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The prostate - specific antigen (psa) level has been an important diagnostic tool for detecting prostate cancer . It has been shown that an abnormal digital rectal examination (dre) has markedly decreased and biochemical findings (elevated psa) have increased over the past 20 years for the indication of prostate biopsy . This means that evaluation of prostate cancer is more and more based on the psa level rather than on suspicious dre . However, cancer detection rates from biopsy in the presence of isolated psa elevation and a normal dre range between 30% and 40% . Also, a high concentration of psa can be found in benign disorders such as benign prostatic hyperplasia (bph), urinary tract infections, and bacterial prostatitis [2 - 5]. Although it has been reported that acute inflammation is necessary to cause psa elevation, there is growing evidence that subclinical inflammation may also contribute to rises in psa levels [6 - 9]. Furthermore, several investigators have shown that treatment of chronic prostatitis, when identified, can decrease psa, which suggests that the use of anti - microbial or anti - inflammatory drugs may reduce the number of men who need prostate biopsies . There is no doubt that symptomatic prostatitis needs to be treated, but uncertainty exists about the appropriate management of asymptomatic patients with elevated psa and normal dre . The aim of our study was to investigate the possibility of reducing the number of prostate biopsies in patients with a high psa level showing a psa decrease or normalization after antibiotic therapy . This approach could be useful in patients for whom it is necessary to postpone biopsy and in patients with previous negative biopsies who are willing to avoid biopsy until further psa increase . This investigation was conducted prospectively among 413 patients with a serum psa level of over 4 ng / ml to under 10 ng / ml from january 2004 to december 2009 . Subjects were excluded if they had been treated by 5-alpha reductase inhibitor for more than 3 months or if they had a history of transurethral resection of the prostate . The psa level was determined by using immunoenzymatic assay before dre and transrectal ultrasonography (trus) to avoid false - positive results . To exclude prostatitis, all patients underwent the expressed prostatic secretion (eps) or voided bladder urine 3 (vb3) test to be classified into two groups . One group had positive findings on the eps or vb3 test and the other group had negative findings . A cover slip was placed over the specimen and it was examined under high - power microscopy . A positive finding was defined as a white blood cell (wbc) count higher than 10 in the prostate secretion after eps or wbc higher than 10 in vb3 urine after eps . The patients with a positive result were treated with quinolone antibiotics for 2 months, and they were asked to return for repeat screening 2 months later . If the psa level was still higher than 4 ng / ml after 2 months, the patients underwent prostate biopsy . In the other cases, the patients avoided prostate biopsy the subjects underwent at least 10 core biopsies with transrectal ultrasound - guided needle biopsy of the prostate . Chicago, il, usa) was used for the statistical analysis, and independent t - tests were used for analysis of the characteristics of both groups . Of the 413 men studied, 215 (52%) patients had positive findings on the eps or vb3 test . Of the 215 patients, 53 men avoided prostate biopsy because their serum psa level had decreased to less than 4 ng / ml . The other patients (162 of 215) still had an elevated serum psa level of more than 4 ng / ml, including 7 in whom the biopsy results revealed cancer . Patients with negative findings on the eps or vb3 test (198 of 413) underwent prostate biopsy immediately (fig . The total prostate cancer detection rate was 11.6% in our subjects, whereas it was 20.7% in the patients with negative findings on the eps or vb3 test and 3.3% in those with positive findings, respectively . The mean age was significantly different between the positive group (65.0) and the negative group (71.2). There were no significant differences in the mean initial serum psa level or prostate volume between the groups (table 1). Since psa was introduced in the 1980s as a variable for detecting prostate cancer, the detection rate of prostate cancer has been increasing . It is true that an abnormal finding on a dre or trus is an indication for prostate biopsy, but in the majority of cases, the psa level is the most important factor in deciding whether we will proceed with prostate biopsy . However, it is reported that 30 to 40% of patients who test positive for prostate cancer in prostate biopsy proceeded when only the psa was increased without any abnormality in the dre or trus . The specificity and sensitivity of psa can result in high values in other positive diseases . The psa may increase not only in bph but also in urinary tract infection and bacterial prostatitis . The psa can increase in acute inflammation and silent infection [7 - 9,12]. Also, some researchers have reported that treatment with antibiotics and anti - inflammatory drugs for chronic prostatitis can decrease the psa and the necessity for prostate biopsy . Nadler et al . Reported that the size and infection of the prostate can be a chief cause of psa increase in the case of patients not diagnosed with prostate cancer . But irani et al . Reported that even if there is an infiltration of inflammatory cells in the prostatic interstitium, it is not related to the psa increase in the blood if the glandular epithelial cell layer is not destroyed at the same time . Furthermore, it has been reported that the existence of inflammatory cells of the interstitium or glandular tissue in the prostate biopsy has no statistical affiliation with the concentration of psa in the blood . Carver et al . Argued that national institutes of health (nih) category iv asymptomatic prostatitis increases the psa concentration in the blood, but its clinical significance is not that greatbecause the mean differences with the normal group are 2.3 ng / ml and 1.4 ng / ml, respectively . Reported that there is an increase of the concentration of psa in the blood in 71% of patients with acute prostatitis, 15% of patients with chronic prostatitis, and 6% of patients with nonbacterial prostatitis, but there was no psa increase in patients who showed only symptoms of chronic prostatitis . Found a relation of the psa concentration in the blood with acute and chronic prostatitis accompanied by clinical symptoms identified histologically, but no relation in the case of deactivated prostatitis . Chang et al . Investigated 223 patients who tested negative in a prostate biopsy by classifying the infection level and reached the conclusion that the whole size of the prostate is an important factor that contributes to the increase in the psa concentration in the blood, but that there was no relation with the prostatitis level . The relationship of prostatitis and the psa increase continues to stimulate dispute, and we suggest that the findings mentioned above are not sufficient to explain the reason for the psa increase in the case of nih category iv asymptomatic prostatitis . Nadler et al . Investigated 421 patients with nih category iii chronic prostatitis and reported recently that the mean psa concentration in the blood of the patients investigated was 1.97 ng / ml . Compared with the level in the normal group of 1.72 ng / ml, this was a statistically significant increase, but it is still below the normal psa range . Also, f - psa, percent f - psa, and the [-2] ppsa isoform show some increase with prostate cancer, but they are inappropriate to be used as biomarkers of prostate cancer diagnosis because of their low specificity and sensitivity . Treatment with antibiotics in the case of chronic prostatitis and increased psa results in a very significant psa decrease . But any special bacteria is found in 90% of cases of prostatitis with or without symptoms . This kind of investigation of the changes in psa after treatment with antibiotics or anti - inflammatory drugs for nonbacterial prostatitis is uncommon . According to the report of potts among 122 patients with an average psa increase of 9.35 ng / ml, 51 (42%) patients were diagnosed with infection by urinalysis after a massage of the prostate or prostatic secretion (eps). The patients were then treated with antibiotics for 4 weeks, and after 6 to 8 weeks they had a psa examination . After the treatment, 22 of the 51 patients had a normal psa concentration (average, 2.9 ng / ml) and they did not need a prostate biopsy . But there was a continuous increase of psa in the remaining 29 patients, and among them, 9 patients were diagnosed with prostate cancer . In comparison with patients diagnosed with cancer in prostate biopsy after treatment with antibiotics, the psa decrease in patients with benign biopsy results was significantly greater (-1.3% vs. -21.3%). In the investigation carried out with 95 patients diagnosed with nonbacterial prostatitis and who showed an increase of psa, bozeman et al . Proceeded with prostate biopsy in patients in whom the psa value did not decrease after 4 weeks of treatment with antibiotics and anti - inflammatory drugs . Among these, the psa of 36.4% of patients decreased from 8.48 ng / ml to 5.39 ng / ml after treatment; in 44 patients (46.3%) the psa decreased below 4 ng / ml, so they avoided prostate biopsy . In the remaining patients who underwent prostate biopsy, 13 patients (25.5%) were diagnosed with prostate cancer, 37 patients (72.5%) with chronic infection, and 1 patient (1.05%) with bph . Among the patients diagnosed with prostate cancer, just 4.8% of patients had shown a psa decrease from 8.32 ng / ml to 7.92 ng / ml, which was not statistically significant . Therefore, the authors argued that chronic prostatitis is the chief cause of psa increase and that treatment for prostatitis can decrease unnecessary prostate biopsy . But hochreiter argued that although the authors of two investigations considered that the patients were not diagnosed with prostate cancer because the psa was decreased to less than 4 ng / ml, it was difficult to conclude that the patients in fact had no cancer . It is not a certainty that psa - normalized patients are cancer free after antibiotic therapy . There are no general guidelines that can be applied to all men with increased psa before prostate biopsy . In cases in which the psa level is increasing, if we first exclude prostatitis and carry out a serial diagnostic procedure, it may help to reduce unnecessary prostate biopsy.
Generation of transgenic lines is described in the supplementary methods . To induce recombination and subsequently genetically label cardiomyocytes, embryos / adults were treated with 4-oht followed by a wash period of at least 1 week prior to amputation . Adult fish were anesthetized in 0.4%tricaine and secured ventral side up in a slotted sponge . Watchmaker forceps were used to remove the surface scales and penetrate the skin, muscle, and pericardial sac . Once exposed, the ventricle was gently pulled at the apex and cut with iridectomy scissors . After surgery, fish were immediately returned to system water . At the specified time points, hearts were removed and fixed in 4% paraformaldehyde overnight at 4c, washed several times in pbs, equilibrated in 30% sucrose in pbs, and frozen for cryosectioning . Fish were anesthetized in 0.4%tricaine, and 0.5ml of a 2.5mg / ml solution of brdu (in pbs) was injected into the abdominal cavity once every 24 h for 7 d. after 14 d, hearts were removed and fixed in 4% paraformaldehyde overnight at 4c, washed several times in pbs, equilibrated in 30% sucrose in pbs, and frozen for cryosectioning . Cyclapolin 9 (sigma c6493) was dissolved in dmso and added to 400ml system water to a final concentration of 3m . Generation of transgenic lines is described in the supplementary methods . To induce recombination and subsequently genetically label cardiomyocytes, embryos / adults were treated with 4-oht followed by a wash period of at least 1 week prior to amputation . Adult fish were anesthetized in 0.4%tricaine and secured ventral side up in a slotted sponge . Watchmaker forceps were used to remove the surface scales and penetrate the skin, muscle, and pericardial sac . Once exposed, the ventricle was gently pulled at the apex and cut with iridectomy scissors . After surgery, fish were immediately returned to system water . At the specified time points, hearts were removed and fixed in 4% paraformaldehyde overnight at 4c, washed several times in pbs, equilibrated in 30% sucrose in pbs, and frozen for cryosectioning . Fish were anesthetized in 0.4%tricaine, and 0.5ml of a 2.5mg / ml solution of brdu (in pbs) was injected into the abdominal cavity once every 24 h for 7 d. after 14 d, hearts were removed and fixed in 4% paraformaldehyde overnight at 4c, washed several times in pbs, equilibrated in 30% sucrose in pbs, and frozen for cryosectioning . Cyclapolin 9 (sigma c6493) was dissolved in dmso and added to 400ml system water to a final concentration of 3m.
The prevention and treatment of ovarian hyperstimulation syndrome (ohss), an iatrogenic and potentially life - threatening disease process, which may occur in healthy young women undergoing controlled ovarian hyperstimulation (coh) for assisted reproduction has been the subject of maximum research and innovation since the inception of assisted reproductive technology (art). As the exact etiopathogenesis of this syndrome is still elusive, the treatment is largely empirical and thus prevention forms the mainstay of management . The preventive strategies aim, to target women at high risk of developing ohss and institution of various pharmacological and non - pharmacological interventions on them . The pharmacological tools being used are: low - dose follicle stimulating hormone (fsh) or gonadotropin - releasing hormone antagonist protocol during stimulation, albumin infusion at the time of oocyte recovery, dopamine agonist cabergoline (cb2) started from the day of ovulation trigger and institution of an insulin sensitizer like metformin, whereas the non - pharmacologic modalities incorporated are: coasting, cycle cancellation, cryopreservation of all embryos for future transfer or use of in vitro maturation . Although these preventive measures have not been able to completely eliminate this iatrogenic complication but have definitely brought about a reduction in the severity of ohss as, it is the, severe ohss, which is the cause of maximum concern . Severe forms of ohss may complicate 0.5 - 5% of in vitro - fertilization (ivf) cycles and can lead to severe morbidity and even mortality if not timely and appropriately intervened . As the search for an ideal preventive therapy continued, a new and innovative therapy in the form of calcium gluconate infusion was introduced in the armamentarium of reproductive specialists to prevent this potentially dreadful complication, however, data and literature on its effectiveness is presently limited . In the present study, we aimed to evaluate the effectiveness of intravenous (iv) calcium infusion in comparison to the dopamine agonist cb2 in preventing ohss in high risk patients undergoing assisted reproductive technique cycles . A total of 202 patients at risk for developing ohss while undergoing ivf cycle at our center during the period of 01 january 2011 - 31 may 2012 were included in the study . It was a comparative study wherein the 202 high risk patients after meeting the strict inclusion and the exclusion criteria, were randomly divided into two groups with 98 subjects in group i and 104 in group ii . The women with even registration numbers were in group i and were administered iv calcium gluconate infusion while the remaining 104 who had odd registration numbers belonged to group ii and received the dopamine agonist cb2 . Evaluation of the subjects for the development of ohss was the primary outcome measure and requirement of hospitalization and abdominal paracentesis or pleural tap in the event of significant fluid collection because of severe ohss was the secondary outcome measure . The inclusion criteria or patients who were at risk for ohss were: known case of polycystic ovarian disease as diagnosed by the rotterdam criteria (2004), development of 18 or more follicles larger than 12 mm in diameter and history of ohss in the previous ivf cycle if any . The patients were excluded from the study if they had other endocrinopathies in the form of diabetes mellitus, hyperprolactinemia or congenital adrenal hyperplasia . Women with systemic diseases like bronchial asthma, hypertension or bleeding disorders were also excluded . If an antagonist cycle was instituted for coh the patients were not included in the study . All 202 patients were administered oral contraceptive pills (tablet loette; pfizer) from the 5 day of their menstrual cycle prior to their down regulation by the luteal long protocol . Leuprolide acetate (lupride; abbott cedex) 1 mg subcutaneously was started from the 21 day of the cycle, i.e., from the 17 day of oral contraceptive pill treatment . This dose was reduced to 0.5 mg / day once down regulation was confirmed both sonologically and by serum estradiol levels (<50 pg / ml). After the documentation of ovarian quiescence, recombinant fsh 150 iu / d (gonal f; merck serono) was administered for next 4 days and doses adjusted thereafter as per the ovarian response as evident on the trans vaginal sonography . Human chorionic gonadotropin (hcg) 10,000 iu i m (pregnyl; organon) was administered if greater than three follicles reached a mean diameter of 18 mm . Conventional ivf or intracytoplasmic sperm injection was performed on the retrieved oocytes depending on the couple's history . The embryos were graded on day 3 according to a 1 - 4 scoring system with 1 being the best, which was based on fragmentation, cell symmetry and blastomere number . Embryo transfer was not carried out in any of the high risk study group patients and all grade 1 and grade 2 embryos were cryopreserved for future transfer while grade 3 and grade 4 embryos were discarded after informing the patients . The patients were informed about the status of their embryos and fertilization failure if any . All the high risk subjects were managed on an out - patient basis with daily clinical and sonological monitoring and hospitalized only if they went into severe ohss or their clinical condition mandated close supervision . A baseline hematological and biochemical profile on the day of pick up and daily thereafter was also carried out for the study group subjects as a part of monitoring for ohss . The 104 patients belonging to group ii were started on tablet cb2 (tab dostinex 0.5 mg; pfizer) 0.5 mg / day from the day of ovulation trigger and continued until the next 8 days . The 98 high risk patients from group i were started on calcium gluconate injection after ovum pick up as per the protocol mentioned by yakovenko et al . It was prepared by dissolving 10 ml of 10% calcium gluconate solution in 200 ml of physiologic saline and instituted over a period of 40 min . This infusion was administered within 30 min of oocyte retrieval on the day of ovum pickup and on day 1, day 2 and day 3 thereafter . Mild ohss was described as the presence of pelvic discomfort, abdominal distension, nausea vomiting and/or diarrhea and enlarged ovaries as seen on sonography (5 - 12 cm). Moderate ohss was described as the presence of features of mild ohss plus ultrasonic evidence of ascites in the pouch of douglas and pelvis and, enlarged ovaries . In the presence of severe ohss, a lady had features of moderate ohss plus evidence of ascites and/or hydrothorax and breathing difficulties . In addition, there were changes in the blood volume, with increased blood viscosity due to hemoconcentration (hematocrit> 45%), coagulation abnormality and diminished renal perfusion and function (low urine output <600 ml/24 h). All the patients (from both the study groups) after the day of oocyte recovery were monitored daily by clinical examination and a transvaginally sonography . Calcium gluconate infusion in the first group was instituted thereafter until the next 3 days . The volume of ascitic fluid drained daily and the changes in hematological parameters in the hospitalized subjects were recorded . The patients even after completion of calcium gluconate infusion protocol or cb2 protocol were monitored on an out - patient basis until complete resolution of signs and symptoms . The primary aim of this study was to evaluate the occurrence of ohss in both the treatment groups . Assuming the incidence of ohss as 5% with 90% power along with 6% permissible error, the minimum required sample size was estimated as 191 to detect the true difference between the two groups . The test and student's t - test were used for statistical analysis with statistical package for the social sciences software, version 18.0 for windows . A total of 202 patients at risk for developing ohss while undergoing ivf cycle at our center during the period of 01 january 2011 - 31 may 2012 were included in the study . It was a comparative study wherein the 202 high risk patients after meeting the strict inclusion and the exclusion criteria, were randomly divided into two groups with 98 subjects in group i and 104 in group ii . The women with even registration numbers were in group i and were administered iv calcium gluconate infusion while the remaining 104 who had odd registration numbers belonged to group ii and received the dopamine agonist cb2 . Evaluation of the subjects for the development of ohss was the primary outcome measure and requirement of hospitalization and abdominal paracentesis or pleural tap in the event of significant fluid collection because of severe ohss was the secondary outcome measure . The inclusion criteria or patients who were at risk for ohss were: known case of polycystic ovarian disease as diagnosed by the rotterdam criteria (2004), development of 18 or more follicles larger than 12 mm in diameter and history of ohss in the previous ivf cycle if any . The patients were excluded from the study if they had other endocrinopathies in the form of diabetes mellitus, hyperprolactinemia or congenital adrenal hyperplasia . Women with systemic diseases like bronchial asthma, hypertension or bleeding disorders were also excluded . If an antagonist cycle was instituted for coh the patients were not included in the study . All 202 patients were administered oral contraceptive pills (tablet loette; pfizer) from the 5 day of their menstrual cycle prior to their down regulation by the luteal long protocol . Leuprolide acetate (lupride; abbott cedex) 1 mg subcutaneously was started from the 21 day of the cycle, i.e., from the 17 day of oral contraceptive pill treatment . This dose was reduced to 0.5 mg / day once down regulation was confirmed both sonologically and by serum estradiol levels (<50 pg / ml). After the documentation of ovarian quiescence, recombinant fsh 150 iu / d (gonal f; merck serono) was administered for next 4 days and doses adjusted thereafter as per the ovarian response as evident on the trans vaginal sonography . Human chorionic gonadotropin (hcg) 10,000 iu i m (pregnyl; organon) was administered if greater than three follicles reached a mean diameter of 18 mm . Conventional ivf or intracytoplasmic sperm injection was performed on the retrieved oocytes depending on the couple's history . The embryos were graded on day 3 according to a 1 - 4 scoring system with 1 being the best, which was based on fragmentation, cell symmetry and blastomere number . Embryo transfer was not carried out in any of the high risk study group patients and all grade 1 and grade 2 embryos were cryopreserved for future transfer while grade 3 and grade 4 embryos were discarded after informing the patients . The patients were informed about the status of their embryos and fertilization failure if any . All the high risk subjects were managed on an out - patient basis with daily clinical and sonological monitoring and hospitalized only if they went into severe ohss or their clinical condition mandated close supervision . A baseline hematological and biochemical profile on the day of pick up and daily thereafter was also carried out for the study group subjects as a part of monitoring for ohss . The 104 patients belonging to group ii were started on tablet cb2 (tab dostinex 0.5 mg; pfizer) 0.5 mg / day from the day of ovulation trigger and continued until the next 8 days . The 98 high risk patients from group i were started on calcium gluconate injection after ovum pick up as per the protocol mentioned by yakovenko et al . It was prepared by dissolving 10 ml of 10% calcium gluconate solution in 200 ml of physiologic saline and instituted over a period of 40 min . This infusion was administered within 30 min of oocyte retrieval on the day of ovum pickup and on day 1, day 2 and day 3 thereafter . Mild ohss was described as the presence of pelvic discomfort, abdominal distension, nausea vomiting and/or diarrhea and enlarged ovaries as seen on sonography (5 - 12 cm). Moderate ohss was described as the presence of features of mild ohss plus ultrasonic evidence of ascites in the pouch of douglas and pelvis and, enlarged ovaries . In the presence of severe ohss, a lady had features of moderate ohss plus evidence of ascites and/or hydrothorax and breathing difficulties . In addition, there were changes in the blood volume, with increased blood viscosity due to hemoconcentration (hematocrit> 45%), coagulation abnormality and diminished renal perfusion and function (low urine output <600 ml/24 h). All the patients (from both the study groups) after the day of oocyte recovery were monitored daily by clinical examination and a transvaginally sonography . Calcium gluconate infusion in the first group was instituted thereafter until the next 3 days . Hematological and biochemical profile was also carried out . Any patient requiring any active intervention or who went into severe ohss the volume of ascitic fluid drained daily and the changes in hematological parameters in the hospitalized subjects were recorded . The patients even after completion of calcium gluconate infusion protocol or cb2 protocol were monitored on an out - patient basis until complete resolution of signs and symptoms . The primary aim of this study was to evaluate the occurrence of ohss in both the treatment groups . Assuming the incidence of ohss as 5% with 90% power along with 6% permissible error, the minimum required sample size was estimated as 191 to detect the true difference between the two groups . The test and student's t - test were used for statistical analysis with statistical package for the social sciences software, version 18.0 for windows . There were 202 women included in the study of which 98 were in group i and 104 belonged to group ii . The mean female age was 28.1 3.3 years in group i whereas it was 28.2 3.3 years, in group ii (p> 0.05). It was observed that there was no significant difference in terms of female age (years), body mass index (kg / m), duration of infertility (years), basal hormone levels (fsh [miu / ml]), basal antral follicle count, length of ovarian stimulation (days), number of follicles on the day of hcg trigger and the number of oocytes retrieved in both the study groups [table 1]. Fertilization rate, cleavage rate and implantation rate among the groups have not been evaluated and included as it was not part of the study as all the high risk patients underwent frozen embryo transfer . Comparison of clinical and laboratory characteristics in calcium gluconate - administered group (group i) and cabergoline group (group ii) statistical analysis also revealed that the number of women undergoing the first or the second ivf cycle were also comparable so was the history of ohss in the previous cycle in patients undergoing second cycle in both the study groups (24.5% vs. 26%) [table 2]. Comparison of cycle characteristics in the two treatment group the occurrence of ohss was seen in only nine patients (9.2%) in the calcium infusion group, as compared to 16 patients (15.4%) who were administered cb2 . However, the rate of occurrence of ohss in both the study group was not found to be statistically significant . Among the nine patients in the calcium infusion group who developed the complication only one had severe ohss, whereas two women were diagnosed as severe ohss belonging to the cb2 arm [table 3]. All the three women (from both the study groups) with severe ohss required hospitalization . Two patients from the cb2 group required ascitic tap due to massive ascites and breathing difficulties and one woman with severe ohss from group i required abdominal paracentesis . These patients' condition improved with supportive therapy and interventions such as abdominal paracentesis and were discharged once asymptomatic . The patients with mild and moderate ohss from both the study groups were monitored on an out - patient basis until the resolution of signs and symptoms . None of the patients who were started on calcium gluconate injection developed any allergic reactions, anaphylaxis, symptoms or signs of hypercalcemia, or other side effects . Severity of ohss in the two study group (n = number of patients with ohss in two arms) of the various pathophysiological mechanisms implicated for the causation of ohss, it is the angiogenic molecule, vascular endothelial growth factor (vegf), which has been found to be the biggest mediator of this potentially dreadful complication . It has been proven that vegf stimulates new blood vessel development and vascular hyper permeability by interacting with its vegf receptor 2 (vegfr-2). Thus various studies were carried out, which have proven that dopamine agonists can inhibit phosphorylation of the receptor vegfr-2 and can thus reduce the vascular permeability and various presentations of ohss in the art cycles . In pursuance, cb2 was therefore extensively studied and was found to bring about a decrease in the severity or incidence or both of ohss . Apart from the increased capillary permeability brought about by vegf which results in a fluid shift from the intravascular space to third space compartments the other contributing factors elucidated in the pathophysiology of ohss are: increased secretion or exudation of protein - rich fluid from enlarged ovaries or peritoneal surfaces, increased follicular fluid levels of prorenin and renin and increased angiotensin - mediated changes in capillary permeability . Thus the effectiveness and safety of dual renin - angiotensin system blockage for prevention of ohss in over stimulated patients undergoing ivf was evaluated in a study . However, this new strategy for use in patients at high risk for ohss did not completely eliminate the development of the syndrome . In addition, there were concerns about the safety of the treatment, as angiotensin - converting enzyme inhibitors and angiotensin receptor antagonists were associated with possible teratogenic effect in humans . Nevertheless, this study did suggest the possible role of an altered renin angiotensin system in the development of ohss . Another observation found in a separate study was the stimulatory role of low intracellular calcium on adenylyl cyclase, which resulted in cyclic adenosine monophosphate (camp) synthesis and thus, renin release . It was also postulated by beierwaltes that renin secretion is inversely related to the extracellular and intracellular calcium concentrations and therefore, calcium may modify the amplitude of camp - mediated renin - signaling pathways . Thus it was inferred that although calcium does not directly control renin secretion, increased calcium inhibits and decreased calcium amplifies camp - stimulated renin secretion . Gurgan et al . In their retrospective study also researched and found that calcium infusion successfully prevents the development of severe ohss and significantly decreases ohss occurrence rates without any major adverse effect when used for high - risk patients such as those with polycystic ovary syndrome (pcos). With the background of success, demonstrated by both cb2 and calcium for the prevention of this iatrogenic complication, we compared their efficacy . Identifying patients who are at - risk is the most critical step in the prevention of ohss as it guides a clinician to make changes to the ovarian stimulation regimen and to add other preventative measures . Predictive factors for ohss can be primary risk factors, which confer an increased risk of ohss on patients and secondary risk factors, which become apparent during ovarian stimulation when patients with no known predisposing factors experience an excessive response to treatment . In this study too, we incorporated these risk factors and targeted them to either of the preventive strategy . It is noteworthy that in this study we compared the two drugs which ultimately targeted the same key molecule: vegf . The pathway of reaching the target and the mode of administration of both the drugs might be different but the preventive mechanism was same, i.e., either antagonizing vegf receptor as in cb2 or decrease vegf levels as with calcium gluconate infusion . It has been hypothesized that calcium infusion for patients with high - risk factors for ohss, initially prevent renin secretion . Reduced renin production results in decreased angiotensin ii synthesis . As a consequence, the stimulatory effect of angiotensin ii on vegf production all these pathophysiologic mechanisms (decreased synthesis of renin, angiotensin ii and vegf) which occurs from calcium gluconate infusion, therefore prevents the development of ohss in such high risk patients undergoing art cycles . Our results also document that calcium infusion can effectively prevent the development of severe ohss and decreases ohss occurrence rates without any major adverse affect when used for high - risk patients such as those with pcos . It also needs to be reiterated that very few studies have been carried out on the efficacy of this new strategy of calcium gluconate infusion and comparison between this novel protocol and the established role of dopamine agonist is the first of its kind . Our observations in this present study was only limited to the occurrence of early onset of ohss in contrast to the findings of carizza et al . As we carried out freezing of all the embryos so late onset of ohss was not evaluated . We can say that even though both the drugs were found to be equally effective for the prevention of ohss and most importantly in decreasing the severity of this potentially life - threatening complication, their effect on the implantation process and the comparison of the pregnancy rate, implantation rate and miscarriage rate was not computed which accounts for the limitation of this study . Larger well designed trials need to be carried out incorporating the aforementioned factors as well as measurements of vegf levels in both the study groups . Nevertheless, as both the drugs are safe, cheap and have comparable success rates either of them can be employed as a treatment strategy for patients with high risk factors for ohss undergoing art cycles.
A 32-year - old man was transferred to an intensive care unit (icu) due to respiration difficulties from a hepatology ward where he had been admitted after a 7-day history of symptoms of acute viral hepatitis a (ha) and a 3-day history of progressive distal and proximal weakness of limbs with additional facial weakness . Initial laboratory tests performed at the ward (results listed in table 1) suggested acute viral ha infection with high igm anti - ha antibody titers . Despite improvement in liver function and hepatitis symptoms, nerve conduction was studied and cerebrospinal fluid (csf) was tapped due to progressive weakness . A diagnosis of guillain - barr syndrome (gbs) was supported by albuminocytologic dissociation (1 white blood cell / mm, 2 red blood cells / mm, 115.0 mg / dl protein, and 58 mg / dl glucose) and multiple motornerve - conduction defects with decreased compound motor action potentials (table 2). On the second day in the icu his heart rate was 85~120 beats / min and his arterial blood pressure was up to 200/110 mmhg . His pupils were dilated to 5 mm and not reactive to light . On day 4 a positive pupil response to a 0.1%-pilocarpine test indicated ciliary postganglionic parasympathetic neuropathy with supersensitivity to acetylcholine . These tests and signs indicated the absence of all brain reflexes, suggesting the presence of peripheral deafferentation . In addition, complementary laboratory tests for vasculitis or other infectious agents were normal except for the presence of latent herpes simplex virus (hsv) infection . A polymerase chain reaction for hsv was negative, and no symptoms of hsv infection were detected . A follow - up nerve - conduction study performed 2 weeks after transfer to the icu suggested severe demyelinating sensorimotor polyneuropathy (table 2). Intravenous gammaglobulin (ivig) (0.4 g / kg / day) was administered for 5 days, but his neurological status had worsened due to fulminant deafferentation . Therefore, pulse therapy with methylprednisolone (500 mg / day) were given for 3 days . After the initial 5-day course of therapy, ivig was added twice weekly 2 weeks later . Three months later he recovered complete power in his upper and lower limbs, except for a residual deficit of left foot weakness (dorsiflexor: grade 4, plantar flexor: grade 4) and bilateral hypopathic sensory change of soles . The development of clinical symptoms of hepatitis, marked increases in bilirubin, aspartate aminotrasferase, alanine aminotrasferase (alt), and -glutamyltransferase levels, and a positive igm - ha virus antibody test supported the diagnosis of acute ha . Cases exhibiting an association between gbs and ha are extremely rare.1 the reported clinical features of nine reviewed cases of gbs following ha were as follows: (1) a uniformly good outcome of the neuropathic symptoms, independent of the level of alt, which corresponds to the severity of liver dysfunction; (2) highest occurrence in men; and (3) the interval between the onset of the hepatitis and the development of neuropathic symptoms is less than 14 days.2 the hsv infection in our patient, in spite of neither symptoms nor signs, might have aggravated the severity of gbs . The initial nerve - conduction study and blink - reflex test in this patient revealed inexcitability of most nerves, which was due to distal pathology of the motor axons: either a distal conduction block or axonal degeneration . The nature of this pathology cannot be predicted by the results of an initial electrophysiological evaluation.3 the very prolonged distal motor latencies in the electromyogram recorded in a subsequent nerve - conduction study suggested the presence of severe demyelinating polyneuropathy and axonopathy . In the case of gbs mimicking cerebral death,4 a sural nerve biopsy indicated that demyelination was the early pathological mechanism . Therefore, the exact physiopathology and whether our patient had distal demyelination and conduction block with secondary axonal loss or axonal degeneration, or both, remained unclear . The various treatments used for fulminant gbs mimicking cerebral death are plasma exchange, ivig, or plasma exchange and corticosteroids.4 our patient was treated with ivig to modulate the immunologic reaction, and with high - dose pulse therapy with methylprednisolone for suppressing the acute severe inflammation in the peripheral nervous system . There is no evidence that conventional doses of corticosteroids (around 60 mg of prednisone daily) are effective in shortening the course of or reducing residual deficits in acute gbs.5 however, high - dose steroids are perhaps currently best applied to patients who cannot tolerate plasma exchange (e.g., due to severe cardiovascular dysautonomia) or when other treatments are unavailable.6 - 8 our patient recovered progressively, and could walk alone with mild weakness of the left foot at 3 months after admission . Gbs mimicking brain death usually has a poor recovery rate and a high mortality, particularly in relation to dysautonomia.9 the timely application of combination pulse therapy with both ivig and methylprednisolone might improve the prognosis of fulminant gbs . In summary, in rare cases gbs presents with signs of coma and absent brainstem reflexes.
Keratoacanthoma is a rapidly evolving cutaneous tumor composed of keratinizing squamous cells originating from the supra - seboglandular portion of the hair follicle . Keratoacanthoma may be solitary or multiple but the most common form is the classical solitary variety, which occurs on the exposed body parts of the elderly person, and involutes by itself even if left untreated . Multiple keratoacanthomas, on the other hand, develop sporadically (e.g. Eruptive keratoacanthomas of the grzybowski type, or the familial syndromes like muir - torre syndrome, or its incomplete form, ferguson - smith syndrome). Keratoacanthoma centrifugum marginatum (kcm) is among the rare varieties and, in contrast to the classical variety, does not show any tendency of spontaneous regression . Lack of spontaneous regression is also a feature of another rare variant of keratoacanthoma, the giant keratoacanthoma, from which kcm is differentiated by the absence of downward vertical spread with destruction of the underlying tissue . Progressive peripheral extension with a raised rolled - out margin and atrophy at the center of the lesion is a characteristic feature of the kcm . The most common site of kcm is reported to be the dorsum of the hands and legs . The etiology of kcm is multifactorial, which includes chronic ultraviolet ray exposure, the smoking habit, and contact with chemical carcinogens like pitch, mineral oil, tar, etc . The role of human papilloma virus (hpv) in keratoacanthoma remains inconclusive, with one study finding evidence of hpv infection by polymerase chain reaction, whereas another study failing to detect any virus material . A 65-year - old healthy male, a cultivator by occupation, presented with a solitary painful plaque involving his left arm and forearm . It started spontaneously four years previously as a hyperpigmented nodule on the extensor aspect of the left forearm, which was operated on by a local doctor . The lesion recurred following the operation and gradually grew peripherally with healing of the center . The patient could not recall any history of trauma preceding the onset of the lesion, neither was there any family history of a similar disorder . Apart from tobacco smoking, there was no significant history of drug addiction, ingestion of halogenated compounds, or contact with chemicals . On examination, a large single plaque 3010 cm in size was found occupying the left forearm (involving both dorsal and ventral aspects), the elbow, and the lower part of the left arm (figure 1). The margin of the lesion was rolled out and interrupted, having hyperpigmented, firm, discrete, tender nodules and a cribriform pattern with comedonal orifices . The nodules had a central depression and foul - smelling cheesy material could be squeezed out on application of firm pressure on these nodules (figure 2). The center of the plaque was atrophic and depigmented with intervening areas of normal skin . The lesion was not found to be adherent to the underlying structures and there was no regional lymphadenopathy or organomegaly . Figure 1plaque with rolled - out interrupted margin and atrophic center with island of normal skin located on the extensor aspect of the left forearm and lower part of the left arm . Plaque with rolled - out interrupted margin and atrophic center with island of normal skin located on the extensor aspect of the left forearm and lower part of the left arm . Figure 2close - up picture showing cheesy material exuding from (after being compressed laterally) a punctum overlying the margin of the lesion . Close - up picture showing cheesy material exuding from (after being compressed laterally) a punctum overlying the margin of the lesion . Hematocrit, platelet, and leukocyte counts, and hepatic and renal function tests were within normal limits . A chest x - ray and ecg showed no abnormality . An x - ray of the left hand including wrist and elbow joints showed no bony involvement . The culture from the cheesy material revealed the presence of a gram - negative organism belonging to proteus species . A biopsy taken from the margin of the lesion showed large irregularly shaped craters filled with keratin (figure 3) and, from the base of the crater, irregular epidermal proliferations extending downward but not extending below the level of the sweat glands (figure 4). At the periphery of the epidermal proliferations there was evidence of a thin layer of basophilic cells with the base of the crater containing epithelial pearls (figure 5) and showing rich keratinization imparting a glassy appearance (figure 6). A mild to moderate degree of mononuclear cellular infiltrate was present within the dermis but mitotic figures were found rarely . There was no evidence of granuloma and no fungal element could be detected with pas staining . Figure 3histopathological section showing a large crater filled with keratin and having irregular epithelial proliferations extending into the papillary dermis . The basal layer is intact with no evidence of cellular atypia (hematoxylin and eosin stain; 100 magnification). Histopathological section showing a large crater filled with keratin and having irregular epithelial proliferations extending into the papillary dermis . The basal layer is intact with no evidence of cellular atypia (hematoxylin and eosin stain; 100 magnification). Figure 4cross - section of the crater showing a large cavity filled with keratin and irregular epidermal proliferations extending downward from its base, but not extending below the level of the sweat gland (hematoxylin and eosin stain; 40 magnification). Cross - section of the crater showing a large cavity filled with keratin and irregular epidermal proliferations extending downward from its base, but not extending below the level of the sweat gland (hematoxylin and eosin stain; 40 magnification). Figure 5histopathological section of the lateral wall of the epithelial proliferation showing a thin uninterrupted layer of basophilic cells and the base of the crater having epithelial pearls and well - keratinized cells giving a glassy appearance . No abnormal mitotic figures are detected and the papillary dermis shows scattered mononuclear cell infiltrates (hematoxylin and eosin stain; 100 magnification). Histopathological section of the lateral wall of the epithelial proliferation showing a thin uninterrupted layer of basophilic cells and the base of the crater having epithelial pearls and well - keratinized cells giving a glassy appearance . No abnormal mitotic figures are detected and the papillary dermis shows scattered mononuclear cell infiltrates (hematoxylin and eosin stain; 100 magnification). Figure 6higher magnification of the epidermal proliferation showing an eosinophilic, glassy appearing cell mass, marginated by a 12 layer of basophilic nonkeratinized cells (hematoxylin and eosin stain; 400 magnification). Higher magnification of the epidermal proliferation showing an eosinophilic, glassy appearing cell mass, marginated by a 12 layer of basophilic nonkeratinized cells (hematoxylin and eosin stain; 400 magnification). Figure 7six months after wide excision and repair of the nodules on the margins of the lesion showing the grafts (near the wrist and above the elbow) satisfactorily taken . Six months after wide excision and repair of the nodules on the margins of the lesion showing the grafts (near the wrist and above the elbow) satisfactorily taken . The clinicopathological correlation confirmed the case to be kcm and the patient was started on acitretin (initially 0.5 mg / kg / day then increasing the dose to 1 mg / kg / day after 15 days). Despite treatment for three consecutive months, the lesion showed no signs of improvement and a few new nodules appeared near the elbow crease . The nodules at the margin of the lesions were excised with a 1-cm margin, up to a depth of the antebrachial fascia . Postoperative graft uptake was satisfactory and the patient has been followed - up for the past six months, showing no signs of recurrence (figure 5). Kcm is a very rare type of keratoacanthoma, and a pubmed search produced 31 entries of kcm in the world literature since its first description in 1962 . Diagnosis of the condition was a real challenge in our case and meticulous clinical and histopathological examinations were required to differentiate it from several other conditions that closely mimic kcm . The clinical differential diagnoses include squamous cell carcinoma (scc), lupus vulgaris (lv), botryomycosis, blastomycosis - like pyoderma (blp), and pseudoepitheliomatous hyperplasia (peh). The fact that the lesion showed resolution in some places was a useful clinical clue in differentiating it from scc . The presence of mitotic figures in our case cannot be taken as a marker of scc as there were very few, although it has been reported that they may be associated with kcm . The large plaque with atrophy at the center of the lesion also prompted consideration of lv in the setting of the indian subcontinent, but the absence of an underlying tubercular granuloma helped us to exclude this condition . The diagnostic dilemma was the finding of the presence of the proteus organism within the lesions, which is a feature of botryomycosis . The absence of tumefaction or purulent discharge, with the histopathological finding failing to reveal any neutrophilic infiltration, grape - shaped granules, or splendore - hoeppli phenomenon, ruled out the possibility of botryomycosis . In fact, bpl may present also with a large verrucous plaque with pseudoepitheliomatous hyperplasia . However, we easily ruled out this condition as there were no pustules demonstrated clinically and no suppuration was found on histopathology; moreover, bpl usually affects immunosuppressed patients . Furthermore, peh could be ruled out by the fact that it was not preceded by any other skin lesion, and by the histopathological finding of epidermal hyperplasia restricted to above the level of the sweat glands and the presence of a large crater, all of which are unlikely in the case of phe . After the exclusion of the clinical mimickers, kcm was diagnosed on the basis of classical clinical and histopathological findings . The appearance of recurrent nodular skin lesions with central clearing and progressive peripheral spreading on the distal extremities, in the setting of the histopathological findings of large irregularly shaped craters filled with keratin, with irregular epidermal proliferation having a glassy appearance and not extending below the level of the sweat glands, was diagnostic of kcm . The findings in our case were similar to those of previous case reports with the presence of large lesions and no tendency to spontaneous resolution . The margin of the lesion showing multiple comedonal orifices giving rise to a cibriform pattern, as seen in our case, may represent a unique phenomenon of kcm . We hypothesized that the typical appearance may arise as a result of sequential involvement of multiple adjacent hair follicles in a centrifugal fashion . It is an interesting finding and, if future case reports of this rare variant of keratoacanthoma document the same feature, then this hypothesis will be validated . However, making the diagnosis was not the end and therapy was also a challenge . There are reports that kcm has been treated successfully with oral retinoids; hence, we started oral acitretin . After three months of high - dose therapy, it had to be abandoned because of no therapeutic improvement . The decision was taken in favor of surgical intervention and wide local excision was planned, considering the fact that the patient showed recurrence following previous inadequate excision . Following our intervention, the patient is showing no recurrence, highlighting that surgical intervention can be adopted as the preferred mode of therapy for kcm, keeping in mind that a wide excision is performed to prevent subsequent recurrence.
When fibrosarcoma is diagnosed during the early years of life, it is called congenital infantile fibrosarcoma (cif), representing less than 1% of all pediatric malignant tumors (1, 2). In contrast with adult fibrosarcoma, infantile fibrosarcoma (if) rarely exhibits distant metastasis and has a good prognosis; greater than 90% of patients may be cured with appropriate management . The tumor usually occurs in soft tissues of the extremities and is often asymptomatic . In such cases, rarely, if may occur in the gastrointestinal tract; a few cases of gastrointestinal fibrosarcoma have been reported in the literature . Necrotizing enterocolitis (nec), which is a primary intestinal wall pathology, has been cited as the major cause of pneumoperitoneum in most published literatures (3, 4). However some conditions, not affected by a primary pathology of intestinal wall itself (e.g. Intestinal atresia, stenosis, meconium ileus, colonic aganglionosis or volvulus), may lead to neonatal pneumoperitoneum . This case was a pneumoperitoneum not associated with a nec also . In this report, we present an extraordinary case of cif of the sigmoid colon causing pneumoperitoneum in a newborn baby . A 2-day - old korean boy, delivered at 37 weeks by cesarean section owing to fetal bradycardia, with a birth weight of 3,420 g, was referred to the neonatal intensive care unit in pusan national university children's hospital because of abdominal distension and free air in the peritoneal cavity at may 1st, 2011 . A plain abdominal radiograph showed normal air - filled loops of bowel; however a large amount of free air occupied the central portion of the abdomen (fig . 1). The patient underwent an emergency laparotomy on the day of admission to the neonatal intensive care unit . A large amount of bile - stained a round mass, about 5 cm in diameter, was observed in the left lower abdominal cavity, and it seemed to wrap around a loop of sigmoid colon (fig . Pathologic analysis report described an infantile fibrosarcoma, measuring about 4.7 2.8 cm, involving mainly the submucosa and muscularis propria with an infiltrative growth pattern (fig . The immunohistochemical stain of the tumor was positive for vimentin and sma, and negative for h - caldesmon, s-100, cd34, c - kit, desmin, and alk-1 (fig . 4). The ki-67 proliferation index was 20% . Because complete surgical resection was performed and the surgical resection margins were free tumor, the patient was observed without adjuvant therapy . Fibrosarcoma is a tumor that arising from mesenchymal cells and composed of malignant fibroblasts within a collagen background . After infantile fibrosarcoma first recognized in 1962 (5), it has been generally studied . Congenital infantile fibrosarcoma (cif) is a very rare type of nonrhabdomyosarcoma soft - tissue sarcomas, usually occurring in the 1st year of life, with approximately 40% of lesions present at birth and more than 80% diagnosed within 1 yr (6). It accounts for 5%-10% of all soft - tissue sarcomas in infants younger than 1 yr and is slightly more common in boys (7, 8). In contrast with adult fibrosarcoma, infantile fibrosarcoma (if) is considered a low - grade malignant tumor and carries an excellent prognosis, with survival rates of 80%-90% (9). If is usually located in subcutaneous tissues at various anatomic sites, most frequently involving the extremities and the axial regions . Especially in older children, if less frequently involves the trunk, head and neck (6). The manifestation of infantile fibrosarcoma in the reported cases is generally a painless swelling, exhibiting steady growth . However, it may rarely involve the retroperitoneum, mesentery, mouth, presacral region, lung, or gastrointestinal tract . A few cases have been reported recently about if involving the gastrointestinal tract (e.g., the duodenum, jejunum, ileum, and hepatic and splenic flexures of the colon (10 - 12). Intestinal obstruction or meconium peritonitis due to bowel perforation was the primary manifestation in the reported cases involving intestine . In our patient, pneumoperitoneum was the presenting problem, but there were no gross findings at surgery implying a definite perforation of the colon or rupture of the mass; neither was there any ischemic change or obstruction, or apparent necrosis of the tumor on histologic examination . We found no obvious explanation for the large amount of free - air in the peritoneal cavity seen on radiologic examination prior to surgery; we assume that a sealed - off microperforation was responsible for the condition . Pneumoperitoneum in neonates accounts for about 1% of total admission at a neonatal intensive care unit . Most cases occur in relation to a primary intestinal wall pathology such as necrotizing enterocolitis; however, pneumoperitoneum may rarely be associated with intestinal atresia, stenosis, meconium ileus, colonic aganglionosis, or volvulus . It is necessary to distinguish if from other stromal tumors, particularly in cases involving the gastrointestinal tract . . Molecular biological analysis may be useful when identifying the presence of specific transcription errors, e.g., the evt6-ntrk3 translocation, but it is not always used (6, 9, 11). Surgical excision remains the principal component of treatment, showing a good result when complete resection with tumor - free margins is achieved . There is no defined role for adjuvant chemotherapy or radiotherapy after complete surgical excision (9, 13). The prognosis is generally favorable with more than 90% long - term survival (9). In our patient, we could not identify any evidence of local recurrence or distant metastasis during a 1-yr follow - up period . In conclusion, cif of the sigmoid colon is a rare soft tissue tumor, with a favorable outcome likely after appropriate surgical resection . It may present with bowel perforation in the early neonatal period; therefore a neonatal pneumoperitoneum not related to necrotizing enterocolitis requires attention for management.
Symmetry transitions are common during embryogenesis of all multicellular organisms [14]. In most cases, the transition is from radial to bilateral symmetry and controlled by hox and decapentaplegic genes in animals [5, 6]. In fact, the echinoderms provide the only reported example in which this order is reversed such that the radially symmetric animal develops from a bilaterally symmetric larvae stage [7, 8]. In the model plant arabidopsis thaliana, the gynoecium is derived from the fusion of two carpels and forms in the center of the flower . During gynoecium development, the apical style becomes radially symmetric with stigmatic papillae arising (figure 1a and figures s1a s1c available online), suggesting the existence of a switch from bilateral to radial symmetry . Given that the arabidopsis gynoecium originates from two fused leaves [11, 12], it is likely that factors involved in specifying leaf margin tissue are also regulated in the gynoecium . Although margin identity genes may have a role in defining margins in the bilaterally symmetric ovary, we would expect such activities to be repressed in the style to achieve radial symmetry . Kluh (klu) is a margin - identity gene expressed in peripheral cells of arabidopsis petals and in the marginal tissue of the gynoecium . Expression of klu::gus was detected along the entire length of developing gynoecia at stage 9 (figure 1b) but lost at the style of the mature gynoecium (stage 12 in figure 1c; developmental stages defined in). Mutations in the spatula (spt) gene lead to a failure in radial symmetry establishment at the style (figures 1d and s1d s1f). Interestingly, in the spt-12 mutant, klu::gus was still expressed in the apical medial part throughout gynoecium development (figure 1e). These results suggest that the bilateral - to - radial transition occurring during style formation requires transcriptional repression of margin - identity genes . When the spt mutant is combined with mutations in the indehiscent (ind) gene, the effect on style and stigma development is significantly enhanced reflecting the synergistic activities of these two basic helix - loop - helix transcription factors (figures 1j and s1g s1i). In the wild - type gynoecium, the ovary has a bilateral symmetry plane in which the septum divides the ovary into two separate locules, whereas the style is a rounded, compact, and radially symmetric structure (figures 1g1i). Spt and ind spt have defects in septum formation but maintain bilateral symmetry in the ovary (figures 1j, 1l, s1j, and s1l). The style in these mutants fails to acquire radial symmetry showing that ind and spt are required to ensure radial symmetry establishment at the gynoecium apex (figures 1k and s1k). Klu expression was found to be significantly upregulated in spt and ind spt mutants (figure 1f) and downregulated in a 35s::ind: gr line induced by dexamethasone (dex) (figure s1n). This is in agreement with a role of ind and spt in promoting radial symmetry, at least partially, by repressing margin identity . We next tested whether ind and spt are sufficient to establish radial symmetry in an alternative developmental context such as a bilaterally symmetric flat leaf . To this end, the dex - inducible 35s::ind: gr line was grown on medium supplemented with dex . After 15 days, completely radialized leaves emerged as rod - like and cup - like structures (figures 1 m, 1o, and s1 m). Notably, the epidermal cell shape of these radialized leaves is reminiscent of the shape of style cells (figure 1s and inset 1s), which is in contrast to the normal jigsaw - shaped leaf epidermal cells from noninduced plants (figure 1q). Conversely, anatomical analyses of the internal cell types in cross - sections suggest that ind overexpression reprograms only the marginal cells (figures s1p, s1r, s1 t, and s1v). The ind - driven organ radialization was completely dependent on the presence of spt function, because the effect was lost in the spt-12 mutant background (figures 1n, 1p, 1r, 1 t, s1q, s1s, s1u, and s1w). Altogether, these results show that both ind and spt are necessary and sufficient for mediating organ radialization . During gynoecium development, auxin distribution two apical foci of the auxin - signaling reporter, dr5::gfp, are established in the lateral apical domains at early stages (5/6) of organ development (figures 2a and 2b). Subsequently, two medial foci emerge at stage 8/9 (figures 2c and 2d; movie s1), and immediately prior to formation of the style (stage 10), all four foci are connected in an auxin ring of radial symmetry (figures 2e and 2f). This pattern mimics the transition of bilateral - to - radial symmetry suggesting a role for the spatiotemporal dynamics of auxin in symmetry establishment . We initially tested if the auxin - signaling foci are established by local auxin production . The tryptophane aminotransferase of arabidopsis1 (taa1) gene encodes an auxin - biosynthesis enzyme and is expressed in the same region as spt during early stages of gynoecium development (figures s2a and s2b). Taa1 and its closest homolog tar2 likely regulate auxin dynamics in the gynoecium, because the taa1 tar2 double mutant exhibits a split - style phenotype . We conducted the expression analysis of a taa1::taa1:gfp line concomitantly with dr5::rfp to correlate the dynamics of auxin production and auxin signaling in vivo . Early in development, expression of these two reporters is nonoverlapping with dr5::rfp in the apical lateral part and taa1::taa1:gfp in the medial region (figure s2b). At stage 9, there is overlap in the medial region with taa1::taa1:gfp expanding to the lateral adaxial side (figure s2c). Because the dr5::rfp signal in the lateral foci appears before the taa1::taa1:gfp signal, it is unlikely that the two lateral auxin - signaling foci are established by local auxin synthesis . Next, we analyzed if auxin transport is involved in establishing the auxin - signaling foci . The pin1 gene encodes a plasma membrane (pm) localized member of the pin auxin efflux family that directs polar auxin transport (pat) via their asymmetric subcellular localization [19, 20]. Pin1 protein is located apically in cells of the ovary presumably to direct auxin flux from the base to the top of the developing gynoecium (figure 2 g). At the apex, pin1 localization becomes apolar primarily in the medial part of the gynoecium (figure 2h). Pin1-mediated auxin transport is therefore likely to contribute to the specific pattern of auxin distribution at the apex . Indeed, in gynoecia from a weak pin1 mutant allele (pin1 - 5), the intensity of the two lateral dr5::gfp foci are severely reduced and apical - basal polarity defects are detected (figures s2d s2f). An identical effect occurs in plants with mutations in the pinoid (pid) gene encoding an agc3-type protein kinase that promotes apical pin localization at the pm by phosphorylating specific serine residues in pin proteins [2124] (figures 2 m, 2n, 2s, and 2u). Indeed, mutations in two of those specific serine residues (pin1:gfp s1,3a) lead to apolar distribution of pin1 along the gynoecium (figure 2i) and apical - basal growth defects similar to the weak pid-8 mutant (figures 2 t and 2u). Moreover, this growth - defective phenotype is reminiscent of treatment with the pat inhibitor npa [26, 27]. Another member of the pin family, pin3 is initially confined to a few laterally positioned apical cells (figure 2j) overlapping with the lateral dr5::gfp foci (figure 2a). Later, pin3 is detected throughout the apex in the same domain as dr5::gfp (figures 2f and 2k) with apolar localization of the protein (figures 2j and 2k). A third pin member, pin7, is localized apolarly in a few medially positioned apical cells from around stage 7 (figure 2l), presumably joining the activity of pin1 in establishing the medial foci . At later stages, pin7 is found throughout the apex sustaining the ring formation similarly to pin3 (figure s2 g). Expression and localization of pin1/pin3/pin7 suggests that pat mediates the transition from a bilaterally to a radially distributed auxin response (figures 2a, 2b, 2e, and 2f). The requirement for apolarly localized pins to establish the radial auxin maximum at the gynoecium apex resembles the apolar localization of pin4 around the quiescent center cells of the root apical meristem and its precursor cells during embryogenesis [28, 29]. In this tissue, pin4 is necessary for the proper positioning of the auxin - response maximum at the embryo stem cell niche . To address the role of the lateral and medial pairs of auxin - signaling foci, we tested dr5 expression dynamics in mutants with defects in either apical - basal growth or style development . Dr5::gfp in pin1 - 5 and dr5::rfp in pid-8 mutants showed a drastically decreased signal in the lateral foci, whereas the auxin ring appeared normally, thus correlating with radial style formation (figures 2 m, 2n, 2p, 2q, 2u, and s2d s2f). As in many organ - development processes, gynoecium growth along the apical - basal polarity axis follows the direction of auxin flux, directing growth toward the two lateral auxin foci providing cell and tissue polarity . In agreement with the reduced lateral dr5 signals, pin1 - 5 and pid-8 mutants show apical - basal growth defects (figures 2u and s2d). Therefore, the two lateral foci are important to ensure apical - basal growth of the two carpels . In mutants with defects in the bilateral - to - radial symmetry transition, the two lateral dr5 foci are correctly established early during gynoecium development, and these mutants have no apparent apical - basal defects (figures 2v, s1j, and s2h). In contrast, the medial dr5 foci were not established in these mutant backgrounds (figures 2o and s2i) and the dr5 ring fails to form (figures 2r and s2j). The lack of dr5::rfp in spt-12 is unlikely to be due to lack of auxin biosynthesis, because taa1::taa1:gfp is still expressed in spt-12 (figure s2k). These results suggest that the medial auxin - signaling foci promote the bilateral - to - radial symmetry switch . In agreement with this, the medial dr5 foci form normally in pid-8 gynoecia with no defect in establishing the dr5 ring and correlating with formation of a radial style (figures 2n and 2q). It was previously shown that spt and ind directly repress pid expression [15, 16]. Accordingly, we found that a pid::gus reporter was ectopically expressed in the style region of the spt-12 mutant compared to wild - type (figures 3a and 3b). The importance of apolar pin1 localization was analyzed by expressing a version of pin1 that mimics constitutive phosphorylation of the three serine residues targeted by pid (pin1:gfp s1,2,3e) in the pin1 mutant background . Gynoecia from this line exhibited a split - style phenotype similar to the spt-12 mutant (figures 1d, 3c, and 3f). Interestingly pin1:gfp s1,2,3e protein could not be detected at the apex as opposed to a nonmutated pin1:gfp version (figures 3d and 3e), suggesting that apical localization renders pin1 unstable in this tissue . Consistent with defective pin1:gfp s1,2,3e protein localization, dr5::rfp was not detected in the medial foci of pin1:gfp s1,2,3e pin1 (figure 3i) but only in the lateral foci, thereby resembling dr5 distribution in spt and ind spt mutants (figures 2o, 2r, and s2h s2j). These results suggest that pid - mediated phosphorylation of pin1 is sufficient to prevent radial symmetry . As expected, loss of pin1 phosphorylation had no effect on radial symmetry establishment, because constitutive apolar localization of the pin1:gfp s1,3a mutant protein sustains apolar auxin flux (figures 3 g, 3h, s3a, and s3b). Together, these results show that apolar localization of pin1 is required for radial style formation . We next tested the developmental relevance of the sequential appearance of the lateral and medial pairs of foci . The gynoecium phenotype resulting from crosses between pid loss - of - function mutants and spt-12 was analyzed to distinguish between two possible scenarios: (1) if activity of the medial foci is sufficient for radial symmetry establishment, complementation of the spt split - style phenotype was expected by eliminating pid function and (2) if the role of lateral foci is functionally upstream of the medial foci, a failure to establish radial style development was expected in the double mutant . Analysis of the pid-8 spt-12 and pid-9 spt-12 double mutants revealed a strong enhancement of the spt-12 phenotype and a complete failure in radial symmetry establishment . This result is in agreement with the second scenario and suggests that the lateral foci are required to support the role of the medial foci during style development (figures 3j, s3e, and s3f). To study whether the split - style phenotype in spt gynoecia is due to a failure of spt in controlling auxin transport in the medial apex, we introgressed the pin1::pin1:gfp s1,3a loss - of - phosphorylation mutant into spt-12 . Here, the background was kept wild - type for the endogenous pin1 gene to sustain formation of the lateral foci and promote apical - basal growth . Gynoecia from this genetic combination exhibited complete restoration of the split defect with perfectly radialized styles (figures 3k, s3c, and s3 g). This was dependent on wild - type endogenous pin1 in the background, because gynoecia from the pin1::pin1:gfp s1,3a spt-12 pin1 triple combination phenocopied spt pid double mutant gynoecia (figures 3j and 3l). As with the spt pid double mutants, this triple combination was unable to sustain the apical - basal growth, thus affecting the activity of the lateral foci and enhancing the spt phenotype (figures 3l, s3d, and s3h). Overall, these results show that spt (and ind) controls radiality at the gynoecium apex by controlling auxin transport, thus governing auxin flux in the medial region of the style . They also reveal that activity of the medial foci is linked to and dependent on the lateral auxin - signaling foci . The functional relation between the lateral and medial auxin - signaling foci described here is closely aligned with the stereotypical stages occurring during gynoecium development . As indicated in figure 4, the early function of the lateral foci is to sustain apical - basal growth allowing to build up the ovary . Subsequently, at stages 8 and 9, in order to obtain a radialized apical style, spt and ind establish the medial foci by directly repressing pid expression [15, 16], thus sustaining apolar pin localization and auxin accumulation (figure 4). It is unknown what stimulates expression of the ind / spt module, but it is an intriguing possibility that a feedback mechanism exists between ind / spt and auxin . Finally, we hypothesize that a long - distance signal is required to connect the different foci in a radial auxin - signaling maximum to achieve a switch in cell polarity and thus orchestrating the coordinated growth of the radial style to facilitate fertilization . Excellent progress has been made in understanding how auxin provides polarity and identity to cells in a range of developmental contexts . The example presented here demonstrates that auxin can also be recruited to coordinate a heterogeneous group of cells to commit to a program, which imposes homogeneous identity to them . This activity leads to an unusual developmental bilateral - to - radial symmetry transition in the arabidopsis style . The radial style is a general feature of the female reproductive organ in angiosperms, which arose during the cretaceous period 100125 million years ago . The early angiosperms underwent a remarkably rapid diversification and have since reached ecological domination in the plant kingdom in terms of number of species (> 300,000) a phenomenon that charles darwin referred to as the abominable mystery . Because a radial style is necessary to facilitate efficient fertilization, radialization of the style may have been a key event in allowing the success of flowering plants . Conceived the hypothesis and planned the experiments, l.m . Carried out the experimental work, and l.m . And l. .
