, leg up vs. head up, p<0.05). Tamv showed no significant difference between the supine position and leg- up position (table 1table 1.time-averaged maximum flow velocity in the common femoral vein in three different positions with and without stockings). Under the with stockings condition, the tamv in the head - up position (13.6 4.4 cm / sec) was also significantly lower than those in the supine (33.1 15.5 cm / sec) and leg - up positions (27.7 9.4 cm / sec) (supine vs. head up, p<0.01, leg up vs. head up, p<0.01). Tamv showed no significant difference between the supine position and leg - up position (table 1). In this study, elastic stockings did not affect the blood flow velocity in the common femoral vein . This can be attributed to the fact that subjects were young health males who had no problem in their venous valves and venous vascular walls . Generally, the venous diameter is wider than the arterial diameter, but the venous vascular wall is thinner than the arterial vascular wall because it has a thinner layer of smooth muscle and fewer elastic components . For this reason, veins have great compliance and thus a high blood volume, and compression can easily change the venous shape11 . In venous incompetence, the number of collagen fibers increases, and the fibers are randomly aligned12, 13 . Furthermore, veins cannot constrict and expand adequately in response to the stimulation of noradrenalin, potassium chloride, angiotensin ii, nitric oxide, calcium ion, etc ., and they do not function normally14 . Additionally, expansion of veins and blood stasis occur easily because dysfunction of venous valves causes the blood pressure to increase in veins . Thus, it is likely that elastic stockings are effective in preventing blood stasis in venous incompetence by compressing veins continuously and preventing them from expanding15 . In this study, therefore, it is suspected that compression by elastic stockings does not lead an increase in blood flow velocity, as their venous valves and venous vascular walls functioned properly . On the other hand, it is known that an increase in hydrostatic pressure disturbs venous return16, thus in venous incompetence, the blood velocity in the common femoral vein can decrease markedly because venous incompetence disturbing venous return may be affected profoundly by gravity . This study has some limitations: (1) the number of subjects in this study was relatively low, and (2) the subjects were only young healthy male volunteers . This could have affected results, and a study for different age groups of people should be performed.
Primary stabbing headache is a short duration headache disorder that is characterized by brief paroxysms of sharp head pain often located in the first division of the trigeminal nerve, occurring at an irregular frequency, lacking any accompanying symptoms, and not attributable to any underlying cause . Stabbing headache has also been reported to occur as a symptom of, or in association with, a variety of underlying secondary disorders, including giant cell arteritis [2, 3], pituitary tumors, meningiomas, ocular pathology, ischemic stroke [6, 7], cavernous hemangioma of the frontal bone, and varicella zoster meningoencephalitis . Stabbing headache has not been reported to manifest as a direct consequence of a small well - circumscribed acute brain lesion, and its pathophysiology remains elusive . Herein, a lesional cause of stabbing headache, a small acute thalamic hematoma, is presented . A 95-year - old right - handed caucasian woman presented to our emergency department with the sudden onset of a constellation of spontaneous neurological symptoms . She first noted repetitive, sharp, 12 s paroxysms of pain in strictly the right frontal and supraorbital region, occurring dozens of times per hour but at irregular and unpredictable frequencies . Her pain was unaccompanied by photophobia, phonophobia, cranial autonomic symptoms, or any visual changes . She did not identify any triggers and tactile stimulation of any area of the head or face did not provoke the pain . These attacks continued until she finally fell asleep in the hospital 12 h later, and upon awakening 6 h later, did not recur . Minutes after the head pain onset, she noted mildly slurred speech, which also resolved by the following morning, and upon attempting to ambulate she felt very imbalanced, which persisted for several days . Her past medical history included hypertension, hyperthyroidism, gastroesophageal reflux, osteoarthritis, and a left intertrochanteric femoral fracture sustained after a fall, which was surgically repaired 4 months previously . Postoperatively she developed cholangitis from choledocholithiasis, an acute pulmonary embolism, and paroxysmal atrial fibrillation, for which she was treated with intravenous antibiotics and placed on long - term anticoagulation . She had no history of any primary headache disorder, denied ever experiencing any focal neurological symptoms . Her medications included warfarin, fosinopril, omeprazole, metoprolol, digoxin, and methimazole . She lived with her husband and was a retired school teacher . On review of systems, for several months she had ongoing right upper quadrant pain and occasional nausea that had been attributed to choledocholithiasis . She denied jaw claudication, episodes of visual loss, myalgias, or neck pain . Her general medical examination revealed a well - appearing woman with normal vital signs, and only mild right upper quadrant tenderness . On neurological examination, deep tendon reflexes were 1 + in the arms, trace at the knees, and she lacked ankle jerks . She had a white blood cell count of 12,000/l, a total bilirubin of 2.3 mg / dl, a direct bilirubin of 1.2 mg / dl, an alkaline phosphatase of 560 u / l, a sgot of 341 u / l, and a sgpt of 292 u / l . Noncontrast computed tomography of the head revealed a rounded hyperdense lesion in the left lateral thalamus abutting the posterior limb of the internal capsule, consistent with an acute small hemorrhage, along with multiple subcortical lacunes and white matter ischemic changes (fig . 1). The hematoma volume was estimated to be 0.12 cm using the abc/2 method .fig . 1axial noncontrast computed tomography of the brain revealed an acute, rounded hyperdense lesion in the region of the left thalamus and likely abutting the posterior limb of the internal capsule (white arrow), corresponding to a small acute hemorrhage axial noncontrast computed tomography of the brain revealed an acute, rounded hyperdense lesion in the region of the left thalamus and likely abutting the posterior limb of the internal capsule (white arrow), corresponding to a small acute hemorrhage the patient was administered fresh frozen plasma and vitamin k to reverse her coagulopathy and ultimately had an endoscopic retrograde cholangiopancreatography, where numerous gallstones were removed . The patient was ultimately discharged to a short - term rehabilitation facility for her imbalance and deconditioning . The correspondence of the acuity of the clinical syndrome with the radiographic demonstration of an acute hematoma strongly supports its causality, although the lack of magnetic resonance imaging makes the exclusion of other acute lesions slightly less definitive . A fixed site of stabbing headache may signify an underlying symptomatic cause of the disorder, as was the case with this patient . Although a stabbing pain character of headache is not uncommon in association with acute cerebrovascular disease, the diagnosis of stabbing headache has not been well described in this population . In a large prospective series of over 2,000 patients with acute ischemic stroke where headache details were captured at stroke onset, 20% of the patients described their pain character as stabbing, although the diagnosis of stabbing headache was not mentioned . Pareja and colleagues mentioned the onset of stabbing headache in a patient with an ischemic stroke to the parietal lobe, but did not describe its laterality and relationship to the site of the stabbing head pain . Piovesan and colleagues reported the occurrence of stabbing headache in a delayed fashion after an ischemic stroke in three patients who all likely sustained acute middle cerebral artery territory infarctions . Regarding hemorrhagic stroke, in a large series of 90 intracranial hemorrhage survivors, six patients experienced stabbing headache de novo after intracranial hemorrhage, only one of whom had no premorbid headache history . Acute thalamic hematomas in particular can lead to headache over half the time, but similar to other deep structures do not lead to headache nearly as often as hemorrhage in the posterior fossa or lobar structures . In this patient, stabbing headache resulted from an acute parenchymal brain lesion in the thalamus, a location vital to the processing of pain . More specifically, the location of the hematoma may have encompassed the ventral posteromedial nucleus of the thalamus, a relay center for sensory input from the trigeminal system . The association of the pain strictly occurring in the right trigeminal distribution with a lesion in the corresponding left thalamus is intriguing . Some authors have proposed the disorder results from spontaneous activation of peripheral nerve branches in the trigeminal or upper cervical distribution [14, 15]. Another theory is that patients with stabbing headache have segmental and fleeting disinhibition of central pain pathways [14, 15]. Perhaps in cerebrovascular disease, such disinhibition occurs in an acute fashion in those patients with contralateral pain to the site of infarction or hemorrhage . In stroke patients with ipsilateral stabbing headache this report demonstrates an association of an acute thalamic lesion with stabbing headache occurring in the contralateral trigeminal distribution . Particularly in an elderly patient or individual taking anticoagulants, a small thalamic hemorrhage should be considered in the differential diagnosis of new - onset stabbing headache, even when fleeting.
When do such problems become abnormal and merit a specific psychiatric diagnostic label, remains a question under study with no clear cut answers . We present a case of a young girl for whom clinical attention was sought for transient eating problems . A 12-year - old young lady belonging to lower socioeconomic rural background was seen by the psychiatry services when referred for problems of eating since past 2 months . The patient had a history of delayed development and achieved milestones slower than other siblings . She would eat what was given to her by her mother, and ask for specific food items occasionally . Around 2 years back, the patient developed generalized tonic clonic seizures during sleep which were followed by myoclonic jerks . The electroencephalograpm (eeg) findings were consistent with a diagnosis of juvenile myoclonic epilepsy (jme). The episodes of generalized tonic clonic seizures were well controlled with valproate, but myoclonic jerks continued to recur . Due to the occurrence of jerks at school, other children would tease her . She became irritable with her family members quite often, and would hit the siblings when provoked . She did not have depressive cognitions or change in sleep or appetite then . Due to behavioral problems of refusal to go to school and irritability, intelligence quotient (iq) assessment was done and reported as 57 with a diagnosis of mild mental retardation . Since around 2 months prior she would eat rice at the previous amounts, but would refuse to eat rotis, the staple diet of the family . If persuaded, she would get irritable and start crying and go to sleep without eating anything . At times she said that she was hungry and would ask for food, and would eat rice but refuse rotis . She did not have any associated loose stools, abdominal pain, vomiting, or regurgitation . She would not demand for specific food items like chocolates, etc . ; would not appear sad, withdrawn or fearful, or have any disturbances in sleep . Over the period of 1 month, the weight reduced by about 1 kg . On further exploration, no preoccupation with body image, of previously fussiness of eating was reported by the family members . The child had a slow to warm up temperament and there was no history suggestive of eating inedible objects, delusions, hallucinations or repetitive behaviors, significant obstinacy, hyperactivity or inattentiveness . The general physical examination was within normal limits, the height and weight was over the 10 percentile and there were no signs of chronic malnutrition . Rapport could be established with difficulty and the child answered briefly only after urging from the mother and therapist . When patient returned in 2 weeks, there was no further decrement in weight and the patient started to eat all kinds of food stuffs again and to the previous amounts . Even on recovery, she did not elaborate the cause of decreased intake of certain foodstuffs and that of resumption . Multiple possibilities can be entertained in this child while evaluating a child presenting with such eating problems . The age appropriateness of such behaviors, development level aptness, sociocultural background, parenting style, and medical conditions need to be ascertained before considering whether such a behavior can be assessed as abnormal . Feeding habits in adolescents reveals deficits in calories and proteins in apparently healthy school - going children from relatively well - to - do backgrounds . With such a premise, is diagnosing the transient problems of eating as a separate disorder in this child a useful and relevant exercise, or whether these problems can be passed off as developmentally appropriate erratic behaviors? Even if a diagnostic label is required, where does the child fit in? Is the diagnosis of mental retardation sufficient to subsume these symptoms or an additional diagnosis is needed? Since the diagnostic categories of affective, psychotic, and anxiety spectrum disorders do not apply in this case, whether the problems can be better characterized as eating disorder not other specified (eating disorder nos)? However, there was no evidence of body image disturbances . Does feeding disorder of infancy and childhood better characterize this case, though the obvious atypicality being age of presentation and transient symptoms in this case; or does this case remain a many researchers have proposed alternate pragmatic classification systems for characterizing the problems of eating in childhood . Fox and joughin have described a variety of eating problems that are encountered in the childhood and adolescence . Among these, the index case exhibits symptoms closest to food avoidance emotional disorder which occurs in the age range of 5 - 16 years and is characterized by avoidance of food and emotionality in the absence of preoccupation of body weight or body image . Many children who present with behavioral problems of eating cannot be pigeonholed into diagnostic class of present nosology, but still benefit with interventions . Hence, we suggest that these disorders should be treated pragmatically after detailed assessment without being excessively concerned about the diagnostic label, at the same time not overlooking the obvious diagnostic possibilities.
Alzheimer's disease (ad) is the most common cause of dementia among the aging population . Early memory deficits and progressive loss of higher cognitive functions are common clinical features of ad patients . Pathologically, ad is characterized by insoluble aggregates of extracellular amyloid - beta (a) peptides (senile plaques) and intracellular filaments composed of hyperphosphorylated tau (neurofibrillary tangles) in the brain . Strong evidence from human genetics and transgenic mouse models has implicated a in the etiology and pathogenesis of ad . A peptides are derived from -secretase- and -secretase - mediated sequential proteolytic cleavage of the amyloid - precursor protein (app), with a140 and a142 being the most abundant species . Many human mutations associated with familial ad, such as those that are found in genes encoding app and the catalytic subunit of -secretase, presenilin (ps1 and ps2), promote amyloidogenic processing of app, leading to enhanced a production . Recent studies have shown that soluble oligomeric forms of a (ranging from dimers and trimers to dodecamers) exert potent and acute neurotoxic effects on the structure and function of synapses, including reduced excitatory synaptic transmission, loss of dendritic spines, and aberrant neuronal network activity [4, 5]. These deleterious effects could contribute to the cognitive deficit and memory loss associated with ad, indicating that synaptic failure is likely to be one of the earliest events that occurs in the pathogenesis of ad prior to neuronal loss [68]. The majority of fast excitatory synaptic transmission in the mammalian central nervous system is mediated by the release of glutamate from the presynaptic terminal and its binding to glutamate receptors on the postsynaptic membrane . The ionotropic glutamate receptors consist of ampa (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), nmda (n - methyl - d - aspartate), and kainate receptors . Among these, ampa receptors (ampars) are the principal receptors that mediate fast excitatory synaptic transmission in the mammalian brain . They are tetrameric assemblies of two dimers of four potential subunits (glua1glua4) encoded by distinct genes, gria1gria4 . The predominant ampars expressed in the hippocampal and cortical pyramidal neurons are composed of glua1/glua2 and glua2/glua3 subunits . Brief periods of high neuronal activity open nmda receptors (nmdars) and induce ca influx, leading to a long - lasting increase in synaptic efficacy, known as long - term potentiation (ltp), which is characterized by an increase in the number of ampars on the postsynaptic membrane and spine growth . In contrast, repetitive low frequency stimulation leads to the removal of synaptic ampars to produce long - term depression (ltd), that is, a decrease in synaptic strength . It has long been postulated that these forms of synaptic plasticity represent a cellular correlate of learning and memory . One of the key mechanisms underlying synaptic plasticity is the tight control of ampar number at synapses . This requires a balance between the biosynthesis (number of receptors being produced), membrane trafficking (the movement of receptors to and from the plasma membrane via exocytosis and endocytosis), and degradation of receptors (receptor turnover), all of which are dynamically regulated by ampar interacting proteins as well as by various posttranslational modifications that occur on their cytoplasmic carboxyl terminal domains [11, 12]. Aberrant trafficking of ampars usually leads to impaired synaptic plasticity and deficits in learning and memory . Importantly, several studies have demonstrated a role for a in promoting ampar endocytosis and hence synaptic depression [1316]. This review focuses primarily on nmdar and metabotropic glutamate receptor- (mglur-) mediated signaling . In particular, we highlight several mechanisms that underlie synaptic ltd as common signaling pathways that are hijacked by the neurotoxic effects of a. several pharmacological agents that target these pathways and are efficacious in inhibiting or reversing the neurotoxic effects of a on glutamatergic neurotransmission and synaptic plasticity are also discussed . The ability of neurons to modulate their synaptic strength is widely believed to be a cellular correlate of learning and memory . Nmdar - dependent ltp and ltd are two major forms of synaptic plasticity that are best studied in the hippocampus, a region of the brain that is both critical for memory formation and highly vulnerable to a toxicity . It is well established that synthetic soluble a oligomers [17, 18] or those secreted from cell lines overexpressing app acutely and potently block hippocampal ltp at high concentration . More recent studies have further shown that soluble a dimers, but not a monomers, either prepared by chemical cross - linking or extracted directly from postmortem ad brains, are extremely potent in inhibiting hippocampal ltp both in vitro and in vivo [4, 20]. Congruent with the ltp hypothesis of long - term memory, injection of these soluble a oligomers into the rat hippocampus disrupts cognitive function and learned behavior [4, 21]. Most transgenic ad mouse models overexpressing different familial ad mutations, such as tg2576 (appswe; k670n / m671l), pdapp (appind; v717f), 3xtg (appswe, tau p301l, and ps1 m146v), and 5xfad (appswe, appflorida; i716v, applondon; v717i, ps1 m146l, and ps1 l286v), generally display impairments in ltp and cognition [2226]. Notably, some ad transgenic mice show abnormal ltp and learning deficits well in advance of plaque formation [22, 27, 28]. Collectively, these results lend support to the idea that soluble oligomeric a plays a key role in disrupting synaptic plasticity . More importantly, studies performed in human subjects have also revealed deficits in ltp - like cortical plasticity in mild - to - moderate ad patients [2931]. Consistent with the fact that a induces an impairment in ltp, soluble a oligomers have been demonstrated to facilitate the expression of ltd in the hippocampus [4, 17, 32]. Although the exact mechanisms underlying a-induced ltd remain equivocal, they have been shown to involve internalization of nmda- and ampa - type glutamate receptors, dendritic spine shrinkage, and eventual synaptic loss [14, 16, 33, 34]. Dynamic trafficking of ampars to and from synapses is a critical mechanism underlying the induction of synaptic plasticity . Defects in the endocytosis and lysosomal trafficking pathways are known to contribute significantly to ad pathogenesis . Consistent with this notion, overexpression of app and a high concentration of soluble oligomeric a are able to induce the removal of surface ampars at synapses, leading to synaptic depression and inhibition of ltp [14, 19, 36, 37]. Mechanistically, these neurotoxic effects of a are mediated by high levels of glutamate at synapses as a result of a disrupted glutamate reuptake process that subsequently leads to aberrant activation of nmdars, mglurs, and the cellular prion protein (prp), as well as elevated levels of ampar ubiquitination . Activation of these signaling pathways in turn promotes synaptic depression, via common pathways shared with ltd as summarized in figure 1, which are discussed in detail in the following sections . Nmdar - dependent ltd induced by low frequency stimulation or by direct application of nmda (chemically induced ltd) triggers ca entry into the postsynaptic compartment and activates protein phosphatase 2b (pp2b, also known as calcineurin), which in turn leads to the activation of protein phosphatase 1 (pp1) [38, 39]. Pp1 and pp2b are known to mediate nmdar - induced ampar internalization by dephosphorylating the glua1 subunit of ampars at ser-845 [40, 41], a protein kinase a (pka) site that is crucial for maintaining the stability of ampars at perisynaptic sites and ltp [4244]. Nmdar - dependent ltd also induces the p38 mitogen activated protein kinase (p38 mapk) signaling pathway via the activation of rap small gtpases, leading to the removal of ampars [45, 46]. Emerging evidence demonstrates that toxic levels of a aberrantly enhance the activity of nmdars in favor of ltd induction, thereby preventing ltp [32, 37, 47]. In cultured neurons and acute brain slices, soluble oligomeric a induces excessive influx of ca through the glun2b - containing extrasynaptic nmdars, which subsequently activates the rap - p38 mapk signaling pathway, as well as the protein phosphatases, pp1 and calcineurin [13, 14, 16, 32, 37, 4850]. One of the consequences of a-induced activation of calcineurin is reduced phosphorylation of ser-845, which induces ampar endocytosis and impairs the synaptic incorporation of these receptors . Consistent with this finding, appswe, ind transgenic mice display lower levels of ser-845 phosphorylation, a phenomenon that correlates well with the loss of ampars on the cell surface and deficits in initial learning and memory . Another key substrate of pp1 the activity of which is required for the expression of nmdar - dependent ltd is glycogen synthase kinase-3 (gsk3). Pp1 can activate gsk3 by a direct dephosphorylation mechanism, as well as via the modulation of the upstream caspase akt signaling pathways, which are also crucial for ampar internalization and ltd [51, 52]. Interestingly, both gsk3 and caspases are enzymes that have been widely implicated in ad . Indeed, it has been demonstrated that inhibition of ltp by a is mediated by the caspase 3, akt1, and gsk3 signaling pathway [32, 53]. Paradoxically, however, gsk3 activity has also been reported to play a role in maintaining ampar synaptic expression under basal conditions as its inhibition leads to the loss of surface ampar expression by controlling the rate of ampar internalization . However, during nmdar - dependent ltd, gsk3 may preferentially phosphorylate other substrates including the key scaffolding protein in excitatory synapses, postsynaptic density-95 (psd-95). Psd-95 stabilizes ampars at synapses through its interaction with transmembrane ampar regulatory proteins (tarps), auxiliary subunits of ampars . Overexpression of psd-95 promotes synaptic maturation and enhances synaptic strength, whereas psd-95 knockdown results in the opposite effects [5660]. It appears that gsk3 phosphorylation of psd-95 at thr-19, following its dephosphorylation at ser-295 by pp1, destabilizes and mobilizes psd-95 away from the psd, resulting in increased ampar internalization [61, 62]. Whether or not the phosphorylation status of psd-95 is modulated by oligomeric a via the gsk3 and pp1 signaling pathways remains to be determined . Gsk3 is also a major kinase that phosphorylates the microtubule - associated protein tau [63, 64]. A causes tau hyperphosphorylation and mislocalization from axons to somatodendritic compartments, where it accumulates and mediates a-induced downregulation of surface ampars [6568]. Recent studies have shown that nmdar - induced gsk3 phosphorylation of tau at ser-396 is required for hippocampal ltd by enhancing the interaction between the glua2 subunits of ampars with the protein interacting with c - kinase 1 (pick1) [69, 70], a process that is fundamental for ampar internalization and/or intracellular retention during ltd [7176]. Furthermore, phosphorylation of pick1 by gsk3 at ser-416 has also been reported to augment this interaction . Glua2 can be phosphorylated by protein kinase c (pkc) at ser-880 and by the protein tyrosine kinase of the sarcoma (src) family at tyr-876, both of which are required for ampar internalization and ltd [7880]. Glua2 phosphorylation at these sites differentially regulates the interaction of the subunit with pick1 and glutamate receptor interacting proteins (grip) 1 and 2 [80, 81]. Grip1 plays an important role in stabilizing ampars at synapses and is essential for ltd [72, 79]. Given that phosphorylation of glua2 weakens the interaction of the subunit with grip1, but not pick1, it has been postulated that ltd involves destabilization and detachment of glua2 from synapses, allowing ampars to be internalized . In accord with the role of a in inducing aberrant ampar endocytosis, one study has observed that oligomeric a increases pkc - mediated phosphorylation of glua2 at ser-880 and subsequently reduces surface expression of ampars in cultured hippocampal neurons . More importantly, several molecular and pharmacological manipulations that inhibit glua2 internalization potently prevent a-induced synaptic depression and rescue memory impairment in ad mice . These include the glua2-r845a mutant, glua2 - 3y peptides, and a small molecule pick1 inhibitor . A new mechanism underlying the pathological action of a that involves the cyclin - dependent kinase 5- (cdk5-) activating peptide, p25, has recently been described by seo et al . . Elevated levels of p25 have been implicated in many neurodegenerative diseases, including ad . In their study, seo et al . Found that a induces calpain - mediated cleavage of p35 into p25 in the hippocampus, a process that requires the activity of glun2b - containing nmdars and ca / calmodulin - dependent protein kinase ii (camkii). The a-induced elevation in p25/cdk5 activity subsequently enhances the phosphorylation of dopamine- and cyclic adenosine monophosphate - regulated neuronal phosphoprotein (darpp-32) at thr-75, thereby inhibiting the activity of pka . In a synergistic manner, a also triggers dephosphorylation of darpp-32 at thr-34, presumably by calcineurin, thereby releasing its inhibition on pp1 [86, 87]. These converging mechanisms eventually lead to the loss of glua1 phosphorylation at ser-845 and induce ampar internalization and synaptic depression . Remarkably, genetic inhibition of p25 generation rescues ltp and memory deficits in 5xfad transgenic mice . In addition to promoting the internalization of ampars, oligomeric a can also act through mechanisms that prevent the forward trafficking of ampars towards the plasma membrane . A has been shown to cause aberrant redistribution of camkii from the synaptic to the cytosolic fraction both in cultured neurons and in the brain of appswe transgenic mice . Camkii can potentiate ampar - mediated transmission via (a) phosphorylation of glua1 at ser-831 to enhance ampar channel conductance, (b) phosphorylation of the tarp, stargazin, to facilitate synaptic recruitment of ampars, and (c) potentiation of the ras - erk (extracellular signal - regulated kinase) pathway to promote ampar insertion into the plasma membrane [45, 8991]. Consistent with the role of camkii in synaptic potentiation, exposure of soluble a oligomers reduces surface glua1 clusters in cultured neurons, concomitant with decreased ampar synaptic responses in cortical pyramidal neurons recorded from acute brain slices of appswe transgenic mice . A has been shown to interact with nmdars [92, 93] and to reduce their surface expression through endocytosis . A-induced internalization of nmdars involves dephosphorylation of the glun2b subunit at tyr-1472 by step61 (striatal - enriched protein tyrosine phosphatase 61), the expression of which is upregulated in several ad mouse models, as well as in the postmortem prefrontal cortex of ad patients [33, 9496]. The fact that a enhances the internalization of nmdars seems counterintuitive given the role of nmdars in mediating ampar endocytosis, spine loss, and ultimately excitotoxicity in neurons . Recent studies on the putative oligomeric a receptor, prp, have provided insights into two potential mechanisms that regulate nmdar function [97, 98]. Firstly, soluble oligomeric a binding to prp activates the tyrosine kinase fyn, which initially phosphorylates glun2b and transiently enhances nmdar function, before the step61 level increases and dephosphorylates glun2b . Secondly, a disrupts the ability of prp to limit excessive nmdar activity in a copper - dependent manner, potentially by chelating copper ions and preventing them from binding to prp, thereby producing large nondesensitizing steady - state nmdar currents . Albeit controversial, loss of prp function has been reported to prevent a-induced ltp and memory impairment in mice [98, 101105]. Despite this, the role of prp in regulating ampar trafficking has not been directly examined . Recent studies by kessels and colleagues have challenged the central role of nmdar - mediated ca influx in a-induced synaptic depression . It is well established that the neurotoxic effects of oligomeric a on synapses can be blocked by the nmdar antagonist, d - apv (d-2-amino-5-phosphonopentanoic acid), which prevents glutamate binding and blocks the activation of nmdars . However, noncompetitive nmdar antagonists that block ion flow through the receptor, such as mk-801, ketamine, and 7-chlorokynurenic acid, are not able to rescue a-mediated synaptic depression [106, 107]. A similar finding consistent with the idea that a operates through shared pathways with ltd, metabotropic, but not ionotropic, nmdar function has been shown to be required for nmdar - dependent ltd in the hippocampus by activating the p38 mapk signaling pathway . In fact, ligand binding to the extracellular domain of nmdars induces conformational change and movement of their cytoplasmic tails, allowing pp1 to dephosphorylate camkii together with other signaling molecules that contribute to synaptic depression [110, 111]. Although the role of metabotropic nmdars remains controversial, it does offer an explanation for the fact that the fda - approved nmdar antagonist, memantine, has poor efficacy in treating early - stage ad . Further research is warranted, as delineating the metabotropic nmdar signaling pathway may shed light on new strategies for the development of future ad drugs . Mglurs belong to the g - protein - coupled receptor superfamily that modulates neuronal excitability, synaptic transmission, and plasticity in the central nervous system . Group i mglurs, which consist of two members, mglur1 and mglur5, predominantly localize to the postsynaptic membrane and are canonically coupled to gq/11 to activate phospholipase c (plc) that catalyzes the hydrolysis of phosphoinositides into inositol 1,4,5-triphosphate (ip3) and diacylglycerol (dag). Subsequently, these second messengers trigger the release of ca from intracellular stores and activate pkc, respectively . Group i mglurs, and more specifically mglur5, are the predominant receptors that mediate mglur - dependent ltd in the hippocampus and have been widely implicated in ad . It is well established that mglur - dependent ltd requires the internalization of glua2-containing ampars, leading to a long - term reduction in the number of surface ampars [115, 116]. One of the mechanisms that regulates mglur - induced ampar endocytosis involves the phosphorylation of glua2 at ser-880 by pkc, a process that is facilitated by pick1 [117119]. However, in the ca1 region of the hippocampus, internalization of ampars does not require pkc but instead relies on the dephosphorylation of glua2 at tyr-876 by step61 [120122]. Dephosphorylation of glua2 stimulates the binding of brag2 (brefeldin resistant arf gef 2), which in turn activates the small gtpase arf6 through augmentation of its gef (guanine - nucleotide exchange factor) activity and promotes ampar endocytosis . In accordance with this model genetic deletion of step61 restores the number of ampars on the postsynaptic membrane, enhances ltp, and improves cognitive function in ad mice [95, 123]. A new small molecule inhibitor of step61, tc-2153, like nmdar - dependent ltd, mglur - mediated ltd also involves the rap1-p38 mapk signal transduction pathway to facilitate ampar internalization via the formation of the gdi - rab5 complex [125127]. In addition, a role for erk in mglur - dependent ltd has also been reported . One unique feature of mglur - dependent ltd is its requirement for rapid translation of preexisting mrnas (local protein translation) in dendrites . Mglur - dependent de novo protein synthesis can be regulated through multiple pathways, including the pi3k - akt - mtor (mammalian target of rapamycin) and erk signaling pathways that converge on the initiation and elongation factors of protein translation . Several mrna encoding proteins that regulate ampar trafficking are locally translated during mglur - dependent ltd, including the activity - regulated cytoskeleton - associated protein (arc), microtubule - associated protein 1b (map1b), and step [121, 131133]. Given that grip1 stabilizes ampars at synapses, the newly synthesized map1b may sequester grip, hence loosening its interaction with glua2 . On the other hand, arc interacts with the endocytic proteins, endophilin and dynamin, and is able to enhance dynamin polymerization and gtpase activity, thereby promoting ampar endocytosis [135, 136]. Interestingly, soluble oligomeric a rapidly induces arc expression in neurons, which may contribute to the loss of ampars from the plasma membrane . Moreover, arc also regulates the endosomal trafficking of app and bace1, as well as ps1, a mechanism that is essential for the activity - dependent production of a in the brain, and genetic deletion of arc reduces the a load in appswe;ps1e9 transgenic ad mice . This may serve as a positive feedback mechanism underlying the overproduction of a in the pathophysiology of ad . Studies from several laboratories have implicated the mglur - dependent signaling pathway in the neurotoxic effects of a on synaptic function [4, 14, 139143]. Notably, genetic and pharmacological inhibition of mglur5 prevents oligomeric a-induced impairment in ltp, spine loss, and cognitive deficits in ad mouse models [139, 142145]. More recently, a seminal study by strittmatter and colleagues identified an interaction between mglur5 and prp, which together act as a coreceptor for oligomeric a . They also revealed an essential role for mglur5 and prp coupling in the pathology of ad . Mechanistically, mglur5 links prp to key intracellular signaling molecules, such as homer1b / c, pyk2, fyn, and camkii, all of which play major roles in synaptic plasticity [144, 146, 147]. When neurons are exposed to oligomeric a, the prp - mglur5 complex mediates the aberrant activation of pyk2, fyn, and camkii, causing altered neuronal states that lead to impaired ltp [99, 144, 146]. It is interesting to note that a also induces a biphasic alteration in camkii activity, resembling that of fyn, in a prp - mglur5-dependent manner, and that this is accompanied by the increased association of mglu5 with camkii . Given that mglur5 activation enhances nmdar forward trafficking through camkii - mediated phosphorylation of glun2b at ser-1303, it is hypothesized that a-induced enhancement of the association between mglur5 and camkii may prevent synaptic potentiation . Furthermore, pharmacological activation of mglur5 in the presence of prp causes a redistribution of camkii into the cytoplasm, which may have an impact on ampar trafficking . Interestingly, the cross talk between mglur5 and nmdar signaling is bidirectional . Not only can mglur5 potentiate nmdar currents through camkii and pkc signaling pathways [148, 149], but also activation of nmdars can potentiate mglur5 responses under physiological conditions [150, 151]. This involves the nmdar - dependent activation of calcineurin that dephosphorylates mglur5 and reduces receptor desensitization . However, a high concentration of nmda can induce pkc - dependent mglur5 phosphorylation and inhibit mglur5 responses . Although the interaction of mglur5 and nmdars has been implicated in synaptic plasticity and various animal behaviors [153156], their alteration in the presence of a binding to prp and how this impacts on ampar trafficking remain unclear . Posttranslational ubiquitination, a regulatory signal that controls protein trafficking and turnover, has recently emerged as an important mechanism that regulates ampar function [157, 158]. All ampar subunits undergo activity - dependent ubiquitination in cultured neurons, a process that is ca - dependent and requires the activity of l - type voltage - gated ca channels [159161]. The primary e3 ligases that catalyze the ubiquitination of glua1 and glua2 subunits are nedd4 - 1 and rnf167, respectively [160, 162]. While the role of protein ubiquitination on the glua1 and glua2 subunits in ligand - induced ampar endocytosis remains controversial, it is well accepted that ubiquitination of ampars regulates the intracellular sorting of receptors into late endosomes for degradation [159161, 163]. Under normal conditions, the degradation of ampars is required for protein homeostasis to ensure turning over of old or used receptors in order to maintain healthy levels of ampars in neurons . However, when the ubiquitin pathway is hijacked (e.g., by elevated levels of a), there is an excessive downregulation of ampars and synaptic depression . Indeed, a new finding has demonstrated a role for naturally secreted and synthetic a in promoting the ubiquitination of ampars by nedd4 - 1 . Interestingly, knocking down nedd4 - 1 rescued a-induced synaptic deficits, including reduced glutamatergic synaptic transmission, decreased levels of surface ampars, and the loss of dendritic spines . These findings have important implications in targeting ubiquitin e3 ligases as potential drug targets for the treatment of ad . Research over the past decade has provided strong evidence that the cognitive deficit associated with ad is caused by the neurotoxic effects of soluble a oligomers on synaptic function . Increasing evidence indicates that the trafficking of ampars, which is essential to multiple forms of synaptic and structural plasticity in the brain, is aberrantly dysregulated by oligomeric a and manifests as impairments in ltp, learning, and memory . It is particularly encouraging to learn that pharmacological and genetic manipulations that block endocytosis or enhance the forward trafficking of ampars can rescue ltp and reverse cognitive deficits in ad mice . Given that a-induced ampar internalization requires the same adaptor proteins as the conventional trafficking pathway, it will be challenging to minimize unwanted side effects . Hence, further research is needed to identify specific targets for improving the memory deficits associated with ad . Rapid progress has been made in delineating the molecular mechanisms and signaling pathways underlying the loss of ampars from the plasma membrane induced by oligomeric a (figure 1). The discovery of prp as a receptor for soluble a oligomers that signals through nmda and mglur5 receptor has underscored the importance of glutamatergic signaling in the etiology of ad . It is likely that these receptors act cooperatively to mediate the synaptotoxic effects of a, highlighting the need for further investigation of the associated signaling mechanisms with a view to developing more effective therapeutic strategies for the treatment of ad.
Chronic suppurative otitis media (csom) is defined as long - standing chronic suppuration of the middle ear cleft and its mucoperiosteal lining resulting in discharging ear and deafness . Chronic suppuration can occur with (tympano - mastoid type) or without (tubo - tympanic type) cholesteatoma . The tubo - tympanic type is the usually safe variety and follows a benign course . It usually occurs as a complication of acute otitis media, where there is perforation of the tympanic membrane . Bacterial pathogens in the tubo - tympanic type csom vary considerably, and can be a combination of both aerobic and anaerobic bacteria . In csom, the bacteria may be aerobic (e.g., escherichia coli, staphylococcus aureus, streptococcus pyogenes, proteus mirabilis, klebsiella species, escherichia species, haemophilus influenzae, etc .) Or anaerobic (e.g., bacteroides, peptostreptococcus, proprionibacterium, etc.). [24] bacteriological cultures may not be needed to establish the diagnosis of csom as exhaustive studies have established that 90100% of chronic draining ears yield two or more isolates consisting of both aerobic and anaerobic bacteria . The choice of the antibiotic agent depends greatly on the knowledge of the type of bacteria most frequently implicated in csom and their sensitivity to antibiotics . Since their introduction, systemically acting fluoroquinolones have opened up new therapeutic possibilities for using an orally administered antibiotic in the treatment of csom . Ciprofloxacin, in particular, has proven to be very active in vitro against a large number of gram - negative and gram - positive organisms . Moreover, ciprofloxacin concentrations within the structures of the middle ear have been shown to exceed the minimum inhibitory concentration (mic) for the microorganisms responsible for csom . Cefpodoxime, an oral third - generation cephalosporin, has good antimicrobial activity against aerobic gram - positive and gram - negative bacteria . There are few studies that have evaluated the effectiveness of cephalosporins like cephalexin and ceftriaxone in acute exacerbation of chronic suppurative otitis media (aecsom), and results have shown that they can be considered as an effective agent for the treatment of aecsom . Against this backdrop, the present study was planned to compare the effectiveness and safety of cefpodoxime with ciprofloxacin in aecsom . The objective of this study was to demonstrate equivalence between cefpodoxime (test drug) and ciprofloxacin (comparator) with respect to their effectiveness in aecsom . The study was approved by the institutional ethics committee and conducted according to the icmr guidelines for biomedical research on human subjects, 2006, and the declaration of helsinki . Subjects were recruited in the ent outpatient department of a tertiary care teaching hospital and the study was conducted between june 2011 and october 2011 . The study was registered under clinical trials registry - india (registration number - ctri/2011/10/002079). The objective of the study was to demonstrate equivalence in the effectiveness between the two treatment groups . A difference of 10% in clinical cure rates was assumed to be the largest clinically acceptable effect for which equivalence could be accepted (equivalence limit). Considering the true mean difference between the two treatment groups as zero and the expected standard deviation of 10% in the study population, 90% power and = 0.05, the number of subjects required in each treatment group was 21 . The sample size was calculated by using primer of biostatistics software (version 5.0). Clinically documented cases of tubo - tympanic - type csom, defined as long - standing chronic suppuration of middle ear cleft and its muco - periosteal lining resulting in discharging ear and deafness presenting with clinical symptoms and signs of acute exacerbation of the disease and baseline otological symptom score [table 1] of> 4 but 8 were included in the study . Severe cases of aecsom for which hospitalization or parenteral antibiotic treatment is required and patients with otological symptom score of 4 and> 8 were also excluded from the study . Patients with foul - smelling discharge and those who received antibiotic in the preceding 4 weeks of screening were left out . Number of subjects achieving treatment success in each treatment group was considered to be the effectiveness parameter . Treatment success was based on changes in the otological symptoms scores at day 14 visit . It was subdivided into two categories: (a) clinical cure if the otological symptom score was <3 at day 14 visit or (b) clinical improvement if the otological symptom score was between 3 and 5 on day 14 . Treatment failure was declared if there was no change or increase in the baseline otological symptom score on day 14 . Patients were evaluated clinically at baseline (day 0) and at subsequent follow - up visits on days 3, 7 and 14 . Mumbai, india) tablet (200 mg) and group b received ciprofloxacin (ranbaxy laboratories ltd . Study medications were dispensed twice during the study period: first during baseline visit for 3 days and next during day 3 visit for the next 4 days . Patients were advised to take each of their respective study medications orally twice daily after food for the first 7 days . Compliance was assessed by the traditional pill count method at each follow - up visit and at the end of the study . All patients were advised to stop smoking and consumption of alcohol during the study period . All aes spontaneously reported by the subjects or elicited by the investigators were recorded and causality analysis was done as per the world health organization - uppasala monitoring centre (who - umc) criteria . Data in ordinal scale were analyzed by friedman's test for intragroup comparison and by mann adults of either sex, aged between 18 and 60, were considered . Clinically documented cases of tubo - tympanic - type csom, defined as long - standing chronic suppuration of middle ear cleft and its muco - periosteal lining resulting in discharging ear and deafness presenting with clinical symptoms and signs of acute exacerbation of the disease and baseline otological symptom score [table 1] of> 4 but severe cases of aecsom for which hospitalization or parenteral antibiotic treatment is required and patients with otological symptom score of 4 and> 8 were also excluded from the study . Patients with foul - smelling discharge and those who received antibiotic in the preceding 4 weeks of screening were left out . Number of subjects achieving treatment success in each treatment group was considered to be the effectiveness parameter . Treatment success was based on changes in the otological symptoms scores at day 14 visit . It was subdivided into two categories: (a) clinical cure if the otological symptom score was <3 at day 14 visit or (b) clinical improvement if the otological symptom score was between 3 and 5 on day 14 . Treatment failure was declared if there was no change or increase in the baseline otological symptom score on day 14 . Patients were evaluated clinically at baseline (day 0) and at subsequent follow - up visits on days 3, 7 and 14 . Mumbai, india) tablet (200 mg) and group b received ciprofloxacin (ranbaxy laboratories ltd . Study medications were dispensed twice during the study period: first during baseline visit for 3 days and next during day 3 visit for the next 4 days . Patients were advised to take each of their respective study medications orally twice daily after food for the first 7 days . Compliance was assessed by the traditional pill count method at each follow - up visit and at the end of the study . All patients were advised to stop smoking and consumption of alcohol during the study period . All aes spontaneously reported by the subjects or elicited by the investigators were recorded and causality analysis was done as per the world health organization - uppasala monitoring centre (who - umc) criteria . Data in ordinal scale were analyzed by friedman's test for intragroup comparison and by mann of the 55 subjects screened, 46 fulfilled the selection criteria and were randomized - 23 to group a (cefpodoxime) arm and 23 to group b (ciprofloxacin). One subject was lost during follow - up in group b and did not attend the hospital after the first visit . The mean age of patients was 35.8 years and 28.1 years in the cefpodoxime and ciprofloxacin groups, respectively . 69.5% were male in the cefpodoxime group and 56.5% were male in the ciprofloxacin group . The consort flowchart there was no statistically significant difference in the baseline demographic profile and baseline otological symptom score . Changes in otological symptom score from baseline have been shown in figures 2 and 3 and table 2 . Changes in otological symptom score in the cefpodoxime group (group a).p <0.05- days 3, 7, 14 vs day 0 changes in otological symptom score in the ciprofloxacin group (group b). P <0.05- days 3, 7, 14 vs day 0 intergroup comparison of otological symptom score intragroup analysis of otological symptom score at baseline (day 0) against day 3, day 7 and day 14 scores showed a highly significant decrease in both groups [figures 2 and 3], and there was a clinically significant improvement in the signs and symptoms of the aecsom . Thus, it can be suggested that both cefpodoxime and ciprofloxacin are effective in the treatment of aecsom . An intergroup analysis of the otological symptom scores showed that there was no statistically significant difference in the baseline, day 3 and day 7 and day 14 otological symptom score [table 2]. Therefore, it can be suggested that both cefpodoxime and ciprofloxacin are equally effective in the treatment of aecsom . Table 3 shows the number and the percentage of patients categorized as treatment success or treatment failure twenty - two subjects of the 23 enrolled in the cefpodoxime group achieved treatment success, i.e. Either clinical improvement or clinical cure, and the remaining one subject was categorized as treatment failure . Similarly, in the ciprofloxacin group, 20 subjects of the 22 evaluated showed treatment success, i.e. Either clinical improvement or clinical cure, and the remaining two were categorized as treatment failure . Intergroup comparison of the percentage of subjects who were categorized as treatment success showed no statistically significant difference (p = 0.25). Comparison of treatment success rates there were one patient in the cefpodoxime group and two patients in the ciprofloxacin group who were categorized as treatment failure and had to be put on other antibiotics after the day 7 evaluation . Safety analysis was carried out as per the intention to treat (itt) analysis . These aes were mild in nature and did not require any dose reduction or withdrawal of the study medications . Therefore, the safety and tolerability profile of both the study drugs were good without any reported cases of serious ae . Several published studies have shown the effectiveness of ciprofloxacin for the treatment of aecsom in adult patients . Few studies have also proved the effectiveness of cephalexin and ceftriaxone in adult patients with aecsom . But, there is no published comparative controlled trial that evaluated ciprofloxacin with an oral third - generation cephalosporin in adult patients with aecsom . The results of our study showed that cefpodoxime and ciprofloxacin are equally effective in clinically diagnosed cases of aecsom, both in terms of effectiveness and in terms of safety . After treatment with a 7-day course, the clinical success rates were comparable, i.e. 95.6% in the cefpodoxime group and 90.9% in the ciprofloxacin group . These aes were nonserious in nature and did not require dose modification or withdrawal of drug therapy . Baba et al . Conducted a clinical trial that showed that ceftriaxone was effective in 65% patients with acute otitis media and 72% patients with aecsom . Another trial by baba et al . Reported that cephalexin has a 35.5% failure rate in patients with aecsom . Clinical cure rate with cefpodoxime in our study is significantly higher compared with those two trials with other cephalosporins . Very few studies have evaluated the effectiveness of oral third - generation cephalosporins in patients with otitis media . A study reported by block et al . Compared the effectiveness of cefdinir with cefprozil, and the results showed that the overall clinical cure rate with cefdinir was 80% versus 82.5% with cefprozil in the treatment of pediatric acute otitis media . Another study by kafetzis showed that the overall clinical cure rate with cefixime was 85% in patients with acute otitis media . Our study suggests that cefpodoxime has higher cure rates (95.6%) compared with other oral third - generation cephalosporins a double - blind study could not be conducted due to financial constraints and logistic problems . Secondly, we did not perform bacteriological culture of the cases as exhaustive studies have established that 90100% of chronic draining ears yield two or more isolates consisting of both aerobic and anaerobic bacteria . Another reason is that, very often, clinicians start antimicrobial therapy at outpatient setting before the bacteriological culture report arrives, which takes about 72 h. therefore, we conducted this study mainly to provide information to clinicians on the comparative effectiveness of these two antibiotics as initial antibiotics for aecsom patients based on clinical assessment scores . The results of this study demonstrated that a 7-day course of cefpodoxime is comparable to ciprofloxacin in terms of both clinical effectiveness and safety for the treatment of aecsom in an outpatient setting . Although cost of drug therapy was higher with cefpodoxime compared with ciprofloxacin, it should not be considered as a drawback . Future trials are warranted to evaluate the bacteriological cure and relapse rates of these two drugs to provide additional supportive scientific evidence.
To report surgical results of canaloplasty surgery for primary open - angle glaucoma (poag) in japanese patients . Three eyes of 3 patients underwent canaloplasty alone and 8 eyes of 6 patients underwent canaloplasty combined with cataract surgery . Canaloplasty was performed with a 10 - 0 polypropylene tensioning suture and an itrack 250a microcatheter . Mean number of antiglaucoma drops was 2.8 0.6 before canaloplasty and decreased to 1.2 0.8 at 12 months after canaloplasty (p <0.01). Mean iop decreased postoperatively, being 13.7 2.8 mm hg at 1 month, 12.8 3.5 mm hg at 3 months, 14.0 4.4 mm hg at 6 months, and 15.0 4.1 mm hg at 12 months . The most frequent postoperative complication was mild hyphema (45.5%), which disappeared within 14 days after surgery . Canaloplasty may be an alternative surgery for poag patients to reduce iop to a value of approximately 15 mm hg . Canaloplasty is a new nonpenetrating glaucoma surgery that is expected to increase physiological aqueous outflow from the anterior chamber through the trabeculo - descemet's window into schlemm's canal [1, 2]. Using the flexible itrack 250a microcatheter (iscience interventional, menlo park, calif ., usa), designed especially for canaloplasty, 10 - 0 polypropylene tensioning suture is installed into schlemm's canal circumferentially . We studied the surgical results of canaloplasty surgery for japanese primary open - angle glaucoma (poag) patients . Nine patients (11 eyes) of poag treated at toyama university hospital between december 2008 and june 2009 were included in this study . Three eyes of 3 patients underwent canaloplasty alone and 8 eyes of 6 patients underwent canaloplasty combined with phacoemulsification / aspiration and intraocular lens implantation (pea + iol). Written informed consent was obtained from each of the patients after they were provided with sufficient information about the procedures . The mean age of the patients was 71.7 8.5 years (mean standard deviation). Surgical techniques were as follows . Briefly, after limbal conjunctival peritomy, a superficial parabolic flap approximately 4 mm wide and 5 mm long was created at the 12 o'clock limbus . After injection of viscoelastic material into the ostia of schlemm's canal, 10 - 0 polypropylene tensioning suture was installed using an itrack 250a microcatheter . The iop was measured at 1, 3, 6, and 12 months postoperatively . Mean iops were 13.7 2.8 mm hg at 1 month, 12.8 3.5 mm hg at 3 months, 14.0 4.4 mm hg at 6 months, and 15.0 4.1 mm hg at 12 months (fig . Statistical analysis was carried out by a paired t test and dunn's multiple comparison analysis . All postoperative mean iops were significantly decreased compared to the preoperative iop of 23.4 5.5 mm hg (p <0.001 or p <2 shows an image of the anterior segment optical coherence tomography (casia; tomey corporation, nagoya, japan), which indicated the tensioned and dilated schlemm's canal (arrows) at 12 months after canaloplasty surgery . A kaplan - meier survival model was used for cumulative probability of qualified success with or without medications (fig . The qualified success rate of an iop of 21, 18, and 16 mm hg or less at 12 months was 81.8, 54.5, and 54.5%, respectively (fig . The mean postoperative number of antiglaucoma drops was significantly reduced to 1.2 0.8 (p <0.01). The most frequent postoperative complication was mild hyphema (45.5%), which disappeared within 14 days after the surgery without detrimental effect . We showed a similar mean postoperative iop of 15.0 4.1 mm hg at 12 months after canaloplasty . Because this was a retrospective study canaloplasty reduced the mean postoperative number of antiglaucoma drops and it may be suitable to reduce the iop to a value of approximately 15 mm hg . Mild hyphema was the most frequent postoperative complication (45.5%). During the installation of the itrack 250a microcatheter into schlemm's canal, mild bleeding from the trabecular tissue into the anterior chamber koch et al . Reported that anterior chamber hemorrhage with hyphema was found in 15 of 21 eyes (71.4%) on day 1 after canaloplasty, and the eyes without hyphema showed higher iop than those with hyphema . It is possible that microperforation may be one of the mechanisms of iop reduction by canaloplasty surgery . Although long - term results of canaloplasty have not been clarified yet, canaloplasty may be an alternative method for japanese patients with poag when the desired pressure is around 15 mm hg.
The studies included in stage 1 were drawn from 11 epidemiological studies of prostate cancer among african american men . These studies included: the multiethnic cohort (mec; 1,094 cases /1,096 controls), the southern community cohort study (sccs, 212/419), the prostate, lung, colorectal, and ovarian cancer screening trial (plco, 286/269), the cancer prevention study ii nutrition cohort (cps - ii, 76/152), prostate cancer case - control studies at md anderson (mda, 543/474), identifying prostate cancer genes (ipcg, 368/172), the los angeles study of aggressive prostate cancer (laapc, 296/303), prostate cancer genetics study (cap genes, 75/85), case - control study of prostate cancer among african americans in washington, dc (dcpc, 292/359), king county (washington) prostate cancer study (kcpcs, 145/81), and the gene - environment interaction in prostate cancer study (gecap, 234/92). Stage 2 included 1,396 cases and 2,383 controls from 7 studies: san francisco bay area prostate cancer study (sfpcs, 86/37), the flint men s health study (fmhs, 135/353), the multiethnic cohort / los angeles county (mec - la, 554/557), north carolina prostate cancer study (ncpcs, 214/249), wake forest university prostate cancer study (wfpcs, 59/66), washington university prostate cancer study (wupcs, 75/153), and the ghana men s health study (ghs, 271/968). Stage 3 included 484 cases and 947 controls from 3 studies: the study of clinical outcomes, risk and ethnicity (score, 152/280), prostate - genetique - recherche - senegal (progrs, 86/414) and prostate cancer in a black population (pcbp, 246/253). Detailed information about the design and organization of each study is provided in the supplementary note . Genotyping in stage 1 (3,621 cases and 3,502 controls) was conducted using the illumina infinium human1m - duo . Samples (n=408) were removed based on the following exclusion criteria: 1) unknown replicates across studies, 2) call rates <95%; 3)> 10% mean heterozygosity on the x chromosome and/or <10% mean intensity on the y chromosome, 4) ancestry outliers, and; 5) samples that were related (discussed below). The concordance rate for 158 replicate samples was 99.99% . Starting with 1,153,397 snps, we removed snps with <95% call rate, mafs <1%, or> 1 qc mismatch based on sample replicates (n=105,411). We used plink to calculate the probabilities of sharing 0, 1, and 2 alleles (z = z0, z1, z2) across all possible pairs of samples to determine individuals who were likely to be related to others within and across studies . We identified 167 pairs of related subjects (mz twin, parent - offspring, full and half - sibling pairs), based on the values of their observed probability vector z being within 1 sd of the expected values of z for their respective relationship . The criterion for removal was such that individuals that were connected with a higher number of pairs were chosen for removal . In all other cases, the eigenstrat software was used to calculate eigenvectors that explained genetic differences in ancestry among samples in the study . We included data from both hapmap populations (ceph (utah residents with ancestry from northern and western europe) (ceu), japanese in tokyo, japan (jpt), yoruba in ibadan, nigeria (yri), and african ancestry in southwestern u.s . (asw)) and our study, so that comparisons to reference populations of known ethnicity could be made . A total of 2,546 ancestry - informative snps from the illumina array were selected based on low inter - marker correlation and ability to differentiate between samples of african and european descent . An individual was subject to filtering from the analysis if his value along eigenvector 1 or 2 was outside of 4 sds of the mean of each respective eigenvector . Eigenvector 1 was highly correlated (=0.997, p<1 10) with percentage of european ancestry, estimated in hapmix . Genotyping in the stage 2 and 3 studies was conducted using the taqman allelic discrimination assay . In stage 2, we removed samples missing data for> 3 snps (n=36). To assess genotyping reproducibility each study included replicate samples; the concordance was> 98% for each snp within each study . Snp rs13116912 deviated from hwe in all but one of the stage 2 studies and was removed from the stage 2 analysis . No other snp deviated from hwe (i.e. P<0.01 in> 2 studies) in stage 1 or 2 . The call rate for rs7210100 was very high in stage 1 (99.9%) and similar in cases (99.9%) and controls (99.9%). The call rate for this snp was also very high in stages 2 (99.8% overall, 99.9% in cases and 99.8% in controls) and 3 (96.1% overall, 97.3% in cases and 95.5% in controls). Bi - directional sequencing of rs7210100 and the 5 coding exons of znf652 was performed in 48 subjects (20 homozygous for the risk variant, 20 heterozygous for the risk variant and 8 homozygous for the wild - type allele .) Primers were designed at least 50 bases upstream and downstream from each exon . In stage 1, we tested the association of each snp and prostate cancer risk using a 1-d.f . Likelihood ratio test from a logistic regression analysis adjusted for age, study and the first 10 eigenvectors estimated by principal components analysis . Over - inflation of the test statistic was examined with and without adjustment for ancestry and visualized with quantile - quantile plots . Lambdas were estimated as the median of the test statistics divided by 0.456 (the median of the 1-d.f . Age - adjusted odds ratios (or) and 95% confidence intervals (95% ci) for each snp were estimated from the same logistic regression model . At each locus and for each participant, local ancestry was defined as the estimated number of european chromosomes (continuous between 02) carried by the participant, estimated via the hapmix program . Local ancestry at the 17q21 locus was evaluated as a confounder in the analysis of rs7210100 . Phased haplotype data from the founders of the ceu and yri hapmap phase 2 samples were used to infer ld patterns in order to impute untyped markers . The rsq metric was used as a threshold in determining which snps to filter from analysis (rsq<0.3). Imputed snps in the 17q21 risk region, as shown in figure 2, were examined in association with prostate cancer risk as described for typed snps above . In stage 2, the snps were analyzed using logistic regression controlling for age and study (in the pooled analysis). Information regarding european ancestry was available for 7 studies included in stages 2 and 3 . As observed in stage 1 (supplementary table 2) the or for rs7210100 was similar with and without adjustment for estimated european ancestry in these studies (supplementary table 4). The results for rs7210100 in stage 2, stage 3 and stages 1 + 2 + 3 are presented without adjustment for ancestry . Association testing in the stage 2 and stage 3 studies was performed using logistic regression, adjusting for age and study . For seven of the replication studies, information about global european ancestry was available and examined as a confounding factor for variant rs7210100 . For rs7210100, effect modification by age and first - degree family history of prostate cancer was assessed in stratified analyses, and significance determined comparing the model with and without the cross - product term using a likelihood ratio test . We also examined the association of rs7210100 genotype with stage, gleason score as well as the combination of stage and grade, with advanced disease defined as gleason score8 or stage 2 (non - localized disease) and non - advanced disease defined as gleason score<8 and stage=1 (localized disease). Case - only analysis was used to test for differences in the association of rs7210100 with disease phenotypes . The association of rs7210100 with least - squares geometric mean psa levels was examined using multiple linear regression adjusting for age, body mass index and study . We estimated the risk ratio between populations of different ancestral origin (african / european) due to rs7210100 as rr= [(1-pa)+2pa(1-pa)rr1+parr2]/(1-pe)+2pe(1-pe)rr1+perr2]. Here pa is the risk allele frequency in african origin populations, pe is the risk allele frequency in european populations and rr1 is the relative risk associated with carrying 1 copy of the risk allele (compared to none) and rr2 is the relative risk associated with carrying 2 copies of the risk allele . We used values pa = 0.05, pe = 0, rr1 = 1.5, and rr2 = 1.5 so that the risk ratio between populations due to the influence of this risk allele was estimated to be equal to 1.050625 . Using the seer incidence rates of prostate cancer in african americans (234.6 per 100,000) and non - hispanic whites (150.4 cases per 100,000) the percentage of greater risk to african americans that may be associated with rs7210100 was estimated as 1-[(1.561.050625)/(1.56 - 1)] 100 . The studies included in stage 1 were drawn from 11 epidemiological studies of prostate cancer among african american men . These studies included: the multiethnic cohort (mec; 1,094 cases /1,096 controls), the southern community cohort study (sccs, 212/419), the prostate, lung, colorectal, and ovarian cancer screening trial (plco, 286/269), the cancer prevention study ii nutrition cohort (cps - ii, 76/152), prostate cancer case - control studies at md anderson (mda, 543/474), identifying prostate cancer genes (ipcg, 368/172), the los angeles study of aggressive prostate cancer (laapc, 296/303), prostate cancer genetics study (cap genes, 75/85), case - control study of prostate cancer among african americans in washington, dc (dcpc, 292/359), king county (washington) prostate cancer study (kcpcs, 145/81), and the gene - environment interaction in prostate cancer study (gecap, 234/92). Stage 2 included 1,396 cases and 2,383 controls from 7 studies: san francisco bay area prostate cancer study (sfpcs, 86/37), the flint men s health study (fmhs, 135/353), the multiethnic cohort / los angeles county (mec - la, 554/557), north carolina prostate cancer study (ncpcs, 214/249), wake forest university prostate cancer study (wfpcs, 59/66), washington university prostate cancer study (wupcs, 75/153), and the ghana men s health study (ghs, 271/968). Stage 3 included 484 cases and 947 controls from 3 studies: the study of clinical outcomes, risk and ethnicity (score, 152/280), prostate - genetique - recherche - senegal (progrs, 86/414) and prostate cancer in a black population (pcbp, 246/253). Detailed information about the design and organization of each study is provided in the supplementary note . Genotyping in stage 1 (3,621 cases and 3,502 controls) was conducted using the illumina infinium human1m - duo . Samples (n=408) were removed based on the following exclusion criteria: 1) unknown replicates across studies, 2) call rates <95%; 3)> 10% mean heterozygosity on the x chromosome and/or <10% mean intensity on the y chromosome, 4) ancestry outliers, and; 5) samples that were related (discussed below). The concordance rate for 158 replicate samples was 99.99% . Starting with 1,153,397 snps, we removed snps with <95% call rate, mafs <1%, or> 1 qc mismatch based on sample replicates (n=105,411). We used plink to calculate the probabilities of sharing 0, 1, and 2 alleles (z = z0, z1, z2) across all possible pairs of samples to determine individuals who were likely to be related to others within and across studies . We identified 167 pairs of related subjects (mz twin, parent - offspring, full and half - sibling pairs), based on the values of their observed probability vector z being within 1 sd of the expected values of z for their respective relationship . The criterion for removal was such that individuals that were connected with a higher number of pairs were chosen for removal . In all other cases, the eigenstrat software was used to calculate eigenvectors that explained genetic differences in ancestry among samples in the study . We included data from both hapmap populations (ceph (utah residents with ancestry from northern and western europe) (ceu), japanese in tokyo, japan (jpt), yoruba in ibadan, nigeria (yri), and african ancestry in southwestern u.s . (asw)) and our study, so that comparisons to reference populations of known ethnicity could be made . A total of 2,546 ancestry - informative snps from the illumina array were selected based on low inter - marker correlation and ability to differentiate between samples of african and european descent . An individual was subject to filtering from the analysis if his value along eigenvector 1 or 2 was outside of 4 sds of the mean of each respective eigenvector . Eigenvector 1 was highly correlated (=0.997, p<1 10) with percentage of european ancestry, estimated in hapmix . Genotyping in the stage 2 and 3 studies was conducted using the taqman allelic discrimination assay . In stage 2, we removed samples missing data for> 3 snps (n=36). To assess genotyping reproducibility each study included replicate samples; the concordance was> 98% for each snp within each study . Snp rs13116912 deviated from hwe in all but one of the stage 2 studies and was removed from the stage 2 analysis . No other snp deviated from hwe (i.e. P<0.01 in> 2 studies) in stage 1 or 2 . The call rate for rs7210100 was very high in stage 1 (99.9%) and similar in cases (99.9%) and controls (99.9%). The call rate for this snp was also very high in stages 2 (99.8% overall, 99.9% in cases and 99.8% in controls) and 3 (96.1% overall, 97.3% in cases and 95.5% in controls). Bi - directional sequencing of rs7210100 and the 5 coding exons of znf652 was performed in 48 subjects (20 homozygous for the risk variant, 20 heterozygous for the risk variant and 8 homozygous for the wild - type allele .) In stage 1, we tested the association of each snp and prostate cancer risk using a 1-d.f . Likelihood ratio test from a logistic regression analysis adjusted for age, study and the first 10 eigenvectors estimated by principal components analysis . Over - inflation of the test statistic was examined with and without adjustment for ancestry and visualized with quantile - quantile plots . Lambdas were estimated as the median of the test statistics divided by 0.456 (the median of the 1-d.f . Age - adjusted odds ratios (or) and 95% confidence intervals (95% ci) for each snp were estimated from the same logistic regression model . At each locus and for each participant, local ancestry was defined as the estimated number of european chromosomes (continuous between 02) carried by the participant, estimated via the hapmix program . Local ancestry at the 17q21 locus was evaluated as a confounder in the analysis of rs7210100 . Phased haplotype data from the founders of the ceu and yri hapmap phase 2 samples were used to infer ld patterns in order to impute untyped markers . The rsq metric was used as a threshold in determining which snps to filter from analysis (rsq<0.3). Imputed snps in the 17q21 risk region, as shown in figure 2, were examined in association with prostate cancer risk as described for typed snps above . In stage 2, the snps were analyzed using logistic regression controlling for age and study (in the pooled analysis). Information regarding european ancestry was available for 7 studies included in stages 2 and 3 . As observed in stage 1 (supplementary table 2) the or for rs7210100 was similar with and without adjustment for estimated european ancestry in these studies (supplementary table 4). The results for rs7210100 in stage 2, stage 3 and stages 1 + 2 + 3 are presented without adjustment for ancestry . Association testing in the stage 2 and stage 3 studies was performed using logistic regression, adjusting for age and study . For seven of the replication studies, information about global european ancestry was available and examined as a confounding factor for variant rs7210100 . For rs7210100, effect modification by age and first - degree family history of prostate cancer was assessed in stratified analyses, and significance determined comparing the model with and without the cross - product term using a likelihood ratio test . We also examined the association of rs7210100 genotype with stage, gleason score as well as the combination of stage and grade, with advanced disease defined as gleason score8 or stage 2 (non - localized disease) and non - advanced disease defined as gleason score<8 and stage=1 (localized disease). Case - only analysis was used to test for differences in the association of rs7210100 with disease phenotypes . The association of rs7210100 with least - squares geometric mean psa levels was examined using multiple linear regression adjusting for age, body mass index and study . We estimated the risk ratio between populations of different ancestral origin (african / european) due to rs7210100 as rr= [(1-pa)+2pa(1-pa)rr1+parr2]/(1-pe)+2pe(1-pe)rr1+perr2]. Here pa is the risk allele frequency in african origin populations, pe is the risk allele frequency in european populations and rr1 is the relative risk associated with carrying 1 copy of the risk allele (compared to none) and rr2 is the relative risk associated with carrying 2 copies of the risk allele . We used values pa = 0.05, pe = 0, rr1 = 1.5, and rr2 = 1.5 so that the risk ratio between populations due to the influence of this risk allele was estimated to be equal to 1.050625 . Using the seer incidence rates of prostate cancer in african americans (234.6 per 100,000) and non - hispanic whites (150.4 cases per 100,000) the percentage of greater risk to african americans that may be associated with rs7210100 was estimated as 1-[(1.561.050625)/(1.56 - 1)] 100.
The incidence of diabetes mellitus is increasing worldwide; this trend is particularly strong for type 2 diabetes mellitus (t2dm)1 . T2 dm is a chronic disease characterized by decreased insulin sensitivity and overall poor glucose control . The systematic review by boule et al . Indicates structured exercise programs have a statistically and clinically significant beneficial effect on glycemic control in patients with t2dm3 . In addition, patients with t2 dm who use insulin, low - intensity exercise significantly reduces the prevalence of hyperglycemia4 . Despite the benefits of physical activity, many people with t2 dm for some individuals, secondary diabetes - related complications such as lower - limb amputations, neuropathies, hypertension, nephropathies, and retinopathies can either contraindicate exercise or make it more difficult . In addition, many elderly people with t2 dm do not have sufficient physical ability to perform aerobic exercise and thus have problems maintaining euglycemia5 . Passive static stretching occurs when sustained tension develops within a person s muscles through external forces . Accordingly, blood glucose levels could decrease following a program of successive sustained muscle stretching . In addition, because passive stretching requires minimum effort by the person performing the stretch, people with t2 dm who are reluctant or unable to exercise may be willing to follow a stretching protocol6 . Therefore, this study determined the effect of passive static stretching on blood glucose levels in people with t2 dm . Fifteen in - patients with t2 dm at a hospital in busan, korea participated in this study . Patients were eligible if they were sedentary (i.e., not participating in regular aerobic or strengthening exercises 6 months before the study) and willing to commit to an 8-week supervised exercise program7 . All patients were diagnosed with t2 dm confirmed by a glycated hemoglobin (hba1c) level 6.5% or higher as a criterion for the diagnosis of diabetes8 . All patients meeting the inclusion criteria were given verbal and written information about this study . Patients were randomized to the control group (n = 7) or passive static stretching group (pss, n = 8). The control group was instructed to maintain their diet and medications for diabetes and not to perform any exercise during the experiment . Meanwhile, patients in the pss group followed the same instructions as the control group but received a 40-minute intervention consisting of 6 lower - body and 4 upper - body static passive stretches . For each stretch, the muscle was held in the stretched position for 30 seconds and was repeated 4 times . Each repetition was separated by a 15-second relaxation period, and different stretches were separated by a minimum of 1 minute . A description of stretch is provided in table 1table 1.descriptions of the stretches used in the interventionstretchdescriptionseated knee flexor (bilateral)the patient sat on the floor with their legs extended and arms above their head . From this position, they lowered their head toward their knees while the experimenter pushed down on their back.seated knee flexor hip adductor (bilateral)the patient sat on the floor in the cross - legged position . From this position, the patient lowered their head toward the floor while the experimenter pushed down on their back.seated shoulder lateral flexor (bilateral)the patient sat in a chair with fingers interlaced behind their head . Keeping their arms in this position, the experimenter stood behind the patient and pulled the elbows back toward the body s midline.supine hip flexor knee extensor (unilateral)the patient lay on their back with their leg hanging over the edge of the table with the knee flexed at approximately 90. the hip was then hyperextended by the experimenter while pushing down on the thigh.seated hip external rotators, extensors (unilateral)the patient sat on the floor with one leg extended . The opposite leg was flexed at the knee, and the foot was placed flat against the extended leg s inner thigh . The patient then lowered their head toward the extended knee while the experimenter pushed down on their back.seated shoulder extensors, adductors, retractors (unilateral)while seated in a chair, the patient extended one arm and placed it horizontally across the front of the chest . The experimenter stood behind the patient, grabbed their wrist, and pulled their arm against the chest as much as possible while keeping the arm parallel to the floor.supine knee flexor plantar flexor (unilateral)the patient lay on their back with the legs extended . The experimenter then raised one leg and simultaneously flexed the hip and dorsiflexed the ankle.prone hip flexor (unilateral)the patient lay on their stomach and flexed one knee at approximately 60. keeping the knee in the flexed position, the experimenter lifted the thigh to hyperextend the hip.seated shoulder flexors, depressors (bilateral)the patient sat on the floor with the legs extended . The experimenter then grabbed their wrists and hyperextended the shoulder by raising the arms behind the back and up toward the head while keeping the back and elbows straight.seated shoulder and elbow flexors (unilateral)the patient sat on the floor with the legs extended, with one elbow flexed and brought up near the ear . From this position, the shoulder was hyperflexed by the experimenter by pushing the upper arm down toward the floor.6 . The pss group performed the stretches 3 times per week for 8 weeks . For outcome measurements, a 10-ml blood sample was collected from each patient to determine blood glucose levels using an hba1c analyzer (variant turbo, bio - rad laboratories, inc . A paired t - test was used to determine whether there were significant changes in blood glucose levels before and after the intervention . Meanwhile, an independent t - test was used to analyze differences between the 2 groups . The level of significance was set at p <0.05 . The baseline characteristics of the patients are shown in table 2table 2.baseline characteristics of the patientscon (n = 7)pss (n = 8)page (years)58.4 1.849.6 5.20.2duration of diabetes (years) 5.2 2.9 5.4 con: control group; pss: passive static stretching group; bmi: body mass index . There were no significant differences in the baseline characteristics between groups (p> 0.05). The results of outcome measures are summarized in table 3table 3.outcome measurescontrol group(n = 7)passive static stretching group(n = 8)pre - intervention post - interventionpre - interventionpost - interventionhba1c (%) 7.4 1.37.4 1.47.4 1.56.8 1.5values are means sd . p <0.05 vs. post - intervention . Hba1c: glycated hemoglobin a1c .. there was no significant difference in hba1c level after the intervention in the control group (p> 0.05). However, hba1c levels decreased significantly in the pss group after the intervention (p <0.05) and were significantly different between groups (p <0.05). Con: control group; pss: passive static stretching group; bmi: body mass index values are means sd . * p <0.05 vs. post - intervention . As mentioned above, this study determined the effect of passive static stretching on blood glucose levels in patients with t2 dm . The results showed hba1c levels decreased significantly after an 8-week passive static stretching intervention . There are several possible mechanisms that could explain how passive stretching of skeletal muscles decreased blood glucose levels . According to a review by dohm9, glucose transport into the skeletal muscles is primarily mediated by a glucose transport protein, glut-4; accordingly, exercise can increase glut-4 levels in the skeletal muscles . Furthermore, increased metabolic activity accompanying passive muscle stretching is related to the glut-4 activation pathway9, 10 . Therefore, passive muscle stretching could induce the incorporation of glut-4 into the stretched skeletal muscles . Other studies also support the possibility of stretching - induced incorporation of glut-4 into the skeletal muscles . First, the activity of protein kinase b controls glut-4 incorporation; accordingly, protein kinase b is activated by passive stretching of isolated muscles11 . Report that ischemia induces the translocation of glut-4 to the plasma membrane of cardiac myocytes12; accordingly, passive stretching of the skeletal muscles can cause ischemia13 . Third, in an experimental study by roberts et al ., exercise - induced increases in nitric oxide levels resulted in increased glucose transport14; accordingly, passive stretching can increase nitric oxide release from excised soleus muscles by 20%15 . Finally, mitogen - activated protein kinase activity stimulates glucose uptake in muscle cells16; martineau et al . Report that the activity of mitogen - activated protein kinase directly reflects the magnitude of mechanical stress (e.g., actively or passively generated tension) applied to the muscle17 . The sample size is insufficient to generalize the results to all patients with t2 dm . In addition, as hba1c reflects the average plasma glucose level over the preceding 23 months, the 8-week study period might have been too short to determine changes in blood glucose levels as a result of stretching18 . Therefore, further studies are required to ascertain the long - term (i.e., more than 3 months) effects of passive static stretching on blood glucose levels in a larger population of patients with t2 dm.
Cardiovascular disease (cvd) is the leading cause of mortality in both men and women in developed countries . Nonetheless, sex - associated differences regarding the age of cvd onset and its progression are observed worldwide . Incidence of cvd in premenopausal women is markedly lower than age - matched men in epidemiological studies (messerli et al ., 1987; bairey merz et al ., 2006; shaw et al after menopause, however, the incidence is comparable or even higher in women than in men (lerner and kannel, 1986; eaker et al ., 1993), making cvd the primary cause of death in postmenopausal women (55 versus 43% in men), exceeding all cancer deaths (rosamond et al ., 2008). The lower cvd risk among fertile women is often attributed to the protective role of estrogens at the vascular level . According to epidemiological observations and extensive basic research, estrogen and other sex steroids estrogen modulates a myriad of molecular pathways that improve vascular function, whether at the physiological level or when administered as hormone replacement therapy (hrt; grodstein et al ., 2000, 2001; miller and duckles, 2008) nevertheless, some clinical studies have questioned the protective value of hrt against vascular disease . Two randomized clinical trials, the women s health initiative (whi; rossouw et al ., 2002) and the heart and estrogen / progestin replacement study (hers i and ii; gambacciani et al ., 2002), indicate that hrt may increase cvd risk and events in postmenopausal women . The reason for this paradox could be attributable to many patient characteristics, including age . Although aging occurs progressively in both men and women, the onset of menopause marks a sudden increase in the appearance of aging - associated signs in women, and more specifically in the progress of vascular aging . Information about the role of age and menopause in the development of cvd in women is scarce . This review of clinical and experimental data on the effects of aging, estrogens, and hrt on vascular function of females aims to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages . The numerous vascular effects of estrogens are triggered by complex genomic and non - genomic mechanisms . They include modulation of vascular function and inflammatory response as well as metabolic and hemodynamic effects . Estradiol, the most abundant and potent estrogen in humans, mainly binds and activates estrogen receptors (ers). Vascular estrogen signaling involves at least three ers identified in both vascular smooth muscle and endothelium, reinforcing the idea that estrogen has a key role in controlling vascular function . The classical subtypes er (soloff and szego, 1969) and er (kuiper et al ., 1996) vary not only in their tissue distributions, but also in their agonist / antagonist profile with respect to several compounds (cano and hermenegildo, 2000). These er subtypes belong to the intracellular receptors classically defined as nuclear ligand - activated transcription factors . Activation of these receptors by the corresponding hormones affects gene expression by acting on estrogen - response elements in the target genes and modulating transcriptional events (beato et al ., 1995). Estrogen binding to er and er regulates gene expression in a time- and tissue - dependent manner, generating controversy about the type of receptor involved in vascular protection (murphy, 2011). In the cardiovascular system, both er and er have been identified in the endothelium, smooth muscle cells, adventitia, and adrenergic nerve endings of arteries from various territories and several species, including humans (karas et al ., 1994; kim - schulze et al ., 1996; although it has been reported that cultured endothelial cells do not express er (toth et al ., 2009), other investigators have demonstrated the presence of both er and er mrna in endothelium (wagner et al ., 2001) and data from our group demonstrate the protein expression of both er and er in huvec (sobrino et al ., 2009, 2010). In addition to their classic nuclear location, er can also target the plasma membrane, enabling estrogen activation of several signaling pathways, including those involved in calcium mobilization (zhang et al . Prakash et al ., 1999) and the phosphatidylinositol-3-kinase (pi3k) pathway (hisamoto et al ., a third type of er, g - protein coupled, and mainly located in the plasma membrane, was initially named gpr30 (takada et al ., 1997), then renamed gper by the international union of basic and clinical pharmacology, iuphar (alexander et al ., 2008). Gper is expressed in both endothelial and smooth muscle cells of human arteries and veins (haas et al ., gper activates rapid signaling cascades such as extracellular signal - related kinase and pi3k (meyer et al ., 2011). Several rapid and non - genomic estrogen effects formerly attributed to er have now been described as gper - mediated (prossnitz and barton, 2011). The vascular protection conferred by estrogen may be mediated indirectly by its influence on the metabolism of lipoproteins or by a direct action on the modulation of molecular pathways in the vessel wall, and more specifically on endothelial cells (hermenegildo et al ., 2002). Vascular endothelium not only regulates vascular tone through flow - mediated mechanisms, but also confers antithrombotic and antiinflammatory properties to the blood vessel . Nitric oxide (no), the primary endothelial - derived mediator, is involved in many physiological processes, including vasodilation and inhibition of thrombosis, cell migration, and proliferation (dudzinski and michel, 2007; lamas et al . Estrogen is known to increase no bioavailability by mechanisms that either directly increase no generation (figure 1) or decrease superoxide anion o2 concentration, thereby attenuating o2-mediated no inactivation . Mechanisms involved in estrogen - induced increases in no availability include: (1) transcriptional stimulation of endothelial no synthase (enos) gene expression (huang et al ., 1997; sumi and ignarro, 2003); (2) non - genomic activation of enzyme activity via a pi3k / phosphokinase b (pkb / akt)-mediated signaling pathway (hisamoto et al ., 2001); (3) increased intracellular free ca concentration ([ca]i) in endothelial cells (rubio - gayosso et al ., 2000); (4) decreased production of asymmetric dimethylarginine (adma), the enos endogenous inhibitor (monsalve et al ., 2007); and (5) attenuated o2 concentrations (wassmann et al . Estradiol effects on enos - mediated nitric oxide (no) production include both genomic and non - genomic effects . Genomic effects include the classical intracellular estrogen receptors (er), which after binding of e2 interact with estrogen - response element (ere) in dna, resulting in an increased enos expression . Moreover, e2 binding to gper leads to activation of different transcriptional factors such as camp response element (cre) which also induces enos expression . Among non - genomic effects, er and gper regulate the e2-induced enos activity (modified from sobrino, 2011). Estrogens such as 17-estradiol, estrone, and estriol have been described to act as reactive oxygen species (ros) scavengers by virtue of the hydrogen - donating capacity of their phenolic molecular structure (halliwell and grootveld, 1987; dubey and jackson, 2001). However, in these studies the direct effect of estrogens as scavengers can only be observed at concentrations above 1 m (arnal et al ., 1996; kim et al ., 1996). Considering that plasma concentrations of estrogen in physiological conditions are within the nanomolar range, it is likely that direct scavenger action is not estrogen s main antioxidant mechanism . Estrogen modulates ros concentration through a mechanism that involves interaction with its estrogenic nuclear receptors to decrease oxidative proteins and/or increase antioxidant enzymes expression . Many studies have associated changes in estrogen levels with altered levels of antioxidant enzymes including glutathione peroxidase, catalase, and superoxide dismutase (capel et al ., 1981; robb and stuart, 2011; sivritas et al ., 2011). Moreover, estrogen modulates nadh / nadph oxidases and at1 receptor gene expression, both of which are major sources of o2 production (wassmann et al ., 2001; dantas et al ., estrogen also has a modulating effect on constrictive factors and positively upregulates the production of endothelium - derived relaxing factors such as pgi2 (sobrino et al ., 2009, 2010) and the endothelium - derived hyperpolarizing factors (golding and kepler, 2001), both of which are important mediators of vascular relaxation in resistance - sized arteries . The beneficial effects of estrogen on the endothelium can be partially explained by an inhibitory effect on the production or action of the cyclooxygenase (cox)-derived vasoconstrictor agents prostaglandin h2, pgh2, and thromboxane a2, txa2 (davidge and zhang, 1998; dantas et al ., 1999; novella et al ., 2010), and of endothelin-1 (et-1; david et al ., 2001). It regulates contractile responses by a direct modulation of ca mobilization into the vascular smooth muscle cells . Direct interaction of estradiol with voltage - gated maxi - k channel subunit beta, which confers higher ca sensitivity, may modulate vascular smooth muscle (valverde et al ., 1999). Estrogen does not inhibit ca release from the intracellular stores (crews and khalil, 1999; murphy and khalil, 1999). However, supraphysiological concentrations of estrogen impede ca influx from the extracellular space (han et al ., 1995; crews and khalil, 1999; murphy and khalil, 1999) by inhibiting ca entry through voltage - gated ca channels (freay et al ., 1997; kitazawa et al ., 1997; crews and khalil, 1999; murphy and khalil, 1999). Expression of the l - type ca channels in cardiac muscle is substantially increased in er - deficient mice (johnson et al ., 1997), suggesting er - mediated regulation of ca mobilization . Estrogen also exerts direct modulation on the components of the renin - angiotensin system (ras), a key regulator of blood pressure and smooth muscle cell growth . Production of angiotensin ii (ang ii), the active hormone of the ras, is reduced by estrogen inhibition of angiotensin - converting enzyme (ace) expression . In animal models of menopause and in postmenopausal women, chronic estrogen replacement reduces ace activity in the circulation and in tissues including the kidney and aorta (brosnihan et al ., 1999; seely et al ., 2004). Furthermore, estrogen attenuates expression of and tissue response to type 1 angiotensin receptor (at1) in the aorta, heart, and kidney (silva - antonialli et al . Vascular aging is associated with endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, defective vascular repair, and an increasing prevalence of atherosclerosis (lakatta and levy, 2003; erusalimsky, 2009). Aging - associated changes in structure and function of large elastic arteries are seen even in the absence of clinical cvd (moreau et al ., 2003). Although aging per se has detrimental effects in the vasculature, the lack of estrogen due to menopause may add an aggravating cvd risk factor in women, compared to arterial aging in men . In middle - aged females, aging - associated vascular dysfunction is potentiated by lack of estrogen due to menopause or ovariectomy and improves with estrogen replacement (harman, 2004; stice et al ., 2009; novella et al ., 2010 unfortunately, the onset of menopause coincides with a time when aging - associated damage may be noted, making it particularly difficult to distinguish between the contributions of aging and the lack of estrogen . Vascular aging is a natural phenomenon that could be simply described as a consequence of physical stress, beginning early in life . Arteries are elastic tissues, susceptible to fatigue, and fracture over time as a consequence of extension - relaxation cycles during heartbeats (avolio et al ., 1983). In cross - sectional studies, postmenopausal females taking hrt have less arterial stiffness than their non - treated peers (moreau et al ., 2003; radial artery distensibility fluctuates in accordance with estrogen levels during menstrual cycles (giannattasio et al ., 1999). Basic research using animal models of estrogen withdrawal and aging suggests a modulatory role for estrogen in the molecular mechanisms to prevent arterial stiffening (zhang et al ., 2000) a recent study reported that hrt improves arterial compliance, an effect related in part to estrogen actions in the control of endothelial - dependent vasodilatory tone (moreau et al ., 2012). Collagen and elastin content of arterial walls is a key factor in arterial thickening and stiffening . It is mostly regulated by matrix metalloproteinases (mmp), enzymes capable of degrading components of the extracellular matrix . During estrogen replacement in ovariectomized rats increases mmp activity and restores aged arteries to structural properties similar to those of younger animals studied (zhang et al ., 2000). Dysfunction of both endothelial and smooth muscle molecular signaling appears during the aging process and favors vasospasm, thrombosis, inflammation, and abnormal cell migration and proliferation (lakatta and levy, 2003; briones et al . Endothelial dysfunction in the elderly has been associated with malfunctioning of vascular tissue, resulting in atherosclerosis, hypertension, and coronary artery disease (lakatta and levy, 2003; herrera et al ., 2010), renal dysfunction (schmidt et al ., 2001; erdely et al ., 2003), and alzheimer disease (price et al ., 2004). In women, a slight age - related decrease in endothelium - dependent relaxation persists until middle age (around 50 years). After that, the declining response to the endothelium - dependent vasodilator hastens, even exceeding the rate experienced by men (taddei et al ., the mechanisms for age - associated endothelial dysfunction are multiple, although most are associated with decreased no bioavailability (hayashi et al ., 2008; santhanam et al ., 2008; erusalimsky, 2009; kim et al ., reduced endothelium - dependent and no - mediated vasodilation has been described in both human and animal models of aging (kim et al ., 2009; virdis et al . Lower no production in the elderly may be based on decreased no synthesis or increased no degradation . Suggested mechanisms to explain reduced no production include: (1) decreased expression of enos (briones et al ., 2005; yoon et al ., 2010); (2) a lack of no precursor (l - arginine; santhanam et al ., 2008) and enos cofactor tetrahydrobiopterin (bh4; yoshida et al ., 2000; eskurza et al ., 2005; meyer et al ., 2011); and (3) increased endogenous enos inhibitor adma (xiong et al ., 2001; kielstein et al ., on the other hand, strong evidences support the hypothesis that age - associated increase in oxidative stress and consequent production of o2 is a potent contributor to lower no bioavailability and increased endothelial dysfunction (jacobson et al ., 2007; rodriguez - manas et al ., there is little information to correlate the progression of aging with the production / degradation of no in women . Although several studies have described decreased expression of enos in senile female rats and mice (wynne et al ., 2004; novensa et al ., 2011), aging - associated effects on enos in women can be easily confounded with the effects of lack of estrogen, since most of these studies grouped women into just two time - points: premenopausal and menopausal groups . Even though the decline in no bioavailability could sufficiently explain most of the changes in the functioning of vascular cells, other molecules crucial to control of vascular function are also modified by aging . In the regulation of vascular tone, cox - derived factors are particularly important as they can induce both vascular relaxation (pgi2) and contraction (txa2 and pgh2). Some studies have reported a prevalence in the production of relaxing cox factors in the vasculature of young and healthy individuals (tang and vanhoutte, 2008). With aging, cox - dependent vasoconstrictors production becomes evident, leading to increased vascular contraction (taddei et al . However, the cox isoform involved in the generation of contractile prostanoids remains unclear . In functional studies developed in femoral arteries of aged rats, oxygen free radicals participate in the augmented endothelium - dependent contractions mediated by cox - derived prostanoids . Both the constitutive and inducible isoforms of cox contribute to this endothelial dysfunction (shi et al . Molecular studies performed in endothelial cells from aged rats showed an increase in mrna levels of cox-1, cox-2, and other enzymes involved in the synthesis of prostanoids (tang and vanhoutte, 2008), demonstrating the importance of the arachidonic acid cox cascade in the endothelial and vascular dysfunction associated with aging . Moreover, functional studies have demonstrated an interaction between no and prostanoids pathways . In aorta from aged female mice, no bioavailability increases when the cox pathway is inhibited; both gene and protein expression of cox-1 are increased (novella et al ., 2011). Furthermore, activation of inflammatory pathways in the vascular wall plays a central role in the process of vascular aging . Even in the absence of traditional risk factors for atherosclerosis, an age - associated shift to a proinflammatory gene expression profile, known as endothelial activation, induces upregulation of cellular adhesion molecules and cytokines, which increases endothelial leukocyte interactions and permeability, mechanisms considered crucial to initial steps in the development of atherosclerosis (herrera et al ., 2010; seals et al ., 2011). Accordingly, a sex - associated difference in inflammatory responses during aging has been proposed . Inflammatory atherosclerosis and associated acute coronary heart disease develop earlier in life in men than in women (roger et al ., 2011) and are associated with earlier death, although men and women present the same overall plaque burden (frink, 2009). In animal models of atherosclerosis, male sex contributes to a faster and more severe progression of lipid deposition, remodeling, and aortic lesions (pereira et al . With the wide - ranging data from experimental research, estrogens might appear to promise protection against the progression of vascular aging and cvd in women . Epidemiological observational studies also suggest that postmenopausal women on hrt are less likely to develop cvd than non - users at the same age (grodstein et al ., 2000, 2001) nevertheless, these studies contrast with the large prospective clinical trials, hers and whi, which failed to show reduced cardiovascular events in postmenopausal women on hrt . In fact, whi suggested that hrt was associated with increased risk to the cardiovascular system (rossouw et al ., 2002). Possible reasons for this discrepancy have been extensively discussed and include the average age of women entering most hrt clinical trials, 65 years and older, which results in a study population with some degree of aging - associated vascular damage . In addition, participants had been estrogen - deficient for an average of 10 years before starting hrt, a relatively late start that could modify the status of ers and molecular signaling so as to attenuate the benefits of estrogen . For instance, during aging ers can undergo posttranslational modifications such as methylation, which decreases their expression and activity . We recently reported that aging contributes to increased dna methylation in female mice aorta, which could be associated with the decrease in the modulatory effects of estrogen (novensa et al ., 2011). A few clinical studies also provide evidence for aging - associated dysregulation of er methylation and suggest that focal epigenetic changes in er could contribute to decreased estrogen activity and to the development of atherosclerosis in elderly women (post et al ., 1999; detailed examination of whi data reveals that early initiation of estrogen replacement produces more favorable results than the later average initiation employed in the whi studies overall (grodstein et al ., 2006; these findings, together with observational studies, have led scientists to create the so - called timing hypothesis that estrogen - mediated benefits to prevent cvd only occur when treatment is initiated before the detrimental effects of aging are established on vascular walls (harman, 2006). These effects include endothelial dysfunction and pathophysiological actions, such as increased vascular calcification and generalized stiffening of the arterial tree that increase the prevalence of hypertension and atherosclerosis (lakatta and levy, 2003; erusalimsky, 2009; kovacic et al ., 2011). Little information is available on whether and how vascular effects of estrogen are modified with aging in females . Aging has been associated with significant reductions in the direct estrogen - mediated mechanisms of vascular relaxation (wynne et al ., 2004; the lack of estrogen responses in these animal studies was not related to age - associated changes in the plasma levels of estrogen or activity of er, but rather to possible age - related changes in estrogen - mediated signaling pathways in the vasculature . Modifications in the ratio between er and er in older female mice are associated with the lack of protective effects of estrogen on no production and with a reversal in its antioxidant effect to a pro - oxidant profile (novensa et al ., 2011). Moreover, clinical studies have revealed that cvd risk factors in postmenopausal women were lower among women aged 5059 years at hrt initiation (manson et al . These studies clearly establish the complexity of estrogen effects, which may be influenced by pathophysiological conditions including aging and subclinical cvd . Despite convincing arguments by the followers of the timing hypothesis the potential extrapolation of the protective effects of estrogen replacement, well described in young females, to older women remains controversial . The field still lacks detailed experimental and clinical research on the long - term effects of estrogen and how it modulates cardiovascular function during aging . Our society is aging progressively, and increased life expectancy enhances the risks for diseases associated with the natural fatigue of the body, including cvd . Despite this undeniable reality, there is evidence that vascular aging in women does not follow the same chronology as in men . The vascular protective effects exerted by estrogens have been proposed as the major reason for reduced signs of vascular aging and cvd risk in premenopausal women, compared to men . When natural estrogen withdrawal occurs and a woman enters her climacteric stage, effects of sudden vascular aging become evident, leading to vascular dysfunction and increased risk of a cardiovascular event . The lack of crucial information from clinical trials and the discrepancies in the available data on the regulation of the female cardiovascular system can lead to inappropriate diagnosis and treatment of cvd in older women . Women have been treated like men, despite the notable sex - associated differences in the elements of aging and disease processes . Much research effort is still needed to understand age- and sex - related differences in cardiovascular control, establish the impact of the menstrual cycle and hrt on vascular function, and propose new therapeutic strategies to improve cvd diagnosis and treatment and the overall management of vascular senescence in women . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Prostatic acid phosphatase (pap), a glycoprotein synthesized by the prostate gland, is traditionally used to diagnose prostate cancer . Pap has two isoforms, secreted and transmembrane isoforms, and, more than in the prostate gland, is also extensively expressed in nonprostate tissue such as nervous tissues .. recent studies have identified pap is molecular identity to thiamine monophosphatase (tmpase) and, historically, tmpase, with the acid phosphatase characteristics, has been widely used for labeling nociceptive dorsal root ganglia (drg) neurons . Pap hydrolyzes adenine monophosphate (amp), particularly in acidic environments, to adenosine that acts as an analgesic effector . Moreover, small nociceptive drg neurons, including peptidergic and nonpeptidergic neurons, coexpress pap, suggesting that pap profiles correlate with pain modulation . For example, a growing body of evidence has shown that intrathecal injection of recombinant pap counteracts inflammatory pain and enhances neuropathic pain in pap - knockout mice . Previously, we have established a mouse model of small - fiber neuropathy, in which small sensory nerves were specifically depleted by resiniferatoxin (rtx), a capsaicin analog acting on the transient receptor potential vanilloid receptor 1 (trpv1). This rtx - induced neuropathy model mimics human small - fiber neuropathy; that is, the model exhibits the degeneration of cutaneous sensory nerves and neuropathic pain . Furthermore, rtx - induced neuropathy with skin denervation markedly increases the expression of activating transcription factor 3 (atf3), a marker of neuronal injury, in nonpeptidergic p2x3 neurons, which is correlated with neuropathic pain . Whether the expression profiles of this subpopulation of pap neurons correlate with injury - induced neuropathic pain should be determined, and the mechanism responsible for modulating the analgesic potential in injury - induced neuropathic pain should be investigated . The compound 4-methylcatechol (4mc) enhances the synthesis of neurotrophins such as nerve growth factor (ngf) and promotes skin reinnervation . Cutaneous sensory nerves function as the peripheral receptors of small drg neurons; thus, inducing atf3 expression in these neurons caused skin denervation and neuropathic pain in our rtx - induced neuropathy model . Ngf is trophic to small drg neurons and modulates neuropathic pain through its high - affinity receptor, trka; a reduction in ngf and trka expression is accompanied by various peripheral neuropathies . Whether 4mc modulates pap expression profiles through ngf - trka signaling is unclear . Rtx - induced neuropathy is an excellent model for determining how the loss of the analgesic effect of pap leads to injury - induced neuropathic pain . In the present study, we investigated the intervention of pap profiles to neuropathic pain and the effect of 4mc on reversing neuropathic pain through ngf - trka signaling . This study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner . Administration of a single dose of rtx (50 g / kg, sigma, st . Louis, mo) (the rtx group). Injections of 4mc (10 g / kg, wako, osaka, japan) immediately after rtx treatment for seven consecutive days (the 4mc group), or an equal volume of vehicle as a control (the vehicle group). To confirm the effect of 4mc, . Administered . Briefly, 2.5s ngf (alomone labs, jerusalem, israel) was dissolved in dulbecco s modified eagle medium, and mice received ngf (1 g/10 g) immediately after rtx treatment (the ngf group). Some mice in the 4mc group received additional anti - ngf antisera (30 g / kg, sigma) to neutralize ngf induced by 4mc (the abngf group). To compare each experimental approach, 2.5s ngf and anti after treatment, the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum . All procedures were conducted in accordance with ethical guidelines for laboratory animals, and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university . All experimental procedures were performed carefully, and all efforts were made to minimize animal suffering . The changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments (somedic sales ab, hrby, sweden) in accordance with our established protocol . Briefly, a series of monofilaments was applied to the plantar region of the hindpaw . If paw withdrawal occurred, a monofilament of a smaller caliber was applied . In the absence of paw withdrawal four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses, and the mechanical thresholds were calculated using a formula published in a previous study . Animals were sacrificed by intracardiac perfusion with 0.1 m phosphate buffer (pb) followed by 4% paraformaldehyde (4 p) in 0.1 m pb . After perfusion, the 4th and 5th (l4 and l5, respectively) lumbar drgs were removed carefully and postfixed in 4p for another 6 h. the drgs were cryoprotected with 30% sucrose in 0.1 m pb overnight, and 8 -m - thick cryosections were obtained using a cryostat (cm1850, leica, wetzlar, germany). For adequate sampling, two ganglia (l4/l5) per mouse and 5 to 8 sections per drg tissue (at 80-m intervals) were immunostained . The following primary antisera were used in this study: anti - pap (chicken, 1:600, aves labs, tigard, or), anti - atf3 (rabbit, 1:100, santa cruz biotechnology, santa cruz, ca), anti - peripherin (rabbit, 1:800, chemicon, temecula, ca), anti - neurofilament smi32 (mouse, 1:600, covance, emeryville, ca), anti - p2x3 (rabbit, 1:400, neuromics, edina, mn), and anti - trka (rabbit, 1:200, santa cruz). Briefly, sections were incubated overnight at 4 with one of the primary antiserum combinations: p2x3/pap, pap / peripherin, pap / smi32, atf3/pap, and pap / trka . Thereafter, the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera (1:100, jackson immunoresearch, west grove, pa) corresponding to the appropriate primary antisera for 1 h. sections were mounted with vectashield (vector, burlingame, ca) for quantification . To quantify the various phenotypic drg neurons, each drg section was systematically photographed at 200 under a fluorescence microscope (axiophot microscope, zeiss, oberkochen, germany) to produce a montage of the entire drg section according to an established procedure . To avoid density bias, only neurons with a clear nuclear profile were counted, and only sections containing neuronal ganglia were measured using imagej version 1.44d (national institutes of health [nih], bethesda, md). For morphometric analysis of pap neuropathology, the diameters of pap(+) neurons and atf3(+)/pap(+) neurons were measured using image pro - plus (media cybernetics, bethesda, md) and plotted as a histogram . To minimize individual bias, five to seven mice were included in each group, and the coding information was masked during the behavioral tests and quantification procedures . All data are expressed as mean standard derivation (sd) of the mean . A t test was performed to analyze the data following a gaussian distribution . For data which did not follow a gaussian distribution, a nonparametric mann this study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner . Administration of a single dose of rtx (50 g / kg, sigma, st . Louis, mo) (the rtx group). Injections of 4mc (10 g / kg, wako, osaka, japan) immediately after rtx treatment for seven consecutive days (the 4mc group), or an equal volume of vehicle as a control (the vehicle group). To confirm the effect of 4mc, . Administered . Briefly, 2.5s ngf (alomone labs, jerusalem, israel) was dissolved in dulbecco s modified eagle medium, and mice received ngf (1 g/10 g) immediately after rtx treatment (the ngf group). Some mice in the 4mc group received additional anti - ngf antisera (30 g / kg, sigma) to neutralize ngf induced by 4mc (the abngf group). To compare each experimental approach, 2.5s ngf and anti after treatment, the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum . All procedures were conducted in accordance with ethical guidelines for laboratory animals, and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university . All experimental procedures were performed carefully, and all efforts were made to minimize animal suffering . The changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments (somedic sales ab, hrby, sweden) in accordance with our established protocol . Briefly, a series of monofilaments was applied to the plantar region of the hindpaw . If paw withdrawal occurred, a monofilament of a smaller caliber was applied . In the absence of paw withdrawal four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses, and the mechanical thresholds were calculated using a formula published in a previous study . Animals were sacrificed by intracardiac perfusion with 0.1 m phosphate buffer (pb) followed by 4% paraformaldehyde (4 p) in 0.1 m pb . After perfusion, the 4th and 5th (l4 and l5, respectively) lumbar drgs were removed carefully and postfixed in 4p for another 6 h. the drgs were cryoprotected with 30% sucrose in 0.1 m pb overnight, and 8 -m - thick cryosections were obtained using a cryostat (cm1850, leica, wetzlar, germany). For adequate sampling, two ganglia (l4/l5) per mouse and 5 to 8 sections per drg tissue (at 80-m intervals) were immunostained . The following primary antisera were used in this study: anti - pap (chicken, 1:600, aves labs, tigard, or), anti - atf3 (rabbit, 1:100, santa cruz biotechnology, santa cruz, ca), anti - peripherin (rabbit, 1:800, chemicon, temecula, ca), anti - neurofilament smi32 (mouse, 1:600, covance, emeryville, ca), anti - p2x3 (rabbit, 1:400, neuromics, edina, mn), and anti - trka (rabbit, 1:200, santa cruz). Briefly, sections were incubated overnight at 4 with one of the primary antiserum combinations: p2x3/pap, pap / peripherin, pap / smi32, atf3/pap, and pap / trka . Thereafter, the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera (1:100, jackson immunoresearch, west grove, pa) corresponding to the appropriate primary antisera for 1 h. sections were mounted with vectashield (vector, burlingame, ca) for quantification . To quantify the various phenotypic drg neurons, each drg section was systematically photographed at 200 under a fluorescence microscope (axiophot microscope, zeiss, oberkochen, germany) to produce a montage of the entire drg section according to an established procedure . To avoid density bias, only neurons with a clear nuclear profile were counted, and only sections containing neuronal ganglia were measured using imagej version 1.44d (national institutes of health [nih], bethesda, md). For morphometric analysis of pap neuropathology, the diameters of pap(+) neurons and atf3(+)/pap(+) neurons were measured using image pro - plus (media cybernetics, bethesda, md) and plotted as a histogram . To minimize individual bias, five to seven mice were included in each group, and the coding information was masked during the behavioral tests and quantification procedures . All data are expressed as mean standard derivation (sd) of the mean . A t test was performed to analyze the data following a gaussian distribution . For data which did not follow a gaussian distribution, a nonparametric mann in a previous study, we developed an rtx - induced small - fiber neuropathy mouse model, in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . In the present study, rtx affected pap expression, as demonstrated by the coexpression of p2x3 and pap (figure 1). Notably, pap and p2x3 showed inverse patterns after neuropathy; specifically, there was an increase in p2x3(+) and decrease in pap(+) neurons, resulting in an increased p2x3/pap ratio (67.4% 2.1% vs. 44.7% 3.4%, p <0.0001) and decreased pap / p2x3 ratio (87.1% 5.4% vs. 76.8% 2.3%, p = 0.001) (figure 1(e)). Furthermore, pap expression was higher in peripherin(+) neurons than in neurofilament (smi32)(+) neurons (figure 2). The diameter histograms confirmed that the pap(+) neurons were small to medium drg neurons (25th75th percentile: 17.123.7 m) (figure 2(f)). Figure 1.colocalization of prostatic acid phosphatase (pap)(+) and p2x3(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a d) double immunofluorescence staining was performed with the following primary antiserum combinations: anti - pap (a, c) and anti - p2x3 (b, d) antisera in the vehicle (a, b) and rtx (c, d) groups . (e) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+) and pap(+)/p2x3(+) neurons in the vehicle (opened bar) and rtx (filled bar) group according to panels (a) to (d).bar, 50 m . * * p <0.01, * * * p <0.001 . Figure 2.expression of prostatic acid phosphatase (pap)(+) by the peripherin(+) and neurofilament (sim32)(+) dorsal root ganglion (drg) neurons . Double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations: (a, b) pap / smi32 and pap / peripherin (c, d). (a, b) the micrographs show extremely low colocalization of pap (a) and smi32 (b). (c, d) the micrographs show high colocalization of pap (c) and peripherin (d). (e) the graph quantifies the colocalized ratios of pap(+)/smi32(+) (open bar) and pap(+)/peripherin(+) neurons (filled bar) according to panels (a) to (d). (f) the graph shows the diameter histogram of pap(+) neurons, indicating that pap(+) neurons are small to medium neurons.bar, 50 m . * * p <0.01 colocalization of prostatic acid phosphatase (pap)(+) and p2x3(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a d) double immunofluorescence staining was performed with the following primary antiserum combinations: anti - pap (a, c) and anti - p2x3 (b, d) antisera in the vehicle (a, b) and rtx (c, d) groups . (e) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+) and pap(+)/p2x3(+) neurons in the vehicle (opened bar) and rtx (filled bar) group according to panels (a) to (d). Bar, 50 m . * * p <0.01, * * * p <0.001 . Expression of prostatic acid phosphatase (pap)(+) by the peripherin(+) and neurofilament (sim32)(+) dorsal root ganglion (drg) neurons . Double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations: (a, b) pap / smi32 and pap / peripherin (c, d). (a, b) the micrographs show extremely low colocalization of pap (a) and smi32 (b). (c, d) the micrographs show high colocalization of pap (c) and peripherin (d). (e) the graph quantifies the colocalized ratios of pap(+)/smi32(+) (open bar) and pap(+)/peripherin(+) neurons (filled bar) according to panels (a) to (d). (f) the graph shows the diameter histogram of pap(+) neurons, indicating that pap(+) neurons are small to medium neurons . Bar, 50 m . * * p <0.01 to understand the expression of pap and its relationship with atf3, an injury marker, we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy (figure 3(a) to (e)). Pap(+) neurons were mildly lower in the rtx group than in the vehicle group (190.6 12.2 vs. 269.5 6.9 neurons / mm, p = 0.0003) (figure 3(f)). By contrast, atf3(+) neurons were markedly higher in the rtx group than in the vehicle group (192.3 18.0 vs. 9.0 13.1 neurons / mm, p <moreover, atf3 was induced preferentially in pap(+) neurons (figure 3(a) to (e)), resulting in an increased ratio of atf3(+)/pap(+) neurons in the rtx group (29.0% 5.6% vs. 0.2% 0.2%, p = 0.0043) (figure 3(h)). Figure 3.phenotypic changes in prostatic acid phosphatase (pap)(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a e) double immunofluorescence staining was performed with anti - activating transcription factor 3 (atf3; a e in red) and anti - pap (a e in green) antisera in the vehicle (a), rtx (b), 4-methylcatechol (4mc) (c), ngf (rtx + 2.5s ngf; d), and (e) abngf (4mc + anti - ngf antisera) groups . (f h) the graphs quantify the density changes in (f) pap(+) and (g) atf3(+) neurons and (h) the ratio changes in atf3(+)/pap(+) neurons according to panels (a) to (e). (i) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments, as described in the materials and methods . The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf (dotted bar) groups . (j, k) the graphs indicate that the mechanical thresholds correlated with pap expression, that is, a linear correlation with (j) pap densities and (k) an inverse correlation with the ratio of atf3(+)/pap(+) neurons . (l, m) the graphs show the diameter histogram of atf3(+)/pap(+) neurons in the rtx (l) and 4mc (m) groups . The diameter histogram shows no difference between the rtx and 4mc groups, but higher numbers of atf3(+)/pap(+) neurons in the rtx group.bar, 50 m . * p <0.05, * * p <0.01, * * * p <0.001 . Phenotypic changes in prostatic acid phosphatase (pap)(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a e) double immunofluorescence staining was performed with anti - activating transcription factor 3 (atf3; a e in red) and anti - pap (a e in green) antisera in the vehicle (a), rtx (b), 4-methylcatechol (4mc) (c), ngf (rtx + 2.5s ngf; d), and (e) abngf (4mc + anti - ngf antisera) groups . (f h) the graphs quantify the density changes in (f) pap(+) and (g) atf3(+) neurons and (h) the ratio changes in atf3(+)/pap(+) neurons according to panels (a) to (e). (i) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments, as described in the materials and methods . The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf (dotted bar) groups . (j, k) the graphs indicate that the mechanical thresholds correlated with pap expression, that is, a linear correlation with (j) pap densities and (k) an inverse correlation with the ratio of atf3(+)/pap(+) neurons . (l, m) the graphs show the diameter histogram of atf3(+)/pap(+) neurons in the rtx (l) and 4mc (m) groups . The diameter histogram shows no difference between the rtx and 4mc groups, but higher numbers of atf3(+)/pap(+) neurons in the rtx group . P <0.05, * * p <0.01, * * * p <0.001 . Mice in the rtx group showed mechanical allodynia compared with the vehicle group (747.1 156.0 vs. 299.6 70.6 mg, p = 0.0025) (figure 3(i)). This behavioral change correlated with the pap profiles; that is, the densities of pap(+) neurons (r = 0.75, p <0.0001; figure 3(j)) and the ratios of atf3(+)/pap(+) neurons (r = 0.60, p = 0.003; figure 3(k)) were inversely linear to the mechanical thresholds . Collectively, these findings indicate that pap expression had a strong influence in mediating pain development . After neuropathy was induced using rtx, 4mc normalized pap expression and relieved mechanical allodynia; specifically, the pap(+) neuronal density (269.7 48.8 neurons / mm, p = 0.61) (figure 3(c) and (f)) and mechanical allodynia (693.8 146.0 mg, p = 0.54) (figure 3(i)) in the 4mc group were comparable with those in the vehicle group . Furthermore, although 4mc reduced the atf3(+) neuronal density (102.4 27.8 neurons / mm, p <0.0001) and ratio of atf3(+)/pap(+) neurons (15.8% 5.9%, p = 0.0095), the ratios of atf3(+)/pap(+) neurons were still higher in the 4mc group than in the vehicle group (p = 0.016; figure 3(c), (g), (h)). Morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+) neurons but did not have a significant effect on their mean diameter (15.5 2.2 vs. 16.5 2.8 m, p> 0.05) (figure 3(l) vs. (m)). To understand the putative mechanisms of pain relief by 4mc, we examined the coexpression of pap(+)/trka(+) neurons (figure 4). For each group, pap(+) neuronal density was similar to trka(+) neuronal density (figure 3(f) vs. 4(k)). The pap / trka ratios correlated linearly with the mechanical threshold (r = 0.62, p = 0.0062; figure 4(m)). To confirm the effect of 4mc, the mice in the ngf group were systemically (i.p .) Administered 2.5s ngf . The ngf group exhibited the same effect as the 4mc group; that is, pap and trka expression profiles were reversed, atf3 induction was normalized, and mechanical allodynia relieved . Those findings, however, were not observed in the abngf group (administered anti - ngf antisera to neutralize ngf) (figures 3 and 4). Figure 4.colocalization of prostatic acid phosphatase (pap) and high - affinity nerve growth factor (trka) receptor after resiniferatoxin (rtx)-induced neuropathy . (a j) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap (a e) and trka (f - j) in the vehicle (a, f), rtx (b, g), 4-methylcatechol (4mc; c, h), (d, i) ngf (rtx + 2.5s ngf), and (e, j) abngf (4mc + anti - ngf antisera) groups . (k, l) the graphs show the changes in (k) trka density and (l) the colocalized ratios of pap(+)/trka(+) neurons in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf groups (dotted bar) according to panels (a) to (j). (m) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+) neurons.bar, 50 m . * p <0.05, * * p <0.01 . Colocalization of prostatic acid phosphatase (pap) and high - affinity nerve growth factor (trka) receptor after resiniferatoxin (rtx)-induced neuropathy . (a j) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap (a e) and trka (f - j) in the vehicle (a, f), rtx (b, g), 4-methylcatechol (4mc; c, h), (d, i) ngf (rtx + 2.5s ngf), and (e, j) abngf (4mc + anti - ngf antisera) groups . (k, l) the graphs show the changes in (k) trka density and (l) the colocalized ratios of pap(+)/trka(+) neurons in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf groups (dotted bar) according to panels (a) to (j). (m) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+) neurons . In a previous study, we developed an rtx - induced small - fiber neuropathy mouse model, in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . In the present study, rtx affected pap expression, as demonstrated by the coexpression of p2x3 and pap (figure 1). Notably, pap and p2x3 showed inverse patterns after neuropathy; specifically, there was an increase in p2x3(+) and decrease in pap(+) neurons, resulting in an increased p2x3/pap ratio (67.4% 2.1% vs. 44.7% 3.4%, p <0.0001) and decreased pap / p2x3 ratio (87.1% 5.4% vs. 76.8% 2.3%, p = 0.001) (figure 1(e)). Furthermore, pap expression was higher in peripherin(+) neurons than in neurofilament (smi32)(+) neurons (figure 2). The diameter histograms confirmed that the pap(+) neurons were small to medium drg neurons (25th75th percentile: 17.123.7 m) (figure 2(f)). Figure 1.colocalization of prostatic acid phosphatase (pap)(+) and p2x3(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a d) double immunofluorescence staining was performed with the following primary antiserum combinations: anti - pap (a, c) and anti - p2x3 (b, d) antisera in the vehicle (a, b) and rtx (c, d) groups . (e) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+) and pap(+)/p2x3(+) neurons in the vehicle (opened bar) and rtx (filled bar) group according to panels (a) to (d).bar, 50 m . * * p <0.01, * * * p <0.001 . Figure 2.expression of prostatic acid phosphatase (pap)(+) by the peripherin(+) and neurofilament (sim32)(+) dorsal root ganglion (drg) neurons . Double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations: (a, b) pap / smi32 and pap / peripherin (c, d). (a, b) the micrographs show extremely low colocalization of pap (a) and smi32 (b). (c, d) the micrographs show high colocalization of pap (c) and peripherin (d). (e) the graph quantifies the colocalized ratios of pap(+)/smi32(+) (open bar) and pap(+)/peripherin(+) neurons (filled bar) according to panels (a) to (d). (f) the graph shows the diameter histogram of pap(+) neurons, indicating that pap(+) neurons are small to medium neurons.bar, 50 m . * * p <0.01 colocalization of prostatic acid phosphatase (pap)(+) and p2x3(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a d) double immunofluorescence staining was performed with the following primary antiserum combinations: anti - pap (a, c) and anti - p2x3 (b, d) antisera in the vehicle (a, b) and rtx (c, d) groups . (e) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+) and pap(+)/p2x3(+) neurons in the vehicle (opened bar) and rtx (filled bar) group according to panels (a) to (d). Bar, 50 m . * * p <0.01, * * * p <0.001 . Expression of prostatic acid phosphatase (pap)(+) by the peripherin(+) and neurofilament (sim32)(+) dorsal root ganglion (drg) neurons . Double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations: (a, b) pap / smi32 and pap / peripherin (c, d). (a, b) the micrographs show extremely low colocalization of pap (a) and smi32 (b). (c, d) the micrographs show high colocalization of pap (c) and peripherin (d). (e) the graph quantifies the colocalized ratios of pap(+)/smi32(+) (open bar) and pap(+)/peripherin(+) neurons (filled bar) according to panels (a) to (d). (f) the graph shows the diameter histogram of pap(+) neurons, indicating that pap(+) neurons are small to medium neurons . To understand the expression of pap and its relationship with atf3, an injury marker, we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy (figure 3(a) to (e)). Pap(+) neurons were mildly lower in the rtx group than in the vehicle group (190.6 12.2 vs. 269.5 6.9 neurons / mm, p = 0.0003) (figure 3(f)). By contrast, atf3(+) neurons were markedly higher in the rtx group than in the vehicle group (192.3 18.0 vs. 9.0 13.1 neurons / mm, p <moreover, atf3 was induced preferentially in pap(+) neurons (figure 3(a) to (e)), resulting in an increased ratio of atf3(+)/pap(+) neurons in the rtx group (29.0% 5.6% vs. 0.2% 0.2%, p = 0.0043) (figure 3(h)). Figure 3.phenotypic changes in prostatic acid phosphatase (pap)(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a e) double immunofluorescence staining was performed with anti - activating transcription factor 3 (atf3; a e in red) and anti - pap (a e in green) antisera in the vehicle (a), rtx (b), 4-methylcatechol (4mc) (c), ngf (rtx + 2.5s ngf; d), and (e) abngf (4mc + anti - ngf antisera) groups . (f h) the graphs quantify the density changes in (f) pap(+) and (g) atf3(+) neurons and (h) the ratio changes in atf3(+)/pap(+) neurons according to panels (a) to (e). (i) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments, as described in the materials and methods . The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf (dotted bar) groups . (j, k) the graphs indicate that the mechanical thresholds correlated with pap expression, that is, a linear correlation with (j) pap densities and (k) an inverse correlation with the ratio of atf3(+)/pap(+) neurons . (l, m) the graphs show the diameter histogram of atf3(+)/pap(+) neurons in the rtx (l) and 4mc (m) groups . The diameter histogram shows no difference between the rtx and 4mc groups, but higher numbers of atf3(+)/pap(+) neurons in the rtx group.bar, 50 m . * p <0.05, * * p <0.01, * * * p <0.001 . Phenotypic changes in prostatic acid phosphatase (pap)(+) dorsal root ganglion neurons in resiniferatoxin (rtx)-induced neuropathy . (a e) double immunofluorescence staining was performed with anti - activating transcription factor 3 (atf3; a e in red) and anti - pap (a e in green) antisera in the vehicle (a), rtx (b), 4-methylcatechol (4mc) (c), ngf (rtx + 2.5s ngf; d), and (e) abngf (4mc + anti - ngf antisera) groups . (f h) the graphs quantify the density changes in (f) pap(+) and (g) atf3(+) neurons and (h) the ratio changes in atf3(+)/pap(+) neurons according to panels (a) to (e). (i) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments, as described in the materials and methods . The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf (dotted bar) groups . (j, k) the graphs indicate that the mechanical thresholds correlated with pap expression, that is, a linear correlation with (j) pap densities and (k) an inverse correlation with the ratio of atf3(+)/pap(+) neurons . (l, m) the graphs show the diameter histogram of atf3(+)/pap(+) neurons in the rtx (l) and 4mc (m) groups . The diameter histogram shows no difference between the rtx and 4mc groups, but higher numbers of atf3(+)/pap(+) neurons in the rtx group . Bar, 50 m . * p <0.05, * * p <0.01, * * * p <0.001 . Mice in the rtx group showed mechanical allodynia compared with the vehicle group (747.1 156.0 vs. 299.6 70.6 mg, p = 0.0025) (figure 3(i)). This behavioral change correlated with the pap profiles; that is, the densities of pap(+) neurons (r = 0.75, p <0.0001; figure 3(j)) and the ratios of atf3(+)/pap(+) neurons (r = 0.60, p = 0.003; figure 3(k)) were inversely linear to the mechanical thresholds . Collectively, these findings indicate that pap expression had a strong influence in mediating pain development . After neuropathy was induced using rtx, 4mc normalized pap expression and relieved mechanical allodynia; specifically, the pap(+) neuronal density (269.7 48.8 neurons / mm, p = 0.61) (figure 3(c) and (f)) and mechanical allodynia (693.8 146.0 mg, p = 0.54) (figure 3(i)) in the 4mc group were comparable with those in the vehicle group . Furthermore, although 4mc reduced the atf3(+) neuronal density (102.4 27.8 neurons / mm, p <0.0001) and ratio of atf3(+)/pap(+) neurons (15.8% 5.9%, p = 0.0095), the ratios of atf3(+)/pap(+) neurons were still higher in the 4mc group than in the vehicle group (p = 0.016; figure 3(c), (g), (h)). Morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+) neurons but did not have a significant effect on their mean diameter (15.5 2.2 vs. 16.5 2.8 m, p> 0.05) (figure 3(l) vs. (m)). To understand the putative mechanisms of pain relief by 4mc, we examined the coexpression of pap(+)/trka(+) neurons (figure 4). For each group, pap(+) neuronal density was similar to trka(+) neuronal density (figure 3(f) vs. 4(k)). The pap / trka ratios correlated linearly with the mechanical threshold (r = 0.62, p = 0.0062; figure 4(m)). To confirm the effect of 4mc, the mice in the ngf group were systemically (i.p .) Administered 2.5s ngf . The ngf group exhibited the same effect as the 4mc group; that is, pap and trka expression profiles were reversed, atf3 induction was normalized, and mechanical allodynia relieved . Those findings, however, were not observed in the abngf group (administered anti - ngf antisera to neutralize ngf) (figures 3 and 4). Figure 4.colocalization of prostatic acid phosphatase (pap) and high - affinity nerve growth factor (trka) receptor after resiniferatoxin (rtx)-induced neuropathy . (a j) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap (a e) and trka (f - j) in the vehicle (a, f), rtx (b, g), 4-methylcatechol (4mc; c, h), (d, i) ngf (rtx + 2.5s ngf), and (e, j) abngf (4mc + anti - ngf antisera) groups . (k, l) the graphs show the changes in (k) trka density and (l) the colocalized ratios of pap(+)/trka(+) neurons in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf groups (dotted bar) according to panels (a) to (j). (m) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+) neurons.bar, 50 m . * p <0.05, * * p <0.01 . Colocalization of prostatic acid phosphatase (pap) and high - affinity nerve growth factor (trka) receptor after resiniferatoxin (rtx)-induced neuropathy . (a j) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap (a e) and trka (f - j) in the vehicle (a, f), rtx (b, g), 4-methylcatechol (4mc; c, h), (d, i) ngf (rtx + 2.5s ngf), and (e, j) abngf (4mc + anti - ngf antisera) groups . (k, l) the graphs show the changes in (k) trka density and (l) the colocalized ratios of pap(+)/trka(+) neurons in the vehicle (open bar), rtx (filled bar), 4mc (grey bar), ngf (slashed bar), and abngf groups (dotted bar) according to panels (a) to (j). (m) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+) neurons . This is the first report documenting the intervention of pap and ngf - trka signaling, which mediates neuropathic pain . We speculate one possible mechanism: neuropathic pain in rtx - induced small - fiber neuropathy develops because of pap neuropathology and the decrease in ngf - trka signaling . Rtx is specifically neurotoxic to trpv1(+) neurons; it also induces small - fiber neuropathy, such as skin denervation, which results in thermal hypoalgesia, small - diameter neuron injury, and peripheral sensitization of p2x3 purinoceptors, leading to mechanical allodynia . Atf3 is both an injury marker and a marker of pain; for example, the ratios of p2x3(+)/atf3(+) neurons correlate linearly with the degree of neuropathic pain . Pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+) and pap(+)/peripherin(+) neurons . Thus, injured pap neurons are critical for modulating neuropathic pain; that is, the ratios of atf3(+)/pap(+) neurons are inversely linear to the mechanical threshold . The balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity, which responded for the cellular pathophysiological homeostasis . The ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine, which acts as an analgesic ligand . Pap downregulation leads to the imbalance of amp / adenosine ratios, inhibiting the analgesic effect of adenosine . Taken together, these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides, resulting in the loss of the analgesic effect of pap . Additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation . A previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . In our rtx - induced neuropathy model, trpv1 was completely depleted, and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation . Furthermore, pain development correlated with trka profiles; for example, the ratios of pap(+)/trka(+) neurons were linearly correlated with the degree of mechanical allodynia . This report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling . In addition to previous studies demonstrating that 4mc promotes skin reinnervation, the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma, as indicated by the reduced ratios of atf3(+)/pap(+) neurons . Conversely, ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . Coincidentally, several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission . The present study shows contrary outcomes; for example, 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+) neuronal profiles coinciding with the normalization of mechanical allodynia . 4mc appears to have a pleiotropic effect in modulating neuronal activity; for example, it also increases the synthesis of the brain - derived neurotrophic factor (bdnf) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . Regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy, bdnf modulates the function of the motor and myelinated sensory neurons, but no effect was observed in this current neuropathic model because (1) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons, and (2) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 (nt3) expression in the peripheral tissues, as determined in our previous study . Regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy, this report provides immunohistochemical evidence of the potential effect of 4mc, as demonstrated by the pap / trka ratios of approximately 70% . Collectively, these data reinforce our speculations: 4mc regulates pap pathology through ngf - trka signaling, which was confirmed through alternative ngf administration and ngf neutralization experiments . Combining with the 4mc reduced the atf3(+)/pap(+) neurons; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . In summary, atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap, and pap is a downstream molecule of ngf - trka signaling, restoring the analgesic effect of pap in a trka - dependent manner (figure 5). Figure 5.diagram of prostatic acid phosphatase (pap) pathology modulates the pain perception in resiniferatoxin (rtx) neuropathy . This diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner, as described in the following steps: (1) rtx sensitizes the transient receptor potential vanilloid subtype 1 (trpv1). (2) rtx then induces activating transcription factor 3 (atf3) upregulation on pap(+) neurons, indicating the pap pathology . (3) the pap pathology reduces the amp hydrolysis . (4) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . (5) nerve growth factor (ngf) binds to high - affinity ngf receptor (trka) and (6) reverses the induction of the atf3 and pap pathology . (7) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . Diagram of prostatic acid phosphatase (pap) pathology modulates the pain perception in resiniferatoxin (rtx) neuropathy . This diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner, as described in the following steps: (1) rtx sensitizes the transient receptor potential vanilloid subtype 1 (trpv1). (2) rtx then induces activating transcription factor 3 (atf3) upregulation on pap(+) neurons, indicating the pap pathology . (3) the pap pathology reduces the amp hydrolysis . (4) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . (5) nerve growth factor (ngf) binds to high - affinity ngf receptor (trka) and (6) reverses the induction of the atf3 and pap pathology . (7) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . Rtx is specifically neurotoxic to trpv1(+) neurons; it also induces small - fiber neuropathy, such as skin denervation, which results in thermal hypoalgesia, small - diameter neuron injury, and peripheral sensitization of p2x3 purinoceptors, leading to mechanical allodynia . Atf3 is both an injury marker and a marker of pain; for example, the ratios of p2x3(+)/atf3(+) neurons correlate linearly with the degree of neuropathic pain . Pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+) and pap(+)/peripherin(+) neurons . Thus, injured pap neurons are critical for modulating neuropathic pain; that is, the ratios of atf3(+)/pap(+) neurons are inversely linear to the mechanical threshold . The balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity, which responded for the cellular pathophysiological homeostasis . The ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine, which acts as an analgesic ligand . Pap downregulation leads to the imbalance of amp / adenosine ratios, inhibiting the analgesic effect of adenosine . Taken together, these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides, resulting in the loss of the analgesic effect of pap . Additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation . A previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . In our rtx - induced neuropathy model, trpv1 was completely depleted, and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation . Furthermore, pain development correlated with trka profiles; for example, the ratios of pap(+)/trka(+) neurons were linearly correlated with the degree of mechanical allodynia . This report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling . In addition to previous studies demonstrating that 4mc promotes skin reinnervation, the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma, as indicated by the reduced ratios of atf3(+)/pap(+) neurons . Conversely, ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . Coincidentally, several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission . The present study shows contrary outcomes; for example, 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+) neuronal profiles coinciding with the normalization of mechanical allodynia . 4mc appears to have a pleiotropic effect in modulating neuronal activity; for example, it also increases the synthesis of the brain - derived neurotrophic factor (bdnf) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . Regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy, bdnf modulates the function of the motor and myelinated sensory neurons, but no effect was observed in this current neuropathic model because (1) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons, and (2) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 (nt3) expression in the peripheral tissues, as determined in our previous study . Regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy, this report provides immunohistochemical evidence of the potential effect of 4mc, as demonstrated by the pap / trka ratios of approximately 70% . Collectively, these data reinforce our speculations: 4mc regulates pap pathology through ngf - trka signaling, which was confirmed through alternative ngf administration and ngf neutralization experiments . Combining with the 4mc reduced the atf3(+)/pap(+) neurons; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . In summary, atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap, and pap is a downstream molecule of ngf - trka signaling, restoring the analgesic effect of pap in a trka - dependent manner (figure 5). Figure 5.diagram of prostatic acid phosphatase (pap) pathology modulates the pain perception in resiniferatoxin (rtx) neuropathy . This diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner, as described in the following steps: (1) rtx sensitizes the transient receptor potential vanilloid subtype 1 (trpv1). (2) rtx then induces activating transcription factor 3 (atf3) upregulation on pap(+) neurons, indicating the pap pathology . (3) the pap pathology reduces the amp hydrolysis . (4) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . (5) nerve growth factor (ngf) binds to high - affinity ngf receptor (trka) and (6) reverses the induction of the atf3 and pap pathology . (7) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . Diagram of prostatic acid phosphatase (pap) pathology modulates the pain perception in resiniferatoxin (rtx) neuropathy . This diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner, as described in the following steps: (1) rtx sensitizes the transient receptor potential vanilloid subtype 1 (trpv1). (2) rtx then induces activating transcription factor 3 (atf3) upregulation on pap(+) neurons, indicating the pap pathology . (3) the pap pathology reduces the amp hydrolysis . (4) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . (5) nerve growth factor (ngf) binds to high - affinity ngf receptor (trka) and (6) reverses the induction of the atf3 and pap pathology . (7) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . For pain development, this study provides evidence of an alternative pathway to conventional small nociceptor sensitization - induced neuropathic pain; the loss of analgesic effect of pap is mediated by neuronal soma irritation and/or injury . Alternatively, restoring the analgesic ligand may be a new direction for targeting painful neuropathy, that is, maintenance of steady - static adenosine content and functional pap ectonucleotidase activity at the neuronal soma level . The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article . The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by grants from the national science council (nsc100 - 2320-b-037 - 018 and nsc102 - 2320-b-037 - 009); ministry of science and technology (most103 - 2320-b-037 - 015-my3), taiwan; and kaohsiung medical university hospital (kmuh104 - 4m19).