Deep vein thrombosis (dvt) remains an underestimated problem in icu patients, despite the findings of many randomized controlled trials performed in the field of dvt prophylaxis after surgery during the past few decades . The canadian survey reported in the present issue of critical care provides a useful snapshot of daily clinical practice in canada with regard to dvt prophylaxis . It strongly suggests that studies dedicated to dvt prophylaxis in icu patients should be performed in order to develop useful recommendations . Furthermore, a great effort would have to be made to educate physicians regarding both dvt screening and pharmacological aspects . Clinicians should be aware that dvt in icu patients has unusual characteristics that make its clinical diagnosis difficult ., in which the clinical signs of dvt (e.g. Oedema, pain and flushing) occurred in less than 1.5% of patients . As a result, physicians are often lulled into an inappropriate sense of security . Moreover, the diagnosis is not always easy to confirm . The insensitivity of doppler ultrasound and the major difficulty in performing venography in icu patients generally lead to blind anticoagulant prophylaxis . Even when a pulmonary embolism leads to death, diagnosis is often difficult to confirm in a patient who has already been treated and ventilated for a pulmonary condition because autopsies are rarely conducted in trauma victims . As stated at the most recent american college of chest physicians consensus conference, however, trauma patients represent a group that is at very high risk for dvt . The discussion should therefore no longer focus on the incidence of thrombosis, but rather on the different methods of prevention that could be used . Most of the canadian respondents in the survey appear to be aware of this, which would explain why up to 34% of icu directors do not consider mechanical prophylaxis at all . It has been strongly suggested, however, that elastic stockings should be combined with lmwh . Combining noninvasive with pharmacological prophylaxis it is also cost - effective and easy to use . In order to improve the benefit from such combinations of mechanical and pharmacological measures, classic intermittent pneumatic compression devices applied directly to the entire leg are often difficult to use because of fractures, immobilization with plaster casts, or external fixation instruments . With the foot pump is designed to overcome the venous stasis that is associated with surgery . It flattens the metatarsal arch, emptying the venous plexus (30 ml blood) and thus reproducing the effect of normal weight - bearing . The efficacy of the foot pump has already been demonstrated in level ii and iii studies . A recent large, prospective, randomized study conducted in 274 patients with total hip replacement compared the safety and effectiveness of the foot pump with those of lmwh prophylaxis . That study showed no significant difference between the two methods; dvt was detected in 24 (18%) patients randomized to foot pump prophylaxis as compared with 18 patients (13%) randomized to receive the lmwh enoxaparin . There was no difference in the transfusion requirements or intraoperative blood losses between the two groups . This new method could be helpful in trauma, neurological, or neurosurgical patients when anticoagulants are contraindicated . In summary, mechanical prophylaxis should systematically be used alone or in combination with pharmacological prophylaxis in icu patients . Although unfractionated heparin (5000 iu administered subcutaneously two or three times per day) is used extensively in the canadian centres, there is considerable evidence that these small doses of heparin are relatively ineffective in comparison with doses used in orthopaedic surgery . In the literature, selection of unfractionated heparin was supported by dvt detection methods, such as echography and duplex scanning . These methods are unacceptable because of their low sensitivity in asymptomatic patients, especially in the icu . Administration of lmwh has been shown to result in significantly better results . In 1996, . Showed that 30 mg enoxaparin given twice daily exhibited superior antithrombotic efficacy as compared with subcutaneous heparin 5000 iu twice daily . The overall venographic dvt rate was reduced from 44 to 31%, and the proximal dvt rate from 15 to 6% in patients receiving heparin and enoxaparin, respectively . Since then, only one relevant study has been reported, which compared lmwh with placebo in icu patients . In that study, nadroparin was able to decrease the dvt rate significantly, but no direct comparison between lmwh and unfractionated heparin was undertaken . Although there is still insufficient data in the icu setting, the large amount of data gathered by surgical trials should allow extrapolation . Lmwh appears to be effective and safe postoperatively, and hence should probably be recommended in icu patients except when renal function is impaired and in very old patients . The optimal duration of treatment has not been defined, but it appears reasonable to suggest that prophylaxis with lmwh should be continued for as long as risk factors are present, such as inflammation, sepsis and immobilization . New compounds such as recombinant hirudin and pentasaccharide should be evaluated in these very high risk patients because those agents have exhibited high efficacy in preventing dvt after total hip replacement surgery . They may be particularly useful in those settings in which thrombotic risk rapidly exceeds haemorrhagic risk . Oral anticoagulants cannot be recommended in trauma patients because some have to undergo multiple surgical procedures . In addition, interactions between vitamin k antagonists and other drugs used in this setting may be hazardous . Thrombotic complications (dvt, pulmonary emboli) are a major concern in icu patients and still occur in a significant number of patients . Antithrombotic agents, and lmwh in particular, should be considered in a systematic manner, except for those cases in which they are contraindicated . Finally, educational programmes should be implemented that include epidemiological and therapeutic aspects of vte prevention . Dvt = deep vein thrombosis; icu = intensive care unit; lmwh = low - molecular - weight heparin; vte = venous thromboembolism.
The european working group on sarcopenia in older people has recently suggested that the definition of sarcopenia should include not only low muscle mass but also low muscle function (strength or performance) because sarcopenia is a syndrome that is characterized by both . The loss of muscle mass and strength are problems that plague many older adults, though the causal and correlative link between diminishing lean mass and strength is not well supported in aging humans . Aging is also characterized by increasing depots of fat both between and within skeletal muscles . This increase in intramuscular adipose tissue (imat), and its lipotoxic effect, has been identified as a potential contributor to declining strength and muscle quality; with some also associating it with mobility limitations in older adults [48]. The recent structural focus on imat content rather than lean muscle mass is important as it could provide a plausible explanation for age - associated strength and mobility deficits . Fatty infiltration into skeletal muscle may alter muscle fiber orientation and hence the force producing capabilities of the whole muscle . Imat may also be a metabolically active component of muscle that secretes inflammatory cytokines leading to systemic inflammation, much like visceral adipose tissue, that can inhibit muscle force production even in the absence of muscle atrophy . Further, imat may be an appropriate therapeutic rehabilitation target as some suggest its presence can blunt the strength response to resistance training . Currently, it is accepted that muscle weakness, loss of lean tissue mass, and imat contribute to functional decline . In order to gain insight and parcel out the contributions of these muscle variables to mobility, this study examined the association of imat of the thigh, along with muscle lean tissue and muscle strength, with mobility measures in older adults . One hundred and nine older (mean = 74.1 years 6.8) adults participated in this study . Recruitment occurred over a 5-year period from 20062011 and consisted of receiving names and identifying information from clinical databases at the university of utah . Each of the subjects received a personal letter providing information about the study, and they were then contacted directly via phone or in - person to assess their interest and to screen for eligibility following receipt of a signed informed consent document . The 109 subjects were community ambulating males (n = 32) and females (n = 77) 60 years of age or older with 2 or more comorbid disease conditions and were at risk for falling due to fatigue, muscle weakness and/or had experienced a fall in the previous 12 months . All scored> 23 on a folstein minimental examination or passed the mini - cog instrument for dementia . Individuals were excluded if they had any of the absolute contraindications for mri, progressive diagnosed neurologic disease (e.g., parkinson's, multiple sclerosis, guillain - barre, and alzheimer's), any dystrophies, or rheumatologic conditions that primarily affect muscle (muscular dystrophy, pmr) or had been participating in regular (3x / week) aerobic or resistance exercise over the past 12 months (table 1). Knee extension strength was determined via a maximum voluntary isometric contraction (mvic) on a kincom dynamometer (chattanooga inc ., hixon, tn) as follows: participants were stabilized by chest and thigh straps and seated with their knees fixed at 60 degrees of flexion with their arms folded across their chest . Prior to testing, participants practiced submaximal contractions at 50 and 75% of their perceived maximal effort prior to one practice maximal contraction trial . After a 2-minute rest period, three separate maximal contractions were performed . The outcome variable muscle force was calculated as the average peak force of three trials . The respective imat and lean tissue cross - sectional areas were calculated from the mri scans . Subjects were placed supine in a 3.0 tesla whole body mr imager (siemens trio, siemens medical, erlangen, germany). The legs were scanned in a coronal plane with a turbo spin echo (tse) t1-weighted sequence to depict the femoral heads and the femoral condyles . The midpoint of the thigh was determined and defined as half way between the superior margin of the femoral head and the inferior margin of the femoral condyles . Axial imaging (5 mm thick slices at 1 cm intervals) of the legs was then performed over 1/2 the length of the femur, centered at the midpoint of the thigh . This was performed with a three - point dixon multislice 2d gradient recalled echo (gre) sequence (tr = 300 ms, te = 5.15/6.4/7.65 ms, and matrix size = 512 288). Field of view was adjusted to the individual subject anatomy to obtain optimal in - plane spatial resolution, typically 1 1 mm or better . Separate fat and water images were created with custom software using the three - point dixon method . A tissue model was then used to calculate estimates of total fat and nonfat volume fractions on a per - pixel basis, which were displayed in image form . Four images from the middle 1/3 of each thigh were used to determine average cross - sectional area (cm) of imat and lean tissue . Manual tracing eliminated subcutaneous fat and bone and isolated the fascial border of the thigh to create a subfascial region of interest (roi). Total imat and lean tissue were calculated by summing the value of percent fat fraction and percent lean tissue fraction over all pixels within the roi using custom - written image analysis software (matlab; the mathworks, natick, massachusetts). This sum was multiplied by the area of each pixel to give total fat and lean tissue csas within the roi . This method accurately measures fat and lean tissue in pixels that contain both by allowing fractional contributions to the fat and lean tissue csa calculations . This allows microscopic fat within muscle tissue as well as thin planes of fat adjacent to fascial planes to be accurately taken into account, even when image resolution is inadequate to delineate these visually (figure 1). The same investigator, blinded to time point of the scan and slice location, performed measurements of individual participants . This technique has demonstrated high levels of intrarater reliability, test - retest reliability [13, 14], and concurrent validity when compared to imaging of a cadaveric phantom limb . Mobility was determined using four tests: (1) a six - minute walk (6mw), (2) stair ascent (stair a), (3) stair descent (stair d), and (4) a timed up and go (tug). These performance tests were chosen to represent mobility function and have been shown to be both valid and reliable in this population [1517]. The 6mw test, a measure of the distance a subject walks in 6 minutes, was used to assess overall mobility . Participants were asked to cover as much distance as possible in six minutes without running . The stair a test required participants to ascend one flight of stairs under close or contact supervision as quickly and safely as possible . Time was recorded to the nearest 0.01 second from a verbal go signal to final foot placement on a standard flight of 10 stairs, and the average of three trials was recorded . The stair d test was performed exactly as the stair a test, except that participants were required to descend one flight of stairs as quickly and safely as possible . The tug test required participants to rise from a seated position, walk out 3 meters, turn around, and return to sitting as quickly and safely as possible . Time was recorded to the nearest 0.01 second from the time the person's buttocks left the chair until return contact with the chair . Data management and statistical analyses were performed with pasw statistics 18.0 (spss, chicago, il). Descriptive data were calculated for demographic variables and dependent measures and are presented as means sd . Pearson correlation coefficients were calculated to determine the bivariate relationship between each muscle and mobility variable . The relative contribution of each muscle variable to explaining the variability in the mobility outcomes were examined using step - wise hierarchical linear regression models . Each mobility test was used as the dependent variable in separate models, with knee extension strength, quadriceps lean tissue, and imat considered for entry in a stepwise manner . For each variable entered in the final model, the part correlation was examined to determine the unique amount of variance in the mobility outcome that was accounted for by the variable . The bivariate correlation of mobility variables with muscle variables revealed moderately strong and significant correlations (r range = 0.230.55, p <0.05) (table 2). The direction of the correlations indicates that as strength and average lean tissue increase, mobility function improves . As average imat increases, mobility function declines . These results support the use of step - wise regression analyses to examine the unique and shared contributions of the muscle variables towards explaining the variance in mobility measures . The multiple regression analysis on the 6mw revealed that the predictors as a group accounted for 34.6% of the variance in walk distance, with strength (p = 0.005), imat (p = 0.002), and lean (p = 0.005) each significantly contributing to the final model (p = 0.005). The part correlation of strength was 0.23, of imat was 0.30, and of lean was 0.23 indicating that strength, imat, and lean explained 5.3%, 9.0%, and 5.3% of the variance in the 6mw score, respectively, with all other variables in the model held constant . The predictor variables as a group accounted for 45.1% of the variance in stair ascent time, with strength (p = 0.001) and imat (p <0.001) contributing significantly to the final model (p = 0.001). The part correlation of strength was 0.54 and of imat was 0.39, indicating that strength and imat explained 29.2% and 15.2% of the variance in the stair ascent scores, respectively, with all other variables in the model held constant . The predictor variables as a group accounted for 37.4% of the variance in stair descent time, with strength (p = 0.001) and imat (p = 0.001) contributing significantly to the final model (p = 0.001). The part correlation of strength was 0.49 and of imat was 0.37, indicating that strength explained 24.0% and imat explained 13.7% of the variance in stair descent with all other variables in the model held constant . The predictor variables as a group accounted for 26.5% of the variance in tug score, with strength (p = 0.001) and imat (p = 0.001) contributing significantly to the final model (p = 0.001). The part correlation of strength was 0.42 and of imat was 0.28, indicating that strength and imat explained 17.6% and 7.8% of the variance in tug scores, respectively, with all other variables in the model held constant (table 3). The novel finding in this investigation is that even when accounting for total body mass, thigh muscle adipose tissue surfaces as a potent muscle variable related to the ability of older adults to move about in their community . Specifically, normalized muscle strength, imat and lean tissue composition measured via mri were tested to determine the contribution of these muscle variables to declining mobility levels in older adults . The link between imat and mobility deficits has been shown in large epidemiological studies; however, this is the first study to target older individuals needing mobility related rehabilitation interventions . Skeletal muscle lipid content in older men and women is independently associated with maximal torque production even after adjusting for muscle size, height, weight, age, and race, accounting for 3236% of the total variance in strength . Greater fat infiltration into muscle is also associated with increased risk of future mobility loss in older men and women . Our results punctuate the potential negative impact of fatty infiltration of muscle on mobility function, an important component of sarcopenia in the elderly and are particularly interesting considering that imat explains a surprisingly high amount of the variance in these mobility tasks . Though by no means causal, the correlations reported here between imat and specific performance tests of mobility function in older adults support, in part, the potential inflammatory pathway linking ectopic fat deposition and deteriorating physical ability of older individuals . Evidence of a direct effect of inflammatory cytokines on muscle catabolism [19, 20], combined with the knowledge that inflammation is often elevated with aging [21, 22] suggests inflammation could be a reason for functional decline and frailty . The current study, however, does not provide any data to support this hypothesized role, and the lack of any biochemical data should be considered a limitation . Recent evidence [23, 24] suggests that an elevated inflammatory milieu is associated with loss of muscle, strength, and function in the older individuals, making it tempting to speculate that a connection between imat, inflammation, and sarcopenia exists, and future interventions should target not only the impact of lean tissue loss, but also of increased imat deposits . There is wide variation in aging populations with respect to mobility levels, comorbid conditions, and body composition . Because of this, strong correlations between these variables are difficult to identify . A recent review of body composition factors and their relationship to mobility in older individuals highlights this fact, emphasizing the importance of our results and suggesting that future studies should examine imat as an important variable with respect to sarcopenia and mobility function in older individuals . Future studies are needed to confirm these findings and to determine whether decreases in imat are associated with concurrent improvement in mobility function . Additionally, the participants in this study, while varying in age between 60 and 93 years, were generally high - functioning individuals . Because individuals functioning at lower levels were not included, this should be considered when generalizing these findings . This newly identified muscle impairment has not traditionally been targeted by clinicians concerned with sarcopenia, but now in addition to the loss of lean tissue and muscle strength, imat should be considered an important factor that may contribute to deficits in mobility in the aging population . Preliminary data show that moderate physical activity in older adults may prevent skeletal muscle fat infiltration and that exercise can decrease imat [27, 28]. This suggests that imat may be amenable to change with exercise countermeasures, though these findings are limited by small sample sizes and nonrandomized designs . Well controlled, randomized studies are needed to determine the best exercise countermeasure to decrease imat and importantly, how this decrease impacts mobility function in mobility limited older adults . Thigh imat contributes a significant amount of variance to the mobility performance of older adults needing rehabilitation, even when accounting for total body mass . Imat has a negative impact on mobility function, an important component of sarcopenia in the elderly . Locomotor muscle fatty infiltration, in addition to lean tissue and muscle strength, should be considered an important factor contributing to mobility deficits in this vulnerable population.
An essential requirement of any orthodontic therapy is the maintenance of a meticulous oral hygiene so as to effectively control the growing of bacterial plaque over tooth surfaces . The fixed orthodontic appliances may lead to an increase in the levels of plaque and alteration of its quality . Early tooth decay around the brackets can cause white or brown marks [demineralized white lesions (dwls)] to appear on teeth during fixed orthodontic treatment . Build - up of dental plaque around these brackets is associated with an increased risk of rapid demineralization of the teeth enamel . A recent study found that the prevalence of dwls might be 74% in patients who have undergone a fixed orthodontic treatment . In another study, it has been seen that, even five years after treatment, orthodontic patients had a significantly higher incidence of dwls than a control group of participants who did not undergo orthodontic treatment . Therefore, in clinical practice, various therapies have been involved to reduce any kind of enamel lesions as much as possible . One of the most known and used is the application of topical fluorinated substances in different stages of orthodontic treatment . Sodium fluoride gels or varnishes at different concentrations can be used safely and effectively to treat demineralization of dental structures as well as to prevent caries . Because of the aqueous oral cavity, the fluoride ions have the ability to precipitate inside the enamel prisms in place of calcium and phosphate, transforming the hydroxyapatite in fluorhydroxyapatite, a more resistant phase to acid attack, promoting the re - mineralization of the tooth surface, and inhibiting the action of the bacterial enzymes that produce the acid . However, tooth pretreatments with such agents act on the enamel interfering with the bond strength of the brackets . Different studies have already been conducted concerning the use of fluoride and its possible action on the shear bond strength of the brackets . The purpose of this study, therefore, is to understand, starting from the analysis of the detachment forces from the enamel surface, the best temporal association between the application of a fluoride varnish on enamel and bonding procedures in order to avoid any negative influence of the fluoride treatment on the bond strength of the brackets . Eighty freshly extracted mandibular bovine incisors, obtained from the same farm in order to have the same level of fluoride concerning their feeding, were used . Teeth were cleaned of debris and then polished with nonfluoridate pumice and rubber prophylactic cups at low speed for 15 s. tooth selection criteria included integrity of the buccal and lingual enamel surfaces under visible light at 4 magnification, absence of traumatic injuries, cavities, enamel erosions, and smooth and flat buccal surface suitable for bonding . They were then stored in distilled water for no more than 2 weeks (iso / tr 11405). Teeth were randomly divided into 4 groups (20 teeth each) by using a random numbers table . Three groups (groups 13) were treated with fluoride varnish (fluor protector, ivoclar vivadent, schaan, liechtenstein) according to manufacturer's instructions, and one (group 4) served as control with no pretreatment . All the teeth were stored in deionized water (37c) and subjected to thermal cycling for 400 (group 1), 800 (group 2), and 2500 (group 3) cycles corresponding, respectively, to 15, 30, and 90 days in order to simulate the three different timing of bracket bonding after the application of the fluoride varnish . Brackets (edgewise standard, 4.4 mm 3.2 mm, leone s.p.a ., florence, italy) were all bonded using the same standard technique; enamel surfaces were treated with 37% phosphoric acid (etching gel 3 m, unitek, monrovia, ca) for 60 s, rinsed with a water spray for 20 s, and air dried . All brackets were bonded with transbond xt (3 m unitek, monrovia, ca) and light - cured with the same led lamp (bluephase polywave, ivoclar vivadent, schaan, liechtenstein) for 40 s. excessive sealant and adhesive were removed from the periphery of the bracket base to keep each bonding area uniform . The exposure was performed from both the mesial and distal sides for 20 s. for shear bond strength (sbs) tests, each tooth was mounted on self - cured acrylic resin blocks with a mounting jig used to align its buccal surface so that it was perpendicular to the bottom of the mold . Specimens were then mounted in the jig and the sbs was measured with an instron universal testing machine (model 3343, instron corp ., continuous shear force was applied at a crosshead speed of 1 mm per minute until bracket failure . The force required to detach the bracket was recorded in newtons (n) and converted to megapascals (mpa) using the following formula: bond strength (mpa) = debonding force (n)/[w l] (mm), where w = width of the bracket base, l = height of the bracket base and, 1 mpa = 1 n / mm . After the detachment, each tooth surface was examined under a stereomicroscope at 10 magnification to assess the amount of adhesive remnant using adhesive remnant index (ari). The ari index was ranked from 0 to 3 as follows: 0 = no adhesive on the enamel; 1 = less than 50% adhesive on the enamel; 2 = more than 50% adhesive on the enamel; 3 = 100% adhesive on the enamel . Description and inferential statistical analyses were performed using medcalc statistical software (medcalc software, ostend, belgium). The data were found to be normally distributed, and there was homogeneity of variance among the groups . One - way analysis of variance (anova) and tukey's honestly significant difference post - hoc test were used for the comparison of sbs values between groups (p <0.05). The chi - square test was used to examine whether there were differences among the groups in the ari scores . Description and inferential statistical analyses were performed using medcalc statistical software (medcalc software, ostend, belgium). The data were found to be normally distributed, and there was homogeneity of variance among the groups . One - way analysis of variance (anova) and tukey's honestly significant difference post - hoc test were used for the comparison of sbs values between groups (p <0.05). The chi - square test was used to examine whether there were differences among the groups in the ari scores . Descriptive statistics, including the mean, standard deviation (sd), and minimum and maximum values of the sbs for each of the four groups are presented in table 1 . One - way anova and tukey post - hoc test showed that the sbs of different groups were significantly different and was impacted by the different timing of bonding (p <0.05). The main differences were between the control group (17.02 6.38 mpa) and the group 1 (6.93 4.3 mpa). Mean, standard deviation (sd), and minimum and maximum values of the shear bond strength for each of the four groups there were no statistical differences between the group 2 and group 3 . The residual adhesive on the enamel surfaces, as indicated by the ari scores, are presented in table 2 . The results of the chi - square comparisons showed that there were no significant differences between the four groups tested . All groups showed a higher percentage of ari scores of 0 and 1, which indicated that debonding distribution failures were mainly at the adhesive orthodontic patients are often at high risk of developing dental lesions during orthodontic treatment, especially when their compliance to oral hygiene instructions is poor . Controlling dental plaque before and during fixed orthodontic treatment, without compromising the sbs of brackets, has always been an area of research in orthodontics . People often wear braces for more than 1 year, and there is a risk that tooth decay will damage the teeth, requiring restorations and fillings . Topically applied sodium fluoride solution causes remineralization, mainly by reducing apatite dissolution by forming less soluble fluorapatite . The effects of fluoride on the prevention of tooth decay and re - mineralization of decalcified enamel have been elaborately described . However, in previous studies, the effect of fluoride application on sbs has been reported with different and controversial results . In general, fluoride pretreatment induces lower sbs values resulting in reduced bond strength of the brackets; topical application of fluoride interferes with the etching effect of phosphoric acid on enamel surfaces, resulting in reduced bond strength of orthodontic brackets . In order to simulate the aging of the tooth after the fluoride treatment, we used the thermocycling technique that is widely used as an artificial aging methodology . A literature review concluded that 10000 cycles corresponds approximately to 1 year of in - vivo functioning . In the current study, 400, 800, and 2500 cycles were used in order to simulate the aging period of 15, 30, and 90 days, respectively . In our results, the sbs of orthodontic bracket was significantly and highly decreased by the application of the fluoride varnish . Group 1 showed the lowest values with a mean sbs of 6.93 mpa that could be considered a satisfactory result even if it is really close to the minimum value . However, in this group, many measurements were lower than 6 mpa with the lowest value of 2.93 mpa, which is really an undesirable clinical situation due to the high possibility of detachment of the bracket . However, fluorides are one of the most favored remineralizing agent and their positive role in prophylaxis treatments should not be limited due to the possible negative effect on the sbs . That is why we tested the hypothesis of bonding brackets in a delayed time expecting that the negative effect on the sbs could be reduced after 15 or 30 days . The results of the test groups, after 30 and 90 days of aging, showed higher values of sbs, with a mean over 12 mpa that could be considered a safety the ari results indicated that more than 85% samples were included between scores 0 and 1 . The most desired clinical condition is a low ari score with less composite remaining on the tooth surface in order to reduce enamel damage during debonding procedures . The worst score to be considered is an ari of 3; in our tests, the samples included in this score were mainly from the groups 1 and 2 . However, considering our results, we can confirm that all the test groups showed a good ari index, between score 0 and 1, suggesting that the bond between bracket and resin was stronger than that between the resin and enamel . The use of bovine tooth is considered a limitation of this study, owing to the difficulties in obtaining human teeth . However, bovine enamel has already been used in several other studies as a substitute model without statistically significant differences in sbs comparing bovine and human enamel . It is acknowledged that the in vitro bond strength testing is not truly representative of the highly demanding intraoral conditions and at best gives only an indication of the possible clinical performance of the material tested . In spite of these limitations, the results can still assist in suggesting the ideal timing of bonding procedure after the use of fluoride varnishes . Difference in the application time, variation in the fluoride concentrations used, the properties of the bonding agents, and/or bracket retention mechanism should always be considered when comparing such results . Based on the results of the present study, the following conclusions can be drawn: the use of fluoride varnish significantly lowered the sbs valuesthe sbs values of bracket bonded after 15 days from the varnish application were just over the minimum value recommended (6 mpa)the results after 30 days returned to an optimal valuethe indication is to wait more than 15 days after the application of the fluoride varnish in order to obtain an optimal bond strength of the bracket to the enamel . The use of fluoride varnish significantly lowered the sbs values the sbs values of bracket bonded after 15 days from the varnish application were just over the minimum value recommended (6 mpa) the results after 30 days returned to an optimal value the indication is to wait more than 15 days after the application of the fluoride varnish in order to obtain an optimal bond strength of the bracket to the enamel.
A 68-year - old, otherwise healthy female presented with a sudden, temporary loss of vision in her left eye, which occurred 1 day prior to the encounter . She stated that it seemed like a black window shade was pulled over the bottom half of her eye . The loss of vision lasted for ~1 minute, which occurred while she was reading a book using reading glasses . She stated that she had no chest pain or palpitations either during the time of visual loss or in the past . Her ocular history included an uncomplicated cataract extraction in both eyes 2 years prior and dry eyes . Ethical permission was granted by the premier health network . Written patient consent was received for case report presentation . Her systemic medications / supplements include baby aspirin (81 mg daily), artificial tears, glucosamine, and vitamin d. her family history was significant for multiple malignancies including bowel, breast, and colon cancers, heart disease, hypertension, stroke, glaucoma, and macular degeneration . She had no history of glaucoma, macular degeneration, or any heart problems such as atrial fibrillation . She was allergic to azithromycin and erythromycin, both of which result in a rash . The patient s blood pressure was 140/80 mmhg, and her pulse was 80 beats per minute, with a regular rate and rhythm . Ocular examination showed a best - corrected visual acuity of 20/25 + 2 in her right eye and 20/20 in her left eye . Anterior slit - lamp examination revealed dry eyes with meibomian gland dysfuncion oculus uetrque, posterior chamber intraocular lenses that were well placed, and was otherwise insignificant . Fundoscopic examination showed clear vitreous and sharp margins of the optic nerve with a cup - to - disc ratio of 0.2 bilaterally . Examination of the macula, vessels, and periphery was normal, showing no signs of a hollenhorst plaque, embolus, or occluded vessels . Based on the patient s clinical history and presenting symptoms, a differential diagnosis was made consisting of amaurosis fugax (afx), transient ischemic attack (tia), atrial fibrillation, papilledema, and migraine . The patient did not demonstrate any cardiac abnormalities at the time of the event, examination, or in the past, and so the diagnostic focus was shifted to vascular pathology . Based on the history of monocular vision loss lasting ~1 minute and not associated with any headache, change in posture, or exertion, and a normal cup - to - disc ratio, afx as a result of retinal hypoperfusion was determined to be the most likely diagnosis . This diagnosis highly raises the suspicion of an ipsilateral carotid atherosclerotic lesion; therefore, computed tomography (ct) angiography was performed to look for a source of an embolism . The most likely source of an atheroembolism in the case of afx is the carotid bifurcation;1 however, an atheroembolus occasionally arises along the length of the common carotid artery, common carotid origin, aortic arch, or calcific aortic valve . For this reason, ct angiography and three - dimensional reconstructions were performed analyzing the aortic arch, great vessels, cervical vessels, and intracranial vessels . The findings of the ct angiography (figure 1) revealed a congenital vasculature anomaly, which was previously undiagnosed . There was a common origin of the left common carotid and left subclavian arteries that coursed behind the trachea and esophagus . At the origin of the left common carotid artery, there was a 1 cm unstable plaque that was causing critical, irregular stenosis, resulting in a pinpoint lumen (depicted by arrow in figure 1). After the origin, it coursed caudally then turned 180 immediately after the stenotic region . Carotid ultrasound was also performed, which confirmed stenosis at the origin of left common carotid artery . After discovery of the atherosclerotic plaque, a lipid panel was performed, which showed a mixed hyperlipidemia with elevated low - density lipoproteins and triglycerides . In a case of afx, pharmacologic or surgical intervention, or a combination or both, may be indicated . Because of the aortic arch anomaly, diminutive size of the left carotid system, and extensive tortuosity of the left common carotid artery, neither endovascular repair nor open surgical repair of the ulcerated, stenotic carotid origin was feasible . The twisted carotid vessel eliminated potential endovascular repair because the plaque could not be approached with this method . This demonstrates that the location of a plaque and the vascular anatomy are important determining factors of whether surgical intervention is appropriate . In addition, the smaller size of the entire left carotid system indicated that there was a limited risk of serious cerebral or ocular embolic events in the future, including future episodes of afx, a tia, or a stroke . Open surgery involving a subclavian - carotid bypass was entertained, but decided against because the patient remained asymptomatic without any recurrences . The patient was placed on an increased daily aspirin dosage of 162 mg, lipitor 40 mg daily, and will be monitored closely with ultrasound to attempt to prevent further embolic events . The patient has shown improvement in low - density lipoprotein and triglyceride levels to a normal range and has not had any recurrences of afx or other cerebrovascular events since her initial visit . Ophthalmic findings suggesting retinal ischemia or retinal artery findings of hollenhorst plaques are of diagnostic importance, but may be absent depending on the timing of examination . In this case, no hollenhorst plaque was seen, and the cause of the event was an embolus from an atherosclerotic plaque at the origin of the common carotid artery . Afx associated with severe atherosclerotic carotid stenosis, as in the present case, typically has a rapid onset within seconds and lasts between 1 and 10 minutes.2 the unique diagnosis of this patient results from two factors . First, this patient displayed a previously undiagnosed congenital defect with a right - sided aorta . The second abnormality was a common origin of the left common carotid and left subclavian arteries with a tortuous common carotid artery . The origin of the left common carotid artery was stenotic, due to the formation of an atherosclerotic plaque in an abnormal location . The tortuosity of the vessel may have contributed to the formation of the plaque at this location by increasing turbulent blood flow at that region of the artery, causing endothelial damage and increasing the risk of an atherosclerotic plaque . The primary concern over this or any other event suggesting afx is the potential for atheroembolization from an ulcerated or unstable arterial plaque . The most common source of an atherosclerotic embolus is from the carotid bifurcation . Among patients who experienced afx or a hemispheric tia, 88% had a plaque identified by angiography at the carotid bifurcation, and 81% of patients had a plaque resulting in stenosis or occlusion.3 the morphology of the plaque is important to consider when assessing the risk of future embolic events . Moore and hall4 demonstrated the relationship between atheroembolization of the carotid artery and tias, and the same relationship has been established with afx . Atherosclerotic emboli are associated with an increased risk of recurrent cerebrovascular events even when no other risk factors for stroke are present.5 this demonstrates the very important principle that afx, along with a tia, is a warning sign for future retinal or cerebral infarction . Afx and tias present very differently but offer the same potential progression to catastrophic ischemic injury . After an episode of afx, repeated episodes of embolization can lead to retinal artery occlusion, causing a 1% risk per year of permanent monocular blindness.5 future episodes are also associated with cerebral arterial embolization with tia or stroke . Large ulcerative lesions in the carotid system without any intervention present an increased risk of a stroke compared to smaller benign lesions.6 after an episode of afx without any intervention, the risk of stroke within 2 weeks is 18.6% and within 90 days is 33%.7 when carotid stenosis is at least 70%, the risk of stroke after afx within a 2-year period is 16.6%.8 of all parameters discussed, a careful and detailed history suggesting afx is the most important, regardless of whether the diagnosis is verified by ophthalmic findings . Because of these findings, we strongly recommend formal vascular imaging with ct angiography of chest, neck, and head as an initial examination to be followed by vascular surgical consultation . Endarterectomy has been proven to effectively limit future embolic events and restore blood flow to the affected areas.911 this case shows that successful identification and treatment of this disease is of paramount importance and can prevent future damage to visual and cerebral function, even when a rare anomaly such as a right - sided aortic arch is identified in the patient.
Multiple primary or secondary malignancies after anticancer therapy were recently reported to be increasing in frequency because of advancement of chemotherapy technology and resultant longer patient survival times . The incidence of multiple primary malignancies is approximately 0.73% to 11.7% of cancer patients6) and incidence of the metachronous second primary cancers was 3.8%.14) intramedullary spinal cord metastasis (iscm) is found in only 1.65% of autopsy studies of patients with lung cancer.11) iscm raises many ethical concerns and challenges in treatment because determination of the most appropriate treatment must be made based on medical conditions and expected survival of the patient . The author experienced a case of metastasis of metachronous basaloid carcinoma of the lung to the brain parenchyma and spinal cord that developed after chemotherapy and radiation therapy for uterine cervical carcinoma . A 40-year - old female visited the gynecology department for evaluation of leg edema . Abdominal computed tomography (ct) showed bilateral hydronephrosis and lymphadenopathy in the pelvic and para - aortic areas . A punch biopsy of the erosive lesion of the uterine cervix revealed a combination of well - differentiated adenocarcinoma and small - cell carcinoma . The histologic findings of the uterine cervix showed adenocarcinoma with an irregular glandular architecture and infiltration of small hyperchromatic tumor cells . Positron emission tomography (pet) showed the cervix and pelvic and para - aortic lymph nodes lesions . Under a diagnosis of stage iiib cervical cancer, weekly cisplatin (40 mg / m) follow - up pet carried out 5 months later showed extensive lymph node metastasis along the left paravertebral lymph nodes on the pelvic side . After 1 year, pet and chest ct revealed multiple metastatic nodules in both lungs and subpleural nodules with vascular connections . A wedge resection of lung was performed . Gross findings of the specimen showed a 2.82.7-cm, well - circumscribed, whitish, firm, lobulated mass with hemorrhage and necrosis . The histologic findings of the lung specimen revealed pulmonary basaloid carcinoma which lobulated solid tumor cell sheets with peripheral palisading and comedo type necrosis (figure 1). Five years later, she visited the clinic again complaining of headache and left hemiparesis . Brain magnetic resonance imaging (mri) showed a huge heterogeneous mass in the right parieto - occipital area (figure 2). After the craniotomy and surgical resection of the lesion, patient recovered consciousness and hemiparesis, and the pathologic findings revealed pulmonary basaloid carcinoma . Whole - brain radiation therapy comprising a total dose of 5,500 cgy in 250 cgy daily fractions was administered postoperatively, and six cycles of chemotherapy with genexol and carboplatin was performed . One year after the brain surgery, the patient began to suffer from right leg weakness followed by severe radiating pain on the same side and voiding difficulty . We checked follow up brain ct which did not showed recurrence and lumbar mri showed an intramedullary tumor at the level of t12-l1 (figure 3). She underwent gross removal of the spinal tumor through a t12-l2 hemilaminectomy (figure 4), and the pathologic findings revealed metastatic pulmonary basaloid carcinoma . Booster radiotherapy with a total dose of 4000 cgy in 300 cgy daily fractions was administered in the spine postoperatively . Unfortunately, five months later, she was transferred to the emergency room due to stuporous consciousness, and brain mri revealed multiple metastatic tumors in the cerebrum, cerebellum, and subependymal area . Reoperation was not feasible, and despite brain radiotherapy, the patient did not recover consciousness and died of brain swelling . It also occurs in the skin, anus, tongue, larynx, and lungs.4) basaloid carcinoma of the lung was first reported by brambilla et al.5) in 1992 . The overall survival rate of patients with cancer has rapidly increased due to the development of intensive multimodal therapies including immunosuppressive and cytotoxic therapies . However, as a result, it has brought about subsequent complications, especially the development of secondary malignant neoplasms, which has increased by 10 to 20 times.5) some authors reported metachronous tumors induced by radiation therapy.121012) the rate was higher among patients who received adjuvant radiation exceeding 40 gy, pointing out the possibility of secondary neoplasm development due to chemotherapy.3) in current case, the cell characteristics, including hyperchromatic nuclei, are the same in the lung, brain, and spinal cord based on the immunohistochemistry study, including cytokeratin 7, etc . It has been stated that the majority of spinal metastasis is connected to brain metastasis, and such seeding could thus easily occur.8) hematogenous spread is a hypothesis commonly accepted in the majority of studies . One noteworthy hypothesis is that intraspinal metastasis is established as portacaval circulation and pulmonary circulation form bypass when coughing and sneezing through spread via a paravertebral plexus of veins (batson's vein plexus).9) in particular, the tumors that develop due to hematogenous spread are well defined and circumscribed such that they are discrete between the tumor and the spinal cord, thereby rendering them easy to eliminate.7) in current case, the border between metastatic tumor and cord was relatively discrete in mri finding and also in surgical field and other intraspinal canal lesion was not observed . Considering that the mean survival time among patients with brain metastasis is 12 to 15 months, surgical resection in patients with iscm and neurologic deficit is a delicate issue . Therefore, in determining the additional operation for the iscm of such patients should be seriously considered for ethical problem . Generally, the survival rate of patients with iscm who receive only radiation therapy is 4 months, whereas the average survival rate of those who receive both surgery and radiation therapy is 6 months.13) however, this is especially true in the patient described herein, who demonstrated a poor response to chemotherapy and radiation therapy . The authors reported a metastasis case of metachronous pulmonary basaloid carcinoma that showed after chemotherapy and radiation therapy for cervical uterine carcinoma . So there's the possibility that a secondary pulmonary neoplasm developed due to the chemotherapy and radiotherapy conducted as cervical cancer treatment.