, it ranked first among both kuwaiti and non - kuwaiti women . During the period from 2000 to 2008, it constituted 36.0% and 39.5% from the newly diagnosed cancer cases in kuwaiti and non - kuwaiti females, respectively . Excision of the primary tumor (by mastectomy or breast conserving surgery) and sentinel lymph node or axillary lymph node dissection are standard procedures for the treatment of most cases . Surgical site infection (ssi) the development of ssi can lead to prolonged hospital stay with increased costs, poor cosmetic results, psychological trauma, and, occasionally, a delay in postoperative adjuvant therapies . To the best of our knowledge, no published studies reported ssi rate after breast cancer surgery in kuwait . Hence, the present investigation was aiming to determine the ssi rate after breast cancer surgeries and the causative microorganisms as a step to improve the management and the outcome of such patients . All patients undergoing breast surgery in kuwait cancer control center which is a comprehensive cancer center devoted exclusively to the care of patients with cancer as a treatment for breast cancer from january 2009 to december 2010 were prospectively followed for the development of ssi . Indirect prospective detection of ssi was used to identify patients with ssis [5, 6]. Patients were followed up to 30 days after surgery if there was no implant and up to one year when there was an implant placed during the operation . Ssis were identified using centers for disease control and prevention / national healthcare safety network (cdc / nhsn) surveillance definition of health care - associated infections . Diagnosis was based on collecting information from patients medical records including reviewing of clinical data (symptoms and signs), investigations (laboratory, histopathology, radiological, etc . ), microbiological culture and sensitivity results, and medication charts in addition to discussions with the treating surgeons [5, 6]. Infections were identified either during the original surgical admission, at readmission to the hospital, or during outpatient follow up of the surgical wound by reviewing of medical records of surgery clinic patients [5, 6]. Ssi rate during the study period was calculated using the following formula: (1)number of ssis in breast cancer surgery100number of breast cancer surgery . The total number of operations performed for breast cancer during the study period was 438 . Females represented the majority of patients, 434 (99.1%), while males constituted only 4 (0.9%) cases . Breast surgeries done were excision of the tumor either by breast conserving surgery or mastectomy accompanied with sentinel lymph node biopsy or axillary lymph node clearance . Two patients 2/10 (20%) required secondary wound closure or incision and drainage of the wound in the operating room . Most of the ssis (8 cases, 80%) were detected after patients discharge, during outpatient follow up of the surgical wound . Out of them 5/8 (62.5%) all patients with ssi except one (90%) received systemic antibiotic therapy for management of their wound infection . Staphylococcus aureus (s. aureus) was the most common pathogen (isolated from 40% of all cases). Details of microbiological results were reported in table 1 . The majority (6/8 (75%)) of empirical therapy it was reported that around 75% of breast cancer cases had undergone surgery as a part of their treatment . However, any surgical procedure has the potential risk of infection . The potential morbidity caused by infection can lead to serious complications . Delaying postoperative radiotherapy results in poorer control of local diseases . A significant increase in metastatic relapse and reduced survival exist when adjuvant chemotherapy is delayed . (2.7%), 4.5% by leinung et al . By olsen et al . Most of ssis were diagnosed after patients' discharges . With the current trends favoring a shortened postoperative hospital stay, it becomes more likely that a significant percentage of surgical site infections will occur after these patients' discharges . Other investigators reported that an estimated 47% to 84% of ssis occur after discharge; most of these are managed entirely in the outpatient setting [13, 14]. Minor infections are usually managed on outpatient basis while complicated wounds require readmission with the possibility of surgical interference . This is usually accompanied by delay in wound healing and consecutively delays of the start of postoperative adjuvant therapy . Most of our patients who developed ssi were readmitted for management of their complicated infections (including reoperation) adding extra cost on the healthcare settings and may worsen the patient outcome . We believe that the rate of ssi in our study may be higher than we reported . As the postoperative length of stay is decreasing, the follow up of the patient is mainly carried out on an outpatient basis . During outpatient visits, when the ssi develops and requires no readmission, surgeons may not document the infection in the patient's records and may not request microbiological sampling of the wound . This is primarily due to fear of medical malpractice claims or negligence especially in a surgery classified as a one like breast surgery . In the present investigation, despite the fact that s. aureus (mssa and mrsa) was the primary pathogen isolated from ssis, gram negative bacteria were isolated in 40% of cases representing a significant finding . However, mukhtar et al . Reported gram negative bacteria as the most common isolated pathogens . These findings support the importance of the use of empirical broad - spectrum antimicrobial (not only targeting s. aureus) coverage (if any) until culture results become available.
Among natural amino acids histidine is one of the strongest metal ion coordinators . Hence, histidine plays an important role in metal ion coordination in proteins and peptides . Histidine is a tridentate ligand, which provides ligands at the imidazole imido nitrogen, the amino nitrogen, and the carboxylate oxygen . The imidazole ring nitrogen of histidine residues often provides the primary coordination site for metal ions . Copper is an essential metal ion for biological functions and is found in bound forms in metalloproteins and in low molecular weight complexes to avoid toxicity associated with free copper . Copper - containing proteins often have binding sites with irregular geometries containing one or more histidine ligands . Copper coordination in amyloidogenic proteins such as amyloid- (a), prion, and -synuclein is achieved through histidine residues . Small changes in the coordination may have an effect on aggregation and other chemical mechanisms in the diseased state . Hence, it is critical to elucidate the structural details of the different coordination modes to completely understand the biological role played by metal protein complexes . In -synuclein two cu(ii) coordination sites at the n - terminal region were identified using mass spectrometry (ms) and nuclear magnetic resonance (nmr). Later circular dichroism (cd) and electron spin resonance (esr) results indicated that these sites are independent of each other . The high affinity site is anchored by the n - terminus, and the second site is anchored by the histidine at position 50 . Other than these two sites, recent nmr and esr results propose a low affinity cu(ii) binding site at the c - terminal region . In prion protein the presence of four binding sites at higher cu(ii) occupancy in each of the phgggwgq octarepeat regions were identified based on esr, cd, nmr, and x - ray absorption spectroscopy (xas) results . Two additional cu(ii) equivalents coordinate via the histidine residues located at positions 96 and 111 . Additionally, the cu(ii) coordination environment in prion octarepeat domain has been extensively studied by ab initio methods . The copper coordination to a is highly heterogeneous, with the specific coordination environment depending on the ph, ionic strength, concentration of the metal ions, and the presence of other metal ions . The metal binding domain of a contains three histidine residues at positions 6, 13, and 14 . The presence of all three histidine residues in cu(ii) coordination spheres was initially proposed by nmr and was later confirmed by esr . Other techniques such as xas, fourier transform infrared spectroscopy (ftir), cd, ultraviolet visible (uv vis), ms, and ab initio calculations also proposed the involvement of histidine residues in cu(ii) coordination . A number of esr studies have suggested an equilibrium between two different cu(ii) coordination modes in an equimolar acu(ii) complex at ph 7.4, known as component i and component ii . Component ii is dominant at higher ph (ph 8.7). These two components are believed to have a different number of histidine residues coordinated to the cu(ii) ion . Component i is believed to have two histidine residues that simultaneously coordinate to cu(ii). Previously we used electron spin echo envelope modulation (eseem) in conjunction with n isotopic labeling, to determine that the a component i consists of three subcomponents, at physiological ph where two of the three histidines are simultaneously coordinated to cu(ii) ion . Interestingly, only two of these subcomponents, are present at lower ph values . Cu(ii) coordinated to his 6his 13 and his 6his 14 are found in equal proportions, while the his 13his 14 coordination is absent at lower ph . These measurements were able to uncover the critical role of his 13his 14 in cu(ii) coordination at physiological ph and provide a possible rationale for the presence of amorphous aggregates, rather than fibrils at high cu(ii) concentrations . The number of histidine residues coordinated to cu(ii) in component ii is controversial . One hypothesis proposes that cu(ii) is coordinated to the co group of the ala 2glu 3 bond and three histidine residues . The second proposition involves the n - terminus, one n atom from one of the three histidine imidazoles, the backbone n from asp 1-ala 2 peptide bond, and the co group of the ala 2glu 3 peptide bond . The coordination of cu(ii) in component ii is believed to be highly dynamic . The elucidation of the coordination in component ii is critical as a recent research shows that, in the presence of zn(ii), cu(ii) coordination moves to a component ii - like coordination . Furthermore, it has been shown that zn(ii) can only displace cu(ii) from component i coordination . One equivalent of zn(ii) ions displaces 25% of the bound cu(ii) from a(116) at physiological ph, while rearranging the rest of the bound cu(ii) in component i. in the presence of one equivalent of zn(ii) ions, components i and ii account equally for cu(ii) coordination in a. however, at excess amounts of zn(ii), component ii becomes the dominant cu(ii) coordination mode . In brain tissues affected by the alzheimer s disease, the concentration of zn(ii) is approximately three times higher than cu(ii). Therefore, component ii cu(ii) coordination may be the most dominant coordination mode in vivo . Shedding light into the coordination of component ii is critical to understand the behavior of cu(ii) in ad etiology . Herein, we propose a method to quantify the number of n nuclei coordinated to a cu(ii) center by the use of integrated intensities of the fourier - transformed eseem . Eseem spectroscopy is a pulsed esr technique that has been used to identify and quantify the number of histidine imidazoles coupled to a cu(ii) center . Spectral simulations of the eseem spectra are used to determine the number of coupled n nuclei . Compared the normalized integrated intensities of the frequency domain eseem spectra to calculate the intensity reduction resulting from the replacement of a n with n in a(116)cu(ii) complexes . Although, the use of integration method was useful in elucidating important structural information, the validity of the method was not tested experimentally . Here we obtained eseem data from simple model complexes with different numbers of imidazoles coordinated to cu(ii). The normalized integrated intensities of the model complexes increased monotonically, when the number of imidazole rings increased in model complexes . Two small peptides with known cu(ii) coordination was used to test the validity of the method . The dahk finally, our method was used to distinguish between the two proposed modes of cu(ii) coordination . Then, in conjunction with n isotopic labeling we quantified the subcomponent proportions in component ii . For the preparation of tetrakisimidazolecopper(ii) sulfate (see figure 1a), 40 ml of a 1 m imidazole (99% sigma - aldrich co., st . Louis, mo) solution was added to 10 ml of 1 m cuso45h2o (98+% sigma - aldrich co., st . After a few days, dark blue colored crystals formed . For the preparation of bis(2-methylimidazole)copper(ii) diacetate (see figure 1b), 0.5 g of cu(ii) acetate hydrate (98% sigma - aldrich co., st . Louis, mo) and 1.0 g of 2-methylimidazole (99% sigma - aldrich co., st . Louis, mo) were added and dissolved in a mixture of chloroform (10 ml) and methanol (2.5 ml). Then, 15 ml of diethyl ether was added to the filtrate and stirred for 10 min . Then another 5 ml of diethyl ether was added and filtered under reduced pressure and washed with diethyl ether and chloroform . The solid was air - dried and recrystallized from methanol / diethyl ether . For dienimidazolecopper(ii) diperchlorate (see figure 1c), 30 ml of 2 mm cu(clo4)26h2o (98% acros organics, new jersey) in a methanol / acetonitrile (5:1) mixture was stirred with 3 ml of 2 mm triethylenediamine (97% fluka, netherlands) in water . Then, 10 ml of 2 mm imidazole in methanol was added to the mixture . After being left overnight, all the solvents used in synthesis were purchased from sigma - aldrich co., st . Crystals were dissolved in water to make 10 mm stock solutions . For esr experiments, samples were prepared in the presence of 25% (v / v) glycerol (99% sigma - aldrich co., st . Louis, mo), with a final cu(ii) concentration of 1.25 mm . Structures of the model complexes with 1, 2, and 4 imidazole rings and their crystal structures . The peptide phgggw, the cu(ii) binding domain of prion, was synthesized at the molecular medicine institute, university of pittsburgh, using standard fluorenylmethoxycarbonyl chemistry . Isotopically enriched [g - n]-n-fmoc - n-trityl - l - histidine was purchased from cambridge isotope laboratory (andover, ma), in which all nitrogen atoms are n enriched . Three different variants of amyloid-(116) (daefrhdsgyevhhqk) with each containing an n enriched histidine at either position 6, 13, or 14 were synthesized at the molecular medicine institute, university of pittsburgh, using standard fluorenylmethoxycarbonyl chemistry . Double - labeled peptides containing two n enriched histidine residues were synthesized in the same manner . All the labeled amyloid- (116) variants were purified by high - performance liquid chromatography and characterized by mass spectroscopy . Nonlabeled amyloid- (116) peptide was purchased from rpeptide (bogart, ga). The peptide fragment dahk, the n - terminus region of the human serum albumin, was purchased from fisher scientific, hanover park, il . (116) peptide was purchased from rpeptide (bograt, ga). For peptide samples, the concentration of the peptide was 1.25 mm and an equimolar amount of cu(ii) was present in both the samples . Samples were prepared in n - ethylmorpholine (nem) buffer at ph 7.4 in 25% (v / v) glycerol and appropriate amounts of hydrochloric acid . X - ray diffraction data for the one - imidazole and two - imidazole model complex structures were collected using a single crystal on a bruker x8 prospector ultra ccd diffractometer with a cuk (= 1.54178 a) imus radiation source . The parameters used during the collection of diffraction data for each structure are summarized in supporting information . Crystals were mounted in fluorolube oil on a mitegen mount and placed in a cold n2 stream (150(1) k) for data collection . X - ray diffraction data for the four - imidazole model complex was collected on a bruker smart apex ccd diffractometer with graphite - monochromated mok (= 0.71073) radiation at room temperature . For each structure, unit - cell dimensions were derived from the least - squares fit of the angular settings of 999 strong reflections from the data collection . Each structure was solved via direct methods, which located the positions of the non - hydrogen atoms . An inspection of fo vs fc values and trends based upon sin, miller index, or parity group failed to reveal any systematic error in the data . All computer programs used in the data collection and refinements are contained in the bruker program package apex2 v.2013.100 . All three crystal structures have been reported previously, and our results are in substantial agreement with those previously reported structures . A 200 l aliquot of complex samples with a concentration of 1.25 mm was transferred into a quartz tube with an inner diameter of 3 mm . All esr experiments were performed on a bruker elexsys e580 x - band or a bruker elexsys e680 x - band ft / cw spectrometer equipped with bruker er4118x - md5 and en4118x - md4 resonators, respectively . The temperature was controlled using an oxford itc503 temperature controller and an oxford cf935 dynamic continuous flow cryostat connected to an oxford llt 650 low - loss transfer tube . Continuous - wave esr experiments were carried out on sample solutions at 80 k and at x - band microwave frequency . The magnetic field was swept from 2600 to 3600 g for 1024 data points . A time constant of 10.24 ms, a conversion time of 20.48 ms, modulation amplitude of 4 g, a modulation frequency of 100 khz, and a microwave power of 0.1993 mw were the other instrument parameters used for the cw experiment . The three - pulse eseem experiments were performed on the sample solutions at 20 or 80 k, by using a /2 /2 t /2-echo pulse sequence with a /2 pulse width of 16 ns . The first pulse separation,, was set at 144 ns, and the second pulse separation, t, was varied from 288 ns with a step size of 16 ns . The experiments were carried out at the magnetic field, where the esr intensity was maximum . After the baseline correction, the spectra were fast fourier - transformed . Then the final spectra were obtained as the magnitude of the fourier transforms . We normalized the eseem spectra using the integrated intensity of h eseem signal (1316 mhz). Possible limitations of the above method are discussed in the results section . For a single n nucleus coupled to an electron spin, the relative modulation depth is1where k is the modulation depth and subscripts 14 and 1 denote the n and h spin, respectively . Superscripts and denote the and spin manifolds of the electron spin, respectively . For two equivalent n nuclei coupled to an electron spin the relative modulation depth becomes2 k14 is the modulation depth of the spin manifolds of the two equivalent n nuclei . The relative modulation depth of n increases with the addition of the n nucleus, and the factor of increase is given by3 if k14 and k14 are much smaller than 1, the factor converges to 2 . The theoretical value for k14 and k14 is approximately 0.15, for a /2 pulse length of 16 ns . If two nonequivalent n nuclei are coupled to the electron spin, the relative modulation depth is given as4k14 is the modulation depth of the spin manifolds of the two nonequivalent n nuclei . The increase in the relative modulation depth with the additional n nuclei is given by5if k14, k14, k14, and k14 are much smaller than 1, eq 5 simplifies to6if two n nuclei are equivalent (i.e., k14 = k14), the factor in eq 6 becomes 2 . However, obtaining modulation depth information from eseem time domain data is difficult as many components are overlaid in the signal . It has been shown that integrated intensities can be used to account for the changes in the modulation depth . In the frequency domain n eseem signal is well separated from the h eseem signal, so it is possible to integrate separately . The n eseem intensity is obtained by integrating between 011 mhz, and the h eseem intensity is obtained for the region between 1316 mhz . In this work we experimentally determine the validity of the use of eseem integration intensities to quantify the number of n nuclei coupled to a cu(ii) center . To this end a series of model complexes with different numbers of imidazole ligands coordinated to copper are used . Then, the eseem analysis was carried out on two small peptides with known cu(ii)histidine coordination, namely dahk and phgggw peptides . The dahk peptide is the n - terminus region of the human serum albumin (hsa) and coordinate cu(ii) in the well characterized atcun motive . The dahk cu(ii) complex contains a single n eseem active nucleus through the histidine imidazole coordination . The peptide phgggw is a truncated fragment of the octarepeat cu(ii) binding domain of the prion protein and has a known cu(ii) coordination including a crystal structure . Cu(ii) complex is specifically used as there are two nonidentical n eseem active nuclei coupled to cu(ii). One n nucleus is from the histidine imidazole coordination and the other from the peptide backbone coordination . Then we used eseem to distinguish between the two proposed modes of coordination for component ii of a. furthermore, the proportions of each histidine residue coordinated to cu(ii) ion as an equatorial ligand were measured with the use of systematic n - labeled histidine residues . In copper imidazole complexes, the imidazole ring has a noncoordinated distal nitrogen, which contributes toward the eseem signal at x - band frequencies (9.5 ghz), with pulse lengths used in this work (/2 = 16 ns). Hence, the n eseem intensity will increase with the addition of imidazole rings to the coordination . In order to quantify the increase in n eseem intensity we synthesized three model complexes (shown in figure 1) containing 1, 2, and 4 imidazoles coordinated to a copper center, respectively . All three complexes were synthesized as crystals, and the structure of the complexes was verified using x - ray crystallography . All these structures contain copper centers coordinated with four equatorial ligands according to the information gained from crystal structures . In the one - imidazole and the four - imidazole complexes, structures of the counterion ligands perchlorate and the sulfate, esr measurements are less sensitive to axial ligands and are not considered in the analysis . The one - imidazole complex contains four directly coordinated nitrogens; three from the tridentate ligand, and one from the imidazole ring . Directly coordinated nitrogens do not contribute to eseem at x - band, with pulse lengths used in this work . The one - imidazole model complex contains just single n eseem active nuclei, which is in the imidazole ring . As shown in figure 2, the one - imidazole complex has g and a values of (2.22 0.005) and (191 1) gauss, respectively . These esr parameters correspond to four nitrogen nuclei coordinated to the cu(ii) center in the equatorial plane, which is consistent with the structure of the one - imidazole complex, as shown in figure 1a . The two - imidazole complex has g and a values of (2.30 0.005) and (158 1) gauss, respectively, which is consistent with a two nitrogen and two oxygen nuclei equatorial coordination . For the four - imidazole complex, g and a values are (2.26 0.005) and (182 1) gauss, respectively, which again is consistent for four directly coordinated nitrogen nuclei . Hence, cw - esr parameters clearly show that model complexes maintain the same cu(ii) coordination environment in solution . Nuclear quadrupole interactions (nqi) of n give rise to features below 2 mhz . The broad feature around 4 mhz is due to the double quantum (dq) transition of the remote nitrogen in an imidazole ring . The intensity of the dq peak increases with the number of imidazole rings coordinated to the cu(ii) center . A peak around 9 mhz (black arrow in figure 3) is also indicative of multiple imidazole coordination . This peak is clearly observed in the two- and four - imidazole complexes . Experimentally obtained three - pulse eseem spectra of the model complexes at the maximum g position . Appearance of a peak around 9 mhz in two- and four - imidazole complexes is indicative of multiple imidazole coordination . The integrated intensity for n - eseem was calculated for the region between 011 mhz and then divided by the h - eseem intensity integrated between 13 and 16 mhz . Details of the error calculation are provided in the supporting information (see figure s3). The normalized n - eseem intensity increases from 8.4 to 21 when going from single imidazole to two imidazoles as shown in table 1 . When there are four imidazoles coordinated to the cu(ii) center, normalized n - eseem intensity is increased to 40 . Hence, there is a monotonic increase in the normalized n - eseem intensity with the increase of number of imidazole rings coordinated . In the text, values in the last column are referred as normalized integrated intensity . In order to test our claim in a biologically relevant system, eseem experiments were conducted on two different peptide fragments with well - known cu(ii) coordination . The four amino acid peptide dahk is the n - terminus fragment of the human serum albumin . In the dahk peptide, the imidazole ring of the histidine residue coordinates to the cu(ii) ion, and the distal nitrogen of the imidazole ring is the only eseem active nuclei as shown in figure 4 . The comparison between the eseem spectra of cu(ii)dahk and one - imidazole complex is shown in figure 4 . The intensity of dq peak reflects the number of imidazoles coordinated to the cu(ii) center . The normalized integrated intensity for cu(ii)dahk is 7.0 0.1 compared to 8.4 0.1 for the one - imidazole complex (table 1). Only one n - eseem active nuclei is present in the reported coordination of dahk . Then we used the phgggw peptide fragment, which is the cu(ii) binding domain of the prion protein . Resolved the cu(ii) coordination environment of the octarepeat fragment using esr experiments and a crystal structure of the cu(ii)phgggw complex . As shown in figure 5, cu(ii) is coordinated to an imidazole nitrogen in a histidine, two backbone nitrogens from two glycine residues, and an oxygen from a carboxylic group . The structure of the cu(ii) bound hgggw pentapeptide was found to be unstable in solution due to the breaking of the axial water coordination also, eseem results have indicated that histidine imidazole coordination and backbone coordination is present in the cu(ii)phgggw complex in solution . The eseem spectra of the cu(ii)phgggw complex and the two - imidazole complex are shown in figure 5 for comparison . Given the different coordination environments the two spectra do not have identical peak positions . The two - imidazole complex has a larger dq peak compared to the phgggw complex . The reported structure for the cu(ii)phgggw structure contains two n - eseem active nuclei from the distal nitrogen of the imidazole histidine and the backbone coordination as shown in figure 5 . Hence, the intensity of dq peak is expected to be different between the two complexes, as dq peak intensity is indicative of the number of imidazoles coordinated . However, the normalized n - eseem integrated intensity obtained for the cu(ii)phgggw complex is similar to the integrated intensity of the two - imidazole complex . This information verifies that the integration method can predict the number of n nuclei coupled to a cu(ii) center . Comparison of eseem spectra of cu(ii)phgggw complex and the two - imidazole complex . The integration analysis was used to determine the number of nitrogens coupled to the cu(ii) ion in a(116) in component ii . As shown in the inset of figure 6, zn(ii) selectively displaces cu(ii) coordinated to component i. at excess amounts of zn(ii) (ten equivalents of zn(ii)), component ii accounts for 65% of the overall coordination (inset of figure 6), where in the absence of zn(ii) the percentage is only 35% . Amyloid aggregates in brain tissues contain approximately three times zn(ii) than cu(ii). Figure 6 shows the comparison between the two - imidazole complex and the a(116)cu(ii) complex at ph 8.7 . At ph 8.7 only the component ii of cu(ii) coordination exists . The integrated intensities tabulated in table 1 suggest that two n nucleus are coupled to the cu(ii) ion . The features of the eseem spectrum clearly illustrate the imidazole histidine coordination as shown in figure 6 . The backbone coordination peak is possibly due to the coupling between the cu(ii) ion with the remote backbone nitrogen nuclei of glu 3, where cu(ii) is coordinated to the carbonyl oxygen of ala 2 . This suggests just a single histidine residue is coordinated to cu(ii) in component ii coordination . Comparison of eseem spectra of cu(ii)a(116) complex at ph 8.7 and the two - imidazole complex . The inset shows the increase of component ii cu(ii) contribution in the presence of zn(ii). The normalized integrated intensity (n - eseem / h - eseem) of a(116) is 17 compared to 21 and 22 for the two - imidazole complex and the phgggw, respectively . The lower value for a(116) is possibly due to the smaller number of protons that interact with the cu(ii) center . The cu(ii) centers in the model complexes are solvent accessible . In a(116) the solvent cu(ii) interaction may be restricted because of the neighboring amino acids . In our analysis we have normalized the integrated area of the h - eseem peak to be the same for all spectra . Therefore, we may be underestimating the n - eseem integrated intensity for a(116). Nevertheless, the normalization method does suggest that cu(ii) is coupled to two n - eseem active nuclei, not one or three . Hence, we can answer the crucial question of the number of histidines coordinated to component ii . Three - pulse eseem spectroscopy was used in conjunction with isotopic substitution to determine the coordination of component ii . Specifically the aim of these experiments was to provide more insight into the proportions of histidine residues involved in component ii coordination . Two histidine residues at a time are isotopically labeled with n. upon n substitution, the modulation depths of the frequencies due to eseem active n nuclei will decrease . This decrease is because the single quantum transition of n nuclei does not substantially contribute to the eseem signal . We compare the integrated intensities of nonlabeled and n - labeled variants . As the modulation depth of h frequency is not affected by the n substitution, the h eseem peak is used to normalize the integrated intensity of n eseem . Because two of the three histidine residues are labeled, the n eseem signal intensity results only from the single nonlabeled histidine . This provides a direct method to determine the extent to which each histidine residue is involved in component ii . All the samples were at ph 8.7, and the experiments were carried out at 3355 g, which corresponded to the maximum signal in the echo detected field sweep . The eseem intensities are integrated between 0 and 11 mhz in all the eseem spectra collected (figure 7). The integrated eseem intensity of the his 6,13-labeled variant was 40% of that of the nonlabeled variant . In the his 6,13 variant, his 14 is the only labeled histidine residue and the n eseem intensity results from only his 14 . Likewise, his 13 contributes 40% and his 6, 20%, toward the component ii cu(ii) coordination (table 2). The integrated intensities are tabulated in the supporting infomation (table s2). To further confirm the proportions of the histidine residues, we performed the experiments using single - labeled variants in which only one histidine is labeled at a time . The decrease in the signal intensity in the n eseem region with respect to the nonlabeled variant indicates the extent of the involvement of the labeled histidine in the coordination . The eseem spectra for single - labeled variants are shown in figure s4, supporting infomation . The integrated n eseem intensities (table s3, supporting infomation) for single - label variants show the similar pattern of his 14 his 13> his 6 as observed with the double - label variants . Three - pulse eseem spectra of the nonlabeled and single n - labeled a(116) variants mixed with equimolar amounts of cu(ii) at ph 8.7 . The decrease in intensity below 8 mhz in n - labeled a(116) variants gives the contribution of each histidine residue for component i in a(116)cu(ii). The inset shows an expanded view of the 06 mhz region for the labeled peptides . This trend can be rationalized by the pka values of the histidine side chains . His 6 has a pka of 7.1, while for his 13 and 14 the pka values are 7.7 and 7.8, respectively . As the imidazole ring in his 6 has a lower pka value, ring nitrogens will be deprotonated at lower ph values than in his 13 and 14 . When the ph is raised, his 13 and 14 rings become more accessible for cu(ii) coordination . Hence the proportions of his 13 and 14 are increased with the increase in ph . These results are in accordance with an x - ray examination of a(116), which suggests that his 13 and his 14 are readily accessible for metal ion coordination . The design of a curcumin scaffold was discussed in this work, which is used to compete for cu(ii) coordination with the his 13his 14 dyad . Hence, we suggest only one histidine residue is involved in component ii, with a preference to his 13 and his 14 over his 6 . The other residues involved in the component ii coordination are the carbonyl oxygen of ala 2, the n - terminus (asp 1), and the amide nitrogen of ala 2 . The peak around 2.8 mhz in the eseem spectra is indicative of backbone coordination and further confirms the involvement of the peptide backbone in component ii coordination . His 13 and his 14 equatorially coordinate to cu(ii) more than does his 6 . Our eseem results performed using both the double and single n - labeled histidine residues indicate that his 13 and his 14 have a higher preference for the equatorial coordination position in cu(ii) component ii coordination . Chemically, component ii cu(ii) coordination is really interesting as zn(ii) biologically, it is important to understand the component ii coordination, as component ii may be the most significant cu(ii) coordination in vivo, as zn(ii) coexists with cu(ii). The insight into the component ii coordination, more importantly the proportions of subcomponents, will shed light on understanding the role of metal ions in alzheimer s disease . We experimentally validated the use of eseem intensities to quantify the number of n nuclei coupled to a cu(ii) ion . A monotonic increase in the n eseem intensities were observed for the model complexes synthesized with different numbers of imidazole rings . Then, the validity of the method was tested with two well - characterized cu(ii) binding peptides . We used our method to solve an important structural problem in acu(ii) complex . We determined that only a single histidine residue is coordinated to cu(ii) ion in component ii in a. finally, in component ii, cu(ii) uses his 13 and his 14 as an equatorial ligand over his 6 . The proportions of the three histidine residues in cu(ii) coordination can be rationalized by the pka values of the histidine side chains . Shedding light into the component ii coordination was critical as component ii might be the dominant cu(ii) coordination mode of a in vivo.
Over the past several decades, numerous studies have described pharmacologic strategies to utilize matrix metalloproteinase - inhibitors (mmp - is) to prevent connective tissue breakdown associated with various inflammatory and other diseases, for example, periodontitis, arthritis, osteoporosis, cardiovascular disease, and cancer [14]. Recently, these have also included less obvious strategies such as (but not limited to) blocking mmp - mediated cleavage of insulin receptors in type-2 diabetics to improve insulin sensitivity and to reduce hba1c levels . However, to date, the only orally (systemically) administered mmp - is approved by the us - fda and other national regulatory agencies (europe and canada) are those based on the surprising nonantimicrobial properties of the tetracycline antibiotics [4, 79]. In this regard, studies on experimental animals and on human subjects have demonstrated the efficacy of nonantimicrobial tetracycline formulations, as pleiotropic mmp - is, in periodontal and other diseases [4, 7, 9, 10]. In addition to demonstrating that these medications, which include two formulations of subantimicrobial - dose doxycycline (both fda - approved), can inhibit collagenolysis, connective tissue destruction, and bone resorption in the diseased periodontal tissues, other therapeutic mechanisms have also been identified . These include suppressed expression of inflammatory mediators such as the cytokines (e.g., il-1, tnf-, and il-6), prostaglandins, reactive oxygen species (e.g., hocl), and nitric oxide, the latter reflecting the inhibition of inducible nitric oxide synthase [11, 12]. Given this background, a search has been underway for new drug molecules which exhibit a similar active site for mmp - inhibition as the tetracyclines but with a different phenolic superstructure . With this strategy in mind, the therapeutic potential of the tetracycline diketonic, metal - ion binding site [8, 9] has been expanded by the recent development of a new series of compounds with a similar zinc - binding moiety, which are bicyclic rather than tetracyclic, that is, the chemically modified curcumins or cmcs . The structures of these compounds, their potency and mechanisms of action as mmp - is, and their zinc - binding (and other) characteristics have been described recently, and a lead this compound, cmc 2.24, is a phenylamino carbonyl curcumin, is triketonic (which enhances its zinc - binding characteristics) in contrast to the diketonic active site on both the tetracyclines and on traditional / natural curcumin compounds, and has shown evidence of efficacy in vitro, in cell and organ culture, and in animal models of chronic inflammatory and other diseases [1315]. As additional background, recent studies have shown that natural / unmodified curcumin administered to rats with experimentally induced periodontal disease was effective in reducing inflammatory mediators and mmps in the gingiva and periodontal ligament but was ineffective in reducing the excessive resorption and loss of alveolar bone . Accordingly, the current report describes the first of a series of studies which examined the efficacy of cmc 2.24 as a pleiotropic mmp - i in several rat models of periodontitis with a particular focus on its ability to inhibit pathologic alveolar bone loss . Moreover, because of the long - standing interest in the link between the oral disease, periodontitis, and systemic inflammation (the latter associated with increased risk for various diseases, notably cardiovascular disease and more severe diabetes [4, 17]), the effects of treatment with this novel compound on biomarkers in the circulation were also examined . Eleven male holtzman rats (rattus norvegicus albinus) weighing 150250 g were maintained under pathogen - free conditions with controlled temperature (21 1c) and humidity (6570%) and a 12 h light - dark cycle . 30 g of lipopolysaccharide (lps) from escherichia coli (strain 055:b5; sigma chem co., st . Louis, mo, usa) diluted in phosphate buffered saline (pbs) was injected into the palatal gingiva (3 l volume per injection) using a hamilton microsyringe (agilent, santa clara, ca, usa) as described by us previously . These lps injections were made into the palatal tissue between the upper 1st and 2nd molars, on the left side of the animal, three times a week for 14 days (a total of 6 injections and 180 g of lps in each site). The opposite side received injections of the same volume of pbs vehicle and served as the control site (split - mouth also at the time of sacrifice, blood samples were collected and the serum and plasma were separated by standard procedure and analyzed for mmps and cytokines as described below . The study protocol was previously approved by the institution's committees (araraquara - unesp, sp, brazil, and stony brook university, ny, usa) for experimental animal use . The effects of cmc 2.24 (a phenylamino carbonyl curcumin) were assessed in a prophylactic model (the efficacy of this compound in a therapeutic model will be assessed in future studies) in which the induction of periodontal disease by lps injections was carried out during the same period of time (14 days) as the daily oral administration of cmc 2.24 (30 mg / kg) or the vehicle - control . The test compound and the vehicle - control (a 1 ml suspension of 2% carboxymethyl cellulose) the rats and their periodontal tissues were randomly distributed into the following experimental groups as illustrated in figure 1 . Group 1gingiva injected with pbs in rats systemically administered vehicle alone (n = 5); group 2gingiva injected with e. coli lps in the vehicle - treated rats (n = 5) (note: with this split - mouth design, group 1 and group 2 tissues involve the same 5 rats); group 3gingiva injected with pbs in rats systemically administered the test medication (cmc 2.24; n = 6); and group 4gingiva injected with e. coli lps in rats systemically administered cmc 2.24 (n = 6) (as above, groups 3 and 4 involve the same 6 rats). However, for the -ct analysis, additional rats were added to each experimental group resulting in n = 10 rats per group . The gingival tissues from the hemimaxilla of each rat were excised and pooled per experimental group (5 - 6 rats per group) as described by us previously [19, 20]. The pooling of gingival tissues for each group was necessary because individual rats do not yield sufficient gingiva for enzyme analyses . The gingival tissues were extracted and the mmps were partially purified as described by us previously [19, 20]. In brief, the samples were homogenized (all procedures at 4c) with a glass grinder (kontes, glass co., vineland, nj) attached to a t - line lab stirrer (model 106 taboys engineering corp ., nj) in 50 mm tris - hcl buffer (ph 7.6) containing 5 m urea, 0.2 m nacl, and 5 mm cacl2 and then extracted overnight and centrifuged at 15,000 rpm for 1 h. the supernatants were collected and dialyzed exhaustively against 50 mm tris buffer (ph 7.8) containing 0.2 m nacl and 5 mm cacl2 . Ammonium sulfate was added to the dialysate to produce 60% saturation, allowed to stand overnight, and the precipitate containing the mmps was collected by centrifugation at 15,000 rpm for 90 min . The pellets were then dissolved in the tris buffer (ph 7.8) containing nacl, cacl2, and 0.05% brij and exhaustively dialyzed against the same buffer . The relative levels of the higher molecular weight proforms and the lower molecular weight activated forms of mmp-2 and mmp-9, in the pooled gingival extracts from each of the four experimental groups (figure 2), were determined by zymography (the gelatin zymography system was purchased from invitrogen corp ., carlsbad, ca). In brief, all samples were run under nonreducing denaturing conditions on the gelatin zymography system containing polyacrylamide copolymerized with gelatin at a final concentration of 1 mg / ml . After electrophoresis, the gels were washed in 2.5% triton x-100 and incubated at 37c overnight in the assay buffer (40 mm tris, 200 mm nacl, and 10 mm cacl2; ph 7.5). Clear zones of lysis against a blue background indicate gelatinolytic activity, as described by us previously [11, 21, 22]. Densitometric analysis of the gelatinolytic bands was carried out using the scientific imaging system (kodak i d 3.5, rochester, ny). Since this is a major outcome in the experimental periodontal disease model and since reducing alveolar bone loss is a key therapeutic goal in treating human inflammatory periodontal disease, two methods were used to assess the effects of cmc 2.24 on this inflammatory - driven bone loss model . As described previously, the soft tissues were carefully dissected to maintain the integrity of the maxillary bone specimens . These were then completely defleshed by immersion in 8% sodium hypochlorite for 4 h followed by gentle mechanical scavenging of the remaining soft tissue . After washing in running water, the specimens were immediately dried with compressed air . To distinguish the cementum - enamel junction (cej), 1% aqueous methylene blue solution (sigma - aldrich, saint louis, mo, usa) was applied to the specimens for 1 min and then washed in running water . The specimens were fixed on 3 mm thick red dental wax with their palatal surface facing up . Standardized orientation was achieved by positioning the specimens with the palatal cusp tip of the first and second molars superimposed on the corresponding buccal cusp tips (i.e., occlusal plane perpendicular to the ground). To validate measurement conversions, the specimens were positioned under a stereomicroscope (leica mz6, buffalo grove, il, usa) and digital images were obtained at 25x magnification using a 6.1-megapixel color digital camera coupled to the microscope . A single examiner, who was not aware of the experimental group allocation of the specimens, carried out all morphometric measurements of alveolar bone loss by delineating the area of exposed root surface of the first and second molars using an image analysis software (leica application suite, v3.8.0, leica microsystems, buffalo grove, il, usa) and the results were converted to mm using measurement of the reference millimeter grid . The area of exposed root surface in each specimen was averaged according to the experimental groups . Intraexaminer calibration was performed by evaluating repeated measurements of 10 nonstudy images presenting alveolar bone loss similar to the present study ., the hemimaxillae of the rats were dissected including teeth and surrounding soft tissues, fixed for 1824 h in 10% neutral buffered formalin at 4c, washed in distilled water, and transferred to 70% ethanol . This procedure allowed us to use these same specimens for the histological assessments used in subsequent studies (guimaraes et al . These samples were scanned on a microcomputer tomograph (skyscan 1176, skyscan, aartselaar, belgium) using 18 m slices . The digital radiographic images of each sample were reconstructed into a three - dimensional model (nrecon software, skyscan, aartselaar, belgium) consisting of a matrix of 18 18 18 m and a standardized gray scale value to visualize only mineralized tissues . Using the software package dataviewerctanctvol (skyscan, aartselaar, belgium), the reconstructed tridimensional matrix of each sample was initially reoriented in a standardized manner on three planes: sagittal, coronal, and transversal . Subsequently, a cubic region of interest (roi) of 9.72 mm was defined using standardized dimensions and anatomical landmarks: cementum - enamel junction of the first molar as the coronal limit extending vertically 1.5 mm apically, an anteroposterior dimension of 3 mm from the distal aspect of the mesial root of the first molar, and the transversal (buccolingual thickness) dimension of 2.16 mm (120 slices of 18 m each). This roi included the first molar, half of the second molar, and also approximately 1 mm from the most palatal aspect of the first molar crown (including the palatal bone adjacent to the first and second molar teeth which was the site of lps injections). We determined the relative volume of this roi occupied by mineralized tissue in each sample . The data was averaged for each experimental group and compared by nonpaired t - tests using welch's correction for unequal variances . Mmp-8 levels in plasma and gingival extracts, the latter prepared as described above, were determined by western blot analysis . In brief, samples were reduced, boiled, subjected to sds / page, and transferred to polyvinylidene difluoride (pvdf) membrane (amersham pharmacia biotech inc ., piscataway, nj). Blots were blocked with 5% nonfat dry milk for 2 h at room temperature . The membranes where then incubated with polyclonal antibodies specific for mmp-8 (abcam plc, cambridge, ma) overnight at 4c . Blots were washed and incubated with secondary antibodies purchased from thermo scientific for 2 h at room temperature . Detection of the bands was carried out on radiographic film by using supersignal west dura extended duration chemiluminescent substrate (thermo fisher scientific inc ., the band densities were quantified by scanning on a laser densitometer . To assess the levels of inactive (proform) and smaller molecular weight active forms of the mmp-8 (collagenase-2), bands corresponding to both molecular weight forms were quantitated, and the data is expressed as densitometric units and as the ratios of inactive / active forms . Recombinant rat mmp-8 (source: mouse myeloma cell line, nso derived) from r&d systems (minneapolis, mn) was used as a standard for western blot analysis of the rat plasma samples . This mmp-8 standard was incubated for 4 hours at room temperature, in the presence or absence of 1 mm amino phenyl mercuric acetate (apma), a known activator of higher molecular weight pro - mmps into the lower molecular weight activated forms . The level of mmp-13 was measured in the gingival tissue extracts and plasma of each rat by enzyme - linked immunosorbent assay (elisa). This assay was performed according to the manufacturer's instructions (tsz scientific llc, framingham, ma). The levels of 3 bone resorptive cytokines (il-1, il-6, and tnf-) were measured in serum and gingival tissue extracts by enzyme - linked immunosorbent assays (elisas). These assays were performed according to the manufacturer's instructions (r&d systems, minneapolis, mn), and the results were normalized to the total concentration of protein in the samples . The levels of both mmp-2 (72 kda progelatinase) and mmp-9 (92 kda progelatinase) were assessed by gelatin zymography in pooled gingival tissue from half - jaws of rats from each experimental group (figure 2). Lps - induced periodontal disease dramatically increased mmp-2 and mmp-9 levels in the pooled gingival tissue, while lower levels of the pro- (higher molecular weight) and activated (lower molecular weight) forms of these gelatinases were seen in the gingival tissue from all of the other experimental groups . Treatment of the rats with systemically administered cmc 2.24 appeared to normalize the pathologically excessive levels of the various molecular weight forms of these gelatinolytic mmps in the lps - injected gingiva assessed either visually (figure 2(a)) or by densitometric analysis of the zymograms (figure 2(b)). Some reduction of these mmp proteinases by cmc 2.24 administration was also seen in the gingiva from the rats without lps injections (figures 2(a) and 2(b)). In a pattern reminiscent of the zymograms described above and based on morphometric analysis of alveolar bone height loss which measured the area of exposed root relative to the cementoenamel junction as a fixed anatomical landmark, lps injections into the gingiva significantly (p = 0.005) increased alveolar bone loss (figure 3). Moreover, when the lps - injected rats were treated by oral administration of cmc 2.24, alveolar bone loss was significantly reduced (p = 0.003) back to the normal level seen in the rats not exposed to gingival lps injections . Note that cmc 2.24 treatment did not affect alveolar bone loss in the control rats receiving injections of pbs vehicle rather than lps (figure 3). To confirm and expand these data on alveolar bone loss in the four experimental groups (figure 1),, these data again demonstrate that lps increased the loss of bone in the aoi and that cmc 2.24 administration reduced this bone loss to the level seen in the control rats in which the gingivae were injected with pbs instead of lps . Analysis of il-1 in extracts of the pooled gingival tissues indicated that lps injections markedly increased the level of this proinflammatory cytokine since it was not detectable in the extracts of the pbs - injected gingival tissue (figure 5(a)). Moreover, cmc 2.24 administration reduced the pathologically excessive levels of il-1 in the gingiva by 93% (figure 5(a)). Similar concentrations of il-6 were detected in the gingival tissues from the different groups of rats; however, the lps injections did not appear to affect these levels and cmc 2.24 treatment only slightly reduced the levels of this cytokine by about 15% (data not shown). Tnf- was undetectable in both gingival extracts and serum (see below). In the experimental protocol used in the current study (a split - mouth design), mmp-8 (neutrophil - type collagenase, collagenase-2) and mmp-13 (collagenase-3) were both detected in the plasma samples from the different groups of rats but neither was detected in the gingiva (see section 4). Based on western blot analysis, the plasma samples from the lps - injected rats (half - jaw only) which were treated by oral administration of cmc 2.24 appeared to exhibit reduced levels of activated, lower molecular weight forms of mmp-8 compared to the plasma from the lps - treated rats administered with the vehicle alone (controls) (figure 6(a)). Based on the densitometric analysis of these western blots (figure 6(a)), the plasma of the cmc 2.24-treated rats with lps - induced periodontitis exhibited a ratio of pro / active mmp-8 of 2.52 0.20 (sem) which was 89.5% higher than the ratio, 1.33 0.05, seen in the plasma from the vehicle - treated lps - periodontitis rats (figure 6(b)), and this inhibition of activation of the precursor (latent) form of mmp-8 by the cmc2.24 treatment was statistically significant (p = 0.024). Note that a 4-hour incubation of the standard recombinant rat mmp-8 with 1 mm apma, a known activator of pro - mmps in vitro, converted the higher molecular weight pro - mmp-8 into the smaller molecular weight activated form of this leukocyte - type collagenase (see figure 6(a)). The plasma levels of mmp-13 assessed by elisa were found to be about 1.1 g / ml . Administration of cmc 2.24 to the lps - periodontitis rats appeared to slightly reduce the levels of this collagenase in the plasma; however, this effect was not statistically significant (data not shown). Regarding the proinflammatory cytokines in the serum (figure 5(b)), because of the split - mouth design (see figure 1), there were no serum samples from rats without gingival lps injection . However, the levels of il-1 in the serum of these lps - exposed rats (about 30 pg / ml) were significantly (p = 0.03) reduced to undetectable levels by cmc 2.24 administration, a pattern similar to that seen in the gingival tissues (figure 5(a)). Il-6 showed higher concentrations in the serum (about 95 pg / ml) than il-1 in the lps - periodontitis rats, and, again, cmc 2.24 appeared to reduce the level of this cytokine . However, this lesser effect (about 18% reduction) was not statistically significant (data not shown). This paper advances a novel therapeutic strategy which uses systemically administered medications as adjunctive therapy to modulate the host response in periodontal disease (periodontal therapy has traditionally only focused on locally suppressing the pathogenic microorganisms in the oral biofilm), with applications for other chronic inflammatory diseases as well (see below). The clinical application of this strategy began with the discovery that tetracyclines (tcs), unexpectedly, can inhibit host - derived mmps, inflammatory mediators (e.g., the cytokine il-1), and collagen degradation including bone resorption; and by mechanisms not dependent on the antibacterial properties of these drugs [4, 710]. Soon thereafter, doxycycline was found to be a more potent mmp - inhibitor than other tetracycline antibiotics, including minocycline and tetracycline itself, and was subsequently developed and approved as a nonantibiotic low - dose formulation for long - term administration to patients with chronic periodontitis and the dermatologic inflammatory disease, rosacea [4, 9]. Based on these earlier and the current studies, the nontetracycline chemically modified curcumin (discussed below) appears to be as, or more, potent an mmp - inhibitor compound compared to doxycycline [4, 8, 9, 13]. As one example, the ic50 (the concentration of the compound required to inhibit 50% of mmp activity in vitro) of doxycycline has been reported to be approximately 15 m [8, 9]. In contrast, recent studies by our group have demonstrated ic50 levels of cmc 2.24 at even lower m levels (25 m) when tested in vitro against mmps such as mmp-8 (leukocyte - type collagenase), mmp-9 (leukocyte - type gelatinase), mmp-12 (macrophage metalloelastase), and mmp-14 (membrane - type mmp). However, a significant disadvantage of the approved subantimicrobial - dose formulations of doxycycline is that no increase in the dose of this tetracycline can be prescribed to the patient (which might be desirable in order to, possibly, enhance the efficacy of this treatment in collagen - destructive diseases, e.g., periodontitis) because the low nonantibiotic blood levels of the drug (<1 g / ml) produced by this formulation cannot be exceeded in order to prevent an important side - effect, namely, the emergence of tetracycline - resistant or pan - antibiotic - resistant bacteria . In contrast, the potential strategy of long - term administration of cmc 2.24, for inflammatory diseases, would not be undermined by this strict, low - dose, limitation because this compound is not an antibiotic like the tetracyclines . As described earlier (see section 1), natural curcumin has a similar active site (i.e., the diketone zinc - binding moiety) as the tetracyclines and can also modulate the host response including mmp - inhibition and suppression of inflammatory mediators [2531], although it is ineffective against alveolar bone loss (see below). However, the chemically modified curcumin, cmc 2.24, tested in the current in vivo study, has a modified active site which is triketonic as detailed by us in previous studies by zhang et al . Recently, newer host - modulating medications have also been investigated as adjunctive treatment for periodontal disease and related medical disorders . These, in particular, have included (1) the resolvins such as the polyunsaturated fatty acids which do not suppress the acute inflammatory response required by the host to combat infection, but which do prevent the tissue - destructive prolongation of this process, and (2) the subject of the current study, the chemically modified curcumins (cmcs). Of importance, the latter have shown improved solubility, zinc - binding, and biological effects in comparison with natural curcumin [13, 14]. Development of these cmcs is based on maintaining a similar active site for mmp - inhibition as that of the tetracyclines but with a different phenolic superstructure, which most recently resulted in the development of a new series of compounds with a triketonic zinc - binding moiety, which are still bicyclic rather than tetracyclic, that is, the chemically modified curcumins or cmcs . A series of these triketonic cmcs have been developed including cmc 2.5 (a methoxy carbonyl curcumin) which, in turn, has been superseded by a more potent mmp - i compound, cmc 2.24, a phenylamino carbonyl curcumin; the latter has shown evidence of efficacy (and safety) in vitro, in cell and tissue culture, and in vivo models of several diseases including arthritis, diabetes, and cancer [1315, 33]. The current study is the first to demonstrate efficacy of this compound, cmc 2.24, in an animal model of experimental periodontitis . Evidence of the onset and progression of this disease, induced by several injections of lps into the gingiva of the rat, included dramatic increases in several forms (both pro- and activated) of connective tissue - destructive mmp-2 (72 kda) and mmp-9 (92 kda) gelatinases, elevated levels of the inflammatory cytokine often associated with periodontitis, il-1, and, most importantly in this model, a significant increase in alveolar bone loss, assessed morphometrically and by -ct, in the same jaws as the increase in gingival inflammatory mediators and mmps (the impact of this local inflammatory disease and this experimental treatment on systemic levels of mediators is discussed below). The potent efficacy of cmc 2.24 was demonstrated by (i) the statistically significant reduction of the lps - induced, pathologically elevated alveolar bone loss down to the levels seen in the healthy controls and (ii) the essentially complete reduction of the pro- and activated, pathologically excessive levels of mmp-2, mmp-9, and il-1, in the inflamed gingival tissues back down to the un- (or barely) detectable levels seen in the control gingiva . As a result of the profound efficacy of this novel compound in this initial study, we now have a rationale to initiate studies using a modified animal model of experimental periodontal disease, which does not use split - mouth design, and in a periodontitis model in which the cmc 2.24 is administered therapeutically (after the disease has been established) rather than prophylactically as in the current study . In a more recent study in which alveolar bone loss was assessed at the cellular level histomorphometrically and histochemically, a similar pattern of change was seen, namely, that lps injection increased osteoclast - mediated bone resorption and that cmc 2.24 inhibited this mechanism of alveolar bone loss (guimaraes et al ., in preparation). The potency of the biological effects of cmc 2.24 at an oral dose of 30 mg / kg is further demonstrated by the fact that, in previous experiments, we have not observed a significant decrease of inflammatory - driven bone resorption with 100 mg / kg dose of natural curcumin . Interestingly, in recent experiments, we found that daily administration of 400 mg / kg of natural curcumin significantly reduced inflammatory - driven bone resorption in this model, but this dose of natural curcumin is more than 10-fold higher than the dose of cmc 2.24 administered in the current study (guimaraes et al ., in preparation). Regarding insights into the mechanisms, plus the impact of this local disease and its treatment on the systemic condition of the host, we also observed the following: (i) the apparent reduction of il-1 by cmc 2.24 treatment in the pooled gingival tissues was paralleled by a dramatic and significant reduction in this inflammatory mediator in the systemic circulation of the same animals, and (ii) for cmc 2.24 treatment, although it did not appear to alter the total levels of mmp-8 (neutrophil - type collagenase) in the blood samples of the lps - injected rats, it did significantly reduce the ratio of the lower molecular weight, activated, collagen - destructive forms of this collagenase relative to the higher molecular weight, inactive, proforms of this mmp (note that, in the current experiment, mmp-8 could not be detected in the pooled gingival tissue). Mechanisms could include the ability of cmc 2.24 to inhibit other neutral proteinases such as plasmin, elastase, and mmp-1 which are known to cleave the amino - terminal propeptide domain of pro - mmp-8, converting it into the smaller molecular weight activated forms [9, 20]. Of relevance to the mechanisms involving cmcs ability to inhibit pro - mmp activation, recent studies (s. simon et al ., unpublished data) indicate that 2.24 can inhibit serine neutral proteinases (i.e., neutrophil elastase) which could explain the reduced conversion of pro- into smaller molecular weight activated mmps which was observed in the current study in the systemic circulation . Still another possible mechanism involves the potential of this compound to inhibit the production of reactive oxygen metabolites (e.g., hypochlorous acid, hocl). These are known to mediate proteinase activation by dissociating the thiol group in the propeptide domain . This mechanism is significant because mmp-8 is largely derived from the degranulation of polymorphonuclear leukocytes, and, in the human periodontal pocket, mmp-8 constitutes about 8090% of the total collagenase in this lesion; mmp-13 is the second most dominant collagenase in the periodontal pocket in humans, contributing about 1020% of the total, and is thought to be derived from the junctional epithelium and bone cells [7, 34]. However, in the rat, mmp-13 is analogous to the constitutive collagenase, mmp-1, in humans and likely plays a role in physiologic turnover of collagen rather than the pathological degradation of collagen during periodontitis . In this regard, mmp-13 also could not be detected in the inflamed gingival tissues in the rats in the current study and, although it was detected in the plasma, was not reduced by cmc 2.24 treatment suggesting a preferential effect of the test compound on pathologically elevated rather than on constitutive levels of these mmps . Additional mechanisms include the ability of natural curcumins to inhibit various signaling pathways and transcription factors involved in the expression of inflammatory mediators (ap-1, mapk, nf - kb, and stat3) resulting in a decrease in the expression of the inactive proforms of the mmps and of inflammatory cytokines and, ultimately, a marked change in the microenvironment [25, 35, 36]. The results of this initial study indicate that the oral administration of a novel, triketonic phenylamino carbonyl curcumin (cmc 2.24), to rats with endotoxin- (lps-) induced periodontitis, is a significant and potent inhibitor of both pathologic alveolar bone loss and its inflammatory and collagen - destructive mediators . Moreover, this chemically modified curcumin appears to have additional benefits by reducing the impact of this local inflammatory disease on systemic biomarkers of the host without (apparently) negatively affecting the mediators of constitutive connective tissue turnover . Studies are now underway to expand these observations in additional rat models of experimental inflammatory periodontal disease with a particular focus on cmc 2.24 effects (i) on the cellular mechanisms of alveolar bone loss; (ii) in a model in which the test medication is administered therapeutically (i.e., after the disease has been established) rather than prophylactically; and (iii) on the pharmacokinetics (such as peak blood levels; serum half - life) of this novel compound.
Peroneal nerve entrapment is the most frequent nerve entrapment in the lower extremities and the third most common entrapment in the body, following the median and ulnar nerves . Peroneal neuropathies often develop after trauma, direct injury or upon entrapment during the course of the nerve proximal or distal to the fibular head . Mechanical entrapment by an osteochondroma in this anatomic region is a rare occurrence . In this study, five patients with a drop foot resulting from osteochondroma of the proximal fibula who did not receive a proper diagnosis in neurosurgery and physical therapy clinics are presented . A 2.5-year - old boy presented to the neurosurgery clinic with the complaint of limping that started shortly after beginning to walk . His neurologic background history and examination included cranial and comprehensive spinal mri for embolisms and congenital anomalies . All test results were negative for etiologic factors and the patient was referred to a physical therapy and rehabilitation clinic for six months . During the physical examination, simple gait analysis revealed missing dorsiflexion of the right foot . According to the medical research council (mrc) scale, further radiologic evaluation by plain x - rays, computerized tomography (ct) and magnetic resonance imaging (mri) displayed a posterolateral exostosis on the fibular neck (fig . 1). Surgery was performed, and an osteochondroma, including its cartilage cap, was removed . Pathologic examination revealed an osteochondroma with a thin, 2-mm cartilage cap . In the early post - operative period, electrostimulation and active and passive range of motion (rom) exercises were initiated . Within three months following surgery, full recovery of motor functions was observed . One year after surgery, electromyogram results indicated normal excitations, and recurrence of the tumor was not noted (table 1). A 15-year - old boy presented to the physical therapy and rehabilitation center (ptrc) complaining of inability to dorsiflex his left toe for five months . No improvement was detected with vigorous ptrc; therefore, the patient was referred to a neurosurgery clinic, where spinal mri studies were conducted . No improvement of the condition was observed, and the patient was referred to our department . The patient was examined, and dorsiflexion of the ankle and toe was determined as mrc grade 1 without any sensory deficit . Radiologic assessment detected compression of the peroneal nerve due to an osteochondroma of the proximal fibula and fatty degeneration of the proximal musculotendiosis area of extensor hallucis longus . Hourglass constriction of the peroneal truncus at the level of the fibular neck by an osteochondroma was observed . Pathologic specimens depicted an osteochondroma . Within the first 24 h following the surgery, electrical stimulation, accompanying active and passive range of motion exercises, was started . At the end of the post - operative first month, 5/5 muscle strength was achieved . An 11-year - old girl presented to the neurosurgery clinic complaining of gradual weakness of foot dorsiflexion, which was first noted 4 months prior to admission . She was evaluated for possible intervertebral disk pathology, but the radiologic studies were normal . She was referred to a physical therapy and rehabilitation clinic . By the time she consulted with our department, her ankle and toe extensors were assessed as mrc grade 2 without any sensory deficit . Radiologic examination showed osteochondroma that was accompanied by a bursitis at the level of the proximal fibula, causing peroneal nerve entrapment . Pathologic evaluation showed osteochondroma . By the third week following surgery, gradual increase of the muscle strength was noted . A 14-year - old boy presented to the neurosurgery clinic with weakness of foot dorsiflexion that was first noticed five months prior . Upon spinal examination for rehabilitation purposes, the ankle and toe extensor was evaluated as mrc grade 2 without sensory deficit . During the electromyogram radiologic examination showed osteochondroma accompanied by a bursitis at the level of proximal fibula, causing peroneal nerve entrapment . A 10-year - old girl presented to the physical therapy and rehabilitation center and neurosurgery clinic with spinal pathologies that started three months prior due to her complaint of right drop foot . The patient was examined and dorsiflexion of the ankle and toe was determined as mrc grade 1 without any sensory deficit . Radiologic examination showed osteochondroma accompanied by a bursitis at the level of the proximal fibula, causing peroneal nerve entrapment . Pathologic examination results indicated osteochondroma . A week after the surgery, the muscle began to gain strength . A 2.5-year - old boy presented to the neurosurgery clinic with the complaint of limping that started shortly after beginning to walk . His neurologic background history and examination included cranial and comprehensive spinal mri for embolisms and congenital anomalies . All test results were negative for etiologic factors and the patient was referred to a physical therapy and rehabilitation clinic for six months . During the physical examination, simple gait analysis revealed missing dorsiflexion of the right foot . According to the medical research council (mrc) scale, further radiologic evaluation by plain x - rays, computerized tomography (ct) and magnetic resonance imaging (mri) displayed a posterolateral exostosis on the fibular neck (fig . 1). Surgery was performed, and an osteochondroma, including its cartilage cap, was removed . Pathologic examination revealed an osteochondroma with a thin, 2-mm cartilage cap . In the early post - operative period, electrostimulation and active and passive range of motion (rom) exercises were initiated . Within three months following surgery, full recovery of motor functions was observed . One year after surgery, electromyogram results indicated normal excitations, and recurrence of the tumor was not noted (table 1). A 15-year - old boy presented to the physical therapy and rehabilitation center (ptrc) complaining of inability to dorsiflex his left toe for five months . No improvement was detected with vigorous ptrc; therefore, the patient was referred to a neurosurgery clinic, where spinal mri studies were conducted . No improvement of the condition was observed, and the patient was referred to our department . The patient was examined, and dorsiflexion of the ankle and toe was determined as mrc grade 1 without any sensory deficit . Radiologic assessment detected compression of the peroneal nerve due to an osteochondroma of the proximal fibula and fatty degeneration of the proximal musculotendiosis area of extensor hallucis longus . Hourglass constriction of the peroneal truncus at the level of the fibular neck by an osteochondroma was observed . Pathologic specimens depicted an osteochondroma . Within the first 24 h following the surgery, electrical stimulation, accompanying active and passive range of motion exercises, was started . At the end of the post - operative first month, 5/5 muscle strength was achieved . An 11-year - old girl presented to the neurosurgery clinic complaining of gradual weakness of foot dorsiflexion, which was first noted 4 months prior to admission . She was evaluated for possible intervertebral disk pathology, but the radiologic studies were normal . She was referred to a physical therapy and rehabilitation clinic . By the time she consulted with our department, her ankle and toe extensors were assessed as mrc grade 2 without any sensory deficit . Radiologic examination showed osteochondroma that was accompanied by a bursitis at the level of the proximal fibula, causing peroneal nerve entrapment . Pathologic evaluation showed osteochondroma . By the third week following surgery, gradual increase of the muscle strength was noted . A 14-year - old boy presented to the neurosurgery clinic with weakness of foot dorsiflexion that was first noticed five months prior . Upon spinal examination for rehabilitation purposes, the patient was referred to the physical therapy and rehabilitation clinic . Upon admission, the ankle and toe extensor was evaluated as mrc grade 2 without sensory deficit . During the electromyogram radiologic examination showed osteochondroma accompanied by a bursitis at the level of proximal fibula, causing peroneal nerve entrapment . A 10-year - old girl presented to the physical therapy and rehabilitation center and neurosurgery clinic with spinal pathologies that started three months prior due to her complaint of right drop foot . The patient was examined and dorsiflexion of the ankle and toe was determined as mrc grade 1 without any sensory deficit . Radiologic examination showed osteochondroma accompanied by a bursitis at the level of the proximal fibula, causing peroneal nerve entrapment . Pathologic examination results indicated osteochondroma . A week after the surgery, the muscle began to gain strength . The increasing number of fascicles in this area, expansion of epineural tissue, and nerve's unprotected surface transition play an important role in this pathology . The peroneal nerve is connected to the fibular head and is subjected to the movements of the fibula . Spur or tumoral structure formation of the tubular bones can potentially apply pressure to the surrounding structures . Flores and koerbel reported a case of peroneal nerve compression resulting from fibular head exostosis . Only a few cases of compressions of the peroneal nerve by cartilaginous exostosis have been reported thus far . However, due to their rare occurrence, lesions such as osteochondromas may be overlooked by non - orthopedic clinics . Osteochondroma is the most common benign bone tumor and can cause a variety of symptoms . Depending on the location of the lesion pain, a palpable mass, puffiness (inflammation), and tinel's sign might be significant clinical symptoms and findings . Furthermore, when an exositosis accompanying stiffness is diagnosed, hereditary multiple exositosis syndrome must be considered . Motor deficits are more common than sensory nerve lesions, which might be explained by the arrangement of the fascicles inside the common peroneal nerve . The exostosis grows from the bone surface to the periphery, compressing the motor fibers earlier . This is most likely due to medial placement of the motor branches within the nerve so that they are more prone to injury by a newly forming osteochondroma . Advanced knowledge of the peroneal nerve anatomy as well as the mri and ct scan assessments of the lesion are key factors for preventing complications . A surgery for an osteochondroma causing peroneal nerve entrapment should be performed within three months; otherwise, the surgical success rate decreases . Although the mean time that passed until the patients consulted with us was 5.7 months (range: 312 months), the loss of muscle strength in our cases fully recovered following surgery . The mean age of our patients was low, which could have played a role in the fast and complete neurological recovery (mean age 10.5 (2.515 years)). In spite of its frequent occurrence, drop foot associated with peroneal nerve entrapment by an osteochondroma is not easily remembered and diagnosed . Especially in pediatric patients, drop foot can be overlooked during physical examination and may even be more difficult to detect in non - orthopedic clinics . With careful physical examination along with radiologic and electrophysiologic studies, nerve entrapment can be easily diagnosed, enabling surgeons to perform surgery in a timely manner, thus preventing further sequelae . Written informed consent was obtained from each patient for the publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal . All authors have been equally involved in the collection of data and drafting of the manuscript.
Pneumothorax is more frequent in the neonatal period than at any other time in life . Symptomatic pneumothorax occurs in 0.08% of all live births and 5% to 7% of infants with birth weight of below 1500 the development of a pneumothorax with ensuing hypoxia and hypercapnia is a potentially life - threatening event . To predict who will survive or die for newborns presenting with pneumothorax traditionally a plain posteroanterior chest radiograph is the most appropriate method in the initial investigation . The calculation of pneumothorax size on chest x - ray or computed tomography is a significant parameter for decision of treatment in adults . However, to our knowledge, the clinical significance of this calculation in neonates is still unknown . Therefore we aimed to investigate whether assessment of pneumothorax size on chest x - ray may be a predictor of prognosis in newborns . This retrospective cohort study was performed on 60 newborns presenting with pneumothorax among 5929 infants admitted to our neonatal intensive care unit (nicu) from january 2007 to april 2011 . Our institution is a tertiary hospital and its nicu has an annual admission of 1500 newborns . The study was approved by the local ethics committee of tepecik teaching and research hospital . Clinical data were collected from patients medical records including gestational age, birth weight, gender, type of delivery, apgar score, side of pneumothorax, mechanical ventilation, surfactant therapy, underlying lung pathology (transient tachypnea of newborn, pneumonia, meconium aspiration syndrome), duration of hospitalization, time of diagnosis and need for chest tube . The chest tube insertion was performed by the nicu physicians as soon as the clinical indication (presence of acceptable respiratory distress, abnormal blood gas levels and cardiovascular instability) was evident . A common protocol for mechanical ventilation was performed by a positive pressure ventilator (babylog 8000 plus, draeger, lubeck, germany). Fio2 was given when it was needed to achieve arterial oxygen saturation between 85% and 95% by pulse oximetry . Inspiratory time was set at 0.35 - 0.40 sec and positive end - expiratory pressure at 4 - 6 cmh2o according to the status of the patient . The peak inspiratory pressure was set by the clinician to keep the blood gasses at the target range . The newborns were switched from the assist / control mode to the simv mode for weaning on the basis of each infant's blood gasses and clinical status . Ventilatory rate was then reduced using the simv mode of the ventilator . If newborns treated with fio2<30% tolerated it at a respiratory rate of 10 bpm, were extubated . The calculation was done on the chest x - rays stored electronically in hospital picture archiving and communication system (pacs). Pneumothorax size was calculated as the percentage of the widest transverse diameter of the pneomothorax area in the posteroanterior view to the widest transverse diameter of the thoracic cavity above the diaphragm . Fischer's exact test was used to compare categorical variables, mann - whitney u test for continuous variables and the wilcoxon rank sum test for ordinal variables . The regression analysis was performed for evaluating the statistical significance of confounding variables . A receiver - operator characteristic (roc) curve was used to explore various cut - off values of pneumothorax size . In order to determine whether the cut - off value was significant, this retrospective cohort study was performed on 60 newborns presenting with pneumothorax among 5929 infants admitted to our neonatal intensive care unit (nicu) from january 2007 to april 2011 . Our institution is a tertiary hospital and its nicu has an annual admission of 1500 newborns . The study was approved by the local ethics committee of tepecik teaching and research hospital . Clinical data were collected from patients medical records including gestational age, birth weight, gender, type of delivery, apgar score, side of pneumothorax, mechanical ventilation, surfactant therapy, underlying lung pathology (transient tachypnea of newborn, pneumonia, meconium aspiration syndrome), duration of hospitalization, time of diagnosis and need for chest tube . The chest tube insertion was performed by the nicu physicians as soon as the clinical indication (presence of acceptable respiratory distress, abnormal blood gas levels and cardiovascular instability) was evident . A common protocol for mechanical ventilation was performed by a positive pressure ventilator (babylog 8000 plus, draeger, lubeck, germany). Fio2 was given when it was needed to achieve arterial oxygen saturation between 85% and 95% by pulse oximetry . Inspiratory time was set at 0.35 - 0.40 sec and positive end - expiratory pressure at 4 - 6 cmh2o according to the status of the patient . The peak inspiratory pressure was set by the clinician to keep the blood gasses at the target range . The newborns were switched from the assist / control mode to the simv mode for weaning on the basis of each infant's blood gasses and clinical status . Ventilatory rate was then reduced using the simv mode of the ventilator . If newborns treated with fio2<30% tolerated it at a respiratory rate of 10 bpm, were extubated . The calculation was done on the chest x - rays stored electronically in hospital picture archiving and communication system (pacs). Pneumothorax size was calculated as the percentage of the widest transverse diameter of the pneomothorax area in the posteroanterior view to the widest transverse diameter of the thoracic cavity above the diaphragm . Fischer's exact test was used to compare categorical variables, mann - whitney u test for continuous variables and the wilcoxon rank sum test for ordinal variables . The regression analysis was performed for evaluating the statistical significance of confounding variables . A receiver - operator characteristic (roc) curve was used to explore various cut - off values of pneumothorax size . In order to determine whether the cut - off value was significant, sensitivity and specificity values were also calculated . Of 60 newborns (1.0%) presenting with pneumothorax among 5929 patients, 46 (76%) were preterm and 14 (24%) term . Pneumothorax was on the right side in 30 (50%) newborns, on the left side in 21 (35%) and bilateral in 9 (15%) patients . Of 46 preterm infants, 41 were on mechanical ventilation and 28 needed chest tube as overall 6 term infants on mechanical ventilation (fig . 1). The flowchart of the study group patients characteristics are summarized in table 1 . The mean gestational age of patients was 33.64.4 weeks and mean birth weight 2282909 g . Of overall 60 newborns, 45 (75%) were male and 15 (25%) female . Forty - three deliveries (71.6%) were by cesarean section, and the remaining by vaginal delivery . Seven patients (11%) had transient tachypnea of the neonate, 10 (17%) pneumonia and 27 (45%) respiratory distress syndrome . The cause of deaths included sepsis in immaturity and respiratory failure in 8 (44.4%) patients, sepsis in 5 (27.7%), severe intraventricular hemorrhage in 3 (16.6%) and hypoxia in 2 (11.1%). Five patients (13.3%) received prophylactic surfactant whereas 21 (35%) received rescue therapy . Demographics and patients characteristics (n = 60) sd: standard deviation results of statistical analysis for prognostic significance of pneumothorax size calculated on chest x - ray and clinical parameters are summarized in table 2 . Pneumothorax size was significantly higher in non - survivors (31.12.8 vs. 16.41.4, p<0.001) and also in patients treated with a chest tube (25.81.9 vs. 14.22.0, p<0.001). In addition, mortality discrimination was quantified by calculation of the area under the roc curve . Area under the curve (auc) was 0.85 (good discrimination by an area is 0.80). The cut - off point of pneumothorax size for predicting survival was determined as 20% . The sensitivity of the calculation of pneumothorax according to the cut - off value was 72% whereas specificity was 83% . Statistical analysis of significance in predicting mortality data are presented as mean (standard deviation) the confounding factors including early gestational age, low birth weight, resuscitation at birth, mechanical ventilation and need for chest tube were of great significance in predicting mortality . However regression analysis revealed that only pneumothorax size was the independent prognostic factor (p=0.02). In contrast, other parameters such as gender, type of delivery, time of diagnosis did not show any statistical significance between survivors and non - survivors . In the present study we aimed to evaluate significance of pneumothorax size in predicting the mortality of newborns . The development of a pneumothorax with ensuing hypoxia and hypercapnia is a potentially life - threatening condition and 30% of the infants in the present study died in nicu . This mortality rate is comparible with data in the literature of the last 20 years . Even if not fatal, morbidity is a significant risk in these patients . The risk for developing pneumothorax is increased in infants with respiratory distress syndrome, meconium aspiration syndrome, and pulmonary hypoplasia and in infants who need resuscitation at birth . Pulmonary disease is the underlying condition in 59 - 61% of neonates with pneumothorax . In our study pulmonary pathology this finding can be explained by the fact that the majority of our cases were preterm . Based on our findings we think that low birth weight and preterm birth are the significant risk factors for developing pneumothorax . Development of pneumothorax is increased in infants resuscitated with positive pressure ventilation and/or chest compression in the delivery room . The pathogenesis is either the rupture of over distended alveoli during resuscitation or lung injury due to rib fracture fallowing chest compression . Three fourth of our cases had a history of mechanical ventilation which was more common in non - survivors like those who needed chest tube . Although the risk factors related to mortality in the neonates presenting with pneumothorax are well - known, any practical method to predict who will survive or die is still of great importance in the clinical setting . The diagnosis of pneumothorax is usually confirmed by imaging techniques which may also yield information about its size as well . The size of pneumothorax on chest x - ray or computed tomography is a significant parameter in adults for treatment of the disease . To the best of our knowledge, there is no study in the english medical literature investigating the prognostic value of pneumothorax size in newborns . However the present study was carried out in a single large unit, therefore decreasing variation in the respiratory therapy was applied . Three dimensional lung volumes or lung pathologies like pneumothorax can be calculated by magnetic resonance imaging or computed tomography . Although these techniques are more reliable than routine chest x - ray, they are less practical especially in clinically unstable patients like those in the nicu . In addition chest x - ray is also a less expensive method . Because anteroposterior chest x - ray is a routine method, it is practical for the calculation of pneumothorax size in the nicu like assessment of cardiothoracic index and also reasonable for a retrospective study . In the present study, calculation of pneumothorax size revealed statistical significance as a prognosticator in the neonatal period . The pneumothorax size was significantly higher in non - survivors and appeared as an independent prognostic factor among the confounding variables . The present study also showed that the neonates who had a pneumothorax size greater than 20% calculated by our method were likely to have worse prognosis . The risk of mortality was 13 times higher in these newborns than in those with smaller pneumothorax size . Categorization of the patients according to the cut - off value appeared to show higher sensitivity and specificity . Although pneumothorax is not a cause of death, patients with pneumothorax usually carry higher risk of death and related complications due to more severe clinical status . In conclusion development of pneumothorax in the neonatal period carries high risk of mortality especially in low birth weight and premature infants . Therefore identifying the risk groups by calculation of pneumothorax size can be a practical and reliable method . However further studies are in need to emphasize whether this method has a rationale to be used in clinical protocols.
, urinary incontinence (ui) affects about one third of healthy elderly people, and up to 90% of nursing home patients . The proportion of the elderly population is increasing worldwide; it is currently around 10%, and is expected to reach 20% within 40 years . Accordingly, the prevalence of conditions such as metabolic syndrome and obesity are increasing annually, and these are key risk factors for voiding dysfunction . Moreover, voiding dysfunction influences patient quality of life, and can contribute to serious morbidities, such as falls and bone fractures . The average age of the south korean population is increasing more rapidly than that in other countries: the proportion of elderly people rose from 3.8% in 1980 to 9.5% in 2006 . Consequently, south korean society is becoming more concerned over voiding problems after middle age . For this reason, it is essential that investigators conduct epidemiologic studies into the prevalence of voiding dysfunctions in south korea . In this review, we aimed to clarify the prevalence and clinical features of voiding dysfunctions among both men and women . Several extensive epidemiologic studies have been performed in western countries since the 1990s; however, in south korea, epidemiologic studies have only been conducted since the start of the 21st century . To advance epidemiologic research, longitudinal designs confer greater statistical power than cross - sectional designs, because longitudinal research uses serial data that reveal substantial realistic prevalences, as well as the potential causes of the disease . On the other hand, more epidemiologic studies involve a cross - sectional design . To ensure significant epidemiologic data, investigators must select a proper target population and adopt proper survey methods; questionnaires should be adjusted to the target population . Early epidemiologic studies into voiding dysfunction were mainly performed by postal survey; this method has been commonly used since the 1960s . This approach involves lower costs and higher efficiency, and it has fewer effects on the patients mood; therefore, the postal survey served as a strong epidemiologic method . More recently, communication devices and multimedia technology have allowed telephone interviews and internet surveys to emerge as practical epidemiologic tools . Telephone interviews can provide a cross section of almost the entire population, and they are cost- and time - effective . Furthermore, the results of telephone interviews are less influenced by the interviewer than are those of personal interviews . Nonetheless, this method is limited, because, in the past, few people in south korea had telephones . With the increased distribution of telephones,, the telephone interview can be affected by social desirability bias, which can arise in cases of delicate personal questions, especially in the field of urology . Furthermore, some people feel their privacy is violated by random - dialing surveys; therefore, the nonresponse rate increases . After high - level communication and multimedia technology were developed, the internet survey was introduced to gather data . However, such an approach confers a coverage problem, because most people are still not capable of accessing surveys via the internet or electrical devices . For this reason, the internet survey must be developed further if it is to become the ideal survey design . In addition, internet surveys require comprehensive programming, which is not true of face - to - face interviews . Therefore, the face - to - face interview is still frequently used in epidemiologic studies, even though more money, time, and manpower are required . The major advantage of the personal interview is that the researcher can explain the intent and exact meaning of questions; this guarantees higher response quality . It is true that every survey method has its own advantages and handicaps, and sometimes various survey methods are used concurrently . Nonetheless, large - scale epidemiologic data are most frequently obtained by telephone interview or internet survey . Furthermore, in both sexes, ui, lower urinary tract symptoms (luts), overactive bladder (oab), and nocturia are highly prevalent, distressing conditions . Herein, we have provided an overview of the epidemiologic studies into the above voiding dysfunctions in south korea . All of the surveys included in this review were conducted after profound consideration of the various aspects . Furthermore, we have reviewed in detail each specific survey method, as well as the methods for obtaining detailed clinical details of voiding dysfunction . Finally, we considered the practical limitations of the survey selection processes, which are influenced by special factors . Its various voiding - related symptoms, enhanced by obstruction of the bladder outlet, include nocturia, tenesmus, urgency, and hesitancy; these symptoms reduce the quality of life in affected men . The prevalence of bph increases with age, and it affects 40% to 70% of men aged between 60 and 70 years . Bph is diagnosed pathologically in approximately 40% of autopsies of men in their 50s, and in 70% of autopsies of men in their 60s . Hence, in this review, we have used based based definitions of bph as a voiding dysfunction . To investigate the real prevalence of bph the demarcation of what constitutes clinical bph is somewhat controversial; to date, clinicians have come to no consensus regarding the definition of bph . In addition, different characteristics of the target population, as well as the methods used to collect information, can influence the results . This diversity in the definition of bph influences the ability of investigators to analyze and compare results across studies . The first study into bph prevalence in the inland area of chungcheongbuk - do was performed in 1999, and 27.7% of 764 men were found to be affected . In this study, bph was defined when the prostate volume (pv), measured by transrectal sonography, was 20 ml, and when the maximal flow rate (qmax) was 10 ml / sec . In another study, bph was defined when the international prostate symptom score (ipss) was 8, when the pv (measured by finger) was 30 ml, and when qmax was 15 ml / sec . The rate of moderate - to - severe ipss was 49.5% (213 in 431 men). But by the definitions in this study, bph prevalence were 4.3% of men in their 50s, 13.2% of men in their 60s, and 16.3% of men in their 70s or older (overall, 11.1% of men aged over 50 years). The population - adjusted prevalence of bph in south korean men aged 50 and over was 8.7% . Another community - based study defined bph in cases of an ipss 8 points and a qmax 10 ml / sec; the study found that the incidence of bph was 25.5% . On the basis of ipss, 36.3%, 49.7%, and 14.0% of the men were mildly (17), moderately (819), and severely (2035) symptomatic, respectively . In 2009, park et al . Found that the prevalence of bph, defined in cases of an ipss> 7 and a pv> 30 ml, was 40% . This study was based on the korean longitudinal study on health and aging, a population - based, prospective study involving a randomly sampled population of 301 men aged> 65 years living in seongnam, south korea . In the latest study, which involved 779 men who lived in yangpyeong county and participated in a prostate examination campaign, the wide range of bph prevalence in previous studies from 11% to 40%is mainly due to methodological differences between studies, such as differences in selection of the target population . Furthermore, the proportion of the population who are elderly is higher in some districts than in others . The prevalence of clinical bph in south korea is similar to that in western and other eastern countries . Specifically, the bph prevalence in scotland was estimated as 25.3% in 705 men . A cross - sectional study involving 8,466 iranian men (> 40 years old) conducted by 74 personal interviewers identified a prevalence of 23.8% when bph was defined as an ipss> 7, a qmax of <15 ml / sec, and a pv> 30 ml . Asian data were collected from china, south korea, singapore, pakistan, the philippines, india, thailand, and taiwan . The prevalences of an ipss 8 points were 18%, 29%, 40%, and 56% among men in their 40s, 50s, 60s, and 70s or older, respectively . These rates are higher than those reported for the united states (12% 17%, 23%, and 29%, respectively). This may be due to differences in the cellular composition of prostatic tissue between asians and caucasian - americans . Some studies have reported that asians have more portions that are glandular and less muscle and connective tissue, as well as a transition zone that occupies a higher volume ratio within the prostate . However, studies use various combinations of parameters to define bph, including symptom score, pv, and urodynamic parameters . Therefore, it remains difficult to determine the exact in prevalence, and to make further progress in the survey method . Even though ui is not life threatening, it is a severe health problem and causes psychological distress, hygiene problems, and social impairment in affected patients and their families . According to the international continence society definition, any involuntary leakage of urine constitutes ui . In general, many women regard ui as a natural consequence of childbirth and aging . Even though ui can be managed in various ways by clinicians, many women tend to manage their ui personally . In addition, due to shame, many people conceal their problem rather than ask for medical help . The incidence of ui increases with aging, and with the rapid growth of the aged population, ui is gradually becoming an enormous social burden in south korea . The earliest prevalence data for ui in south korea were reported in 2004; the study comprised an internet survey involving 3,372 respondents, and the prevalence of ui in women was 21% . An additional large telephone survey reported that the overall prevalence of ui was 40.8% among 1,301 women . With regard to ui type, 22.9% of cases were stress - type, 3.1% were urgetype, and 14.9% were mixed . In 2008, the korean national survey on urinary incontinence yielded nationwide representative data; in this survey, 13,345 households were interviewed . The prevalence of ui was 24.4%, and stress ui was the most prevalent type . A further population - based cross - sectional telephone survey of south koreans included 888 men and 1,112 women . The most predominant types were other (1.3%) in men and stress (20.7%) in women . Data from 9,873 women participants in the korea national health and nutrition examination survey were reported in 2014 . This study reported that the overall prevalence of ui among women was 7.9% . By age, the prevalences were 10.1% and 10.3% in women over 40 and 60 years of age, respectively . The reported prevalences were 0.8%, 4.6%, 9.3%, 10.8%, 10.9%, and 9.7% among women in their 20s, 30s, 40s, 50s, 60s, and 70s or older, respectively . The 2008 actual living conditions of the elderly and welfare need survey obtained data by personal interview; this survey attempted to provide longitudinal population information using a cluster sampling method . The 8,961 women studied, 6.5% had self - reported ui and 2.3% had been medically diagnosed with ui . The most recent population - based, cross - sectional study randomly sampled 500 korean women who reside in the seoul, incheon, and gyeonggi - do areas . The survey comprised a computer - aided telephone interview, and used 16 questions to collect data; the questions sought information on demographic characteristics, information sources, disease insights, and general health - seeking behavior . Among the responders, 23.8% of women experienced ui . Western data report a prevalence range of 3.9% to 24.4% in women, while asian data have shown variable results: 53.7% of japanese women aged over 40 years, and 27.7% of taiwanese women aged 65 years or older . Korean data on ui have also varied widely; however, the prevalence is comparable with that of worldwide data, irrespective of region or race . Because korea is a conservative country, and koreans are relatively passive in coping with ui problems, ui may be more common among south korean women than is reported therefore, assessment of the real ui prevalence is the first step to creating public awareness and increasing public knowledge regarding this problem . In this way oab is a broad terminology that refers to a complex of symptoms such as urinary urgency, urge incontinence with nocturia, and frequent urination . Investigation into the prevalence of oab is still in its infancy, because the definitive characters of oab are obscure in that there is no clear metabolic or histologic explanation for the symptoms . Nonetheless, oab is a common voiding dysfunction, especially among older people . In addition to causing physical problems, oab may have a negative effect on the person s emotions and quality of life; furthermore, the disorder may eventually place a severe financial burden on society not only in medical terms, but also through loss of productivity . The reported incidence of oab has varied significantly among epidemiologic studies, because the diagnostic criteria with regard to each individual urological symptom are different . In south korea, epidemiologic studies into oab that had consistent diagnostic criteria began relatively recently, as did studies into the impact of the condition on the individual and society . We summarized the south korean studies into oab in table 3 . In 2004, 3,757 participants completed a questionnaire via an internet survey . Among the 3,372 women who responded, 429 suffered from oab (12.7%). In another study, nationwide community samples were obtained from south koreans aged 40 to 89 years . Through a telephone survey that used allocation sampling, choo et al . Reported that the prevalence rates in men and women were 13.3% and 16.3% for dry - type oab, and 7.5% and 15.0% for wet - type oab, respectively . In 2011, lee et al . Performed a population - based survey using the international continence society (ics) definition of oab the study was cross - sectional in design and used a telephone survey method to gather data from 2,005 persons . They found that 12.2% of individuals had oab, with similar rates among men (10.0%) and women (14.3%). In a regional study in guri city and yangpyeong county, gyeonggi - do, the general oab prevalence was 14.1% (12.2% in men and 15.5% of women). The survey involved face - to - face interview of 926 residents, and defined oab when the urgency score was> 2 points and the oab symptom score was> 3 points . In general, south korean prevalence data range from 10% to 16%, with a higher rate in women than in men . This is similar to the prevalence found in western countries; furthermore, the prevalence of oab in south korea increases with age, just as in western countries . For instance, in a survey involving 5,204 americans, the prevalence of oab was 16.0% in men and 16.9% in women . Data from six european countries revealed that the overall prevalence of oab among 16,776 people was 16.6% . Referring again to asia, the prevalence among 4,570 japanese respondents was 12.4%, and it was 20.9% among 1,247 taiwanese women . Oab patients are known to have a lower quality of life; however, investigators require a deeper insight into the condition s harmful influence in the community, especially in south korea . Proper epidemiologic data will be a valuable resource for educating the south korean population about oab a term unfamiliar to the public at the current time . According to the 2002 ics definition, the term luts encompasses various types of storage, voiding, and postmicturition symptoms . Similar to other voiding dysfunctions, luts are a common condition, and their prevalence has positive correlations with age . One problem with luts prevalence studies is that the condition is difficult to define, and most studies use criteria that overlap with those of bph, ui, or oab . Until recently, when investigation into luts prevalence began, one innovative study involving south korean men was performed in yeoncheon county . This community - based epidemiologic study assessed the severity of urinary symptoms in 514 men over 50 years of age . The overall incidence was 23.2%: 17.7% in those aged 5059 years, 23.3% in those aged 6069 years, and 35.3% in those aged over 70 years . In 2001, cho et al . A total of 1,356 men between 40 and 79 years old living in the seoul area were selected using stepwise random sampling and surveyed with a questionnaire that included questions about ipss . About 16% had moderate - to - severe luts, defined as an ipss> 7 . The prevalence of moderate - to - severe luts increased significantly with age, 10%, 16%, 29%, and 45% in the age groups 4049, 5059, 6069, and 7079 years, respectively . One population - based telephone survey involving 2,000 individuals reported that the overall frequency of luts was 61.4% (53.7% in men, 68.9% of women). 64.4% of women, voiding luts affected 28.5% of men and 25.9% of women, and postmicturition luts affected 15.9% of men and 13.9% of women . Kim et al . Surveyed a random sample of men older than 40 years . Responses from 1,842 men were investigated, and the overall prevalence of luts was 83.4% . Storage luts (70.1%) were more common than voiding (60.4%) or postmicturition luts (38.3%). As can be seen in the above summary, the reported prevalences of luts vary widely, from 16% to 83% . Nonetheless, the south korean prevalence data regarding luts are comparable to those in reports from across the world . One study in europe found that the prevalences of luts were 16.2%25.1% in men and 12.6%23.7% in women . Moderate - to - severe luts were reported by 36% of men aged 5059 years, 50% of men aged 6069 years, and 60% of men aged 7079 years . However, there have been fewer epidemiologic studies into luts in asian countries than in western countries, and prevalence data focus mainly on men rather than on the general population; there are few data regarding the prevalence in women . As shown in table 4, the prevalence varies widely between studies worldwide, mainly because investigators still have no clear definition of luts . The condition is complex, and this is a severe obstacle to researchers who wish to obtain the true epidemiology of luts in south korea . Nocturia is defined by the ics as the complaint that the individual has to wake at least once at night to void . Getting up at night to void urine increases the risk of individual physical or mental injuries in elderly people . Nocturia reduces the quality of life and is associated with sleep fragmentation, reductions in productivity, reduced general health, and falls or fractures . Most urologists consider a single episode to be nocturia, but many epidemiologic studies have defined nocturia as more than two episodes of voiding per night . Only a few studies into nocturia have been conducted in south korea (table 5). In 2008, choo et al . Conducted a clinically validated, computer - assisted telephone survey . Among the 2,005 respondents (1,005 women and 1,000 men), 33.5% complained of once - per - night voiding, and 48.2% stated that they had to void twice or more per night . Interestingly, even though the prevalence of nocturia was high, the negative influence on everyday life was reported to be minimal . In 2000, data were published involving 2,000 participants (888 men and 1,112 women). According to this cross - sectional telephone survey, the prevalences of nocturia (2 voids per night) were 36.6% in men and 48.2% of women . In 2012, lee et al . Investigated the prevalence of nocturia (2 voids per night) using a face - to - face interview method . The prevalence was 56.0% in this community - based study involving 439 elderly south korean men in a single city (seongnam). It is true that not all patients suffer from nocturia, and the degree of impact is influenced by the severity of the condition . To understand the exact epidemiologic nature of nocturia in south korea, it will be useful to examine to what degree cultural background has an effect on the psychological impact of nocturia . The prevalence of nocturia in south koreans appears to be higher than that in other communities . The prevalences of nocturia (2 voids per night) among japanese community - dwelling adults were 28.1% in men and 27.1% in women . A multistage, cluster sampling study with a personal interview - type design was used to obtain data from 15,988 people in the united states . The prevalences of nocturia (2 voids per night) were 9% in men and 16% in women . Data collected from 1,247 women and 1,221 men in austria showed that 37.2% of men and 43.1% of women experienced 1 voids per night, and that 7.5% of men and 8.1% of women experienced 2 voids per night . These lower prevalences in other populations may have been affected by the population distributions studied, as well as by the definition of nocturia used . Nonetheless, the probable causes of the high prevalence of nocturia among south koreans remain to be clarified . Epidemiologic surveys are essential for understanding the potential causes and characteristics of related medical disorders . In addition, they are the necessary first steps in evaluating psychosocial impacts of a disorder . In general, most of the estimated prevalences of voiding dysfunctions in south korea were similar to those in other countries; they varied widely, primarily due to different definitions of the respective disorder . In addition to the lack of solid definitions of voiding dysfunctions, inconsistencies in integrated questionnaires and variances among target populations render population - based surveys difficult to compare . Most investigations were based on self - reported symptoms, so recall bias and person - to - person social bias may have been present, especially in personal interviews . Nonresponse bias could also be a problem in that nonresponders to a survey may have been significantly different from responders in terms of the respective symptoms . Population - based surveys are carried out because diagnosis by strict medical assessment is not practical in such large - scale prevalence studies . Nonetheless, researchers need to develop other ways to obtain more proper, valuable epidemiologic data . We think that investigators should focus on unifying all of these disease entities and definitions . More efficient investigational processes and methodologies are also warranted . From a methodological point of view, 14 of the studies (58.3%) included in the present report, involved personal interview data . Despite considering the advantages of the personal interview, however, the bashful and conservative nature of koreans may prevent investigating the epidemiology of voiding dysfunction in simpler, less time - consuming ways . As a result, relatedly, the random digital dialing in telephone surveys is often perceived as violating personal privacy; for this reason, telephone surveys will not be easy to conduct in the future . Due to the noteworthy progress in multimedia technology in south korea, epidemiologic studies using the internet and other applications are a promising method . Using web services an internet survey can gather enormous amounts of data from large groups within a relatively short time . Furthermore, videos or pictures can be used easily, at reasonable cost, and with less social desirability bias than the other study methods . Mobile communication techniques may improve methothologic efficiency in various social and natural science fields, as well as in medicine . For example, bladder diary apps may exclude the disadvantages of pen - and - paper diaries in clinical application . Currently, the apps available vary in quality, but there is room for medical associations to collaborate with developers in this area . Even though some of the public are not familiar with surveys by internet or electrical devices, much progress will be made in epidemiologic studies due to technology, especially with regard to methodology . This review constituted a nationwide representative summary of studies on the prevalence of voiding dysfunctions in south korea . We hoped to contribute to the understanding of voiding dysfunctions by providing the epidemiologic characteristics of each condition . However, much work remains to be done in this field, because few surveys had a longitudinal design, and more investigations are needed that consider the whole range of age, sex, and location in the south korean population . Given the influence of voiding dysfunction on quality of life, as well as the socioeconomic burden of the condition, more studies on the optimal approaches to epidemiologic study will be needed in the future.
However, graft dysfunction remains a problem affecting up to one - third of the recipients, despite reports of good to excellent long - term outcome . The organ dysfunction is considered multifactorial, but ischemia and reperfusion (i / r) injury is probably the most important contributing factor, although the detailed steps of pathogenesis are controversially debated . I / r injury is a major cause of liver graft dysfunction resulting in adenosine triphosphate (atp) decrease, evidence of intracellular acidosis, and cell swelling of the hepatocytes . This aspect is also accompanied by or linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the posttransplant liver biopsy . However, biliary marker levels increase usually only 57 days after transplantation stimulating several fields of research in the last couple of decades . Cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation, and the steps reaching this state are not completely understood . We reviewed the canine liver transplantation model as i / r injury model to delineate in detail the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes, which seems to be a post - transplant event occurring in the liver at an earlier stage than frank cholestasis . Here, we speculate that i / r liver injury through a phenotypical switch of the liver cells may contribute to the poor outcome of the liver graft . Liver transplantation is widely known as the most effective therapy for both acute and chronic liver failure . In 1963, thomas starzl, an american surgeon, northwestern university medical school graduate with degrees in anatomy, neurophysiology, and medicine, was the first to perform the liver transplantation on a child suffering from biliary atresia . Despite the significant success of liver transplantation, infection, poor graft function, and rejection are major problems that may still contribute to death of the transplanted patient or to deterioration of the graft . Reperfusion following long ischemia of liver during liver transplantation causes severe injury, which is now universally indicated as i / r injury . I / r injury of liver is a major cause of morbidity and mortality in patients undergoing liver transplantation . Several mechanisms and distinct pathways may involve i / r leading to both initial poor function and primary nonfunction of the liver allograft . Three cytoskeletal proteins form a network that provide the cellular structure and fundamental integrity of the eukaryotic cells: microfilaments, microtubules, and intermediate filaments . The intermediate filament proteins have an important role in liver protection against mechanical and nonmechanical injury, which have been demonstrated in animal models . Overexpression of proteins and mutations of the corresponding keratin genes have been reported to contribute to several human oncological and nononcological diseases . Eukaryotic cells have a unique cytoplasmic structure labeled as cytoskeleton, which consists of three distinct kinds of cytoskeletal filaments, including microfilaments, microtubules, and intermediate filaments . Besides giving the rigidity of the cell and maintaining cell shape and borders or cell scaffolding, cytoskeleton plays important and probably crucial roles in intracellular transport, cell division, gene regulation, and signal transduction of the genetic information [6, 7]. Actin, which is widely accepted to be a highly conserved structure among different species, is the major protein that constitutes the microfilaments . There are three known classes of human actin gene that have been identified as -, -, and -actin . Cell - cell interaction, signal transduction, cell shape maintenance, and cell motility are the central functions of actin . Microtubules, which have a diameter of around 23 nm, are protofilaments that are important for the intracellular transport, movement of cilia and flagella, mitotic spindle, and cell wall synthesis . Both microfilaments and microtubules have several groups of binding proteins that modulate their stability and biological functions, such as signal transduction . Intermediate filaments with around 10 nm of diameter are more stable than actin filaments and function in the maintenance of cell shape by carrying tension . Intermediate filaments, which include, for example, vimentin, glial fibrillary acidic protein, neurofilament proteins, keratins, and nuclear lamins, organize the internal 3d cell structure anchoring cyto - organelles and serving as structural scaffolds of the nucleus and cytoplasm . Cytokeratins (ks) are the main protein family that constitutes the intermediate filaments with more than fifty members that have been identified . Polypeptides of the ks are variants and have been divided into type i and type ii, or acidic and basic, respectively . Studies have shown that each epithelial cell has a distinct content of cytokeratins (i.e., expression of keratins is described as tissue - specific manner) playing a major role in the tissue identification of metastasis of unknown primary carcinomas . Ks usually present in cells as heteropolymers pairs composed of type i (k9k20) and type ii (k1k8). K8 (mw 52kd) and k18 (mw 45kd) are considered as the only ks that are normally expressed in human hepatocytes . Moreover, both k8 and k18 have been found to be expressed in early development stage of mouse embryos . Immunohistochemical studies have revealed that cholangiocytes (bile duct cells) express k7 (54 kd) and k19 (40 kd) in addition to k8 and k18, and those ks have been used as valid markers for both studies on development of the intrahepatic biliary system and for assessment of bile duct damage . It has also been well established that cholangiocytes are derived from hepatoblasts found around portal vein . These cells show a particularly intense expression of k8 and k18 . As indicated above, k7 and k19 have been linked to development of the intrahepatic bile duct system in both humans and animal models . The expression of k19 is useful to identify primitive biliary cells, while the expression of k7 appears after 20 weeks of gestation in humans . Both k7 and k19 are consistently expressed in the development of the intrahepatic bile duct with elevated levels until one month of postnatal age . Thus, in order to phenotypically switch hepatoblasts into cholangiocytes, these cells should firstly express k19 followed by expression of k7 . However, it has been shown that hepatocytes might express k7 in response to different conditions, such as ductopenia and cholestasis, and, interestingly, it has been found that hepatocellular carcinoma cells expressing k19 are significantly associated with reoccurrence of neoplasm after transplantation . A recent study has demonstrated that hepatocytes are frequently expressing k7 in case of chronic liver allograft rejection . Have demonstrated that hepatocytes intensively expressed k7 and k19 early following cold ischemia in a canine isolated perfused liver transplantation model . Furthermore, they demonstrated that bile duct cytokeratins are very useful markers to diagnose an early sign of cholestasis . It has become clear that cholestasis is significantly associated with high morbidity and mortality in patients undergoing liver transplantation . Therefore, investigations that lead to discover the mechanisms of the cholestasis are dramatically required . I / r injury is a phenomenon that occurs when blood flow and oxygen delivery return to reperfused tissue . It is considered one of the major causes of morbidity and mortality among patients undergoing liver transplantation . I / r injury has been demonstrated in several diseases such as cerebrovascular diseases, peripheral vascular diseases, sepsis, myocardial infarction, and organ transplantation . It involves several mechanisms, which lead to organ failure, circulatory dysfunction, and, finally, death of the transplanted patient . Cellular mechanisms of i / r injury include a few essential cell cascades that suggest the role of activation of endothelial cells, kupffer cells, reactive oxygen species (ros), and polymorphonuclear leukocytes (pmn) or neutrophils in the pathogenesis of i / r injury . There may also be mechanisms involving t lymphocytes that seem to have a key role in short- and long - term damage during i / r injury . T lymphocytes act as mediator in the subacute inflammatory phase of neutrophilic recruitment following i / r injury . Another important constituent of tissue injury following i / r injury that needs to be emphasized is the production of oxygen free radicals (ofrs). These ofrs can arise from different sources, such as kupffer cells, pmn, and xanthine oxidase (xo) that is the most significant source . Several etiological factors are involved in i / r injury, including atp reduction, activation of proteases, and alteration in the intracellular concentration of cytokines and chemokines, and cell swelling . Investigations of posttransplantation surgery have indicated that reperfusion plays an essential role in primary graft nonfunction, which is one of the most serious complications of liver transplantation . I / r injury also causes early organ failure up to 10% as well as increases the acute and chronic rejection . Several studies demonstrated that during i / r injury, morphological changes of liver tissues occur, and these changes can have a prognostic significance . The intermediate filament (if) cytoskeletal protein is one of the three major cytoskeletal proteins whose result is important in maintaining both cellular structure and integrity of eukaryotic cells . The other two filament proteins are microfilaments (mf) and microtubules (mt). Beside their cellular functions like cell motility, division, and stress responses, they also have an essential role in human diseases due to mutations in filament proteins . If proteins consist of five types of keratins according to structure of genome and composition of amino acid, if proteins types i iv are cytoplasmic, and type v if contain nuclear lamins . The keratins are obligating noncovalently heteropolymers because they consist of one of each type i and type ii keratins as pairs . Neutral to basic keratins are the largest group of if proteins classified as type i (k9k20) and type ii (k1k8). Another type of if proteins is type iii if that includes vimentin, peripherin, glial fibrillary acidic protein, and desmin of mesenchymal cells, peripheral neurons, glia cells and astrocytes, and muscle cells, respectively . Type iv if proteins include neurofilament proteins (nf - l, nf - m, and nf - h), synemin, nestin, syncoilin, and -internexin; lamins a as indicated above, the adult hepatocytes express k8 and k18 only compared to other epithelial cells that express 2 or more type i or type ii keratins such as bile duct, which expresses additional keratins k7 and k19 . The first disease to be discovered related to keratin mutation was epidermolysis bullosa simplex (ebs) with mutations in k5 and k14 . Additionally, mutations in k8 and/or k18 lead to acute or chronic liver diseases . Normally in hepatocytes, further studies indicated that reperfusion alone causes alteration of theses microfilaments (f - actin) leading to contraction of canaliculi, relocalization of enzymes and transporters of canaliculi, and increasing permeability of cellular tight junction . Cholestatic liver disease occurs when there is a decrease in bile flow, which is an abnormal physiologic state, and retention of toxic bile acids . Clear cytoplasmic hepatocytes (cholate stasis) presented in cirrhotic nodules and showed a decrease in keratin if network in the cytoplasm . Studies on mice reported that keratin was overexpressed in epithelia of bile duct due to ligation of bile duct and in hepatocytes . In cholestatic liver disease, bile duct epithelial - type keratin (k7 and k19) intermediate hepatocytes express k7, while reactive bile ductules express k7 and k19 . In post - transplant period, keratins of biliary type were detected in the hepatocytes in the early period of i / r injury . Moreover, bile canaliculus progressively dilate after ischemia, and a change of the microvillous integrity is demonstrable . The liver is one of the organs with an incredible capacity for in vivo tissue engineering which allow restoration of the liver architecture and reestablishment of certain vital functions . The study of liver regeneration in humans arise ethical issues, and it is difficult to carry out because of a plethora of heterogeneous liver lesions . Accordingly, using experimental animal models is more useful and helpful for studying liver regeneration . In vitro studies need to be followed by in vivo ones to simulate the interaction between liver cell populations . In our opinion, the use of large animals such as dog, pig, or sheep is more advantageous than small animals like rat or mouse; large animals are similar to human beings in their physiology and anatomy, and techniques of microsurgery are not necessary to carry out determinate experiments . Only few studies have been carried out on i / r animal models in liver . The canine liver model following i / r injury has been used as a liver transplantation model to study excretion of bile and intrahepatic intermediate filaments expression involved in morphological changes of the biliary system . To the best of our knowledge, in a canine isolated and perfused liver model, there was the first clear - cut evidence of cholestatic changes starting early following cold ischemia despite prompt recovery of the bile flow . According to some other authors, the use of the canine liver transplantation model seems to allow a better vascular perfusion under ex vivo conditions in contrast to the rat model . 's study revealed an ischemia - dependent impairment of an experimental biliary dye excretion during early stage of reperfusion and a progressive cytokeratin expression of biliary type in the hepatocytes despite a prompt recovery of the bile flow after i / r . The recovery of the bile output after i / r represents a key event in lt . The nonstimulated bile flow rate was unaffected by cold ischemia up to 10 h in our canine isolated perfused liver model, but the decrease of the biliary dye excretion during early stage of reperfusion correlated with the peak output rate of the dye across the canalicular membrane . Remarkably, the dye uptake at the sinusoidal membrane was not affected by cold ischemia, leading to a possible accumulation of toxic compounds in the hepatocytes . Hepatic elimination of the dye following i / r has been analyzed mainly by plasma clearance . Very importantly was the study of the hepatic elimination of the dye after a bolus injection by compartmental spectrophotometric analysis both in the perfusate by transhepatic sampling and in the bile . Differential mechanisms can be suggested for the impaired biliary excretion of organic anions following i / r . Previously, a positive correlation between biliary dye excretion, viability of the graft, and hepatic atp content has been found in cholestatic liver disease, and reduced biliary dye excretion has been attributed to the lack of atp . Atp content does not seem to be the main limiting factor for the hepatocellular transport following i / r . An impairment of the intracellular transport or a decreased transport rate across the canalicular membrane might play a major role as contributing factor . The step across the canalicular membrane generally represents the rate - limiting one in the hepatocellular transport . In the canine liver transplantation model, the flow in the portal vein was continuous, because roller pump, oxygenator, and heat exchanger in the perfusion line were connected in series with the roller pump upstream . The oxygen content of the saline reperfusion solution was 2% in volume (10% of arterial hepatic blood and about 15% of the portal venous blood). Thus, one can expect to have reactive - oxygen - species- (ros-) linked damage at the beginning of the reperfusion after 8 or 10 h of ischemia . However, the excellent reperfusion results of the control group and data of the 2 h ischemia group indicate that this is not a general problem, but may be an ischemia - time - linked problem . High o2 partial pressure in the liver stimulates ros formation after extensive long ischemia times, and this may be one of the most important limiting factors of the ischemia tolerance . Electron microscopy studies in hepatocytes revealed microfilaments, which are distributed along the plasma membrane and in the region of the bile canaliculus . As indicated above, cytoskeleton filaments are also essential for the maintenance of cell shape, bile canalicular architecture, and integrity of the cellular tight junctions . The microfilaments network surrounding the canalicular pole and extending into the canalicular microvilli is damaged during cholestasis . Reperfusion, but not ischemia, has been considered faulty, inducing an alteration of f - actin microfilaments, suggesting an impairment of the canalicular contraction, an increase of the tight junction permeability, and a probable relocalization of canalicular enzymes and transporters . Bile canalicular size change as detected in our study may be coincident with the reorganization of the pericanalicular filaments and the colocalization of actin and myosin . In fact, contraction in this study has been associated with shortening and/or twisting of bile canaliculi . However, graft dysfunction remains a problem affecting up to one - third of the recipients, despite good to excellent long - term outcome . The organ dysfunction is considered multifactorial, but i / r injury is probably the most important contributing factor . The detailed steps of the pathogenesis of i / r injury continue to be a topic of vivid debate . I / r injury is a major cause of liver graft dysfunction resulting in atp decrease, intracellular acidosis, and cell swelling of the hepatocytes . This feature is also linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the liver biopsy . However, the plasmatic levels of biliary markers increase usually only 57 days after transplantation prompting on - going research to address new markers of early liver damage . This could also be a stimulus to support more grant approvals for liver posttransplantation studies . Cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation, but the steps reaching this state are still deemed elusive . We reviewed the canine lt model as an i / r injury model and reviewed the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes . In fact, this phenotypic switch of the liver cells, which is also sometimes called pseudo- or neoacinar transformation in diseases with cholestasis, seems to occur at a stage earlier than frank cholestasis . We emphasized that i / r injury through this peculiar phenotypic switch of the hepatocytes may be a milestone to investigate the pathways contributing to the poor outcome of the liver graft . Reperfusion injury following ischemia affects graft function in liver transplantation by induction of phenotypical changes in hepatocellular keratins . Keratin phenotype of hepatocytes leads to hepatocellular damage that is intimately connected to several intracellular processes, which are under intense investigation in our laboratory, and the appropriate choice of an animal model is crucial in forwarding research in very complex areas requiring not only human, but also comparative (veterinary) pathology knowledge.
The indications for laparoscopic pyeloplasty are flank pain, infection, stone formation, and decreased renal function . Many studies have reported that laparoscopic pyeloplasty has an excellent success rate ranging from 9098% [15]. Success is usually defined as improvement in hydronephrosis, as determined by ultrasound (us) or intravenous pyelography (ivp), and the improvement of function on diuretic renography along with a decrease in washout time . Although diuretic renography may be the most accurate modality with which to evaluate surgical outcome, renal us is more commonly used for long term follow up because it is less invasive and more cost effective . Although there have been some published reports on the longitudinal renal sonographic changes after this procedure in children [68], there is no similar report in adults . We evaluated the improvement of hydronephrosis longitudinally using us and ivp after retroperitoneal laparoscopic dismembered anderson between january 2006 and june 2012, 16 patients with upjo were treated by retroperitoneal laparoscopic dismembered anderson the group included 8 females (50%) and 8 males (50%) with a mean age of 30 years . All 16 patients had upjo as confirmed by renal us, ivp, retrograde pyelography, and a diuretic renal scan . The patients had the disease on the right side in 4 cases and on the left in 12 cases . Four patients (25%) had renal calculi associated with upjo (table 1). A 2cm incision was first made at the midpoint between the 12 rib and the iliac crest in the middle axillary line . After a working space was created in the retroperitoneum by balloon dissector, a 12mm blunt a second 12mm trocar was placed 7 cm dorsal to the camera port under laparoscopic guidance . A third 5mm trocar was placed in the anterior axillary line at the level of the iliac crest, and a fourth 5mm trocar was placed in the subcostal region at the anterior axillary line . The lateroconal fascia was incised along the quadratus lumborum muscle, which allowed for exposure of the psoas muscle and the posterior lamina of gerota's fascia . The ureter was identified and dissected in the cephalad direction toward the ureteropelvic junction (upj). After dissection and clear visualization of the upjo, a stay suture was placed in the medial edge of the renal pelvis and was pulled out through the abdominal wall using endo close. The medial portion of the redundant pelvis was partially excised, and the proximal ureter was laterally spatulated with a 1.0cm longitudinal incision . The renal pelvis was then dismembered with the proximal ureter and the stenotic segment of the upj was resected . The first suture, a 40 polydioxanone suture, was placed from the most interconnected portion of the renal pelvis to the most inferior point of the ureteral spatulation . After the anterior anastomosis was completed, the same suture was applied to the posterior wall of the anastomosis . If a crossing vessel was identified during dissection of the ureter, it was preserved and the ureter was transposed anterior or posterior to the vessel . After hemostasis was confirmed, a 5mm suction drain was placed in the retroperitoneum, and all wounds were closed . After the renal pelvis was transected, stones were removed individually under direct laparoscopic vision with the use of standard laparoscopic graspers . Renal us was performed at 3, 6, 12, 18, and 24 months and ivp was performed at 6 and 12 months after surgery . The degree of hydronephrosis was graded as 0 to 3 according to ellenbogen's grading system . The procedure was considered successful when patients experienced the complete resolution of pain, improvement of hydronephrosis on us and ivp, and decreased half time clearance (t1/2) as measured during a diuretic renal scan . Between january 2006 and june 2012, 16 patients with upjo were treated by retroperitoneal laparoscopic dismembered anderson the group included 8 females (50%) and 8 males (50%) with a mean age of 30 years . All 16 patients had upjo as confirmed by renal us, ivp, retrograde pyelography, and a diuretic renal scan . The patients had the disease on the right side in 4 cases and on the left in 12 cases . Four patients (25%) had renal calculi associated with upjo (table 1). A 2cm incision was first made at the midpoint between the 12 rib and the iliac crest in the middle axillary line . After a working space was created in the retroperitoneum by balloon dissector, a 12mm blunt a second 12mm trocar was placed 7 cm dorsal to the camera port under laparoscopic guidance . A third 5mm trocar was placed in the anterior axillary line at the level of the iliac crest, and a fourth 5mm trocar was placed in the subcostal region at the anterior axillary line . The lateroconal fascia was incised along the quadratus lumborum muscle, which allowed for exposure of the psoas muscle and the posterior lamina of gerota's fascia . The ureter was identified and dissected in the cephalad direction toward the ureteropelvic junction (upj). After dissection and clear visualization of the upjo, a stay suture was placed in the medial edge of the renal pelvis and was pulled out through the abdominal wall using endo close. The medial portion of the redundant pelvis was partially excised, and the proximal ureter was laterally spatulated with a 1.0cm longitudinal incision . The renal pelvis was then dismembered with the proximal ureter and the stenotic segment of the upj was resected . The first suture, a 40 polydioxanone suture, was placed from the most interconnected portion of the renal pelvis to the most inferior point of the ureteral spatulation . After the anterior anastomosis was completed, the same suture was applied to the posterior wall of the anastomosis . If a crossing vessel was identified during dissection of the ureter, it was preserved and the ureter was transposed anterior or posterior to the vessel . After hemostasis was confirmed, a 5mm suction drain was placed in the retroperitoneum, and all wounds were closed . After the renal pelvis was transected, stones were removed individually under direct laparoscopic vision with the use of standard laparoscopic graspers . Renal us was performed at 3, 6, 12, 18, and 24 months and ivp was performed at 6 and 12 months after surgery . The degree of hydronephrosis was graded as 0 to 3 according to ellenbogen's grading system . The procedure was considered successful when patients experienced the complete resolution of pain, improvement of hydronephrosis on us and ivp, and decreased half time clearance (t1/2) as measured during a diuretic renal scan . The degree of hydronephrosis as determined based on us was identical to that determined based on ivp . The postoperative improvement of hydronephrosis was 63%, 100%, 100%, 100%, and 100% at 3, 6, 12, 18, and 24 months, respectively . The postoperative improvement of hydronephrosis by one grade was 56%, 73%, 67%, 50%, and 40% at 3, 6, 12, 18, and 24 months, respectively . Improvement in the degree of hydronephrosis by two grades was observed in 6%, 27%, 33%, 50%, and 60% of patients at 3, 6, 12, 18, and 24 months, respectively (table 2). Complete resolution of the hydronephrosis was observed in 8 of 16 patients (50%). Hydronephrosis was still improving even after 12 months in 5 of 12 patients (42%) (figure 1). T1/2 improved in 9 of 12 patients (75%) as determined during the diuretic renal scan at 12 months (table 3). Three patients experienced no changes in the results of the diuretic renal scan 1 year postoperatively . Among these patients, the degree of hydronephrosis decreased to grade 1 and grade 2 at 6 months, respectively, but no further improvement of hydronephrosis was observed at 24 months . In the third patient who had grade 3 hydronephrosis preoperatively, the degree of hydronephrosis decreased to grade 2 at 3 months and continued to decrease gradually until complete resolution was obtained at 24 months . This case was regarded as successful, although improvement could not be confirmed on the diuretic renal scan . Rate of improvement in hydronephrosis as determined on us and ivp after laparoscopic pyeloplasty us = ultrasound; ivp = intravenous pyelography the change of t1/2 of the diuretic renal scan after laparoscopic pyeloplasty laparoscopic pyeloplasty was introduced in 1993 by schussller and was developed worldwide as the first minimal option, which has been shown to reduce hospital stay while offering success rates equivalent to those of the open pyeloplasty [1, 11, 12]. An advantage in adopting retroperitoneal laparoscopic approach compared with transperitoneal counterpart for pyeloplasty is related to a more direct path to the upj and the low risk of injury to intraperitoneal organ . Conversely, the retroperitoneal approach has a limited working space that may increase the difficulty of reconstruction . Whereas the magnitude of pelvic enlargement correlates statistically with the likelihood of obstruction, us cannot be used to diagnose obstruction because the degree of pelvic dilatation does not specifically indicate the presence or absence of obstruction, or predict whether the hydronephrosis will improve or worsen . Therefore, the success of laparoscopic pyeloplasty is usually demonstrated radiographically by an improvement in drainage and/or function as visualized by the postoperative diuretic renogram and by decreased hydronephrosis on renal us . In children amling reported that only 38% of the kidneys improved during the first 6 months of follow up, while 81% were improved 2 years postoperatively, and ultimate improvement to grade 0 or 1 was noted in only 19% of cases . Kis et al . Reported that 102 babies who underwent pyeloplasty were investigated by us at 6 and 12 months postoperatively . One year after surgery, the renal pelvis was smaller in 76% of cases, and renal parenchyma was normal or had increased in 92% of cases . The authors reported that the resolution of hydronephrosis after surgery was quite slow in the first postoperative year, while the increase in the renal parenchyma was relatively rapid . In the series by neste et al ., postoperative us and diuretic renograms were reviewed in patients monitored an average of 26 months after pyeloplasty . Improvement as determined using us was much more gradual than the change in renographic pattern with renographic improvement observed well in advance of that observed on the us . There has been no report on the longitudinal assessment of hydronephrosis after pyeloplasty in adults . In our adult series, an improvement of hydronephrosis was observed in 10 of 16 patients (63%) at 3 months, and all cases showed improvement at 6 months after surgery . All patients were without symptoms or infection after 3 months, although hydronephrosis was still present in many patients for months . In 5 of 12 patients (42%), hydronephrosis was still improving gradually even after 12 months postoperatively, and the complete resolution of hydronephrosis was observed in 8 of 16 patients (50%). The results suggest that hydronephrosis starts to improve relatively earlier in adults than in children and continues to improve gradually over the long term . In children, hydronephrosis may be found as an abdominal mass . On the contrary, in adults, flank pain or urinary tract infection since most adult upjos are symptomatic, adult patients are diagnosed relatively early when hydronephrosis has increased . For this reason, hydronephrosis may improve rapidly and remain decreased for a long time after the obstruction was treated . In most institutions, diuretic renogram is usually performed at 3 months and annually thereafter [1, 2, 11]. In our series, patients were followed up with us (with or without ivp) at 3, 6, 12, 18 and 24 months to assess morphologically, and diuretic renogram was performed at 12 months to assess functionally . Since the improvement in hydronephrosis and the improvement observed on the diuretic renal scan were not necessarily seen simultaneously, we recommend that patients who underwent laparoscopic dismembered pyeloplasty should undergo a diuretic renal scan at 12 months postoperatively and be followed up at least 2 years postoperatively . Concerning the relation between us, ivp, and renal scan, the degree of hydronephrosis as determined based on us was identical to that determined based on ivp . There were three patients who experienced no improvement in the diuretic renal scan at 12 months . Among these patients, one patient had gradual improvement of hydronephrosis even after 12 months and had complete resolution at 24 months . In this patient it could have been confirmed that t1/2 decreased if the diuretic renal scan was performed at 24 months . These results showed that serial us showing improvement of hydronephrosis may be a good alternative to ivp or diuretic renal scan in the follow up of upjo patients who underwent laparoscopic pyeloplasty . Longitudinal analysis of larger numbers of patients with hydronephrosis after laparoscopic pyeloplasty will be necessary . Although hydronephrosis had not changed in 37% of patients at 3 months, all cases showed improvement at 6 months, and continuous improvement was found in 40% of patients who were followed up for more than 12 months . Hydronephrosis improves relatively more rapidly and for a longer period of time in adults compared to pediatric patients . However, a one grade improvement in the degree of hydronephrosis does not necessarily represent true therapeutic success . Follow up by diuretic renal scan as well as us is needed if improvement cannot be confirmed by diuretic renal scan at 12 months postoperatively.
Breast carcinoma is the most frequent cancer among women with considerable invasive and metastatic behavior . The recent increased knowledge in molecular mechanisms of this cancer and consequent targeted treatments have improved its outcome . It belongs to the family of d - type cyclins, which regulate cell cycle progression from g1 to s phase by regulating the activity of cyclin - dependent kinases (cdks). Cyclin d1 and its close relatives, cyclin d2 and cyclin d3, all appear to associate with the same kinase partners . Binding of cyclin d1 to cdk4 and cdk6 induces hyperphosphorylation of retinoblastoma protein (rb). This leads to the activation of e2f and transcription of several genes required for the g1 to s phase transition, thereby promoting cellular proliferation . Recent findings have revealed further roles of cyclin d1 in promoting cell cycle progression through cdk - independent mechanisms such as interaction with and modulation of transcription factor activities . The well - known oncogene ccnd1, which encodes cyclin d1 is amplified in a substantial proportion of human cancers including parathyroid carcinoma, colon cancer, lymphoma, melanoma, prostate cancer and breast cancer . However, the impact of cyclin d1 overexpression on behavior of breast cancer remains controversial . Although cyclin d1 has a pivotal role in promoting cell cycle progression and its overexpression in breast cancer is expected to be associated with poor prognosis, recent studies have shown both positive and negative prognostic impacts . Besides inducing cell cycle progression through regulation of cdks activity, cyclin d1 promotes other regulatory molecules by cdk - independent mechanisms . Cyclin d1 represses the transcriptional activity of signal transducer and activator of transcription 3 (stat3) in vitro . Cyclin d1 is the intermediary molecule in other cell cycle pathways such as nuclear factor-b (nfb), rac1 and 5 adenosine monophosphate - activated protein kinase (ampk) signaling pathways . Decline in rac1 levels causes inhibition of nfb signaling and induces downregulation of cyclin d1 . Active ampk leads to loss of cyclin d1 messenger ribonucleic acid and protein . Only a minority of breast cancers with cyclin d1 overexpression show amplification of the cyclin d1 gene, indicating that pathogenic transcriptional activation of this gene by factors such as estrogen receptor (er) could be another important mechanism triggering its overexpression . Using antibody against cyclin d1 protein, overexpression of the protein can be detected even in the absence of any apparent increase in copy numbers . Therefore, immunohistochemical staining with the specific monoclonal antibody provides an accurate method for detection of deregulated cyclin d1 expression . Er is another regulatory molecule with critical roles in proliferation of cancer cells in reproductive organs such as breast and uterus . Most er positive breast cancers are low - grade and have lower metastatic potential while er negative ones demonstrate poor differentiation . Er expression is in turn influenced by the expression of other genes such as mta1 . Silencing mta1 gene results in er expression in er negative cells . Genetically modified mouse models reveal the necessity of cyclin d1 expression for postnatal mammary development in response to the sex steroids . Indeed, cyclin d1 expression in mammary epithelial cells is induced by estrogen and progesterone . The present study aims to find the frequency of cyclin d1 overexpression and the relationship between cyclin d1 overexpression and some well - known clinicopathologic prognostic determinants in breast invasive ductal carcinoma . In this descriptive - analytical and cross - sectional investigation, 89 patients with breast invasive ductal carcinoma hospitalized in alzahra hospital, isfahan, iran from 2003 to 2008 enrolled in the study . Patients had undergone mastectomy and axillary lymph node dissection and the status of er, progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2)-neu had been determined immunohistochemically using the formalin fixed and paraffin embedded tissue samples of the primary tumor . The studied variables included age, tumor grade, tumor stage, er, pr, her2-neu and cyclin d1 status . Grading was carried out based on the nottingham modification of bloom and richardson system, which considers the three parameters of tubule formation, nuclear pleomorphism and mitotic count as determining factors . Staging was carried out based on the tnm classification of malignant tumours (tnm staging system) for breast carcinoma . Expression of er and pr was considered as negative if lesser than 1% of nuclei were stained and as positive if 1% or higher of nuclei were stained . The antibodies used were against er (monoclonal mouse anti - human; clone: 1d5; isotype: igg1, kappa; dakocytomation, denmark) and pr (monoclonal mouse anti - human; clone: 1a6; isotype: igg1, kappa; dakocytomation, denmark). The antibody used for her2-neu study was polyclonal rabbit antihuman antibody against c - erbb-2 oncoprotein, dakocytomation, denmark . Her2-neu expression was scored as 3 + (positive) when strong complete staining of the membrane was present in more than 30% of the neoplastic cells, 2 + (positive) if weak to moderate complete staining of the membrane was seen in more than 10% of the neoplastic cells, 1 + (negative) when faint staining was detected in a portion of the circumference of the cytoplasmic membrane in more than 10% of the neoplastic cells and 0 (negative) when no membranous staining was identified or membrane staining was observed in less than 10% of the tumor cells . Antibody used was monoclonal mouse anti - human antibody, clone cyclin d1 antibody (dcs-6) (isotype: igg2a, kappa), dakocytomation, denmark . Four micron sections were prepared from each formalin - fixed and paraffin - embedded tissue sample to be stained with antibody against cyclin d1 using the envision method . Sections were placed on poly - l - lysine slides and dried in an oven at 60 c for 60 min . Subsequently, they were deparaffinized, rehydrated and rinsed in tap water before antigen retrieval . After inactivation of endogenous catalase by 3% hydrogen peroxide, the sections were incubated with primary antibody for 1 h and secondary antibody for 30 min . After each step of antibody treatment, slides were drained with phosphate buffer to eliminate excess antibody . The validity of immunohistochemistry staining was provided by using positive and negative controls in each set of staining . The intensity and distribution of cyclin d1 immunoreactivity were semiquantitatively scored using the allred scoring method . The intensity of immunohistochemical reaction by light microscopy was recorded as 0 (negative) when no staining of the nuclei was seen even at high magnification, 1 (weak) if staining was visible only at high magnification, 2 (moderate) when staining was readily visible at low magnification and 3 (strong) if staining was strikingly positive even at low power magnification . The proportion of tumor cell nuclei showing positive staining was also scored as either 0 (none), 1 (<1/100), 2 (1/100 - 1/10), 3 (1/10 - 1/3), 4 (1/3 - 2/3) and 5 (> 2/3). The intensity and proportion scores were then added to obtain a total score ranging from 0 to 8 and the tumors were categorized into four groups based on their total score: negative expression: total score of 0weak expression: total score of 1 or 2intermediate expression: total score of 3 - 5strong expression: total score of 6-8.only nuclear staining was considered specific . Negative expression: total score of 0 weak expression: total score of 1 or 2 intermediate expression: total score of 3 - 5 strong expression: total score of 6 - 8 . Only nuclear staining was considered specific . Data was collected in prepared checklist and analyzed by version 16 of statistical package for the social sciences (spss) software (spss corp, chicago, usa) using anova and chi - square tests . The mean age of patients (sd) was 51.38 11.37 years (range: 26 - 83 years). 18%, 41.6% and 40.4% of the tumors were grade i, ii and iii, respectively . 7.9%, 40.4%, 18%, 30.3%, 1.1% and 2.2% of the cases fitted into stages 1, 2a, 2b, 3a, 3b and 3c, respectively . Er, pr and her2-neu were positive in 60.7%, 58.4% and 36% of the cases, respectively . Cyclin d1 was strong (s), intermediate (i), weak (w) and negative (n) in 19.1%, 44.9%, 14.6% and 21.3% of cases, respectively [figures 1 and 2]. Negative cyclin d1 status was mostly seen in grade iii strong cyclin d1 status was more frequent in grades i & ii there was a statistically significant reverse relationship between cyclin d1 and tumor grade (p = 0.009). The n status of cyclin d1 was mostly seen in grade 3 while s, i and w states were most frequent in grade 2 [table 1]. The relationship between cyclin d1 and tumor grade the relationship between cyclin d1 and er was also statistically significant (p = 0.0001). The s and i states were mostly seen in patients with positive er while the n status was most frequent in patients with negative er [table 2]. The relationship between cyclin d1 and estrogen receptor a statistically significant relationship was found between cyclin d1 and pr (p = 0.0001). The s, i and w states were mostly seen in patients with positive pr while the n status was most frequent in patients with negative pr [table 3]. The relationship between cyclin d1 and progesterone receptor there was no statistically significant relationship between cyclin d1 on one hand and age, stage and her2-neu on the other (p> 0.05). The present study was conducted to determine the relationship between cyclin d1 overexpression and well - known clinicopathologic prognostic determinants in breast invasive ductal carcinoma . It has been shown in previous studies that cyclin d1-deficient mice are susceptible to mammary carcinomas induced by c - myc or wnt-1, but not those induced by c - neu and v - ha - ras . These findings suggest a pivotal role for cyclin d1 in a subset of breast cancers . The oncogene ccnd1 is amplified in 10 - 20% of breast carcinomas while the overexpression of its product cyclin d1 is more frequent and seen in about 34 - 81% of breast carcinomas . In our study, cyclin d1 expression was seen in 78.6% of the cases . The frequency of strong (s), intermediate (i), weak (w) and negative (n) states of cyclin d1 were found to be 19.1%, 44.9%, 14.6% and 21.3%, respectively . Many researches have shown that overexpression of cyclin d1 in tumors is related to an unfavorable outcome, but others have yielded different results . In our study, there was no statistically significant relationship between cyclin d1 overexpression and tumor stage . This finding may be explained by the fact that cyclin d1 overexpression cannot always be attributed to gene amplification . Activation of other mitogenic pathways such as -catenin, stat 5, stat 3, nuclear factor kappa b, c - jun, e2f-1, ppar y, calveolin-1 and ras signaling may provide alternate routes to disturb regulation of cyclin d1 . Moreover, the promotion provided by cyclin d1 to proceed the cell through the cell cycle notwithstanding, degradation of cyclin d1 is essential for replication of deoxyribonucleic acid (dna). Cyclin d1 level rises early in g1 phase of the cell cycle and continues to accumulate followed by a rapid decline by the entrance to the s phase . Some studies have demonstrated that acute overexpression of cyclin d1 in fibroblasts prevents s - phase entry . Cyclin d1 has been shown to repress dna replication by binding to proliferating cell nuclear antigen and cdk2 . In the present study, a statistically significant reverse relationship was found between cyclin d1 and tumor grade as evidenced by the observation that negative cyclin d1 status was mostly seen in grade iii while strong, and intermediate states of cyclin d1 were more frequent in grades i and ii . This observation is the same as the finding of some other studies in this field . The grade of invasive ductal carcinoma is estimated by histological evaluation of tubular formation, mitosis and pleomorphism . Low grade tumors are well - differentiated and show histological features closer to their original tissue . The reverse relationship observed between cyclin d1 overexpression and tumor grade suggests that higher expression of cyclin d1 may directly or indirectly result in maturation of tumor cells . S and i states of cyclin d1were mostly seen in patients with positive er while n status was most frequent in patients with negative er . S and i states were most frequent in patients with positive pr while n status was mostly seen in patients with negative pr . These findings further confirm the results of some previous studies in this field, which have stated a positive relationship between hormone receptor status and cyclin d1 overexpression in breast carcinoma . This is in favor of the effect of cyclin d1 on cell maturation and differentiation . However, overexpression of her2-neu has been reported to be associated with the high expression of cyclin d1 . The reverse relationship between cyclin d1 overexpression and tumor grade as well as the positive relationship between the overexpression of this marker and hormone receptor status in breast carcinoma suggest a regulatory role of cyclin d1 overexpression in cell maturation and differentiation.
Patients with ibd may have disabling symptoms such as frequent diarrhea, often with blood or mucus discharge, abdominal pain, weight loss, malabsorption, malnutrition, and fatigue 1 . Moreover, patients may be affected by extraintestinal manifestations involving other organs such as the joints, eyes, skin, liver, and bile ducts 2 . The cause of ibd is unknown and there is no medical cure, although several therapeutic advances have been made in recent years; medical and surgical treatment for ibd is complex . Current treatment paradigms recommend the use of immunomodulators with or without biological therapy aiming at maintaining clinical and endoscopic remission to reduce the inflammatory burden, minimize complications, and the need for surgery, and as a result achieve an improved quality of life for the patient 3,4 . There is an increasing interest in and use of complementary and alternative medicine (cam) in patients with chronic diseases, including those with ibd 58 . Patients with ibd may turn to cam for various reasons: for example, when conventional therapies are inadequate 9 or associated with adverse side effects, or for symptomatic relief and to regain control over their disease 10 . The amount of steroid medication may be a predictor of cam use 11; moreover, cam use may indicate psychosocial distress in patients with ibd 12,13 . The terms complementary medicine and alternative medicine refer to a broad set of healthcare practices that are not part of a country s own tradition and are not fully integrated into the dominant healthcare system . Tm has a long history and is the sum of the knowledge, skills, and practices on the basis of the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not in the maintenance of health 14 . There are different types of cam: whole medical systems (homeopathic medicine, traditional chinese medicine, ayurveda), mind body medicine (meditation, prayer, healing), natural products (herbs, also known as botanicals, vitamins and minerals, and probiotics, often sold as dietary supplements), manipulative and body - based practices (chiropractic or osteopathic manipulation, massage), and energy medicine (qi gong, reiki, therapeutic touch, the use of magnetic fields) 1517 . Overall, 30% of the world s population do not have access to conventional medicine and for these patients, herbal medicines and tm are the main options 14 . A review of the who in 142 countries showed that, in 99 countries, cam, that is, natural products (herbal products and dietary supplements) are sold over the counter without prescriptions 17 . Cam is mostly used for self - care 18, and is often recommended by friends . Many cam treatments are available in our present - day society and the quality of the information on cam, often provided by the media and the internet, is variable . In general, a wide range of cams are recommended for many conditions, and a variety of treatments are recommended for the same conditions . Today, some cam treatments are supported by evidence from randomized - controlled trials, meta - analyses, and systematic reviews 1922, and there are several interesting studies on cam for the treatment of ibd 19 . A recent review of clinical trials of various herbal therapies for ibd 23 presents the most important studies on aloe vera gel 24, polyphenols (green tea) 2527, wheat grass juice 28, bilberry 29, wormwood 30,31, boswellia serrata 19,32, cannabis 33, and chinese herbal medicine 34 . Probiotics have been shown to increase the clinical response and remission rate in mild to moderate uc 37,38 and to prevent pouchitis 39 . Considering the mounting evidence that dietary changes influence gut microbiome, dietary intervention studies have been attempted 40, and as patients are becoming more interested in and are using specific diets to better control the disease 41, diets might be considered cam . Studies using psychological interventions comprising relaxation techniques, patient education 44, and psychotherapy, however, showed that psychotherapy had no effect on disease activity, health - related quality of life, or emotional status 45 . However, a recently published study showed improved anxiety, quality of life, and mindfulness after a stress - reduction program on the basis of mindfulness in patients with cd 46 . There are safety aspects because some herbal - based cams may be associated with adverse side effects and may cause interactions with conventional therapy 48,49 . It is noteworthy that there is emerging evidence that cam therapies may modulate or disrupt the immune system 32 . Thus, the use of cam in patients with ibd needs to be considered in daily practice when making clinical decisions . This multicenter survey was conducted to determine the extent of cam use, the reason for cam use, and perceived positive or negative effects from cam in patients with ibd in sweden . Eight hundred and fifty - four patients with ibd from 12 swedish hospitals were invited to participate in the study . A control group matched for age and sex, urban or rural, and geographic area was recruited . Ten of the ibd centers were university based; one was a large teaching hospital, one was a private clinic, and one was a nonprofit hospital . The inclusion criterion was an established diagnosis of ibd according to medical records being treated at the clinic . The patients were contacted at the ibd centers by an ibd nurse or a physician who provided oral and written information on the study . If the patients were willing to participate, they filled in a questionnaire either at the clinic or at home using a prestamped, addressed reply envelope . All data sampling was performed at each ibd center between august 2008 and june 2009 . The individuals in the control group were selected randomly from a residence registry, statens personadressregister (spar). Spar includes all individuals who are registered as residents in sweden and the data are updated continuously from the swedish population register . An age, sex, and residence match was performed after the first 300 patients with ibd had been included . The questionnaire was sent by post to 1400 individuals together with an informative letter explaining the study, and a stamped, addressed reply envelope . The questionnaire was developed from a previously used questionnaire from an international survey, in which two of the authors (l.o . After updating the previous questionnaire with the help of an expert group on integrative care and cam 50, a final list of 24 different cams was extracted . The respondents were asked to indicate the type and frequency of cam use (use in the past year, use in the last 2 weeks), perceived positive and negative effects of cam, and their source of cam information . Others if the particular cam used was not listed . Further data on demographic characteristics such as age, sex, education, marital status, employment status, urban versus rural residence, annual income, diet, and lifestyle habits (tobacco and alcohol use) were collected . The questionnaire also included questions on disease characteristics, type of ibd, current symptoms, year of diagnosis, conventional medication use, and perceived adverse events from conventional medication . For comparison between two groups, fisher s exact test was used for dichotomous variables, the mantel odds ratio and confidence interval (ci) (adjusted for age, sex, residence, and diet) were calculated for the association of cam use between ibd patients and controls . All participators were informed that participation was voluntarily and that they could withdraw at any time without consequences . The study was approved by the ethical committee for all participating sites (dnr 2008/4:6, 2009/85232). Eight hundred and fifty - four patients with ibd from 12 swedish hospitals were invited to participate in the study . A control group matched for age and sex, urban or rural, and geographic area was recruited . Ten of the ibd centers were university based; one was a large teaching hospital, one was a private clinic, and one was a nonprofit hospital . The inclusion criterion was an established diagnosis of ibd according to medical records being treated at the clinic . The patients were contacted at the ibd centers by an ibd nurse or a physician who provided oral and written information on the study . If the patients were willing to participate, they filled in a questionnaire either at the clinic or at home using a prestamped, addressed reply envelope . All data sampling was performed at each ibd center between august 2008 and june 2009 . The individuals in the control group were selected randomly from a residence registry, statens personadressregister (spar). Spar includes all individuals who are registered as residents in sweden and the data are updated continuously from the swedish population register . An age, sex, and residence match was performed after the first 300 patients with ibd had been included . The questionnaire was sent by post to 1400 individuals together with an informative letter explaining the study, and a stamped, addressed reply envelope . The questionnaire was developed from a previously used questionnaire from an international survey, in which two of the authors (l.o . After updating the previous questionnaire with the help of an expert group on integrative care and cam 50, a final list of 24 different cams was extracted . The respondents were asked to indicate the type and frequency of cam use (use in the past year, use in the last 2 weeks), perceived positive and negative effects of cam, and their source of cam information . Others if the particular cam used was not listed . Further data on demographic characteristics such as age, sex, education, marital status, employment status, urban versus rural residence, annual income, diet, and lifestyle habits (tobacco and alcohol use) were collected . The questionnaire also included questions on disease characteristics, type of ibd, current symptoms, year of diagnosis, conventional medication use, and perceived adverse events from conventional medication . The inclusion criterion was an established diagnosis of ibd according to medical records being treated at the clinic . The patients were contacted at the ibd centers by an ibd nurse or a physician who provided oral and written information on the study . If the patients were willing to participate, they filled in a questionnaire either at the clinic or at home using a prestamped, addressed reply envelope . All data sampling was performed at each ibd center between august 2008 and june 2009 . The individuals in the control group were selected randomly from a residence registry, statens personadressregister (spar). Spar includes all individuals who are registered as residents in sweden and the data are updated continuously from the swedish population register . An age, sex, and residence match was performed after the first 300 patients with ibd had been included . The questionnaire was sent by post to 1400 individuals together with an informative letter explaining the study, and a stamped, addressed reply envelope . The questionnaire was developed from a previously used questionnaire from an international survey, in which two of the authors (l.o . After updating the previous questionnaire with the help of an expert group on integrative care and cam 50, a final list of 24 different cams was extracted . The respondents were asked to indicate the type and frequency of cam use (use in the past year, use in the last 2 weeks), perceived positive and negative effects of cam, and their source of cam information . Further data on demographic characteristics such as age, sex, education, marital status, employment status, urban versus rural residence, annual income, diet, and lifestyle habits (tobacco and alcohol use) were collected . The questionnaire also included questions on disease characteristics, type of ibd, current symptoms, year of diagnosis, conventional medication use, and perceived adverse events from conventional medication . For comparison between two groups, fisher s exact test was used for dichotomous variables, the mantel haenszel -test was used for ordered categorical variables, and the mann odds ratio and confidence interval (ci) (adjusted for age, sex, residence, and diet) were calculated for the association of cam use between ibd patients and controls . All participators were informed that participation was voluntarily and that they could withdraw at any time without consequences . The study was approved by the ethical committee for all participating sites (dnr 2008/4:6, 2009/85232). Of the 854 patients with ibd who were invited to participate, 164 did not return the questionnaires (despite two reminders), 40 patients declined participation, and two were excluded owing to incomplete questionnaires . In total, 648 patients with ibd were included, yielding a response rate of 76% . Fourteen hundred individuals were invited to participate in the control group, of whom 440 responded, yielding a response rate of 32% . Twenty individuals declined participation, 33 letters were returned because of unknown address, one individual had died, and 906 did not return the questionnaires despite two reminders . The patients with ibd who did not respond had a mean age of 41.6 years; 48.5% were men, 39.2% had uc, and 42.9% had cd . The nonresponders in the control group had a mean age of 40.8 years and 56.5% were men . Sociodemographic and disease data are listed in table 1 . Of the 648 patients with ibd included in the study, 324 (50%) had uc, 319 (49.2%) had cd, and five (0.8%) had ibdu . The mean disease duration was 13.3 years and the mean age of the ibd patients with ibd was 42.7 years . The individuals in the control group were significantly older than the patients with ibd (mean age 45.9 years; p=0.0004). In the ibd group, 48.3% of the patients were women and 58.1% of the controls were women (p=0.002). Significantly more of the controls were cohabiting compared with the patients with ibd (p=0.04). Patients with ibd lived significantly more often in urban areas (p=0.001) compared with controls . Patients with ibd used various kinds of diets (e.g. Lacto vegetarian, lacto ovo vegetarian, vegan, and other types of diets) more often than the controls who used more normal diets (p<0.0001). Sociodemographic and disease data, comparison between groups the level of education was similar in the patients and controls . There were also no differences between patients with ibd and controls in occupation . In all, 28% of the patients with ibd were active tobacco users, 13.6% of them smoked and 14.8% used other tobacco (e.g. Snuff tobacco), the differences were not significant compared with controls . Current alcohol use was significantly higher among the controls than the patients with ibd (p=0.005). Overall, 93% of the patients with ibd reported the use of conventional medicine for ibd and 39.8% reported having experienced an adverse drug event from conventional medicine . The controls often did not reply to the question on conventional medication or adverse events . Differences between patients with ibd and controls were adjusted for when comparing cam use between groups . Patients with ibd and individuals in the control group used different kinds of cam (table 2). Of the patients with ibd, 48.3% had used some kind of cam during the past year compared with 53.5% of the controls (p=0.11). However, after adjusting for age, sex, geographic residence, and diet in a multivariate analysis, a statistically significant difference was observed [p=0.025, odds ratio 1.16 (95% ci 1.021.32)]. The most frequently used cam among patients with ibd was massage, used by 21.3%, compared with 31.4% of the controls (adjusted p=0.0003). The second most frequently used cam was herbal products, which were used by 18.7% of the patients with ibd compared with 22.3% of the controls (adjusted p=0.018). The most commonly used natural products used by patients were omega 3, probiotics, aloe vera, vitamins, arctic root, and other herbal products ., kan yang, siberian ginseng, arctic root, and herbal products (data not shown). Other cams used to a similar extent by patients with ibd and the controls were yoga, acupuncture, counseling, chiropractic, and meditation . More controls used naprapathy than did ibd patients (adjusted p=0.0055), reflexology, and healing (unadjusted p=0.026). Type of complementary and alternative medicine used in patients with ibd within the last year, comparison between groups patients sought cam treatments to reduce pain, mainly pain from back, neck, joints, and bowel but also as strategies to handle their disease in order to decrease bowel symptoms and improve well - being . Ibd patients used cam primarily on their own initiative, but patients were also referred to cam practitioners or recommended cam use by healthcare professionals . They obtained information on different cams mainly from friends and their next of kin, but also from the media, the internet, and the literature and from health food stores (fig . Sources of information on cam (%). Other comprised literature (scientific articles, textbooks), own initiative, and wellness at workplace . The perceived experiences of patients with ibd of cam are presented in table 3 . In all, 83% of the patients with ibd who had used any cam during the past year perceived the cam as a positive experience, whereas 14.4% of them had experienced a negative effect (or effects) of the cam treatment . The majority of the patients who used massage found it to be positive (i.e. Relaxing, providing pain relief, and well - being) and 5.8% experienced negative effects (pain, unease, or ill - being). Natural products were used by 18.7% of the patients with ibd; 66.1% of these patients perceived positive effects, improved disease symptoms, well - being, and general improvement . There were no negative experiences of relaxation; yoga was experienced as a means to achieve well - being, relaxation, and improved mobility . Patients with ibd who used acupuncture experienced pain relief, well - being, and improved disease symptoms . Perceived effects of cam in patients with ibd (n=648) the patients with ibd who did not respond had a mean age of 41.6 years; 48.5% were men, 39.2% had uc, and 42.9% had cd . The nonresponders in the control group had a mean age of 40.8 years and 56.5% were men . Sociodemographic and disease data are listed in table 1 . Of the 648 patients with ibd included in the study, 324 (50%) had uc, 319 (49.2%) had cd, and five (0.8%) had ibdu . The mean disease duration was 13.3 years and the mean age of the ibd patients with ibd was 42.7 years . The individuals in the control group were significantly older than the patients with ibd (mean age 45.9 years; p=0.0004). In the ibd group, 48.3% of the patients were women and 58.1% of the controls were women (p=0.002). Significantly more of the controls were cohabiting compared with the patients with ibd (p=0.04). Patients with ibd lived significantly more often in urban areas (p=0.001) compared with controls . Patients with ibd used various kinds of diets (e.g. Lacto vegetarian, lacto ovo vegetarian, vegan, and other types of diets) more often than the controls who used more normal diets (p<0.0001). Sociodemographic and disease data, comparison between groups the level of education was similar in the patients and controls . There were also no differences between patients with ibd and controls in occupation . In all, 28% of the patients with ibd were active tobacco users, 13.6% of them smoked and 14.8% used other tobacco (e.g. Snuff tobacco), the differences were not significant compared with controls . Current alcohol use was significantly higher among the controls than the patients with ibd (p=0.005). Overall, 93% of the patients with ibd reported the use of conventional medicine for ibd and 39.8% reported having experienced an adverse drug event from conventional medicine . The controls often did not reply to the question on conventional medication or adverse events . Differences between patients with ibd and controls were adjusted for when comparing cam use between groups . Patients with ibd and individuals in the control group used different kinds of cam (table 2). Of the patients with ibd, 48.3% had used some kind of cam during the past year compared with 53.5% of the controls (p=0.11). However, after adjusting for age, sex, geographic residence, and diet in a multivariate analysis, a statistically significant difference was observed [p=0.025, odds ratio 1.16 (95% ci 1.021.32)]. The most frequently used cam among patients with ibd was massage, used by 21.3%, compared with 31.4% of the controls (adjusted p=0.0003). The second most frequently used cam was herbal products, which were used by 18.7% of the patients with ibd compared with 22.3% of the controls (adjusted p=0.018). The most commonly used natural products used by patients were omega 3, probiotics, aloe vera, vitamins, arctic root, and other herbal products ., kan yang, siberian ginseng, arctic root, and herbal products (data not shown). Other cams used to a similar extent by patients with ibd and the controls were yoga, acupuncture, counseling, chiropractic, and meditation . More controls used naprapathy than did ibd patients (adjusted p=0.0055), reflexology, and healing (unadjusted p=0.026). Type of complementary and alternative medicine used in patients with ibd within the last year, comparison between groups patients sought cam treatments to reduce pain, mainly pain from back, neck, joints, and bowel but also as strategies to handle their disease in order to decrease bowel symptoms and improve well - being . Ibd patients used cam primarily on their own initiative, but patients were also referred to cam practitioners or recommended cam use by healthcare professionals . They obtained information on different cams mainly from friends and their next of kin, but also from the media, the internet, and the literature and from health food stores (fig . Sources of information on cam (%). Other comprised literature (scientific articles, textbooks), own initiative, and wellness at workplace . The perceived experiences of patients with ibd of cam are presented in table 3 . In all, 83% of the patients with ibd who had used any cam during the past year perceived the cam as a positive experience, whereas 14.4% of them had experienced a negative effect (or effects) of the cam treatment . The majority of the patients who used massage found it to be positive (i.e. Relaxing, providing pain relief, and well - being) and 5.8% experienced negative effects (pain, unease, or ill - being). Natural products were used by 18.7% of the patients with ibd; 66.1% of these patients perceived positive effects, improved disease symptoms, well - being, and general improvement . There were no negative experiences of relaxation; yoga was experienced as a means to achieve well - being, relaxation, and improved mobility . Patients with ibd who used acupuncture experienced pain relief, well - being, and improved disease symptoms . Perceived effects of cam in patients with ibd (n=648) a high percentage of the patients with ibd (48.3%) had used some kind of cam within the last year, which is in line with previous research (3268%) in other western countries 5,8,10,5154 . The most common cam use in the current study was massage, followed by natural products, relaxation, yoga, acupuncture, and counseling . The use of cam was significantly higher in the control group (53.1 vs. 48.3%); however, this high use of cam in the control group must be interpreted with caution because of the low response rate . Patients with ibd may be so used to conventional medication that they dare not use cam to a greater extent or they may be influenced by healthcare professionals showing a disparaging attitudes toward patients cam use 55 . It has been argued that in the absence of critical assessment of cam, gastroenterologists could simply be supportive, cautious, and open - minded about widely available cams 6 . Overall, 93% of the patients with ibd in the present study reported the use of conventional medication and as many as 40% of these reported adverse event from conventional medicine . This high figure of adverse drug events could be because of a selection bias because the patients responding to the questionnaires could be those explicitly interested in cam and/or those who had experienced adverse effects from conventional medicine . However, patients on ibd medication often report adverse drug reactions, mainly from steroid therapy . A review showed that adverse events lead to cessation of medication in up to 55% of patients being prescribed steroids, and 1011% of patients prescribed antitumor necrosis factor therapy and immunomodulators, 56 . The ibd patients in our study sought cam treatments to reduce pain and to handle stress and symptom related to their disease . The majority of the patients perceived positive effects from cam treatments, but interestingly, no major improvement in disease symptoms was observed . A significantly higher proportion of the controls (22.3%) used natural products compared with patients with ibd (18.7%), controlled for age, sex, residence, and diet . This was to some degree surprising because patients with a chronic disease were expected to use more cam compared with a control group . This difference may possibly be explained by the fact that the controls were significantly older, more of them were women, more were living in urban areas / cities, and the controls were following more normal diets . With respect to patient safety, it is notable that as many as 18.7% of the patients with ibd in our study used natural products . The patients also used other cams (but to a lesser extent): anthroposophy (0.5%), ayurveda (0.3%), homeopathy (2.3%), and traditional chinese medicine (0.5%), which are not included in the the definition of cam used in this study in terms of natural products includes a variety of products, herbals (botanicals), vitamins and minerals, and probiotics (often sold as dietary supplements) 16 . Research on the importance of vitamin d is increasing and clinicians may recommend ibd patients such products as vitamins, omega 3, and probiotics 5759; however, the patients in the present study did identify these products as cams, indicating that they were not recommended these by their physicians . A review showed that herbal therapy appeared to be effective in ibd, but the safety profile and long - term efficacy require further research 60 . Certain herbal therapies have been reported to have anti - inflammatory properties, and the use of these cams may theoretically cause interactions with conventional medicines . Herbal treatments may have toxic side effects, and some treatments are contraindicated and may be dangerous 49 . Liver toxicity has been described in the literature, for example, in relation to the consumption of noni juice 61,62; none of the ibd patients in the present study had used noni . The results from the present study highlight the importance of healthcare professionals being aware of the potential effects, potential side effects, and interactions of such therapies and the fact that our patients are using these cams . A number of herbal - based and traditional medical products are registered and controlled by the swedish medical products agency 63 . An european union directive incorporated into the who has a strategy that encourages countries to incorporate cams into conventional healthcare 14 . Legalization in sweden guides swedish healthcare professionals on how to relate to and recommend these herbal products . This and the fact that patients with ibd do use these natural - based cams should be considered when making decisions in clinical care . However, there are some practical issues in sweden . There is no general cam policy and cams have not been officially approved in healthcare or within the educational system; thus, policy development is essential 64 . We conclude that patients with ibd in sweden are using cam treatment to a large extent (48.3%), but the control group used cam to a greater extent (53.1%). This should be interpreted with caution because of the low response rate in the control group . Patients with ibd might very well be influenced by healthcare professionals showing disapproving attitudes toward patients cam use 55, thus hindering cam use . The majority of the patients experienced positive effects from cams, mainly well - being, whereas no major improvement in disease symptoms was observed . In all, 40% of the patients had experienced adverse events from conventional medication, which may be a reason for cam use; furthermore, the patients in this study experienced predominantly positive effects from cam therapies . A high level of use of natural products was noted, more common in controls (22.3%) compared with patients with ibd (18.7%), but still used by about one - fifth of the patients . The possible risk of interaction with cam must be considered when prescribing conventional medication . Consequently, an open - minded dialog with our patients is necessary to determine their cam use.
Cytokines are small proteins initially thought to be components of the immune system, but have since been found to play a much broader role in physiology . Interleukin-6 (il-6) is a cytokine originally identified as a b - cell differentiation factor (bsf-2) in 1985 1, as a factor that induced the maturation of b cells into antibody - producing cells . Human il-6 2 and il-6 receptor 3 were cloned soon thereafter . As with many other cytokines, it was soon realized that il-6 was not a factor only involved in the immune response . In early 1990s it was clear that besides controlling other immune cells such as t cells, il-6 was also important in the regulation of hepatocytes, hematopoietic progenitor cells, the skeleton, the cardiovascular system, the placenta and the nervous and endocrine systems 4 . Structural analysis has allowed the grouping of these proteins into different structural classes such as the helical cytokines, the trimeric tumor necrosis factor (tnf) family, the cysteine knot growth factors and the -trefoil growth factors 5 . Cytokines can also be grouped according to the type of receptor they bind, which comprise six major families: class i cytokine receptors (the largest family), class ii cytokine receptors, tnf receptors, tyrosine kinase receptors, and chemokine receptors 5 . Cytokine families may be named differently according to other aspects such as the sharing of a receptor subunit (i.e. The gp130 family) or its physiological roles (i.e. Neuropoietic family, for its effects on hematopoietic and nervous system). Il-6 is a prototypical four - helix bundle cytokine that is the founder member of the neuropoietins, a group of cytokines structurally related, that include il-6, il-11, il-27, il-31, leukemia inhibitory factor, oncostatin m, cardiotrophin-1, neuropoietin and cytokine cardiotrophin - like (also known as new neurotrophin 1 and b cell stimulatory factor-3), and two viral analogs of il-6 5 - 10 . These cytokines bind to class i cytokine receptors, membrane proteins with a characteristic modular architecture that do not have intrinsic enzymatic activity, and that for signaling often need to recruit additional receptor proteins shared by different cytokines: gp130, c or c . This protein has also a modular architecture, part of which has features typical of the cytokine receptors (two fibronectin type iii modules containing conserved cysteine residues and a wsxws motif) 4 . For il-6 specifically, a hexamer forms (two il-6, two il-6r and two gp130) that can activate intracellular tyrosin - kinases such as janus kinase (jak) and, to a lesser extent, tyk, which, in turn, activate a number of proteins including the stat (signal transducer and activator of transcription) family of transcription factors, or the ras - raf - mapk pathway, pi3 (phosphatidyl inositol-3) kinase, or irs (insulin receptor substrate) 11 . The sharing of gp130 explains at least in part the redundancy of the actions of these cytokines . It was soon established that the expression of il-6r is restricted to some tissues, while that of gp130 is ubiquitous, and that il-6 may upregulate gp130 expression 12 . The same study already provided evidence that extracellular, soluble il-6r (sil-6r) in the presence of il-6 could activate cells expressing gp130, and stated that naturally occurring sil-6r could be found in the murine serum . It is now widely accepted that sil-6r is formed physiologically, either by limited proteolysis of the extracellular domain of membrane il-6r (mil-6r) by metalloproteases such as adam10 and adam17, or by alternative splicing of il-6r mrna, and that sil-6r can bind both il-6 and gp130 and signal in cells with or without endogenous il-6r expression, a mechanism known as trans - signaling 13 . To complicate things further, it is also known that a soluble form of gp130 (sgp130) is also formed 14, in this case only by alternative splicing of gp130 mrna; sgp130 can inhibit trans - signaling but does not affect normal signaling by mil-6r (figure 1). Soon after its discovery, it was demonstrated that some astrocytoma and glioma lines expressed il-6 when stimulated with il-1, which prompted speculation that il-6 could have a role in the cns 2 . The same group demonstrated that il-6 indeed was capable of inducing the neuronal differentiation of the rat pheochromocytoma pc12 cell line, to some extent similarly to the prototypical neurotrophin ngf 15, 16 . It was therefore not surprising the finding that both glial and neuronal cells expressed il-6 and il-6r to various degrees throughout the brain 17 - 24 . In vitro, microglia, astrocytes and the neuronal line n18, but not oligodendrocytes, expressed il-6r 25; in vivo, nevertheless, oligodendrocytes may express il-6 and il-6r 26 . Also, il-6 and il-6r were expressed in sympathetic and sensory ganglia, predominantly in neurons 27, as well as in adrenal chromaffin cells 28, of adult rats . In line with these results, in vitro studies demonstrated that dissociated sympathetic neurons and pc12 cells expressed il-6 and the two receptors, il-6r and gp130 29, 30 . Besides neurons and glial cells, endothelial cells produce copious amounts of il-6, which can act on surrounding cells but also in an autocrine manner regulating a number of adhesion proteins but also il-6 synthesis, particularly in the presence of sil-6r 31 - 33 . Thus, both the central and the peripheral nervous system appear to express il-6 and the corresponding receptors (figure 1). Many cytokines and inflammatory factors as well as neurotranmitters and neuropeptides have been shown to affect il-6 regulation in brain cells; we will comment here some of them studied in in vitro assays . Both virus - infected microglial cells and il-1 and tnf induced il-6 in cultured cortical neurons 36 and astrocytes 37, 38, in the latter involving nfb 39 and the pkc pathway 40 . It is likely that membrane depolarization is one of the main mechanisms for neuronal upregulation of il-6, where ca currents (such as those elicited by the glutamate agonist nmda) and ca / calmodulin - dependent kinases are critical factors 43, 44 . The major bacterial pathogen, lps, normally induces il-6 in both astrocytes and microglia 45 - 47, but tnf- induces il-6 in astrocytes but not microglia 45 . There may be species - specific effects since in human cells in vitro lps mostly affect microglia rather than astrocytes (obtained from brains at second trimester of gestation) regarding tnf, il-1 and il-6 production, although il-1 is a potent stimulator of il-6 production in astrocytes (in microglia the 3 cytokines are upregulated) 46, 48 . Gm - csf stimulates microglial il-6 but not that of astrocyte 49, whereas ifn- induces il-6 (and no) in the murine microglial cell line 6 - 3 50; this cytokine does not induce il-6 in astrocytes unless it is coincubated with il-1 37 . Il-6 production by astrocytes is subjected to autocrine regulation by il-6, and the addition of sil-6r synergizes dramatically with il-1 and tnf to induce il-6 52 . Oncostatin m (osm) induced il-6 alone and synergized with tnf to induce il-6 52 . Il-17 functioned in a synergistic manner with il-6 (+ sil-6r) to induce il-6 expression in astrocytes 53 . Norepinephrine, vasoactive intestinal peptide (vip) and pituitary adenylate cyclase activating polypeptide (pacap38) stimulate il-6 in astrocytes, and may synergize with il-1 and tnf 54 - 56; in contrast any of these factors have a major effect in microglia 54 . Vip may induce il-6 through the pka pathway and independently of prostaglandins 57, 58 . Prostaglandin e1 (pge1) and pge2, but not pgd2 and pgf2 alphae2, induce il-6 in human astrocytoma cells; pge2 potentiates il-1 induction of il-6 59, 60 . The synthetic ceramides c2- and c6-ceramide as well as the enzyme sphingomyelinase were able to induce il-6 in astrocytes 61 . Il-1, substance p and histamine induced il-6 expression in human astrocytoma cells 41, 62, through nf-b for il-1 and through nf - il-6 for sp and histamine 63 . Serotonin and adenosine agonists were also effective inducers in human astrocytoma cells 65 66; in mouse astrocytes, adenosine induces il-6 through activation of pka and nf - il-6 67 . Tgf- inhibits microglia proliferation and activation, including il-6 production 68; in contrast, it stimulates il-6 production in astrocytes 69 . Il-6 may affect neuronal functionality, for instance it induces the cholinergic phenotype of sympathetic neurons 30, 70, 71 . Sensory neurons are particularly affected by il-6 deficiency, since in normal conditions il-6 ko mice show a 60% reduction of the compound action potential of the sensory branch of the sciatic nerve and a dramatic decrease of temperature sensitivity in the frontpaw withdrawal time in the hot - plate assay 72; these neurons are also highly dependent on il-6 for functional recovery following injury 72, 73 . Results with il-6 ko mice imply a role for il-6 on sympathetic sprouting induced by nerve injury 74, 75 . It also promotes sprouting and functional recovery of organotypical cultures of hippocampus 76, and causes a dose - dependent decrease of post - tetanic potentiation (ptp) and long - term potentiation (ltp) in the ca1 region of rat hippocampus 77 . Il-6 also has a notorious role in adult neurogenesis 10, 78, the process of creating new neurons and glial cells from neural stem cells (nscs) discovered almost 50 years ago 79, which is now known to be dramatically affected by a myriad of factors such as exercise, environmental enrichment, stress, or aging . Not surprisingly, neurogenesis is also altered in many neuropathological situations like stroke, status epilepticus, mechanical damage, and alzheimer, parkinson and huntington diseases; in all these cases a detrimental role of inflammation has usually been suggested 80, 81, and, as stated above, il-6 will be upregulated and could have a role on neurogenesis . Gfap - il6 mice, indeed, show a diminished hippocampal neurogenesis 82, and moreover, in vitro il-6 clearly decreases the differentiation of neural stem / progenitor cells into neurons 83, 84 . In contrast, il-6 is involved in oligodendrogliogenesis 85, 86 and astrogliogenesis 84 . Yet, other studies claimed that il-6 promotes both gliogenesis (through the stat-3 pathway) and neurogenesis (mapk / creb pathway) 87, 88 . Besides effects on neurons, il-6 also displays a number of effects in glial and endothelial cells . Early in vitro assays showed that both virus - infected microglia and astrocytes secreted il6, and that il-6 induced the secretion of the major neurotrophin ngf by the latter 34 . In vitro there is some conflicting results whether or not il-6 does have proliferative effects on astrocytes 89, 90, probably it does but synergizing with other factors 91 . Microglia in culture consistently proliferate when stimulated with il-6 92il-6-il-6/sil-6r may alter the factors produced by astrocytes: for instance it induces specific patterns of neurotrophins 93, inhibits tnf 94, and together with sil-6r and il-17, it may shift chemokine production to that favoring t cell recruitment to the cns 95 . Differences in the pattern of cytokines secreted by astrocytes of il-6 ko mice are readily noticeable in vitro 96 . In vivo experiments demonstrate that il-6 exerts profound effects on their differentiation, which is dependent on the brain area from which they are isolated 93 . Transgenic mice overexpressing il-6 show prominent astrogliosis and microgliosis 97 - 100, whereas the opposite is usually observed in il-6 ko mice in different models of injury 101 - 105 . Il-6 alone does not affect intercellular adhesion molecule-1 (icam-1) gene expression, but dramatically inhibits the activating effect of ifn-, il-1 and tnf in rat astrocytes, and that of ifn- in microglia, very much alike il-10 106 . In human astrocytes, il-6, sil-6r or both do not affect vcam-1 or icam-1, but il-6/sil-6r complex (and h - il-6) inhibits tnf--induced vcam-1 gene expression; sil-6r upregulates endogenous il-6 production 107 . This inhibitory effect of il-6 in astrocytes is in sharp contrast with the stimulatory effect it has on endothelial cells, where again sil-6r greatly affects the activation of endothelial cells, not only upregulating the adhesion proteins e - selectin, icam-1 and vcam-1 but also il-6, il-8 and monocyte chemotactic protein 1 (mcp-1); this constitutes a model where neutrophils may retrogradely signal inflammation to endothelial cells by shedding sil-6r, which can then recruit leukocytes and contribute to their extravasation 32, 108 . Regarding oligodendrocytes, in addition to promoting oligondendrogenesis il-6 promotes survival and myelin production of oligodendrocytes 85, 86, 109, 110 . Although il-6 is often related to inflammatory and pathological situations, it is a factor that contributes decisively in the normal function of the brain . Thus, il-6 is involved in the control of body weight, food intake and energy expenditure 111 - 115, stimulates the pituitary - adrenal axis 116 117, induces fever 111, 118, 119 and is for instance very important in the control of body temperature following recovery from stroke 120 . Results with il-6 ko mice imply a facilitatory role for il-6 in pain 74, 75, effects on sleep - wake beavior 121, emotional reactivity 122, 123, sickness behaviour 124 and learning and memory 125 - 127 . As stated above, soon after its discovery il-6 was shown to induce the neuronal differentiation of pc12 cells 15, 16 . After these initial studies, a flurry of papers demonstrated that il-6 affected in many different ways neurons and glial cells . Thus, it was shown to promote the survival of cultured basal forebrain and septal cholinergic neurons and mesencephalic catecholaminergic neurons 128, 129, retinal ganglion cells 130, sympathetic neurons and dorsal root ganglia (drg), particularly if sil-6r was added to the culture 30, 131 . It has been suggested that a survival mechanism could be the inhibition of neuronal activity and release of glutamate 132 . In vivo, il-6 increases with development in drg, but levels are low in adults; however, following sciatic nerve transection its expression increases dramatically, not only in the drg but also in many neurons in the corresponding motor nucleus or sympathetic ganglion 133 . Studies in a drop weight model of cortex lesion (closed skull) also indicate that il-6 expression increases following axonal damage, mainly in neurons 134, thus probably neuronal induction of il-6 in response to injury is a general response . In the sciatic model, il-6 expression increases potently within large and medium - sized axotomized neurons 133, whose survival is decreased by 50% in il-6 ko mice 135 . Il-6 probably promotes survival through inducing bdnf, and indeed in vivo il-6 ko mice do not upregulate this neurotrophin in drg following nerve injury 136 . Experiments after injury of the hypoglossal nerve in mice also demonstrated a nerve regenerating role of il-6 137 . In contrast, in the facial nerve axotomy model no difference in neuronal survival was observed 104, whereas il-6 was shown to be detrimental in a model of optic nerve injury 138 . Cns il-6 is upregulated whenever neuroinflammation is expected, such as following cns infection or injury or in a number of cns diseases (figures 2 and 3). Early studies demonstrated that il-6 was expressed and produced in cns during viral meningitis, in encephalitis mouse models, and in csf of patients with acute viral infections 139 . Il-6 was also found to be upregulated in mouse experimental cerebral malaria (ecm) 140 . Moreover, il-6 was highly found in csf of patients with systemic lupus erithematosus (sle) with cns involvement 141 or in those in advanced stages of patients with human immunodeficiency virus 142 infections 143 . Il-6 levels in csf were also significantly higher than plasma levels in patients who had suffered traumatic brain injury 144, and recently an il-6 polymorphism (-174c / g) has been associated with fatal outcome in patients with severe traumatic brain injury 145 . As expected, il-6 is upregulated in several animal models of brain injury and shows a myriad of actions as suggested by studies in il-6 ko mice which show a compromised inflammatory response, increased oxidative stress, impaired neuroglial activation, decreased lymphocyte recruitment and a slower rate of recovery and healing 47, 101, 102, 104, 134, 146 - 148 . Of note, il-6 is a critical cytokine controlling the transition from innate to acquired immunity, which is imperative for dealing properly with injured (and infected) cns tissue, and where il-6 trans - signalling is dramatically important 149 . In line with the results with il-6 ko mice, gfap - il-6 mice (which overexpress il-6 in the cns) showed more rapid healing and recovery after traumatic brain injury because of extensive revascularization 148, 150 . The transcriptomic analysis of il-6ko mice versus wt mice 151 and that of gfap - il-6 mice 152 in a model of brain cortex cryoinjury revealed that il-6 modulates the expression of many genes involved in inflammation, apoptosis and oxidative stress among others . Stroke patients also show significant elevations of csf and serum il-6 shortly after the ischemic event that correlate with brain infarct volume, and il-6 haplotype affects both infarct size and il-6 levels 153 - 157 . Ischemic brain injury involves inflammation, excitotoxicity, oxidative damage and apoptosis, and thus to some extent it is a similar scenario as that following traumatic brain injury . It is therefore not surprising that il-6 may be an important factor orchestrating the responses elicited by stroke . In animal models of cerebral ischemia it has been a consistent finding an upregulation of il-6, mostly in neurons but also in glial cells and vascular endothelium 158 - 161 . In line with the in vitro studies detailed above, neuronal il-6 upregulation following cerebral ischemia is likely mediated by glutamate - induced neuronal depolarization 44, 162, 163 . Several of these studies clearly demonstrated a neuroprotective role of exogenously administered il-6, as did experiments injecting anti - mouse il-6 receptor monoclonal antibody 164 and with il-6 ko mice provided body temperature is controlled 120, 165 . Controlling oxidative stress 166 and angiogenesis 165 are among the attributed functions of il-6 during stroke . Epileptic seizures often involve excitotoxicity, and thus it was expected to find that patients suffering of epilepsy show increased csf levels of il-6 after seizures 167, and that well - known animal models of epilepsy such as the glutamate analog kainic acid (ka) upregulate cns il-6 162 . Il-6 ko mice are more susceptible to various convulsant stimuli including several glutamate analogs and show clear signs of increased hippocampal damage 168 - 170 . Yet, this is a complex system, since intranasal administration of il-6 to rats 171 and transgenic il-6 expression in the brain 172 are proconvulsive . Also, some authors also claim a damaging role of il-6 on neuron development in culture and on the response to nmda 173 174 . The reasons for such dual effects of il-6 remain uncertain, but probably reflect that the context has a dramatic effect as often is the case with growth factors . As a prototypical cytokine with roles in the control of inflammation, il-6 it is therefore not surprising that il-6 expression is altered in the brains of alzheimer's disease (ad) patients, being increased around amyloid plaques and in cerebrospinal fluid 175 - 179 . Il-6 stimulated the synthesis of the ad beta - amyloid precursor protein 180, 181, and, conversely, il-6 is upregulated in cultured glial cells upon stimulation with the carboxy - terminal 105 amino acids of app 182 . Moreover, il-6 also enhances neuronal damage induced by beta - amyloid peptide in cultured rat cortical neurons 174 . Despite this detrimental role of il-6 seen in vitro, in vivo studies with ad transgenic mouse models rather show a beneficial role of il-6, in principle due to a massive gliosis which attenuated beta - amyloid peptide deposition and enhanced plaque clearance 183 . This is not unexpected as activated microglia can efficiently phagocytize beta - amyloid peptide and delay pathology course in transgenic models 184 - 186 . Astrocytes also may be involved in the clearance of beta - amyloid peptide 187, also by regulating microglial phagocytosis 188 . In humans, attempts to find important interactions between polymorphisms in specific alleles, such as the il-6 - 174 g / c promoter allele, and genotype frequencies, are not conclusive 189 - 191 . The -572c / g polymorphism of il-6 gene promoter region is another polymorphism that might be associated with ad 192 . Multiple sclerosis (ms) is an inflammatory demyelinating autoimmune disease of cns mediated by cd4+t cells and soluble inflammatory mediators, in which il-6 has an important role . While there is significant controversy on whether or not ms is correlated with either plasma or csf il-6, sil6r and sgp130 levels 193 - 196, it has been demonstrated the presence of il-6 in acute and chronic active plaques of ms patients, mainly associated with astrocytes rather than macrophages or mononuclear infiltrating cells 197 . Yet, the association of ms with il-6 polymorphisms is again not conclusive 198, 199 . One of the most common animal model of ms is experimental autoimmune encephalomyelitis (eae) 200 . Il-6 is upregulated in cns during eae 201, and different approaches have demonstrated a major role of this cytokine . Thus, it was soon established that neutralization of il-6 with antibodies led to a reduced disease 202, which later on was seen to be due to the suppression of the mog - induced differentiation of naive t cells into th17 and th1 cells 203 . A seminal series of early studies demonstrated that il-6 deficient mice were resistant to eae 204 - 207, highlighting the essential role of this cytokine . Absence of infiltrating cells in the cns, reduction of lymphocyte proliferation, change of the cytokine profile and failure to stimulate endothelial vcam-1 were some of the reasons suggested to be responsible for resistance to eae at the time . Trans - signaling is also crucial for eae induction as its blockade with gp130-fc fusion protein delayed the onset of adoptively transferred eae compared to controls due to a reduction in vcam-1 expression on spinal cord microvessels 208 . Since the discovery of two novel subsets of t helper cells, named treg and th17 cells, and its importance in ms, it has been shown that il-6 has a major role in th17 cell differentiation from naive cd4 t cells, particularly in the eae model 203, 209, 210 . Th17 cells produce il-17 (among other cytokines) which enhances il-6 production by astrocytes, which in turn induces differentiation of th17 cells in a positive feedback loop between il-17 and il-6 53, 211 . Although eae is considered a disease mostly induced peripherally, the fact is that the cns local milieu may have dramatic effects . Thus, the gfap - il6 mouse model of chronic transgenic il-6 expression 97 presents an atypical eae due to a retargeting of the immune attack, with no signs of spinal cord damage while showing a prominent cerebellar damage 212 . The mechanisms responsible for these responses to il-6 in the cerebellum are complex and likely to include an increased vascular activation, loss of integrity of the bbb and the induction of specific cytokines and chemokines . Parkinson's disease (pd) is a degenerative disorder of the cns with severe motor impairment, like shaking or rigidity among many others, due to the death of dopaminergic neurons in the substantia nigra . Il-6 is upregulated in pd 213, 214, and il-6 levels in the csf of pd patients are elevated 215, although an inverse correlation between severity of pd and il-6 levels is observed 216 . Il-6 ko mice show increased vulnerability to the mptp, a molecule that is metabolized into the complex i inhibitor mpp+ by astrocytes and is a selective dopaminergic toxin 217, whereas il-6 is capable of protecting rat dopaminergic neurons from the neurotoxicity of mpp 218 . Altogether, the results with these animal models of pd suggest that il-6 could be exerting a neuroprotective role during pd . Huntington's disease 208 is an inherited neurodegenerative disorder with both neurological and systemic abnormalities, characterized by abnormalities in the huntingtin gene . Il-6 expression is dramatically elevated in the striatum of hd patients, and in general these patients show clear signs of abnormal immune activation, il-6 being one of the cytokines affected and monocytes, macrophages and microglia (from both hd and mouse hd models) being overactive upon stimulation 219 . Il-6 is suggested to be neuroprotective as concluded from results with the quinolonic acid rat model of hd 220 . Some studies have pointed out that il-6 levels in csf but not in plasma were increased in combat veterans with posttraumatic stress disorder (ptsd) in comparison with those of controls 223, and that il-6 and/or sil-6r levels in plasma but not sgp130 were significantly higher in ptsd patients respect to controls, and higher in ptsd patients with concurrent major depression than in ptsd patients or controls 224 . Il-6 has also been related with schizophrenia (sz), a complex neurological disorder characterized by a breakdown of thought processes and by poor emotional responsiveness, commonly manifested with hallucinations, delusions, paranoid and mental deterioration 225, 226 . Thus, a single maternal injection of il-6 during pregnancy causes schizophrenia - like behavioral abnormalities in wt mice but not in il-6 ko mice 227 . Finally, il-6 has been found to be increased in the cerebellum of autistic brain 228 and has been suggested to mediate autism - like behaviors 229 . Many of its effects are caused by trans - signaling, while others are mediated by the membrane receptor; both can be essentially considered an integrated, unique system . The relevance of trans - signaling in vivo in a number of peripheral and cns diseases is widely recognized 111, 149, 230 - 234, and not surprisingly therapeutic approaches aiming to counteract il-6 effects not only focus in il-6 membrane receptor 235 - 238 but also in il-6/sil-6r complex 13, 239 - 241 . Likewise, sgp130 is also of an invaluable utility to specifically block diseases states in which trans - signaling responses exist 232, 234, 241, to the extent that the molecule has recently been structurally - optimized to better antagonize trans - signaling pathologic effects 242 . Still, we must keep in mind that il-6 has a role in the normal brain, and interfering with them may be a significant source of concern . Finally, we need to understand in vivo what the role(s) of il-6 are to be due to specific cellular production of and response to il-6; hopefully the production of floxed mice for il-6 will help to answer these questions.
Meigs syndrome is defined as the coexistence of benign ovarian fibroma, hydrothorax, and ascites . By contrast, pseudomeigs syndrome is limited to other ovarian or pelvic tumors associated with hydrothorax and ascites 1 . Both are treated by resectioning of the tumors, which could control the fluid accumulation in the thorax and abdomen 2 . Although many different types of operative methods are chosen to treat rectal prolapse, rectopexy is considered the most appropriate in terms of curability, but its use is limited because of its invasiveness . Laparoscopic rectopexy is expected to expand its indication even to elderly patients; however, it is unclear whether patients with a large volume of ascites due to pseudomeigs syndrome would be candidates for laparoscopic rectopexy . We herein report a case of rectal prolapse caused by ascites from pseudomeigs syndrome that was successfully managed with laparoscopic surgery . A 64yearold female presented to our surgical outpatient department complaining of discomfort caused by rectal prolapse and abdominal distension attributable to ascites . She had been receiving outpatient chemotherapy to treat breast cancer with metastasis for 13 years, which she had commenced after a leftsided mastectomy, and her survival prognosis was estimated to be more than 1 year according to previous reports 3, 4, 5 . Computed tomography (ct) revealed complete rectal prolapse; disseminated masses, including an ovarian metastasis; pleural effusion; and large amounts of ascitic fluid (especially in the pelvic cavity) (fig . Approximately 3500 ml of slightly yellow transparent ascitic fluid was aspirated . As large ovarian metastases covered the pelvic cavity and impeded the procedure, we first performed a bilateral oophorectomy . The rectum was then dissected in the mesorectal plane to the level of the levators and subsequently stapled to the sacral bone around the region of the promontory (fig . The patient was discharged on day 10 after surgery with no apparent complications and continued to receive outpatient chemotherapy . Pathological examination revealed that the ovarian tumors were compatible with breast cancer metastases . At the 1year followup, no relapse was evident in terms of abdominal or thoracic fluid or rectal prolapse (fig . 1). Thus, laparoscopic surgery was useful to treat both the rectal prolapse and pseudomeigs syndrome . (left) preoperative computed tomography (ct) scan: enlarged bilateral ovaries (white arrows) and a large amount of ascites fluid are evident in the pelvic cavity . (right) postoperative ct scan taken 2 months after surgery: both ovaries have been resected and the ascites has disappeared . (a) both ovaries were enlarged within the pelvic cavity and covered the rectum . (b) the ovarian artery and vein were ligated, and both ovaries were then resected using an ultrasonic coagulation device . (c) the rectum was completely mobilized down to the level of the levators . This case emphasizes that laparoscopic rectopexy can be used to treat rectal prolapse in the presence of comorbidities and that treatment of pseudomeigs syndrome improves quality of life and reduces the rectal prolapse relapse rate . A comparative study demonstrated that laparoscopic suture rectopexy is a safe and feasible procedure and has comparable results with regard to operative time, morbidity, bowel function, and recurrence; it even has slightly better results than mesh rectopexy . Furthermore, the cost of the mesh used in mesh rectopexy is absent in suture rectopexy 8, and it is unclear whether mesh rectopexy might be safe and feasible for patients with multiple peritoneal dissemination . A recent systematic review showed that laparoscopic colorectal surgery in the elderly afforded significant advantages in terms of shortterm outcomes 9 . As the incidence of rectal prolapse increases with age, the use of laparoscopic methods might be able to extend surgical indications to elderly, fragile patients . One small randomized clinical trial showed that laparoscopic rectopexy was associated with less postoperative pain, an earlier return to alimentation, an earlier discharge from the hospital, and lowerlevel relapse, at the expense of needing a longer operation time compared to open methods 10, 11, 12 . Additionally, laparoscopy permits visualization of the entire abdominal cavity, facilitating simultaneous treatment of several pathological lesions . In our present case, laparoscopic management was chosen not only to reduce the relapse rate but also to prevent the development of an abdominal incisional hernia attributable to increased intraabdominal pressure from ascites . As large ovarian metastases covered the pelvic cavity and impeded the procedure, we first performed a bilateral oophorectomy . This effectively treated the pseudomeigs syndrome . Additionally, mitigation of the ascites prevented prolapse recurrence . Palliative treatment (i.e., the miwagant procedure) would not be expected to prevent early relapse because of the abundant ascites associated with pseudomeigs syndrome . In this case, neither pleural effusion nor ascites were clinically evident; therefore, they did not affect the decision to perform the oophorectomy . If the patient were obese, it may have impeded the ability to clinically identify the presence of large ascites . However, the ascites contributed to the rectal prolapse, and the oophorectomy was thus useful . To the best of our knowledge, this is the first case in which rectal prolapse and pseudomeigs syndrome have been simultaneously treated and successfully managed by laparoscopic surgery . Such surgery may thus be considered as the first choice when treating patients with rectal prolapse and pseudomeigs syndrome . Tk, mt, hh, ko, ks and yk: designed the research; tk, mt, and th: collected clinical information; tk and mt: wrote the paper.
It has been well established that elevated serum cholesterol levels are associated with increasing cardiovascular risk as the population ages.13 meta - analyses have suggested significantly higher cardiovascular mortality rates in older individuals compared with their younger cohorts, with a 10-fold higher risk seen with every 38.6 mg / dl increase in total cholesterol (tc) seen.2 trials with statins in older patients (> 65 years of age) with clinical atherosclerotic cardiovascular disease show significant reductions in total mortality (relative risk 0.78, 95% confidence interval 0.65 to 0.89).4 thus, prescribing cholesterol - lowering therapy in the form of statins to reduce cardiovascular risk in older patients is of considerable benefit . With the practice - shifting changes made with the most recent american college of cardiology / american heart association guidelines for treating blood cholesterol, more elderly patients may be prescribed statin therapy.5 these guidelines expand the use of statins for primary prevention; a change that is most notable in older persons . Statins are recommended in patients who have 10-year atherosclerotic cardiovascular disease risk of 7.5%.6 this means that most men over 60 years and women over 70 years of age will be eligible to receive statin therapy.5 of concern to some is the safety of cholesterol - lowering drug therapy in the aging population; an issue likely to increase in prevalence with these expanded use guideline recommendations . This can be particularly troublesome with the development of frailty which increases patients susceptibility to adverse events.1,6 therefore, it is imperative that practitioners have not only a working knowledge of information related to statins, but more specifically to their efficacy and safety in older populations . Pitavastatin (livalo; kowa pharmaceuticals america, montgomery, al, usa) is the most recent statin to receive regulatory approval . It is indicated for the treatment of primary hyperlipidemia or mixed dyslipidemia as an adjunctive therapy to diet.7,8 the aim of this article is to review the pharmacologic, pharmacokinetic, efficacy, and safety data of pitavastatin specifically in older populations . Pitavastatin mimics other statins by inhibiting hmg - coa (3-hydroxy-3-methylglutaryl - coenzyme a) reductase to reduce low - density lipoprotein cholesterol (ldl - c), elevated tc, apo b, and triglycerides (tg) and to increase high - density lipoprotein cholesterol (hdl - c).7 in contrast with other statins, pitavastatin contains a cyclopropyl group that fits within the hydrophobic areas of the hmg - coa reductase enzyme, partially explaining its potent inhibitory activities.8 studies have described pitavastatin to have a 2.4- to 6.8-fold greater potency than simvastatin and pravastatin, respectively.9 further, pitavastatin inhibits cholesterol synthesis greater than simvastatin (2.9-fold) and atorvastatin (5.7-fold), and its inhibitory effects on sterol synthesis appear to be liver selective and significantly stronger than simvastatin.10,11 pitavastatin is a potent and synthetic hmg - coa reductase inhibitor, with a structure especially similar to fluvastatin and rosuvastatin.12 comparison of pharmacokinetic attributes (table 1) shows that pitavastatin has the highest bioavailability amongst all statins, and that it reaches peak plasma concentration (cmax) within 1 hour after oral administration, the fastest of the seven approved statins in the united states.8,13 administration of pitavastatin with a high - fat meal decreases the cmax by 43%; however, the area under the curve (auc) is not significantly reduced.7 further, the auc of pitavastatin remains unchanged whether it is administered in the morning or evening.7 pitavastatin is highly protein bound, more than 99%, predominantly to albumin and alpha1-acid glycoprotein.7 pitavastatin is rapidly metabolized primarily via hepatic glucuronidation with udp glucuronyltransferase (ugt1a3 and ugt2b7), forming the major inactive metabolite pitavastatin lactone with minimal cyp2c9 and 2c8 metabolism of clinical concern.13 pitavastatin is rapidly taken up into the liver through oatp, including oatp1b1 and oatp2.14,15 the 24-hour auc of pitavastatin was increased 4.6-fold when it was concurrently administered with a potent oatp2 inhibitor (eg, cyclosporine), and is thought to be the major rate - limiting step in the hepatobiliary clearance of pitavastatin.16 the plasma elimination half - life of pitavastatin is between 11 and 12 hours, with approximately 15% of the dose excreted in the urine, and a mean of 79% of the dose excreted in the feces within 7 days.7,13 to date, pharmacokinetic studies have not demonstrated clinically significant differences based either on sex (men versus [vs] women) or age (younger vs elderly, age> 65 years). Morgan et al studied the effects of severe renal impairment not on hemodialysis on pitavastatin metabolism, transport, and clearance.17 the study looked at age, body mass index, and sex - matched healthy adult subjects, using an us food and drug administration (fda)-approved cut - off of healthy estimated gfr of 80 ml / min/1.73 m to align the study with a previous pitavastatin analysis conducted in europe.17 elevations in auc and cmax were not found to be significantly different from their healthy comparators . They concluded that the relationship between renal function and pitavastatin exposure may not be evident, partly due to limited urinary excretion, though they admitted that there was a small sample size (n=16) and single dosing (4 mg) in determining that pitavastatin was safe and well tolerated in patients with severe renal impairment not on hemodialysis.17 pharmacokinetic studies have not demonstrated clinically significant differences based either on age (younger vs elderly age> 65 years) or sex (men vs women).7 significant increases in cmax have been seen in cirrhotic patients when compared with healthy subjects.18 similarly, the elimination half - life was 8.3 hours and 14.4 hours in patients with child - pugh a and child - pugh b classes, respectively . Pitavastatin mimics other statins by inhibiting hmg - coa (3-hydroxy-3-methylglutaryl - coenzyme a) reductase to reduce low - density lipoprotein cholesterol (ldl - c), elevated tc, apo b, and triglycerides (tg) and to increase high - density lipoprotein cholesterol (hdl - c).7 in contrast with other statins, pitavastatin contains a cyclopropyl group that fits within the hydrophobic areas of the hmg - coa reductase enzyme, partially explaining its potent inhibitory activities.8 studies have described pitavastatin to have a 2.4- to 6.8-fold greater potency than simvastatin and pravastatin, respectively.9 further, pitavastatin inhibits cholesterol synthesis greater than simvastatin (2.9-fold) and atorvastatin (5.7-fold), and its inhibitory effects on sterol synthesis appear to be liver selective and significantly stronger than simvastatin.10,11 pitavastatin is a potent and synthetic hmg - coa reductase inhibitor, with a structure especially similar to fluvastatin and rosuvastatin.12 comparison of pharmacokinetic attributes (table 1) shows that pitavastatin has the highest bioavailability amongst all statins, and that it reaches peak plasma concentration (cmax) within 1 hour after oral administration, the fastest of the seven approved statins in the united states.8,13 administration of pitavastatin with a high - fat meal decreases the cmax by 43%; however, the area under the curve (auc) is not significantly reduced.7 further, the auc of pitavastatin remains unchanged whether it is administered in the morning or evening.7 pitavastatin is highly protein bound, more than 99%, predominantly to albumin and alpha1-acid glycoprotein.7 pitavastatin is rapidly metabolized primarily via hepatic glucuronidation with udp glucuronyltransferase (ugt1a3 and ugt2b7), forming the major inactive metabolite pitavastatin lactone with minimal cyp2c9 and 2c8 metabolism of clinical concern.13 pitavastatin is rapidly taken up into the liver through oatp, including oatp1b1 and oatp2.14,15 the 24-hour auc of pitavastatin was increased 4.6-fold when it was concurrently administered with a potent oatp2 inhibitor (eg, cyclosporine), and is thought to be the major rate - limiting step in the hepatobiliary clearance of pitavastatin.16 the plasma elimination half - life of pitavastatin is between 11 and 12 hours, with approximately 15% of the dose excreted in the urine, and a mean of 79% of the dose excreted in the feces within 7 days.7,13 to date, pharmacokinetic studies have not demonstrated clinically significant differences based either on sex (men versus [vs] women) or age (younger vs elderly, age> 65 years). Morgan et al studied the effects of severe renal impairment not on hemodialysis on pitavastatin metabolism, transport, and clearance.17 the study looked at age, body mass index, and sex - matched healthy adult subjects, using an us food and drug administration (fda)-approved cut - off of healthy estimated gfr of 80 ml / min/1.73 m to align the study with a previous pitavastatin analysis conducted in europe.17 elevations in auc and cmax were not found to be significantly different from their healthy comparators . They concluded that the relationship between renal function and pitavastatin exposure may not be evident, partly due to limited urinary excretion, though they admitted that there was a small sample size (n=16) and single dosing (4 mg) in determining that pitavastatin was safe and well tolerated in patients with severe renal impairment not on hemodialysis.17 pharmacokinetic studies have not demonstrated clinically significant differences based either on age (younger vs elderly age> 65 years) or sex (men vs women).7 significant increases in cmax have been seen in cirrhotic patients when compared with healthy subjects.18 similarly, the elimination half - life was 8.3 hours and 14.4 hours in patients with child - pugh a and child - pugh b classes, respectively . Information on the lipid - lowering efficacy of pitavastatin in elderly patients comes either from subgroup analysis of age - diverse studies19,20 or those performed specifically in older adults.2123 a phase 3, randomized, double - blind, parallel - group, active - controlled study of 355 patients by eriksson et al compared pitavastatin (4 mg / day) to simvastatin (40 mg / day) over a 12-week period.19 patients 65 years of age represented 23% of the population . While no significant difference in percent lowering of ldl - c from baseline was seen between pitavastatin (44%) and simvastatin (42%) in those <65 years, a greater reduction was shown with simvastatin (48%) vs pitavastatin (43%; p=0.024) in those 65 years of age although the overall reduction between age groups was similar.19 a recently published study by sponseller et al randomized 328 subjects with primary hyperlipidemia or mixed (combined) dyslipidemia to either pitavastatin 4 mg / day or pravastatin 40 mg / day for 12 weeks.20 the reduction in ldl - c from baseline in the overall population was 38.1% with pitavastatin and 26.4% with pravastatin (p<0.001). In subjects <65 years of age, the median percent change in ldl - c from baseline was 36.3% with pitavastatin and 25.5% with pravastatin while the reduction in those 65 years of age was 41.9% with pitavastatin and 27.6% for pravastatin (figure 1). The exact rationale for the greater response in the older population is unknown . Whether differences in baseline ldl - c concentrations could have resulted in varying reductions the magnitude of within - treatment effect for pravastatin was similar between those <or 65 years of age, the age effect on reduction in ldl - c was greater for pitavastatin vs simvastatin (pinteraction = 0.02).20 the efficacy of pitavastatin exclusively in a population 65 years of age was evaluated by stender et al in 942 patients with primary hypercholesterolemia or combined (mixed) dyslipidemia.21 participants, who had a mean age of 70 years (range 6589), were randomized to receive either pitavastatin (1, 2, or 4 mg / day) or pravastatin (10, 20, or 40 mg / day) for 12 weeks in a double - blind, double - dummy fashion . The primary efficacy outcome was the percent change in ldl - c from baseline with analyses performed to demonstrate the noninferiority of pitavastatin versus pravastatin . Additional lipoprotein parameters of interest included changes in tc, hdl - c, tg, and apo - b . The following dosage comparisons were made: pitavastatin 1 mg vs pravastatin 10 mg; pitavastatin 2 mg vs pravastatin 20 mg; and pitavastatin 4 mg vs pravastatin 40 mg . The mean baseline ldl - c across the various dosing groups ranged from 163 mg / dl to 167 mg / dl . The mean decrease in ldl - c from baseline decreased across all doses in both arms in a dose - related fashion (figure 2). The percent ldl - c reduction was approximately 10% greater with pitavastatin versus pravastatin, which met the criteria for noninferiority (p<0.001). Pitavastatin nearly uniformly improved secondary outcome parameters vs pravastatin, including tc, hdl - c (except pitavastatin 1 mg vs pravastatin 10 mg; p=0.425), tg (except pitavastatin 2 mg vs pravastatin 20 mg; p=0.063), apo - b, and non - hdl - c (p<0.05 for all, except where specified). The same author group concurrently published results of an open - label, 60-week extension study for those who had completed the 12-week core study.22 all patients, irrespective of their original randomization group, were started on open - label pitavastatin 2 mg / day, then up - titrated to 4 mg / day after 8 weeks if they did not attain their ldl - c target . The specific ldl - c target was not provided, but was based on the individual patients risk and followed the national cholesterol education program adult treatment panel iii guidelines . Following 60 weeks of pitavastatin treatment, the ldl - c was 43% lower than at baseline of the 12-week core study.21 all other secondary lipid parameters were also lower after 60 weeks compared with baseline . While 85.8% and 72.9% of pitavastatin and pravastatin (respectively) patients had attained their ldl - c goals after the 12-week core study,21 the value increased to 93.8% after 60 weeks of pitavastatin.22 comparative efficacy information for pitavastatin as well as other statins in patients <65 years and 65 years of age was included when the agent was submitted for approval to the fda.23 the percent ldl - c reductions in ldl - c for patients in the 65 versus <65 years groups are shown in figure 3.23 it should be noted that direct statistical comparisons between these groups were not conducted, nor inferred . Information on the non - lipid characteristics of pitavastatin in elderly patients is also available . The effect of pitavastatin on prevention of atrial fibrillation as well as cardiac structure and function was evaluated in 220 patients 65 years of age.24 individuals with hypertension and established left - ventricular (lv) hypertrophy and preserved systolic function (lv ejection fraction 50%) were included . Patients who required lipid lowering therapy (n=110) according to japanese guidelines were started on pitavastatin (12 mg / day) in addition to antihypertensive treatment, while the control group (n=110) continued on their baseline antihypertensive regimens . All baseline clinical, laboratory, and echocardiographic parameters were similar between the two groups, with the exception of ldl - c . Patients receiving pitavastatin had a significantly lower rate of developing new - onset atrial fibrillation (5/110, 4.5%) than the non - statin group (15/110, 13.6%; p=0.019). In a multivariate cox regression model, pitavastatin use (p=0.002) and an echocardiographic finding (left atrial peak strain; p=0.013) were independent predictors of reduced atrial fibrillation risk while prior coronary artery disease (p<0.001) was associated with elevated risk . Moreover, pitavastatin use resulted in a 6% reduction in lv mass index from baseline with no difference seen in the non - statin group (p<0.05). This study shows that pitavastatin not only reduces lipid parameters, but also improves echocardiographic parameters in patients with hypertension and lv hypertrophy and lowers their risk of developing new - onset atrial fibrillation . The manufacturer reports that of the 2,800 patients randomized to pitavastatin 1 mg to 4 mg in controlled clinical studies, 1,209 (43%) were 65 years of age . No significant differences in efficacy or safety were observed between elderly patients and younger patients when pitavastatin was approved by the fda.7 stender et al reported that 82% of pitavastatin patients in their 60-week open - label extension trial developed adverse events; however, only 14.3% of these were deemed to be specifically related to drug treatment.22 the most common treatment - related adverse events included myalgia (2.6%), elevated serum creatine kinase (1.9%), and nausea (1.5%). Two patients died during the study, although neither death was attributed to pitavastatin therapy . Stender et al reported 48 cases of myalgias; however, only 14 of these were considered treatment related, and none were severe (21 were moderate, 27 were mild).22 only four patients discontinued pitavastatin due to myalgia, and no cases of myopathy, myositis, or rhabdomyolysis were identified . Patients receiving pitavastatin 4 mg / day had a higher incidence of elevations in liver enzymes (ast and alt) by week 60.22 one patient had an ast elevation three times the upper limit of normal (uln), and one more than five times the uln . Two patients had alt increases more than three times the uln, one more than five, and one more than ten . Two patients were said to have discontinued the study due to increases in alt and ast, both of which were assessed as mild intensity and only one identified as treatment - related . Taken together, despite elevations in liver function tests (lfts) in a handful of patients, significant liver injury requiring treatment discontinuation was rare . Other studies have included elderly patients in their assessments of pitavastatin safety and tolerability; however, limited subgroup analysis data exist specific to those patients aged 65 years and older.2527 the significant majority of adverse events were reported as mild and less frequent than with other potent statins, and compare similarly to the data from stender et al.22 older patients who have been deemed frail may be at increased risk for adverse events.1 this includes individuals with a combination of weakness, slowness, exhaustion, inactivity, and shrinking.1 unfortunately, data on the safety of pitavastatin in frail older patients are lacking and requires additional study . Information on the impact of pitavastatin on humanistic outcomes, such as health - related quality of life, patient satisfaction, and treatment adherence is limited . No data on the impact of pitavastatin on health - related quality of life could be found . Patient satisfaction was evaluated in a spanish study of 6,489 patients from primary or specialized health clinics in spain who had been using pitavastatin for at least 12 weeks.28 the mean age of the population was 60.911.2 years, with no age - related subgroup data provided . Patients who completed their prescribed treatment course (65% of the included population) showed improved satisfaction with the drug and with the drug efficacy than those who did not finish the treatment (p<0.001 for both). Treatment compliance in elderly patients was reported in an extension study by stender et al.22 compliance, defined as taking between 80%120% of the prescribed treatment regimen, was achieved in 97.5% of the 60-month follow - up population . The overall body of evidence for the efficacy and safety of pitavastatin in elderly patients is small . The available data suggest that the ability of pitavastatin to lower ldl - c in elderly patients is at least similar, and may be greater than that seen in comparatively younger cohorts . A similar trend was seen with other statins, including atorvastatin, pravastatin, and simvastatin . Percent reductions ranging from 31%45% were seen with pitavastatin compared with 22%34% with pravastatin, 38%48% with simvastatin, and 40%46% with atorvastatin in patients 65 years of age.23 studies using rosuvastatin 10 mg / day in patients 65 years of age have shown 50% reductions in lcl - c from baseline, proportionally greater than pitavastatin.29 real - world studies of elderly patients have shown rosuvastatin to result in greater ldl - c reductions from baseline compared with other drugs in the class, including atorvastatin, pravastatin, and simvastatin.30 pitavastatin was not available at the time of this investigation; thus, the comparative efficacy of it with rosuvastatin is not currently known . Clinical trials have shown that statins significantly reduce all - cause mortality, nonfatal myocardial infarction, and stroke in elderly patients.4 the study assessing goals in the elderly (sage) of patients 6685 years of age showed that more aggressive lipid lowering with atorvastatin reduced cholesterol and mortality to a greater extent than moderate intensity therapy with pravastatin.31 unfortunately, data on the impact of pitavastatin on terminal outcomes such as mortality and myocardial infarction are not available . Pitavastatin has been shown to have similar pleiotropic effects to other statins, including decreasing platelet activation and improving cardiac and endothelial function.32 thus, it is plausible that its use may confer similar clinical benefits, but these have yet to be elucidated . It should, therefore, be selected based on its lipid lowering ability and safety profile alone . The safety of statins in elderly patients is a question frequently encountered by clinicians and patients alike . One of the biggest concerns, from a patient perspective, is the risk of statins adversely affecting cognition . A systematic review suggested that no causal link existed between statin use and development of alzheimer disease or dementia.33 similarly, no decline in cognitive performance was seen with statin use . A task force commissioned by the national lipid association concluded that no association with the statin class and adverse effects on cognition are known and routine screening should not be performed.34 while no information on the specific effects of pitavastatin on cognition in elderly patients is currently available, this adverse effect should not be of concern . Muscle issues have also been a concern with statins.35 meta - analyses of observational studies in general populations have reported greater than a two - fold increase in the odds of myopathy with statin use (odds ratio 2.63, 95% confidence interval 1.50 to 4.61).35 while statin - associated myalgia may be difficult to differentiate from that caused by other sources, tests can be run on individuals who have exhibited symptoms to provide a definitive diagnosis.36 stender et al reported that myalgia (2.6%) and increased serum creatine kinase (1.9%) did occur in elderly patients receiving pitavastatin.22 this is compared with an approximate 3% incidence with moderately - potent comparators.23 liver toxicity is another safety issue routinely encountered with statin use.35 few of the elderly patients given pitavastatin by stender et al exhibited signs or symptoms suggestive of liver damage.22 elevations in lfts are seen at a similar rate between pitavastatin and other agents in the class.23 while lfts are not recommended to be routinely screened following statin initiation, they can be checked in individuals with symptoms suggestive of liver damage.37 taken together, the limited available data suggest that pitavastatin is effective at improving lipid parameters in elderly patients with a similar safety profile to other agents in the class . Current guidelines recommend its use when moderate - intensity statin therapy is indicated.6 a drawback to its use is likely to be its cost . Since pitavastatin is still available only as a branded product in the united states, other agents with similar efficacy and safety and lower cost are likely to be used with greater frequency . Until data become available distinguishing pitavastatin from the other available options, its ultimate role in the hyperlipidemia treatment armamentarium remains unclear.
Advanced glycation end - products (ages) have been shown to accumulate in various tissues under diabetic conditions, and they participate in the development of vascular complications such as diabetic retinopathy [1, 2]. Our recent results showed that even a low concentration of ages, for example, 10 g / ml, can induce neuronal apoptosis in retinal neurons and decrease the number of regenerating neurites in cultures . Ages accomplish their effects by binding to specific cellular receptors [4, 5], such as receptors for ages (rages). These receptors have been found on neurons, mesangial cells, smooth muscle cells, and endothelial cells [68]. The binding of rage to ages precursors generates intracellular oxidative stress which then induces receptor - mediated production of reactive oxygen species . This then results in the activation of the free radical - sensitive transcription factor nuclear factor-b (nf-b) [9, 10]. The activated nf-b translocates into the nucleus and causes pathological changes in gene expression [9, 1113]. Specificity protein 1 (sp1) is a transcription factor that either activates or represses transcription in response to physiological and pathological stimuli . It regulates the expression of a large number of genes involved in a variety of processes such as cell growth, apoptosis, differentiation, and immune responses . Several genes can be regulated by a combination of nf-b and sp1, and in specific cases by direct interaction between the nf-b protein and sp1 protein . Tanaka et al . Found that ages can activate the rage gene through nf-b and sp1, causing enhanced age - rage interactions in human vascular endothelial cells . However, there are no reports demonstrating the relationship between nf-b / sp1 expression and regeneration in age exposed retinal neurons . Growing evidence indicates that neuronal abnormalities such as neuronal cell death and vascular abnormalities are associated with the development of early diabetic retinopathy . Because neuronal cell death is an irreversible change and can affect visual function of diabetic eyes, neuroprotective and regenerative therapies need to be determined . However, no reports have been simultaneously compared the neuroprotective and regenerative effects of several neurotrophic factors including neurotrophin-4 (nt-4), hepatocyte growth factor (hgf), glial cell line - derived neurotrophic factor (gdnf), and tauroursodeoxycholic acid (tudca) in age exposed retinas cultured in the same system . The purpose of this study was to examine the effect of high doses of ages on neuronal cell death and neurite regeneration in isolated rat retinas . We also determined the neuroprotective and regenerative effects of four neurotrophic factors, namely, nt-4, hgf, gdnf, and tudac in age exposed retinas . In addition, we also examined whether the expressions of nf-b and sp1 were correlated with the neuroprotective and regenerative effects of different neurotrophic factors in ages exposed rat retinas . Seven - week - old male sprague - dawley (sd) rats (japan slc co., hamamatsu, japan) were used . All of the procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research . The retinas were isolated under sterile conditions and cut into square pieces of 0.16 mm with sharp razor blades . Then the retinal explants were cultured on three - dimensional collagen gels as described in detail [1823]. The retinal explants were incubated in 6 different types of media; (1) serum - free control culture media, (2) 100 g / ml glucose - age - bsa (cyclex co., nagano, japan) or glycolaldehyde - age - bsa (cyclex co) or glyceraldehyde - age - bsa (cyclex co) media, (3) glucose - age or glycolaldehyde - age or glyceraldehyde - age + 100 ng / ml nt-4 (r&d systems, minneapolis, mn) media, (4) glucose - age, glycolaldehyde - age, or glyceraldehyde - age + 100 ng / ml hgf (r&d systems) media, (5) glucose - age, or glycolaldehyde - age, or glyceraldehyde - age + 100 ng / ml gdnf (r&d systems) media, and (6) glucose - age, or glycolaldehyde - age, or glyceraldehyde - age + 100 m tudca (wako, osaka, japan) media . The serum - free media contained 7.5 mm glucose, 5 g / ml insulin, 16.1 g / ml putrescine, 10% bovine serum albumin, 3.7 mg / ml nahco3, 5.2 mg / l na2seo3, and 3.6 mg / ml hepes in minimum essential medium as described [21, 23, 24]. One hundred g / ml bsa was added to the control medium (n + a) as a control for the concentration of age - bsa . To determine whether apoptosis had occurred, the retinal explants were fixed in 4% paraformaldehyde after 7 days in culture and sectioned on a cryostat . Then, tdt - dutp terminal nick - end labeling (tunel) staining was carried out with an apoptosis detection kit (chemicon international, temecula, ca) according to the manufacturer's instructions . Sections were costained with 4,6-diamidino-2-phenyl indole (dapi, polyscience inc ., warrington, pa). For quantitative analyses, the ratio of the number of tunel - positive cells to the total number of dapi - staining nuclei in the ganglion cell layer (gcl) was determined . A total of 18 sections from the 6 explants / group were studied, and the results were used for the statistical analyses . The total number of nuclei counted was 405 (n), 215 (n + a), 816 (ages), 472 (nt-4), 396 (hgf), 512 (gdnf), and 494 (tudca). The retinal explants were fixed as described and cryosections were cut . After blocking the sections in 5% goat serum and 3% bovine serum in 0.1 m phosphate buffer saline, they were incubated with antibodies against rabbit anti - phosphorylated nf-b (p - nf-b) and sp1 transcription factor (santa cruz biotechnology, santa cruz, ca) at 4c overnight . Then, the sections were incubated with fluorescein isothiocyanate - conjugated anti - rabbit igg for one hour . The number of p - nf-b- and sp1-positive cells in the gcl was counted . For quantitative analyses, the number of immunopositive cells in the gcl was expressed relative to the total number of dapi - stained nuclei . The total number of nuclei counted was 301 in n, 234 in ages, 198 in nt-4, 254 in hgf, 186 in gdnf, and 276 in tudca . The number of neurites regenerated from the explants was counted under a phase - contrast microscope after 7 days in culture when the number of regenerating neurites was very high [3, 18, 21, 23, 24]. Branched neurites were counted as one . The number of explants examined was 101 in the control group including that in serum - free media (n, n + a, n + nt-4, n + hgf, n + gdnf, n + tudca), 11 in the glucose - age - bsa group, 19 in the glycolaldehyde - age - bsa group, 18 in the glyceraldehyde - age - bsa group, 14 in the glucose - age - bsa + nt-4, 12 in the glycolaldehyde - age - bsa + nt-4, and 11 in the glyceraldehyde - age - bsa + nt-4 groups, 12 in the glucose - age - bsa + hgf, 14 in the glycolaldehyde - age - bsa + hgf, 13 in the glyceraldehyde - age - bsa + hgf groups, 12 in the glucose - age - bsa + gdnf, 13 in the glycolaldehyde - age - bsa + gdnf, 14 in the glyceraldehyde - age - bsa + gdnf groups, 10 in the glucose - age - bsa + tudca, 11 in the glycolaldehyde - age - bsa + tudca, and 10 in the glyceraldehyde - age - bsa + tudca groups . To determine whether ages were toxic to the retinas in culture, the number of tunel - positive cells in the gcl was counted . The majority of the tunel - positive cells were detected in the gcl because all of the retinal ganglion cells (rgcs) were axotomized to isolate the retina [3, 18, 21, 2426]. In retinas added 100 g / ml bsa more to the control medium (n + a), the number of tunel - positive cells was not significantly different from the control medium (n) (9.3 2.3% versus 9.7 6.8%, p = 0.244). In retinas cultured in glucose - age - bsa, glycolaldehyde - age - bsa, and glyceraldehyde - age - bsa, the number of tunel - positive cells in the gcl was significantly higher than that in the serum - free control medium (38.1 6.2% versus 9.7 6.8%, p <0.0001; 36.1 4.9% versus 9.7 6.8%, p <0.0001; and 39.9 9.4% versus 9.7 6.8%, p <0.0001, resp . ; figure 1). The addition of nt-4 decreased the number of tunel - positives cells more than in glucose - age - bsa without nt-4 (17.8 7.2% versus 38.1 6.2%; p = 0.0002), in glycolaldehyde - age - bsa without nt-4 (12.8 4.2% versus 36.1 4.0%; p = 0.0046), and in glyceraldehyde - age - bsa without nt-4 (13.4 3.6% versus 39.9 9.4%; p = 0.0078; figure 1). Addition of hgf in age - bsa did not decrease the number of tunel - positives cells compared to the number in glucose - age - bsa without hgf (34.0 5.5% versus 38.1 6.2%; p = 0.7861) and glyceraldehyde - age - bsa without hgf (29.1 11.0% versus 39.9 9.4%; p = 0.1722) but it significantly decreased in glycolaldehyde - age - bsa without hgf (25.4% 7.3% versus 36.1 4.0%; p = 0.0039; figure 1). Addition of gdnf did not decrease the number of tunel - positives cells compared to the number in glucose - age - bsa without gdnf (32.4 3.0% versus 38.1 6.2%; p = 0.0935) and in glycolaldehyde - age - bsa without gdnf (35.2% 5.1% versus 36.1 4.0%; p = 0.7373) and in glyceraldehyde - age - bsa without gdnf (31.2 9.78% versus 39.9 9.4%; p = 0.1293; figure 1). The addition of tudca decreased the number of tunel - positives cells more than that in glucose - age - bsa without tudca (28.2 6.5% versus 38.1 6.2%; p = 0.0184) and in glyceraldehyde - age - bsa without tudca (26.6 8.3% versus 39.9 9.4%; p = 0.0294) but it did not decrease in glycolaldehyde - age - bsa without tudca (31.6 3.3% versus 36.1 4.0%; p = 0.45; figure 1). The sections were immunostained for p - nf-b to determine whether nf-b was activated in retinas exposed to ages . The effects of the neurotrophic factors on the activation of nf-b were also examined . In retinas cultured with glucose - age - bsa, the number of p - nf-b immunopositive cells was higher than in serum - free control medium (53.2 7.2% versus 31.6 16.1%; p = 0.0146; figures 2 and 3). Addition of nt-4 decreased the number of immunopositive cells more than in the serum - free media (11.3 6.9% versus 31.6 16.1%; p = 0.0315) and more than in glucose - age - bsa without nt-4 (31.5 5.3% versus 53.2 7.2%; p = 0.0133; figures 2 and 3). Addition of hgf decreased the number of immunopositive cells more than in serum - free media (26.8 5.3% versus 31.6 16.1%; p = 0.049) and in glucose - age - bsa without hgf (42.6 2.7% versus 53.2 7.2%; p = 0.0114; figures 2 and 3). Addition of gdnf decreased the number of immunopositive cells more than in serum - free media (13.3 5.0% versus 31.6 16.1%; p = 0.0495) and in glucose - age - bsa without gdnf (25.8 5.3% versus 53.2 7.2%; p = 0.0011; figures 2 and 3). Addition of tudca decreased the number of immunopositive cells more than in serum - free media (18.6 3.97% versus 31.6 16.1%; p = 0.0024) and in glucose - age - bsa without tudca (36.2 7.2% versus 53.2 7.2%; p = 0.0034; figures 2 and 3). The sections were immunostained for sp1 transcription factor to determine whether it was expressed in retinas exposed to ages . We also examined the effect of neurotrophic factors on the expression of sp1 transcription factor . In retinas cultured with glucose - age - bsa, the number of sp1 immunopositive cells was higher than in serum - free control medium (32.2 6.8% versus 6.3 1.2%; p <0.0001; figures 4 and 5). Addition of nt-4 increased the number of immunopositive cells more than in serum - free media (13.0 4.3% versus 6.3 1.2%; p = 0.0315) but did not decrease the number of immunopositive cells in glucose - age - bsa without nt-4 (30.2 8.4% versus 32.2 6.8%; p = 0.6753; figures 4 and 5). Addition of hgf increased the number of immunopositive cells more than in serum - free media (19.7 5.16% versus 6.3 1.2%; p = 0.0001) but did not decrease the number of immunopositive cells in glucose - age - bsa without hgf (24.0 6.7% versus 32.2 6.8%; p = 0.0926; figures 4 and 5). Addition of gdnf increased the number of immunopositive cells more than in the serum - free media (12.3 1.4% versus 6.3 1.2%; p <0.0001) but it decreased compared to in glucose - age - bsa without gdnf (19.7 6.4% versus 32.2 6.8%; p = 0.0007; figures 4 and 5). Addition of tudca increased the number of immunopositive cells more than in serum - free media (10.2 3.9% versus 6.3 1.2%; p = 0.0454) but it decreased compared to in glucose - age - bsa without tudca (19.5 5.5% versus 32.2 6.8%; p = 0.0075; figures 4 and 5). In retinas added 100 g / ml bsa more to the control medium (n + a), the number of neurites was not significantly different from the control medium (n) (94.6 24.0/mm versus 97.5 34.9/mm, p = 0.948). In retinas incubated with ages (glucose - age, glycolaldehyde - age, and glyceraldehyde - age), the number of regenerating neurites was less than in retinas without age (45.0 27.5/mm versus 97.5 34.9/mm, p = 0.0046; 29.4 29.4/mm versus 97.5 34.9/mm, p = 0.0003; 25.0 15.6/mm versus 97.5 34.9/mm, p <0.0001; figures 6 and 7). All of the retinas incubated in the neurotrophic factors (nt-4, hgf, gdnf, and tudca) had an increase in the number of regenerating neurites in serum - free media (541.9 77.5/mm versus 97.5 34.9/mm, p <0.0001; 207.5 49.1/mm versus 97.5 34.9/mm, p <0.0001; 211.3 70.6/mm versus 97.5 34.9/mm, p <0.0001; 229.4 33.8/mm versus 97.5 34.9/mm, p <0.0001; figures 6 and 7). In addition, all of the neurotrophic factors increased the number of regenerated neurites in ages exposed retinas, but the most significant regenerative effect was found in the nt-4 group: 379.4 178.0/mm versus 45.0 27.5/mm, p <0.0001, in nt-4 group; 281.3 100.6/mm versus 45.0 27.5/mm, p <0.0001, in hgf group; 148.8 35.0/mm versus 45.0 27.5/mm, p <0.0001, in the gdnf group; 247.5 56.9/mm versus 45.0 27.5/mm, p <0.0001, in tudca group supplemented with glucose - age incubated retinas; 440.0 165.0/mm versus 29.4 29.4/mm, p <0.0001, in nt-4 group; 196.3 89.4/mm versus 29.4 29.4/mm, p <0.0001, in hgf group; 161.1 53.9/mm versus 29.4 29.4/mm, p <0.0001, in gdnf group; 238.1 33.1/mm versus 29.4 29.4/mm, p <0.0001, in tudca group supplemented to glycolaldehyde - age incubated retinas, 230.6 116.9/mm versus 25.0 15.6/mm, p <0.0001, in nt-4 group; 123.8 29.4/mm versus 25.0 15.6/mm, p <0.0001, in hgf group; 137.5 48.8/mm versus 25.0 15.6/mm, p <0.0001, in gdnf group; 178.8 39.4/mm versus 25.0 15.6/mm, p <0.0001, in tudca group supplemented to glyceraldehyde - age incubated retinas (figures 6 and 7). The importance of ages in the pathogenesis of diabetic complications has been shown in animal models . Two unrelated age inhibitors were found to partially block some functional and structural changes in retinas, neuronal tissues, and kidneys of diabetic animal models [2729]. Our previous study showed that even a low concentration of ages, for example, 10 g / ml, induced neuronal apoptosis in the gcl and decreased the number of regenerated neurites in culture . Higher doses (100 g / ml) of ages had similar effects as low concentrations and decreased the number of tunel - positive cells and significantly blocked neurite regeneration . The results of this study confirmed that the apoptosis in the gcl were caused by ages and also that neurite regeneration was significantly suppressed . All examined neurotrophic factors were able to increase the number of regenerative neurites; however the most significant regenerative effect was observed with nt-4 . In addition, all examined neurotrophic factors suppressed the expression of nf-b expression in ages exposed retinas . Nf-b is a primary transcription factor that plays an important role in regulating cellular responses, that is, a transcription factor that is present in cells in an inactive state and does not require new protein synthesis to become activated . Other members of this family include transcription factors such as c - jun, stats, and nuclear hormone receptors . Some aspects of the mechanism by which nf-b protects cells against toxins have been identified . For example, tumor necrosis factor- has been shown to protect hippocampal neurons against excitatory amino acid toxicity through nf-b activation by inducing bcl-2 and bcl - x expression . Sp1 transcription factor belongs to a group of factors that are associated with gc - rich promoters that are involved in basal promoter activity . Sp1 regulates the expression of different genes, including the vascular endothelial growth factor, fibrogenic cytokine, and many matrix genes . Several studies reported that the interactions between age and rage cause phenotypic changes in the microvascular endothelial cells and pericytes [3238]. Our results showed that ages increase the expression of the transcription factor nf-b and sp1 in retinal neurons . This suggests that ages enhanced the expression of the rages gene in retinal neurons through the increased expression of nf-b and sp1 . An upregulation of nf-b and sp1 proteins in retinal neurons suggests that different protective mechanisms are important for the protection of these cells against ages - mediated cell death . Tudca is a member of a group of compounds that modulate the endoplasmic reticulum (er) function, protecting the cells against er stress - induced apoptosis . Earlier studies showed that tudca had protective effects on damaged retinal neurons under diabetic stress as an anti - er reagent . The neuroprotective effect of tudca was correlated with the suppression of phosphorylated jnk and phosphorylated c - jun expression [18, 26]. It is also a potent mitogen and differentiation factor for endothelial cells, and it had neurotrophic and neuroprotective activity for central nervous system neurons [41, 42]. . Used an optic nerve axotomy model and demonstrated that hgf prevented rgc apoptosis in vivo in a concentration - dependent manner . Found that hgf promoted long - term survival and axonal regeneration of rgc after optic nerve injury . Recently, gdnf was found to be a growth and survival factor for neuronal cells, and it promoted the survival of peripheral sensory and sympathetic neurons and also motor neurons . Koeberle and ball studied the effects of gdnf on rgc survival and apoptosis after optic nerve transection . Nt-4 is a member of the nerve growth factor family which acts on different types of nerve cells, for example, sensory, cortical, and hippocampal neurons ., we investigated the neuroprotective and regenerative effects of nt-4 on retinal neurons under diabetic condition [3, 18, 21, 26]. Our results showed that nt-4 promoted the survival and the regeneration of neuronal cells in the retinas incubated in high glucose media . The neuroprotective and regenerative effects of nt-4 were correlated with the reduction in the activation of caspase-9 and -3, expression of perk and chop, and c - jun and jnk expression . The results of the present study showed that all examined neurotrophic factors decreased the number of nf-b immunopositive cells in glucose - age - bsa exposed retina, but nt-4 had the highest significant effect . However, the number of sp1 immunopositive cells was increased by the addition of neurotrophic factors in serum - free media and more significantly in the hgf and nt-4 groups . Thus after the addition of neurotrophic factors to glucose - age - bsa media, the level of sp1 transcription factor remained high in the nt-4 and hgf group . Kanda et al . Found that prostaglandin e2 promoted innervation in skin lesions by the induction of nt-4, and the induction was mediated by sp1 . Their results showed that the ep3, g - protein - coupled receptors, mediated by prostaglandin e2 transcription of nt-4 was dependent on the activity of sp1 . Thus, our findings suggest that sp1 may be related to neuronal survival and regeneration . It is possible that the increased expression of sp1 may result from an enhanced phosphorylation of sp1, because the sp1 promoter contains several sp1-binding elements and is positively regulated by its own gene product, sp1 protein . Further studies are needed to identify the connection between sp1 expression and nt-4 transcription . To find a possible decrease in the effect of the ages on the retina is important . Our findings that a suppression of nf-b expression in retinal neurons by several neurotrophic factors can result in neuroprotection, and reducing inflammation and oxidative stress suggests therapeutic potential of neuroprotective therapy in various ocular pathologies associated with ages accumulation . In conclusion, high - dose ages inhibit neurite regeneration which is correlated with increased expression of nf-b and sp1 . Nt-4 enhances neurite regeneration in ages exposed retinas more than other neurotrophic factors such as hgf, gdnf, and tudca . Our results indicate the therapeutic potentials of the neurotrophic factors as axoprotectants in ages exposed retinas.
The strong quantum confinement effect of colloidal semiconductor nanocrystals (ncs) has made these materials the subject of extensive research [1 - 5]. In the past decades, binary semiconductor ncs, identified as groups ii vi and iii v, have been studied intensively because of their size - dependent photoluminescence (pl) covering the spectrum range from ultraviolet to near infrared [6 - 10]. The importance of cds ncs is manifested in their potential application as fluorescent materials with uv blue pl emission [11 - 17]. The typical synthesis recipes for cds ncs are based on a process that involves the injection of cd - oleic acid (oa) into batch reactors . Nearly, monodispersed cds ncs have been prepared using octadecene (ode) as the non - coordinating solvent, and subsequent treatment of these ncs resulted in considerable improvement of pl efficiency . In these cases, the inefficient heat and mass transfer of batch reactors constrained the economical synthesis of cds ncs . In order to overcome the energy barrier to form active monomers, a high reaction temperature (250300c) is generally required . Meanwhile, the temperature and concentration gradients involved in large reactors lead to poor quality and low process reproducibility . Furthermore, according to the international commission on illumination chromaticity diagram, the ideal wavelength for blue light is 460480 nm . To achieve cds ncs with blue pl emission, large cds ncs with diameters greater than 5.2 nm are required . However, the preparation of these cds ncs is limited by the high reaction temperature and long reaction time, where the advent of ostwald ripening leads to wide size distributions . The enhanced heat and mass transfer properties in the microenvironment could provide highly homogeneous conditions for the chemical synthesis of ncs [18 - 24]. Compared with the batch reactor, a microreactor offers a controllable way to synthesize ncs continuously in a steady fashion [9,20 - 23]. Meanwhile, the incorporation of novel channel structures and flow patterns facilitated the synthesis of monodisperse ncs in an accelerated manner . However, the limited research conducted to date on the microfluidic synthesis of cds ncs has been based on aqueous processes, and the resulting ncs generally had wide size distributions and deep - trap luminescence that overwhelmed the visible range [19,25 - 27]. Droplet microfluidics has been demonstrated as a powerful tool for achieving efficient mixing, while realizing highly homogeneous environment for the synthesis of monodisperse cds ncs, but its low throughput limits its use to commercial - scale synthesis . In this study, a capillary microreactor was used for the accelerated synthesis of monodisperse cds ncs . In order to obtain high - quality samples, systematic investigations of temperature and residence time requirements also, a quantitative investigation of the influence of oa on size distribution and growth kinetics was conducted to achieve the size - controlled synthesis of cds ncs . The influence of oa on the reaction kinetics was evidenced by the collected absorption spectra . In addition, the high quality of the resulting cds ncs was confirmed by the pl spectra, powder x - ray diffraction (xrd), and high - resolution transmission electron microscopy (hrtem). Cadmium oxide (cdo, scr, 99.9%), sulfur (s, scr, 99.5%), 1-octadecene (ode, fisher, 90%), oleic acid (oa, scr, 90%), and analytic grade acetone and chloroform (scr) were used directly without further processing . Cds ncs were prepared using a recipe similar to the one previously reported by yu et al . . Typically, 1 mmol cdo, 4.5 mmol oa, and ode (5 ml in total) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear, yellow, cadmium - precursor solution . Meanwhile, a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode (5 ml in total) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system, a convective micromixer (50 l), and a length of polytetrafluoroethylene (ptfe) capillary (i d = 462 m, length = 50 cm), as shown in fig . Equal volume solutions of cd and s precursors were delivered by a syringe pump (harvard 22, usa) at the same flow rate, and the precursors were combined and mixed in a magnetic micromixer . Then, the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c . A thermally stable oil bath was used as a heat source, and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration, various amounts of oa were replaced by ode, while maintaining a constant volume of solution . Schematic representation of the capillary microfluidic reactor uv vis absorption spectra were recorded on a cary 50 uv vis spectrometer (varian, usa) at room temperature . Pl spectra were measured at room temperature with a cary eclipse spectrofluorometer (varian, usa) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm . Quantum yields (qy) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye (rhodamine 6 g). To obtain samples for tem and xrd characterization, the formed cds ncs were precipitated by adding acetone into their chloroform solution; the ncs were isolated and purified by repeated centrifugation and decantation . Xrd patterns were obtained using a d / max-2550 diffraction meter (rigaku, japan) with a cu anode . During the preparation of samples for xrd analysis, the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem (jeol, japan). The samples for hrtem observations were obtained by dipping a carbon copper grid in a dilute, chloroform - dispersed solution of ncs . Cadmium oxide (cdo, scr, 99.9%), sulfur (s, scr, 99.5%), 1-octadecene (ode, fisher, 90%), oleic acid (oa, scr, 90%), and analytic grade acetone and chloroform (scr) were used directly without further processing . Cds ncs were prepared using a recipe similar to the one previously reported by yu et al . . Typically, 1 mmol cdo, 4.5 mmol oa, and ode (5 ml in total) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear, yellow, cadmium - precursor solution . Meanwhile, a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode (5 ml in total) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system, a convective micromixer (50 l), and a length of polytetrafluoroethylene (ptfe) capillary (i d = 462 m, length = 50 cm), as shown in fig . Equal volume solutions of cd and s precursors were delivered by a syringe pump (harvard 22, usa) at the same flow rate, and the precursors were combined and mixed in a magnetic micromixer . Then, the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c . A thermally stable oil bath was used as a heat source, and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration, various amounts of oa were replaced by ode, while maintaining a constant volume of solution . Uv vis absorption spectra were recorded on a cary 50 uv vis spectrometer (varian, usa) at room temperature . Pl spectra were measured at room temperature with a cary eclipse spectrofluorometer (varian, usa) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm . Quantum yields (qy) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye (rhodamine 6 g). To obtain samples for tem and xrd characterization, the formed cds ncs were precipitated by adding acetone into their chloroform solution; the ncs were isolated and purified by repeated centrifugation and decantation . Xrd patterns were obtained using a d / max-2550 diffraction meter (rigaku, japan) with a cu anode . During the preparation of samples for xrd analysis, the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem (jeol, japan). The samples for hrtem observations were obtained by dipping a carbon copper grid in a dilute, chloroform - dispersed solution of ncs . In batch reactions, the long response time for temperature stabilization and the vibration at reaction conditions make it difficult to achieve precise control of the sizes of cds ncs . On the contrary furthermore, the enhanced heat and mass transfer in a microreactor greatly improves the concentrations of reactive monomers, resulting in reductions in the reaction temperature and residence time required to form high - quality ncs . In order to achieve a balance between process stability and the reactivity of precursors,, the reaction temperature is an important parameter that determines both the ligand and reaction kinetics . Low temperatures are insufficient for overcoming the energy barrier for highly reactive monomers, while high temperatures that exceed a threshold lead to the early advent of ostwald ripening . In this study, the reaction temperature was adjusted from 180 to 260c at a constant residence time of 68 s. figure 2 shows the absorption and pl spectra as well as the kinetics data of cds ncs prepared at various reaction temperatures . Generally, the sharp band - edge absorbance with a half width at half maximum (hwhm) value of 12 nm and several higher transitions are clearly resolved for cds ncs that were synthesized at temperatures above 200c . Whereas, due to the weak reactivity, the magic - size cluster was observed at 180c, indicated by the appearance of an absorption peak with a wavelength of almost 320 nm . In addition, pure band - gap emissions with very narrow full width at half maximum (fwhm~17 nm) were observed at 220c . The earlier mentioned results clearly point to a narrow size distribution and well - passivated surfaces of the cds ncs . When the temperature was increased from 180 to 240c, a 44-nm redshift in the pl peak was observed, indicating that the growth rate of the ncs improved at the higher temperatures . However, impurities in the technical grade ode resulted in the evolution of gas at high reaction temperatures above 240c . The presence of the gas decreased the residence time and induced the blueshift of the pl peak . A absorption, b pl spectra, and c relative kinetics of cds ncs prepared at various temperatures at a constant residence time of 68 s (oa: 15 . 6 vol%) fwhm of pl is an indirect measure for the size distribution of ncs . In fig . 2c, it can be seen that fwhm of the ncs tended to decrease when the temperature was changed from 180 to 220c . Further increases in the temperature led to wider fwhm values . During the diffusion - controlled synthesis of spherical ncs, the burst of nucleation that separated from the growth is the prerequisite for achieving monodisperse products . Temperatures below 180c cannot overcome the energy barrier to form the reactive monomer species, which restricted the burst of nucleation . The nucleation in the whole reaction phase led to poor size distribution . With the increase in reaction temperature from 180 to 220c, the continuously increased reactivity of the monomer allowed the burst of nucleation, leading to gradual narrowing of the size distribution . The narrowest fwhm was observed at 220c, indicating a balance of nucleation and growth at this temperature, which is a threshold after which the widened fwhm was induced by further increases in temperature . The underlying kinetics for the growth of ncs played an important role in the observed fwhm in fig . 2c . For the diffusion - controlled growth of spherical ncs, the size(r)-dependent growth rate (dr / dt) of ncs has a maximum at a critical radius rcr . When r> rcr, the smaller ncs grow faster than the larger ones, resulting in a narrower size distribution . When r <rcr, the smaller ncs grow slower than the larger ones or even dissolve in the solution (ostwald ripening), which led to a broad size distribution of the final products . In our experiment, temperatures above 220c resulted in the rapid depletion of monomers, and the low concentration of monomers led to a larger critical size . As a result, large ncs grew from the monomers formed by the dissolution of small ncs, and broad fwhm was observed with the increase in temperature . The microfluidic reaction facilitated the process of optimization using a minimal amount of precursors . In this study, the optimization process for the reaction temperature was achieved within an hour, while consuming less than 1 ml of the precursors . Furthermore, the enhanced heat and mass transfer in the microreactor resulted in a reduction in the temperature required to achieve the synthesis of high - quality ncs . In our work, monodisperse cds ncs were synthesized at a fairly low temperature of 220c, which is among the lowest values reported based on other studies using similar recipes . The evaluation of residence time was achieved by changing the flow rate from 17.8 to 2.22 ml h. figure 3 compares the absorption and pl spectra, as well as relative kinetic data, of the samples prepared at residence times ranging from 17 to 136 s at a constant reaction temperature of 220c . The sharp band - edge absorbance and three higher transitions, as well as band - gap emissions, can be clearly observed, displaying the high quality of the as - formed ncs . As shown in fig . 3b, the increased residence time induced a 30-nm redshift of the pl peak, elucidating the growth of ncs . Concerning the fwhm of pl, the optimal residence time was observed to be 68 s when a narrow fwhm of only 17 nm was observed . With the increase in residence time from 17 to 68 s, the fwhm of pl continuously decreased from 20 to 17 nm, indicating the focusing of the size distribution in this phase, whereas increasing the residence time beyond 68 s resulted in increased fwhm values . In the early reaction stage, the high monomer concentration led to a small rcr, which is smaller than the normal size of the ncs presented in the solution . In this phase, small ncs exhibited a more rapid growth rate than large ncs, and the size distribution of the ncs continuously focused . Meanwhile, the velocity of the cds ncs had a parabolic profile inside the capillary, while induced residence time distribution (rtd) for cds ncs . Numerical simulation and experimental results demonstrated that the rtd effect becomes trivial at low flow rates (corresponding to long residence time in this study). As a result, the prolonged growth time resulted in low monomer concentration, which led to large rcr, and the size of some cds ncs fell below rcr while the advent of ostwald ripening led to increased fwhm of pl for cds ncs . A absorption, b pl spectra, and c relative kinetics of cds ncs synthesized at different residence times (with a constant reaction temperature of 220c, oa: 15 . 6 vol%) in this paper, oa was utilized as the ligand for the formation of cationic precursors and passivation of the ncs, while ode was chosen as the non - coordinating solvent . Size and size distribution can be simply tuned by varying the concentration of ligands in the solution . The temporal evolutions of uv vis spectra at various residence times were recorded at different volume percentages of oa, as shown in fig . The discrete reactions were performed under identical conditions, except for the concentration of oa in the reaction mixture . During the variation of volume ratio for oa over a wide range (10.551.2 vol%) and the variation of residence time from 17 to 136 s, monodisperse cds ncs were obtained in different size ranges, which were clearly shown by the observation of a sharp absorption peak (hwhm~12 nm) and up to three excitonic transition states . With the increase in residence time and the variety of ligand concentrations, a 64-nm shift of absorption peaks (from 389 to 453 nm) was obtained . Absorption spectra of the samples monitored during the evolution of residence time and oa volume (oa only in cd side, t = 220c) the wavelength of the first excitonic absorption peak and the corresponding absorption intensity, as well as hwhm, was utilized to calculate the mean particle diameter and particle number of the obtained cds ncs, using the empirical fitting function reported by yu . In fig . 5, the data obtained appear to show a significant influence of oa on the mean size and size distribution of cds ncs . At the same residence time, a high volume percentage of oa resulted in large particle sizes and low particle concentration . Taking the residence time of 68 s as an example, tuning the volume percentage of oa from 10.5 to 51.2% led to a significant increase in the mean diameter of the ncs from 3.8 to 5.3 nm, accompanied by dramatic decrease in particle concentration from 2.20 10/l to 0.20 10/l . Since oa restrains the formation of nuclei during the nucleation process, the low particle concentration was observed at high oa concentrations . Furthermore, the analysis conducted by peng et al . Indicated that the depletion rate of monomers was basically constant after the primal stage of the reactions, even though different initial oa concentrations were used . As a result, for cds ncs that were prepared by utilizing high oa concentrations, less nuclei were formed while the depletion rate of the cd - oa monomers kept the same, compared with low oa concentrations . The combination of the above two factors led to a rapid growth rate of ncs, resulting in large particle sizes and low particle concentrations . At the same time, the rapid growth negated the annealing process and led to higher concentrations of surface defects, which led to weak pl intensity . A mean diameter and b particle concentration of the cds samples prepared during the evolution of residence time and volume percentage of oa (oa only in cd side, t = 220c) in batch reactions, cds ncs prepared using high oa concentrations generally demonstrated wide size distribution however, monodisperse (hwhm~11 nm) cds ncs can be obtained with a high volume ratio of oa of 51.2%, and the size of the cds ncs were in the range of 4.55.3 nm in the microreaction . The efficient mixing and heat transfer, as well as uniform reaction conditions, achieved by the microreactor contributed to the formation of the high - quality products . In addition, the narrow diffusion length along the radius of the microchannels created homogeneous reaction conditions for the uniform growth of cds ncs, even at rapid growth rate that was induced by high oa concentrations . The hwhm and pl spectra of the cds ncs prepared using various volume percentages of oa are shown in fig ., the absorbance of the samples at the excitation wavelength was adjusted to be the same . Figure 6a shows that a narrower hwhm of approximately 13 nm can be obtained for different oa content, indicating the narrow size distribution of the resulting ncs . However, with the increase in the volume ratio of oa, a significant improvement of pl intensity was observed and reached the highest value when applying volume ratio of oa as 15.6 vol% (fig . 6b), while further increases in the volume ratio resulted in decreased pl intensity . For cds ncs prepared using a volume ratio of 51.2%, the deep - trap emission made the samples demonstrate red color under an uv lamp . The pl efficiency of the ncs was mainly governed by the surface state of the ncs . The slow growth rate induced by the low oa concentration and the optimal ligand capping at 15.6 vol% resulted in the best pl efficiency, while the improved growth rate that accompanied the increase in oa concentration resulted in increased defects on the surface of ncs, which justified the low pl intensity under high oa concentration above 35 vol% . The qy could be improved by the surface capping with zns shell, and this research is under active investigation in our group . A hwhm of uv vis and b pl intensity of cds samples prepared at various volume percentage of oa (t = 220c, t = 68 s) just by varying the volume ratio of oa and residence time, various sizes of high - quality cds ncs ranging from 3.0 to 5.4 nm were obtained at the fairly low reaction temperature of 220c (fig . The band - gap emission was observed when the particle size was smaller than 5 nm . Therefore, due to the high quality of the as - prepared cds ncs, zns capped cds core shell structure ncs with excellent blue luminescence efficiency, color purity, and a narrow size distribution can easily be obtained . Uv vis absorption and pl spectra of the as - prepared cds ncs with various mean sizes . (a absorption intensity, ipl = pl intensity) figure 8 shows tem and hrtem images of cds ncs, which clearly display the narrow size distribution and fairly spherical morphology of the cds ncs with an average diameter of 5.2 nm . Statistical calculations for the 50 dots in a selected regime figure 9 shows the xrd spectra of 5.2-nm cds ncs, which exhibited a typical pattern of the zinc - blende structure with three distinct features . The first diffraction peak at 2 = 27 corresponded to the (111) reflection, and the other two features, which appeared at 2 = 44 and 52, corresponded to the (220) and (311) reflections, respectively . A tem and b hrtem images of cds ncs (band - gap absorption of 449 nm with an hwhm value of 11 nm) synthesized in open air via microreaction xrd pattern of 5.2-nm cds ncs synthesized at 68 s (oa: 51.2 vol%) in conclusion, a capillary microreactor was used for the continuous synthesis of cds ncs, and the influences of reaction temperature and residence time were investigated systematically to obtain the optimal synthesis parameters . The enhanced mass and heat transfer involved in microchannels facilitated the rapid synthesis of high - quality cds ncs at the low reaction temperature of 220c . The combined effect of rtd and ostwald ripening resulted in the optimal residence time as 68 s. the continuous operation and low sample consumption involved in microreaction allowed an accelerated study of the kinetics involved . Variations of oa concentration and residence time were demonstrated as an effective way to control the size of cds ncs produced . With an increase in residence time from 17 to 136 s and an increase in the volume percentage of oa from 10.5 to 51.2%, cds ncs in the size range from 3.0 to 5.4 nm were obtained, with corresponding pl peak wavelength from 391 to 463 nm . With the careful selection of reaction parameters, a moderate qy as 14.5% was obtained . Furthermore, excellent size distributions were obtained over a wide range of oa concentrations, and narrow hwhm absorption peaks (from 11.6 to 14.0 nm) were maintained during the entire process . Authors appreciated the financial supports from the nsfc (50772036), the focus of scientific and technological research projects (109063) and the state key laboratory of chemical engineering at ecust (skl - che-08c09).
A 70 year - old male presented to the emergency department of our institution after being involved in a motor vehicle accident . He had sustained a minor injury of the head (glasgow coma scale 15/15), the chest (seat belt burn) and lower extremities (bruises). His past medical history was significant for diabetes mellitus controlled with an oral hypoglycemic (metformin) for 6 years with satisfying control of blood glucose levels . The patient's medical history did not reveal any previous high - energy damage of the right ankle and clinical examination of other joints was negative for hypermobility ., there was moderate edema of the right ankle region and obvious anterior - lateral dislocation of the talus with the forefoot in line . The right foot had normal skin temperature, pedal pulses were palpable and there was slight hypoesthesia of the dorsal aspect in comparison to the contralateral foot . The initial plain radiographs (anteroposterior and lateral views of the ankle joint) confirmed the diagnosis of talar dislocation, but also the absence of any fracture (fig . 1). Owing that there was no fracture and major neurovascular trauma, an attempt of closed reduction was performed at the emergency department at approximately 1 hours after the injury . Radiographic ankle views at the initial presentation showing the anterior - lateral dislocation of the ankle (a, b). The closed reduction was achieved by axial traction of the calcaneus with one hand while the other hand was placed at the dorsal aspect of the midfoot with the foot held in slight plantarflexion for a couple of minutes, followed by lateral compression and external rotation of the talus and, finally, dorsiflexion of the foot in a way to redirect the articular line of the ankle joint . The attempt was successful and confirmed with radiographic imaging after the closed reduction (fig . 2). Maintenance of reduction was accomplished with a posterior ankle splint while the patient remained at the hospital for observation . General measures for pain and edema of the closed reduction included elevation of the right lower extremity, ice therapy, oral analgesic and non - weight bearing status to the right foot . Ten days later, the edema had subsided significantly, the splint was changed to a cast and partial weight - bearing was allowed for 5 weeks followed by full weight bearing for the next 3 weeks . After 8 weeks, the lower extremity cast was removed and the patient was followed with standard and stress radiographic views . At that time, there were no signs of instability and the range of motion of the right ankle was satisfactory and painless . The patient was then allowed to full weight - bearing status without the use of additional cast applications . One year after the initial injury, the radiological findings were negative for any signs of osteonecrosis or early signs of ankle arthritis (fig . 3). The right ankle was pain - free with similar range of plantarflexion and dorsiflexion as compared to the left ankle [plantarflexion: 044 (right), 046 (left)/dorsiflexion: 015 (right), 016 (left)]. The first case of tibiotalar dislocation without an accompanying fracture of the ankle was described by peraire in 1913 (4). Wilson et al . Reported the first large series of 14 cases collected from the literature and added two cases of their own (4, 12). Although multiple cases of ankle dislocations have been reported in the literature, there is rare mentioning of an anterior - lateral dislocation of the ankle, especially in diabetic patients . Closed ankle dislocation without an associated fracture or disruption of the tibiofibular syndesmosis is rare and only a small series have been reported in the literature (1, 2, 4, 7, 11, 15, 16). The ligaments are stronger than the malleoli, so that ankle dislocation is usually accompanied by fracture (4, 9). Cases of open dislocation of the ankle without fracture have been also reported and are common (1, 4, 6). Ankle dislocations are caused primarily by motor vehicle accidents, with the second most common cause being sports trauma and these usually occur in young athletes (17). Pertinent information about the mechanism of trauma injury is paramount in order to make an accurate and timely diagnosis (4, 18). Tibiotalar dislocation is classified by fahey and murphy into five types, based on the direction of the dislocation (19). The ankle dislocation can be anterior, posterior, medial, lateral, upward or a combination of these (1). The wider anterior talus wedging results in forced widening of the joint and could be accompanied by either disruption of the tibiofibular syndesmosis or fracture of the lateral malleolus . Anterior dislocation occurs when a posterior force is applied to the tibia with forced dorsiflexion . Lateral and medial dislocations occur from forced eversion, inversion or rotation of the ankle (4, 12, 13). According to a study on cadavers (20), the author was able to manually dislocate the ankle medially or laterally without associated tibia or fibular fracture by placing an inversion or eversion stress on a maximally plantarflexed foot . The supporting structures that failed were the anterolateral part of the joint capsule followed by the anterior talofibular and calcaneofibular ligaments . The author assumed that once the ankle had been dislocated, the talus and the foot were pulled posteriorly by the achilles tendon . The talus has a rhomboidal shape when viewed from its superior aspect and its posterior portion is narrower than the anterior portion . Plantarflexion places the narrowest portion of talus in the ankle mortise which is the most unstable position . An inversion force causes ligamentous and capsular failure which causes the potential for ankle dislocation with or without fracture . The published literature supports the non - operative treatment of closed ankle dislocation without fracture . Closed injuries have been treated conservatively with prompt reduction and cast immobilization and the results were reported to be good to excellent (1, 4, 5, 7, 8, 15, 16, 21). It is absolutely necessary to check the neurovascular status of the foot before and after manipulation . After achieving a satisfactory reduction, the integrity of the ankle ligaments, especially the deltoid ligament, consideration must also be given to other anatomical anomalies, especially hypoplasia of the medial malleolus, which must be addressed as part of the final treatment plan . Complications, such as the loss of ankle range of motion, stiffness or residual instability, and early arthritis, were reported following closed treatment of ankle dislocations (7). In lateral ankle dislocations, as presented in this case report, rupture of the anterior and posterior talofibular ligaments, as well as rupture of the calcaneofibular ligament have been reported as probable causes (8, 22). The association of the lateral with the anterior type of ankle dislocation could be the result of a posterior force applied to the tibia with the ankle in dorsiflexion and rupture of the talofibular and calcaneofibular ligaments . The anatomical characteristics of the talus and the positioning of the ankle mortise during dorsiflexion make this injury rare . Early diagnosis and treatment of this rare type of anterior - lateral ankle dislocation is paramount in the overall patient's successful outcome . Our rare case was reported in a diabetic patient that was treated conservatively with closed reduction and cast immobilization . Continuous post - operative care is essential in this type of population with known diabetes related complications . The authors have not received any funding or benefits from industry to conduct this study.
Bacteria of the genus actinomyces are gram - positive bacilli which generally colonize mouth, colon and vagina ., if actinomycosis occurs, it often presents as an indolent, slowly progressive infection characterized by abscess formation, which can progress across tissue boundaries, can present with mass like features which simulate malignancy, can develop into a sinus tract and/or develop clinically into a refractory or relapsing infection after a short course of therapy . A 43-year old woman with no significant medical history presented with complaints of an unilateral slowly growing painful mass in the left breast . Mammography (fig . 1) and ultrasonography were conducted and showed a small abscess - like density retroareolarly.fig . . 1 mammography of the left breast showing a confined density in the retroareolar area . Species identification was performed using matrix - assisted laser desorption ionization - time - of - flight mass spectrometry (maldi - tof ms; bruker). Susceptibility testing for a. neuii was performed using e - tests (biomerieux). At the time that culture findings became available, therefore, the cultured actinomyces was considered as skin commensals and a wait and see policy was installed . After several months, the patient returned to the outward clinic with complaints of pain in the breast . Psychical examination of the breast showed mild erythema and ultrasonography showed a minimal increase of the lesion . The abscess was surgically drained and new purulent fluid from the abscess was sent to the laboratory . The material was processed as described above . In contrast to initial culture findings, determination by the maldi - tof ms showed peptostreptococcus magnus (score value 2.056). After four days there was also growth observed in the brewer thioglycollate medium . The isolate was shown to be susceptible to penicillin, amoxicillin, clindamycin, meropenem and vancomycin . Histopathological examination of the obtained tissue showed abscess formation in an infection with a sinus tract . Subsequently, the patient was treated for six weeks of intravenous penicillin followed by six months of oral amoxicillin successfully . Secondly, a 73-year old woman with a history of bilateral breast cancer was seen at with severe pain and swelling of the left breast ten days after her ablation which was complicated by a large hamatoma treated conservatively . Gram strain showed many leukocytes, some gram - positive cocci and many gram - positive rods . Determination using maldi - tof ms identified a staphylococcus aureus after 2 days there were colonies visible on the anaerobically incubated schaedler agar . Determination by the maldi - tof ms revealed an a. neuii susceptibility testing was performed using e - tests . Unfortunately, patient returned four days after discharge with a relapse of the hematoma which was surgically evacuated . A month later at the outward clinic she showed full recovery.fig . Other isolates that have been identified as pathogens causing primary breast actinomycosis include actinomyces viscosus, actinomyces turincensis and actinomyces radingae . It differs from the other actinomyces species because of its aerobic growth and its microscopic morphology without branching . We were able to retrieve only three former cases of a. neuii causing primary actinomycosis . In the first case, treatment with antibacterials was unsuccessful and the lesion had to be surgically removed . In the second case, patient was successfully treated with amoxicillin for 21 days . In the third case, actinomycosis, regardless of species, should be treated with high doses of antibacterials for a prolonged period of time although therapy needs to be individualized . Current guidelines recommend high dose of penicillin intravenously for two to six weeks, followed by oral therapy with penicillin or amoxicillin for 6 to 12 months . Alternative antibacterial treatments include doxycycline, erythromycin or clindamycin . If actinomycosis presents with well - defined abscess then surgical drainage followed by long - term antibacterial therapy is indicated . We describe this combination of treatment in our cases . In the first case initial cultures showed islolated a. neuii . Because signs of infection had decreased this finding was interpreted as skin flora and not as a pathogen . Therefore medical treatment was considered unnecessary . In retrospect, if antibacterial therapy had been given at first presentation recurrence of the infection might have been prevented . Actinomyces species morphologically resemble diphtheroids and could be dismissed as skin commensals, even when isolated from an abscess sample . In both of our presented cases we found different organisms in the culture additional to a. neuii . In the first case we found p. magnus and in the second case s. aureus was present . Although a. neuii infection is known for its chronicity, the influence of these mixed organisms on the severity of infections remains unclear . Further research on the effect of mixed organism on the severity of an a. neuii infection is required.
Acute megakaryoblastic leukemia (amkl) with t(1;22)(p13;q13) represents <1% of all acute myeloid leukemias (aml), and is associated with the rbm15 (ott)-mkl1 (mal) fusion gene . It occurs at higher incidence in the pediatric population and accounts for approximately 70% of infants with amkl 1,2 . In contrast to infants with 11q23-associated leukemia who frequently exhibit hyperleukocytosis, those with rbm15-mkl1 fusion - positive amkl usually present with decreased leukocyte count, severe anemia, and thrombocytopenia . The pathological findings show fibrosis along with the leukemic cells in the bone marrow and lymph node 3 . Amkl with t(1;22)(p13;q13) is a rare disease that is often associated with massive organomegaly . Neuron - specific enolase (nse), the -subunit of enolase, is found not only in neuroendocrine cells but also in other cells including erythrocytes and subsets of lymphocytes 4,5 . Increased levels of serum nse, we report on a neonate who presented with massive hepatomegaly and increased nse levels and was misdiagnosed with congenital neuroblastoma . The patient was the second daughter of healthy and non - consanguineous japanese parents, born at 38 weeks of gestation following an uncomplicated pregnancy, and her body weight was 3164 g. at birth, she was found to have abdominal distension . Peripheral blood analysis demonstrated thrombocytopenia with a platelet count of 32 10/l . Laboratory investigations revealed the following: total bilirubin of 8.8 mg / dl, direct bilirubin of 0.7 mg / dl, lactate dehydrogenase of 2942 iu / l, aspartate aminotransferase of 248 iu / l, alanine aminotransferase of 66 iu / l, -glutamyl transpeptidase of 222 iu / l, blood urea nitrogen of 4 mg / dl and creatinine of 0.3 mg / dl . Serum nse level was extremely high (1050 ng / ml; normal value <25 ng / ml) and -fetoprotein was 59,540 ng / ml (within the normal range of age - matched controls). Immunophenotyping and karyotyping of the bone marrow sample could not be examined because of the very small size of the sample . Arrows indicate blast cells with large nuclei and basophilic cytoplasm (may giemsa staining, original magnification 1000). An initial diagnosis was made of neuroblastoma stage 4s with an unknown primary lesion, although urine vma and hva levels were normal . However, she died of pulmonary hemorrhage 1 day after therapy . Abdominal mri imaging . Reverse transcriptase - polymerase chain reaction (rt - pcr) assay for the detection of rbm15-mkl1 fusion transcripts was performed in a not bone marrow, but liver sample with forward primer rbm15-s (5-ttcctcagctgcatcagaca-3) located in rbm15 exon 1 (genbank accession no . Nm_022768.4) and reverse primers mkl1-as (5-tcattgaggtcatcggctag-3) and mkl1-as2 (5-tcattgaggtcatcggctag-3) located in mkl1 exon 7 (genbank accession no . Pcr amplifications were performed using the qiagen one - step rt - pcr kit (qiagen gmbh, hilden, germany), in accordance with a previously published protocol 7 . Sequence analysis was directly performed on the amplified rt - pcr product by using bigdye terminator cycle sequencing chemistry (applied biosystems, foster city, ca) on an automated sequencer abi prism 310 genetic analyser (applied biosystems), in accordance with the manufacturer's instructions . The patient was the second daughter of healthy and non - consanguineous japanese parents, born at 38 weeks of gestation following an uncomplicated pregnancy, and her body weight was 3164 g. at birth, she was found to have abdominal distension . Peripheral blood analysis demonstrated thrombocytopenia with a platelet count of 32 10/l . Laboratory investigations revealed the following: total bilirubin of 8.8 mg / dl, direct bilirubin of 0.7 mg / dl, lactate dehydrogenase of 2942 iu / l, aspartate aminotransferase of 248 iu / l, alanine aminotransferase of 66 iu / l, -glutamyl transpeptidase of 222 iu / l, blood urea nitrogen of 4 mg / dl and creatinine of 0.3 mg / dl . Serum nse level was extremely high (1050 ng / ml; normal value <25 ng / ml) and -fetoprotein was 59,540 ng / ml (within the normal range of age - matched controls). Immunophenotyping and karyotyping of the bone marrow sample could not be examined because of the very small size of the sample . Arrows indicate blast cells with large nuclei and basophilic cytoplasm (may giemsa staining, original magnification 1000). An initial diagnosis was made of neuroblastoma stage 4s with an unknown primary lesion, although urine vma and hva levels were normal . However, she died of pulmonary hemorrhage 1 day after therapy . Abdominal mri imaging . Reverse transcriptase - polymerase chain reaction (rt - pcr) assay for the detection of rbm15-mkl1 fusion transcripts was performed in a not bone marrow, but liver sample with forward primer rbm15-s (5-ttcctcagctgcatcagaca-3) located in rbm15 exon 1 (genbank accession no . Nm_022768.4) and reverse primers mkl1-as (5-tcattgaggtcatcggctag-3) and mkl1-as2 (5-tcattgaggtcatcggctag-3) located in mkl1 exon 7 (genbank accession no . Pcr amplifications were performed using the qiagen one - step rt - pcr kit (qiagen gmbh, hilden, germany), in accordance with a previously published protocol 7 . Sequence analysis was directly performed on the amplified rt - pcr product by using bigdye terminator cycle sequencing chemistry (applied biosystems, foster city, ca) on an automated sequencer abi prism 310 genetic analyser (applied biosystems), in accordance with the manufacturer's instructions ., there were multiple nodules composed of diffuse proliferation of small round cells in the liver (fig.3a). Immunohistochemically, the proliferating cells were positive for cd41, cd42b, cd43, and nse, but negative for myeloperoxidase, tdt, cd7, cd68, cd163, pgp9.5, and tyrosine hydroxylase (fig.3b). Fluorescence in situ hybridization revealed chromosome 21 disomy, indicating that the patient did not have down syndrome . Atypical cells are observed in the liver (hematoxylin and eosin staining left panel: original magnification 200; right panel: original magnification 1000). The lesion is positive for nse (left panel) and cd41 (right panel) (original magnification 200). Lanes 1, 2 and 4 indicate a 100-bp molecular marker, an internal control, and a negative control, respectively . (b) sequence analysis of the amplified rt - pcr product revealed an in - frame fusion between rbm15 exon 1 and mkl1 exon 5 . Leukemia occurs much less frequently in the perinatal period than in later childhood . Although not as common as neuroblastoma, leukemia is the leading cause of death due to neoplastic disease in neonates 8 . Many cases of congenital neoplasms do not present with overwhelming signs and symptoms, and rapid and accurate diagnosis may be difficult . Amkl is a rare variant of aml, and the leukemic blasts show characteristic morphologic and phenotypic features, indicating megakaryocytoid differentiation . A distinct entity characterized by t(1; 22)(p13;q13), resulting in the rbm15-mkl1 fusion oncogene, has recently been identified . Of the recognized subtypes of amkl, the disease associated with t(1;22) has clinicopathologic features that present in neonates and infants and is frequently associated with abdominal organomegaly 3,9 . The liver was involved as the primary site in some patients with congenital leukemia 9 . Neonates with down syndrome sometimes present transient myeloproliferative disorder mimicking amkl 10; however, this patient was not diagnosed with down syndrome . At the initial evaluation,, the elevation of serum nse level and a few and absent blastic cells in the bone marrow and the peripheral blood might have led to a clinical misdiagnosis . Nse is usually synthesized by neurons and neuroendocrine cells, and it is a useful marker for the diagnosis and monitoring of patients with neuroendocrine tumors such as neuroblastoma and small cell lung cancer 11 . However, nse is found not only in neuroendocrine cells but also in other cells including erythrocytes and subsets of lymphocytes 4,5 . Some of the hematopoietic cell lines including t - cell leukemia and epstein barr virus - immortalized b lymphoblastoid cell lines produce nse . Thus, nse is not exclusively expressed in neuroendocrine tumor cells 12 . In the present case, the fibrosis might have been caused by the production and secretion of transforming growth factor beta from the megakaryoblasts, which stimulates collagen synthesis in bone marrow fibroblasts 13 . There are very few documented cases of congenital leukemia in which the primary site of involvement was shown to be the liver, without initial bone marrow findings . A case of congenital anerythremic erythroleukemia was reported to present with hepatic failure and have a fetal outcome of multivisceral involvement 14 . An infant with amkl and a complex translocation demonstrated only 520% blasts on the initial bone marrow aspirate and core biopsy . Therefore, myelodysplastic syndrome was initially diagnosed; however, only later did blasts become apparent in the peripheral blood 15 . In the present case, amkl might have occurred in the fetal period because hematopoietic cells are mainly produced in the fetal liver . Here, we report on a neonate who presented with massive hepatomegaly, increased serum nse levels, and was misdiagnosed with congenital neuroblastoma.