The formation of solvated and in particular hydrated forms, in which solvent molecules occupy regular positions in the crystal structure, is widespread for pharmaceutical materials . Recent statistical surveys based on the results of 245 polymorph screens of organic compounds and our own evaluation of the literature on 960 organic drug compounds present in the european pharmacopoeia revealed that ca . Furthermore, according to our statistics, more than 40% of all compounds that are known to form hydrates are used and specified as water adducts in the pharmacopoeia . During the manufacturing process, the drug compound is often in contact with water (for example during crystallization, freeze - drying, wet granulation, aqueous film - coating, spray drying, storage, etc . ). Consequently, knowledge of hydration and dehydration conditions is essential, as the presence of water in the crystal lattice may lead to very different physicochemical properties, such as solubility, dissolution rate, chemical stability, and bioavailability . Thus, finding and characterizing the range of possible hydrates is fundamental to drug development; however, it is time - consuming and requires effective strategies combining a variety of techniques . The widely used, experimental screening approaches for solid forms, which include solvent crystallization and slurry conversions in aqueous solvents as well as dynamic moisture sorption studies, may quickly indicate the formation of hydrate(s). However, they do not provide insight into the structural and thermodynamic reasons for hydrate formation . X - ray diffraction and spectroscopic methods, combined with calorimetric (differential scanning calorimetry, solution calorimetry, etc .) And solubility measurements can generate a complete structural and thermodynamic description of a hydrate / anhydrate system . However, if it is difficult to obtain large amounts of the pure phases and representative crystals suitable for single crystal x - ray diffraction, then this picture will be incomplete . Hence the computational generation of thermodynamically feasible crystal structures offers a complementary technique, and provides a molecular level understanding of the related compounds that often cannot be achieved by experimental techniques alone . For example, crystal structure prediction (csp) techniques have been used to rationalize the formation and structures of various stoichiometric hydrates of dihydroxybenzoic acids, some diastereomeric salt pairs, and racemic and enantiopure naproxen . Phloroglucinol (1,3,5-trihydroxybenzene, phg, figure 1) is a small, symmetrical organic molecule whose dihydrate (hy2) and anhydrous (ah) forms have been widely investigated . Phg is used as a precursor for the synthesis of a variety of industrial products such as pharmaceuticals, dyes, and explosives . It is the parent compound for a high number of derivatives, called phloroglucinols, which are present in a number of plants and algae . The compound exhibits a broad spectrum of pharmacological activities: antibacterial, antiviral, antifungal, antioxidant, and antidepressant . Only the enol tautomer of phg has to be considered, as it is far more stable than the keto form . The two low energy conformers of the phg molecule and the atom numbering used throughout this study . The intramolecular degrees of freedom (torsions and h o c angles) that were optimized in the lattice energy minimizations are indicated with arrows (1: c2c1o1h, 2: c2c3o2h, and 3: c4c5o3h) and emboldened on the less stable cs conformer . There have been several x - ray crystallographic studies on phg because it readily crystallizes in the hy2 form, but with variable morphologies and crystal quality, with even the best quality crystals showing diffuse scattering effects . Banerjee and ahmand, bose and sen, and chorgade independently determined the cell constants, and they proposed possible space groups and a potential crystal structure . Wallwork and powell reported the hy2 structure (cambridge structural database (csd) refcode: phgloh) and proposed a model for the observed diffuse thermal reflections and disorder streaks, but they did not locate the polar protons . Subsequently, singh and singh reinvestigated the growth and disorder of the hy2 crystals, confirming the results observed by wallwork and powell . Singh and singh proposed a link between the crystal morphologies and surface features, which they found to depend on growth conditions, to the diffuse scattering effects . (csd refcode: phgloh01) redetermined the hy2 structure, including all proton positions . Very recently, thomas et al . Also investigated hy2 and proposed a model for the hy2 diffuse scattering based on first - principles solid - state quantum mechanical calculations . Other joint experimental and theoretical studies on phg include investigations into the dipole moment of phg in ethanol, and vapor pressure and lattice energy calculations . The interactions of each of the two phg conformers with water molecules were modeled computationally in vacuo and in water solution . The less readily obtained anhydrate structure has also been determined at room temperature by maartmann - moe (csd refcode: phglol) and redetermined at 105 k (csd refcode: phglol01) by goerbitz et al . The present study develops a consistent quantitative account of the solid state properties and the structural and thermodynamic features of the ah / hy2 system . A broad range of analytical techniques are applied to resolve and characterize the complexity of the phg solid forms emerging from a polymorph screen, including hot - stage microscopy (htm), differential scanning calorimetry (dsc), thermogravimetric analysis (tga), relative humidity (rh)-perfusion and solution calorimetry, x - ray diffractometry (powder and single crystal), infrared spectroscopy (ir), and dynamic moisture sorption analysis . The relative enthalpy of ah and hy2 could only be established using innovative approaches of solution (rh - perfusion) and dsc measurements, as recently demonstrated for barbituric acid . The computational work sought to establish the stability of the ah and hy2 experimental crystal forms relative to other possible structures, thus investigating the disorder observed in the hy2 structure and the relative stability difference of the hydrate and anhydrate phase . The relative humidity dependence of the phase change between hy2 and ah and the temperature / composition phase diagram are also determined . Hence we provide a complete stability picture, including a variety of new thermo - physical data, of this complex hydrate / anhydrate system, which should be useful in the design of an industrial production process . Anhydrous phg (purity 99.0%) was purchased from sigma - aldrich . A phase pure hy2 sample was obtained by crystallizing the compound from a hot saturated water: ethanol (1:1) solution . Ah was prepared by drying the hy2 in a drying oven at 100 c for about 5 h. for thermo - microscopic investigations, a reichert thermovar polarization microscope equipped with a kofler hot - stage (reichert, vienna, a) was used . Microphotographs were taken with a digital camera (color view iiiu, olympus optical co. ltd ., the phg hy2 morphologies were observed under a hirox kh-7700 3d video microscope (hirox co. ltd, japan). Dsc thermograms were recorded on a dsc 7 or diamond dsc equipped with a controlled cooling accessory (cca 7), operated with the pyris2.0 software (perkin - elmer, norwalk, ct, usa). A few milligrams of accurately weighed (mettler um3 ultramicrobalance) sample were heated in perforated al - pans or sealed, gold plated stainless steel high - pressure capsules (30 l). For the construction of the temperature / composition phase diagram, different hy2ah mixtures were prepared by dehydrating hy2 samples isothermally at 40 c in a tga oven until a desired mass loss was obtained . The hy2water mixtures were produced by placing precisely weighed amounts of hy2 and pure water with the aid of a hamilton syringe and a second accurate weight measurement in a um3 ultramicrobalance (mettler, greifensee, ch) into high - pressure dsc pans . The sealed sample pans were stored for at least 4 h before the dsc runs were started to equilibrate the mixture . Ah and hy2 samples, and hy2-ah mixtures were heated from 25 to 250 c at a rate of 5 c min and the hy2water mixtures were scanned, after equilibrating for 30 min at room temperature and 5 min at 20 c, from 20 to 250 c (5 c min). The two instruments were calibrated for temperature with pure benzophenone (mp 48.0 c) and caffeine (236.2 c), and the energy calibration was performed with indium (mp 156.6 c, heat of fusion 28.45 j g). The errors on the given temperatures (extrapolated onset temperatures) and enthalpy values are stated as 95% confidence intervals (cis) based on at least five measurements . Tga was carried out with a tga7 system (perkin - elmer, norwalk, ct, usa) using the pyris 2.0 software . Two - point calibration of the temperature was performed with ferromagnetic materials (alumel and ni, curie - point standards, perkin - elmer). For dynamic temperature scans, a heating rate of 5 to 10 c min was applied, and dry nitrogen was used as a purge gas (sample purge: 20 ml min, balance purge: 40 ml min). The enthalpy of solution (solh) of hy2 was measured with the precision solution calorimeter of the tam iii thermal activity monitor (ta instruments inc . ). The measurement temperature was 25 0.0001 c, the volume of the vessel was 100 ml, and the stirrer speed was 500 rpm . The calorimeter was calibrated with kcl (analytical grade,> 99.5%, merck). The measured standard solution enthalpy was solh = 17.51 0.02 kj mol, which is in agreement with the nist value of solh = 17.584 0.017 kj mol . The tam assistant software v0.9 and solcal version 1.2 was used for the data analysis . Rh perfusion calorimetry experiments were performed with the tam iii nanocalorimeter unit in a 4 ml stainless steel rh perfusion ampule . The relative humidity was controlled with two mass flow controllers, and dry n2 was used as carrier gas at a constant flow rate of 100 ml h. the measurement was performed with 58.9 mg anhydrous phg and the humidity profile (% rh vs time) was executed as follows: 10% (2.5 h), 20% (5 h), 50% (31.5 h), 20% (1 h). The rh perfusion cell was calibrated with saturated solutions of nacl (73.5% rh), mg(no3)2 (52.8% rh), and licl (11.3% rh). The heat flow of the empty rh perfusion ampule (baseline run with the same humidity steps) was subtracted from the heat flow of the sample measurement . Dynamic moisture sorption and desorption studies were performed with the automatic multisample (gravimetric) moisture sorption analyzer (sps11 - 10, project - messtechnik, ulm, d). The experiment started at 40% rh with a desorption cycle (decreasing humidity) followed by a sorption cycle (increasing humidity) up to 90% rh . In order to assess the dehydration and hydration step with high precision, the rh changes were set to 2% below 40% rh, whereas above this humidity more coarse changes of 10% were chosen . The powder x - ray diffraction (pxrd) patterns were obtained using an xpert pro diffractometer (panalytical, almelo, the netherlands) equipped with a / coupled goniometer in transmission geometry, programmable xyz stage with well plate holder, cu k1,2 radiation source with a focusing mirror, a 0.5 divergence slit and a 0.02 soller slit collimator on the incident beam side, a 2 mm antiscattering slit and a 0.02 soller slit collimator on the diffracted beam side, and a solid state pixcel detector . The patterns were recorded at a tube voltage of 40 kv and a tube current of 40 ma, applying a step size of 2 = 0.013 with 40 s per step in the 2 range between 2 and 40. samples were prepared by mortar and pestle grinding to reduce preferred orientation effects . A rectangular parallelepiped shaped crystal of hy2 showing no significant surface features, obtained from slow cooling crystallization from water, was chosen for the single crystal diffraction experiment . The x - ray data were collected at room temperature (20 c) on a stoe ipds - ii diffractometer using mo k radiation (= 0.71073). The structure solution and refinement were carried out using the sir04 and shelxl97 programs incorporated in the wingx program suite . All non - h atoms were refined anisotropically, and h atoms were located and refined isotropically . The o(1)h(1) proton and one water proton (h6) are disordered over two positions and both modeled with occupancies of 50:50 each . The disordered h positions of the water molecule were refined by applying geometrical restraints on the hh distance . Crystal energy landscapes were generated using the two low energy conformers of phg, which differ in the mutual orientation of the oh groups, c3h and cs (figure 1), held rigid as obtained by ab initio optimization of the isolated molecule structure at the scf/6 - 31g(d, p) level of theory using the program gaussian 03 . Using the program crystalpredictor, 100 000 z = 1 anhydrate and 500 000 dihydrate crystal structures were randomly generated in 25 space groups, p1, p1, p21, p21/c, p21212, p212121, pna21, pca21, pbca, pbcn, c2/c, cc, c2, pc, cm, p21/m, c2/m, p2/c, c2221, pmn21, pnna, pccn, pbcm, pmmn, and pnma . Each crystal structure was relaxed to a local minimum in the intermolecular lattice energy, calculated from the fitexp-6 repulsion - dispersion potential and atomic charges that had been fitted to electrostatic potential around the mp2/6 - 31g(d, p) charge density using the chelpg scheme . The 15 000 anhydrate and 20 000 hydrate lowest energy structures were reminimized using dmacrys with a more realistic, distributed multipole model for the electrostatic forces that had been derived using gdma2 to analyze the mp2/6 - 31g(d, p) charge density . Thus the intermolecular lattice energy (uinter) includes the highly directional electrostatic interactions arising from the lone pair and electrons . The optimal proton positions (figure 1) in all crystal structures within 10 kj mol of the global minimum for the anhydrates and 8 kj mol for the hydrates this was done by minimizing the lattice energy (elatt), calculated as the sum of the intermolecular contribution (uinter) and the conformational energy penalty paid for distortion of the molecular geometry to improve the hydrogen bonding geometries . Conformational energy penalties (eintra, with respect to c3h), and isolated molecule charge densities were computed at the scf/6 - 31g(d, p) and mp2/6 - 31g(d, p) levels, respectively, for each conformation considered in the minimization of elatt . To approximate the polarization of the molecular charge distribution in the crystal, as has been found necessary in csp studies of peptides, the charge density used in the final evaluation of elatt was generated in a dielectric constant, = 3, a value typical of organic crystals . Thus, the final mp2/6 - 31g(d, p) electron density calculations used the polarizable continuum model (pcm) implemented in gaussian 03 . The intramolecular energy penalty eintra was calculated from the scf energies in the same pcm ab initio calculations, excluding the interaction energy between the molecule and the polarizable continuum . All calculated hydrate structures were run through the addsym function of platon in order to determine the true space group and z value . Structural comparisons were made using mercury, including the evaluation of the difference in the optimal root - mean - square overlay of all non - hydrogen atoms in a 15 molecule coordination cluster (rmsd15). The experimental screen from 26 solvents resulted in the two known solid forms (anhydrate and dihydrate), and novel solvates from methanol and dimethyl sulfoxide . The methanol solvate is unstable and transforms quickly to the anhydrate if removed from the mother liquor . This solvate shows an inhomogeneous melting process (a peritectic / melting decomposition of the solvate that overlaps with the crystallization of the anhydrate) at 87 c upon heating . The surface features and morphology of the hy2 crystals were observed to depend on the solvent and crystal growth temperature (as previously reported). Solvent evaporation experiments predominantly gave rhombic and six - sided plates exhibiting characteristic spiral growth steps, i.e., lozenge - shaped steps on the rhombic plates or rhombic to six - sided shaped steps on the six - sided plates (figure 2). Slow cooling crystallization experiments predominantly gave rhombic to rectangular shaped plates or blocks with no surface features . Phg dihydrate crystals representing the range of morphologies: (a) six - sided crystal exhibiting closed loop spiral growth steps obtained from evaporation from a saturated etoh: water (1:1) solution at room temperature . (b) rhombic, plate - like crystal with lozenge - shaped steps obtained from evaporation from a saturated water solution at room temperature . (c) block - like dihydrate crystal from cooling crystallization of a saturated water solution . The moisture sorption / desorption isotherm of phg (figure 3) shows that the anhydrate is stable (i.e., does not absorb water) up to 32% rh . At higher moisture conditions, the sample takes up water, and the transformation to hy2 occurs . The measured mass gain of 27.9% corresponds to 1.95 mol water, which is only slightly below the theoretical value of 2 mols of water per mole of phg (28.6% referred to the anhydrous mass). On decreasing the humidity, the dehydration to the distinct steps and hysteresis between the sorption and desorption isotherms are characteristic for a stoichiometric hydrate, but the hysteresis in the phg system is rather small compared to 2,4-dihydroxybenzoic acid hemihydrate and aripiprazole monohydrate . The sorption / desorption isotherms show that hy2 is a very stable hydrate, which releases its water only at low relative humidities, and conversely the ah is a rather unstable anhydrate phase, which absorbs water (moisture) above 32% which is below ambient conditions (40 to 60% rh). This information is crucial for handling of the ah, and therefore all experiments performed on the ah were done with samples that had been stored over a desiccant . The circles present data points that fulfill the present equilibrium condition (mass change), whereas crosses mark measurement values that did not reach the equilibrium within the allowed time limit (100 h). The single x - ray crystal structure of the anhydrate deposited in the csd as phglol01 was used for structural comparisons . The phg molecule is essential planar, c3h point symmetry, with the oh protons slightly out of plane of the benzene ring forming more linear hydrogen bonds . The molecules pack in a folded molecular aggregation pattern, consisting of almost perpendicular hydrogen - bonded planes . Hy2 crystallizes in the orthorhombic space group pnma with half a phg and one water molecule in the asymmetric unit . [crystal data ofhy2: c6h6o32(h2o), mr = 162.14, orthorhombic, space group pnma, t = 20(2) c, size [mm]: 0.35 0.3 0.1, a = 6.6209 (17), b = 13.561 (3), c = 8.0462 (15), v = 722.4 (3), z = 4, calc = 1.491 mg m, 4393 reflections measured, 732 independent reflections, 650 observed reflections, range for data collection: 2.94 26.05 h, k, l, range: 8 <h <8, 15 <k <16, 8 <l <9, data: 732, restraints: 2, parameters: 81, r[f> 2(f)]= 0.037, wr(f) = 0.093, r int = 0.037, goodness of fit on f= 1.10, max = 0.23, min (e) = 0.24 .] The stoichiometry is in agreement with the moisture sorption / desorption (section 3.2) and tga results (section 3.5) as well as previous structure determinations . The average x - ray structure exhibits a mirror plane located down center of the benzene ring of the phg molecule (figure 4a), which dictates a 50:50 positional disorder of one of the three ho protons, and, consequently, one of the water protons is also 50:50 disordered over two positions . In contrast to the ah structure, the hy2 oh protons are nearly coplanar with the benzene ring (max . Hydrogen bonds through the water molecules, with each of the three phg oh groups forming two hydrogen bonds with water and each water molecule forming three hydrogen bonds: two with phg and one with an adjacent water molecule . The strongly hydrogen - bonded phg and water molecules form corrugated layers parallel to (100). The corrugated layers, as defined by wallwork and powell and singh and singh, are formed by the phg / water molecules located at x 1/8 and x 3/8, and the phg / water molecules at x 5/8 and x 7/8, respectively (figure 4b and c). The hydrogen bond formed between a pair of water molecules links the different layers (figure 4d). Compared to the high number of interactions within each corrugated layer (six hydrogen bonds for each phg molecule), the interlayer interactions involve only waterwater interactions . There is an alternative set of layers defined by the molecules at x 5/8 and x 3/8 (figure s2, supporting information), with the phg molecules in different orientations . Interactions within the layer are through water molecules and the interlayer interactions through a waterphg interaction . Perpendicular to either definition of layers, parallel to (010), there are sheets of oxygen atoms, formed of water and phg oxygens (figure 4c), and each sheet is linked through o phg and water oxygen atoms within the same sheet but different layers have approximately the same y and z coordinates and differ in their x coordinates by approximately a/2 (figure 4c). (a) left: phg water complex, showing the hydrogen bonds on a local level; right: proton disorder as observed from the average x - ray structure . (b) one hydrogen bonded layer of the structures as seen in projection on (100). (c) structure of phg dihydrate projected along, showing the corrugated layers formed by the phg molecules (indicated in green and blue) and sheets of oxygen atoms (red dotted lines). (d) dihydrate structure projected along . The lattice energy landscape of the phg anhydrate (figure 5a, table 1) has two structures that are more stable than any others, both of which correspond to the experimental structure if proton positions are ignored . The most stable structure overlays a 15-molecule coordination sphere with an rmsd15 of 0.39 including the hydrogen atoms . The other low energy structure has the same packing of all c and o atoms (figure 5b), but differs in some proton positions, giving a different cs molecular symmetry and different space group (pna21 instead of p212121). The two structures exhibit the same intermolecular interactions, differing in the directionality of the o ho hydrogen bonds (figure 5b), with the deviation of the polar hydrogen atoms from planarity well reproduced by the modeling technique . The small lattice energy difference and iostructurality of the calculated structures indicates that proton disorder cannot be excluded as domains of the alternate structure may exist within the known form . However, the x - ray diffraction studies did not report any proton disorder or diffuse scattering . (b) hydrogen bonding of the calculated experimental (a_c3h_1) and second lowest calculated structure (a_cs_3), showing the isostructurality apart from some hydroxyl protons . A full list of the structures shown in figures 5 and 6 is given in the supporting information (tables s3 and s4). I d: experimental (exptl .) And identification codes for the computed structures . Proton ordered, z = 1 space group, which platon addsym classifies as pnmaz = 0.5 . Proton ordered, z = 1 space group, which platon addsym classifies as c2/cz = 0.5 . The optimal 15-molecule coordination cluster overlay with the experimental structure (non - hydrogen atoms only). The calculated ah lattice energy of 126.2 kj mol is in good agreement with de wit et al.s experimentally determined heat of sublimation of 127.9 kj mol using a torsion - effusion technique and 126.0 kj mol by mass loss effusion, measured in the temperature range of 100 to 133 c . Since the lattice energy corresponds to infinitely separating the molecules in a static crystal at absolute zero (273 c), the agreement implies that the empirical parametrization of the repulsion dispersion potential has partially absorbed the thermal (heat capacity) and crystal zero - point energy effects . The phg dihydrate lattice energy landscape (figure 6a) shows a much higher density of favorable structures than for the anhydrate (figure 6), but all low - energy dihydrate structures contain the same infinitive linear chains (figure 6b, right). The experimental structure is the most stable, but there are two groups of low energy structures (groups a and b, table 1) which are closely related to the experimental structure . The hydrogen boding motifs are identical for these eight structures if the directionality of the o ho hydrogen bonding is ignored (figure 7). Green triangles: structures that match all non - hydrogen atoms with the experimental structure (table 1, group a); blue diamonds: match two stacks of adjacent phg water chains of the experimental structure (table 1, group b); gray dots: structures with just the same infinitive linear chains as experimental structure . The red ellipsoid encompasses proton - ordered versions of the proton - disordered x - ray structure, the red dotted circle encloses the structures that may account for the diffuse scattering effects . (b) packing comparison between group a and b structures, defined in table 1 . Phg molecules, forming the corrugated / cascaded layers are shown in the same color . Hydrogen bonding connectivity (within one layer) of the four group a and b dihydrate structures related to the experimental phg dihydrate: (a) h_cs_1613 and h_cs_29, (b) h_cs_6 and h_cs_27, (c) h_c3h_4696 and h_c3h_19, and (d) h_cs_14 and h_cs_64 . Four of the low energy structures (group a, table 1, figure 6), including the global minimum, show the packing observed in the experimental hy2 structure, (i.e., carbon and oxygen atom positions are essentially the same,) although three of these structures have the cs proton conformation, and all differ in the directionality of the o hydrogen bonds (figure 7). Only the two most stable group a structures exhibit phg and water oh proton positions observed as the disorder components of the experimental structure (figure 4b). The difference between these two lowest - energy group a structures is the directionality of every alternate o(1)ho hydrogen bond, leading to p212121 and pna21 space groups . The other two less stable group a structures (both space group p212121, elatt <7 kj mol with respect to most stable hy2) differ in the o(2)h and o(3)h proton positions from those determined in the x - ray diffraction experiment . These two higher energy structures cannot be ignored in discussions of possible proton disorder . Checking for higher (pseudo)symmetry using platon showed that all four group a structures can be expressed as proton disordered pnma structures . The two lowest - energy group a structures show in their common maximal nonisomorphic supergroup (pnma) the 50% occupancy proton disorder in the (average) experimental x - ray hy2 structure . On a local level, the proton must adopt one position, and this will determine the hydrogen bonding directionality, resulting in p212121 and pna21 domains within the crystal . Four other calculated low energy structures (group b, figure 6) have the same sheets of oxygen atoms as group a (figure 4c). The two groups differ in the displacement of half of the oxygen sheets by a/2, leading to cascaded hydrogen - bonded layers in group b structures in contrast to the corrugated hydrogen - bonded layers present in group a (figure 6b). If one sheet of oxygen molecules is displaced by a/2 with respect to the other, the oxygen atoms of the displaced sheet are still in a position that allows them to form the same number of strong hydrogen bonds with the oxygen atoms of the adjacent sheet . This is possible because the phg oxygen atoms take approximately the same position as the water oxygen atom, and vice versa . Such a displacement of oxygen sheets would also give rise to diffuse scattering effects in x - ray diffraction experiments . The similarities in structure and energy of the group a and group b structures imply a high probability of stacking - fault - like defects of the oxygen sheets and possible intergrowth of group a and group b domains, illustrated in figure 8, as observed for aspirin, in addition to the p212121 and pna21 domains within group a. the intergrowth of group a and group b domains (figure s8, supporting information) adds the range of possible proton positions to the defect structure anticipated by wallwork and powell and singh and singh from the experimental hy2 structure . The orthorhombic packing motif (group a) seems to be preferred for phg hy2, as none of the crystallization experiments resulted in a phase where the monoclinic domains (group b) dominated the structure . Hypothetical packing of phg dihydrate showing the possible intergrowth of corrugated and cascaded layers . Selected symmetry elements are shown: black in common to both layers; green for the corrugated layers in the p212121 and pna21 group a structures that have pnma symmetry if proton positions are ignored; blue for the cascaded layer in the p21/c and cc group b structures which have c2/c symmetry if proton positions are ignored . We have compared the pxrd measurements of hy2 batches obtained from different solvents and crystallization rates with the simulated powder patterns of the computer generated structures . Figure 9a contrasts the pxrd pattern for hy2 produced by crystals obtained from cooling crystallization (sample 1, did not possess any significant surface features) with that of crystals obtained from solvent evaporation from a petri dish (sample 2, irregular six sided platelet with spiral steps). Several of the diffraction peaks are broadened in the pattern of sample 2 but sharp for sample 1 . The most notable differences are in reflections originating from the (111) and (121) planes, which are formed by phg oxygen (figure 9b) and water oxygen atoms (figure 9c). The experimental powder pattern for sample 1 (the slow crystallization product) is in good agreement with those of the group a crystal structures . The only differences in the powder patterns arise from the temperature disparity (thermal expansion), while the impact of the proton disorder is negligible . Similarly, as discussed in the supporting information, varying proportions of the stacking faults described by figure 8 are compatible with the variations in the powder patterns . (a) experimental pxrd patterns for different phg dihydrate crystallization batches (sample 1: cooling crystallization from water; sample 2: solvent evaporation from ethanol) contrasted with the simulated patterns of a computationally generated group a and group b dihydrate structures (table 1) and of a p1 cell corresponding to a combination of the two structures (group a+b, figure 8). Note that the computationally generated structures are perfectly ordered structures and therefore exhibit no diffuse scattering . The area highlighted in yellow shows the key differences corresponding to the packing diagrams showing (b) the (111) and (c) the (121) planes . Oxygen atoms lying on the planes are shown as balls . The dehydration process of hy2 between 55 and 90 c, as observed with htm (figure 10), is governed by a nucleation and growth mechanism, which is indicated by the appearance and growth of dark spots upon heating . These spots, emerging at the surface and macroscopic defects of the hy2 crystals, represent the nucleation centers of the ah . Although the number of the nucleation centers increases with temperature, the overall dehydration process is dominated by the high growth rate of a limited number of nuclei . The process results in the formation of aggregates of homogeneously sized ah crystals (<10 m) with the original shape of the hy2 crystals (called pseudomorphosis), which is characteristic for the desolvation of stoichiometric solvates . Between 217 and 220 c, the sublimed ah crystals decompose and melt . By embedding the hy2 crystals in high viscosity silicon oil and applying a heating rate of about 10 c min, the incongruent melting (peritectic decomposition) of the hy2 can be determined at 118 the hy2 crystals fuse partially, and at the same time nucleation and growth of ah occurs, accompanied by the release of water vapor as indicated by the formation of bubbles . All dehydration experiments resulted in only one anhydrous phase (ah), which was confirmed by ir - spectroscopy and/or pxrd . The removal of the water from hy2 is connected with a disruption of all o ho hydrogen bonds and rearrangement of the phg molecules . The observed nucleation at the surface and macroscopic defects is likely to be associated with the escape of the water . The considerable rearrangement of the phg molecules into the anhydrate structure could be relatively facile, as it can be simulated by lattice energy minimization from the dihydrate structure with the water molecules computationally removed and a proton transferred to give a c3h conformation (figure 11). A model for the rearrangement of the phg dihydrate structure to the anhydrate after computational removal of water molecules . (a) phg dihydrate, with arrows indicating the direction of movement of the molecules when the water molecules (position indicated in blue) are removed and the resulting lattice energy minimized; (b) an intermediate structure emphasizing the tilting of the molecules; and (c) the anhydrate structure that results . Hydrogen atoms are omitted, but the simulation form required the c3h molecular geometry (c3h_4696, table 1). The tga curve of hy2 shows (figure 12) a one - step dehydration process, which corresponds to the loss of 2 mols of water per phg molecule (measured mass loss: 22.3%, theoretical value for 2 mols water relative to the hydrate is 22.22%). The second step observed in the tga curve occurring around the melting temperature of ah corresponds to the decomposition of the phg . In a pin - holed dsc capsule, the dehydration process of hy2 is observed as a broad endothermic peak, followed by the melting process of ah at 221 c . The dsc curve of the ah exhibits only the melting process, indicated by an endothermic peak with a heat of fusion (fushah) of 34.4 mol . The decomposition process that starts before melting of the ah does not produce a strong change in the dsc signal, and so the thermolysis (thermal decomposition) will contribute to the wide range of ah melting points reported in textbooks, the beilstein reference database, and other literature sources . One can also find melting points of phg reported in the range from 113 to 118 c, which corresponds to the peritectic decomposition range of hy2 and not the melting point of the anhydrous substance . Dsc and tga thermograms of phg anhydrate and dihydrate (pin - holed sample pans and a heating rate of 5 c min was used for all thermograms). The temperature / composition phase diagram of phg / water (figure 13) shows the typical behavior of an incongruent melting hydrate with a peritectic temperature at 120 c and a eutectic (monotectic) between hy2 and water at 0 c . The peritectic temperature (<120 c, at roughly ambient pressure) may be defined as the temperature up to which a hydrate is thermodynamically stable in the presence of its own saturated solution and vapor pressure . Thus a slurry (suspension) of the hydrate in water would contain only anhydrous phg above 120 c . (a) selected dsc thermograms of the phg water system (sealed capsules and heating rates of 5 c min were applied) used for the construction of the (b) temperature / composition phase diagram . The enthalpy of the hy2/ah transition can be estimated from the results in table 2 . The dehydration process, dehyhhy2-ah, measured in open dsc pans (pin holed lid, figure 12), can be subdivided (application of hess s law) into the enthalpy of hydrate to anhydrate transformation, trshhy2-ah, and the vaporization of the expelled water.1 melting and decomposition occur in the same temperature range at the heating rates applied . No detailed experimentation was carried out to study the thermal decomposition and its impact on the melting temperature and heat of fusion . If we subtract the known enthalpy value for the vaporization of water at the dehydration temperature (onset, tdehy 75 c at which vaphh2o = 41.81 kj mol) from the measured enthalpy of dehydration according to eq 1, we can estimate the enthalpy of the hydrate to anhydrate phase change as 19.3 kj mol . With isothermal calorimetry (t = 25 c, rh - perfusion cell) an enthalpy of hydration (hyhah - hy2) of 107.04 kj mol was obtained . Since the magnitude of the heat of condensation of water (condhh2o) is equal to the heat of vaporization, we can use eq 2 to calculate the transition energy of ah to hy2 (trshah - hy2):2using a value of vaphh2o (25 c) = 43.99 kj mol = condhh2o (25 c) gives a transition energy of 19.1 kj mol, which is in excellent agreement with the value obtained from dsc experiments (19.3 kj mol) measured at a higher temperature . The order of magnitude of the experimental enthalpic transition energy is in the range of values measured for other stoichiometric dihydrates . The simplest estimate for the hydrate to anhydrate transition is to calculate uhy2-ah from ah, hy2, and ice lattice energies, elatt.3using the lattice energy of the experimental structures (the global anhydrate and hydrate search minima (table 1)) and the energy range for the ordered ice polymorphs (elatt(ice) = 57.54 to 60.02 kj mol, supporting information table s2) gives uhy2-ah of 20.0 to 25.0 kj mol . The experimental measurements and computational estimates compare the separation of the hydrate into anhydrate and water into different phases at different temperatures . Computationally, we break the hydrate into infinitely separated anhydrate and water molecules (ideal gas) at absolute zero (273 c), neglecting thermal contributions and zero - point vibrational effects . In contrast, experimentally we transform the hydrate into solid anhydrate and water vapor, assuming that water evaporates from the liquid state . The experimental thermodynamic arguments are complicated by consideration of whether the heat of vaporization of water or the heat of sublimation of ice are more appropriate, influenced by whether the water within the hydrate structure is isolated or liquid - like . The transition enthalpy of hydrate to anhydrate can, in theory, also be calculated from the differences of the heats of solution (solh, eq 3) of ah and hy2:4 for hy2 we measured a solhhy2 of 39.7 1.4 kj mol; however, a meaningful determination of solh by solution calorimetry in water for ah was not possible, since the transformation of ah to hy2 is too rapid . We can estimate solhah by the use of the trshah - hy2 value obtained with the rh perfusion technique and application of eq 3 (see also supporting information, figure s4) as approximately 21 kj mol at 25 c . The crystallographic disorder is associated with sufficiently similar water environments within the crystal and very small energy differences (by calculation). Hence we would not expect the crystallographic disorder to affect rate and dehydration, which was consistent with any difference being far too subtle to be detected by the analytical methods . The experimental search for solid state forms of phg has resulted in only two practically relevant solid phases: the dihydrate and anhydrate . A highly metastable methanol solvate and a dimethyl sulfoxide solvate that could arise in processing pholoroglucinol have also been detected . Confidence that all stable anhydrate and dihydrate forms have been found is provided by the crystal energy landscapes which have the experimental anhydrate and a proton ordered version of the dihydrate as the global minima in lattice energy . These landscapes show that closely related structures are so similar in energy, well within the likely energy range for possible polymorphs, that they can account for the proton disorder and stacking faults in the dihydrate . These two groups of low - energy hy2 structures provide a valuable starting point for further disorder modeling and experiments to explain the complex diffuse scattering in the hydrate . The layer models giving rise to the diffuse scattering proposed by wallwork and powell and singh and singh may therefore be improved upon on a molecular scale by a combination of the local ordering suggested by thomas et al . (group a) and the layer intergrowth models (groups a and b) proposed here . The dihydrate loses water below 16% rh, and this relatively small hysteresis, close to ambient conditions, makes this otherwise straightforward hydration behavior practically problematic . The temperature / composition phase diagram under saturated water vapor pressure, the thermal desolvation behavior, and the moisture sorption / desorption studies provide the thermodynamic and kinetic data needed for controlling the handling, processing, and storing of phg . The dihydrate is enthalpically stabilized by approximately 19 kj mol as derived from two experimental approaches . Monitoring the loss of water by thermal microscopy revealed that nucleation was associated with macroscopic defects on the surface, consistent with the loss of water not being significantly affected by the stacking faults / domains and surface morphology variations, and the relationships between the crystal structures at the atomic level . This novel experimental and computational strategy has provided deeper understanding of this hydrate system, with a reassuring consistency between the different techniques . Phg represents another example where the computed crystal energy landscape calculations can assist in proposing feasible models for the experimentally observed disorder . In this case, the small energy differences between structures differing only in polar proton positions adds to the confidence that the nature of the disorder, both in proton position and stacking defects, does not affect the processing.
The vestibular system, or the system of balance, provides information about the motion, equilibrium, and spatial orientation of the body . Lesions of the vestibular system result in postural and visual deficits accompanied by dizziness, vertigo, and changes in cardiorespiratory and gastrointestinal functions . Signs of the disorder are classified as static and dynamic symptoms and many, but not all are regained spontaneously in the process of vestibular compensation (dieringer, 1995; vidal et al ., 1998; hitier et al ., the vestibular compensation incorporates modifications in a number of processes, like changes in discharge properties of bilateral vestibular neurons (dieringer, 1995, 2003; vibert et al ., 1999b; straka et al ., 2005; dutia, 2010), in the efficacy of synaptic inputs from the existing non - labyrinthine pathways to the deafferented vestibular neurons and in remodeling of synaptic connections through axonal sprouting and synaptogenesis (dieringer, 1995, 2003; vibert et al ., based on previous results suggesting the modifications of extracellular matrix components in other parts of the nervous system after injuries, we suppose that these interrelated events of vestibular compensation have influence on the molecular assembly of the extracellular matrix also in the vestibular nuclear complex . In the central nervous system, the major form of extracellular matrix, the perineuronal net, emerges in condensed form around the perikarya, proximal dendrites and axon initial segment (carulli et al ., 2006; bruckner et al ., 2008; dityatev et al ., 2010; frischknecht and seidenbecher, 2012; lendvai et al ., 2012; blosa et al ., principal molecular constituents of perineuronal net are the hyaluronan, chondroitin sulfate proteoglycan lecticans, tenascin - r and various link proteins (celio et al ., 1998; zimmermann and dours - zimmermann, 2008; kwok et al ., 2011). The constituents of perineuronal net is activity dependent and its molecular assembly, as the parts of the synaptic machinery (dityatev and rusakov, 2011), can modify the synaptic transmission (bukalo et al . Although the role of extracellular matrix in the lesion - induced plasticity and regeneration was confirmed in various parts of the central nervous system (dityatev and schachner, 2003; moon et al ., 2003; galtrey and fawcett, 2007; lin et al ., 2009; dityatev et al ., 2010; alilain et al ., 2011; dityatev and rusakov, 2011; michaluk et al ., 2011), similar works on the vestibular system were published only from our lab (halasi et al . We observed that unilateral labyrinthectomy is accompanied by the radical decrease of hyaluronan and chondroitin sulfate proteoglycans expression in the lateral vestibular nucleus during the compensatory period . The reorganization of extracellular matrix assembly in the perineuronal net and neuropil correlated with the time course of recovery from the postural deficits and reappearance of normal resting discharge of vestibular neurons (dieringer, 1995; curthoys, 1996; vidal et al ., 1998; curthoys and halmagyi, 1999; darlington and smith, 2000), suggesting the involvement of hyaluronan and the lecticans in the process of vestibular plasticity (deak et al ., 2012). Here, we extend this study to the tenascin - r as the third major contributor of the perineuronal net integrity, forming the ternary network with the hyaluronan and lecticans (koppe et al ., 1997; bruckner et al ., 2000; pesheva and probstmeier, 2000; dityatev and schachner, 2003; carulli et al ., 2006;, 2006; zimmermann and dours - zimmermann, 2008; kwok et al ., 2010; wang and fawcett, 2012). Other studies on various species indicated that tenascin - r is an important modulator of neural plasticity and repair processes in various parts of the central nervous system (apostolova et al . Based on these findings, we hypothesized that the tenascin - r expression in the superior, medial, lateral, and descending vestibular nuclei varied following unilateral vestibular lesion and subsequent compensation . The experiments were carried out in accordance with european community guidelines and state regulations and with the approval of the university animal care committee (demb, 11/2011/de mab). All efforts were made to minimize animal discomfort and reduce the number of animals used . Adult female wistar rats, aged 1214 weeks old, weighing 250300 g, were used in the experiments (n = 12). The animals were anesthetized using an intramuscular injection of 2% xylazine (10 mg / kg, cp pharma handels gmbh, germany) and 10% ketamine (100 mg / kg, cp pharma handels gmbh). A 1.5 cm - long skin incision was made behind the left external acoustic meatus; the cervical muscles, posterior belly of the digastric muscle, the stylohyoid and their nerves were spared . The ventral wall of the tympanic bulla was carefully opened and the labyrinth containing the vestibular sensory organs was approached by breaking the promontory . The left vestibular sensory organs were mechanically destroyed with special care taken on keeping the stapedial artery and facial nerve intact . After a period of 1, 3, 7 or 14 days of survival, the rats (three animals at each time point) were re - anesthetized with intraperitoneal administration of 10% urethane (1.3 mg/100 g, reanal, budapest, hungary) and perfused transcardially with physiological saline . The brainstem was removed and immersed into sainte - marie's fixative (99% absolute ethanol and 1% glacial acetic acid) for 1 day at 4c . The specimens were embedded in paraffin and transverse sections of 8 m thickness were made . The tenascin - r was detected by incubating the samples in polyclonal goat anti - tenascin - r antibody (r&d systems, minneapolis, mn, usa; af 3867) diluted in 1% bovine serum albumin + 3% normal rabbit serum overnight at 4c . The primary antibody incubation was followed by repeated rinse steps in pbs, and then biotinylated rabbit - anti - goat igg (vector laboratories, burlingame, ca, usa) was used as the secondary antibody . Visualization of labeling was performed by incubating the samples with extravidin peroxidase complex (sigma - aldrich) diluted in pbs for 1 hour at room temperature, followed by 3,3-diaminobenzidine - tetrahydrochloride (dab; sigma - aldrich) with h2o2 . After dehydration, sections were coverslipped with dpx mounting medium (sigma - aldrich). Specificity of tenascin - r antibody was assessed previously in our lab and the details were already published (gal et al ., 2014; rcz et al ., images were recorded by using nikon eclipse e800 (nikon corporation, tokyo, japan) conventional light microscope and processed by photoshop cs4 v11.0 (adobe systems inc ., san jose, ca, usa) with minimal adjustments of contrast and background . For the semiquantitative assessment of tenascin - r reaction, pictures from identical cross sectional levels of each individual vestibular nucleus of three animals were captured using the same magnification, contrast, and brightness . On the images, the staining intensity of tenascin - r reaction was evaluated on the same computer screen by using four - grade scaling:: no staining, +: weak staining, + +: moderate staining, + + +: strong staining (table 1). The subjective grading was performed by two authors (bg and iw) and checked by the other (cm), independently . The optical density measurement would fail to detect the clear - cut distinction between the diffuse ecm and its condensed forms (carulli et al ., 2006, 2007; costa et al ., 2007; galtrey et al ., 2008; gati et al ., 2010; lendvai et al . 2013) and this method was successfully applied in the inferior olive (kecskes et al ., 2014). Semiquantitative assessment of the staining intensity of tenascin - r in the perineuronal nets of individual vestibular nuclei on the operated versus unoperated sides on survival days 1, 3, 7, and 14 following unilateral labyrinthectomy as the exact quantification of our data was difficult because there are no objective distances between the grades of staining intensity established by semiquantitative assessment, we considered these data as ordinal variables allowing application of nonparametric statistical methods to confirm our conclusions . The first statistical analysis was devoted to test the differences in staining intensities of perineuronal nets between the operated and unoperated sides in the vestibular nuclei of the same animal . The variables for this analysis were determined by calculating the median values of the intensity scales of tenascin - r provided by the three independent investigators . For the statistical analysis, the wilcoxon signed rank test was applied . In the second statistical analysis, we examined whether the staining intensity has been changed during the postoperative period in the individual vestibular nuclei by using kruskal - wallis analysis of variance . In those vestibular nuclei where changes were detected, the subsequent days were compared with mann - whitney u test . In each statistical analysis, the statistical analysis was performed using spss 21.0 software (spss, chicago, il, usa). The experiments were carried out in accordance with european community guidelines and state regulations and with the approval of the university animal care committee (demb, 11/2011/de mab). All efforts were made to minimize animal discomfort and reduce the number of animals used . Adult female wistar rats, aged 1214 weeks old, weighing 250300 g, were used in the experiments (n = 12). The animals were anesthetized using an intramuscular injection of 2% xylazine (10 mg / kg, cp pharma handels gmbh, germany) and 10% ketamine (100 mg / kg, cp pharma handels gmbh). A 1.5 cm - long skin incision was made behind the left external acoustic meatus; the cervical muscles, posterior belly of the digastric muscle, the stylohyoid and their nerves were spared . The ventral wall of the tympanic bulla was carefully opened and the labyrinth containing the vestibular sensory organs was approached by breaking the promontory . The left vestibular sensory organs were mechanically destroyed with special care taken on keeping the stapedial artery and facial nerve intact . After a period of 1, 3, 7 or 14 days of survival, the rats (three animals at each time point) were re - anesthetized with intraperitoneal administration of 10% urethane (1.3 mg/100 g, reanal, budapest, hungary) and perfused transcardially with physiological saline . The brainstem was removed and immersed into sainte - marie's fixative (99% absolute ethanol and 1% glacial acetic acid) for 1 day at 4c . The specimens were embedded in paraffin and transverse sections of 8 m thickness were made . The tenascin - r was detected by incubating the samples in polyclonal goat anti - tenascin - r antibody (r&d systems, minneapolis, mn, usa; af 3867) diluted in 1% bovine serum albumin + 3% normal rabbit serum overnight at 4c . The primary antibody incubation was followed by repeated rinse steps in pbs, and then biotinylated rabbit - anti - goat igg (vector laboratories, burlingame, ca, usa) was used as the secondary antibody . Visualization of labeling was performed by incubating the samples with extravidin peroxidase complex (sigma - aldrich) diluted in pbs for 1 hour at room temperature, followed by 3,3-diaminobenzidine - tetrahydrochloride (dab; sigma - aldrich) with h2o2 . After dehydration, sections were coverslipped with dpx mounting medium (sigma - aldrich). Specificity of tenascin - r antibody was assessed previously in our lab and the details were already published (gal et al ., 2014; rcz et al ., images were recorded by using nikon eclipse e800 (nikon corporation, tokyo, japan) conventional light microscope and processed by photoshop cs4 v11.0 (adobe systems inc ., san jose, ca, usa) with minimal adjustments of contrast and background . For the semiquantitative assessment of tenascin - r reaction, pictures from identical cross sectional levels of each individual vestibular nucleus of three animals were captured using the same magnification, contrast, and brightness . On the images, the staining intensity of tenascin - r reaction was evaluated on the same computer screen by using four - grade scaling:: no staining, +: weak staining, + +: moderate staining, + + +: strong staining (table 1). The subjective grading was performed by two authors (bg and iw) and checked by the other (cm), independently . The optical density measurement would fail to detect the clear - cut distinction between the diffuse ecm and its condensed forms (carulli et al ., 2006, 2007; costa et al ., 2007; galtrey et al ., 2008; gati et al ., 2010; lendvai et al ., 2012; rcz et al ., 2013) and this method was successfully applied in the inferior olive (kecskes et al ., semiquantitative assessment of the staining intensity of tenascin - r in the perineuronal nets of individual vestibular nuclei on the operated versus unoperated sides on survival days 1, 3, 7, and 14 following unilateral labyrinthectomy as the exact quantification of our data was difficult because there are no objective distances between the grades of staining intensity established by semiquantitative assessment, we considered these data as ordinal variables allowing application of nonparametric statistical methods to confirm our conclusions . The first statistical analysis was devoted to test the differences in staining intensities of perineuronal nets between the operated and unoperated sides in the vestibular nuclei of the same animal . The variables for this analysis were determined by calculating the median values of the intensity scales of tenascin - r provided by the three independent investigators . For the statistical analysis, the wilcoxon signed rank test was applied . In the second statistical analysis, we examined whether the staining intensity has been changed during the postoperative period in the individual vestibular nuclei by using kruskal - wallis analysis of variance . In those vestibular nuclei where changes were detected, the subsequent days were compared with mann - whitney u test . In each statistical analysis, the statistical analysis was performed using spss 21.0 software (spss, chicago, il, usa). In the unoperated animals, as shown in our previous work (racz et al ., 2014), the tenascin - r immunoreactivity was intense in the perineuronal nets of each vestibular nucleus (figure 1a, c, e, g, i, k, m, o and figure 2a, c, e, g, i, k, m, o; table 1), except for the caudal part of the descending vestibular nucleus which showed weaker staining in the pericellular area (figure 2q, s, u, w). The neuropil showed a diffuse, reticular appearance presenting strongly stained areas in the superior, medial and lateral nuclei, as well as in the rostral parts of the descending vestibular nucleus, whereas the staining intensity was weaker in the caudal part of the descending vestibular nucleus . Distribution of tenascin - r staining intensity in the perineuronal nets of the superior (svn) and medial vestibular nuclei (mvn) on the operated versus unoperated sides (a p) on postoperative days 1, 3, 7 and 14 . In the svn, decreased tenascin - r staining was seen in the perineuronal nets on the operated side on postoperative days 1 and 3 (b, d). In the mvn, weak tenascin - r staining in the perineuronal nets was only seen on postoperative day 1 on the operated side (j). Distribution of tenascin - r staining intensity in the perineuronal nets of the lateral (lvn), rostral (dvn rostral) and caudal (dvn caudal) parts of the descending vestibular nucleus (dvn) on the operated versus unoperated sides (a x) on postoperative days 1, 3, 7, and 14 . In the lvn, decreased tenascin - r staining was seen on the operated side on postoperative day 1 (b), and lesser difference was seen on postoperative days 3 and 7 (d, f). In the rostral part of dvn, weak tenascin - r staining in the perineuronal nets was only seen on postoperative day 1 on the operated side (j); in the caudal part of the dvn, no difference was observed throughout the postoperative period . Scale bar: 20 m . In the superior vestibular nucleus, the tenascin - r staining of perineuronal nets completely disappeared on the side of labyrinthectomy on the first postoperative day, whereas the pericellular staining did not change on the intact side (figure 1a, b; table 1). In the neuropil, the intensity of reaction was similar to that of the control animals, bilaterally . On survival day 3, the tenascin - r staining pattern was similar to day 1 with the exception of the lighter staining of ipsilateral neuropil (figure 1c, d). On survival days 7 and 14, perineuronal nets were recognizable at both sides, showing minor decrease of staining in the perineuronal nets of the operated side (figure 1e h; table 1). The staining intensity of neuropil was stronger on the unoperated side compared to postoperative day 3 (figure 1e h). In the magnocellular part of the medial vestibular nucleus, the staining pattern of perineuronal nets and neuropil showed similar appearance to that of superior vestibular nucleus bilaterally on postoperative day 1 (figure 1i, j). On postoperative day 3, the staining of perineuronal nets no longer showed recognizable difference on the operated side and it was also similar on the following survival days (figure 1k p; table 1). Neuropil remained the same in intensity on both sides . In the lateral vestibular nucleus, the staining of perineuronal nets and the neuropil was the same as in case of the superior and medial vestibular nuclei on postoperative day 1 (figure 2a, b). On postoperative day 3, the staining intensity of perineuronal nets occurred slightly weaker on the operated side, but was considerably increasing after postoperative day 1 on the side of labyrinthectomy (figure 2c, d; table 1). The perineuronal nets showed the same intensity bilaterally on postoperative day 7 and the staining of ipsilateral pericellular areas was lighter compared to that on postoperative day 3 (figure 2e, f). By postoperative day 14, the staining of pericellular area showed no bilateral differences (figure 2 g, h; table 1). The staining of neuropil remained unchanged during the postoperative periods . In the rostral part of the descending vestibular nucleus, the perineuronal nets were not recognizable bilaterally on postoperative day 1, and the staining intensity of neuropil was weaker compared to the control animals at both sides (figure 2i, j). On postoperative day 3, the staining intensity of perineuronal nets and neuropil appeared bilaterally at the level of control animals (figure 2k, l; table 1). The same pattern of tenascin - r immunoreactivity was shown on postoperative day 7 (figure 2 m, n). On postoperative day 14, the staining intensity decreased bilaterally both in the perineuronal nets and neuropil (figure 2o, p). In the caudal part of the descending vestibular nucleus, moderately stained pericellular areas, similar to that of control animals, were observed bilaterally during the postoperative periods (figure 2q x). Statistical analysis revealed that in the superior vestibular nucleus, the changes of staining intensity of tenascin - r reaction were non - significant between days 1 and 3 and days 7 and 14, whereas they were statistically significant between days 3 and 7 (p <0.001). In the magnocellular part of the medial vestibular nucleus, the statistical analysis did not show any significant changes in the staining intensity of tenascin - r reaction during the postoperative period . In the lateral vestibular nucleus and the rostral part of the descending vestibular nucleus, the staining intensity was statistically significant between days 1 and 3 (p <0.001) and no significant changes were detected between the other survival days (table 1). Unilateral labyrinthectomy results in elimination of sensory inputs from the vestibular receptors and the subsequent deafferentation - induced plasticity contributes to the restoration of vestibular function . Our results demonstrated for the first time that unilateral labyrinthectomy and subsequent compensation is accompanied by the modification of tenascin - r staining pattern in the vestibular nuclei of the rat . The modification of tenascin - r expression showed regional differences in the vestibular nuclear complex which may be associated with the morphological and functional heterogeneity of the individual vestibular nuclei and with their different roles in the compensatory processes . The tenascin - r has versatile, sometimes opposite functions in the central nervous system depending on its location, the type of targeted cells, receptors, signaling pathways, the molecular composition of surrounding extracellular matrix as well as the embryonic and postembryonic periods of life (pesheva and probstmeier, 2000; anlar and gunel - ozcan, 2012). Experimental studies showed that tenascin - r restricts functional recovery from spinal cord injury, and in agreement with this finding the tenascin - r - deficient mice recovered better than wild - type controls after spinal cord compression (apostolova et al ., 2006). Therefore, it appears reasonable that the temporary decrease in the tenascin - r expression presented in our study plays a role in the recovery from the vestibular disorder . In the lack of data on the role of tenascin - r in the vestibular system, we can merely state, at present, that the tenascin - r expression is changing after unilateral labyrinthectomy and during the subsequent compensation . However, based on results of earlier experiments related to the role of tenascin - r in various parts of the central nervous system, we may suggest the following possible involvements of tenascin - r in the mechanisms of vestibular compensation (dieringer, 1995; dutia, 2010; lacour and tighilet, 2010). First, the tenascin - r is known to activate the microglia cells which, in response, secrete cytokines and growth factors including brain - derived neurotrophic factor and nerve growth factor (liao et al ., 2005). In the deafferented vestibular nuclei of the labyrinthectomized rat, intense microglial reaction was detectable as early as day 1 after lesion and it persisted several weeks afterwards (campos torres et al ., 1999). This microglial reaction constitutes one of the signals responsible for astroglial reaction observed in the vestibular nuclei during the 1 - 3 postoperative days (de waele et al ., 1996). As suggested by campos torres et al . (2005), growth factors as well as pro- and anti - inflammatory cytokines produced by activated astroglial cells could promote the survival of deafferented vestibular neurons and contribute to recovery of their resting discharge (dieringer, 1995; vibert et al ., 1995; li et al ., 1999; straka et al ., 2005; dutheil et al . Lacour and tighilet (2010) confirmed bilateral up - regulation of brain - derived neurotrophic factors along with its trkb receptor, both of which appeared as early as 1 day after unilateral labyrinthectomy and peaked at postoperative day 3 in the descending and lateral vestibular nucleus . As a result of glial reactions described above the increased staining of tenascin - r in the neuropil from postoperative day 3 (sometimes from postoperative day 7) might be associated with the microglial activation thereby the tenascin - r may promote the plasticity of vestibular nucleus and contribute to the repair of vestibular disorders . Second, the extracellular matrix in physiological conditions, by stabilization of synapses, creates a barrier against the formation of new synaptic contacts and restricts the synaptic plasticity (galtrey and fawcett, 2007). In our present study, the common feature of the tenascin - r expression was the decrease or disappearance of tenascin - r immunoreactivity from the perineuronal nets in each vestibular nucleus . Decreased staining intensity of non - permissive tenascin - r may stimulate the formation of new synaptic contacts, as one of the possible mechanisms during the restoration of vestibular function (dieringer, 1995; li et al ., 1999; de waele et al ., 2000; lacour and tighilet, 2010). The third possible involvement of tenascin - r in the vestibular compensation might be related to inhibitory commissural pathways existing between the bilateral vestibular nuclei (holstein et al . Unilateral labyrinthectomy results in severe imbalance in the gabaergic commissural system and it is regarded as a key cause of the static oculomotor and postural symptoms . Similarly, the asymmetry in spontaneous resting activity between the intact and deafferented vestibular neurons is due to the imbalance of gabaergic interaction between the ipsilateral and contralateral sides (gliddon et al ., 2004). The re - balancing of commissural inhibition occurs in parallel with the restoration of impaired resting activity and with the subsequent behavioral recovery during vestibular compensation (gliddon et al ., 2004; straka et al ., 2005; tighilet et al ., 2007; the tenascin - r regulates, via interactions of its hnk-1 (human natural killer cell) carbohydrate epitope, the gabab receptor mediated perisomatic inhibition and thus influences synaptic transmission and plasticity in the hippocampus (saghatelyan et al . Dityatev and schachner, 2003; brenneke et al ., 2004). As the unilateral labyrinthectomy resulted in marked downregulation of the functional efficacy of gabab receptors in the cells of the ipsilesional medial vestibular nucleus (yamanaka et al ., 2000; 2003) similar regulatory function of tenascin - r is possible in the vestibular system . Fourth, the tenascin - r is detectable around the nodes of ranvier predominantly at the large, myelinated axons (apostolova et al ., 2006; bekku et al ., 2009). Here, the tenascin - r is binding to the voltage - gated sodium channels and initiates the clustering of channels and then stabilizes the clusters after they have formed thereby it is regarded as a functional modulator of sodium channel beta subunits (srinivasan et al ., 1998; xiao et al ., 1999). In the tenascin - r - deficient mice, there was a significant decrease in conduction velocity of myelinated axons (weber et al ., 1999). As the vestibular nuclei have large caliber myelinated axons (sotelo and palay, 1970), similar function of the tenascin - r might be suggested here, as well . Although the individual vestibular nuclei are different from each other in their morphological, physiological and biochemical characteristics associated with their different functions in balance, vestibulo - ocular reflexes, spatial cognition and automatic responses (babalian and vidal, 2000; birinyi et al ., 2001;, 2005; eugene et al ., 2011; mccall and yates, 2011; kodama et al ., 2012; racz et al ., 2014) their specific role in the compensatory processes is not yet determined . Most of the experiments on vestibular lesion and subsequent compensation have been performed on the medial vestibular nucleus . This nucleus is engaged mostly in the postural reflexes, and restoration of which is known as the initial event during the vestibular compensation (yamanaka et al ., 2000; beraneck et al ., 2003; beraneck and idoux, 2012). The earliest re - establishment of tenascin - r reaction in the perineuronal nets around the neurons of medial vestibular nucleus might support these findings . The only part of the vestibular nuclear complex where the tenascin - r reaction remained almost unchanged following unilateral labyrinthectomy is the caudal part of descending vestibular nucleus . This subnucleus is involved in the rapid modulation of the cardiovascular, respiratory and digestive systems in response to locomotion and postural adjustments (ruggiero et al ., 1996; matesz et al ., 1997; porter and balaban, 1997; holstein et al ., 2011). In the light of our results, it is tempting to assume that the role of extracellular matrix is less important in the compensatory processes of impaired vestibulo - autonomic function . The presented results contribute to our earlier findings on the spatially and temporally specific alterations of hyaluronan and chondroitin sulfate proteoglycans during vestibular compensation (deak et al ., 2012). The reduction of the immunostaining of tenascin - r also suggests the extracellular facilitation of plastic modifications of the vestibular circuit after lesion . The results may also assist in developing new therapeutic strategies for the treatment of symptoms of vestibular lesion.