From may through october 2008, we trapped small mammals at 5 sites in southern sweden (figure 1). Our primary objective was to obtain samples from bank voles, myodes glareolus, but other species were also sampled when captured . A nested pcr was performed with primers for anaplasmataceae specific for the 16s rrna gene (10). Prevalence of infection: hglinge, n = 45 infections, 0% candidatus n. mikurensis, 44.4% bartonella spp . ; revinge, n = 623 infections, 9.3% candidatus n. mikurensis, 33.7% bartonella spp . ; istaby, n = 53 infections, 3.8% candidatus n. mikurensis, 34% bartonella spp . ; hemmestr, n = 64 infections, 4.7% candidatus n. mikurensis, 39.1% bartonella spp . ; herseby, n = 49 infections, 12.5% candidatus n. mikurensis, 45.0% bartonella spp . All amplified fragments were sequenced, and a blast (http://blast.ncbi.nlm.nih.gov/blast.cgi) search showed that 68 animals were infected by candidatus n. mikurensis . The primers were chosen to be specific for bacteria belonging to the families rickettsiaceae and anaplasmataceae (10), but they also amplified bartonella spp . Under the given pcr conditions . In total,, as indicated by double peaks on the sequencing chromatogram, occurred in 12 cases . To further characterize the obtained candidatus n. mikurensis, we sequenced 1,426 bp of the 16s rrna and 1,233 bp of the groel gene (1). The obtained 16s rrna candidatus n. mikurensis sequences in this study were identical (1,426/1,426 bp) to sequences obtained from human patients in germany and switzerland (2,3). The groel sequence was identical to the isolate from germany (1,233/1,233 bp) but differed slightly from the isolate from switzerland (1,072/1,084 bp, 98.9% pairwise identity). A phylogenetic network containing unique 16s rrna sequences from candidatus n. mikurensis available at the national center for biotechnology information was made with the program network 4.5.1.6 (www.fluxus-engineering.com) by using the median - joining algorithm (11) (figure 2). Phylogenetic network of 16s rrna sequences (1,231 bp) from candidatus neoehrlichia mikurensis, southern sweden, 2008 . Numbers on branches represents mutations, numbered according to nucleotide position in the alignment . The sequence obtained in this study is shown in boldface and is identical with sequences from human patients in germany (3) and switzerland (2). The japanese reference strain tk4456 (7) showed 99.2% similarity with our sequence, and the north american candidatus n. lotori strain (12) showed 98.3% similarity . The prevalence of candidatus n. mikurensis at the 5 sites ranged from 0% to 12.5%, and bartonella spp . Occurred in 33.7% to 45.0% of the animals (figure 1). Candidatus n. mikurensis occurred in all 4 rodent species, but not in the shrews; the difference in prevalence between rodents and shrews was statistically significant (p = 0.011, by fisher exact test). In the present field survey of anaplasmataceae in swedish rodents, we have amplified 16s rrna and groel sequences identified as candidatus n. mikurensis identical to sequences obtained from humans . This organism has been amplified from humans with febrile illness on 4 occasions (13), demonstrating that candidatus n. mikurensis can cause human infections, at least occasionally . Apart from the first human case, to our knowledge, candidatus n. mikurensis has not been detected in sweden . However, we identified candidatus n. mikurensis at 4 of 5 sites, indicating that this organism is widespread in southern sweden . Identical or similar sequences have been detected in ixodes ricinus ticks from several european countries (36,13), showing that it is distributed over a large area of europe . I. ricinus is the dominating tick species in sweden, and animals at all our study sites were heavily infested with larvae of this species and occasionally with nymphs (m. andersson and l. rberg, unpub . Reservoir hosts are essential for several tick - borne pathogens that lack the capacity for transovarial transmission from female ticks to larvae, such as a. phagocytophilum (14). Approximately 70% of the tick larvae in sweden engorge on small mammals, such as voles, mice, and shrews (15). The prevalence of candidatus n. mikurensis was similar in all investigated rodent species in our study with a mean value of 8.8% . These results are consistent with several studies in japan, which also found candidatus n. mikurensis in several different rodent species (7,10). This finding indicates that rodents play a role in the natural cycle of candidatus n. mikurensis in europe and that rodents are likely to be competent reservoir hosts . In contrast, the closely related candidatus n. lotori, found in the united states, seems to use raccoons rather than rodents as hosts (12). Whether the european variant of candidatus neoehrlichia is capable of infecting animals other than rodents and humans remains to be investigated . We found the organism was completely absent in shrews, which suggests that they might not be competent hosts for these bacteria . We conclude that candidatus n. mikurensis is geographically widespread in southern sweden and that several rodent species, the main source of blood meals for larvae of i. ricinus in sweden (15), can be infected . The relatively high prevalence in common rodent species suggests that the risk for humans and domestic animals to encounter infected i. ricinus nymphs and adults is substantial . Thus, candidatus n. mikurensis should be considered when diagnosing bacterial infections associated with tick bites.
Central venous catheters (cvcs) have an essential part to play in the management of a critically ill patient . They are useful for hemodynamic monitoring, for administration of specific medications like vasoactive drugs, parenteral nutrition and for hemodialysis . These are associated with substantial risks of complications, which can be mechanical, septic and thrombotic . Upper limb dvt is a well known complication of thrombosis associated with cvcs, which, in its fatal form, can sometimes lead to life - threatening pulmonary embolism . The incidence of thrombosis varies from 1.9% in subclavian cvcs to 21.5% in femoral catheters . The risk of thrombosis associated with ijv catheters is estimated to be four - times higher than that with subclavian catheters . Sixty - six percent of the patients with internal jugular vein (ijv) catheters have evidence of thrombus formation either on ultrasound or on autopsy . The aim of this study was to determine the incidence of thrombosis associated with right - sided ijv catheters, the extent of thrombosis, its clinical implications and to relate it with the pharmacological prophylaxis for dvt . One hundred consecutive adult patients admitted to our critical care unit, who had right - sided ijv catheters placed for various indications, were included in the study . Cvc insertion was performed by intensivists or doctors having at least 5 years of experience in cvc insertion . Color doppler sonography was done on the 3 and 6 days of catheter placement . Local signs of inflammation and any clinical signs of upper limb dvt or pulmonary embolism, if any, were noted . Presence of pharmacological prophylaxis for dvt, presence of parenteral nutrition and other medications like mannitol and hypertonic saline were noted . Patients with diagnosed or suspected malignancy, those with diagnosed prothrombotic states and those having hemodialysis catheters in ijv were excluded from the study . Among the patients enrolled in our study, there were 70 males and 30 females . There was evidence of thrombus in 33 patients in color doppler duplex sonography (33.0%). In males, thrombus was found in 23 of 70 patients (32.86%) and in females thrombus was present in 10 of 30 patients (33.33%). The incidence in males was 158.62 per 1000 catheter - days and in females 185.18 per 1000 catheter - days . We categorized the thrombus arbitrarily into three groups: small, medium and large, depending on the size of the thrombus . Small thrombus was just around the catheter, medium - sized thrombus was up to 4 mm in diameter and the large - sized thrombus was more than 4 mm in diameter . In 15 patients the thrombus was small, in nine figures 1a, b and c show the different sized thrombi on vascular doppler studies . The incidence of different sizes of thrombus in males and females has been shown below [table 1]. The incidence of small - sized thrombus was significantly higher in males (p = 0.05) and the incidence of large - sized thrombus was significantly higher in females (p = 0.05). Small thrombus in the right internal jugular vein medium - sized thrombus in the right internal jugular vein large - sized thrombus in the right internal jugular vein incidence of cvc - related thrombus in males and females of 33 patients found to be having thrombus, in 21 (63.64%) it was detected on the third day after cvc insertion, whereas in 12 patients (36.36%) it was detected on the sixth day . If the thrombus was small or medium sized, then it was observed and followed - up with a color doppler sonography after 3 days . Of 33 patients, 24 had low molecular weight heparin as part of the dvt prophylaxis and nine did not have the same due to some medical contraindications . We did not find any significant effect of hypertonic solutions including mannitol and 3% saline as a risk factor for thrombus formation . We did not find diabetes mellitus, hypertension or smoking as increasing the risk of thrombus formation . They provide an important invasive tool for hemodynamic monitoring . They also allow delivery of certain medications and nutrition to the patient safely . As a result, cvc insertion is one of the most frequently performed invasive procedures in the intensive care unit . Unfortunately, their use is associated with many untoward complications, which increases the morbidity of the patient and can be life threatening . Hirsch and coworkers detected the prevalence of dvt in critically ill patients to be 33% with the help of ultrasonography and color doppler imaging . Merrer and colleagues found the incidence of cvc - associated thrombosis to be 1.9% in the subclavian route and 21.5% in the femoral route . A review showed that in such patients, the incidence of asymptomatic cvc - related thrombosis varied from 1.5 to 34.1% and that of symptomatic thrombosis from 1.2 to 13% . Another review found that the prevalence of cvc - related upper torso deep venous thrombosis in asymptomatic cancer patients varies from 11.7 to 44%, whereas in symptomatic patients it varies from 6.7 to 48% . There have been some studies on catheter - related thrombosis in patients having hemodialysis catheter in situ . Terrence and colleagues found the evidence of right ijv thrombus in 25.9% of the patients . Apart from patients with hematological and other malignancies as well as those having hemodialysis cannula, the incidence of cvc - related thrombosis and its clinical impact have not been studied well . In this study, we found the incidence of catheter - related thrombosis in right - sided ijv catheter to be 33.0% . The etiology of thrombosis associated with cvcs can be explained on the basis of virchow's triad of endothelial damage, altered blood flow and hypercoagulability . Vessel endothelium damage can be due to various factors including mechanical injury during the process of cvc insertion, number of vein punctures as well as irritation of endothelium by hypertonic solutions and drugs . Within hours after insertion of the catheter there is deposition of platelets around the cvcs, reaching its peak in 34 h. this is followed by formation of a sleeve around the cvc, which is an adherent coating of fibrin and collagen . The formation of sleeve is reported to occur in up to 47% of the catheters . The fibrin sleeve in itself is benign, but promotes infection and may lead to thrombus formation . Mural thrombosis can lead to subtotal stenosis or occlusion of vessel lumen leading to clinical manifestation of thrombosis and its complication . Uncomplicated or noninfected cases present with pain and swelling in the neck and a cord can be palpated beneath the sternocleidomastoid muscle . Described the following clinical manifestation in a large series of patients with septic ijv thrombosis: fever (83%), leucocytosis (78%), cervical pain (66%), neck swelling (72%), cord sign (39%), sepsis syndrome (39%), pleuropulmonary complications (28%), superior vena cava syndrome (11%), chylothorax (5%) and jugular foramen syndrome (6%). They are thrombogenecity of the catheter material, circumferential size of the catheter, catheter tip position, side of insertion, puncture site of insertion, multiple venipuncture attempts, composition of infusate, thrombophilic abnormalities, cvc - related infection and duration of catheter placement . Borow and crowley studied the adhesion of chromium-51-labeled platelets to the different cvc materials and found that the least thrombogenic catheters were hydromer - coated polyurethane catheters . The incidence of cvc - related thrombosis is found to be higher in patients in whom the catheter tip is placed in the innominate vein or proximal superior vena cava as compared with the distal superior vena cava / right atrial junction . Showed a 2.6-fold higher risk when the catheter was located in the superior vena cava compared with the right atrium . Other important risk factors are side of insertion of the catheter and puncture site of cvc . Tesselaar et al . Also showed that the placement of cvc on the left side was associated with a 3.5-times higher risk of thrombosis as compared with the right side . In children, the incidence of catheter - related thrombosis was 44% in subclavian vein cvcs compared with 20% in jugular vein cvcs . This difference in the relative risk can be possibly explained by the anatomy of the venous system in the upper torso . As compared with the right side, the left brachiocephalic vein is longer and has a more horizontal course, leading to a sharper angle to the superior vena cava . Further, compared with the jugular cvcs, the subclavian cvcs follow a sharper curve into the superior vena cava, facilitating wall adherence . The subclavian cvcs enter where the vein passes between the clavicle and the first rib, which may cause vein compression and kinking of the cvc . Cvc - related infection is also an important risk factor that can lead to increased propensity for thrombus formation . Van rooden et al . Showed an increased incidence of cvc - associated thrombosis in patients with cvc - related infection as compared with those without infection in a population of patients with hematological malignancy . The major contributing factor for both of them is the formation of a fibrin sheath around the catheter . They also produce a coagulase enzyme that enhances the thrombogenic process . On the other hand other modalities used to diagnose upper limb dvt are computed tomography scan with contrast, magnetic resonance imaging and nuclear medicine scan . Color doppler duplex sonography is an noninvasive, safe and convenient means of diagnosing upper limb dvt and cvc - related thrombus . A systematic review of studies reported a sensitivity of compression ultrasound ranging from 56 to 100% and a specificity of 94100% for the diagnosis of upper limb dvt . It still remains a matter of debate whether antithrombotic prophylaxis is effective in preventing cvc - related thrombosis . There have been studies using unfractionated heparin, minidose warfarin and low molecular weight heparin for antithrombotic prophylaxis but, due to lack of well - designed prospective studies, it still remains a matter of debate . Treatment strategies consist of thrombolytic therapy, initiation of systemic anticoagulation, removal of the catheter or both . For cvc - related thrombosis, the preferred treatment is a combination of low molecular weight heparin followed by oral anticoagulant for 36 months, but no prospective randomized studies have been published on this subject . It appears to be more common in patients with hematological and other malignancies and in patients having hemodialysis cannula in situ . Many risk factors have been identified in different studies that have a strong association with thrombus formation . In this study, those patients who had cvc in the right ijv were only included . Males had a significantly higher incidence of small - sized thrombus . In 63.64% patients, the low molecular weight heparin used for dvt prophylaxis was not effective in preventing the cvc - related thrombus . The risk of thrombosis was not increased with the use of hypertonic solutions or with the presence of factors like diabetes mellitus, hypertension and history of smoking . There is no consensus regarding management of asymptomatic cvc - related thrombus, and more prospective randomized studies are required . Till then, knowledge of the different risk factors for the thrombus, its prevention and effective treatment of cvc - related infection remains the mainstay to avoid cvc - related thrombosis and its dreaded complications.
Colorectal adenocarcinoma (crc) is one of the most common cancers worldwide and the third leading cause of death in the united states . Crc can be histologically subtyped into adenocarcinoma (ac), which accounts for the large majority of cases; mucinous ac; signet - ring cell carcinoma (srcc), and other even less frequent subtypes . Primary srcc of the colon is a rare entity, accounting for less than 1% of all crc . Srcc is associated with a higher - grade tumor associated with young age, female patients, and distinct molecular patterns, such as microsatellite instability (msi) and activating mutations of the braf gene compared to mucinous ac . However, since clinical symptoms tend to occur late in the course of srcc, most cases are usually detected at an advanced stage with a poor overall survival rate . Primary srcc at an early stage is rare, and only 27 cases have been reported . We report a case of a 36-year - old female who presented with progressive right lower quadrant pain and was diagnosed with primary srcc of the cecum . The patient responded well without any adverse reactions to an adjuvant fluropyrimidine (5-fu)-based therapy with oral capecitabine . A 36-year - old female presented to her primary care physician with progressive right lower quadrant abdominal pain without any significant past medical and family history . Computed tomography (ct) scan of the abdomen and pelvis with contrast showed a 4.9 3.5 3.1 cm, lobulated, septated cystic mass arising from the cecum . The mass demonstrated wall enhancement with multiple coarse calcifications and ascites inferior to the mass (fig . The specimen revealed organ - confined disease with final pathological staging iia (pt3, pn0, pmx). High - grade mucinous carcinoma with signet - ring cells invaded the muscularis propria into the subserosal adipose tissue (fig . The cells clustered into various sizes of clusters and gland - like structures at 40 magnification . At 400 magnification, the moderately differentiated malignant cells formed classical columnar strips (fig . 3). At 600 magnification, the cells floated within the abundant extracellular mucin pools as either clusters or isolated single cells (fig . 4). Molecular study specific for msi showed the preserved protein expression of mlh1, pms2, msh2 and msh6 . Carcinoembryonic antigen was within the normal limit (<0.3 ng / ml). Due to the patient's young age, early - stage disease, and aggressive high - grade mucinous carcinoma with microsatellite stability, an adjuvant 5-fu - based therapy with oral capecitabine the patient has completed 4 of the planned 8 cycles and has tolerated and responded to the therapy without any significant hematological or nonhematological toxicities . Based on several reports including a large population - based study of 197,757 crc patients, srcc is a distinct entity based both on clinical presentation and pathology . Srcc occurs in the younger population with female predominance, usually less than 40 years of age . More than 96% of cases of srcc arise in the stomach with the rest occurring in the colon, rectum, gallbladder, pancreas, urinary bladder, and breast . Srcc usually presents at an advanced stage with node - positive disease and metastatic spread, which results in poor prognosis . A unique feature of srcc is its fibrotic appearance and preference for peritoneal metastasis over the liver . Pathologically, srcc demonstrates unique features that distinguish the cells from routine ac of the colon and confer aggressiveness to these cells . The signet - ring, malignant cells are seen floating in abundant extracellular mucin pools either as clusters or isolated cells . These cells have a slightly lower prevalence of kras mutation but a higher braf mutation rate as compared to classical ac, and the prognostic implication is unknown . Loss of e - cadherin expression has been reported and contributes to the high - grade and invasive nature of srcc as the cells acquire stem cell - like characteristics . There is some evidence that srcc patients with stage ii disease do not benefit from adjuvant therapy, whereas stage iii patients with high msi benefited from single - agent 5-fu adjuvant therapy . The overall prognosis of srcc in general is similar to other cancers in that staging and grade are important determinants . The available literature suggests that the tumor staging is the best predictive factor for the prognosis of srcc of the colon where higher tumor staging means poorer prognosis . The 5-year survival rate in srcc of the colon ranges from 0 to 12%, and disease recurrence is more frequent in srcc of the colon compared to ac . Absence of lymphovascular invasion, lack of lymph node metastasis, and lower tnm stage had a favorable effect on the survival of srcc patients . Patients with srcc can definitely benefit from closer follow - up or even intensified adjuvant therapy due to their high rates of local and distant recurrence . In a study by hugen et al ., adjuvant chemotherapy for srcc stage iii of the colon was associated with improved survival . Other than presenting with abdominal pain and being microsatellite stable, her srcc occurred in the cecum and had invaded the subserosal fat . Instead of having a fibrotic appearance, her cancer is more cystic in appearance than the generally reported srcc . Due to early disease of stage iia and microsatellite stability in our patient, an adjuvant 5-fu - based therapy with oral capecitabine primary srcc is considered a distinct entity based on the clinical and pathological features . In this case report, we have shown a rare case of primary srcc of the cecum . Given the poor prognosis of these patients, early disease of stage ii in microsatellite - stable patients should be considered for adjuvant 5-fu - based therapy in an attempt to prevent recurrence.
Digital impression systems using intraoral scanning have been available since the introduction of cerec 1 (sirona s bluecam) as part of the single - sitting dental appointment concept . These systems offer remarkable benefits compared with conventional impression methods, including easier data storage, smaller storage requirements, more time - efficient treatment procedures, and an increase in patient comfort . However, restorations fabricated with the first digital impression systems available showed relatively poor marginal adaptation compared to the ones made with conventional impression methods; therefore, some authors expressed concerns on the longevity of the restorations, since adequate marginal adaptation is a decisive factor for clinical longevity . On the other hand, technical advances have significantly improved the adaptation of restorations fabricated with digital impression systems; a recent study demonstrated that a complete crown fabricated using intraoral scanning resulted in a better marginal fit than crowns created with conventional techniques . The process of fabricating restorations using digital impressions and computer - aided - design / computer - aided - manufacturing (cad / cam) technology comprises the following steps: data acquisition, data processing, and manufacturing . These steps are equally important for the fabrication of well - fitting restorations, and errors in any of these steps may result in distortions of the restorations . Therefore, the scanning procedure, the specific software, and the milling machine used have all been suggested as factors that could have a detrimental effect on the fit of restorations fabricated with the cad / cam technology . As a consequence, a number of studies have been performed to compare marginal and internal adaptation of restorations scanned with different intraoral scanners or fabricated made with different milling machines . However, to perform accurate comparisons it is also essential to take into account the software version used with each cad / cam system and the specific parameter settings (i.e. Spacer settings) selected . Despite the importance of these factors, therefore, the purpose of this study was to evaluate marginal and internal adaptation of restorations fabricated with different versions of the cad / cam software and the effect of different parameter settings . An acrylic model of the maxillary left second molar was prepared to receive a single ceramic crown restoration (nissin dental model, nissin dental prod . The preparation featured a 1-mm - deep chamfer finish line with a total convergence angle of 12 degrees . To verify a standardized tooth reduction, an initial impression was taken with a vinyl polysiloxane material (aquasil soft putty, dentsply detrey gmbh, konstanz, baden - wrttemberg, germany) before preparation . This impression was sectioned vertically (at a width of 2 mm), and set in the place of the prepared model . The depth needed for tooth preparation was estimated by comparing the impression body and the prepared model . For repeated measurements, a titanium replica of the acrylic model was made using a customized cad / cam system (myplant, addtech co., seoul, seoul, korea). A thin uniform layer of an antireflection powder (cerec optispray, sirona dental systems gmbh, bensheim, hesse, germany) was sprayed on the reference model . Digital impressions were taken using a cerec ac with bluecam (sirona s bluecam, sirona dental systems gmbh, bensheim, hesse, germany) system according to the scanning protocol of ender and mehl (2013), including occlusal views with scans at an angle of 30 degree from the buccal and lingual surfaces . One single dentist trained in taking digital impressions acquired 40 digital impressions of the reference model . The 40 digital impressions acquired were first divided into two groups (n=20 each), depending on the software version that was used in the processing (i.e., cerec version 3.8 or version 4.2). Each group was further divided into two subgroups (n=10 each), depending on the spacer parameter settings (i.e., 40 or 80 m) (table 1). The acquired datasets were transmitted to a milling unit (cerec mcxl, sirona dental systems gmbh, bensheim, hesse, germany), and crowns were fabricated using lavaultimate restorative milling blocks (3 m espe, st . Table 1two - way anova analysis showing that software version and spacer parameter significantly affected the fit of the crowns (p<.05)independent variablesignificancesoftware versionp<0.05space parameterp<0.05 the replica technique previously described by molin and karlsson (1993), and validated by rahme, et al . The crowns were filled with a low viscosity silicone material (fit checker, gc dental, tokyo, tokyo, japan), and placed in the titanium reference model using a shimadzu universal tester (ag-10knx, shimadzu co, tokyo, tokyo, japan) with a 20 n load . After 5 minutes, the silicone material attached to the internal surface of the crowns was removed and then stabilized with the impression material of higher viscosity (examixfine regular type, gc dental, tokyo, tokyo, japan). All replicas were cut along the abutment axis both in the buccolingual and in the mesiodistal direction, using a razor blade, yielding four fragments per crown . For each cross section, four points were measured, thus 16 thickness points of the titanium replica were measured (figure 1). The points from 1 to 8 were included in the buccolingual direction section, and the points from a to h were included in the mesiodistal direction section . The measuring points were divided into 4 categories considering the location of tooth: margin (1, 8, a, h), lower axial wall (2, 7, b, g), upper axial wall (3, 6, c, f), and occlusal surface (4, 5, d, e). Figure 1groups defined for the study all analyses were performed using a double - blind protocol . Replica film thickness was measured with a video measuring system (optical video measuring system, seven ocean optical technology, donnguan, guangdong, china) at a 10x magnification with external light source . The mean and the standard deviation of the fit accuracy were calculated for each group . The influence of independent variables, including the ones from the software and from the spacer parameters, were analyzed using a friedman two - way analysis of variance (anova) (p=.05). The groups within each category (margin, lower axial wall, upper axial wall, occlusal surface) were merged, and the mean and the standard deviation of each category were calculated . The comparisons between groups within each category were conducted using paired t - tests (p<05). All statistical analyses were carried out with medcalc version 12.5.0 (medcalc software, ostend, vlaanderen, belgium). An acrylic model of the maxillary left second molar was prepared to receive a single ceramic crown restoration (nissin dental model, nissin dental prod . The preparation featured a 1-mm - deep chamfer finish line with a total convergence angle of 12 degrees . To verify a standardized tooth reduction, an initial impression was taken with a vinyl polysiloxane material (aquasil soft putty, dentsply detrey gmbh, konstanz, baden - wrttemberg, germany) before preparation . This impression was sectioned vertically (at a width of 2 mm), and set in the place of the prepared model . The depth needed for tooth preparation was estimated by comparing the impression body and the prepared model . For repeated measurements, a titanium replica of the acrylic model was made using a customized cad / cam system (myplant, addtech co., seoul, seoul, korea). A thin uniform layer of an antireflection powder (cerec optispray, sirona dental systems gmbh, bensheim, hesse, germany) was sprayed on the reference model . Digital impressions were taken using a cerec ac with bluecam (sirona s bluecam, sirona dental systems gmbh, bensheim, hesse, germany) system according to the scanning protocol of ender and mehl (2013), including occlusal views with scans at an angle of 30 degree from the buccal and lingual surfaces . One single dentist trained in taking digital impressions acquired 40 digital impressions of the reference model . The 40 digital impressions acquired were first divided into two groups (n=20 each), depending on the software version that was used in the processing (i.e., cerec version 3.8 or version 4.2). Each group was further divided into two subgroups (n=10 each), depending on the spacer parameter settings (i.e., 40 or 80 m) (table 1). The acquired datasets were transmitted to a milling unit (cerec mcxl, sirona dental systems gmbh, bensheim, hesse, germany), and crowns were fabricated using lavaultimate restorative milling blocks (3 m espe, st . Table 1two - way anova analysis showing that software version and spacer parameter significantly affected the fit of the crowns (p<.05)independent variablesignificancesoftware versionp<0.05space parameterp<0.05 the replica technique previously described by molin and karlsson (1993), and validated by rahme, et al . The crowns were filled with a low viscosity silicone material (fit checker, gc dental, tokyo, tokyo, japan), and placed in the titanium reference model using a shimadzu universal tester (ag-10knx, shimadzu co, tokyo, tokyo, japan) with a 20 n load . After 5 minutes, the silicone material attached to the internal surface of the crowns was removed and then stabilized with the impression material of higher viscosity (examixfine regular type, gc dental, tokyo, tokyo, japan). All replicas were cut along the abutment axis both in the buccolingual and in the mesiodistal direction, using a razor blade, yielding four fragments per crown . For each cross section, four points were measured, thus 16 thickness points of the titanium replica were measured (figure 1). The points from 1 to 8 were included in the buccolingual direction section, and the points from a to h were included in the mesiodistal direction section . The measuring points were divided into 4 categories considering the location of tooth: margin (1, 8, a, h), lower axial wall (2, 7, b, g), upper axial wall (3, 6, c, f), and occlusal surface (4, 5, d, e). Figure 1groups defined for the study all analyses were performed using a double - blind protocol . Replica film thickness was measured with a video measuring system (optical video measuring system, seven ocean optical technology, donnguan, guangdong, china) at a 10x magnification with external light source . The mean and the standard deviation of the fit accuracy were calculated for each group . The influence of independent variables, including the ones from the software and from the spacer parameters, were analyzed using a friedman two - way analysis of variance (anova) (p=.05). The groups within each category (margin, lower axial wall, upper axial wall, occlusal surface) were merged, and the mean and the standard deviation of each category were calculated . The comparisons between groups within each category were conducted using paired t - tests (p<05). All statistical analyses were carried out with medcalc version 12.5.0 (medcalc software, ostend, vlaanderen, belgium). The accuracy of the fit at each measuring point for each experimental group is summarized in figures 2 to 6 . The results drawn from the average values of the same categorized measuring points are shown in figure 9 . The two - way anova analysis showed the software version and the spacer parameter significantly affected the fit of crowns (p<.05) (table 1). The points 1 to 8 are positioned on the buccolingually sectioned surface, and a to h are on the mesiodistally sectioned surface . Points of 1, 8, a, h are positioned on the margin, 2, 7, b, g are on the lower axial wall, 3, 6, c, f are on the upper axial wall, and 4, 5, d, e are on the occlusal surface figure 3accuracy of fit of each measuring point in group 1 (cerec sw 3.8, 40 mm). The top of box and bottom of box indicate the 75% and 25% values of results . The midline in the box means the average of the results figure 4accuracy of fit of each measuring point in group 2 (cerec sw 3.8, 80 mm). The top of box and bottom of box indicate the 75% and 25% values of results . The midline in the box means the average of the results figure 5accuracy of fit of each measuring point in group 3 (cerec sw 4.2, 40 mm). The top of box and bottom of box indicate the 75% and 25% values of results . The midline in the box means the average of the results figure 6accuracy of fit of each measuring point in group 4 (cerec sw 4.2, 80 mm). The top of box and bottom of box indicate the 75% and 25% values of results . The midline in the box means the average of the results figure 7mean internal fit (mm) of each measuring points on buccolingually sectioned surface . * indicates statistically significant differences figure 8mean internal fit (mm) of each measuring points on mesiodistally sectioned surface . * indicates statistically significant differences figure 9the results of each average values of measuring points categorized as margin (1, 8, a, h), lower axial wall (2, 7, b, g), upper axial wall (3, 6, c, f) and occlusal suface (4, 5, d, e). * indicates statistically significant differences the accuracy of the fit of all measuring points on the margin (1, 8, a, h) showed no statistical significant difference between groups, and the average values of measuring points categorized as the margin also showed no significant difference (p>.05). On the lower and on upper axial wall, some specific measuring points (i.e., points 2 and g of the lower axial wall; points 3 and f of the upper axial wall) showed a significantly larger gap when data were processed using the version 3.8 of the software compared with the version 4.2 of the software, regardless of the spacer settings used . Measuring points 6 (upper axial wall) and 7 (lower axial wall) showed a significantly larger gap using the version 3.8 of the software compared with the 4.2 of the software when the spacer parameter was set at 40 m (p<.05). The comparison of the average values of the measuring points categorized as lower axial wall and upper axial wall showed a significantly larger gap using the version 3.8 of the software, regardless of the spacer parameter used (p<.05). In the occlusal surface, measuring points d and e showed a significantly larger gap when the data were processed using the version 3.8 of the software compared with the version 4.2 of the software, regardless of the spacer parameter used; while measuring point 4 showed the same result only when the spacer parameter was set at 80 m (p<.05). The comparison of the average values of the occlusal surface showed the group processed with the version 3.8 of the software had a larger gap compared with the group processed with the version 4.2 of the software only when the spacer parameter was set at 40 m (p<.05). Although we expected the larger spacer parameter setting would have caused a larger gap between crown and tooth, the use of 40 m as the spacer parameter setting in the version 3.8 of the software produced a larger gap when compared to the setting of the spacer at 80 m for the six measuring points, including 5 (occlusal surface), 6 (upper axial wall), a (margin), b (lower axial wall), c (upper axial wall), and h (margin). On the contrary, for the version 4.2 of the software, one measuring point (i.e., point 6 of upper axial wall) showed a smaller gap with a 40 m spacer setting when compared with the group selecting 80 m, while the group selecting 80 m in the version 4.2 of the software showed the least variations among the results . The process of dental restorations using cad / cam technology involves data acquisition, data processing, and manufacturing . The aim of this study was to evaluate the effect of data processing on the adaptation of crown restorations . Variables such as different versions of software and space parameters were analyzed in the four groups of crown impressions, and the fit of the crown was measured using the replica technique . As the results showed, the crowns designed using the version 4.2 of the software produced a better fit than those designed using the version 3.8 of the software, particularly in the axial wall . The spacer parameter was more accurately represented in the version 4.2 of the software than in the version 3.8 . In addition, the use of the version 4.2 of the software combined with the spacer parameter set at 80 m showed the least variation . On the other hand, such parameters indicate the setting factors to determine particular features of a restoration in cad / cam system . The cement space, the contact strength, the occlusion strength, and the minimum thickness of a restoration can be easily changed as providing different parameter in the cad / cam system . Space parameter can be set from -100 to 100 m in the version 3.8, and from 0 to 100 m in the version 4.2 . Among the overlapped space parameter (from 0 to 100 m), forty m is commonly used as space parameter because resin cements range from 20 to 35 m concerning the thickness of their film; the 80 m is the double value of 40 m to evaluate the effect of space parameter . To fabricate well - fitting dental crowns using cad / cam systems in the clinic, the optimal combination involving the intraoral scanner, the software version, and although many studies have compared the fit of crown restorations fabricated with different intraoral scanners and milling machines, there have been few studies evaluating the effect of different versions of the software . The results of this study showed the fit of a restoration could be affected by the software version and the spacer parameter setting, even when the same scanner and milling machine were used . Compared to the version 3.8 of the cad / cam software, the version 4.2 of this software can be recommended for the fabrication of well - fitting crown restorations, and for the appropriate regulation of the spacer parameter . Even though the 80-m spacer setting produced a larger gap, this setting could be recommended for the version 4.2 of the cad / cam software because it showed a good repeatability . Adequate marginal gaps and internal fits are decisive factors for the clinical longevity of crown restorations . A large marginal gap can cause discoloration, disintegration of cement, plaque retention, and gingival inflammation . The recommended marginal gap for the prevention of the above mentioned complications is below 100 m . The results obtained in this study showed a clinically acceptable marginal fit could be achieved regardless of the software version and of the spacer parameter settings, with an average gap of 35 m . A larger internal gap could produce restorative fractures and postoperative sensitivity . The results of this study demonstrated the version 3.8 of the software and the spacer parameter set at 40 m produced a relatively larger internal gap when compared with the version 4.2 of the software and with the spacer parameter set at 80 m . The use of a sufficient amount of cement with harder mechanical properties is recommended in these cases to avoid the complications caused by large internal gaps . Although zirconia is one of the most popular materials used in the fabrication of crowns by utilizing the cad / cam system, the additional process of sintering may introduce variability among crowns . For the accurate evaluation of the effect of the version of the software and of the spacer parameter settings, lava ultimate restorative, a resin of nano ceramic material that does not require the sintering process was selected as the restoration material in this study . Compared with previous studies, the smaller marginal and internal gaps reported in this study could be due to the use of a different restoration material . Although measurements using a digital 3d scanner are precise, convenient, and easily visualized, the replica technique is the appropriate measurement tool for the accurate assessment of multiple points in the internal surface of restorations fabricated using the cad / cam system . The generally recognized improvement introduced by the version 4.2 of the software compared with the version 3.8 is an increased convenience for the use of the cerec cad / cam system . The version 4.2.5 is already available these days, being a supplementary version of the 4.2 and it does not seem to have big functional differences with the version 4.2 . The results of this study showed that a smaller internal gap could be obtained using the version 4.2 of the software, particularly at the axial wall, despite the fact the marginal gap was almost the same . The improvement of the adaptation obtained with the version 4.2 of the software could be in part due to differences in the pre - set space, basically providing space regardless of the parameter setting at the internal part between software versions . It has been shown that a greater internal space resulted in less marginal gap leading to less internal premature contact; a greater internal space might be necessary for achieving an adequate marginal gap in crown restorations, taking into account the accuracy and reproducibility of intraoral scanners and milling machines that are currently used with the version 3.8 of the software . Another possible explanation could be the improvement in resolution of the version 4.2 of the software during the processing of the acquired data through the intraoral scanner . The application of the cad / cam technology in dentistry have been producing improvement not only in dental clinics but also in dental laboratories . Many studies are using the ad / cam technology to compare it with conventional methods or to manage it as a tool to measure specimens . Our results suggest that, to achieve accurate comparisons across studies, all reports should specify the software version and the space parameter settings used (additional studies of reference on the effect of various software and parameters will be necessary). The fit of a crown restoration can be affected by the specific cad / cam software version and by the parameter settings selected, even if the same scanner and milling machine were used . In comparison with the version 3.8 of the cad / cam software, the version 4.2 of this software can be recommended for the fabrication of well - fitting crown restorations, and for the appropriate regulation of the spacer parameter . Even though the 80-m spacer setting produced a larger gap, this setting can be recommended for the version 4.2 of the cad / cam software because it has shown a good repeatability.
In united states, about 595,800 establishments make up the healthcare industry . According to the american hospital association, in 2009 about 21 percent of hospital jobs are in service occupations, such as nursing, psychiatric, and home health aides, or cleaners . The international labour organization's (ilo) key indicators of the labor market suggest that wages for work such as performed by cleaners is low in comparison to most other occupations . More specifically, employment statistics on jobs in the us show that the mean annual wage estimates of housekeepers are approximately one - half of the mean annual wage estimates for all occupations . In 2008, the total number of nonfatal occupational injuries in hospitals (private industry, state government, and local government) was 323,200 . The rate of occupational injuries and illnesses among cleaners was 3.9 per 100 full - time workers . Such injuries and illnesses affect the job performance of the cleaners thus affecting their efficiency . Due to the reduced efficiency and absenteeism, the employers have to incur losses and the treatment and rehabilitation of these employees are costly to society . Many of the cleaners develop conditions like arthritis, tendonitis, carpel tunnel syndrome, and other musculoskeletal conditions . The tasks performed by cleaners are labor intensive and most of the cleaners have to work under time constraints increasing the physical and mental stress . A study carried out in four countries of the european union showed that the prevalence of health problems like musculoskeletal problems, skin problems, and psychosomatic disorders among cleaners is high compared to the average wage earners in general . Occupational physical risk factors responsible for musculoskeletal morbidity include repetitive work, working with hands above shoulder height or below knee height, carrying heavy loads, and operating vibrating tools [8, 9]. All of the above - mentioned risk factors are prevalent in cleaners and may therefore contribute toward musculoskeletal morbidity in the cleaning occupation . The bureau of labor statistics provides the number of nonfatal occupational injuries and illnesses for cleaners; however, there is no record of these among cleaners specific to hospitals . It is apparent from the review of literature that little work has been done on the cleaning workers specific to hospitals . A large number of studies have investigated the association of musculoskeletal disorders and physical workload [1012]. Most studies have suggested the evidence of physical work factors as determinants of musculoskeletal disorders of the upper extremity and the low back [10, 13, 14]. Association between physically demanding work tasks and disorders of the lower extremity is evident in some studies [1517]. Therefore, the specific objectives of this study were to (a) determine the rates of nonfatal occupational injury and illness in hospital cleaners in one hospital in texas and (b) investigate the prevalence of musculoskeletal symptoms in this population in one texas hospital . The focus of this study is on injuries related to slip / trip / fall, material handling, and work - related musculoskeletal aches, pains and discomfort . This study explores information about incidence rates among hospital cleaners categorized by various factors such as type of injury and source of injury . The study sample comprised of 106 of 108 housekeeping employees of a hospital located in texas, united states giving a response rate of 98.14% . The mean age of the sample was 46.36 years (sd 13.89 years), and median age was 48.5 years . The cleaners worked for an average of 8 h per day in one of three shifts . 51% worked during the first shift (7 am to 3 pm), 35% worked during the second shift (3 pm to 11 pm), and 14% worked during the third shift (11 pm to 7 am). The number of years they worked in cleaning jobs ranged from 1 year to 40 years with a mean of 14.2 years (sd 9.84 years). The cleaners perform repetitive cleaning of assigned patient rooms, dismissals, units, x - ray rooms, surgical unit, recovery rooms, all offices, and other areas using standard cleaning supplies and disinfectants . As part of their job profile, they spend 40% of their time cleaning patient rooms, clinics, and offices, 25% of their time emptying trash and linen, and 10% of their time vacuuming and shampooing carpets . They spend 5% of their time on mopping floors, cleaning furniture, refinishing floors, moving furniture, and responding to hospital emergency drills, respectively . The cleaners carry supplies of linens, towels, toilet items, and cleaning materials, using carts and keep storage areas clean and tidy and carts well stocked . Cleaning patient rooms, clinics, and offices involves pushing / pulling beds, turning the mattress, moving furniture, dusting furniture, sweeping and mopping the floor . Moving trash involves lifting trash bags and dumping them into a cart, walking long distances pushing the cart to the dump station, and emptying the trash into the electric dumpster . Cleaning bathrooms involves bending over and stooping to clean toilets and tubs, washing sinks, mopping floors, refilling soap, and loading toilet paper . Water from the mop is squeezed by wringing the washcloths using a wringer on the mop bucket . Washing windows, walls, ceilings, woodwork, and waxing and polishing as necessary is also done . Cleaning rugs, carpets, upholstered furniture, and/or draperies, using vacuum cleaners and/or shampooers are common housekeeping tasks . The cleaners are also required to prepare rooms for meetings, arrange media equipment, and furniture for social or business functions in auditoria of the hospital . The linen room has three employees who sort, count, fold, and store linen in closets; they also move linen carts to various units of the hospital ., employee injury and illness reports were obtained from the occupational health services of the hospital . The report consisted of information about 117 incidents among cleaners from 2004 to 2008 . The second part consisted of a cross - sectional survey using an interview - based modified nordic musculoskeletal questionnaire to study the extent of musculoskeletal symptoms of different body parts among the housekeeping employees . Responses to the questionnaire were noted . The questionnaire comprised of four sections: demographic information, information on pain and discomfort, types of work tasks, and work organization . The demographic information included age, gender, height, body weight, number of hours worked each day, areas of the hospital assigned to clean, number of years working in the hospital, and total number of years in the cleaning work . Subjective complaints of pain and discomfort from neck, shoulders, elbows, wrist / hands, upper back, lower back, hips / thighs / buttocks, knees, and ankles / feet during the last 12 months were obtained . The employees who reported pain or discomfort were asked to identify the painful body parts on a body map showing the body regions and the level of pain on a scale of 0 to 10 ranging from no pain at all to worst imaginable pain . The level of pain was categorized as mild (score: 1 to 3), moderate (score: 4 to 7), and severe (score: 8 to 10). The third section included questions on buffing, vacuuming, lifting equipment, mopping floors, carrying garbage and linen, cleaning bathrooms, making beds, moving furniture, carrying heavy loads, and working with the back, neck, or arms in awkward postures . In the study, the employees were asked about the frequency of their work tasks . The answers were categorized into three classes (1 = frequently, 2 = sometimes, 3 = never). The work organization section asked participants if they had to work fast, work intensively and if they had enough time to do their work . Participants were grouped as per gender, age, number of years in cleaning occupation, race, body mass index, and shift work . The prevalence of musculoskeletal aches, pains and discomfort among cleaners in the past 12 months in association to the above - mentioned demographic variables and selected body parts was calculated . Incident rates among the housekeeping staff and the rest of the hospital staff were extracted . The incidence rates in this sample represent the number of injuries per 100 full - time employees (fte). The incidence rates were calculated as (n / h) 200,000 where n = number of injuries and illness, h = total hours worked by all employees during period covered, and 200,000 = represents the equivalent of 100 employees working 40 h / week, 50 weeks / year . The incidence rates for all injuries, for injuries due to slip / trip / fall, and injuries due to material handling among cleaners and rest of the hospital staff were compared . The 117 incidents among cleaners were categorized as strains, contusion / abrasion, laceration / scratch, puncture wound, chemical exposure, others and the number of cases in each category was calculated . The frequency of execution of different tasks performed by housekeepers was extracted thus calculating the percentage of housekeepers performing tasks frequently, sometimes, and never . Background demographic characteristics and the prevalence of musculoskeletal pain for the study population are shown in table 1 . Female cleaners had a higher prevalence of musculoskeletal symptoms in the last 12 months than male cleaners . Cleaners in age group from 39 to 58 years, housekeepers working in housekeeping jobs for over 31 years, housekeepers with body mass index (bmi) less than 18.5, and those working in the first shift had the highest prevalence of musculoskeletal pain in the last 12 months . This being a hospital setting, the workload for cleaners during the first shift is a lot more as compared to other shifts . This could be attributed to the influx of patient admissions during the first shift and the cleaning of surgical units and other units where the workload is highest in the morning hours . The incidence rate for total occupational injuries in cleaners for each year from 2004 to 2008 was significantly higher than that for all other employees of the hospital with the highest incidence rate during 2005 (75.18 per 100 fte) (table 2). The drop in the incidence rates in the subsequent years can be attributed to preventive measures that were taken by the occupational health services of the hospital to address the high incidence rate . Mandatory slip resistant footwear, lowering of threshold plates for those areas where carpet to tile transitions (to reduce pushing / pulling injuries), ergonomic mops, soiled laundry receptacles that emptied from the bottom, department - specific education about ergonomics, and enforcement of use of appropriate personal protective equipment were some of the measures taken to reduce the injuries and illnesses among cleaners . Nursing services were also included in training so that laundry hampers were not overfilled, which left an msd hazard for the cleaners . The five - year incidence rate of total injuries for the housekeeping employees was found to be 28.32 per 100 fte and that for rest of the employees of the hospital was 13.54 per 100 fte (p <.05). The incidence rate of slips / trips / falls among the housekeeping employees and rest of the employees of the hospital was 4.39 per 100 fte and 2.37 per 100 fte, respectively (p <.05). The incidence rate of injury due to material handling in the housekeeping employees and rest of the employees of the hospital was 5.45 per 100 fte and 1.08 per 100 fte, respectively (p <.05) (table 2). The most common type of injury among housekeepers was due to strains followed by contusion / abrasion (table 3). The most common cause of injury was being struck by an object (table 3). Symptoms in the lower back were most prevalent (49%) followed by the right wrist (43%), ankle, left knee, left wrist (35% each), right knee (34%), right shoulder (25%), and other selected body parts (table 4). Similarly, current pain and discomfort was reported by 42% of the cleaners . In this sample, 82% of those who complained of pain in the last 12 months reported that the pain was related to work . 4% of the participants complained of mild pain in the wrists, whereas 16% had moderate pain and 11% had severe pain in the wrists . The frequency of execution of different tasks by housekeepers was expressed as percent (table 5). The prevalence of musculoskeletal pain was highest in participants who worked with the back in the awkward posture (65%), followed by employees who worked with their arms and neck in the awkward posture, respectively (64%). The employees who cleaned bathrooms had a prevalence of 64%, followed by those who mopped floor and carried / emptied garbage (63%). The prevalence of musculoskeletal pain in the participants who carried heavy loads, made beds, moved furniture, and used the vacuum ranged between 61% and 63% (table 6). In order to determine the number and incidence rate of nonfatal occupational injuries and illnesses among hospital housekeepers, the authors used the injury data from the housekeeping employees of a hospital to extrapolate possible number of injuries in this occupation in healthcare industry . As per the 2008 statistics, in the united states, cleaners held about 1.5 million jobs . Hospitals employed about 17 percent of these workers, thus making it approximately 255 thousand cleaners in hospitals . In 2008, there were 22 injuries among 108 housekeeping employees studied . Considering approximately 255,000 cleaners in the hospitals, in 2008, cleaners would have over 52,000 injuries . Since musculoskeletal disorders (msds) affect a significant proportion of the workforce, they are a major problem in several economic activity sectors in industrialized countries this includes an estimated $155 billion for occupational injuries . In this study, for 108 housekeeping employees the cost incurred by the hospital in 2008 for occupational injuries and illnesses was $127,955, thus extrapolation of this rate makes it approximately over $302 million for claim costs for all the hospital cleaners in us in 2008 . The true economic burden of work - related musculoskeletal disorders is likely to be even greater because many cases are not reported to the workers' compensation system . A study of individuals diagnosed with work - related musculoskeletal disorders of the upper extremity, neck, or low back reported that only 25% filed a workers' compensation claim . A population survey in connecticut, estimated that one - tenth of working - age adults have a work - related musculoskeletal condition, but only 10% of these workers file compensation claims . The cleaners in this study had higher rates of injury due to slip / trip / fall and material handling . Slips and falls were reported as the leading cause of death in the workplace and source of more than 20% of all disabling injuries . Occupational injuries associated with slip and fall accidents pose a significant problem to industry both in terms of human suffering and economic losses . In terms of the labor costs, over one - quarter of overall fall - related injuries resulted in 31 days or more workdays being lost, costing us economies nearly $10 billion / year . In the self - reported work - related illness in 1995 survey, musculoskeletal disorders were by far the most commonly reported class of work - related illness, and it was estimated that 52% of the 1.2 million reported a work - related musculoskeletal disorder due to manual handling associated with their job . Manual handling is not only one of the frequent workplace activities, but is also one of the most common causes of workplace injury . The number of sprains and strains for all private industries put together (416,620 in 2008) was nearly 4 times the number of bruises and contusions (93,650 in 2008), the second highest type of injury of interest to ergonomists . Injuries to the back were the highest category of injuries in the service occupations (about 31%). These findings are similar to those noted in this study . In terms of their contribution to total workers' compensation costs, at the workplace, back problems are the single most costly injury . In 1992, back cases represented 24% of us workers' compensation claims and 31% of costs . After low back pain, the next common body part affected in this sample was the wrist . In a similar study it was found that wrist / hand, elbow and knee pain were more prevalent among cleaners; neck, shoulder, low back, hip, and ankle pain / discomfort was similar to other referent groups, while upper back pain was reported less by cleaners . An occupational injury profile of the american industry states that upper extremity injuries, including injuries to the wrists, the hands, and the fingers, were the next highest category of injuries after back injuries . This correlates to the presence of pain in the back and wrist in descending order in the participants of this study . The prevalence of musculoskeletal pain and the trend in incidence rates in this sample were similar to the prevalence in other studies [24, 30, 31]. This can be explained by the high prevalence of pain among participants who worked with their back in an awkward posture . As it is seen in other studies, it is evident from this study too that the cleaning occupation has a high prevalence of musculoskeletal pain which can predispose the cleaners to musculoskeletal disorders and injuries . Several studies have indicated the various risk factors for musculoskeletal ill - health, high workload, repetitive motion, speed and intensity of work, and weak organizational strategies to name a few . There is evidence that exposure to each of these ergonomic factors causes msds in one or more body regions: repetitive upper extremity motion patterns; forceful exertions, nonneutral body postures; and vibration . The risk is especially pronounced when a job includes exposure to a combination of two or more of these risk factors . All authors found that the most significant risk factors associated with the physical work of cleaners are static muscle loads (much of which involves bending and twisting of the back) and repetitive movements of the arms and hands using a high output of force . These types of prolonged static and repetitive muscle activities cause muscle fatigue and may lead to musculoskeletal disorders . A sizable proportion of msds among exposed workers are preventable, and protective action is both warranted and necessary . Concentrating only on physical ergonomic factors like equipment design, reduction in forces, and improvement in postures may not achieve as much benefits in terms of reduction in sickness rates as a more holistic approach would, which also takes account of work organizational risk factors . A holistic approach is essential to address these issues and reduce the incidence rates of occupational injuries and illnesses . Health and safety researchers and practitioners are constantly trying to control workplace injuries by trying to understand the causal mechanisms underlying workplace injuries and by designing safer working conditions through job, equipment and workplace design, educating employees, and matching employees to jobs . A review by bigos et al . On preventing episodes of low back pain suggests a strong evidence that exercise programs are effective, whereas other interventions including education alone (ergonomic, back school, and stress management), back supports (back belts), and shoe inserts were not effective daily sessions of exercise and relaxation techniques before the employee starts work will help reduce the tension in the muscles thus preparing the body for various work related postures . A study by toivanen et al . Showed that regular relaxation training at the workplace to provide stress management diminished tension in the neck - shoulder region efficiently and decreased absenteeism . Inter correlations were found between the neck - shoulder tension, psychosocial factors, depression, and the absentee rate . Thus a continued application of a combination of regular exercise, relaxation training, participatory ergonomic training programs, team work, work organization changes, and effective communication between the cleaners and the supervisors may help in curbing musculoskeletal ill - health among the housekeepers . First, the findings may not be generalizable as they are not based on representative random sample drawn from hospital cleaners around the country . However, a response rate of 98.1% clearly indicates the validity of findings at that institution . Another limitation is that our study assessed work - relatedness of pain or injury (and the severity of these conditions) by self - report . Data were not available to validate doctor visits for work - related pain or workers' compensation claim reporting and acceptance rates . However, the self - report data was supported by medical or administrative records making it more reliable, and also self - reports can be a more reliable source for determining the frequency of work - related pain . The questions were answered based on the memory of the participant thus the possibility of recall bias exists . Recall may have been influenced by such factors as presence of pain or negative affect . Further research is required to study the risk factors associated with musculoskeletal pain and discomfort in each body part to develop better intervention strategies.
The classification of ehlers - danlos syndrome (eds) is based on the extent of clinical and pathological features . Vascular eds (type iv eds, mim #130050) is the most malignant form and owes its bad reputation to a susceptibility to sudden death from spontaneous catastrophic bleeding or the rupture of blood vessels or hollow organs (1). Type iv eds is characterized by its clinical manifestations: easy bruising, thin skin with visible veins, acrogeria, and the rupture of arteries, uterus, or intestines (2). Vasculopathy involves the rupture, dissection, or aneurysms of the aorta or large arteries, carotid - cavernous fistula (ccf), and intracranial aneurysms . Arterial complications are the leading cause of death in vascular eds because they are unpredictable and surgical repair is difficult because of tissue fragility (3). In a recent review of 419 patients, 70% of deaths were the result of arterial rupture, mostly of thoracic and abdominal vessels, but also of cerebral vessels (1). However, bruisability is very striking, and' ecchymotic - type eds' is a synonym for type iv eds . Type iv eds is inherited in an autosomal dominant fashion but approximately 50% of patients represent new mutations . Most patients have a defect in either the synthesis or structure of type iii procollagen, with a genetic mutation in col3a1 . However, it has been suggested that eds type iv is a genetically heterogeneous disease . Pepin et al . Have reported that only 61% of patients with abnormal type iii procollagen molecules have mutations in the col3a1 gene, whereas the others have no mutation in col3a1 (1). In a recent study, mutations in the genes encoding transforming growth factor receptors 1 and 2 (tgfbr1 and tgfbr2, respectively) have been identified in patients with a provisional diagnosis of eds type iv without the characteristic type iii collagen abnormalities . This new syndrome was designated loeys - dietz syndrome, type 2 (4, 5). Here, we report our experience of three korean patients, in whom a clinical diagnosis of eds type iv was made based on clinical findings . However, a genetic study identified col3a1 mutations in two of these three patients . In the other one patient the diagnoses of these patients were made based on clinical presentation, physical examinations, and radiological findings . Information about deceased family members and other family members who were suspected of being affected was obtained from the probands . Genomic dna was extracted from peripheral blood leukocytes with the wizard genomic dna purification kit (promega, madison, wi, u.s.a . ), following the manufacturer's instructions . The coding exons and their flanking introns of the col3a1 (total 51 exons) on chromosome 2q31, and tgfbr1 and tgfbr2 on chromosome 9q33-q34 and 3p22, respectively, were amplified using primers designed by the authors (available upon request). Direct sequencing was performed with the bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca, u.s.a .) On the abi 3100 genetic analyzer (applied biosystems). A 48-yr - old woman was admitted with sudden severe epigastric pain radiating to the back one day before admission . A spontaneous retroperitoneal hematoma had been treated with percutaneous catheter drainage three years previously . On first presentation, her blood pressure was 121/78 mmhg, her pulse rate was 80 beats per minute, and her heartbeat was regular . A clinical examination showed tenderness in the left lower abdominal quadrant, very thin skin with visible vessels on the thorax, which bruised easily with minimal trauma (fig . The results of a neurological examination were normal . Computed tomography (ct) demonstrated an abdominal aortic dissection with extension from the proximal superior mesenteric artery to both common iliac arteries (fig . Because of the high surgical mortality rate in patients with eds type vi, conservative medical treatment was given . Follow - up ct angiography revealed a progression of the aortic dissection with hemothorax, hemoperitoneum, and retroperitoneal hemorrhage (fig . 2c, a family history revealed that her younger brother had died suddenly from cerebral hemorrhage (fig . 3a). Genetic analysis revealed a g to t transition (c.2195g> t) leading to the substitution of the glycine at position 732 (p.gly732val) of the triple helix of type iii protocollagen with valine (fig . A 36-yr - old woman presented with left ocular pain, headache, diplopia, and tinnitis, which had started one month before presentation . On physical examination, her skin showed a bruise at a trauma site (fig . A family history revealed that her daughter had a chronic subluxation of the proximal interphalangial joint of the left hand (fig . 3b). Magnetic resonance imaging revealed a large ccf, and the diagnosis was confirmed by angiography (fig . The ccf was successfully treated with detachable balloons using a femoral arterial approach . During the procedure, a pseudoaneurysm of the left proximal internal carotid artery occurred, and a stent was inserted . Then, surgical evacuation of a massive hematoma was performed, followed by the insertion of a stent graft into the punctured femoral artery, with arteriotomy to control bleeding . The postoperative course was uneventful, except for ophthalmoplegia and the loss of visual acuity due to a neurotropic corneal ulcer . A mutation analysis by dna sequencing of the col3a1 gene revealed a duplication of 15 base pairs (c.3221_3235dup), which resulted in an interposition of five amino acids (p.gly1074_pro1078dup) and a subsequent formation of a repeat unit composed of only two amino acids (gly - x) (fig . Some of the clinical findings of this patient have been reported previously (6). A 21-yr - old woman presented with left ocular pain, severe pulsatile headache, and vomiting after her left eye was hit with a champagne cork . On physical examination, her skin was so thin that the subcutaneous blood vessels were visible (fig . 7b). There was no significant hyperelasticity of the skin or hypermobility of the joints of the hand . Carotid angiography showed a large ccf and an aneurysm in the cervical portion of the left internal carotid artery (fig . Computed tomography of the thoracoabdominal aorta demonstrated a large ovoid aneurysm of the left renal artery and a medium - sized aneurysm of the splenic artery (fig . Seven years later, she developed a spontaneous hematoma of the left calf, which was relieved by conservative management . A 48-yr - old woman was admitted with sudden severe epigastric pain radiating to the back one day before admission . A spontaneous retroperitoneal hematoma had been treated with percutaneous catheter drainage three years previously . On first presentation, her blood pressure was 121/78 mmhg, her pulse rate was 80 beats per minute, and her heartbeat was regular . A clinical examination showed tenderness in the left lower abdominal quadrant, very thin skin with visible vessels on the thorax, which bruised easily with minimal trauma (fig . The results of a neurological examination were normal . Computed tomography (ct) demonstrated an abdominal aortic dissection with extension from the proximal superior mesenteric artery to both common iliac arteries (fig . Because of the high surgical mortality rate in patients with eds type vi, conservative medical treatment was given . Follow - up ct angiography revealed a progression of the aortic dissection with hemothorax, hemoperitoneum, and retroperitoneal hemorrhage (fig . 2c, a family history revealed that her younger brother had died suddenly from cerebral hemorrhage (fig . 3a). Genetic analysis revealed a g to t transition (c.2195g> t) leading to the substitution of the glycine at position 732 (p.gly732val) of the triple helix of type iii protocollagen with valine (fig . A 36-yr - old woman presented with left ocular pain, headache, diplopia, and tinnitis, which had started one month before presentation . On physical examination, her skin showed a bruise at a trauma site (fig . A family history revealed that her daughter had a chronic subluxation of the proximal interphalangial joint of the left hand (fig . 3b). Magnetic resonance imaging revealed a large ccf, and the diagnosis was confirmed by angiography (fig . The ccf was successfully treated with detachable balloons using a femoral arterial approach . During the procedure, a pseudoaneurysm of the left proximal internal carotid artery occurred, and a stent was inserted . Then, surgical evacuation of a massive hematoma was performed, followed by the insertion of a stent graft into the punctured femoral artery, with arteriotomy to control bleeding . The postoperative course was uneventful, except for ophthalmoplegia and the loss of visual acuity due to a neurotropic corneal ulcer . The patient is alive and doing well at the seven - year follow up . A mutation analysis by dna sequencing of the col3a1 gene revealed a duplication of 15 base pairs (c.3221_3235dup), which resulted in an interposition of five amino acids (p.gly1074_pro1078dup) and a subsequent formation of a repeat unit composed of only two amino acids (gly - x) (fig . Some of the clinical findings of this patient have been reported previously (6). A 21-yr - old woman presented with left ocular pain, severe pulsatile headache, and vomiting after her left eye was hit with a champagne cork . 7b). There was no significant hyperelasticity of the skin or hypermobility of the joints of the hand . Carotid angiography showed a large ccf and an aneurysm in the cervical portion of the left internal carotid artery (fig . Computed tomography of the thoracoabdominal aorta demonstrated a large ovoid aneurysm of the left renal artery and a medium - sized aneurysm of the splenic artery (fig . Seven years later, she developed a spontaneous hematoma of the left calf, which was relieved by conservative management . Here, we report three patients with a provisional diagnosis of eds type iv . The first and second patients had mutations of the gene for type iii procollagen (col3a1). Although the other patient showed clinical features compatible with eds type iv, genetic analysis of col3a1 did not reveal a mutation . Absence of mutations in patients 3 might be either due to a deletion of single or multiple exons in the col3a1 gene or due to a genetic heterogeneity . Description of patients with eds type iv - like features in korea is quite rare (7). To the best of our knowledge, patient 1 and 2 are the first korean patients with a molecular diagnosis of type iv eds . The mutation found in patient 1 is a novel mutation rather than a genetic variation because most of known missense mutations of col3a1 involves glycine residue and the same variation of the patient was not found in either other family members with normal phenotype or fifty control subjects . Also, any genetic alteration at the duplicated location of patient 2 had not been previously reported in eds type iv . The nature of the mutation found in patient 2 was quite different from the vast majorities of other previously reported mutations on the col3a1 gene, which usually were point mutations affecting the glycine residue of the normal gly - x - y repeats and caused classical eds type iv (1). As a result of duplication of 15 base pairs, an interposition of five amino acids among the triple repeats has occurred, and a subsequent formation of a repeat unit consisting of two amino acids (gly - x) just before the duplicated segment was observed . These atypical properties of the mutation in this patient might have had an effect on her phenotypic features that lacked one of the cardinal manifestations of classical eds type iv, i.e. Thin transparent skin . We previously reported patient 3 as having eds type iv (6), demonstrating that a genetic diagnosis is mandatory for a final diagnosis in patients with the clinical features of eds type iv . Recent developments in genetic diagnosis have broadened the clinical spectrum of type iv eds . In the largest series of patients with eds iv, pepin et al . Analyzed the col3a1 gene in 220 index patients who had biochemical defects in type iii collagen (1). They identified causative mutations in the col3a1 gene in only 135 index cases (61%). In other words, 39% of patients with eds iv, with abnormal type iii collagen, had a mutation in a gene other than col3a1, which indicates that even eds iv with a proven defect in type iii collagen is a genetically heterogeneous disease . Furthermore, in a recent study, mutations in the tgfbr1 and tgfbr2 genes have been reported in loeys - dietz syndrome type 1, which is characterized by the triad of arterial tortuosity and aneurysms, hypertelorism, and bifid uvula or cleft palate (5, 6, 8). Mutations in the gene encoding tgfbr1 or tgfbr2 were also found in 12 (30%) of 40 patients with eds type iv without the characteristic type iii collagen abnormalities . This new disease was designated loeys - diets syndrome type 2, distinguishing it from type 1, which has additional craniofacial abnormalities . Based on these findings, we propose new categories of patients who have eds - type - iv - like phenotypes (table 2). Patient 1 and 2 belongs to category i. patients 3 could be classified into category ii or iv . However, there is a possibility that a causative genetic abnormality in patients 3 could be a deletion of single or multiple exons in the col3a1, tgfbr1 or tgfbr2 gene . The first patient had the most severe phenotype of the three patients and died of rapidly progressive aortic dissection with rupture . Some authors have suggested that the nature of a mutation relates to the patient's phenotype (9). Mutations in the middle or at the 5' end of a gene produce more variable phenotypes and milder variants compared with those caused by mutations around the 3' end, which have severe manifestations . However, pepin et al . (1) demonstrated no apparent relationship between the type of complication and the locations of mutations . Even though patient 1 had a substitution in the middle part of the col3a1 gene product (732nd codon of 1,466 amino acids) and patient 2 had a duplication in the 3' part of the col3a1 gene (1,074th to 1,078th residues of 1,466 amino acids), patient 1 had relatively severe and full - blown clinical manifestations compared to those of patient 2 . Pepin et al . Reported that, among 183 pregnancies with 167 deliveries of live - born infants at term, 12 women died (1). However, it is not known whether the use of elective cesarean section reduces mortality . The age at death of our patient was 48, which is consistent with a previous report in which the calculated median survival of the entire cohort of 220 index patients was 48 yr (1). Ccf is exceedingly rare in the general population, among whom the vast majority is posttraumatic . Spontaneous ccf is one of the most common central nervous system vascular complications of eds iv . North et al . Reviewed these cerebral vascular complications in 202 individuals with biochemically confirmed type iv eds (10). Of these, one of the striking features of patients 2 and 3 was their vascular complications after invasive vascular procedures . The arteries of both patients were so fragile that they were easily dissected during catheter and guidewire interventions or the ballooning of arteries . They also showed severe complications at transfemoral puncture sites, including giant groin hematoma or severe ecchymosis, which required surgical evacuation of the hematoma and insertion of a stent graft into the injured artery . Therefore, as far as possible, conventional arteriography should be avoided because of the high risk of massive hematoma and/or arterial dissection, as noted in our patients . In an attempt to avoid arterial dissection or rupture, ccf could be closed via a venous rather than the more standard arterial route (11). In conclusion, we emphasize that patients with a presentation suggestive of eds iv should undergo biochemical analysis of their type iii collagen and dna analysis of the col3a1, tgfbr1, and tgfbr2 genes . However, these laboratory tests cannot identify the genes causing the category ii or iv disease, which await further progress and understanding of the molecular pathogenesis of patients with eds - iv - like phenotypes.
Lead poisoning is one of the most serious environmental health hazards, with a particularly acute effect on young children . The u.s . Centers for disease control and prevention estimated that approximately 2.5% of children aged 15 years in the u.s . Have elevated blood lead levels . The percentage of affected children in other countries is expected to be even higher . Lead poisoning causes learning disabilities, behavioral problems, and, at very high levels, seizures, coma, and even death . The mechanism of pb toxification mainly involves binding of pb to proteins and the subsequent inhibition of the proteins physiological functions in blood and tissues . The documented proteins targeted by pb include several zinc enzymes or proteins (such as -aminolevulinic acid dehydratase (alad), acetylcoline esterase, cys2his2 zinc - finger proteins, and acid phophatases) and calcium - binding proteins (calmodulin, calbindin, and troponin c). Pb can replace zinc and calcium at the oxygen / nitrogen / sulfur - rich active sites of these proteins, thereby inhibiting the protein functions by altering their coordination chemistry and native structures . For example, the function of alad, an enzyme involved in the second step of heme biosynthesis, is altered by pb binding at the active site via a trigonal pyramidal geometry . Consequently, the hemoglobin synthesis is blocked, leading to anemia . To reduce the pb - induced toxicity, organisms have developed various defensive mechanisms with species such as metallothioneins (mts), glutathione, phytochelatins, and lead - binding proteins (pbbps, which are non - mt acidic proteins that have not been fully characterized). Mts, a class of thiol - rich (up to 30% of its amino acid residues), low - molecular - weight proteins whose abundance is particularly high in the liver and kidneys of mammals, are perhaps the most important species for lead detoxification . Mts is crucial to a better understanding of the chemical stabilities, biological functions, and detoxification mechanism of pb mts . However, due to the absence of single crystals of pb mts, information about the pb the coordination and binding stoichiometry between mts and metals are dependent on the type of metal ions . Usually, divalent metal ions, such as cd and zn, are tetrahedrally coordinated by four cysteine sulfurs and bind to mts with a stoichiometry of 7:1 . It can be complexed by a combination of s, o, n, and p - donor ligands with a coordination number ranging from 2 to 9 . When pb binds to sulfur - rich proteins, three sulfurs in a trigonal pyramidal geometry and the pb 6s lone - pair electrons occupying the axial position (hemidirected) constitute the coordination sphere . A number of complexes containing the pb s3 coordination have been observed using spectroscopic and mass spectrometry (ms) methods . In the presence of biomolecules possessing several distinct donor ligands (n, o, and s), the pb coordination chemistry is diverse and includes formation of pbsxoy and pbsxny . Owing to the presence of o- and s - donor ligands in mts and the unique electronic configuration of pb, coordination of pb with mt is of higher complexity than that of zn or cd . Visible (uv vis) spectra and microcalorimetry have suggested that different pb palacios et al . Demonstrated with electrospray ionization mass spectrometry that the metal content in pb mt2 complexes is dependent on the solution ph (neutral or 4.5). Using extended x - ray absorption fine structure (exafs), vasak et al . However, crucial questions such as the difference of pb coordination and the chemical stability and physiological function of the two pb theoretical calculations have been widely used for the prediction of structures and spectra of metalloproteins . The combination of quantum mechanics and molecular mechanics - based hybrid (qm / mm) method allows two or more computational methods to be performed in a single calculation, making it possible to investigate the chemistry of complex systems with high precision . In this context, the oniom (our own n - layered integrated molecular orbital + molecular mechanics) scheme is a general approach because it can combine any number of molecular orbital and molecular mechanics methods . The oniom method has been successfully utilized in the elucidations of structural and functional properties of many metalloproteins such as cyt c and azurin . In our study, optical methods (uv vis absorption and circular dichroism (cd) spectrometry) and nmr were used in tandem with the two - layer oniom method to investigate the effect of ph on the structures of two different pb the differences in the coordination chemistry of the metal centers and the protein structures at various ph were deciphered . We also investigated the chemical stabilities and structural flexibility of these two complexes in proteolytic processing to gain insight into the lead detoxification process involving mts . Zn - containing mt2, isolated from rabbit liver, was purchased from hunan lugu biotechnology co. (changsha, china). Individual metal - free domains (apo-mt2 and apo-mt2) of both rabbit liver and human mt2s, and the corresponding mutants (d25n - apo-mt2 and d2n - apo-mt2) were synthesized by shanghai apeptide co. (shanghai, china). All the domains and their mutations were confirmed by mass spectrometry performed by the vendor, and the corresponding purity values (> 95%) were determined with hplc - ms . Pb (99.1%) was acquired from isoflex usa (san francisco, ca). Lead nitrate, 5,5-dithio - bis-(2-nitrobenzoate) (dtnb), and cathepsin b were purchased from sigma - aldrich (st . Deionized water with resistivity of 18.2 m cm was collected from a millipore simplicity 185 system (millipore co., billerica, ma). All solutions were prepared with deionized water and degassed with nitrogen for at least 30 min . A 3 kda cutoff millipore (ym-3) membrane (millipore, billerica, ma), equilibrated with 0.01 mol l hcl, was used to separate zn from zn7mt2 . After spinning at 13 000 rpm for 30 min at room temperature in an eppendorf 5417r centrifuge (eppendorf, hamburg, germany) complete removal of zn from zn7mt2 was confirmed by the disappearance of the characteristic absorption of zn7mt2 at 220 nm . The apo - mt2 concentration was determined by assaying thiol groups with ellman s reagent, dtnb . Mt2 solution was stored in a nitrogen - saturated flask to avoid thiol oxidation in apo vis absorption experiments were carried out on a uv-2450 spectrophotometer (shimadzu, japan) in quartz cuvettes (1 cm path lengths). Cd data were obtained with a jasco-810 spectrophotometer (jasco corporation, japan). Zeta potentials of individual apo - mt2 domains or their mutants were measured at room temperature in a folded capillary cell with a zetasizer nano zs instrument (malvern instruments, southborough, uk). Pb was dissolved with 0.15 m trace metal grade nitric acid (fisher scientific) at 250 c, and the pb(no3)2 precipitate was collected, dried, and weighed . Appropriate amounts of mt2 (250 m), kcl (10 mm), and d2o were mixed under n2 atmosphere . Both pb7mt2 complexes were prepared by adding pb(no3)2 to obtain an mt2/pb stoichiometry of 1:7 at corresponding ph (7.0 or 4.0). The resultant solutions were allowed to incubate for 1 h, and the solution ph was brought up to ph 7.0 with koh . D2o was added to a final volume of 1 ml, and the solution was transferred to an nmr tube . All pb nmr spectra were recorded on a bruker advance drx-400 mhz spectrometer at 25 c using 60 pulses, a 2 s relaxation delay, and a 0.12 s acquisition time (spectral width of 555.6 khz). A linear prediction was performed to remove the noise, and the real free induction decay (fid) was determined before data processing . After zero - filling, the data (128 000 data points) were processed with an exponential line broadening of 5 hz using the software topspin nmr . The pb nmr chemical shifts are reported downfield from tetramethyllead (= 0 ppm; toluene) using 1.0 m pb(no3)2 salt (fisher) as an external standard (= 2990 ppm, d2o, 25 c; relative to tetramethyllead). Both pb7mt2(i) and pb7mt2(ii) complexes were freshly prepared and diluted in 10 mm kcl solution (ph 7.0) at 37 c to desired concentrations . Cathepsin b (1.8 ng), with a specific activity of 3000 pmol min g, was mixed with 504 pmol of pb7mt2(i) or pb7mt2(ii) (the final concentration of pb was 57 m) in 10 mm kcl solution (ph 5.0). Oniom calculations were performed to predict the optimal geometries and electronic absorption spectra of pb7mt2 complexes . The initial atomic coordinates of pb4mt2 and pb3mt2 were taken from the corresponding pb substituted cd4mt2 and cd3mt2, respectively . The structures of cd4mt2 and cd3mt2 were retrieved from the rcsb protein databank (pdb i d: 1 mrb for the -domain and 2 mrb for the -domain), in which the absent hydrogen atoms were added using gaussview 4.0 . The protonation states of titratable residues (e.g., aspartic acid and lysine) of pb4mt2 and pb3mt2 at ph 4.0 and 7.0 were determined using pka values estimated with propka 2.0 . The electronic structures of pb4mt2 and pb3mt2 were modeled with the inclusion of the protein environment, using the two - layer oniom (qm / mm) model . The qm region comprises the active sites of pb4mt2 and pb3mt2, and the mm region contains the protein environment (cf . Spin unrestricted density functional theory (dft) with the becke s three - parameter hybrid exchange functional and the lee yang parr correlation functional (b3lyp) or pure functional bp86 were used for the qm system, and the dunning basis set aug - cc - pvtz - pp was used for pb and 6 - 31+g * basis set for other atoms for the qm calculations . For the mm region, the protein molecule was treated using the universal force field (uff). An electronic embedding scheme was adopted to deal with the electrostatic interactions between the qm and mm regions in the qm / mm calculations . On the basis of the optimized geometries, vertical excitation energies were computed within the oniom scheme by employing the time - dependent density functional theory (tddft). Tddft calculations were carried out with the density functional b3lyp and bp86 . The trizeta basis set aug - cc - pvtz - pp was used for pb, and 6 - 31+g * * was used for others . The results were transformed via the swizard program (version 4.6) into each uv spectrum using gaussian functions with half - widths of 3000 cm . Uv vis absorption and cd spectra were recorded during the titration of rabbit liver apo - mt2 with pb at ph 7.0 (figure 1). For simplicity, we termed the pb mt2(i) exhibits a characteristic peak centered at 330 nm (figure 1a). The time - resolved absorbance changes (inset) indicates that the complexation reaction is fast (completed in less than 10 min). During the titration of apo mt2 with pb at neutral ph, some pb(oh)2 (ksp = 1.4 10) precipitate was formed and affected the absorbance value of pb7mt(i) at 330 nm, as evidenced by the small fluctuation even after 30 min . The precipitation can be avoided by lowering the solution ph below 6.0 . As shown in the inset of figure s1 in the supporting information, the absorbance value of pb7mt(i) remains stable after the complexation reaction is completed . As indicated by the mt2/pb stoichiometry (figure 1b), the maximum binding stoichiometry is 1:7, consistent with results reported from the substitution experiment of zn7mt2 with pb . The absorption wavelength (330 nm) and extinction coefficients (3500 m cm) of pb7mt2(i) are analogous to values reported for several pbs3 complexes . The secondary structural variation from apo - mt2 to pb7mt2(i) is shown in figure 1c . This is in contrast with the intense cd peaks of cd7mt2 and zn7mt2 between 210 and 290 nm . In zn7mt2, the characteristic cd band centering at 244 nm is attributed to the zn(sr)4 chromophore . Similarly, the cd bands of cd7mt2 at 242 and 262 nm can be attributed to the excitation coupling between adjacent pairs of the cd(sr)4 chromophore . Since the m(sr)4 chromophore leads to the appearance of these cd peaks, the absence of any obvious cd peaks in figure 1c suggests that pb7mt2(i) adopts a different metal coordination geometry from those of cd7mt2 and zn7mt2, as alluded to in the introduction . The individual - and -domains display uv vis and cd spectral features (data not shown) similar to those of apo - mt2 during the titration with pb, indicating that the metal centers in the two different domains have similar coordination spheres . Quantitative analysis confirms that the pb / mt2 stoichiometric ratios are 4:1 and 3:1 in the - and -domain, respectively . Time - dependent (a) uv vis absorption and (c) cd spectra in 10 mm kcl solution (ph 7.0) containing 7.20 m rabbit liver apo - mt2 and 20 mol equiv of pb . (b) dependence of uv absorption peak at 330 nm upon addition of pb to apo - mt2 . The pb7mt2(ii) complex displays dramatically different uv vis absorption and cd spectra (figure 2) from those of pb7mt2(i). In the uv vis absorption spectra, two intense peaks at 325 and 375 nm appear, with the latter having a shoulder peak at 400 nm . In the cd spectra (figure 2c), a strong envelope with maxima at 240 (+), 265 (+), 320 (), 350 (+), 370 (+), and 395 () is produced isodichroically (280, 340, and 375 nm). Positions of all cd peaks are invariant with the pb / apo mt2 ratio (increased stepwise from 1:1 to 7:1), but the peak intensity increases with the ratio . The cd spectra of cd7mt2 have two bands at 240 and 260 nm split from the 250 nm band, which corresponds to the conversion of isolated cd(sr)4 to the (sr)3cd sr thus, the unsplittable pb7mt2(ii) cd peak is indicative of the absence of excitation coupling between adjacent chromophores . The multiple peaks in the uv vis absorption and cd spectra suggest that pb in the pb7mt2(ii) complex has binding modes that are distinctively different from cd in cd7mt2 . Similar to pb7mt2(i), the pb / mt2 stoichiometric ratios in the pb7mt2(ii) complex are 4:1 and 3:1 in the - and -domains, respectively . The uv vis absorption peaks of pb4mt2(ii) are slightly shifted, with higher intensity than those of pb3mt2(ii) (figure 3d). These differences suggest that the metal centers in the two domains have different coordination geometries . To pinpoint the ligands responsible for coordination of pb in the two different domains at neutral and acidic ph time - dependent (a) uv vis absorption and (c) cd spectra in 10 mm kcl solution (ph 4.5) containing apo - mt2 (7.20 m) and 20 mol equiv of pb . (inset) the time - resolved absorbance changes at 325 and 375 nm, respectively . (b) the dependence of uv absorption peaks at 325 and 375 nm on the addition of pb to apo - mt2 . Structures and electronic absorption spectra of the - and -domains in (a, c) pb7mt2(i) and (b, d) pb7mt2(ii) obtained by experimental and oniom methods . In panels a and b, the dark gray spheres are pb, yellow spheres are s, red spheres are o, gray spheres are c, and white spheres are h atoms . The oniom method has been successfully used to predict metalloprotein structures . To validate the method for the studies of mt2 structures, structural optimization of cd4mt2 and cd3mt2 models compared to the nmr results, the computed structures of the metal clusters display little deviation (e.g., the bond lengths have a root - mean - square deviation of only 0.040.05 when compared to the experimental data) (figure s2 and table s1 in supporting information). Therefore, we conclude that the oniom method is viable for the studies of the two pb7-mt2 complexes . The theoretical calculations were performed separately on the - and -domains on the basis that the two domains are structurally independent . The initial atomic coordinates of each domain in pb7mt2 were respectively adopted from the pb - substituted cd4mt2 and cd3mt2 because a well - established model of apo - mt2 is not available . The protonation states of titratable residues (e.g., asp and lys) at different ph were determined from the pka values estimated with propka 2.0 . In the two - layer oniom model of pb7mt2(i), the qm region consists of four pb atoms, and the side chains consist of eleven cysteine residues in the -domain . As for the -domain, the three pb atoms and the side chains of nine cysteine residues constitute the qm region . Figure 3a displays the optimized molecular geometries of the - and -domains in pb7mt2(i), which are quite different from the well - characterized cd7mt2 and zn7mt2 structures . In cd7mt2 and zn7mt2, each metal adopts the tetrahedral coordination with terminal and bridging thiolates to form a metal ligand six - membered ring in the -domain and two fused six - membered rings in the -domain. (cf . Figure s2 in supporting information) however, all seven pb ions in pb7mt2(i) are trigonally coordinated by three cysteine sulfurs (pb s3) without any metal these significant differences can be attributed to the pb 6s lone - pair electrons, which disrupt the tetrahedral coordination by occupying the axial position with a significant stereochemical activity . This point is in line with the report by godwin and co - workers, who stated that pb s4 is not a preferred coordination and that trigonal pyramidal geometry in all - sulfur coordination is predominant . Moreover, due to the pb s3 coordination, only one bridging cysteine (pb6s18pb7 in the -domain) remains, preventing a pb according to the corresponding structural parameters (table 1), the average length of the pb s bonds is 2.68, close to the reported exafs value (2.65). The electronic absorption spectra (red line curves), simulated by tddft using the b3lyp functional, are overlaid with the experimental results (black line curves) in figure 3c . The agreement between the simulation and experimental data in the oscillator strengths indicates that the optimized molecular geometries of pb4mt2(i) and pb3mt2(i) are reasonable . Detailed analysis of vertical excitation energies, oscillator strengths, and molecular orbital contributions (table s3 in supporting information) indicated that four transitions contribute to the absorption peak at 330 nm . These transitions are homolumo, homo-1lumo, homo-2lumo, and homo-1lumo+1 . From the frontier molecular orbitals (figure s3 in supporting information), lumo and lumo+1 (the final state of the transitions) s bonds, while homo and homo-1 (the initial states of the transitions) are largely localized at the sulfur atoms . Therefore, the electronic absorption band at 330 nm can be attributed to the spb ligand - to - metal charge transfer (lmct). In the two - layer oniom model of pb7mt2(ii), we initially assigned the qm region of each domain to be the same as that of pb7mt2(i). However, the optimized molecular geometries revealed two unexpected short pb / o distances (5.52 for pb1 with the carbonyl oxygen of asp2 in the -domain and 4.31 for pb6 with the carbonyl oxygen of asp56 in the -domain). These short distances should result from the electrostatic interaction between the protein surface (the mm region) and the negatively charged pb acidic ph conditions, the protein surface is neutral or positively charged, causing the mm and qm regions to move closer, thereby shortening the distance between the pb moreover, based on the pka values of asp (4.00), both asp residues are neutral in weakly acidic solution, which are more favorable than the negatively charged (deprontonated) form to the positioning of the negatively charged pb these pb / o distances are close to the sum of the van der waals radius of pb and o (3.54), indicating the pbo interaction must be taken into account . We therefore modified the initial qm region in each domain by including the respective asp residue . As shown in figure 3b, the optimized molecular geometry of pb7mt2(ii) shows an entirely different coordination sphere from that of pb7mt2(i). The overall coordination sphere includes the trigonal pyramidal pb s3 mode, the distorted trigonal pyramidal pb s2o1 mode in the -domain, and the distorted quadrilateral pyramidal pb s3o1 in the -domain . The pb 6s lone pair electrons occupy the axial position with a significant stereochemical activity, resulting in hemidirectionality in the pb ligand coordination . Moreover, due to protonation of the peptide side chain, several cysteine sulfurs (especially in the -domain) are closer to the protein exterior and increase the effective radius of the metal center and the pb s bond length (cf . The loosened structure of the metal center renders a higher flexibility to pb7mt2(ii). The simulated oscillator strengths are in good agreement with the experimental data (figure 3d), indicating that the optimized molecular geometries of pb4mt2(ii) and pb3mt2(ii) are reasonable . The small deviation between the electronic absorption spectra of the two domains (cf . Detailed vertical excitation energies and molecular orbital contributions (table s5 and figure s4 in supporting information) indicate that all three bands are primarily associated with the spb lmct . Note that all of the above calculations are based on b3lyp, a hybrid functional to fit data primarily for main - group elements . Because of the large exact exchange component (20%), b3lyp is known to favor loose electron densities and low transition energies . To confirm the reliability of b3lyp, the pure functional bp86, a generalized - gradient approximation (gga) class with zero exact exchange, is used as a control study on the geometry optimization and calculations of the excited states for both pb7mt2 complexes . The simulated structures of both pb7mt2 complexes from bp86 pure functional (table s69 in supporting information) are similar to those from b3lyp hybrid functional . However, the spectra from bp86 pure functional display a significant deviation from the experimental data (figure s5 in supporting information), indicating that the b3lyp hybrid functional is a better choice . The b3lyp hybrid functional produces better spectral accuracy, which can be attributed to its increased amount of hartree local minima in the qm optimization . In the qm / mm calculation of biomolecules, the vast size of the available configuration space may cause the qm optimization to stop at local minima . One way to circumvent this problem is to use a reliable structure (e.g., a structure deduced from nmr or x - ray crystallography) at the beginning of the qm optimization . Have used the x - ray structure of azurin as the initial structure in the qm / mm calculation of metal - substituted azurins and obtained noticeable structural changes on the active sites when cu in azurin was substituted by metal ions such as co, ni, or zn . We adopted this approach by using the pb - substituted cd4mt2 and cd3mt2 structures deduced from the nmr experiments as the initial structures . The theoretical spectra agree well with the experimental results, suggesting a high level of reliability of the calculation and in the predicted structures . To provide more experimental evidence to our computational results about the two different pb7mt2 complexes, we conducted pb nmr in solutions of the two complexes (figure 4). Pb7mt2(i) displays one pb peak at 5679 ppm, while pb7mt2(ii) exhibit two peaks at 5820 and 4348 ppm . Thus it is clear that the pb coordination in these two complexes are different . Furthermore, the pb signal of pb7mt2(i) at 5679 ppm is well within the chemical shift region (from 5600 to 5800 ppm) where pbs3 species with the trigonal pyramidal coordination are observed . Thus we conclude that pbs3 is the binding mode in pb7mt2(ii). For pb7mt2(ii), the peak at 5820 ppm is also assigned to the pbs3 coordination, given its close vicinity to the 56005800 ppm region . Compared to the pb7mt2(i) peak, the shift by 141 ppm can be attributed to the changes of the aforementioned pb s bond length and the s pb s bond angle in the pbs3 coordination . The lower intensity is indicative of the decreased number of pbs3 clusters in pb7mt2(ii). The conversion of metal centers in the pbs3 coordination to a different binding mode contributes to the appearance of the peak at 4348 ppm . It is well - known that the nmr signal of pb bound to o - containing ligands is shifted upfield with respect to that bound to s - containing ligands . Pb nmr spectra of (red) pb7mt2(i), (blue) pb7mt2(ii), and (black) pb(no3)2 at ph 7.0 . The carbonyl group of asp is the only o - donor ligand in mt2 . To further verify the formation of the pb o bond in pb7mt2(ii), a titration was performed by adding pb into a peptide solution whose asp residue had been mutated with asn (i.e., d25n - apo-mt2 and d2n - apo-mt2). Zeta potential and cd measurements did not show discernible changes in the surface charge and structure of both mutants (figure s6 and s7 in supporting information), confirming the viability of these mutants for coordination studies . At neutral ph, both uv vis absorption and cd spectra of the two mutants show features similar to those in the same spectra of pb4mt2(i) and pb3mt2(i), verifying that asp does not participate in the pb coordination in pb7mt2(i). At acidic ph (ph <5), the uv vis absorption spectra of both mutants display only a single peak at 330 nm, and the cd spectra did not reveal any changes . While these features are rather different from the corresponding spectra of pb4mt2(ii) and pb3mt2(ii), they are analogous to those of pb4mt2(i) and pb3mt2(i). We therefore conclude that asp is involved in the coordination of pb in pb7mt2(ii). The unique uv vis absorption spectra of pb7mt2(ii), with multiple peaks and the presence of a new pb nmr peak at 4348 ppm, are well - correlated with the mutational study . Another piece of evidence for the formation of pb o bond is the transition from a single peak to three peaks in the time - dependent spectra of a mixture of pb / apo - mt2 at ph 4.55.0 as well as the appearance of multiple peaks in the cd spectra (cf . However, because of the negative impact of acidity on the formation of the pb s bond (cf . The proton - releasing process shown in reaction 1) and the rearrangement of the peptide chain, the pb therefore, under more acidic conditions (ph <4.5), the time - dependent peak transition disappears, and the spectra show features typical of pb7mt2(ii).1 time dependence of (a) uv vis absorbance and (b) cd spectra in a kcl solution after the addition of 7 mol equiv of pb to an apo - mt (7.20 m) solution at ph 5.0 . Reaction times from bottom to top: 0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 175, and 180 min . To study the acid tolerance of both pb7mt2 complexes, a series of spectrophotometric titrations at different ph was performed . The corresponding complex was confirmed by the appearance of characteristic uv vis absorption peaks of pb7mt2(i) or pb7mt2(ii). The results show that pb7mt2(i) is formed above ph 5.0, whereas pb7mt2(ii) is produced at more acidic ph . Pb7mt2(i) can be transformed to pb7mt2(ii) by adjusting the solution ph to 5.0 or lower . However, once pb7mt2(ii) is formed, it remains stable at neutral ph . Our demetalation experiments (figure s8 in supporting information) indicate that ph 2.5 is sufficiently low for the complete removal of pb from pb7mt2(i), but stripping pb completely of pb7mt2(ii) requires a ph as low as 2.0 . These results (summarized schematically in figure 6) indicate that pb7mt2(ii) has a higher tolerance toward an acidic environment than pb7mt2(i) has . Moreover, pb7mt2(ii) remains stable even in the presence of apo - mt at neutral ph (data not shown). We believe that the greater acid tolerance and higher structural stability of pb7mt2(ii) results from the pb o bond . Such a finding has a significant implication to the lead detoxification process in physiological milieu . The pb - inflicted toxicity stems from its tight binding to a variety of sulfur - rich proteins, such as gata proteins and the steroid receptor dna - binding domains . Mt2 reduces the pb - inflicted toxicity by seizing free pb or sequestering pb from these proteins to recover the native protein function . Higher acid tolerance and greater structural stability render mt2 a greater power in effectively scavenging pb in different environments . As shown in reaction 1, mt2 accompanies the release of h, which in turn increases the acidity in a highly localized region (e.g., in cytosol). Moreover, it has been reported that elevated pb concentrations induce the stress level of various organisms, and acidity is also correlated with the stress level . We posit that pb7mt2(ii) is more effective than pb7mt2(i) in lead detoxification, given its greater structural stability and acid tolerance . Elucidation of the metabolism of the pb - mt complexes is vital for understanding the lead detoxification by mts . Some reports have suggested that exogenous mt is processed mainly by the lysosomal protease, and the rate of mt degradation is dependent on the types of metals bound by mts . Four different cathepsins have been identified in lysosomes, and the cysteine protease (cathepsin b, l, and h) is the principal protease for mt degradation . Variations in the concentrations of the two pb7mt2 complexes in the presence of cathepsin b were measured by uv vis absorption spectrometry . In figure 7, the pb7mt2 concentrations were normalized with respect to their initial concentrations . Within 120 min, pb7mt2(ii) such a higher degradation rate can be attributed to the more flexible structure of the pb7mt2(ii) complex . Cathepsin b is an endopeptidase that cleaves internal peptide bonds and favors a large hydrophobic side chain in the substrate protein . Side chains on the amino acids dock into the cathepsin s subsites, whose interaction with the protein substrate is dependent on the flexibility of substrate protein . In mts, the existence of bridging cysteine sulfurs compacts the metal center, which dominates the protein folding . In pb7mt2(ii), due to the lack of bridging cysteine sulfurs, the metal center is loosened, which improves the flexibility of the protein . Moreover, when the pb s3o1 become distorted . Consequently, pb ions are positioned farther from the cysteine sulfurs, and the pb s bonds are weakened . Both processes facilitate the conformational adjustment of pb7mt2(ii). The significantly improved flexibility of pb7mt2(ii) facilitates the protein in the induced - fit model with cathepsin b. as a result, the proteolytic processing of pb7mt2(ii) is greatly accelerated . In living organisms, the acidity in lysosome is about 5, which is sufficiently low to cause the structural conversion from pb7mt2(i) to pb7mt2(ii) and to accelerate the degradation of pb mt2 complexes . Time - dependent proteolytic processing of (black) pb7mt2(i) and (red) pb7mt2(ii) by cathepsin b. the coordination of pb with individual human apo mt2 or apo mt3 domains at different ph was also studied by uv vis absorption and cd spectrometry . Mutational studies revealed that the asp residue is also essential for the ph - dependent structural variation . In line with the data observed for the rabbit liver mt2, the structure - dependent chemical and biological activities of pb7hmt2(ii) formed between human mt2 and pb have higher acid tolerance, more coordination stability, and faster proteolytic processing than pb7hmt2(i) (figure s10 in supporting information). Our results suggest that there exists a commonality in the pb coordination chemistry among mammalian mt2s . In this work, the ph - dependent coordination chemistry between pb and mt2 was systematically studied . The combination of spectroscopic studies and oniom calculations provided a detailed description of the two different pb7mt structures . The results and structures were further verified by pb nmr and mutational experiments . The similar structural, chemical, and biological properties between rabbit liver pb7mt2(ii) and human pb7mt2(ii) suggest a commonality in the pb coordination chemistry among mammalian mt2s . The higher acid tolerance, greater coordination stability, and faster degradation rate of pb7mt2(ii) have significant implications for the pb detoxification process . Specifically, mt2 reduces the pb - inflicted toxicity by seizing free pb in the cellular milieu or by sequestering pb from pb - inflicted proteins . The unique properties of pb7mt2(ii) render mt2 a greater power to effectively scavenge pb in different environments (e.g., in a localized acidic cytosol region). Moreover, the greater flexibility of pb7mt2(ii), resulting from the absence of bridging cysteine sulfurs, helps to accelerate its processing by lysosomal protease . The structural conversion from pb7mt2(i) to pb7mt2(ii) is likely to occur in the acidic environment of lysosome, facilitating the effective detoxification and metabolism of pb.
We applied the blastp program (5) to identify, for each predicted protein sequence in the h37rv genome (genbank accession no . Ae000516). Following genbank annotations, we compared 3,972 predicted proteins in h37rv with 4,187 predicted proteins in cdc1551 . We used a strict search criterion (e = 10 - 50) to identify truly orthologous gene pairs . We aligned (6) the putative orthologous pairs of amino acid sequences (n = 3,428), then imposed this alignment on the dna sequences . Visual inspection of amino acid alignments showed that certain alignments, usually near the n - terminus or c - terminus, had regions of very low sequence identity . Examination of the dna sequences of the corresponding genes showed that these regions of low identity were typically caused by a frameshift in one of the two genomes relative to the other . Whether these frameshifts are biologically real or result from sequencing error was uncertain; therefore, we eliminated 119 such gene pairs from our data set . For the remaining gene pairs (n = 3,309), we computed the proportion of synonymous substitutions per synonymous site (ps) and the proportion of nonsynonymous substitutions per site (pn) by using nei and gojobori s method (7). Because values of ps and pn were very low in most cases, we did not correct for multiple hits . Because ps values appeared to fall into two groups (see results), we used a simple probabilistic model to separate these two sets of gene pairs . We assumed that the probability of synonymous substitution followed two separate binomial distributions, designated models a and b, with probabilities of success (i.e., of a synonymous difference) designated pa and pb, respectively . Using the bayes equation, for each gene pair with a given ps value, we computed the probability that model a applies, given the observed ps: p(a\|ps) = (psa) fa /[(psa) fa + (psb) fb], where fa is the frequency of cases to which model a applies, fb the frequency of cases to which model b applies, psa is the binomial probability of obtaining the observed ps, given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pa; and psb is the binomial probability of obtaining the observed ps, given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pb . Of 3,309 pairs of putatively homologous protein - coding genes in the h37rv and cdc1551 genomes of m. tuberculosis, 2,662 (80.5%) showed no synonymous or nonsynonymous nucleotide differences between the two genomes, and 3,010 (91.0%) showed no synonymous differences between the two genomes . However, in a small number of gene pairs, the proportion of synonymous differences per synonymous site (ps) was surprisingly high . In 13 (0.4%) gene pairs, ps was> 0.01, and in 3 gene pairs ps exceeded 4% . These extreme ps values seen in a small number of gene pairs are much higher than generally observed between alleles at neutrally evolving loci in eukaryotes (8). Thus, the comparison of protein - coding genes between the two m. tuberculosis genomes suggested the existence of two distinct groups of gene pairs: a large group having few or no synonymous differences and a much smaller group with a substantial degree of synonymous divergence . We used a simple probabilistic model (see methods) to separate these two sets of gene pairs, designated group a and group b, respectively (figure). The application of this method showed 11 loci with unusually high ps values and probabilities of assignment to group a of <50% (figure). A plot of p(a\|ps), the probability of assignment of a locus to group a given the observed ps value, as a function of ps at 3,309 loci compared between the h37rv and cdc1551 genotypes of mycobacterium tuberculosis . The plot shows the bimodal nature of the distribution of ps values, with overall higher values of ps at the 11 loci having p(a\|ps)> 50% . We assumed that group a members are truly orthologous gene pairs that diverged at the time of the common ancestor of the h37rv and cdc1551 genomes . Group a included 3,298 pairs, with mean ps for all genes of 0.000328 0.000022 standard error . When ps was estimated for the 3,298 genes concatenated together (a total of 934,413 synonymous sites), an estimate of ps = 0.000348 0.00019 was obtained . The range of ps values in group a was between zero and 0.012; a total of 288 loci in group a had ps values other than zero . These results show a substantial level of nucleotide diversity, approximately half the level of nucleotide diversity in humans (9). Rates of nucleotide substitution per unit time are difficult to estimate in bacteria given the lack of calibration from the fossil record (10). To obtain an estimate of the rate of synonymous nucleotide substitution, we used published data on comparisons of escherichia coli and salmonella typhimurium (11,12), which are believed to have diverged approximately 100 million years ago (13,14) (table 1). This procedure yielded estimates for the last common ancestor of h37rv and cdc1551 in the range of 34,00038,000 years (table 1). These estimates are approximately twice previous estimates of the age of the common ancestor of worldwide m. tuberculosis (2,3). To obtain the observed mean ps value between h37rv and cdc1551 within 15,00020,000 years would require a rate of synonymous substitution approximately twice that observed in enterobacteria . Based on synonymous substitutions between escherichia coli and salmonella typhimurium, assumed to have diverged 100 million years ago (13,14). Estimates are shown standard error, based on standard error of mean ps . Group b consisted of 11 gene pairs with mean ps of 0.0286 0.0050 (table 2). Paired sample t - test of the hypothesis that ps = pn, p<0.01 . The quantities ps and pn are the proportion of nucleotide difference per synonymous site and per nonsynonymous site, respectively . In enterobacteria, a negative correlation exists between observed proportions of synonymous difference and codon bias (11). In the case of mycobacterium, codon bias results mainly from the very high third position g+c content of most genes (15). In our data, however, we observed no correlation between ps and proportion g+c at third codon positions (r = -0.010; not significant). A number of additional possibilities may explain the occurrence of gene pairs with higher than expected ps values: 1) balanced polymorphism . Selectively maintained polymorphisms are expected to be much older than neutral polymorphisms and may even predate speciation events (16). In the case of haploid organisms such as bacteria, balancing selection would take the form of frequency - dependent selection rather than overdominant selection . 2) differential deletion . In a multi - gene family, if one member of an orthologous pair of genes were deleted in one genotype, the gene pairs would involve paralogous, not orthologous comparisons . 3) horizontal gene transfer . A gene obtained by one of the two genotypes from another bacterial species one indication of a balanced polymorphism is a higher rate of nonsynonymous than synonymous substitution (8). There was no strong evidence of such selection in the present case; ps was greater than pn at 10 of the 11 loci, and pn exceeded ps only slightly at one locus (table 2). In addition, we compared ps and pn in sliding windows of 30 codons along the length of these genes . No regions were observed in which pn was greater than ps (data not shown). Thus, there was no evidence of positive selection acting on specific regions of these genes . On the other hand, differential deletion can probably explain some cases, most notably members of the pe multi - gene family (11) (table 2). The remaining gene pairs are possibly cases of horizontal gene transfer (table 2), for which there is some recent evidence in m. tuberculosis (17). Our results did not support the hypothesis that the common ancestor of m. tuberculosis was relatively recent (2). Rather, the pattern of nucleotide substitution at synonymous sites suggested a divergence time for the two available genotypes of this species approximately 35,000 years ago . Since h37rv and cdc1551 represent two genotypes sampled from within the species, they are probably not the most divergent genotypes possible . Thus, the last common ancestor of m. tuberculosis likely occurred considerably earlier than 35,000 years ago . While the difference between an estimate of 15,00020,000 years and one of 35,000 years is not large on an evolutionary time scale, such a difference is substantial on the scale of human history . For example, the existence of two genotypes in the current population of m. tuberculosis with a common ancestor at 35,000 years is evidence against the hypothesis that m. tuberculosis arose, presumably from m. bovis, at the time of human domestication of cattle (18). Our result is thus consistent with phylogenetic analyses based on insertion - deletion events, which suggest that the m. tuberculosis lineage was a human pathogen well before the origin of m. bovis (19). Thus, along with recent evidence of an ancient origin and extensive polymorphism in the malaria parasite plasmodium falciparum (20,21), our study provides evidence against the long - held view that virulent pathogens are invariably evolutionarily recent (22). Our estimate is conservative because the rate of synonymous substitution may actually be lower in mycobacterium than in enterobacteriaceae, given the highly skewed g+c content in the former . Furthermore, our estimate of the mean proportion of synonymous difference was conservative because we excluded 119 loci with potential frameshifts between the two genotypes as well as a set of 12 loci with unusually high ps values . In addition to the 12 loci assigned to our group b, certain other loci might also have originated from horizontal gene transfer . However, even if horizontal gene transfer has occurred at other loci besides those in group b, eliminating further loci with relatively high ps values from group a will not affect the results greatly . For example, if we eliminate the 10 loci with highest ps values from group a, mean ps will be reduced only to 0.000299, and the estimated age of the common ancestor will be barely affected . The degree of polymorphism observed in this study is unlikely to have been substantially influenced by sequencing errors . The error rate for finished sequences from the institute for genomic research (where cdc1551 was sequenced) has been independently estimated at <1 in 88,000 bases (23). Assuming a similar error rate for both 4.4 mega - bp m. tuberculosis genomes, we would expect to see approximately 100 differences between them due to sequencing errors . Approximately 21 such differences would be expected in the 938,778 synonymous sites in group a and group b genes . In fact, 411 synonymous differences were observed at these sites; thus, even if present, sequencing errors are likely to have made up only a small fraction (approximately 5%) of the total synonymous polymorphism . At such a rate, sequencing errors would have little effect on our estimates of nucleotide diversity at synonymous sites or the age of the common ancestor of the two genomes . In addition, the hypothesis that the single nucleotide polymorphisms (snps) observed between these genotypes are real received strong support from a recent study that observed a number of the same snps in clinical isolates (24). Moreover, since sequencing errors are expected to occur at random with respect to the reading frame of coding sequences, the fact that mean ps exceeded mean pn in both group a and group b was strong evidence against the hypothesis that a substantial proportion of the observed polymorphism was due to sequencing error . Simple considerations of probability can explain why earlier studies produced relatively low estimates of this species age . If we assume that the per - site probability of a synonymous difference between two m. tuberculosis genomes is equal to the mean ps observed between h37rv and cdc1551 (0.000328), then the probability is approximately 95% that no synonymous differences will be seen in a gene with 150 synonymous sites . The probability that no synonymous differences will be seen in 10 such loci chosen at random is approximately 60%, and the probability that no synonymous differences will be observed at 20 such loci is approximately 37% . On the other hand, the probability that no synonymous differences will be seen at 100 these calculations emphasize the need to examine a very large number of nucleotide sites to obtain a reliable estimate of nucleotide diversity and thus of the age of the most recent common ancestor in cases where the frequency of substitution is less than one in a thousand . Even when the frequency of substitution is between one in a thousand and one in a hundred, substantial stochastic error is possible if the number of loci examined is small . Thus, any study that estimates population parameters from nucleotide sequence data needs to survey a substantial number of loci . These considerations are particularly important in the case of pathogenic microorganisms, where a number of factors (including both natural selection and horizontal gene transfer) may lead to substantial differences among loci with respect to the level of nucleotide diversity . Comparison of two complete genomes of m. tuberculosis showed a greater extent of sequence polymorphism than would be expected on the basis of previous studies, in turn suggesting that analysis of additional genomes will likely show further polymorphism . Polymorphism in any species of pathogen may complicate therapeutic strategies because it implies the existence of variation on which selection can act, including selection imposed by human vaccines and pharmacologic agents (20). On the other hand, known polymorphisms may prove useful to investigators in reconstructing the evolutionary relationships among clinical isolates and in providing markers for understanding the genetic basis of complex phenotypic traits.
The consequences associated with professional burnout affect both the health care professionals and recipients . Increased professional burnout is associated with absenteeism, physical illness, emotional problems, poor job performance and negative attitudes for the health care professionals in general . Health care recipients can experience a decrease in quality of care provided and poor communication from healthcare providers as a result of the healthcare professional's burnout . Variations in the prevalence and severity of professional burnout have been reported across specialties by both doctors and nurses . Doctors and nurses who work in intensive care units (icu) are thought to have higher levels of burnout because of their stressful work demands associated with caring for critically ill patients . Guntupalli and fromm, first studied burnout among icu physicians who were members of the section of internal medicine of the society of critical care medicine . The authors indicated that icu physicians experience high levels of ee, dp and decreased personal achievement . They found 29% of respondents scored high in ee, 20.4% scored high in dp and with 59% had low personal achievement scores on the maslach burnout inventory - human service survey (mbi - hss). Gender status, workload and conflict with other employees appear to be significant factors that increase their level of professional burnout . Professionals who have dedicated personality, over - committed personality and authoritarian personality are more susceptible to the effects of burnout . Common causes leading to burnout are overwhelming and hard work with no clear goals, being powerless to change something important and forcing oneself to make the impossible happen . Early warning signs for burnout syndrome nurses are also influenced by professional burnout but appear to be impacted by different factors . Nurses, who are younger in age, separated or divorced, do not receive days off as requested, experience conflict with patients and co - workers, make the end of life decisions regarding patient care and work full time in icus are more prone to severe burnout . Nursing burnout has been documented in the literature for critical care, oncology, palliative care, emergency care and other areas . In a french study evaluating 2497 nurses working in 165 different icus found four groups of nurses to have high burnout syndrome; younger age, participation in icu work groups, quality of working relationships and end of life related factors . The determinants and predictors of professional burnout in an icu setting vary according to professions . Based on earlier studies, it is erroneous to assume that similar factors perpetuate professional burnout in icu staff . The literature currently places little attention on the prevalence of burnout for respiratory therapists (rt) working in icu . Burnout is a term used for a long - term exhaustion with diminished interest usually in the work context . Certain professions such as entrepreneurs, teachers and athletes in addition to healthcare professionals are susceptible to work - related burnout . Burnout syndrome is reported to have three components (1) ee (2) dp and (3) low pa . The focus of this study was healthcare professionals, specifically registered nurses (rn) and rt, involved in the care of icu patients . The professional staff in the icu setting are often required to spend considerable periods of intense involvement with other people . Rn and rt are frequently involved with patients and families having serious physical, psychological and social problems creating interactions charged with feelings of anxiety, fear, embarrassment, anger and despair . Solutions to most of the problems are not always obvious, straight forward or easily obtained, thus adding to ambiguity and frustrations of the situation . For the helping professionals who work continually under such circumstances, this chronic stress can be emotionally draining and pose the risk of burnout . The consequences of burnout are potentially serious to staff, clients and institutions where they interact . Initial research on this syndrome suggests that it can lead to deterioration in the quality of care or services provided by the staff . It is believed to be a factor in job turn over, absenteeism and low morale . Maslach and jackson first identified the construct burnout in the 1970's and developed a measure that weighs the effects of ee, dp and reduced sense of personal achievement . This inventory has become the standard tool for measuring burnout in research performed on the syndrome . Few studies have been done on health care professionals involved in the care of icu patients . This study was conducted in the united states to explore burnout among nurses and rt in icu . This study was conducted in the icus of the harris county hospital district houston, a safety net hospital for the 700,000 indigent populations residing in harris and neighboring counties of texas . The survey was conducted at ben taub general hospital containing 120 icu beds and lbj hospital using 16 icu beds . Study approval was obtained from the baylor college of medicine institutional review board and the harris county hospital district . The survey consisted of a two - part handout placed in an envelope and given to the icu rns and rts . Part 1 addressed demographic information including gender, age, credentials, race, years in practice, employment status, work schedule, hours worked / week and departmental position . Part 2 of the handout was the mbi - hss; an inventory consisting of 22 questions to assess three components of burnout, ee, dp and lack of personal achievement . These items were written in the form of statement about personal feelings and attitudes and answered on a 7-point scale from 0 as never to 6 as every day . The 5-item dp subscale assesses personal and caring attitudes and the 8-item pa subscale assesses feelings of competence and successful achievement when working with people . For ee and dp subscales the higher mean scores on the ee and dp subscales and a lower mean score on the pa subscale are consistent with burnout [table 1]. Burnout scores classification statistical analysis was performed using microsoft excel and spss (ibm; armonk, new york) statistical package . This study was conducted in the icus of the harris county hospital district houston, a safety net hospital for the 700,000 indigent populations residing in harris and neighboring counties of texas . The survey was conducted at ben taub general hospital containing 120 icu beds and lbj hospital using 16 icu beds . Study approval was obtained from the baylor college of medicine institutional review board and the harris county hospital district . The survey consisted of a two - part handout placed in an envelope and given to the icu rns and rts . Part 1 addressed demographic information including gender, age, credentials, race, years in practice, employment status, work schedule, hours worked / week and departmental position . Part 2 of the handout was the mbi - hss; an inventory consisting of 22 questions to assess three components of burnout, ee, dp and lack of personal achievement . These items were written in the form of statement about personal feelings and attitudes and answered on a 7-point scale from 0 as never to 6 as every day . The 5-item dp subscale assesses personal and caring attitudes and the 8-item pa subscale assesses feelings of competence and successful achievement when working with people . For ee and dp subscales, a higher mean score corresponded to a higher degree of burnout . The higher mean scores on the ee and dp subscales and a lower mean score on the pa subscale are consistent with burnout [table 1]. Burnout scores classification statistical analysis was performed using microsoft excel and spss (ibm; armonk, new york) statistical package . This study was conducted in the icus of the harris county hospital district houston, a safety net hospital for the 700,000 indigent populations residing in harris and neighboring counties of texas . The survey was conducted at ben taub general hospital containing 120 icu beds and lbj hospital using 16 icu beds . Study approval was obtained from the baylor college of medicine institutional review board and the harris county hospital district . The survey consisted of a two - part handout placed in an envelope and given to the icu rns and rts . Part 1 addressed demographic information including gender, age, credentials, race, years in practice, employment status, work schedule, hours worked / week and departmental position . Part 2 of the handout was the mbi - hss; an inventory consisting of 22 questions to assess three components of burnout, ee, dp and lack of personal achievement . These items were written in the form of statement about personal feelings and attitudes and answered on a 7-point scale from 0 as never to 6 as every day . The 5-item dp subscale assesses personal and caring attitudes and the 8-item pa subscale assesses feelings of competence and successful achievement when working with people . For ee and dp the higher mean scores on the ee and dp subscales and a lower mean score on the pa subscale are consistent with burnout [table 1]. Statistical analysis was performed using microsoft excel and spss (ibm; armonk, new york) statistical package . Univariate frequencies were performed [table 2] and included 213 total subjects 151 (71%) rns and 62 (29%) rts . Age was evenly distributed, with 20 - 30 years being the smallest category with 32 (15%) respondents . Years of work in critical care was also distributed as expected with a decline between 21 and 25 years; nearly 25% of the sample had been working in critical care <5 years and 21%> 21 years . 38 respondents (18%) reported managerial or charge duties and approximately half of the sample reported working overtime in the past month . Demographic data and univariate frequencies of the subjects results of the ee subscale revealed 54% scored moderate or notably, 40.6% scored low on the pa subscale [table 3]. Odds ratios were performed on rns and rts for other variables [table 3]. No difference was found between gender, managerial or non - managerial position, years of experience, overtime worked and rotating shifts . Scatter plots of the raw data showed worsening trends for ee, dp and pa [figures 13] in the subject group . Frequencies of scores for emotional exhaustion, depersonalization and personal achievement emotional exhaustion correlated with depersonalization emotional exhaustion correlated to personal achievement depersonalization correlated to personal achievement linear regression revealed surprising trends in the univariate analysis model expecting the night shift to have higher ee and dp scores with lower pa scores when compared to the day shift which was not supported in this study . In the final model, moderate and high categories for ee, dp and accomplishment were collapsed due to the presence of small cells (n <8) among some variables . The model was tested for years of experience and age separately since these variables was not identified as independent for all study participants . These variables were also tested as originally stratified and then collapsed yielding no significance . In the analysis of night shift and day shift, night shift nurses had less degree of burnout when compared with the day shift nurses and regression analysis showed the night shift to have a protective effect although with a wide confidence interval (ci) (p <0.05; ci: 0.256 - 0.976). Managerial and non - managerial positions had no significant effect on ee (p = 0.28; ci: 0.695 - 3.594). Moderate or high dp (p 0.05; ci: 2.587 - 10.14) and pa scores (p 0.5; ci: 1.236 - 4.622) had a significant effect on moderate or high ee scores rns had also higher burnout rate when compared with rts (p <0.05; ci: 1.085 - 6.937). In this study, 54% of the staff suffered from moderate to severe ee, 41% suffered from moderate to severe dp and 40% had low personal achievements . Study results showed gender, managerial position and years of experience, had no effect on burnout and no difference was identified based on shift; consistent with the ilhan et al . Study surprisingly, no difference in burnout was found between staff with or without overtime hours . Rns had higher overall burnout when compared to rts with dp being higher among the rns . Night shift may have some protective effect possibly attributed to survival of the initial phase of burnout . Those unable to tolerate night shift have likely changed shifts or possibly even changed professions . Icu staff (rn / rt) results differed from previous studies on surgical residents, radiologists and chinese nurses . This study's icu staff had higher ee scores, lower dp scores yet similar pa scores when compared to surgical residents and a burnout rate when compared to radiologist and chinese nurses . Gender, managerial position and years of experience, had no effect on burnout and no difference in burn out on the basis of shift rotation consistent with the study by ilhan et al . Surprisingly, no difference in burnout was identified between the staff working over - time and those with no over - time hours . Nursing staff were shown to have higher burnout syndrome and more specifically dp when compared to rt . In the comparison by shift rotation it may be possible that nurses or therapists working the night shift have already survived the initial phase of burnout and those who were not able to tolerate the night shift have moved to the day shift or changed professions . This study identified important components of burnout among icu staff by using the mbi - hss the limitations of this study was the exclusion of confounding variables such as the nursing shortage, increased patient complexity, pressure to comply with increasing regulations and higher consumer demands . The lower rate of burnout among rt may be explained by their well - defined scope of practice and area of patient care allowing less involvement with a minute - to - minute decisions . It was conducted in a single institution and did not account for nurses and therapist who may have dropped out or changed the shifts, or profession . It was conducted in a single institution and did not account for nurses and therapist who may have dropped out or changed the shifts, or profession . It was conducted in a single institution and did not account for nurses and therapist who may have dropped out or changed the shifts, or profession . The syndrome identified as burnout is seen in across healthcare professionals including residents, physicians, surgeons, nurses and rt . Increased demand on healthcare workers due to the shortage of providers, limited resources and increasing consumer load can lead to burnout of these providers and attrition . Further multi - center prospective studies are warranted to assess the effect of burnout on attrition further contributing to the workforce shortage.
The typical clinical presentation of poi is pain in the right side of the abdomen, and this may lead to a misdiagnosis of acute appendicitis or cholecystitis . Here, we present a case of poi, with pain in the left lower quadrant . A 50-year - old woman admitted to the emergency room with a history of worsening abdominal pain for 5 days . The pain began in the upper abdomen and later localized to the left lower quadrant, and the pain intensity exacerbated with activities . The pain was sharp and constant in nature without nausea or vomiting . On physical examination, the patient was febrile with a temperature of 38.1c and had severe left lower quadrant tenderness and abdominal rigidity . The previous history included an open ectopic pregnancy surgery in 1996, and no atrial fibrillation, no venous thrombosis, no contraceptive, or aspirin usage . The laboratory test showed white blood cell 9.71 10/l, d - dimer 1.58 mg / l, and no other abnormalities . An abdominal computed tomography (ct) was performed and showed a left ovarian cyst measuring 6.0 cm 4.5 cm with no other remarkable findings [figure 1a]. After about 1 day, the patient complained of worsening abdominal pain and absence of passage of feces and gas . (a) axial image showed a left ovarian cyst measuring 6.0 cm 4.5 cm . Median sagittal section showed an increased fat - density mass consequently, we performed laparoscopic exploration . During the procedure, we found a segment of congested necrotic omentum adhering to the abdominal wall with a segment of small intestine [figure 2a], bloody ascites (200 ml) accumulated in the abdominal cavity [figure 2b], and an ovarian cyst measuring 6.0 cm 4.5 cm in pelvic cavity [figure 2c]. We resected the nonviable omental segment, and the gynecologist removed the ovarian cyst using laparoscopic procedures . (b) a dark purple mass on the left side in the lower part of the omentum and bloody ascites in the left abdomen . (c) a huge ovarian cyst by reanalyzing the preoperative ct, a segmental fat mass with an increased density was noted in the left lower quadrant which was consistent with the intraoperative findings [figure 1b]. Final pathological examination revealed hemorrhagic infiltrations, thrombosis of the tissue, acute inflammatory cellular infiltrate, and fibrinoid necrosis, and poi was diagnosed [figure 3]. Histological examination of the resected nonviable omentum revealed hemorrhagic infiltrations, thrombosis of the tissue, acute inflammatory cellular infiltrate, and fibrinoid necrosis up till now, only a few hundred cases have been published in the english literature . Susceptible factors include (1) anatomic malformations such as a bifid or accessory omentum causing the spontaneous torsion, (2) sudden movement, violent exercise, and hyperperistalsis, and (3) obesity . Most authors support the hypothesis that it is associated with an anomalous and fragile blood supply of the right lower portion of the omentum, which is consequently susceptible to infarction . Thus, 90% of all the cases have initial pain in the right side of the abdomen . Our patient had pain in the left lower quadrant, so we did not include poi in the differential diagnosis spectrum . Poi has no early specific signs and is often characterized by progressive, persistent abdominal pain . Clinical features include unspecific gastrointestinal symptoms, slightly febrile, and mild leukocytosis . In the present case, d - dimer can be used as an objective parameter in the differential diagnosis of poi as it is used in other thromboembolic diseases . The most diagnostic finding is an ill - defined heterogeneous fat density with surrounding inflammatory changes . By retrospective reanalysis of the preoperative ct in this study, ultrasound is specific but not sensitive for diagnosing poi; suspected imaging features include hyperechoic, incompressible, ovoid mass, and detectable in <50% of cases, even when reviewed retrospectively . Two patients underwent surgery, one because of the absence of spontaneous regression and the other because of extremely severe clinical symptoms . In the other four patients, conservative management was given and successful . If a confirmed diagnosis of poi can be made with the typical clinical signs and imaging studies, conservative treatment is the first - line therapy during the first 2448 h while resuscitation and antibiotics are initiated . However, if the diagnosis is in doubt or if conservative treatment fails, laparoscopy should be performed without delay . As in the present case, the pain was not relieved after conservative treatment for 24 h. intestinal obstruction occurred as a segment of small intestine adhered to the abdominal wall . Poi should be considered in the differential diagnosis of any patient presenting with acute abdominal pain . Laparoscopic exploration should be performed if new symptoms occur or do not relieve within 2448 h. there are no conflicts of interest.
Early childhood caries (ecc) is one of the most common chronic diseases of the childhood . According to the american academy of pediatric dentistry (2003) ecc may be defined as the presence of one or more decayed, missing (due to caries) or filled tooth surfaces in any primary tooth in a child 71 months of age or younger . The prevalence is 1 - 12% in developed countries and 70%, while in india, a prevalence of 44% has been reported for caries in 8 - 48 months old children . The early loss of primary anterior teeth may result in reduced loss of vertical dimension, masticatory efficiency, development of parafunctional habits (tongue thrusting, speech problems), esthetic functional problems such as malocclusion, psychological problems that can interfere in the personality and behavioral development of the child . This case report describes the challenging task of treating a 3-year - old child with severely decayed maxillary anterior teeth that were restored using modified omega loop post followed by strip crown . A 3-year - old child reported to the department of pedodontics and preventive dentistry, with a complaint of severely decayed upper front teeth . The child was shy, and behavior of the child was definitely negative according to frankl behavior rating scale . Intraoral examination revealed a complete set of deciduous and caries involving with 61 (maxillary left primary central incisor). Coronal portions of 61 were severely damaged, and most of the tooth structure was lost with pulpal involvement . 61 . After taking parents, consent diet analysis, counseling, and oral prophylaxes were performed . 61 using metapex, followed by custom - made half omega - shaped post with 0.9 mm stainless steel orthodontic wire using no.130 to make omega loop, and serrations were done to increase the stability of the esthetic restoration and the mechanical retention of the core . About 5 mm of the metapex was removed from the coronal end of the root canal, and 1 mm of glass ionomer cement (gic) was placed . The incisal end of the wire projected 3 - 4 mm above the remaining root structure . After gic set, the canal was prepared to get a space of about 3 mm [figure 1]. (a - f) pulpectomy procedure with modified omega loop post followed by strip crown i.r.t . 61 the root canal was etched with 35% phosphoric acid for 20 s, followed by bonding agent was placed and cured for 20 s. the flowable composite was injected into the root canal along with the loop . The composite was light - cured for 40 s. crown was reconstructed using strip crown . Finishing and polishing treatment of preschool children and restoration of primary anterior teeth with the severe loss of coronal structure is a challenging task for the pediatric dentists . The main aim is to preserve teeth and restore them so that child is able to perform normal like mastication, speech, and esthetics . The failure rate is high in such type of cases due to the absence of tooth structure, poor adhesion of bonding agent of primary teeth, limited availability of material and technique . After the successful endodontic treatment and placement of intracanal retainers, the remaining coronal structure can be restored with indirect or direct technique or single tooth prostheses, such as strip crown, stainless steel crown, metal plastic crown, porcelain veneers, polycarbonate crowns, and acrylic resin crown . Nowadays, esthetic crowns are introduced in pediatric dentistry like pedo jacket crowns, fuks crowns, new millennium crowns, preveneered crowns, cheng crowns, dura crowns, pedo pearl crowns and kinder crowns to overcome the disadvantages of strip crown like low retention and poor adhesive of bonding agent . Many studies advocate the use of nonmetallic posts such as ceramic post, polyethylene glass fibers, carbon fibers, etc ., but in such kind of cases, it has some disadvantages like technique sensitive, time - consuming, multiple steps, and expensive . A simpler and effective method is to use an omega loop that was introduced by mortada and king . In this technique, omega loops wire extensions are placed at the depth of around 3 - 4 mm inside pulp chamber and the projected portion of the loop is used for retention of the coronal restoration . The biggest advantage is that the wire does not cause any internal stresses in the root canal as it is incorporated in the restorative material mainly, and it can be done with minimal chair side time . In the present case report, a simple and effective method for reconstruction of severely destroyed primary anterior teeth has been used . This technique can be done directly in the oral cavity without involving any laboratory procedures . The complete procedure can be completed in one appointment, easy for the pediatric patient and less cooperation required from the child . As the core length of the omega loop, which is placed intracanal, is around 3 mm thus occupies only the cervical one - third of the canal and does not interfere with deciduous tooth root resorption and permanent tooth eruption . Omega loop technique can be an easier, simpler, and inexpensive treatment of choice for severely damaged primary anterior teeth . The modified omega loop with serration used in this case report demonstrated good retention, good esthetics, and masticatory function to the child . However, it is a long time success, and its durability in children having parafunctional habits like bruxism, deep bite, etc.
Cytomegalovirus (cmv) is a significant cause of morbidity among solid organ transplant recipients . Active infection can result in the well - defined direct effects of either cmv syndrome or tissue - invasive disease, while the indirect effects include potential immune - mediated injury of the allograft as well as an increased propensity for coinfections . Ganciclovir and its prodrug, valganciclovir, each have been effective in both prevention and treatment of cmv disease [24]. However, the emergence of ganciclovir - resistant (gcv - r) cmv has posed a more significant threat due to an aggressive disease course and a greater mortality risk . Treatment options for gcv - r cmv are limited, with foscarnet being recommended as the initial treatment option followed by cidofovir . Although these agents are known to have activity against cmv, both are associated with substantial side effects, the most notable of which is nephrotoxicity . Significant renal injury with these agents has been reported in 3060% of patients and may even occur after only 1 - 2 doses [7, 8]. Furthermore, cidofovir is contraindicated in patients with an estimated creatinine clearance of <55 ml / min or a serum creatinine of> 1.5 mg / dl, which are common findings among the solid organ transplant population . Herein, we report a successful strategy of reduced - dose cidofovir in combination with cmv - hyperimmune globulin (cmv - igg) in four consecutive kidney transplant recipients with varying degrees of renal impairment and mild cases of genotypically confirmed gcv - r cmv . We chose to use cidofovir instead of foscarnet in these cases based on our own disappointing experience with the latter agent, having observed a high rate of acute renal failure requiring dialysis, in patients with severe gcv - r cmv disease . The rationale for reduced - dose cidofovir was the impaired baseline renal function observed in our patients as well as the ability to use probenecid and hydration as nephroprotective measures . Cmv - igg was provided as an adjunct to antiviral therapy, as well as for its potential immunomodulatory properties . Use of this regimen resulted in viral clearance, preservation of graft function, and avoidance of rejection in all four cases . Four consecutive kidney transplant recipients with documented ganciclovir - resistant cmv were included in this review . Immunosuppression was administered per center protocol: antithymocyte globulin (atg) induction was administered to three patients based on high immunologic - risk status (panel reactive antibody greater than 20% and/or african american race) while basiliximab induction was given to one patient with low - immune risk status . All four patients were high serologic risk for cmv (donor igg positive / recipient igg negative) and were to receive valganciclovir 450 mg daily for 6 months with routine cmv polymerase chain reaction (pcr) screening at prespecified time points (1, 3, 6, 9, and 12 months) after transplant and when clinically warranted . Treatment of cmv disease in clinically stable patients consisted of a valganciclovir 900 mg twice daily (adjusted for renal function) induction period for 2128 days, followed by a 900 mg / day maintenance phase for an additional 28 days or until two consecutive negative pcrs (defined as <300 copies / ml) were achieved two weeks apart . Interestingly, all four patients included in this report developed breakthrough viremia in conjunction with symptoms during the period of prophylaxis . Therefore, valganciclovir dosages were increased to 900 mg twice daily (adjusted for renal impairment). Genotypic analysis for ul97 or ul54 mutations was performed upon failure to eradicate detectable viremia on treatment dosages of valganciclovir . All patients demonstrated confirmation of ul97 mutations and absence of ul54 mutations with documented resistance only to ganciclovir . Each patient was then initiated on combined therapy with intravenous (iv) cidofovir 1 - 2 mg per kg in 5001000 ml of normal saline and cmv - igg (cytogam, csl behring ag, king of prussia, pa) 100 mg per kg . Oral probenecid was administered at a dosage of 2 grams, 1 hour prior to and 4 hours after the completion of each cidofovir infusion . In addition, maintenance immunosuppressants were reduced to attain tacrolimus levels of 48 ng / ml and mycophenolate mofetil dosages of 500 mg twice daily . Combination therapy and pcrs were repeated every 2 weeks until two consecutive negative pcrs were achieved, at which point treatment was discontinued and pcr monitoring became less frequent . The patients were young, with a mean age of 33 years, and all were male . Three of four patients were african american and the same three received deceased donor kidneys . One patient (patient 1) had an episode of acute rejection in the first posttransplant month requiring atg treatment prior to cmv detection . The mean time from transplant to the first detectable cmv pcr was 114 days . Because low - dose valganciclovir in some patients may actually be appropriate dosing based on the presence of renal dysfunction, we evaluated renal function using both estimated 4-variable glomerular filtration rate (gfr) and cockcroft - gault equations . At the time of detectable cmv replication, only one patient (patient 4) had a gfr greater than 60 ml / min/1.73 m. however, 3 of 4 patients had a creatinine clearance greater than 60 ml / min using the cockcroft - gault method . Therefore, according to approved labeling of valganciclovir, three of the four patients were indeed receiving low - dose genotypic assessment and diagnosis of gcv - r cmv were made at a mean of 103 days from the initial cmv pcr detection date, after persistence of detectable pcrs despite treatment dosages of valganciclovir . The average duration of valganciclovir exposure at the time of resistance diagnosis, including posttransplant prophylaxis, was therefore 217 days . All patients experienced cmv syndrome, with a predominance of fever, leukopenia, or malaise . In addition, patient 1 had gastrointestinal symptoms, although a biopsy was not performed to evaluate for tissue invasion . Two consecutive negative pcrs were attained in all patients, occurring after an average of 5 treatments and a median of 42 (range 14 to 105) days from the date of the first infusion (figure 1). Following discontinuation of therapy, patients 1 and 4 each developed recurrent viremia . In each patient, the recurrence was not associated with symptoms; however, treatment was reinitiated . Viremia subsequently cleared again after 2 additional courses of cidofovir / cmv - igg in patient 1 and after 10 doses of cidofovir alone in patient 4 . In the latter patient, a repeat genotype was obtained, demonstrating the same ul97 mutation site and conferring resistance only to ganciclovir . The mean change in gfr from the time of gcv - r diagnosis to the completion of therapy was 1 ml / min . At the time of this writing and after a mean follow - up of 21 months since the final dose of cidofovir, all patients are currently alive with functioning grafts . None of the patients developed recurrent disease and none experienced acute rejection throughout the follow - up period . Available data on the use of cidofovir for treatment of cmv in solid organ transplant recipients is scarce . While cidofovir is contraindicated in patients with reduced renal function, several reports describe its safety at lower dosages for the treatment of polyomavirus - induced nephropathy . Furthermore, based on limited pharmacokinetic data in renally impaired patients, reduced dosages of cidofovir may provide adequate antiviral concentrations . This led us to investigate the ability to use reduced - dose cidofovir to eliminate mild cases of gcv - r cmv disease . In addition, each patient received cmv - igg for two reasons: first, to augment antiviral treatment by providing cmv antibodies in previously seronegative patients and, second, to take advantage of the potential immunomodulatory effects in an effort to prevent rejection . In all four patients, this regimen led to resolution of gcv - r disease without deterioration of renal function or rejection . Antiviral - resistant cmv has been reported in up to 14% of transplant recipients, with the highest rates occurring in high - risk serostatus recipients and lung transplant recipients [5, 1214]. Ganciclovir, a guanosine analogue, requires three stages of phosphorylation in order to exert its inhibitory effect on cmv dna polymerase . The initial phosphorylation occurs via viral ul97 phosphotransferase, which is followed by two subsequent phosphorylations by host cellular kinases . Resistance can be conferred by mutations at the ul97 kinase and ul54 polymerase genes [15, 16]. Mutations in ul97 alone can result in resistance to ganciclovir and other nucleoside analogues by inhibition of the initial phosphorylation step . Mutations in ul54, albeit much less common, can confer resistance to cidofovir and foscarnet . Inadequate drug exposure and prolonged duration of antivirals have been attributed to the development of resistant cmv strains . In addition to its significant morbidity and mortality, treatment options for gcv - r cmv are also associated with severe adverse outcomes . Recommended options such as cidofovir and foscarnet are associated with a high incidence of nephrotoxicity, often resulting in renal failure in both kidney and non - kidney transplant recipients [12, 13, 18, 19]. Additionally, rejection may also occur as a result of reduced immunosuppression and/or indirect effects associated with cmv infection . While foscarnet is considered the preferred treatment for gcv - r, our own experience with the agent has been disappointing due to a very high incidence of acute renal failure requiring hemodialysis in patients with more severe disease . Given our familiarity in using cidofovir as a treatment option for polyomavirus nephropathy and the ability to use probenecid and hydration as nephroprotective measures, we chose this agent in these four cases of milder disease . Cidofovir is approved in the united states for cmv retinitis in patients with acquired immunodeficiency syndrome (aids). The drug requires phosphorylations to monophosphate and diphosphate (active) forms by pyrimidine nucleoside diphosphate kinase and nucleoside diphosphate kinase, respectively . The final metabolite, cidofovir diphosphate, inhibits cmv dna polymerase, a product of gene ul54 . While each of our patients demonstrated mutations only in ul97, development of resistance to cidofovir was an obvious concern . However, due to the relatively mild nature of disease and the presence of renal dysfunction in each patient, we felt that a reduced dose would be appropriate . A genotype was repeated in one patient with a slight rise in pcr after resuming cidofovir and was confirmed negative for the ul54 mutation . An interesting finding in our cohort was that each patient developed their initial infection while on valganciclovir prophylaxis . This suggests that breakthrough cmv viremia during prophylaxis should warrant testing for antiviral resistance . Treatment of gcv - r cmv in solid organ transplant recipients is challenged by dose - limiting toxicities of effective agents and potential graft injury due to indirect viral effects or rejection associated with lowering of immunosuppressive agents . In our experience, 4 consecutive patients with documented ul97 mutations conferring resistance to gcv were successfully treated with a regimen of reduced - dose cidofovir and cmv - igg . It is important to highlight that all patients were diagnosed relatively early in the course of their infections, making this conservative approach more plausible . Undoubtedly, a more aggressive treatment would be required in patients with greater disease severity . We also cannot rule out the impact and importance of reduction of immunosuppression in the management of patients with gcv - r cmv . Nevertheless, all patients experienced viral eradication, and, importantly, none developed nephrotoxicity or rejection . We conclude that this regimen in addition to reduction of immunosuppression may serve as a treatment option in patients with mild gcv - r cmv disease.
A pure ground - glass nodule (ggn) in the lung may represent an early lung malignancy, such as adenocarcinoma in situ (ais) or minimally invasive adenocarcinoma (mia) (1). Approximately 20 - 30% of patients with adenocarcinoma and a predominantly pure ggn who undergo surgery have additional ggn lesions, either in the same or different lung lobes (2). In such cases, multiple limited resection may be an appropriate treatment if a few pure ggn lesions are scattered in multiple lobes or peripherally in one lobe, and the patient has sufficient pulmonary functional reserve (3). However, alternative options should be considered if several ggn lesions are scattered in both lungs with multiple lobes involved, and there is a risk for insufficient pulmonary functional reserve after resection . Chemotherapy has been suggested as an additional therapeutic option, if surgery is not possible due to a lack of pulmonary functional reserve (4). Another strategy is to rely on serial computed tomography (ct) scans until the nodule develops a definite solid portion . If curative therapy is not performed, there remains a significant chance for progression (1). Cryoablation has been explored as an option for treating metastatic lesions in patients with non - small cell lung cancer who cannot undergo surgery or chemotherapy (5). However, no clinical reports of cryoablation techniques applied to multiple ggns in the lung have been published . Here, we describe a patient with lung cancer and a pure ggn in the left lower lung lobe that persisted after several limited resections and was successfully treated with cryoablation . A 59-year - old woman visited our hospital due to abnormal chest radiography findings on a health maintenance exam at another hospital . All pre- and post - procedural ct scans were performed using a multi - detector ct scanner (somatom definition flash, enlargen siemens, forchheim, germany). The scanning parameters were: 120 kvp, 200 mas, 0.75-mm collimation, 2-mm reconstruction intervals, smooth algorithm, and pitch of 1 . Several ggns were detected by ct scan: a 12-mm - sized, partially solid nodule in the right middle lobe (fig . 1a), a 7-mm - sized pure ggn in the right upper lobe, and two 5-mm - sized pure ggns in each of the lower lobes (fig . The patient underwent a right middle lobectomy and wedge resection of the right upper lobe using video - assisted thoracoscopic surgery . The partially solid nodule in the right middle lobe was diagnosed as adenocarcinoma, and the pure ggn in the right upper lobe was diagnosed as a mia . The two pure ggns in both lower lobes remained without significant change on a 10-month follow - up ct scan . An additional wedge resection was performed for the pure ggn in the right lower lobe, and this nodule was diagnosed as ais . After these surgeries, the patient did not have sufficient pulmonary functional reserve to tolerate any additional surgery . Thus, we decided to perform cryoablation on the pure ggn in the left lower lobe . The patient fasted for 6 hours before the procedure, and her blood coagulation parameters were checked . We used an argon and helium gas - based system (seednet, galil medical, yokneam, israel), a 1.5-mm 17-g cryoprobe, and a thermal sensor . The procedure was performed in a fluoroscopy room by an interventional radiologist . After placing the patient in the prone position on the table, a cross - shaped radio - opaque skin marker was applied to determine the needle insertion site under fluoroscopy . Subsequently, a c - arm cone - beam ct (cbct) scan (dynact, enlargen siemens) was taken (fig . 1d - f) to obtain 1.5-mm thick axial, coronal, and sagittal images . Based on these images, we measured the distance from the center of the skin marker to the target ggn . The area of the skin marker was draped, and a 1% lidocaine solution was injected into the skin down to the pleural surface . After localizing the needle tip, a cbct scan was performed to confirm the probe insertion site . A 17-g cryoprobe (seednet, galil medical) was carefully introduced into the pure ggn under fluoroscopic guidance . We used a fluoroscope and cbct to visualize the size of the ice ball during the procedure . We took a ct scan to confirm that the ice ball had reached sufficient size, as indicated by a low attenuation region with an envelope of ground - glass opacity and a margin of 1 cm beyond the ggn . A small pneumothorax was noted after removing the cryoprobe; however, the patient was asymptomatic with stable vital signs . We performed a follow - up ct scan 1 day after cryoablation to assess any initial changes in the ablated lesion and procedural - related side effects (fig . The scan showed a 3-cm sized ablated zone in the left lower lobe with no major procedure - related complications, such as hemothorax . The size of the ablated zone had decreased markedly on a follow - up ct scan 2 months later . The pure ggn in the left lower lobe was judged to have been successfully ablated without recurrence on a 6-month follow - up ct scan (fig . Our results show the potential of cryoablation as a new treatment method for ggn, particularly in patients in whom surgery is contraindicated . Some controversy exists as to whether some nodules should be resected or followed by serial ct scans . Kim et al . (6) noted that several pure ggn lesions detected in patients undergoing surgery for bronchioloalveolar carcinoma did not change in size or features during follow - up . They suggested that surgical resection should be considered in selected cases in which nodules exhibit significant changes in size or appearance during follow - up . (7) found that all ggn lesions> 10 mm were carcinomas and insisted that these lesions should be resected rather than followed . Partially solid ggn lesions should be resected regardless of size because they represent more invasive lesions than pure ggns . Thus, a non - surgical alternative is needed for patients with insufficient pulmonary functional reserve or in patients in whom surgery is technically not feasible . Minimally invasive ablation techniques, such as radiofrequency ablation (rfa) and cryoablation, may be useful adjunctive treatment options for lung cancer or metastatic lesions and preserve pulmonary function (5, 9, 10). Minimally invasive ablation techniques for lung cancer have several advantages, including selective damage, minimal morbidity and mortality, minimal loss of lung function, repeatability, low cost, excellent monitoring during treatment, less pain, and shorter hospital stay (11). Ablating lung tumors is currently used for curative treatment of primary lung cancer and metastatic lung malignancies or cytoreduction (12). Ablation is also an option in patients with lung metastases from colorectal and renal cell carcinoma, melanoma, hepatocellular carcinoma, or primary sarcoma (13). Although rfa is a contemporary tumor ablation technique, it may cause serious complications, such as air embolism or uncontrolled pain (9, 14). Cryoablation is a thermoablational technique that consists of alternating cycles of decreasing (freezing) and increasing (thawing) temperatures, leading to direct cellular and vascular injury . Cryoablation works by forming an ice ball, which causes increased extracellular osmolarity and, as a consequence, water diffuses from the intracellular space into the extracellular space . Thus, cryoablation may be a better option for patients with extensive emphysema (10). However, percutaneous or surgical biopsy was difficult due to the small size and multiplicity of the ggn lesions . Nevertheless, localized ggns can include invasive disease, such as ais or mia (1). In our case, two similar nodules in the right upper and right lower lobes were identified during surgery as mia and ais, respectively . Cryoablation may reduce the likelihood that a remnant ggn will progress to advanced cancer and does not require additional surgery . Thus, long - term follow - up may be necessary to determine the outcome of cryoablation for treating pure ggns . In conclusion
Treatment of metastatic kidney cancer has changed dramatically over the past years with the use of vegf - targeted therapies and mtor inhibitors . However, resistance occurs . We report here two cases of patients who benefited from the addition of bevacizumab to temsirolimus . A 48-year - old man presented with several episodes of loss of consciousness in 2005 . The brain metastases were treated with gamma - knife radiosurgery . In 2006, he developed lung metastases and mediastinal lymph nodes . In march 2007, the mediastinal lymph nodes increased and he was started on sunitinib (50 mg 4 weeks on treatment and 2 weeks off). The lesions were stable but the lymphangitic infiltration was extensive and the patient still experienced dyspnoea . He improved dramatically, his oxygen consumption decreased . On the ct scan the mediastinal lymph nodes and the lymphangitic carcinomatosis lesions improved . A 54-year - old man presented with hematuria revealing a left kidney tumour in january 2004 . The pathological examination showed a pt2 clear cell carcinoma, furman grade ii tumour . In march 2005, he progressed with liver and lung metastases and he was enrolled in the phase iii study comparing interferon to sunitinib . He had slowly progressive disease on interferon and crossed over to sunitinib in march 2006 . He had a partial response . In july 2008, the lung and liver lesions were stable but he developed peritoneal carcinomatosis with ascitis . He was started on temsirolimus: ascitis was less abundant, the other lesions were stable . There were probably several causes to his anaemia (a side effect of temsirolimus and the disease). In january 2009, the liver lesions increased and he was started on a combination of bevacizumab (10 mg / kg every 2 weeks) and temsirolimus (20 mg iv weekly). His general condition improved, the anaemia disappeared, the lesions were stable and the ascitis much less abundant . He felt that he had fewer side effects on the combination treatment than on temsirolimus alone . These two cases show the effect of adding bevacizumab to temsirolimus for patients who were progressing on the mtor inhibitor . Treatment of metastatic kidney cancer has changed dramatically in the past years with the use of vegf - targeted therapies and mtor inhibitors . The vegfr tyrosine kinase inhibitor, sunitinib, is usually used in the first - line setting . Another tyrosine kinase inhibitor or an mtor inhibitor can be used in the second - line setting, after progression on initial treatment . Resistance to vegf pathway inhibition can be caused by upregulation of alternative pro - angiogenic factors (fgf, angiopoietin), inadequate target inhibition or enhanced receptor signalling . There are a few cases in the literature of patients who were progressing on sunitinib and who benefited from the adjunction of bevacizumab; however, the first data show that these combinations are very toxic . This could drive upregulation of mtorc2 and further activation of akt and hif2. This could therefore lead to increase of expression of hif2 target genes, such as vegf . Combining vegf - blocking agents with mtor inhibitors could contribute to reversing resistance when used in a sequential manner . However, results from the torava phase ii randomized trial show no improvement of non - progression rates with the upfront combination of bevacizumab and temsirolimus but increased toxicity leading to a high drop - out rate . There is no data in the literature on why patients should feel fewer side effects when adding bevacizumab to temsirolimus . However, some reports already mention the development of polyglobulia with anti - angiogenesis agent . This effect may correct a pre - existing anaemia [4, 5, 6].
The inflammatory bowel diseases (ibds) are chronic illnesses affecting the gastrointestinal tract: they predominantly comprise crohn's disease (cd) and ulcerative colitis (uc). Although cd and uc share some features, they have distinctive endoscopic and histological characteristics and disease patterns . Furthermore, the clinical manifestations and outcomes of these two conditions vary . At present, there are no specific medical cures for cd and uc: current therapies aim to control the disease and prevent adverse outcomes . Although ibd is uncommon in the first decade of life, presentation in infancy is well recognized . Ibd is more common in the second decade, particularly in adolescence [2, 3]. There are clear demonstrations of increasing incidence of ibd around the world over the last decades, with increased rates also noted in areas with previous very low rates [38]. The changing incidence of ibd has also been noted in paediatric populations, with particular increases in cd [9, 10]. A history of weight loss or poor weight gain is commonly recorded at the time of diagnosis with ibd in children and adolescents . Poor linear growth may also be evident at the initial assessment, leading to growth failure in some . Impaired nutrition may lead onto short- and long - term consequences that include delayed puberty, micronutrient deficiencies, impaired adult height, and bone disease . Regular monitoring and detailed attention to nutritional support remains a central facet of the management of children and adolescents with ibd . This paper will focus on the impact of ibd upon the nutritional state of children and adolescents and will highlight recent advances in this area . Numerous cohort studies have demonstrated weight loss or poor weight gains at the time of initial diagnosis of ibd . Reports indicate that most children with cd have a history of weight loss or absent weight gains prior to diagnosis . In a cohort of 386 canadian children diagnosed with cd over a ten - year period, 80% had a history of weight loss . A retrospective chart review of a cohort of 61 children diagnosed with cd at sydney children's hospital, sydney, australia showed that 51% had a history of weight loss at the time of diagnosis . In a prospective uk study of 379 paediatric cd patients, 58% had weight loss at diagnosis and 27% had weight weight loss tends to be seen less commonly in children with uc up to 31% of 172 children in the same uk cohort had a history of weight loss at diagnosis . Maintaining appropriate weight gain also continues to be an ongoing issue after diagnosis . In a cohort of 41 children with longstanding cd recruited to assess dietary intakes and nutrition, the 18 children with active cd had lower mean z scores for weight, height, and bmi than the 23 children currently in remission . The reasons for altered weight gains are likely multifactorial, with decreased caloric intake being the most common [1419]. Poor intake may be the consequence of the anorexic effects of proinflammatory mediators, such as interleukin-1 or tumour necrosis factor (tnf)- [20, 21] or result from early satiety, pain, or nausea . Pain associated with eating, leading to poor tolerance of foods, and the fear of diarrhoea following meals are additional reported reasons for reduced intake . Small bowel involvement may lead to disaccharide intolerance resulting in shorter gut transit times, pain, and exacerbation of diarrhoea . Malabsorption of food components and the diversion of calories to sites of gut inflammation may be further contributing factors leading to impaired growth . In addition to presentation with loss of body weight or poor weight gains, children also commonly have altered patterns of linear growth . Decreased height velocity was noted in up to 88% of a group of 50 children at the time of diagnosis with cd a number of these children had impaired linear growth prior to the onset of gut symptoms . Impaired linear growth tends to be more pronounced in boys than girls this is principally related to the male pubertal growth spurt occurring later and lasting longer . Furthermore, the limited duration of puberty limits the time before impaired linear growth becomes irreversible [23, 24]. Cd is commonly associated with impaired linear growth either at diagnosis or as a long - term outcome [2229]. Some earlier studies reported that despite growth retardation occurring during teenage years, many of these patients eventually achieved heights within the normal range for the general population [30, 31]; however, the genetic component of linear growth was not considered in these studies . Markowitz et al . Retrospectively assessed the growth patterns of 48 adults with ibd (38 with cd and 10 with uc: 73% male) who had previously been diagnosed during childhood . These investigators used two height prediction methods and employed the american national centre for health statistics growth charts: 31% of this group were found to have permanently impaired linear growth . In their study of 132 children and young adults aged between 525 years, sentongo and colleagues argued that height adjusted for genetic potential was a more powerful way of assessing height status in children with cd . In these children, the z scores based on adjusted heights were significantly lower than z scores based on measured heights . Again, males in this group were most severely affected, with the average adjusted height for age z score one standard deviation less than expected . Lee et al . Recently undertook a prospective assessment of height in 295 children with cd and uc who had been diagnosed between 2002 and 2008 in boston, usa . Twenty - two percent of these children (90% of whom had cd) had linear growth impairment (height for age z - score of 1.64 or less) documented on one or more measurements from the time of diagnosis . The final mean adult height z score in this cohort was 0.39.the main predictors of final adult height in this cohort were found to be lower parental height and the patient's lowest height z score . A recent british study also assessed the final adult height of a group of 23 individuals who had been diagnosed with cd prior to their 16th birthday . This group had a final adult height z score of 0.29: very similar to that observed in the north american study . The mean final height of these patients was 2.4 cm less than the parental or target height (range of 20.0 to + 9.0 cm). Twenty percent of the group had a final adult height greater than 8.0 cm below their target height . Jejunal disease and the length of symptoms prior to diagnosis (diagnostic delay) were predictive factors of final adult height in this cohort . In this cohort, exposure to corticosteroids, surgical intervention, and parental height jejunal disease location has previously been suggested to be associated with linear growth impairment [32, 33]. A danish population - based assessment of growth in children with ibd highlights the differential effects of cd and uc upon growth . Forty - four children with cd were included in this study, along with 50 with uc and four with ibd unclassified (ibdu). The children with uc had similar height and bmi scores for age to control data . In contrast, the children with cd had height for age z score of 0.77: also they were shorter than the children with uc (p = .005). Fifty percent of the children with cd had a height less than the 25th centile for age . Elegant studies in rodent models show that approximately 40% of growth impairment is consequent to the effects of inflammatory proteins, especially il-6 [38, 39]. Further, tnf- and il-6 both independently suppress levels of insulin - like growth factor (igf)-1, a critical mediator of the local actions of growth hormone . Male gender, age at diagnosis, disease location, disease severity, and treatment modality may also all affect final height acquisition . Severity of disease (defined by various measures such as steroid requirements, use of immunosuppressives, and cumulative period of hospitalisation) was correlated with impaired weight and height gains in a cohort of israeli children . Interestingly, the presence of the nod2 genotype was not associated with impaired weight or linear growth, despite associations between ileal location and mutations in this gene . A recent report highlighted the importance of genetic influences upon linear growth in children with ibd . In a cross - sectional study, 951 subjects, comprising 317 cd patient - parent trios, linear growth impairment in the children with cd was associated with a polymorphism in the dymeclin gene dym (odds ratio 3.2). Delayed puberty is also frequently observed in children with ibd, especially those with cd [43, 44]. In one cohort of children diagnosed with cd prior to the onset of puberty, menarche was noted to occur at or later than 16 years of age in almost three - quarters of the girls . Delayed pubertal development is more common in children who have never achieved remission or who have had repeated disease relapses, emphasizing the interactions between disease control and growth in children . Although the most concern in regard to the nutritional impact of ibd in children is towards under nutrition, some children with ibd may be overweight (bmi> 85%) and/or obese (bmi> 95%). In a cohort of 166 children with newly diagnosed ibd from wisconsin, usa, 12.1% of those with cd and 17.6% of those with uc were overweight / obese . In a group of 1598 american children with known ibd overweight / obesity in this multicentre cohort was associated with african - american ethnicity and medicaid insurance status . In addition, a prior surgical intervention was linked with overweight / obese status in the children with cd, suggesting that the presence of over nutrition may be associated with a more severe disease course . Corticosteroids, for example, can lead to numerous side effects, including nutritional consequences . Increased appetite and fluid retention are commonly seen after commencement of steroids . Consequently, apparent improvements in weight during a course of corticosteroids may reflect fluid retention and not an improvement in the underlying nutritional status . Steroids also lead to enhanced bone resorption and decreased new bone formation, adversely affecting bone health [48, 49]. Further, daily corticosteroid therapy can suppress igf-1 activity, contributing to inhibition of linear growth and impaired final height . Sulphasalazine may lead to folate malabsorption; however, daily folate supplementation does not appear necessary . Folate supplementation is required, however, when methotrexate is used to avoid folate deficiency due to the inhibition of the conversion of folate to the active moiety tetrahydrofolate . Cholestyramine, when required for bile - salt diarrhea, may impede absorption of vitamin b12 . In contrast, some interventions, such as exclusive enteral nutrition (een) and biological therapies, have numerous positive nutritional effects . Een is a specific nutritional therapy that involves the administration of a liquid formula as sole nutritional intake (with exclusion of normal diet) for up to 8 weeks . This therapy is efficacious in active cd, with remission rates similar to those seen with corticosteroids . Een leads to positive improvements in weight and linear growth, normalization of igf-1 levels, and stabilization of bone turnover . Een is also associated with improved vitamin d status compared to children treated with corticosteroids . Biological therapies in children with cd examined the growth patterns of 32 children treated with infliximab (28 responders) for severe cd . These children had improved height velocity, and height centile increases after infliximab, with timing prior to early puberty being important . . Found that improvements in linear growth after infliximab can be independent of pubertal status and reduction in corticosteroids . These effects of infliximab in children with cd may be due to improved inflammation with mucosal healing or due to direct effects upon growth hormone and igf-1 signaling . In a recent report, cromb et al . Demonstrated that the height z scores of a group of 41 french children responding to infliximab improved from 0.57 1.18 to 0.25 0.99 (p = .04) over the period of followup . In contrast, those children not responding to this drug had no catch up growth . Given the frequency and patterns of disturbed growth in children with ibd (and especially those with cd), the management of such children must include a clear and constant focus upon growth and nutrition . Resumption of normal growth patterns should be seen as an element of the success of therapy . Baseline assessment of nutritional status at the time of diagnosis should include detailed standard anthropometry, along with clear documentation of preceding growth patterns and familial growth patterns (especially parental heights). Furthermore, close monitoring of growth during followup must be considered a mandatory part of the multidisciplinary care of children and adolescents with ibd . There is now plentiful evidence demonstrating the significant impact of ibd upon growth and nutrition in children . Recent studies have helped to demonstrate the importance of nutritional, inflammatory, and genetic factors in growth outcomes . Consequently, a key aspect of the ongoing management of children and adolescents with ibd should be the close and constant attention to growth.
Piroxicam (pxm), an enolic benzothiazine and potent member of the oxicam series, suppresses the synthesis of proinflammatory enzymes, reducing lipid mediators such as prostaglandins (pgs) and thromboxane (txs), and also inhibits ornithine decarboxylase (odc) induction,1 both pathways participating in carcinogenesis . Further effects of pxm are the inhibition of metalloproteinase-2 activity and induction of apoptosis, determined to have an antiaging effect on skin texture.2 pxm belongs to a class of nonsteroidal anti - inflammatory drugs (nsaids) indicated for the symptomatic treatment of inflammatory and degenerative rheumatic diseases (rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis). On the basis of the documented effect of pxm in skin tumorigenesis, through the block of cyclo - oxygenases-1 and -2 (cox-1 and -2) activity,3 we reported preliminary studies of its usefulness in the treatment of actinic keratoses (aks).4 cox inhibitors, diclofenac and celecoxib, have also been used for the treatment of aks and squamous cell carcinomas (sccs).5 the effectiveness of nsaids is attributable to the inhibitory effect on the increased cox-1 and -2 enzyme activity in skin tumors produced after chronic exposure to uvb (ultraviolet b) (accompanying skin inflammation). Aberrant overexpression of cox-2 in both ak and scc upon uvb irradiation and uvb - induced p53 tumor suppressor gene mutations are early events responsible for the progression of sun - exposed nonmelanoma skin cancers (nmscs).5 topical nsaid treatment works as a nonspecific cox inhibitor and is an effective and well - tolerated treatment for ak . Cox inhibitors have been examined with respect to their role in cancer prevention and cancer treatment in both animal models and humans.6 in humans, topical application of nsaids is also potent in inhibiting the uv light - induced acute sunburn reactions such as erythema and peeling of superficial layers, if applied immediately after uvb exposure.7,8 an exhaustive meta - analysis on pxm made by richy et al9 on controlled clinical trials pointed out a more favorable efficacy and safety profile of pxm compared to other topical nsaids (diclofenac, naproxen, tenoxicam, indomethacin, etodolac, meloxicam, ibuprofen, salicylates, nabumetone, aceclofenac, droxicam, flurbiprofen, ketoprofen, nimesulide, and diflunisal). Here, we described the different modalities of action of pxm on the pathogenesis of nmsc . The inhibitory effects of pxm on cox-1 and -2 activity have been investigated by analyzing its binding mode . We also reported our clinical experience about the effectiveness of local use of pxm as chemopreventive agent on aks and field cancerization . Skin cancer is the most common cancer in humans . The skin of the head and neck accounts for less than 10% of the body s surface area, but this region accounts for 70%80% of skin cancer cases . Although mortality from nmsc is low, its high incidence leads to a significant public health burden, making them a suitable target for chemoprevention and long - lasting research . Nmsc includes scc and basal cell carcinoma (bcc).10 carcinogenesis occurs in two steps: initiation and promotion.11 the promotion phase, an important target for the design of potential chemoprevention studies, is temporally prolonged and potentially reversible.11 a variety of model systems have demonstrated an increase in tissue polyamine levels, including putrescine, during tumor promotion.12 mammalian polyamine biosynthesis is at least partially controlled via induction of odc, which makes the inhibition of this enzyme a potential target for chemoprevention.13 -difluoromethylornithine is an enzyme - activated irreversible inhibitor of odc that has been shown to prevent tumors in experimental animal systems.14 in addition, accumulation of genetic events within cells leads to a gradually dysplastic cellular manifestation, deregulated cell growth, and finally, carcinoma . An upregulation of cox-2 plays a significant role in pg and vascular epidermal growth factor (vegf) production for the tumor proliferation.15 scc of the head and neck showed little response to cox-2 inhibition . Therefore, a cotreatment of cox-1 and -2 determined a meaningful inhibition of vegf, as observed by park et al.15 increased levels of prostaglandin e2 and f2 (pge2 and pgf2) in pre - malignant and/or malignant cutaneous tumors are favored by upregulation of cox-2 and downregulation of tumor suppressor gene 15-hydroxy - prostaglandin dehydrogenase (15-pgdh).16 chemoprevention can be a hopeful approach to inhibit carcinoma occurrence before an invasive tumor develops . The chemopreventive effect of nsaids on nmsc has been established in animals1720 and in vitro studies.21 experimental studies have proposed the topical or oral use of nsaids in human subjects for the regression of cutaneous tumors.22 findings from observational cases studies or clinical trials documented a defensive effect of nsaids against colorectal cancer.2325 the hypothesis that nsaids might prevent the occurrence of colorectal cancer arose from studies showing that pge2 levels were higher in colorectal cancer than in surrounding normal tissue . On the other hand, few epidemiological studies have investigated the efficacy of nsaids in the prevention of skin cancer.2628 it is well known that uv irradiation is the major cause of induction and development of ak and nmsc . Normally, sun - damaged cells undergo, through p53 activation, apoptosis for self - destruction.5 skin cells can acquire tumorigenic mutations by uv irradiation or chemical stimulation and loss of the normal ability of cell differentiation and apoptosis . The stepwise evolution of ak with an increased expression of antiapoptotic bcl-2 favors the progression to scc.29 as a matter of fact, retinoid - induced modulation of apoptosis in nmsc is an efficacious therapeutic approach.3032 in addition, the dermal response with inflammation, characterized by erythema and edema, mediated by pgs is a critical component of tumorigenesis, promoting tumor growth, tissue invasion, angiogenesis, and metastasis.29 the action of pxm plays a role in early stages of carcinogenesis by blocking the phases of inflammation and thus angiogenesis; the evidence of its effectiveness for the treatment of aks has been described in our previous study.4 also, prolonged use of pxm acts on photodamaged areas which is expression of field cancerization.4 to date, a higher occupational sun exposure of men compared with women justified the different incidence of nmsc.33 a study of johannesdottir et al34 provides an alternative elucidation, which attributes the reduced incidence to the chronic use of analgesics for menstrual cycle related symptoms . Several epidemiological studies confirm that aspirin can act as a chemopreventive agent and can decrease the incidence of various cancers, including skin tumors . The major inhibitor effect of aspirin on nmsc and precursors is related to its targeting elevated cox-2 levels in scc . Most studies also examined the use of pxm in different disorders, especially for analyzing absorbance, tolerance, and efficacy in animal models,3537 but the efficacy of pxm in humans was analyzed considering the general effects of nsaids, as in colon rectal carcinoma38,39 or in nmsc.4042 rathore et al43 recently reported that cotreatment of pxm with masitinib significantly inhibited cell proliferation of oral squamous cell carcinoma (oscc) as compared to either drug alone, through the c - kit and akt signaling pathways . Therefore, targeting these two signaling pathways simultaneously was more efficient for inhibition of osccs across these species.43 a few studies evidenced the chemopreventive use of pxm for nmsc,4,44 but its interaction with cox-2 is well known;43 however, more studies provided evidences for efficacy of pxm in melanoma treatment.4547 in vertebrates, two isoforms of active cox enzymes have been identified, which display similar enzymatic properties, but differ in their expression and regulation.48 the best - known mechanism of action of nsaids is the inhibition of cox isoforms, in particular, cox-1 and cox-2, both implicated in inflammation and promotion of tumorigenesis.16 chronic inflammation correlates with the increased risk of developing cancer in the affected organ and can be caused by acute uv radiation and other factors.49,50 the metabolism of arachidonic acid, the predominant precursor of pgs and the major control point for their synthesis, can be triggered by uv effect on the skin cell membranes, thus starting the inflammatory process.51 arachinoid acid metabolism and pg synthesis through the cox - related pathway promotes cancer progression . Cox is the key enzyme in prostanoid biosynthesis, which catalyzes the conversion of arachidonic acid to prostaglandin h2 (pgh2), which is subsequently converted to biologically active lipids such as tx, pge2, and prostacyclin (pgi2) by different enzymes.52 the skin is a major site of pg synthesis . All skin cells are pgs producers: keratinocytes, melanocytes, langerhans cells, mast cells, fibroblasts, and endothelial cells.53 while cox-1 is constitutively expressed, cox-2 is induced by tumor promoters, growth factors, and cytokines.54 cox-1 is responsible for maintenance of the gastric and platelet functions, vascular homeostasis, as well as renal blood flow, and it also seems to contribute to pg synthesis during tumor progression in various tissues, including skin.55,56 the relative contribution of the two cox isoforms to skin inflammation is evaluated by combining studies with genetic knockout models and pharmacological intervention with isozyme - selective inhibitors.57,58 the carcinogenic actions of cox-2 should be distinguished from those of cox-1 in the application of pg - blocking therapy for cancer treatment.59 the proliferative effects of cox-2 are due primarily to the increased synthesis of pgs, which directly influence cell growth after binding to specific cell surface receptors, including pg e, f, and i classes of receptors.60,61 the protumorigenic effect of prostaciclins can be mediated by specific receptors as ep2, and the different forms of pgs mediated their biological effects through ep, fp, and ip receptors.62 pgs may also promote the retention of uv - damaged cells through the inhibition of apoptosis.63 a study of kuzbicki et al64 demonstrated that differently from scc, neither benign epithelial skin lesions nor bcc are associated with enhanced cox-2 expression . Both cox isoenzymes are strongly expressed in scc, deriving from a more differentiated epidermal layer.64 a high cox-2 expression could be considered a risk factor of bcc recurrence, determining a role in bcc prognosis.65 in general, nsaids are effective chemopreventive agents for skin cancer, and they function by acting on cox pathway specifically during the treatment of ak and scc . The inhibitory effects of pxm on cox-1 and -2 activity have been investigated by analyzing its binding mode . Ligand molecular docking has been used to evaluate binding modes and energies between cox-1 or cox-2 and the pxm drug (figures 1 and 2). The docking simulations have been performed by using the autodock vina 1.1.2 program,66 through the autodock / vina pymol plugin (http://wwwuser.gwdg.de/~dseelig/adplugin.html; the pymol molecular graphics system version 1.5.0.4 . The pxm sdf file,67 downloaded from the pubchem compound database (https://pubchem.ncbi.nlm.nih.gov), has been converted into mol2 file and filled with hydrogens using the open babel program.68 in the absence of the human enzymes, the x - ray structures of ovis aries cox-1, pdb i d code 1cqe,69 and mus musculus cox-2, pdb i d code 1pxx,70 deposited in the protein data bank (pdb, http://www.rcsb.org/pdb), have been selected as receptors since their sequence identity with human enzymes is very high (~95%) and the residues comprising the active sites fully conserved . In cox-1 and cox-2, the active sites are composed of the same group of amino acids with the exception of the residue in position 523, which is located at the border of the substrate pocket: that in cox-1 is an isoleucine, while in cox-2 is a valine.71 the cocrystallized ligands flurbiprofen (1cqe) and diclofenac (1pxx) have been removed before starting the simulations that have been performed using the genetic algorithm with local gradient optimization.66 the docking boxes (dimensions x=22.5; y=22.5; z=22.5) have been centered according to the x - ray ligand positions and include all the residues forming the binding sites . As previously reported,4 the molecular docking analysis confirms that pxm is a nonselective drug that blocks the activity of cox-1 and -2, detecting the same binding energy (7.6 kcal / mol) in both the enzymes, even if some slight differences in the binding mode are present . In cox-1 (figures 1a and 2a), the ser530 residue forms a hydrogen bond with the carboxamide group of the pxm, while a second hydrogen bond involves the tyr355 hydroxyl group and the benzothiazine moiety of the drug . In cox-2 (figures 1b and 2b), the same hydrogen bond occurs between tyr355 and benzothiazine group, while the ser530carboxamide interaction is lost and a new hydrogen bond, involving the arg120 side chain and the benzothiazine group, occurs . The remaining interacting residues show a similar distribution pattern, leading to a very comparable binding mode of the pxm molecule in the two binding sites . These results are in line with previous site - directed mutagenesis experiments that identified arg120 as the key residue for selective inhibition of cox-2, while identifying ser530 and tyr355 as nonspecific binding determinants.72 moreover, this structural analysis, determining the same binding energy, confirms that pxm is a nonselective cox-1 and cox-2 inhibitor . Ak is a chronic, progressive disease, which is expected to develop into scc at a rate of 10% over 10 years,73 the latter potentially metastasizing in 5% of cases . Although it is not possible to predict which specific aks will progress to scc, histologic evidence indicates the majority (60%70%) of sccs arise from aks lesions.74 the entire sun - damaged skin is a result of uv - induced field cancerization with multiple visible and subclinical lesions.75 aks are typically seen on fair - skinned population on chronic sun - exposed areas such as the face, arms, dorsum of the hands, bald scalp, and upper back.76 uvb radiation, considered the most important factor implicated in ak pathogenesis, cause mutations in the telomerase gene and tumor suppressor gene p53.5 increased telomerase activity delays programmed cell death, making the mutated cells immortal . The tumor suppressor gene p53 leads to the arrest of cell cycle, allowing the repair of damaged dna . If mutated, the proliferation of damaged cells will give rise to potentially neoplastic cells.77 several other factors increase the prevalence of ak, like skin type i and ii, advanced age, genetic predisposition, photodamage in outdoor workers, sun exposure in childhood, use of artificial tanning beds, puva (psoralens ultraviolet radiation) therapy, administration of x - rays, and immunosuppressive therapies.76 human papilloma viruses are also implicated in etiopathogenesis.78 many patients with epidermodysplasia verruciformis are immunosuppressed patients, ie, renal transplant recipients and develop hpv - associated ak and nmscs . The viral e6 protein of hpv promotes proteolytic degradation of the proapoptotic bak - protein, promoting skin cancerogenesis.79 other molecular markers that may indicate an increased likelihood of malignancy include the expression of p16, the cd95 ligand, tumor necrosis factor - related apoptosis - inducing ligand (trail) and trail receptors, and loss of heterozygosis.80,81 skin inflammation is due to chronic uvb irradiation, which promotes the production of eicosanoids and cox-1 and -2 produce pgs from arachidonic acid . In fact, pgs promote tumor growth by stimulating cell proliferation, invasion, and angiogenesis.82,83 increased levels of pge2 and pgf2 in premalignant and/or malignant epithelial skin cancers are due to the constitutive upregulation of enzymes involved in pg biosynthesis, such as cox-2, and downregulation of tumor suppressor gene 15-pgdh.16 a schematic representation of pxm effect on ak after uvb radiation is shown in figure 3: after chronic uvb exposure (290320 nm), several genes underwent mutations, including p53, with alterations in the arrest of cell cycle . The local use of pxm blocked the action of cox, resulting in blocking the biosynthesis of pgs and in 15-pgdh increased expression . Finally, a reduction of proliferation, tumor progression, and angiogenesis occurred, whereas there was an increase in apoptosis . After topical treatment of ak with pxm, normal epidermal architecture was restored (figure 3). In addition, cox-1 seems to contribute to tumor progression in various tissues including skin, and its inhibition lowers cell proliferation in some cancers, including ovarian and colon cancer.84,85 as recently reported, cox-1 and -2 are involved in vegf production in scc of head and neck.86 the coinhibition of cox-1 and -2 could block vegf synthesis . Treg - inducing vegf reduction counteracted pathological angiogenesis.87,88 the efficacy of pxm is related to its activity on both cox enzymes.89 in a preliminary open - label trial, we evaluated the efficacy and tolerability of pxm 1% gel in the treatment of patients affected by aks . Ten patients affected by multiple aks were treated twice daily for a period of 3 months.4 to measure the efficacy of the therapy, we evaluated the lesions clinically and by means standard error of mean of dermoscopy analysis at an initial baseline visit, at intermediate visits, and after 3 months.4 the modifications during treatment were evaluated using a new scoring system, named akesa, based on the clinical presence of erythema, scale, and atrophy on a target lesion.4 we observed improvement either in the typical features of the aks, as confirmed by the akesa score, or in the perilesional photodamaged skin area in terms of skin softening and a significant reduction in dermal vessels, also documented by dermoscopic investigation.4 on the basis of our clinical experience, we observed a healing response in more than 50% in aks with the use of pxm, a reduction in the appearance of new lesions in patients with multiple aks . In the preliminary trial, we showed that pxm exerts a significant antitumorigenic effect, and new studies with increased number of patients validated our results.4 ak is considered as a precursor to scc, and its eradication is mandatory in affected patients . The drug with a nonspecific cox-1 and cox-2 inhibitor action works on the early stages of skin carcinogenesis, and for this reason it is eligible for the treatment of aks and the field cancerization . The lack of side effects and its efficacy allow the use of pxm for a longer time even up to 1 year . Pxm appears to be safe, effective, and well tolerated, although its use in aks is still off - label . Further studies need to validate the use of pxm on nmsc, exploring its activity on different tumorigenesis pathways and on different histologic subtypes.
Optical coherence tomography (oct) is a noninvasive method that evaluates biological tissues by in vivo imaging.1 since its introduction, oct has undergone several improvements and revolutionized the diagnostic, monitoring, and therapeutic approaches to many retinal diseases and glaucoma . Computerized algorithms can be used on the high - resolution images obtained by modern oct devices in order to identify and measure the thicknesses of discrete retinal layers, including the retinal nerve fiber layer (rnfl), macular ganglion cell complex (gcc), and choroid.2,3 the choroid is the most vascular tissue in the eye and is known to have an important role in ocular nutrition, volume regulation, and temperature control.4 choroidal abnormalities are critical to the onset and progression of many chorioretinal disorders such as age - related macular degeneration (amd), polypoidal choroidal vasculopathy, vogt - kayanagi - harada disease, and central serous chorioretinopathy.5 with the recent development of enhanced depth imaging (edi), in vivo assessment of the choroid has become an area of interest . Edi allows better visualization and quantitative evaluation of the choroid, which was not possible before . Information about the choroidal thickness (ct) is useful in many clinical situations for decision making regarding the management and monitoring of disease progression.6 neurodegenerative diseases such as glaucoma preferentially affect the gcc, which is the sum of the three innermost layers: the rnfl, the retinal ganglion cell layer, and the inner plexiform layer . These contain the axons, cell bodies, and the dendrites of the retinal ganglion cells, respectively.7,8 recent studies have shown that measurement of macular gcc thickness has the same diagnostic potential for glaucoma as rnfl thickness.911 moreover, it has been reported that measurements of average gcc thickness had better sensitivity in detecting changes in retinal thickness in multiple sclerosis neurodegeneration than rnfl thickness measured by oct.12 both ct and gcc thickness measurements can be affected by several factors such as pupil size, scan quality, and cataract . In this study, we aimed to evaluate the effect of uneventful cataract surgery on subfoveal ct and gcc thickness measured by edi - oct . This prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . The present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . Exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus, hypertension, glaucoma, amd, diabetic retinopathy, retinal vein occlusion, uveitis, or other inflammatory and vascular pathologies . Patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure (iop) or cystoid macular edema were also excluded . All patients had a full ophthalmic examination, including best - corrected visual acuity (bcva) assessment, as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation, and iop measurement by goldmann applanation tonometry . Axial length (al) was measured by immersion echography (axis ii; quantel medical inc ., cedex, france) and central corneal thickness (cct) was measured by ultrasonographic pachymetry (pacline; optikon 2000 spa, rome, italy). Prior to the operation, tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . Phacoemulsification was performed by one experienced surgeon (ec) using infiniti vision system (alcon laboratories, inc . ). A clear corneal incision of 2.8 mm, 600 mmhg vacuum, an aspiration flow rate of 30 ml / min, bottle height of 75 cm, and quick - chop technique were used in all surgeries . Two different dispersive viscoelastics (viscoat and bivisc) and two different cohesive viscoelastics (healon and bio - hyalur) were used during cataract surgery . One - piece hydrophobic acrylic intraocular lens (sensar model: aab00; abbott medical optics inc ., santa ana, ca, usa) was used in all surgeries . Viscoelastics were cleaned out with marked attention, even behind the intraocular lenses . The procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . The operative times (ots) and effective phaco times (epts) were recorded in each case . Postoperatively, all patients were treated with topical antibiotics (ofloxacin 0.3%) for 1 week and topical steroid drops (prednisolone acetate 1%) for 4 weeks . Cirrus edi - oct (carl zeiss meditec ag, jena, germany; software version 6.5.0.772) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis (macular cube 512128) scan protocols, respectively . Scans with misalignment, segmentation failure, decentration of the measurement circle, and poor illumination, or those out of focus, were also excluded . The one - line raster is a 6 mm line consisting of 4,096 a - scans . Scan 3 of the five scans taken, which passes through the fovea, was used for all measurements . Ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . To avoid the effects of mydriatic agents or diurnal changes, the ct was measured from the subfoveal region by using the cirrus linear measurement tool . When there was a discrepancy of> 10% of the mean of the two values between the observers in two eyes in the study, observers discussed and repeated the measurements, and the average results from the two observers were determined as the final ct readings . The cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers (gcipl) in six circular sectors centered in the fovea . It also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . Macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans, each composed of 512 a - scans . A built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . The annulus has an inner vertical diameter of 1 mm, which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately, and an outer vertical diameter of 4 mm, which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . The ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . The difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . Similar to other cirrus printouts, it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . Those that are abnormal at the <5% and at the <1% level are represented by yellow and red backgrounds, respectively . The primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes; however, central macular thickness (cmt) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols, respectively, using the cirrus oct device . Statistical analysis was performed using spss statistics for windows, version 20.0 (ibm corporation 2011, armonk, ny, usa). Paired - samples t - test was used for equality of means, and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . This prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . The present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . Exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus, hypertension, glaucoma, amd, diabetic retinopathy, retinal vein occlusion, uveitis, or other inflammatory and vascular pathologies . Patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure (iop) or cystoid macular edema were also excluded . All patients had a full ophthalmic examination, including best - corrected visual acuity (bcva) assessment, as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation, and iop measurement by goldmann applanation tonometry . Axial length (al) was measured by immersion echography (axis ii; quantel medical inc ., cedex, france) and central corneal thickness (cct) was measured by ultrasonographic pachymetry (pacline; optikon 2000 spa, rome, italy). Prior to the operation, tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . Phacoemulsification was performed by one experienced surgeon (ec) using infiniti vision system (alcon laboratories, inc . ). A clear corneal incision of 2.8 mm, 600 mmhg vacuum, an aspiration flow rate of 30 ml / min, bottle height of 75 cm, and quick - chop technique were used in all surgeries . Two different dispersive viscoelastics (viscoat and bivisc) and two different cohesive viscoelastics (healon and bio - hyalur) were used during cataract surgery . One - piece hydrophobic acrylic intraocular lens (sensar model: aab00; abbott medical optics inc ., santa ana, ca, usa) was used in all surgeries . Viscoelastics were cleaned out with marked attention, even behind the intraocular lenses . The procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . The operative times (ots) and effective phaco times (epts) were recorded in each case . Postoperatively, all patients were treated with topical antibiotics (ofloxacin 0.3%) for 1 week and topical steroid drops (prednisolone acetate 1%) for 4 weeks . Cirrus edi - oct (carl zeiss meditec ag, jena, germany; software version 6.5.0.772) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis (macular cube 512128) scan protocols, respectively . Scans with misalignment, segmentation failure, decentration of the measurement circle, and poor illumination, or those out of focus, were also excluded . The one - line raster is a 6 mm line consisting of 4,096 a - scans . Scan 3 of the five scans taken, which passes through the fovea, was used for all measurements . Ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . To avoid the effects of mydriatic agents or diurnal changes, the ct was measured from the subfoveal region by using the cirrus linear measurement tool . When there was a discrepancy of> 10% of the mean of the two values between the observers in two eyes in the study, observers discussed and repeated the measurements, and the average results from the two observers the cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers (gcipl) in six circular sectors centered in the fovea . It also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . Macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans, each composed of 512 a - scans . A built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . The annulus has an inner vertical diameter of 1 mm, which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately, and an outer vertical diameter of 4 mm, which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . The ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . The difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . Similar to other cirrus printouts, it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . Those that are abnormal at the <5% and at the <1% level are represented by yellow and red backgrounds, respectively . The primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes; however, central macular thickness (cmt) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols, respectively, using the cirrus oct device . Statistical analysis was performed using spss statistics for windows, version 20.0 (ibm corporation 2011, armonk, ny, usa). Paired - samples t - test was used for equality of means, and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . A total of 30 eyes of 30 patients (16 male and 14 female), with a mean age of 60.24.2 years were included in this study . Mean bcva improved in all eyes, and this improvement was statistically significant (0.460.15 logarithm of minimum angle of resolution [logmar] units preoperatively and 0.060.49 logmar units postoperatively; p<0.001). Mean preoperative al was 22.80.6 mm (range: 21.524.1 mm) and mean cct was 551.522.1 m . Mean ot was 8.62.7 minutes and mean ept was 6.92.5 seconds (table 1). The mean subfoveal ct was 294.439.2 m (range: 201356 m) preoperatively and 301.439.9 m (range: 231367 m) postoperatively . The mean gcc thickness was 85.04.4 m (range: 7894 m) preoperatively and 89.25.3 m (range: 8199 m) postoperatively . The mean cmt was 247.917.6 m preoperatively (range: 211280 m) and 249.017.8 m (range: 213278 m) postoperatively . The mean rnfl thickness was 97.45.4 m preoperatively (range: 87110 m) and 101.75.6 m (range: 91113 m) postoperatively . Regression analysis showed that age, sex, al, cct, ot, and ept were not associated with ct changes (p=0.834, p=0.129, p=0.203, p=0.343, p=0.547, and p=0.147, respectively) and gcc thickness changes (p=0.645, p=0.542, p=0.152, p=0.664, p=0.448, p=0.268, respectively) after cataract surgery . In this study, we investigated the effect of cataract surgery on subfoveal ct and gcc thickness, as measured by edi - oct, and we found that both subfoveal ct and gcc thickness increased slightly after cataract surgery . Cmt and peripapillary rnfl thicknesses were also evaluated as a secondary objective, and we observed statistically significant increases in cmt and rnfl thickness . Our results indicate that cmt, subfoveal ct, as well as rnfl and gcc thicknesses are slightly affected after phacoemulsification surgery . Although our study included a small group (only 30 eyes), we assume that our data reliability is high because of the homogeneous nature of the patients (similarities of age, cataract type and severity, cct, al, ot, ept and so on) and the careful / detailed oct examinations performed by two observers, avoiding the effects of diurnal changes and mydriatic agents . Zhao et al13 revealed significant diurnal variations of the ct at the macular region in young healthy female individuals . Cataract surgery by phacoemulsification is one of the most common intraocular surgeries that improve the quality of vision . However, it is an invasive procedure leading to an inflammatory response mostly induced by the release of prostaglandins in the retina and choroid and, in many patients, can lead to worsening of preexisting retinal diseases such as diabetic macular edema.15,16 recent studies have shown subclinical increase of cmt and peripapillary rnfl thickness after uneventful cataract surgery.1719 however, the morphologic changes that this inflammatory insult produces in the choroid have not been studied well because of the lack of detailed imaging techniques for the choroid . After the introduction of edi, changes in ct in several pathologic (central serous chorioretinopathy, choroidal neovascularization, and high myopia) and physiologic (age, sex, and al) conditions can be easily assessed.2022 therefore, ct is being measured increasingly often and is becoming an accepted procedure for clinical and research applications . Falco et al23 evaluated the effect of uneventful phacoemulsification on the subfoveal ct and cmt, and they reported that phacoemulsification induced nonpathologic increases in cmt; however, these changes were not accompanied by significant changes in ct . They claimed that the inflammatory insult due to phacoemulsification mainly affects at the retinal level and seems to be independent of ct changes.23 by contrast; our results support the recent studies by ohsugi et al24 and noda et al,25 which report significant increases in ct after uneventful phacoemulsification . Ohsugi et al24 evaluated 100 eyes and emphasized that the al and changes in iop were critical for evaluating the changes in ct after cataract surgery . Noda et al25 evaluated 29 eyes and observed that the increase in subfoveal ct did not subside to baseline even at 6 months postoperatively . They also found that male sex and thicker baseline ct predicted a larger magnitude of increase in ct after cataract surgery.25 in our study, interestingly, the parameters of age, sex, al, baseline ct, and cct did not influence the postoperative ct statistically in regression analysis . We also analyzed the effect of ot and ept on postoperative ct and we did not find any relationship . This may be explained by the short / similar results in terms of ots and epts, as well as the experience of the surgeon conducting a quick surgery with less anterior segment manipulation, thus reducing the inflammatory response after cataract surgery . A speculation is that it may be related to postoperative inflammation because proinflammatory prostaglandins and cytokines are considered to explain macular edema after cataract surgery, and inflammatory disorders are also known to increase the ct.26,27 another speculation is that cataract surgery introduces more light into the eyes, leading to greater metabolic activation in the retinal pigment epithelium, causing angiogenesis and inflammation.28,29 in the beaver dam and blue mountains eye studies,3032 it has been suggested that cataract surgery is associated with the onset of amd, resulting in visual impairment due to neovascularization originating from the choroid . Noda et al25 report that cataract surgery influences the ct and this influence persists for as long as 6 months postoperatively and may affect the process of amd . Choroidal abnormalities are central to chorioretinal disorders such as amd, polypoidal choroidal vasculopathy, and central serous chorioretinopathy . Furthermore, it has been claimed that pathologic choroidal blood supply is an important factor in open - angle glaucoma leading to optic nerve damage.33,34 cts of glaucomatous eyes have been reported to be thinner in postmortem histologic studies . In addition, significant increases in choroidal extravascular volume have been confirmed in many primary angle - closure glaucoma eyes and abnormal ct has been hypothesized to be a contributing feature in these eyes.35,36 however, it is unclear whether these findings represent a risk factor or a consequence of these diseases . The hallmark of glaucoma is the loss of the retinal ganglion cell axons, which leads to a typical optic neuropathy . Qualitative and quantitative evaluations of the optic nerve head and rnfl have been used to detect evidence of glaucomatous damage.911 in a recent study,8 it has been suggested that the gcc thickness may be the most relevant parameter to measure in glaucoma . Nouri - mahdavi et al37 also reported that regional gcc measurements derived from cirrus hd - oct performed as well as regional rnfl outcomes for the detection of early glaucoma . Moreover, macular gcc thickness has been found to have better structure function correlations than rnfl thickness with both visual function and central nervous system findings in multiple sclerosis neurodegeneration.12 as oct uses near - infrared light and is based on interferometry, its image quality is influenced by opacities in the optical path . Loss of oct image quality is caused by attenuation of the light in the oct scanning spot on the retina.17,18 several studies have shown that lens opacity decreased image quality and postoperative rnfl thickness measurements increased significantly.3840 the more advanced the cataract, the less is the signal quality and the thinner are the recorded rnfl and gcc thicknesses . The coexistence of neurodegenerative diseases such as glaucoma and cataract among elderly patients is not unusual, and cataract may decrease the quality of fundus images, leading to an incorrect evaluation in the diagnosis and follow - up of these patients . Nakatani et al7 examined the effect of cataract on the measurements of gcc and rnfl thicknesses, and they reported that all thickness parameters in gcc and rnfl increased slightly after phacoemulsification surgery . Therefore, we decided that the presence of cataract may lead to an underestimation of the thicknesses of gcc and rnfl, and this should be taken into account when analyzing progression in glaucoma and/or neurodegenerative diseases such as multiple sclerosis . After cataract surgery, the examiners should consider obtaining new baseline measurements, as suggested in previous studies . This study has many limitations such as short follow - up time, relatively small sample size, and lack of eyes with glaucoma and neurodegenerative diseases . In addition, we analyzed just mean subfoveal ct and mean thickness of gcc and rnfl, leading to lack of segmentation of these parameters . Scleral interface and measured ct with cirrus linear measurement tool . Although we used scans with signal strength 6 for analysis and excluded poor images, the effect of signal strength index on measurements was not investigated . Furthermore, we used postoperative topical steroids after cataract surgery; thus, the true effect of phacoemulsification on oct parameters could not be evaluated and we could not determine whether and when the increased ct can return to baseline measurements . Phacoemulsification surgery increased ct and it should be kept in mind that the small changes in ct may affect the onset of amd, which is a serious disease that results in central visual impairment . Although the mechanism is unclear, it has been postulated that ct changes may also play a role in glaucoma . We suggest establishment of new baseline measurements after cataract surgery for monitoring patients with glaucoma and neurodegenerative diseases . Further studies with a greater number of patients and longer duration of follow - up and conducted using new computerized oct algorithms are needed.
Prolonged - release (pr) fampridine tablets (known as sustained / modified - release fampridine in some countries and extended - release dalfampridine in the united states) are approved for improving walking in ms based on results from two pivotal trials that demonstrated consistent improvements in walking speed on the timed 25-foot walk and on patients self - assessment of ambulatory disability on the 12-item ms walking scale (msws-12). Walking is a complex activity influenced by many variables, including impairments in balance, which are frequent among patients with ms and correlate with slower walking speed . Furthermore, balance impairment is a strong predictor for perceived difficulty in mobility in ms . This highlights the need for studies that assess effects of pr - fampridine on balance . Pr - fampridine improved standing balance in a small (n = 8) 14-week study; however, randomised, placebo - controlled studies assessing the effect of pr - fampridine on balance have not previously been performed . The timed up and go (tug) test and berg balance scale (bbs) measure mobility / balance and have demonstrated high reliability in patients with ms . The objective of mobile was to explore the effect of pr - fampridine on endpoints related to patients self - assessed walking disability and dynamic / static balance, assessed using the bbs and tug, as well as to subjective impression of well - being and patients impression of change in walking . This trial was conducted in compliance with the declaration of helsinki, the international conference on harmonisation good clinical practice guidelines, the european union clinical trials directive and local regulatory requirements . Written informed consent was obtained from each patient before any evaluations were conducted for eligibility . The trial is registered on clinicaltrials.gov (identifier nct01597297) and the european union clinical trials register (eudract number 2012 - 000368 - 90). Mobile was a randomised, double - blind, exploratory, multi - centre, placebo - controlled trial in patients with ms (electronic supplementary materials, figure 1). Eligible patients were randomised (1:1) to receive pr - fampridine 10 mg tablets or matching placebo twice daily every 12 hours for 24 weeks . Scheduled visits took place at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24 . A post - dosing follow - up visit was conducted two weeks after the end of treatment . Randomisation was determined by a centralised system on day 1 of the trial and treatment assignments were made through an interactive web response system . All patients and trial staff, including the principal investigator, were blinded to patient treatment assignments . Treatment kits were prepared centrally and labelled with unique numbers, which were used to maintain the blind when drug supplies were dispensed . Eligible patients were male or female, aged 1870 years with an expanded disability status scale score of 4.07.0 and diagnosis of primary - progressive ms, secondary - progressive ms, progressive - relapsing ms or relapsing - remitting ms per revised mcdonald criteria of 3 months duration . Key exclusion criteria included: treatment with 4-aminopyridine or 3,4-diaminopyridine in any formulation 30 days before screening; known allergy to pyridine - containing substances; any history of seizure, epilepsy or other convulsive disorder; renal impairment (creatinine clearance <80 ml / min); onset of ms exacerbation 60 days before screening; and a body mass index 40 kg / m . Outcome measures were assessed at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24 (on - treatment period) and at the week 26 follow - up visit (except where noted otherwise). Because this was an exploratory study, there were no pre - specified primary / secondary endpoints and walking ability was assessed using the msws-12 and the seven - item patient global impression of change (pgic) scale . The msws-12 is a 12-item questionnaire that asks patients to rate limitations of their mobility owing to ms during the preceding two weeks on a five - point scale (from 1 = not at all to 5 = extremely). Total score ranges from 160 and was transformed to a scale of 0100; reduced score indicates improvement in walking . The pgic is a global assessment of the patient s impression of how the study drug affected their overall walking during the preceding seven days . The pgic was assessed at weeks 2, 4, 8, 12, 16, 20 and 24, and scored on a seven - point scale (from 1 = very much worse to 7 = very much improved). Mobility and dynamic balance were assessed using the tug test, which measures the time / speed it takes for a patient to stand up from a seated position, walk three metres out, turn around and walk back and return to the seated position . Static and dynamic balance were measured using the bbs, which is comprised of 14 balance - related tasks scored from 0 (unable to perform) to 4 (able to perform independently). The bbs is the sum of scores across these tasks and ranges from 0 (poor balance) to 56 (good balance). The tug and bbs, which have demonstrated high reliability in patients with ms, were chosen to further assess the impact of pr - fampridine treatment on mobility, specifically on static and dynamic balance . Balance has not been previously evaluated with pr - fampridine treatment in a randomised, placebo - controlled trial but may impact patients walking ability and mobility . The impact of ms on the patient and quality of life (qol) were assessed using the 29-item multiple sclerosis impact scale (msis-29) physical subscale (phys) and the euroqol-5 dimensions 5-level (eq-5d-5l). The self - administered msis-29 questionnaire contains a 20-item phys and a nine - item psychological subscale . For a particular visit, the msis-29 phys score was calculated by summing the 20 items and transforming the score to a scale with a range of 0 (no impact of ms) to 100 (extreme impact of ms). Negative change on the msis-29 phys indicates improvement in physical health . The eq-5d-5l is a generic instrument comprised of five questions and a visual analogue scale (vas). A utility score is derived from the five questions addressing mobility, self - care, usual activities, pain / discomfort and anxiety / depression, with responses / scores ranging from 1 (no problem) to 5 (severe problem). The vas score ranges from 0 (worst imagined health state) to 100 (best imagined health state). The eq-5d was assessed at day 1 and weeks 4, 8, 12, 16, 20 and 24, and a positive change in both the utility and vas scores indicates an improvement in health state . Safety and tolerability were assessed by monitoring adverse events (aes), serious aes (saes) and concomitant medications and by performing physical examination, vital sign measurements and 12-lead electrocardiograms . A sample size of 120 patients (60 patients per group) was chosen based on bootstrapping that sampled data from a previous study to create 1000 datasets of 60 patients each, and repeated for sample sizes of 70 and 80 patients . For each sample, the proportion of patients with a six - point change or more in msws-12 score was calculated, and the percentage of samples that fell within an interval of the actual percentage in the study was calculated to explore whether increasing the sample size increased the precision . Using the sample size of 60 patients for both treatment groups, 84% of samples were within 8% of the true proportion and increasing the size did not appreciably increase precision . The intent - to - treat population consisted of all patients who were randomised, received at least one dose of treatment and had at least one post - baseline assessment for a given parameter . Efficacy analyses were based on an intent - to - treat analysis with missing data imputed by the last observation carried forward method when at least one post - baseline value was available . Baseline values were not carried forward and were defined as the mean over the screening and baseline visits . Mean changes in each efficacy endpoint, except for the pgic, were calculated over 24 weeks using the last observation carried forward values to calculate the average . Because the pgic was designed to assess change since the previous visit, week 2 was the only time point that was valid for between - group comparisons of treatment effect . Based on the distribution of changes from baseline across patients, all by - visit analyses were summarised using median change from baseline on the outcome . Mean changes from baseline on the msws-12 were categorised using the following thresholds: a less than one - point improvement / no change / worsening and then increasing thresholds of improvement from 1 point to 10 points in one - point increments . Mean percentage changes from baseline in tug speed were categorised using the following thresholds: 0% (worsening / no change),> 0% (any improvement) and then increasing thresholds of improvement from 10% to 40% in 5% increments . These categories were summarised as the number / percentage of patients meeting each threshold . In order to characterise magnitude of the observed treatment effect relative to variance in the outcome measures, post hoc statistical testing compared multiple thresholds of improvement between treatment groups for msws-12 and tug using a logistic regression adjusted for baseline . Multiple msws-12 thresholds of change were examined based on ranges previously identified as clinically meaningful (46 points based on a 100-point scale) over three months . A post hoc analysis using the chi - square test compared the percentage of patients in each treatment group who reported any improvement in pgic after two weeks of treatment . To assess magnitude of treatment effect over time, changes from baseline to each visit also were summarised for each efficacy endpoint, except for pgic . For missing data in msws-12, a visit in which 50% of the component questions were answered but at least one question was missing msws-12 score was considered missing for a visit in which 50% of the questions were unanswered . For the tug test, two trials of the tug were conducted at each visit and speed for any particular visit was calculated as the average for trials 1 and 2 . If either trial was missing, then speed from the completed trial was used . For the bbs, if at least two questions were missing at a visit, the score was set to missing . If two or fewer questions were missing, these were imputed using the respondent - specific mean score . For msis-29 phys, if a patient had missing data for <10 of the 20 items, the mean of the non - missing items was used to impute the missing items . For eq-5d-5l, no imputation was used for missing values for the five summary scores or for vas . A summary utility index value was calculated for patients with non - missing data for each of the five questions at a visit . The crosswalk method was used to map eq-5d-5l to the eq-5d 3-level united kingdom value set, because value sets for eq-5d-5l are still under development . Utility index value ranges from 0.594 (worst health state) to 1.000 (best health state). This trial was conducted in compliance with the declaration of helsinki, the international conference on harmonisation good clinical practice guidelines, the european union clinical trials directive and local regulatory requirements . Written informed consent was obtained from each patient before any evaluations were conducted for eligibility . The trial is registered on clinicaltrials.gov (identifier nct01597297) and the european union clinical trials register (eudract number 2012 - 000368 - 90). Mobile was a randomised, double - blind, exploratory, multi - centre, placebo - controlled trial in patients with ms (electronic supplementary materials, figure 1). Eligible patients were randomised (1:1) to receive pr - fampridine 10 mg tablets or matching placebo twice daily every 12 hours for 24 weeks . Scheduled visits took place at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24 . A post - dosing follow - up visit was conducted two weeks after the end of treatment . Randomisation was determined by a centralised system on day 1 of the trial and treatment assignments were made through an interactive web response system . All patients and trial staff, including the principal investigator, were blinded to patient treatment assignments . Treatment kits were prepared centrally and labelled with unique numbers, which were used to maintain the blind when drug supplies were dispensed . Eligible patients were male or female, aged 1870 years with an expanded disability status scale score of 4.07.0 and diagnosis of primary - progressive ms, secondary - progressive ms, progressive - relapsing ms or relapsing - remitting ms per revised mcdonald criteria of 3 months duration . Key exclusion criteria included: treatment with 4-aminopyridine or 3,4-diaminopyridine in any formulation 30 days before screening; known allergy to pyridine - containing substances; any history of seizure, epilepsy or other convulsive disorder; renal impairment (creatinine clearance <80 ml / min); onset of ms exacerbation 60 days before screening; and a body mass index 40 kg / m . Outcome measures were assessed at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24 (on - treatment period) and at the week 26 follow - up visit (except where noted otherwise). Because this was an exploratory study, there were no pre - specified primary / secondary endpoints and walking ability was assessed using the msws-12 and the seven - item patient global impression of change (pgic) scale . The msws-12 is a 12-item questionnaire that asks patients to rate limitations of their mobility owing to ms during the preceding two weeks on a five - point scale (from 1 = not at all to 5 = extremely). Total score ranges from 160 and was transformed to a scale of 0100; reduced score indicates improvement in walking . The pgic is a global assessment of the patient s impression of how the study drug affected their overall walking during the preceding seven days . The pgic was assessed at weeks 2, 4, 8, 12, 16, 20 and 24, and scored on a seven - point scale (from 1 = very much worse to 7 = very much improved). Mobility and dynamic balance were assessed using the tug test, which measures the time / speed it takes for a patient to stand up from a seated position, walk three metres out, turn around and walk back and return to the seated position . Static and dynamic balance were measured using the bbs, which is comprised of 14 balance - related tasks scored from 0 (unable to perform) to 4 (able to perform independently). The bbs is the sum of scores across these tasks and ranges from 0 (poor balance) to 56 (good balance). The tug and bbs, which have demonstrated high reliability in patients with ms, were chosen to further assess the impact of pr - fampridine treatment on mobility, specifically on static and dynamic balance . Balance has not been previously evaluated with pr - fampridine treatment in a randomised, placebo - controlled trial but may impact patients walking ability and mobility . The impact of ms on the patient and quality of life (qol) were assessed using the 29-item multiple sclerosis impact scale (msis-29) physical subscale (phys) and the euroqol-5 dimensions 5-level (eq-5d-5l). The self - administered msis-29 questionnaire contains a 20-item phys and a nine - item psychological subscale . For a particular visit, the msis-29 phys score was calculated by summing the 20 items and transforming the score to a scale with a range of 0 (no impact of ms) to 100 (extreme impact of ms). Negative change on the msis-29 phys indicates improvement in physical health . The eq-5d-5l is a generic instrument comprised of five questions and a visual analogue scale (vas). A utility score is derived from the five questions addressing mobility, self - care, usual activities, pain / discomfort and anxiety / depression, with responses / scores ranging from 1 (no problem) to 5 (severe problem). The vas score ranges from 0 (worst imagined health state) to 100 (best imagined health state). The eq-5d was assessed at day 1 and weeks 4, 8, 12, 16, 20 and 24, and a positive change in both the utility and vas scores indicates an improvement in health state . Safety and tolerability were assessed by monitoring adverse events (aes), serious aes (saes) and concomitant medications and by performing physical examination, vital sign measurements and 12-lead electrocardiograms . A sample size of 120 patients (60 patients per group) was chosen based on bootstrapping that sampled data from a previous study to create 1000 datasets of 60 patients each, and repeated for sample sizes of 70 and 80 patients . For each sample, the proportion of patients with a six - point change or more in msws-12 score was calculated, and the percentage of samples that fell within an interval of the actual percentage in the study was calculated to explore whether increasing the sample size increased the precision . Using the sample size of 60 patients for both treatment groups, 84% of samples were within 8% of the true proportion and increasing the size did not appreciably increase precision . The intent - to - treat population consisted of all patients who were randomised, received at least one dose of treatment and had at least one post - baseline assessment for a given parameter . Efficacy analyses were based on an intent - to - treat analysis with missing data imputed by the last observation carried forward method when at least one post - baseline value was available . Baseline values were not carried forward and were defined as the mean over the screening and baseline visits . Mean changes in each efficacy endpoint, except for the pgic, were calculated over 24 weeks using the last observation carried forward values to calculate the average . Because the pgic was designed to assess change since the previous visit, week 2 was the only time point that was valid for between - group comparisons of treatment effect . Based on the distribution of changes from baseline across patients, all by - visit analyses were summarised using median change from baseline on the outcome . Mean changes from baseline on the msws-12 were categorised using the following thresholds: a less than one - point improvement / no change / worsening and then increasing thresholds of improvement from 1 point to 10 points in one - point increments . Mean percentage changes from baseline in tug speed were categorised using the following thresholds: 0% (worsening / no change),> 0% (any improvement) and then increasing thresholds of improvement from 10% to 40% in 5% increments . These categories were summarised as the number / percentage of patients meeting each threshold . In order to characterise magnitude of the observed treatment effect relative to variance in the outcome measures, post hoc statistical testing compared multiple thresholds of improvement between treatment groups for msws-12 and tug using a logistic regression adjusted for baseline . Multiple msws-12 thresholds of change were examined based on ranges previously identified as clinically meaningful (46 points based on a 100-point scale) over three months . A post hoc analysis using the chi - square test compared the percentage of patients in each treatment group who reported any improvement in pgic after two weeks of treatment . To assess magnitude of treatment effect over time, changes from baseline to each visit also were summarised for each efficacy endpoint, except for pgic . For missing data in msws-12, a visit in which 50% of the component questions were answered but at least one question was missing, scores from unanswered questions were imputed using the respondent - specific mean score . Msws-12 score was considered missing for a visit in which 50% of the questions were unanswered . For the tug test, two trials of the tug were conducted at each visit and speed for any particular visit was calculated as the average for trials 1 and 2 . If either trial was missing, then speed from the completed trial was used . For the bbs, if at least two questions were missing at a visit, the score was set to missing . If two or fewer questions were missing, these were imputed using the respondent - specific mean score . For msis-29 phys, if a patient had missing data for <10 of the 20 items, the mean of the non - missing items was used to impute the missing items . For eq-5d-5l, no imputation was used for missing values for the five summary scores or for vas . A summary utility index value was calculated for patients with non - missing data for each of the five questions at a visit . The crosswalk method was used to map eq-5d-5l to the eq-5d 3-level united kingdom value set, because value sets for eq-5d-5l are still under development . Utility index value ranges from 0.594 (worst health state) to 1.000 (best health state). A total of 132 patients were randomised at 24 sites in belgium, canada, italy, the netherlands, sweden and the united kingdom . The first patient was treated on 30 august 2012 and the trial ended on 8 august 2013 . All randomised patients were treated and included in the analysis: 64 were treated with placebo and 68 with pr - fampridine . In each group, 81% of patients completed treatment and 19% of patients discontinued treatment (figure 1). Ae was the most common reason for discontinuation and one patient in each treatment group discontinued owing to lack of efficacy (figure 1). Ae: adverse event; crcl: creatinine clearance; itt: intent - to - treat; pr: prolonged - release . Bbs: berg balance scale; edss: expanded disability status scale; eq-5d-5l: euroqol-5 dimension 5-level; msis-29: 29-item multiple sclerosis impact scale; msws-12: 12-item multiple sclerosis walking scale; nd: not determined; phys: physical subscale; ppms: primary - progressive multiple sclerosis; pr: prolonged - release; prms: progressive - relapsing multiple sclerosis; rrms: relapsing - remitting multiple sclerosis; spms: secondary - progressive multiple sclerosis; tug: timed up and go; vas: visual analogue scale . Pr - fampridine therapy resulted in greater median improvements from baseline in tug speed, bbs total score and msws-12 score compared with placebo during the 24-week treatment period (figure 2(a), (c) and (d)). After treatment discontinuation at week 24, improvements declined and approached zero by the week 26 follow - up visit in the pr - fampridine group . Median changes from baseline and corresponding 95% confidence intervals in efficacy measures by study visit . Outcome measures: (a) 12-item multiple sclerosis walking scale (msws-12); (b) 29-item multiple sclerosis impact scale (msis-29) physical subscale (phys); (c) timed up and go (tug) test; and (d) berg balance scale (bbs) were assessed at baseline (mean over screening and day 1) and weeks 2, 4, 8, 12, 16, 20, 24 and week 26 (off - treatment visit; not assessed for msis-29). Error bars denote non - parametric 95% confidence interval for the median change at each visit . When changes from baseline were summarised as a change at pre - specified thresholds of improvement, a higher proportion of patients receiving pr - fampridine versus placebo met each threshold of improvement in the tug test (> 0% and from 10% to 40%), with statistically significant differences at thresholds 10% (p = 0.0021) and 15% (p = 0.0262; figure 3). Similar results were observed for the msws-12; a higher proportion of patients receiving pr - fampridine versus placebo met each threshold of improvement in msws-12 (1 to 10 points) with statistically significant differences at thresholds 7 (p = 0.0275), 8 (p = 0.0153) and 9 (p = 0.0088; figure 3). Cumulative percentage of patients with mean improvement in 12-item multiple sclerosis walking scale (msws-12) and timed up and go (tug) speed over 24 weeks . Msws-12 (upper panel): cumulative percentage of patients with increasing levels of improvement on the msws-12 over the on - treatment period (weeks 224) across multiple thresholds (thresholds 1 to 10 represent improvements in 1 to 10 points). Tug speed (lower panel): cumulative percentage of patients with average percent increase from baseline in tug speed over the on - treatment period (weeks 224) across multiple thresholds . Pr - fampridine versus placebo: * p = 0.0275; * * p = 0.0153; * * * p = 0.0088; * * * * p = 0.0021; * * * * * p = 0.0262 . Hoc analysis, a significantly greater proportion of patients (n (%)) treated with pr - fampridine (31 (46%)) versus placebo (16 (26%)) also reported improvement on pgic at the week 2 visit (p = 0.023). Pr - fampridine therapy also resulted in greater improvements from baseline in msis-29 phys score versus placebo (figure 2(b)). However, no apparent differences between treatment groups were observed in eq-5d-5l results; median treatment difference (95% confidence interval) for pr - fampridine versus placebo for eq-5d-5l vas was 0.00 (4.17, 4.67) and for utility index was 0.00 (0.04, 0.04). The proportion of patients with any ae was similar in placebo - treated patients (49 (77%)) versus patients receiving pr - fampridine (51 (75%)). Nasopharyngitis and urinary tract infections (utis) were the most frequently reported aes in patients treated with pr - fampridine and placebo, respectively (table 2). Incidence of falls was higher in patients receiving placebo versus pr - fampridine, whereas incidences of balance disorder, gait disturbance and dizziness were higher in patients receiving pr - fampridine . A lower proportion of patients receiving pr - fampridine (3%) versus placebo (8%) reported saes . In the pr - fampridine group, saes were considered unrelated to treatment and included moderate ms relapse in one patient and moderate ms relapse and severe paraparaesis (worsened ms symptoms) in the second patient . Ae: adverse event; pr: prolonged - release; sae: serious adverse event . In this trial, pr - fampridine therapy resulted in greater / sustained improvements in mobility / balance over the six - month trial duration compared with placebo as measured by the tug test and bbs . Benefits were seen as early as two weeks after treatment initiation and were maintained throughout the trial . Upon discontinuation of pr - fampridine therapy, improvements in outcome measures reversed and approached pre - treatment levels within two weeks . Early benefits also were seen in walking ability measured by msws-12, and although msws-12 score fluctuated between weeks 4 and 12, it stabilised after week 16 and benefits were maintained to the end of the study in patients receiving pr - fampridine therapy . In addition to greater median improvements in tug speed and msws-12 score, a higher cumulative proportion of patients receiving pr - fampridine versus placebo met each threshold of improvement in tug speed (> 0% and 10% to 40%) and msws-12 score (1 to 10 points), including thresholds in a range (4 to 6 points) previously identified as clinically meaningful for msws-12 with different data sources . The mobile study was the first randomised, placebo - controlled study that assessed the effect of pr - fampridine on dynamic and static balance . Walking is a complex activity and individual patients with ms may improve in one or more domains, such as speed and/or balance . The findings of mobile suggested that the previous definition of pr - fampridine responder that was based on walking speed alone may be too narrowly defined and may not identify some patients who received treatment benefits associated with improved balance . Pr - fampridine therapy also demonstrated improvements on msis-29 phys, a patient - reported measure of the physical impact of ms, but no clear differences between treatment groups were observed in eq-5d-5l vas and utility index results over time . The discrepancy observed in this trial between the msis-29 and eq-5d-5l may be related to the insensitivity of generic measures to assess ms - related changes in qol . The msis-29 phys results in this trial supported those observed in the enable study, in which pr - fampridine therapy demonstrated significant / clinically meaningful improvements on qol / health state, measured by a broad range of ms - specific and generic patient - reported endpoints, over 48 weeks in> 600 patients who received pr - fampridine . Safety findings from mobile were consistent with the known safety profile of pr - fampridine, with the exception that incidence of utis was higher in placebo - treated patients compared with patients receiving pr - fampridine . This is in contrast to that observed in the pivotal studies, in which incidence of utis was higher for the pr - fampridine group . In the current study, utis were confirmed by culture . In contrast, the pivotal trials did not require confirmation of utis, which may have resulted in an overestimation of the rate of utis . No seizures were observed in mobile and although more patients receiving pr - fampridine reported balance disorders, gait disturbance and dizziness versus placebo, the incidence of falls was higher in the placebo group . Balance disorders, gait disturbance and dizziness are broad, non - specific aes, and increases in these events may not result in an increase in falls, as was the case in the mobile study . Furthermore, the improvement in tug and bbs could have manifested in fewer falls among the patients treated with pr - fampridine . The findings of mobile confirm and expand findings of previous controlled studies with a longer treatment period, geographically different study population and broader range of objective and patient - reported measures of mobility and balance to assess different domains of walking . Mobile was exploratory in design and its findings require confirmation in a prospective trial that is underway . Nevertheless, the results of mobile support that pr - fampridine therapy provides significant and sustained improvements in walking characteristics beyond walking speed; in particular, pr - fampridine therapy resulted in early and sustained benefits on measures of dynamic and static balance and mobility as well as patient - reported walking disability . Overall, these findings provided additional support for the potential of pr - fampridine to result in clinically meaningful improvements in walking quality / ambulatory function in patients with ms with walking disability.
Delusional disorder is an uncommon psychiatric condition characterized by nonbizarre delusions of a single theme, in the absence of other mood or psychotic symptoms . It is differentiated from schizophrenia by the absence of bizarre delusions and impairment of functioning only in relation to the delusional belief . The lifetime prevalence rate is reported to be about 0.2% in the united states, whereas in india rates of about 1% have been reported in a psychiatric clinic population . The disorder is divided into erotomanic, grandiose, jealous, persecutory and somatic subtypes based on the content of the delusion . The most commonly reported subtypes are the persecutory subtype in western literature, whereas delusional parasitosis is most commonly reported in indian literature . Although delusions of a sexual nature are not unusual in schizophrenia and affective disorder, reports of this presenting as a delusional disorder are uncommon . We report a case of a young male who presented with a single delusion regarding his sexual physiology . Mr . A is a 19-year - old single engineering student, from a middle socioeconomic background . Over the previous year and a half, he firmly believed that every time he assumed an upright posture, he attained penile erection . He believed that others were able to identify his physiological state by the appearance of his groin and therefore were laughing at him and making derogatory comments . He had made attempts to mask these perceived bodily changes by changing the way he dressed . Secondary to these beliefs he had also become socially withdrawn, was frequently absent from class and had had significant academic decline . He also avoided situations, which required him to stand upright, such as crowded buses, elevators, and shops . He had attempted self - harm a year earlier by slashing his wrist due to the distress related to his beliefs . The patient had been on fluvoxamine and risperidone for about 7 months at the time of presentation . Physical examination, including a detailed neurological and genital examination did not reveal any abnormalities . Mental status examination revealed a well groomed young man with normal psychomotor activity and speech . He was distressed by the sexual problems that he perceived he had, but denied suicidal ideation . He had a fixed belief that he attained penile erection whenever in an erect posture . The degree of conviction with which the patient held his belief despite evidence to the contrary suggested it to be a delusion, upon which he was acting . A diagnosis of delusional disorder was considered in view of the single delusional theme in the absence of first rank symptoms or affective features . The management focused on establishing rapport with the patient along with eliciting and understanding the explanatory model for his beliefs . Socratic questioning was used to identify negative thoughts and dysfunctional assumptions, which were reflected to the patient . A hierarchy of situations based on distress and avoidance were charted down and he was asked to expose himself to the least anxiety provoking situation . The patient was also encouraged to do some behavioral experiments in these situations to confirm or disprove his assumptions . The antidepressant medication was tapered and stopped, while the dose of risperidone was gradually increased to 4 mg / day . Delusions with sexual content, although not as common as other delusions, have been reported in psychotic disorders such as schizophrenia, depression, and organic conditions . The content of these delusions include that of self - mutilation, delusional pseudo - transsexualism, bizarre sexual mechanisms, threat to genitalia, variations of erotomania, pregnancy, procreation, and homosexuality . In a qualitative review of bizarre delusions among a sample of patients with schizophrenia in india, de et al . Found that delusions with sexual content included false beliefs about sexual relationship between humans, animals and/or supernatural beings; forceful change in sexual gender or identity and threats to genitalia . While sexual delusions are encountered as one among other types of delusions in schizophrenia, it is not commonly reported in the form of a delusional disorder . In this case, organic illness was ruled out and the delusional nature of the belief was confirmed . In the absence of other psychotic symptoms, the patient reported distress secondary to the belief and its consequences, and there was no evidence of a primary mood disorder or other psychotic symptoms . Delusional disorder is managed with pharmacological and nonpharmacological strategies . In this case treatment with antipsychotic agents was combined with supportive measures along with cognitive and behavioural strategies, to allay the patient's anxiety, acknowledge his distress, explore the consequences of his beliefs and modify them . Isolated delusions with a sexual theme are rare . As with any other delusional disorder, the treatment is challenging and involves both psychopharmacology and psychotherapy . Given the chronic nature of this condition, treatment strategies should be tailored to the individual needs of the patients with a focus on maintaining social function and improving quality of life.
Bleomycin a2 (blm, 1, figure 1) is the major component (70%) of the clinical anticancer drug blenoxane, which is currently used for the treatment of hodgkin s lymphoma, melanomas, head and neck carcinomas, and testicular cancers . Blm exerts its biological effects through a metal ion and oxygen dependent dna cleavage that occurs selectively at 5-gc or 5-gt sites . Although capable of producing single - strand and double - strand breaks, the latter is believed to be the most biologically relevant event . Despite being an effective therapy for numerous malignancies, blm treatment is often limited by dose - dependent pneumonitis, lung fibrosis, and skin toxicities, some of which have been attributed to the c - terminal bithiazole appendage . Thus, there remains an interest in identifying synthetic blm - based analogs that lack off - target toxicity and maintain efficacy, which can be used as single component oncology drugs . Each structural unit of blm contributes an important role in the dna binding and cleavage cascade . Systematic evaluation of its structure through single site deep - seated modifications prepared by total synthesis has delineated the function of each subunit and the role of their individual substituents . These studies, in conjunction with nmr - derived structural models of dna bound blm, continue to define the remarkable combination of functional, structural, and conformational properties integrated into the natural product . The pyrimidoblamic acid subunit not only participates in the metal chelation and o2 activation but also is responsible for the dna cleavage selectivity via triplex - like hydrogen bonding to guanine at the 5-gc and 5-gt cleavage sites . In such studies, we have additionally shown that removal of the pyrimidine c5 methyl group, enlisting the functionalized pyrimidine found in p-3a, has no impact in the functional activity of the resulting blm analog . From these studies, ()-pyrimidoblamic acid (2, figure 2) and the related peptide - derived natural product, p-3a (3, figure 2), have been identified as key subunits for the continued preparation of improved or simplified bleomycin analogs . Total syntheses of ()-pyrimidoblamic acid were originally described by umezawa, hecht, and later by us . Although elegant and pioneering in their own regards, these previous efforts have suffered from the inability to fully control the stereochemistry at the benzylic pyrimidine c2 tertiary center (scheme 1). Similarly, our initial total synthesis of p-3a, whose pyrimidine core differs from that of pyrimidoblamic acid by only the lack of the c5 methyl group, relied on a late stage diastereoselective introduction of the benzylic pyrimidine c2 tertiary center that proceeded with modest diastereoselectivity (87:13). Recently, we defined the cycloaddition scope and productive reactivity of substituted electron - deficient 1,2,3-triazines with various dienophiles, including the first report of their ability to participate in previously unexplored [4 + 2] cycloaddition reactions with amidine or imidate heterodienophiles to provide highly substituted pyrimidines . These latter studies provided the basis for the potential development of second generation asymmetric total syntheses of ()-pyrimidoblamic acid and p-3a, in which all necessary stereochemistry is installed prior to a late stage [4 + 2] cycloaddition introduction of the pyrimidine, addressing the common limitation of previously reported routes, permitting the late stage divergent modification of their structures, and further expanding the scope of the 1,2,3-triazine inverse electron demand diels we envisioned that the pyrimidine core of ()-pyrimidoblamic acid would arise from an inverse electron demand diels alder reaction between the highly functionalized amidine 4 and 1,2,3-triazine 5a (scheme 2). Additionally, the same amidine 4 and its cycloaddition with 1,2,3-triazine 5b would produce the pyrimidine core found in p-3a.this late stage convergent assemblage of the pyrimidine cores not only allows the early stage synthesis of the stereochemically rich c2 side chain common to both pyrimidoblamic acid and p-3a but also permits the late stage divergent synthesis of the natural products utilizing two different 1,2,3-triazines . Amidine 4 was expected to arise from the reductive alkylation product of n-(triphenylmethyl)-l - asparagine (6) with aldehyde 7 . Key to the construction of 4 is the manipulation of intermediates, especially the desired free - based amidine, without sacrificing the stereochemical integrity of the -stereocenter, as similar amidines have demonstrated a propensity to epimerize . This concern dictated the late stage amidine introduction in order to minimize the potential impact of amidine epimerization . 1,2,3-triazines 5a and 5b, the electron - deficient heterocyclic azadienes for use in the key cycloaddition reaction, were anticipated to arise from an oxidative ring expansion of n - aminopyrazoles 9a and 9b . A key caveat to the approach is that although the two electron - withdrawing substitutents on the 1,2,3-triazines would be expected to enhance their cycloaddition reactivity, it was unknown whether their placement at the c4 and c6 positions would sterically slow the reaction and electronically redirect the mode of cycloaddition (c4/n1 vs c5/n2) with amidines . The requisite n - aminopyrazole 9a for oxidative ring expansion to the 1,2,3-triazine 5a was prepared from 8a that was accessed by a palladium - mediated tandem cross - coupling / electrocyclization reaction of commercially available ethyl diazoacetate and the vinyl triflate derived from t - butyl acetoacetate (scheme 3).n - amination of 8a was accomplished upon treatment with o-4-nitrobenzoylhydroxylamine in the presence of potassium t - butoxide (t - buok) in n - methylpyrrolidone (nmp), which yielded n - aminopyrazole 9a in 73% yield in a near 1:1 mixture of inconsequential regioisomers . In an analogous fashion and with similar yield (72%), the requisite n - aminopyrazole 9b used to access 1,2,3-triazine 5b was prepared from pyrazole 8b, which was generated from a [3 + 2] dipolar cycloaddition between ethyl diazoacetate and t - butyl propiolate . Although a range of oxidants have been reported for transforming n - aminopyrazoles into 1,2,3-triazines, we have found that the optimal reagent for this oxidative ring expansion reaction is substrate dependent . The ring expansion is often suggested to occur through a nitrene intermediate, which initiates n n bond migration and inserts the central nitrogen of the 1,2,3-triazine . Adaptation of conditions developed by ohsawa and colleagues, in which an n - aminopyrazole is treated with iodine (i2) in the presence of aqueous potassium bicarbonate (khco3), provided the 1,2,3-triazine 5a in excellent yield (75%). These conditions also proved effective for the production of 5b, yielding the 1,2,3-triazine in 68% yield . Notably, 1,2,3-triazines 5a and 5b proved stable to extended storage at room temperature and purification by flash chromatography using silica gel . As summarized in figure 3, the most commonly used oxidants for this transformation (i.e., pb(oac)4, ni2o3, and mno2) proved unsuccessful in converting 9a to the desired 1,2,3-triazine 5a, instead resulting in n silver(i) nitrate, a commonly utilized nitrene transfer reagent in aziridination, alone failed to provide 5a but produced 5a in modest yield (34%) when used in the presence of phenyliodine diacetate (pida) as a co - oxidant . Similarly, sodium periodate (naio4), which promotes the oxidative ring expansion efficiently for 4-substituted n - aminopyrazoles, afforded the 1,2,3-triazine 5a in moderate yield (28%) with a majority (70%) of the material isolated being a mixture of unreacted starting material (9a) and free pyrazole (8a). Addition of a phase transfer catalyst improved the conversions to as high as 70%, but was less consistent, and the use of tetrabutylammonium periodate failed to generate any desired compound . Amidine 4 was prepared from commercially available n-(triphenylmethyl)-l - asparagine (6) and aldehyde 7 (scheme 4). Reductive alkylation of 6 with 7 in the presence of excess nabh(oac)3 provided 10 in 84% yield . Treatment of 10 with 1-hydroxybenzotriazole (hobt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edci) followed by nh3/thf, provided the primary amide 11 . Dehydration of 11 to provide the nitrile 12 was accomplished by treatment with n - propanephosphonic acid anhydride (t3p) in the presence of hunig s base (i - pr2net). All intermediates up to this stage, including 13, exhibited a robust stability and no epimerization was observed throughout the reaction sequence . Treatment of 13 with raney nickel (ra ni) in the presence of acetic acid (acoh) in meoh generated the acetate salt of the desired amidine, which could be filtered through celite, redissolved in ch2cl2, and treated with 20% aqueous naoh to yield the free - based amidine 4 . Chromatographic purification of the amidine 4, as either the acetate salt or the free base, resulted in partial epimerization . As a result, the crude amidine was used without purification and produced immediately prior to its use . Notably, the free - based amidine epimerized much more readily than the protonated amidine in solution, and if the adjacent secondary amine was protected as the t - butylcarbamate (boc), even more significant slow epimerization of the -stereocenter occurred upon amidine formation . Therefore, the free secondary amine is essential to the observed stereochemical stability of the adjacent amidine chiral center . Effective access to the requisite intermediates 4 and 5a set the stage for examination of the key inverse electron demand diels alder reaction . Consistent with the electron - deficient character of the 1,2,3-triazine, the cycloaddition reaction was found to proceed at room temperature or lower . Significantly, the intrinsic regioselectivity of the amidine cycloaddition and 1,2,3-triazine cycloaddition mode were unaltered by the c4/c6 substitution with electron - withdrawing groups, and the amidine was found to add exclusively across c4/n1 (c6/n3) with no evidence of a redirected c5/n2 mode of cycloaddition . By design and because of the pseudo - c2-symmetric nature of the product pyrimidine, the cycloaddition across c4/n1 versus c6/n3 are indistinguishable, permitting the differential ester protection . Although not all conditions surveyed are included, figure 4 summarizes representative examples in the optimization of the reaction between 4 and 5a to provide the desired pyrimidine 14 . In accordance with our previous studies, which utilized 2.0 equiv of amidine, as summarized in figure 4, an increase in reaction time and temperature significantly increases the overall yield (entries 14). However, increasing the reaction temperature diminished the diastereomeric purity of the product, resulting from competitive amidine epimerization . Notably, we found that the reaction produces a single diastereomer of 14 when conducted at 5 c in acetonitrile (entry 5). Because the 1,2,3-triazine 5a is the simpler of the two reaction components, we focused on identifying conditions that employed amidine 4 as the limiting reagent (entries 8 and 9 vs 6 and 7). When the reaction was conducted with 1.0 equiv of amidine 4 and 2.0 equiv of the 1,2,3-triazine 5a, the yield increased to a respectable 46% . Finally, if the reaction was allowed to stir at 5 c for 14 h and then warmed to 25 c for 6 h, 14 was obtained as a single diastereomer in 54% yield . Optimization of the [4 + 2] cycloaddition reaction . With an effective route to 14 in hand, the secondary amine of 14 was protected as the t - butylcarbamate to provide 15 to eliminate oxidation risks later in the synthesis . Chemoselective hydrolysis of 15 with 1 m aqueous naoh in thf: meoh (3:1) was followed with a curtius rearrangement to provide 16 (78% yield over two steps). Removal of the acetonide protecting group with p - toluenesulfonic acid monohydrate (p - tsohh2o) in meoh yielded the boc - protected amino alcohol 17, which was subsequently treated with jones reagent to give the carboxylic acid 18 . Conversion of 18 to amide 19 was accomplished upon treatment with hobt and edci followed by nh3/thf, which provided the fully functionalized and protected ()-pyrimidoblamic acid 19 in 96% yield . Although attempts to promote a global deprotection with 4 m hcl in etoac proved unsuccessful, as the tritylated carboxamide exhibited an unusual stability to these traditional reaction conditions, treatment of 19 with a 3:1 mixture of trifluoroacetic acid (tfa) and ch2cl2 followed by deliberate counterion exchange with a 1 m aqueous hcl workup provided ()-pyrimidoblamic acid (2) in quantitative yield and identical in all respects with authentic material . Although the similarity in the pyrimidine cores of ()-pyrimidoblamic acid and p-3a is striking and may suggest that extrapolation of the approach to p-3a is straightforward, it was not clear what the impact of the c5 substituent might be . With electron - withdrawing groups at the c4- and c6-positions of 5a and 5b, it was still unknown whether the c5 methyl group affects the efficiency of the cycloaddition reaction of 5a and if its absence in 5b would alter the mode of cycloaddition . Thus, before embarking on the synthesis of p-3a, we first examined the reactions of the two 1,2,3-triazines 5a and 5b in parallel . Remarkably, the less substituted 1,2,3-triazine 5b was even more reactive than 5a, providing the product pyrimidine at a faster rate and in a higher yield in its reaction at room temperature with the aliphatic amidine substrate, where good conversion to the product was observed even within 5 min (figure 5). Slower and a more comparable reactivity between 5a and 5b were observed with the aryl amidines . Most significant in these studies is the fact that 5b showed no evidence of a potentially competitive cycloaddition across c5/n2 (vs c4/n1) despite the lack of a c5 substituent . With this knowledge in hand, comparison of the cycloaddition reactions between 5a or 5b and amidines . In accordance with the model substrates, the cycloaddition reaction between 4 and 5b (25 c, ch3cn, 12 h) yielded the desired pyrimidine 26 in 76% yield as a single diastereomer (scheme 6). In contrast to the reaction between 4 and 5a, which requires 5 c for 12 h prior to warming to 25 c to prevent competitive epimerization and preserve the amidine stereochemical integrity, the faster reaction between 4 and 5b can be conducted at 25 c without compromising the diastereomeric purity of the product and provided the stereochemically pure pyrimidine 26 in even higher yield (76%). Following a synthetic route modeled on that used to complete the synthesis of ()-pyrimidoblamic acid, compound 26 was advanced (scheme 6). Boc - protection of the secondary amine provided compound 27, which was subjected to saponification of the ethyl ester . However, attempts to selectively hydrolyze the ethyl ester by treatment with 1 m aqueous naoh in thf / meoh provided a mixture of compounds, which included those derived from transesterification or hydrolysis of the t - butyl ester . By substituting t - butanol (t - buoh) for meoh as the reaction cosolvent the competitive reactions were avoided and the reaction cleanly provided the requisite carboxylic acid, which was subjected to curtius rearrangement conditions to provide 28 in good yield (82% over two steps). Removal of the acetonide protecting group with p - tsohh2o in meoh afforded the amino alcohol 29, which could be transformed to the primary carboxamide 31 in two steps . Global deprotection of 31 was effected by treatment with tfa: ch2cl2 (3:1) to provide 32, a key analog of ()-pyrimidoblamic acid lacking only the c5 methyl group, in quantitative yield . Additionally, treatment of 31 with 1 m aqueous naoh in thf: meoh (3:1) results in t - butyl ester hydrolysis and provided carboxylic acid (33), which was coupled with n - boc - l - his - l - ala - o(t - bu) to provide the fully assembled but protected p-3a (34). Notably, this coupling reaction enlisted a more highly protected derivative of 32 than our prior efforts and proceeded more smoothly, providing higher yields of the product 34 . Global deprotection of 34 with tfa: ch2cl2 (3:1) followed by counterion exchange with a 1 m aqueous hcl workup provided p-3a (3), in quantitative yield and spectroscopically identical in all comparable respects with authentic material . Convergent total syntheses of ()-pyrimidoblamic acid and p-3a were detailed based on the early stage preparation of the chiral highly functionalized c2 side chain and subsequent late stage, divergent construction of the pyrimidine cores through use of a powerful inverse electron demand amidine/1,2,3-triazine [4 + 2] cycloaddition reaction conducted at 25 c . In addition to permitting full control of the natural product stereochemistry, the approach provides the opportunity for the late stage divergent synthesis of modified analogs bearing deep - seated changes in either the pyrimidine core (c4 and c5) or the highly functionalized c2 side chain . Such investigations are in progress and will be reported in due course . The examination of the key 1,2,3-triazine cycloaddition reaction with amidines defined nonobvious 1,2,3-triazine substituent effects that maintain or enhance the heterocyclic azadiene reactivity without altering the intrinsic regioselectivity or mode of cycloaddition . These observations with 1,2,3-triazines extend the utility of the inverse electron demand cycloaddition reactions of electron - deficient heterocyclic azadienes that includes the complementary 1,2,4- and 1,3,5-triazines, 1,2,4,5-tetrazines, and 1,2-diazines in the synthesis of highly substituted and functionalized heterocycles found in complex natural products.
The process of hydrated excess proton solvation and transport in aqueous systems displays many unique characteristics due the unique nature of the net positive charge defect that an excess proton creates . By altering the covalent bonds and hydrogen bonds of surrounding solvent molecules, the hydrated excess proton charge defect is strongly delocalized and creates a series of dynamically interchanging structures (i.e., the zundel h5o2 and eigen h9o4 cations). Proton (or more accurately stated, the charge defect) is also able to hop between neighboring water molecules by structural diffusion via successive hopping events involving the rearrangement of the local bonding topologies ., is crucial to a number of fundamental processes in chemistry, physics, biology, and materials science . In biology, protons are widely used for the transduction of signals and energy (e.g, in channels, transporters, and enzymes). They are transported through both protonatable residues and buried water molecules via grotthuss shuttling . Hence, studies on proton transport (pt) in biological systems have almost always started with the assumption that pt follows aqueous (already hydrated) pathways, which can be obtained from experimental data or by computational predictions . However, hydrophobic regions are commonly found in proteins, which complicates the interpretation of pt . Carbon nanotubes (cnts) have provided insight into the solvation and ion transport properties of homogeneous hydrophobic spaces . Experiments and computer simulations have shown that water molecules can stably occupy the interior of cnts . Theoretical studies and recent experiments have further shown that proton diffusion through nanoconfined spaces, such as hydrophobic channels and nanotubes, can be facile (and possibly even faster than in bulk water). These results provide support for the supposition that the hydrophobic spaces in biomolecules may also transiently contain water molecules capable of proton conduction . However, a common assumption is that hydrophobic spaces must be solvated prior to pt . This logic has led to numerous mechanistic predictions based on the existence of a quasi - stable water wires (e.g., see refs (3133)). However, it has also been clearly demonstrated that the presence of an excess proton greatly influences the local water solvation structure (see ref (4) for a discussion). In the present work, it is in fact found that as soon as a charge defect associated with a hydrated excess proton charge nears a hydrophobic space, the associated solvating water can experience a strong driving force to fill that space . Moreover, this finding naturally leads to the possibility that when an excess proton deprotonates from a peripheral amino acid residue or is solvated outside a hydrophilic (or amphiphilic) region, it can initiate additional solvation of such a region, which will in turn be coupled to the pt process through it . Herein, multiscale reactive molecular dynamics (ms - rmd) is used to study pt through a cnt penetrating a graphene sheet . As described in the previous paragraph, a surprising phenomenon is revealed in which an excess proton charge defect creates its own aqueous transport pathway by shuttling water molecules through it into the hydrophobic nanoconfined space . This process can be described as a variant of grotthuss shuttling, wherein water molecules travel through a hydrated excess proton charge defect . The induced wetting is shown to be excess proton specific; it does not happen when the excess proton (h) is replaced by k or even a the two - dimensional free energy surface reveals a three - step mechanism by which the protonic charge defect is transiently stabilized at the nanotube entrance, facilitates nanotube wetting via grotthuss - facilitated water migration through the charge defect, and then traverses a lower free energy barrier for grotthuss shuttling proton permeation via activated (infrequent event) dynamics . This finding has widespread implications for pt through hydrophobic (and likely other) regions in molecular systems such as proteins, by demonstrating that protons can dynamically create their own solvation pathways that would not be detected by experimental or computational means in absence of an explicit protonic charge defect . The system studied in this work consists of a 29.4 (z - dimension) armchair - type (6,6) single walled carbon nanotube (cnt) with a single layer of graphene and bulk water on either side (figure 1a). The space between the graphene layers was left empty (it is merely intended to provide a low dielectric environment). The graphene layers extend 41.82 and 42.5 in the x and y dimensions and are replicated under periodic boundary conditions . The two slabs of water molecules on either side of the graphene sheets contain seven pairs of k and cl ions collectively . This type of (6,6) cnt has been the focus of previous computational work, partially because its 8 diameter accommodates a single - file chain of water molecules, which is similar to the solvation structure found in some biological channels . As previously reported, the use of standard force field parameters enables spontaneous wetting of a cnt of this diameter . Thus, to mimic hydrophobic environments, the lj lj parameter for the cnt carbon atoms was reduced to provide a mostly dry (hydrophobic) cnt in unbiased molecular dynamics simulations, with only two to four water molecules transiently entering the mouth regions (figure 1a). (a) construct of the simulation system . The armchair - type (6,6) cnt structure is assembled between two graphene single layers that separate the bulk water . (b) overview of the proton induced wetting process along with the motion of the excess proton from bulk tube interface into about 4 of the nanotube . (c) real - time densities traces of the channel water molecules starting from a partially dry nanotube with the existence of the excess proton . Each bright line can represent the trace of the oxygen atom in a water molecule . (d) simulation with the k inside the nanotube, which remains mostly dry . The hydrated excess proton was treated explicitly with the multiscale reactive molecular dynamics (ms - rmd) method developed by the voth group . The multistate empirical valence bond version 3 (ms - evb3) model and the spc / fw water model were used . All other parameters (for ions and carbon atoms) were taken from the standard charmm22 force field . The depth of the lennard - jones potential well for cnt carbon atoms is scaled to 80% of the standard value to make the cnt more hydrophobic as noted above and hence dry in the absence of ions restrained to be in the cnt . The simulations were run with raptor, an in - house extension of the lammps software . The particle particle, particle mesh method was used to treat long - range electrostatics . The nos hoover thermostat at 300 k and a time step of 1.0 fs were used . To construct the free energy surface for the wetting process inside the cnt, we defined a water occupancy collective variable:1where nh2o is total number of the water molecules and ni is the occupancy of the ith water molecule in a defined rectangular box:2the dimensionless coordinate ri, is defined as3where r0 and ri are the positions of the center of the cnt and the oxygen atom of the ith water molecule, respectively . A box similar to the shape of the cnt was defined by bx = by = 4.0 and bx = 14, while d was chosen to be 5.0 to allow a smooth transition of the water occupancy from zero (when the molecule is at the graphene water interface) to 1 (when the molecule is inside the cnt). The umbrella - sampling technique and the wham method were used with the bias potential defined by4applied to two independent reaction coordinates: the z - axis position of the positive charge (h, k, or h3o) and the water occupancy number in the cnt . The resulting 2d free energy surface was constructed from 325 sampling windows covering the range of charge locations from 9.0 to 21.0 in 1.0 intervals and for water occupancies ranging from 2 to 14 with an interval of 0.5 . The bias force constants were chosen to be 10.0 kcal mol and 5.0 kcal mol, respectively, to ensure sufficient window overlap . As described in the section methods, the cnt studied herein was modified to be more hydrophobic and hence mostly dry . In unbiased md simulations only one to two water molecules transiently enter the mouth regions on either side of the cnt (figure 1a). These waters are partially stabilized by interactions with the graphene atoms, which were simulated with the standard lj parameters . The hydrated excess proton (or more accurate the net positive charge defect associated with an excess proton) was described with the ms - rmd method, which as noted earlier has been shown to successfully model pt in numerous aqueous and biomolecular systems . Since there is a large free energy penalty for any ion to shed its solvation shell and enter a nanoconfined hydrophobic region, umbrella sampling was used to calculate the free energy profiles for ion transport through the cnt as described in methods . Figure 1 shows how the charge defect associated with the hydrated excess proton strongly influences the cnt hydration . We note that hereafter this net positive charge defect will be referred to as just the hydrated excess proton or the proton even though it is in fact a positively charged defect having more than one proton and water molecule involved in its definition (see ref (5) for more information). When the proton is far from the mouth of the nanotube, no significant change in the hydration can be observed . As the proton approaches the entrance (within a few), the number of pore water molecules increases from 2 to 4 . As the proton enters the channel, it drags a few waters with it . Surprisingly though, once the proton has entered the mouth of the nanotube, the number of water molecules in the cnt continues to increase by having the waters shuttle through the excess proton charge defect . In other words, the proton shoots waters into the nanotube, thus creating its own water wire for subsequent transport . By the time the excess proton is 23 into the cnt, the tube transitions to the fully hydrated or wet state (figure 1b). To analyze the origin of water molecules that wet the cnt relative to the position of the excess proton, we traced the positions of the water oxygen atoms over the course of a simulation (figure 1c). This demonstrates that water molecules originate on the same side of the graphene sheet as the excess proton defect and hence have to pass through it to enter the cnt . The length of the water wire ahead of the proton fluctuates until it connects with waters from the other side to fully wet the nanotube . Therefore, the transport of water molecules into the nanotube is enabled by an unusual manifestation of the grotthuss shuttling mechanism involving the rearrangement of covalent bonds as water molecules pass through the relatively stationary protonic charge defect near the mouth of the channel . To check whether other monatomic ions can also induce a similar wetting process, the same simulations were carried out with k in place of an excess proton . Although k pulls four water molecules into the nanotube, two on each flanking side, to form its hydration structure, it does not induce a full wetting transition (figure 1d). The free energy profiles for the ion permeation (figure 2a) are also different for h and k. the energy barrier for k to penetrate 5 into the cnt is over 30 kcal / mol, while that for the excess proton is less than 15 kcal / mol (see figure 2a). The large difference in these two free energy profiles is consistent with the principal that the free energy barrier for an ion to enter a nanoconfined region is strongly influenced by the cost of dehydration . Since the excess proton can effectively keep part of its solvation shell as dynamical h5o2 and h7o3 structures, it has a lower barrier for cnt penetration . Free energy profiles of the ion permeation and the water occupancy in different simulation systems . The free energy of h (the hydrated excess proton) has much lower energy barrier than the others, displaying the unique features of the excess h. (b) free energy profiles of the water occupancy with the h charge defect (green), k (red), or classical h3o (yellow) fixed at z = 12.0 (23 inside the mouth of the nanotube), compared to the result without the ions present (blue). The induced wetting process is only seen in the system with h. the average errors are (a) 0.45 kcal / mol and (b) 0.30 kcal / mol calculated from 500 ps block averages . Moreover, the excess proton charge defect can transiently delocalize the net positive charge over even more water molecules and therefore reduce the dehydration penalty in this fashion . Similar to the situation with k, replacing the excess proton with a classical approximation of a hydronium cation (a simple ion with no possibility of grotthuss shuttling and charge defect delocalization) also results in a large free energy cost for cnt penetration (> 25 kcal / mol; see figure 2a). Thus, the excess proton s ability to distribute the charge defect to surrounding water molecules within the cnt clearly also contributes to its decreased translocation barrier . To further study the underlying mechanism of this proton induced cnt wetting process, a water occupancy reaction coordinate (see methods, eqs 13) was defined to calculate the free energy profiles for nanotube wetting as well as those for ion transport at different hydration levels . First, we obtained the free energy profile of the wetting process in the absence of an ion (figure 2b, blue curve). Similar to previous work, the free energy profile of water occupancy has dry and wet minima (at nw = 4 and nw = 13, respectively) separated by a barrier (near nw = 12). Consistent with a dry cnt, the fully wet (water occupied) state is 1 kcal / mol less stable than the dry state . However, when an excess proton is located in the region around z = 12 (just 23 inside the cnt), the wetting free energy profile changes dramatically, as seen in figure 2b, green curve . The wet minimum becomes 6 kcal / mol more stable such that wetting becomes spontaneous, and the transition along this wetting coordinate is nearly barrierless . When a k ion is located in the same location (z = 12), the barrier for wetting is increased to 5 kcal mol and the wet minima is destabilized by 2 kcal / mol (figure 2b, red curve). The case for the classical h3o ion is even more dramatic, replacing the wet minimum with a 6 kcal / mol barrier (figure 2b, yellow curve). The collected results described above highlight the excess proton s unique ability to self - solvate and project outward a water wire into a hydrophobic space, thus wetting a dry region and thereby decreasing the barrier it has to overcome to transport through it . Such a phenomenon may have significant implications for understanding pt through hydrophobic regions of biomolecules and materials . The free energy profiles of figure 2 indicate that the excess proton can induce the wetting of the hydrophobic nanotube and also that the proton has higher conductance (i.e., lower transport barrier) compared to k. are the two processes related? To answer this, two independent collective variables, the water occupancy number and the position of the excess proton charge defect, were sampled from 325 simulation windows to construct a two - dimensional (2d) free energy surface . From the 2d surface (figure 3), a fascinating coupled, stepwise mechanism is clearly seen for the wetting and proton permeation . First, the excess proton is trapped close to the mouth of the nanotube in a free energy minimum 1 kcal / mol more stable than bulk . Hummer and collaborators have reported similar results with a proton minimum at the entry of the cnt . This result is also consistent with the amphipathic nature of the hydrated proton, as reported in several theoretical studies . The vacuum and nanotube wall are hydrophobic while bulk water and the graphene surface are more hydrophilic creating an amphipathic interface where the excess proton is stabilized . Second, after being trapped at the entry of the nanotube, the proton initiates the wetting transition described earlier by greatly lowering the free energy barrier for water penetration and stabilizing waters in the previously dry space (cf . When the proton is 23 into the cnt (zcec 1213 in figure 3), the relative free energy for wetting becomes very favorable with a negligible barrier (figure 4a, blue, green, and red curves), transitioning the hydrophobic nanotube to the fully wet state . As the cnt hydration level increases, the barrier for the proton transport into the nanotube also greatly decreases (figure 4b) and the proton can progress further into the cnt and eventually through via activated (barrier surmounting) dynamics . Indeed, the free energy penalty for the proton moving into the nanotube is greatly reduced from when the nanotube is dry . In fact, the free energy for proton permeation goes from> 36 kcal / mol for the dry cnt, which is close to the energy barrier for k, to 18 kcal / mol for the fully wet cnt (figure 4b). It is interesting to note that the lowest barrier for a constrained solvation state is 18 kcal / mol for nc = 13 (i.e., the fully wet state), while the free barrier with no solvation constraints is only 15 kcal / mol (figure 2a). Thus, the proton induced wetting and proton permeation are dynamic and inherently coupled . The horizontal axis represents the z - position of the hydrated excess proton charge defect, while the vertical axis represents the level of cnt water occupancy . The white dotted line (z = 14.7) indicates the position of the graphene layer and the mouth of the nanotube . A stepwise but coupled mechanism can be observed from the 2d free energy surface, in which three steps, trapping wetting permeation, are highlighted by yellow dashed arrows . 1d free energy profiles extracted from the 2d free energy surface in figure 3 . (a) free energy profiles of the wetting process when the excess proton charge defect is located at different positions along the nanotube axis . (b) free energy profiles of proton permeation while the nanotube is in different hydration states where nc is defined as the number of confined waters in the nanotube . The 2d free energy surface in figure 3 reveals the sensitivity of the cnt s solvation structure and stability to the position of the excess proton charge defect . The excess proton changes the water occupancy in the cnt even before it enters . Although the excess proton is weakly attracted to the entrance to the nanotube, it does not block water penetration as other ions will do . Instead the excess proton defect shuttles waters through it into the nanoconfined space via an unique manifestation of the grotthuss shuttling mechanism, thereby lowering its own barrier for subsequent permeation . The 2d free energy surface for k permeation versus nanotube hydration (figure 5a) highlights two important differences between h and k. first, there is no energy minimum that indicates trapping at the mouth of the cnt . This is consistent with the common finding that k prefers to be fully solvated in the bulk rather than at an interface . In fact, the wetting free energy shift (and barrier) remains positive no matter where the k ion resides and is highest (least favorable) when k is at the mouth of the cnt . Consistent with previous studies, the water molecules in the cnt are highly mobile in the absence of ions, entering and leaving the tips of the nanotube frequently . However, when k is near the entry of the nanotube, it blocks the water flux, which is entropically unfavorable . In contrast, water molecules in the presence of the excess proton charge defect at the entrance of the nanotube retain their ability to move in and out of the channel through the grotthuss mechanism . Thus, delocalization of the excess proton charge defect is essential to decreasing the cost of wetting and ion permeation . 2d free energy surfaces for (a) a k cation and (b) a classical h3o (non - grotthuss shuttling) cation showing the free energy for ion permeation relative to nanotube water occupancy and prepared in the same way as for figure 3 . To confirm this hypothesis, we conducted another set of free energy surface calculations with a classical hydronium model (simple h3o cation), in which the charge defect delocalization and grotthuss shuttling are disabled . As discussed above, the free energy profiles for h3o permeation and wetting (shown in figure 2) are more similar to the results for k than to those for h. the 2d free energy surface (figure 5b) reveals that h3o also has a sort of trapping state near the mouth of the cnt as seen for h. however, the classical hydronium does not exhibit the favorable wetting transition (compare figure 3 to figure 5b). In fact, just as with k, water molecules are blocked from entering and leaving the cnt . In order to quantify the degree of charge defect delocalization of the excess proton in the cnt, we analyzed the magnitude of excess positive charge owned by each water molecule in the simulations with h restricted at z = 12.5 . This is the case when the proton defect center of excess charge coincides with the second cnt water molecule . When the nanotube is dry (nw = 4), about 99% of the positive charge is distributed on three water molecules with the percentages 67%, 20%, and 12%, respectively . When the nanotube is fully wet (nw = 14), the distribution shifts to 55%, 33%, and 10% . In the bulk, eigen cation, h9o4, with 62% of the charge on the central water molecule and the three surrounding waters that possess 19%, 10%, and 5% of the excess charge . Thus, although the charge defect delocalization in the cnt is weaker than it is in the bulk system, because of the strong spatial confinement, it is still significant . Moreover, it shifts to a more zundel - like delocalized species in the fully wet nanotube, which will contribute to the stabilization of the fully wet state . Thus, charge defect delocalization is again confirmed to be essential to the wetting mechanism and decreases the free energy barrier for ion permeation . For many years, studies of pt in biological systems have largely focused on the identification and the analysis of aqueous pathways interlaced with protonatable residues through which excess protons might migrate (especially via grotthuss shuttling). Great effort has been devoted to characterizing internal hydration structures that connect protonatable residues to form such pathways . When hydrophobic cavities are encountered, i.e., those lacking crystallographically resolved water molecules, simulations have often been used to try to identify states of the system (e.g., via oxidation state or conformational changes) that induce wetting . In this manner, mechanisms of pt have been proposed based on the existence and stability of hydrogen - bonded water wires . However, the simulations presented herein suggest that the excess proton itself is strongly coupled to the solvation structure and stability in nanoconfined spaces and hence must be explicitly included in the analysis of internal solvation . By simulating pt through a nanotube penetrating a graphene sheet with ms - rmd, we have discovered that a hydrated excess proton charge defect can induce wetting into a previously dry hydrophobic space . This is a novel wetting process in that water molecules actually pass through the protonic charge defect via grotthuss - like shuttling . Other ions have the opposite effect, blocking the diffusion of water into the nanotube when they are close to the nanotube s entrance . Thus, this wetting process, which relies on charge defect delocalization and grotthuss shuttling, is unique to a hydrated excess proton (though something similar may be possible for the hydroxide anion). Although the present simulations have focused on pt through a cnt, our findings have broad implications for pt in biomolecular and materials systems . Just as our simulations have demonstrated in a cnt, an excess proton may actually transiently induce wetting in biomolecular hydrophobic cavities when it is located near a peripheral residue or water cluster . In this manner, the excess proton can create its own aqueous pathway for subsequent charge transport . As shown in this work, water rearrangement around a confined excess proton can be fast (from hundreds of picoseconds to several nanoseconds) relative to the rare events of biomolecular pt (typically microseconds or longer). Thus, with the strong correlation between solvation rearrangement and the position of a hydrated excess proton, the two processes of pt and wetting are likely to be strongly coupled (as demonstrated in the cnt). Indeed, the concept that pt requires an existing water wire, e.g., one seen in an x - ray crystal structure or md simulations without an explicit excess proton, should be questioned . What is more, computer simulations probing pt mechanisms should include an explicit treatment of the hydrated excess proton (along with its full physics of grotthuss shuttling and charge defect delocalization) to properly capture the coupling between water dynamics, hydration, and pt . Using solvation structures to interpret pt mechanisms in the absence of an explicit excess proton can quite possibly lead to incorrect conclusions.
A 40-year - old man presented with involuntary movement of his right hand and left foot . The symptom started when he was approximately 35 years of age and had slowly worsened . His disability progressed over the following years and was restricted to his right hand and left foot . He had no significant medical history and no history of exposure to neuroleptic drugs, hypoxic environments, or toxic metabolites, except for the intermittent inhalation of silica dust at his workplace . There was no family history of neurological disorders . On examination, the patient had dystonic posture of his right hand, which caused alternating dorsiflexion and neutralization with supination of the forearm and flexion of the metacarpophalangeal joint . An ophthalmologic evaluation showed no kayser - fleischer rings, optic atrophy, or pigmentary retinopathy . Serum ferritin and copper levels were normal, and a blood smear was negative for acanthocytes . The results of other laboratory tests, including serum electrolytes, erythrocyte sedimentation rate, thyroid function test, liver function test, autoimmune antibodies, ferritin, copper, ceruloplasmin, and a blood smear were normal, except for borderline - low 24-hour urine copper (10.2 g / dl, normal 1530 g / dl). A t1-weighted brain mri study showed hyperintensity bilateral anteromedial globus pallidi (figure 1a). A t2-weighted mri showed hypointensity in the bilateral globus pallidi with hyperintense core, the typical eye - of - the - tiger sign (figure 1b). Genomic dna was extracted from peripheral blood, and polymerase chain reactions (pcr) were preformed . The dyt1 gag deletion (dele302/303) automated dna sequence analyses revealed two single - base variants in exon 3 and exon 4 . Both pathological variants result in changes to conserved amino acids in the pank2 protein: asp268 gly (p.d268 g) and arg330prp (p.r330p). To our knowledge, this is the first patient identified with the mutation p. r330p and the second patient with the mutation p.d268 g . The dystonia of the patient s right hand and left foot did not respond to levodopa / carbidopa (100/25 mg t.i.d). Treatment with diazepam (5 mg t.i.d) and trihexyphenidyl (2 mg t.i.d) slightly improved the dystonia . However, his dystonia worsened after one year, and he could not walk without a cane . Our patient had an atypical phenotype of the pank2 gene: compound heterozygous mutations in exon 3 (p.d268 g) and exon 4 (p.r330p). To our knowledge, our patient is the first to be identified with mutation p.r330p and the second to be identified with mutation p.d268 g . The onset of atypical pkan is known to be 1st three decades (mean age 13.6).2 in a previous study, 23 patients with atypical pkan were analyzed, and the mean age at onset was 13.7 years (range 128 years). Patients with atypical pkan had variable clinical features, including parkinsonism, corticospinal tract involvement, speech disturbances such as palilalia, and psychiatric symptoms.3 our patient presented with dystonia and gait disturbance, which are the predominant symptoms of classical pkan . The implication of the eye - of - the - tiger sign on a brain mri is controversial.3,4 the eye - of - the - tiger sign on mri is not pathognomic of pkan, and some patients with pank2 mutations do not have this sign and sometimes, it may appear only transiently . This sign has also been reported in corticobasal degeneration, progressive supranuclear palsy, anoxicischemic leukoencephalopathy, and early - onset levodopa - responsive parkinsonism.4 however, a previous study reported that all patients clinically suspected of nbia with the eye - of - tigersign have mutations in pank2 and that all patients without the typical mri finding do not have these mutations.3 in our patient, the mri finding and presenting symptoms were consistent with classic pkan, while the age at onset and the absence of other features such as retinal degeneration, optic atrophy, and delayed development were consistent with atypical disease or other subtypes of nbia . Genetic studies identified compound heterozygous missense mutations (p.d268 g, p.r330p) in exons 3 and 4 of pank2 . To our knowledge, this is the first description of the p.r330p mutation . The mutation p.d268 g was reported previously in a chinese patient with heterozygous missense mutations (p. d268 g, p.i391n).5 others have attempted to establish a correlation between the genotype and phenotype . Although the linkage between mutations and residual enzymatic activities related to the age of onset has been partially demonstrated, the phenotype is still difficult to predict based on the presence of pank2 mutations.3,68 there were no overlapping clinical features between our patient and the chinese patient with the p.d268 g mutation, except for the typical mri finding . The chinese patient s disease began at 17 years of age, and his predominant symptoms were tremor and rigidity . It is presumed that modifier effects of allelic genes and environmental factors played a roll in the phenotypic differences between these two patients.3 the pank2 gene codes for pantothenate kinase, which is an essential regulatory enzyme in coenzyme a (coa) biosynthesis, catalyzing the cytosolic phosphorylation of pantothenate (vitamin b5) the enzyme of the initial and rate - limiting step in coa biosynthesis: the phosphorylation of pantothenate, n - pantothenoyl - cysteine and pantetheine.9 to compensate for the partial enzymatic deficiency in patients with pkan, supplement of pantothenate (vitamin b5) have been tried without conclusive benefit.3 previous case reports using pallidal deep - brain stimulation reported improvement of dystonia in patients with pkan, but it was also restrictive effect.10 further study is needed for early diagnosis of pkan, correct prediction of disease progress, and rational treatment.