The mechanosensitive channel of small conductance (mscs) is a ubiquitous osmolyte release valve present in all phyla of walled cells, from bacteria to higher plants (pivetti et al ., 2003; balleza and gmez - lagunas, 2009). In bacteria, mscs and three other mechanosensitive channels (mscm, msck, and mscl) comprise a membrane tension driven osmolyte efflux system adjusting turgor in a wide range of osmotic downshifts . Among them, mscs mediates the bulk of osmolyte efflux opening at moderate tensions (58 mn / m), which is above the threshold for mscm (schumann et al ., 2010) but considerably below nearly lytic tensions (1014 mn / m) that open mscl (sukharev et al ., 1999; moe and blount, 2005; belyy et al ., clamp experiments, mscs readily responds to abrupt pulses of tension, but under slow ramps, only a fraction of channel population opens (akitake et al ., analysis of responses to prolonged pressure steps revealed that in excised patches, mscs first undergoes reversible adaptation, and then enters a tension - insensitive inactivated state (akitake et al . Adaptation is a gradual shift of the activation curve midpoint toward higher tensions (by 1020%) ascribed to mechanical stress redistribution in inside - out patches, which is not observed in whole spheroplast mode (belyy et al . Inactivation, in contrast, renders channels completely tension insensitive; it is present in all recording modes and appears to be an intrinsic property of the channel . Our recent data showed that inactivation occurring in wild - type (wt) mscs with a relatively slow (30 s) kinetics provides substantial advantage to bacteria in terms of osmotic survival under different regimes of osmotic shock in vivo when compared with noninactivating or fast inactivating mutants (boer et al ., 2011). This further suggested that mscs inactivation observed in electrophysiology is not an artifact of patch clamp recording, but rather a functional trait excluding unnecessary activity and leakage under persisting but not threatening tensions . Although previous data suggested that inactivation occurs at tensions above the activating threshold from the closed - adapted state (akitake et al ., 2005, 2007), it was unclear whether closed - state inactivation is the only mechanism or it can also occur from the open state as in many voltage - gated channels (aldrich and stevens, 1983; patlak, 1991; armstrong, 2006). Most previous studies addressed the mechanism of tension - driven activation of mscs, in which the crystal structures of wt (steinbacher et al ., 2007) or a106v mscs (wang et al ., 2008, 2008a, b), or unitary conductance and thermodynamically estimated in - plane protein expansion (akitake et al ., 2007; anishkin et al ., 2008a, b), combined with computational techniques were used to envision the opening transition . The tension- and voltage - dependent mechanisms of inactivation (koprowski and kubalski, 1998; vsquez and perozo, 2004; akitake et al ., 2005), however, were not fully addressed in structural terms . Inactivation was linked to formation of the crystallographic kink at g113 in tm3 (akitake et al ., 2007) and strongly facilitated by mutations that hydrophilize the reconstructed hydrophobic interface between tm2 and the gate region on tm3 (belyy et al . The latter study suggested that inactivation may be caused by some displacement of tm1tm2 pairs from the gate, although the spatial scale of this tension - driven transition has never been estimated . The tension dependence of the recovery process (reverse of inactivation) has not been characterized either . Here, we study mscs inactivation with a special set of pressure protocols including preconditioning pressure steps and saturating test pulses and show that opening does not enhance, but rather excludes, inactivation . Analysis of soft and stiff mutants with different opening thresholds illustrates that the opening and inactivation are two competing transition pathways from the same closed state . Tension dependencies of both inactivation and recovery rates suggest the spatial scale of the inactivating transition, which guides the modeling of the inactivated state . Wt escherichia coli mscs was expressed from the pb10b vector (okada et al ., 2002) in mjf465 (kefa, mscs, and mscl) e. coli cells (levina et al ., 1999) or pb113 e. coli strain, which is a reca variant of mjf429 (mscs and msck) (levina et al ., 1999; li et al ., 2002) carrying the chromosomal copy of mscl . Preparation of giant e. coli spheroplasts and patch - clamping procedures were conducted as described previously (blount et al ., 1999; akitake et al ., 2005). Population channel recordings were conducted at + 30 mv (pipette) on excised inside - out patches in symmetrical buffers containing 200 mm kcl, 10 mm cacl2, 40 mm mgcl2, and 10 mm hepes, ph 7.4 . Traces were recorded under programmed pressure stimuli delivered from an hspc-1 pressure clamp machine (ala scientific instruments) using the clampex 10.2 software (molecular devices). After seal formation and excision, each patch was tested with a saturating 1-s pressure ramp to determine the activation midpoint (p0.5). P0.5 was then used as a subsaturating pressure stimulus in step protocols and as a reference point for pressure normalization (fig . 1). Dependence of mscs inactivation on the preconditioning saturating pulse at different tensions . (a) ramp responses of channel population in a patch characterized by the midpoint of 153 mmhg and saturating pressure of 180 mmhg . Responses to the step pulse protocol with the increasing amplitude of a 30-s step (b). (c) responses of the same patch to pulse - step - pulse stimuli, where the first short pulse opens the entire mscs population, and the pulse at the end reveals the part of the population remaining active in both protocols (arrows). Inactivated fractions of channel population plotted as a function of normalized pressure during the 30-s step (d) are similar regardless of the presence of the first pulse . The data were collected on a single representative patch and were qualitatively reproduced on four separate patches . In d, pressures are normalized to the ramp response midpoint p0.5 (a). Here, we further developed protocols that allowed us to separate the kinetically intertwined processes of adaptation and inactivation . In these protocols, the channel populations were subjected to prolonged subsaturating pressure steps with interspersed short test pulses of saturating pressure (fig . 3 a and legend). The current observed during the steps reflected adaptation, and test pulses monitored the noninactivated population of channels . Segments of traces reflecting different stimuli (step vs. pulse) were digitized and fitted in mathcad 13 using analytical solutions of the standard set of differential equations (houston, 2001) describing a three - state kinetic model (ocai):o(t)=o0ek1t;ca(t)=k1k2k1o0(ek1tek2t);i(t)=o0o(t)ca(t),where k1 and k2 are the rate constants for the sequential transitions oca and cai, respectively . The discretization of the traces and isolation of the open (o), closed - adapted (ca), and inactivated (i) populations are illustrated in fig . 1 a shows a typical response to a trapezoidal stimulus that we used to calibrate the pressure sensitivity of the mscs population in a particular patch, which in this case exhibits a midpoint of p0.5 = 153 mmhg . Two protocols are then used: (1) a prolonged (30-s) conditioning step, and then short saturating pulse testing for channel availability (fig . 1 b); and (2) a short saturating pulse that opens the entire population, and then a conditioning step and a short test pulse again (fig . We studied the degree of inactivation at different amplitudes of the conditioning step, which were chosen to be slightly below p0.5 . As indicated by the level of population current to the saturating test pulse at the end of the first protocol (fig . Responses of the same patch to the second protocol with the same amplitude of steps are shown in fig . 1 c. in the first protocol, the conditioning step activates only a fraction of channels, whereas in the latter case, the first saturating pulse obligatorily opens the entire mscs population . In both cases, the population gradually closes with a characteristic time that depends on the amplitude of the step . The second protocol forces the entire population through the opening cycle (oco), yet the fraction of inactivated channels is equal in both protocols (fig . 1 d), indicating that opening does not aid inactivation . The data presented in fig . 2 each of the two pressure protocols includes a saturating step that keeps the population open, an intermediate step at which the channels are given a chance to close, and a final saturating test pulse probing for the remaining active part of the population . In experiments presented in fig . 2 a, the length of the saturating step varied, whereas the second sub - saturating step was kept constant (10 s). As shown by arrows, the responses to the test pulse at the end are the same with an accuracy of 15%, indicating that the degree of mscs inactivation is independent of the open period . In fig . 2 b, the total duration of the stimulus was kept constant, and the length of each step varied reciprocally . When the first step is short (0.1 s), the population is allowed to close gradually under moderate pressure for the entire 30-s duration of the second step, and the inactivation was profound (76%). Extending the length of the first saturating step from 0.1 to 20 s and reducing the time of the second step from 30 to 10 s, respectively, increased the number of active channels . The fraction of inactivated channels progressively decreased with the decrease of time spent at sub - saturating pressure when the channels had time to close . The inactivated fraction plotted as a function of sub - saturating step duration shows a nearly linear dependence (fig . The experiment was repeated four times on different patches, and the error bars in fig . The protocol demonstrates that opening does not help inactivation, but rather locks mscs in a state from which it cannot inactivate . The degree of mscs inactivation is independent of the open period but increases with the time spent in the closed - adapted state . (a) current responses to a double - step protocol in which the first saturating step (105 mmhg) of varied length is followed by a 10-s subsaturating step (75 mmhg). The responses to the short test pulse at the end indicate the fraction of active channels (numbers by the arrows). (b) the 30-s stimulus includes saturating (160 mmhg) and subsaturating (125 mmhg) pressure steps of reciprocally varying duration . The percentage of inactivation averaged over four patches studied with this protocol is shown in c (bars represent sd). The test ramp experiment (d) indicated the midpoint of 125 mmhg for the patch examined in b. the conclusion above is consistent with our previous results obtained with two - step protocols where the channels were first exposed to a prolonged conditioning step above the threshold, and then the population was tested with a saturating pulse at the end . During the prolonged step, the rate of adaptive current decline decreases monotonously as tension increases . The fraction of channels remaining active at the end of the step exhibits a nonmonotonous tension dependence with a minimum at the pressure corresponding to the activation midpoint on 1-s saturating ramp (akitake et al ., 2005 pressures below p0.5 are not strong enough to elicit full mscs inactivation, whereas above p0.5, mscs undergoes very slow adaptive closure being trapped in the open state . Thus, initially tension facilitates inactivation, but once the population of open channels increases, the inactivated fraction declines (akitake et al ., 2005, 2007; the above data suggests that only nonconductive (closed or closed - adapted) channels can inactivate . Previous study (belyy et al ., 2010b) has shown that adaptation of mscs and mscl in excised patches is a consequence of tension redistribution in the membrane, which is essentially the adaptation of the stimulus . Thus, the adaptive current decline should be considered as closure . Under moderate supra - threshold stimuli, when most of the open channels undergo adaptive closure and finally inactivate, the entire process can be presented as a linear three - state scheme: ocai . The pressure protocol we used here is a prolonged (30-s) pressure step with short saturating test pulses interspersed evenly from the beginning to the end (fig . 3 a). The initial test pulse preconditions the entire population to the o state . The pressure - dependent rate of the following current relaxation, reflecting closure oca, can be directly obtained by fitting the time course of current decay (kamaraju and sukharev, 2008). In fig . 3 (b and c), the remaining open fraction (o) is designated by open circles . The current spikes in response to saturating test pulses reveal the fraction of channels that remain active (noninactivated), both open and closed (o+ca). This combined fraction, complementary to the inactivated fraction (i = 1(o+ca)), is shown with diamonds for two different conditioning steps (fig . The intermediate closed - adapted fraction (ca) can be calculated from the analytical solutions of the standard system of differential equations describing two sequential reactions (see materials and methods). B shows the trace - fitting quality obtained at relatively low pressure featuring fast current decay and slow inactivation, and at intermediate pressure (c), at which the adaptation and inactivation rates become comparable . Fitting a series of such traces allowed us to extract the tension dependence of the inactivation rate plotted in fig . 3 e (filled circles). The inactivation rate becomes appreciable at tensions near 5 mn / m and increases with the slope corresponding to the area of 6.1 1.5 (n = 3) nm . This area can be interpreted as the area from the bottom of the closed - state well to the top of the rate - limiting barrier separating the closed (c) and inactivated (i) states (acb). The dependencies of the rates of mscs inactivation and recovery on tension in excised patches . (a) the current traces and the stimulus protocols that involve 30-s steps to varied amplitude (from 25 to 150 mmhg in 25-mmhg increments) with interspersed short saturating (180-mmhg) pulses testing for the availability of channels . The numbers 1, 2, and 3 correspond to the pressure steps of 100, 125, and 150 mmhg, respectively . (b) fitting of mscs transition kinetics recorded at a 100-mmhg pressure step to the three - state model: ocai . The circles designate the position of the continuous trace (a) representing open population (o), the diamonds represent noninactivated population (o + ca), and the intermediate closed - adapted (ca) fraction (dashed line) is calculated by fitting the two other populations to the model (see materials and methods). (c) a similar fit of the trace obtained at higher background tension (125 mmhg) showing slower adaptation but faster inactivation rate . Mscs population was inactivated by a 60-s conditioning step (the last second is shown), and the degree of inactivation was tested by the short saturating test pulse at the end of the step . The pressure was then dropped to different levels, and four saturating test pulses were applied at different time points . The current responses, reflecting the recovered fraction of the population, were then fitted with monoexponential functions . The slopes dlnk / d = a / kt gave estimations of acb = 4.6 nm and aib = 2.6 nm; the protein area changes from the bottoms of the closed (c) or inactivated (i) well to the transition barrier (b). The values averaged over three independent experiments and their sum representing total protein expansion associated with the cai transition are given in results . To find the remaining part of the expansion on the other side of the barrier separating c and i states, we measured the recovery rate from inactivation at different tensions . The protocol combined a long 60-s step driving the entire population into the inactivated state, followed by a drop to a desired subthreshold pressure with a series of test pulses monitoring the return of the population . 3 d. the current values at the tips of the test pulse responses were plotted against time and fitted with monoexponential functions . The rates of mscs recovery were then plotted as a function of tension in semi - logarithmic scale (fig . The slope of the decreasing recovery rate consistently indicated abi = 2.5 0.1 nm (n = 3). In the simplifying assumption of a single rate - limiting barrier separating the closed and inactivated states (because both inactivation and recovery kinetics are close to monoexponential), the two area estimates added together suggest that the total protein in - plane expansion of 8.5 1.6 nm is associated with the process of inactivation . This area is smaller than the area change associated with the opening transition (1315 nm; akitake et al ., 2005; kamaraju et al ., 2010). The slight deviation from the monoexponential kinetics (fig . 3 d) can be a result of noise in a population of 200 channels . It can also be a consequence of non - uniformity of the population (chiang et al ., 2004) because individual channels may reside in a slightly different environment in the patch . The larger spatial scale of the co transition as compared with ci makes the open state more stable under high tension where the channels are trapped with no tendency to inactivate . 3 (d and e) it is clear that tension also strongly stabilizes the inactivated state . Note that these estimations of area changes were made under the assumption that the tension midpoint for mscs 0.5 = 7.8 mn / cm, as recently measured in whole spheroplast experiments (belyy et al . If we use 0.5 = 5.5 mn / cm as previously measured for mscs in liposomes (sukharev, 2002), the area estimates are slightly larger, acb = 8.6 2.2 nm and acb = 3.5 0.2 nm, totaling 12.1 nm, which becomes comparable to the expansion area associated with opening . Previous data have indicated that 10-s steps of pressure at p0.5 (as determined on ramp test) typically cause full transient activation of 90% of mscs population, which adapts and simultaneously inactivates by 50% (akitake et al ., 2005; belyy et al . 4 a shows the response of the soft (gain - of - function) a98s mutant to a series of 10-s pressure steps ending with a 0.5-s saturating test pulse . The test pulse invoked currents invariably show maximal activity indicative of no inactivation . The stiff (loss - of - function) l111s mutant shows complete inactivation at pressures considerably below its midpoint, but also substantial inactivation just below the activation threshold (see trace 3 in fig . 4 b). In other words, l111s mscs inactivates silently, bypassing the opening closing cycle . Wt mscs and the two mutants were expressed in pb113 cells carrying native mscl used as an internal gauge to calibrate their activation midpoints (p0.5) as described previously (akitake et al . The a98s p0.5 was estimated to be 0.75 wt p0.5, and the l111s p0.5 was 1.72 wt p0.5 . 4 c shows the three activation curves, and the bottom panel presents the inactivation rate for wt mscs plotted on the same tension scale . Under the assumption that the a98s and l111s mutations affect primarily the threshold and midpoint for activation but not the process of inactivation the soft a98s mutant opens completely before the inactivation rate becomes appreciable, and the population becomes locked in the open state with no ability to inactivate . We found it practically impossible to even estimate the rate of inactivation for this mutant, as inactivation was undetectable in the entire range of tensions . L111s, in contrast, becomes active at tensions where inactivation rate reaches maximal measurable values, and even before that it quickly inactivates without opening . We have attempted determination of the inactivation rates for l111s, but the extremely high threshold for this mutant made the patches prohibitively unstable under saturating pulses . The comparison of activation and inactivation thresholds for wt mscs, soft a98s, and stiff l111s mutants . (a) a series of traces obtained in response to step pulse protocol shows that the soft a98s mscs mutant does not inactivate . (b) the stiff l111s mutant partially inactivates silently without opening, as illustrated by trace 3 (arrows). (c) positions of activation curves for a98s (0.5 = 5.8 mn / m), wt (0.5 = 7.8 mn / m), and l111s (0.5 = 13.4 mn / m), and the tension dependence of the inactivation rate of wt mscs (data from fig . 1 a shows a typical response to a trapezoidal stimulus that we used to calibrate the pressure sensitivity of the mscs population in a particular patch, which in this case exhibits a midpoint of p0.5 = 153 mmhg . Two protocols are then used: (1) a prolonged (30-s) conditioning step, and then short saturating pulse testing for channel availability (fig . 1 b); and (2) a short saturating pulse that opens the entire population, and then a conditioning step and a short test pulse again (fig . We studied the degree of inactivation at different amplitudes of the conditioning step, which were chosen to be slightly below p0.5 . As indicated by the level of population current to the saturating test pulse at the end of the first protocol (fig . Responses of the same patch to the second protocol with the same amplitude of steps are shown in fig . 1 c. in the first protocol, the conditioning step activates only a fraction of channels, whereas in the latter case, the first saturating pulse obligatorily opens the entire mscs population . In both cases, the population gradually closes with a characteristic time that depends on the amplitude of the step . The second protocol forces the entire population through the opening cycle (oco), yet the fraction of inactivated channels is equal in both protocols (fig . 1 d), indicating that opening does not aid inactivation . The data presented in fig . 2 each of the two pressure protocols includes a saturating step that keeps the population open, an intermediate step at which the channels are given a chance to close, and a final saturating test pulse probing for the remaining active part of the population . In experiments presented in fig . 2 a, the length of the saturating step varied, whereas the second sub - saturating step was kept constant (10 s). As shown by arrows, the responses to the test pulse at the end are the same with an accuracy of 15%, indicating that the degree of mscs inactivation is independent of the open period . In fig . 2 b, the total duration of the stimulus was kept constant, and the length of each step varied reciprocally . When the first step is short (0.1 s), the population is allowed to close gradually under moderate pressure for the entire 30-s duration of the second step, and the inactivation was profound (76%). Extending the length of the first saturating step from 0.1 to 20 s and reducing the time of the second step from 30 to 10 s, respectively, increased the number of active channels . The fraction of inactivated channels progressively decreased with the decrease of time spent at sub - saturating pressure when the channels had time to close . The inactivated fraction plotted as a function of sub - saturating step duration shows a nearly linear dependence (fig . The experiment was repeated four times on different patches, and the error bars in fig . The protocol demonstrates that opening does not help inactivation, but rather locks mscs in a state from which it cannot inactivate . The degree of mscs inactivation is independent of the open period but increases with the time spent in the closed - adapted state . (a) current responses to a double - step protocol in which the first saturating step (105 mmhg) of varied length is followed by a 10-s subsaturating step (75 mmhg). The responses to the short test pulse at the end indicate the fraction of active channels (numbers by the arrows). (b) the 30-s stimulus includes saturating (160 mmhg) and subsaturating (125 mmhg) pressure steps of reciprocally varying duration . The percentage of inactivation averaged over four patches studied with this protocol is shown in c (bars represent sd). The test ramp experiment (d) indicated the midpoint of 125 mmhg for the patch examined in b. the conclusion above is consistent with our previous results obtained with two - step protocols where the channels were first exposed to a prolonged conditioning step above the threshold, and then the population was tested with a saturating pulse at the end . During the prolonged step, the rate of adaptive current decline decreases monotonously as tension increases . The fraction of channels remaining active at the end of the step exhibits a nonmonotonous tension dependence with a minimum at the pressure corresponding to the activation midpoint on 1-s saturating ramp (akitake et al ., 2005 pressures below p0.5 are not strong enough to elicit full mscs inactivation, whereas above p0.5, mscs undergoes very slow adaptive closure being trapped in the open state . Thus, initially tension facilitates inactivation, but once the population of open channels increases, the inactivated fraction declines (akitake et al ., 2005, 2007; the above data suggests that only nonconductive (closed or closed - adapted) channels can inactivate . Previous study (belyy et al ., 2010b) has shown that adaptation of mscs and mscl in excised patches is a consequence of tension redistribution in the membrane, which is essentially the adaptation of the stimulus . Thus, the adaptive current decline should be considered as closure . Under moderate supra - threshold stimuli, when most of the open channels undergo adaptive closure and finally inactivate, the entire process can be presented as a linear three - state scheme: ocai . The pressure protocol we used here is a prolonged (30-s) pressure step with short saturating test pulses interspersed evenly from the beginning to the end (fig . The initial test pulse preconditions the entire population to the o state . The pressure - dependent rate of the following current relaxation, reflecting closure oca, can be directly obtained by fitting the time course of current decay (kamaraju and sukharev, 2008). In fig . 3 (b and c), the remaining open fraction (o) is designated by open circles . The current spikes in response to saturating test pulses reveal the fraction of channels that remain active (noninactivated), both open and closed (o+ca). This combined fraction, complementary to the inactivated fraction (i = 1(o+ca)), is shown with diamonds for two different conditioning steps (fig . The intermediate closed - adapted fraction (ca) can be calculated from the analytical solutions of the standard system of differential equations describing two sequential reactions (see materials and methods). B shows the trace - fitting quality obtained at relatively low pressure featuring fast current decay and slow inactivation, and at intermediate pressure (c), at which the adaptation and inactivation rates become comparable . Fitting a series of such traces allowed us to extract the tension dependence of the inactivation rate plotted in fig . 3 e (filled circles). The inactivation rate becomes appreciable at tensions near 5 mn / m and increases with the slope corresponding to the area of 6.1 1.5 (n = 3) nm . This area can be interpreted as the area from the bottom of the closed - state well to the top of the rate - limiting barrier separating the closed (c) and inactivated (i) states (acb). The dependencies of the rates of mscs inactivation and recovery on tension in excised patches . (a) the current traces and the stimulus protocols that involve 30-s steps to varied amplitude (from 25 to 150 mmhg in 25-mmhg increments) with interspersed short saturating (180-mmhg) pulses testing for the availability of channels . The numbers 1, 2, and 3 correspond to the pressure steps of 100, 125, and 150 mmhg, respectively . (b) fitting of mscs transition kinetics recorded at a 100-mmhg pressure step to the three - state model: ocai . The circles designate the position of the continuous trace (a) representing open population (o), the diamonds represent noninactivated population (o + ca), and the intermediate closed - adapted (ca) fraction (dashed line) is calculated by fitting the two other populations to the model (see materials and methods). (c) a similar fit of the trace obtained at higher background tension (125 mmhg) showing slower adaptation but faster inactivation rate . Mscs population was inactivated by a 60-s conditioning step (the last second is shown), and the degree of inactivation was tested by the short saturating test pulse at the end of the step . The pressure was then dropped to different levels, and four saturating test pulses were applied at different time points . The current responses, reflecting the recovered fraction of the population, were then fitted with monoexponential functions . The slopes dlnk / d = a / kt gave estimations of acb = 4.6 nm and aib = 2.6 nm; the protein area changes from the bottoms of the closed (c) or inactivated (i) well to the transition barrier (b). The values averaged over three independent experiments and their sum representing total protein expansion associated with the cai transition are given in results . To find the remaining part of the expansion on the other side of the barrier separating c and i states, we measured the recovery rate from inactivation at different tensions . The protocol combined a long 60-s step driving the entire population into the inactivated state, followed by a drop to a desired subthreshold pressure with a series of test pulses monitoring the return of the population . The latter part of it is shown in fig . 3 d. the current values at the tips of the test pulse responses were plotted against time and fitted with monoexponential functions . The rates of mscs recovery were then plotted as a function of tension in semi - logarithmic scale (fig . The slope of the decreasing recovery rate consistently indicated abi = 2.5 0.1 nm (n = 3). In the simplifying assumption of a single rate - limiting barrier separating the closed and inactivated states (because both inactivation and recovery kinetics are close to monoexponential), the two area estimates added together suggest that the total protein in - plane expansion of 8.5 1.6 nm is associated with the process of inactivation . This area is smaller than the area change associated with the opening transition (1315 nm; akitake et al . 3 d) can be a result of noise in a population of 200 channels . It can also be a consequence of non - uniformity of the population (chiang et al ., 2004) because individual channels may reside in a slightly different environment in the patch . The larger spatial scale of the co transition as compared with ci makes the open state more stable under high tension where the channels are trapped with no tendency to inactivate . On the other hand, from fig . 3 (d and e) it is clear that tension also strongly stabilizes the inactivated state . Note that these estimations of area changes were made under the assumption that the tension midpoint for mscs 0.5 = 7.8 mn / cm, as recently measured in whole spheroplast experiments (belyy et al ., 2010b). If we use 0.5 = 5.5 mn / cm as previously measured for mscs in liposomes (sukharev, 2002), the area estimates are slightly larger, acb = 8.6 2.2 nm and acb = 3.5 0.2 nm, totaling 12.1 nm, which becomes comparable to the expansion area associated with opening . Previous data have indicated that 10-s steps of pressure at p0.5 (as determined on ramp test) typically cause full transient activation of 90% of mscs population, which adapts and simultaneously inactivates by 50% (akitake et al ., 2005; 4 a shows the response of the soft (gain - of - function) a98s mutant to a series of 10-s pressure steps ending with a 0.5-s saturating test pulse . The test pulse invoked currents invariably show maximal activity indicative of no inactivation . The stiff (loss - of - function) l111s mutant shows complete inactivation at pressures considerably below its midpoint, but also substantial inactivation just below the activation threshold (see trace 3 in fig . 4 b). In other words, l111s mscs inactivates silently, bypassing the opening closing cycle . Wt mscs and the two mutants were expressed in pb113 cells carrying native mscl used as an internal gauge to calibrate their activation midpoints (p0.5) as described previously (akitake et al . The a98s p0.5 was estimated to be 0.75 wt p0.5, and the l111s p0.5 was 1.72 wt p0.5 . 4 c shows the three activation curves, and the bottom panel presents the inactivation rate for wt mscs plotted on the same tension scale . Under the assumption that the a98s and l111s mutations affect primarily the threshold and midpoint for activation but not the process of inactivation the soft a98s mutant opens completely before the inactivation rate becomes appreciable, and the population becomes locked in the open state with no ability to inactivate . We found it practically impossible to even estimate the rate of inactivation for this mutant, as inactivation was undetectable in the entire range of tensions . L111s, in contrast, becomes active at tensions where inactivation rate reaches maximal measurable values, and even before that it quickly inactivates without opening . We have attempted determination of the inactivation rates for l111s, but the extremely high threshold for this mutant made the patches prohibitively unstable under saturating pulses . The comparison of activation and inactivation thresholds for wt mscs, soft a98s, and stiff l111s mutants . (a) a series of traces obtained in response to step pulse protocol shows that the soft a98s mscs mutant does not inactivate . (b) the stiff l111s mutant partially inactivates silently without opening, as illustrated by trace 3 (arrows). (c) positions of activation curves for a98s (0.5 = 5.8 mn / m), wt (0.5 = 7.8 mn / m), and l111s (0.5 = 13.4 mn / m), and the tension dependence of the inactivation rate of wt mscs (data from fig . The data presented above show that (a) wt mscs inactivates from the closed (resting) state, whereas opening prevents inactivation; (b) inactivation, as activation, is also driven by tension and is accompanied by 8.5-nm in - plane protein expansion; and (c) it appears that gain- or loss - of - function mutations that alter the position of the activation curve influence inactivation indirectly, simply by shifting the activation and inactivation curves relative to one another, thus either abolishing inactivation or producing silent inactivation without activation . The experiments presented in figs . 1 and 2 a illustrate that full opening of the mscs population does not increase the fraction of inactivated channels at the end of the pressure protocol . With the increase of the duration of the saturating step and reciprocal shortening of the subsaturating step in the fixed - length protocol (fig . 2 b), the degree of inactivation diminishes . Inactivation increases when the channels are given a chance to close and spend more time at moderate tensions . Applying pressure step protocols with interspersed saturating test pulses delivered from a computer - driven pressure clamp machine critically helped us to characterize the processes of adaptation, inactivation, and recovery . 3 shows that the kinetically intertwined processes of adaptation and inactivation are separable and have opposite tension dependencies . (akitake et al ., 2005), slows down with tension, whereas the inactivation rate increases . Thus, at higher conditioning tensions, when more channels tend to be open, fewer channels are recruited into the inactivation path, but the rate of inactivation increases . 3 (d and e), has an opposite tension dependence from the inactivation process, although with a lower slope . In the simplifying assumptions that recovery is inactivation in reverse and the closed or adapted (c and ca) and inactivated (i) states are separated by a single rate - limiting barrier (b), the sum of acb and abi calculated from the slopes of respective tension dependencies predicts the total expansion of 8.5 nm accompanying the ci transition . Therefore, both processes of opening and inactivation originate in the closed or closed - adapted states, with essentially coinciding thresholds (akitake et al ., 2005). However, larger expansion associated with opening (1215 nm) and correspondingly steeper dependence on tension sets a higher rate and probability for opening at higher tensions that permits a transient response . The schemes illustrating possible transitions between main functional states in wt mscs the major feature is the presence of the adapted closed state (ca) in excised patches, from where inactivation occurs . The ca state may be a result of some pre - expansion of the channel complex under asymmetrically distributed tension in the surrounding membrane because of relaxation of the inner leaflet not attached to the pipette (belyy et al ., 2010b). The possible branching pathways connecting main functional states visited by mscs in excised inside - out patches . (a) the channel undergoes a direct co transition, adaptive closure (oca) because of midpoint shift, from where the channels inactivate in a tension - dependent manner . Upon tension release, (b) under tension, especially gradually imposed, channels first adapt (cca) and then open (cao). Channels remaining in the ca state (not open) gradually escape to the i state . Further adaptive shift of activation curve will add to the ca population from the o state (oca). Because the recovery rate is strongly tension dependent and channels refuse to recover unless tension is very low, it is conceivable that there is more preferential return to the c state as opposed to the ca state . Note that the ca state may not be just a pre - stressed state of the channel by tension, but rather a state of the surrounding membrane with a distorted tension profile (belyy et al ., 2010b). The positions of the state wells on the expansion coordinate are shown by letters (c, ca, o, and i), and the approximate positions of rate - limiting barriers are designated by asterisks . The area change for the opening transition is taken from belyy et al . The estimated in - plane expansion associated with the ci transition provides guidance for the modeling of the inactivated state . Our previous studies have shown that the two nonconductive states are likely characterized by different conformations of tm3 helices predicted to be bent either at g121 in the resting state or at g113 (crystallographic kink) in the inactivated state (akitake et al ., 2007). In both conformations, the tm3 bundle is predicted to be relatively narrow, with a tightly closed dehydrated gate . The estimated 8.5-nm expansion suggests that it is probably not the tm3s but rather the lipid - facing tm1tm2 pairs that change their orientation from a compact tm3-aligned position (anishkin et al ., 2008a; belyy et al ., 2010a) to a wider conformation, taking more space in the bilayer . In this wider state, the lipid - facing helices no longer convey force sufficient to separate the closed dehydrated gate (anishkin and sukharev, 2004). Our recent study suggests that it is unlikely that the tm1tm2 pairs assume a wide splayed crystal - like conformation in the inactivated state, as the tm2tm3 crevices do not seem to be hydrated in any state, yet there must be a mechanism of disengagement of the peripheral lipid - facing helices from the gate . The presence of flexible points in tm1 (gly41) and tm2 (gly76) suggests that there might be a bending / twisting motion of the tm1tm2 pairs that would disjoin the critical f68 (tm2) from l111 and l115 (tm3), thus uncoupling the gate from the stimulus . Our preliminary data already suggest that the g76a mutant lacks inactivation (unpublished data), supporting the idea of tm1tm2 twisting, which will be a topic of separate publication . The estimated in - plane area increase will constrain the mutual positions of the transmembrane helices and the degree of twisting . The inactivation path in mscs starting from the closed state may have its own biological meaning . Most of the ion - permeation mechanisms that carry a signaling function, such as na- or ca - based action potentials, are strictly transient, and intrinsic inactivation in these channels sets limits for the duration of the spike (hille, 2001). Inactivation mechanisms are diverse and often involve not only special inactivation gates (hoshi et al ., 1990; yellen, 1998; panyi and deutsch, 2006) but also selectivity filters (ogielska and aldrich, 1999; cordero - morales et al ., 2007 have been well documented, including the slippage mechanism in which the same gate that produces activation becomes uncoupled from the voltage - sensing domains (shin et al ., 2004; bhring and covarrubias, 2011). It appears that mscs inactivates through a similar mechanism of gate uncoupling from the peripheral stimulus - receiving domains (akitake et al . Mscs does not seem to be involved in signaling, but acts as a relatively large (1-ns) nonselective osmolyte release valve in a rather small bacterial cell with internal volume of only 0.04 m (loferer - krssbacher et al ., when the cell is subjected to an abrupt osmotic downshock, and the population of mscs channels opens, it takes 100 ms for the osmotic equilibration to complete (boer et al ., 2011). If the cell remains in a hypoosmotic medium for an extended period of time, residual tension may persist, and because the activation threshold is not high (45 mn / m), mscs would constantly flicker, dissipating vital gradients and preventing recovery . Inactivation thus ensures that under constant nonthreatening tension, mscs is silent and the cell is allowed to function . The mechanism that prevents inactivation from the open state ensures that mscs will not shut as long as tension is close to saturating and the channel will fulfill its protective role . The requirement of closing before inactivation implies that tension must drop to a level below or near the threshold, from where the channel can be safely disengaged . Return of the cell to a normal or hyperosmotic medium would release tension completely and permit fast recovery to the resting ready - to - fire state.
For the first time in 1911, it was found as a fungal yeast in human stool specimen; then, it was identified as a nonpathogenic protozoan and was forgotten for decades (2, 3). Between 1970 and 1980 with several studies conducted on blastocystis, the first spark of attraction was paid in relation to biology and clinical features of this parasite (4). In recent decades, the results of epidemiological studies, in vitro studies, and research on laboratory animals has shown that this parasite is potentially pathogenic (3). Several factors such as parasite load, secreting enzymes such as cysteine protease, parasite subtypes, parasite proteins and even host conditions are involved in the pathogenesis of parasite (5 - 8). Blastocystis has a worldwide distribution and is transmitted by cysts via contaminated food and water (9, 10). Its prevalence in developing countries is more than in developed countries, which is a result of poor health (11). Isolates of the parasite separated from human are called blastocystis hominis and those from animals are generally called blastocystis sp . In addition, some classifications may be based on the relevant hosts (12). To recognize the genotypes of blastocystis sp ., a pcr was performed using seven pairs of sequence - tagged sites (sts) primers (13). For example, human is the main host of st3, pig and cattle are the main hosts of st5, and rodents are the main hosts of st4; but these hosts are not specific for blastocystis and the parasite has been reported in human too, which is the sign of zoonotic blastocystis (3). Its microscopic diagnosis is difficult so that it is sometimes even ignored by experienced people . Various methods such as direct wet - mount, lugol's iodine staining, formaldehyde - ether sedimentation, dedicated staining, culture and pcr have been used for its diagnosis; pcr is the most sensitive method with high specificity (14, 15). The aim of this study was to determine the subtype of blastocystis in infected cattle in khorramabad city, iran, using seven pairs of sts primers . It was performed for the first time in iran as an introduction to future studies of subtype prevalence in other animals, also assessing the effect of subtypes on pathogenicity of parasites in animals, particularly in cattle . This descriptive cross - sectional study was performed on 196 isolates from cattle stool samples collected from slaughterhouse in khorramabad city, iran, in 2012 . The samples were collected in disposable containers with no fixative . To eliminate waste, 2 g of stool was added to 10 ml of normal saline solution and the mixture was passed through an 80-micron filter . Afterwards, it was centrifuged for 10 minutes at 200 rpm and 200 mg of the sediment was added to a 1.5 ml microtube (16). Dna was extracted using qiaamp dna stool mini kit (qiagen, hilden, germany) according to the manufacturer's protocol . First, primary primers were used according to previous studies for blastocystis identification (14), b11400 forc (5`-gga atc ctc tta gag gga cac tat aca t-3 `) and b11710 revc (5`-tta cta aaa tcc aaa gtg ttc atc gga c-3). The pcr was performed in a thermocycler (corbett, australia) with the following conditions: one initial denaturing cycle at 94c for five minutes, followed by 30 cycles of 94c for one minute, 58c for one minute, and 72c for one minute, and finally one cycle of 72c for five minutes (14). At the end the expected pcr product was 310 bp . To determine the subtype of blastocystis in positive samples, seven pairs of sts primers the pcr program was conducted with an initial denaturation at 94c for five minutes, followed by 30 cycles of 94c for 40 seconds, 57c for 40 seconds, 72c for 40 seconds, with a final extension at 72c for five minutes (13, 17). The dna was prepared by pcr, using seven pairs of sts primers and was sent to bioneer corporation, korea, for sequencing . Abbreviations: f, forward; r, reverse; st, sequence - tagged . Of 198 specimens, 19 (9.6%) were infected with blastocystis (figure 1). Three subtypes including st3, st5 and st6 were found by pcr analysis of the positive samples using the sts primers (figure 2). Among the 19 positive samples, the most common subtype was st5 (47.36%), followed by st3 (10.53%) and st6 (10.53%) (figure 2). St1, st2, st4, and st7 were not identified in the samples under study . The obtained sequences were compared to the sequences reported in gene bank and the results showed high homology with blastocyctis sp . Nucleotides sequence data with accession numbers cjx524459, cjx483863, and cjx524460 respectively for st3, st5 and st6 have been submitted to the genbank database . More studies on this parasite have been performed within the recent 10 years and many indices such as morphology, mode of transmission, parasite dissemination, reservoir hosts, and medical importance have been found, while unknown issues like pathogenicity have remained . Studies have been performed on pig and dog in iran to evaluate gastrointestinal parasites such as blastocystis by the microscopy method (18, 19). This study was the first to investigate the blastocystis subtype in cattle using molecular methods in iran . In the present study, the highest prevalence was for st5 which is so important for epidemiology and risk of human infection . As has been reported in similar studies, there is a higher risk of st5 existence in animals and humans who live close to them (20 - 22). The main hosts of st5 are pig and cattle; most of the studies have been conducted on pig for its use in food worldwide (23). In iran, due to islamic issues therefore, people's contact with cattle is more common than pig . In some studies, it has been reported that being in contact with animals has provided the transmission of animal subtypes to human (24). The reports of high prevalence of st5 in studies on humans in iran can confirm that this subtype is zoonotic (20 - 22, 25). However, some researchers believe that st5 is not zoonotic and is found only in pig and cattle (23, 26). On the other hand, st3 reports in cattle as a subtype in human shows the risk of infection transfer between human and cattle . Since many of the cattle in this study grazed on farms contaminated with human stool, the zoonotic nature of blastocystis was reconfirmed . Another important point in this study st6 has been reported with mixed infections with st7 in birds . According to this subtype report in this study, further researches and revision of the division of the subtype host are necessary (27). The lack of determination for four subtypes of the 19 positive samples, as reported in previous studies, may be due to genotype diversity, implying that only some of them are known (28). Finally, it seems that gathering epidemiological data, ie, identification of zoonotic isolates at the subtype level, is needed for a better understanding of the potential animal reservoirs for human infection.
Pelvic exenteration (pe) describes a radical surgery involving the en bloc resection of the pelvic organs, including the internal reproductive organs, bladder, and rectosigmoid . Indications include advanced primary or recurrent pelvic malignancies, most commonly centrally recurrent cervical carcinoma, but also other gynecologic tumors and urologic and rectal cancers . As the best chance for disease - free survival is surgical resection of regional disease, this procedure is an opportunity to cure advanced and recurrent cancers confined to the pelvis . Pe has also been used for palliation of symptoms related to radiation necrosis or extensive tumor burden . Both total and partial pe require extensive reconstruction and surgical recovery with significant associated morbidity and mortality . Careful patient selection is required to balance the potential goal of cure or symptom palliation with surgical risk . The first cases of total pe were described by brunschwig in 1948 as a palliative procedure for symptoms caused by locally advanced gynecologic cancers . This demonstrated proof of concept for pe, with a postoperative survival of up to 8 months, and a 23% surgical mortality rate . Subsequent data demonstrated that the technique could offer a chance of cure for centrally located tumors, not just palliation, and the focus of the surgery shifted to one of curative intent . Various surgical approaches both for sparing uninvolved pelvic organs and removing extraperitoneal structures such as the sacrum were attempted . Major breakthroughs included separate stomata for urine and fecal diversion and the use of omentum to protect the empty and denuded pelvic space and reduce abscess formation and intestinal obstruction [2, 3]. More recently, techniques to resect tumor involving the pelvic sidewall, previously a contraindication to pe, have been described offering more patients a chance at curative surgery . Pe may also be combined with intra - operative radiation therapy for improved disease control at the pelvic sidewall or possible positive margins [5, 6]. Since 1948 several developments in perioperative care and surgical technique have improved survival, morbidity, and mortality, with recent mortality rates quoted <5% . Development of continent urinary conduits and orthotopic neobladders, as well as low rectal anastomoses has led to the completion of pe without formation of stomata . Various techniques for functional neovaginas have been described, allowing patients to maintain sexual function if they desire . Advances in laparoscopic and robotic assisted technology applied to pe have improved operative recovery . Despite these significant advances and five - year survival rates of approximately 50%, pe remains a radical procedure with significant complications (3192%; see table 1), both physical and psychological . Traditionally pe has been used for centrally recurrent cervical carcinoma, both squamous and adenocarcinoma, with well - documented salvage potential . Up to 25% of women with figo stage ib - iia cervical cancer may recur after initial therapy . Frequently, these recurrences may be treated with radiotherapy; however, radical surgery may offer an alternative for curative treatment . Survival rates ranging from 16 to 60% are reported for these patients [10, 11]. Long - term survival is directly correlated with complete tumor resection [12, 13], so establishing resectability is a key aspect of preoperative planning . Time from primary treatment, with radiation or chemoradiation, to time of pe has also been shown to be related to survival and disease - free interval, with women requiring pe for recurrence less than 2 years following primary therapy demonstrating an 8-month survival versus 33 months in women who recurred more than 2 years following initial treatment in one study, though this has not been shown in all series . Pe has also been utilized as a potentially curative primary treatment for locally advanced cervical cancer (figo stage iva), a practice exercised more frequently in germany than the united states . For example, in their series, marnitz et al . Reported a 52.5% five - year survival . Cases of pe for a variety of histologic types of uterine cancer have been reported, with outcomes similar to pe for other indications . Most recurrent uterine cancers spread beyond the pelvis, given their propensity for diffuse abdominal or heterogenous spread, making pe appropriate intervention for only a select group of patients with recurrent uterine malignancies . Women with only loco - regional recurrence, however, may be candidates for pe with curative intent . Described a series of 21 women with recurrent uterine cancers who underwent pe and demonstrated a five - year survival of 40% . The study also noted varying outcomes dependent on histology, with endometrioid adenocarcinoma (50% five - year survival rate) and sarcoma (66% five - year survival rate) demonstrating improved survival over a group of women with tumors with serous, mixed, and carcinosarcoma - histology (14% five - year survival rate). Morris et al . Reported a five - year survival rate of 45% following pe for recurrent endometrial cancer . Given the similarity of complication rates (4860%) and survival to pe for cervical cancer, patients with locally recurrent uterine cancer may be considered candidates for the procedure . Vulvar cancer has a propensity for regional metastases . For patients with advanced primary or recurrent vulvar cancer who do not have the option of treatment with radiation therapy, pe may be appropriate . The authors demonstrated results similar to other gynecologic malignancies, with a five - year survival of 62% . Complete resection with no evidence of residual disease was associated with improved outcomes, a five - year survival rate of 74%, compared to 21% in patients without complete resection . Absence of tumor lymph node invasion was also associated with an improved five - year survival rate (83% versus 36%). In contrast, combination therapy with vulvectomy and radiotherapy has been described for locally advanced vulvar cancer with the goal to spare the pelvic organs, with five - year survival in two series of 45% and 72%, and sparing of the pelvic organs in 62.5% and 89% of patients [19, 20]. Given the propensity of ovarian cancer to spread throughout the abdomen, women with this disease are rarely candidates for pe with curative intent . Supralevator pe has been reported when needed for optimal cytoreduction, combined with standard staging procedures and for recurrent disease . Two series of modified posterior pe for ovarian cancer demonstrated median survival 33 and 37.4 months after initial surgery . Optimal cytoreduction was achieved in 46% and 58% of patients in the series [21, 22], demonstrating this technique may be used to achieve optimal cytoreduction in patients with disease requiring rectosigmoid resection . As vaginal cancer is rare, this review could not identify any literature specifically addressing pe for this indication . Several cases of vaginal cancer, both primary and recurrent, undergoing pe have been included in larger studies, most frequently including the results for these patients combined with results for cervical cancer [10, 23]. It may be hypothesized, that results following pe for vaginal carcinoma would be similar to those for cervical cancer provided the same other parameters for patient selection apply . Pe has been described for palliation rather than for curative intent, most frequently in the setting of severe radiation necrosis . Both morbidity and mortality were shown to be higher in this group of patients as opposed to those undergoing pe with curative intent, though improvements in quality of life are reported [24, 25]. Pe is thus only considered for palliation if there is no reasonable alternative, though with the development of minimally invasive surgical technology, pe may become a more feasible option . Pe is a major surgery with significant morbidity, and as such selecting appropriate patients is essential . If the surgery is undertaken with curative intent, the tumor should be fully resectable with negative margins . Classically, disease burden was required to be limited to the central pelvis, but with new surgical developments candidates for curative pe may now also include patients with positive lymph nodes, pelvic sidewall involvement, and local bone invasion . Regardless of the indication, patients undergoing pe must be in otherwise good medical health to be able to tolerate a long surgical procedure with extensive fluid shifts and prolonged hospital stay . Preoperative evaluation includes a complete history, physical exam, and, if necessary, an exam under anesthesia, biopsy of any suspicious lesion such as an enlarged lymph node, evaluation of specific patient concerns suggesting metastatic spread, such as unilateral leg pain, chest radiograph or computed tomography (ct). In general, cystoscopy and sigmoidoscopy are not necessary unless the bladder or rectum is to be spared . In this case, laboratory tests should include a complete blood count, platelet count, comprehensive metabolic panel, including hepatic and renal function, as well as clotting factors and urinalysis . Patients should be offered testing for human immunodeficiency virus, which may be a contraindication to pe . Given the nature of the surgery, patients should be counseled about changes in body image and function . Specifically, patients should have an understanding of anatomical changes involving creation of colostomy and urinary conduit and need to be accepting of major changes in body image even in the setting of reconstructive surgery . Patients require significant family support, intact mental capacity and access to continued and long - term medical care . We recommend sharing printed literature and illustrations depicting ostomies and conduits, as well as offering patients the opportunity to speak with other women who have undergone the procedure . Patients should meet with ostomy nursing staff to begin the education process preoperatively and gain confidence with management of the ostomy and conduits . During these visit part of the counseling sessions should focus on sexual function and how this will change for both patients choosing to have creation of a neovagina, as well as for those declining this part of the reconstruction . Patients should be informed of all possible perioperative complications, including infectious, thromboembolic, gastrointestinal, urinary, psychiatric, readmission, and reoperation . Women being considered for pe should be informed of a 35% risk of operative mortality . Some of these complications occur more frequently in the remote postoperative period (days 3190) than in the immediate one (030 days). Patients should expect frequent visits to the hospital during this time given the risk not only of immediate but also delayed complications . Of note, part of the preoperative counseling should include the impact aborting the operation for unexpected surgical findings may have on the patient . That is, patients should be informed that even with the use of state - of - the art, preoperative imaging, the possibility of finding metastatic disease continues to exist, and a minority of exenterative procedures are aborted at the time of surgical exploration . It is critical that patients have sufficient medical and emotional support to manage the physical and psychological challenges central to the operation . Therefore, the goal of diagnostic techniques is to find evidence of unresectable or metastatic disease; thus, making the woman an unsuitable candidate for pe . A number of diagnostic techniques can aid in assessing unresectable disease in a patient who is believed to have central pelvic disease . Computed tomography (ct) and magnetic resonance imaging (mri) can be helpful in assessing the presence of lateral pelvic wall invasion or liver metastasis . However, major limitations of ct and mri lie in their inability to assess minimally enlarged nodes to detect microscopic peritoneal disease and to distinguish fibrosis from tumor in recurrent disease, and the fact that most patients have usually received extensive radiation makes distinguishing radiation fibrosis from malignant tumor extremely difficult . F - fluorodeoxyglucose positron emission tomography (fdg pet) has been shown to perform better in this population . The only prospective study to date in which all patients underwent surgical exploration with curative intent and in which almost all pet positive sites were biopsied showed that sensitivity and specificity of pet imaging in metastatic disease in patients being considered for pe was 100 and 73%, respectively . Despite a negative predictive value of 100%, the high false positive rate found in this study makes surgery obligatory for all pe candidates . Bone scans are usually not indicated unless there is a history of recent bone pain and concern for bony metastases . The procedure begins with the patient in low lithotomy position to allow for abdominal and perineal portions of the surgery . Pe is traditionally performed as an open abdominal procedure, but recent developments in laparoscopy and robotics have allowed for the minimally - invasive adaptation of the technique . The abdomen is opened with a vertical midline incision to allow for maximum ability to explore the upper abdomen as well as the pelvis . Any suspicious lesion is biopsied and sent for frozen section to exclude the possibility of distant metastatic disease that would preclude complete resection or alter the surgical plan . For recurrent cervical cancer, low para - aortic and pelvic lymph node dissection once the disease has been confirmed to be resectable, the operation may proceed [8, 30, 31]. The round ligaments are divided, and the paravesical and pararectal spaces are developed . At this time the pelvic sidewalls may again be examined . At this point, the extent of pe must be determined . Total pe includes removal of the bladder and distal ureters, portions of rectum and sigmoid colon, internal reproductive organs (if still present), and vagina . In well - selected patients, occasionally, if the anatomic location of the recurrence is only the anterior or posterior compartment of the pelvis, the colon or bladder may be spared and only an anterior exenteration or posterior exenteration may be necessary . Removal of the specimen begins with the ligation and division of the fibrovascular pedicle containing the uterine vessels, cardinal ligament, and the ureter bilaterally . The uterine artery is ligated at is origin from the hypogastric, lateral to the ureter . The sigmoid is then mobilized and transected with a gastrointestinal anastomotic stapler (gia), and the sigmoid vessels are identified and ligated . Care must be taken to preserve blood flow to the remaining colon usually the sigmoid artery is left intact and the superior hemorrhoidal artery is ligated . The avascular plane between the sigmoid and the sacrum is developed to the level of the levator ani muscles . The prevesical space is extended bluntly . At this point, the specimen should be freely mobile in the pelvis . The perineal portion of the procedure is then performed (or may be performed synchronously with an additional surgeon). An incision is marked to include the urethra, vaginal opening, anus, and possibly the vulva . The pubococcygeal and anococcygeal ligaments are identified and divided . Upon completion of the dissection, the entire specimen is free to be removed . Anterior pe involves the removal of the bladder and internal reproductive organs but spares the gastrointestinal tract . The rectosigmoid, anus, and lower portion of the posterior vagina are left intact . After division of the cardinal ligaments, uterine vessels, and ureters, the rectum is separated from the upper vagina . The rectum is retracted posteriorly by rectovaginal bimanual exam to ensure the space is clear of tumor and resectable . An incision is made into the peritoneum of the cul - de - sac, and the rectum is dissected sharply off the upper vagina . Posterior pe removes the internal reproductive organs and the rectosigmoid but spares the anterior vagina, urinary bladder, and ureters . In previously irradiated pelves, it is important to consider the possibility of urinary fistulae developing following a posterior pe given the possibility for devascularization . The ureters are identified and dissected as in a radical hysterectomy, and the uterine arteries and cardinal ligaments ligated . Modified posterior pe, as for cytoreduction in ovarian cancer, is a supralevator dissection . There is no perineal phase to the operation . If enough rectum remains (more than 6 centimeters), a low rectal anastomosis may be made, sparing the patient a stoma . If the tumor does not involve the vulva or lower third of the vagina, the patient may be a candidate for a supralevator pe . After the specimen is mobilized as in a total pe, an incision is made into the posterior vaginal wall below the tumor, ensuring an adequate margin . The rectum is isolated and divided with a stapling device, leaving an anorectal stump and possibility for low rectal anastomosis . Brunschwig initially designed reconstruction after pe with an ureterosigmoidostomy, known as a wet colostomy, with urine and feces emptying through one stoma . In current practice, both incontinent ileal and colonic conduits are used, as well as a variety of continent urinary reservoirs, most commonly the miami pouch [30, 31]. The standard ileal conduit is formed by an isolated segment of distal ileum with its vasculature . The ureters are anastomosed directly to the ileum at one end, and the other end is brought to the skin as a stoma . The miami pouch was first reported by bejany and politano in 1988, a modification of prior continent colonic pouches designed to reduce incontinence . The ileum is transected 10 to 15 centimeters proximal to the ileocecal valve, and the transverse colon is transected distal to the middle colic artery . An appendectomy is performed . To form the bulk of the pouch, the colon is opened along the tenia, and the open edges are approximated by folding the colon segment into a u - shape conduit and the edges closed with a stapling device . This formation of the colon creates a reservoir and interrupts the ability of the bowel to peristalse and increase the pouch pressure . For the ureteral anastomoses, the distal ends of the ureters attention is then turned to the ileum, which is tapered distally to support the ileocolic valve and prevent reflux . The free end of the ileum is brought to the skin surface as a stoma . The patient will be required to self - catheterize this stoma, but she is spared a drainage device if the procedure is successful . In a follow - up study of the miami pouch reported 92% continence and reservoir volume of average 650 ml, allowing for a reasonable catheterization interval . For patients whose disease requires infralevator dissection posteriorly, permanent end colostomy will be required because the anal sphincter is compromised or excised . If the sphincter and enough rectum may be spared without compromising the chance at complete disease resection, low rectal anastomosis may be considered to restore continence . Direct end to end anastomosis with circular staplers is a reasonable option if enough healthy tissue remains . To improve frequency of stooling by improving the reservoir of the rectum, a colonic j - pouch may be used, particularly in patients with very little rectum remaining (less than 5 centimeters). Some authors, however, cite the frequency of recurrence of disease near the site of rectal anastomosis (45%) as a reason to perform complete resection and end colostomy in all patients . Other authors strongly support low rectal anastomosis for a chance at preserved function and avoidance of undesirable colostomy for the patient . After vaginectomy, construction of a neovagina for restoration of sexual function should be offered to patients undergoing pe . Several options exist for the creation of a neovagina, including split - thickness skin grafts, myocutaneous grafts, and colon . Rectus abdominus myocutaneous (ram) flaps have been reported routinely in the literature, with 93% viability in the series from ucla . The ram flap may be harvested from the same midline vertical incision used for the pe, improving cosmetic outcomes for the patient . Consideration must be made in the selection of the flap, such as previous maylard incision or other compromise to the inferior epigastric artery . A transverse or vertical flap may be constructed, at least 10 to 12 centimeters in length, maintaining the blood supply from the inferior epigastric artery . The flap is freed, elevated, and sutured into a tube with the skin at the interior, which will serve as the neovagina . The tube is then secured in the pelvis at the vaginal introitus . A mold with estrogen cream is left in the vagina to maintain the lumen for 5 to 7 days . Patient satisfaction and coitus rates are quoted as 5878% [34, 35]. If a neovagina has been created with a myocutaneous flap, such as a ram flap, this graft is usually sufficient to fill the pelvic dead space and ensure adequate vascularity . If no such procedure has been performed, it minimizes complications such as bowel obstruction and maximizes hemostasis to close the pelvic dead space . The omentum is detached at the greater curvature of the stomach while preserving its origin containing the left gastroepiploic artery, which will supply the flap . Other options, such as mesh and pelvic packing, were attempted with poor outcomes . The indications for its use have widened, and the superseding of open surgery seems inevitable in many areas of surgery . This revolution in surgery is in part associated with the technological advancement and a concomitant acquisition of advanced minimally invasive surgical skills by many gynecologic oncologists . Laparoscopy is now a well - accepted tool in the armamentarium of the treatment of gynecological cancer, and data have been published by various centers [3639]. Minimal invasive surgery is generally associated with less intraoperative blood loss, postoperative pain, and shorter hospital stay . [40, 41] were the first group to report two cases of laparoscopic pe for gynecological cancer . Both patients enjoyed the other well - known advantages of laparoscopy including minimal blood loss and quick ambulation, all contributing to a better postoperative quality of life . Subsequently, lin et al . Reported a case of laparoscopy - assisted transvaginal total pe . In addition, ferron et al . Published a series of five patients that underwent a laparoscopic assisted vaginal pe . Their series reports the first application of a rational combination of laparoscopic, perineal, and hand - assisted surgery, with the goal of limiting the potentially long laparoscopic time to a strict minimum . Of note, the authors elected to perform a hand - assisted miami pouch through a minilaparotomy (5 cm) in order to reduce the operative time, safely perform the ureteral anastomosis, restore bowel continuity and, in addition, build the omental cylinder for vaginal reconstruction . The use of a perineal or vaginal approach allowed to quickly and safely free the specimen well above the pelvic floor . In a subsequent report by the same authors, with a mean follow - up of 14 months, four patients died of the disease (three were metastatic), one patient presented a local recurrence, and two patients are disease free . Reported in a series of 16 consecutive patients, the technique, feasibility, and safety of laparoscopic anterior pe as primary treatment for locally advanced pelvic cancers . Thirteen patients underwent anterior pe with ureterosigmoidostomy, while two patients required total pe with wet colostomy . Of note, after a mean follow up of 15 months, all patients were disease free . Also described the feasibility of doing a laparoscopic total pe for palliation in advanced cervical cancer . Of the 7 patients included in their series, no patients required conversion to open surgery . The mean followup was 11 (424) months and mean symptom free period was 8 (324) months . The mean followup of the patients was 11 months (range 4 to 24 months); and the mean symptom free survival period was 8 months (range 3 to 24 months). Three patients are now disease - free for more than a year . The development of robotic technology has facilitated the application of minimally invasive techniques for the treatment and evaluation of patients with gynecological cancers . Robotic surgery offers several advantages over laparoscopy: a three - dimensional vision system, wristed instrumentation, and ergonomic positioning for the surgeon while performing surgical procedures . The enhanced visualization gives the gynecologic surgeon an improved ability to identify tissue planes, blood vessels, and nerves while performing the surgical procedure [4749]. Since the first report of robotic - assisted radical hysterectomy by sert and abeler in 2006 for cervical cancer, there have been some reports of robotic - assisted laparoscopic pe [5052]. The first cases of robotic - assisted laparoscopic pe were described by pruthi et al . In 12 women for clinically localized bladder cancer . Urinary diversion was performed extracorporeally (9 ileal conduit diversion, 3 orthotopic neobladder). Lim reported the first case report of robotic assisted total pe with an ileal loop urinary diversion and an end colostomy for treatment of recurrent cervical cancer . The authors reported that concerning hospital stay, there was no benefit comparing to laparotomy, essentially due to urinary diversion management (catheterization) and to self catheterization patient's autonomy . Despite the apparent encouraging early results suggesting an advantage of minimally invasive surgery for pe, questions remain about the surgical effectiveness of this approach . Further study of minimally invasive techniques to perform a pe is needed prior to widespread clinical application of these techniques . As it is a radical surgery performed in the setting of advanced tumor growth and frequently on irradiated tissue, pe is associated with a significant rate of complications, quoted about 4050% for major complications and about 80% for minor complications . Mortality is quoted from 116%, with disparate causes including sepsis, thromboembolic disease, and cardiopulmonary failure . Despite significant advances in the last fifty years, the extensive nature of the surgery, including blood loss, fluid shifts, and operative time, have led to unavoidable risks . Infection is the most frequent morbidity (1986%), with urinary infections and wound infections most commonly reported . Anastomotic leaks and fistulae from either diverting system are also relatively frequent, cited at 836% . Most of these complications can be managed conservatively, but significant numbers of patients require operative revision [10, 11, 55]. Death in the perioperative period occurs in fewer than 5 percent of patients, with women over the age of 65 at highest risk . Given the radical and prolonged nature of this procedure, patients and providers must be prepared for a long and potentially complicated hospital course . Many patients require a stay in the intensive care unit immediately postoperatively for close monitoring, particularly in the setting of potentially dramatic fluid shifts . Special attention to thromboembolism prophylaxis, respiratory care, and nutrition is required . While no longer routine, some patients will require total parenteral nutrition due to prolonged inability to eat postoperatively, as ileus is relatively common . A team - based approach, including case managers, dedicated nurses, and social workers, may help patients as they heal both mentally and physically postoperatively . As a portion of preoperative counseling and postoperative support, the changes in a woman's body image following pe must be reviewed . Some patients, particularly those undergoing this surgery for palliative management of pain or fistulae, do report improved quality of life following surgery, with decreased narcotic requirements and malodorous discharge . Most women, however, note a decline in specific areas of quality of life . Notably, body image, physical ability, and social function have all be reported decreased in questionnaires compared to patients' preoperative answers . These changes are more pronounced in younger patients and those who do not undergoing vaginal reconstruction . Interestingly, overall function and mental and emotional quality of life are comparable [5759]. Pe is a radical operation, involving en bloc resection of pelvic organs, including reproductive structures, bladder, and rectosigmoid . In gynecologic oncology, it is most commonly indicated for the treatment of advanced primary or locally recurrent cancer . Patients need to be carefully selected and counseled about risks and long - term issues related to the surgery . Total pe is associated with significant surgical morbidity, a fact that underscores the importance of careful patient selection and counseling . The emergence of minimally invasive surgery and application of this technology to radical pelvic surgery including pe may result in a reduction operative morbidity and mortality . Further studies are necessary prior to a widespread adoption of this technology to exenterative procedures.