Most of the reported cases have been associated with the superior mesenteric artery, and to our knowledge, only 25 cases of the spontaneous isolated celiac artery dissection can be found in the literature (1). Of the reported cases, only 2 cases of spontaneous celiac artery dissection awareness of the visceral artery dissection is important because of its life - threatening complications causing extensive hemorrhage or ischemia to the supplied organs . Although a review of the medical literature suggests that the natural history of spontaneous celiac artery dissection may not be fatal, it is difficult to predict . All of the following can potentially occur: spontaneous resolution, ischemia due to occlusion of the vessel, aneurysm formation, and rupture (2). The management of spontaneous celiac artery dissection includes conservative medical treatment, endovascular therapy, and surgery (1). In complicated cases, the optimal management of spontaneous celiac artery dissection remains controversial because this condition is rare . The medical literature does not provide enough information to assess the superiority of one treatment over another . Herein, we report 2 cases of the complicated spontaneous dissection of the celiac artery, both of which were successfully treated using an endovascular stent graft . A 47-year - old man, who had presented with acute abdominal pain to a different healthcare facility, was transferred to our emergency room for further evaluation and treatment of dissection of the splanchnic arteries . The patient had a history of uncomplicated dissection of the superior mesenteric artery, and had been taking anti - hypertension medication for 1 year . However, he had stopped taking his prescription medication 10 months prior to admission to the emergency room . On arrival to the emergency room of a local hospital, his blood pressure was 95/60 mm hg and the pulse rate was 110 beats per min . After blood transfusion and fluid therapy in local hospital, the patient's blood pressure had increased to 150/100 mm hg, and there were no obvious abnormalities on the physical examination or in the laboratory findings on admission to our hospital . A contrast - enhanced ct scan showed a newly developed saccular dissecting aneurysm in the celiac artery with surrounding retroperitoneal hematoma, measuring 2.8 7.9 12.0 cm (fig . The dissection of the superior mesenteric artery had not changed, compared to the previous ct scan . One day after admission, the patient's hemoglobin level had decreased to 9.6 g / dl again, after cessation of blood transfusion . The patient was offered a less invasive endovascular procedure to avoid surgical repair . After puncturing of his right common femoral artery, an 8f guiding catheter (vista brite tip, cordis, miami, fl, usa) was inserted into the orifice of the celiac artery, which allowed for celiac angiography that revealed a dissecting aneurysm at the entry point (fig . The proximal splenic artery was occluded, and the true lumen of the left gastric artery and common hepatic artery were narrowed by intramural hematoma . We placed the 4 - 9 38 mm peripheral stent graft (jostent, abbott vascular, rangendingen, germany), which was mounted over a 6 40 mm savvy balloon (cordis, miami, fl, usa) from the origin of the celiac artery to the common hepatic artery . Post - procedure angiography showed complete coverage of the dissecting aneurysm with the use of a stent graft (fig . Follow - up ct scan, obtained 1 week after the procedure, showed a complete obliteration of the dissecting aneurysm in the celiac artery with preservation of the hepatic arterial flow and marked reduction of the retroperitoneal hematoma . Although occlusion of the proximal splenic artery and proximal left gastric artery after stent graft placement persisted, no abnormalities (i.e., splenic infarction, gastric ischemia, or signal of irritated peritoneum) were noted due to an abundance of collateral blood flow . A 57-year - old man, presented with acute abdominal pain, was admitted to the emergency room . The patient had a previous medical history of spontaneous celiac artery and of the superior mesenteric artery dissection . An abdominopelvic ct scan, obtained 3 months prior to admission, demonstrated a 7 mm - sized saccular aneurysm, with a mural hematoma along the celiac artery to the common hepatic artery and a dissection of the superior mesenteric artery with a mural hematoma (fig . Infiltration of the fat surrounding the celiac artery was noticed as a secondary sign of acute spontaneous celiac artery dissection . Other than the acute abdominal pain, the patient was healthy and had no risk factors associated with the occurrence of visceral artery dissection . He had been treated with an anticoagulant to prevent a thromboembolic complication for 3 months, because he had no major complication associated with the dissection of the celiac artery and superior mesenteric artery . Contrast enhanced ct revealed a progressive dissecting aneurysm along the celiac artery, which had a maximal inner diameter of 2 cm (fig . The aneurysm originated 2 cm distal from celiac artery orifice without compromised distal flow to the distal hepatic artery . In addition, the patient's previous superior mesenteric artery dissection with mural thrombus had improved completely without associated complications . The patient was transferred to an interventional suite for endovascular treatment, because the risk of rupture was high, due to the progressive enlargement of an aneurysm sac . Celiac angiography, obtained after an insertion of the 8 fr guiding catheter (cordis), showed a dissecting aneurysm, which obviously defined the celiac artery, proximal to splenic artery and left gastric artery orifice (fig . We placed the 4 - 9 38 mm peripheral stent graft (jostent, abbott vascular, rangendingen . Germany), which was mounted over a 6 mm 40 mm savvy balloon (cordis) from the origin of celiac artery to the bifurcation of the common hepatic artery and the splenic artery . There was no noticeable compromise in the blood flow to the distal hepatic artery, gastroduodenal artery, and splenic artery . The follow - up ct scan, obtained 3 months after the procedure, showed a complete disappearance of the dissecting aneurysm . A 47-year - old man, who had presented with acute abdominal pain to a different healthcare facility, was transferred to our emergency room for further evaluation and treatment of dissection of the splanchnic arteries . The patient had a history of uncomplicated dissection of the superior mesenteric artery, and had been taking anti - hypertension medication for 1 year . However, he had stopped taking his prescription medication 10 months prior to admission to the emergency room . On arrival to the emergency room of a local hospital, his blood pressure was 95/60 mm hg and the pulse rate was 110 beats per min . After blood transfusion and fluid therapy in local hospital, the patient's blood pressure had increased to 150/100 mm hg, and there were no obvious abnormalities on the physical examination or in the laboratory findings on admission to our hospital . A contrast - enhanced ct scan showed a newly developed saccular dissecting aneurysm in the celiac artery with surrounding retroperitoneal hematoma, measuring 2.8 7.9 12.0 cm (fig . The dissection of the superior mesenteric artery had not changed, compared to the previous ct scan . One day after admission, the patient's hemoglobin level had decreased to 9.6 g / dl again, after cessation of blood transfusion . The patient was offered a less invasive endovascular procedure to avoid surgical repair . After puncturing of his right common femoral artery, an 8f guiding catheter (vista brite tip, cordis, miami, fl, usa) was inserted into the orifice of the celiac artery, which allowed for celiac angiography that revealed a dissecting aneurysm at the entry point (fig . The proximal splenic artery was occluded, and the true lumen of the left gastric artery and common hepatic artery were narrowed by intramural hematoma . We placed the 4 - 9 38 mm peripheral stent graft (jostent, abbott vascular, rangendingen, germany), which was mounted over a 6 40 mm savvy balloon (cordis, miami, fl, usa) from the origin of the celiac artery to the common hepatic artery . Post - procedure angiography showed complete coverage of the dissecting aneurysm with the use of a stent graft (fig . Follow - up ct scan, obtained 1 week after the procedure, showed a complete obliteration of the dissecting aneurysm in the celiac artery with preservation of the hepatic arterial flow and marked reduction of the retroperitoneal hematoma . Although occlusion of the proximal splenic artery and proximal left gastric artery after stent graft placement persisted, no abnormalities (i.e., splenic infarction, gastric ischemia, or signal of irritated peritoneum) were noted due to an abundance of collateral blood flow . A 57-year - old man, presented with acute abdominal pain, was admitted to the emergency room . The patient had a previous medical history of spontaneous celiac artery and of the superior mesenteric artery dissection . An abdominopelvic ct scan, obtained 3 months prior to admission, demonstrated a 7 mm - sized saccular aneurysm, with a mural hematoma along the celiac artery to the common hepatic artery and a dissection of the superior mesenteric artery with a mural hematoma (fig . Infiltration of the fat surrounding the celiac artery was noticed as a secondary sign of acute spontaneous celiac artery dissection . Other than the acute abdominal pain, the patient was healthy and had no risk factors associated with the occurrence of visceral artery dissection . He had been treated with an anticoagulant to prevent a thromboembolic complication for 3 months, because he had no major complication associated with the dissection of the celiac artery and superior mesenteric artery . Contrast enhanced ct revealed a progressive dissecting aneurysm along the celiac artery, which had a maximal inner diameter of 2 cm (fig . The aneurysm originated 2 cm distal from celiac artery orifice without compromised distal flow to the distal hepatic artery . In addition, the patient's previous superior mesenteric artery dissection with mural thrombus had improved completely without associated complications . The patient was transferred to an interventional suite for endovascular treatment, because the risk of rupture was high, due to the progressive enlargement of an aneurysm sac . Celiac angiography, obtained after an insertion of the 8 fr guiding catheter (cordis), showed a dissecting aneurysm, which obviously defined the celiac artery, proximal to splenic artery and left gastric artery orifice (fig . We placed the 4 - 9 38 mm peripheral stent graft (jostent, abbott vascular, rangendingen . Germany), which was mounted over a 6 mm 40 mm savvy balloon (cordis) from the origin of celiac artery to the bifurcation of the common hepatic artery and the splenic artery . There was no noticeable compromise in the blood flow to the distal hepatic artery, gastroduodenal artery, and splenic artery . The follow - up ct scan, obtained 3 months after the procedure, showed a complete disappearance of the dissecting aneurysm . Spontaneous dissection of the celiac artery, which is not associated with aortic dissection, is extremely rare . Approximately one half of the patients with celiac artery dissection are asymptomatic, due to a good collateral flow, via the superior mesenteric artery (3). The symptoms are usually non - specific epigastric pain or post - prandial abdominal pain . These symptoms are considered to be the result of the dissection itself, or to ischemic signs of the abdominal organ (4). However, various clinical presentations, such as liver ischemia, splenic infarction, may occur according to the involvement of the branch vessels, and life threatening hemorrhage due to a rupture of the aneurysm (1, 4). The initial diagnosis of the visceral artery dissection is rarely established by symptoms . This disease entity is found incidentally during imaging studies for the purpose of evaluating other symptoms . There have been reported cases in which the ct findings of isolated dissection of the celiac artery were described (4, 5). The intimal flap with double lumen, thrombosis of false lumen or intramural hematoma, enlarged involved vessels were the findings of visceral artery dissection . Infiltration of the fat surrounding the affected vessel is a well - known secondary sign of acute spontaneous dissection of a visceral artery . Complications of the celiac artery dissection include hemorrhage or ischemic change of the distal organs, propagation of dissection into adjacent vessels, expansion of a false lumen, and the development of an aneurysm . The hepatic artery aneurysm could be presented as a complication of the celiac artery dissection . Expansion of a false lumen may cause either a rupture or poor perfusion to the supplied organ, secondary to the mechanical compression of the true lumen flow (6). However, the inability to predict either regression or expansion of a false lumen mandates close, life - long follow - up . The management of celiac artery dissection depends on the hemodynamic status of the patient, response to conservative treatment, and development of complications (ischemia or hemorrhage). In asymptomatic stable dissection, current treatment recommendations are conservative treatment with anticoagulation or antiplatelet agents under close observation to prevent thromboembolic complications . Blood pressure control is necessary to minimize the progression of a dissection (4, 5). The indications for surgical or endovascular treatment include visceral ischemia, rupture of the dissection, progressing aneurysm (1, 2, 6 - 8). In the era of improved radiologic intervention, percutaneous endovascular treatment has been used as a surgical alternative with increasing frequency for the management of complicated splanchnic artery dissection because of its less invasiveness . The specific application of any interventional technique should be tailored to the needs of the individual patient, because each technique offers different advantages and disadvantages (6 - 8). (6) reported placement of a bare metal stent in a patient with celiac artery dissection as a treatment of poor perfusion secondary to mechanical compression of the true lumen by an expansion of the false lumen, following a failed conservative treatment . But, there was no evidence of a rupture in that case . In our cases, dissection of the celiac artery resulted in a rupture of aneurysm with decreased hemoglobin level in the first case, and progressing aneurysm with high risk of rupture in the second case . Conservative treatment, including anticoagulation or placement of a bare stent, was not amenable to treat or prevent bleeding in our cases because these therapies could not limit the aneurysm . 8) reported isolated dissecting aneurysm of the celiac artery, extending into the proper hepatic artery that was successfully treated by transcatheter arterial embolization . Fortunately, in this reported case, intrahepatic branches were visualized after embolization through collateral pathways of the pancreatic arcade and epicholedochal arterial plexus from the superior mesenteric artery and dorsal pancreatic artery . However, proper collateral flow is essential for the use of transcatheter arterial embolization to treat a dissecting celiac artery to avoid organ infarction . Therefore, a stent - graft could be considered to control the bleeding from a ruptured aneurysm without the inadvertent development of complications caused by arterial embolization in poor collateral channels . Several reports have suggested the stent - graft as a valid alternative endovascular treatment for ruptured sma dissection or celiac artery aneurysm (9, 10). A stent - graft that better preserves the distal flow might provide a safe, efficacious alternative to coil or plug embolization . In our cases, the involved vessels showed a smooth curvature to allow an easy navigation of a stent graft . In the first case, the patient had experienced a previous dissection of the superior mesenteric artery without complications . In addition, he had been treated with anticoagulant and hypertensive medication . The patient had discontinued anticoagulation after 6 months, and no serious complications concerning a superior mesenteric artery dissection was noted on a follow - up ct angiogram . The patient had stopped his hypertension medication arbitrarily . In addition, 10 months later, a ruptured dissecting aneurysm of the celiac artery developed with a concomitant decrease in hemoglobin . Most likely, arbitrary discontinuation of medication could explain the episode of celiac artery dissection . We successfully treated the dissecting aneurysm of the celiac artery that had ruptured with the retroperitoneal hematoma by the use of a stent graft . In the second case, although the urgent complication had not been noticed, the progressive enlargement of the aneurysm is at the greatest risk from rupturing . The patient was managed successfully with the placement of a stent graft . To our knowledge, we are reporting the first case of a spontaneous celiac artery dissection treated with a stent graft . Percutaneous stent graft placement for the treatment of complicated spontaneous dissection of celiac artery is safe and feasible . However, studies with a large group of patients and long - term follow - up will be needed to validate this procedure.
Hepatitis c virus (hcv) is a major public health problem all over the world; it is currently estimated that about 85% of those infected with hepatitis c virus will become a chronic carrier and may develop severe end stage liver diseases including cirrhosis and hepatocellular carcinoma . Currently, the most important risk factor for infecting by hepatitis c is intravenous drug use (idu) which is the most associated with the sharing of drug injection equipments especially needles, syringes and other paraphernalia . Infection with hcv among prison inmates is usually higher than that among the general population mainly because of past history of intravenous drug use and possibly high risk addiction - related behaviors in prison . During the last few years, much attention has been given to the prevalence of blood born diseases including hepatitis c among prisoners . According to limited research studies in iran, prevalence of hcv infection among prisoners with a history of drug injecting varies between 31.5% to 47% in different parts of the country. [68] however, a study in a local prison in fars province revealed 78% prevalence rates of hcv infection among incarcerated drug users . To address the often hidden phenomenon of hcv infection in prisons, considering the limited data on the epidemiology of hcv infection and related risk behaviors in our region, in this study we have focused on this infection and transmission risk factors among prisoners with history of idu in isfahan that could potentially be incorporated into current and future harm reduction initiatives us in iran . In a cross - sectional study, according to self - report and confirm by head of prison's healthcare, the prison inmates who had intravenous drug history or current idus entered into study at march 2009 . Since the most of our enrollees had sclerosis in peripheral vessels, blood sampling was mostly taken from the femoral vein by the health personnel of the prison setting . Blood samples were then sent to the laboratory of infectious diseases research center during 3 hours in cold box to be tested by eilsa (diapro - italy) for the presence of hcv antibody . Incarcerated with idu histories were also asked about their demographic characteristics and hcv - related risk behaviors with an interviewer assisted questionnaire . Face and content validity of the questionnaire were evaluated by specialists and its reliability was confirmed by chronbach alpha = 0.74 . The research protocol was approved by the ethical committee of isfahan university of medical sciences in iran . All available means were used to guarantee privacy during interviews and confidentiality . Instead of a block for the patient's name, each questionnaire and test tube had an anonymous identification code, which was used for reporting laboratory results, too . Statistical analysis was performed using spss for windows (version 16.0, 2007, spss inc, chicago, il, usa). Univariate analysis was used to assess association between being hcv positive and related risk factors . The variables that were significant in the univariate analysis (p - value <0.05) were included in the multiple logistic regression to estimate adjusted odds ratio (aor) and 95% confidence intervals (ci). A total of 943 incarcerated idus (938 male and 5 female) participated in the study . The median age of participants was 32.6 years (range: 18 - 67). The majority of participants (98.6%) were iranian and 523 (55.5%) had ever been married . Among the married persons 451 (92%) had been married once, 35 (7%) had been married twice and 5 (1%) had been married more . 143 (15.1%) cases mentioned a history of traveling to another countries . The socio - demographic characteristics of participants overall, the prevalence of hcv - ab seropositivity was 41.6% [table 1]. The median frequency of iv injections was 30 times per month (range 1500), the median duration of addiction was 12 years (range 0.5 - 57), the median frequency of incarceration was 3 times (range 1 - 37), and the median of total duration of incarceration was 3.66 years (range 0.08 - 35). Frequency of hcv transmission related factors within study population 71.76% of the participants had illegal sex (contact with other than spouse). Among whom, 43% of men had history of having sex with another man (msm), 64.2% had an intercourse with commercial sex workers and 30.9% of them had idu sexual partner . According to self reports, only 31% of them had ever used a condom during sex . Within all studied samples with idu history, 13.3% had a single sexual partner and the others had 2 or more, in their lifetime . Table 3 shows the odds ratio for some risk characteristics of idus using the logistic regression model . The results of this study indicated that overall prevalence of hcv ab seropositivity among idu inmates in isfahan province is 41.6% . In similar studies in iran, similar results were observed . In zakizad study, the seroprevalence of hepatitis c infection in sari addict prisoners has been reported as 30.8% . In another study by khani et al . The prevalence of hcv was 47.7% among drug addict prisoners in zanjan . In a study which was done in 3 provinces in iran (isfahan, chahamahal bakhtiary and lorestan) in 2003, 34.7% of male prisoners who had been imprisoned for various drug - related offenses including purchase or sale and consumption, were hcv ab positive . In mashhad, the seroprevalence of hcv in incarcerated idus had been reported as 59.4% . In mohammad alizadeh study, the hcv antibody positivity among drug abusers in the central prison of hamedan was 30% . In a study on drug abusers admitted to prison in guilan province, of 460 inmates, 45.4% were hcv antibody positive which in intravenous drug abusers the prevalence reached to 88.9% . The prominent aspect of our study in comparison with other similar studies in iran, is the high number of participants whom all of them had history of intravenous addiction . As a matter of fact, this is one of the first large prevalence studies of blood- born infections among incarcerated idus in iran . In this study, the history of shared drug injection inside prison was one of the main incarceration - related risk factors . This association, has also been reported in other studies in the world. [1315] it seems that, the lack of access new needle / syringe in prisons and consequently needle sharing practice is the main reason of the hepatitis c epidemic in prisoners . Our data, highlight for comprehensive and integrated interventions for incarcerated idus to prevent hcv transmission among idu population and community . Effectiveness of needle programs in reducing needle sharing among idus has been shown in many countries . In our country, an outreach program for blood born infections prevention was supported by the united nation and the ministry of health of iran in 2003 . Multiple incarcerations was the another independent risk factor in our subjects which show that the high rates of arrests and incarcerations lead to drug - related offences or other confounding factors inside prison, such as violence . Our findings suggest that preventive interventions are compelling now for idus to ensure their safe passage throughout incarceration . In our study, other important modes of transmission were frequency and length of drug injection . According to these results, harm reduction strategies need to be expanded to prevent new hcv infections, particularly among young injectors. [1920] in our study, history of marriage was a protective factor for hcv - infection in the subjects after controlling for all other variables in the logistic - regression model . Sexual transmission could play a role in sporadic or community - acquired hcv infections . However, there is contradictory finding regarding the association between marriage and hcv infection in the world . Vandelli and colleagues indicated that the risk of sexual transmission of hcv within heterosexual monogamous couples is extremely low or even null . It is shown that, those who have multiple sexual partners, including female sex workers, men having sex with men, and attendees of sexually - transmitted diseases (stds) clinics are the main risk groups for hepatitis c. in the acute hepatitis surveillance study in usa, 18% of newly infected individuals reported sexual contact with an hcv - infected person or multiple sexual partners as their only risk factor for hcv acquisition . For the individual with chronic hcv infection, the estimated risk of sexual transmission of virus was 0% to 0.6% per year for those in monogamous relationships, and 1% per year for those with multiple sexual partners . So, it will be useful to encourage young people to get married to prevent sexually transmitted diseases in our society . In conclusion, according to progressive reported prevalence rates of hcv in the world and the importance of the current and potential burden of hcv - related complications, it is important to primary prevention of hcv infection that will be addressed through implementation of safe injection practices . The mortality associated with hepatitis c is expected to double in the next 10 - 20 years . It is conservatively estimated that the direct costs related to hepatitis c will be $10.3 billion during the years 2010 - 2020 . The indirect costs of hepatitis c (eg, the loss of productivity during that era) are estimated to total another $54.3 billion due to premature death and $20.6 billion due to disability . In addition, the total cost of therapy for hepatitis c is estimated to be $10,000-$12,000 . So, primary prevention will be more cost - effective than secondary prevention of morbidity and mortality from hcv infection through provision of interferon - based therapy . It is good opportunities in prison to have large number of idus over longer period to do specific preventive activities such as needle / syringe exchange and counsel imprisoned idus.
Radiologically, they are similar to ependymomas, and histopathologically, they are similar to neuroendocrine tumors . Most of them are non - secreting tumors, although they are of sympathetic origin . Back pain is the most common symptom, and they are not associated with tachycardia, flushing, or hypertension . The treatment of choice is total surgical resection, and no additional treatment is needed with total resection . Most of them are benign, but, recurrence can occur in cases involving a failure of the total resection . We describe a rare case of a paraganglioma that was adherent to the cauda equina that presented with low back pain and radiating pain in both the lower extremities . A 76-year - old female visited our department with complaints of low back pain and radiating pain in both the lower extremities for more than two months . Magnetic resonance (mr) imaging revealed a lobulated sausage - like intradural mass (fig . 1). She underwent a total laminectomy and near - total tumor removal . During the operation, after opening the dura, a reddish, friable, and well - marginated mass was observed (fig . We removed the mass near - totally except for the lesion that was adherent to the cauda equina . Extradural paragangliomas may compress the spinal cord, resulting in paraparesis5). From a pathologic point of view, paragangliomas are similar to parasympathetic tumors . However, functional paragangliomas are rare6). Histopathologically they are similar to parasympathetic tumors that occur in the carotid body or glomus region . Althogh they are also similar to catecholamine - secreting tumors, symptoms of flushing, tachycardia, or hypertension are rare . However, a case in which hypertension was controlled after thoracic paraganglioma removal has been reported12). Preoperative diagnosis is difficult with radiological tools . Mr images of paragangliomas in the cauda equina region are non - specific and are relatively isointense on t1-weighted images and hyperintense on t2-weighted images . Immunohistochemically, neuron - specific enolase, synaptophysin, and chromogranin staining are positive2). In the cauda equina region, it is very difficult to distinguish paragangliomas from ependymomas . Cauda equina paragangliomas are classified as grade i on the world health organization (who) classification . Recurrence after surgery on a paraganglioma in the cauda equina region, especially if it is encapsulated, is rarely encountered2). The surgical goal is complete total resection with preservation of the surrounding nerve roots1). Thus, we did not treat the patient any additional therapy, and conducted a close follow - up . After incomplete surgical resection, prolonged postoperative observations are mandatory because of the slow evolution of paragangliomas4). Distant metastases very rarely occur5), and metastatic paragangliomas of the spine have been reported7,11). Total resection was difficult in this case due to adhesion to the cauda equina, and close follow - up may therefore be needed.
The procedure included augmentation with transcortical wiring of an onlay - type prosthesis in twelve knees (nine patients) after it had been determined that conventional resurfacing was inadequate . This technique was indicated when the thickness of the remnant patella was less than 8 mm and the cortical rim was not intact . There were 9 patients (12 knees) with a mean age of 68 years (range, 53 to 80 years) at the time of surgery . The patellae were revised because of infection (five knees), loosening of the patellar component (four knees) and polyethylene wear in the presence of a metal - backed patella (three knees). An onlay - type, all polyethylene prostheses (universal dome patella, scorpio, stryker, nj, usa) which are compatible with the femoral component were used . After arthrotomy using a medial parapatellar approach, the patella is everted laterally and fixed in this posture by grasping the quadriceps tendon or patella tendon with towel clips . Cement stuck to the undersurface of the prosthesis is meticulously removed with an oscillating saw and a high - speed burr after implant removal . The new, properly sized patellar prosthesis is then placed on the undersurface, implant location is decided and the margin of the prosthesis and the contact points for each peg are marked . Bone bed preparation is performed with a burr according to marking lines and contact points . On fitting the patellar prosthesis, a check is made to determine whether it matches the concave portion of the undersurface and confirm the peg sites . Three points are then drilled for the pegs and bone holes are placed for wire passage . The next steps involve connecting the wires to the pegs and passing through drill holes . A wire is connected to one peg by winding it around the narrow part of the neck under the peg head and fixing it to the peg . The three wires on the pegs are passed from the undersurface of the patella to the outer surface through the bone holes . Before the prosthesis is brought into contact with the patella, the space between the bone bed and the polyethylene is filled with cement (fig . 2). After approximating the patellar prosthesis to remaining bone as closely as possible, the prosthesis is compressed with a compressor and the cement is given time to harden . Subsequently, sufficient tension is placed on the three wires on the outer surface with wire holders and the wires are twisted together immediately above the cortex . After checking that the wires are under sufficient tension, they are cut and their ends are pressed with an impactor to prevent irritation (fig . After arthrotomy using a medial parapatellar approach, the patella is everted laterally and fixed in this posture by grasping the quadriceps tendon or patella tendon with towel clips . Cement stuck to the undersurface of the prosthesis is meticulously removed with an oscillating saw and a high - speed burr after implant removal . The new, properly sized patellar prosthesis is then placed on the undersurface, implant location is decided and the margin of the prosthesis and the contact points for each peg are marked . Bone bed preparation is performed with a burr according to marking lines and contact points . On fitting the patellar prosthesis, a check is made to determine whether it matches the concave portion of the undersurface and confirm the peg sites . Three points are then drilled for the pegs and bone holes are placed for wire passage . The next steps involve connecting the wires to the pegs and passing through drill holes . A wire is connected to one peg by winding it around the narrow part of the neck under the peg head and fixing it to the peg . The three wires on the pegs are passed from the undersurface of the patella to the outer surface through the bone holes . Before the prosthesis is brought into contact with the patella, the space between the bone bed and the polyethylene is filled with cement (fig . 2). After approximating the patellar prosthesis to remaining bone as closely as possible, the prosthesis is compressed with a compressor and the cement is given time to harden . Subsequently, sufficient tension is placed on the three wires on the outer surface with wire holders and the wires are twisted together immediately above the cortex . After checking that the wires are under sufficient tension, they are cut and their ends are pressed with an impactor to prevent irritation (fig . If the patellar component can be retained during revision tka, this may offer the best option in terms of morbidity . However, if the prosthesis is malpositioned, damaged or mechanically loose, removal is probably the advisable option . The onlay - type all polyethylene patellar prosthesis is an efficient tool when patellar bone stock is sufficient, being over 10 to 12 mm.5) but it is not easy to determine an optimal procedure if bone stock is poor . According to maheshwer et al.,2) patellar resurfacing using an all polyethylene biconvex patellar prosthesis is successful when the remnant patellar thickness is less than 10 mm and the peripheral rim is intact . When the bone stock is too thin or the peripheral rim has been destroyed, it is highly likely to fail when a prosthesis is only cemented to bone . A biconvex patellar prosthesis may also not be likely to be effective . In our series, the remnant patellar thickness averaged 5.6 mm (range, 3.2 to 7 mm) and variable amounts of the peripheral patellar rim were damaged . With regard to these cases, some authors advocated alternative options including a porous tantalum component, a bone grafting and soft tissue flap procedure or a gull - wing osteotomy . Nelson et al.4) evaluated short - term results following patellar resurfacing with a trabecular metal patella in the setting of marked patellar bone loss . Hanssen6) suggested a bone grafting and soft tissue flap procedure . At final follow - up of nine patients, it was found that the mean patellar thickness increased from 8 to19.7 mm and their functional outcomes were improved . Klein et al.7) performed a gull - wing osteotomy in a nonresurfacable patella and reported that there was a significant improvement in the range of motion and knee society scores . Though several shortcomings were encountered during follow - up period, the above techniques showed reasonable outcomes.3,4,6,7) however, further follow - up is required to assess the longer term benefits of these methods . The deficient patella was revised using this technique when the thickness of the remnant patella was less than 8 mm and the cortical rim was not intact . Moreover, it is easily reproducible and does not need a long learning curve, in our experience . Because it protects the extensor mechanism and provides the same contact during patellofemoral articulation as primary tka, painless articulation is possible and hereby, the range of motion is improved . However, there are a few concerns about the use of this procedure in thin patellae . It may not be indicated if the osseous cortical shell is too thin for wiring, so the use of transcortical wires place the patella at risk to fracturing . Also, the problems related with prominent hardware also may occur as is oft en seen with internal fixation of patellar fractures . At a mean 10.3 months follow - up of our series, there was one patellar fracture arising during flexion exercise one week after surgery that was treated with partial patellectomy . Our described technique includes connecting wires to the three pegs of the patellar component, passing the wires from the undersurface of the patella to the outer surface through the bone holes and, after compression of a cemented prosthesis, twisting the wires under sufficient tension . We believe that this technique provides a good alternative option in managing the deficient patella in revision total knee arthroplasty, although any prolonged benefits will require long - term follow - up.
One of the most important functions of proteins is serving as enzymes in catalyzing biochemical reactions . Enzyme nomenclature (1) is available through the nomenclature committee of the international union of biochemistry and molecular biology (nc - iubmb) enzyme list (2) and the enzyme database (3). Information on the functions of enzymes is provided by brenda (4), which contains an extensive collection of facts related to enzymes, including reaction specificity, functional parameters, substrates, products and inhibitors . Intenz (5) is a relational database integrating enzyme data from all three of these sources . The current explosion of structural data has emphasized the need to relate enzyme structures to functions (6). Databases addressing this need are procat (7) and the more recent csa (8) that provides catalytic residue annotation . However, catalytic sites generally only consist of a few, highly conserved amino acid residues, whereas there are usually 1020 amino acid positions in an enzyme that directly interact with various ligands, including substrates, products, cofactors and inhibitors . No resource has been developed that permits the systematic study of all residues involved in ligand binding, although the substrate specificity of an enzyme is determined by these rather than only by the catalytic residues . The best tool to obtain information on specific enzyme ligand interactions is the sequence annotated by structure (sas) facility of the pdbsum website (9). Sas and pdbsum use the programs hbplus (10), to generate lists of hydrogen bonds and non - bonded contacts from the three dimensional (3d) coordinates in a pdb file, and the ligplot program to display the interactions (11). The shortcoming is that each enzyme ligand complex should be analyzed separately, and then the results be collected to form a summary which provides the consensus characterization of the binding site in a homologous family of enzymes . Given a query enzyme, the main function of precise is to find all relevant structure files in the pdb (12), align the corresponding sequences, extract all enzyme ligand interactions and construct the consensus binding site, i.e. Identify all residue positions that contribute to ligand binding in any of the structures, ranked on the basis of the frequency of such interactions found for the residues at each position . Future releases of the database will also include interactions predicted by solvent mapping, a novel method for determining protein binding sites based on their structure (13). While this explains the origin of pre in the name of precise, the current version is based exclusively on interactions extracted from the pdb structure files . As mentioned above, the broadly defined binding or interaction site of an enzyme means the collection of all amino acid residues that interact with any ligand related to enzyme function, including substrates, products, cofactors and inhibitors . The expressions of functional site and recognition site are also used in the literature . The binding or interaction site generally includes 1020 amino acid residues, and should be distinguished from the strict catalytic site that is responsible for the enzyme action and generally comprises only three or four residues . We recall that the procat and csa databases (7,8) provide catalytic residue annotation . These residues obviously interact with the substrate, and hence are part of the binding site . Since the catalytic mechanism cannot be determined from the structures of complexes alone (although co - crystallized transition state analogs provide useful information), in precise we do not attempt to distinguish the catalytic residues from the rest of the binding site . The basis of enzyme classification is the assignment of a specific numerical identifier, the enzyme classification (ec) number, which identifies the enzyme in terms of its function . The second digit of the ec number refers to the subclass, which generally contains information about the type of compound or group involved . The third digit, the sub - subclass, further specifies the nature of the reaction and the fourth digit is a serial number that is used to identify the individual enzyme within a sub - subclass . Enzymes with the same ec number, e.g. Lysozyme (ec 3.2.1.17) have the same catalytic function, but may include several families (e.g., hen egg - white lysozyme and t4 lysozyme) that significantly differ both in sequence and structure . Collecting consensus information, we group enzymes that have the same ec number and also have very high sequence similarity . Such highly homologous proteins share the same tertiary fold, but can still have localized differences in their binding sites and hence in substrate specificity . However, since the sequences can be reliably aligned, we can assess the role of each amino acid position in forming the binding site . An important step in the construction of precise is the clustering of enzymes such that members of each cluster have the same ec number and have high sequence similarity to each other . To assure the second property, we have considered the clusters in the non - redundant pdb chain set (http://www.ncbi.nlm.nih.gov/structure/vast/nrpdb.html), also known as the nrpdb database, maintained by the ncbi . The clusters in nrpdb have been constructed by comparing all chains available from pdb (12) with each other using the blast algorithm (14). The chains are then clustered into groups of sequence - similar entries using a single - linkage clustering procedure . Chains within a sequence - similar group thus derived are also ranked according to the precision and completeness of their structural data . The following measures of the structural quality are used in this order of priority: (i) lower percentage of residues with unknown amino acid type; (ii) lower percentage of residues with incomplete coordinate data; (iii) lower percentage of residues whose coordinate data are missing; (iv) lower percentage of residues with incomplete side - chain coordinate data; (v) higher resolution; (vi) larger number of chains (subunits) contained in the pdb entry; (vii) larger number of heterogens contained in the pdb entry; (viii) larger number of different types of heterogens; (ix) larger number of residues; and (x) alphanumerical order of their pdb codes . To form the clusters in precise we consider the enzymes belonging to the same ec number, and group them into clusters of sequence - similar elements that are members of the same nrpdb cluster obtained using the blast p - value of 10 . The top - ranked chain is generally chosen as the representative of the group . In some cases, however, a lower - ranked chain was chosen . For example, if the top - ranked chain was a mutant protein and there was a native protein with reasonably comparable structural quality, then that lower - ranked native protein might replace the mutant as the representative . Representatives from all of the groups together form a non - redundant set of enzymes with different binding sites . First, the interaction frequencies at each amino acid position are projected onto the representative sequence . Second, we will apply computational solvent mapping onto representatives of clusters with no enzyme ligand complexes available in order to predict putative interaction . While the current version of precise does not include predicted interactions, the availability of consensus interaction sites for a large fraction of enzymes is very useful in a variety of applications . Precise version 1.0 is based on the august 31, 2004 version of the nrpdb (12) and the september 17, 2004 version of ec - to - pdb databases, the latter containing 12 532 files . The total number of enzyme chains in these files is 23 872, belonging to 1176 unique ec numbers . Thus, in the pdb the average number of enzyme chains per ec number is 20.29 . However, the distribution is very uneven, i.e. The majority of ec numbers is represented with less than 10 chains, whereas a few have very many members (figure 1a). Clustering chains with the same ec number and pairwise blast p - value of 10 or less yields 2280 clusters, i.e. On average, 1.93 clusters per enzyme number, with 10.47 chains in each cluster . Figure 1b and c show the distributions for the number of clusters per ec number, and for the number of enzyme chains per cluster, respectively . According to figure 1b, . More generally, over 90% of ec numbers include only five or fewer clusters of chains that are not homologous to each other . It is interesting to note that the frequencies of ec numbers with even number of chains are always somewhat higher than the frequencies of ec numbers with similar but odd number of chains (figure 1a). The same observation applies to the distributions of clusters with even and odd number of chains (figure 1c). While the first appears to be strange, the rule becomes easy to understand when we take into account that many enzymes form homodimers and homotetramers, which increases the fraction of ec numbers and clusters with even number of chains . Since the selected p - value of 10 implies high - sequence identity and similarity within clusters (see figure 1d), the alignment of sequences within each cluster is very simple . A semi - global dynamic programming algorithm with the gonnet matrix (15), gap penalty of 8, and extension penalty of 0.25 was used to align each sequence to the representative of the cluster . The pairwise alignments within a cluster are stacked upon each other to generate a multiple sequence alignment of all sequences relative to the representative sequence . Because the only concern is that interactions to a residue position in a member of the cluster are related to the correct residue position of the representative, there is no need for a more sophisticated multiple sequence alignment that best aligns every cluster member to each other . For each chain, the interactions between the protein and the ligand were extracted using the hbplus program with the default parameter values to define non - bonded and hydrogen bond interactions . The attributes stored for each interactions are the pdb code and chain identifier of the protein; the name, heteroatom code and type of the ligand; the interacting residue and atom in the protein, the interacting atom in the ligand and the type of the interaction (non - bonded or hydrogen bond). The ligand types we distinguish are peptide, nucleotide, cofactor, metal ion, other inorganic ion and other, the latter representing the molecules in none of the above categories . The number of interactions for each residue in each chain was obtained by summing the interactions in which the atoms belonging to that residue participate, without any upper bound on the number of interactions . The number of interactions at a particular amino acid position of the aligned sequences was calculated by summing the interactions found at that position for all members of the cluster . Precise currently contains a total of 5 435 107 atom atom interactions that are distributed among 1533 clusters, i.e. 747 of the 2280 clusters do not have any interactions . The numbers of clusters with different ligand types are as follows: peptides 262, nucleotides 67, cofactors 554, metal ions 733, inorganic ions 673 and figure 2 shows the distributions of the percentages of residues with a given number of interactions . According to this figure, the distribution has a robust peak at 12% of the residues containing 2030 interactions . This confirms that the information contained in our database is specific enough, i.e. The ligands interact with a relatively small fraction of the residues rather than having interactions broadly distributed over long stretches of the sequence . Precise is implemented as a relational database using ms sql server 2000 as the backend . The database structure can be classified into three sections, enzyme information, cluster information and interaction information . The three sections are related but are independent enough to allow for the update of one without affecting the others . Asp.net is used for the computationally intensive functions of the site and to provide the database connectivity layer, in the form of ado.net . Javascript is used throughout the site to enhance the usability and responsiveness within a web browser . The online version of precise is available at http://precise.bu.edu/. Queries can be made using pdb code or ec number . If there are non - homologous chains present in structure for the query pdb code, a subsequent page will let users select any of the chains . The main output page (figure 3a) shows the color - coded sequence of the representative of the cluster . The blue to red color - scheme indicates the residues that belong to the binding site, as well as the total number of interactions found at each amino acid position in all chains of the cluster . Colored residue (i.e. On a residue that has at least one interaction) displays a separate panel with a detailed list of interactions for the selected residue (figure 3b). For each interaction, the list shows the pdb code and chain identifier of the protein; the name, heteroatom code and type of the ligand; the interacting residue and atom in the protein, and the type of the interaction (non - bonded or hydrogen bonds). It is important that the list shows both the interaction position, i.e. The original sequence number of the interacting residue in the pdb file, and the aligned position, which is the sequence number of the same residue in the alignment of sequences for the entire cluster . On the right - hand side of the sequence in the main output page, separate panels show all the pdb codes and chain identifiers of the entries that form the cluster . The user may select any subset of these entries and recalculate the list of interactions . Additional panels permit the users to restrict the set of interactions to selected interaction types (i.e. Non - bonded or hydrogen bond) and to selected ligand types (i.e. Peptides, nucleotides, cofactors, metal ions, other inorganic ions or we are in the process of investigating the origin of some irregularities . In particular, our alignment yields low sequence identity for a limited number of clusters (figure 1d), in spite of low blast p - values, indicating potential problems . The alignment in these clusters is being inspected manually.as we describe, the 1176 ec numbers in the pdb are subdivided into 2280 sequence - similar clusters . We are in the process of separately annotating these clusters, i.e. Identifying the reasons why the same enzymatic action is achieved by non - homologous proteins . The analysis includes the comparison of binding site structures of enzymes that have the same ec number but are in different clusters, and will involve merging some of the clusters.we will provide a rotatable 3d representation of the binding site in any chain using the java - based jmol molecular viewer . The atom set selected for display will include all atoms of the ligand(s), and the side chains of all amino acids that are part of the consensus binding site for the given cluster, including the ones that may not interact with the ligand in the particular chain.at this point, updating precise to account for new structures in the pdb requires rerunning all our scripts that have been used to generate the database . We will develop scripts that align new pdb entries with the existing representatives and either add them to an existing cluster or create a new cluster, thereby facilitating regular updates without the need for recreating the entire database.the major addition to precise will be the inclusion of interactions predicted by computational solvent mapping . The latter is a powerful tool for the identification and characterization of binding sites of enzymes (13,16,17). The method moves molecular probes small organic molecules containing various functional groups around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations and ranks the clusters on the basis of the average free energy . A very important result is that using at least six different solvents as probes, the consensus sites found by the mapping are always in major subsites of the enzyme binding site, and as a result, the amino acid residues that interact with the probes also bind the specific ligands of the enzyme (13). Thus, the method provides detailed and reliable information on the important amino acid residues in the binding site (13). We have already mapped the surface of over 50 enzymes for binding sites . This will permit the comparison of predicted and observed interactions sites if the latter can be determined from the available x - ray structures of complexes . The precise webpage will also contain an email server, and thus users will be able to request the mapping of a specific enzyme by sending an email to the website . Since the mapping runs will be started manually, the response time will be about two days . Once the mapping is finished, the results will be added to the precise database, and an email will be sent to the user . In particular, our alignment yields low sequence identity for a limited number of clusters (figure 1d), in spite of low blast p - values, indicating potential problems . The alignment in these clusters is being inspected manually . As we describe, the 1176 ec numbers in the pdb are subdivided into 2280 sequence - similar clusters . We are in the process of separately annotating these clusters, i.e. Identifying the reasons why the same enzymatic action is achieved by non - homologous proteins . The analysis includes the comparison of binding site structures of enzymes that have the same ec number but are in different clusters, and will involve merging some of the clusters . We will provide a rotatable 3d representation of the binding site in any chain using the java - based jmol molecular viewer . The atom set selected for display will include all atoms of the ligand(s), and the side chains of all amino acids that are part of the consensus binding site for the given cluster, including the ones that may not interact with the ligand in the particular chain . At this point, updating precise to account for new structures in the pdb requires rerunning all our scripts that have been used to generate the database . We will develop scripts that align new pdb entries with the existing representatives and either add them to an existing cluster or create a new cluster, thereby facilitating regular updates without the need for recreating the entire database . The major addition to precise will be the inclusion of interactions predicted by computational solvent mapping . The latter is a powerful tool for the identification and characterization of binding sites of enzymes (13,16,17). The method moves molecular probes small organic molecules containing various functional groups around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations and ranks the clusters on the basis of the average free energy . A very important result is that using at least six different solvents as probes, the consensus sites found by the mapping are always in major subsites of the enzyme binding site, and as a result, the amino acid residues that interact with the probes also bind the specific ligands of the enzyme (13). Thus, the method provides detailed and reliable information on the important amino acid residues in the binding site (13). We have already mapped the surface of over 50 enzymes for binding sites . This will permit the comparison of predicted and observed interactions sites if the latter can be determined from the available x - ray structures of complexes . The precise webpage will also contain an email server, and thus users will be able to request the mapping of a specific enzyme by sending an email to the website . Since the mapping runs will be started manually, the response time will be about two days . Once the mapping is finished, the results will be added to the precise database, and an email will be sent to the user . Precise provides a summary of interactions between the amino acid residues of an enzyme and its various ligands (substrate and transition state analogs, cofactors, inhibitors and products), thereby complementing other databases that contain enormous wealth of data on enzymes, but do not provide information on the binding site . Brenda (4) currently has information on 3600 different ec numbers, including nomenclature, isolation and preparation, stability, reaction specificity, functional parameters and references . In particular, brenda lists the different ligands (substrates, products, inhibitors, cofactors and metals / ions) but without any type of data on the interactions between these ligands and the protein . The catalytic site atlas (csa) (8) provides catalytic residue annotation for enzymes in the pdb . Unlike the catalytic residues that are highly conserved, residues that participate in ligand binding but do not directly contribute to the catalytic activity can change through evolution, and are responsible for changes in substrate specificity . Thus, information on the entire ligand binding site is required for understanding the energetic contributions to substrate binding, for developing modified enzymes using methods of protein engineering or directed evolution, and for the design of enzyme inhibitors . Once residues of the binding site are identified and their importance is determined using precise, the subset of catalytic residues can be found by csa . This research has been supported by grants dbi-9904834 from the national science foundation, gm64700 from the national institute of health and p42 es07381 from the national institute of environmental health sciences.
Thyroid eye disease (ted) is a chronic inflammatory disease of the eye that can occur in patients with systemic thyroid disorder . It is the most common cause of bilateral and unilateral proptosis with a female preponderance and peak incidence in the 5th decade of life.1) although thyroid ophthalmopathy is not uncommon, its pathogenesis has not been thoroughly studied and remains unclear . A consistent pathogenic link between graves' disease and ophthalmopathy has yet to be identified.2) the pathogenesis involves immunoglobulin g antibody and an organ - specific autoimmune reaction . T - cell lymphocytes and thyroid follicular cells with shared antigenic epitopes are thought to react in the retro - orbital space . Sight - threatening compressive optic neuropathy is one of the most feared complications of thyroid ophthalmopathy . However, the occurrence of optic neuropathy is rare and constitutes about 4% to 8% of all complications.34) management of thyroid eye disease is based on the consensus statement produced by the european group on graves' orbitopathy . Thyroid eye disease is usually self - limiting; its frequency is declining; and 3% to 5% cases are sight threatening.5) treatment for graves' disease is directed towards alleviating symptoms, managing hyperthyroidism, and addressing issues such as smoking and other aggravating factors . The following cases illustrate the atypical and subtle presentations of sight - threatening thyroid eye disease that can be missed during an initial visit . They also illustrate the worsening of thyroid eye disease and its relationship to radioactive iodine (rai) therapy . A 67-year - old malay man presented with gradual onset of bilateral eye redness and tearing for 2 months after receiving a second rai therapy for thyrotoxicosis without oral steroid coverage . Ocular examination revealed a poor visual acuity of 6/60 and n24 bilaterally with an absence of relative afferent pupillary defect (rapd). Extraocular muscle examinations revealed generalized restriction in all positions of gaze particularly on downgaze (figure 1). Anterior segment examination of both eyes revealed fullness and redness of the eyelids, both of which appeared ptotic . Conjunctival injection and chemosis, as well as caruncle swelling and redness were also present . There were minimal nuclear sclerosis cataracts present bilaterally and intraocular pressures were normal in both the primary gaze and upgaze . Further color vision, hess chart, binocular single vision, and bjerrum visual fields tests were performed, all of which revealed a derangement of color vision with d15 testing and constricted visual fields consistent with aspects of compressive optic neuropathy in both eyes . Magnetic resonance imaging (mri) of the orbit (figure 2) revealed crowding of the orbital apex consistent with thyroid ophthalmopathy . A thyroid function test revealed that the patient, although taking oral thyrosine, was biochemically hypothyroid . The patient was admitted, and a regimen of high - dose pulse intravenous methylprednisolone was commenced . After the fourth week of treatment, his visual acuity improved to 6/9 bilaterally, and a gradual resolution of the clinical signs and symptoms of thyroid eye disease was noted (figure 3). A 48-year - old malay woman presented with diplopia on upward gaze for 2 months following rai therapy without oral steroid coverage for her thyrotoxicosis . She was newly diagnosed with thyrotoxicosis 4 months prior to the rai therapy . Upon presentation, ocular examination of the right eye revealed a visual acuity of 6/36, 6/12 with a pinhole occluder, and a near vision of n36, while examination of the left eye revealed a visual acuity of 6/12 with a pinhole occluder and a near vision of n6 . Both eyes appeared proptosed, and extra - ocular muscle examination revealed restrictions in all gaze positions, especially in the upgaze (figure 4). Anterior segment examination revealed conjunctiva chemosis and injection particularly at the site of recti - muscle insertion . Nuclear sclerosis cataracts affecting the patient's vision were present bilaterally, and were more pronounced over the right eye . Her intraocular pressures (iop) were high in both the primary gaze and upgaze . Fundus examination revealed a slightly hyperemic and swollen superior edge of the right optic disc, while the optic disc over the left eye was normal . Binocular single vision testing revealed diplopia at all gazes except the inferior gaze (figure 5). Hess chart, color vision, and bjerrum visual field tests were normal . Computed tomography of the orbit with contrast showed a crowding of the right orbital apex with hypertrophy of all the extraocular muscles . These results are consistent with thyroid ophthalmopathy . Considering the clinical evidence for optic neuropathy, a 67-year - old malay man presented with gradual onset of bilateral eye redness and tearing for 2 months after receiving a second rai therapy for thyrotoxicosis without oral steroid coverage . Ocular examination revealed a poor visual acuity of 6/60 and n24 bilaterally with an absence of relative afferent pupillary defect (rapd). Extraocular muscle examinations revealed generalized restriction in all positions of gaze particularly on downgaze (figure 1). Anterior segment examination of both eyes revealed fullness and redness of the eyelids, both of which appeared ptotic . Conjunctival injection and chemosis, as well as caruncle swelling and redness were also present . There were minimal nuclear sclerosis cataracts present bilaterally and intraocular pressures were normal in both the primary gaze and upgaze . Further color vision, hess chart, binocular single vision, and bjerrum visual fields tests were performed, all of which revealed a derangement of color vision with d15 testing and constricted visual fields consistent with aspects of compressive optic neuropathy in both eyes . Magnetic resonance imaging (mri) of the orbit (figure 2) revealed crowding of the orbital apex consistent with thyroid ophthalmopathy . A thyroid function test revealed that the patient, although taking oral thyrosine, was biochemically hypothyroid . The patient was admitted, and a regimen of high - dose pulse intravenous methylprednisolone was commenced . After the fourth week of treatment, his visual acuity improved to 6/9 bilaterally, and a gradual resolution of the clinical signs and symptoms of thyroid eye disease was noted (figure 3). A 48-year - old malay woman presented with diplopia on upward gaze for 2 months following rai therapy without oral steroid coverage for her thyrotoxicosis . She was newly diagnosed with thyrotoxicosis 4 months prior to the rai therapy . Upon presentation, ocular examination of the right eye revealed a visual acuity of 6/36, 6/12 with a pinhole occluder, and a near vision of n36, while examination of the left eye revealed a visual acuity of 6/12 with a pinhole occluder and a near vision of n6 . Both eyes appeared proptosed, and extra - ocular muscle examination revealed restrictions in all gaze positions, especially in the upgaze (figure 4). Anterior segment examination revealed conjunctiva chemosis and injection particularly at the site of recti - muscle insertion . Nuclear sclerosis cataracts affecting the patient's vision were present bilaterally, and were more pronounced over the right eye . Her intraocular pressures (iop) were high in both the primary gaze and upgaze . Fundus examination revealed a slightly hyperemic and swollen superior edge of the right optic disc, while the optic disc over the left eye was normal . Binocular single vision testing revealed diplopia at all gazes except the inferior gaze (figure 5). Computed tomography of the orbit with contrast showed a crowding of the right orbital apex with hypertrophy of all the extraocular muscles . These results are consistent with thyroid ophthalmopathy . Considering the clinical evidence for optic neuropathy, therefore, detection of this condition during clinical examination is highly important so that it can be efficiently managed . Progression of ophthalmopathy was more common in patients treated with rai than in those who received anti - thyroid drugs or surgery.16) one study proposed that the development or progression of ophthalmopathy after rai therapy may be related to the release of thyroid antigens as a result of radiation injury and to the subsequent enhancement of autoimmune responses directed toward antigens shared by the thyroid and orbit.7) rai for graves' hyperthyroidism was followed by the development, or more often, the progression of ophthalmopathy in patients treated with rai alone, but not in those treated with rai and prednisolone.8) in these two cases, both patients were not administered oral steroids prior to or during rai therapy . In case 1, the patient's eyes appeared not proptosed, but ptotic . There was evidence of optic neuropathy confirmed by constriction of the visual field, and mri of the orbit showed crowding of the orbital apex due to enlargement of the extraocular muscles . This pathogenesis is due to the accumulation of glycosaminoglycans, which leads to a retention of water, increasing the intraorbital soft tissue volume and pushing the eye forward, resulting in proptosis . Therefore, the absence of proptosis in active ted increases the risk of optic nerve compression, because the swollen soft tissue within the confined space of the orbit prevents forward decompression of the optic nerve . Diagnosis of optic nerve dysfunction might be missed if there is no obvious proptosis as highlighted in case 1.9) it also stated that an increase in orbital pressure will precipitate dysthyroid optic neuropathy when there is lack of proptosis . The lack of rapd detection may suggest that there was equal and bilateral involvement of the optic nerves by compression . In case 2, both eyes appeared proptosed and there was grade 1 rapd over the left eye with secondary high iop in both eyes . The subtle signs of optic neuropathy must be detected quickly, so that a treatment plan can be initiated to prevent further vision loss . Both patients were treated with 500 mg pulse intravenous methylprednisolone for 6 weeks and then 250 mg once weekly for another 6 weeks (total period of 12 weeks with total dose of 4,500 mg). This treatment regime was adapted according to the recommendation by kahaly et al.10) in patients receiving radiotherapy for ted, modifying the treatment can slow the development of ophthalmopathy.2) the administration of glucocorticoids prior to rai is generally expected to limit the progression of ophthalmopathy.11) in conclusion, active thyroid eye disease often presents as pseudoptosis instead of the classical lid retraction with' starry' gaze (kocher sign). Although rai therapy is one of the primary treatment modalities for ted, it can also have a disastrous effect on the vision . Therefore, it is imperative for the patient to be administered glucocorticoids prior to treatment with rai for hyperthyroidism . Family medicine practitioners and physicians need to anticipate the possibility for the development or worsening of thyroid eye disease post - rai treatment, as timely intervention can prevent the vision loss that results from compressive optic neuropathy in active thyroid eye disease.
An important parameter necessary to achieve a proper endodontic treatment is the quality of root canal filling . The success rate of root canal treatments (rcts) is reported to be> 90% . The success of this treatment depends on many factors, and the technical quality of rct is one of the most important . On the other hand, numerous studies have been reported in several countries on the high prevalence of poor fillings in association with periapical radiolucency, leading to increasing number of public health problems . Amongst the several risk factors corresponding to progressive periapical radiolucency, the quality of root filling was one of the most important factors . Therefore, efforts are needed for improving the quality of rcts . To improve the quality of clinical performance, some factors such as knowledge, training, ability and utilization of technology are necessary . Of these gates - glidden (gg) drills are low - speed rotary instruments that have been used for over one hundred years without major changes in design . Some advantages of such instruments include safety, ease of use, reduction in working time and low cost . In spite of the advantages of rotary instruments the decision as to whether new techniques and materials should be incorporated into the undergraduate curriculum can pose problems for teachers of endodontology . The study of quality of rcts and prevalence of endodontic procedural accidents can help improve educational programs, thus leading to improvement in oral health - related quality of life in society . Therefore, for improving the quality of endodontic performance of practitioners in clinical practice, their basic, preclinical performance and knowledge must be taken into consideration . Until date, very few studies have focused on the preclinical abilities of students . The aim of this study was to evaluate the technical quality of molar rcts performed by undergraduate practitioners at a dental teaching school during the preclinical course . In addition, the effect of gg drills use on the final quality of the performed rcts was evaluated . In this retrospective cross - sectional study, 315 roots of 105 endodontically treated molar teeth in preclinical practice in 2010 were obtained from the undergraduate program in endodontics of the school of dentistry, isfahan university of medical sciences, isfahan, iran . All the undergraduate students underwent extensive laboratory training on the instruments and filling techniques on all groups of teeth . The obtained teeth consisted of 64 maxillary and 41 mandibular permanent molar teeth . For mounting the teeth, the students had used different materials with different concentrations; therefore prior to evaluation, the teeth were removed from the mounts and then assessed . There were two equal groups of endodontically treated teeth: one group had been prepared using k - files along with gg drills (gg group) and the other using only hand instruments (k - files) (no gg group). All the rcts were performed by fourth grade undergraduate dental students, who possessed the same level of knowledge and practical experience of rct, using the step - back technique . They were taught by the instructors to use at least files number 15 for initial length determining via conventional radiographs . After determination of working length, the master apical file was set at minimum of number 30 . In the gg group, gg drills number 1 - 3 were used after the root canals were instrumented by files number 20 - 25 . Using patency file (#10 or 15) and copious canal lavage was recommended to the students . All the teeth were obturated using the cold lateral condensation filling technique using the gutta - percha (ariadent, tehran, iran) and ah-26 sealer (dentsply, detrey, konstanz, germany). Although the students had taken conventional radiographs of completely treated teeth to pass their educational course, the radiation environments were different . Therefore, to calibrate the images and prevent bias, new digital images were obtained . In addition, all the teeth were held in a plaster block containing a very thin layer of wax to ensure immobility . The distances from the x - ray tube to the teeth and the teeth to the receptor were 2 and 0.5 cm, respectively . The radiographs of maxillary and mandibular molar teeth were obtained in the buccolingual direction using paralleling technique . Moreover, as recommended, a 20 horizontal mesial angulation of x - ray tube was used for the mandibular molars to prevent the superimposition of roots . The radiographic procedure was performed using a dental x - ray unit (planmeca intra planmeca oy, helsinki, finland) at 50 kvp, 8 ma and 0.01 s exposure time, and digital sensor (mps, progeny dental, buffalo grove, usa). The selected exposure environments (i.e. Kvp, ma and exposure time) were based on a pilot exposure prior to the final imaging confirmation of an endodontist and a maxillofacial radiologist . In the present study, a total of 146 images including 64 of maxillary molars and 82 of mandibular molars (from two directions) the results were then compared, and a final consensus was reached . In the case of disagreement, a third observer with more experience was asked to interpret the images for final agreement . The observers used the same monitor (lg flatron l1755s 17-inch, manufactured by madiran, iran, under license of lg electronics inc . ). The observers were free to use and perform all the options in the software, such as brightness, contrast adjustment and magnification . The observers were asked to evaluate the radiographic quality of the fillings according to a criteria defined in table 1 . As in other similar studies, the filling quality was assessed on the basis of three parameters: filling length, density (homogeneity) of filling and taper of root canals [table 1]. Apart from the above characteristics, overextension and coke bottle shaped root canals root filling beyond the apex and also the canal that has not been filled adequately within its confines was diagnosed as overextension . The coke bottle shape was diagnosed when a bottle - like widening produced by the over action of gg drills was observed in the root canals . Criteria followed for evaluation of root fillings as shown in table 1, a specific score was assigned to each parameter, which was similar to that in the study of santos et al . The root canal was considered as a sampling unit, and the quality of the root filling was evaluated according to the following parameters: length, density andtaper, which were scored (s) as s0, s1 and s2 . S2 corresponded to an adequate standard whereas s1 and s0 referred to slight and considerable deviation from the adequate standard, respectively table 1 . A root canal filling was considered acceptable (total score: 6) only when the length, density and tapering of the filling were adequate (three scores of 2). Fillings with total scores of 0 - 2 and 3 - 5 were considered to be poor and moderate, respectively . Descriptive analysis was performed separately for each type of root canal, and the sum of all endodontic errors was calculated . Chi - square test was used to compare the gg drill (gg group) with hand instrument (no gg group) with respect to taper of root canal (adequate, under - shaped and over - shaped), length of fillings (adequate, under - fill, over - fill and tip - to - tip) and scoring (poor, moderate and acceptable). To compare the coke bottle shaped errors between the groups, whitney test was used for comparison of frequency of voids within each root canal and the scores of the two groups were obtained . All the aforementioned analyses used for comparing the gg and no gg groups were also used to compare maxillary and mandibular root canals . In this retrospective cross - sectional study, 315 roots of 105 endodontically treated molar teeth in preclinical practice in 2010 were obtained from the undergraduate program in endodontics of the school of dentistry, isfahan university of medical sciences, isfahan, iran . All the undergraduate students underwent extensive laboratory training on the instruments and filling techniques on all groups of teeth . The obtained teeth consisted of 64 maxillary and 41 mandibular permanent molar teeth . For mounting the teeth, the students had used different materials with different concentrations; therefore prior to evaluation, the teeth were removed from the mounts and then assessed . There were two equal groups of endodontically treated teeth: one group had been prepared using k - files along with gg drills (gg group) and the other using only hand instruments (k - files) (no gg group). All the rcts were performed by fourth grade undergraduate dental students, who possessed the same level of knowledge and practical experience of rct, using the step - back technique . They were taught by the instructors to use at least files number 15 for initial length determining via conventional radiographs . After determination of working length, the master apical file was set at minimum of number 30 . In the gg group, gg drills number 1 - 3 were used after the root canals were instrumented by files number 20 - 25 . Using patency file (#10 or 15) and copious canal lavage was recommended to the students . All the teeth were obturated using the cold lateral condensation filling technique using the gutta - percha (ariadent, tehran, iran) and ah-26 sealer (dentsply, detrey, konstanz, germany). Although the students had taken conventional radiographs of completely treated teeth to pass their educational course, the radiation environments were different . Therefore, to calibrate the images and prevent bias, new digital images were obtained . In addition, all the teeth were held in a plaster block containing a very thin layer of wax to ensure immobility . The distances from the x - ray tube to the teeth and the teeth to the receptor were 2 and 0.5 cm, respectively . The radiographs of maxillary and mandibular molar teeth were obtained in the buccolingual direction using paralleling technique . Moreover, as recommended, a 20 horizontal mesial angulation of x - ray tube was used for the mandibular molars to prevent the superimposition of roots . The radiographic procedure was performed using a dental x - ray unit (planmeca intra planmeca oy, helsinki, finland) at 50 kvp, 8 ma and 0.01 s exposure time, and digital sensor (mps, progeny dental, buffalo grove, usa). The selected exposure environments (i.e. Kvp, ma and exposure time) were based on a pilot exposure prior to the final imaging confirmation of an endodontist and a maxillofacial radiologist . In the present study, a total of 146 images including 64 of maxillary molars and 82 of mandibular molars (from two directions) were evaluated . The results were then compared, and a final consensus was reached . In the case of disagreement, a third observer with more experience was asked to interpret the images for final agreement . The observers used the same monitor (lg flatron l1755s 17-inch, manufactured by madiran, iran, under license of lg electronics inc . ). The observers were free to use and perform all the options in the software, such as brightness, contrast adjustment and magnification . The observers were asked to evaluate the radiographic quality of the fillings according to a criteria defined in table 1 . As in other similar studies, the filling quality was assessed on the basis of three parameters: filling length, density (homogeneity) of filling and taper of root canals [table 1]. Apart from the above characteristics, overextension and coke bottle shaped root canals were detected . Root filling beyond the apex and also the canal that has not been filled adequately within its confines was diagnosed as overextension . The coke bottle shape was diagnosed when a bottle - like widening produced by the over action of gg drills was observed in the root canals . Criteria followed for evaluation of root fillings as shown in table 1, a specific score was assigned to each parameter, which was similar to that in the study of santos et al . The root canal was considered as a sampling unit, and the quality of the root filling was evaluated according to the following parameters: length, density andtaper, which were scored (s) as s0, s1 and s2 . Taper, which were scored (s) as s0, s1 and s2 . S2 corresponded to an adequate standard whereas s1 and s0 referred to slight and considerable deviation from the adequate standard, respectively table 1 . A root canal filling was considered acceptable (total score: 6) only when the length, density and tapering of the filling were adequate (three scores of 2). Fillings with total scores of 0 - 2 and 3 - 5 were considered to be poor and moderate, respectively . Descriptive analysis was performed separately for each type of root canal, and the sum of all endodontic errors was calculated . Chi - square test was used to compare the gg drill (gg group) with hand instrument (no gg group) with respect to taper of root canal (adequate, under - shaped and over - shaped), length of fillings (adequate, under - fill, over - fill and tip - to - tip) and scoring (poor, moderate and acceptable). To compare the coke bottle shaped errors between the groups, whitney test was used for comparison of frequency of voids within each root canal and the scores of the two groups were obtained . All the aforementioned analyses used for comparing the gg and no gg groups were also used to compare maxillary and mandibular root canals . The distributions of the evaluated parameters, that is, length, density and taper of rcts are shown in table 2 . A significant relationship was found between the type of teeth (maxillary or mandibular) and the length of fillings, with more number of under - fillings in mandibular root canals (p = 0.001). A significant more number of voids (lower density) were seen in maxillary root canals (p <0.001). The chi - square test showed a significant number of under - shapings in mandibular root canals (p = 0.007). Only one (0.3%) palatal root of the maxillary molar was over - shaped [table 2]. Length, density and taper of rcts in different root canals fisher's exact test did not reveal a significant relation between coke bottle shape mishap and type of the teeth (p = 0.212). The exact distribution of this mishap is demonstrated in table 2 . Table 3 shows the length, density and taper of rcts based on the type of instrument used (whether gg drills were used). No relationship was found between the type of instrument used and the length (p = 0.499) and taper of fillings (p = 0.238). On the other hand, the mann whitney test showed that the root instrumented with gg drills had a significantly higher density when compared with those for which gg drills were not used (p = 0.004). Moreover, gg drills did not increase the rate of the coke bottle shaped canals (p = 0.467). Length, density and taper of rcts based on the types of instruments used by students table 4 shows the technical quality scores of rcts performed in different root canals . A root canal filling was considered acceptable only when the length, density and tapering of the filling were adequate . The percentages of rcts with acceptable, moderate and poor quality were 35.6, 39 and 24.1%, respectively . Mandibular root canals had significantly better quality mean score (p = 0.024) when compared with maxillary root canals . Technical quality scores of performed rcts in different root canals the number of poor, moderate and acceptable fillings with gg drills were 27 (17.3%), 64 (41%) and 65 (41.7%), respectively . On the other hand, the distributions of poor, moderate and acceptable fillings performed using only k - files were 49 (31.4%), 59 (37.8%) and 47 (30.1%), respectively . The mann - whitney test showed that the mean score of root fillings quality was significantly higher for the gg group (4.15 0.12) than the no gg group (3.61 0.14) (p = 0.008). In the present study, the technical quality of root fillings in a preclinical course as well as the influence of using gg on occurrence of endodontic mishaps was evaluated . It should be noted that although the radiographic technical quality of fillings was evaluated in several studies, only in a few studies the quality of preclinical course was considered and that was via questionnaires filled by students . Recently, one study radiographically assessed the quality of rcts performed in preclinical setting . Although it is difficult to compare the results of the present work with other studies because of the differences in the design of the studies, other similar works looked into the quality of rcts performed by undergraduate dental students . In most of the studies, over - filling was considered to be equal to tip - to - tip filling . However, similar to the study of santos et al ., we considered them to be different and assigned a better score to tip - to - tip filling . In this study, the root fillings that were adequate, short, tip - to - tip and over - filled were 52.1, 18.7, 14.3 and 13.7%, respectively . The number of fillings with adequate length was more than those of the lupi - pegurier et al . The percentages of teeth with adequate, short and over - fillings were reported to be 61.3, 34.5 and 4.2%, respectively . Peak et al . Evaluated the technical quality of fillings performed by royal air force dental students and found the percentages of adequate, short and over - filled root canals to be 50, 32 and 18%, respectively . These aberrations can be due to several reasons, the most important being the radiographic technique used . In real clinical practice, the radiographs are commonly obtained using bisecting - angle technique, whereas the paralleling technique was used in the current study . The results of the present study showed that there was a significant under - filling in mandibular molars . It is reported that most of the under - filled root canals are observed in mandibular molars . This can be explained by the anatomy of multi - canalled curved roots in these teeth . In the current study, a significant percentage of adequately tapered root canals (85.3%) were reported in barrieshi - nusair et al . Study . As shown in table 2, most of the poorly tapered root canals were, in fact, under - shaped (41%), and only a few were over - shaped (0.3%). This may be due to the inadequate consideration of undergraduate students to completely clean and shape the canals . Further, 64.4% of root fillings had an adequate density, and 35.6% had at least one visible void . The number of root fillings with adequate density was somewhat less than those reported in the studies of barrieshi - nusair et al . Did not find a significant difference between the density of maxillary and mandibular root fillings . In contrast, in our study, most of the fillings with poor density were observed in maxillary molars when compared with the mandibular molars . Preclinical students were divided into two groups: gg group (used k - files along with gg drills) and no gg group (used only k - files). No significant difference was observed between the two groups with respect to length of fillings and taper of root canals; however, usage of gg drills significantly increased the final density of fillings . In addition, the technical quality scores of root canals were significantly higher in the gg group . This may be attributed to the fact that gg drills provide a better coronal enlargement and shaping of the root canals, leading to a straight - line access to the root apex . The better straight - line access in the properly shaped teeth may lead to a further depth of insertion of spreaders during gutta - percha administration, thus resulting in a better homogeneity of the filling . Coronal enlargement, adequate shaping and straight - line access are the factors responsible for the greater number of acceptable fillings in the gg group in this study . Unexpectedly, coke bottle shape incidence was not significantly related to usage of gg drill and only occurred in 18 (5.7%) of the root canals . This may be because the students, who were taught by preclinical instructors, did not apply too much force when using gg drills . As can be clearly observed from the results, gg drills not only reduced practitioners errors but also significantly increased the quality mean score of rcts when compared with the no gg group . However, wu et al . Concluded that gg drills can increase the risk of perforation; therefore, they must be used with caution . In this study, the percentage of acceptable root fillings reported in this study was nearly in accordance with the studies of er et al . This percentage is much higher than those of the studies of hayes et al . And tarim ertas et al . The low percentage of acceptable root fillings found in the current study could be attributed to several reasons, including the design of the study, criteria followed, rct techniques used and most importantly, the endodontic curriculum of the school . According to the european society of endodontology, an important criterion is the time allocated to the subject matter; however, in this faculty, 55 h are devoted to preclinical endodontic teaching, which seems to be insufficient . The staff to student ratio was 1:15, which should be improved when compared with some other studies . In summary, to improve the technical quality of rcts performed by the undergraduate dental students, the endodontics curriculum can be revised in some aspects . Furthermore, students must be taught new techniques and instruments to ensure that they are up - to - date . The clinical training course must be designed in such a way that the students are provided with proper skills in endodontics, starting with the basic principles of real clinical endodontics . The teeth radiographs taken by students were not used because of the different radiation angles used, and image recapturing was performed by one examiner in order to calibrate the data . Digital radiographs were used to develop the images and to ensure that small endodontic errors were not missed . In addition, other accurate tools such as scanning electron microscopy, stereomicroscopy, cone beam computed tomography, etc . Could be used instead of digital imaging . However, another limitation of this study was that the survey was done in a preclinical setting, and some procedural accidents such as strip perforation was not assessed because the students do not deliver these cases to their evaluators . This fact can lead to a study bias when evaluating the role of gg drills in the final quality of fillings . The technical quality of preclinical molar rcts performed by undergraduate dental students using step - back technique and cold lateral condensation was considered to be acceptable in 35.6% of the cases . When gg drills were used along with k - files, the technical quality of rcts was enhanced.
The basic helix - loop - helix (bhlh) family of transcriptional regulators are key players in a wide array of developmental processes in metazoans, including neurogenesis, myogenesis, hematopoiesis, sex determination and gut development (reviewed in). The bhlh domain is approximately 60 amino acids long and comprises a dna - binding basic region (b) followed by two helices separated by a variable loop region (hlh) (reviewed in). The hlh domain promotes dimerization, allowing the formation of homodimeric or heterodimeric complexes between different family members . Over 400 bhlh proteins have been identified to date in organisms ranging from the yeast saccharomyces cerevisiae to humans (see, for example). In previous work, we took advantage of the complete sequencing of the nematode and fly genomes to extract a large, and possibly complete, set of bhlh genes from these two organisms . A phylogenetic analysis of the amino acid sequences of these bhlhs, together with a large number (> 350) of bhlh from other sources, in particular from mouse, led us to define 44 orthologous families (that is, groups of orthologous sequences that derive from the duplication of a common ancestor), among which 36 include bhlh from metazoans only, and 2 have representatives in both yeasts and metazoans (table 1). We also identified two bhlh motifs present only in yeast, and four that are present only in plants . The 44 families of animal bhlh defined by our phylogenetic analyses families have been named according to the name (or its common abbreviation) of the first discovered or best - known member of the family . The number of members per family in worm, fly and human (complete genomes) as well as in mouse, sea squirt, and pufferfish (uncompleted genomes) is reported . Each family has been tentatively assigned to a high - order group using the classification of atchley and fitch and ledent and vervoort . Genes that cannot be assigned to any families are categorized as' orphan' genes . Beta3 and oligo are closely related families, one c. elegans gene (f38c2.2) belongs to the beta3 family while another (dy3.3) is equally related to both beta3 and oligo families . Mad and mnt are closely related families, one ciona gene (not7) belongs to the mad family while another (lqw20007) is equally related to both mad and mnt families . Tf4 and mlx are closely related families, one c. elegans gene (t20b12.6) is equally related to both families . The hif, sim, and trh families form a strongly supported monophyletic group (bootstrap value, 95%). A few genes that are included in this group cannot be clearly related to one of the three families (see additional data for details). The hey, hairy and enhancer of split families genes form a well - supported monophyletic group (group e; see figure 1). Two clear families (hairy and hey families) with high bootstrap support emerge from this group . All the remaining sequences have been grouped in a single family (named enhancer of split), which has no real phylogenetic support . In addition, we defined higher - order groups which include several evolutionarily related families that share structural and biochemical properties . The different groups were named a, b, c, d, e and f, in agreement with the nomenclature of atchley and fitch . Figure 1 shows the phylogenetic relationships between animal families and their tentative inclusion into the different higher - order groups . A neighbor - joining (nj) tree showing the evolutionary relationships of the 44 animal bhlh families listed in table 1 is shown . We used one gene (usually from mouse) per family to construct this tree . Although there are strong theoretical reasons for preferring the unrooted tree, we show a rooted tree because it is easier to display compactly and more clearly represents the relationships at the tip of the branches . This tree is just a representation of an unrooted tree with rooting that should be considered arbitrary ., we show a tree in which branch lengths are not proportional to distances between sequences . Some of these groups (a and e) are monophyletic groups, others (d and f) correspond to only one family, and yet others (b and c) are paraphyletic (the last common ancestor of the different families that constitute the group is also that of bhlhs that do not belong to that group). A subgroup of group a families (the atonal' superfamily') is also highlighted and is displayed in more detail in figure 2 . Some examples of phylogenetic relationships among human and mouse bhlh . Rooted nj trees are shown . Numbers above branches indicate per cent support in bootstrap analyses (1,000 replicates). As in figure 1, the rooting should be considered arbitrary . Mouse genes are shown in red, human genes in blue, and other species in black . Species abreviations are as followed: br, brachydanio rerio; ce, caenorhabditis elegans; ci, ciona intestinalis; dm, drosophila melanogaster; gg, gallus gallus; tr, takifugu rubripes; xl, xenopus laevis . (a) evolutionary relationships among atonal' superfamily' members (see figure 1). The different constituting families are pointed out . For sake of simplicity, only mouse, this tree is rooted using the closely related twist gene from mouse (see figure 1) as outgroup . In all cases, a human and a mouse sequence cluster together with high bootstrap values, indicating orthology relationships . This tree is rooted using the closely related math1 gene from mouse (see figure 1) as outgroup . Whereas one human and one mouse bhlh (n015926 and pmeso1, respectively) are clearly orthologs, there is no one - to - one relationship between two mouse bhlh (mesp1 and mesp2) and three human bhlh (n010356a, b, c), although these bhlh cluster together with a high bootstrap value . This tree is rooted using the closely related mitf gene from mouse (see figure 1) as outgroup . Two human genes have clear mouse orthologs but two others (q9hap2 and n005106) have no such orthologs . This tree is rooted using the closely related hen1 gene (nscl family) from mouse (see figure 1) as outgroup . The lyl1 and lyl2 mouse genes are collectively orthologs to one human gene (p12980), indicating a probable gene duplication specific to mouse . In brief, groups a and b include bhlh proteins that bind core dna sequences referred to as e boxes (canntg), respectively cacctg or cagctg (group a) and cacgtg or catgttg (group b). Group c corresponds to the family of bhlh proteins known as bhlh - pas, as they contain a pas domain in addition to the bhlh . Group d corresponds to hlh proteins that lack a basic domain and are hence unable to bind dna . Group e includes proteins related to the drosophila hairy and enhancer of split bhlh (her proteins). These proteins bind preferentially to sequences referred to as n boxes (cacgcg or cacgag). They also contain two characteristic domains in addition to the bhlh, the' orange' domain and a wrpw peptide in their carboxy - terminal part . Group f corresponds to the coe family, which is characterized by the presence of an additional domain involved both in dimerization and in dna binding, the coe domain . The completion of the human genome sequencing project now allows us to derive the complete set of bhlh present in a vertebrate genome . Tblastn searches on the human genome draft sequence enabled us to identify 125 different human bhlhs . After exhaustive searches with blastp in protein databases and the use of the smart database (simple modular architecture research tool) we also identified additional fly, worm and mouse bhlh sequences (total number: 58 in fly, 39 in worm, and 102 in mouse). In addition, we made tblastx searches on the incompletely sequenced genomes of the pufferfish takifugu rubripes and the sea squirt ciona intestinalis and retrieved 84 and 18 different bhlhs, respectively . We also retrieved, through blastp searches, eight different bhlh genes from the completely sequenced yeast genome . Phylogenetic analysis of all these sequences allowed us to define 44 orthologous families of bhlh proteins in metazoans (the 38 families defined in our previous report plus 6 additional ones, arising out of the additional sequences used in this analysis). Our work now enables comparison of the putative complete repertoires of bhlhs in metazoans belonging to the two main subdivisions of bilaterian animals (the bilateria; see for a recent overview of the classification of metazoans) - the deuterostomes (human) and the protostomes (fly and worm). This comparison gives us the opportunity to analyze evolution of the diversity of the bhlhs on a metazoan - wide scale, thus giving useful insights into the evolution of multigenic families . In addition, our results allow us to reconstruct the minimum complement of bhlh genes that were present in the bilaterian common ancestor . To isolate human bhlh genes, we made tblastn searches on the human genome draft sequence, as described in materials and methods . We eventually got 125 different human bhlh sequences, which are listed in table 2 . All retrieved sequences were used to make blastp searches against protein databases in order to detect those sequences that were already identified . We found that 80 sequences were already present in protein databases; 45 of the retrieved sequences from the human genome correspond to previously uncharacterized genes . We similarly retrieved, by tblastn, 84 and 18 different bhlh sequences from the incompletely sequenced genomes of the pufferfish t. rubripes and the sea squirt c. intestinalis, respectively (see additional data files). In addition, we retrieved the complete set of bhlh genes present in the fly (total 58), worm (39), and yeast (8) genomes, as well as all the cloned mouse bhlh genes to date (102), as described in materials and methods . These sequences with their accession numbers and some information (genomic localization and orthology relationships) the complete list of bhlh genes from homo sapiens human sequences are identified using their accession number from swissprot, trembl, smart or ncbi genome project sequences . In the latest case, this accession number nt_xxxxxx.y (xxxxxx identifies the contig and y the version of the draft) has been abbreviated as nxxxxxxx . Gene names are those reported in protein databases or have been assigned by us on the basis of the orthology relationships with mouse genes (these names are marked by an asterisk). The identification of the contig in which each of the bhlh gene is included is also given . In a few cases (marked with a question mark), we were unable to retrieve, in the genome sequence, previously cloned genes . This may be due to the fact that these genes lie in still unsequenced regions of the genome, or to some limitations of the current version of blast (see text for details). Chromosomal localizations are given as reported in the ncbi human genome sequence database (locuslink and/or omim). The complete list of bhlh genes from drosophila melanogaster gene names (with commonly used synonyms in some cases) and their usual abbreviation are as reported in flybase, except those marked by an asterisk . In these cases, we propose names based on the orthology relationships with well - characterized vertebrate genes . Sequences are listed with the family in which there are included (or stated as orphan genes), their chromosomal localization (position on the polytene chromosomes map as found in flybase), and their accession number . The' orphan' gene delilah clearly belongs to the high - order group a and is most probably a highly divergent neurod family gene (see for discussion). The complete list of bhlh genes from caenorhabditis elegans gene identifications are those of the c. elegans genome project . The localization of the genes referred to the worm recombination genetic map as found in wormbase . Sequences marked with an asterisk form a well supported monophyletic group and encode proteins with two bhlh (see text for details). The complete list of bhlh genes from mus musculus mouse genes are listed with the family in which they are included, the identification of their human ortholog(s) (? Indicates that no clear ortholog was found, see text for details), and their accession number . In most cases, we report here only one name; synonyms can be found in the protein databases using the reported accession numbers . The complete list of bhlh genes from saccharomyces cerevisiae yeast genes are listed with the family in which they are included and their accession number . To carry out evolutionary analyses of multigene families requires one to distinguish orthologs, which have evolved by vertical descent from a common ancestor, from paralogs, which arise by duplication and domain shuffling within a genome . Failure to do so can result in functional misclassification and inaccurate molecular evolutionary reconstructions . The overall similarity (as determined by the blast e - value) is often used as a criterion to determine orthology relationships within large data sets such as complete genomes, but there is evidence that more rigorous phylogenetic reconstructions are required to confidently determine orthologies . We therefore constructed phylogenetic trees to define groups of orthologous sequences, as we did previously (see materials and methods). We determined 44 orthologous families that contain most of the metazoan bhlh families (table 1 and additional data). The criterion we used to define orthologous families was as in; that is, orthologous families are monophyletic groups found in the gene trees constructed by different phylogenetic methods and whose monophyly is supported by bootstrap values larger than 50% . We named each family according to its first discovered member or, in a few cases, its best - characterized member . This analysis gave similar results to that described in, except that the additional sequences included in the present phylogenetic analyses led us to define six additional families of bhlhs from metazoans, compared with our previous report . We found a total of 125 and 102 different bhlh sequences in human and mouse, respectively (tables 2 and 5). These sequences were used to make phylogenetic reconstructions as described above and in materials and methods . This allows us to infer orthology relationships between mouse and human sequences . Two sequences were considered as orthologs if they are more closely related to each other than to any other mouse or human sequences . This can be easily detected in the phylogenetic trees, as the two sequences will form an exclusive monophyletic group (figure 2a). Among the 125 human sequences, 94 can be accurately related to 1 (or in a few cases 2, see below) mouse genes (table 2) and, conversely, human orthologs can be confidently assigned to 93 of the 102 mouse genes (table 5). Among the 31 human genes and 9 mouse genes that do not show clear orthology relationships to any mouse or human genes, respectively, 8 human genes and 6 mouse genes are members of families in which phylogenetic relationships are uncertain - the mesp, e12 and coe families (figure 2b and additional data). The mesp family contains four human genes and three mouse genes, the e12 family seven human and four mouse genes, and the coe family four human and four mouse genes . Some of these genes cannot be clearly linked to each other (see figure 2b for an example). It is, however, conceivable that such relationships do exist but that phylogenetic reconstruction methods fail to detect them . We therefore consider that, in the mesp family for example (figure 2b), three of the four human genes correspond to the three mouse genes, and so, to date, one human gene lacks an ortholog among the cloned mouse genes . Applying the same reasoning to the e12 and coe families leads us to conclude that at least 26 human genes (20% of the total) do not have orthologs among the mouse bhlh genes cloned to date and only 3 mouse bhlhs (3%) have no orthologs in the bhlh set we derived from the human genome sequence draft . Figure 2c shows a typical phylogenetic tree of a family containing human genes that lack mouse orthologs . The fact that only three mouse genes lack human orthologs strongly argues that, although our analysis was made on a draft version of the human genome sequence, the set of bhlh we retrieved is likely to be almost complete, and hence gives a highly accurate view of the bhlh repertoire of a human being . Additional blast searches for human orthologs of the three mouse bhlhs that lacked orthologs (scleraxis, dermo-1 and s - myc) were unsuccessful, suggesting that these orthologs either do not exist in humans or are not in the draft sequence . We were recently made aware that there is some incompatibility between the current version of blast and the human genome sequence (probably due to the large number of ns (unassigned nucleotides) in the sequence), which makes blast unable to locate some of the best or even exact matches of small query sequences (j.a.m . This may explain why we missed the four genes cited above, and also why, in a few cases, we were unable to find known cloned human genes in the genome sequence (see table 2). We also found eight cases in which two human genes group together (with high statistical support) to the exclusion of any other genes and are often orthologs of a single mouse gene (figure 2b and additional data). Conversely, we found two cases in which two mouse genes are, collectively, orthologs of a single human gene (figure 2d). This may reveal relatively recent duplications specific to the human or mouse lineage . In agreement with this, in all cases amino - acid identity between the two duplicates is high and is not confined to the bhlh . In addition, we found that in two cases (human sequences q9uh92/n005106 and q02363/n005999), one of the two duplicates lacks introns . This strongly suggests that the duplications have occurred by retrotransposition, a type of event that appears to be rather frequent in humans . In both cases, the copy lacking introns has stop codons in the bhlh, suggesting that it is a pseudogene . Among the 39 bhlh from the worm, 6 cannot be assigned to any family (orphan genes; see tables 1 and 4). Five of these have an unusual architecture in they contain two bhlh domains (see also). Phylogenetic analysis of these proteins indicates that they result from the duplication of an ancestral gene that already contained two bhlhs (figure 3). Both bhlh domains are loosely related (on the basis of overall similarity) to her proteins (group e; figure 1), but their inclusion in group e is not supported by phylogenetic reconstruction (figure 3). In addition, they lack the orange domain, which is characteristic of most her proteins and provides them with functional specificity . They also lack the wrpw motif found in the carboxy - terminal region of almost all her proteins and which allows interaction with the groucho represser protein . Moreover, they lack a conserved proline in the basic domain that confers dna - binding specificity on the her proteins . A rooted nj tree is shown that depicts the phylogenetic relationships of the five worm proteins with two bhlh domains . Mouse genes representative of some of the animal families have been included in this analysis . Numbers above branches indicate per cent support in bootstrap analyses (1,000 replicates). As in figure 1 the sequences of the first bhlh of each worm proteins are shown in blue, the second in red . Both form monophyletic groups with high bootstrap values, indicating that these proteins originate from an ancestral protein that already had two bhlh domains . There is, furthermore, a weaker support (40% bootstraps) for an association of the two bhlh domains into a monophyletic group (not shown in the figure, as only nodes with 50% or more support are shown), suggesting that the ancestral protein may have acquired its two bhlh domains through tandem duplication rather than by association of unrelated bhlh domains . No other protein with two bhlhs has been reported in other metazoans and we were unable to find such proteins in the fly and human genomes . A protein with two bhlh domains is found in rice (oryza sativa; protein p0498b01.20; accession number bab61947) but its sequence is completely unrelated to that of the worm protein . Several bhlh proteins do contain other dna - binding and/or dimerization domains in addition to their bhlh, such as the pas domain, leucine zippers or the coe domain . It is conceivable that these domains may cooperate and thereby confer particular functions on the proteins containing them . Similarly, the presence of two bhlhs might modify the specificity of the proteins containing them . Bhlh genes are found in all major subdivisions of the eukaryotes: metazoans, fungi and plants . It seems, therefore, that the bhlh motif was established in early eukaryote evolution . We have found eight different bhlh genes in the unicellular eukaryote, the yeast s. cerevisiae . Most of these genes were already cloned and have been functionally characterized (reviewed in). These genes often regulate biochemical pathways (such as phosphate utilization, phospholipid and amino - acid biosynthesis, glycolysis) through the transcriptional activation of more - or - less large sets of genes involved in these pathways . Orthologs of these genes are found in other distantly related yeasts such as schizosaccharomyces pombe and kluyveromyces lactis (our unpublished observations), indicating an ancient origin for the different bhlh genes among yeasts . The relatively small number of bhlh genes found in the unicellular yeast contrasts with the large number found in multicellular eukaryotes such as animals and plants . We report here the existence of 39 different bhlh genes in c. elegans, 58 in d. melanogaster, and 125 in humans . Preliminary analysis of plant genomes, in particular of arabidopsis thaliana and o. sativa, similarly indicates a large number of bhlh genes (more than 100 in the completely sequenced genome of a. thaliana, our unpublished observations). This important diversification of the bhlh repertoire in animals and plants has occurred independently, as plant and animal bhlh genes are never found in a same family . The current view of eukaryote phylogeny suggests that fungi and animals are more closely related to each other than to the plants . Nevertheless, we found that only two families contain both yeast and animal genes (see table 1), suggesting that the common ancestor of fungi and animals may have possessed even fewer bhlh genes than the present - day yeasts . In the near future, the genome projects currently underway on various' basal' eukaryotes (see) may give important insights into the very early evolutionary history of the bhlh family . We suggest that the diversification of bhlh genes is directly linked to the acquisition of multicellularity and hence to the recruitment of genes involved in cell functions such as metabolism into the developmental processes required to build multicellularity . Indeed, in animals, bhlh genes are generally involved in development and in tissue - specific gene regulation (reviewed in) a similar situation may exist in plants, although very few bhlh genes have been functionally characterized . In addition, in both animals and plants, the diversification of bhlh genes seems to have occurred early in the evolution of these lineages . Indeed, our phylogenetic analysis of animal bhlh genes shows that most belong to 44 different orthologous families . Of these families, 43 contain representatives from both protostomes and deuterostomes, and must therefore be represented in their common ancestor (often called urbilateria), which lived in pre - cambrian times (600 million years ago). In addition, the few bhlh genes that have been cloned from cnidarians, which are not bilaterians, are clearly included in families (see the twist, myod and asc families in additional data), suggesting that the establishment of at least some families predates the divergence of bilaterians and non - bilaterians . Further analyses of bhlh genes in cnidarians, sponges and slime molds will help to resolve the issue of the early evolution of bhlh genes in animals . Our preliminary analyses of plant bhlh genes are consistent with an early diversification in plants, as in animals . Indeed, many a. thaliana bhlh genes have clear orthologs in a distantly related plant, o. sativa, whose genome has been partially sequenced (our unpublished observations). Arabidopsis is a eudicotyledon and oryza a member of the liliopsida (a monocotyledon), and given the phylogenetic relationships of these clades this suggests that the possession of numerous bhlh genes might be ancestral to angiosperms . Further analysis of the evolution of bhlh in plants will require the completion of the genome projects currently underway on rice and tomato (a eudicotyledon of a different lineage from arabidopsis), as well as the isolation of bhlh in a broader spectrum of plant species, in particular in basal angiosperms and non - angiosperms . Comparison of the bhlh repertoires found in the protostomes and the deuterostomes gives important insights in to the evolution of the bhlh family in metazoans . The conclusions that can be drawn are completely consistent with those presented in our previous work but the inclusion of the probable complete set of bhlh from a vertebrate strengthens these conclusions . Most families (43/44) contain genes from protostomes (fly and/or nematode) and deuterostomes, indicating that these families were already present in the last common ancestor of both protostomes and deuterostomes, that is, of all bilaterians . The fact that most families contain both protostome and deuterostome genes also suggests that there was no addition of new bhlh types in the corresponding lineages, and therefore no important diversification of the ancestral repertoire . This may represent the emergence of new bhlh types in the vertebrate lineage, or alternatively a loss of ancestral types in both fly and nematode . The analysis of bhlh genes from molluscs or annelids might help to settle this question . It is now widely believed that the bilateria (the triploblastic metazoans) are composed of three main lineages: deuterostomes (which include vertebrates and echinoderms) and protostomes, which themselves include two large groups, the ecdysozoans (for example, arthropods and nematodes) and the lophotrochozoans (for example, annelids, molluscs and flatworms) (reviewed in). Therefore, the finding of ortholog genes in vertebrates and lophotrochozoans but not in fly and nematode would strongly suggest that gene loss(es) has (have) occurred in the ecdysozoan lineage . Similarly, the case of families that contain vertebrate and either worm or fly genes is best explained by gene losses that occurred, inside the ecdysozoan clade, in either lineage after the arthropod / nematode divergence . This occurred in the fly lineage for very few families (4/44), suggesting the existence of a strong pressure to maintain the entire bhlh repertoire . The much larger number of families (13/44) that have vertebrate and fly members but no nematode representative suggests that extensive bhlh gene losses have occurred in the worm lineage . Strikingly, the worm lacks the important cellular and developmental regulator myc . A similar absence of important developmental regulators, such as hedgehog, toll / il-1 and jak / stat pathway elements has also been reported in the nematode . In addition, a large number of nematode genes (6/39) cannot be clearly assigned to specific families (orphan genes). This is probably due to the high divergence rate reported for nematode genes in general and which we found within our specific data set (and data not shown). Interestingly, however, some nematode sequences have diverged very little from their fly or mouse counterparts . These include the few functionally characterized c. elegans bhlh genes that show overall functional conservation with their vertebrate and/or fly orthologs; for example, the c. elegans orthologs of twist and myod are involved in muscle formation, and the orthologs of atonal and neurod (lin-32 and cnd-1) have a role in nervous - system development . The genetic control of developmental processes such as neurogenesis and myogenesis relies on small sets of interacting genes (syntagms). The function of syntagms crucially relies on specific molecular interactions among their members, hence imposing strong structural constraints on them and preventing structural diversification (for discussion on syntagms and evolution, see). This may explain why such networks are strongly conserved throughout metazoan evolution and why nematode genes involved in such networks have been subject to special constraints . An extensive increase of bhlh family complexity has occurred in vertebrates: the most frequent number of different bhlh genes per family is one in fly (30/44) and worm (27/44), and two in human (14/44; but 20/44 human families do in fact contain more than two genes). Most bhlh families (32/44), as with other gene families, have more members in vertebrates than in other phyla (table 1). Of these families, 14 (32%) contain four or more vertebrate genes (table 1) and hence may reveal the occurrence of two whole - genome duplications (the 2r hypothesis) in early vertebrate evolution . In the most popular version, this is thought to have occurred by one duplication at the root of the vertebrates and a second in the gnathostomata lineage, after its divergence from agnatha (reviewed in). Several recent analyses, however, tend to refute (at least, do not support) this hypothesis (reviewed in). For example, the current mammalian gene number estimations based on the human draft sequence, ests and comparisons with other vertebrates propose that the human genome would contain no more than 35,000 genes; that is, about twice the number of fly and worm . This might, however, result from gene loss during or after the rounds of duplication . In addition, phylogenetic analyses of gene families that comprise four members cast doubt on the 2r hypothesis . As pointed out by hughes, the presence of four members in a vertebrate gene family by itself does not support the genome duplication hypothesis . Support may only come from families whose phylogenetic tree shows a topology of the (ab) (cd) form, that is, two pairs of two closely related paralogs . Hughes discussed the phylogenies of 13 protein families important in development, and found that only one of them shows an (ab) (cd) topology . Similar results were recently obtained by martin and hughes et al . On several other families with much more rigorous phylogenetic tests . These results have led to the alternative hypothesis that the abundance of duplicated genes in vertebrates compared to invertebrates may be due to a high rate of local duplications, rather than entire genome duplications (reviewed in). Phylogenetic trees of the 14 bhlh families that contain four or more members do not clearly show such (ab) (cd) topologies (see additional data). We have, however, to note that the phylogenies inside families often have only poor resolution and it is therefore difficult to draw firm conclusions from them . To isolate human bhlh genes, we made tblastn searches on the human genome draft sequence, as described in materials and methods . We eventually got 125 different human bhlh sequences, which are listed in table 2 . All retrieved sequences were used to make blastp searches against protein databases in order to detect those sequences that were already identified . We found that 80 sequences were already present in protein databases; 45 of the retrieved sequences from the human genome correspond to previously uncharacterized genes . We similarly retrieved, by tblastn, 84 and 18 different bhlh sequences from the incompletely sequenced genomes of the pufferfish t. rubripes and the sea squirt c. intestinalis, respectively (see additional data files). In addition, we retrieved the complete set of bhlh genes present in the fly (total 58), worm (39), and yeast (8) genomes, as well as all the cloned mouse bhlh genes to date (102), as described in materials and methods . These sequences with their accession numbers and some information (genomic localization and orthology relationships) the complete list of bhlh genes from homo sapiens human sequences are identified using their accession number from swissprot, trembl, smart or ncbi genome project sequences . In the latest case, this accession number nt_xxxxxx.y (xxxxxx identifies the contig and y the version of the draft) has been abbreviated as nxxxxxxx . Gene names are those reported in protein databases or have been assigned by us on the basis of the orthology relationships with mouse genes (these names are marked by an asterisk). The identification of the contig in which each of the bhlh gene is included is also given . In a few cases (marked with a question mark), we were unable to retrieve, in the genome sequence, previously cloned genes . This may be due to the fact that these genes lie in still unsequenced regions of the genome, or to some limitations of the current version of blast (see text for details). Chromosomal localizations are given as reported in the ncbi human genome sequence database (locuslink and/or omim). The complete list of bhlh genes from drosophila melanogaster gene names (with commonly used synonyms in some cases) and their usual abbreviation are as reported in flybase, except those marked by an asterisk . In these cases, we propose names based on the orthology relationships with well - characterized vertebrate genes . Sequences are listed with the family in which there are included (or stated as orphan genes), their chromosomal localization (position on the polytene chromosomes map as found in flybase), and their accession number . The' orphan' gene delilah clearly belongs to the high - order group a and is most probably a highly divergent neurod family gene (see for discussion). The complete list of bhlh genes from caenorhabditis elegans gene identifications are those of the c. elegans genome project . The localization of the genes referred to the worm recombination genetic map as found in wormbase . Sequences marked with an asterisk form a well supported monophyletic group and encode proteins with two bhlh (see text for details). The complete list of bhlh genes from mus musculus mouse genes are listed with the family in which they are included, the identification of their human ortholog(s) (? Indicates that no clear ortholog was found, see text for details), and their accession number . In most cases, we report here only one name; synonyms can be found in the protein databases using the reported accession numbers . The complete list of bhlh genes from saccharomyces cerevisiae yeast genes are listed with the family in which they are included and their accession number . To carry out evolutionary analyses of multigene families requires one to distinguish orthologs, which have evolved by vertical descent from a common ancestor, from paralogs, which arise by duplication and domain shuffling within a genome . Failure to do so can result in functional misclassification and inaccurate molecular evolutionary reconstructions . The overall similarity (as determined by the blast e - value) is often used as a criterion to determine orthology relationships within large data sets such as complete genomes, but there is evidence that more rigorous phylogenetic reconstructions are required to confidently determine orthologies . We therefore constructed phylogenetic trees to define groups of orthologous sequences, as we did previously (see materials and methods). We determined 44 orthologous families that contain most of the metazoan bhlh families (table 1 and additional data). The criterion we used to define orthologous families was as in; that is, orthologous families are monophyletic groups found in the gene trees constructed by different phylogenetic methods and whose monophyly is supported by bootstrap values larger than 50% . We named each family according to its first discovered member or, in a few cases, its best - characterized member . This analysis gave similar results to that described in, except that the additional sequences included in the present phylogenetic analyses led us to define six additional families of bhlhs from metazoans, compared with our previous report . We found a total of 125 and 102 different bhlh sequences in human and mouse, respectively (tables 2 and 5). These sequences were used to make phylogenetic reconstructions as described above and in materials and methods . Two sequences were considered as orthologs if they are more closely related to each other than to any other mouse or human sequences . This can be easily detected in the phylogenetic trees, as the two sequences will form an exclusive monophyletic group (figure 2a). Among the 125 human sequences, 94 can be accurately related to 1 (or in a few cases 2, see below) mouse genes (table 2) and, conversely, human orthologs can be confidently assigned to 93 of the 102 mouse genes (table 5). Among the 31 human genes and 9 mouse genes that do not show clear orthology relationships to any mouse or human genes, respectively, 8 human genes and 6 mouse genes are members of families in which phylogenetic relationships are uncertain - the mesp, e12 and coe families (figure 2b and additional data). The mesp family contains four human genes and three mouse genes, the e12 family seven human and four mouse genes, and the coe family four human and four mouse genes . Some of these genes cannot be clearly linked to each other (see figure 2b for an example). It is, however, conceivable that such relationships do exist but that phylogenetic reconstruction methods fail to detect them . We therefore consider that, in the mesp family for example (figure 2b), three of the four human genes correspond to the three mouse genes, and so, to date, one human gene lacks an ortholog among the cloned mouse genes . Applying the same reasoning to the e12 and coe families leads us to conclude that at least 26 human genes (20% of the total) do not have orthologs among the mouse bhlh genes cloned to date and only 3 mouse bhlhs (3%) have no orthologs in the bhlh set we derived from the human genome sequence draft . Figure 2c shows a typical phylogenetic tree of a family containing human genes that lack mouse orthologs . The fact that only three mouse genes lack human orthologs strongly argues that, although our analysis was made on a draft version of the human genome sequence, the set of bhlh we retrieved is likely to be almost complete, and hence gives a highly accurate view of the bhlh repertoire of a human being . Additional blast searches for human orthologs of the three mouse bhlhs that lacked orthologs (scleraxis, dermo-1 and s - myc) were unsuccessful, suggesting that these orthologs either do not exist in humans or are not in the draft sequence . We were recently made aware that there is some incompatibility between the current version of blast and the human genome sequence (probably due to the large number of ns (unassigned nucleotides) in the sequence), which makes blast unable to locate some of the best or even exact matches of small query sequences (j.a.m . This may explain why we missed the four genes cited above, and also why, in a few cases, we were unable to find known cloned human genes in the genome sequence (see table 2). We also found eight cases in which two human genes group together (with high statistical support) to the exclusion of any other genes and are often orthologs of a single mouse gene (figure 2b and additional data). Conversely, we found two cases in which two mouse genes are, collectively, orthologs of a single human gene (figure 2d). This may reveal relatively recent duplications specific to the human or mouse lineage . In agreement with this, in all cases amino - acid identity between the two duplicates is high and is not confined to the bhlh . In addition, we found that in two cases (human sequences q9uh92/n005106 and q02363/n005999), one of the two duplicates lacks introns . This strongly suggests that the duplications have occurred by retrotransposition, a type of event that appears to be rather frequent in humans . In both cases, the copy lacking introns has stop codons in the bhlh, suggesting that it is a pseudogene . Among the 39 bhlh from the worm, 6 cannot be assigned to any family (orphan genes; see tables 1 and 4). Five of these have an unusual architecture in they contain two bhlh domains (see also). Phylogenetic analysis of these proteins indicates that they result from the duplication of an ancestral gene that already contained two bhlhs (figure 3). Both bhlh domains are loosely related (on the basis of overall similarity) to her proteins (group e; figure 1), but their inclusion in group e is not supported by phylogenetic reconstruction (figure 3). In addition, they lack the orange domain, which is characteristic of most her proteins and provides them with functional specificity . They also lack the wrpw motif found in the carboxy - terminal region of almost all her proteins and which allows interaction with the groucho represser protein . Moreover, they lack a conserved proline in the basic domain that confers dna - binding specificity on the her proteins . A rooted nj tree is shown that depicts the phylogenetic relationships of the five worm proteins with two bhlh domains . Mouse genes representative of some of the animal families have been included in this analysis . Numbers above branches indicate per cent support in bootstrap analyses (1,000 replicates). As in figure 1 the sequences of the first bhlh of each worm proteins are shown in blue, the second in red . Both form monophyletic groups with high bootstrap values, indicating that these proteins originate from an ancestral protein that already had two bhlh domains . There is, furthermore, a weaker support (40% bootstraps) for an association of the two bhlh domains into a monophyletic group (not shown in the figure, as only nodes with 50% or more support are shown), suggesting that the ancestral protein may have acquired its two bhlh domains through tandem duplication rather than by association of unrelated bhlh domains . No other protein with two bhlhs has been reported in other metazoans and we were unable to find such proteins in the fly and human genomes . A protein with two bhlh domains is found in rice (oryza sativa; protein p0498b01.20; accession number bab61947) but its sequence is completely unrelated to that of the worm protein . Several bhlh proteins do contain other dna - binding and/or dimerization domains in addition to their bhlh, such as the pas domain, leucine zippers or the coe domain . It is conceivable that these domains may cooperate and thereby confer particular functions on the proteins containing them . Similarly, the presence of two bhlhs might modify the specificity of the proteins containing them . Bhlh genes are found in all major subdivisions of the eukaryotes: metazoans, fungi and plants . It seems, therefore, that the bhlh motif was established in early eukaryote evolution . We have found eight different bhlh genes in the unicellular eukaryote, the yeast s. cerevisiae . Most of these genes were already cloned and have been functionally characterized (reviewed in). These genes often regulate biochemical pathways (such as phosphate utilization, phospholipid and amino - acid biosynthesis, glycolysis) through the transcriptional activation of more - or - less large sets of genes involved in these pathways . Orthologs of these genes are found in other distantly related yeasts such as schizosaccharomyces pombe and kluyveromyces lactis (our unpublished observations), indicating an ancient origin for the different bhlh genes among yeasts . The relatively small number of bhlh genes found in the unicellular yeast contrasts with the large number found in multicellular eukaryotes such as animals and plants . We report here the existence of 39 different bhlh genes in c. elegans, 58 in d. melanogaster, and 125 in humans . Preliminary analysis of plant genomes, in particular of arabidopsis thaliana and o. sativa, similarly indicates a large number of bhlh genes (more than 100 in the completely sequenced genome of a. thaliana, our unpublished observations). This important diversification of the bhlh repertoire in animals and plants has occurred independently, as plant and animal bhlh genes are never found in a same family . The current view of eukaryote phylogeny suggests that fungi and animals are more closely related to each other than to the plants . Nevertheless, we found that only two families contain both yeast and animal genes (see table 1), suggesting that the common ancestor of fungi and animals may have possessed even fewer bhlh genes than the present - day yeasts . In the near future, the genome projects currently underway on various' basal' eukaryotes (see) may give important insights into the very early evolutionary history of the bhlh family . We suggest that the diversification of bhlh genes is directly linked to the acquisition of multicellularity and hence to the recruitment of genes involved in cell functions such as metabolism into the developmental processes required to build multicellularity . Indeed, in animals, bhlh genes are generally involved in development and in tissue - specific gene regulation (reviewed in). A similar situation may exist in plants, although very few bhlh genes have been functionally characterized . In addition, in both animals and plants, the diversification of bhlh genes seems to have occurred early in the evolution of these lineages . Indeed, our phylogenetic analysis of animal bhlh genes shows that most belong to 44 different orthologous families . Of these families, 43 contain representatives from both protostomes and deuterostomes, and must therefore be represented in their common ancestor (often called urbilateria), which lived in pre - cambrian times (600 million years ago). In addition, the few bhlh genes that have been cloned from cnidarians, which are not bilaterians, are clearly included in families (see the twist, myod and asc families in additional data), suggesting that the establishment of at least some families predates the divergence of bilaterians and non - bilaterians . Further analyses of bhlh genes in cnidarians, sponges and slime molds will help to resolve the issue of the early evolution of bhlh genes in animals . Our preliminary analyses of plant bhlh genes are consistent with an early diversification in plants, as in animals . Indeed, many a. thaliana bhlh genes have clear orthologs in a distantly related plant, o. sativa, whose genome has been partially sequenced (our unpublished observations). Arabidopsis is a eudicotyledon and oryza a member of the liliopsida (a monocotyledon), and given the phylogenetic relationships of these clades this suggests that the possession of numerous bhlh genes might be ancestral to angiosperms . Further analysis of the evolution of bhlh in plants will require the completion of the genome projects currently underway on rice and tomato (a eudicotyledon of a different lineage from arabidopsis), as well as the isolation of bhlh in a broader spectrum of plant species, in particular in basal angiosperms and non - angiosperms . Comparison of the bhlh repertoires found in the protostomes and the deuterostomes gives important insights in to the evolution of the bhlh family in metazoans . The conclusions that can be drawn are completely consistent with those presented in our previous work but the inclusion of the probable complete set of bhlh from a vertebrate strengthens these conclusions . Most families (43/44) contain genes from protostomes (fly and/or nematode) and deuterostomes, indicating that these families were already present in the last common ancestor of both protostomes and deuterostomes, that is, of all bilaterians . The fact that most families contain both protostome and deuterostome genes also suggests that there was no addition of new bhlh types in the corresponding lineages, and therefore no important diversification of the ancestral repertoire . This may represent the emergence of new bhlh types in the vertebrate lineage, or alternatively a loss of ancestral types in both fly and nematode . The analysis of bhlh genes from molluscs or annelids might help to settle this question . It is now widely believed that the bilateria (the triploblastic metazoans) are composed of three main lineages: deuterostomes (which include vertebrates and echinoderms) and protostomes, which themselves include two large groups, the ecdysozoans (for example, arthropods and nematodes) and the lophotrochozoans (for example, annelids, molluscs and flatworms) (reviewed in). Therefore, the finding of ortholog genes in vertebrates and lophotrochozoans but not in fly and nematode would strongly suggest that gene loss(es) has (have) occurred in the ecdysozoan lineage . Similarly, the case of families that contain vertebrate and either worm or fly genes is best explained by gene losses that occurred, inside the ecdysozoan clade, in either lineage after the arthropod / nematode divergence . This occurred in the fly lineage for very few families (4/44), suggesting the existence of a strong pressure to maintain the entire bhlh repertoire . The much larger number of families (13/44) that have vertebrate and fly members but no nematode representative suggests that extensive bhlh gene losses have occurred in the worm lineage . Strikingly, the worm lacks the important cellular and developmental regulator myc . A similar absence of important developmental regulators, such as hedgehog, toll / il-1 and jak / stat pathway elements has also been reported in the nematode . In addition, a large number of nematode genes (6/39) cannot be clearly assigned to specific families (orphan genes). This is probably due to the high divergence rate reported for nematode genes in general and which we found within our specific data set (and data not shown). Interestingly, however, some nematode sequences have diverged very little from their fly or mouse counterparts . These include the few functionally characterized c. elegans bhlh genes that show overall functional conservation with their vertebrate and/or fly orthologs; for example, the c. elegans orthologs of twist and myod are involved in muscle formation, and the orthologs of atonal and neurod (lin-32 and cnd-1) have a role in nervous - system development . The genetic control of developmental processes such as neurogenesis and myogenesis relies on small sets of interacting genes (syntagms). The function of syntagms crucially relies on specific molecular interactions among their members, hence imposing strong structural constraints on them and preventing structural diversification (for discussion on syntagms and evolution, see). This may explain why such networks are strongly conserved throughout metazoan evolution and why nematode genes involved in such networks have been subject to special constraints . An extensive increase of bhlh family complexity has occurred in vertebrates: the most frequent number of different bhlh genes per family is one in fly (30/44) and worm (27/44), and two in human (14/44; but 20/44 human families do in fact contain more than two genes). Most bhlh families (32/44), as with other gene families, have more members in vertebrates than in other phyla (table 1). Of these families, 14 (32%) contain four or more vertebrate genes (table 1) and hence may reveal the occurrence of two whole - genome duplications (the 2r hypothesis) in early vertebrate evolution . In the most popular version, this is thought to have occurred by one duplication at the root of the vertebrates and a second in the gnathostomata lineage, after its divergence from agnatha (reviewed in). Several recent analyses, however, tend to refute (at least, do not support) this hypothesis (reviewed in). For example, the current mammalian gene number estimations based on the human draft sequence, ests and comparisons with other vertebrates propose that the human genome would contain no more than 35,000 genes; that is, about twice the number of fly and worm . Consistent with this, many gene families in vertebrates have fewer than four genes . This might, however, result from gene loss during or after the rounds of duplication . In addition, phylogenetic analyses of gene families that comprise four members cast doubt on the 2r hypothesis . As pointed out by hughes, the presence of four members in a vertebrate gene family by itself does not support the genome duplication hypothesis . Support may only come from families whose phylogenetic tree shows a topology of the (ab) (cd) form, that is, two pairs of two closely related paralogs . Hughes discussed the phylogenies of 13 protein families important in development, and found that only one of them shows an (ab) (cd) topology . Similar results were recently obtained by martin and hughes et al . On several other families with much more rigorous phylogenetic tests . These results have led to the alternative hypothesis that the abundance of duplicated genes in vertebrates compared to invertebrates may be due to a high rate of local duplications, rather than entire genome duplications (reviewed in). Phylogenetic trees of the 14 bhlh families that contain four or more members do not clearly show such (ab) (cd) topologies (see additional data). We have, however, to note that the phylogenies inside families often have only poor resolution and it is therefore difficult to draw firm conclusions from them . We identified the probable full complement of bhlh in three different metazoans that are representative of the two major subdivisions of the animal kingdom, the protostomes (c. elegans and d. melanogaster) and the deuterostomes (humans). Most of these families (43/44) have protostome and deuterostome members, and must therefore have been represented in their common ancestor before the cambrian radiation which saw the emergence of all present - day phyla, and many extinct ones . Morphologically, these ancestors (also called urbilateria) were probably coelomates with antero - posterior and dorso - ventral polarity, rudimentary appendages, some form of metamerism, a heart, sense organs such as photoreceptors and a complex nervous system . Genetically, they possessed numerous homeobox genes (among which are at least seven hox genes), several intercellular signaling pathways (tgf-, hedgehog, notch, egf), at least four pax genes, and 38 c2h2 zinc - finger proteins . The functional conservation that is often observed between protostome and deuterostome orthologs indicates that some of the developmental functions associated with the present - day genes were already established in urbilateria, further indicating the genomic and developmental complexity of these ancient ancestors . The full set of bhlh sequences in the fly, worm, and yeast were obtained mostly by blastp searches against the new releases of the complete genomic sequences of c. elegans, d. melanogaster, and s. cerevisiae . Mouse bhlh genes were obtained by blastp searches against the more recent versions of the non - redundant database at ncbi and the sanger protein databases . In addition, we retrieved and analyzed all the bhlhs from these organisms that are listed in the smart database . The comparison with the lists of bhlhs found in the smart database and published by other groups strongly suggests that we retrieved the full set of bhlh genes present in the fly, yeast, and worm genomes, as well as all the cloned mouse bhlh genes to date . Tblastn searches were done at the ncbi on the human genome and at the doe joint genomic institute (university of california and the us department of energy) for the pufferfish and sea squirt genomes . We used as query two different sequences (usually one from mouse and one from fly or worm) of each of the families we defined previously . Searches were done at two stringencies, e <1 and e <0.01, with all other parameters set to default . The blast searches detected some sequences that display only low overall similarity with the query, or similarities only to a part of the bhlh domain . We checked these sequences by hand and found that in all cases they did not correspond to bona fide bhlh domains . We hence did not include these sequences in our subsequent analyses . During the course of our work, the data presented in this paper come from the third version (april 2001). Careful examination of the fourth version (july 2001) did not give additional data . A final check has been done on the latest release (version 6) in november 2001 with no significant changes, except that some contigs have been renamed and two sequences were no longer found . We do not include these two sequences (which were closely related duplications of existing bhlh genes) as they may represent artifacts of the genome sequence assembly process . We cannot exclude the possibility, however, that they are bona fide bhlh genes that were no longer detected as a result of limitations of the current version of blast (see results and discussion). Protein alignments were made using clustalw with no adjustment of the default parameters and were subsequently edited and manually improved in genedoc multiple sequence alignment editor and shading utility (version 2.6.001). The evaluation of percentage conservation of residues in multiple sequence alignments was done using the blosum62 similarity scoring table . Only the bhlh motif (determined as in), plus a few flanking amino acids, was used in most of our analyses because the remaining parts of proteins from independent clades are either not homologous or have diverged so much that the alignments are meaningless . The facilities of the belgian embnet node were used for sequence analysis using genedoc software and for most of the protein alignments using clustalw . Distance trees were constructed with the neighbor - joining (nj) algorithm using paup 4.0 based on a dayhoff pam 250 distance matrix . The resultant trees were bootstrapped (1,000 bootstrap replicates) to provide information about their statistical reliability . Bootstraps were made with paup 4.0, parameters set to default values . Given the large number of sequences (> 300), we were unable, because of computer calculation limitations, to perform maximum - parsimony (mp) and maximum - likelihood (ml) analyses on the multiple alignment that contains all sequences . We made several additional alignments that include only those bhlh sequences that belong to a particular high - order group (figure 1). Nj, mp and ml trees were constructed from these alignments and were fully congruent with the nj trees constructed from the general alignments . The mp analysis was performed using paup 4.0 with the following settings: heuristic search over 100 bootstrap replicates, maxtrees set up to 1,000 due to computer limitations, other parameters set to default values . Maximum likelihood (ml) was done using treepuzzle 4.0.2 . The ml was performed using the quartet - puzzling tree - search procedure with 25,000 puzzling steps, using the jones - taylor - thornton (jtt) model of substitution, the frequencies of amino acids being estimated from the data set, with an uniform rate of substitution . The trees were displayed with the tree view program (version 1.5), saved as pict files, converted into jpeg files using graphic converter, and then annotated using adobe photoshop and adobe illustrator . The full set of bhlh sequences in the fly, worm, and yeast were obtained mostly by blastp searches against the new releases of the complete genomic sequences of c. elegans, d. melanogaster, and s. cerevisiae . Mouse bhlh genes were obtained by blastp searches against the more recent versions of the non - redundant database at ncbi and the sanger protein databases . In addition, we retrieved and analyzed all the bhlhs from these organisms that are listed in the smart database . The comparison with the lists of bhlhs found in the smart database and published by other groups strongly suggests that we retrieved the full set of bhlh genes present in the fly, yeast, and worm genomes, as well as all the cloned mouse bhlh genes to date . Tblastn searches were done at the ncbi on the human genome and at the doe joint genomic institute (university of california and the us department of energy) for the pufferfish and sea squirt genomes . We used as query two different sequences (usually one from mouse and one from fly or worm) of each of the families we defined previously . Searches were done at two stringencies, e <1 and e <0.01, with all other parameters set to default . The blast searches detected some sequences that display only low overall similarity with the query, or similarities only to a part of the bhlh domain . We checked these sequences by hand and found that in all cases they did not correspond to bona fide bhlh domains . We hence did not include these sequences in our subsequent analyses . During the course of our work, the data presented in this paper come from the third version (april 2001). Careful examination of the fourth version (july 2001) did not give additional data . A final check has been done on the latest release (version 6) in november 2001 with no significant changes, except that some contigs have been renamed and two sequences were no longer found . We do not include these two sequences (which were closely related duplications of existing bhlh genes) as they may represent artifacts of the genome sequence assembly process . We cannot exclude the possibility, however, that they are bona fide bhlh genes that were no longer detected as a result of limitations of the current version of blast (see results and discussion). Protein alignments were made using clustalw with no adjustment of the default parameters and were subsequently edited and manually improved in genedoc multiple sequence alignment editor and shading utility (version 2.6.001). The evaluation of percentage conservation of residues in multiple sequence alignments was done using the blosum62 similarity scoring table . Only the bhlh motif (determined as in), plus a few flanking amino acids, was used in most of our analyses because the remaining parts of proteins from independent clades are either not homologous or have diverged so much that the alignments are meaningless . The facilities of the belgian embnet node were used for sequence analysis using genedoc software and for most of the protein alignments using clustalw . Distance trees were constructed with the neighbor - joining (nj) algorithm using paup 4.0 based on a dayhoff pam 250 distance matrix . The resultant trees were bootstrapped (1,000 bootstrap replicates) to provide information about their statistical reliability . Bootstraps were made with paup 4.0, parameters set to default values . Given the large number of sequences (> 300), we were unable, because of computer calculation limitations, to perform maximum - parsimony (mp) and maximum - likelihood (ml) analyses on the multiple alignment that contains all sequences . We made several additional alignments that include only those bhlh sequences that belong to a particular high - order group (figure 1). Nj, mp and ml trees were constructed from these alignments and were fully congruent with the nj trees constructed from the general alignments . The mp analysis was performed using paup 4.0 with the following settings: heuristic search over 100 bootstrap replicates, maxtrees set up to 1,000 due to computer limitations, other parameters set to default values . Maximum likelihood (ml) was done using treepuzzle 4.0.2 . The ml was performed using the quartet - puzzling tree - search procedure with 25,000 puzzling steps, using the jones - taylor - thornton (jtt) model of substitution, the frequencies of amino acids being estimated from the data set, with an uniform rate of substitution . The trees were displayed with the tree view program (version 1.5), saved as pict files, converted into jpeg files using graphic converter, and then annotated using adobe photoshop and adobe illustrator . Additional data files are available with the online version of this paper as follows: multiple alignments (in rich text format) on which the trees displayed in the figures are based . A list of all bhlh sequences from human in rich text format multiple alignments for each family of bhlh proteins: the achaete - scute a family, the achaete - scute b family, the ahr family, the ap4 family, the arnt family, the atonal family, the beta3 and oligo families, the bmal family, the clock family, the e12/e47 family, the extramacrochaete family, the enhancer of split family, the fig alpha family, the hairy family, the hand family, the hey family, the mad and mnt families, the max family, the mesp family, the mist family, the mitf family, the myc family, the myod family, the myor family, the net family, the neurod family, the neurogenin family, the nscl family, the paraxis family, the ptf a and b families, the scl family, the src family, the srebp family, the tf4 and mlx families, the trh, hif, and sim families, the twist family, and the usf family . Representative phylogenetic trees (nj trees bootstrapped 1,000 times to provide statistical support to the nodes, usually rooted with a sequence from a closely related family . In a few cases, closely related families are shown in the same phylogenetic tree) of each family of bhlh proteins: the achaete - scute a family, the achaete - scute b family, the ahr family, the arnt family, the atonal family, the beta3, mist, and oligo families, the bmal family, the clock family, the e12/e47 family, the extramacrochaete family, the enhancer of split family, the fig alpha family, the hairy family, the hand family, the hey family, the mad and mnt families, the max family, the mesp family, the mitf family, the myc family, the myod family, the myor family, the net family, the neurod family, the neurogenin family, the nscl family, the paraxis family, the ptf a and b families, the scl family, the src family, the srebp family, the tf4 and mlx families, the trh, hif, and sim families, the twist family, and the usf family . Species name abbreviations are as in the figure legends and as below: av, asteris vulgaris; avim, avian myelocytomatosis virus cmii; bb, branchiostoma belcheri; bf, branchiostoma floridae; bm, bombyx mori (domestic silkworm); caebr, caenorhabditis briggsae; cc, ceratitis capitata; cp, cynops pyrrhogaster; cs, cupiennius salei; cyca, cyprinus carpio; ds, drosophila simulans; dv, drosophila virilis; dy, drosophila yakuba; hr, halocynthia roretzi; hv, hydra vulgaris (hydra attenuata); ilo, ilyanassa obsoleta; jc, juonia coenia (precis coenia) (peacock butterfly); kl, kluyveromyces lactis; lv, lytechinus variegatus (green urchin); nv, notophtalmus viridens; ol, oryzias latipes (japanese medaka); om, oncorhyncus mikis; pc, podocorine carnea; pv, patella vulgata (common limpet); rn, rattus norvegicus; sb, spermophilus beecheyi; sc, saccharomyces cerevisiae (baker's yeast); sp, schizosaccharomyces pombe; spu, strongylocentrotus purpuratus (purple urchin); st, silurana tropicalis; tc, tribolium castaneum; tricho, trichinella spiralis multiple alignment on which figure 1 is based click here for additional data file a list of all bhlh sequences from human click here for additional data file multiple alignment of the bhlh of achaete - scute a family proteins click here for additional data file multiple alignment of the bhlh of achaete - scute b family proteins click here for additional data file multiple alignment of the bhlh of ahr family proteins click here for additional data file multiple alignment of the bhlh of ap4 family proteins click here for additional data file multiple alignment of the bhlh of arnt family proteins click here for additional data file multiple alignment of the bhlh of atonal family proteins click here for additional data file multiple alignment of the bhlh of beta3 and oligo families proteins click here for additional data file multiple alignment of the bhlh of bmal family proteins click here for additional data file multiple alignment of the bhlh of clock family proteins click here for additional data file multiple alignment of the bhlh of e12/e47 family proteins click here for additional data file multiple alignment of the bhlh of extramacrochaete family proteins click here for additional data file multiple alignment of the bhlh of enhancer of split family proteins click here for additional data file multiple alignment of the bhlh of fig alpha family proteins click here for additional data file multiple alignment of the bhlh of hairy family proteins click here for additional data file multiple alignment of the bhlh of hand family proteins click here for additional data file multiple alignment of the bhlh of hey family proteins click here for additional data file multiple alignment of the bhlh of mad and mnt families proteins click here for additional data file multiple alignment of the bhlh of max family proteins click here for additional data file multiple alignment of the bhlh of masp family proteins click here for additional data file multiple alignment of the bhlh of mist family proteins click here for additional data file multiple alignment of the bhlh of mitf family proteins click here for additional data file multiple alignment of the bhlh of myc family proteins click here for additional data file multiple alignment of the bhlh of myod family proteins click here for additional data file multiple alignment of the bhlh of myor family proteins click here for additional data file multiple alignment of the bhlh of net family proteins click here for additional data file multiple alignment of the bhlh of neurod family proteins click here for additional data file multiple alignment of the bhlh of neurogenin family proteins click here for additional data file multiple alignment of the bhlh of nscl family proteins click here for additional data file multiple alignment of the bhlh of paraxis family proteins click here for additional data file multiple alignment of the bhlh of ptf a and b families proteins click here for additional data file multiple alignment of the bhlh of scl family proteins click here for additional data file multiple alignment of the bhlh of src family proteins click here for additional data file multiple alignment of the bhlh of srebp family proteins click here for additional data file multiple alignment of the bhlh of tf4 and mlx families proteins click here for additional data file multiple alignment of the bhlh of trh, hif, and sim families proteins click here for additional data file multiple alignment of the bhlh of twist family proteins click here for additional data file multiple alignment of the bhlh of usf family proteins click here for additional data file representative phylogenetic tree of the achaete - scute a family of bhlh proteins click here for additional data file representative phylogenetic tree of the achaete - scute b family of bhlh proteins click here for additional data file representative phylogenetic tree of the ahr family of bhlh proteins click here for additional data file representative phylogenetic tree of the arnt family of bhlh proteins click here for additional data file representative phylogenetic tree of the atonal family of bhlh proteins click here for additional data file representative phylogenetic tree of the beta3, mist, and oligo families of bhlh proteins click here for additional data file representative phylogenetic tree of the bmal family of bhlh proteins click here for additional data file representative phylogenetic tree of the clock family of bhlh proteins click here for additional data file representative phylogenetic tree of the e12/e47 family of bhlh proteins click here for additional data file representative phylogenetic tree of the extramacrochaete family of bhlh proteins click here for additional data file representative phylogenetic tree of the enhancer of split family of bhlh proteins click here for additional data file representative phylogenetic tree of the fig alpha family of bhlh proteins click here for additional data file representative phylogenetic tree of the hairy family of bhlh proteins click here for additional data file representative phylogenetic tree of the hand family of bhlh proteins click here for additional data file representative phylogenetic tree of the hey family of bhlh proteins click here for additional data file representative phylogenetic tree of the mad and mnt families of bhlh proteins click here for additional data file representative phylogenetic tree of the max family of bhlh proteins click here for additional data file representative phylogenetic tree of the mesp family of bhlh proteins click here for additional data file representative phylogenetic tree of the mitf family of bhlh proteins click here for additional data file representative phylogenetic tree of the myc family of bhlh proteins click here for additional data file representative phylogenetic tree of the myod family of bhlh proteins click here for additional data file representative phylogenetic tree of the myor family of bhlh proteins click here for additional data file representative phylogenetic tree of the net family of bhlh proteins click here for additional data file representative phylogenetic tree of the neurod family of bhlh proteins click here for additional data file representative phylogenetic tree of the neurogenin family of bhlh proteins click here for additional data file representative phylogenetic tree of the nscl family of bhlh proteins click here for additional data file representative phylogenetic tree of the paraxis family of bhlh proteins click here for additional data file representative phylogenetic tree of the ptf a and b families of bhlh proteins click here for additional data file representative phylogenetic tree of the scl family of bhlh proteins click here for additional data file representative phylogenetic tree of the src family of bhlh proteins click here for additional data file representative phylogenetic tree of the srebp family of bhlh proteins click here for additional data file representative phylogenetic tree of the tf4 and mlx families of bhlh proteins click here for additional data file representative phylogenetic tree of the trh, hif, and sim families of bhlh proteins click here for additional data file representative phylogenetic tree of the twist family of bhlh proteins click here for additional data file representative phylogenetic tree of the usf family of bhlh proteins click here for additional data file we are grateful to lionel christiaen who made us aware of the takifugu and ciona genome projects, and to marc colet and robert herzog for comments on the manuscript . This work has been supported by the federal office for scientific, technical and cultural affairs (v.l .) And the centre national de la recherche scientifique, the institut franais de la biodiversit, and the universit paris - sud (m.v . ).