, a bilateral breast cancer recurrence was diagnosed and cured with demolition surgery and cmf chemotherapy for 8 cycles . Given that the patient was 67 years old at the time of brca mutation diagnosis, a bilateral oophorectomy was proposed, and in june 1998, she underwent a bilateral oophorectomy showing no evidence of disease in the ovaries and tubes . During surgery, massive peritoneal adhesions were detected which complicated the surgical procedure possibly due to a previously undiagnosed pelvic inflammatory disease syndrome (pid). In october 2004, after an increase in serum ca-125 levels was registered, an abdominal computed tomography scan was performed evidencing a pelvic 8-cm complex mass reaching the hepatic flexure and the transverse colon . The results of a colonoscopy and a gastroscopy were negative, so the patient underwent an abdominal longitudinal midline laparotomy with removal of the mass and right hemicolectomy . During surgery, it became evident that the mass was originating from a residual of the right fallopian tube which, inaccurately, had not been removed during the previous prophylactic oophorectomy, probably because of the significant scarring due to the previous pid . The histological report documented a poorly differentiated (g3) serous carcinoma derived from the right residual tube with presence of tumor at the tubal fimbria, complete absence of ovarian stroma, and involvement of the peritoneum of the right colon without muscle and mucosa infiltration (figo stage iiic). After surgery, the patient received 6 cycles of carboplatin auc6-paclitaxel 175 mg / mq chemotherapy which was completed in april 2005 . Fifteen to 40% of women with brca1/2 mutations present an increased risk of ovarian cancer . The suggested prophylactic surgical approach for women with a family history of ovarian carcinoma or brca1/2 mutation is bilateral salpingo - oophorectomy with the intent to reduce the risk of ovarian cancer . The history of this patient seems to suggest that much attention should be paid during prophylactic salpingectomy to carefully and completely remove the fallopian tubes, even taking away the intrauterine portion of the tubes, as suggested by several authors, due to the risk that a part of the organ might be erroneously left in place and then would represent the originating site of a subsequent neoplastic transformation . Moreover, a more intriguing scenario is opening up concerning the pathogenesis of ovarian cancer . Recently, kurman proposed a hypothesis on ovarian cancer origin which suggests that ovarian cancer may represent the metastatic lesion of a tumor with tubal origin . In 2001, dutch investigators first described tubal intraepithelial carcinomas, later designated as serous tubal intraepithelial carcinomas (stics), and occult invasive high - grade serous carcinomas (hgscs) in the fallopian tube of women with a genetic predisposition to ovarian cancer that closely resembled ovarian hgsc, in the absence of ovarian lesions . It was later proposed that secondary implantation of malignant cells from the tubal carcinoma to the ovary develops into a tumor mass resembling cancer originating in the ovary . The authors proposed that the failure to identify the precursors of ovarian cancer in the past was due to the research of these lesions being concentrated on the ovaries where, logically, they were expected to be, thus explaining why a careful and systematic examination of the fallopian tubes had been neglected . Additional studies, in which the fallopian tubes were completely sectioned using the sectioning and extensively examining the fimbria (see - fim) protocol, confirmed that stics and small early invasive tubal carcinomas occurred not only in women with a genetic predisposition to the development of ovarian cancer, but also in 5060% of women without recognized brca mutations . Moreover, in most cases, the tumor lesions were detected in the fimbria, and it has been proposed that the earliest neoplastic changes begin in the secretory - type cells . Further evidence supporting this hypothesis comes from the detection of identical tp53 mutations in stics and concomitant ovarian hgscs, indicating a clonogenic relationship between the two lesions . In a series of 342 consecutive gynecologic cancers, stics were present in 18.8% of women with serous ovarian cancer, while it was not possible to identify these precursor lesions in any other histological subtype . A gene profiling study showing that the gene expression profile of hgsc is more closely related to fallopian tube epithelium than to the ovarian surface epithelium and immunohistochemical studies emphasizing that hgsc expresses pax8, a mllerian marker, but not calretinin, a mesothelial marker, lend further support to kurman's hypothesis . There are no features according to which it is possible to distinguish between primitive tubal carcinomas and ovarian carcinomas originating in the tube and secondarily implanted in the ovary; this aspect further supports the theory that these tumors are not different entities . The ovary appears not to be the originating site also of endometrioid and clear - cell tumors . Data support the theory that endometrioid and clear - cell tumors arise from endometrial tissue passing through the fallopian tube and being secondarily implanted into the ovary and that mucinous tumors also develop from the tubal - peritoneal junction . We speculate that the detached clusters of endometrial epithelium pass through the tube and are implanted on the ovarian surface where they can develop into atypical proliferative serous tumors (apsts) or are implanted on the pelvic and abdominal peritoneum to produce noninvasive implants . The history of our patient is very intriguing: she had both ovaries and the left fallopian tube removed during the prophylactic surgery, while the right tube was erroneously left attached to the uterus, possibly because of the scarring and adhesions related to a previous pid which made it difficult to detect the tube . Six years later, a hgsc originating from the residual tube was diagnosed and completely removed during surgery . The patient was treated with conventional platinum - based chemotherapy and is still alive and disease free . The impressive extensive disease - free interval of our figo stage iiic patient positively fits the surgical radicality obtained during cytoreductive surgery and the documented increased platinum sensitivity in brca mutation carriers . Kurman's theory and our clinical case report possibly suggest that salpingectomy or fimbriectomy alone with ovarian sparing might be sufficient to reduce the risk of ovarian cancer in patients with brca mutation and preservation of fertility and hormonal status . At present, this approach needs to be evaluated in randomized clinical trials.
The common iliac arteries start from the aortic bifurcation and terminate by dividing into the external and internal iliac arteries (figure 1). The length of the common iliac artery constitutes the distance between its more variable origin and its more stable termination [13]. It is of note that these 2 points indicate an age - related shift in relation to the spine . During prenatal life, both the origin and termination of the common iliac arteries apparently ascend in relation to the spine (pseudoascensus). On the contrary, in adults the aortic bifurcation undergoes a downward shift (pseudodescensus) due to osteoporosis of the vertebrae, degenerative changes of the intervertebral discs, and reduction in the length of the spine . In adults the left common iliac artery is shorter than the right one, because it begins on the left side of vertebra l4 . The common iliac arteries develop from proximal segments of the primary umbilical arteries, in direct extension of the dorsal aortas . As reported by mansfield and howard, in case of complete absence of the common iliac arteries, the abdominal aorta is divided directly into 4 branches: the 2 internal and the 2 external iliac arteries . Presented aplasia of the right common iliac artery in an asymptomatic patient, in whom both the right pelvis and lower limb were supplied by an anomalous branch from the left internal iliac artery . Some authors therefore argued that congenital absence of the common iliac arteries should be included in the differential diagnosis for intermittent claudication of the legs (leriche syndrome). Advances in perinatal medicine have required an extensive knowledge of fetal aorto - iliac quantitative morphology . The normative morphometric data of the common iliac arteries in human fetuses may be useful as a reference for future doppler studies in the prenatal diagnosis and monitoring of congenital abnormalities (aneurysms, idiopathic infantile arterial calcification) that include discordant diameters of the aorta and its branches [1114]. Although morphometric values of the common iliac arteries in human fetuses have been presented by gocicka et al ., and zgner and sulak, to data, growth curves for the common iliac artery dimensions have not been reported in the professional literature . Because of this, in the present study we aimed to determine: age - specific reference intervals for length, external diameter and volume of the 2 common iliac arteries at varying gestational age, and the normal growth curves for each morphometric feature . The examinations were carried out on 124 human fetuses of both sexes (60 males, 64 females) derived from spontaneous abortions or stillbirths in the years 19892001 . Legal and ethical considerations had been approved by the university research ethics committee (kb/217/2006).on macroscopic examination, both internal and external anatomical malformations were ruled out in all the included specimens, which were diagnosed as normal . In no case was the cause of fetal death related to congenital cardiovascular or non - cardiovascular anomalies . The sample included fetuses which were the outcome of causes of intra - uterine growth restriction . Gestational ages were determined from measurements of the crown - rump length on the basis of iffy tables . For statistical analysis, the fetuses were divided into 6 monthly groups, related to the 49 months of gestation . The arterial bed was filled with white latex lbs 3060 through a catheter stericath (diameter of 0.51 mm), which was introduced by lumbar access into the abdominal aorta . The fetal arteries were filled under a controlled pressure of 5060 mm hg, using a syringe infusion pump sep 11s (ascor sa, medical equipment, warsaw 2001). All specimens were immersed in 10% neutral buffered formalin solution for 424 months for preservation, and then dissected under 10-fold magnification using a stereoscope with huygens ocular . In each fetus, the dissected common iliac arteries in situ with a millimeter scale were placed vertically to the optical lens axis, then recorded using a camera (nikon coolpix 8400), and digitalized to tiff images (figure 1). Next, digital pictures of the common iliac arteries were assessed by 1 researcher using digital image analysis (leica qwin pro 16, cambridge), which semi - automatically estimated length, external diameter and volume of the marked common iliac arteries . Each measurement was performed 3 times and the mean of them was then used . Briefly, measurements of the parameters examined were derived by assuming that the filled common iliac arteries constituted a flexible cylinder . Diameter measurements were derived by assuming that the filled vessels were circular in cross - section . In order to calculate the arterial volume from a 2d image, it was assumed that each vessel of varying diameter can be divided into a large number of small irregular cylinders [1719] with both varying diameter (d) and height (h). Their volumes could be described by the following equation: (v volume, 3.14, d the sum of the volumes of such cylinders approximating the vessel was given in mm as the common iliac artery volume . For each fetus, on both sides, the 3 following parameters of the common iliac artery were assessed: length in mm distance between the aortic bifurcation and the common iliac artery bifurcation, proximal external diameter in mm - immediately below the aortic bifurcation, and the length, proximal external diameter and volume of the common iliac arteries were correlated to fetal age so as to establish their growth . The relative growth of the common iliac arteries was expressed as the length - to - proximal external diameter ratio . As the first step in the statistical analysis, student s t test was used to examine the influence of sex on the values of the parameters studied . The morphometric results were evaluated by one - way anova test for unpaired data and post - hoc rir tukey test . Regression analysis was used to derive the line of best fit for each morphometric feature of the common iliac arteries and gestational age . No significant differences were found in the values of morphometric parameters of the common iliac arteries according to sex (p>0.05). Therefore, the values obtained for the right (table 2) and left (table 3) common iliac arteries have been summarized without regard to sex . By contrast, there were significant correlations between all the parameters studied and gestational age (p=0.0000). Although the right - left differences for the whole group were not found to be statistically significant (p>0.05), the results for each common iliac artery are presented separately because of their great inter - individual variability (tables 2 and 3) and a strong trend towards greater values on the right side . The values of the common iliac artery length increased from 4.761.05 to 15.381.60 mm on the right, and from 4.921.33 to 14.911.25 mm on the left for the 4-month group and 9-month group of gestation, respectively . In 75 (60.5%) specimens the individual values of the common iliac artery length were greater on the right side . With regard to fetal age, the lengths of the right (figure 2a) and left (figure 2b) common iliac arteries increased according to the linear functions: y=3.598 + 0.585age 1.522 (r=0.83) and y=3.107 + 0.554age 1.444 (r=0.83), respectively . The proximal external diameter of the common iliac artery took the values from 0.660.19 to 2.300.42 mm on the right, and from 0.660.14 to 2.160.42 mm on the left at the fetal ages of 4 and 9 months, respectively . The individual values of the proximal external diameter of the right common iliac artery were greater in 79 (63.7%) fetuses . Their growth followed the quadratic models: y=1.3920.110age+0.004age 0.285 (r=0.77) and y=1.2830.099age+0.004age 0.238 (r= 0.81) for the right (figure 3a) and left (figure 3b) common iliac arteries . Nevertheless, on the right and left sides both the length and external diameter of the common iliac artery did not rise proportionally during the study period, because they did not have the same rate of growth through the analyzed fetal ages . This change of rate is illustrated in figure 4, in which the length - to - proximal external diameter ratio of the common iliac arteries was found to increase until the age of 6 months, after which its value decreased . In fetuses aged 4 and 9 months of gestation, the values of the common iliac artery volume were increasing from 1.931.74 to 66.9529.31 mm on the right, and from 1.911.65 to 56.8625.17 mm on the left, respectively . In 84 (67.7%) fetuses the individual values of the common iliac artery volume were greater on the right side . The volumetric growth of the common iliac artery generated the quadratic functions: y=99.6910.60age+0.287age 14.40 (r=0.67) on the right (figure 5a), and y=82.628.86age+0.242age 11.60 (r= 0.71) on the left (figure 5b). After reviewing the professional literature on the common iliac arteries in human fetuses we managed to find only limited reference data for their dimensions . The existing data in the literature has focused on the length and diameter, with no information about their growth curves . Therefore, the present anatomical, digital and statistical study provides reference values for length, external diameter and volume of the common iliac arteries in normal human fetuses . In fact, because these spontaneous abortions and stillbirths were related to intra - uterine growth restriction, the measurements of the common iliac arteries may be somewhat smaller than normal . However, the aortic root diameter was reported to remain normal in most cases of intra - uterine growth retardation . The lack of such quantitative information in the literature concerning the common iliac arteries limits discussion on this subject . Furthermore, tissue shrinkage related to neutral buffered formalin fixation has little influence on the measurements of the filled common iliac arteries in situ, the wall of which is mainly composed of elastic connective tissue . The autopsy findings of szpinda showed only 0.51% shrinkage in elastic fetal arteries in situ, which were filled with latex and then immersed in 10% neutral buffered formalin solution for 424 months . In the current study, no significant male - female differences concerning the 3 morphometric parameters of the common iliac arteries were found, in keeping with the results of some authors concerning both the length and diameter of the common iliac arteries in fetuses and adults . Although no laterality differences for the 3 parameters within the whole group were observed, the individual anatomical parameters of the right common iliac artery were found to be greater than those of the left one in relation to its length (in 60.5% of individuals), external diameter (in 63.7% of fetuses), and volume (in 67.7% of cases). Similarly, zgner and sulak demonstrated no laterality differences in both the length and external diameter of the common iliac arteries . Our findings are in disagreement with gocicka et al ., who emphasized that the left common iliac arteries were always both thinner and longer than the right ones . As pointed out in the introduction, the vertebral levels at which the common iliac arteries start and bifurcate obviously influence the length of the common iliac arteries . In cases having both high aortic bifurcation and low bifurcation of the common iliac arteries, the common iliac artery tends to be longer . Studied skeletopy of the common iliac arteries in relation to the spine in 70 fetuses aged 49 months . Pseudoascensus of the common iliac arteries was found with advanced fetal age . During the study period, the origin of the common iliac artery was displaced upwards half the distance of the vertebral body and projected from the level of the upper half of vertebra l5 to the lower half of vertebra l4 . At the same time, the termination of the common iliac artery shifted from the level of the lower border of vertebra s1 to the lower border of vertebra l5 . According to these authors, the common iliac artery bifurcation displaced upwards in a double fashion due to the development of both the longitudinal dimension of the spine and the transverse pelvic dimension . Some authors [23, 25, 26] revealed that the abdominal aorta length was negatively related to the length of the common iliac arteries, therefore the shorter common iliac arteries were correlated with the longer abdominal aortas . We found that the common iliac artery length rose from 4.761.05 to 15.381.60 mm, and from 4.921.33 to 14.911.25 mm, and generated the linear patterns: y = 3.598 + 0.585age 1.522 and y=3.107 + 0.554age 1.444 for the right and left sides, respectively . The mean values for the length of the common iliac arteries recorded by gocicka et al . Were slightly greater in fetuses aged 4 and 9 months, and much smaller in fetuses aged 58 months, when compared to our results . These differences may be attributed in part to the inter - individual variability and methodological differences in measurements . According to findings of zgner and sulak, the lengths of the common iliac arteries in 3 trimesters (912 weeks, 1325 weeks, 2637 weeks) and in full - term (3840 weeks) fetuses were 2.100.4 mm, 6.131.8 mm, 12.721.6 mm and 19.602.3 mm on the right, and 1.920.4 mm, 6.001.8 mm, 12.71.8 mm and 19.441.8 mm on the left side, respectively . Of note, we found that proximal external diameters of the right and left common iliac arteries varied from 0.660.19 to 2.300.42 mm, and from 0.660.14 to 2.160.42 mm for fetuses at the ages of 4 and 9 months, which were given by the quadratic patterns: y=1.3920.110age+0.004age 0.285 and y=1.2830.099age+0.004age 0.238, respectively . These quadratic functions were the best models for the diameter growth, because the coefficients of determination between the diameter and fetal age attained the greatest values for the right (r=0.77) and left (r=0.81) common iliac arteries . Although the mean values for external diameters of the common iliac arteries reported by gocicka et al . Correspond reasonably well to our numerical data, these authors presented a quasi - linear relationship . To our knowledge, no other angiometric study dealt with a quadratic pattern for any diameter growth, which was always presented in a linear fashion [3,11,12,15,1820,2729]. In the material of zgner and sulak with the 4 above - mentioned groups of fetuses, external diameters of the common iliac artery attained the following values: 0.920.1 mm, 1.690.3 mm, 3.060.5 mm and 4.130.3 mm for the right one, and 0.920.7 mm, 1.630.3 mm, 2.940.5 mm and 4.070.3 mm for the left one . Because the size of the specimens varied, we took into account the relative growth of each common iliac artery, expressed as the length - to - proximal external diameter ratio . As indicated in figure 4, their relative growth turned out to be not proportional . First, until the age of 6 months, the length of the common iliac artery grew faster than its external diameter, being expressed by the positive increment of the length - to - proximal external diameter ratio . Later, in contrast, the external diameter grew faster than the length of the common iliac artery, which was indicated by the decrement of the value of the length - to - proximal external diameter ratio . In our view, because the length and proximal external diameter of the common iliac arteries do not grow at the same rate all the time, the specific individual variations in growth may produce the specific adult forms in particular individuals . Thus, in adults the origin of the common iliac arteries was most often (80%) noted at the level of the lower (48%), middle (12%) or upper (12%) part of vertebra l4, rarely (12%) at the level of vertebra l5, and occasionally (8%) between l3 and l4 vertebrae . The common iliac arteries were found to start as low as the upper part of s1, and as high as the level of l2 . According to kornreich et al ., the position of the aortic bifurcation showed an age - related downward shift with increasing age, which was more pronounced in women (r=0.26) than in men (r=0.13). With each increasing decade of life there was a decline equivalent to 17.5% and 9.5% of the vertebral height for women and men, respectively . In turn, the bifurcation of the common iliac arteries in 60% of specimens projected on the lower margin of vertebra l5, in 30% of fetuses it projected slightly above, and in 10% of the remaining fetuses it projected slightly below this level . In the only previous report related to the common iliac artery volume in human fetuses, gocicka et al . Did not present any growth curves for its development . According to these authors, the mean volume of the common iliac arteries in fetuses aged 49 months varied from 2.14 to 74.82 mm on the right, and from 2.08 to 60.82 mm on the left . Thus, when compared to theirs, the volumetric data in the material under examination turned out to be smaller on both sides in fetuses aged 4 and 9 months, similar on both sides in fetuses aged 5 months, and on the left side in fetuses aged 6 months, and greater in the remaining fetuses . Our findings demonstrated that the common iliac artery volume increased from 1.931.74 to 66.9529.31 mm on the right, and from 1.911.65 to 56.8625.17 mm on the left . It is noteworthy that several transformations were generated to find the most precise mathematical model for the volumetric growth of the common iliac artery . We have proved that the best fit correlation between the volumetric growth of the common iliac artery and gestational age is a parabola . This fact was confirmed by the quadratic regressions: y=99.6910.60age+0.287age 14.40 and y=82.628.86age+0.242age 11.60 in relation to the right and left common iliac arteries, respectively . Of note, we did not come across any study dealing with the growth curves for the common iliac artery volume . In the fetuses examined, the common iliac artery volume rose 35-fold on the right, and 30-fold on the left . These data were related to the product of the length and the squared external diameter, which increased approximately 3.2- and 3.5-fold for the right common iliac artery, and 3.0- and 3.3-fold for the left one . The present study describes the normal growth of the length, external diameter and volume of the common iliac arteries, providing mathematical models for their growth curves . A particular strength of this study is the large number (n=124) of normal specimens used to generate the growth curves . In spite of the fact that this study was performed on autopsy material, the detailed morphometric data as a database for intra - uterine examination of the common iliac arteries may be useful in the early diagnosis, monitoring and management of aorto - iliac malformations in obstetrics, perinatology, and fetal pathology departments . There are no significant differences between sexes for the morphometric parameters of the common iliac arteries . The common iliac arteries grow linearly in length, and parabolically in both diameter and volume . The right common iliac artery constitutes a predominant vessel in relation to its length (60.5%), external diameter (63.7%) and volume (67.7%). The morphometric data on the common iliac arteries may serve as a useful reference to professionals working in the area of congenital aorto - iliac abnormalities and morphologists teaching human developmental anatomy.
Left main coronary artery arising from the right sinus of valsalva as a single coronary ostium is an extremely rare anatomic anomaly occurring in approximately 0.06% of angiographic series . Single coronary artery is encountered more frequently with other congenital cardiac malformations such as persistent truncus arteriosus, tetralogy of fallot, transposition of the great arteries, or pulmonary atresia . Some single coronary ostium variants have been reported to carry a significant risk of severe cardiac events including myocardial infarction (mi) and sudden cardiac death, especially during exercise . We present a case of single coronary artery from right sinus with left main coronary artery originating from the same ostium, which underwent successful angioplasty with stenting to right coronary artery through the radial route . A 60-year - old male was referred to our tertiary care institute for coronary angiography . Patient suffered acute coronary syndrome (acs) - inferior wall mi 3 days prior to admission in our institute . He was thrombolysed with streptokinase at a peripheral center and referred to our institute on the 4 day post thrombolysis in view of post mi angina . Transthoracic echocardiogram showed regional hypokinesia in the right coronary artery (rca) territory with adequate left ventricular ejection fraction . Coronary angiography (cag) by radial approach using tiger 5f catheter (terumo corp ., cag revealed blunt left sinus, with no artery originating from the left sinus [figure 1 and video 1]. Right coronary artery originated from the usual location and a long left main coronary artery arose from the same ostium [figures 2, 3 and video 2]. We exchanged the tiger catheter with judkin's right coronary catheter (medtronic, inc ., minneapolis, mn, usa) as it was not possible to selectively cannulate the ostium with the tiger catheter . There was significant tubular stenosis in the mid - rca region and non - critical plaque in the proximal left coronary artery (lca) [figures 2, 3 and videos 2, 3]. As it is important to know the course of anomalous coronaries before any intervention, ct angiogram was done which revealed retroaortic course of the left coronary [figures 4 and 5]. It was decided to do percutaneous coronary intervention (pci) with stenting to the mid - rca in view of post mi angina and was performed on the 5 day post thrombolysis . Rca was selectively cannulated with judkin's right coronary catheter (medtronic, inc . ). The lesion was crossed with 0.014 190 cm bmw wire (abbott vascular, santa clara, ca, usa) and predilated with 2.5 12 mm maverick balloon (boston scientific, florida, usa) [figure 6 and video 4]. During predilatation, guide sucked in the vessel and pressure damping was noted due to obstruction of supply to the left coronary and this was managed by meticulous decannulation and avoiding deep intubation . A 3.25 33 mm xience v stent (abbott vascular, florida, usa) was deployed at 14 atm [figure 7 and video 5]. 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - ap view fluoroscopy shows blunt left sinus (arrow) with no coronary origin . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - right anterior oblique angiographic view shows both right and left coronaries originating from the right sinus (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - lateral angiographic view shows both rca and lca originating from same ostium (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - right anterior oblique angiographic view shows both right and left coronaries originating from the right sinus . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - lateral angiographic view shows both rca and lca originating from same ostium . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Ct coronary angiography - ct image with 3d reconstruction shows retroaortic course of lca (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography shows left anterior oblique fluoroscopic view of rca during predilatation (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiograph shows left anterior oblique fluoroscopic view of rca during stent deployment (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . 60-year - old male with acs - inferior wall mi post thrombolysis and post mi angina . Coronary angiography - left anterior oblique angiographic view of rca shows end result post stenting (arrow). 60-year - old male with acs - inferior wall mi, post thrombolysis and post mi angina . Coronary angiography - left anterior oblique angiographic view of rca shows end result post stenting . Lipton et al ., classified coronary variations based on origin and anatomical course relating to the ascending aorta and pulmonary trunk . Type l represents an rca originating from the left main stem and type r indicates that the coronary artery originates from the rca . Class ii indicates one coronary artery arising from the proximal part of the normally located opposite coronary artery . In class iii, the left anterior descending (lad) and left circumflex (lcx) arise separately from the proximal part of a normal rca . Classes ii and iii are then designated as anterior (type a) to pulmonary artery or posterior (type p) to aorta, or interarterial (type b) if it courses between the ascending aorta and the pulmonary trunk . Type b morphology has been associated with a high risk of clinical consequences when associated with an intramural course . Angelini et al ., proposed a slightly different classification according to the anatomical course within the interventricular sulcus and atrioventricular groove, as well as the location of penetrating side branches . According to lipton's classification, our patient had r ii p subgroup (single coronary artery from the right sinus with lca arising from the proximal part of rca and a posterior course to aorta). Lipton's classification has been modified by others, adding to this classification the s though there are several case reports of pci in single coronary artery, most are through the femoral route . To the best of our knowledge, we did not have much difficulty during pci as we were using right radial artery approach and judkin's right catheter . Other catheters like internal mammary artery (i m a), multipurpose, and amplatz left (al) can also be used according to the situation . The present case merits mention because of several points: 1) intervention in a single coronary artery through radial approach has been rarely reported and in type r ii, it is all the more rare . 2) ct 3d reconstruction is a useful tool to understand the ostial configuration and course of anomalous coronary . 3) radial pci is as good as femoral pci for anomalous coronaries, provided good hardware is selected and operator has experience in radial interventions . 4) meticulous attention should be given to pressure damping while doing pci in cases with both coronaries originating from single ostium . 5) we should take non - selective shoot if there is no coronary originating from either sinus (left / right).
Acommon problem in clinical laboratories is maintaining the stability of serum analytes during sample storage . Samples are usually stored in the door (48c) of a refrigerator for short durations or in a deep freezer (20c) for longer time periods . Thus, the temperature at which the samples are stored constitutes an important preanalytical variable that may affect analysis results in the clinical biochemistry laboratory setting . These laboratories face many challenges including equipment breakdown and the lack of reagents, which can prevent same - day processing of samples . In such cases, the only option is to preserve the samples in a deep freezer (20c). In addition, samples are sometimes stored for an extended duration until subjected to routine batch analysis for research purposes . Previous studies have provided information regarding the stability of analytes in serum using a number of methods that have since become obsolete . Although many blood analytes have been shown to deteriorate within hours in unseparated samples kept at ambient temperature, the few studies examining unseparated samples stored at low temperatures involved prolonged contact between serum and cells, which could have caused erroneous test results . Moreover, the stability of 72 analytes following prolonged serum - cell contact has been previously described . The effects of prolonged storage on the stability of 31 analytes in plasma and serum separated from cells with a gel barrier have also been reported . A number of studies have described approximately thirty analytes in serum immediately separated from cells . In addition, several studies have examined the effect of storage conditions on the stability of various serum components . However, limited information is available regarding the stability of commonly used clinical biochemical analytes in human serum including the effect of storage temperatures as low as 20c on blood - separated serum . Therefore, the present study examined the stability of 17 routine chemistry analytes in immediately cell - separated serum following storage at a designated temperature (20c) for different periods (0, 7, 15, and 30 days) using the previously described standard guidelines for blood sample handling and separation . This hospital - based study included ten random samples from outpatients being treated at the hospital clinics . The samples collected from each patient were for physician - ordered laboratory testing; no additional blood was taken from the subjects . The institutional ethical committee approved the study, and informed consent was obtained from all the participants . All procedures were conducted in accordance with the guidelines of the helsinki declaration on human experimentation . Fasting venous blood (total of 6 ml blood) was collected in the morning using a vacuette standard tube holder and vacuette 22ga 1 (0.7 mm 25 mm) multisample needle (becton, dickinson and company, usa). The blood specimens were drawn into 7.5 ml plastic vacuette serum tubes (bd vacutainer serum; bd, franklin lakes nj, usa). The sample tubes were left in an upright position for 30 min at room temperature followed by centrifugation at 3500 rpm for 10 min . The serum samples of each subject were pooled into a plain tube and then aliquoted into 1.5 ml eppendorf tubes (eppendorf, milano, italy); four aliquots per patient samples were kept (three for storage at 20c) and the remaining serum was used for the baseline measurement (t1d). The serum aliquots were stored frozen at 20c for either 7 (t7d), 15 (t15d), or 30 (t30d) days and then analyzed separately for stability . The following analytes were examined: [table 1] methods used for measuring biochemical parameters metabolites: na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubinproteins: total protein and albuminlipids: total cholesterol and triglyceridesenzymes: alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and amylase . Metabolites: na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubin proteins: total protein and albumin lipids: total cholesterol and triglycerides enzymes: alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and amylase . All measurements were performed at the hospital laboratory services using an olympus au 400 auto analyzer, except for serum electrolyte levels, which were obtained with a roche avl electrolyte analyzer . To determine time - dependent changes in cell - separated serum analytes, the mean value from all ten subject samples was calculated for each analyte at each time point . Clinically significant changes were determined using the significant change limit (scl) approach, defined as: scl = initial value 3.0 usual standard deviation (usd). This is based on the assumption that the usd is representative of the inherent day - to - day variability of the method . In our study, the usd was obtained by averaging the standard deviation of the quality control data of the previous 2 months for each analyte . The quality - control reference serum with the target mean most closely matching the t1d mean for each analyte was used to determine the usd . For simplicity, the scl was computed for each analyte by establishing the range (3.0 usd) from the subject mean at t1d . This hospital - based study included ten random samples from outpatients being treated at the hospital clinics . The samples collected from each patient were for physician - ordered laboratory testing; no additional blood was taken from the subjects . The institutional ethical committee approved the study, and informed consent was obtained from all the participants . All procedures were conducted in accordance with the guidelines of the helsinki declaration on human experimentation . Fasting venous blood (total of 6 ml blood) was collected in the morning using a vacuette standard tube holder and vacuette 22ga 1 (0.7 mm 25 mm) multisample needle (becton, dickinson and company, usa). The blood specimens were drawn into 7.5 ml plastic vacuette serum tubes (bd vacutainer serum; bd, franklin lakes nj, usa). The sample tubes were left in an upright position for 30 min at room temperature followed by centrifugation at 3500 rpm for 10 min . The serum samples of each subject were pooled into a plain tube and then aliquoted into 1.5 ml eppendorf tubes (eppendorf, milano, italy); four aliquots per patient samples were kept (three for storage at 20c) and the remaining serum was used for the baseline measurement (t1d). The serum aliquots were stored frozen at 20c for either 7 (t7d), 15 (t15d), or 30 (t30d) days and then analyzed separately for stability . The following analytes were examined: [table 1] methods used for measuring biochemical parameters metabolites: na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubinproteins: total protein and albuminlipids: total cholesterol and triglyceridesenzymes: alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and amylase . Metabolites: na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubin proteins: total protein and albumin lipids: total cholesterol and triglycerides enzymes: alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and amylase . All measurements were performed at the hospital laboratory services using an olympus au 400 auto analyzer, except for serum electrolyte levels, which were obtained with a roche avl electrolyte analyzer . To determine time - dependent changes in cell - separated serum analytes, the mean value from all ten subject samples was calculated for each analyte at each time point . Clinically significant changes were determined using the significant change limit (scl) approach, defined as: scl = initial value 3.0 usual standard deviation (usd). This is based on the assumption that the usd is representative of the inherent day - to - day variability of the method . In our study, the calculated mean for each analyte at t1d represented the initial value . The usd was obtained by averaging the standard deviation of the quality control data of the previous 2 months for each analyte . The quality - control reference serum with the target mean most closely matching the t1d mean for each analyte was used to determine the usd . For simplicity, the scl was computed for each analyte by establishing the range (3.0 usd) from the subject mean at t1d . The analysis results for 17 biochemical parameters measured in serum samples under different storage conditions are shown in table 2 . Statistical analysis of selected analytes; serum samples were stored at -20c for 7 (t7d), 15 (t15d), or 30 (t30d) days and then analyzed at room temperature no significant statistical or clinical differences were found among most of the metabolites (na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubin) under the different storage conditions . Albumin also demonstrated statistically significant variation on t30d compared with fresh sera on t1d; however, this variation was not clinically significant . Aside from amylase, none of the enzymatic parameters exhibited clinically significant reduction inactivity . Sera stored for different periods at 20c showed a statistically significant decrease in alp; however, this trend was not clinically significant . The effect of storage at lower temperatures on serum amylase was statistically as well as clinically significant . The changes in serum cholesterol and serum triglyceride were statistically significant on t15d and t7d, respectively, compared to t1d; however, they were not clinically significant . The results of our study indicate that nearly all of the examined metabolites were stable even after 30 days of storage at 20c . In agreement with the findings of zhang et al ., no clinically significant differences were found for sodium levels following different storage durations compared to fresh samples; however, our serum na findings are not consistent with the findings of studies investigating serum with prolonged contact with cells at room temperature . Serum potassium was found to be stable up to t30d when the serum samples were separated from cells and stored in aliquots, whereas previous studies have demonstrated an increase in k after 24 h due to serum - cell contact at room temperature . The increase in k after 24 h is most likely caused by malfunction of the na / k atpase pump, resulting in diffusion of k from the erythrocytes driven by the intracellular extracellular concentration gradient . Moreover, our results showed a clinically insignificant increase in na level at t15d and in urea, calcium, total bilirubin, and direct bilirubin at t30d . However, no significant statistical or clinical differences were observed between the levels of the metabolites (na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubin) in fresh samples and in samples stored at 20c for 7, 15, and 30 days . These results are in agreement with those of a previous study reporting that serum calcium, total bilirubin, and direct bilirubin showed clinically equivalent levels, but that serum urea levels exhibited an appreciable increase in bun values over time; however, bun instability, indicated by a substantial decrease (15.6% on average) in levels, has been reported for samples stored at 20c . Consistent with a previous study, we did not detect any statistically or clinically significant change in creatinine levels . However, according to boyanton and blick, the increase in serum creatinine levels after 24 h is due to serum - cell contact at room temperature . In contrast to previous studies demonstrating that serum uric acid concentrations were unstable after 48 h of storage at 4c, our results showed a serial decrease in uric acid concentration over time; however, these changes were neither statistically nor clinically significant . Furthermore, our results demonstrating a slight increase in phosphorus concentration (not statistically or clinically significant) disagree with previous studies showing that serum phosphorus concentrations increased after 24 h. no differences in serum protein (total protein and albumin) were detected at any of the three time points compared to fresh samples . Moreover, our results are similar to a previous study reporting clinically equivalent levels of total protein and albumin . Total cholesterol and triglyceride concentrations were stable up to t30d, in agreement with the findings of cuhadar et al . And paltiel et al . A decrease in the concentration of serum alt, ast, and alp was observed; however, these changes were not statistically or clinically significant . In addition, according to paltiel et al ., ast activity remains stable for 1015 freeze - thaw cycles following storage at 80c . Significant statistical or clinical differences were observed between serum amylase levels in fresh samples and samples stored at 20c for 7, 15, and 30 days; serum amylase levels decreased with prolonged storage . To the best of our knowledge, this is the first report regarding the effects of storage at low temperatures on serum amylase levels . The results of our study indicate that nearly all of the examined metabolites were stable even after 30 days of storage at 20c . In agreement with the findings of zhang et al ., no clinically significant differences were found for sodium levels following different storage durations compared to fresh samples; however, our serum na findings are not consistent with the findings of studies investigating serum with prolonged contact with cells at room temperature . Serum potassium was found to be stable up to t30d when the serum samples were separated from cells and stored in aliquots, whereas previous studies have demonstrated an increase in k after 24 h due to serum - cell contact at room temperature . The increase in k after 24 h is most likely caused by malfunction of the na / k atpase pump, resulting in diffusion of k from the erythrocytes driven by the intracellular extracellular concentration gradient . Moreover, our results showed a clinically insignificant increase in na level at t15d and in urea, calcium, total bilirubin, and direct bilirubin at t30d . However, no significant statistical or clinical differences were observed between the levels of the metabolites (na, k, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, and total bilirubin) in fresh samples and in samples stored at 20c for 7, 15, and 30 days . These results are in agreement with those of a previous study reporting that serum calcium, total bilirubin, and direct bilirubin showed clinically equivalent levels, but that serum urea levels exhibited an appreciable increase in bun values over time; however, bun instability, indicated by a substantial decrease (15.6% on average) in levels, has been reported for samples stored at 20c . Consistent with a previous study, we did not detect any statistically or clinically significant change in creatinine levels . However, according to boyanton and blick, the increase in serum creatinine levels after 24 h is due to serum - cell contact at room temperature . In contrast to previous studies demonstrating that serum uric acid concentrations were unstable after 48 h of storage at 4c, our results showed a serial decrease in uric acid concentration over time; however, these changes were neither statistically nor clinically significant . Furthermore, our results demonstrating a slight increase in phosphorus concentration (not statistically or clinically significant) disagree with previous studies showing that serum phosphorus concentrations increased after 24 h. no differences in serum protein (total protein and albumin) were detected at any of the three time points compared to fresh samples . Interestingly, zhang et al . Noted similar observations for albumin and total protein in serum specimens . Moreover, our results are similar to a previous study reporting clinically equivalent levels of total protein and albumin . Total cholesterol and triglyceride concentrations were stable up to t30d, in agreement with the findings of cuhadar et al . And paltiel et al . A decrease in the concentration of serum alt, ast, and alp was observed; however, these changes were not statistically or clinically significant . In addition, according to paltiel et al ., ast activity remains stable for 1015 freeze - thaw cycles following storage at 80c . Significant statistical or clinical differences were observed between serum amylase levels in fresh samples and samples stored at 20c for 7, 15, and 30 days; serum amylase levels decreased with prolonged storage . To the best of our knowledge, this is the first report regarding the effects of storage at low temperatures on serum amylase levels . All common clinical chemistry analytes examined, aside from serum amylase, showed adequate stability following up to 30 days storage at 20c . These results indicate that deep freezing at 20c could serve a useful tool for additional analyses at later time points as well as for research purposes, which require that samples be stored for longer periods until batch analysis can be conducted.
The tree of life (tol) is a widely used metaphor to describe the history of life on earth . While darwin argued that the' coral of life' may be a more apt description (since only the surface remains alive, supported by the dead generations beneath it), relationships between organisms based on shared characters are best organized using the schematic representation of a tree . Use of molecular markers, in particular small - subunit ribosomal rna, have allowed this metaphor to be extended to microorganisms; however, this has also presented unique challenges for notions of phylogeny and evolution . One of the most significant challenges is the impact of horizontal gene transfer, which causes genes that coexist in a genome to have different molecular phylogenies . Despite these challenges, the increasing ease with which genomes can be sequenced has reinvigorated attempts to use genomic information to reconstruct the tol . Therefore, a vertical line of descent exists, and could theoretically be reconstructed as a purely bifurcating tree (that is, an organismal or cytoplasmic tree). However, while evolution presupposes and requires descent via reproduction, the two are not analogous . Evolution is, by definition, the change in the genetic material within a population of organisms across generations; therefore, any process by which genetic material within a population changes that is unrelated to the reproduction of individuals will show a history that is unrelated to the organismal vertical line of descent . The sum effect of these other genetic processes may completely obfuscate vertical descent, leaving only some measure of' relatedness' based on overall genetic similarity . Two common approaches in constructing a genome - based tol are supermatrix analyses, in which sequence alignments for individual gene families are concatenated into a single dataset that is then used to construct a tree, and supertree analyses, in which a consensus phylogeny is constructed from multiple gene trees . In some cases, datasets are generated by finding orthologous genes in all organisms and removing all genes whose conflicting phylogenetic topologies seem to indicate horizontal gene transfer, and then using the remaining genes to reconstruct the presumed vertical lines of descent of the genomes (see, for example, [4 - 6]). This approach has an obvious shortcoming in that gene transfer and the resulting phylogenetic conflicts can only be inferred if each individual gene has retained sufficient phylogenetic information to enable its origin to be correctly assigned . Furthermore, the absence of evidence for gene transfer does not constitute evidence for the absence of gene transfer . Thus, combining genes with different histories into a single data set will almost certainly result in a phylogeny that represents neither the history of any individual gene, nor the history of the organism as a whole . Another problem with supermatrix and supertree analyses is that they often give equal weight to genes that have different histories of horizontal gene transfer . This results in an average or median phylogeny that may not represent organismal history; if there are' highways' of gene sharing that is, large numbers of genes have, for some reason, been shared between specific groups of otherwise phylogenetically distinct organisms this can easily be mistaken for a consistent signal supporting an organismal tree . For example, because of such highways of gene sharing these types of analyses group members of the order thermotogales with the firmicutes, and the members of the aquificales with the -proteobacteria . In contrast, 16s rrna gene phylogenies and concatenated ribosomal protein phylogenies strongly support these two orders as deeply branching bacterial lineages (figure 1). (a) extensive horizontal gene transfer at all phylogenetic levels combine to produce a' web of life' that often obscures the lines of descent between groups (modified from). Copyright (2008) national academy of sciences, usa . (b) major microbial groups as defined by 16s ribosomal rna phylogeny . Bands represent some avenues of extensive gene sharing involving thermotogales, aquificales, and firmicutes . (c) impact on relationships between thermotogales and aquificales of genome content changes due to extensive horizontal gene transfer . Grey clouds represent groups of shared genes between clades that are non - monophyletic in the 16s tree . The phylogeny based on these' gene content' clouds is quite distinct from that of 16s or other ribosome - based trees . If stringent criteria are applied to remove or down - weigh transferred genes from supertree or supermatrix analyses, the resulting trees at best represent the history of only a minor fraction of the genome, largely consisting of ribosomal proteins, effectively a' tree of one percent' . Even if this remaining' genome core' retains a strong signal of vertical descent, this does not capture the true evolutionary history of genomes; that is, a web where different strands depict the history of different genes . A ribosomal tree of life has other shortcomings, in that within taxonomic orders many recombination and lineage sorting events may occur, and ribosomal genes are so highly conserved that such events at the tips of the tree may not be detectable . However, it can still provide a useful backbone for a reticulated genomic or organismal phylogeny, especially with respect to sets of genes that clearly have undergone horizontal transfer between more distantly related groups . While ribosomal protein and rna encoding genes have been transferred in the past (see discussion in), these genes are resistant to transfer, with most transfers occurring between close relatives . These properties make a phylogenetic reconstruction using ribosomal rna and proteins an ideal scaffold upon which to map horizontal gene transfers, clearly depicting their distinct contribution to genomic (and organismal) evolution . Several attempts have been made to capture this web - like genome history (see, for example, using ribosomal rrna as a backbone (figure 1). Conceptually, this method is distinct from any' tree of one percent' or genome averaging approach in that rather than being discarded, genes undergoing horizontal transfer are included in the final reconstruction without obscuring the vertical signal, even if that vertical signal is preserved only in a minority of genes . In this issue, puigbo, wolf and koonin present an approach for salvaging the tol that is a variant on other supertree methods, in which nearly 7,000 phylogenetic trees of prokaryotic genes (a' forest of life') are compared in order to determine a central tendency in their topologies . The trees are built from clusters of orthologous groups of proteins (cogs), and the central tendency is deduced from a set of nearly universal trees (nuts), defined by puigbo et al . As those trees generated from a set of cogs that are represented in> 90% of the analyzed prokaryote taxa . What distinguishes their approach from earlier supertree analyses apart from the very large number of genes included in the comparison is that it does not depend on a concatenation of highly conserved proteins or rrnas, or on a supertree generated by' pruning' down to those genes giving a consistent topology, to determine a central tendency . Instead, calculate an' inconsistency score' that is a measure of how representative a particular topology of each tree is to the rest of the trees in the forest of life . In reconstructing the central tendency in such a broad distribution of gene phylogenies, the work by puigbo et al . Also shows the difficulty in resolving deep branches, which often simply collapse into radiations without any topological structure . In confronting this problem, they show that the relationship between phylogenetic depth and resolution supports a tree - like structure for these deep branches . This result is significant in that it suggests that there is no need to postulate exotic' big bang' radiations early in evolution; rather, deep phylogenies can still be represented as bifurcating evolutionary events, albeit with extremely short branches that can prove difficult (or sometimes impossible) to resolve . Integrating the vertical descent of organisms and their genomes with the myriad phylogenetic patterns produced by horizontal gene transfer is essential for a truly comprehensive understanding of evolution . A new method that acknowledges and promotes this integration, even if falling short of fully encompassing the intricate details of a complex genome - based biological reality, represents progress towards this goal, and it now appears that a vertical signal can be discerned, if not clearly resolved . Work in the authors' lab is supported through the nsf assembling the tree of life (deb 0830024) and nasa exobiology (nag5 - 12367 and nnx07ak15 g) programs.
Glycaemic variability is considered a risk factor for diabetic complications, over and above raw glycaemic levels (as measured through fasting blood glucose or glycosylated haemoglobin) [13]. However, there is still controversy about which metric should be used to assess these dynamic aspects . Conventional statistics (standard deviation, coefficient of variability) have the pitfall of considering every measure as independent, thus overlooking an essential part of the time series: its sequentiality . Mean amplitude of glycaemic excursions (mage) takes sequentiality into account but fixes an arbitrary threshold of significant excursions, thus overlooking the fine - grain regulation . Complexity analysis of glucose time series, measured by means of detrended fluctuation analysis (dfa), has emerged as a useful alternative and is increasingly being used as a standard to measure glucose dynamics, especially in diabetic patients [515]. In all of these papers, there is a consistent correlation between loss of complexity (i.e., increased dfa) and glucoregulatory dysfunction . Ogata et al . Have also described a crossover point in dfa, located approximately in the 2-hour time window . Furthermore, they observed a decrease in long - range negative correlations (i.e., decreased complexity in large time windows) in patients with diabetes . Although dfa has mainly been used in patients with diabetes, several papers suggest that there is a progressive fall in complexity (i.e., increase in dfa) as a patient walks his way from health, through the prediabetes states to full - blown type 2 diabetes mellitus (t2 dm) [6, 912, 1517]. The present study intends to analyse the characteristics of the dfa crossover point in a population with high risk of becoming diabetic and to find out if these characteristics may have any influence on the risk of developing t2 dm . A sample of 262 patients from the internal medicine outpatient clinic and the vascular risk unit of the university hospital of mostoles were selected based on an assumed increased risk of developing t2 dm . The inclusion criteria were an hba1c> 5% and <6.5% and any of the following: essential hypertension;bmi 30 kg / m;a first - degree relative with a diagnosis of t2dm.patients were excluded if they had a diagnosis of dm or were on drugs that could interfere with glucose regulation (e.g., glucocorticoids). After an interview, physical exam, and routine biochemical tests, a 3-day glucometry was performed by means of a continuous glucose monitoring system device (ipro, medtronic minimed, northridge, ca, usa). The glucometry was obtained in an ambulatory setting, while the patient followed his normal life, with no special dietary restrictions . The patient was thereafter followed up every 6 months with a clinical visit and routine biochemical tests . The main outcome was a diagnosis of t2 dm (basal glycaemia 7.0 mmol / l, glycosylated haemoglobin (hba1c) 6.5%, or starting on antidiabetic drugs). From the glucometry obtained at admission, a clean, 24-hour - long time series was selected for each patient . Whenever possible, the selected 24-hour sequence started at 08.00 am the day after the device insertion, to avoid the stressful hours in the hospital . If there were missing values, these were obtained by interpolation as long as the missing string was <3 consecutive values . If there were three or more consecutive missing values, another 24-hour period was selected . If no adequate 24-hour period was found, the time series was considered unsuitable and discarded . Each selected series was thus composed of 288 consecutive measures of interstitial glucose, sampled every 5. each time series was submitted to detrended fluctuation analysis, without previous integration . A full description of dfa may be consulted in . A brief description can be found in, and a basic introductory video is available at http://www.complexity - at - the - bedside.org / complexity / tutorials/. In essence, dfa estimates the degree of long - range correlations within a signal, analysing how the time series and its linear regression diverge as the time window considered increases (figure 1). Metaphorically, one could consider the linear regression of each time window as a map of a certain territory . As the time windows increase, the regression's fitness deteriorates, and thus the map - to - territory gap increases . The rate at which this gap increases reflects how the informational content of the time series is distributed . A high - complexity time series will have comparatively more information encoded in the small windows . Conversely, low - complexity time series will have more information encoded in the large time windows, and therefore the map - to - territory gap will be increasing at a steady pace well into larger time windows . Specifically, we submitted the time series (without pretreatment by integration) to detrending with a windowing sequence of 3, 4, 6, 8, 9, 12, 16, 18, 24, 32, 36, 48, 72, 96, 144, and 288 points (corresponding to time windows of 15, 20, 30, 40, 45, 60, 80, 90, 120, 160, 180, 240, 360, 480, 720, and 1440). For each time window, a map - to - territory gap was calculated:(1)fn=1nk=1nykynk2.a log(fn) ~ log(time window) was drawn for each glucometry, with 16 points (the aforementioned time windows). Next, a set of pairs of linear regressions was built for several combinations of points (i.e., points 14 for the first limb and 516 for the second, then 15 and 616, then 16 and 716, etc . A combined weighted r was obtained for each pair of regression lines, and the best - fit pair was selected as the best representation of the time series . The abscissa of the intersection of both limbs, expressed in minutes, was considered the crossover point, and the angle was from the difference between the slopes of the two limbs . The slope of the first and second limb was assumed to be the dfa for the short and long time windows, respectively . Comparison between admitted and excluded patients was performed by means of t - test (for quantitative variables) or chi - square test (for qualitative variables). The effect of the various variables was analysed by means of a multivariate cox proportional hazard survival analysis . Significance was set at two - tailed p <0.05, although p <0.10 were also displayed . Of the 262 patients initially included, 40 were finally excluded because we were not able to obtain a suitable glucometry . 15 patients had no follow - up visits, and one patient was excluded because she started on high - dose glucocorticoids due to a facial palsy . Except for a slightly lower diastolic blood pressure (73.9 mmhg versus 78.1 mmhg, p = 0.01), there were no major differences between admitted and excluded patients regarding anthropometric, physical exam or analytical parameters . The 206 patients finally included were followed up for a mean of 18 months (iqr 15) (table 1). There were 18 events (t2 dm new diagnoses), for an incidence of 58.2 cases/1000 patients - year . The median to the crossover point was 114 min (iqr 64.7 min), and the median angle between the first limb (small time windows, before the crossover point) and the second limb (large time windows) was 0.64 radians (iqr 0.17 rad). In a cox proportional hazard ratio model, the crossover point was a significant risk factor for the development of t2 dm (= 0.015, p <0.001). These results did not change significantly when adjusting for other relevant variables, whether anthropometric (gender, age, body mass index, and waist circumference), clinical (blood pressure and first - degree relatives with diabetes), or analytical (hba1c, insulin, mean glucose, glucose standard deviation, mage, homa, or global dfa). When adjusting for basal glycaemia, the effect of crossover did not attain significance, although it persisted as a trend (p = 0.08). The crossover angle had no significant influence on the development of t2 dm when considered alone (= 2.43, p = 0.15) but became significantly protective when the model considered also the crossover point (= 4.172, p = 0.005). Similarly, neither the dfa of the first nor the second limb (before and after the crossover) alone had significant influence on the hazard rate of developing t2 dm (dfa1: = 0.475, p = 0.735; dfa2: = 1.452, p = 0.279), but they both became significant when adjusting for the crossover point (dfa1: = 4.876, p = 0.018; dfa2: = 4.050, p <0.001). Dfa analyses how the correlation between successive points evolve as the time - window considered increases . Following the conventional homeostatic paradigm, therefore, it is reasonable to expect a deterioration in the fit of the regression line as the time windows increase . Furthermore, one would expect that as the physiological system becomes old or dysfunctional, its response will become sluggish, and the decorrelation will be slower . This loss of sharpness (i.e., loss of complexity) is displayed as an increase in dfa . Indeed, there is ample evidence relating diabetes mellitus with an increase in glucose time series' dfa [6, 7, 912, 16]. An important advantage of dfa with respect to other conventional dynamic metrics (i.e., coefficient of variability or mage) is that it considers the time series as a whole, not as a set of independent measurements (as with the coefficient of variability) nor does it make any assumptions on the significance of each glycaemic excursion (as with mage). Glucoregulation is a rather asymmetric system: while there are at least four main counterregulatory hormonal systems in charge of fighting hypoglycaemia (glucagon, alpha - sympathomimetics, glucocorticoids, and growth hormone), there is only one strictly antihyperglycaemic hormone, namely, insulin . This has obvious evolutionary justifications (short - term hypoglycaemia is far more dangerous than hyperglycaemia) but may cause significant differences in the counterregulatory dynamics . While the hyperglycaemic drift may be a swift, multisystem driven reaction, the antihyperglycaemic push is mainly a one - man job and may therefore have more abrupt characteristics . Arguably this may explain the dynamic change underlying the crossover point described by ogata et al . . If this were the case, it would be reasonable to expect a progressive delay (and fading) of this dynamic change as the beta - function deteriorates, long before its failure allows for the diagnosis of diabetes . Our findings of a delay in the crossover point and a blunting of the angle between both limbs as prognostic factors for the development of t2 dm in patients at increased risk are congruent with this hypothesis . Arguably, this may represent both a delay and a dampening of the insulin kick - in and may reveal an early dysfunction of glucoregulation . However, our model has significant advantages over other experimental evaluations of beta - function: it may be applied in real - life situations rather than in the laboratory, it is much simpler, and it displays the functioning of the glucoregulatory system as a whole, not as the specific response to a certain glycaemic load or insulin infusion . We may be starting to have drugs available that can delay or prevent the evolution to t2 dm in subjects at risk [1923]. It will be crucial to identify those patients who would eventually walk all their way to diabetes in order to better target therapeutic interventions . Classic variables (basal glycaemia, oral glucose tolerance test, and hba1c) are probably insufficient, and it is not through fine - tuning thresholds that this problem will be solved . Arguably, glucodynamic techniques studying how glucose levels fluctuate in time may afford a fresh, new insight into this problem . It should be mentioned that, contrary to most studies with dfa in glycaemia, we have not preprocessed the time series through integration before performing the sliding - windows fluctuation analysis . This arguably takes us out of the conventional random - walk model and the standard 1.5 threshold of brown noise (integrated random series) dfa cannot be applied . Our model is therefore only a tool to compare different time series (within similar series length and time windows). However, integrating the time series erases important information (e.g., figure 3 displays the same time series, with and without pretreatment through integration), and we believe that preserving this information is worth the loss of standardization caused by omitting the conversion to a random - walk model . Dfa measures the complexity of a time series by evaluating how the map - territory gap enlarges (i.e., how the linear regression and the curve diverge) as the time window increases and thus provides a useful measure of the series' entropy even omitting the random - walk model . We have run the same analysis before treating the time series through integration, and although the same tendencies persist, the crossover effects are much less obvious and often do not reach statistical significance . Oral glucose tolerance tests were not performed, and thus neither impaired glucose tolerance nor insulin response to ogtt could be analysed . The notion that a delay in crossover represents a dampening of beta - function is only a hypothesis and needs confirmation through conventional experimental tests . The characteristics of the crossover phenomenon have predictive value for the development of t2 dm in patients at risk and may provide a sensitive and easy way to explore the earliest signs of glucoregulatory failure.
Thrombotic thrombocytopenic purpura (ttp) is a rare disorder with an incidence of 45 cases per million per year . Timely diagnosis and availability of effective treatment such as plasma exchange therapy have reduced the morbidity and mortality significantly . Usually, it is idiopathic or autoimmune, but several predisposing factors have been described to cause secondary ttp . In this case report, we discussed a very rare presentation of ttp secondary to dengue virus infection and how the timely diagnosis along with the immediate institution of appropriate management helped us to cure the patient . A 35-year - old female with no other comorbidities was admitted to the hospital with complaints of fever and vomiting over 1 week . Her investigations revealed a low platelet count and dengue igg, and igm antibodies were positive . She was symptomatically treated at a local hospital and was transfused 16 units of random donor platelets over a period of 6 days . But despite this, platelet count was remaining low . The patient was drowsy, arousable to verbal commands only, and had decreased limb movement on the right side . Laboratory investigations were showing anemia, thrombocytopenia, and increased lactate dehydrogenase (ldh) level as shown in table 1 . Her peripheral blood smear revealed numerous schistocytes and computed tomography (ct) brain was unremarkable . Ultrasonography abdomen was suggestive of bulky kidneys, and she was retrovirus negative . With this clinical picture and laboratory findings, she was diagnosed as a case of ttp and was advised urgent plasma exchange therapy . Although there is no firm recommendation for administration of steroids in ttp, we started her on low - dose injection methylprednisolone (100 mg daily) considering its autoimmune etiology . On the 2 day of the admission, she had an episode of convulsion which was treated with anticonvulsive medication . She required intubation and ventilatory support because of postconvulsion low glasgow coma scale (gcs). She convulsed again on 3 day with drop in hemoglobin and platelets [table 1], so injection rituximab 500 mg was added to treatment in addition to plasma exchange . Platelet count improved, serum ldh level showed decline over a period [table 1]. She underwent total eight plasmapheresis cycles, and before discharge, her platelet counts were 152 mm, serum ldh was 511 we followed her up in the outpatient department, and at 6 months, she is well with normal hemogram . This patient had dengue viral infection confirmed by serology which was complicated by ttp during the acute phase of dengue viral infection . We diagnosed this by clinical features and ruling out other differential diagnosis by laboratorial and radiological tests . The patient responded well to the plasma exchange therapy with complete recovery from symptoms and no recurrence after 6 months follow - up . Usually, it presents as mild / asymptomatic type of infection; however, at times, it may manifest in more severe forms such as dengue hemorrhagic fever, or dengue shock syndrome . Many unusual complications have been observed with dengue viral infection such as hepatic damage, cardiomyopathy, and encephalopathy . The presence of ttp with dengue viral infection is very rare presentation and has been reported in very few case reports . Ttp itself is a rare clinical syndrome characterized by fever, microangiopathic hemolytic anemia, thrombocytopenia, central nervous system involvement, and renal impairment . Secondary ttp can occur after infections, medications (e.g., clopidogrel, cyclosporine), autoimmune diseases, malignancies, pregnancy, and bone marrow transplantation . Infections known to cause thrombotic microangiopathy include hepatitis c, parvovirus b19 virus infection, hiv infection as well as patient with dental foci or streptococci infection . Pathophysiology of ttp includes deficiency of protease enzyme (adamts13) that cleaves von willebrand factor (vwf) in small segments . In the absence of adamts13 enzyme, large vwf cause platelet aggregation and fibrin deposition in small vessels and the patient was diagnosed as having dengue viral infection by the presence of igm and igg antibodies and treated accordingly with fluid resuscitation and platelet transfusion . But later, the patient showed features such as fever, thrombocytopenia, hemolytic anemia, and neurological involvement in the form of decreased consciousness, hemiparesis, and seizures (ct brain - normal). All these features were suggestive of ttp and patient responded well to plasma exchange therapy, steroid therapy, and injection rituximab . Although the exact incidence of ttp in dengue viral infection is not known, such cases are reported in the recent past . Hence, special attention must be given to dengue virus infection patients presenting with signs and symptoms of ttp in the future . The prompt clinical recognition of such complication and early initiation of specific therapy with plasma exchange is likely to improve the patient's outcome.
Olfaction plays a critical role in the daily life of vertebrates, such as prey detection, predator avoidance, mating, and territoriality (mombaerts 1999). Two distinct nasal olfactory systems exist in most terrestrial vertebrates: the main olfactory system (mos) and the vomeronasal system (vns) (dulac and torello 2003; grus and zhang 2006). Although partially overlapping in function, the mos appears to be mainly responsible for recognizing environmental odorants, whereas the vns primarily detects pheromones, which constitute a poorly defined class of chemicals that are emitted and sensed by individuals of the same species to elicit sexual / social behaviors and physiological changes (restrepo et al . The mos and the vns are anatomically and neurologically separated; they use different receptors and have distinct signal transduction pathways (dulac and torello 2003; grus and zhang 2006). The vns is of particular interest to evolutionists because of its high diversity in complexity among species (grus et al . 2005; young et al . 2005, 2010; shi and zhang 2007; grus and zhang 2008). Although the morphological components of the vns are believed to first emerge in the common ancestor of tetrapods, its genetic components have been inferred to exist in the common ancestor of all vertebrates (grus and zhang 2006, 2009). Among tetrapods, the vns varies from completely absent in birds, catarrhine primates (humans, apes, and old world monkeys), most bats, and many cetaceans to rudimentary in amphibians to highly complex in murids, opossums, and the platypus (zhang and webb 2003; grus et al . 2005, 2007; grus and zhang 2006; shi and zhang 2007, 2009; zhao et al . Vomeronasal sensitivity is mediated by two families of g - protein coupled receptors known as v1rs and v2rs (mombaerts 2004). In the genome of the laboratory mouse, there are about 190 putatively functional v1r and 70 v2r genes (shi et al . 2005; yang et al . 2005). Note that despite the availability of genome sequences, the gene numbers are only approximate due to among - strain variations and/or incomplete genomic sequencing (zhang et al . 2004). The v1r and v2r gene repertoires, especially the former, have been examined in many mammalian genomes (rodriguez and mombaerts 2002; rodriguez et al . 2002; grus and zhang 2004, 2008; grus et al . 2005, 2007; young et al . It was reported that the among - species size variation in v1r and v2r gene repertoires is among the highest of all mammalian gene families (grus et al . This variation is not random, at least in the case of v1rs, because a clear positive correlation exists between the morphological complexity of the vns and the number of putatively functional v1r genes (grus et al . An evolutionary hallmark of v1rs and v2rs is the exceptionally rapid gene turnover that results in lineage - specific receptors . For example, between 187 mouse and 106 rat v1rs examined, only 18 are one - to - one orthologous (grus and zhang 2004, 2008), in sharp contrast to the genome - wide estimate that 8694% of rat genes have one - to - one mouse orthologs (gibbs et al . 2004). Despite the striking macroevolutionary diversity of v1rs and v2rs, the evolutionary forces acting on these genes are unclear due to the lack of knowledge about the population genetic dynamics of v1r and v2r genes specifically, it would be interesting to test whether v1rs and v2rs evolve by divergent selective pressures in sibling species because pheromones are by definition species specific (brennan and keverne 2004). In this work, we study the microevolution of vomeronasal receptor genes in two closely related mouse species, mus musculus (abbreviated as mm) and mus domesticus (md). Mm is distributed from eastern europe to japan, across russia and northern china, whereas md is common in western europe, africa, and the near - east and was transported by humans to the americas and australia (guenet and bonhomme 2003). The two species form a narrow zone of hybridization through central europe that extends from the jutland peninsula to the bulgarian coast of the black sea (sage et al . 1993; tucker 2007). Mice from the center of the hybrid zone have higher parasite loads than those from the edges of the hybrid zone (sage et al . 1986; moulia et al . 1991, 1993), indicative of reduced fitness due to hybrid inviability . There is clear evidence of hybrid male sterility between the two species (forejt and ivanyi 1974; forejt 1996; alibert et al . 1997; storchova et al . 2004; britton - davidian et al . 2005; trachtulec et al . 2005; vyskocilova et al . 2005). There is also evidence for limited female sterility in some crosses but not others (forejt and ivanyi 1974; britton - davidian et al . Evidence for partial premating isolation is also ample (laukaitis et al . 1997; smadja and ganem 2002; smadja et al . Although some authors regard mm and md as two subspecies of the species m. musculus (tucker 2007), for simplicity, we treat them as two species that are in an early stage of divergence with a low degree of gene flow . First, the laboratory mouse, a mosaic of mm, md, and mus castaneus (frazer et al . 2007; yang et al . 2007), is a model organism for studying vomeronasal sensitivity . A substantial amount of genetic, neurological, and behavioral data related to vomeronasal sensitivity is available for the laboratory mouse, allowing a more accurate interpretation of the evolutionary and population genetic data that we collect . Second, the genome sequence of the laboratory mouse is known, making the experimental design much easier . Third, mice represent those vertebrates with a relatively high level of vomeronasal sensitivity (takami 2002; grus et al . 2005). Thus, their vomeronasal sensitivities may be more important in determining organismal fitness and under stronger natural selection . Avoiding the hybrid zone, we trapped seven wild mm and seven wild md in czech republic and france, respectively . Our present study focuses on v1rs because they have only one coding exon, making dna amplification and sequencing much easier . Here, we report the microevolution of 44 v1rs and 25 presumably neutral noncoding regions in these 14 mice . Seven m. musculus and seven m. domesticus individuals were collected from czech republic and france, respectively . Although the mice from each species were sampled from restricted geographic areas (supplementary table 1, supplementary material online), it should not affect our results because mice have little geographic differentiation (salcedo et al . 2007). The identity of the mice was confirmed by sequencing a 683-nt segment of the mitochondrial cytochrome c oxidase subunit i (coxi) gene that is commonly used as a barcode for identifying animal species . The liver genomic dnas of the mice were extracted using the puregene genomic dna purification kit (gentra systems, minneapolis, mn), following the manufacturer's instruction . Gene - specific primers for amplifying 44 v1r genes were designed according to the mus musculus reference sequence from genbank (supplementary table 2, supplementary material online). The protein - coding region of each v1r gene studied has 8701,104 nt, which were completely amplified in our experiments . Polymerase chain reactions (pcrs) were performed with gotaq dna polymerase (promega corp, madison, wi) under conditions recommended by the manufacturer . Samples showing duplicated electropherograms due to insertions / deletions were cloned with topo pcr cloning kit (invitrogen, carlsbad, ca) and sequenced with universal t7 and m13 primers using the sanger method on an automatic dna sequencer . Otherwise, the pcr products were enzymatically processed using calf intestinal phosphatase and exonuclease i (exo i) (new england biolabs, ipswich, ma) before being sequenced bidirectionally with the gene - specific primers . Sequencher (genecodes, ann arbor, mi) and mega4 (tamura et al . Twenty - five presumably neutral noncoding regions (supplementary table 3, supplementary material online), most with 1,000 nt, were also amplified and directly sequenced in the same 14 mice . Watterson's, nucleotide diversity, tajima's d, and fu and li's d * were computed using dnasp (librado and rozas 2009). Tajima's test (tajima 1989) and fu and li's test (fu and li 1993) were conducted by 10,000 coalescent simulations in dnasp . The sequences reported in this article have been submitted to genbank (accession numbers jf782602jf783819, jf782044jf782601, and jf783820jf783959). 2005), there are at least 188 putatively functional v1r genes in the mouse genome (fig . We carefully selected 44 of them for an in - depth study in 14 mice . These 44 genes were chosen to represent major lineages of mouse v1rs, to include genes with (14) and without (30) rat one - to - one orthologs, and to allow gene - specific amplification and sequencing (fig . 1). For comparison, we also sequenced 25 presumably neutral noncoding regions in these 14 mice . Five of these 25 noncoding regions were from a previous study (baines and harr 2007), and the sequenced segments are either in introns or in intergenic regions that are> 5 kb upstream of coding regions . The remaining 20 noncoding regions were randomly picked from the genome, with the criteria that the regions are at least 200 kb away from any known gene . The average length of the noncoding regions sequenced (936 nt) is similar to the average length of the v1rs sequenced (934 nt). Branches denoted with * have putatively functional rat v1r orthologs (grus and zhang 2008). The tree was reconstructed using the neighbor - joining method (saitou and nei 1987) with poisson - corrected protein distances . The basic population genetic parameters of individual v1rs and noncoding regions are presented in table 1 and table 2, respectively . 2007), we found nucleotide diversity per site () at the 25 noncoding regions to be higher in md (0.0021) than in mm (0.0013), although the difference is not statistically significant (p = 0.15, two - tailed paired t - test; table 2). However, the opposite is found for v1rs, although the difference is again not significant (p = 0.09, two - tailed paired t - test; table 1). Compared with the noncoding regions, v1rs show an overall higher in mm (p = 0.24, two - tailed mann whitney test) but a lower in md (p = 0.014, two - tailed mann species is there a significant difference in between v1rs with one - to - one rat orthologs and those without such orthologs (p> 0.2, two - tailed mann intra- and interspecific sequence variations of 44 mouse v1r genes intra- and interspecific sequence variations of 25 noncoding regions in mice note.most noncoding regions do not have standard names, and the names listed are the idendification numbers used in our laboratory . Nucleotide positions in the mouse genome sequence of national center for biotechnology information build 37 . Length in m. musculus . We applied tajima's test of neutrality (tajima 1989) to each of the v1rs (table 1) and noncoding regions (table 2). Note that the null hypothesis in tajima's test is the wright fisher model of strict neutrality . Thus, rejection of the null hypothesis may indicate one or more of the following: purifying selection, positive selection, and demographic changes . For both v1rs and noncoding regions, several loci show significantly negative or positive tajima's d. for example, in mm, five v1rs and two noncoding regions show significantly positive d (nominal p <0.05), whereas five v1rs and one noncoding region show significantly negative d. in md, zero v1r and two noncoding regions show significantly positive d, whereas two v1rs and one noncoding region show significantly negative d. in neither species is there a significant difference between v1rs and noncoding regions in the fraction of loci with significantly positive or negative d (p> 0.1 in all cases, test). We also compared the frequency distribution of tajima's d between v1rs and noncoding regions (fig . 2a and 2b) but found no significant differences (p = 0.44 for mm and 0.12 for md, kolmogorov we found similar results from comparing the distribution of fu and li's d * between v1rs and noncoding regions (p = 0.74 for mm and 0.30 for md, kolmogorov frequency distributions of tajima's d and fu and li's d * among 44 v1rs and 25 noncoding regions in mouse . (a) tajima's d in m. musculus; (b) tajima's d in m. domesticus; (c) fu and li's d * in m. musculus; and (d) fu and li's d * in m. domesticus . The mean number of nucleotide difference per site between mm and md is 0.00442 for the v1rs the mean nonsynonymous nucleotide difference per nonsynonymous site (dn) of the 44 v1rs divided by the mean synonymous difference per synonymous site (ds) of the same set of genes is 0.315 . Seven v1rs have dn / ds> 1, but none of them significantly exceeds 1 by fisher's exact test (zhang et al . The fraction of v1rs with a dn / ds ratio below 1 is significantly greater than 50% (p = 3 10, binomial test). These results indicate that the evolutionary divergence of v1rs is overall governed by purifying selection . Combining the polymorphism and divergence data, we conducted several mcdonald kreitman tests (mcdonald and kreitman 1991) by varying the consideration of the polymorphic data from one or both species (table 3). In all cases, nonsynonymous / synonymous ratio is lower for divergence than for polymorphism, although the differences are not statistically significant (table 3). These findings suggest that the evolution of mouse v1rs is largely neutral, with the presence of only weak purifying selection that hampers the fixation of some nonsynonymous changes . For instance, the nonsynonymous / synonymous ratio is 102/56 = 1.82 for intraspecific polymorphisms in md but 32/31 = 1.03 for interspecific divergences (p = 0.068, fisher's exact test). Consistent with the above interpretation, we found the nonsynonymous / synonymous ratios for polymorphisms and divergences to be more similar to each other when only derived alleles with frequencies equal to or greater than 2/14 are considered for polymorphisms . For instance, the nonsynonymous / synonymous ratio now becomes 62/44 = 1.41 for polymorphisms in md, closer to the ratio of 1.03 for interspecific divergences (p = 0.76, fisher's exact test). Numbers of synonymous and nonsynonymous sequence variations in v1rs note.variations in the 25 noncoding regions . Because of the relatively small numbers of synonymous polymorphisms and substitutions in our v1r data, we augmented this dataset with the 25 noncoding regions to enhance the statistical power of the mcdonald kreitman test . That is, we lumped synonymous and noncoding changes and compared them with nonsynonymous changes (table 3). The results are consistent with those from the comparison between synonymous and nonsynonymous changes, but the difference between polymorphism and divergence becomes statistically more significant (table 3). For example, the nonsynonymous/(noncoding+synonymous) ratio is 0.510 for intraspecific polymorphisms in md, significantly higher than that (0.227) for interspecific divergences (p <0.001, fisher's exact test). Together, the various mcdonald kreitman tests demonstrate the overall action of purifying selection hampering the spread and fixation of nonsynonymous changes in v1rs . Kreitman tests for individual v1rs because of the low numbers of synonymous and nonsynonymous changes in each v1r and the consequent low statistical power . We examined d/ for each v1r in each species, where d is the average nucleotide difference per site between an mm allele and an md allele and is watterson's estimate of polymorphism per site in a species (table 4). Because some sequences have no polymorphic sites, we used the actual number of polymorphic site plus 1 in calculating for each v1r gene or noncoding region . In mm, the mean d divided by mean is 2.59 for v1rs, whereas the corresponding ratio is 7.30 for the noncoding regions . In md, thus, overall, v1rs have lower divergence - to - polymorphism ratios than noncoding regions, indicative of purifying selection on v1rs . When each gene is examined separately by the hka test (hudson et al . 1987), however, three genes (a9, b8, and c5) show significantly greater d/ than the 25 noncoding regions in both mm and md and three additional genes (f3, f5, and j3) show significantly greater d/ than noncoding regions only in md (table 4). Because 44 tests were conducted in each species, some of the significant cases (on average 2.2 cases per species) may be artifacts of multiple testing . After examining the fixed differences between mm and md, we believe that f3, f5, and j3 are probably false positives because they lack any fixed differences, whereas a9, b8, and c5 are likely to be true positives because they each contain at least five fixed nonsynonymous differences between the two species, and the statistical significance of the hka test is high (p <0.0025 for each gene in each species). Even after the conservative bonferroni correction thus, it is likely that a small fraction of v1rs has been subject to positive selection in the divergence of mm and md . Comparison between v1rs and the 25 noncoding regions by the hka test note.interspecific divergence per site divided by watterson's polymorphism per site . The actual number of polymorphc site plus 1 was used in calculating watterson's polymorphism per site . Values of d/ that are significantly greater than expected from the 25 noncoding regions are underlined . We observed a large number of v1r genes that have segregating null alleles in either one or both mouse species based on the occurrences of single nucleotide polymorphisms and/or insertions / deletions (indels) that introduce premature stop codons . For two v1r genes (c28 and f3), amplification was unsuccessful in some but not all mouse individuals even after extensive experimentation with multiple primer sets including those within coding regions, suggesting that the two genes may have been partially or entirely deleted in these individuals . We thus regard these cases as null alleles as well . In total, 14 v1rs harbor null alleles in mm and 7 in md (table 5). Given these numbers, we should expect 14 7/44 = 2.23 v1rs to have segregating null alleles in both species if v1r pseudogenization in the two species is independent . Consistent with this expectation, two v1rs harbor segregating null alleles in both species, and the pseudogenization events were independent because the null alleles in the two species were generated by different open reading frame (orf)disrupting mutations (table 5). In addition to the prevalence of pseudogenized v1rs, the frequencies of the null alleles are not particularly low (table 5), especially in mm, suggesting the lack of strong selection preventing the null alleles from spreading through the populations . This finding is consistent with an overall low purifying selection acting on v1rs and provides a microevolutionary explanation for the rapid gene turnover observed at the macroevolutionary time scale . Mouse v1rs with segregating null alleles note.numbers in the table are the numbers of null alleles (of 14 per species) for v1rs that harbor null alleles in the species . 2005), there are at least 188 putatively functional v1r genes in the mouse genome (fig . We carefully selected 44 of them for an in - depth study in 14 mice . These 44 genes were chosen to represent major lineages of mouse v1rs, to include genes with (14) and without (30) rat one - to - one orthologs, and to allow gene - specific amplification and sequencing (fig . 1). For comparison, we also sequenced 25 presumably neutral noncoding regions in these 14 mice . Five of these 25 noncoding regions were from a previous study (baines and harr 2007), and the sequenced segments are either in introns or in intergenic regions that are> 5 kb upstream of coding regions . The remaining 20 noncoding regions were randomly picked from the genome, with the criteria that the regions are at least 200 kb away from any known gene . The average length of the noncoding regions sequenced (936 nt) is similar to the average length of the v1rs sequenced (934 nt). Branches denoted with * have putatively functional rat v1r orthologs (grus and zhang 2008). The tree was reconstructed using the neighbor - joining method (saitou and nei 1987) with poisson - corrected protein distances . The basic population genetic parameters of individual v1rs and noncoding regions are presented in table 1 and table 2, respectively . 2007), we found nucleotide diversity per site () at the 25 noncoding regions to be higher in md (0.0021) than in mm (0.0013), although the difference is not statistically significant (p = 0.15, two - tailed paired t - test; table 2). However, the opposite is found for v1rs, although the difference is again not significant (p = 0.09, two - tailed paired t - test; table 1). Compared with the noncoding regions, v1rs show an overall higher in mm (p = 0.24, two - tailed mann whitney test) but a lower in md (p = 0.014, two - tailed mann species is there a significant difference in between v1rs with one - to - one rat orthologs and those without such orthologs (p> 0.2, two - tailed mann intra- and interspecific sequence variations of 44 mouse v1r genes intra- and interspecific sequence variations of 25 noncoding regions in mice note.most noncoding regions do not have standard names, and the names listed are the idendification numbers used in our laboratory . Nucleotide positions in the mouse genome sequence of national center for biotechnology information build 37 . Length in m. musculus . We applied tajima's test of neutrality (tajima 1989) to each of the v1rs (table 1) and noncoding regions (table 2). Note that the null hypothesis in tajima's test is the wright fisher model of strict neutrality . Thus, rejection of the null hypothesis may indicate one or more of the following: purifying selection, positive selection, and demographic changes . For both v1rs and noncoding regions, several loci show significantly negative or positive tajima's d. for example, in mm, five v1rs and two noncoding regions show significantly positive d (nominal p <0.05), whereas five v1rs and one noncoding region show significantly negative d. in md, zero v1r and two noncoding regions show significantly positive d, whereas two v1rs and one noncoding region show significantly negative d. in neither species is there a significant difference between v1rs and noncoding regions in the fraction of loci with significantly positive or negative d (p> 0.1 in all cases, test). We also compared the frequency distribution of tajima's d between v1rs and noncoding regions (fig . 2a and 2b) but found no significant differences (p = 0.44 for mm and 0.12 for md, kolmogorov we found similar results from comparing the distribution of fu and li's d * between v1rs and noncoding regions (p = 0.74 for mm and 0.30 for md, kolmogorov frequency distributions of tajima's d and fu and li's d * among 44 v1rs and 25 noncoding regions in mouse . (a) tajima's d in m. musculus; (b) tajima's d in m. domesticus; (c) fu and li's d * in m. musculus; and (d) fu and li's d * in m. domesticus . The mean number of nucleotide difference per site between mm and md is 0.00442 for the v1rs (table 1) and 0.00804 for the noncoding regions (table 2). The mean nonsynonymous nucleotide difference per nonsynonymous site (dn) of the 44 v1rs divided by the mean synonymous difference per synonymous site (ds) of the same set of genes is 0.315 . Seven v1rs have dn / ds> 1, but none of them significantly exceeds 1 by fisher's exact test (zhang et al . The fraction of v1rs with a dn / ds ratio below 1 is significantly greater than 50% (p = 3 10, binomial test). These results indicate that the evolutionary divergence of v1rs is overall governed by purifying selection . Combining the polymorphism and divergence data, we conducted several mcdonald kreitman tests (mcdonald and kreitman 1991) by varying the consideration of the polymorphic data from one or both species (table 3). In all cases, nonsynonymous / synonymous ratio is lower for divergence than for polymorphism, although the differences are not statistically significant (table 3). These findings suggest that the evolution of mouse v1rs is largely neutral, with the presence of only weak purifying selection that hampers the fixation of some nonsynonymous changes . For instance, the nonsynonymous / synonymous ratio is 102/56 = 1.82 for intraspecific polymorphisms in md but 32/31 = 1.03 for interspecific divergences consistent with the above interpretation, we found the nonsynonymous / synonymous ratios for polymorphisms and divergences to be more similar to each other when only derived alleles with frequencies equal to or greater than 2/14 are considered for polymorphisms . For instance, the nonsynonymous / synonymous ratio now becomes 62/44 = 1.41 for polymorphisms in md, closer to the ratio of 1.03 for interspecific divergences (p = 0.76, fisher's exact test). Numbers of synonymous and nonsynonymous sequence variations in v1rs note.variations in the 25 noncoding regions . Because of the relatively small numbers of synonymous polymorphisms and substitutions in our v1r data, we augmented this dataset with the 25 noncoding regions to enhance the statistical power of the mcdonald kreitman test . That is, we lumped synonymous and noncoding changes and compared them with nonsynonymous changes (table 3). The results are consistent with those from the comparison between synonymous and nonsynonymous changes, but the difference between polymorphism and divergence becomes statistically more significant (table 3). For example, the nonsynonymous/(noncoding+synonymous) ratio is 0.510 for intraspecific polymorphisms in md, significantly higher than that (0.227) for interspecific divergences (p <0.001, fisher's exact test). Together, the various mcdonald kreitman tests demonstrate the overall action of purifying selection hampering the spread and fixation of nonsynonymous changes in v1rs . Kreitman tests for individual v1rs because of the low numbers of synonymous and nonsynonymous changes in each v1r and the consequent low statistical power . We examined d/ for each v1r in each species, where d is the average nucleotide difference per site between an mm allele and an md allele and is watterson's estimate of polymorphism per site in a species (table 4). Because some sequences have no polymorphic sites, we used the actual number of polymorphic site plus 1 in calculating for each v1r gene or noncoding region . In mm, the mean d divided by mean is 2.59 for v1rs, whereas the corresponding ratio is 7.30 for the noncoding regions . In md, thus, overall, v1rs have lower divergence - to - polymorphism ratios than noncoding regions, indicative of purifying selection on v1rs . When each gene is examined separately by the hka test (hudson et al . 1987), however, three genes (a9, b8, and c5) show significantly greater d/ than the 25 noncoding regions in both mm and md and three additional genes (f3, f5, and j3) show significantly greater d/ than noncoding regions only in md (table 4). Because 44 tests were conducted in each species, some of the significant cases (on average 2.2 cases per species) may be artifacts of multiple testing . After examining the fixed differences between mm and md, we believe that f3, f5, and j3 are probably false positives because they lack any fixed differences, whereas a9, b8, and c5 are likely to be true positives because they each contain at least five fixed nonsynonymous differences between the two species, and the statistical significance of the hka test is high (p <0.0025 for each gene in each species). Even after the conservative bonferroni correction thus, it is likely that a small fraction of v1rs has been subject to positive selection in the divergence of mm and md . Comparison between v1rs and the 25 noncoding regions by the hka test note.interspecific divergence per site divided by watterson's polymorphism per site . The actual number of polymorphc site plus 1 was used in calculating watterson's polymorphism per site . Values of d/ that are significantly greater than expected from the 25 noncoding regions are underlined . We observed a large number of v1r genes that have segregating null alleles in either one or both mouse species based on the occurrences of single nucleotide polymorphisms and/or insertions / deletions (indels) that introduce premature stop codons . For two v1r genes (c28 and f3), amplification was unsuccessful in some but not all mouse individuals even after extensive experimentation with multiple primer sets including those within coding regions, suggesting that the two genes may have been partially or entirely deleted in these individuals . We thus regard these cases as null alleles as well . In total, 14 v1rs harbor null alleles in mm and 7 in md (table 5). Given these numbers, we should expect 14 7/44 = 2.23 v1rs to have segregating null alleles in both species if v1r pseudogenization in the two species is independent . Consistent with this expectation, two v1rs harbor segregating null alleles in both species, and the pseudogenization events were independent because the null alleles in the two species were generated by different open reading frame (orf)disrupting mutations (table 5). In addition to the prevalence of pseudogenized v1rs, the frequencies of the null alleles are not particularly low (table 5), especially in mm, suggesting the lack of strong selection preventing the null alleles from spreading through the populations . This finding is consistent with an overall low purifying selection acting on v1rs and provides a microevolutionary explanation for the rapid gene turnover observed at the macroevolutionary time scale . Mouse v1rs with segregating null alleles note.numbers in the table are the numbers of null alleles (of 14 per species) for v1rs that harbor null alleles in the species . In this study, we characterized the intra- and interspecific sequence variations of 44 v1r genes and 25 noncoding regions in two closely related mus species . Both intraspecific polymorphisms and interspecific divergences are generally reduced in v1rs compared with the noncoding regions, suggesting that the overall force in v1r evolution is purifying selection . This is reflected by a ratio of approximately 0.32 between the mean nonsynonymous substitution rate and mean synonymous substitution rate of the 44 v1rs . A similar ratio (0.34) is obtained when only fixed differences between mm and md are considered . In comparison, this ratio is on average 0.11 when all 11,503 mouse rat orthologous genes have a dn / ds ratio greater than 0.28, but we found that 57% of v1rs belong to this category, the difference being highly significant (p <10, binomial test). The overall weak purifying selection is also reflected by the high fraction of v1r loci that have segregating null alleles in either one or both mus species examined . It is likely that a sizable proportion of v1r genes are not functionally constrained in each mouse species, consistent with the previous observation of virtually neutral variations of v1r gene copy number within and between species (nozawa et al . In other words, the seemingly rapid v1r gene turnover is at least in part caused by neutral genomic drift (nozawa et al . We did not find any v1r that has been duplicated in one of the two mus species since their separation, but this is attributable to our intentional avoidance of studying v1rs with closely related paralogs to ease gene - specific amplification . In fact, in our preliminary study, one gene (e13) appeared to be duplicated in some individuals, but it was removed from the subsequent study due to the difficulty in designing gene - specific primers . Our findings of weak purifying selection in the microevolution of v1rs is generally consistent with a recent interspecific comparison of a subset of v1rs between the laboratory mouse and m. spretus (kurzweil et al . 2009), which diverged from each other much earlier than the separation between mm and md . Kurzweil et al . Sequenced a genomic segment of m. spretus that harbors two subfamilies of v1rs, including 18 genes . They observed that 1 of 11 genes in subfamily a and 2 of 7 genes in subfamily b have been lost in m. spretus, whereas 2 genes in subfamily a are becoming pseudogenes in m. musculus . However, they did not analyze population genetic dynamics of v1rs because no intraspecific polymorphism data were collected . Our data thus complement theirs in providing necessary information for inferring the microevolutionary forces acting on v1rs . It should be noted that, across mammals, there is a strong positive correlation between the morphological (and presumably physiological) complexity of the vns in a species and the number of intact v1r genes the species has (grus et al . 2005, 2007). Furthermore, during the evolutionary transition of vertebrates from water to land, there was a 50-fold increase in the ratio of the number of v1rs, which likely bind to airborne ligands, to that of v2rs, which likely bind to water - soluble ligands (shi and zhang 2007). Thus, it is likely that the evolution of the v1r repertoire is also subject to positive selection . Indeed, using the hka test, we detected positive selection for nucleotide substitutions in 5% of the mouse v1rs surveyed after controlling for multiple testing . Extrapolating this result to all v1rs suggests that there are 10 v1rs that have adaptive differences between the two mus species compared . Although we have no knowledge about the number of pheromonal differences between the two species, it is not unlikely that they differ by no more than a dozen pheromones . Recently, karn et al . Suggested that a particular v1r gene, vmn1r67 (or e10 in our nomenclature), experienced adaptive divergences among mus species and that it might be responsible for detecting the androgen - binding protein, a species - specific cue for species recognition (karn et al . Although this gene is not included in our 44 v1rs, positive selection on this gene would not be inconsistent with our estimate of 5% of positively selected v1rs in the divergence between mm and md . In the future, it will be interesting to confirm their finding in the wild mice used here . An earlier paper compared two assemblies of mouse genome sequences, which were acquired from different mouse inbred lines, and reported an overall dn / ds ratio between the two assemblies to be 1.13 for v1rs (zhang et al . The authors interpreted this finding as evidence for positive selection acting on most v1rs without actually testing whether the dn / ds ratio is significantly greater than 1, which is required for establishing positive selection . In our study, we found the mean dn / ds ratio for polymorphisms to be 0.5 when all v1r polymorphisms in the 14 mice sequenced here are considered . It remains to be seen whether this disparity is due to the difference between the genes we sampled and the rest of v1rs, sequencing errors in draft genome sequences, or the differences between the wild mice and inbred laboratory mice . Furthermore, it will be important to reexamine the polymorphism and divergence of mouse v1rs at sites important for binding to ligands when such sites are identified . It is important to note that the sample size (seven mice per species) is relatively small in our study, making population genetic tests of positive selection less powerful . Nonetheless, multiple observations from our data are consistent with one another in supporting the conclusion that weak purifying selection is the predominant force in mouse v1r evolution . Furthermore, the mcdonald kreitman test also strongly rejects the strict neutrality in support of purifying selection rather than positive selection . Thus, it is unlikely that the observed paucity of positive selection in v1rs is an artifact of our small sample . We focused our analysis exclusively on coding regions rather than on regulatory regions because all v1rs are expressed in the vns and are not expected to have important evolutionary changes in gene regulation . A previous analysis of the promoter regions of v1rs supports this view (stewart and lane 2007). One limitation of our study is that we focused exclusively on point mutations and small indels, whereasthe macroevolution of v1rs is also known to be characterized by gene duplication, deletion, and possibly gene conversion . In the future, it would be especially interesting to examine the dynamics of copy number variations for v1rs as has been analyzed for odorant receptor genes (nozawa et al . Evolutionary changes of large gene families appear to contribute disproportionately to genomic evolution because several of the largest gene families in eukaryotic genomes evolve rapidly (shiu et al . 2004; nei and rooney 2005; niimura 2009; shi and zhang 2009). It will be interesting to study whether our findings on the microevolution of v1rs extend to other large gene families.
Percutaneous coronary intervention (pci) revolutionized treatment of coronary artery disease (cad); to date, it is the most commonly used myocardial revascularization method in cardiology [1 - 3]. Drug - eluting stents (dess) can markedly reduce restenosis and have become the most commonly used devices in interventional cardiology for treatment of coronary stenosis . Although this technique does not completely abolish restenosis, a drastic reduction due to significant decrease in neointimal hyperplasia has been reported . On the other hand, the rate of restenosis bare - metal stent (bms) varies according to clinical setting, patient and angiographic characteristics, with rates as high as 60% [5, 6]. Oral drugs for reduction of restenosis are not a practice in interventional cardiology because studies performed did not support it [6, 7]. Several studies that evaluated oral administration of sirolimus or immunosuppressants after bms deployment have reported reduced neointimal hyperplasia compared with bms alone; however, this reduction was not sufficient to allow their clinical use [7, 8]. Increased knowledge of restenosis pathophysiology after stent implantation in the past decade has helped to explain why certain drugs were not effective when administered orally . Methotrexate (mtx) is a folate antagonist that blocks the s - phase of cell division, consequently blocking mitosis . This drug was initially developed for cancer treatment; however, it has been used to treat rheumatic diseases including rheumatoid arthritis (ra) and psoriasis . In this study, the primary objective was to evaluate the safety of oral administration of mtx to patients with severe cad and the secondary goal was to evaluate the possibility that mtx has an impact on restenosis after bms deployment . This was a transversal, prospective and descriptive study that recruited 16 patients in whom pci was planned from january to december 2016 . This study followed the ethical principles in clinical research and was approved by ethics committee and all patients signed the informed consent . The inclusion criteria were: age> 18 years, one vessel disease with de novo lesion> 70% and moderate or severe ischemia according to myocardium scintigraphy for which optimal medical therapy was taken, and one of the following: diabetes mellitus (dm); reference vessel diameter <2.5 mm; or chronic kidney disease stages iii associated with reference diameter of the target vessel <2.75 mm . The exclusion criteria were: contraindications to mtx administration; severe lung disease; liver or kidney disease; and inability to undergo cine coronary angiography at follow - up . Patients with stenosis 70%, requiring pci, were potentially candidates to be enrolled and the study was explained to them and those that accepted assigned the informed consent . Mtx was administered to patients at a dose of 5 mg / week for 2 weeks before pci and 8 weeks after pci . Patients were monitored clinically every 15 days during the first 2 months after the procedure and monthly until 9 months after pci . Clinical restenosis was defined as the occurrence of acute coronary syndrome or stable coronary disease with positive ischemia detection test (moderate or severe ischemia). Angiographic restenosis was defined as 50% in - stent stenosis or within 10 mm proximal or distal to stent [6, 10]. The complications associated with the procedures evaluated included hematoma, allergic reaction, pseudoaneurysm, infections, acute myocardial infarction (ami), stroke, emergency surgery, retroperitoneal bleeding, arteriovenous fistula, and death . The following complications associated with the use of mtx were evaluated: erythematous rash, pruritus, urticaria, photosensitivity, depigmentation, alopecia, ecchymosis, acne, furunculosis, bone marrow depression, leukopenia, thrombocytopenia, anemia, hypogammaglobulinemia, hemorrhage, septicemia, gingivitis, pharyngitis, stomatitis, anorexia, vomiting, diarrhea, hematemesis, melena, gastrointestinal ulceration, enteritis, liver disease, fatty change, renal failure, azotemia, cystitis, hematuria, menstrual dysfunction, abortion, birth defects, severe nephropathy, interstitial pneumonitis, headache, drowsiness and blurred vision, hemiparesis, aphasia, paresis, and convulsions . A descriptive analysis was performed and the numerical variables were presented as mean and standard deviation after the normal distribution of the data has been tested by shapiro - wilks . There was a predominance of male participants, the average age was 62.4 8.3 years, and majority of the patients had an elementary school level of education . Analysis of coronary angiograms revealed 26 severe, 21 moderate, and 18 mild stenosis cases . Of note, eight patients had single - vessel disease, five had two - vessel disease, and three had multi - vessel disease . Coronary artery stenosis was more prevalent in the left anterior descending artery (lda) (table 3). The average diameter and length of the stents were 3.0 0.4 and 18.1 5.9 mm, respectively, as shown in table 4 . Adverse events and side effects due to use of mtx occurred in three patients (prevalence 18.7%). Of note, two patients had skin desquamation on the extremities of the upper limbs and one patient had dryness of the oral mucosa . Only one patient had angiographic restenosis (prevalence was 6.2%; however, her myocardial perfusion imagining did not show ischemia). Based on the few side effects observed, this study demonstrated the safety of mtx administration in this study population (patients with severe cad). In addition, these complications did not result in treatment withdrawal . Mtx has been used for treatment of patients with rheumatic disease and the rate of side effects is low . There were no reported cases of clinical restenosis . These results are quite satisfactory considering the clinical and angiographic characteristics of the patients enrolled . Vascular injury occurs, followed by a reparative process, in which migration of vascular smooth muscle cells (vsmc) occurs from the medial layer into the intima, cellular multiplication, and cellular secretion of molecules . Dess have been used as a platform to carry and release drugs at the site of injuries resulting from pcis . The drugs most commonly used in clinical practice are sirolimus (and its analogues) and paclitaxel . These types of des reduce the chance of restenosis up to 70% compared to bms [12 - 18]. Dess with some cell mitosis inhibitors, such as dexamethasone and tacrolimus, have not shown sufficiently positive effects in pre - clinical and clinical studies to allow their use in clinical practice . The prevalence of angiographic restenosis after bms deployment is up to 60% . Despite many attempts to reduce restenosis rates, only reduction of the stent structure thickness reduction of restenosis was attempted by oral drug administration . However, the results of the drugs tested were not satisfactory enough to warrant their incorporation into clinical practice [7, 20]. Some studies, including the oral sirolimus to inhibit recurrent in - stent stenosis (osiris), oral rapamune to inhibit restenosis (orbit), and prospective, randomized oral rapamycin in argentina (orar) studies, as well as other investigations, evaluated oral use of sirolimes and reported an intermediate efficacy however, these drugs caused many major side effects, which may have contributed to the lack of interest for phase - iii clinical trials [20 - 23]. The high prevalence of side effects reported in these studies was surprising considering that sirolimus (rapamycin) has been used in transplanted patients and this high frequency of side effects had not been previously reported . Authors evaluated the neointimal hyperplasia of transplanted kidney patients (taking immunosuppressive therapy) that underwent bms implantation and reported that neointimal hyperplasia was minimal and determined 9% of vessel obstructions . Some studies evaluated the use of mtx and restenosis after pci . In this context, in 1992, an animal study evaluated, among other aspects, whether mtx would have any effect on neointimal hyperplasia after stent deployment . The authors concluded that the amount of neointima did not differ from the amount obtained after using stents alone [25, 26]. Two decades after this study, some considerations need to be made about these findings . The stent used in this study was a coil . Nowadays, this type of stent is out of market because it did not have good outcomes in clinical studies . The kinetic of drug release was not appropriate because the majority of the drug had been released from the stent during the first hour after deployment . According to stent design, these release kinetics are considered unacceptable considering recent standards for any experiment involving dess . In this study, it is known that this kind of model is related to higher possibility of great amount of hyperplasia . Therefore, the large amount of drug delivered in the first hour, the small amount of drug delivered to the vessel wall, and the balloon - artery ratio are factors that may raise doubts about the authors conclusions . In 2004, a study was published that compared mtx - eluting stents to sae - coated stents which was done in pigs . In this experiment, the stent - artery ratio was 1:1 and 50% of the drug had been released by 24 h. the results showed that mtx - eluting stents reduced neointimal hyperplasia (1.22 0.34 vs. 2.25 1.28 mm, p <0.01) and in - stent obstruction (21 8% vs. 36 21%, another aspect that deserves attention was the finding that mtx had no effect on cell proliferation in vitro . However, this conclusion was based on an experiment that used cells derived from rabbit aortas and not from stent - treated pigs, which were the basis of the study . We should imagine how neointimal hyperplasia could have been reduced in this study if mtx had no effect on vsmc proliferation . Therefore, we hypothesize that other mechanisms leading to hyperplasia reduction, e.g. Inactivation of the secretory function of vsmc or the effect of mtx on the proliferation of vsmc from rabbit aortas was different from that on pig cells may justify this matter . One case - control study evaluated 228 lesions in patients with ra taking mtx and 677 lesion controls treated by pci . Both groups were followed for a mean of 3.8 years . In the ra group, davis et al evaluated the relationship between polymorphisms in the enzyme methylenetetrahydrofolate reductase (mthfr) and cardiovascular events in 1,047 subjects . They did not find any type of association between mthfr and cardiovascular events in ra, while mtx use was protective against these events . In addition, our results allow us to hypothesize that mtx may have beneficial effects on restenosis . It is of note that administration of mtx for 15 days before pci and its maintenance for 60 days aimed to block inflammation, considered one of the triggers for formation of the neointimal hyperplasia, and mitosis . At the time of the previous studies on mtx, the efficacy of des is high and its safety has improved in second- and third - generation devices . However, high costs have limited the introduction of this type of material on a large scale in underdeveloped or developing countries [29 - 31]. Therefore, the search for low - cost therapies that have a positive effect on restenosis is extremely important for patients living in countries with limited financial resources . Furthermore, the feasibility of effective and safe therapies that are not used in the population due to their high cost should be addressed . Mtx was safe in the study population and raised the possibility that a low - cost drug may have positive effects on restenosis after bms implantation . However, studies with larger sample sizes and other imagine modalities (intravascular ultrasound and/or optical coherence tomography) are required to confirm this hypothesis . Mtx was safe in the study population and raised the possibility that a low - cost drug may have positive effects on restenosis after bms implantation . However, studies with larger sample sizes and other imagine modalities (intravascular ultrasound and/or optical coherence tomography) are required to confirm this hypothesis.