Stainless steel alloys have remained the material of choice despite the emergence of the more recent titanium, composite and polycarbonate orthodontic brackets . Stainless steel alloy contains 8%-12% nickel, 17%-22% chromium and other elements such as copper, iron molybdenum, manganese, silicon and sulfur[3 - 5] in the oral environment, orthodontic brackets are subjected to mechanical and chemical damaging which results in susceptibility to corrosion . Corrosion leads to loss of substance from the material, change in its structural characteristics, or loss of structural integrity . Due to the electrolytic capabilities of saliva various types of brackets are commercially available and each demonstrates a unique pattern of corrosion . In soldered brackets, this corrosion is due to the presence of dissimilar metals (i.e. The silver solder and the stainless steel), a phenomenon termed galvanic corrosion . Metal injection molding (mim) brackets are manufactured as a single unit and therefore do not demonstrate galvanic corrosion . Corrosion can have detrimental effects on the surface of stainless steel brackets due to the continuous loss of metal ions . Corrosion can increase the surface roughness of the bracket which leads to elevated friction forces between the bracket and the archwire . This increase in friction results in unfavorable distribution of forces and reduces the effectiveness of archwire guided orthodontic tooth movement. [7 - 8] moreover, by means of increased stress, the friction would further accelerate the corrosion process . The release of metal ions following the corrosion of brackets has concerned clinicians and has instigated research in this field . Among these metal ions, furthermore, direct and prolonged contact of orthodontic appliances and the resulting corrosion products have been shown to cause local pain and swelling in the adjacent tissues . Edema, gingivitis, gingival hyperplasia, perioral stomatitis, dna instability and altered cellular metabolism are among other reported side effects . Corrosion also has detrimental effects on the teeth, and permanent staining around the bracket base has been reported . Due to the wide diversity of options regarding bracket selection, orthodontists look for brackets which possess satisfactory biomechanical properties while presenting with a reasonable price . It was mentioned that corrosion has a detrimental effect on the physical properties of brackets and causes unwanted biological side effects; therefore, brackets with minimal corrosion tendencies are more desirable . The present study aimed to investigate five different brands of stainless steel brackets and compare their tendency towards corrosion by measuring ion release in an in - vitro setting . Five different brackets (dentaurum, 3 m, ortho organizer, cobas and o.r.g) were selected based on their popularity among faculty members of the orthodontic department . (table 1) ten central incisor brackets were selected from each brand . In order to simulate conventional orthodontic treatment, 8 mm of 0.016 stainless steel archwire (dentaurum, germany) was tied in each bracket using 10 mm of 0.25 mm ligature wire (dentaurum, germany). Once the brackets were prepared, they were placed in poly - ethylene capped vials containing 10 ml of artificial saliva at a ph of 7.2 . The vials were incubated at 37c for 6 weeks and then they were subjected to thermocycling with 500 temperature cycles from 5c to 55c to simulate the effect of temperature changes in the oral cavity . The brackets were immersed in each bath for 30 seconds with 2 seconds at air temperature in - between the immersions . The details of the brackets selected for the study after thermal - cycling the solutions from the vials were analyzed to determine the amount of nickel, chromium, manganese, and iron using an inductively coupled plasma (icp) spectrometer (icp - oes; varian, vista - pro model, mulgrave, victoria, australia; 1400w applied power). For the purpose of analysis, the icp device had to be calibrated for each of the metal ions . Calibration was performed using standard stock solutions (100 mg / ml) prepared by dissolving nitrate salts of the aforementioned ions in distilled deionized water . The standard stock solutions were then diluted to render the concentrations necessary (0.1- 10 mg / ml). Once the icp device was calibrated and a standard calibration curve obtained, the samples were analyzed and the readings were recorded . Before each reading a drop of 65% nitric acid the addition of nitric acid facilitates the stabilization of the released ions by producing nitrate salts . A vial containing only the artificial saliva was used as the device blank so that the ions present in the saliva itself do not compromise the readings . Statistical analyses were performed using spss software for windows (version 11; spss inc, chicago, iii). The kruskal - wallis test was used to analyze the differences among mean ion concentrations in the 5 groups . The median levels of ion release were measured for chromium, iron, manganese and nickel using an icp device (figure 1). The overall amount of ion release was greatest in the cobas bracket, while ortho organizer and org brackets demonstrated minimal corrosion . Ion release from brackets made by 5 different manufacturers measured in milligram per liters (mg / l) the level of chromium release was highest in the cobas bracket followed by 3 m, dentaurum, org and ortho organizer . It should be noted that the difference between cobas and 3 m was statistically insignificant (p> 0.05) as was the difference between org and ortho organizer (p> 0.05). Iron release presented the same order that was observed for chromium; however, the levels were generally higher for iron especially in the cobas bracket . Considering manganese, it can be observed that the levels are higher in 3 m followed by cobas, dentaurum, org and ortho organizer . The differences between all brackets except dentaurum and org were statistically significant (p <0.05). Nickel release however was more pronounced in cobas followed by 3 m, org, ortho organizer and dentaurum . Only the cobas bracket demonstrated statistically significant differences with the other brackets (p> 0.05). From the ions that were analyzed, iron was released the most, followed by nickel, manganese and chromium (figure 2). A: total iron release, rendered by adding the median values for the analyzed brackets . B: percentage of total ion release attributed to each bracket if we consider looking at the brackets separately, iron release was found to be greater than the other ions in every bracket . Due to the importance of the biocompatibility of orthodontic armamentaria, in the present study we chose five popular brackets among orthodontists and determined their susceptibility to corrosion by measuring ion release in anin - vitro environment . The brackets and wires in this study were all manufactured from 18 - 8 stainless steel (18% chromium, 8% nickel). Based on the results of this study, we found that except for manganese, the cobas bracket had the highest level of ion release . Furthermore, we observed that among the studied brackets, iron was released more than the other ions . And in contrast with the findings of huang et al . In which nickel release was more profound . Moreover the amount of ion release in our samples was higher compared to similar studies. [3,16 - 17] this difference in ion release may be due to the fact that we chose to tie a segment of archwire to the bracket to better simulate conventional orthodontic treatment . First, by increasing the amount of alloy available to be subjected to corrosion and second, the potential for galvanic corrosion increases since the alloy for the archwire is different from the alloy used in the bracket . Another reason for greater levels of ion release may be due to the technique used for detection of ions . The values obtained in the present study closely match the values presented by kuhta et al . Who elected to use an icp device for ion detection . The results of the present study indicated that soldered brackets do not necessarily demonstrate less resistance to corrosion when compared to brackets made by metal injection molding (mim). When the org (soldered) and the dentaurum (mim) bracket are compared we observed that, except for iron which was released more by the dentaurum bracket, the other ions were not significantly different . While it has been argued that soldered brackets present a tendency towards galvanic corrosion, mim brackets have increased porosity rendering them susceptible to another kind of chemical breakdown, termed pitting corrosion . This may be the reason why similar studies also found no measurable advantage regarding the utilization of mim brackets . The presence of an archwire may be a confounding factor which may induce galvanic corrosion to mim brackets that cancels out any possible differences . The results of the present study indicated that iron was released more than the other ions, followed by nickel . While this was the overall pattern, in the 3 m bracket, it was observed that manganese was released more than nickel . This finding is in contrast to the findings of karnam et al . Who reported that nickel was the major ion released from orthodontic brackets . The findings of this study also suggest that there were no statistically significant differences between the 3 m and dentaurum brackets in any of the ions studied . These differences could be due to study design, addition of an archwire element, testing conditions and measurement methods . It is noteworthy that in the present study, an icp - ms was used to measure the released ions . Icp - ms has shown lower detection limit compared to atomic absorption spectrometry used in similar studies . Another advantage of the icp device over atomic absorption is that it can measure several ions simultaneously without interference from other ions . This study in agreement with previous findings declares that the values of ions released from orthodontic brackets falls short of the permissible daily doses determined by the world health organization . The maximum tolerable dose of nickel, iron, manganese and chromium for a 60-kg individual is 1.2, 15, 10 and 50 - 200 mg / day, respectively . However the chronic nature of metallic exposure resulting from orthodontic treatment is a cause for concern . It has been reported that chronic exposure to metal ions can cause alterations in cell morphology and metabolism leading to inflammation and dna instability. [21 - 26] iron ions in particular have been demonstrated to cause mitochondrial dysfunction in cells with active mitochondrial activity . They have also been linked with lysosome instability which could result in cell death and apoptosis . Regarding irreversible damage to oral tissues resulting from orthodontic treatment however, hafez et al . Concluded that 6 months after the removal of the orthodontic appliance, no difference was observed between the orthodontic patients and the controls . The findings of this study are based on an in - vitro experimental design, and therefore suffer from the inadequacies related to the simulation of the in - vivo environment . A bracket in the oral environment is subjected to many kinds of chemico - thermal insults including rapid changes in ph, the intake of hot and cold beverages and mechanical challenges . While in many senses the in - vitro design is a drawback, in measuring ion release such a setup is rather useful . Not all of the ions released from orthodontic brackets are found in the oral fluids . Some of the ions are absorbed into the oral and gingival tissues, while others are ingested and could disperse into distant organs . The advantage of the in - vitro design is that it presents the total ions released from the bracket, which could better show the true effect of this phenomenon . Another drawback to this study is that we did not evaluate the effects of different wire - bracket combinations and only used a single wire for all of the brackets . Based on the findings of the present study, regarding ion release levels, the org and ortho organizer brackets were superior while dentaurum and 3 m did not demonstrate any significant differences.
It is well known that the stress transfer between an implant device and a bone is not homogeneous when young's moduli of the implant device and the bone are different; this is defined as stress shielding . In such conditions, bone atrophy occurs and leads to the loosening of the implant and refracturing of the bone . Therefore, it is desirable if the stiffness (young's modulus) is not too high compared to that of bone . Implant devices are mainly made from metallic biomaterials such as stainless steels, co - cr alloys, and titanium (ti) and its alloys . Young's moduli of these metallic biomaterials are generally much greater than that of the bone . Young's moduli of the most widely used stainless steel for implant devices, sus316l stainless steel and co - cr alloys, are around 180 gpa and 210 gpa, respectively . Young's moduli of ti (pure titanium) and its alloys are generally smaller than those of stainless steels and co - cr alloys . For example, ti and its alloy, ti-6al-4v eli, which are widely used for constructing implant devices, have a young's modulus of around 110 gpa . However, this value is still higher than that of the bone, that is, 1030 gpa . Ti alloys are grouped into -, (+)-, and -type alloys . Young's moduli of - and (+)-type titanium alloys such as ti and ti-6al-4v eli are higher than those of -type titanium alloys . Therefore, -type titanium alloys are advantageous for the development of titanium alloys with low young's modulus for biomedical applications . Researchers have been focusing on reducing young's moduli of -type titanium alloys for use in biomedical applications because they are composed of toxicity- and allergy - free elements . A number of -type titanium alloys mainly composed of toxicity- and allergy - free elements and with low young's moduli have been developed or are still being developed . Their young's moduli are approximately below 80 gpa in solution - treated conditions . Young's modulus of a material can be different depending on the type of measurement methods used, such as tensile tests, three - point bending tests, and free resonance methods . The lowest value of young's modulus reported for the polycrystal -type titanium alloy, ti-35nb-4sn, or ti-24nb-4zr-7.9sn, subjected to severe cold working, is around 40 gpa . The authors have also developed a -type titanium alloy, ti-29nb-13ta-4.6zr, referred to as tntz, that is composed of toxicity- and allergy - free elements and that has a low young's modulus . Young's modulus of tntz subjected to solution treatment and measured by a resonance method has been found to be around 60 gpa . This value is lowered to around 55 gpa by severe working such as severe cold rolling and cold swaging . The strength as well as young's modulus of titanium alloys is a very important factor for their long - term use in implants for biomedical applications . In particular, dynamic strength such as fatigue strength is highly important . Developing the fatigue strength and simultaneously lowering young's modulus is somewhat difficult because they are opposite natures when the fatigue strength of -type titanium alloys used in biomedical applications with maintaining the young's modulus as low as possible is currently being developed . Concerning young's modulus, the level of the value of young's modulus, which is effective to prevent the stress shielding between the implant made of the low - young's modulus -type titanium alloy and bone should be proved . The effect of young's modulus on bone atrophy has previously been investigated using implants made of titanium alloys with different young's moduli . This paper chiefly describes the simultaneous improvement of the dynamic strength and lowering young's modulus of the -type titanium alloy, tntz . The effect of young's modulus on bone atrophy and bone remodeling has also been discussed in this paper . Improving static strength such as tensile strength can be achieved by employing strengthening mechanisms such as work hardening, grain refinement strengthening, precipitation strengthening, and dispersion strengthening . One of the best ways to increase tensile strength while maintaining a young's modulus low is to generally introduce many dislocations by ordinal severe cold working such as severe cold rolling and swaging and special severe cold working such as high pressure torsion (hpt), accumulative roll - bonding (arb), and equal channel angular pressing (ecap). Figure 1 shows the relationships between the tensile properties and working ratio of tntz subjected to cold working by general cold rolling or swaging . The relationships between young's modulus and working ratio of tntz subjected to severe cold working by general severe cold rolling or swaging are shown in figure 2 . The tensile strength and 0.2% proof stress increase with an increase in the working ratio and become almost equal to those of ti-6al-4v eli (having tensile strength of around 800 mpa) with good elongation of tntz subjected to both cold rolling and cold swaging . Young's modulus of tntz subjected to cold rolling or cold swaging is almost constant with increasing working ratio . Young's modulus of tntz subjected to cold rolling tends to decrease when the working ratio is high because the trend of the formation of the texture becomes significant . Figure 3 shows the tensile properties of tntz subjected to hpt as a function of the number of rotations, n. it also shows the tensile properties of tntz subjected to solution treatment and severe cold rolling . The tensile strength under hpt increases significantly with an increase in the number of rotations, in contrast, the elongation decreases with an increase in the number of rotations . Young's modulus of tntz subjected to hpt is almost constant with increasing the number of rotations although it decreases a little with increasing the rotation number . As shown in figure 4, the dynamic strength, that is, fatigue strength of severe cold worked tntz is not high as compared to that of tntz subjected to solution treatment . The fatigue strength is significantly improved by conducting aging treatment after solution treatment or thermomechanical processing including severe cold working and aging treatment . The phase or the phase precipitates in the matrix phase by aging treatment . Therefore, the fatigue strength can be significantly improved by precipitation strengthening caused by the precipitation of the or phase . However, as seen in figure 5, young's modulus increases by the precipitation of the or phase because young's moduli of these phases are much higher than that of the matrix phase . The phase precipitation significantly increases the strength and young's modulus of the alloy as compared to the phase precipitation although the phase enhances its brittleness . Therefore, a small amount of phase precipitation is expected to improve the fatigue strength of tntz while maintaining its young's modulus fairly low . For this purpose, short - time aging at a fairly low temperature, which enhances phase precipitation by a small amount, is effective . Figure 6 shows young's moduli of tntz subjected to solution treatment (st), severe cold rolling (cr), and aging after solution treatment at a temperature of 573 k(at) as a function of the aging time . Up to an aging time of around 10.8 ks, young's modulus is below 80 gpa, which is a tentative target value for low young's modulus . Figure 7 shows fatigue properties (s - n curves) of tntz subjected to solution treatment (st), severe cold rolling (cr), and aging for 3.6 ks (at3.6) and 10.8 ks (at10.8) at a temperature of 573 k. the fatigue strength of tntz is improved by aging treatment for 10.8 ks (at10.8), whereas young's modulus is lower than 80 gpa . Thus, employing the proper precipitation method for the phase, that is, short - time aging at a relatively low temperature improves the fatigue strength of tntz while keeping young's modulus low . The addition of a small amount of ceramic particles in the matrix is also expected to improve the fatigue strength of -type titanium alloys while maintaining low young's modulus . Figure 8 shows young's modulus of tntz with y2o3 additions subjected to severe cold rolling as a function of y concentration . Young's modulus is nearly constant at around 60 gpa with increasing y concentration . Figure 9 shows the s - n curves of tntz with 0.2 mass% and 0.5 mass% (tntz-0.2ycr and 0.5ycr, resp .) Subjected to severe cold rolling after solution treatment and the s - n curves of tntz subjected to solution treatment or cold rolling after solution treatment . Relationships between tensile strength and elongation of tntz added with different amounts of y2o3 subjected to severe cold rolling (0.05ycr: tntz-0.05ycr, 0.1ycr: tntz-0.1ycr, 0.2ycr: tntz-0.2ycr, 0.5ycr: tntz-0.5ycr, and 1.0ycr: tntz-1.0ycr) and tntz subjected to severe cold rolling after solution treatment (tntcr) are shown in figure 10 . The balance of the tensile strength and elongation of tntz with y2o3 additions is excellent . While using low - modulus titanium alloys, some surgeons specializing in spinal diseases, such as scoliosis, spondylolisthesis, and spine fracture, pointed out that the amount of spring back in the implant rods should be small so that the implant offers better handling ability during surgeries . The implant rods undergo bending when they are manually handled by surgeons within the small space inside the patient's body for in situ spine contouring . It is considered that the amount of spring back depends on both the strength and young's modulus of the implant rod . If two implant rods having the same strength but with different young's moduli are used, the implant rod having lower young's modulus shows greater spring back . Implant rods made of low modulus titanium alloys exhibit lower young's modulus, resulting in greater spring back . Thus, low young's modulus, which is one of the key features of -type titanium alloys such as tntz as a metallic biomaterial, is obviously a desirable property for patients but becomes an undesirable property for surgeons . Titanium alloys, which satisfy the requirements of both surgeons and patients with regard to young's modulus of the implant rod, are currently being developed . The amount of spring back is considered to be small for the alloy having higher young's modulus as compared to that of one having low - young's modulus as schematically shown in figure 11 . Therefore, the low - young's modulus -type titanium alloy, whose young's modulus varies and becomes high only at the deformed part, is considered to reduce spring back and satisfy the low - young's modulus condition as well . This concept is called self - adjustment of young's modulus . In general, the young's modulus of metals and alloys does not drastically change by deformation . However, in the case of certain metastable -type titanium alloys, nonequilibrium phases such as,, and phases appear in the matrix during deformation . If young's modulus of the deformation - induced phase is higher than that of the original phase, young's modulus of only the deformed part of the implant rod increases, whereas that of the nondeformed part remains low . In orthopedic operations performed for the treatment of spinal diseases, the implant rod is bent by the surgeons so that it corresponds to the curvature of the spine . Therefore, if a suitable titanium alloy is employed as the implant rod material, spring back can be suppressed by the deformation - induced phase transformation that occurs during bending in the course of operation, while low young's modulus can be retained for patients . In general, young's modulus of phase is much greater than those of the,,, and phase . Among these phases, the,, and phase one of the candidate alloys with self - adjustable young's modulus for biomedical applications has been reported to be ti-12cr . Figure 12 shows young's moduli of ti-12cr subjected to solution treatment (ti-12cr - st) and severe cold rolling (ti-12cr - cr) along with those of tntz subjected to solution treatment (tntz - st) and severe cold rolling (tntz - cr). Ti-12cr - st exhibits low young's modulus of <70 gpa; this value is comparable to that of tntz - st, which has been developed as a biomedical -type titanium alloy having low young's modulus . Tntz - cr also shows low young's modulus almost equal to that of tntz - st . However, in the case of ti-12cr, young's modulus increases by cold rolling and that of ti-12cr - cr is> 80 gpa . The deformation - induced phase was detected in ti-12cr, but no induced phase was detected in tntz . Therefore, the increase in young's modulus of ti-12cr is probably the deformation - induced phase transformation . Figure 13 shows the tensile properties of ti-12cr - st, ti-12cr - cr, tntz - st, and tntz - cr . The tensile strengths of both ti-12cr - st and tntz - st show an increase, but the elongation due to cold rolling tends to decrease . Further, the tensile strengths of ti-12cr - st and ti-12cr - cr can be higher than those of tntz - st and tntz - cr, respectively . Moreover, the elongations of ti-12cr - st and ti-12cr - cr are> 10% and ~10%, respectively . High strength is an essential requirement from the viewpoint of practical application, although such high strength could lead to undesirable spring back . Therefore, the fundamental composition of ti-12cr makes it one of the preferred candidates for use in spinal fixation devices as a biomedical titanium alloy with the ability to self - adjust its young's modulus . In the case of some types of internal fixation devices implanted into the bone marrow such as femoral, tibia, and humeral marrow, in the case of screws used for bone plate fixation, and in the case of implants used for children, which otherwise would grow into the bone, it is essential to remove the internal fixation device after surgery owing to certain specific indications; these indications include significant local symptoms such as palpable hardware, wound dehiscence / exposure of hardware, or athletes returning to contact sport [16, 17]. The assimilation of removable internal fixation device with the bone due to precipitation of calcium phosphate might cause refracture of the bone during the removal of the fixation device . Therefore, in these cases, it is essential to prevent the adhesion of the alloys with the bone tissues . Hence, considering this requirement, it is essential to inhibit the precipitation of calcium phosphate . It is reported that zr, which is nontoxic and allergy - free element, has the ability to prevent precipitation of calcium phosphate and ti alloys with zr contents exceeding 25 mass% prevent the formation of calcium phosphate, which is the main component of human bones . Thus, ti-30zr - mo has been proposed as low young's modulus titanium base biomaterials for use in removable implants . Figure 14 shows young's moduli of ti-30zr - xmo alloys subjected to solution treatment and those of the alloys considered for comparison . Young's modulus of ti-30zr - xmo is lower than that of the alloys considered for comparison except tntz . Young's modulus of 6mo shows a minimum value of around 60 gpa, and tntz also shows low young's modulus . In orthopedic applications, ideal biomedical implant materials are required to have high strength and low young's modulus . The elastic admissible strain, defined as the strength - to - modulus ratio, is a useful parameter considered in orthopedic applications . The higher the elastic admissible strain is, the more suitable are the materials for such applications . Figure 15 shows the distribution of the as - solutionized ti-30zr - xmo alloys and the alloys considered for comparison in the plot of elastic admissible strain against elongation . 6mo and 7mo exhibit larger elongation and higher elastic admissible strain than those of the other ti-30zr - xmo and sus316l, cp ti, ti64 eli, and tntz . Figure 16 shows the density of the cell cultured in 7mo and the alloys considered for comparison, for 24 h. 7mo has the highest value of cell density . Therefore, 6mo and 7mo show promising potential to be new candidates for use in biomedical applications . It is very important to prove that the implant has young's modulus similar to that of the bone, which will inhibit bone atrophy and good bone remodeling [1, 4]. Of course, the geometry of implant is another factor to control young's modulus . Studies have been conducted on the implantation of intramedullary rods and bone plates made of low - young's modulus -type titanium alloy (tntz), conventional practical (+)-type titanium alloy (ti-6al-4v eli), and conventional stainless steel (sus316l) into fracture models made into the tibiae of rabbits [8, 22]. Young's moduli of tntz, ti-6al-4v eli, and sus316l stainless steel used for intramedullary rods, which were measured by three - point bending tests, were 58, 108, and 161 gpa, respectively . In both cases, the implantation of intramedullary rod and the implantation of bone plate, bone atrophy, and bone remodeling have been reported to be the least and the best, respectively, for tntz . Figure 17 [8, 22] shows the x - ray photographs of the fracture models implanted with intramedullary rods made of tntz and sus316l stainless steel at 24 weeks after implantation . Bone atrophy can be observed at the upper rear portion of the tibia for the intramedullary rod made of sus316l stainless steel, but no bone atrophy is seen for the intramedullary rod made of tntz . Large bone formation can be observed at the front part of the tibia for the intramedullary rod made of sus316l stainless steel, but very small bone formation can be observed at the front part of the tibia for the intramedullary rod made of tntz . Therefore, low young's modulus is effective in inhibiting bone atrophy and leads to excellent bone remodeling . Further study on the effect of young's modulus on the bone remodeling has been done by implanting bone plates made of tntz, ti-6al-4v eli, and sus316l stainless steel into the fracture models made in tibiae of rabbits . Only for the case of the bone plate made of tntz, the increase in the diameter of the tibia and the double - wall structure in the intramedullary bone tissue has been reported to be observed as shown in figure 18 . For figure 18, the inner wall bone structure is the original cortical bone, namely, the remained old cortical bone and the outer wall bone structure is newly formed one . This is the possible result of the bone remodeling with the low - young's modulus bone plate . Furthermore, it is necessary to understand the level of young's modulus, which is effective for inhibiting bone atrophy and enhancing bone remodeling . Figure 19 shows the profiles of the extracted bone plates made of tntz subjected to solution treatment (tntz - st), tntz subjected to aging after solution treatment (tntz - at), and sus316 l stainless steel (sus316l) attached to the tibiae of rabbits at 52 weeks after implantation . Young's moduli of tntz - st, tntz - at, and sus316l, which were measured by three - point bending tests, were 58, 78, and 161 gpa, respectively . The upper and side surfaces of each bone plate made are covered by newly formed bone, but a fairly large amount of newly formed bone can also be observed on the heads of the screws made of tntz - st and tntz - at, which are surrounded by circle marks . Figure 20 shows the optical micrographs of the bone state beneath the bone plates made of tntz - st, tntz - at, and sus316l . Bone atrophy can be observed in all cases but becomes more distinct with increasing young's modulus; it is the highest for sus316l, followed by tntz - at and tntz - st . It is expected that titanium alloy with even lower young's modulus will be advantageous in inhibiting bone atrophy and lead to much better bone remodeling . Titanium base alloys with low young's modulus have been proved to be effective for inhibiting bone atrophy and enhancing bone remodeling by conducting animal tests in rabbits . Therefore, low young's modulus titanium alloys are expected to be useful in practical applications such as implant devices used for replacing failed hard tissue . However, considering the ease of the operation, other properties such as small spring back, which is effective in maintaining the bending shape of the implant in the body, and low yielding stress and high ultimate strength, which lead to ease in achieving permanent deformation of the implant in the narrow space in the body, are also important . Metallic biomaterials, which satisfy the demands of both patients and surgeons, are highly required.
, neurological side effects of carcineurin inhibitor are becoming controversial recently . The neurological change related to tacrolimus was first presented as posterior reversible leukoencephalopathy in 1996 . Until now, there have been many clinical symptoms reported and they are referred to various names including posterior reversible encephalopathy syndrome . We report an 11-year - old girl's reversible cerebral infarction and cerebral vascular stenosis after using tacrolimus for her kidney transplantation . An 11-year - old girl with end - stage renal disease had underwent a transplantation of kidney from her mother . Pre - transplant donor - specific t- and b - cell cross - matches were negative . Initial immunosuppressive treatment consisted of tacrolimus (0.2 ng / kg / day orally, target level 15 to 20 ng / ml), mycophenolate mofetil (1,000 mg twice a day orally), and methylprednisolone (25 mg twice a day orally). Renal function was quite good after kidney transplantation . On post - operative day 12, hypertension (160/100), headache, and left motor weakness (grade i) suddenly occurred . The brain - magnetic resonance imaging (mri) and magnetic resonance angiography (mra) findings showed acute cerebral infarction of subcortical white matter of the right hemisphere and multiple stenosis of both anterior cerebral artery (aca) and middle cerebral artery (mca) (fig . Conservative treatment was done for several days and tacrolimus continued with dosage adjusted to maintain a serum level of 5 to 10 ng / ml . The repeated brain - mri and mra scaning the findings showed newly developed acute cerebral infarction on the subcortical white matter of the left hemisphere, cortex of the left parietal lobe and mildly improving status of stenosis in both aca and mca (fig . 2). This neurological change seems to be acute cerebral infarction due to cerebral vascular stenosis caused by either vasculitis or vascular spasm, which is highly suspected to be the consequence of tacrolimus . Afterwards, neurologic symptoms improved and follow - up (post - operative day #29) brain mri and mra findings showed an improving status of multifocal acute infarction and no stenosis of either aca or mca (fig . In 1996, hinchey et al . Described a syndrome of acute but reversible clinical findings including headache, mental status alteration, seizures, hypertension, and acute visual changes associated with abnormalities seen on mri of symmetric white matter lesions, usually in bilateral parietal and occipital lobes . Although it was initially thought to affect the subcortical white matter only, additional studies supported by improved radiologic imaging modalities have revealed that the cortical gray matter may also be involved . The term " posterior reversible encephalopathy syndrome (pres) " proposed by casey et al . Is widely used recently because it expresses its clinical manifestation and radiologic findings appro priately . There have been many reports of this syndrome in the literature since its initial definition . As pres for example, reports have linked pres to hypertension, immunosuppressive / chemotherapeutic agents, eclampsia, porphyria, and renaldysfunction . The incidence of neurotoxicity, which is one of the major adverse events of calcineurin inhibitors, was higher in patients receiving tacrolimus rather than cyclosporine . As both sensory and motor functions may be adversely affected, patients thus present with a wide range of neurological and psychiatric disorders . Severe symptoms could manifest as psychoses, hallucinations, cortical blindness, seizures, cerebellar ataxia, motor weakness, or pres . Fluid - attenuated inversion recovery is the most sensitive sequence for recognition of cortical and subcortical edema in pres where hyperintense signal alterations are more prevalent than in conventional sequences . This girl showed slightly different radiologic findings from those of typical pres what causes vasogenic edema with vasculopathy, such as vasoconstriction and vasodilation . Thus, the neurological complication of this patient seems to have been caused by the use of tacrolimus . Pres induced by tacrolimus can usually be diagnosed on the basis of a characteristic clinical and radiographic pattern and is usually reversible by reducing the dosage or with holding the drug for a few days . Therefore, careful examination of patients receiving immunosuppressive agents was needed to discover this uncommon but good prognostic complication.
Morphine and codeine are naturally occurring alkaloids in opioid plants, have long been used as a drug, and are also abused . While the presence of illicit drugs or their metabolites in urine is an evidence of intake, their concentrations in blood are expected to correlate with their effects on the central nervous system . Codeine is a potent -opioid receptor agonist which is used for the treatment of adult cough . Simultaneously, there have been athletes in sports competitions who use a larger dose in order to improve performance . This practice is contrary to the principle of fair competition and also harmful to the health of the athletes' body . Heroin as one of the most widely abused drug, rapidly metabolized to 6-monoacetylmorphine (6-mam) once inside the human body . This specific heroin metabolite 6-mam is detected at a higher concentration usually within 2 to 4 hours, and after six hours, has not been detected in the urine . The absence of 6-mam in urine, however, morphine is both a well - known pharmaceutical agent and an important metabolite of codeine and heroin which have relatively long a detection time . Morphine and codeine analysis of urine is used in forensic toxicology to study drug addiction . There are numerous papers published about the simultaneous determination of morphine and codeine in human fluids, including the micellar electrokinetic chromatography (mekc) method, disposable pipette extraction (dpx) method, high performance liquid chromatography method, liquid chromatography - mass spectrometry, and liquid chromatography / triple quadrupole tandem mass spectrometry (lc / ms / ms) method [68]. Several gas chromatography - mass spectrometry (gc - ms) methods have been developed for the analysis of codeine, morphine, or other opiates . Much attention has been directed to the confirmation of morphine and codeine in urine by gc - ms . A few methods have been developed specifically for the analysis of 6-acetylmorphine (6-am) with morphine and codeine because all three drugs are often present after heroin use . Assays of morphine and codeine by gc - ms are capable of high sensitivity, specificity, and selectivity . Gc - ms is superior to other analytical methods which provide important diagnostic value to study the drug abuse . The aim of this study was to establish methods and seek out more reliable identification and quantitation of morphine and codeine for detection addicts sample . Although through in saliva is another approach; the reliability of saliva analysis is limited by the fact that analyte levels, and even the availability of required sample volume, are again dependent on several physiological factors, nutrition and fluid intake, while the biological effects of the consumed illicit substance may also be a significant factor . The identification of chronic consumers or the late verification of a single intake is feasible using hair as a matrix, but it is not suitable for the early verification of consumption . Urine is a preferable matrix for analytical purposes in comparison with saliva because of the minimal discomfort caused to sampled individuals, so it is widely available . The simple and effective ethyl acetate extraction was employed in our work, and ethyl acetate was adopted because of its high extraction efficiency . Propionic anhydride was chosen as the derivatization reagent because it exhibited better effect than acetic anhydride or trifluoroacetic acid anhydride, which could provide preferable stability, and the disadvantage of acetyl derivatives indistinguishable from morphine and the 6-am can be avoided . . Evaluated propionic anhydride, mbtfa, hfaa, and bstfa for gc - ms analysis of 6-am . They concluded that propionic anhydride gave accurate, precise, and sensitive results while providing compatibility with other methods on the same gc - ms instrument . Residual derivatization reagent in the injector will react with drugs in other methods not intended for derivatization . The derivatization procedure accommodates the analysis of opioids commonly requiring gc - ms confirmation in urine . Concentrations of the analytes in the samples were expected to be smaller than the low end of the therapeutic range (25 ng / ml), which highlighted the importance of efforts aimed at increasing the sensitivity of detection . The relative standard deviation of the retention parameters of the target compound was required not to exceed 5% relative standard deviation . Morphine [10 g / ml in methanol] and codeine [10 g / ml in methanol] solutions were obtained from the institute of forensic science under the ministry of justice (shanghai, china). Sodium hydroxide (purity> 98.0%) was purchased from sigma - aldrich trading co (shanghai, p.r ., china), and ethyl acetate (purity> 98.0%) was purchased from siyou chemical reagent co., ltd (tianjin, china), and propionic acid anhydride (purity> 98.0%) was purchased from sinopharm chemical reagent co., ltd (beijing, china). Pyridine was from shenbo chemical co., ltd (shanghai, china). While methanol was obtained from siyou chemical reagent co., ltd (tianjin, china). Ultrapure water was prepared by a milli - q purification system from millipore (bedford, usa). Analysis was performed on an agilent 6890n gas chromatograph (gc) coupled with an agilent 5975b mass spectrometer (ms, agilent technologies, wilmington, de, usa). The capillary column used was a hp-1ms [30 m 0.25 mm, 0.25 m]. Helium was the carrier gas at a flow rate of 1.0 ml / min . The temperature program was: initial temperature, 100c for 1.5 min; ramp at 25c / min to 280c and held for 15 min; injection temperature, 250c; and transfer line, 280c . Electron impact ionization was performed at 70 ev energy and at a 230c ion source temperature . Sim mode was applied to quantify analyzes using target ions at m / z 341, 397, and 268 for morphine propionyl compound and m / z 229, 355, and 282 for codeine propionyl compound (figure 1). The primary standard stock solutions of morphine (100 g / ml) and codeine (100 g / ml) were separately prepared in 10 ml volumetric flasks with urine; 10% naoh was added dropwise until ph 9.09.2 was reached, and 1.0 ml of borax buffer solution was added . To this, 3 ml of extraction solvent (ethyl acetate) was added and vortex - mixed on a vortexer for 2.0 min, followed by centrifugation at 3000 r / min for 5 min . The supernatant organic layer was transferred into a 5 ml glass test tube and dried under air stream at 60c . The dried residue was reconstituted in 50 l of propionic anhydride and 20 l of pyridine . All reagents were vortex - mixed, then heating for 3 min at 80c and dried under air stream at 60c . The dried residue was reconstituted in 50 l of methanol, and 1 l of this solution was injected into gc - ms . Specificity was determined by analysis of blank urine, without addition of morphine and codeine to determine possible interference with these compounds . To evaluate the linearity, the calibration curves were generated using the analyte peak area by linear regression on three consecutive days . The lloq was estimated in the process of calibration curve construction and was defined as the lowest concentration for which precision (rsd) was better than 20% . Qc samples at three concentration levels (50, 200, and 1600 ng / ml for morphine and codeine) were analyzed to assess the accuracy and precision of the method . Again, the assays were performed on three separate days, and on each day six replicates of the qc samples at each concentration level were analyzed . The assay precision for each qc level was determined as the relative standard deviation (rsd) of the measured concentrations . The intra- and interday precisions were required to be below 15%, and the accuracy was required to be within 15% . Stability in urine was assessed in the autosampler at room temperature for 12 h. the effect of three freeze - thaw cycles was also investigated . Figure 2 shows the typical chromatograms of a blank urine sample spiked with morphine and codeine . No interfering endogenous substances were observed at the retention times of the morphine and codeine . Calibration curves for morphine and codeine were generated by linear regression of peak area ratios against concentrations, respectively . The regression equation for the calibration plot were y = 2270.9c + 202.3 with r = 0.9974 for morphine, and y = 3099.0c + 31625.7 with r = 0.9958 for codeine (y is the peak area of analyte, and c is the concentration of analyte in human urine), and concentrations are in the range 252000 ng / ml for morphine and codeine, respectively . The lloq for morphine in human urine was 25 ng / ml and the precision and accuracy at lloq were 10.5% and 87.6%, respectively . The lloq for codeine in human urine was 25 ng / ml and the precision and accuracy at lloq were 13.8% and 88.9%, respectively . The precision of the method was determined by calculating rsd for qcs at three concentration levels over three validation days . Intraday precision was 12% or less and the interday precision was 13% or less at each qc level . The aforementioned results demonstrate that the values are within the acceptable range and the method is accurate and precise . The recovery of morphine and codeine was evaluated by comparing peak area ratios of extracted qc samples with those of reference qc solutions reconstituted in blank urine extracts . All the stability studies of morphine and codeine in human urine were conducted at three concentration levels (50, 200, and 1600 ng / ml for morphine and codeine) with three replicates for each concentration . The stability results showed that morphine and codeine in human urine were stable during three freeze - thaw cycles . Stability of morphine and codeine extracts in the sample solvent on autosampler was also observed over a 12 h period . A stable, selective, and sensitive gc - ms method has been developed for the simultaneous determination of codeine and its metabolite morphine in human urine . This developed method with derivatization for sample preparation was successfully applied for the determination of morphine and codeine in human urine for methodological study.
Disporopsis pernyi (hua) diels, which belongs to disporopsis genus of liliaceae family, mainly grows in south asia such as vietnam, laos, thailand, and yangtze river basin of china . It was a well - known traditional chinese medicine which has been widely used for the treatment of abnormal sweating, chronic cough, women postpartum weakness and irregular menstrual cycle, and so forth . The medical value of disporopsis genus plants has not got much attention until the beginning of 21st century . The roots of the plant are rich in bioactive substances which are possible to have considerable medicinal value and can be used for hypertension cough, inflammation, and tumor . Study suggests that a total of 4 polyphenolic compounds are found in extract including rutin, luteolin, quercetin, and betulinic acid, and the phenolic compounds contribute significantly to the antioxidant and antimicrobial activities [2, 3]. Rutin has both antihypertensive effect and antidiabetes effect [4, 5]. Luteolin which has anti - inflammatory, antibiosis, and anticancer properties has been used for relieving cough and eliminating phlegm . In addition, it has potential anti - hiv effect [6, 7]. Quercetin can be used for relieving cough and eliminating phlegm and for hypertension and hyperlipemia; also, it has neuroprotective and antiproliferative activities [8, 9]. Acid can kill human melanoma cell without hurting healthy cell and inhibit the hiv-1 infection . Additionally, recent study shows that it also has the inhibition effects of cerebroma and leukocythemia [10, 11]. The four compounds are major bioactive constitutes in the extract of disporopsis pernyi (hua) diels roots . So far, there is no report on the content of the 4 polyphenolic compounds in disporopsis pernyi (hua) diels . Uplc is a simple and quick tool for the quantitative determination of the bioactive constituents in pharmaceutical industry [1214]. As rutin, luteolin, quercetin, and betulinic acid the hplc - grade methanol and acetonitrile used were purchased from caledon (canada) and formic acid was obtained from dima company (beijing, china). The rutin, luteolin, quercetin, and betulinic acid were purchased from the national institute for the control of pharmaceutical and biological products in china . The purity of the standard compounds was 98%; their chemical structures are shown in figure 1 . Disporopsis pernyi (hua) diels were collected from songtao which is in guizhou province of china . Standard stock solutions of rutin, luteolin, quercetin, and betulinic acid were dissolved in methanol, at concentration of 1.0 mg / ml . All standard solutions were filtered through 0.22 m syringe filter . The extraction was carried out using 5.0 g of powdered roots . It was dissolved in 50 ml of 70% ethanol solution and back - flow for 60 min . After filter and rotary evaporation to no ethanol smell, 50 ml acetic ether was added and extraction was done three times . The extract and washing liquid were combined and filtered and then evaporated to dryness under reduced pressure in a rotary evaporator . The dried extract was dissolved in methanol and diluted to a 5 ml volumetric flask . The hplc system used was a shimadzu nexera x2 uplc (kyoto, japan) chromatograph equipped with a solvent delivery unit (lc-30ad), an autosampler (sil-30ac), a column oven (cto-20a), a degasser (dgu-20a5r), and a photodiode array detector (spd - m20a). Separation was conducted on a shim - pack xr - ods column (2.0 75 mm, 1.6 m; shimadzu cooperation, japan). The column temperature was set at 30c . The mobile phase consisted of water containing 0.1% formic acid (a) and acetonitrile (b). The composition of the mobile phase was 5% (b) for 02 min, 5%10% (b) for 24.5 min, 10%40% (b) for 4.511 min, 40%60% (b) for 1113 min, 60%70% (b) for 13 - 14 min, 70%80% (b) for 1416 min, 80%90% (b) for 16 - 17 min, 90% (b) for 1720 min, and it was held for 3 min and then reequilibrated to 5% (b) until the end of the analysis . The flow rate was 0.2 ml / min and the injection volume was 5 l . The detection wavelengths of all standards and samples were in the uv at 210, 254, and 280 nm . The 4 standard compounds were accurately weighed and dissolved in methanol to prepare stock solutions at a concentration of 1.0 mg / ml . Stock solutions of the compounds were serially diluted to construct calibration curves . The diluted concentrations of compounds were plotted against the peak area on the calibration curves and the linearity was measured from the correlation coefficient . Blank samples were analyzed in triplicate and the area of the noise peak was calculated as the response . The lod and loq were calculated as lod = 3.3 sd / s and loq = 10 sd / s, where sd is the standard deviation of the response and s is the slope of the calibration curve . The precision was calculated by analyzing sample extracts containing low and high concentrations of the compounds . The precision was represented by the relative standard deviation (rsd), which was calculated using the equation rsd = (standard deviation / mean) 100 . The precision was measured three times in a single day (intraday precision) and over three consecutive days (interday precision). The recovery was calculated as follows: recovery (%) = ((detected concentration initial concentration)/spiked concentration) 100 . We used pda detector in this experiment, which could select each wavelength of chromatogram . In our study, we took into account the question that most of the components we studied also have good absorption at wavelength of 350 nm and 370 nm [15, 17]. By comparing the resolution and response at different wavelength, the results showed that the resolution and response of the four components at 350 nm and 370 nm are not as good as the wavelengths 210, 254, and 280 nm we chose . Also, we chose 210 nm to detect betulinic acid for its good response . Combined with the literature reports, methanol - water, acetonitrile - water, and methanol - acetonitrile - water were examined as mobile phase as well as the type (formic acid and glacial acetic acid) and concentration (0.01%, 0.05%, and 0.1%) of the acid . A shim - pack xr - ods column was employed for the simultaneous determination of the 4 compounds, as it has been the most frequently used technique in the chemical analysis of herbal medicines by uplc . Peak resolution and shape of the compounds were considered better indicators when 0.1% formic acid was used as a modifier . Taking peak shape, degree of separation, the symmetrical factor, and other factors into consideration, acetonitrile-0.1% formic acid water solution was determined as gradient elution process . The linearity of the calibration curve was measured by the correlation coefficient (r), which ranged in value from 0.9992 to 0.9997 for each compound . The lod and loq were 0.1370.264 g / ml and 0.4560.881 g / ml, respectively (table 1). The intra- and interday precision, which were represented by the rsd values, were rsd <2.0% . The recoveries of the 4 compounds were in the range 96.2%102.6%, with rsd <2.0% (table 2). The results indicate that the developed analytical method was accurate and precise for the analysis of the 4 compounds in disporopsis pernyi (hua) diels . The method we developed was applied to determine the 4 compounds in disporopsis pernyi (hua) diels successfully . The calculated contents of the four compounds were 5.63 g / g for rutin, 2.51 g / g for luteolin, 3.87 g / g for quercetin, and 2.41 g / g for betulinic acid . The uplc method mentioned here represented an excellent technique for simultaneous determination of rutin, luteolin, quercetin, and betulinic acid in the extract of disporopsis pernyi (hua) diels roots, with good sensitivity, precision, and reproducibility . The method gives a good resolution among the four components with the analysis time (25 min). Furthermore, the method can be used as quality control of polyphenolic compounds in disporopsis pernyi (hua) diels roots and will play a reference role on the determination of polyphenolic compounds in other medicinal plants or pharmaceutical preparations.
Maple syrup urine disease (msud) is a rare inherited autosomal recessive disorder of branched - chain amino acid (bcaa) metabolism presenting with life - threatening cerebral edema and dysmyelination in affected individuals . Msud affects approximately 1 out of 180,000 infants and has a much higher prevalence in children of amish, mennonite, and jewish descent . We present a case report of a baby boy born to consanguineous married parents, who presented on the 8 postnatal day with history of poor feeding, lethargy, and tonic - clonic seizures, since 2 days . The child was born at term (39 weeks) after an uneventful pregnancy and normal vaginal delivery . The baby was apparently normal in the first week of postnatal life . On 8 day, clinical systemic examination showed hypotonia and poor neonatal reflexes . Routine biochemical evaluation including serum electrolytes: sodium 138 (133 - 145 meq / l), potassium 4.2 (3.5 - 5.3 meq / l), chloride 102 (98 - 106 meq / l), calcium 8.6 (7.6 - 10.4 mg / dl), and magnesium levels 1.6 (1.40 - 2.55 mg / dl) were within normal limits . Non - enhanced computed tomography (nect) scan of the brain showed bilateral symmetrical white matter hypodensities in the posterior limb of internal capsule, thalami, midbrain, and cerebellar white matter [figure 1]. Nect of the brain, axial images shows bilaterally symmetrical hypodensities in the posterior limb of internal capsule (blue arrow in figure 1a) and in the midbrain (blue arrow in figure 1b) with compressed ventricles and gyral swelling conventional magnetic resonance imaging (mri) showed bilateral symmetric white matter hyperintensites in the posterior limb of internal capsule, thalami, midbrain, corticospinal tracts, and cerebellar white matter on t2-weighted magnetic resonance (mr) sequence [figure 2a and b]. Dwis with corresponding adc maps demonstrate restricted diffusion in the posterior limbs of the internal capsules (blue arrows in figure 2a and 2b), pons, corticospinal tracts (blue arrow in figure 2c), and cerebellar white matter (figures 2c and 2d) diffusion - weighted imaging (dwi) showed characteristic pattern of bilateral symmetrical restricted diffusion within the myelinated areas in the posterior limb of the internal capsule, centrum semiovale, corona radiata, corticospinal tract, thalami, posterior aspect of the mid brain, pons, middle cerebellar peduncle, medulla, and cerebellar white matter, attributed to intramyelinic edema [figure 3a and b]. Axial t2-weighted mr images shows bilateral symmetrical hyperintensities in posterior limb of internal capsule (blue arrow in figure 3a), central cerebellar white matter, and in the corticospinal tracts (blue arrow in figure 3b) multivoxel proton mr spectroscopy showed the presence of bcaas and branched - chain -keto acids resonating at 0.9 - 1.0 ppm, which are seen especially during a metabolic crisis [figures 4 and 5]. Three days later tandem mass spectroscopy revealed increased levels of bcaas (valine, leucine, and isoleucine), confirming the diagnosis of msud . Multivoxel proton mr spectroscopy showed the presence of bcaas and branched - chain -keto acids resonating at 0.9 - 1.0 ppm, which are seen especially during a metabolic crisis [figures 4 and 5]. Three days later tandem mass spectroscopy revealed increased levels of bcaas (valine, leucine, and isoleucine), confirming the diagnosis of msud . It is a rare autosomal recessive disorder, associated with defects in the branched- chain ketoacid dehydrogenase complex . It is divided into four major categories: (1) classic, (2) intermediate, (3) intermittent, and (4) thiamine responsive, which carry differing symptoms and prognostic factors . The exact cause for brain injury is not clearly understood . According to a study by zinnanti et al ., they suggest two converging mechanisms of brain injury in msud including: (i) neurotransmitter deficiencies and growth restriction associated with bcaa accumulation and (ii) energy deprivation through krebs cycle disruption associated with branched - chain ketoacid accumulation . Neonates will be normal at birth, presents after disease - free interval, usually within the 4 - 7 days of life with poor feeding, vomiting, poor weight gain, and increasing lethargy . In crisis, patient's urine smells like maple syrup, secondary to the large accumulation of isoleucine . Classic appearing msud edema involving: cerebellar white matter, brain stem, globus pallidus, thalamus, cerebral peduncles, and corticospinal tracts . Dwi shows marked restriction and decreased apparent diffusion coefficient (adc) which indicates msud edema is an intracellular oedema (cytotoxic oedema). Dwi is more sensitive than conventional mri in detecting msud brain alterations and it can become a useful tool for early diagnosis and follow - up of metabolic diseases in neonates . Kilicarlsan et al ., reported six cases with dwi in which the changes in all patients were reversed with treatment without evidence of volume loss or persistent tissue damage . Acute metabolic rescue to reverse cerebral edema may require hemodialysis during acute crisis to limit neurotoxicity / damage . Metabolically appropriate diet (protein - modified) minimizes severity and prevent deficiencies of essential amino acids . If treated with low - bcaa diet and peritoneal dialysis within a few days from the onset of the symptoms, most patients survive and develop only minimal or no neurological deficits . The changes in cell osmolarity and metabolism can reverse completely after metabolic correction in metabolic decompensated msud with clinical neurological improvement . It is a rare autosomal recessive disorder, associated with defects in the branched- chain ketoacid dehydrogenase complex . It is divided into four major categories: (1) classic, (2) intermediate, (3) intermittent, and (4) thiamine responsive, which carry differing symptoms and prognostic factors . The exact cause for brain injury is not clearly understood . According to a study by zinnanti et al ., they suggest two converging mechanisms of brain injury in msud including: (i) neurotransmitter deficiencies and growth restriction associated with bcaa accumulation and (ii) energy deprivation through krebs cycle disruption associated with branched - chain ketoacid accumulation . Neonates will be normal at birth, presents after disease - free interval, usually within the 4 - 7 days of life with poor feeding, vomiting, poor weight gain, and increasing lethargy . In crisis, patient's urine smells like maple syrup, secondary to the large accumulation of isoleucine . Classic appearing msud edema involving: cerebellar white matter, brain stem, globus pallidus, thalamus, cerebral peduncles, and corticospinal tracts . Dwi shows marked restriction and decreased apparent diffusion coefficient (adc) which indicates msud edema is an intracellular oedema (cytotoxic oedema). Dwi is more sensitive than conventional mri in detecting msud brain alterations and it can become a useful tool for early diagnosis and follow - up of metabolic diseases in neonates . Kilicarlsan et al ., reported six cases with dwi in which the changes in all patients were reversed with treatment without evidence of volume loss or persistent tissue damage . Acute metabolic rescue to reverse cerebral edema may require hemodialysis during acute crisis to limit neurotoxicity / damage . Metabolically appropriate diet (protein - modified) minimizes severity and prevent deficiencies of essential amino acids . If treated with low - bcaa diet and peritoneal dialysis within a few days from the onset of the symptoms, most patients survive and develop only minimal or no neurological deficits . The changes in cell osmolarity and metabolism can reverse completely after metabolic correction in metabolic decompensated msud with clinical neurological improvement . Early imaging diagnosis of this condition can prevent the progress of neurological deficits and help in appropriate management of the disease.
Osteoarthritis is the most common joint disorder in the united states and many other industrialized countries (1). In adult individuals aged 60 years and above, the prevalence of symptomatic knee osteoarthritis is estimated at about 10% in men and 13% in women (2). However, the prevalence of osteoarthritis varies according to the definition used, the specific joints under investigation, and the characteristics of the study population (2). The age - standardized prevalence of radiographic knee osteoarthritis in adults aged 45 years or above was 19.2% among the participants in the framingham study and 27.8% in the johnston county osteoarthritis project (3). In addition, in the third national health and nutrition examination survey (nhanes iii), about 37% of participants aged 60 years and above exhibited radiographic knee osteoarthritis (2, 3). Furthermore, the age- and sex - standardized incidence rates of symptomatic hip, knee, and hand osteoarthritis were estimated at 88, 240 and 100 (per 100,000 person - years), respectively, according to a study conducted in massachusetts, usa (4). The etiology of osteoarthritis is multi - factorial and is generally considered as a product of interaction between systemic and local factors (1, 2). From this point of view, certain individuals may have a genetic predisposition to develop osteoarthritis, but this condition (osteoarthritis) will be established only if an injury to the joint takes place . Essentially, the systemic risk factors for osteoarthritis include age (1,3,5), sex (6, 7), race and ethnicity (8, 9), genetics (10, 11), congenital and other developmental factors (12, 13) and diet (14, 15). On the other hand, local factors consist of obesity (1, 2), injuries (2, 16), occupation (2, 17), physical exercise (2, 18), mechanical factors, alignment, or laxity (2, 19, 20). Yet, the relative importance of each risk factor may vary for different joints affected, for different stages of osteoarthritis, for the development (establishment) versus the progression of disease, and for radiographic compared to symptomatic disease (2). It should be noted that the number of individuals who develop osteoarthritis is expected to increase due to the aging of the population and the obesity epidemic (2). A similar aging and obesity situation is evident also in albania, a post communist country in south eastern europe which is experiencing a significant socioeconomic transition in the past twenty five years . Due to the demographic transition in the past couple of decades, in albania, the proportion of individuals aged 65 years and above has increased up to 11% (21). This proportion is going to increase further given the decline of fertility rates (21), a gradual increase in life expectancy (21, 22) and the ongoing emigration of young people (22). In addition, overweight and obesity pose a serious public health concern in view of the rapid changes in lifestyle / behavioral patterns, where processed foods are increasingly replacing traditional foods in albania (22). According to the world health statistics 2014 report, the prevalence of obesity in albania is 21.7% in males and 20.5% in females (23). The burden of musculoskeletal disorders has also increased in albania in the past two decades . Overall, the share of these disorders accounted for 8.5% of the total burden of disease in 1990, whereas in 2010 it increased up to 11.0% (22, 24). In relative terms, there was evidence of a stronger increase in females (with 3.7% increase in the proportional burden of disease) compared to males (only 2.0%) (22, 24). However, specific information on osteoarthritis including its prevalence and associated risk factors is scarce for albania . In this context, we aimed to describe the distribution of the main risk factors among primary health care users diagnosed with osteoarthritis in albania, which was the most isolated communist country in europe until 1990 . Our study involved all individuals who were diagnosed with osteoarthritis over a two - year period (from january 2013 to december 2014) in several primary health care centers in tirana, the albanian capital . Selection of the primary health care centers included in this study was based on the probability proportional to size principle . On the whole, during this two - year period, 1179 adult individuals were diagnosed with osteoarthritis (521 men aged 60.110.6 years and 658 women aged 58.19.6 years). According to the recommendations of the american college of rheumatology for the clinical diagnosis of osteoarthritis, the diagnosis of osteoarthritis was based on the following criteria (25, 26): (i) history of the disease: self - reported presence of pain, aching, stiffness, or other symptoms in the joints affected by osteoarthritis; (ii) physical examination: difficulties in flexion / extension and rotation of the joints (range of motions), tenderness, crepitations, or enlargement of the joints; (iii) laboratory findings: erythrocyte sedimentation rate, rheumatoid factor, c - reactive protein and uricemia, and; (iv) radiological findings: joint space narrowing and presence of osteophytes . Fisher s exact test was used to compare the distribution of the major risk factors (smoking, alcohol intake, body mass index, genetic factors, major trauma, weight lifting, heavy physical exercise and preexisting inflammatory diseases) among primary health care men and women diagnosed with osteoarthritis . Conversely, binary logistic regression was used to assess the sex - differences regarding the main risk factors among primary health care users diagnosed with osteoarthritis in tirana . Initially, crude (unadjusted) odds ratios (ors) and their respective 95% confidence intervals (cis) were calculated . Next, multivariable - adjusted ors and their respective 95% cis were calculated in logistic regression models controlling (adjusting) simultaneously for all the main risk factors for osteoarthritis (smoking, alcohol intake, body mass index, genetic factors, major trauma, weight lifting, heavy physical exercise and preexisting inflammatory diseases). Statistical package for social sciences (spss, version 17.0) was used for all the statistical analyses . Table 1 presents the distribution of risk factors among primary health care users diagnosed with osteoarthritis in tirana during the period 2013 - 2014 . It was substantially higher in men than in women (37.2% vs. 13.1%, respectively; p<0.001). Similarly, the prevalence of alcohol intake was remarkably higher in men compared to women (78.1% vs. 19.6%, respectively; p<0.001). There was no significant difference in the prevalence of overweight or obesity among male and female patients with osteoarthritis (p=0.111). On the other hand, the prevalence of the predisposing genetic factors was significantly higher in women compared to men (35.1% vs. 23.6%, respectively; p<0.001). Major trauma experienced in life (different types of accidents) were somehow equally distributed between men and women (p=0.195). Conversely, the proportion of men reporting weight lifting was significantly higher compared to women (17.5% vs. 7.0%, respectively; p<0.001). Likewise, the prevalence of self - reported heavy physical exercise (at work, at home, or at leisure time) was considerably higher in male patients compared to their female counterparts (36.3% vs. 18.5%, respectively; p<0.001). On the other hand, the prevalence of preexisting inflammatory conditions (including rheumatoid arthritis, ankylosing spondylitis, metabolic arthropathy, or other inflammatory diseases) was significantly higher in women than in men (11.9% vs. 8.1%, respectively; p=0.033) (table 1). Distribution of risk factors among primary health care users diagnosed with osteoarthritis in tirana during 2013 - 2014 . Table 2 presents the sex - differences with regard to selected risk factors between primary health care men and women diagnosed with osteoarthritis in tirana during the period 2013 - 2014 . In crude (unadjusted) logistic regression models, there was an inverse association of female gender with smoking (or=0.25, 95%ci=0.19 - 0.34) and especially with alcohol intake (or=0.07, 95%ci=0.05 - 0.09). On the other hand, there was evidence of a positive association between female gender and constitutional (genetic) factors (or=1.75, 95%ci=1.35 - 2.27). There was no significant association with past major trauma experiences (p=0.341), but an inverse association of female gender with weight lifting (or=0.36, 95%ci=0.24 - 0.52) and heavy physical exercise (or=0.40, 95%ci=0.31 - 0.52). Sex - differences regarding risk factors among primary health care users diagnosed with osteoarthritis in tirana during 2014 - 2015 . * odds ratios (or: women vs. men), 95% confidence intervals (95%cis) and p - values from binary logistic regression . Overall p - value and degrees of freedom (in parentheses). Upon simultaneous adjustment for all the risk factors presented in table 2, female gender was inversely associated with smoking (or=0.39, 95%ci=0.27 - 0.56), alcohol intake (or=0.08, 95%ci=0.06 - 0.10), overweight but not obesity (or=0.65, 95%ci=0.46 - 0.91 and or=0.74, 95%ci=0.46 - 1.18, respectively), weight lifting (or=0.38, 95%ci=0.22 - 0.66) and heavy physical exercise (or=0.69, 95%ci=0.46 - 1.03). On the other hand, female gender was positively related to genetic factors (or=2.17, 95%ci=1.55 - 3.04) and preexisting inflammatory diseases (or=1.53, 95%ci=0.93 - 2.53) (table 2). Our main findings relate to a negative (inverse) association of female gender with lifestyle / behavioral factors including smoking and alcohol intake, overweight (not obesity though), weight lifting and heavy physical exercise . On the other hand, female gender in this study conducted in tirana similar to our findings, women have been largely shown to be at a higher risk for osteoarthritis compared to men including also development of more severe forms of this condition (2, 6, 7). Regarding the genetic predisposition, several studies have demonstrated that osteoarthritis is inherited and this tendency may vary by joint site (2). In addition, some studies have reported an inverse (negative) association between general joint hypermobility, a lone benign trait, with hand and knee osteoarthritis and serum cartilage oligometric matrix protein levels (2). The association with obesity in our study is compatible with prior international reports (1, 2). Obesity and overweight have long been recognized as important risk factors for osteoarthritis, especially of the knee (1, 2). According to the arthritis, diet, and activity promotion trial, weight loss combined with exercise, but neither weight loss nor exercise alone, were effective in decreasing pain and improving function in obese elderly individuals with symptomatic knee osteoarthritis (27). As for the role of physical activity, there are conflicting findings in the international literature . Interestingly, the overall level of physical exercise may in itself increase the risk of osteoarthritis (2). Findings from the framingham study indicate that older individuals who engaged in high levels of physical exercise (consisting of leisure time walking and gardening) had a threefold greater risk of developing radiographic knee osteoarthritis than their sedentary counterparts (2, 18). Similar results were obtained in another study in which women who had a high lifetime level of physical exercise had also a high prevalence of hip osteoarthritis (28). Conversely, some other studies indicate that, in the absence of acute injury, recreational (but moderate) long distance running and jogging does not increase the risk of osteoarthritis (29). Our study may have some limitations, mainly related to the recruitment of participants and the information obtained through the interview . In our study, we included several primary health care centers (on a probability proportional to size basis) which are assumed to be representative of the overall primary health care facilities in tirana . Our data collection period lasted for two years and included all male and female adults diagnosed with osteoarthritis in the primary health care centers under investigation . Hence, the sample of patients with osteoarthritis included in our study is assumed to be representative of the overall primary health care users in tirana . Nonetheless, representativeness of our study sample cannot be assumed for the overall adult population of albania given the fact that we did not include in our study health centers from other districts of albania . From this point of view, our findings should be interpreted with caution and should be limited to adult primary health care users of the albanian capital namely tirana . In our study, the diagnostic criteria for osteoarthritis were based on the instruments and criteria recommended by the american college of rheumatology (25,26), which is a clear strength pointing to employment of standardized and well - validated tools for diagnosis of osteoarthritis in the albanian population . On the other hand, in our study, data on behavioral / lifestyle determinants was based on interview, which may have been subject to different information biases . Thus, male and female participants may have tended to report differently about their lifestyle characteristics . If so, we cannot entirely exclude the possibility of reporting bias for the self - reported information about lifestyle / behavioral factors including smoking, alcohol intake and physical exercise . Potential limitations aside, this study offers useful evidence about the distribution of the main risk factors for osteoarthritis in adult individuals diagnosed with osteoarthritis in albania . This information may support health professionals and decision - makers in albania for evidence - based health planning and policy formulation in order to control the toll of osteoarthritis in this transitional society.
The world health organization considers leishmaniasis as one of the most neglected tropical diseases which has received little attention and resources despite its serious impacts on both the economic developments and quality of life (who 2012). Leishmaniasis is one of the most important vector - borne disease and public health problem in iran that transmitted by sandflies to human and other animals . The main visceral foci are located in ardabil (northwest) and fars (southwest). Cutaneous leishmaniasis is the main vector - borne disease in the country with an average of more than 22,000 cases in the last decade (karimi et al . 2014), about 80% of them are zoonotic cutaneous leishmaniasis (zcl), the endemic foci of this type are in rural areas of 17 out of 31 province (yaghoobi - ershadi 2012, karimi et al . Zcl is a disease that primarily uses animals such as rodents as reservoir hosts (mirzaei et al . Humans are an accidental host that can be involved in the transmission cycle of leishmania parasites (rouhani et al . Geographically, zcl is widely distributed in africa, the middle east, central asia, and the rajasthan area of india (rouhani et al . 2014). In iran based on animal reservoir host, there are four foci of disease in our country: the first one has been located in central and northeast of iran, where rhombomys opimus and phlebotomus papatasi play important roles as reservoir and vector of the disease . The second focus of zcl is located in the west and southwest of iran, where tatera indica replaced with r. opimus as a reservoir and p. papatasi as a vector . Baluchistan province, in the southeast of iran is considered as the third focus of zcl . In this region meriones hurrianae has been approved as a natural reservoir host . From the reported evidences, it is apparent that the most rural areas of provinces in central and southern iran can be considered as the zcl focus where m. libycus is the primary and main reservoir host of the disease, while r. opimus and t. indica were absent and p. papatasi is considered as the proven vector of zcl (yaghoobi - ershadi et al . Golestan province is one of the well - known foci of zcl in iran and two counties of this province are famous: gonbad - e - kavoos and maraveh tapeh that located in north and northeastern of this province . In one study carried out in gonbad - e - kavoos county, 4% of population have acute ulcer and 78% scar and in other study in maraveh tapeh county the prevalence of acute ulcer and scare rate were 3.03% and 63.7% respectively (sofizadeh 2007, cherabin et al . Many researchers have argued that r. opimus and m. libycus are reservoir hosts of zcl and p. papatasi is the main vector of this disease in golestan province (rassi et al . The main aim of this study was to determine relation between rodents active burrows and incidence of zcl in golestan province using spatial analysis . A cross - sectional study was carried out in golestan province from february 2013 to march 2014 . Golestan province is located (3738n and 5458 e) in northeastern of iran and is bounded by caspian sea and mazandaran province in the west, semnan province in the south, north khorasan province in the east and a borderline with turkmenistan in the north (fig 1). Most parts of golestan province are plain and more than 2/3 of the plain area has arid and semiarid climates and 1/3 of others have a mild climate . The area of the province is 20437.74 km (1.3 areas of iran) with 1823117 population . There are 14 counties, 27 districts, 60 rural districts, 25 cities and 1732 villages in golestan province . Maximum and minimum of temperatures were recorded as 40.8 and 02 c respectively and the mean annual relative humidity was recorded as 74% . The total annual rainfall was 772 mm and the minimum precipitation in august and maximum in february . Location of study area, golestan province in iran three villages were randomly selected in all rural districts (60) of golestan province and the number of active rodents burrows were counted in area of one hectare . Furthermore, all villages with positive human cases in 2013 were obtained regardless of the number of cases reported from each village maps of the spatial distribution of the disease were prepared using gis software . The number of human positive cases in each rural districts of the province, as well as the incidence of disease were calculated . For calculating, the incidence rate of the disease, we used population at risk in the denominator . The relationship between the number of rodents active burrows with incidence rate and the number of positive cases of disease in rural districts were calculated using spss software version 13 and sperman test . A cross - sectional study was carried out in golestan province from february 2013 to march 2014 . Golestan province is located (3738n and 5458 e) in northeastern of iran and is bounded by caspian sea and mazandaran province in the west, semnan province in the south, north khorasan province in the east and a borderline with turkmenistan in the north (fig 1). Most parts of golestan province are plain and more than 2/3 of the plain area has arid and semiarid climates and 1/3 of others have a mild climate . The area of the province is 20437.74 km (1.3 areas of iran) with 1823117 population . There are 14 counties, 27 districts, 60 rural districts, 25 cities and 1732 villages in golestan province . Maximum and minimum of temperatures were recorded as 40.8 and 02 c respectively and the mean annual relative humidity was recorded as 74% . The total annual rainfall was 772 mm and the minimum precipitation in august and maximum in february . Three villages were randomly selected in all rural districts (60) of golestan province and the number of active rodents burrows were counted in area of one hectare . Furthermore, all villages with positive human cases in 2013 were obtained regardless of the number of cases reported from each village maps of the spatial distribution of the disease were prepared using gis software . The number of human positive cases in each rural districts of the province, as well as the incidence of disease were calculated . For calculating, the incidence rate of the disease, we used population at risk in the denominator . The relationship between the number of rodents active burrows with incidence rate and the number of positive cases of disease in rural districts were calculated using spss software version 13 and sperman test . Among all counties of golestan province, the highest incidence rate of the disease were observed in moravehtappeh (121.5/100000) and gonbad kavoos (99.3/100000) respectively (table 1). These two counties were endemic foci of cl in golestan province . In the rest of counties, demographic characters of zcl in golestan province (2013) the highest (53.8%) and lowest (11.5%) of cl cases were observed in the age groups of 14 and 1 years old respectively (table . It should be noted that, 60.2% of patients were male . In the assess the existence of rodent's active burrows in a radius of 300 meters from the villages, they were found only in the rural districts of gonbad kavoos and moraveh tappeh counties . In the rest of studied districts, there were not rodent's active burrows, or were located at a distance of one kilometer of the villages . However, the maximum number of rodent's active burrows was observed in rural district of korand in gonbad kavoos county (fig . 2). Distribution of rodent's active burrows in golestan province, 2013 the number of positive cases as well as the incidence of disease were calculated 0100/100000 and 035/100000 in the different rural districts of golestan province respectively . It must be mentioned, similar to rodent's active burrows, the most positive cases and the highest incidence of disease were found in rural district of korand in gonbad kavoos county (fig . 2, 3). Number of positive cases of zcl in golestan province, 2013 based on the statistical analysis of data, there were significant difference between the number of rodent's active burrows with the number of positive cases (0.465, p <0.001) as well as, the incidence of disease (0.470, p <0.001) (table 1). The most positive cases were found in the northern villages of plain areas in october and november (figs . 4, 6). Incidence of zcl in golestan province, 2013 trend of zcl cases in golestan province (2013) spatial distribution of zcl cases in golestan province (2013) there was a significant positive correlation between the number of rodent's active burrows and incidence of disease as well as the number of positive cases in studied areas (0.470, p <0.001). According to fig . Based on literature review of cutaneous leishmaniasis in golestan province, the causative agent of disease (zcl) was l. major and p. papatsi has been reported as the principal vector to human . Two rodents of r. opimus and m. lybicus were main and secondary reservoir host of disease respectively (rassi et al . 2008, parvizi and hedayati 2010, roshanghalb and parvizi 2012, bordbar and parvizi 2014). Since the sand flies are often active in a distance of 200 meters from its habitats (rassi and hanafi bojd 2006), therefore, the presence of rodents burrows, one kilometer from the village, are considered as one of the risk factors of disease transmission . Based on the results of our study, the active rodents burrows were only observed in rural districts of maraveh tappeh and gonbad - e kavoos counties . The previous studies have demonstrated that two species of r. opimus and m. libycus, are chosen the plains with soft soil to build their colonies under bushes and along streams (nadim et al . 2009), so, in the rural district of palizan to centroid qazanqaya village (561528.686e 375531.132n) due to the rocky nature of area as well as, in the rural districts of shalami to centroid arab qari haji village (55458.727e 374123.994n) and golidagh to centroid golidagh village (555641.944e 373841.094n) due to the mountainous nature of the region, no rodent burrows were observed . We found several active burrows with high population of rodents in both rural districts of the korand to centroid korand village (55315.842e 37 5751.47n) and atrak to centroid dashliburun village (544845.653e 373757.254n) located in gonbad - e kavoos county . In the rural districts of agh - abad to centroid agh - abad village (551426.891e 371814.929n) and sultan ali to centroid sultan ali village (5537.15e 37144.863n) in some villages, the rodents were active with low abundance . In the tamran rural district to centroid tamer ghare ghozi village (552955.568e 372951.35n) located at the kalaleh county, there was no rodent burrows at a distance of one kilometer from the villages . Also, in the rest counties of golestan province, no rodent's active burrows were observed nearby villages . Based on our study, there were high correlation between the number of rodent's active burrows with the number of positive cases (0.465, p <0.001) as well as, the incidence of disease (0.470, p <0.001). This was due to the abundance of active wild rodents as the reservoir of disease and vicinity of their colonies to human settlements . Therefore, high number of positive cases as well as high incidence of disease were observed in the rural districts of moravehtappeh (moravehtappeh county) and korand and also atrak in gonbad kavoos county . It should be noted that, two rural districts of agh - abad and sultan - ali in gonbad kavoos county were in the second rank of positive human cases . All positive cases in other rural districts of the province were imported cases, due their business trip to endemic areas . Evaluation forms for these patients revealed that in most counties, the patients had a history of traveling to endemic areas of disease including rural districts of maraveh tapeh, atrak or korand . Some patients had also at least one travel to the provinces of semnan, khorasan and isfahan . For example, the number of 20 cases of cutaneous leishmaniasis had been reported from the district of south fenderesk to centroid dar kalateh village (545738.738e 365758.499n) located at the county of ramian, they were workers of brick kilns, who had traveled to the province of semnan in the early spring and had returned to their homes in the early autumn of 2013 . The highest incidences of the disease were found in the counties of maravehtappeh and gonbad kavoos counties respectively, while the high number of positive cases were observed in the second county . Because the most population of the maraveh tappeh county (43.1%) were living in endemic areas and were at greater risk of the disease, while in the county of gonbad - e kavoos county about 15% of the population were living in endemic areas . The hightest (53.8%) and lowest (11.5%) of cl cases were observed in the age groups of> 14 and <1 respectively (table 1). Whereas most patients (53.8%) were adults over 14 years old, these results were similar to studies conducted in maraveh tapeh and gonbad - e kavoos countis in this province (cherabin et al . 2012, sofizadeh et al . 2013) and in the rural districts of damghan, and kasahan in the provinces of semnan and isfahan respectively (doroodgar et al . 2009, mohammadi azni et al . 2010). The lowest morbidity rate of disease (11.5%) was observed in age group of 1 years old . This age group had the lowest presence in outdoors and considering to hypoendemic of disease in studied areas (sofizadeh et al . Our study showed, the males are infected more than females (60.2%). Because, the males are busy in agriculture tasks outside of home during the nights of summer and attending to active peak of sand flies (sofizadeh et al . 2009) they have received more infected bites . The greatest number of cases were occurred in the months of october and november and coincided with the second peak of sand flies in september (sofizadeh 2009). Due to direct relation between the number of rodent's burrows and the incidence of disease, we propose to use different methods of reodent control as complementary measure for the control of disease . Results of current study indicated the direct relationship between the burrows of rodents and zcl prevalence . Therefore the presences of rodents as the reservoir host of disease play an important role on prevalence as well as incidence rate of disease . Considering this, control of rodents will have an important role in controlling the disease . The other point that we need to pay attention, do actions that take more than a kilometer distance between human and the rodent's life places.
To evaluate neuroradiological features of mps, including brain abnormal signal intensity and atrophy . The mucopolysaccharidoses (mps) are a group of heritable lysosomal storage disorders caused by a deficiency in glycosaminoglycan (gag)-degrading enzymes . Dermatan sulphate, heparan sulphate, keratan sulphate and chondroitin sulphate in lysosomes causes progressive damage of affected tissues, including heart, respiratory system, bones, joints and central nervous system . Seven distinct clinical types of mps have been identified and described in the literature, caused by 11 different enzymatic deficiencies . Even if each type of mps presents a rare incidence, transmission occurs in an autosomal recessive fashion, except for mps ii, which is x - linked . The ubiquitous nature of gags within the connective tissue of the body results in a wide range of clinical effects . The type of gags stored and the classification of disease depend on the specific enzyme deficiency . Table 1 outlines the latest classification, with details of the accumulating compounds and enzymes deficiencies, as well as the eponym used for each condition.table 1summary of epidemiological, pathophysiological and clinical features characterising different types of mpstypename of syndromeincidencedeficient enzymeaccumulated productsmain symptomsihurler (i h)1/100.000alpha - l - iduronidasehscorneal clouding; dysostosis multiplex; organomegaly; heart disease; mental retardation; death in childhooddscorneal clouding; stiff joints; normal intelligence and life spanscheie (i s)intermediate phenotype, between mps ih and mps ishurler - scheie (i h - s)iihunter1/250.000iduronate sulphatasehsdysostosis multiplex; organomegaly; short stature; death before 15 years (severe);survival to 20s to 60s (mild)dsiiisanfilippo a1/150.000heparan sulphamidasehsrelatively mild somaticmanifestations; hyperactivity; profound mental deteriorationsanfilippo bn - acetyl - glucosaminidasesanfilippo cacetyl - coa: alpha - glucusaminide acetyltranferasesanfilippo dn - acetylglucosamine 6-sulphataseivmorquio a1/75.000galactose-6-sulphate sulphataseksdysostosis multiplex; short stature; motor dysfunctionmorquio bchbeta - galactosidaseksvthis designation is no longer used; the phenotype, which was first classified as mps v, was found to be mps i svimaroteaux - lamy<1/250.000n - acetylgalactosamine-4-sulphatasedsdysostosis multiplex; short stature; motor dysfunction; kyphosis; heart defects; survival to teens in severe formviisly<1/250.000beta - glucuronidasehshepatomegaly; dysostosis multiplex; short stature; corneal clouding; developmental delay; wide spectrum of severity including fetal hydrops and neonatal formdsch summary of epidemiological, pathophysiological and clinical features characterising different types of mps like most genetic disorders, there is a continuous spectrum of phenotype from the very severe to the most mildly affected; many mutations are responsible for these phenotypic differences . The typical symptoms, encountered in the majority of mps, include organomegaly, dysostosis multiplex, mental retardation and developmental delay . Otological (otitis media) and respiratory (airway obstruction) problems can also be present . Other manifestations include impaired vision (corneal clouding and photophobia) and cardiovascular involvement (myocardial hypertrophy, systolic dysfunction and valve dysfunction) [57]. The course of mps is variable from very severe forms, with an average expected life span of 1 or 2 decades (neuronopathic forms in particular are usually fatal diseases) to attenuated or slowly progressing ones that allow the patient to reach adulthood [1, 3, 8]. A combination of clinical picture and analysis of urinary gags is usually performed to achieve the diagnosis of mps, even though this method cannot recognise subtypes; definitive diagnosis is usually possible through measuring enzyme activity in cultured fibroblasts or leukocytes [1, 2]. Treatment consists mainly of symptomatic and supportive care, including decompression of the craniocervical narrowing, tracheostomy insertion and corneal transplantation . In recent years the development of new therapies represented by enzyme replacement therapies, substrate inhibition therapy and haematopoietic cell transplantation have changed the treatment of these patients, with a fundamental shift in the approach from symptomatic management to therapeutic intervention; in many cases, the introduction of these new therapies has significantly improved the duration and quality of life for patients [9, 10]. In patients with mps, the most important radiological findings occur in the skeletal system with multiplex dysostosis (table 2), a complex of anomalies involving several bones [1214], dominating the clinical picture in mps iv and mps vi.table 2radiological skeletal manifestations of mps: dysostosis multiplexdysostosis multiplexskullmacrocephaly with dolicocephalyvertical frontal crestabnormal j - shaped sella turcicathickened cortical bonefacial anomalies lack of pneumatization of mastoid process cells and of paranasal cavities obtuse mandibular angle with prognatism teeth widely spacedthoraxpaddle - shaped or oar - shaped ribs (widened anteriorly and tapered posteriorly)short and thickened claviclesspinecraniovertebral junction: atlantoaxial instability, stenosis and compression of the spinal cordthoracolumbar spine: gibbusmalformations of the vertebral bodiespelvisrounded iliac wingsinferior tapering of the ileumhip dysplasia poorly developed acetabulum underdevelopment of the medial portion of the proximal femoral epiphysis coxa valgalong bonesmildly hypoplastic epiphysesproximal humeral notchinglong and narrow femoral neckkneesgenu valgumhands and feetv - shaped deformity of the hypoplastic distal ulna and radiushypoplastic and irregularly shaped carpal and tarsal bonesproximal pointed metacarpals and metatarsalsbullet - shaped phalanges radiological skeletal manifestations of mps: dysostosis multiplex the abnormal storage of gag in the mouth, nose, pharynx and larynx results in several otorhinolaryngological disorders; in view of this, otorhinolaryngological imaging findings have been described . Regarding neuroradiological features, the involvement of the central nervous system is predominant in mps i, ii, iii and mps vii [15, 16]. In this pictorial review we focus on skeletal, otorhinolaryngological and neuroradiological imaging findings encountered in patients with mps . The most important radiological findings in the axial skeleton regard the skull, thorax, spine and pelvis . The skull is often characterised by an abnormal j - shaped conformation of the sella turcica, seen on the lateral view of the cranium (fig . 1). The sella turcica is normally a saddle - shaped depression in the sphenoid bone, while in mps patients it is usually wide with long clinoid apophyses and horizontal orientation: the tuberculum sellae is flattened and the dorsum sellae is rounded, respectively, forming the straight edge and the loop of the j . This configuration may represent a normal anatomic variant and may be associated with neurofibromatosis or with a slow - growing tumour adjacent to the sella, such as an optic chiasm glioma, so the j - shaped skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) magnified views of lateral skull radiographs . Skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) the cortical bone of the skull is thickened . The premature closure of the sagittal suture is responsible for the development of macrocephaly with dolicocephaly, plus the metopic perisutural hyperostosis causes a vertical frontal crest . The most important facial anomalies are represented by the lack of pneumatization of mastoid cells and paranasal cavities; the mandible is short and broad, mandibular condyles are undeveloped and the temporomandibolar joint may exhibit limited motion; unerupted and widely spaced teeth can also be found (fig . 1). Concerning the thorax, the main abnormality concerns the ribs, which can be paddle - shaped or because of the widening of the anterior archs and of the tapering of the posterior ones . Other common modifications are small scapulae, usually with flattening of the glenoid cavities, a short sternum and short and thickened aspect of the clavicles (fig . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) thoracic abnormality . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) issues affecting the spine are extremely common (figs . 3 and 4), and mps patients may risk important complications due to compression on the spinal cord and emerging nerve roots.fig . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seenfig . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities spine abnormality . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seen deformation of the spine . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities at the craniovertebral junction level, the most important abnormalities are: odontoid process displasia - hypoplasia;atlantoaxial instability or subluxation;periodontoid tissue and ligaments thickening;spinal stenosis . Odontoid process displasia - hypoplasia; atlantoaxial instability or subluxation; periodontoid tissue and ligaments thickening; these features represent a critical aspect in mps (particularly in mps iv), because if the spinal cord is compressed, cervical myelopathy may result . Modifications of the shape of vertebral bodies are very common, resulting in flattened and rounded vertebrae (fig . 5). At the thoraco - lumbar level the vertebral body can show a deficiency in its anterosuperior corner and, as a consequence, an apparent prolongation of the anteroinferior one, resulting on the lateral x - ray in an a 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients x - ray of multiplex dysostosis of the spine . A 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients these vertebral morphologic changes may progressively evolve to gibbus deformity (fig . 6), particularly in mps i. scoliosis is usually not bad enough to require surgery; however, myelopathy can represent an indication of the need for surgical treatment .fig . Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal a 45-year - old female suffering from mps iv (morquio disease). Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal the most common radiological features in the pelvis are rounded iliac wings and inferior tapering of the ileum (fig . The alterations of the hip joint can lead to hip dysplasia because of the poor development of the acetabulum and the underdevelopment of the medial portion of the proximal femoral epiphysis . This alteration has not been shown to respond to medical therapy, so for these children surgical reconstruction is often required; the target of this treatment is the optimisation of hip mechanics .fig . Images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b d multiplex dysostosis of the pelvis . Images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b diaphyses are shortened and curved in the distal part; the epiphyses are slightly hypoplastic and thinned cortically with osteoporosis . Other features that can be found are the notching of the proximal part of the humerus, the long and narrow aspect of the femoral neck (fig . 8), and the hypoplasia of the lateral tibial hemiplate, resulting in genu valgum.fig . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal appearances of multiplex dysostosis . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal knees can also be involved, particularly in children with mps iv and mps i, developing genu valgum (fig . This condition is sometimes severe enough to require surgery . In adulthood an advanced state of arthrosis anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Diffuse cortical thinning and osteopenia are also observed (b) on mri images, the growth plate is irregularly enlarged, with multiple defects and erosions well depicted on coronal fast spin - echo proton - density - weighted images (fig coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions dysostosis of the knee . A 15-year - old female affected by mps vi . Coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions almost all forms of mps show distortion of the hand (fig . Carpal and tarsal bones are hypoplastic and irregularly shaped; the metacarpal bones are proximally pointed, shortened and thickened . The functionality of the fingers is compromised because of the bone alterations and the thickening of the subcutaneous tissues, resulting in a failure of a complete extension of the fingers with a claw hand dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) dysostosis multiplex of long bones . Dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) distal ulna and radius can be hypoplastic and have a v - shaped appearance; this oblique deformity of the terminal part of both bones results in the alteration of the carpal angle . The alterations in the skeletal anatomy, combined with an excessive gag deposition in the connective tissue, cause the constriction of the median nerve resulting in carpal tunnel syndrome . Abnormal secretions and infiltration of gag in the mouth, nose, pharynx and larynx result in several otorhinolaryngological manifestations, sometimes the first to appear; ear, nose, throat and respiratory disorders are very common . The most important otorhinolaryngological problems are rhinitis, otitis media, and upper and lower airway obstruction . Upper airway obstruction may be the consequence of macroglossia, adenotonsillar hypertrophy and chronic nasal discharge in combination with a decreased temporo - mandibular joint mobility . Eustachian tube dysfunction due to enlarged adenoids and inflammation as a consequence of abnormal storage of gag in the temporal bone are responsible for chronic otitis media and conductive hearing loss . A thickened, retracted tympanic membrane and an increased attenuation of the tympanic cavity and mastoid cells can be observed on multidetector computed tomography (mdct) images (fig . Hearing impairment can also have a sensorineural component because of gag deposition in the inner ear or central nervous system . The surgical removal of the tonsils and adenoids is often required, even though it is only a temporary solution.fig . Axial scans presented from caudal to cranial (a - b - c) demonstrate nasopharyngeal airway and eustachian tube narrowing (arrow); thickened tympanic membrane (arrowhead); opacification of mastoid cells and left middle air cavity (curved arrow). Other abnormalities, including mucosal thickening of ethmoidal air cells, poor pneumatization of sphenoidal bone and maxillar sinus cavities and impacted teeth, are also depicted . Coronal view of temporomandibular joints (d) shows flattened and deformed mandibular condyles (empty arrow) otorhinolaryngological imaging findings . Axial scans presented from caudal to cranial (a - b - c) demonstrate nasopharyngeal airway and eustachian tube narrowing (arrow); thickened tympanic membrane (arrowhead); opacification of mastoid cells and left middle air cavity (curved arrow). Other abnormalities, including mucosal thickening of ethmoidal air cells, poor pneumatization of sphenoidal bone and maxillar sinus cavities and impacted teeth, are also depicted . Coronal view of temporomandibular joints (d) shows flattened and deformed mandibular condyles (empty arrow) lower airway obstruction, due to tracheomalacia and narrowing of the tracheal lumen, may lead to pneumonia and acute respiratory insufficiency . The radiologist should know that multiplanar reconstruction of a mdct scan, usually performed in these patients for the evaluation of the atlanto - axial instability, may be an important tool to assess the status of the whole airway passage . Sometimes it may be necessary to perform a tracheostomy in order to ensure airway safety [24, 29]. The pathogenetic substrate of central nervous system involvement lies in the abnormal accumulation of mucopolysaccharides within perivascular spaces and neuro - axonal units with an adversely affected myelin turnover . On the basis of mri findings we describe the main neuroradiological features: abnormal signal intensity in the white matter and in the basal ganglia, dilatation of periventricular spaces, widening of cortical sulci, brain atrophy and enlargement of extraventricular spaces, and spinal cord compression . One of the typical neuroradiological imaging findings is represented by focal or diffuse white matter lesions, detected as areas of high signal intensity on t2-weighted sequences . Diffuse white matter lesions usually show a symmetrical periventricular distribution, although they can also be found as patchy lesions in the subcortical region (fig . They derive from the delay of myelination in young children and progressive demyelination in the course of the disease; these lesions can also reflect gliosis [32, 33]. Focal white matter lesions consist of multiple small spot - like areas isointense to the cerebrospinal fluid (csf) on fluid attenuated inversion recovery (flair), t2-weighted and t1-weighted sequences (fig . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) neuroradiological imaging findings . A 12-year - old female affected by mps vi . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . 13d) neuroradiological imaging findings . A 15-year - old female affected by mps vi . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) the corpus callosum, best depicted on sagittal images, is sometimes the only location of these lesions, although they can also be encountered in the parietal and occipital lobe, in the basal ganglia, in the thalamus and at the grey - white matter junction level [3234]. A typical imaging feature in the brain of patients with mps is the honeycomb - like appearance in the basal ganglia and thalami (fig . 15); this imaging finding has been observed in patients with mps i, ii and iiib [35, 36]. Two theories have been proposed to explain the pathogenesis of basal ganglia involvement; the first concerns the accumulation of gags in neurons and astrocytes, leading to neuronal loss and demyelination; another theory maintains that the basal ganglia appearance could be related to an increase of fluid content in the periventricular spaces.fig . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), this typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter neuroradiological imaging findings . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), this typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter the pathogenesis of enlargement of perivascular spaces is not entirely clear . Two main hypotheses have been formulated: accumulation of gag around vessels and impairment of cerebrospinal fluid reabsorption (caused by the deposit of mucopolysaccharides in the leptomeninges). In addition, in the white matter of parietal and occipital lobes, multifocal variable - sized areas of high signal intensity which does nt match that of cerebrospinal fluid may be detected on flair and t2-weighted sequences; these lesions, probably due to gliosis, tend to become extensive and confluent . Ventricular enlargement, with or without involvement of the subarachnoid spaces, is a common finding in patients with mps (figs . 13, 14, 15, 16 and 17). Communicating hydrocephalus may be the consequence of either diffuse brain atrophy or reabsorption failure of cerebrospinal fluid due to dysfunction of the arachnoid granulations . A cystic - appearing enlargement of the cerebellomedullary and/or suprasellar cisterna has been described .fig . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of lifefig . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). The brain atrophy does not show significant progression through the different mr examinations . Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment neuroradiological imaging findings . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life neuroradiological imaging findings . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). The brain atrophy does not show significant progression through the different mr examinations . Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment the atrophy of the brain, probably due to neuronal death and myelin loss, is a common feature in mps and is seen as widened subarachnoid spaces and enlargement of the cortical sulci . The neuronal death could be explained by ischaemic damages due to the progressive accumulation of gag in blood vessels; on the other hand, the neuronal death could be caused by direct intracellular storage of gag . Brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life; on the contrary, patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life . The different degree of brain atrophy has been recently evaluated for monitoring disease progression and response to therapy [15, 33]; these authors reported a classification using the method introduced by lee et al . . Brain atrophy could be: absentmild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved)moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern)severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). Mild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved) moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern) severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). A strong correlation was found between severity of brain atrophy and cognitive impairment in mps [33, 39], whereas other authors did not find the same correlation . Compression of the spinal cord most frequently occurs at the atlanto - axial (c1-c2) joint (figs . Many of these patients suffer from several craniocervical junction abnormalities due to structural defects involving the spine . The most important one is atlanto - axial subluxation, which is the result of several causes: laxity of the transverse ligament, dural thickening resulting from deposition of collagen and gags, hypoplasia or absence of the odontoid, anterior soft tissue mass of the odontoid (representing a combination of unossified fibrocartilage and reactive changes) and indentation of the posterior arch of c1 . The anomaly of the odontoid process ranges in severity from total aplasia to a triangular - shaped configuration with a loss in vertical height and a broad - based tip . Chronic subluxation of the c1-c2 level may lead to a ligamentous hypertrophy and to further narrowing in the region of the craniocervical junction with the consequence of additional cord compression . Another cause of cord compression in these patients is gibbous formation in the thoracic spine, resulting from the malformations of vertebral bodies (most common in morquio disease). Compressive cervical myelopathy is a critical problem since the involvement of the bulbar - spine junction may lead to central respiratory failure .fig . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation involvement of the craniocervical junction . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Note also the abnormal j - shaped sella (white arrow, d) involvement of the craniocervical junction: recurrence after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation mrs (fig . 20) is a non - invasive imaging technique able to provide information about tissue molecular structures . Brain application leads to revealing the concentration of brain metabolites and can be used to monitor biochemical changes in brain diseases.fig . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio mrs . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio numerous studies have suggested a contribution of mrs to mucopolysaccharidoses assessment [42, 43]. [42, 43] found a reduction of the white matter n - acetylaspartate / creatine (naa / cr) ratio in mps iva and ii proportional to cognitive indices and to disease progression [42, 43], while vedolin did not find a significant difference in the naa / cr ratio between patients with and without cognitive impairment; vedolin found instead an elevated myo - inositol / creatine (mins / cr) ratio in patients with cognitive impairment . The increase in the mins / cr ratio is interpreted as a possible marker of glial activity (fig . Takahashi et al . Measured presumptive mucopolysaccharides in white matter lesions and in white matter without lesions and compared the presumptive mps / cr ratios before and after bone marrow transplantation (bmt). They found a reduction in mucopolysaccharides in white matter without lesions after the bmt; they also found a correlation between mucopolysaccharide accumulation and neuronal damage resulting in a decreased naa / cr ratio . In summary, new evidence is needed in order to understand the real value of mrs in the evaluation of neurological involvement . Another commonly found sign in mps patients is macrocephaly, resulting from hydrocephalus combined with abnormal accumulation of incompletely broken down gags within the brain, meninges and skull . Macrocephaly is diagnosed when the circumference of the head exceeds normal values by more than two standard deviations . Other dysmorphic features, such as frontal prominence, scaphocephaly, a short neck and enlarged tongue, can be observed . Several orbital abnormalities can be found: thickening of the sclera and of the optic nerve sheath, optic canal narrowing and optic nerve atrophy (fig . Optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve is also observed finally, another imaging features reported in literature is the closed encephalocele (figs . 22 and 23); regarding this point, the presence of parenchymal / meningeal herniation at the level of the anterior or middle cranial fossa has been described as a characteristic neuroradiological feature of patients affected by mps ii . Instead of the term meningoencephalocele, which indicates the herniation of brain and/or meningeal tissue through a bone defect, the term closed cephalocele has been proposed to describe this skull abnormality because the protrusion of intracranial structures occurs without a detectable bone defect . The etiopathogenesis of this further manifestation seems to be related to an increased intracranial pressure and/or an altered bone maturation process . Ct and mri reveal a variable - sized pouch filled with brain parenchyma and cerebrospinal fluid, delimited by a bone wall and usually located in the anterior cranial fossa at the level of the lamina cribrosa as a weak area of the skull . The radiologist should look out for the presence of a closed cephalocele because it may interfere with rhinosurgery and cause epilepsy .fig . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows)fig closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma closed encephalocele in mps vi . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows) closed encephalocele in mps vi . Closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma the most important radiological findings occur in the skeletal system with multiplex dysostosis (table 2), a complex of anomalies involving several bones [1214], dominating the clinical picture in mps iv and mps vi.table 2radiological skeletal manifestations of mps: dysostosis multiplexdysostosis multiplexskullmacrocephaly with dolicocephalyvertical frontal crestabnormal j - shaped sella turcicathickened cortical bonefacial anomalies lack of pneumatization of mastoid process cells and of paranasal cavities obtuse mandibular angle with prognatism teeth widely spacedthoraxpaddle - shaped or oar - shaped ribs (widened anteriorly and tapered posteriorly)short and thickened claviclesspinecraniovertebral junction: atlantoaxial instability, stenosis and compression of the spinal cordthoracolumbar spine: gibbusmalformations of the vertebral bodiespelvisrounded iliac wingsinferior tapering of the ileumhip dysplasia poorly developed acetabulum underdevelopment of the medial portion of the proximal femoral epiphysis coxa valgalong bonesmildly hypoplastic epiphysesproximal humeral notchinglong and narrow femoral neckkneesgenu valgumhands and feetv - shaped deformity of the hypoplastic distal ulna and radiushypoplastic and irregularly shaped carpal and tarsal bonesproximal pointed metacarpals and metatarsalsbullet - shaped phalanges radiological skeletal manifestations of mps: dysostosis multiplex the abnormal storage of gag in the mouth, nose, pharynx and larynx results in several otorhinolaryngological disorders; in view of this, otorhinolaryngological imaging findings have been described . Regarding neuroradiological features, the involvement of the central nervous system is predominant in mps i, ii, iii and mps vii [15, 16]. In this pictorial review we focus on skeletal, otorhinolaryngological and neuroradiological imaging findings encountered in patients with mps . The most important radiological findings in the axial skeleton regard the skull, thorax, spine and pelvis . The skull is often characterised by an abnormal j - shaped conformation of the sella turcica, seen on the lateral view of the cranium (fig . 1). The sella turcica is normally a saddle - shaped depression in the sphenoid bone, while in mps patients it is usually wide with long clinoid apophyses and horizontal orientation: the tuberculum sellae is flattened and the dorsum sellae is rounded, respectively, forming the straight edge and the loop of the j . This configuration may represent a normal anatomic variant and may be associated with neurofibromatosis or with a slow - growing tumour adjacent to the sella, such as an optic chiasm glioma, so the j - shaped . Normal skull, presenting a regularly shaped sella (a). Skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) magnified views of lateral skull radiographs . Normal skull, presenting a regularly shaped sella (a). Skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) the cortical bone of the skull is thickened . The premature closure of the sagittal suture is responsible for the development of macrocephaly with dolicocephaly, plus the metopic perisutural hyperostosis causes a vertical frontal crest . The most important facial anomalies are represented by the lack of pneumatization of mastoid cells and paranasal cavities; the mandible is short and broad, mandibular condyles are undeveloped and the temporomandibolar joint may exhibit limited motion; unerupted and widely spaced teeth can also be found (fig . 1). Concerning the thorax, the main abnormality concerns the ribs, which can be paddle - shaped or oar - shaped because of the widening of the anterior archs and of the tapering of the posterior ones . Other common modifications are small scapulae, usually with flattening of the glenoid cavities, a short sternum and short and thickened aspect of the clavicles (fig . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) thoracic abnormality . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) issues affecting the spine are extremely common (figs . 3 and 4), and mps patients may risk important complications due to compression on the spinal cord and emerging nerve roots.fig . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seenfig . 4deformation of the spine . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities spine abnormality . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seen deformation of the spine . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities at the craniovertebral junction level, the most important abnormalities are: odontoid process displasia - hypoplasia;atlantoaxial instability or subluxation;periodontoid tissue and ligaments thickening;spinal stenosis . Odontoid process displasia - hypoplasia; atlantoaxial instability or subluxation; periodontoid tissue and ligaments thickening; these features represent a critical aspect in mps (particularly in mps iv), because if the spinal cord is compressed, cervical myelopathy may result . Modifications of the shape of vertebral bodies are very common, resulting in flattened and rounded vertebrae (fig . 5). At the thoraco - lumbar level the vertebral body can show a deficiency in its anterosuperior corner and, as a consequence, an apparent prolongation of the anteroinferior one, resulting on the lateral x - ray in an a 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients x - ray of multiplex dysostosis of the spine . A 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients these vertebral morphologic changes may progressively evolve to gibbus deformity (fig . 6), particularly in mps i. scoliosis is usually not bad enough to require surgery; however, myelopathy can represent an indication of the need for surgical treatment .fig . Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal a 45-year - old female suffering from mps iv (morquio disease). Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal the most common radiological features in the pelvis are rounded iliac wings and inferior tapering of the ileum (fig . The alterations of the hip joint can lead to hip dysplasia because of the poor development of the acetabulum and the underdevelopment of the medial portion of the proximal femoral epiphysis . This alteration has not been shown to respond to medical therapy, so for these children surgical reconstruction is often required; the target of this treatment is the optimisation of hip mechanics .fig . Images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b diaphyses are shortened and curved in the distal part; the epiphyses are slightly hypoplastic and thinned cortically with osteoporosis . Other features that can be found are the notching of the proximal part of the humerus, the long and narrow aspect of the femoral neck (fig . 8), and the hypoplasia of the lateral tibial hemiplate, resulting in genu valgum.fig . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal appearances of multiplex dysostosis . A 6-year - old boy with mps ii . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal knees can also be involved, particularly in children with mps iv and mps i, developing genu valgum (fig . 9). This condition is sometimes severe enough to require surgery . In adulthood an advanced state of arthrosis anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Diffuse cortical thinning and osteopenia are also observed (b) on mri images, the growth plate is irregularly enlarged, with multiple defects and erosions well depicted on coronal fast spin - echo proton - density - weighted images (fig . Coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions dysostosis of the knee . Coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions almost all forms of mps show distortion of the hand (fig . Carpal and tarsal bones are hypoplastic and irregularly shaped; the metacarpal bones are proximally pointed, shortened and thickened . The functionality of the fingers is compromised because of the bone alterations and the thickening of the subcutaneous tissues, resulting in a failure of a complete extension of the fingers with a claw hand dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) dysostosis multiplex of long bones . Dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) distal ulna and radius can be hypoplastic and have a v - shaped appearance; this oblique deformity of the terminal part of both bones results in the alteration of the carpal angle . The alterations in the skeletal anatomy, combined with an excessive gag deposition in the connective tissue, cause the constriction of the median nerve resulting in carpal tunnel syndrome . The most important radiological findings in the axial skeleton regard the skull, thorax, spine and pelvis . The skull is often characterised by an abnormal j - shaped conformation of the sella turcica, seen on the lateral view of the cranium (fig . 1). The sella turcica is normally a saddle - shaped depression in the sphenoid bone, while in mps patients it is usually wide with long clinoid apophyses and horizontal orientation: the tuberculum sellae is flattened and the dorsum sellae is rounded, respectively, forming the straight edge and the loop of the j . This configuration may represent a normal anatomic variant and may be associated with neurofibromatosis or with a slow - growing tumour adjacent to the sella, such as an optic chiasm glioma, so the j - shaped . Normal skull, presenting a regularly shaped sella (a). Skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) magnified views of lateral skull radiographs . Normal skull, presenting a regularly shaped sella (a). Skull of a 2-year - old patient affected by mps vi (b); the abnormal j - shaped sella (white arrow), is clearly recognisable . Skull of a 17-year - old patient affected by mps vi (c), presenting a j - shaped sella (white arrow) and some molars unerupted and angulated in both the jaws (white arrowheads) the cortical bone of the skull is thickened . The premature closure of the sagittal suture is responsible for the development of macrocephaly with dolicocephaly, plus the metopic perisutural hyperostosis causes a vertical frontal crest . The most important facial anomalies are represented by the lack of pneumatization of mastoid cells and paranasal cavities; the mandible is short and broad, mandibular condyles are undeveloped and the temporomandibolar joint may exhibit limited motion; unerupted and widely spaced teeth can also be found (fig . 1). Concerning the thorax, the main abnormality concerns the ribs, which can be paddle - shaped or oar - shaped because of the widening of the anterior archs and of the tapering of the posterior ones . Other common modifications are small scapulae, usually with flattening of the glenoid cavities, a short sternum and short and thickened aspect of the clavicles (fig . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) thoracic abnormality . Frontal radiographs showing a normal chest in a 10-year - old girl (a) and a chest of a 10-year - old girl affected by mps iv (b), the latter presenting ribs (white arrowheads) tapered proximally and wider distally; broad and short clavicles (white arrow) can be seen in the magnified view of the clavicles (c) issues affecting the spine are extremely common (figs . 3 and 4), and mps patients may risk important complications due to compression on the spinal cord and emerging nerve roots.fig . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seenfig . 4deformation of the spine . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities spine abnormality . Sagittal t2-weighted fast spin - echo image (a); sagittal t1-weighted fast spin - echo image (b). The figures show the entire spine of a 14-year - old female with mps vi: narrowing of the craniocervical junction (white arrow), vertebral bodies deformities (curved white arrow) and nucleus pulposus hypotrophy (white arrowhead) are well depicted; gibbus at thoracolumbar region is also seen deformation of the spine . Sagittal t2-weighted (a), sagittal t1-weighted (b) and coronal t2-weighted (c) fast spin - echo mri acquisitions demonstrate marked kypho - scoliotic deformation of the spine, with disc hernias and vertebral bodies deformities at the craniovertebral junction level, the most important abnormalities are: odontoid process displasia - hypoplasia;atlantoaxial instability or subluxation;periodontoid tissue and ligaments thickening;spinal stenosis . Odontoid process displasia - hypoplasia; atlantoaxial instability or subluxation; periodontoid tissue and ligaments thickening; these features represent a critical aspect in mps (particularly in mps iv), because if the spinal cord is compressed, cervical myelopathy may result . Modifications of the shape of vertebral bodies are very common, resulting in flattened and rounded vertebrae (fig . 5). At the thoraco - lumbar level the vertebral body can show a deficiency in its anterosuperior corner and, as a consequence, an apparent prolongation of the anteroinferior one, resulting on the lateral x - ray in an a 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients x - ray of multiplex dysostosis of the spine . A 4-year - old child with hurler syndrome shows vertebral bodies rounded (white arrow, a and b). Aspect (white arrowhead, c) with posterior scalloping and the platyspondylia with wedge - shaped deformity (curved white arrow, d) are observed in other radiographs of different mps patients these vertebral morphologic changes may progressively evolve to gibbus deformity (fig . 6), particularly in mps i. scoliosis is usually not bad enough to require surgery; however, myelopathy can represent an indication of the need for surgical treatment .fig . Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal a 45-year - old female suffering from mps iv (morquio disease). Sagittal mri t2-weighted (a) and t1- weighted (b) fast spin - echo acquisitions of the cervico - thoracic spine show severe kypho - scoliosis with narrowing of the spinal canal the most common radiological features in the pelvis are rounded iliac wings and inferior tapering of the ileum (fig . The alterations of the hip joint can lead to hip dysplasia because of the poor development of the acetabulum and the underdevelopment of the medial portion of the proximal femoral epiphysis . This alteration has not been shown to respond to medical therapy, so for these children surgical reconstruction is often required; the target of this treatment is the optimisation of hip mechanics .fig . Images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b images of the pelvis of mps patients (b d) showing typical imaging findings of disease: rounded iliac wings, inferior tapering of the ilea with a poorly developed acetabulum, underdeveloped medial portion of the proximal femoral epiphysis, increased coxofemoral joint space and coxa valga are well depicted in b d diaphyses are shortened and curved in the distal part; the epiphyses are slightly hypoplastic and thinned cortically with osteoporosis . Other features that can be found are the notching of the proximal part of the humerus, the long and narrow aspect of the femoral neck (fig . 8), and the hypoplasia of the lateral tibial hemiplate, resulting in genu valgum.fig . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal appearances of multiplex dysostosis . A 6-year - old boy with mps ii . Radiographs show several morphological appearances of multiplex dysostosis in long bones, with proximal humeral notching (white arrow, a), long and narrow femoral neck (curved white arrow, b), and frayed and flared tibial metaphyses (c). All segments, particularly those of the upper limb, are short and squat; they also have hypoplastic epiphyses, cortical thinning and flaring of the diaphyseal canal knees can also be involved, particularly in children with mps iv and mps i, developing genu valgum (fig . 9). This condition is sometimes severe enough to require surgery . In adulthood an advanced state of arthrosis anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Anteroposterior femoral x - ray image (a) showing bilateral genu valgum; proximal and distal epiphyses are flared and irregular . Diffuse cortical thinning and osteopenia are also observed (b) on mri images, the growth plate is irregularly enlarged, with multiple defects and erosions well depicted on coronal fast spin - echo proton - density - weighted images (fig . Coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions dysostosis of the knee . A 15-year - old female affected by mps vi . Coronal mri fast spin echo image (a) and spoiled gradient echo image (b) show an irregularly enlarged growth plate with multiple defects and/or erosions almost all forms of mps show distortion of the hand (fig . Carpal and tarsal bones are hypoplastic and irregularly shaped; the metacarpal bones are proximally pointed, shortened and thickened . The functionality of the fingers is compromised because of the bone alterations and the thickening of the subcutaneous tissues, resulting in a failure of a complete extension of the fingers with a claw hand dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) dysostosis multiplex of long bones . Dysostosis multiplex of hands and wrists in a 5-year - old boy (b) and in a 15-year - old female (d), both affected by mps vi (radiographs are compared with normal hands and wrists belonging to subjects of the same age, respectively a and c). The main imaging findings encountered in these areas are reported: the v - shaped hypoplastic distal ulna and radius (white arrow), the presence of small irregular carpal bones (curved white arrow), the broad and proximally pointed short metacarpals (white arrowhead) and the bullet - shaped phalanges (empty white arrow) distal ulna and radius can be hypoplastic and have a v - shaped appearance; this oblique deformity of the terminal part of both bones results in the alteration of the carpal angle . The alterations in the skeletal anatomy, combined with an excessive gag deposition in the connective tissue, cause the constriction of the median nerve resulting in carpal tunnel syndrome . Abnormal secretions and infiltration of gag in the mouth, nose, pharynx and larynx result in several otorhinolaryngological manifestations, sometimes the first to appear; ear, nose, throat and respiratory disorders are very common . The most important otorhinolaryngological problems are rhinitis, otitis media, and upper and lower airway obstruction . Upper airway obstruction may be the consequence of macroglossia, adenotonsillar hypertrophy and chronic nasal discharge in combination with a decreased temporo - mandibular joint mobility . Eustachian tube dysfunction due to enlarged adenoids and inflammation as a consequence of abnormal storage of gag in the temporal bone are responsible for chronic otitis media and conductive hearing loss . A thickened, retracted tympanic membrane and an increased attenuation of the tympanic cavity and mastoid cells can be observed on multidetector computed tomography (mdct) images (fig . Hearing impairment can also have a sensorineural component because of gag deposition in the inner ear or central nervous system . The surgical removal of the tonsils and adenoids is often required, even though it is only a temporary solution.fig . Axial scans presented from caudal to cranial (a - b - c) demonstrate nasopharyngeal airway and eustachian tube narrowing (arrow); thickened tympanic membrane (arrowhead); opacification of mastoid cells and left middle air cavity (curved arrow). Other abnormalities, including mucosal thickening of ethmoidal air cells, poor pneumatization of sphenoidal bone and maxillar sinus cavities and impacted teeth, are also depicted . Coronal view of temporomandibular joints (d) shows flattened and deformed mandibular condyles (empty arrow) otorhinolaryngological imaging findings . Axial scans presented from caudal to cranial (a - b - c) demonstrate nasopharyngeal airway and eustachian tube narrowing (arrow); thickened tympanic membrane (arrowhead); opacification of mastoid cells and left middle air cavity (curved arrow). Other abnormalities, including mucosal thickening of ethmoidal air cells, poor pneumatization of sphenoidal bone and maxillar sinus cavities and impacted teeth, are also depicted . Coronal view of temporomandibular joints (d) shows flattened and deformed mandibular condyles (empty arrow) lower airway obstruction, due to tracheomalacia and narrowing of the tracheal lumen, may lead to pneumonia and acute respiratory insufficiency . The radiologist should know that multiplanar reconstruction of a mdct scan, usually performed in these patients for the evaluation of the atlanto - axial instability, may be an important tool to assess the status of the whole airway passage . Sometimes it may be necessary to perform a tracheostomy in order to ensure airway safety [24, 29]. The pathogenetic substrate of central nervous system involvement lies in the abnormal accumulation of mucopolysaccharides within perivascular spaces and neuro - axonal units with an adversely affected myelin turnover . On the basis of mri findings we describe the main neuroradiological features: abnormal signal intensity in the white matter and in the basal ganglia, dilatation of periventricular spaces, widening of cortical sulci, brain atrophy and enlargement of extraventricular spaces, and spinal cord compression . One of the typical neuroradiological imaging findings is represented by focal or diffuse white matter lesions, detected as areas of high signal intensity on t2-weighted sequences . Diffuse white matter lesions usually show a symmetrical periventricular distribution, although they can also be found as patchy lesions in the subcortical region (fig . They derive from the delay of myelination in young children and progressive demyelination in the course of the disease; these lesions can also reflect gliosis [32, 33]. Focal white matter lesions consist of multiple small spot - like areas isointense to the cerebrospinal fluid (csf) on fluid attenuated inversion recovery (flair), t2-weighted and t1-weighted sequences (fig . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) neuroradiological imaging findings . A 12-year - old female affected by mps vi . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . 13d) neuroradiological imaging findings . A 15-year - old female affected by mps vi . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) the corpus callosum, best depicted on sagittal images, is sometimes the only location of these lesions, although they can also be encountered in the parietal and occipital lobe, in the basal ganglia, in the thalamus and at the grey - white matter junction level [3234]. A typical imaging feature in the brain of patients with mps is the honeycomb - like appearance in the basal ganglia and thalami (fig . 15); this imaging finding has been observed in patients with mps i, ii and iiib [35, 36]. Two theories have been proposed to explain the pathogenesis of basal ganglia involvement; the first concerns the accumulation of gags in neurons and astrocytes, leading to neuronal loss and demyelination; another theory maintains that the basal ganglia appearance could be related to an increase of fluid content in the periventricular spaces.fig . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), this typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter neuroradiological imaging findings . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), these areas (white arrow and white arrowhead) are hypointense . This typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter the pathogenesis of enlargement of perivascular spaces is not entirely clear . Two main hypotheses have been formulated: accumulation of gag around vessels and impairment of cerebrospinal fluid reabsorption (caused by the deposit of mucopolysaccharides in the leptomeninges). In addition, in the white matter of parietal and occipital lobes, multifocal variable - sized areas of high signal intensity which does nt match that of cerebrospinal fluid may be detected on flair and t2-weighted sequences; these lesions, probably due to gliosis, tend to become extensive and confluent . Ventricular enlargement, with or without involvement of the subarachnoid spaces, is a common finding in patients with mps (figs . 13, 14, 15, 16 and 17). Communicating hydrocephalus may be the consequence of either diffuse brain atrophy or reabsorption failure of cerebrospinal fluid due to dysfunction of the arachnoid granulations . A cystic - appearing enlargement of the cerebellomedullary and/or suprasellar cisterna has been described .fig . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of lifefig . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). The brain atrophy does not show significant progression through the different mr examinations . Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment neuroradiological imaging findings . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life neuroradiological imaging findings . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). The brain atrophy does not show significant progression through the different mr examinations . Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment the atrophy of the brain, probably due to neuronal death and myelin loss, is a common feature in mps and is seen as widened subarachnoid spaces and enlargement of the cortical sulci . The neuronal death could be explained by ischaemic damages due to the progressive accumulation of gag in blood vessels; on the other hand, the neuronal death could be caused by direct intracellular storage of gag . Brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life; on the contrary, patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life . The different degree of brain atrophy has been recently evaluated for monitoring disease progression and response to therapy [15, 33]; these authors reported a classification using the method introduced by lee et al . . Brain atrophy could be: absentmild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved)moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern)severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). Mild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved) moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern) severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). A strong correlation was found between severity of brain atrophy and cognitive impairment in mps [33, 39], whereas other authors did not find the same correlation . Compression of the spinal cord most frequently occurs at the atlanto - axial (c1-c2) joint (figs . Many of these patients suffer from several craniocervical junction abnormalities due to structural defects involving the spine . The most important one is atlanto - axial subluxation, which is the result of several causes: laxity of the transverse ligament, dural thickening resulting from deposition of collagen and gags, hypoplasia or absence of the odontoid, anterior soft tissue mass of the odontoid (representing a combination of unossified fibrocartilage and reactive changes) and indentation of the posterior arch of c1 . The anomaly of the odontoid process ranges in severity from total aplasia to a triangular - shaped configuration with a loss in vertical height and a broad - based tip . Chronic subluxation of the c1-c2 level may lead to a ligamentous hypertrophy and to further narrowing in the region of the craniocervical junction with the consequence of additional cord compression . Another cause of cord compression in these patients is gibbous formation in the thoracic spine, resulting from the malformations of vertebral bodies (most common in morquio disease). Compressive cervical myelopathy is a critical problem since the involvement of the bulbar - spine junction may lead to central respiratory failure .fig . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation involvement of the craniocervical junction . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Note also the abnormal j - shaped sella (white arrow, d) involvement of the craniocervical junction: recurrence after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation mrs (fig . 20) is a non - invasive imaging technique able to provide information about tissue molecular structures . Brain application leads to revealing the concentration of brain metabolites and can be used to monitor biochemical changes in brain diseases.fig . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio mrs . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio numerous studies have suggested a contribution of mrs to mucopolysaccharidoses assessment [42, 43]. Davison et al . [42, 43] found a reduction of the white matter n - acetylaspartate / creatine (naa / cr) ratio in mps iva and ii proportional to cognitive indices and to disease progression [42, 43], while vedolin did not find a significant difference in the naa / cr ratio between patients with and without cognitive impairment; vedolin found instead an elevated myo - inositol / creatine (mins / cr) ratio in patients with cognitive impairment . The increase in the mins / cr ratio is interpreted as a possible marker of glial activity (fig . 20). Takahashi et al . Measured presumptive mucopolysaccharides in white matter lesions and in white matter without lesions and compared the presumptive mps / cr ratios before and after bone marrow transplantation (bmt). They found a reduction in mucopolysaccharides in white matter without lesions after the bmt; they also found a correlation between mucopolysaccharide accumulation and neuronal damage resulting in a decreased naa / cr ratio . In summary, new evidence is needed in order to understand the real value of mrs in the evaluation of neurological involvement . Another commonly found sign in mps patients is macrocephaly, resulting from hydrocephalus combined with abnormal accumulation of incompletely broken down gags within the brain, meninges and skull . Macrocephaly is diagnosed when the circumference of the head exceeds normal values by more than two standard deviations . Other dysmorphic features, such as frontal prominence, scaphocephaly, a short neck and enlarged tongue, can be observed . Several orbital abnormalities can be found: thickening of the sclera and of the optic nerve sheath, optic canal narrowing and optic nerve atrophy (fig . Optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve is also observed finally, another imaging features reported in literature is the closed encephalocele (figs . 22 and 23); regarding this point, the presence of parenchymal / meningeal herniation at the level of the anterior or middle cranial fossa has been described as a characteristic neuroradiological feature of patients affected by mps ii . Instead of the term meningoencephalocele, which indicates the herniation of brain and/or meningeal tissue through a bone defect, the term closed cephalocele has been proposed to describe this skull abnormality because the protrusion of intracranial structures occurs without a detectable bone defect . The etiopathogenesis of this further manifestation seems to be related to an increased intracranial pressure and/or an altered bone maturation process . Ct and mri reveal a variable - sized pouch filled with brain parenchyma and cerebrospinal fluid, delimited by a bone wall and usually located in the anterior cranial fossa at the level of the lamina cribrosa as a weak area of the skull . The radiologist should look out for the presence of a closed cephalocele because it may interfere with rhinosurgery and cause epilepsy .fig . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows)fig closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma closed encephalocele in mps vi . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows) closed encephalocele in mps vi . Closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma one of the typical neuroradiological imaging findings is represented by focal or diffuse white matter lesions, detected as areas of high signal intensity on t2-weighted sequences . Diffuse white matter lesions usually show a symmetrical periventricular distribution, although they can also be found as patchy lesions in the subcortical region (fig . They derive from the delay of myelination in young children and progressive demyelination in the course of the disease; these lesions can also reflect gliosis [32, 33]. Focal white matter lesions consist of multiple small spot - like areas isointense to the cerebrospinal fluid (csf) on fluid attenuated inversion recovery (flair), t2-weighted and t1-weighted sequences (fig . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) neuroradiological imaging findings . A 12-year - old female affected by mps vi . Axial t2-weighted fast spin - echo (a), t2-weighted flair (b) and coronal t2-weighted fast spin - echo (c) images of the brain show symmetrically diffuse increased signal intensity of periventricular white matter, with enlargement of subarachnoid spaces in the middle cranial fossa and ventriculomegaly . 13d) neuroradiological imaging findings . A 15-year - old female affected by mps vi . Axial t2-weighted fast spin - echo images (a and c) and t2-weighted flair acquisition (b) show cribriform focal lesions of periventricular white matter due to enlarged perivascular spaces with ventricular enlargement . White matter lesions in the corpus callosum are well depicted by the mid - sagittal t2-weighted fast spin - echo acquisition (white arrowheads, d) the corpus callosum, best depicted on sagittal images, is sometimes the only location of these lesions, although they can also be encountered in the parietal and occipital lobe, in the basal ganglia, in the thalamus and at the grey - white matter junction level [3234]. A typical imaging feature in the brain of patients with mps is the honeycomb - like appearance in the basal ganglia and thalami (fig . 15); this imaging finding has been observed in patients with mps i, ii and iiib [35, 36]. Two theories have been proposed to explain the pathogenesis of basal ganglia involvement; the first concerns the accumulation of gags in neurons and astrocytes, leading to neuronal loss and demyelination; another theory maintains that the basal ganglia appearance could be related to an increase of fluid content in the periventricular spaces.fig . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), this typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter neuroradiological imaging findings . Involvement of basal ganglia in an mps vi patient; the mr exam was acquired when the patient was 17 . Axial t2-weighted fast spin - echo (a) brain image obtained at the level of the third ventricle with magnification focused on basal ganglia (b) demonstrating symmetrical focal areas of hyperintensity in the lenticular nucleus (black arrow) and in the thalamus (black arrowhead) because of enlarged perivascular spaces . On corresponding t2-weighted flair images (c and d), this typical imaging described in the brain of patients with mps is the honeycomb - like appearance . Note also ventriculomegaly and diffuse high signal intensity in the periventricular white matter the pathogenesis of enlargement of perivascular spaces is not entirely clear . Two main hypotheses have been formulated: accumulation of gag around vessels and impairment of cerebrospinal fluid reabsorption (caused by the deposit of mucopolysaccharides in the leptomeninges). In addition, in the white matter of parietal and occipital lobes, multifocal variable - sized areas of high signal intensity which does nt match that of cerebrospinal fluid may be detected on flair and t2-weighted sequences; these lesions, probably due to gliosis, tend to become extensive and confluent . Ventricular enlargement, with or without involvement of the subarachnoid spaces, is a common finding in patients with mps (figs . 13, 14, 15, 16 and 17). Communicating hydrocephalus may be the consequence of either diffuse brain atrophy or reabsorption failure of cerebrospinal fluid due to dysfunction of the arachnoid granulations . A cystic - appearing enlargement of the cerebellomedullary and/or suprasellar cisterna has been described .fig . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of lifefig . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). The brain atrophy does not show significant progression through the different mr examinations . Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment neuroradiological imaging findings . Axial (a) and coronal (d) t2-weighted fast spin - echo brain images of a 2-year - old male with mps ii show almost normal subarachnoid and ventricular spaces . The t2-weighted acquisitions (b and e) in a second mps patient (a 45-year - old female affected by morquio disease) reveal mild enlargement of subarachnoid spaces . Brain images (c and f) in a third mps patient (an 18-year - old female affected by mps vi) demonstrate higher enlargement of subarachnoid spaces, with associated dilatation of ventricles . Patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life; on the contrary, brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life neuroradiological imaging findings . Brain atrophy in a patient affected by mps vi; the patient started treatment with enzyme replacement therapy (ert) in 2005 . The studies were collected from 2005 (when she was 11 years old) to 2012 (at the age of 17). The patient shows a moderate degree of brain atrophy, with mild enlargement of the sylvian fissure on axial images (a, c, e, g); on sagittal acquisitions there are some focal lesions along the corpus callosum and an enlargement of cisterna magna (b, d, f, h). Despite encouraging results observed in patients with mps ii, the ert therapy does not seem to improve the appearance of neuroradiological imaging findings; the enzymes do not cross the haematoencephalic barrier . However, new evidence is needed in order to investigate the progression of neuroradiological imaging findings after treatment the atrophy of the brain, probably due to neuronal death and myelin loss, is a common feature in mps and is seen as widened subarachnoid spaces and enlargement of the cortical sulci . The neuronal death could be explained by ischaemic damages due to the progressive accumulation of gag in blood vessels; on the other hand, the neuronal death could be caused by direct intracellular storage of gag . Brain atrophy usually develops earlier in mps i, ii, iii and vii, becoming visible during the first few years of life; on the contrary, patients with mps iv and vi typically have normal intelligence and do nt show signs of atrophy until the second decade of life . The different degree of brain atrophy has been recently evaluated for monitoring disease progression and response to therapy [15, 33]; these authors reported a classification using the method introduced by lee et al . . Brain atrophy could be: absentmild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved)moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern)severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). Mild (widening of the sylvian and interhemispheric fissures <3 mm, but not all sulci are involved) moderate (widening of all fissures and sulci between 35 mm with visualisation of the entire mesencephalic cistern) severe (widening of all fissures and sulci> 5 mm with definite loss of cortex and white matter). A strong correlation was found between severity of brain atrophy and cognitive impairment in mps [33, 39], whereas other authors did not find the same correlation . Compression of the spinal cord most frequently occurs at the atlanto - axial (c1-c2) joint (figs . Many of these patients suffer from several craniocervical junction abnormalities due to structural defects involving the spine . The most important one is atlanto - axial subluxation, which is the result of several causes: laxity of the transverse ligament, dural thickening resulting from deposition of collagen and gags, hypoplasia or absence of the odontoid, anterior soft tissue mass of the odontoid (representing a combination of unossified fibrocartilage and reactive changes) and indentation of the posterior arch of c1 . The anomaly of the odontoid process ranges in severity from total aplasia to a triangular - shaped configuration with a loss in vertical height and a broad - based tip . Chronic subluxation of the c1-c2 level may lead to a ligamentous hypertrophy and to further narrowing in the region of the craniocervical junction with the consequence of additional cord compression . Another cause of cord compression in these patients is gibbous formation in the thoracic spine, resulting from the malformations of vertebral bodies (most common in morquio disease). Compressive cervical myelopathy is a critical problem since the involvement of the bulbar - spine junction may lead to central respiratory failure .fig . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation involvement of the craniocervical junction . Sagittal t2-weighted fast spin - echo mri acquisitions at the craniocervical junction in a patient affected by mps vi before (a) and after (c) the decompressive surgical procedure . Magnifications (b and d) show a t2 hypointense lesion surrounding the odontoid process (white arrowhead, b) with marked narrowing of the foramen magnum and cord compression; the stenosis improved moderately after surgery . Note also the abnormal j - shaped sella (white arrow, d) involvement of the craniocervical junction: recurrence after surgery . Craniocervical junction appearance in an mps vi patient before (a) and after (a) the decompressive surgical procedure: note the improvement in size of the craniocervical junction, with enlargement of subarachnoid spaces after the surgical procedure . B and b show recurrence of stenosis of the craniocervical junction in an mps iv patient 3 and 12 years respectively after surgery; the recurrence is due to progression of gag accumulation mrs (fig . 20) is a non - invasive imaging technique able to provide information about tissue molecular structures . Brain application leads to revealing the concentration of brain metabolites and can be used to monitor biochemical changes in brain diseases.fig . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio mrs . (legend: mins = myo - inositol; cho = choline; cr = creatine; naa = n - acetyl - aspartate). Multivoxel mrs performed on an mps vi patient; after post - processing analysis, two spectroscopic graphics are shown in a and c (corresponding voxels in b and d). The mins / cr ratio was found to have slightly increased in the white matter lesion and in the gray matter area (respectively values of 0.68 and 0.59). As reported by vedolin et al ., cerebral gag deposition induces changes in glial cells, with a consequent increase in the mins / cr ratio numerous studies have suggested a contribution of mrs to mucopolysaccharidoses assessment [42, 43]. Davison et al . [42, 43] found a reduction of the white matter n - acetylaspartate / creatine (naa / cr) ratio in mps iva and ii proportional to cognitive indices and to disease progression [42, 43], while vedolin did not find a significant difference in the naa / cr ratio between patients with and without cognitive impairment; vedolin found instead an elevated myo - inositol / creatine (mins / cr) ratio in patients with cognitive impairment . The increase in the mins / cr ratio is interpreted as a possible marker of glial activity (fig . Takahashi et al . Measured presumptive mucopolysaccharides in white matter lesions and in white matter without lesions and compared the presumptive mps / cr ratios before and after bone marrow transplantation (bmt). They found a reduction in mucopolysaccharides in white matter without lesions after the bmt; they also found a correlation between mucopolysaccharide accumulation and neuronal damage resulting in a decreased naa / cr ratio . In summary, new evidence is needed in order to understand the real value of mrs in the evaluation of neurological involvement . Another commonly found sign in mps patients is macrocephaly, resulting from hydrocephalus combined with abnormal accumulation of incompletely broken down gags within the brain, meninges and skull . Macrocephaly is diagnosed when the circumference of the head exceeds normal values by more than two standard deviations . Other dysmorphic features, such as frontal prominence, scaphocephaly, a short neck and enlarged tongue, can be observed . Several orbital abnormalities can be found: thickening of the sclera and of the optic nerve sheath, optic canal narrowing and optic nerve atrophy (fig . Optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve optic nerve involvement in the same mps patient as fig . 14: axial (a), coronal (b) and sagittal (c) t2-weighted fast spin - echo acquisitions show widening of the optic nerve sheath, with enlargement of the perineural csf space; angulation of the right optic nerve is also observed finally, another imaging features reported in literature is the closed encephalocele (figs . 22 and 23); regarding this point, the presence of parenchymal / meningeal herniation at the level of the anterior or middle cranial fossa has been described as a characteristic neuroradiological feature of patients affected by mps ii . Instead of the term meningoencephalocele, which indicates the herniation of brain and/or meningeal tissue through a bone defect, the term closed cephalocele has been proposed to describe this skull abnormality because the protrusion of intracranial structures occurs without a detectable bone defect . The etiopathogenesis of this further manifestation seems to be related to an increased intracranial pressure and/or an altered bone maturation process . Ct and mri reveal a variable - sized pouch filled with brain parenchyma and cerebrospinal fluid, delimited by a bone wall and usually located in the anterior cranial fossa at the level of the lamina cribrosa as a weak area of the skull . The radiologist should look out for the presence of a closed cephalocele because it may interfere with rhinosurgery and cause epilepsy .fig . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows)fig . Closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma closed encephalocele in mps vi . Axial t2-weighted (a), coronal t2-weighted (b), sagittal t2-weighted (c) and sagittal t1-weighted (d) acquisitions demonstrate a closed cephalocele (white arrows) closed encephalocele in mps vi . Closed encephalocele in patient affected by mps vi (same patient as in fig . Coronal mdct image of middle cranial fossa (a), obtained at the level of the pterygoid processes, revealing a bone depression of the right great wing of the sphenoid . The corresponding coronal t2-weighted mri image (b) demonstrates the pouch filled with cerebrospinal fluid and brain parenchyma skeletal x - ray and mri may show specific features in mps patients, although it is not possible to accurately differentiate between mps types based on skeletal and neurological characteristics . The evaluation of these imaging findings is useful for suggesting and supporting mps as a possible diagnosis, usually obtained by laboratory analysis, for monitoring the chronic and progressive course of the disease, for surgical and medical planning and for assessing the impact of therapy.