Laparoscopic wedge resection has been widely accepted and is now considered to be a minimally invasive surgery for small gastrointestinal stromal tumor (gist) in the stomach . Simple resection using a linear stapler is technically easy, although unnecessary excessive resection of unaffected gastric wall is generally unavoidable . Laparoscopic and endoscopic cooperative surgery (lecs) [2, 3] and laparoscopic intragastric surgery (ligs) have been advocated in efforts to minimize the area to be resected . In fact, these procedures minimize the surgical specimen and provide better outcomes [5, 6]. However, these methods also carry inherent risks of peritoneal infection because of the necessity of gastric perforation . Although the exact incidence of peritoneal contamination with these procedures has yet to be determined, it has become evident that iatrogenic gastrotomy leads to seeding of bacteria in animal models [7, 8] and in humans as well . Although gastrotomy and the resultant bacterial spillage are not associated with severe septic complications [9, 10], avoidance of contamination is undoubtedly preferable . We previously demonstrated that a new technique of gastric full - thickness resection is technically feasible and safe, in an ex vivo model and an in vivo survival model . This procedure, at least theoretically, would minimize the resected tissue volume as well as prevent peritoneal contamination . We herein report a small series of patients with suspected gastric gist treated by this new technique, termed non - exposed endoscopic wall - inversion surgery (news). Between july 2011 and september 2012, we performed news on six patients with suspected small gastric gist . Tumors of the exophytic growth type were excluded . The protocol was approved by the institutional ethics review board of our university, and informed consent was obtained from all the patients . A surgeon, a first assistant, a laparoscopist, and an endoscopic operator were positioned as shown in fig . 1) one camera port was primarily inserted in the umbilical portion, and pneumoperitoneum was established . Then, 5-mm trocars were placed in the left upper, left lower, and right upper quadrants and a 12-mm trocar in the right lower quadrant, five trocars in total.fig . 1position of the study participants in the operating room: s surgeon, a assistant, l laparoscopist, e endoscopist position of the study participants in the operating room: s surgeon, a assistant, l laparoscopist, e endoscopist the tumor location was confirmed employing a flexible endoscope with a carbon dioxide supplier . Markings were made on the mucosa around a lesion with the tip of a dual knife (kd-650l; olympus medical systems, tokyo, japan). Accordingly, serosal markings were made laparoscopically on the side opposite the mucosal markings with a hook knife, guided by pressing the gastric wall using the tip of a flex knife, or the fiberoptic probe of a diode laser system (udl-60; olympus) (fig . A 0.4% sodium hyaluronate solution with a small amount of indigo carmine dye was endoscopically injected into the submucosal layer circumferentially.fig . 2procedures of non - exposed endoscopic wall - inversion surgery . A laparoscopic markings on the serosal surface guided by light from the fiberoptic probe shining through the gastric endoscope . B circumferential seromuscular dissection outside the serosal markings . G flipped tissue to be resected . H dissected lines of the mucosal surface were spontaneously combined and closed using clipping devices procedures of non - exposed endoscopic wall - inversion surgery . A laparoscopic markings on the serosal surface guided by light from the fiberoptic probe shining through the gastric endoscope . H dissected lines of the mucosal surface were spontaneously combined and closed using clipping devices a circumferential seromuscular incision was laparoscopically made around the serosal markings with the hook knife or an energy surgical device (harmonic ace; ethicon endo - surgery) (fig . The seromuscular layer was continuously sutured using 3 - 0 absorbable braided suture (figs . The mucosubmucosal layer was circumferentially incised outside the mucosal markings with a dual knife and an it knife2 (kd-611l; olympus) using endoscopic submucosal dissection (esd) techniques (fig . 2f, g). After the lesion removed, we closed the mucosal layer optionally by the endoscopic clipping device even when seromuscular anastomosis has been established [13, 14]; (fig c mucosubmucosal layer is cut by the endoscopic device scheme of the procedure . A seromuscular layer suture after submucosal injection and seromuscular cutting . C mucosubmucosal layer is cut by the endoscopic device the specimen was extracted using an endoscopic retrieval device (roth net retriever - polyp; us endoscopy, oh, usa). Between july 2011 and september 2012, we performed news on six patients with suspected small gastric gist . Tumors of the exophytic growth type were excluded . The protocol was approved by the institutional ethics review board of our university, and informed consent was obtained from all the patients . A surgeon, a first assistant, a laparoscopist, and an endoscopic operator were positioned as shown in fig . 1) one camera port was primarily inserted in the umbilical portion, and pneumoperitoneum was established . Then, 5-mm trocars were placed in the left upper, left lower, and right upper quadrants and a 12-mm trocar in the right lower quadrant, five trocars in total.fig . 1position of the study participants in the operating room: s surgeon, a assistant, l laparoscopist, e endoscopist position of the study participants in the operating room: s surgeon, a assistant, l laparoscopist, e endoscopist the tumor location was confirmed employing a flexible endoscope with a carbon dioxide supplier . Markings were made on the mucosa around a lesion with the tip of a dual knife (kd-650l; olympus medical systems, tokyo, japan). Accordingly, serosal markings were made laparoscopically on the side opposite the mucosal markings with a hook knife, guided by pressing the gastric wall using the tip of a flex knife, or the fiberoptic probe of a diode laser system (udl-60; olympus) (fig . A 0.4% sodium hyaluronate solution with a small amount of indigo carmine dye was endoscopically injected into the submucosal layer circumferentially.fig . 2procedures of non - exposed endoscopic wall - inversion surgery . A laparoscopic markings on the serosal surface guided by light from the fiberoptic probe shining through the gastric endoscope . B circumferential seromuscular dissection outside the serosal markings . H dissected lines of the mucosal surface were spontaneously combined and closed using clipping devices procedures of non - exposed endoscopic wall - inversion surgery . A laparoscopic markings on the serosal surface guided by light from the fiberoptic probe shining through the gastric endoscope . B circumferential seromuscular dissection outside the serosal markings . H dissected lines of the mucosal surface were spontaneously combined and closed using clipping devices a circumferential seromuscular incision was laparoscopically made around the serosal markings with the hook knife or an energy surgical device (harmonic ace; ethicon endo - surgery) (fig . The seromuscular layer was continuously sutured using 3 - 0 absorbable braided suture (figs . The mucosubmucosal layer was circumferentially incised outside the mucosal markings with a dual knife and an it knife2 (kd-611l; olympus) using endoscopic submucosal dissection (esd) techniques (fig . 2f, g). After the lesion removed, we closed the mucosal layer optionally by the endoscopic clipping device even when seromuscular anastomosis has been established [13, 14]; (fig 3scheme of the procedure . A seromuscular layer suture after submucosal injection and seromuscular cutting . C mucosubmucosal layer is cut by the endoscopic device scheme of the procedure . A seromuscular layer suture after submucosal injection and seromuscular cutting . C mucosubmucosal layer is cut by the endoscopic device the specimen was extracted using an endoscopic retrieval device (roth net retriever - polyp; us endoscopy, oh, usa). The mean diameters of the specimen and tumor were 34.8 mm (range 2845 mm) and 22.7 mm (range 1726 mm), respectively . 4ac.table 1clinicopathological characteristics of the submucosal tumorscase no.age (years)genderlocation circumference specimen (mm)tumor (mm)pathology158mmgre45 35 2224 23 19schwannoma259mupost33 27 1319 16 11gist361mupost30 30 2026 26 17gist471fugre38 23 2325 23 23gist579fuless35 32 2025 20 20gist649muant28 19 1817 17 17gist the three portions of the stomach: u upper third, m middle third the four equal parts of the gastric circumference: . Less lesser curvature, gre greater curvature, ant anterior wall, post posterior wallgist gastrointestinal stromal tumorfig c cross section clinicopathological characteristics of the submucosal tumors the three portions of the stomach: u upper third, m middle third the four equal parts of the gastric circumference: . Less lesser curvature, gre greater curvature, ant anterior wall, post posterior wallgist gastrointestinal stromal tumor representative resected tissue . A view from mucosal side . B serosal side . C cross section operative data for news are shown in table 2 . The cause of the perforation was muscle injury by the endoscopic knife during mucosal cutting in case 1 and laparoscopic mucosal injury during seromuscular cutting in case 3.table 2operative data of our seriescase no.en bloc resectionperforationoperation time (min)blood loss (ml)postoperative hospital stay (days)complications1yesyes397307none2yes(conversion)2922507none3yesyes3572508none4yesno265508none5yesno1901007none6yesno14007none operative data of our series in case 2, we converted the procedure, because of poor recognition of the tumor margin, to endoscopic full - thickness resection with subsequent laparoscopic suture closure of an iatrogenic gastric defect . After the initial three cases, we introduced the optical fiber to identify the outer portion of the tumor via endoscopy, dissected the seromuscular layer as well as the deeper layer of the submucosa laparoscopically, and doubled the amount of hyaluronate solution . In the latter three cases, the entire procedure was carried out successfully . The mean operation time and blood loss were 349 min and 177 ml in the first three cases; in the latter three cases, these were 198 min and 50 ml, respectively . None of our cases experienced postoperative complications such as hemorrhage, anastomosis insufficiency, delayed gastric emptying, or surgical site infection . All patients started oral intake on postoperative day 2 . During the mean follow - up period of 8 months (range 216 months), none of our patients exhibited any symptoms, and there were no changes in dietary habits . Although an en bloc full - thickness resection with a minimal margin was successful, we were not able to carry out this procedure without perforation in the first three patients . The endoscopic view was quite different from that of esd, and the cutting line was stereoscopic and varied according to tumor size and shape . Laparoscopic cutting of the submucosa to the fullest extent possible during the laparoscopic procedure was beneficial in terms of entering the right space between the sutured muscular layer and the lifted lesion (fig . Identification of the tumor margin via pressing with the endoscopic forceps was not essential and was limited according to the tumor location . Because the endoscopic forceps moved only along the line tangential to the wall in case 2 and misalignment of the serosal marking seemed to be highly associated with pseudo - capsule injury and tumor rupture, we converted the procedure . After this case, we used the light from the fiberoptic probe of a diode laser through the gastric endoscope for guidance . The light allowed clear identification with no limitation from tumor location, and we were able to confirm the tumor margin in the latter four cases based on the illumination provided . One mucosal micro - perforation was observed during the laparoscopic seromuscular cutting in case 3 . Muscle layer thickness is known to differ according to location [15, 16]. Similarly, doubling the amount of hyaluronate solution, as well as the sequential additive injections during the procedure, were effective for avoiding mucosal tearing . As a result, we accomplished news without perforations in the latter three patients after these modifications of the procedure . Continuous suturing of the seromuscular layer was safe and feasible as previously reported [13, 14]. Although this is a preliminary report and an additional larger cohort treated employing this procedure is needed to evaluate this technique before it can be considered feasible and valid, this non - opened technique for the digestive tract theoretically provides major benefits . First, postoperative inflammatory responses as well as the surgical site infection rate might show positive effects . Second, this method enables us to perform full - thickness resection while avoiding possible tumor dissemination into the peritoneal cavity, and thus it may have potential as a treatment modality even for patients with ulcerated gist or gastric cancer with minimal risk of lymph node metastasis . Third, upper limit of tumor size safely extracted orally should be meticulously evaluated . In conclusion, a new laparoendoscopic technique, news,
Neuroendocrine tumors (nets) originate from neural crest cells which belong to the amine precursor uptake and decarboxylation (apud) lineage and have both neural and endocrine cell features . These tumors are generally seen in the gastroenteropancreatic tract and lungs and rarely in ovary . These tumors can be imaged using metaiodobenzyl guanidine (mibg) tagged to 131-iodine; which enters by a specific energy - dependent uptake mechanism competing with norepinephrine and majority of it is trapped in the intracellular granule fraction . This tracer has shown better sensitivity in sympathoadrenomedullary tumors as compared to the other net though the uptake is heterogeneous . Over - expression of somatostatin receptors (sstr) is noted in these tumors and this patho - physiology is exploited in radioimmunoscintigraphy (ris). Sstr imaging in net is indicated for detection of the primary, staging, monitoring response to therapeutic somatostatin and treatment planning for sstr directed radionuclide therapy . All the subtypes of sstr expressed by net have affinity for the native peptide but vary in their affinity for the somatostatin analogues; hence, the sensitivity of the study depends on the density of the sstr in the tumor and the type of analogue used in the study . Indium 111 (in-111) tagged somatostatin analogues were the commonly used tracers and majority of the literature related to somatostatin receptor scintigraphy (srs) had been done using this tracer . Studies have revealed the sensitivity of in-111 labeledsrsto be in the range of 80 - 90% . It has shown superiority to other diagnostic imaging methods (such ascomputed tomography [ct] and magnetic resonance) in identifying and assessing the staging of net, except for insulinoma (density of sstr is very low). The disadvantages of long half - life, physiological uptake in abdominal organs, and a higher energy of in-111 warranted research in use of a technitium-99 m (99mtc) labeled agent for somatostatin receptor imaging, which is better suited for single photon emission computed tomography (spect) imaging . Tc labeled tyrosine-3 octreotide (toc) has been identified as a suitable tracer which uses hydrazinonicotinic acid (hynic) as a complexing ligand . The pharmacokinetic properties of tc - hynic toc were found to be better than those of in - octreotide . Higher target - to - non - target ratios and higher absolute tumor uptake values were observed for tc - hynic toc and the optimal acquisition time for imaging was identified as 4 h after injection . Srs has low sensitivity for lesions that are present in organs having physiological tracer concentration like the liver and lesions smaller in size due to the limitation of the mechanics and tracers used in spect . Imaging with pet (positron emission tomography) has higher resolution of the lesions, an inherent property of the modality . Initial data showed the tracer gallium 68 (ga 68) dota toc to have a good pharmacokinetic and imaging characteristic as compared to conventional nuclear medicine procedures . A large prospective study also demonstrates a higher accuracy of ga 68 dota toc in comparison to the anatomical imaging modality, ct, and conventional srs . However, the pet / ct modality is not often available and spect imaging is still the feasible option for imaging of net . Our pictorial will try to demonstrate the utility of sstr imaging using tc hynic toc in various clinical settings and project its role in prognostication when done in conjunction with 18f flouro- deoxy glucose (fdg)_pet / ct . Patients receiving cold somatostatin therapy were asked to refrain from the therapy for 4 weeks, whereas those patients who had undergone a surgery had their imaging done after the 3 post - operative week . Patients were injected with 20 mci (740 mbq) of the tracer; a whole body planar image was obtained at 30 min post - injection (p.i) on a dual head gamma camera (infinia hawkeye, ge, milwaukee). A repeat whole body planar image and spect of the abdomen and regions with abnormal tracer uptake were performed 2 hafter injection in majority of the cases; a pilot study of 15 cases revealed the 2-h images to be as sensitive as a 4-h image (as suggested in literature). In cases with a doubtful lesion in the2-hp.i image, a delayed image at 4 h p.i . Fdg pet / ct was done 60 - 90 min after intravenous injection of 18 fdg, with the patient in a fasting state within a week of the srs . Acquisition was done as per the snm guidelines, from base of skull to mid - thigh on a dedicated pet / ct scanner (discovery st, ge, milwaukee). The normal distribution of the tc hynic toc tracer is seen in thegall bladder, kidneys, liver, spleen [figure 1], and sometimes in the pituitary and thyroid . This image ((a) anterior and (b) posterior) depicts the normal distribution of the radiotracer, 99 m tc hynic toc . Note the uptake in the thyroid (small arrow), the liver and spleen . The kidney and urinary bladder are seen due to the part excretion through this system staging of histologically proven neuroendocrine malignancies.the management of net depends on the stage of the disease, i.e., whether it is localized or metastatic . Surgery is offered as an option to patients who have a non non - metastatic primary mass lesion . Patients with locally advanced disease generally undergo a debulking surgery with the residual disease being treated with targeted therapies . Cytoreduction followed by targeted therapies or specific local therapies like radioablation is the treatment option for a local disease with a solitary metastatic site . A disseminated disease is tried to control with targeted therapies.the conventional staging for net is done with a cect of the suspected local site with ct of abdominopelvic and thorax regions . Ris is now incorporated in the staging of net as it helps trace the extent of the primary disease and also the spread of the malignancy in a single setting as seen in figure 2.netsshow unusual site of metastases less frequently though not uncommon . Ris helps locate the odd sites of disease as seen in figure 3.initial detection and localization of suspected net and potential metastases in presence of a clinical or biochemical suspicion or to locate primary in a case identified to have a solitary metastatic lesion on conventional imaging.patients with netsmore often present with symptoms due to high endocrine secretion rather than the pressure effect caused by the primary mass . Conventional imaging modalities are able to map the metastatic sites but tracing the primary site is difficult at times . Figures 4 and 5 depict the utility of srs in this indication.treatment response assessment of net: patients with metastatic disease are treated with medical line of treatment and the treatment response assessment is generally done with biochemical markers and clinically . Reduction of the symptoms with a decline in tumor markers is noted with responsive tumors . Imaging studies are used to document treatment response; however, it is difficult to differentiate between functional and non - functioning residual tissue . The ability to identify residual functioning tissue by a non - invasive procedure is useful to plan continuation of therapy . Pre- and post - therapy srsis a helpful tool in this respect as shown in figures 6 and 7 . The management of net depends on the stage of the disease, i.e., whether it is localized or metastatic . Surgery is offered as an option to patients who have a non non - metastatic primary mass lesion . Patients with locally advanced disease generally undergo a debulking surgery with the residual disease being treated with targeted therapies . Cytoreduction followed by targeted therapies or specific local therapies like radioablation is the treatment option for a local disease with a solitary metastatic site . The conventional staging for net is done with a cect of the suspected local site with ct of abdominopelvic and thorax regions . Ris is now incorporated in the staging of net as it helps trace the extent of the primary disease and also the spread of the malignancy in a single setting as seen in figure 2 . Initial detection and localization of suspected net and potential metastases in presence of a clinical or biochemical suspicion or to locate primary in a case identified to have a solitary metastatic lesion on conventional imaging . Patients with netsmore often present with symptoms due to high endocrine secretion rather than the pressure effect caused by the primary mass . Identifying conventional imaging modalities are able to map the metastatic sites but tracing the primary site is difficult at times . Treatment response assessment of net: patients with metastatic disease are treated with medical line of treatment and the treatment response assessment is generally done with biochemical markers and clinically . Reduction of the symptoms with a decline in tumor markers is noted with responsive tumors . Imaging studies are used to document treatment response; however, it is difficult to differentiate between functional and non - functioning residual tissue . The ability to identify residual functioning tissue by a non - invasive procedure is useful to plan continuation of therapy . Pre- and post - therapy srsis a helpful tool in this respect as shown in figures 6 and 7 . Whole body planar images (a) of a 99 m tc hynic toc study of a recently detected case of pancreatic net reveals the uptake in the primary (arrow) and the metastatic lesions which are localized on the transaxial spect / ct images to correlate with the lesion in the skull (b), a enlarged prevascular node (c) and a tiny pleural based pulmonary nodule (d) which is identified on the correlative ct image (triangulated in e). Another pulmonary nodule is seen as a focus of uptake in the left hemithorax on the whole body planar image (a) a diagnosed case of small cell carcinoma of the lung referred for staging using sstr imaging revealed a large uptake in the upper abdomen on the whole body planar images (a), better visualized in the anterior aspect (arrow). A spect / ct of the abdomen shows a large peritoneal mass (b and c) with focal tracer uptake in the primary in the left lung mass (d) and an unusual subcutaneous metastases in the posterior chest wall in paravertebral region (e). No fdg pet / ct study was done for this patient patient with diarrhea evaluated for net, conventional ct imaging revealed hepatic metastases and was referred for somatostatin receptor scintigraphy . Planar wb images show avid tracer concentration in the known sites of hepatic metastasis (bold arrow in a and b). A small focus of uptake to the right of the midline in the abdomen (small arrow in a) corresponded to site of the primary in the duodenum well depicted on the spect / ct images (triangulated in c) patient who presented with metastatic left supraclavicular node from an unknown net with multiple hepatic metastases on cect of the abdomen was investigated with srs . The wb image in lesser intensity showed multiple hepatic lesions (b), while the darker intensity images (a) showed focal uptake in the left supraclavicular node (thin arrow a) and an uptake in the left aspect of the upper abdomen (thick arrow a) which correlates on the transaxial spect ct image to the stomach wall (arrow head in c) suggesting gastrinoma as a possible primary, later confirmed histologically responder: a metastatic case of small bowel net; the baseline planar whole body image (a) shows uptake in the primary (thin arrow) and the multiple hepatic metastases (thick arrow). Whole body planar sstr imaging (b) after 3 cycles of somatostatin therapy shows complete regression of the tracer uptake at the primary and the metastatic liver lesions depicting the suppression of somatostatin receptors due to the therapy . He was documented to be df on his last follow up, 1.5 year post last radioimmunoscintigraphy non responders: a case of net of the duodenum with hepatic metastases, the pre treatment wb planar images show multiple abnormal uptakes in the liver with no obvious focal uptake at the primary site (a), the hepatic lesions showed partial regression in the post therapy scan (b). A small focus of tracer in the right aspect of the abdomen corresponding to the primary (arrow b and d) of the post therapy scan which was not appreciated in the pre therapy scan (c) probably due to masking . The combined studies suggest suboptimal suppression of the somatostatin receptor pathway change in biology of the tumors is a known phenomenon and is attributed to either a change in the tumor receptor density or expression of a new receptor . Delineating these patients on follow - up with clinical or biochemical suspicion of a recurrence evaluated with ris with poor to absent sstr expression raise the probability of altering receptor status . Net is a well - differentiated pathology and does not express glucose transporter (glut) receptors and hence a fdg pet / ct study is not utilized in the work up . Dedifferentiating tumors show an increase in the glut receptor expression with a decline in the somatostatin receptor density; hence, a fdg pet / ct study would be efficacious in locating sites of tumor spread . Combination receptor imaging will help in staging the disease as per the who classification which is based on the histology type 1a: well - differentiated benign, type 1b: welldifferentiated with low - grade malignancy, and type 2 poorly differentiated . The prognosis of the tumor is dependent on the differentiation of the tumor, poorly differentiated having a bad prognosis . Netscan be categorized depending on the pattern of somatostatin and glut receptors expression with type i at one end of the spectrum suggestive of a well - differentiated tumor and type iv which depicts a dedifferentiated tumor with poor to absent sstr at the other end [table 1]. Categorisation of neuroendocrine tumors based on somatostatin receptor scintigraphy and flouro deoxy - glucose uptakes . Figures 8 - 13 illustrate the various combinations of ris and fdg scans depicting the varied biologies of nets confirming the utility of conjugate receptor imaging . Type i uptake pattern: the wb planar images of 99 m tc hynic toc study show multiple hepatic metastases (a). The transaxial spect / ct images show focal concentration in the primary in head of pancreas (b) and the fused image of the hepatic metastases (c). The mip image of the fdg pet / ct study (d) of this patient does not show abnormal focal fdg concentration either in the primary or the hepatic metastases . The combined srs and fdg images in this patient portray a type i pattern type ii uptake pattern: the anterior and posterior whole body planar image of ris (a) with avid uptakes seen in the liver metastases and right aspect of the midline region, the transaxial spect / ct images show the focal uptake in the right of the midline correlating with the primary in the body of the pancreas (b) and the multiple hepatic metastases (c) type ii uptake pattern (contd): the fdg pet / ct study of the patient in fig 9 revealed an area of minimal increased tracer uptake in the right lobe of the liver on the mip image (arrow a) which on transaxial images corresponds to the largest hepatic lesion in the right lobe (triangulated in b) with no glut expression in the primary lesion at the junction of the body and head of the pancreas (arrow c) or the other larger hepatic lesions (d), these tumors may have a propensity for alteration of tumor biology type iii uptake pattern: tumors with poor differentiation show reduction in density of sstr with increase in density of glut receptors; these are visualized as fdg and srs avid lesions categorized as type iii pattern . This is a case of recurrent net of the tail of the pancreas evaluated with ris, the planar and the correlative fused transaxial spect / ct images show somatostatin avid recurrent lesion in tail of pancreas (thin arrow a and triangulation in (b). Note the intense focal uptake in the gall bladder (arrow head a), confirmed in the spect / ct image (c) type iii uptake pattern (contd): the recurrent pancreatic tail lesion seen in fig 11 showed avid fdg uptake seen on mip image of the 18 f fdg pet / ct (arrow head a) and the transaxial image (b) suggestive of dense glut receptor expression . A low grade fdg uptake is seen in a hepatic lesion on the fdg pet / ct study (arrow a and c) with no hynic toc concentration in the liver on correlative transaxial spect image of the ris (d). The metastatic lesions demonstrate altered biology, no somatostatin receptor expression but expression of glut receptor type iv uptake pattern: dedifferentiated tumors show poor somatostatin receptor expression with high expression of glut receptor which shows a type iv pattern of uptake -fdg avid and poor sstr lesions . A net of the lung with mediastinal nodal metastases shows poor somatostatin expression as seen by low uptakes on the whole body planar images of 99 m tc hynic toc study (a) with intense fdg concentration in the primary and the nodal disease seen on the mip image (b) and the transaxial images of the thorax (c and d) of the 18 f fdg pet / ct it would be appropriate to suggest that in combined srs and fdg pet / ct studies, an increasing fdg uptake with declining sr uptake would convey loss of tumor differentiation and predict a poor prognosis . Peptide receptor radionuclide therapy (prrnt) is emerging as a promising therapeutic option in view of the specific targeting of tumor receptors . The consensus report of the net clinical trials planning meeting mentions the need for a randomized phase iii trial with use of prrnt in one arm which is based on the somatostatin receptor expression . Sstr would be useful in this setting to identify the differentiation of the tumor, its spread, and will also be used for tailored dosimetry . It would be worthy to note the advantage of ris in that it provides all the necessary treatment planning information in a single study . Change in biology of the tumors is a known phenomenon and is attributed to either a change in the tumor receptor density or expression of a new receptor . Delineating these receptor changes assists in prognosticating the disease and alter management . Patients on follow - up with clinical or biochemical suspicion of a recurrence evaluated with ris with poor to absent sstr expression raise the probability of altering receptor status . Net is a well - differentiated pathology and does not express glucose transporter (glut) receptors and hence a fdg pet / ct study is not utilized in the work up . Dedifferentiating tumors show an increase in the glut receptor expression with a decline in the somatostatin receptor density; hence, a fdg pet / ct study would be efficacious in locating sites of tumor spread . Combination receptor imaging will help in staging the disease as per the who classification which is based on the histology type 1a: well - differentiated benign, type 1b: welldifferentiated with low - grade malignancy, and type 2 poorly differentiated . The prognosis of the tumor is dependent on the differentiation of the tumor, poorly differentiated having a bad prognosis . Netscan be categorized depending on the pattern of somatostatin and glut receptors expression with type i at one end of the spectrum suggestive of a well - differentiated tumor and type iv which depicts a dedifferentiated tumor with poor to absent sstr at the other end [table 1]. Categorisation of neuroendocrine tumors based on somatostatin receptor scintigraphy and flouro deoxy - glucose uptakes . Figures 8 - 13 illustrate the various combinations of ris and fdg scans depicting the varied biologies of nets confirming the utility of conjugate receptor imaging . Type i uptake pattern: the wb planar images of 99 m tc hynic toc study show multiple hepatic metastases (a). The transaxial spect / ct images show focal concentration in the primary in head of pancreas (b) and the fused image of the hepatic metastases (c). The mip image of the fdg pet / ct study (d) of this patient does not show abnormal focal fdg concentration either in the primary or the hepatic metastases . The combined srs and fdg images in this patient portray a type i pattern type ii uptake pattern: the anterior and posterior whole body planar image of ris (a) with avid uptakes seen in the liver metastases and right aspect of the midline region, the transaxial spect / ct images show the focal uptake in the right of the midline correlating with the primary in the body of the pancreas (b) and the multiple hepatic metastases (c) type ii uptake pattern (contd): the fdg pet / ct study of the patient in fig 9 revealed an area of minimal increased tracer uptake in the right lobe of the liver on the mip image (arrow a) which on transaxial images corresponds to the largest hepatic lesion in the right lobe (triangulated in b) with no glut expression in the primary lesion at the junction of the body and head of the pancreas (arrow c) or the other larger hepatic lesions (d), these tumors may have a propensity for alteration of tumor biology type iii uptake pattern: tumors with poor differentiation show reduction in density of sstr with increase in density of glut receptors; these are visualized as fdg and srs avid lesions categorized as type iii pattern . This is a case of recurrent net of the tail of the pancreas evaluated with ris, the planar and the correlative fused transaxial spect / ct images show somatostatin avid recurrent lesion in tail of pancreas (thin arrow a and triangulation in (b). Note the intense focal uptake in the gall bladder (arrow head a), confirmed in the spect / ct image (c) type iii uptake pattern (contd): the recurrent pancreatic tail lesion seen in fig 11 showed avid fdg uptake seen on mip image of the 18 f fdg pet / ct (arrow head a) and the transaxial image (b) suggestive of dense glut receptor expression . A low grade fdg uptake is seen in a hepatic lesion on the fdg pet / ct study (arrow a and c) with no hynic toc concentration in the liver on correlative transaxial spect image of the ris (d). The metastatic lesions demonstrate altered biology, no somatostatin receptor expression but expression of glut receptor type iv uptake pattern: dedifferentiated tumors show poor somatostatin receptor expression with high expression of glut receptor which shows a type iv pattern of uptake -fdg avid and poor sstr lesions . A net of the lung with mediastinal nodal metastases shows poor somatostatin expression as seen by low uptakes on the whole body planar images of 99 m tc hynic toc study (a) with intense fdg concentration in the primary and the nodal disease seen on the mip image (b) and the transaxial images of the thorax (c and d) of the 18 f fdg pet / ct it would be appropriate to suggest that in combined srs and fdg pet / ct studies, an increasing fdg uptake with declining sr uptake would convey loss of tumor differentiation and predict a poor prognosis . Peptide receptor radionuclide therapy (prrnt) is emerging as a promising therapeutic option in view of the specific targeting of tumor receptors . The consensus report of the net clinical trials planning meeting mentions the need for a randomized phase iii trial with use of prrnt in one arm which is based on the somatostatin receptor expression . Sstr would be useful in this setting to identify the differentiation of the tumor, its spread, and will also be used for tailored dosimetry . It would be worthy to note the advantage of ris in that it provides all the necessary treatment planning information in a single study . The ability of the modality to delineate the somatostatin receptor expression gives explicit information of the biology of the net, both at primary and metastatic site and helps in treatment planning . Srs in conjunction with glut receptor imaging helps locate change in tumor receptor expression and thus helps in prognostication of the disease . This can stratify patients who would benefit from somatostatin analogue or peptide therapy, which is the emerging treatment option for net . Ris will be an effective method to monitor response to radioimmunotherapy, which will identify a non - responder early and help alter treatment in such patients.
Before ecd can be used for analysis, they must first be prepared so that they accurately represent the health risks and outcomes of the patients studied, as well as the care provided by health care system . Davis and colleagues report on a retrospective observational study in veterans with rheumatoid arthritis to validate diagnostic and procedural codes for identification of acute cardiovascular events . Beyond the specific population and set of outcomes in this example, reimer and madigan demonstrate how the development of a fully integrated medical transport record for patients undergoing medical transport as an example will offer the ability to address complex questions related to patients clinical outcomes in a real - world clinical setting while providing an electronic data infrastructure that can enable high - quality, clinically rich, prospective, and multisite data collection to support cer . Stuart and colleagues outline the challenges in estimating causal effects using electronic health data generally, and offer some solutions, with particular attention to propensity score methods . The methods are illustrated with a case study showing how medicare and medicaid administrative data can be used to estimate the effect of the medicare part d prescription drug program among individuals with serious mental illness . Zurovac and colleagues demonstrate how the concept of multifactorial experiments, drawn from the evaluation literature, offers promise for cer with ehrs . They examine some of the unique aspects of such missing data, present some statistical advice about how to handle these issues, and provide some suggested areas for future methodological research . A more specific challenge in using ecd is that observations are not scheduled as they would be in a designed randomized clinical trial, but rather occur at irregular intervals coincident with patient visits, which potentially depend on outcomes . Luo and colleagues show how discrete - time hidden markov models can be used to estimate transition rates in this context, using chronic kidney disease as an example . A final paper in this issue provides an example of how methods for preparing and analyzing ecd can come together to enable rigorous cer . Written in the form of a protocol for a prospective, longitudinal cohort study, sills and colleagues demonstrate how survey methodologies and secondary analysis of existing structured clinical, administrative, and claims data can be used to estimate the effects of patient - centered medical home characteristics on asthma control in adults and children . The setting for this research is the scalable architecture for federated translational inquiries network (saftinet), an ahrq - funded safety net - oriented practice - based research network, which is designed to measure these variations in delivery system characteristics . As useful as they are, these papers only begin to address the methodological developments needed to advance the national dialogue on the use of ecd to conduct cer, support qi, and generally to improve outcomes in a learning health care system . These papers offer a beginning snapshot of some critical ideas and innovations shaping the field . Egems remains interested in publishing researcher perspectives on methodological challenges resulting from using ecd and lessons learned from facing these challenges, and welcomes future methods - related submissions to our general issue . For more information on submitting papers i encourage you to read the papers in this collection and think about whether your organization has developed similar approaches or radical alternatives and write about them . You might also try some of the approaches discussed here, and let us know how they work out . We are particularly interested in papers that address key topics raised in prior edm forum discussions, including strategies for evaluating and addressing missing data and other data quality issues; analytic strategies that can account for granular temporal or spatial information; and new, rigorous methodological approaches to maximizing the use of big data to generate new evidence and determine what works best for whom and under what conditions.
The pubmed search string cardiac surgical procedures (mesh) and randomized controlled trial (publication type) was used to identify cardiac surgery randomized controlled trials published between january 1, 2010, and june 30, 2014 . Our search was limited to 5 high - impact journals in general medicine, cardiovascular medicine, and cardiothoracic surgery6: new england journal of medicine, circulation, journal of the american college of cardiology, annals of thoracic surgery, and the journal of thoracic and cardiovascular surgery . Studies were included if the study design was randomized and the study population was undergoing coronary artery bypass grafting and/or heart valve repair or replacement surgery . Studies were excluded if the primary outcome was not a clinical event (eg, the primary outcome was a biomarker) or if the study population was pediatric or focused on congenital heart disease . Case reports, case series, editorials, reviews, and post hoc analyses of randomized controlled trial data were also excluded . For each article meeting inclusion criteria, the following variables were extracted: first author, journal, year of publication, trial name and registration, sample size, study population, intervention, control, primary outcomes, secondary outcomes, and duration of follow - up . In addition, the operational definitions used to ascertain mi, stroke, prolonged ventilation, acute renal injury, and bleeding were extracted . Patient - centered outcomes included postoperative pain, quality of life, mood, neurocognitive function, and new york heart association functional class . Health care resource utilization included hospital and intensive care unit length of stay and cost analyses . Studies were reviewed, and data were extracted in duplicate by 2 independent observers (m.g ., l.d . ); disagreements were resolved by consensus . The primary and secondary outcomes were represented in tabular format and summarized according to the number and proportion of randomized controlled trials reporting each outcome measure . The stata 13 software package (statacorp) was used to organize the extracted data and to prepare summary statistics . The pubmed search string cardiac surgical procedures (mesh) and randomized controlled trial (publication type) was used to identify cardiac surgery randomized controlled trials published between january 1, 2010, and june 30, 2014 . Our search was limited to 5 high - impact journals in general medicine, cardiovascular medicine, and cardiothoracic surgery6: new england journal of medicine, circulation, journal of the american college of cardiology, annals of thoracic surgery, and the journal of thoracic and cardiovascular surgery . Studies were included if the study design was randomized and the study population was undergoing coronary artery bypass grafting and/or heart valve repair or replacement surgery . Studies were excluded if the primary outcome was not a clinical event (eg, the primary outcome was a biomarker) or if the study population was pediatric or focused on congenital heart disease . Case reports, case series, editorials, reviews, and post hoc analyses of randomized controlled trial data were also excluded . For each article meeting inclusion criteria, the following variables were extracted: first author, journal, year of publication, trial name and registration, sample size, study population, intervention, control, primary outcomes, secondary outcomes, and duration of follow - up . In addition, the operational definitions used to ascertain mi, stroke, prolonged ventilation, acute renal injury, and bleeding were extracted . Patient - centered outcomes included postoperative pain, quality of life, mood, neurocognitive function, and new york heart association functional class . Health care resource utilization included hospital and intensive care unit length of stay and cost analyses . Studies were reviewed, and data were extracted in duplicate by 2 independent observers (m.g ., l.d . ); disagreements were resolved by consensus . The primary and secondary outcomes were represented in tabular format and summarized according to the number and proportion of randomized controlled trials reporting each outcome measure . The stata 13 software package (statacorp) was used to organize the extracted data and to prepare summary statistics . Of 190 potentially relevant trials, 34 met the selection criteria and were included in our systematic review (figure 1). Sample sizes ranged from 57 to 4752 participants (median 351; quartiles 1 to 3: 198 to 699). Overall, 26 trials involved coronary artery bypass grafting only, 5 involved valve repair or replacement only, and 3 involved a combination . The maximum duration of follow - up for outcome surveillance ranged from 5 to 14 days (median 7.5 days) in 6 trials, from 30 days to 1 year (median 365 days) in 19 trials, and was> 1 year (median 1825 days) in 9 trials . Summary of trials meeting inclusion criteria af indicates atrial fibrillation; aki, acute kidney injury; ards, acute respiratory distress syndrome; ats, annals of thoracic surgery; cabg, coronary artery bypass grafting; circ, circulation; co, cardiac output; cva, cerebrovascular accident; dswi, deep sternal wound infection; gi, gastrointestinal; iabp, intra - aortic balloon pump; iv, intravenous; jacc, journal of american college of cardiology; jtcs, journal of thoracic and cardiovascular surgery; mi, myocardial infarction; mr, mitral regurgitation; mv, mitral valve; nejm, new england journal of medicine; pci, percutaneous coronary intervention; revasc ., repeat revascularization; savr, surgical aortic valve replacement; tavr, transcutaneous aortic valve replacement . Flow diagram for search strategy . Mortality (all - cause and/or cardiovascular) was reported as an individual end point or as part of a composite end point in 28 trials (82%), mi was reported in 23 trials (68%), need for repeat revascularization or reoperation was reported in 22 trials (65%), stroke or transient ischemic attack was reported in 18 trials (53%), acute kidney injury was reported in 11 trials (32%), and bleeding complications were reported in 8 trials (24%) (table 2). Health care resource utilization was reported in 12 trials (35%). Graphical representation of primary and secondary outcomes in included trials with overview of commonly used combined endpoints in cardiovascular research composite end points were used as the primary outcome measure in 19 trials and as a secondary outcome measure in 4 trials . Overall, 14 different composite end points were used, of which 6 were variants of the macce composite, 3 were variants of the mace composite, and none were based on the sts composite . Mi was defined based on world health organization criteria in 2 studies, european society of cardiology and/or american heart association criteria in 4 studies, varc criteria in 1 study, creatinine kinase elevation greater than the upper limit of normal in 2 studies, creatinine kinase or troponin elevation> 3 times the upper limit of normal in 1 study, creatinine kinase or troponin elevation> 5 times the upper limit of normal in 4 studies, and creatinine kinase or troponin rise to various levels depending on time after surgery in 3 studies . Stroke was defined based on focal neurological deficit with imaging findings in 3 trials and on acute focal neurological deficit lasting 24 hours with or without confirmatory imaging in 11 trials; no diagnostic criteria were provided in 6 trials . Acute kidney injury was defined based on need for renal replacement therapy in 5 trials, need for renal replacement therapy or prespecified elevation in creatinine (each with different thresholds, ranging from 221 mmol / l [3.5 mg / dl]) in 3 trials, and prespecified elevation of twice the preoperative creatinine level with or without oliguria in 2 trials; no diagnostic criteria were provided in 2 trials . Prolonged ventilation or intubation was defined as> 24 hours in 2 trials and> 48 hours in 1 trial . The definition of postoperative bleeding differed in each of the 8 trials in which it was reported . To our knowledge, this review of adult cardiac surgery trials is the first to examine the current state of outcome reporting . We found that mortality and mi were most frequently reported as individual or composite end points and, conversely, that the sts composite was not used as an outcome measure . First, the decision to use or not use a composite as the primary outcome measure was evenly split between trials . Third, the operational definitions of events were ill defined, particularly the thresholds used to dichotomize continuous metrics such as troponin rise for mi or ventilation duration . Mace and macce also had varied definitions in the trials, similar to prior reports in general cardiology.7 heterogeneity in cardiac surgery outcome reporting limits the ability to synthesize and meta - analyze results across trials to generate guidelines with the highest level of evidence.8 this is relevant, given the shift toward evidence - based practice derived from randomized controlled trials in cardiac surgery and other surgical subspecialties.9 in addition, nonstandardized outcome measures limit the ability to directly compare the effectiveness of various surgical techniques, perioperative interventions, and providers.10 as new competing techniques and technologies emerge, it is increasingly important that surgical outcome reporting be comparable among trials . Due to the lack of consensus outcome measures in cardiac surgery, investigators often default to using mortality as the end point of choice, despite being grossly underpowered to do so (as was apparent in the many of the trials reviewed and in an even greater proportion of observational studies). Similarly, with the exception of the sts models, most risk scores use mortality as the sole end point, neglecting the importance of other complications and patient - centered outcomes . The use of composite end points in cardiac surgery may be beneficial for several reasons . Composite end points avoid the arbitrary choice of a single outcome when several may be of clinical importance for the cardiac surgery patient45 and allow for estimation of the net clinical benefit of the intervention when risks and benefits are both considered.46 composite end points encompass postoperative complications, which are important determinants of functional recovery and quality of life.47 patient recruitment in cardiac surgery trials has historically been difficult48 and continues to be challenging despite the emergence of collaborative research networks.49 composite end points yield an increased number of events and a smaller required sample size, resulting in improved statistical efficiency and precision.50 this is especially true if event rates are low and efforts to analyze individual outcomes or to perform meta - analyses lead to false - negative and false - positive conclusions.51 presenting a clear sample size calculation matched to the primary outcome of interest, as was done in most reviewed trials, is critical in this regard . The breadth of adverse events encountered after cardiac surgery is not captured by generic composite outcome measures traditionally used in cardiology, such as mace or macce . Acute kidney injury and deep sternal wound infections, for example, are postoperative events that are associated with considerable morbidity . Mi, the usual driver of mace - type end points in cardiology trials, has different connotations and prognostic impact in the postoperative setting and concerns pertaining to measurement errors if ascertaining mi soon after surgery . The use of composites may be justified only if each component is of similar importance to the patient,2 whereas it may be questionable if components are empirically different in impact (eg, deep sternal wound infection and stroke).2 assigning weights to the components may circumvent this caveat and help increase the validity of conclusions.52 quality of life, physical performance, cognitive function, and dependency for activities of daily living have been broadly categorized as patient - centered outcomes because they reflect domains that are crucial to the patient but are extrinsic to traditional domains of mortality and pathophysiology that are emphasized by physicians and researchers . There is increasing awareness in the cardiovascular community that these data should be collected and reported, particularly when studying elderly populations in which the priority of care may have shifted from longevity to quality of life . Postoperative length of stay, stroke, and readmission have been identified as important indicators of quality of care,53 strengthening the rationale for also reporting these end points.54 for a criterion to be a useful part of a composite end point, it should be clinically important and reliably ascertainable . In the cardiology literature, consensus efforts have been made to standardize adjudication of mi,55,56 renal injury,3 and bleeding.4 these consensus documents are not necessarily portable to the specific context of cardiac surgery, for which the mechanism, magnitude, and clinical implication of certain events are fundamentally different . The sts composite does not include perioperative mi, which is in part due to its low ascertainment reliability.1 recommended definitions of perioperative mi vary considerably, from a highly restrictive approach requiring evidence of an acute coronary embolus57 to a multifaceted approach incorporating clinical and biomarker criteria.55 other potentially important cardiac surgery outcomes, such as prolonged postoperative mechanical ventilation, have not been uniformly defined or adopted for use . Varc is a context - specific consensus document focused on transcatheter aortic valve replacement; it provides standardized end points with clearly defined criteria for reporting.5 a meta - analysis showed that the varc end points were frequently being implemented to report clinical outcomes.58 although there has been an initial attempt to develop a similar document focused on pediatric cardiac surgery,59 there has yet to be an attempt in adult cardiac surgery . Because our search was limited to 5 scientific journals (for feasibility purposes), we did not capture trials published in other journals . The selected journals represent the highest ranked impact factors in their respective subspecialties of cardiothoracic surgery, cardiology, and general medicine, and we expect that the heterogeneity of outcome reporting could have been more pronounced if we had included smaller lower ranked studies . Conversely, the selected trials encompassed a wide variety of interventions and comparators (surgery versus surgery, surgery versus transcatheter procedure, surgery plus adjunctive medical therapy versus surgery alone), such that the heterogeneity of outcome reporting could have been less pronounced if we restricted our selection criteria to one of these types of trials . We excluded trials that did not report a clinically driven primary outcome, and this also led to underrepresentation of smaller studies that were underpowered to assess clinical events . We chose to focus on clinical events because these will likely form the basis of future efforts to develop standardized guidelines for reporting outcomes . Because our search was limited to 5 scientific journals (for feasibility purposes), we did not capture trials published in other journals . The selected journals represent the highest ranked impact factors in their respective subspecialties of cardiothoracic surgery, cardiology, and general medicine, and we expect that the heterogeneity of outcome reporting could have been more pronounced if we had included smaller lower ranked studies . Conversely, the selected trials encompassed a wide variety of interventions and comparators (surgery versus surgery, surgery versus transcatheter procedure, surgery plus adjunctive medical therapy versus surgery alone), such that the heterogeneity of outcome reporting could have been less pronounced if we restricted our selection criteria to one of these types of trials . We excluded trials that did not report a clinically driven primary outcome, and this also led to underrepresentation of smaller studies that were underpowered to assess clinical events . We chose to focus on clinical events because these will likely form the basis of future efforts to develop standardized guidelines for reporting outcomes . Outcome reporting in the cardiac surgery literature is heterogeneous, and efforts should be made to standardize the outcomes reported and the definitions used to ascertain them . Measures of functional status and resource utilization are currently underreported and should be integrated in standardized reporting schema . The development of standardizing outcome reporting is an essential step toward strengthening the process of evidence - based care in cardiac surgery.
A total of 130 consecutive p. aeruginosa isolates recovered from different clinical specimens at sanjay gandhi postgraduate institute of medical sciences in lucknow, uttar pradesh, india, during november 2013april 2014 were included in the study; all specimens were collected from within the state (figure 1). Aeruginosa isolates were identified by standard microbiological techniques (6) and further confirmed by phoenix automated identification and sensitivity systems (bd biosciences, san jose, ca, usa). The drug susceptibility profile was interpreted by using clinical and laboratory standards institute breakpoints (7). These isolates were subjected to pcr by using blandmspecific primers (8) followed by amplicon sequencing . The isolates were further screened for high - level aminoglycoside resistance by their ability to grow on muller hinton agar containing amikacin and gentamicin 256 mg / l each as a marker for 16s rmtase (3). A total of 33 (86.84%) isolates were positive for high - level aminoglycoside resistance . Each of these isolates were further subjected to pcr for detection of 16s rmtases (arma and rmta rmth) by using primers and conditions described previously (25); 17 (51%) isolates were positive for 16s rmtases . Their distributions were as follows: arma in 6 (18%); rmtb in 4 (12%); arma + rmtb in 4 (12%); rmtc in 2 (knpa1a and knpa1c) (6%); and rmtc + rmtf in 1 (knpa1b) (3%). Knpa1a, knpa1b, and knpa1c were further characterized; sequence analysis of amplicons confirmed rmtc with 100% nucleotide identity originally described in proteus mirabilis strain ars68 from japan (9) and assigned embl / genbank nucleotide accession nos . Mics of the 3 isolates for different aminoglycosides, -lactams, -lactam/-lactamase inhibitor combinations, carbapenems, and colistin are provided in the table . Location of uttar pradesh state, india, showing geographic location of patients infected with 16s rrna methyl transferase positive pseudomonas aeruginosa (gray shading) and rmtc - positive isolates knpa1a, knpa1b, and knpa1c (black dots); knpa1b was also positive for rmtf . Knpa1a was isolated from surgical drainage from a woman, 59 years of age, who had hypertension and underwent an abdominal hysterectomy for cervical carcinoma, followed by external beam radiotherapy . Her condition stabilized, and she was discharged with advice for repair of the fistula, but she did not return for further treatment . During hospitalization, she received multiple antimicrobial drugs . Knpa1b was isolated from endoscopic nasobiliary drainage (enbd) collected from a man, 57 years of age, who had extrahepatic biliary obstruction as a complication of hilar cholangiocarcinoma . Stent block and fever occurred, necessitating a repeat of the procedure and drainage of fluid . He was discharged with the drainage tube in situ and was advised to return for surgery . Knpa1c was isolated from a man, 68 years of age, who had diabetes mellitus and hypertension . He had stricture of the urethra and meatal narrowing after having a transurethral prostate resection . A urinary tract infection was diagnosed, and p. aeruginosa (knpa1c) was recovered from urine . The patient received piperacillin / tazobactam and colistin combination therapy; urine culture was sterile on day 3 posttreatment . Resistance genes such as metallo--lactamases (e.g., imp, vim, sim, gim, spm) and extended - spectrum -lactamases such as tem, shv, ctx - m, and ampc were detected by using pcr (8) (table). To study genetic relatedness among the 3 isolates, genomic dna in agarose blocks was separated on 1.0% agarose gels in 0.5 tris - borate - edta buffer with the chef ii d - mapper xa pulsed - field gel electrophoresis system (bio - rad, hercules, ca, usa) following standard conditions (10). Multilocus sequence typing (mlst) was done according to protocols described in the pseudomonas aeruginosa mlst database (http://pubmlst.org/paeruginosa). Seven chromosomal genes were pcr amplified and sequenced; the sequences were compared with those on the mlst database to determine allele numbers and sequence types (sts). Knpa1a, knpa1b, and knpa1c belonged to st764, st902, and st880, respectively . * caz, ceftazidime; ctx, cefotaxime; fep, cefepime; atm, aztreonam; cps, cefoperazone / sulbactam; tzp, piperacillin / tazobactam; ipm, imipenem; mer, meropenem; col, colistin; ak, amikacin; g, gentamicin . Lane 1, ladder; 2, knpa1a; 3, knpa1b; 4, knpa1c . B) chromosomal location of rmtc, rmtf, and blandm-1 genes by i - ceui - digested genomic dna of p. aeruginosa isolates . Lane 5, knpa1a; 6, knpa1b; 7, knpa1c; smears show southern blot analysis of genomic dna with probes specific to 16s rrna, rmtc, rmtf, and ndm-1 genes . Isecp1 was previously shown to promote both expression and transposition of rmtc (11); hence, to assess association of isecp1 with rmtc, pcr was performed on the 3 isolates with primer pairs isecpir - f and rmtc - down and isecp15 and rmtc - r, as described (12). Sequence analysis of amplicons revealed association of an intact isecp1 element with rmtc in knpa1a; however, complete isecp1 could not be amplified in knpa1b and knpa1c, corroborating earlier observations of either partial deletion of this element or role of a different isecp1-like element in the spread of rmtc in gram - negative bacteria (13). Attempts to transfer rmtc from all 3 isolates to rifampin - resistant escherichia coli 20r764 and ciprofloxacin - resistant p. repeated attempts to obtain amikacin - resistant (mic 16 g / ml) transformants of e. coli dh5 and p. aeruginosa pa01 by electroporation with plasmid preparation by using the kado and liu method (14) were also unsuccessful, despite successful transfer of control plasmids . To determine the location of rmtc, rmtf, and blandm-1, genomic dna from the 3 isolates was digested separately with restriction enzyme i - ceu-1 (new england biolabs, beverly, ma, usa), separated by pfge, and subsequently assayed with 16s rrna, rmtc, rmtf, and blandm-1 probes (13). All these probes were hybridized with chromosomal dna (figure 2) and not with plasmid extract . This result shows that rmtc, rmtf, and blandm-1 were located and stabilized on the chromosome of p. aeruginosa . We describe an occurrence of 16s rmtases rmtc and rmtf in clinical isolates of p. aeruginosa co - producing blandm-1 . The rmtc and rmtf genes might have been acquired from plasmids as part of mobile genetic elements and finally integrated and stabilized on the chromosome, but the underlying mechanism of transmission needs to be elucidated . Further, spread of multidrug - resistant p. aeruginosa strains expressing rmtc with and without an intact isecp1 element and ndm-1 is of major clinical concern and calls for further studies to limit the spread of such strains.
The field of small - molecule organocatalysis via noncovalent interactions has seen rampant growth over the past decade . This area, which aims to mimic the mechanisms used by nature in enzyme catalysis, is attractive due to its potential for high catalyst tunability and substrate specificity, as well as obviating the use of metals . While such research has resulted in many catalysts operating through bifunctional mechanisms, the primary interaction responsible for electrophile activation occurs through hydrogen - bonding to an acceptor moiety . The consequent lumo - lowering results in rate enhancement . In comparison to metal - based systems (i.e., lewis acids), current metrics to estimate the reactivity of hydrogen bonding catalysts are ineffective . Although pka values of the donor and acceptor may be used to infer hydrogen - bond strengths, this analysis fails to account for several important secondary interactions, including sterics, dual - activation, and binding geometry . As a result, the discovery of reactions compatible with hydrogen - bond catalysis is far outpacing understanding of catalyst interaction and mechanism . Indeed, while certain privileged organocatalyst motifs have been identified to be successful for several reaction types, rational design of these structures remains limited, relying on trial and error to achieve optimal reactivity and selectivity . In a previous communication, we described preliminary results showing the utility of small organic chromophore s for the detection of hydrogen - bonding interactions by uv herein, we assess the general utility of this colorimetric sensor as a predictive gauge for the relative reactivity of a broad range of organocatalysts, including several widely used motifs, with the goal of encompassing many different hydrogen - bonding arrays . An additional goal was to validate the sensor measurements across significantly different reaction profiles, particularly those involving noncarbonyl electrophiles . The lack of comprehensive rate data for a range of catalysts in different reactions is a barrier to understanding the factors controlling catalytic activation . As a consequence, the relative rates have been measured for an array of catalysts in two reactions with different groups that interact with the catalysts . The sensor signal has been analyzed with respect to reaction profile, catalyst structure, acidity, and acceptor preference . Our findings establish the sensor as a useful substrate surrogate for probing and gauging catalyst performance . Moreover, the data unequivocally establish that catalyst structure and binding mode are far more relevant to catalytic activity than acidity . A key consideration in designing a method for measuring hydrogen - bond strengths is the very broad range of these noncovalent interactions (0.240 kcal / mol). Observing the very weak range of these interactions is a challenge with commonly employed spectroscopic techniques . For example, despite successful application in measuring lewis acid binding effects, preliminary nmr studies proved too insensitive for detecting the interactions of weak hydrogen - bonding catalysts with a carbonyl acceptor . We proposed an alternative approach using the sensitivity of uv vis absorption profiles, in which a change in electronic excitation of an acceptor chromophore occurs upon binding to a hydrogen - bond donor (scheme 1). Specifically, imidazopyrazinone s displays solvatochromism with protic solvents as well as color changes with a small number of lewis acids . We postulated that upon treatment with various hydrogen - bond donors, the carbonyl moiety of s would act as an acceptor moiety . The resulting hydrogen - bonding interaction would alter the electronic transition of the chromophore, detectable by simple uv vis spectroscopy . In line with this reasoning, treatment of sensor s in dichloromethane with various hydrogen - bond donors resulted in visible hypsochromic (blue) shifts (figure 1). Importantly, compounds anticipated to be weaker donors, such as diphenylthiourea (1), yielded significant changes in sensor signal . Variation of binder concentration resulted in titration - like behavior, with a measurable end point upon saturation of sensor with catalyst . An array of catalysts (chart 1), varying in structure and anticipated strength, was examined with the colorimetric sensor, and the max upon saturation was determined . Response in the uv vis spectrum of s upon increasing amounts of 12 . [s] = 2.22 10 m in ch2cl2, = 0 to 1.78 10 m. dft molecular orbital calculations were performed on bound and unbound sensor for selected hydrogen - bonding agents to gauge the orbital perturbation (table 1). The calculated lowest energy transition accurately predicts the observed absorbance maximum for the free sensor . Lumo energy gap was larger for all bound complexes, in accord with the empirically observed hypsochromic shift in figure 1 . An increased shift (lower max) is predicted for binders of ostensibly greater strength (e.g., proton> benzoic acid> phenol). The uv absorption behavior of the sensor with the hydrogen - bonding agent can be represented as shown in figure 2a . The lowest energy electronic transition may be ascribed to the n (homo) to * (lumo) transition, e1, corresponding to the measured max . As supported by the above calculations, addition of a hydrogen - bonding agent stabilizes the ground state (homo) to a greater extent than the excited state (lumo), i.e., e3> e2 . As a consequence, a hypsochromic shift is observed upon interaction of the sensor with the hydrogen - bond donors . For comparison, figure 2b illustrates the energy diagram for a typical reaction with a hydrogen - bonding catalyst, in which catalysis is effected by lumo - lowering of the electrophile (ea). E2 is proportional to ea, i.e., the wavelength shift of the bound sensorcatalyst is proportional to the rate enhancement afforded in a reaction with the hydrogen - bonding catalyst . (a) proposed energy diagram of the lowest energy electronic transition of the sensor upon interaction with catalysts of increasing strength, corresponding to the hypsochromic wavelength shift (max). (b) lumo - lowering of reactants via hydrogen - bonding catalysts, corresponding to increased reaction rates (krel). As shown in figure 1, a continuous wavelength shift was revealed upon saturation of the sensor with the catalyst . The lack of two distinct peaks in intermediate measurements containing both bound and unbound sensor indicates a rapid equilibration . Thus, plots of absorbance vs [catalyst] (see figure 3 for an example with bisamidinium 12) were used to determine the binding constants (keq) for the sensorcatalyst complex . A significant range of blue shifts was observed for the different catalyst donors, ranging from 490 to 465 nm (max 1030 nm). In general, catalysts with larger max values possessed much stronger binding constants . Since e1 is proportional to 1/max, the energetics of the interaction of the sensor with the catalysts (e3 e2) is proportional to 1/max(sensorcatalyst) 1/max(sensor). Indeed, a good correlation of this inverse wavelength shift with ln(keq) was found (figure 4). Note that in this plot, both axes are linearly proportional to energy terms: to the e of the sensor electronic absorption, and ln(keq) to g of sensorcatalyst formation . Importantly, this relationship establishes the observed wavelength shift as a reliable gauge for binding affinity of a catalyst to the sensor molecule . Uv - titration curve of catalyst 12 in ch2cl2 using [s] = 2.22 10 m. inlay: visible color change of sensor before (red) and after addition of 12 (yellow; = 1.78 10 m). All titrations were performed with [s] = 2.22 10 m in ch2cl2 . Using the sensorcatalyst wavelength shift as predictors of catalyst reactivity yields several noteworthy observations . Diol - based 30 (taddol) and silanol catalysts 31 and 32 afforded very weak shifts, despite application in numerous transformations, including rawal s seminal report on the asymmetric hetero diels alder reaction . The greater max shift of 32 compared to the related monosilanol 31 mirrors the increased reactivity of this silanediol scaffold, as elegantly reported by mattson and franz . Benzoic acids and phenols spanned the intermediate range of sensor shifts, with trends clearly based on the electronic effects of aromatic substitution . Although these structures are not as commonly incorporated as hydrogen - bond catalysts, schafmeister and co - workers have recently demonstrated the spiroligozyme catalyst 24, containing a carefully arranged carboxylic acid and phenol, as an effective ketosteroid isomerase mimic for the aromatic claisen rearrangement.n, n-diaryl thioureas and ureas, particularly those with multiple trifluoromethyl substituents such as schreiner s catalyst 4, afforded some of the largest sensor shifts, indicative of the immense utility of these structures in various organocatalysts . The internally activated bf2-urea 9 provided the largest shift within this class, in line with experimental reactivity data reported by mattson and co - workers . Finally, formally cationic species, including guanidinium, amidinium, and takenaka s azaindolium 14(22) were the strongest binders, with wavelength shifts ranging from 26 to 34 nm (max = 473465 nm). Interestingly, one of the strongest noncationic binders was thiophosphoramide 16, possessing a pocket of three potential n h donors . Squaramide - containing scaffolds have yielded excellent results as hydrogen - bond activators; however, these compounds possess limited solubility, and are typically employed as heterogeneous catalysts . Representative squaramide 11, containing the common n-3,5-(cf3)2aryl and n-alkyl array, was synthesized, and gave an apparent sensor end point of 480 nm . Due to its relative insolubility, an accurate binding equilibrium value could not be determined . It is worth noting the experimental ease with which the sensor metric can be obtained . Compound s itself is easily obtained in 2 steps from commercial materials, and very little sensor or catalyst (particularly for strong catalysts) is necessary to obtain the wavelength shift . The titration experiment is largely insensitive to moisture, as illustrated by the poor binding observed in the sensor titration with water . Applying the method of continuous variation to the sensor with catalyst 12 revealed a 1:1 binding stoichiometry with the sensor molecule (figure 5). This observation is significant, since several other binding situations may be postulated, including donation of one catalyst molecule to several sensors (4 equivalent n job plot analysis of catalyst 12 with sensor s showing 1:1 binding stoichiometry . Benzoic acids and phenols offer useful templates to study electronic effects on sensor signal due to availability and well - understood behavior of aromatic substitution . Due to solubility limitations, ortho - substituted benzoic acids were studied rather than the para - substituted analogs . The electronic effects from substitution on the sensor interaction can be illustrated via a hammett - type plot, as shown in figure 6 . As may be anticipated from the brnsted catalysis law (see section 2.6 for further discussion), increasingly electron - withdrawing substituents on these structures correlate with larger hypsochromic shifts of the sensorcatalyst complex . For both catalyst sets, highly linear relationships are evident with substituent parameters indicating that the wavelength shift provides an accurate readout of electronic perturbation on the hydrogen - bonding ability . Correlation of hammett parameters for o - benzoic acids (ortho) and p - phenols (para) with sensorcatalyst wavelength shifts . Notably, the wavelength shifts seen with the sensor do not correspond directly with pka either in water (figure 7a, r = 0.0007) or dmso (figure 7b, r = 0.0950). However, correlations are observed for closely related catalyst structures, wherein electronic perturbations modify the acidity of the donor moiety without introducing significant secondary effects . This observation provides potential for the sensor to estimate pka values within a series of related compounds . Persubstituted phenol 29 deviates from other phenolic catalysts, which may be attributed to the increased steric demand around the donating o plot of catalyst acidity in water (a) or dmso (b) vs sensorcatalyst wavelength shifts . The correlation of the observed blue shift with the binding strength across a large range of hydrogen - bond donors proved the metric to be able to quantitatively detect these interactions . However, this finding does not necessitate a correlation with catalyst reactivity . In order for this correlation to occur, the sensor must be a good facsimile of the substrate that is undergoing reaction . Other factors, including alternate binding modes and steric effects, might come into play when a substrate interacts with hydrogen - bonding agent in a catalyzed reaction . To be a useful metric for the community, the sensor signal must correlate to empirically obtained rate enhancement via hydrogen - bond catalysis (scheme 2). Myriad reaction profiles have been reported that are established to proceed via hydrogen - bond activation of the electrophile (lumo - lowering activation). In order to best isolate the reactivity enhancement offered by the catalysts strictly due to hydrogen - bonding, we first targeted a reaction where the electrophile has only one possible point of interaction with the catalyst, and the nucleophile does not contain binding points (i.e., no heteroatoms). Additionally, the reaction should have minimal background rate and a method to easily analyze starting material and/or product concentrations . The reaction of methyl vinyl ketone (mvk) with cyclopentadiene (cp) offers a useful reaction platform that fulfills these criteria (scheme 3), and has been used to gauge the relative strength of thiourea and bisphenol catalysts previously . Importantly, the binding in the sensorcatalyst complex is very similar to that of the mvkcatalyst intermediate as both interactions arise from a carbonyl acting as a hydrogen - bond acceptor . Systematic investigation of the diels alder reaction of mvk and cp with a variety of catalysts was performed under pseudo - first order conditions as described in scheme 3 . Kinetic data was acquired via continuous sampling (5 min intervals) by h nmr spectroscopy, and each rate measurement was performed in triplicate . Relative rate constants, krel, were calculated as described in eq 1 from the observed pseudo - first order rate constant kobs and background rate kbackground, and were normalized for catalyst concentration n. the resulting values directly provide the rate enhancement afforded by the catalyst.1 as displayed in figure 8, a plot of ln(krel) against the inverse sensor wavelength shift of 18 catalysts shows an excellent correlation . Catalysts with greater blue shifts when treated with the sensor show greater activity in the diels alder reaction via correspondingly greater lumo lowering of the ketone in the dienophile . More precisely, the change in energy of the sensor upon binding with the catalyst is proportional to the change in activation energy of the hydrogen - bond catalyzed diels alder reaction . Correlation between the sensorcatalyst wavelength shift and catalyst rate enhancement in the diels alder reaction between mvk and cp . The observed correlation establishes that, at least in this class of reaction, the sensor signal is a good indicator of lumo - lowering ability of these small molecules as hydrogen - bond catalysts . Importantly, the results also indicate that the binding interaction of the sensor with catalysts is similar to that of methyl vinyl ketone with catalyst, i.e. The sensor is a useful gauge of carbonyl activation . The addition of various nucleophiles into nitroalkenes is one of the most widely used reaction motifs in hydrogen - bonding catalysts; it is often used as a measure of reactivity when developing and comparing novel catalyst structures . To test the effectiveness of our sensor metric beyond the diels alder reaction crafts addition of n - methylindole (34) into nitrostyrene 35 (scheme 4). Deuterated 35 was easily prepared via henry condensation using d3-nitromethane with the corresponding aldehyde . Nucleophile 34 provides a reasonably active coupling partner while minimizing potential catalyst interactions [pka(h2o) n - methylindolium = 1.8]. Thus, the effects of hydrogen bonding catalysts on the activation of the styrene electrophile via binding to the nitro acceptor can be cleanly delineated . Significantly, the catalyzed rates of this reaction allow comparison of the effects of hydrogen - bonding catalysts on carbonyl acceptors (the sensor and mvk) versus nitro acceptors (nitrostyrene 35) as outlined in scheme 5 . While binding geometries to carbonyl groups are anticipated to be similar, an analogous correlation may not be automatically presumed for a nitro group . In particular, the nitro group contains a formally delocalized negative charge across three atoms, and has been suggested to form -activated intermediates with certain catalyst structural types such as squaramides and thioureas . As illustrated in figure 9, kinetic reaction data was acquired using h nmr spectroscopy . Although initial h nmr spectroscopic studies with proteo-35 provided usable data, employment of a deuterium label provides exceptional signal isolation . Moreover, overlap of signals from the catalyst is completely mitigated, since only substrate, product, and internal standard exhibit appreciable resonances . As a result, any catalyst can be readily analyzed . (a) stacked h nmr plot and (b) kinetic profile for the friedel crafts reaction shown in scheme 4 using catalyst 12 . Conditions: = 1.33 m, = 0.133 m, = 0.133 m, = 2.67 10 m. the cdcl3 peak arises from natural abundance in chcl3 . Crafts reaction was studied under pseudo - first order conditions using the broad series of hydrogen - bond catalysts shown in chart 1 . The relative catalytic strength of each catalyst was calculated according to eq 1, using averaged kobs values from duplicate trials . A plot of ln(krel) and inverse sensor wavelength shift is shown in figure 10a . Similar to the diels alder reaction, and predicted based on sensor shifts, cationic binders proved to be the most effective catalysts, followed by electron - deficient ureas and thioureas . Squaramide 11, employed here as a heterogeneous catalyst, provided moderate rate enhancement as predicted by its sensor wavelength shift . The krel values reported for these catalysts and 4 align closely with the values measured in this work . (a) correlation observed between the sensorcatalyst wavelength shift and catalyst rate enhancement in the friedel crafts reaction . The overall correlation of the sensor wavelength shift with catalyst strength for the friedel crafts reaction is good (r2 = 0.84), but not as strong as that found in the diels alder reaction (r2 = 0.95). Analysis of this data suggests that the binding interaction of a hydrogen - bonding catalyst with the sensor carbonyl does not fully mimic that of a hydrogen - bonding catalyst with the activated nitroalkene . Closer inspection reveals that the wavelength shift is correlated even more strongly to catalyst strength within an isostructural catalyst series (figure 10b). Specifically, the catalysts can be placed in four groups based on their general structure: benzoic acids, phenols, y - type binders, and other n h binders . The y - type group consists of catalysts possessing two n h donor groups separated by a single atom, such as ureas, thioureas, guanidines, bis - sulfonamides, etc . The other n h binders include those catalysts that have more than one atom separating the donor array (squaramide 11, bisamidinium 12) or can only donate one n interestingly, the silanediol catalyst 32 exhibits reactivity falling nicely in line with benzoic acids, possibly due to a similar o h geometry . This analysis quantitatively shows that y - type structures are superior for nitroalkene activation . In contrast, phenols provided the least activation relative to their binding interaction with the sensor . Overall, the sensor provides a good assessment of relative reactivity of hydrogen - bond catalysts in the friedel crafts reaction . The presence of stronger correlations within catalyst structural classes is consistent with some catalysts activating nitroalkenes via a different mode (e.g., -binding) that is not completely captured by the interactions of the catalysts with the carbonyl of the sensor molecule . This analysis underlines the complex nature of hydrogen - bonding, emphasizing that caution must be exercised in generalizing catalyst reactivity or selectivity from one reaction to another . A general equation to describe catalyst strength for reaction r based on sensor response () is presented in eq 2 . Parameter rr (slope) represents the responsiveness of the rate per unit catalyst strength as determined by the sensor measurement . Parameter cr (y - intercept) corresponds to inherent complementarity of the catalyst to the electrophilic reaction partner.2 the parameter values (table 2) for the reactions shown in scheme 3 (diels alder) and scheme 4 (friedel crafts) were obtained using the kinetic data from figures 8 and 10, respectively . Comparison of the rda and rfc values reveals that the friedel crafts is more sensitive to catalyst strength . In other words, the same catalyst produces a greater relative rate enhancement for the friedel crafts reaction than for the diels alder reaction . Similarly, a given wavelength shift of the sensor by a catalyst will cause a greater reactivity change in the friedel crafts vs the diels alder reaction . On the other hand, the cda and cfc values indicate the inherent complementarity of the electrophilic substrate with catalysts; greater complementarity translates to greater reactivity . Notably, the c values represent reactivity when there is no wavelength shift (no perturbation of the sensor by the catalysts) and represent the lower limit of lumo activation afforded by the catalyst . Catalyst group - specific coefficients rr and cr can be introduced (eq 3) to account for variation if the sensor binds the catalyst differently than the reaction electrophile . Due to the strong correlation of the sensor shift to relative rates independent of catalyst structure, coefficients are unnecessary for the diels alder reaction (rda cda 1). As discussed in section 2.4, the sensor does not completely model catalyst binding to the nitrostyrene acceptor of the friedel crafts reaction . Accordingly, the slope and intercept data from figure 10b were combined with the r and c values from table 2 to afford coefficient values for rfc and cfc, respectively, as provided in table 3.3 the coefficient values in table 3 reveal general trends between the different catalyst structural types and rate in the friedel again, rfc values are a measure of responsiveness of a given catalyst architecture to a perturbation in sensor binding . For y - type and phenolic catalysts, rfc is noticeably higher than the other n h binders, and particularly benzoic acids . Thus, for the same amount of wavelength shift, the phenol and y - type catalysts provide greater increases in reactivity relative to the other n h binders and benzoic acids . This observation indicates that the sensor can assess electronic effects in a catalyst series . Comparison of the hammett effects on (figure 6; acid = 5.8, phenol = 4.4) with those on ln(krel) (acid = 0.60, phenol = 0.33) provides support for this assertion; both measures show a stronger electronic effect for the carboxylic acid series . On the other hand, lower cfc values indicate the inherent complementarity of a given catalyst architecture . For example, the y - type binders activate nitro electrophiles to a greater extent at a given wavelength shift relative to phenols or the other n h binders . Interestingly, the cfc value for benzoic acids would predict high catalytic activity relative to y - type binders, but only in the weak binding regime (left side of plot). Due to the low rfc value for benzoic acids, the trends invert such that y - type binders are superior in the strong binding regime (right side of plot). Considering both terms together, the y - type binders are both more complementary to the nitroalkene and more efficient at lumo lowering, thereby providing superior reactivity . Acidity values have widely been used as a guiding principle in hydrogen - bond catalyst design, under the premise that a more acidic donor will form a stronger interaction and stabilize the buildup of anionic charge in the transition state to a greater extent . Indeed, several reports have observed increased activity with judicious electronic tuning of the donor hydrogen . However, even ostensibly subtle changes to catalyst structure can cause secondary factors to override the reliability of pka as a predictive measure, as demonstrated by cheng s recent study on thiourea derivatives and even noted in the seminal work by hine on mono- and bis - phenols . Having proved the effectiveness of the sensor signal as a gauge for catalyst strength, we undertook a comparison with acidity to determine the similarities and differences between the two metrics . Aggregate data for all catalysts spanning 3 orders of magnitude in reactivity for the diels alder reaction and 4 orders in the friedel crafts reaction is organized by increasing sensor wavelength shift in table 4 . Recent efforts by schreiner and others provided accurate acidity values of common hydrogen - bond donors . The brnsted catalysis equation (eq 4), which describes the relationship for the rate of an acid - catalyzed reaction with the pka of the acid, was applied to this data.4 value for 2-naphthol . Figures 11 and 12 display the results for selected catalyst series in the diels alder and friedel crafts reactions, respectively . These plots prove the linear free energy relationships (lfers) between catalyst acceleration and acidity among catalysts of very similar structure . In general, these lfers exhibited a narrow range of values (0.390.47; table 5), indicating a similar degree of hydrogen - bonding in the transition states and that these reactions are not proceeding through formal protonation (= 1). Slightly higher values found for benzoic acids in the diels alder (= 0.57) indicate a greater degree of proton transfer in the transition states consistent with the ionic nature of benzoic acids . Lower values for ureas in the friedel crafts (= 0.31) indicate a lesser degree of proton transfer in the transition states in line with the covalent nature of the n h bonds . Brnsted catalysis plot for the diels alder reaction, demonstrating lfers for closely related catalyst groups . More significantly, these figures clearly demonstrate the inherent limitations of estimating catalyst strength using acidity metrics . Based solely on pka measurements, thioureas would be predicted to provide much higher activity . In practice, the opposite is observed where ureas exhibited greater (6, 7 vs 2, 3) or similar (8 vs 4) activation of the nitro group compared to their thiourea analogues, despite the much greater acidity of the thioureas (45 orders of magnitude difference). Highly reactive catalysts not belonging to a clearly defined series, including common brnsted acids, are included in figure 11 and further highlight the disparity between acidity and activity . Interestingly, takenaka s azaindolium catalyst 14 displayed much higher activity than ppts, despite similar acidities and similar pyridinium in conclusion, a sensor is described that provides an assessment of the reactivity over 34 orders of magnitude for 33 hydrogen - bonding catalysts . Useful correlations are obtained between the wavelength shifts that catalysts cause to the pyrazinone sensor and the rate - determining steps in diels alder and friedel crafts reactions . As a result, only the wavelength shifts upon saturation of the sensor with catalysts need to be measured vs the more time - consuming titration studies . Consequently, the sensor may also find use as a rapid means for measuring pka values in series of related compounds . In contrast to established acidity (pka) values, the sensor wavelength shift is a highly predictive metric for the relative reactivity of catalysts encompassing a broad range of structures and strengths . Notable acidity - activity disparities include cationic catalysts (low acidity, high reactivity), and benzoic acids (high acidity, low reactivity), the strengths of which are more accurately gauged by their interaction with the sensor . Overall, the sensor is a superior surrogate for the diverse electrophiles (enone and nitroalkene) used compared to water and is better able to assess secondary interactions . The data collected was used to formulate the relationship described in eq 3, which provides a direct means of assessing the reactivity of a catalyst in a given reaction using the sensor signal . The resulting parameters also reveal relationships between substratecatalyst binding, catalyst - induced lumo - lowering, and catalyst structure . Investigation of additional catalyzed reactions with the sensor, empirically or computationally, has the potential to expand eq 3 to achieve quantitative predictive power across a large range of reaction platforms and catalysts (hydrogen bonding, brnsted acid, and lewis acid).