Patients with cardiovascular disease (cvd) are three times more likely to experience depression than other members of the community.1 depression is more frequently observed in ambulatory cardiac patients (9.3%) than in the general population (4.8%), and in patients undergoing treatment in hospital.2,3 large, international, epidemiologic studies have shown that many patients treated by physicians, including cardiologists and neurologists, have some form of clinical depression that requires antidepressant therapy.4 depression affects around one - fifth of heart failure patients (21%) and between 15% and 20% of patients admitted to hospital for myocardial infarction.5,6 patients with cvd diagnosed with depression have an increased risk of poor cardiovascular outcomes.7,8 however, it is not clear whether treatment with antidepressants improves or worsens this risk.9,10 tricyclic antidepressants and monoamine oxidase inhibitors are contraindicated in many patients with cvd as they are considered cardiotoxic, while selective serotonin reuptake inhibitors (ssris) are considered effective for treating depression in cvd patients and may even improve patient prognosis.11,12 however, the sadhart - chf (sertraline against depression and heart disease in chronic heart failure) study showed that while sertraline was safe, it was not effective in treating depression and had no impact on short- or long - term cardiovascular events or survival.13 examination of data from the regards (reasons for geographic and racial differences in stroke) study in stroke patients using an antidepressant at baseline found antidepressant use was associated with a small increase in risk of all - cause mortality.14 furthermore, antidepressants have been associated with an increased risk of stroke in two other studies.15,16 nonetheless, a large cohort study found no association between ssri use and poor cardiovascular outcomes, and even reported a reduction in risk of myocardial infarction.17 there is a real need for an effective and safe agent for the treatment of depression in patients with cvd . It is estimated that approximately 15%25% of depressed cardiac patients stop taking antidepressants due to adverse events within 6 months of treatment initiation, highlighting the importance of selecting an antidepressant with good tolerability.18 treatment of depression in cvd patients requires careful monitoring, and drugs that are known to be well tolerated and safe should be the first choice of the prescriber . Agomelatine has been shown to demonstrate antidepressant efficacy in both short- and long - term studies19,20 as well as in clinical practice,2123 and is unique among antidepressive agents in its ability to relieve anhedonia early in treatment.24,25 due to its novel pharmacology (melatonergic receptor agonist and 5-hydroxytryptamine 2c [5ht2c] receptor antagonist) and good tolerability profile,19,26 agomelatine could be a good candidate for the treatment of depression in patients with cvd, but no specific studies have evaluated agomelatine in cvd patients so far . An increase in liver transaminases has been observed in some patients treated with agomelatine, and hence, liver function tests are required . Pulse was therefore to obtain data on the efficacy and tolerability of agomelatine at standard doses for the treatment of mild and moderate depressive disorders in a range of patients with cvd in attending cardiologists in russia . This study included men and women with cvd, between 18 and 65 years old, having a score of 11 points on the depression subscale of the hospital anxiety and depression scale (hads), and fulfilling the criteria of the international classification of diseases, tenth edition, for depressive episodes of mild or moderate severity without psychotic symptoms, suicidal thoughts and intentions, and seasonal changes of state . For women with intact reproductive function, patients who were under psychiatrist supervision and/or were taking any psychotropic drugs, who had alcoholism or a history of drug addiction, a history of idiosyncrasy, or had been previously treated unsatisfactorily with agomelatine (administered at an adequate dose [2550 mg / day] and for adequate duration [at least 4 weeks]) were not included . Patients were also excluded if they had severe somatic disorders including oncologic, hepatologic, and neurologic diseases, were taking inhibitors of cytochrome p450 1a2 (eg, ciprofloxacin, fluvoxamine), were pregnant or breastfeeding, or had lactase deficiency, galactosemia, or glucose galactose malabsorption . Patients received a once - daily treatment of agomelatine for depression, at either 25 mg or 50 mg always at bedtime, over a period of 12 weeks . During this time, patients were examined by a doctor at four mandatory visits: enrollment visit (w0), and after 3 weeks (w3), 6 weeks (w6), and 12 weeks (w12). The following range of psychometric instruments was used for patient assessment at each visit: hads questionnaire, clinical global impression severity (cgi - s) and clinical global impression improvement (cgi - i) scales, a visual analog scale (vas), reactive anxiety subscale of spielberger khanin anxiety scale, whitely hypochondria index, and short form 36 (sf-36) quality - of - life questionnaire . Treatment tolerability was assessed by the spontaneous reporting of complaints by patients and by changes in the main clinical parameters . A standardized, routine physical examination included assessment of blood pressure, heart rate, body weight, and biochemistry at each visit . Liver enzyme monitoring (aspartate aminotransferase and alanine aminotransferase) was performed following the recommendations of the agomelatine summary of product characteristics including the following: performing baseline liver function tests in every patient before starting treatment with agomelatine, not starting treatment if serum transaminases exceeded three times the upper limit of normal (uln), and monitoring liver function at 3, 6, and 12 weeks . Student s t - test for paired samples was used for within - group comparisons of continuous variables that were normally distributed, and wilcoxon s test for those that were not normally distributed . Student s t - test for independent samples was used for between - group comparisons of continuous variables, and the mann all tests were two - sided, and the type i error rate was 5% . A total of 293 cardiologists from 46 regions in russia enrolled 896 patients between october 2012 and april 2013 . Of these, 20 patients (2.2%) dropped out prematurely, leaving 876 patients eligible for analyses . Depression of mild or moderate severity occurred predominantly in women aged around 50 years (51.49.9 years) who were married, employed, and had a level of higher education (table 1). In addition to cvd, patients typically had concomitant disorders affecting the endocrine, digestive, respiratory, and nervous systems (table 2). A list of the most prescribed non - psychotropic therapy, administered to> 5% of patients, is presented in table 3 . Just under two - thirds of patients (60%) were diagnosed with depression of moderate severity, with the remainder having a diagnosis of mild depressive episode . The most prevalent symptoms of depression diagnosed by cardiologists in addition to depressed mood, included sleep disturbances, fatigue, cognitive impairment, and anhedonia (figure 1). Cardiologists were less likely to identify ideas of insignificance, decreased libido, and changes in appetite . The majority of patients (758 [86.5%]) included in the analyses were receiving agomelatine as a 25 mg daily dose, with only 118 patients (13.5%) receiving 50 mg per day . The hads total score was significantly reduced by 12 weeks and at each visit compared with the previous (p<0.0001, table 4). The majority of patients (84.6%) were in remission by week 12 (hads total score <7). There was a statistically significant decrease in anxiety (spielberger khanin questionnaire) at each subsequent visit compared with the previous visit, with the number of patients with severe reactive anxiety decreasing from 840 (95.9%) to 131 (15%) by week 12 (p<0.0001) (figure 2). The whiteley index for hypochondria also decreased significantly (p<0.0001) over the course of 12 weeks of treatment with agomelatine, with the number of patients with severe hypochondria decreasing from 784 (89.5%) to 176 (20.1%) (figure 3). Physicians reported an increase in the overall effectiveness of agomelatine as measured using the cgi - i scale, with 69.3% of patients classed as showing a significant improvement compared with baseline . The overall assessment of the severity of the patient s condition on the cgi - s scale also showed a steady increase in the proportion of patients in remission or borderline state furthermore, by the end of therapy, the number of patients graded as moderately and substantially ill had significantly reduced from 92.9% to 20.9% . The subjective assessment of patient health on the vas improved significantly during 12 weeks of agomelatine treatment, dropping from 7.0 to 2.9 points (p<0.0001). The improvements observed were also statistically significant compared with the previous visit (p<0.0001). In general, the assessment of agomelatine efficacy by cardiologists and patients was similar and was described as good to excellent by 96% of physicians and 97% of patients who completed the study . Results from the sf-36 quality - of - life questionnaire indicated that agomelatine was highly efficacious and safe when treating depression in patients with cvd (figure 5). Eight quality - of - life parameters allowed two integral parameters of the sf-36 scale to be characterized: physical and mental components of health . The values of both components increased early in the study and continued to increase until study completion with statistically significant improvements compared with baseline (p<0.0001) (figure 6). The tolerability of agomelatine for the treatment of mild and moderate depression in patients with cvd was considered good . Subjectively intolerable adverse events (increased anxiety and agitation, increased insomnia and dizziness) led to the withdrawal of only five out of the 20 patients who prematurely dropped out of the study . The physical characteristics of patients obtained at weeks 0 and 12 before and after receiving the combined antidepressive and non - psychotropic (cardiotropic) therapy are summarized in table 5 . Patients heart rate as well as systolic and diastolic blood pressure values were significantly lower at 12 weeks (p<0.0001). Patient body weight after 12 weeks of therapy was lower than before therapy (p<0.0001), but the difference did not exceed 5% of body weight at baseline . Transaminase levels were increased to below or equal to the uln in around 3%6% of patients after 6 weeks of treatment, but there were no cases where liver function parameters were threefold more than the uln range at baseline . At baseline, 0.2% and 0.7% of patients, respectively, had values of alkaline phosphatase and gamma - glutamyl transpeptidase superior to normal, but less than or equal to three times the uln . After treatment, no liver function parameters were threefold more than the uln (table 6). A total of 293 cardiologists from 46 regions in russia enrolled 896 patients between october 2012 and april 2013 . Of these, 20 patients (2.2%) dropped out prematurely, leaving 876 patients eligible for analyses . Depression of mild or moderate severity occurred predominantly in women aged around 50 years (51.49.9 years) who were married, employed, and had a level of higher education (table 1). In addition to cvd, patients typically had concomitant disorders affecting the endocrine, digestive, respiratory, and nervous systems (table 2). A list of the most prescribed non - psychotropic therapy, administered to> 5% of patients, is presented in table 3 . Just under two - thirds of patients (60%) were diagnosed with depression of moderate severity, with the remainder having a diagnosis of mild depressive episode . The most prevalent symptoms of depression diagnosed by cardiologists in addition to depressed mood, included sleep disturbances, fatigue, cognitive impairment, and anhedonia (figure 1). Cardiologists were less likely to identify ideas of insignificance, decreased libido, and changes in appetite . The majority of patients (758 [86.5%]) included in the analyses were receiving agomelatine as a 25 mg daily dose, with only 118 patients (13.5%) receiving 50 mg per day . The hads total score was significantly reduced by 12 weeks and at each visit compared with the previous (p<0.0001, table 4). The majority of patients (84.6%) were in remission by week 12 (hads total score <7). There was a statistically significant decrease in anxiety (spielberger khanin questionnaire) at each subsequent visit compared with the previous visit, with the number of patients with severe reactive anxiety decreasing from 840 (95.9%) to 131 (15%) by week 12 (p<0.0001) (figure 2). The whiteley index for hypochondria also decreased significantly (p<0.0001) over the course of 12 weeks of treatment with agomelatine, with the number of patients with severe hypochondria decreasing from 784 (89.5%) to 176 (20.1%) (figure 3). Physicians reported an increase in the overall effectiveness of agomelatine as measured using the cgi - i scale, with 69.3% of patients classed as showing a significant improvement compared with baseline . The overall assessment of the severity of the patient s condition on the cgi - s scale also showed a steady increase in the proportion of patients in remission or borderline state furthermore, by the end of therapy, the number of patients graded as moderately and substantially ill had significantly reduced from 92.9% to 20.9% . The subjective assessment of patient health on the vas improved significantly during 12 weeks of agomelatine treatment, dropping from 7.0 to 2.9 points (p<0.0001). The improvements observed were also statistically significant compared with the previous visit (p<0.0001). In general, the assessment of agomelatine efficacy by cardiologists and patients was similar and was described as good to excellent by 96% of physicians and 97% of patients who completed the study . Results from the sf-36 quality - of - life questionnaire indicated that agomelatine was highly efficacious and safe when treating depression in patients with cvd (figure 5). Eight quality - of - life parameters allowed two integral parameters of the sf-36 scale to be characterized: physical and mental components of health . The values of both components increased early in the study and continued to increase until study completion with statistically significant improvements compared with baseline (p<0.0001) (figure 6). The tolerability of agomelatine for the treatment of mild and moderate depression in patients with cvd was considered good . Subjectively intolerable adverse events (increased anxiety and agitation, increased insomnia and dizziness) led to the withdrawal of only five out of the 20 patients who prematurely dropped out of the study . The physical characteristics of patients obtained at weeks 0 and 12 before and after receiving the combined antidepressive and non - psychotropic (cardiotropic) therapy are summarized in table 5 . Patients heart rate as well as systolic and diastolic blood pressure values were significantly lower at 12 weeks (p<0.0001). Patient body weight after 12 weeks of therapy was lower than before therapy (p<0.0001), but the difference did not exceed 5% of body weight at baseline . Transaminase levels were increased to below or equal to the uln in around 3%6% of patients after 6 weeks of treatment, but there were no cases where liver function parameters were threefold more than the uln range at baseline . At baseline, 0.2% and 0.7% of patients, respectively, had values of alkaline phosphatase and gamma - glutamyl transpeptidase superior to normal, but less than or equal to three times the uln . After treatment, no liver function parameters were threefold more than the uln (table 6). In this prospective, observational study, agomelatine proved effective for the treatment of depressive disorders in patients with cvd evaluated using a range of psychometric measures, and was well tolerated . Results were observed from the first post - baseline visit at week 3 and continued to improve throughout the study with statistically significant changes at each evaluation compared with the previous visit and with baseline . Early symptom relief is important, particularly in patients with cvd, as comorbid depression worsens cvd prognosis.27 depression has also been associated with other behaviors that impact on cvd, such as medication non - adherence, that reduce the likelihood of successful disease management.27 an ideal treatment for depressive disorders would provide symptom relief followed by the restoration of normal functioning and prevention of relapse . In this study, hads scores for anxiety and depression decreased from 13.1 and 13.9, respectively, at baseline to 3.7 and 3.9, respectively, at week 12 with agomelatine . Furthermore, the majority of patients (84.6%) were in remission by week 12 . These results are in agreement with those obtained with agomela - tine in clinical trials and clinical practice . The efficacy of agomelatine in patients with major depressive disorder has been extensively evaluated in a number of randomized, head - to - head clinical trials . Long - term treatment with agomelatine demonstrates a sustained duration of action.28 a meta - analysis of published and unpublished short - term studies involving agomelatine in the treatment of depression that included 20 trials and around 7,500 participants suggested similar efficacy to standard antidepressants including paroxetine, fluoxetine, escitalopram, sertraline, and venlafaxine.19 furthermore, those randomized to agomelatine were also less likely than those receiving comparator antidepressants to discontinue treatment because of adverse effects . A pooled analysis of four published 24-week studies suggested that treatment with agomelatine was at least as effective as treatment with ssris in the long term.29 cvd and depression often have overlapping symptoms such as fatigue, low energy, and difficulty in sleeping and carrying out daily routines of life . It is therefore not surprising that the symptoms of depression are sometimes thought to be due to cvd alone . Considering the prevalence of depression among cvd patients, it is important that cardiologists easily identify the key symptoms of depression such as hypothymia, anhedonia, fatigue, sleep disturbances, and cognitive impairments . One of the most common symptoms in this study was hypochondria, which could be caused by the presence of both serious somatic disease and affective (anxious depressive) disorders, and was significantly reduced by the end of the study . Agomelatine was also effective at relieving other key symptoms of depression including anxiety and hypochondria, both of which improved significantly at each visit compared with the last, throughout the study . Anxiety within depression is common and associated with worse prognosis, increased disability, and higher use of medication . The ability of agomelatine to improve anxiety in patients suffering from depression and cvd confirms findings from a pooled analysis of nearly 2,000 patients with major depressive disorder from six studies of 68 weeks duration: three placebo - controlled and three versus comparator antidepressants.30 in this analysis, agomelatine reduced the hamilton depression rating scale (ham - d) score as early as the second week (p<0.004) compared with placebo, and the reduction remained significant over the entire study (p<0.001). Compared with the other drugs in the study, agomelatine proved more effective in reducing anxiety symptoms with a substantial difference on the hamilton anxiety rating scale of 1.39 points (p=0.006). Quality of life is significantly decreased in patients with cvd suffering from depression, and improvement or restoration of quality of life is an important aspect of disease management . In the current study, agomelatine was associated with an improvement in both physical and mental quality - of - life components, which occurred early in the study and continued to improve until study completion . Although this is the first observational study of agomelatine in an exclusively cvd population, several other observational studies have examined the treatment effects and tolerability of the drug for depressive symptoms in a broad range of patients in clinical practice . In the large chronos observational study conducted in russia, patients demonstrated statistically significant changes in the ham - d during treatment.23 a german observational study, which included severely depressed patients and elderly patients with comorbid conditions, including cvd, also reported improvements throughout the study.21 the efficacy of agomelatine in depression is thought to be due to its pharmacological profile, acting as both an agonist to melatonergic receptors and an antagonist to 5ht2c receptors . These receptors act in synergy to increase dopaminergic and noradrenergic neurotransmission, and there is a selective release of both neurotransmitters in the prefrontal cortex.31,32 furthermore, it has no effect on extracellular serotonin in the brain, and thus, there is no dampening effect of serotonin in the release of dopamine, as may occur with the ssris and serotonin norepinephrine reuptake inhibitors (snris).33,34 this potentiation of noradrenaline and dopamine with no effect on serotonin release is consistent with the known clinical actions of agomelatine in depression including improvement of anhedonia and reduction of emotional blunting . This receptor profile is also responsible for the good adverse event profile of agomelatine, which differs from ssri and snri antidepressants, especially with regard to gastrointestinal side effects, headache, sexual dysfunction, and psychomotor agitation.35 even though no head - to - head studies with agomelatine and comparator treatments have been conducted in patients with cvd and depression, agomelatine may prove particularly beneficial in these patients because of its favorable cvd safety profile . In addition to efficacy, the side - effect profile of antidepressants and their potential for significant drug interactions is important in patients with cvd as they are likely to be suffering from other comorbidities and taking a number of concomitant medications . In this study, agomelatine was well tolerated, the side effects being those already documented in the summary of product characteristics for agomelatine . There were no cases of liver function parameters exceeding threefold the uln during the study . The observed changes in somatic symptom disorders suggest that there was a positive effect of combining agomelatine and a non - psychotropic agent (cardiotropic) that was not accompanied by the development or potentiation of significant adverse events . The improvements in heart rate, and systolic and diastolic blood pressure values could have been due to an effective cardiotropic therapy, or the result of an effective combination of therapies including agomelatine . The ability of agomelatine to normalize patients mental state may have resulted in a decrease in distressing thoughts, feelings, and behaviors regarding their somatic symptoms, including the frequency and severity of exacerbations of somatic symptoms . Agomelatine therapy was associated with a decrease in body weight after 12 weeks, but this did not exceed 5% of body weight at baseline . While the weight loss is unlikely to have been related to the pharmacological properties of agomelatine, it may have been a consequence of the improvements in many of the somatic symptom disorders that were assessed in this study . As such, this is an important finding as weight loss is often considered a positive step in patients with cvd . A number of antidepressants have unwanted cardiovascular effects, and when treating depression in cvd, the choice of treatment requires a careful balance of safety and efficacy . Other treatments have been associated with dose - dependent effects on repolarization interval, characterized by lengthening of the qt interval on an electrocardiogram, a marker of arrhythmic risk.37 in a pharmacovigilance study, using health records from 38,397 patients treated with antidepressants, statistically significant evidence of modest qt prolongation was identified for the tricyclic antidepressant amitriptyline, as well as for citalopram and escitalopram.38 in contrast, agomelatine up to 400 mg has been shown to have no effect on the qtc interval in healthy volunteers.39 some antidepressant agents with demonstrated efficacy in major depressive disorder may be less effective in patients with certain cardiac conditions . For example, both the sadhart - chf study with sertraline13 and the recently published mood - hf study (effects of selective serotonin re - uptake inhibition on morbidity, mortality, and mood in depressed heart failure patients) with escitalopram40 showed that while the drugs were safe, they did not provide a greater reduction in depressive symptoms than placebo or have a greater impact on cardiovascular events or survival . This was despite the fact that mood - hf followed patients for over 18 months.40 these findings led both sets of study authors to suggest that the pathophysiology of depression in patients with heart failure may have unique characteristics that may be less responsive to some antidepressant therapies . In the current observational study, agomelatine was effective in patients presenting with a range of cvds including hypertension, angina, myocardial infarction, atherosclerosis, rhythm and conduction disturbances, as well as chronic heart failure . This study was subject to the inherent limitations of observational studies including susceptibility to selection bias and confounding . In addition, the number of patients per investigator was small, which could overemphasize the significance of the results . The lack of a control group also limits the conclusions that can be drawn from the study . Real - world observational studies nevertheless remain valuable for evaluating efficacy and safety of a treatment in a broad range of patients who more closely reflect routine clinical practice than those found in the restricted populations of randomized controlled trials . Depression and cvd are both widespread among the general population and often occur simultaneously in the same individual . The results of this multicenter, observational study show that agomelatine provides a statistically significant improvement in depressive symptoms, anxiety, and hypochondria in depressed patients with a range of cvds and demonstrates good tolerability . Quality of life was significantly improved, and the majority of patients and cardiologists reported agomelatine efficacy as good to excellent . The results of this observational study suggest that agomelatine is an effective and safe choice for the treatment of depression in patients with cvd.
The genus flavivirus currently includes 86 viruses, of which 73 are grouped into 53 species (1). More than 40 of these flaviviruses are known to be pathogenic for humans and other vertebrates, in which they cause a variety of clinical diseases from mild febrile illness to severe encephalitis and/or hemorrhagic fever . Flaviviruses are small enveloped viruses with positive - sense single - stranded rna genomes consisting of an open reading frame (orf) flanked by 5 and 3 noncoding regions (ncr). The polyprotein encodes three structural proteins (the capsid [c], membrane [m], and envelope [e] proteins) and seven nonstructural (ns) proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) (2, 3). Flaviviruses have extensive geographic distributions and diverse arthropod vectors, and many of them infect vertebrate hosts (4). Among the arthropod - borne flaviviruses there is a correlation between phylogenetic relationships and virus - vector - host interactions (58). On the basis of virus neutralization studies and, separately, the association of arthropod vectors with vertebrates, 4 major groups of flaviviruses are recognized: the tick - borne flaviviruses (tbfvs), the mosquito - borne flaviviruses (mbfvs), no - known - vector flaviviruses (nkvs), and no - known - vertebrate - host flaviviruses (5, 6, 9, 10). The mosquito- and tick - borne borne flaviviruses contain important animal and human pathogens, including yellow fever virus (yfv), dengue virus (denv), west nile virus (wnv), st . Louis encephalitis virus (slev), japanese encephalitis virus (jev), and tick - borne encephalitis virus (tbev), which, in total, annually cause millions of human infections worldwide . Subsequently, on the basis of phylogenetic analysis of a relatively limited number of viral envelope gene sequences, the mosquito - borne flaviviruses were subdivided into the aedes- or culex - associated viruses . Some of the aedes - transmitted viruses induce hemorrhagic fevers in humans and primates, whereas many of the culex - transmitted viruses are associated with encephalitic infection in avian species (5). In addition to nkvs, sokoluk virus (sokv), entebbe bat virus (entv), and yokose virus (yokv), which share ancestral roots with the aedes - associated mbfvs, were isolated from vertebrates but not from arthropods . They form a clade normally referred to as the entebbe bat virus group, which is closely related to the yfv and edge hill virus (ehv) clades . It was previously suggested that these viruses may have lost their association with arthropods during their evolution and divergence (11). In addition to these three groups of flaviviruses which infect vertebrates, additional groups have been isolated only from mosquitoes or sand flies and under experimental conditions appear to infect and replicate only in insect cell lines . Accordingly, they have tentatively been defined as insect - specific flaviviruses, until appropriate taxonomic criteria are devised and approved . The cell - fusing - agent virus (cfav) was the first of many insect - specific flaviviruses (classical insect - specific flaviviruses [clsfs]) to be identified . Cfav was first isolated from an aedes aegypti cell line in 1975 (12), and its genomic sequence was characterized in 1992 (13). Cfav and a subsequently identified heterogeneous group of related clsfs form a distinct lineage in flavivirus phylogenies . These viruses have subsequently been isolated from a wide range of mosquito species in many countries throughout the world (1422). An additional separate group of flaviviruses that do not appear to infect vertebrate cells currently consists of nine viruses: lammi virus (lamv) (23), ilomantsi virus (ilov) (24), marisma mosquito virus (mmv) (19), donggang virus (donv) (unpublished data; genbank accession number nc_016997), chaoyang virus (chaov) (25, 26), nounane virus (nouv) (27), barkedji virus (bjv) (28), nhumirim virus (nhuv) (29), and nanay virus (nanv) (30). These nine viruses form a distinct clade that sits within the mbfv group of viruses . Moreover, flavivirus - like genomic sequences integrated within the genomes of aedes mosquitoes (21, 31) have also been identified ., tamana bat virus (tabv), ngoye virus (ngov), and mogiana tick virus (mgtv), are also considered to be flaviviruses because they share similar genome organizations with the recognized flaviviruses (1, 32). The only representative of a sand fly - borne flavivirus, saboya virus (sabv), sits in the group of mosquito - borne flaviviruses primarily associated with aedes mosquitoes in africa (5, 33). Flavivirus rna has also been discovered in phlebotomine sand flies from algeria (34) and portugal (unpublished data; genbank accession number hm563684). We report here on the detection, isolation, complete genome sequence, and phylogenetic assignment of a novel sand fly - borne flavivirus in psathyromyia abonnenci (pa . Abonnenci) sand flies . We propose the name ecuador paraiso escondido virus (epev), based on the village where the sand flies were collected . We also discuss the possible significance of the discovery of a new world (nw) sand fly - associated virus that shares a common ancestral lineage with nonvectored old world (ow) flaviviruses . Sand flies were trapped during march 2011 in the locality of paraiso escondido (008503n, 791749w), pichincha province, ecuador . The identified sand flies were pooled on the basis of species and sex with up to 50 individuals per pool and placed in 1.5-ml tubes for storage at 80c . Pools of sand flies were ground in 600 l of eagle minimal essential medium (supplemented with 7% fetal bovine serum, 1% penicillin - streptomycin, and 1% l - glutamine [200 mm]) in the presence of 3-mm tungsten beads using an mm300 mixer mill (qiagen, courtaboeuf, france) as described previously (37). A 200-l aliquot was used for viral nucleic acid (na) extraction with a biorobot ez1-xl advanced virus extraction minikit (qiagen) and eluted in a 90-l volume . Five microliters of this solution was used for sybr green reverse transcription (rt)-pcr and seminested pcr assays with the primers and by the protocol described previously (34, 38). Bands of the expected size were purified (amicon ultra centrifugal filters; millipore) and directly sequenced . A 100-l volume of the pcr - positive sand fly pool was inoculated onto c6/36 cell monolayers in 25-cm tissue culture flasks after being mixed with 900 l of l15 medium enriched with 1% penicillin - streptomycin, 1% l - glutamine (200 mm), 5% kanamycin, 3% amphotericin b (fungizone), and 5% tryptose phosphate broth solution . After incubation at room temperature for 1 h, 5 ml fresh medium containing 5% fetal bovine serum (fbs) was added . The flasks were incubated at 28c and examined daily for the presence of a cytopathic effect (cpe). Cell lines of different vertebrate species, including human (sw13), hamster (bhk), monkey (vero), and amphibian (xtc), were inoculated with the supernatant medium of epev - infected c6/36 cells obtained at passage 6 . Two flasks were inoculated for each cell line and incubated at either 32c or 37c . A 100-l volume of the pcr - positive sand fly homogenate was also inoculated onto vero cells . In the absence of a cpe, regardless of the absence of a cpe, 5 serial passages were performed, and each was tested by real - time rt - pcr (38) for the presence of epev rna . A total of 15 l of undiluted epev - containing supernatant medium (passage 4) or 15 l of epev - containing supernatant medium (passage 4) diluted 1:10 with minimal essential medium was injected intracerebrally into 2-day - old newborn of1 mice . Nucleic acids were purified from the brain tissues and used for the detection of epev rna by a specific real - time rt - pcr assay (38). Additional mice were injected with supernatant medium containing a pool of infected brain tissue from the previously infected mice . They were observed for 14 days and then euthanized, and nucleic acids were purified from the brain tissues and used for detection of epev rna by a specific real - time rt - pcr assay (38). Veterinary services of the ministry of agriculture has approved animal experimentation under the number a1301309 . The epev strain (passage 6 in c6/36 cells) and the original homogenate of the epev - positive sand fly pool were used independently for complete genome characterization through next - generation sequencing (ngs). Briefly, 140 l of each sample was incubated at 37c for 7 h in 30 u of benzonase endonuclease (catalog number 70664 - 3; novagen) to eliminate cellular dna and rna and preserve encapsidated viral particles . The encapsidated viral particles were then processed for rna extraction using a biorobot ez1-xl advanced viral rna minikit (qiagen) without an rna carrier . Random amplification was performed using a tagged random primer for rt and using tag - specific and random primers for pcr amplification (applied biosystems). The pcr products were purified (amicon ultra centrifugal filters; millipore), and quantification was done using a qubit fluorometer . Two hundred nanograms of the sample was processed for sequencing using an ion pgm sequencer (life technologies sas, saint aubin, france) (39). Analyses of the sequencing data were performed using the clc genomics workbench program (v6.5; qiagen). The clarified supernatant medium of epev - infected c6/36 cells was incubated at 4c overnight in a polyethylene glycol 6000 (sigma - aldrich) solution, to concentrate the virions . The concentrate was centrifuged at 3,000 rpm for 30 min, and the pelleted sediments were resuspended and ultracentrifuged at 30,000 rpm for 3 h using a fiberlite f14 - 6 250y fixed - angle rotor with a thermo scientific heraeus multifuge x3r ultracentrifuge . The phase 4 fraction of the centrifuge tube was collected to provide the sample that was then used for viral rna extraction . A 400-l volume was used for viral rna extraction as mentioned above and treated with 10 u/l tobacco acid pyrophosphatase (epicentre); circularization was performed with 5 u/l t4 rna ligase (ambion) at 10c overnight . Specific primers were designed to perform rt - pcr and to amplify the extremities using an access rt - pcr one - step kit (promega). The positive samples were gel purified using a qiaquick gel extraction kit (qiagen), and both strands were sequenced . Flavivirus sequences available in the genbank database were collected to obtain a data set including a representative of at least one sequence for each species of the genus flavivirus available as an amino acid sequence of the complete orf . Alignments of the amino acid sequences of the complete orf were generated using both the clustal w2 (40, 41) and muscle (42) programs, available at the embl server (http://www.ebi.ac.uk/tools/), and refined manually for comparison . The effect of mass removal of regions of ambiguous alignment by use of the gblocks algorithm (43) was also investigated . Phylogenetic trees were reconstructed using markov chain monte carlo (mcmc) analysis implemented in the mrbayes software program (v3.1.2) (44). The analysis was performed using the wag substitution model with a gamma - distributed rate variation among sites and default priors . Five independent markov chains were run for 10 million generations, with the first 10% of samples being discarded as burn - in . Stationarity was confirmed on the basis of effective sample sizes of> 400 using the tracer program (v1.4.1) (45). A maximum clade credibility tree was summarized using the treeannotator program, which annotates all nodes with posterior probability support values . The amino acid distances among the representatives of the flaviviruses were calculated for the complete orf and ns5 protein by use of the p - distance method in mega software (v5) (46). Nucleotide distances were also calculated for the 1-kb region in the ns5 protein reported by kuno et al . Putative cleavage sites from amino acid sequence alignments that included epev and other flaviviruses were deduced and compared according to the proteolytic processing cascade pattern previously described for the flavivirus orfs (47). Predicted glycosylation and conserved cysteine residue sites were determined for epev and compared with those of known flaviviruses . Conserved enzymatic patterns of epev were also analyzed and compared with those of known flaviviruses . Possible ribosomal frameshifting sites on the epev genome were investigated using the methods described previously (4850). The complete genomic sequence of epev has been submitted to the genbank database and may be found under accession number kj152564 . One thousand three hundred sand flies (1,150 females and 150 males) were collected in paraiso escondido village, and the following species were identified morphologically: nyssomyia trapidoi, psychodopygus panamensis, psathyromyia aragoi, pa . Abonnenci, lutzomyia hartmanni, trichophoromyia reburra, and a trichophoromyia sp . Twenty - six pools were tested by pcr for the presence of flavivirus rna . Abonnenci sand flies contained flavivirus rna when the pan - flavivirus rt - pcr (38) was used, and on the basis of quantitative real - time pcr, the pool contained> 10 genome copies . Importantly, this epev - positive pool consisted exclusively of females that were neither gravid nor engorged, thus precluding the possibility that a blood meal from a vertebrate host might be the source of epev in the tested sample . On the basis of the fact that all the other sand fly pools were negative and the positive pool contained nonengorged sand flies, it seems reasonable to assume that a single sand fly might have been positive for epev rna . Thus, the evidence strongly supports the concept that epev must have infected and reproduced efficiently in the positive sand fly . However, we cannot rule out the possibility that epev might have been detected in mosquitoes if they had been collected from the same area where the sand flies were trapped . C6/36 cells infected with the sand fly homogenate from the pcr - positive pool showed a cytopathic effect within 3 days and 1 day at the 4th passage and 6th passage, respectively (fig . Amplification of the virus using the pan - flavivirus rt - pcr assay (38) for the first three passages was also confirmed, with threshold cycle values being 18.32, 16.21, and 19.01, respectively . In order to test whether or not epev could infect human and/or other vertebrate cells in culture, vero, bhk, sw13, and xtc cell cultures moreover, no cpe was detected following five serial passages of vero cells inoculated with the original epev - positive sand fly homogenate . Viral rna detection tests using the pan - flavivirus rt - pcr assay also produced negative results, indicating that epev did not replicate in any of the vertebrate cell lines tested . Finally, newborn mice inoculated with the original epev - positive sand fly homogenate showed no signs of disease, and epev rna could not be detected in the brains of these newborn mice . Thus, under the conditions employed in these experiments, epev did not infect or replicate in vertebrate cells or newborn mice . Initially, an almost complete sequence of c6/36 cell - derived virus was obtained by ngs . Unfortunately, the sand fly homogenate - derived virus gave very poor sequence readouts after ngs . Reads with minimum lengths of 30 nucleotides and with a minimum quality of 99% per base were trimmed using the clc genomic workbench program (v6.5) and mapped to reference sequences previously obtained by sanger sequencing . Parameters were set to ensure that each accepted read mapped to the reference sequence for at least 50% of its length and had a minimum of 80% identity to the reference sequence . Thus, the reported sequence was derived from ngs of the c6/36 cell - derived virus and sanger sequencing of the gaps . Missing regions, including segments of the capsid protein, the pr protein, and the 5 and 3 noncoding regions, were successfully amplified after cyclization of the genome and sequencing of the concatenated 3 and 5 termini . This resulted in determination of the complete genomic sequence of epev (genbank accession number kj152564), which was 10,761 nucleotides (nt) in length and which had an orf of 10,323 nt (positions 120 to 10,442). The single complete orf was predicted to encode a polyprotein of 3,441 amino acids (aa). The lengths and positions of the genes or genomic regions are shown in table 1 . Genome organization of epev at each terminus of the genome, 2 nucleotides which are conserved among members of the entire flavivirus genus (5153), i.e., 5-ag and ct-3, were detected . Mutagenesis studies have previously shown that ct-3 is probably a recognition site for the replication complex to initiate rna synthesis (54, 55). The 5 and 3 ncrs are thought to function as recognition sites for virus translation, replication, and possibly, assembly (56). The lengths of the epev 5 and 3 ncrs were 119 and 316 nt, respectively . On the basis of comparative alignment of the nucleotide sequences of all flaviviruses, the epev 316-nt 3 ncr is significantly shorter than that of any of the previously studied flaviviruses and lacks a number of conserved sequences found in the mosquito - borne flaviviruses . The only conserved sequence detected, conserved sequence 1 (cs1), was 5-catattgacaccagggaaaagac-3, which is located near the 3 terminus of the 3 ncr . Cs1 is complementary to a conserved sequence within the n - terminal coding region of the capsid protein . This complementarity results in a long - range rna interaction that promotes cyclization of the flavivirus genome, forming a panhandle - like rna structure required for viral rna synthesis (57). In the epev genome, the first 12 nucleotides of cs1 are complementary to a region in the capsid protein . Conserved sequence 2 (cs2) is also found in all mosquito - borne flaviviruses . The nucleotides in a short stretch prior to cs1 in epev showed very poor homology with the nucleotides at the equivalent positions of other flaviviruses, and the degenerate reverse primer 3utr - mos (5-ggtctccwmtaacctctag-3), which anneals to cs2 (58), was unable to anneal to this related region in the epev 3 untranslated region . The highly conserved pentanucleotide motif 5-cacag-3 at the terminus of the 3 ncr was identified . This pentanucleotide is conserved among almost all mosquito- and tick - borne flaviviruses (the exception is 5-caccg-3 in murray valley encephalitis virus [mvev]), and it has been reported to be an element essential for viral rna synthesis, whereas its functional role is not known (5961). The predicted pentapeptide cleavage sites and conserved domains of epev were compared with the corresponding sites of all available flavivirus polyprotein sequences generated using amino acid sequence alignments . A comparison of these cleavage sites with those of other flaviviruses is shown in table s1 in the supplemental material . The similarities detected are consistent with the phylogenetic position of epev, which is explained below in phylogenetic analysis and genetic distances . Putative polyprotein cleavage sites of epev polyprotein the mature virion capsid protein (virc) is cleaved from the nascent capsid protein (anchc) after a dibasic amino acid sequence at positions 101 and 102 and before a c - terminal hydrophobic domain (cthd) by the viral serine protease (vsp) in the cytoplasm of the host cell . For epev the proposed residues arg - arg were also identified in other flaviviruses . However, asn at position 1 has not been reported for virc / anchc but it was reported for other cleavage sites . The cthd protein is cleaved from the prm polyprotein after cys (at position 119 for epev) by a host signalase (hs) in the lumen of the endoplasmic reticulum (er). Cys at position 1 was not reported for flaviviruses, but it is consistent with the (3, 1) rule of von heijne (62). Pr is cleaved from the m protein by the host cell furin or a similar enzyme (63, 64) in the lumen of the er . A specific pattern of arg - x-(arg / lys)-arg was reported for this cleavage site (65). For epev, we propose the signature his - arg-(arg / arg)-ser after the amino acid at position 230 or ser - pro - arg / ser - ile after the amino acid at position 234 . The m protein is cleaved from the envelope protein after gly at position 308 by an hs in the lumen of the er, which is consistent with the rule of von heijne (62). The envelope protein is cleaved from the nonstructural ns1 protein after ala at position 799 by a signalase (hs) in the lumen of the er, which is consistent with the rule of von heijne (62). The ns1/ns2a cleavage site occurs after ala at position 1153, which is consistent with the rule of von heijne (62). The ns2a / ns2b cleavage site occurs after arg at position 1384, which is cleaved by the viral serine protease, but thr at position 2 is not consistent with the dibasic requirement at positions 1 and 2 . Experimental evidence is required to determine whether the proposed cleavage site is functional or not . The ns2b / ns3 cleavage site occurs after two arg residues at positions 1514 and 1515, which are cleaved by the viral serine protease . The ns3/ns4a cleavage site occurs after a double arg at positions 2138 and 2139, which are cleaved by the viral serine protease . The ns4a/2k cleavage site occurs after gln and arg at positions 2264 and 2265, respectively, which are cleaved by the viral serine protease . The 2k / ns4b cleavage site occurs after ala at position 2288, which is cleaved by a host signalase . The ns4b / ns5 cleavage site occurs after a double arg at positions 2537 and 2538, which are cleaved by the viral serine protease . The pr gene region contains 1 possible n - linked glycosylation site and 6 cys residues which are conserved among other flaviviruses except the clsfs, the e - gene region contains 2 possible n - linked glycosylation sites and 12 cys residues which are conserved in all other flaviviruses except the clsfs, and the ns1 protein contains 2 possible n - linked glycosylation sites and 12 cys residues which are highly conserved . Cysteine residues are required for stabilizing disulfide bridges in all mosquito - borne viruses (3). A sequence homologous to the fusion peptide, a 14-aa motif thought to be involved in virus fusion with cellular membranes (66), is present at positions 406 to 419 in the ns1 protein . It conforms with the asp - arg - gly - trp - x - x-(gly / his)-cys - x - x - phe - gly - lys - gly motif observed for all mosquito - borne flaviviruses (67, 68). The n - terminal sequence of the flavivirus ns3 protein contains conserved regions (boxes 1 to 4) that have significant similarity to serine proteases and belong to the trypsin superfamily (69, 70). This protease activity was reported to be required for the polyprotein processing of flaviviruses . On the other hand, the c - terminal sequence contains conserved regions (motifs i, ia, and ii to vi) which are similar to rna helicases of the dead family (71). Compared with the sequences of other flaviviruses, these patterns are highly conserved in the epev genome . The epev ns5 protein contains the conserved sequences (motifs a to d) associated with rna - dependent rna polymerase activity (72) and also the gly - asp - asp sequence of motif c, which is the active site of the polymerase . In the n - terminal domain, two conserved motifs, viz . The numbers 1 to 4 indicate the conserved enzymatic motifs in the proteins encoded by the ns3 and ns5 genes . Slashes, gaps; residues in yellow, nonconserved positions . With the ultimate objective of generating a phylogenetic tree, the nucleotide and amino acid sequences of the entire genome of epev were aligned with the corresponding sequences of all available flaviviruses . Despite the fact that epev was genetically significantly different from the other flaviviruses, the results of the alignment clearly identified epev to be a flavivirus . Nevertheless, the epev genome was distinct from the genomes of all other recognized flaviviruses in that the capsid protein contained a unique sequence of 17 amino acids from positions 141 to 157 . Use of the blast search engine revealed no equivalent sequence in any known virus or cellular sequences . However, fragments from this sequence of up to 10 amino acids matched fragments of sequences found in a variety of bacterial enzymes . We then prepared a truncated flavivirus alignment containing the amino acid sequences of only the capsid and prm, and we attempted to align manually a 26-aa fragment that contained the 17-aa fragment of the unique epev sequence . This truncated alignment was manually adjusted as described previously when the untranslated regions of the flaviviruses were analyzed (7376). Using this method, residual amino acids within the 26-aa fragment aligned with amino acids in the upstream region of the prm protein, particularly with those of yfv, sepik virus (sepv), and wesselsbron virus (wslv), i.e., the most closely related viruses (fig . Strain names and genbank accession numbers are given after the names of the viruses, which are abbreviated as follows: kunv, kunjin virus; kouv, koutango virus; yaov, yaounde virus; usuv, usutu virus; alfv, alfuy virus; cpcv, cacipacore virus; itv, israel turkey meningoencephalitis virus; bagv, bagaza virus; ntav, ntaya virus; tmuv, tembusu virus; stwv, sitiawan virus; dedsv, duck egg drop syndrome virus; rocv, rocio virus; ilhv, ilheus virus; iguv, iguape virus; aroav, aroa virus; bsqv, bussuquara virus; njlv, naranjal virus; kokv, kokobera virus; strv, stratford virus; nmv, new mapoon virus; denv_1 to denv_4, dengue virus serotypes 1 to 4, respectively; zikv, zika virus; spov, spondweni virus; kedv, kedougou virus; dgv, donggang virus; ugsv, uganda s virus; liv, louping ill virus; ssev, spanish sheep encephalitis virus; tsev, turkish sheep encephalitis virus; ggev, greek goat encephalitis virus; ohfv, omsk hemorrhagic fever virus; lgtv, langat virus; ahfv, alkhurma virus; kfdv, kyasanur forest disease virus; rfv, royal farm virus; ksiv, karshi virus; ggyv, gadgets gully virus; kadv, kadam virus; meav, meaban virus; srev, saumarez reef virus; tyuv, tyuleniy virus; ppbv, phnom penh bat virus; bcv, batu cave virus; mmlv, montana myotis leukoencephalitis virus; rbv, rio bravo virus; jutv, jutiapa virus; modv, modoc virus; mosfv, mosquito flavivirus virus; qbv, quang ninh virus; cv_theileri, culex theileri flavivirus; nakv, nakiwogo virus; pcv, palm creek virus; krv, kamiti river virus; hankv, hanko virus . The definitions of the other abbreviations for viruses are provided in the text . The topology of the phylogenetic tree based on the genomic sequence is in agreement with that in previously published analyses (11, 23, 33, 58, 77, 78) and clearly demonstrates the segregation of the major clusters consisting of the mosquito - borne, tick - borne, and no - known - vector flaviviruses with 100% support . The mbfvs showed a divergence of the two major clades, i.e., the viruses primarily associated with either aedes or culex mosquitoes (as delineated in fig . Interestingly, the most recent common ancestor for epev was shared with the nonvectored entv clade and the larger mbfv clade that includes sepv, yfv, and several other viruses . It is also important to note that, apart from epev, in these three clades that share an ancestor, only yfv is found in the nw, where it is believed to have been introduced during the slave trade . Nevertheless, epev diverged from these other lineages and was clearly distinct from these other flavivirus species, the closest of which was wslv, with which it had a genome sequence homology of 52.8% . In view of the discovery of a unique sequence fragment in the epev capsid region and the knowledge that some flavivirus genes possess regions with ambiguous, highly variable sequences, it was decided to conduct two phylogenetic analyses . In the first, a tree was constructed in which all ambiguous / highly variable sequence regions, including the unique epev capsid sequence, were retained in the alignment . In the second tree, these ambiguous regions were removed using gblocks trimming . Despite this cautious approach, the overall topology, branching patterns, and support for the trees were closely similar (data not shown). Moreover, the relative position of epev remained the same in both trees with 100% support . Independent phylogenetic analyses based on the complete orf and, separately, the e - gene sequence also resulted in a similar position of epev, in which it appeared outside the entv and yfv clusters (data not shown). However, in trees based on the ns3 and ns5 gene sequences, epev was positioned between the entv and yfv clusters (data not shown). The pairwise distances of the amino acid sequences between epev and other flaviviruses are shown in table s2 in the supplemental material . More than 15 years ago, in a pioneer study, it was proposed that virus species and clades within the genus flavivirus should be based on nucleotide distances using a 1-kb region of the ns5 gene (4), and cutoff values were set at 69% and 84% for discriminating clades and species, respectively . Using these values, epev, which is at best 68% similar to the 13 most closely related viruses (sokv, entv, yokv, sepv, wslv, yfv, ehv, bouboui virus [bouv], banzi virus [banv], uganda s virus, jugra virus [jugv], potiskum virus [potv], and sabv), would represent a new species . The maximum 68.0% pairwise nucleotide sequence identity of epev with these viruses was <69.0%, which was the cutoff value that grouped viruses in the same clade, and <84%, which was the cutoff to consider two viruses to be the same species . Moreover, the maximum pairwise amino acid sequence identities of the complete orf and ns5 gene (52.8% and 65.4%, respectively) between epev and the yfv - related clade were lower than the minimum pairwise amino acid identities of the complete orf and the ns5 gene (61% and 69.4%, respectively) between the clades defined at that time (4). In a separate analysis, the cutoff value based on complete orf sequences was recorded to be <55% amino acid sequence identity for a number of arthropod - borne flaviviruses (77), which is also higher than the maximum identity of 52.8% with epev . Many rna virus genomes contain specific sequences that direct a proportion of ribosomes into an alternative reading frame . Where functionally utilized, this is known as programmed ribosomal frameshifting (prf). The most common type of prf involves 1 tandem slippage of the p- and a - site trnas on a slippery heptanucleotide sequence with the consensus motif x_xxy_yyz, where x_xx represents any three identical nucleotides (with some exceptions, such as u_cc and g_ga); y_yy represents a_aa or u_uu; z represents a, c, or u; and underscores separate zero - frame codons . For efficient 1 prf to occur, an extra stimulatory element is required, and this normally takes the form of a downstream rna stem - loop or pseudoknot structure separated from the heptanucleotide shift site by a spacer sequence of 5 to 9 nt (79). Ribosomal 1 frameshifting in the gene expression of jev serogroup viruses (33, 48, 80) and the clsfs (i.e., cfav, culex flavivirus [cxfv], and relatives) has been described previously . On the basis of comparative genomic analysis, 1 prf has also been predicted to be utilized by viruses in the chaov - lamv - donv - ilov and wslv sepv flavivirus clades (33, 49). These frameshift sites occur within the region of the genome encoding ns2a - ns2b, with the site being conserved within a clade but not obviously conserved between clades, suggesting that 1 prf in the flaviviruses may have evolved independently on several occasions . On the one hand, the genome contains several slippery heptanucleotide motifs, a few of which have potential downstream rna structures . However, predictions based on a single sequence are inadequate, as such motifs have a high probability of occurring in random sequences . None of the potential 1 prf sites identified aligned with the previously predicted 1 prf site in the related wslv sepv clade . Moreover, the high degree of divergence of epev from all other sequenced flavivirus species precluded robust comparative genomic analyses to identify (or tentatively rule out) the occurrence of purifying selection (i.e., evidence for functionality) at other sites with a potential 1 prf . During the past 10 years, thousands of sand flies (phlebotomus and sergentomyia spp .) Have been collected in europe, africa, and the middle east and analyzed for the presence of flavivirus rna . None of these analyses produced evidence of infection by epev or any other flavivirus, suggesting that the well - known flaviviruses are rarely (if at all) vectored by sand fly species, at least in these regions of the ow . In the current study, we analyzed pools of sand flies collected in ecuador for the presence of flavivirus - related rna . Abonnenci sand flies yielded a pcr product, the sequence of which suggested a genetic relationship with viruses in the genus flavivirus . This pcr - positive pool of sand flies was also inoculated onto c6/36 cells, and a cytopathic virus was subsequently isolated . However, attempts to isolate the virus using vero cells failed, implying that epev is another example of an insect - specific flavivirus . Thus, epev appears to be unique, in that it was isolated in ecuador, i.e., the nw, from pa . Detailed comparative analysis of the entire viral rna sequence of epev revealed a genomic organization very similar to that of viruses in the genus flavivirus (2, 3). However, alignment of the corresponding regions of epev and other flaviviruses revealed in epev a unique insert of 51 nucleotides (17 amino acids) between amino acid positions 141 and 157 (as determined from the 5-terminal starting codon), i.e., near the boundary between the viral capsid and prm genes . The lack of hydrophobic amino acids in this region indicates that this unique sequence is more representative of the newly formed n - terminal segment of the prm protein than the hydrophobic transmembrane (tm) domain of immature c protein . The tm domain is proteolytically cleaved to generate mature c and prm proteins following coordinated signalase- and ns2b/3-mediated cleavage (81, 82). By manually adjusting the alignment as described previously (7376), residual amino acids within the region unique to epev aligned with amino acids in the upstream region of the prm protein, particularly but not exclusively with those of yfv, sepv, and wslv . Importantly, no similar nucleotide sequences were identified among other virus and cellular sequences when the blast search engine was used . This unique epev sequence therefore appears to contain ancestral elements of the flaviviral prm sequence which have presumably survived during the insertion and deletion process of rna evolution, as described previously (7376). The unique single polyprotein with conserved cleavage sites, the 5-to-3 orientation of structural and nonstructural proteins, and the highly conserved amino acid sequence domains in the virus genome - encoded enzymes justify the inclusion of epev in the genus flavivirus . The cysteine residues, 6 in the prm, 12 in the envelope, and 12 in the ns1, proteins which play an important role in protein folding through disulfide bridges, are all conserved among other flaviviruses . There are also n - glycosylation motifs which were speculated to be very important for viral replication, virulence, and maturation of viral particles (8386) in the prm, envelope, and ns1 proteins, some of which are conserved in epev . It contains only conserved sequence 1, which is found in all mbfvs, whereas conserved sequence 2 was highly variable . This conserved sequence 2 was reported to have a minor effect on viral rna synthesis but seems to decrease viral pathogenicity (8790). The presence of a given virus in an arthropod does not confirm that the corresponding vector species plays a role in the natural cycle of the virus . However, in the case of the epev - positive sand fly pool, the pa . Abonnenci females were neither gravid nor engorged, and an extremely high viral load was detected . Thus, pa . Abonnenci and possibly other nw sand flies may play a direct role in the natural infectious life cycle of epev . Epev is not the first flavivirus isolated from sand flies, although few viruses have been found in phlebotomine flies until now . Saboya virus was isolated from sand flies in senegal (91), although it was originally isolated from the gerbil species tatem itempi in saboya village in senegal (92) and was later recovered from rodents of the mastomys, aivicantis nilaticus, and mus musculus species (93, 94) and chiropters in the republic of guinea (95). In addition, two flavivirus sequences were detected in phlebotomus perniciosus sand flies in algeria . These formed a monophyletic group more closely related to culex - associated insect - only flaviviruses (34). Flavivirus rna has also been discovered in phlebotomine sand flies from portugal (unpublished data; genbank accession number hm563684). The phylogenetic analysis showed that epev diverges from the common ancestral lineage of aedes - associated mosquito - borne flaviviruses that include yfv and also the nkv - like flaviviruses (yokv, entv, sokv). Epev is more distantly related to the other group of aedes - borne flaviviruses that include dengue viruses and the culex - associated group that includes wnv, slev, and zika virus . Interestingly, epev is also only distantly related to the clsfs (cfav, kamiti river virus, hanko virus, and cxfv) and dual - host - affiliated isfs (disfs) (nouv, lamv, chaov, donggang virus (dgv) and ilov) and the nkvs that include bat- and rodent - associated flaviviruses, such as modoc virus and montana myotis leukoencephalitis virus . Epev appeared alone as a lineage which was separated from a major phylogenetic clade that included the yfv, ehv, and entv groups . The phylogenetic analysis showed that epev appears at the root of these groups with 100% bootstrap support . However, the phylogenetic position among the mosquito - borne viruses of epev conflicts with its lack of an ability to infect and replicate in vertebrate cell cultures or newborn mice . The insect - specific flaviviruses nouv and lamv also appear to infect and replicate only in insect cell lines (23, 27), although they are phylogenetically grouped with mosquito - borne flaviviruses . These data therefore support the identification of the first representative of a novel group of nw sand fly - borne flaviviruses . When the origins of epev and the other flaviviruses are considered, most of the earliest phylogenetic lineages of the mosquito - borne viruses, tick - borne viruses, and nkvs are found in europe, asia, australia, and, in particular, africa . On the basis of the close relationships of the nw and ow mbfvs, the current opinion is that these viruses were introduced to the nw at various times before, during, or after the period of slave trading (33). The only representative of the tick - borne flaviviruses in the nw is powassan virus (powv) and its relatively recent descendant variant, deer tick virus (dtv). On the basis of historical, ecological, anthropological, and molecular epidemiological evidence, it was proposed that powv was introduced into the nw between 15,000 and 11,000 years ago, possibly during the tick and mammalian migrations, when the bering land bridge connected asia and north america (78, 96). Among the nkvs, the ow virus apoi virus (apoiv), which roots all other nkvs, is the only rodent - associated virus related to the nkv group, whereas the bat - associated nkvs are found in both the ow and the nw . This supports the idea of a single dispersion into the nw, most likely through bats (11, 33). It has recently been suggested that the introduction of viruses into the nw occurred several times during two evolutionary time periods (33). It seems highly probable that the most recent introductions occurred from the period of increasing slave and commercial trading across the atlantic ocean from the 16th to the 19th centuries . The most convincing evidence of such dispersions includes the mosquito - borne viruses yfv, denv, and, in 1999, wnv . For each of these viruses there are historical records and robust phylogenetic data (97102). In the case of the potentially earlier introductions to the nw, which include aroa virus, ilheus virus, slev, cacipacore virus, and the nkvs, their dispersion appears to have occurred thousands of years before slave and commercial trading (33). Whether or not these introductions to the nw occurred during the period when the bering land bridge connected asia and north america cannot be determined . However, it seems unlikely, since there are no recognized ow sand flies in the nw . On the other hand, it is recognized that estimation of the time of occurrence of such assumed introductions is highly dependent on the choice of dates used for calibration (33, 78). As analytical methods improve and more viruses are identified and characterized, it should become possible to provide more robust estimates for these virus dispersion patterns . The genus flavivirus comprises both vectored and nonvectored viruses, raising the possibility that arthropod - mediated transmission is either a derived trait or a secondary loss . The former traditional hypothesis is supported extensively (4, 5, 10, 77, 103, 104), but the alternative hypothesis should not be discarded at this stage of our comprehension . The insect - specific flavivirus group consisting of nouv, bjv, donv, ilov, chaov, lamv, and the newly discovered nanv from peru clusters phylogenetically with the mbfvs, but none of these insect - specific viruses have been found to replicate in mammalian cells (23, 24, 104). In contrast, the other nonvectored viruses, entv and yokv, replicate in mosquito cells . This reflects the need for more in - depth field investigations to identify the possible arthropod vectors of these viruses . For example, the isolation of sokv, a close relative of yokv, from argasidae ticks, birds, and bats and serological evidence of human infections by yokv were recently reported (105). Our phylogenetic analysis places epev at the root of the yokv, sokv, and entv clade, which itself roots the aedes - associated mosquito - borne flaviviruses (ehv, bouv, uganda s virus, banv, jugv, potv, sabv, wslv, sepv, and yfv). In contrast to epev, all of these viruses have been found only in the ow . As previously elaborated (106), sampling issues should be taken into consideration in generating these apparently conflicting results . The situation with epev is further complicated when one considers that it was isolated from a pool of nw sand flies . However, it replicates in mosquito cells but does not appear to replicate in mammalian cells, and it is ancestral to the entv and yfv clades . Since epev roots clades of ow viruses, is closely related to yfv, and has also been shown to replicate to high titers in mosquito cells, we can speculate that the vectors for the transmission cycle of epev in the ow may include mosquitoes . In this case, the introduction of epev might then be attributable to the slave trade, in common with several other flaviviruses (33). This argument is further supported by the compelling evidence that the alphavirus chikungunya virus (chikv) caused many disease outbreaks in humans about 200 years ago in the americas (107). It therefore seems highly likely that chikv was also introduced into the nw during the period of slave trading across the atlantic ocean . In this case, however, the introduced chikv does not appear to have become permanently established in nw reservoir hosts . The alternative possibility, that epev was introduced into the nw via infected sand flies from the ow, seems unlikely, since ow sand fly species have never been found in the nw . Clearly, mosquito trapping and screening are required in the region where the epev - positive sand fly pool was collected to discover whether or not epev circulates among the indigenous mosquito species . If this proves to be the case, it would support the hypothesis of an early divergence of the vector - borne viruses during the evolution of the flaviviruses . Moreover, the insect - specific flaviviruses (lsfs) also replicate only in mosquito cells, and the number of viruses in this group has been rapidly increasing . Most flaviviruses are associated with mosquitoes and vertebrates, but the isf group is associated only with mosquitoes, appearing at the root of the mbfv, nkv, and tbfv branches . The highly divergent tabv, isolated in 1973 from an insectivorous bat in trinidad (108), is now included as a tentative flavivirus (1). Recently, it appears to be highly divergent but associated with the genus flavivirus on the basis of a partial ns3 and ns5 analysis . Tabv and mgtv are highly divergent from each other, but they were both isolated in the nw . Nevertheless, they are related, albeit distantly, to the other flaviviruses . Thus, together with epev, tabv, and mgtv in the nw, viruses in the genus flavivirus appear to have evolved following their introduction into the nw from the ow . However, the wide variety of different flaviviruses and their attributes are potentially biased by the small number of representatives of different lineages that have currently been identified (24). At this stage, we cannot rule out the possibility that epev may be vectored by mosquitoes . However, the results in this study suggest that epev represents a new flavivirus species and could be the first recognized representative of a novel clade including other yet - to - be - discovered sand fly - borne flaviviruses . There is support for this proposal: (i) epev has been isolated from pa . Abonnenci, a species of nw sand flies that is known to be present in bolivia, brazil, colombia, french guiana, panama, peru, suriname, venezuela, and ecuador (cipa: computer - aided identification of phlebotomine sand flies of the americas, http://cipa.snv.jussieu.fr), (ii) pa . Abonnenci is not listed among the species of medical interest (110), and (iii) the absence of viral replication in various vertebrate cells and in the brains of newborn mice suggests that epev could be an insect - only flavivirus rather than an arbovirus . Further studies are required to understand if there are other sand fly species infected with epev in the region where the pa . Alternatively, if mosquitoes are the natural vectors, it would seem sensible to look first in the region of ecuador where epev was discovered.
Odontogenic myxoma (om) is considered to be a relatively rare benign tumor of mesenchymal origin . It is found in the skin and subcutaneous tissue, heart and also in various sites of the head and neck region . Myxomas of the jaw bones have been traditionally considered to have an odontogenic origin due to the close relation to teeth . According to the literature histologically, it is composed of spindle- or stellate - shaped cells in an abundant mucous intercellular substance, with little collagen . Those cases with higher amounts of collagen om exhibits aggressive infiltration of the adjacent tissue as it is not encapsulated and complete surgical removal is difficult . It has a high tendency to recur and can transform into malignant lesion; hence, radiographic and histopathological interpretation are important to establish appropriate surgical management . The treatment options can include curettage with peripheral ostectomy, segmental resection and radical resections for the more aggressive lesions . A 17-year - old male patient visited us with a complaint of swelling in the left maxillary molar region, which enlarged to the present size within a span of 3 months . Extraoral swelling was evident in the left side of the maxilla [figure 1]. Multilocular radiolucency extending from the distal aspect of the canine to the maxillary tuberosity region was observed on panoramic [figure 2a] and occlusal radiographs [figure 2b]. The computed tomographic (ct) scan showed swelling with bony expansion and thinning of the cortical plates with strong enhancement of the mass lesion in the anterior maxilla [figure 3]. The microscopic examination of hematoxylin and eosin (h and e)-stained section showed fine fibrillar mucoid stroma with evenly spaced spindle- and stellate - shaped cells, and a mild to moderate amount of collagen was observed [figure 4]. The mucoid nature was confirmed with a positive reaction with alcian blue stain [figure 5], and periodic acid - schiff stain was negative . Subsequently, the lesion was diagnosed as om and surgical resection followed by prosthetic reconstruction was proposed . Swelling present in the left maxilla (a) orthopantomogram showing multilocular appearance in the left maxilla, (b) occlusal radiograph showing multilocular radiolucency extending from the distal aspect of the canine to the third molar region computed tomography scan showing the extent of the lesion hematoxylin and eosin section showing stellate - shaped cells in the fine fibrillar stroma (100) alcian blue - positive reaction (40) the histogenesis of om is related to the odontogenic ectomesenchyme of a developing tooth or undifferentiated mesenchymal cells in the periodontal ligament . The odontogenic origin has been supported by the following reasons: exclusive occurrence in the tooth - bearing areas of the jawsassociation with an unerupted tooth or a developmentally absent toothfrequent occurrence in young individualshistological similarity between om and pulpal ectomesenchymeoccasional presence of sparse amounts of odontogenic epitheliumits uncommon occurrence in other parts of the skeleton . Exclusive occurrence in the tooth - bearing areas of the jaws association with an unerupted tooth or a developmentally absent tooth frequent occurrence in young individuals histological similarity between om and pulpal ectomesenchyme occasional presence of sparse amounts of odontogenic epithelium its uncommon occurrence in other parts of the skeleton . The majority of cases are reported in the second and third decades of life . According to kaffe om is usually a central lesion, and the radiographic appearance is important to establish the diagnosis . Multilocular radiolucency with either distinct or poorly defined margins is observed in adults and in the posterior part of the jaw . Zang et al . Examined the radiographic appearances of 41 om that were divided into six types . Multilocular (including honeycomb, soap bubble and tennis racquet patterns); type iii involvement of local alveolar bone; type iv involvement of the maxillary sinus; type v osteolytic destruction; and type vi a mix of osteolytic destruction and osteogenesis . Kaffe et al . In his radiographic study revealed an interesting correlation between size and locularity; unilocular lesions were smaller than 4 cm and multilocular lesions were larger than 4 cm . In the present case, it is difficult to differentiate solid ameloblastoma, odontogenic keratocyst and om using radiographs as all these lesions exhibit multiloculation . Dental radiographs are a bidimensional projection of a tridimensional structure, and therefore superimposition of anatomic landmarks can masquerade important findings . Asuami et al . Examined the dynamic magnetic resonance imaging (mri) features to differentiate these lesions; solid areas of ameloblastoma showed an earlier enhancement than the whole areas of the om . These results indicated that the dynamic mri features of the tumor substance of ameloblastoma differs from om . Because of the scarcity of studies using mri, the characteristics of the om have not been established satisfactorily . They found that the tumor borders were generally well defined with a smooth margin both for bony and soft tissue structures . Cortical plate continuity was lost in numerous patients and intralesional trabeculations were observed . In the present case, thinning and erosion of the cortical plates was present in the anterior and posterior regions of the maxilla and intralesional trabeculations were seen . Macdonald - jankowski suggested that both ct and radiographs should be used in the investigation of an om . Ct assesses perforation and pattern of septa while radiographs allow a better assessment of the degree of definition of the lesion's margins with the adjacent normal bone . On gross examination microscopically, it is made up of loosely arranged spindle- and stellate - shaped cells, many of which have long fibrillar processes that tend to intermesh . In cases of myxofibroma, the amount of collagen in the mucoid stroma is more prominent . The mucoid nature was confirmed with a positive reaction with alcian blue staining and negative periodic acid - schiff staining . Epithelial islands are not commonly observed in the myxomas of the jaws that do not play a significant role in om . Akihiro kimura et al . Reported a case of om, in which the interesting feature was the presence of active - looking and irregularly proliferating epithelial islands with a microcystic appearance . Immunohistochemical positivity with ck 19 supports the odontogenic origin and high labelling index for ki-67 indicates active epithelium, which has never been reported . Oms are extensively described as case reports; however, the invasive behavior of these lesions has not been explained . Surgical procedures vary from currettage, enucleation, local excision and partial and total jaw resection . The lack of a capsule and infiltrative growth pattern is responsible for a high rate of recurrence when conservative enucleation and curettage are performed . Boffano et al . Proposed the protocol to perform conservative surgery by enucleation and curettage when lesions were smaller than 3 cm, whereas a segmental resection with immediate reconstruction is preferred in patients affected by a bigger tumor . Resection of the jaw was planned for this patient as the lesion in the maxilla is in close relation to vital structures; resection procedures minimize the risk of involvement of these structures and also reduce the recurrence rate . Om and other odontogenic tumors share common features on conventional radiographs that lead to a diagnostic dilemma . In order to establish a treatment protocol,
Peroxisome proliferator - activated receptor gamma (pparg) gene is a member of nuclear hormone receptor superfamily, and it regulates the transcription of several genes involved in glucose metabolism, adipocyte differentiation, lipid oxidation, angiogenesis, and inflammation . Ppar displays different protein isoforms generated by different promoters and alternative splicing [2, 3]. The functional role of ppar is well documented, and its alterations have been widely associated with metabolic diseases, such as type 2 diabetes and obesity [4, 5]. Indeed, the most analyzed pparg polymorphism, pro12ala in the exon b, exerts an unclear effect on receptor structure, and it seems to reduce the receptor binding affinity with the responsive elements, also inducing a decrease in transcriptional activity both with and without ppar agonists [7, 8]. The common pro12ala polymorphism has been associated with increased risk of developing type 2 diabetes, even though a recent hypothesis suggests pro12ala could be involved in the aetiology of metabolic disorders not by itself, but because of linkage disequilibrium with other single nucleotide polymorphisms (snps) in the pparg promoter [9, 10]. Moreover, pparg gene polymorphisms have also been associated with micro- and macrovascular complications of diabetes, although the few available studies do not unambiguously confirm the role of pparg in the pathogenesis of these complications, especially diabetic retinopathy [1113]. Indeed, recent studies have demonstrated that ppar ligands could both inhibit intraocular angiogenesis and upregulate vegf gene expression in several cell types . To date, most of the mutation analyses focused on the coding region of the pparg gene, and only few studies have analyzed snps in the regulatory region of pparg possibly associated with metabolic disorder susceptibility [15, 16]. A novel functional variant in the ppar2 promoter, c-2821 t, that associates with predictors of type 2 diabetes and obesity, was recently reported in pima indians population . In attempt to analyze this variant in italian individuals affected by type 2 diabetes, we have performed an snp analysis on 211 unrelated type 2 diabetic patients from campania county, southern italy . Subsequently, we have extended this analysis to a cohort of 205 unrelated obese patients and 254 control individuals . Furthermore, we have genotyped the above - mentioned cohort (diabetic, obese, and control individuals), for two extensively studied snps in high linkage disequilibrium, pro12ala and c1431 t . Snp analysis was performed on genomic dna from 211 unrelated italian type 2 diabetic patients (102 of which suffered from proliferative retinopathy), 205 unrelated obese patients, and 254 control individuals . All the subjects were recruited from the department of geriatrics and metabolic diseases, second university of naples, italy, and from the department of neuroscience, federico ii university of naples, italy . All procedures were approved by the ethics board of the participating institutes and adhered to the tenets of the declaration of helsinki . The diabetes and obese status were determined according to the criteria of world health organization . Patients with type 2 diabetes were eligible for the study if they had background or proliferative diabetic retinopathy, similar age (1 year) and similar duration of diabetes (1 year). The clinical characteristics (age, sex, duration of t2 dm, 2h - plasma glucose, hba1c and bmi) of the 102 diabetic patients with proliferative retinopathy were not different from those of the 109 diabetic patients with background retinopathy (see table 1). Pro12ala snp analysis was performed with specific primers pairs (pro12alaf 5-ttt taa cgg att gat ctt ttg c-3 and pro12alar 5-acc ctt aca taa atg ccc cc-3) amplifying a fragment of 214 bp . C1431 t snp in the exon 6 of pparg gene was analyzed by pcr amplification of a 358 bp fragment using specific primers pairs (c1431tf 5-ctg aac ccc ctg ttg tgt tt-3 and c1431tr 5-ggt gtc aga ttt tcc ctc aga-3). The screening of c-2821 t and a-2819 g snps was carried out on a 453 bp genomic region of ppar2-specific promoter, pcr amplified with specific oligonucleotides (c-2821tf 5-tac acc aat ggg ttg tca-3; c-2821tr 5-gta gcc aaa gac agg ttc tg-3) by muller et al . Pcr reactions set - up with amplitaq gold (applied biosystems, calif, usa) were performed using an abi 9700 automatic thermal cycler under the following conditions: 95c for 10 minutes; then 35 cycles of 95c for 1 minute, at annealing temperature (ta) for 1 minute, of 72c for 2 minutes; and a final step of 72c for 10 minutes . Pcr products were analyzed by denaturing high performance liquid chromatography (dhplc), and those with a dhplc pattern different from controls were sequenced using the abi prism big dye terminator cycle sequencing v2.0 (applied biosystems). Statistical analysis was performed using standard chi - square, fisher, and student's t - tests . For continuous variables, the general estimating equation procedure was used to adjust for covariates . The association of polymorphisms with type 2 diabetes, obesity, and retinopathy was analyzed by exact chi - square test, comparing snps frequencies . Allele and genotype frequencies (pearson statistics), o.r ., and p values, as well as dominant and recessive genetic models, were analyzed using finetti (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Hardy - weinberg equilibrium of tested groups and armitage's trend test (att) were also calculated using finetti . Att takes into account genotypes rather than alleles, avoiding a possible bias due to doubling of sample size . It assumes additive (or codominant) disease model where all disease allele are independent and have the same contribution to the disease risk . Continuous clinical data, such as duration of type 2 diabetes (years), hba1c and bmi (body mass index - kg / m), different between background and proliferative retinopathy populations, were compared by unpaired student's t - test (two - tailed test); a resulting p value less than 0.05 (c.i . To investigate whether snps in the pparg gene could be a risk factor for type 2 diabetes and obesity, an extensive polymorphism analysis in a population of 670 individuals from campania county, in southern italy, was performed . The diabetes and obese status were determined according to the criteria of world health organization . We genotyped three snps, pro12ala, c1431t in the coding region of pparg gene and c-2821 t in the regulatory region, in 211 individuals with type 2 diabetes, 205 suffering from obesity, and 254 control individuals . Our analysis revealed that t-2821 allele was not found in 670 individuals, and consequently, c-2821 allele frequency was 100% . Allelic frequencies of pro12ala and c1431 t variants (listed in table 2) are in agreement to the data previously reported by doney et al ., 2004, although 1431 t allele was shown to be less frequent in obese than in control individuals in the analyzed population . Moreover, during the snp analysis of the c-2821 t snp, we have characterized a novel snp, a-2819 g, located two base pair downstream, included within the same putative e2 box . This novel snp was genotyped on the entire cohort of 670 individuals using the same standard procedures . The -2819 g minor allele frequency, observed in 211 individuals with type 2 diabetes, was about 2-fold than the frequency of the risk allele in the control and obese groups (see table 2). In logistic regression analyses, aimed to test the hypothesis of a-2819 g as risk factor for type 2 diabetes under the assumption of a codominant, recessive and dominant model, the dominant model best fitted the data (p = 1.9 10, table 3). According to this model, -2819 g allele carriers (ag and gg genotype) showed an o.r . 1.463.44) of developing type 2 diabetes compared to the noncarriers (table 3). The a-2819 g allele is in hardy - weinberg disequilibrium (dhw) in the control group (p = .01 see table 3). The newly identified risk allele -2819 g was not found to be associated with obesity (table 2) and it was estimated to be not in linkage disequilibrium with pro12ala and c1431 t snps . The possible association of a-2819 g snp with some specific clinical features of both type 2 diabetes and obesity was also considered . Specifically, bmi in obese individuals, blood glucose, and ha1c levels and the presence of proliferative retinopathy in type 2 diabetic patients were evaluated . The analysis revealed that -2819 g frequency in diabetic females with proliferative retinopathy was 2-fold increased with respect to background retinopathy female (p = .02; see table 4 and figure 1). In logistic regression analyses, the dominant model best fitted the data (p = .03; see table 5). According to this model, type 2 diabetes female with -2819 g allele (ag and gg genotype) showed an o.r . = 1.0575.56) of developing proliferative retinopathy compared to the noncarrier diabetic female (table 5). The a-2819 g allele is in hardy - weinberg equilibrium in both background and proliferative groups (table 5). Moreover, diabetic females with proliferative retinopathy had more than 3-fold risk compared to control females (data not shown). In conclusion, our results show that a novel nucleotide variant a-2819 g, in the regulatory region of pparg, is associated with type 2 diabetes and with proliferative retinopathy in females in an italian cohort . Previous studies have shown that nucleotide variations, both point mutations and snps, in the pparg coding region, are associated with an increased susceptibility of developing type 2 diabetes and obesity . Particularly, the well - known pro12ala substitution has been widely associated with both type 2 diabetes and obesity, or predictors of these diseases, in different populations [10, 12]. The pro12ala polymorphism has been recently predicted to confer protection, through unknown mechanisms, from type 2 diabetes and nephropathy [12, 15, 21, 22]. Conversely, it has also been shown that pro12ala does not associate with diabetic retinopathy [11, 13]. The pro12ala snp has been described in high linkage disequilibrium with c1431 t polymorphism, located in exon 6 of pparg gene . Several studies have shown t minor allele to be associated, in obese individuals, with low plasmatic leptin levels . The same variation has been associated with high bmi values in obese female from finland in coassociation with ala12 allele . In the present study, we demonstrated that pro12ala and c1431 t variants are not associated with type 2 diabetes, obesity, bmi variation, and also microangiopatic complication of diabetes, in a cohort of 670 individuals from campania county, in the southern italy . We also found that -2821 t allele, described in the pima indians population from arizona, was not present in the analyzed population . In contrast, a novel nucleotide variation, a-2819 g, two base pair downstream the c-2821 t snp described by muller et al ., was identified . We demonstrated that the a-2819 g nucleotide variant is associated with type 2 diabetes, but not with obesity or bmi variations (tables 2 and 3). Dhw may be due to several factors such as genotyping errors, population stratification, selection, inbreeding, the presence of a causative allele or simply by the chance . We can therefore exclude with high confidence that dhw in the control group could be due to genotyping errors . For a-2819 g snp, as well as for all the examined snps, we cannot exclude that dhw is due to other confounding factors; however, at least for the autosome genes, the presence of dhw in controls is compatible with the segregation of a causative variant according to a dominant model . G snp, revealed statistical significant association with a common and detrimental microvascular complication of type 2 diabetes, namely, the diabetic proliferative retinopathy, in the diabetic females of the examined population (o.r . = 2.43; c.i . 1.094.83; p = 0.02). This microvascular complication represents a leading cause of blindness, characterized by an increased vascular permeability, haemostatic abnormalities, increased tissue ischemia, and also neoangiogenesis . The newly identified a-2819 g snp and the c-2821 t are both located in a binding site for a transcriptional repressor ef1 . The polymorphisms described by muller et al ., 2003, leading to higher levels of pparg transcription, has been associated with an increased risk of development of type 2 diabetes and obesity in pima indians . This finding is supported by the notion that supraphysiological activation of ppar by thiazolidinedione (tzd) agonists increases triglyceride (tg) content of white adipose tissue, thereby decreasing tg levels in the liver and muscles, resulting in increased insulin sensitivity and type 2 diabetes . In this study, we demonstrated that a novel polymorphism, in the regulatory region of pparg, associates to proliferative retinopathy in diabetic females, suggesting a possible involvement of the hormonal female component, such as the estrogen receptor signaling that interferes with the physiological ppar intracellular pathway . We can reject sex differences due to genotype bias, since males and females were analyzed together, in blind fashion, and the gender was assigned after the analysis . We postulate that ppar may be involved in the development and progression of diabetic retinopathy via a plethora of mechanisms, although its pathogenic effect on the development of the microvascular complications in type 2 diabetic patients is not yet known . Indeed, muller et al . (2003) concluded that -2821 t allele, via its effect on ppar expression, may exert functional consequences on different ppar2-activated pathways and that its association with the well - known pro12ala may contribute to ppar2-related phenotypes . Full and partial tzd agonists can induce distinct receptor combinations, leading to selective gene expression . Ligand - receptor complexes, assuming different three - dimensional conformations, show unique and differential interactions with cofactors and other transcription factors . Particularly, ppar2 is highly and heterogeneously expressed in the human eye, where its ligand - dependent activation evokes potent and robust inhibition of corneal angiogenesis . Moreover, altered lipid oxidation in the retina supporting cells (retinal pigment epithelium, rpe) and in the eye vessels, and an increased glucose uptake from rpe cells, has pathological effects on eye vessels [28, 29]. However, it was already shown that lipid peroxidation is influenced by c1431 t polymorphism, also involved in the susceptibility to premature myocardial infarction . G nucleotide variation possibly altering ppar2 transcript abundance may affect the complex and highly regulated gene expression network in the eye, conferring in turn the susceptibility to diabetic retinopathy . Further studies enrolling a greater number of subjects, both patients and controls, would be useful to better elucidate the involvement of this novel snp in the susceptibility of developing diabetic retinopathy . However, these findings strengthen the hypothesis that ppar could be a novel pharmacological target of angiostatic agents in the treatment of diabetic retinopathy
Intravascular lymphomatosis (ivl) is an uncommon systemic disease in which malignant proliferation of lymphocytes occurs exclusively in the vessels . The clinical symptoms are nonspecific in most cases, making an early diagnosis difficult [1, 2]. A unique characteristic of this disorder is that the proliferation of malignant lymphocytes occurs only within the vessels, in contrast to primary central nervous system lymphoma (pcnsl). We herein report a patient with ivl who presented with fever of unknown origin (fuo) and in whom a brain mass developed, mimicking pcnsl . A 75-year - old right - handed female patient was admitted to our hospital for the evaluation of recurrent fevers . She had been well until 3 weeks before admission, when fever and fatigue developed . She had a history of cholecystectomy, spinal compression fracture, and an old cerebral infarction of the left middle cerebral artery . After the brain infarction, she developed aphasia and right hemiplegia and became dependent on a wheelchair . She showed paroxysmal atrial fibrillation, and warfarin was prescribed to prevent a second stroke . On examination, her blood pressure was 150/70 mm hg, her pulse 90 beats per minute, and her body temperature was 37.5c . There was a small decubitus ulcer on the sacrum and a nonpitting edema in both legs . Her blood levels of lactate dehydrogenase (ldh), c - reactive protein, and soluble interleukin-2 receptor (sil-2r) were high at 458 iu / l (normal <229 iu / l), 16.8 mg / dl (normal <0.8 mg / dl), and 3,458 u / ml (normal <515 u / ml), respectively . Her blood levels of electrolytes, glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, amylase, brain natriuretic protein, and vitamin b1 were normal, as were her renal and thyroid function . Lumbar puncture was performed; there was no pleocytosis in the cerebrospinal fluid, but the protein and igg levels were increased to 85 mg / dl (normal <50 mg / dl) and 17 mg / dl (normal <4 mg / dl), respectively . A contrast - enhanced ct of the chest, abdomen, and pelvis failed to reveal any abnormal lesions that could have caused the fever . A brain mri obtained 3 weeks after admission showed an old infarction but was otherwise normal . Tests for antibodies to hepatitis b, hepatitis c, human immunodeficiency virus, human t - lymphotropic virus, and the treponema pallidum were negative . Antibiotic treatments including ciprofloxacin at 600 mg / day, panipenem / betamipron at 1 g / day, and ceftazidime at 2 g / day combined with arbekacin at 200 mg / day were undertaken, but all were ineffective . The fever continued for more than 3 weeks and fulfilled the criteria for fuo . Because the serum levels of ldh and sil-2r remained high, we suspected a hematogenous tumor such as myeloma, and bone marrow puncture was performed, but the result was normal . Although there were no skin lesions, we strongly suspected ivl and thus performed a random skin biopsy . The biopsy was obtained from 1 lesion of the purpura on the abdomen and 2 regions of the skin with a normal appearance on the abdomen and left thigh . Histological examination revealed atypical cells lodged in the intravascular lumens in the subcutaneous tissue of the normally appearing left thigh region (fig . Higher magnification revealed vessels filled with large malignant lymphoid cells and eosinophilic nucleoli (fig . 1b); the cells were stained with cd20 antibodies but not with cd3 (fig . The patient was thus diagnosed with ivl (intravascular large b - cell lymphoma). An mri obtained 7 weeks after admission showed a mass lesion involving the right basal ganglia, thalamus, corona radiata, uncus, and crus cerebri (fig . 2). The mass was isointense on a t1-weighted image, hyperintense on a t2-weighted image, and exhibited mild restricted diffusion on a diffusion - weighted image (dwi). An mri obtained 9 weeks after admission showed an enlargement of the mass (fig . Ivl is an uncommon systemic disease in which malignant proliferation of lymphocytes occurs exclusively in vessels . Studied 10 cases of ivl in a single institution and reported that fuo and a mental status change were the most common signs, being present in 60% of cases . They reported that the duration of fuo ranged from 2 to 6 months . In our patient, fuo had been present for 7 weeks until the diagnosis of ivl was made . In patients with long - standing fuo histological confirmation of the malignant proliferation of lymphocytes in vessels is required for the diagnosis of ivl . Although the number of reported cases of ivl in which antemortem diagnosis was achieved by biopsy is small, such biopsies reportedly involved the brain, adrenal gland, kidney, liver, spleen, lung, bone marrow, muscles, and the skin . Because dermatological manifestations including nodules, plaques, and macules are present in up to one third of patients with ivl, skin biopsies should be conducted when any skin lesions are found in patients in whom ivl is suspected . The involvement of clinically unaffected skin has been reported in autopsy findings, and this involvement serves as the basis for a random skin biopsy . In our case, we performed a random skin biopsy because of the strong suspicion of ivl based on the high serum levels of ldh and sil-2r . The mri findings of ivl have not been well characterized because the number of reported cases is relatively small . However, the most commonly reported mri findings are multifocal lesions that are hyperintense on t2-weighted images, mimicking small infarctions . Baehring et al . Recently evaluated 5 patients with ivl in whom serial diffusion - weighted mris were obtained . They reported that multifocal dwi lesions are the most common observations, suggesting the presence of small - vessel ischemia that is probably caused by the occlusion of vessels by malignant lymphomatous cells . A brain mass developed in the present patient . The mass was isointense on t1-weighted images and hyperintense on t2-weighted images and it showed a restricted diffusion on dwi and homogeneous gd enhancement . The differential diagnoses for a brain lesion appearing hyperintense on t2-weighted images with gd enhancement may include pcnsl, toxoplasmosis, glioblastoma multiforme, abscess, metastasis, tumefactive multiple sclerosis, and primary angiitis of the central nervous system (pacns). Of these etiologies, homogeneous gd enhancement and restricted diffusion on dwi make pcnsl most likely, and the serological findings suggesting a lymphomatous disease could have allowed for a diagnosis of pcnsl in the present case if the brain biopsy had not been performed . Moreover, when the mass appeared, there were no definitive ischemic lesions, which are common radiological findings of ivl . Antibiotic treatment was not effective and blood culture was negative in this case; however, there was still a probability of developing a brain abscess after admission . Despite this, the lack of a characteristic thick rim of contrast enhancement around the lesion was considered to make a brain abscess unlikely . Pacns is a rare vasculitis of unknown etiology that exclusively involves the central nervous system . Commonly reported mri findings of pacns are multifocal deep gray and subcortical hyperintensities on t2-weighted images . Recently, some cases of pacns have been reported to present as brain masses, mimicking brain tumors . The low sensitivity of the characteristic angiographic signs and the lack of specific findings in general medical laboratory investigations make a brain biopsy necessary for the diagnosis of pacns . The findings in our case suggest that if pacns is suspected in a patient with a brain mass, a random skin biopsy prior to brain biopsy should be considered to allow for the avoidance of a more invasive brain biopsy . However, some authors have reported that extravasation of malignant lymphocytes can occur to some degree in the late stage of ivl [1, 7]. To the best of our knowledge, there have been 4 cases of ivl in which postmortem examination revealed a brain mass and 5 cases of tissue - proven ivl in which mri showed a brain mass (table 1) [8, 9, 10, 11, 12, 13, 14]. Our case is notable because it is the first description of dwi of a brain mass in a patient with ivl . Dwi is a series of t2-weighted sequences that detect the movement of protons in water . The causes of restricted diffusion on dwi include acute ischemia, cerebral abscesses, diffuse axonal injury, active demyelination, and highly cellular tumors such as lymphoma . Dwi change in acute ischemia is commonly known to occur, and marked restricted diffusion in acute ischemia generally remains for 35 days . In our case, the restricted diffusion enlarged over 2 weeks, supporting the high cellularity of the mass as the cause of restricted diffusion . Although there are no pathognomonic presenting symptoms or signs in pcnsl, focal neurological deficits such as personal changes or cerebellar signs are reportedly rather common . Patients with pcnsl may infrequently present with fuo as the initial clinical manifestation . The majority of patients with pcnsl reportedly develop a solitary supratentorial tumor, and the most common sites are the frontal lobe, temporal lobe, parietal lobe, deep nuclei, occipital lobe, cerebellum, or the brain stem . Considering the fact that there are no specific symptoms or signs of pcnsl and that the brain mass noted in our case was a solitary mass located in the basal ganglia, which is a common site of pcnsl, the present case could have been misdiagnosed as pcnsl instead of ivl if the random biopsy had not been performed . A combination of cyclophosphamide, doxorubicin, vincristine, and prednisone with the recombinant anti - cd20 antibody rituximab (r - chop) is the most commonly employed treatment in patients with ivl . On the contrary, chop is either ineffective or toxic in the treatment of pcnsl, and methotrexate with brain radiation therapy is usually employed . If the skin biopsy had not been performed, this patient could have been treated by methotrexate, which is not effective against ivl . Mri showed a brain mass mimicking pcnsl; however, a random skin biopsy confirmed the diagnosis of ivl . Although postmortem examination was not performed, the brain mass noted on mri was considered to be a collection of malignant lymphomatous cells invading from the vessels.
The number of cardiovascular implantable electronic device (cied) implantation has increased over the past few years . The conventional access of lead implantation is the subclavian vein, which is often accompanied by high complication rate, including pneumothorax, hemopneumothorax, inadvertent artery puncture, local hematoma, subclavian crush syndrome, and difficulty in lead operating . Thus, the axillary vein puncture has been proposed as an alternative technique to the conventional subclavian vein access . However, the comparison between subclavian and axillary vein access through large - size sampled and randomized clinical trial is still limited . Here, we proposed a randomized, two - armed, open - label study to analyze the efficacy and safety of optimized axillary vein puncture versus conventional subclavian puncture in cied implantation . Patients aged 18 years or older with the indication of permanent pacemakers, cardiac resynchronization therapy pacemakers / defibrillators (crt - p / crt - d), and implantable cardioverter defibrillators (icd) were included in the study . All eligible patients were asked to sign and date an informed consent document in person before randomized into different groups . Patients undergoing lead replacement were excluded from the study . The study was reviewed and approved by the clinical research ethics board at china - japan friendship hospital and was registered to www.clinicaltrials.gov with trial number nct02358551 . According to a computer - generated randomization list, patients were randomized to the optimized axillary vein puncture group or the conventional subclavian vein puncture group at a ratio of 1:1 using an 8-sized block . The pulse generators were implanted on the surface of pectoralis major on the same side . Operated chest walls were locally strapped for 24 h and pressed with sandbags for 4 h. operations in both groups were performed consistently by one skilled electrophysiology physician . Is usually fixed, we used the 21-g needle to search for the axillary vein with x - ray fluoroscopy at the point where the first rib meets the clavicle . The direction of needle was toward the first anterior rib, with an angle of about 4560 between the needle and the skin . After the venous blood was drawn, a 0.018 inch j - shaped guidewire was put into the 21-g needle, and the 5 fr sheath (cook group incorporated, bloomington, indiana, usa) was inserted into the vein [figure 1]. Subsequently, both the 0.018 inch guidewire and 5 fr sheath were replaced by the standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath (biotronik, berlin, germany). If five attempts of puncture failed, an ipsilateral cubital vein angiography will be performed to observe the passage of axillary vein [figure 2] and subsequently the same method of vein puncture should be used as stated above . The left arrow indicates the axillary vein and the right arrow indicates the subclavian vein . Subclavian vein puncture ipsilateral cubital vein trocar was retained before the procedure . Pacemaker pocket was created below the clavicle . We used the 18-g needle to search for the subclavian vein with x - ray fluoroscopy at the point of clavicle . The entry point of the needle was a one - third point to the sternoclavicular joint, and direction of the needle was toward the suprasternal fossa, with an angle of about 1530 between the needle and the skin . After the venous blood was drawn, a standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath were inserted into the vein . If five attempts of puncture failed, an ipsilateral cubital vein angiography will be performed to observe the passage of subclavian vein and the same method of vein puncture should be used as stated above . Success of puncture was defined as drawing of venous blood and insertion of guidewire and sheath . One - time success was defined as minor adjustment of needle direction and point of puncture, without withdrawing the needle . Number of puncture needed, length of time in puncture and x - ray exposure, parameters of pacemakers including threshold, impedance, and p / r wave amplitude were recorded . Patients were followed up at 1, 3, 6, and 12 months after the operation and for every 12 months afterward . Continuous variables were used to compare between groups by t - test for normally distributed values; otherwise, mann - whitney u - test should be used . Patients aged 18 years or older with the indication of permanent pacemakers, cardiac resynchronization therapy pacemakers / defibrillators (crt - p / crt - d), and implantable cardioverter defibrillators (icd) were included in the study . All eligible patients were asked to sign and date an informed consent document in person before randomized into different groups . Patients undergoing lead replacement were excluded from the study . The study was reviewed and approved by the clinical research ethics board at china - japan friendship hospital and was registered to www.clinicaltrials.gov with trial number nct02358551 . According to a computer - generated randomization list, patients were randomized to the optimized axillary vein puncture group or the conventional subclavian vein puncture group at a ratio of 1:1 using an 8-sized block . The pulse generators were implanted on the surface of pectoralis major on the same side . Operated chest walls were locally strapped for 24 h and pressed with sandbags for 4 h. operations in both groups were performed consistently by one skilled electrophysiology physician . Is usually fixed, we used the 21-g needle to search for the axillary vein with x - ray fluoroscopy at the point where the first rib meets the clavicle . The direction of needle was toward the first anterior rib, with an angle of about 4560 between the needle and the skin . After the venous blood was drawn, a 0.018 inch j - shaped guidewire was put into the 21-g needle, and the 5 fr sheath (cook group incorporated, bloomington, indiana, usa) was inserted into the vein [figure 1]. Subsequently, both the 0.018 inch guidewire and 5 fr sheath were replaced by the standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath (biotronik, berlin, germany). If five attempts of puncture failed, an ipsilateral cubital vein angiography will be performed to observe the passage of axillary vein [figure 2] and subsequently the same method of vein puncture should be used as stated above . The left arrow indicates the axillary vein and the right arrow indicates the subclavian vein . Subclavian vein puncture ipsilateral cubital vein trocar was retained before the procedure . Pacemaker pocket was created below the clavicle . We used the 18-g needle to search for the subclavian vein with x - ray fluoroscopy at the point of clavicle . The entry point of the needle was a one - third point to the sternoclavicular joint, and direction of the needle was toward the suprasternal fossa, with an angle of about 1530 between the needle and the skin . After the venous blood was drawn, a standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath were inserted into the vein . If five attempts of puncture failed, an ipsilateral cubital vein angiography will be performed to observe the passage of subclavian vein and the same method of vein puncture should be used as stated above . Success of puncture was defined as drawing of venous blood and insertion of guidewire and sheath . One - time success was defined as minor adjustment of needle direction and point of puncture, without withdrawing the needle . Number of puncture needed, length of time in puncture and x - ray exposure, parameters of pacemakers including threshold, impedance, and p / r wave amplitude were recorded . Patients were followed up at 1, 3, 6, and 12 months after the operation and for every 12 months afterward . Continuous variables were used to compare between groups by t - test for normally distributed values; otherwise, mann - whitney u - test should be used . A total of 247 patients were enrolled in this study between january 2013 and november 2015, with no loss during follow - ups . Among all the patients, 125 were randomized into the axillary vein group and 122 were randomized into the subclavian vein group . The sex and age distribution, body mass index, and types of cied were similar between the two groups . Baseline characteristics of the study population * normally distributed continuous variables are presented as mean sd . : value;: t value . Cied: cardiovascular implantable electronic device; crt - p / crt - d: cardiac resynchronization therapy pacemakers / defibrillators; icd: implantable cardioverter defibrillator; sd: standard deviation . Patients in the axillary vein group were implanted with 231 pacing / defibrillation electrode leads, of which 221 punctures succeeded . Patients in the subclavian vein group were implanted with 227 pacing / defibrillation electrode leads, of which 218 punctures succeeded . One - time success rate of both groups was 68.4% (158/231) and 66.1% (150/227), respectively . The mean time spent in puncture of the axillary vein group was 45.9 14.1 s, whereas in the subclavian vein group, it was 28.7 13.9 s (t = 9.679, p <0.001). The duration of x - ray exposure was 150.1 33.6 s and 145.5 34.9 s, respectively [table 2]. Success rate and duration of the procedure * normally distributed continuous variables are presented as mean sd . The vein punctures failed in 11 patients, who all received cubital vein angiographies to observe the passage of axillary veins or subclavian veins . Among them, three patients in the axillary vein group had total occlusion of veins and two had shifting veins; two patients in the subclavian vein group had total occlusion of veins, one had vein stenosis, and three had shifting veins . We successfully performed the puncture under the guidance of angiography or performed the procedure on the opposite side . In the subclavian vein group, pneumothorax occurred in three patients . Three patients subclavian gaps were too tight to allow operation of the electrode lead (all of them had puncture repeated once to complete the operation). There were no puncture - associated complications in the axillary vein group . In either group, none had pocket hematoma or other severe complications . In a mean follow - up of 24.1 7.4 months, lead dislocations occurred in one patient in both groups, both of whom underwent lead replacements . Pocket infection occurred in one patient in both groups, both of whom underwent pocket debridement and later were implanted with the cieds in the opposite side . Two patients in the subclavian vein group had subclavian crush syndrome, both of whom underwent lead replacements . The remaining patients had no complications, and threshold, p / r wave amplitude, and impedance of their cieds were all in the normal range during the follow - ups . The complication rate in perioperative period and follow - ups of the axillary and subclavian vein group was 1.6% (2/125) and 8.2% (10/122), respectively (= 5.813, p = 0.016) [table 3]. Complications in perioperative period and follow - ups a total of 247 patients were enrolled in this study between january 2013 and november 2015, with no loss during follow - ups . Among all the patients, 125 were randomized into the axillary vein group and 122 were randomized into the subclavian vein group . The sex and age distribution, body mass index, and types of cied were similar between the two groups . Baseline characteristics of the study population * normally distributed continuous variables are presented as mean sd . : value;: t value . Cied: cardiovascular implantable electronic device; crt - p / crt - d: cardiac resynchronization therapy pacemakers / defibrillators; icd: implantable cardioverter defibrillator; sd: standard deviation . Patients in the axillary vein group were implanted with 231 pacing / defibrillation electrode leads, of which 221 punctures succeeded . The overall success rate was 95.7% . Patients in the subclavian vein group were implanted with 227 pacing / defibrillation electrode leads, of which 218 punctures succeeded . One - time success rate of both groups was 68.4% (158/231) and 66.1% (150/227), respectively . The mean time spent in puncture of the axillary vein group was 45.9 14.1 s, whereas in the subclavian vein group, it was 28.7 13.9 s (t = 9.679, p <0.001). The duration of x - ray exposure was 150.1 33.6 s and 145.5 34.9 s, respectively [table 2]. Success rate and duration of the procedure * normally distributed continuous variables are presented as mean sd . : the vein punctures failed in 11 patients, who all received cubital vein angiographies to observe the passage of axillary veins or subclavian veins . Among them, three patients in the axillary vein group had total occlusion of veins and two had shifting veins; two patients in the subclavian vein group had total occlusion of veins, one had vein stenosis, and three had shifting veins . We successfully performed the puncture under the guidance of angiography or performed the procedure on the opposite side . Three patients subclavian gaps were too tight to allow operation of the electrode lead (all of them had puncture repeated once to complete the operation). Group, none had pocket hematoma or other severe complications . In a mean follow - up of 24.1 7.4 months, lead dislocations occurred in one patient in both groups, both of whom underwent lead replacements . Pocket infection occurred in one patient in both groups, both of whom underwent pocket debridement and later were implanted with the cieds in the opposite side . Two patients in the subclavian vein group had subclavian crush syndrome, both of whom underwent lead replacements . The remaining patients had no complications, and threshold, p / r wave amplitude, and impedance of their cieds were all in the normal range during the follow - ups . The complication rate in perioperative period and follow - ups of the axillary and subclavian vein group was 1.6% (2/125) and 8.2% (10/122), respectively (= 5.813, p = 0.016) [table 3]. Common vein access for cied lead implantation was cephalic, subclavian, and axillary veins . Subclavian vein puncture has faster learning curve, and the success rate is generally high . However, due to its anatomic characteristics, complications are relatively common, including pneumothorax, hemopneumothorax, inadvertent subclavian artery puncture, brachial nerve plexus injury, subclavian crush syndrome, and electrode lead fracture . A large - scale retrospective study found that cephalic vein access was related with lower rate of lead failure . However, it requires vein incision which renders the operation more complicated and time consuming . Moreover, the size of the axillary vein is relatively small, thus it often suffers from a high failure rate, especially with multiple leads . The number of implantations of icds and crt - ps / crt - ds has increased in the past few years, and cephalic vein puncture may not be an optimal procedure . Anatomically, the axillary vein terminates at the lateral margin of the first rib and becomes the subclavian vein . Its passage is outside the clavicle and far from the cupula pleurae, and the diameter is larger compared with the cephalic vein with little variation . Therefore, the axillary vein puncture avoids nerve or pleura injury and subclavian crush syndrome, which ensures a high success rate . However, these maneuvers are complicated and have steep learning curves, which limit the clinical application . Belott reported an approach based on anatomic landmark on body surface and radiograph, where the axillary vein terminates and becomes the subclavian vein with little variation . Overall, axillary vein punctures guided by fluoroscopic landmark, contrast venography, or ultrasound have shown high success rate and low complication rate . In this trial, furthermore, we used the 21-g needle instead of the routine 18-g needle, which reduced tissue injury as well as the risk of artery injury and pneumothorax . In this study, the overall success rate and one - time success rate of axillary and subclavian vein puncture were similar . No pneumothorax or difficulty on lead operating occurred in the operation and no subclavian crush syndrome occurred in the follow - ups, which confirmed the efficacy and safety of this technique . No pocket hematoma or other severe complications occurred in either group, probably due to the operator's experience and the use of electrotome . The duration of procedure in the axillary vein group is longer than the subclavian group, probably because the axillary vein puncture requires more fluoroscopy guidance and searching in local area . In addition, the axillary vein puncture included an extra procedure: exchange of guidewire and sheath, which was time consuming . The relatively steep learning curve of axillary vein puncture is an issue, and innovations in the axillary vein puncture have been proposed recently, such as using the cephalic vein as anatomic landmark, introducing guidewire retrogradely from the femoral vein up to the left axillary vein as roadmap, and utilizing the caudal fluoroscopic view . First, the relatively short follow - up period is not sufficient to evaluate long - term efficacy . Second, it was a single - centered trial and all the procedures were performed by one operator, thus interpretation and extrapolation of the results should be cautious . A multi - centered trial with larger scale and longer follow - ups is necessary to present more evidence of the advantage of the axillary vein puncture technique . In conclusion, optimized axillary venous approach may be superior to conventional subclavian vein approach for cied lead placement . This work was supported by grants from fund for clinical study of capital characteristic (no . This work was supported by grants from fund for clinical study of capital characteristic (no.
For more than 40 years, metal ceramic restorations (mcrs) have been widely used in the fabrication of fixed partial dentures (fpds) and still represent the gold standard nowadays.1 the success of these mcrs depends on the effective bonding between the veneering ceramic and metal substructure, which is believed to develop from chemical bond of the metal oxides.2 however, although the conventional mcrs have demonstrated superior fracture resistance, metal - free restorations have gained importance and their development has been accelerated by increasing interest in aesthetic dentistry.3 the introduction of zirconium dioxide as a framework structure or coping for ceramic restorations has initiated lengthy discussions on the design and limitations of these restorations . Furthermore, the bonding mechanism between zirconia framework and veneering ceramic is not yet well understood since no documented evidence of the bonding mechanism between these materials is available.4 however, a few studies have looked into methods to increase the surface roughness of the zirconia.56 as the surface roughness increases, the bonding between zirconia coping and veneering ceramic increases, thus leading to success of the restorations . Various surface treatments (e.g. Sandblasting, acid etching, glazing, heat treatment, and application of liner onto coping materials) have been recommended to enhance the bonding efficiency between veneering ceramic and coping material . However, none of these treatments have been determined to produce the highest bond strength . Airborne - particle abrasion or sandblasting, an important treatment procedure for achieving strong adhesion of veneering ceramics, works by increasing surface roughness and providing undercuts.7 for ceramic surface treatment, acid reacts with the glassy matrix that contains silica and forms hexafluorosilicates.8 as a result, the surface of the ceramic becomes rough, which is advantageous for micromechanical retention on the ceramic surface . Combination of surface treatments, such as sandblasting with alumina oxide - particle and acid etching may substantially increase the surface area for micromechanical retention.9 this will subsequently increase the bond strength . Therefore, the purpose of this study is to investigate shear bond strength (sbs) of veneering ceramic to different types of coping material (metal alloy, zirconia oxide, and lithium disilicate) after various pre - surface treatments such as sandblast and acid etch . The null hypothesis is that the shear bond strength of these veneering ceramic will not be different after various pre - surface treatments . Ninety - six disc specimens measured 14 4 0.2 mm were prepared from three different type of coping materials (n = 32): i) metal alloy (ma), ii) zirconia oxide (zo), and iii) lithium disilicate (ld) pre - cast wax patterns were prepared using silicone mold (metrosil, metrodent, huddersfield, england) and were invested with investment materials ., ramat - gan, israel) pre - heated to 850 with alumina plunger for 90 minutes . For ma group, the mold was filled with molten ni - cr alloy (4all, ivoclar vivadent ag, schaan, liechtenstein) using a casting machine (fornax, bego gmbh, bremen, germany). For ld group, ips e.max press (ivoclar vivadent ag, liechtenstein) ingots were softened at 920 and were automatically pressed into the mold in a furnace (ep 600, ivoclar - vivadent, liechtenstein). After pressing and cooling to room temperature finally, the specimens were cleaned in an ultrasonic bath with distilled water for 15 minutes and air - dried for 10 seconds . For zo group, the specimens were machined out of a block of pre - sintered zirconia (cercon, degudent gmbh, hanau - wolfgang, germany). The specimens were milled using cercon brain expert (degudent gmbh, germany) machine in an enlarged proportion according to the data installed from manufacturer's software . The milled specimens were then sintered in the cercon heat plus furnace (degudent gmbh, germany) at 1,350 for six hours, producing final specimens sized 14 4 mm . The specimens from each coping material group were randomly divided into 4 subgroups with 8 specimens in each subgroup (n = 8). Four pre - surface treatments were prepared for each subgroup; a) no treatment or control (c), b) sandblasted (sb), c) acid etched (ae), and d) combination of sandblasted and acid etched (sbae). The specimens in subgroup c were grounded with diamond disc with no further treatment applied to the surface . The specimens in subgroup sb were treated with 50 m alumina (al2o3) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen . The specimens in subgroup ae were etched with 5% hydrofluoric acid (hf) (ips ceramic etching gel, ivoclar vivadent ag, liechtenstein) for 20 seconds . The specimens in subgroup sbae were sandblasted with 50 m alumina (al2o3) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen . All specimens were cleaned in an ultrasonic bath containing acetone, alcohol, and distilled water for 10 minutes . Two thin layers of opaque veneering ceramic in paste - liquid form were applied to the prepared surfaces of the specimens from group ma (ips inline, ivoclar vivadent ag, liechtenstein) and zo (ceram kiss, degudent gmbh, germany). The specimens were fired in the furnace (programart p500, ivoclar vivadent ag, liechtenstein) according to the recommendations of the manufacturers . A silicone mold (duplicone, shofu inc ., kyoto, japan) was fabricated with a slit measured 4 4 mm for the placement of veneering ceramic . The silicone mold was placed on top of the specimen disc . Veneering ceramic for specimens ma (ips inline, ivoclar vivadent ag, liechtenstein), zo (ceram kiss, degudent gmbh, germany) and ld (ips emax ceram, ivoclar vivadent ag, liechtenstein) in the study were manipulated as recommended by the manufacturer . Liquid was added to veneering ceramic powder until paste consistency was obtained and condensed in the mold . After condensation was completed and excess moisture was removed with absorbent paper, the silicone was carefully removed . After the first firing cycle, another layer of dentin ceramic was applied and excess water was removed using absorbent paper . The specimens were fired for the second time to compensate for the shrinkage generated during the first cycle . At the end of these firing cycles, the veneering ceramic surface was ground flat and parallel to the coping surface using diagen turbo grinder (bredent gmbh, senden, germany). A universal testing machine (utm) (autograph ag - x, shimadzu, kyoto, japan) was used for the sbs test at a crosshead speed 0.5 mm / min . A shear load was applied until failure occurred and failures fractures were determined from the chart recorder . The data were compiled and analyzed using the statistical package for social science (spss) software version 20.0 (ibm inc ., armonk statistical analyses using two - way anova and tukey's multiple comparisons tests were used . The mean sbs values for all groups of coping materials ranged from 11.61 4.84 mpa to 27.89 5.64 mpa (table 1). Shapiro - wilk normality test showed that the data had a normal distribution (p>.05). Two - way anova revealed that there were statistically significant interactions for the types of coping material groups, pre - surface treatment groups, and among the groups (p <.05) (table 2). Furthermore, there was a statistically significant interaction between the effect of coping materials and pre - surface treatments on shear bond strength, as shown by non - parallel lines within the graph (p <.05) (fig . Tukey's multiple comparisons showed a statistically significant difference in sbs of veneering ceramic within the coping material groups and the pre - surface treatment groups as shown in table 3 and table 4 (p <.05). Among various types of coping materials tested, group zo produced significantly higher sbs than group ma, while group ma produced significantly higher sbs than group ld . On the other hand, the specimens treated with sbae showed significantly higher sbs than the specimens treated with sb alone or with no treatment . The specimens treated with ae were also showed significantly higher shear bond strength than the specimens treated with sb . For relationship between coping material and pre - surface treatment, tukey's comparisons test showed a statistical significant interaction between the groups with p <.05 (table 5). In group ma, the specimens treated with ae showed significantly higher shear bond strength than other pre - surface treatments, while, in group zo and ld, the specimens treated with sbae showed significantly higher shear bond strength than other pre - surface treatments . International standard of organization (iso) has standardized the bond strength measurement of metal ceramic system through the schiwickerath crack initiation test; the mean debonding strength should be greater than 25 mpa . However, due to the brittleness of all - ceramic coping materials, this test cannot be applied to all - ceramic multilayered system.10 to date, there is no standardized test or minimum bond strength requirement for all - ceramic system.11 according to some authors, sbs values of 10 mpa is the minimum value for clinical flaw to happen between metal and ceramic.1213 therefore, sbs values more than 10 mpa indicate excellent bonding clinically . Assuming that shear stresses are generally responsible for the clinical failure of the coping - ceramic interface, sbs test was adopted in this present study . In addition, this test required simple preparation of specimens and the testing could be performed easily . Important aspects should be considered, including storage condition, type of substrate, specimen preparations, rate of load application, cross - sectional surface area, and experience of the researcher.11 in the present study, when the ceramic was veneered to ma, sbs values ranged from 14 to 24 mpa with mean value of 19.00 6.39 mpa . This finding was lower than sbs of base metal group recorded by al - dohan et al .. 11 the specimen's size could be accounted for the differences . They used smaller diameter of veneering ceramic, which could have given higher bond strength value as the formula was calculated by dividing the maximum applied force by the bonded cross - sectional area . In other studies, sbs between nickel - chromium or cobalt - chromium metal alloy were reported higher than the sbs finding in the present study.141516 however, it is difficult to compare the results of the present study with those obtained in other studies because different methods were used to evaluate the sbs . Furthermore, some authors suggested that the failure of the bonded interface occurred when a crack propagated from a flaw of a considerable size found in an area subjected to high tensile stresses.10 the sbs values reported for zo group in this study ranged from 22 to 27 mpa with mean value of 24.45 5.14 mpa . This finding was slightly lower than the sbs values obtained from other similar studies.1718 the other studies used the circular interface test, which was different from the sbs test in the present study . Guess et al.19 found the mean sbss of veneering ceramic to zr to be ranged from 9.4 to 12.5 mpa, which were lower than the values in the present study . The different findings were attributed to different testing method, particularly related to the size and form of the specimens tested . The mean sbs value for ld group was 13.62 5.12 mpa, which the lowest sbs value obtained among all coping materials . The present study is in agreement with study done by umer et al.20 the finding of the current study showed that application of veneering ceramic onto the coping material lowered the strength of the bilayer specimens . However, several authors have reported that the mean sbs value for ld group was higher than zirconia and base metal groups in their studies, which contradicted the current finding.1121 there are several methods used for surface grinding, which include grinding using an abrasive paper or wheels (sic or al2o3), particle air - abrasion using al2o3 or other abrasive particles ranging in size from 50 to 250 m, and grinding using a diamond bur.22 the highest mean sbs value for sandblasting treatment was obtained from group zo, followed by ma and ld groups . This finding is in agreement with other studies.2324 the other studies reported that sbs value for sandblasting group was higher than the other groups and concluded that sbs of veneering ceramic on zirconia treated with sandblasting was significantly higher than that subjected to other pre - surface treatments . Contrary to the current finding above, some researches have shown that surface grinding techniques have no significant effect on increasing the bond strength of zirconia to veneering ceramic.25 another possible problem with sandblasting is that it can create surface microcracks that can initiates bigger crack . These cracks later can decrease strength and cause fracture toughness of the material.26 since acid etching was first suggested as a ceramic presurface treatment for resin bonding, many different etching periods have been advocated and used . The most profound ceramic surface roughness and the highest bond strength data at the ceramic resin interface have been obtained by 2-minute acid etching, as reported by chen and suh in 1998.27 the present study found that the mean of sbs value for acid etching treatment was highest in ma group, followed by zo and ld group . The application of hf acid to metal was capable of roughening the surfaces, therefore increasing mechanical retention . The hf acid can also cause diminishing of oxide layer to the degree that would not affect the bonding.28 the statement might explain why the sbs value of ma group was still the highest after acid etching treatment was done . On the contrary, smielak and klimek found that etching zirconia copings with 5% hf showed no significant difference to the surface roughness, as the nature of the material was not glass - like.5 the present study found that the mean of sbs value for the combined treatment of sandblasting and acid etching produced the highest sbs value in ma and ld group compared with other pre - surface treatment . Sandblasting with 50 m alumina particles changed the surface by increasing the number of pits per unit area . Application of hf acid following acid etch was able to remove the glass matrix and the second crystalline phase, thus creating irregularities within the ld crystals.29 zirconia had the highest shear bond strength value, while lithium disilicate had the lowest shear bond strength value among the coping materials tested . Combination of sandblasting and acid etching produced the highest sbs value in zirconia and lithium disilicate.
International organization for standardization (iso) had published an international standard for medical laboratories, iso:15189 medical laboratories particular requirements for quality and competence (1). It was not feasible to implement the requirements of iso:15189 all at once in medical laboratories in different sectors and in different provinces, so it had been decided to define the minimal quality requirements that could be mandatory for all clinical laboratories throughout the country . To do so reference health laboratory (rhl) of ministry of health organized expert committees, and finally in september 2007 national standard manual was finalized and officially announced as the minimal quality requirements for all medical laboratories in the country (2, 3). With the finalization the aim of the workshops was training capable and knowledgeable pool of auditors in accordance with national standards and its respective checklists . Apart from auditing laboratories, rhl has performed benchmarking auditing of medical laboratory network (surveys) in provinces . Seasonally, laboratory auditors gather in one selected province to audit a noticeable fraction of its total laboratories based on the national standard checklist (3). 12 benchmarks performed in tehran and alborze provinces in 2010 (on that time these two provinces were merged as one province named tehran province). Regarding too many active laboratories in these two provinces, this benchmark performed in three stages: public hospitals: 84 public hospitals were audited by 60 auditorsprivate hospitals: 54 private hospitals were audited by 70 auditorsoutpatient, private medical laboratories public hospitals: 84 public hospitals were audited by 60 auditors private hospitals: 54 private hospitals were audited by 70 auditors outpatient, private medical laboratories the results of these audits are the main interest of this study . Through the discussion and analyzing process we try to compare different processes, their quality and accordance with national standard measures between public and private hospital laboratories . The assessment of the medical laboratories in tehran and alborz provinces were performed by the trained auditors and based on the national standard and its respective checklist . The questions were categorized based on the requirements in national standard . There were four options for each question: yes, no, corrective action is required and not applicable . Yes suggested that the requirement is completely fulfilled; no showed that the requirement has not been fulfilled yet, corrective action is required showed that an attempt to accomplish the requirements was tried but it is not totally completed and further action is required, and not applicable showed that the requirement is irrelevant in this precise laboratory . The results obtained from each respective checklist with the laboratory s informational data were entered in software for further statistical analysis . The reason for this study was to compare the assessment results of 84 public hospital laboratories with 54 private hospital laboratories . The comparison was done on the personnel management, biosafety, equipment management, environment, pre - analytical, analytical and post - analytical processes, quality assurance processes, purchasing and inventory, referral and referee laboratories relations, non - conformity management . We tried to define the weakness and threats in both settings and compare their success ratio . The results of the questionnaire with respecting ethical issues are presented in tables 1 to 3 . Several studies comparing the health service of public and private sector, as some of them are as follows: a systematic review of comparative quality of private and public ambulatory health care in low and middle income countries show both public and private sectors, scored low on infrastructure, clinical competence and practice, nevertheless private sector performed better with regard to responsiveness and effort . Synthesis of qualitative components indicates the private sector is more client centered (4). Other study in south korea revealed public hospital workers were stereotyped as lazy and incompetent, and public hospitals were portrayed as poorly managed and of low quality, unlike the claims made by the government (5). In a study in bangladesh about public and private hospitals, private hospitals were evaluated better on responsiveness, communication, and discipline (6). In combination with other evidence on health service delivery in china, it was suggested that changes in ownership type alone are unlikely to dramatically improve or harm overall quality (7). Comparison of different processes in two sectors: - personnel managementprivate sector shows a 10% better implementation ratio in comparison to the public sector . During discussion with the public laboratory directors it was noticed that they encounter certain managerial restrictions, which has affected their field of action as the laboratory director . In some of the laboratories in the public sector, the directors were supposed to only supervise the technical issues and the personnel management was not one of their responsibilities . This issue had apparently affected many aspects of the personnel management such as trainings or related corrective actions . Due to our observation the training was one of the most affected areas.on the other hand as some of the legal regulations such as the permit requirements do not include the public sector, the directors did not feel the necessary responsibility toward the standard implementation.who in one of its reports brought highly trained individuals tend to move to more stable (and available) jobs in the private sector (8).- biosafetyno regulation or guideline can ensure safe practices . Individual and organizational attitudes regarding safety will influence all aspects of safe practice, including willingness to report concerns, response to incidents, and communication of risk (9).private laboratories showed 12% better implementation rate . The detailed analysis of the questions showed that the public laboratories problem area rests in waste management and providing the personnel protective equipment . We concluded that this part is more due to the budget.- equipment managementpublic laboratories showed less achievement in this field . The personnel are neither well convinced to the importance of documentation nor have received the required sufficient training . The failure is most apparent at usage of control material and also at generating the records of all procedures.- environmentone series of five papers shows the private sector performs significantly better than public sector institutions . This series examines this variation in performance with the objective of identifying successful approaches to the management of medical equipment (10).public laboratories showed 10% less achievement in environment requirements . This was mainly due to the old structure of a few hospitals that has affected the total percentage dramatically.class ii biological safety cabinet only existed in 40% of laboratories irrespective of the group that they belong to . Class ii biological safety cabinet is mandatory for the laboratories that are supposed to perform blood culture . Reference health laboratory should pursue this issue for dedication of necessary budget.- pre - analytical, analytical and post analytical procedurespublic laboratories showed less achievement in these fields . Fortunately the weakness was not at the technical aspect of the laboratories but as mentioned earlier it was on documentation management.- quality assuranceat this category, which we believe is one of the most fundamental and essential parts for assuring the quality of the results, unfortunately both groups showed weaknesses in these areas and achieved almost 35% . The personnel were not able to analyze the result of quality control procedures and implement the corrective action.we also find out that to empower our assessing tool, we should increase the number of our questions in this field.- purchasing and inventorydue to the centralized purchasing department in hospital, laboratory director had minimal say in choosing the supplier . This problem was more highlighted in public hospitals.- referral and referee laboratorieswe noticed that only a handful of public laboratories were a referral laboratory with full capacity . In other cases though the laboratory was not capable of performing all the desired tests but they do not participate in sending the samples . It should be noted that this issue is not in accordance with national standards, and may cause serious problems especially in terms of biosecurity . It also may affect the quality of specimen because of improper condition of specimen transport . It is strongly recommended that there should be determined policy and documented procedures concerning the test performing and their turnaround times in hospital laboratories . Requests of clinicians of different wards of the hospital should take in to the account . The president of hospital should approve the list of performing tests and provide the needed resources.if for any reason the laboratory could not perform a certain groups of tests, the specimen could be sent to a referral laboratory according the related documented standards.- non - conformity managementnone of the groups were able to show any credibility in management of non - conformities and also corrective and preventive actions . Since management of the nonconformities is considered as a more mature step in establishing quality management system, the results of evaluation indicated that all laboratories in public and private sectors had difficulties in detecting the nonconformities and take proper corrective and preventive actions . Training in this field and performing internal audit as well as documentation of the detected nonconformities and taking corrective actions should be strongly considered . - personnel management private sector shows a 10% better implementation ratio in comparison to the public sector . During discussion with the public laboratory directors it was noticed that they encounter certain managerial restrictions, which has affected their field of action as the laboratory director . In some of the laboratories in the public sector, the directors were supposed to only supervise the technical issues and the personnel management was not one of their responsibilities . This issue had apparently affected many aspects of the personnel management such as trainings or related corrective actions . Due to our observation on the other hand as some of the legal regulations such as the permit requirements do not include the public sector, the directors did not feel the necessary responsibility toward the standard implementation . Who in one of its reports brought highly trained individuals tend to move to more stable (and available) jobs in the private sector (8). Individual and organizational attitudes regarding safety will influence all aspects of safe practice, including willingness to report concerns, response to incidents, and communication of risk (9). The detailed analysis of the questions showed that the public laboratories problem area rests in waste management and providing the personnel protective equipment . The personnel are neither well convinced to the importance of documentation nor have received the required sufficient training . The failure is most apparent at usage of control material and also at generating the records of all procedures . One series of five papers shows the private sector performs significantly better than public sector institutions . This series examines this variation in performance with the objective of identifying successful approaches to the management of medical equipment (10). This was mainly due to the old structure of a few hospitals that has affected the total percentage dramatically . Class ii biological safety cabinet only existed in 40% of laboratories irrespective of the group that they belong to . Class ii biological safety cabinet is mandatory for the laboratories that are supposed to perform blood culture . - pre - analytical, analytical and post analytical procedures public laboratories showed less achievement in these fields . Fortunately the weakness was not at the technical aspect of the laboratories but as mentioned earlier it was on documentation management . At this category, which we believe is one of the most fundamental and essential parts for assuring the quality of the results, unfortunately both groups showed weaknesses in these areas and achieved almost 35% . The personnel were not able to analyze the result of quality control procedures and implement the corrective action . We also find out that to empower our assessing tool, we should increase the number of our questions in this field . - purchasing and inventory due to the centralized purchasing department in hospital, laboratory director had minimal say in choosing the supplier . - referral and referee laboratories we noticed that only a handful of public laboratories were a referral laboratory with full capacity . In other cases though the laboratory was not capable of performing all the desired tests but they do not participate in sending the samples . It should be noted that this issue is not in accordance with national standards, and may cause serious problems especially in terms of biosecurity . It also may affect the quality of specimen because of improper condition of specimen transport . It is strongly recommended that there should be determined policy and documented procedures concerning the test performing and their turnaround times in hospital laboratories . Requests of clinicians of different wards of the hospital should take in to the account . The president of hospital should approve the list of performing tests and provide the needed resources . If for any reason the laboratory could not perform a certain groups of tests, the specimen could be sent to a referral laboratory according the related documented standards . - non - conformity management none of the groups were able to show any credibility in management of non - conformities and also corrective and preventive actions . Since management of the nonconformities is considered as a more mature step in establishing quality management system, the results of evaluation indicated that all laboratories in public and private sectors had difficulties in detecting the nonconformities and take proper corrective and preventive actions . Training in this field and performing internal audit as well as documentation of the detected nonconformities and taking corrective actions should be strongly considered . Although in most cases implementing the standard requirements are performed in the private laboratories, there is still a long way to absolute fulfillment of the requirements . Probably lack of motivation, plays a key role in obtaining less desirable results in laboratories in public sectors . In public laboratories there is no financial competition, and no need for licenses renewal through accreditation process, so the pace of progression toward implementation of standards are much slower than what it is in the private sector . Taking in to account that public laboratories are providing service to a large number of people, it is strongly recommended that motivation, responsibility and commitment of laboratory directors and technical staff should be increased through incentive programs . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
Eq . 2 can be generalized to the case where the charge moves continuously, corresponding to an infinite number of steps . If we makezi = qmax / n, i=1 n, vi = vo, i=1 n, then all i =, and we can write eq . 4 can also be written as(5)qnor(v)=12[1+coth[qmax(vvo)2kt]2ktqmax(vv0)],which is of the same form of the classical equation of paramagnetism (see kittel, 2005). Typically, the experimental value of the charge plotted is normalized to its maximum because there is no knowledge of the absolute amount of charge per molecule and the number of molecules . The normalized q - v curve, qnor, is obtained by dividing q(v) by the sum of all the partial charges . 1 shows qnor computed using eq . 2 for one, two, three, four, and six transitions and for the continuous case using eq . 5 (squares) with superimposed fits to a two - state boltzmann distribution (eq . 1, lines). The computations were done with equal charge in each step (for a total charge qmax = 4e0) and also the same vi = 25 mv value for all the steps . It is clear that fits are quite acceptable for cases up to four transitions, but the fit significantly deviates in the continuous case . Examples of normalized q - v curves for a qmax = 4 computed with eq . 2 for the cases of one, two, three, four, and six transitions and the continuous case using eq . 5 (squares). All the q - v curves were fitted with eq . 1 (lines). The insets show the fitted valence (qmax) and half - point (v1/2). Considering that experimental data normally have significant scatter, it is then quite likely that the experimenter will accept the single - transition fit even for cases where there are six or more transitions (see fig . 1). In general, the case up to four transitions will look as a very good fit, and the fitted qmax value may be inaccurately taken and the total charge transported might be underestimated . To illustrate how bad the estimate can be for these cases, we have included as insets the fitted value of qmax for the cases presented in fig . It is clear that the estimated value can be as low as a fourth of the real total charge . The estimated value of v1/2 is very close to the correct value for all cases, but we have only considered cases in which all vi s are the same . It should be noted that if i of the rightmost transition is heavily biased to the last state (vi is very negative), then the qmax estimated by fitting a two - state model is much closer to the total gating charge . In a three - state model, it can be shown that the fitted value is exact when v1 and v2 because in that case, it converts into a two - state model . Although these values of v are unrealistic, the fitted value of qmax can be very close to the total charge when v2 is much more negative than v1 (that is, v1>> v2). On the other hand, if v1 <<v2, the q - v curve will exhibit a plateau region and, as the difference between v1 and v2 decreases, the plateau becomes less obvious and the curve looks monotonic . These cases have been discussed in detail for the two - transition model in lacroix et al . We conclude that it is not possible to estimate unequivocally the gating charge per sensor from a single - boltzmann fit to a q - v curve of a charge moving in multiple transitions . The estimated qmax value will be a low estimate of the gating charge qmax, except in the case of the two - state model or the case of a heavily biased late step, which are rare occurrences . It is then safer to call apparent gating charge the fitted qmax value of the single - boltzmann fit . The most general case in which transitions between states include loops, branches, and steps can be derived directly from the partition function and follows the general thermodynamic treatment by sigg and bezanilla (1997), chowdhury and chanda (2012), and sigg (2013). The reaction coordinate is the charge moving in the general case where it evolves from q = 0 to q = qmax by means of steps, loops, or branches . In that case, the partition function is given by(6)z=iexp(qi(vvi)kt).we can compute the mean gating charge, also called the q - v curve, as(7)q(v)=q=ktzz = ktdlnzdv=iqiexp(qi(vvi)kt)iexp(qi(vvi)kt).the slope of the q - v is obtained by taking the derivative of q with respect to v:(8)dq(v)dv=(kt)2d2lnzdv2 . The charge fluctuation will depend on the number of possible conformations of the charge and is expected to be a maximum when there are only two possible charged states to dwell . As the number of intermediate states increases, the charge fluctuation decreases . Now, a measure of the charge fluctuation is given by the variance of the gating charge, which can be computed from the partition function as:(9)q2=q2q2=(kt)2(zz(zz)2)=(kt)2d2lnzdv2.but the variance (eq . This implies that the slope of the q - v is maximum when there are only two states.

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