Olfactory neuroblastoma or esthesioneuroblastoma is a rare malignant neoplasm of neuroectodermal origin arising from olfactory membrane of the sinonasal tract . It was originally described in french literature by berger et al . In 1924 who coined the term the various synonyms used for this entity are olfactory esthesio - neuroepithelioma, esthesio - neurocytoma, esthesio - neuroepithelioma, esthesio - neuroblastoma, olfactory neuroblastoma and olfactory placode tumor . The first case in american literature this tumor has broad histologic spectrum and is often histologically confused with peripheral neural ectodermal tumors and is notorious for its wide clinical behaviors . The common clinical symptoms include nasal obstruction, recurrent epistaxis, hyposomia, rhinorrhea, headache and visual disturbances . Here we report a rare case of esthesioneuroblastoma in a female with presenting symptom as tooth pain . A 50-year - old female reported to the department of oral medicine with the complaint of pain in the left side of the upper jaw since seven months, which was spontaneous, dull and reduced on taking analgesics . She visited a general dentist five months ago and maxillary left second molar was extracted as it was considered as the causative tooth for pain, however, the patient did not get relief and the pain continued . She noticed swelling of left side of the face, which gradually increased in size and there was a concomitant decrease in size of her left eye opening and the eyeball was pushing superiorly since two months . Her medical and family history was noncontributory and upon general physical examination, all her vital signs were within normal limits . On extraoral examination, a diffuse swelling was seen on the left maxillary area extending from infraorbital margin to mid of the cheek supero - inferiorly and ala of nose to outer canthus of eye antero - posteriorly, which was soft in consistency and tender on palpation [figure 1a]. Intraorally, swelling was evident on palpation extending from maxillary tuberosity to the vestibule causing vestibular obliteration till the posterior aspect, which was tender and soft in consistency on palpation [figure 1b]. Clinical photograph showing swelling on maxillary region and decrease in the size of the left eye (a). Intraorally, vestibular obliteration was evident (b) on the basis of history and clinical examination, a provisional diagnosis of malignant neoplasm of maxillary sinus was considered . The coronal ct revealed a large mass originating in left maxillary sinus with destruction of its medial, superior and postero - lateral wall . Medially, this mass was extending into left nasal fossa leading to marked luminal compromise on the left side and superiorly extension into left orbit was noticed . Posteriorly it was extending into infra - temporal fossa, left masseteric space and left pterygomaxillary fissure . Partial erosion of anterior maxillary wall, hard palate and lateral pterygoid plate on left side and extension of this mass from lateral maxillary wall into left cheek was also noticed [figure 2a and b]. Coronal computed tomographic scan showing extension of lesion into sinus and nasal fossa incisional biopsy was performed and on histopathological examination, hematoxylin and eosin stain showed basophilic cells arranged in large lobar sheets with intertwining connective tissue septa . Cells had large round nucleus with minimal pleomorphism and a thin rim of cytoplasm . Pronounced vascularity and formation of pseudorosettes (homer - wright type) were seen in some areas, suggestive of esthesioneuroblastoma [figure 3a and b]. Immunohistochemistry revealed strongly positive neuron - specific enolase among the tumor cells and s-100 showed weak association [figure 4a and b]. So based on clinical examination and the investigations performed, a final diagnosis of esthesioneuroblastoma was given . The case was referred to the oncology institute for the management and the patient was started with chemotherapy, cisplatin 50 mg (per day) and 5 fluorouracil 1 mg (per day), and recalled after 15 days; there was improvement in the symptoms after which the patient was lost to follow up . Photomicrograph showing cells arranged in sheets and intervening connective tissue stroma (a) (h&e stain, 40). Photomicrograph showing cells with large round nucleus, thin rim of cytoplasm arranged in large sheets with intertwining connective tissue septa and formation of pseudorosettes (homer - wright type) (b) (h&e stain, 100) immunohistochemistry showing strongly positive neuronspecific enolase among the tumor cells (a) (ihc stain, 200) and weak association with s-100 protein (b) (ihc stain, 200) esthesioneuroblastoma is a rare malignant tumor arising from olfactory epithelium and its neural origin was established by trojanowaski et al . In 1982 who demonstrated the presence of neurofilament proteins (nfp) in the tumor cells . Though, it has been reported in patients as young as two years to 90 years, a bimodal age distribution has been noted in the second and sixth decades of life . There are no known etiologic agents for human olfactory neuroblastoma, however, injection of diethylnitrosamine in syrian hamsters and n - nitrosopiperidine in rats has produced tumors histologically identical to human olfactory neuroblastoma . The common symptoms associated are unilateral nasal obstruction (70%) and epistaxis (46%), and less common manifestations are anosmia, headache, pain, excessive lacrimation and ocular disturbances . In the present case, the patient complained of pain in left maxillary molar teeth, which is a rare symptom and also, there was decrease in size of left eye, which may be due to superior extension of tumor in the left orbit . The most common site of origin is in the upper nasal cavity in the region of the cribriform plate; other primary sites such as maxillary sinus and nasopharynx have also been reported . Esthesioneuroblastoma is generally believed to be derived from the neurosensory receptor cells of olfactory mucosa . Other sources of origin suggested include sphenopalatine ganglion by escat, organ of jacobson, ganglion of luci by martin et al . And olfactory placode . The tumor in the present case was seen to arise from left maxillary sinus and extended to involve nasal fossa, infra - temporal fossa, masseteric space, cheek and the left orbit . According to this classification, stage a tumors are confined to the nasal cavity, stage b tumors have paranasal sinus extension and stage c tumors have extra - paranasal extension including the involvement of the cribriform plate, base of skull, orbit or intracranial cavity . This classification was modified by morita et al . In 1993 who established stage d in the classification for tumors with metastasis to cervical lymph nodes or distant site . Kadish's classification has been used by various authors and appears to correlate well with the clinical outcome . The present case can be categorized as belonging to stage c of kadish's classification . In evaluation of esthesioneuroblastoma, the extent of disease is best determined by pre- and post - contrast mr imaging in which there is intense signal in t2-weighted images with marked enhancement of t1-weighted images after gadolinium injection . Details of bone erosion (lamina papyracea, cribriform plate and fovea ethmoidalis) are better demonstrated by ct scan . Tumor is presented as homogenous density mass, equal or greater to the surrounding soft tissue and no tumor cysts or calcifications . Coronal images were of value in evaluating extension to the orbit and through the cribriform plate and the anterior cranial fossa . (2001) demonstrated the usefulness of technetinium-99m - ethyl cysteinate dimer (tc - ecd) spect in detection of olfactory neuroblastoma . (1960) stated that the characteristic histologic features of olfactory neuroblastoma are plexiform intercellular fibrils, round - to - oval - shaped nuclei with scanty cytoplasm, distinct and sharply defined chromatin, which may be coarse or fine, compartmentation of sheets of neoplastic cells into lobules by slender vascular fibrous septa, true neural rosettes (flexner - wintersteiner type) and pseudorosettes (homer - wright type). They concluded that if fibrils were absent, then the tumor cannot be classified as neuroblastoma with certainity . Histologically neuroblastoma has been classified as olfactory neurocytoma (pattern i of mendeloff), a tumor with a sheet of round small cells separated by connective tissue septa and occasionally pseudorosettes; olfactory neuroepithelioma (pattern ii of mendeloff), a tumor containing round to oval nuclei clear nuclear membrane, scant cytoplasm and rosettes; and a tumor similar to neuroblastomas found elsewhere in the body . Immunologically, it is most reactive with neuron - specific enolase (nse) as seen in the reported case . It has shown reactivity with s-100, synaptophysin, nfp, class iii beta - tubulin and microtubule - associated protein . Light microscopy can usually establish the diagnosis, but highly undifferentiated tumors make this difficult . In case when light microscopy fails to establish diagnosis, electron microscopy is helpful . Esthesioneuroblastoma must be distinguished from lymphosarcoma, transitional cell carcinoma, plasmacytoma, reticulum cell carcinoma, small cell undifferentiated carcinoma and ewing's sarcoma . Small cell carcinoma typically is a submucosal hypercellular proliferation growing in sheets, cords and ribbons; the distinct lobular pattern of olfactory neuroblastoma is absent . The cells are small and hyperchromatic with oval- to spindle - shaped nuclei, absent nucleoli and minimal cytoplasm . Although uncommon, neural - type rosettes similar to those seen in olfactory neuroblastoma can be seen in association with small cell carcinoma . In contrast to olfactory neuroblastoma, nse reactivity in small cell carcinoma is more likely to be focal than diffusely positive and the s-100 protein staining, if present, is dispersed throughout the cellular proliferation and not limited to sustentacular cells . Ewing's sarcoma is composed of solid sheets or masses of solid round cells with very little stroma, scanty cytoplasm and ovoid large nuclei; necrosis is common . The characteristic cells of plasmacytoma and so - called cylinders in cylindromas readily help in identifying these tumors . Small cell undifferentiated carcinomas does not contain fibrils and the distinct cytoplasmic borders can be seen . The current accepted standard of treatment is craniofacial resection followed by adjuvant radiotherapy to a dose of 55 - 65 gy for kadish stages b and c. small kadish stage a disease is treated by surgery alone in some situations by some groups, but most suggest adjuvant radiotherapy for these lesions also . Neoadjuvant chemotherapy has been used in advanced disease and where complete resection is not possible, with cyclophosphamide, vincristine ifosphamide, etoposide and cisplatin combined with preoperative radiotherapy . The overall five, 10 and 15-year survival rates have been reported to be 78%, 71% and 68%, respectively . Initial multimodality therapy is associated with five - year survival of 80% for low - grade tumors and 40% for high - grade tumors . The most hazardous and often fatal complication is intracranial extension of tumor through destruction of cribriform plate and orbital plates and secondly due to distant metastasis . Prognosis has been correlated to clinical staging with five - year survival of 75 - 91%, 68 - 71% and 41 - 47% for stages other factors that have been implicated in prognosis include histologic grading, proliferation rate and ploidy . Histologically lower grade tumors (grades i and ii) have been reported to have a better five - year survival than higher grade tumors (grades iii and iv). The tumor metastasizes widely by both hematogenous and lymphatic routes, approximately 10 - 60% will experience distant metastasis . The most common site for metastasis spread are cervical lymph nodes, and less frequent are lungs, brain, bone, spinal column, breast and abdominal viscera . Metastasis to the central nervous system is infrequent and in spinal cord 80% of metastasis is in cauda equine ., this tumor has been difficult to recognize and diagnose pathologically and the wide spectrum of presentation of this tumor has resulted in its frequent misdiagnosis . Thus, the diagnosis of esthesioneuroblastoma demands a specialized and experienced head and neck pathologist, especially as incidence rate is low . Also, esthesioneuroblastoma may present, only as tooth pain as in the present case . So, it may be considered as a possible differential diagnosis, though rare in cases of dento - facial pain of idiopathic cause.
Accidents and injuries are part of daily events, and many of these injuries are initially treated by untrained personnel due to lack of health care providers on the accident site . Therefore, implementing correct first aid measures is vital for victims in emergency cases and helps to improve the overall outcome of the first aid process in emergency situations . The national first aid science advisory board defined first aid as making an assessment and implementing interventions that can be performed by a bystander (or by the victim) with minimal or no medical equipment . Several studies have been conducted around the world to evaluate the level of knowledge about first aid among different groups including university students . Some studies showed that a high percentage of students in different countries lack the appropriate first aid knowledge . Similarly, different studies showed that the immense majority of peoples had little or no first aid training . Many factors had been shown to be associated with better knowledge, including taking a first aid course during school, having a driving license, or having a higher level of education . Few reports assessing the first aid knowledge were carried out in arab countries and most of them reported that university students have poor levels of first aid knowledge . To the best of researcher's knowledge and experience, no studies have been conducted about general first aid knowledge in jordan . This gives the research a unique importance in building the cornerstone of the research background in the jordanian context . Therefore, first aid basics will equip persons around the injured person to reduce the danger posed by the accident and can make the difference between life and death in these situations . Hence, the current study has aimed at evaluating the level of knowledge about first aid process among university students in jordan . This cross - sectional study was conducted among university students in the large public university in the north of jordan using simple random sampling and involved administering a questionnaire . The study included 1500 students from the total 14 colleges at yarmouk university, jordan between september and december of 2014 . Inclusion criteria for participation involved: be an enrolled student at yarmouk university and be a jordanian citizen . The newly drafted questionnaire was presented to a reviewer panel composed of three members for refinement and re - wording of questions to ensure that statements were understandable and meaningful to the participants, and to ensure that the questionnaire consistently measured what it was intended to measure . Reliability of the surveying instrument was determined by using the internal consistency method . After administering the questionnaire used in this study, cronbach's alpha coefficients for all domains were produced and ranged from 0.77 to 0.91 indicating that the items within each domain are consistent in measuring the same attribute . The final questionnaire was composed of four sections: demographic characteristics of students (7 items), general first aid knowledge (6 items), first aid knowledge in various emergency situations (17 items), and attitudes toward first aid education (2 items). The total number of questionnaire items was 25 questions in addition to the 7 demographic characteristics . To ensure maximum representativeness, questionnaires were handed out to students during the general university requirement classes in which students from all faculties and specializations are registered after getting permission and obtained 15 min out to fill the questionnaire . Participants completed a multiple - choice questions and questions required short answers on their level of knowledge about first aid . All data were analyzed using spss (version 20 for windows) (spss statistics for windows, version 20.0 . Armonk, ny: ibm corp). Frequency distribution and descriptive criteria were calculated . The study was approved by the ethics committee at the faculty of medicine / yarmouk university, jordan and granted the number 30/2014 . This cross - sectional study was conducted among university students in the large public university in the north of jordan using simple random sampling and involved administering a questionnaire . The study included 1500 students from the total 14 colleges at yarmouk university, jordan between september and december of 2014 . Inclusion criteria for participation involved: be an enrolled student at yarmouk university and be a jordanian citizen . The newly drafted questionnaire was presented to a reviewer panel composed of three members for refinement and re - wording of questions to ensure that statements were understandable and meaningful to the participants, and to ensure that the questionnaire consistently measured what it was intended to measure . Reliability of the surveying instrument was determined by using the internal consistency method . After administering the questionnaire used in this study, cronbach's alpha coefficients for all domains were produced and ranged from 0.77 to 0.91 indicating that the items within each domain are consistent in measuring the same attribute . The final questionnaire was composed of four sections: demographic characteristics of students (7 items), general first aid knowledge (6 items), first aid knowledge in various emergency situations (17 items), and attitudes toward first aid education (2 items). The total number of questionnaire items was 25 questions in addition to the 7 demographic characteristics . To ensure maximum representativeness, questionnaires were handed out to students during the general university requirement classes in which students from all faculties and specializations are registered after getting permission and obtained 15 min out to fill the questionnaire . Participants completed a multiple - choice questions and questions required short answers on their level of knowledge about first aid . All data were analyzed using spss (version 20 for windows) (spss statistics for windows, version 20.0 . Armonk, ny: ibm corp). Frequency distribution and descriptive criteria were calculated . The study was approved by the ethics committee at the faculty of medicine / yarmouk university, jordan and granted the number 30/2014 . A total of 1500 questionnaires were distributed to students of which 1116 (74.4%) were returned . About 20% of the returned questionnaires were incomplete for which they had to be excluded, leaving a total of 883 questionnaires valid for statistical analysis . Of the 883 questionnaires included in the final analysis, 34.1% participants (n = 301) were males and 65.9% (n = 582) were females . The average (standard deviation) age of members of the sample group was 19.9 (2.6) years . Most of the respondents were undergraduates (99.3%, n = 877) compared with only 0.7% (n = 6) postgraduate students . The majority of students were studying at literary colleges (59.3%), 31.4% of scientific colleges, 6.3% of physical education college, and the remaining 2.9% were studying in health sciences . In the general knowledge domain, which asked about the correct civil defense call number, vital signs normal values or limits, and the normal blood sugar, students were more knowledgeable about normal body temperature and the civil defense call number . The percentage of students who reported correct answers about vital signs and other general information are described in table 1 . Frequency and percentage of students who reported correct answers about vital signs and other general information as per gender about 80% of students gave correct responses of the normal body temperature, however, less than half of students knew the normal pulse rate (48.2%) and normal blood sugar values (46.5%). Only a small percentage of students knew the respiration rate and normal blood pressure values . The overall knowledge of first aid in different emergency situations has revealed that participants were more knowledgeable when asked about the position of patients in cardiopulmonary resuscitation (cpr) and care in case of bleeding; however, they were least knowledgeable when asked about the correct position in coma situations and care of stabbing wounds . Participants correct responses about the care of victims in various emergency situations are shown and ranked in table 2 . Frequency and percentage of students who reported correct answers about the care of victims in various emergency situations as per gender only gender, faculty, and having previous first aid experience were significantly associated with first aid knowledge . Female students were more knowledgeable than male students in all aspects of first aid knowledge which revealed significant statistical associations . Table 3 describes the significant associations between the previous experience of students and correct responses of their level of first aid knowledge . Significant associations between previous experience of students and correct responses of their level of first aid knowledge having previous first aid experience was significantly associated with a better level of first aid knowledge among students when they were asked about vital signs (body temperature, pulse, respiration, and blood pressure), correct position in case of coma, correct site of doing cardiac messages, how to check pulse in case of coma, burn care, and care in stabbing wounds . With respect to faculty background, several significant statistical associations have been revealed . Significant associations between faculty of students and percentages of correct responses of their level of first aid knowledge remarkably, health sciences students were more knowledgeable about first aid care in all emergency situations . However, students from the scientific colleges were more knowledgeable than students from other colleges when asked about the correct emergency call civil defense number . The last two questions in the questionnaire asked students whether the media in jordan offer enough information about the care or first aid for the situations mentioned in the questionnaire and whether students think that first aid course and training should be handled at secondary schools in jordan . About three - quarters of students thought that the media does not offer enough first aid information, and about 97% of students believed that first aid course and training should be handled at secondary schools . No similar studies evaluating the level of first aid knowledge among university students in jordan exist so far . Results of the current study reveal the inability of the majority of the students surveyed to provide efficient first aid in emergency cases . Even with respect to simple questions (e.g., normal values of the pulse, respiration rate, the number of mouth - to - mouth ventilation or chest compressions in cpr), there are a large number of students, even among those trained in first aid, presenting serious lacks of basic first aid knowledge . Interestingly, students were less knowledgeable about the correct respiratory rate in an adult in 1 min . Only about 11% of the participants knew the correct respiratory rate . However, respiration is one of the most crucial vital signs of an individual . Similarly, al - khamees (2006) reported that university students in kuwait have poor levels of first aid knowledge . This result is supported by the results of an indian study, which revealed that 11.5% of male students and 15.4% of female students had a good level of knowledge on first aid measures . Having previous first aid experience was strongly associated with better first aid knowledge of students . Furthermore, in greece, hatzakis et al . Supports this result showing that trained industry workers on first aid were more knowledgeable than nontrained workers . A similar trend was noted among university students in pakistan . Moreover, a study conducted in austria demonstrated that the monstrous greater part of individuals had next to zero first aid preparing and that there was an immediate relationship between the level of emergency treatment preparing and the nature of first aid measures taken by the general population who attended accidents . Students from the health sciences and scientific colleges had better first aid knowledge compared to students of literary and sport colleges . In jordan, joining health sciences or scientific colleges requires students to get higher averages in the high school which indicates the better academic performance of those students . This notion may explain the difference in knowledge between the health sciences and scientific colleges from one side and students from other colleges on the other side . However, in fact, health sciences college is just starting at yarmouk university, and the last batch of students is still in the 1 year during which general university requirements are taught rather than core health sciences courses . Therefore, the low study year may explain this lack of knowledge among students of health sciences college . Despite the relatively large sample size (n = 883), the researcher acknowledges the limitation of this study regarding reliable generalization of results to the whole population of university students in jordan as the total participants in this particular study were sampled from a single university . Despite the relatively large sample size (n = 883), the researcher acknowledges the limitation of this study regarding reliable generalization of results to the whole population of university students in jordan as the total participants in this particular study were sampled from a single university . Overall, first aid knowledge among students at yarmouk university was considered to be insufficient . To decrease the early mortality and morbidity of accidents and emergencies, first aid should be a standard component and separate course of educational programs introduced at secondary school and college levels as well as in the media . Furthermore, first aid course should be updated at regular intervals throughout different study levels . The knowledge deficit of first aid measures among university students is evocative that only a minority of people have adequate emergency treatment preparing . It is suggested that all adults should be able to administer first aid since everyone is expected to experience emergency situations at any time.
There are over 50 million epileptic patients all over the world according the data from world health organization (1). In the world, there are approximately 2.4 million new epileptic patients each year, of which at least 50% begin in childhood and adolescence (1). In the treatment of partial and tonic - clonic seizures in children carbamazepine (cbz) is reported to be effective equal to phenobarbitone, phenytoin and sodium valproate (1). Clinical recommendations in europe and the usa suggest use of cbz as first choice of treatment for partial epilepsies (2). However, cbz was reported to increase lipid peroxide level and erythrocyte osmotic fragility (3). Free radicals and lipid peroxides are involved in many physiological processes and pathogenesis of a number of diseases (4). The effects of antiepileptic drugs on generation of oxidative stress have also been investigated (5, 6). Free radicals attached irreversibly to proteins and deoxyribonucleic acid (dna) macromolecules that are the primary targets for alkylation agents (6). They also cause degradation of dna, nucleotide and structural coenzymes in cells and tissues . Furthermore, they can bind covalently to proteins, lipids and enzymes; consequently alter enzyme activities; corrupt cell membranes; and damage transport systems (7). The membrane lipid peroxidation due to an increase in free radicals or decrease in activities of antioxidant defense mechanisms has been suggested to be causally involved in some forms of epilepsy (7). On the other hand, malondialdehyde (mda) values, an indicator of membrane lipid peroxidation, were found to be unchanged in children with epilepsy (8). Oxidative stress index (9) is reported as a valuable parameter indicating oxidative stress level . Furthermore, previously conducted studies revealed that oxidative stress index (osi) rate indicates oxidative injury, as well (10 - 12). Total antioxidative status (tas) determines the balance between the oxidative stress and antioxidant status . Measurement of total antioxidant capacity reflects the cumulative effect of antioxidants of plasma and body (13). Therefore the aim of this study was to evaluate the oxidative and antioxidative status in cbz - treated epileptic children to improve the data regarding the connection between cbz treatment and oxidative stress . The study is composed of 40 epileptic children (22 males, 18 females) and 31 healthy children (17 males, 14 females), with an age range between 4 and 12 years . All randomized subjects were selected from those who visited relating departments of mustafa kemal university faculty of medicine and antakya state hospital for checkup or medical treatment . Patients that were included in the study were chosen among those with epileptic seizures receiving cbz treatment for 1 year . The patients with systemic diseases (malignancy, diabetes mellitus, hypertension, autoimmune diseases, cardiac, metabolic, and central nerve ischemia), who use systemic and/or local corticosteroids were excluded . Serum cbz level was assessed using a biochemical auto analyzer (architect c8000 clinical chemistry analyzer, abbott, u.s ., japan). Total oxidative status (tos) (9) and tas of serum was determined using a novel automated measurement method developed by erel (10, 11). Oxidants present in the sample oxidize the ferrous ion - o - dianisidine complex to ferric ion . The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium . The percent ratio of tos to tas yields the osi, an indicator of the degree of oxidative stress (14). Percent rate of tos level to tas level was accepted as osi (15). To apply the calculation, the result unit of tas, mmol trolox equivalent / l, was changed to mmol trolox equivalent / l and the osi value was calculated using following formula: analysis of obtained data was performed using spss 13.0 for windows, a statistical software package . Pearson correlation test was used for the correlation between blood cbz level and blood tos value . Correlation coefficient (r) and significance values (p) was determined . A p value of <0.05 was accepted as significant . Serum cbz level was assessed using a biochemical auto analyzer (architect c8000 clinical chemistry analyzer, abbott, u.s ., japan). Total oxidative status (tos) (9) and tas of serum was determined using a novel automated measurement method developed by erel (10, 11). Oxidants present in the sample oxidize the ferrous ion - o - dianisidine complex to ferric ion . The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium . The percent ratio of tos to tas yields the osi, an indicator of the degree of oxidative stress (14). Percent rate of tos level to tas level was accepted as osi (15). To apply the calculation, the result unit of tas, mmol trolox equivalent / l, was changed to mmol trolox equivalent / l and the osi value was calculated using following formula: analysis of obtained data was performed using spss 13.0 for windows, a statistical software package . Pearson correlation test was used for the correlation between blood cbz level and blood tos value . Correlation coefficient (r) and significance values (p) was determined . A p value of <0.05 was accepted as significant . Tas, tos and osi levels of each group were compared, and statistically significant differences observed . Tas levels of cbz treated group were significantly lower compared to those of the control group (p <0.0001). Conversely, tos and osi levels were found to be significantly higher in cbz treated group (p <0.004 and p <0.0001, respectively) (table 1). In cbz treated epileptic patients group, correlation between blood cbz level and tas - tos levels was examined . A positive correlation was found between cbz and tos levels (r = 0.472, p <0.01) (figure 1). Abbreviations: cbz, carbamazepine; osi, oxidative stress index; tas, total antioxidant status; tos, total oxidant status . Cbz: carbamazepine; tos: total oxidant status . Our study showed that cbz treatment increased oxidative stress in patients with epilepsy . In the literature, there is a limited number of clinical case studies investigating the relation between oxidative stress and cbz treatment in patients with epilepsy (16 - 20). Secondly, we firstly evaluated the relationship between oxidative stress and cbz treatment with tas, tos and osi measurement in epileptic patients . Li et al . (21) investigated the effects of cbz on the oxidative stress of common carp spermatozoa in vitro . They reported that the oxidative stress was apparent and the significant inhibition of antioxidant enzymes activities including superoxide dismutase (sod) and glutathione peroxidase (gsh - px) after 2 hours exposure of cbz at higher concentration (2.0 or 20 mg / l). Their results suggested that cbz can induce reactive oxygen species (20) stress and could impair the antioxidant defense system . Li et al . (22) also investigated the effect of long - term exposure to cbz on the antioxidant system in brain tissue of rainbow trout . The fish were exposed to sublethal concentrations of cbz (1.0 g / l, 0.2 mg / l or 2.0 mg / l) for 7, 21, and 42 days . Crz exposure at 0.2 mg / l led to significant increases of oxidative stress indices after 42 days and, at 2.0 mg / l, after 21 days . Activities of the antioxidant enzymes including sod, cat, and gpx in cbz - treated groups slightly increased during the first period (7 days). However, activities of antioxidant enzymes were significantly inhibited at 0.2 mg / l exposure after 42 days and after 21 days at 2.0 mg / l . After 42 days, the content of glutathione (gsh) in fish brain was significantly lower in groups exposed to cbz at 0.2 mg / l and 2.0 mg / l . Prolonged exposure to cbz resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain . The authors emphasized that a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long - term exposure to cbz could lead to serious oxidative damage in fish brain . Oxidative stress is the condition with an imbalance between generation and elimination of ros and reactive nitrogen species, creating the potential for organic damage . Oxidative stress is blamed for the pathogenesis of epilepsy as a potential mechanism (23). Several studies on animal models and genetic studies have demonstrated an increase in mitochondrial oxidative stress and subsequent cell damage after persistent seizures (24 - 26). It has been reported that the increased amount of active oxygen metabolites or reduced activity of antioxidative defense mechanisms may cause greater frequency of seizure (7, 27). The role of antioxidant action in the effect of antiepileptic drugs (aeds) is controversial . Hamed and abdellah (28) reviewed the relation between membrane lipid peroxidation, antioxidants, neuronal excitotoxicity, and aeds . The authors identified that cbz was found to be a better anti - epileptic for the control of free radical - related seizures . On the other hand, solowiej (29) reported that sod activity decreased, gsh - px, glutathione disulfide (gssg - r) activities and mda level increased in the serum of children and adolescents with epilepsy . According niketic (30), the activity of antioxidant enzymes were decreased by 40% in epileptic children on cbz compared to healthy individuals . However, increased tos was observed in the same study . Certain studies noted that phenytoin (31) and valproic acid treatment (32) increase lipid peroxidation in epileptic children . Increased tos and decreased tas oxidative damage caused by cbz was revealed by some previously conducted studies (16, 17). Different from the literature, our findings are based on tas, tos and osi measurements for the first time . Therefore, we discussed our results in the light of related literature . Both the role of antioxidant action in epilepsy pathogenesis and the effect of aeds, particularly cbz, on the oxidant - antioxidant status in epilepsy patients are controversial . This could be due to complex pathogenic mechanisms of epilepsy and methodological issues . Yet oxidative stress may originate from various sources in the body and peripheral measurements might not necessarily accurately reflect the oxidative stress in the central nervous system . Another contribution of our study is that the relationship between the cbz levels and oxidative damage was firstly evaluated in the present study . This finding might be an explanation for oxidative damage secondary to cbz treatment, and for efficacy of antioxidants (17). Epilepsy has a multifactorial mechanism and it is difficult to isolate a single cause of the disease . Therefore the mechanism of action of aeds could not be fully understood yet . Based on our results, antioxidant mechanism could not be playing any role in antiepileptic effect of cbz . Furthermore, increased oxidative stress induced by cbz could be the cause of carbamazepine - induced seizures, a complication of cbz treatment.
Prostaglandin e2 (pge2) is mainly produced by osteoblasts during bone resorption associated with inflammation and acts as a potent stimulator of bone resorption . Inflammatory cytokines, such as interleukin (il)-1 and tumor necrosis factor (tnf) which induces pge2 production by osteoblasts, and pge2 are able to induce the expression of receptor activator of nf-b ligand (rankl) on the surface of osteoblasts . Two types of cyclooxygenase (cox), cox-1 and cox-2, are expressed in osteoblasts, and the expression of cox-2 is markedly induced by inflammatory stimulants . The blockage of pge2 synthesis by nonsteroidal anti - inflammatory drugs (nsaids) could suppress rankl - dependent osteoclastic bone resorption associated with inflammation [2, 3]. Membrane - bound pge synthase-1 (mpges-1) is also essential for pge2 synthesis in osteoblasts and pge - mediated osteoclast differentiation [2, 3]. An mpges-1-null mouse demonstrated that pge2 production by osteoblasts is essential for inflammatory bone loss induced by lipopolysaccharide (lps) in vivo . The transient receptor potential (trp) subfamily of ion channels contains six members (trpv1trpv6), most of which were thermal - sensitive cation channels . Trpv1 has been cloned from a cdna library of the dorsal root ganglion in sensory neurons, and the trpv1 signal is suggested to be a potential regulator of pain associated with thermal stimulation [5, 6]. Capsaicin is a natural ligand for trpv1 that regulates nerve - related pain - sensitive signals, inflammation, and cancer growth . Mouse osteoblasts express trpv1, and capsaicin suppresses il-1-induced osteoclast differentiation in the cocultures of mouse bone marrow cells and osteoblasts . Rossi et al . Reported that human pre - osteoclasts express trpv1 and its signal modulates rankl - mediated osteoclast differentiation . Recent studies suggesting some of the possible roles of trpv1 in bone metabolism are controversial, and the physiological and pharmacological effects of trpv1 signals are not known in bone tissues . The present study examined the effects of capsaicin, a natural trpv1 ligand, on inflammatory bone resorption in vitro and in vivo . These results showed that the trpv1 signal suppressed osteoclastic bone resorption associated with pge2 production in vitro and attenuated inflammatory bone loss induced by lps in vivo . Pge2 was obtained from sigma - aldrich co. llc (st louis, mo). Newborn (2-day - old and 5-day - old) and adult (6-week - old) ddy mice were obtained from japan slc inc . Primary osteoblastic cells were isolated from newborn mouse calvariae after five routine sequential digestions with 0.1% collagenase (wako pure chemical) and 0.2% dispase (godo shusei co. ltd, tomakomai, japan) as previously described . Osteoblastic cells collected from fractions 24 were combined and cultured in -modified mem (mem) supplemented with 10% fetal calf serum (fcs) at 37c under 5% co2 in air . The cells adhering to the well surface were stained for tartrate - resistant acid phosphatase (trap), and trap - positive multinucleated cells containing three or more nuclei per cell were counted as osteoclasts as previously described . The concentrations of pge2 in the cultured medium were determined using an enzyme immunoassay, as previously described . Mouse calvariae were collected from 5-day - old mice and then were cultured for 24 h in bgjb medium containing 1 mg / ml bovine serum albumin (bsa). The calvaria was transferred into new medium, with or without lps after 24 h, and then was cultured for 5 days at 37c under 5% co2 in air . The bone - resorbing activity was determined by measuring the concentration of calcium in the conditioned medium using a calcium kit (calcium c test; wako pure chemical) as reported previously . The bone - resorbing activity was expressed as an increase in medium calcium, which is consistent with the osteoclastic bone resorption as shown in the previous studies . Primary mouse osteoblastic cells were cultured for 24 h in mem containing 1% fcs then treated with lps for 3 h. the total rna was extracted from mouse osteoblasts using the acid guanidium - phenol - chloroform method . Cdna was synthesized from 10 g of total rna by reverse transcriptase (superscript ii preamplification system; invitrogen life technologies co., carlsbad, ca) and amplified using pcr . Primers for the mouse cox-1, cox-2, mpges-1, and glyceraldehydes-3-phosphate dehydrogenase (gapdh) genes were used in pcr as reported previously . The pcr product was separated on a 1% agarose gel and stained with ethidium bromide . Six - week - old mice were i.p . Injected with lps (10 mg / kg body weight) on days 0 and 4 . Some mice were i.p . Injected with capsaicin (1 mg / kg body weight). The femurs were collected 8 days after the first injection of lps or phosphate - buffered saline (pbs). For each group, was measured by dual x - ray absorptiometry (model dcs-600r; aloka, co. ltd, tokyo, japan) as reported previously . The bmd was calculated by dividing the bone mineral content of the measured area by the area . Lps markedly induced osteoclast differentiation in cocultures of mouse bone marrow cells and osteoblasts on day 7, while 30 m of capsaicin completely suppressed the osteoclast formation induced by lps (figure 1(b)). The conditioned coculture medium was examined to elisa to determine the effects of capsaicin on pge2 production induced by lps . The level of pge2 in the conditioned medium treated with lps was higher than that of the control and was clearly suppressed by adding 30 m capsaicin in the coculture (figure 1(c)). Bone - resorbing activity was measured by the increase in calcium in the conditioned medium . The addition of 30 m capsaicin significantly suppressed the bone - resorbing activity induced by lps (figure 1(d)). These results indicate that capsaicin clearly suppressed the osteoclastic bone resorption associated with inflammation in vitro . The expression of cox-2 and mpges-1 is essential for pge2 production by osteoblasts treated with bone - resorbing cytokines such as il-1 and lps, and the pge2 production is essential for inflammatory bone resorption . An rt - pcr analysis showed the expression of cox-2 and mpges-1 mrnas to be markedly induced by lps in mouse primary osteoblasts at 3 h, and simultaneous addition of capsaicin clearly suppressed both the cox-2 and mpges-1 expression (figure 2(a)). A low level cox-1 mrna expression was detected in the osteoblasts, and this expression was not influenced by lps or capsaicin . Capsaicin significantly suppressed lps - induced pge2 production in mouse primary osteoblast culture (figure 2(b)). Lps administration induces the severe loss of trabecular bone in distal femurs in mice, and mpges-1 knockout mice are resistant to the bone loss induced by lps . Injected with capsaicin with or without lps, and the femurs were collected on day 8 for measurement of femoral bmds . Distal femoral bmd was markedly suppressed by the treatment with lps, and simultaneous treatment with capsaicin significantly restored the lps - induced bone loss in mice (figure 3). The present study showed that capsaicin, a trpv1 ligand, suppresses lps - induced osteoclastic bone resorption associated with inflammation by inhibiting pge2 production by osteoblasts . We have reported that pge2 binds to the ep4 receptor, one of the pge receptor subtypes ep1-ep4, and induces the rankl expression to stimulate bone resorption, using agonist and antagonist of eps and respective ep - knockout mice [2, 11]. Pge2 produced by osteoblasts binds to ep4 in osteoblasts and induces rankl expression via ep4-mediated signals . Lps markedly induces the expression of cox-2 and mpges-1 mrnas in osteoblasts, and the pge2 production is essential for rankl - dependent osteoclast formation . The transcriptional regulation is important to understand the biological significance in target tissues because mpges-1 is an inducible terminal enzyme associated with pge2 biosynthesis . The mrna expression of mpges-1 is coupled with cox-2, and the induction of cox-2 mrna proceeded mpges-1 after adding lps . The mouse cox-2 gene promoter possesses functional regulatory elements for nf-b, nfil-6, ap-1, and c / ebp . In contrast, the mouse mpges-1 gene promoter possesses ap-1 and c / ebp, but not nf-b . Capsaicin clearly suppressed the lps - induced expression of cox-2 and mpges-1 mrnas in osteoblasts in the present study . Further studies are needed to define the transcriptional regulation of cox-2 and mpges-1 by capsaicin in osteoblasts . Capsaicin, a typical ligand for trpv1, is derived from chili peppers, which elicit a burning sensation through trpv1 [5, 6]. Mouse primary osteoblasts and osteoblast cell line mc3t3-e1 express trpv1, but expression of trpv1 mrna is not detected in bone marrow macrophages, and that capsaicin suppresses il-1-induced osteoclast differentiation in the cocultures of mouse bone marrow cells and osteoblasts . In addition, both resiniferatoxin, a natural trpv1 agonist, and olvanil, a synthetic trpv1 agonist, suppress osteoclast formation induced by il-1 in the cocultures (miyaura and inada; unpublished data). The present study found that capsaicin suppressed the pge2 production induced by lps in osteoblasts and suppressed lps - induced osteoclast formation (figures 1 and 2). These results indicate that osteoblasts are main target cells for trpv1 ligand in bone tissues and regulate osteoclast differentiation associated with inflammation . Chen et al . Reported that capsaicin acts on the macrophage cell line raw264.7 to attenuate lps - induced cox-2 expression, but raw 264.7 cells do not express trpv1 . On the other hand, rossi et al . Have shown that human mature osteoclasts express trpv1 isoforms and trpv1 agonist enhances the expression and the activity of trap and cathepsin k, two specific osteoclast biomarkers . The addition of capsaicin to bone marrow macrophages cultures in the presence of soluble rankl and macrophage colony stimulating factor (m - csf) does not influence osteoclast differentiation from macrophages (miyaura and inada; unpublished data). Idris et al . Have shown that capsazepine, a trpv1 antagonist, acts on preosteoclasts and suppresses the differentiation into mature osteoclasts . Therefore, the roles of trpv1 signals still remain controversial in macrophages, preosteoclasts, and mature osteoclasts . The current study showed that capsaicin significantly restored inflammatory bone loss in the femur induced by lps in mice (figure 3). Therefore, trpv1 ligands possess potential as clinical drugs targeting bone diseases associated with inflammatory bone resorption . Sancho et al . Reported that capsaicin has the potential to protect against inflammatory bowel diseases associated with severe inflammation, thus indicating the anti - inflammatory effects of trpv1 signal in intestinal diseases . Idris et al . Reported that the trpv1 antagonist capsazepine inhibits bone loss due to estrogen deficiency in ovariectomized (ovx) mice . However, the mechanisms of bone loss in ovx animals differ from that in lps - treated animals accompanied with severe inflammation . In addition, clark et al . Reported that trpv4, a calcium permeable ion channel, possess chondroprotective role and deletion of trpv4 gene resulted in severe osteoarthritis using trpv4 knockout mice . Further studies using several animal models for bone and cartilage diseases are needed to define the possible roles of trpvs signals in bone.
S. aureus is commonly present in diabetic foot infections [14], particularly limb - threatening infections [57], as well as more invasive infections like endocarditis . Additionally, hyperglycemia is associated with an increased risk of death from s. aureus bacteremia . Thus, diabetes and hyperglycemia appear to increase the risk and severity of infection by s. aureus by inhibiting normal host defenses . Currently, these mechanisms are only partially understood . To date, immunological insufficiency related to hyperglycemia has primarily focused on inhibition of neutrophil responses [1012]. We have recently shown in vitro that hyperglycemic conditions (> 6 mm glucose) inhibit complement effectors against s. aureus including opsonization and anaphylatoxin generation . Hyperglycemic inhibition of complement - mediated opsonization resulted in decreased phagocytosis efficiency by euglycemic neutrophils, such that neutrophil function was not directly inhibited by excess glucose . Previous investigators had shown that the central component of complement, c3, could be slowly glycated (i.e., 20% glycation over 24 hours). However, our data demonstrated the effects of elevated glucose on complement activation on the surface of s. aureus occurring in minutes . By mass spectrometry we showed that hyperglycemic conditions produced no changes in glycation over one hour, but hyperglycemic conditions did produce changes in the tertiary structure of c3, likely altering its function . These in vitro results suggested that the complement system, a major contributor to innate immune host defenses against s. aureus [1416], was significantly inhibited by hyperglycemic conditions in responding to s. aureus . Our results were consistent with prior reporting that diabetic patients have a decreased ability to fix complement by igg . In order to measure the extent to which a hyperglycemic environment altered complement - mediated immune effectors against s. aureus infection in vivo a peritonitis model was chosen as a pertinent clinical model, as well as an excellent model for evaluating complement - mediated effectors [15, 18, 19] by analyzing recovered peritoneal lavage fluid . Previous investigators have evaluated s. aureus foot - pad infection in a nod diabetic mouse model, but the mice were c5-deficient limiting the ability to evaluate complement - mediated effects . Streptozocin (sigma aldrich) was dissolved in saline at 0.5 mg / ml . Nph insulin (eli lily) was 10 u / ml . S. aureus strain reynolds was grown in columbia 2% nacl broth overnight at 37c to stationary phase . Rats were provided acetaminophen in their drinking water (1.6 mg / ml) for analgesia . Rats were monitored for weight, appearance, and behavior, but no animals demonstrated moderate or severe distress during the experiments . Pilot studies were performed with 65 mg / kg streptozocin i.p ., which induced stable hyperglycemia after 12 hours and was reversible with 2 units of nph insulin s.c . Dosing pilot studies were performed with streptozocin - treated rats using 10, 10, or 10 cfu of s. aureus i.p ., evaluated by peritoneal wash at 6 hours . The timing of immune - pathogen interactions in streptozocin - treated animals (10 cfu s. aureus i.p .) Demonstrated optimal evaluation of early events at 2 hours and late events at 24 hours after infection . Four rats were untreated and underwent peritoneal wash to measure baseline (0 hour) parameters . Ten (10) rats received sham streptozocin (stz) injections and 30 rats received stz 65 mg / ml i.p . Stable hyperglycemia was achieved (figure 1(b)) for all but one stz - treated rat, which was excluded from subsequent analyses . Thirty - nine (39) rats were inoculated with 10 cfu s. aureus i.p . This amount of s. aureus is consistent with established s. aureus infections . At 2 hours after infection, 19 rats were euthanized and had peritoneal lavage . Two (2) peritoneal wash samples demonstrated particulate matter suggestive of fecal matter and were excluded from subsequent analyses . At 2 hours after infection 10 of the remaining rats were injected with insulin (2 u nph s.c . ), which was repeated (2 u nph s.c .) At 8 hours after infection . At 24 hours after infection pellets were resuspended to their original volume in cold pbs and divided for subsequent analyses . Cytospins (100 l) were performed to generate multiple slides for each sample . Other aliquots of resuspended pellets were washed with water to lyse neutrophils and then serially diluted for colony count assays or stripped of opsonic c3-fragments and c4-fragments . Wright stains (diff - quik) were performed and 5 random high powered fields were counted for each slide by a blinded observer and averaged . Phagocytosis was evaluated by fluorescence microscopy after acridine orange staining of bacteria followed by crystal violet quenching of extracellular bacteria, as previously described . One hundred neutrophils were observed in random high powered fields by a blinded observer and evaluated for the percentage of neutrophils phagocytizing bacteria and the total number of bacteria phagocytized . Bacterial suspensions were generated from peritoneal wash samples pelleted and resuspended to their original volume in water to lyse neutrophils . The suspensions were then serially diluted and multiple dilutions were plated for colony counting, as previously described . S. aureus recovered from the peritoneal washes were divided for analysis by flow cytometry, elisa, or western blot . S. aureus for flow cytometry analysis were incubated with fitc - labeled anti - c3 antibody (mp biomedicals). Negative controls included (1) unstained peritoneal bacteria and (2) uninjected bacteria incubated with anti - c3 antibody; both yielded minimal fluorescence values below the threshold for detection used for data collection . Fluorescence intensities were measured on a fluorescence activated cell sorter facscalibur (bd biosciences) for 10,000 events . Flowjo software (tree star) was used to measure the area under the curve (auc) for c3-fragments bound to the bacterial surface . Bound c3-fragments and c4-fragments were stripped from s. aureus using methylamine, the most commonly used and accepted methodology, as previously described . The rat c3 elisa was performed using a rabbit anti - rat c3 antibody for capture (bethy lab) and probed with mouse anti - rat c3 antibody (hycult) followed by goat anti - mouse hrp antibody (sigma). Western blot analysis was performed using rabbit anti - rat c3 followed by a goat anti - rabbit hrp antibody . Quantitation was calculated from standard curves using purified c3 and c4 proteins (comptech). Total igg concentration in peritoneal wash samples was measured by sds - page and coomassie staining to minimize backgrounds found with elisa assay . Peritoneal wash samples and a titration of purified igg (gammagard, baxter) were assayed by sds - page with coomassie brilliant (biorad) staining . Images were captured digitally and optical densitometry measurements were performed with quantity one (biorad). Peritoneal wash samples were measured for total c4 and total c3 by elisa and assayed by c3 western blot, as described above . C3a concentration in peritoneal wash samples was measured by quantitative western blot to minimize backgrounds found with elisa . Samples were blotted along with a titration of pure c3a (comptech) to generate a standard curve . A rabbit anti - human c3a antibody (comptech) was followed by a goat anti - rabbit hrp antibody (sigma). C5a concentration was measured by elisa (r&d systems) as per the manufacturer's instructions . Free dna in the peritoneal wash supernatants was assayed using quant - it picogreen (molecular probes). Briefly, 1.0 ml of peritoneal wash was combined with 1.0 ml of quant - it picogreen reagent and fluorescence (excitation 480 nm, emission 520 nm) was measured on a spectrofluorometer (perkin elmer). Myeloperoxidase activity was measured using tmb substrate solution (thermo scientific). In a 96-well plate, 10 l of peritoneal wash sample was combined with 100 l of tmb . The plates were allowed to incubate at room temperature for 30 min and then absorbance values were read at 450 nm . All data are shown as mean standard error of the mean . Comparisons between two groups were made by student's t - test with p values 0.05 considered statistically significant . In order to evaluate hyperglycemic effects on s. aureus - induced influx of humoral immune components critical in the control of s. aureus infection, we assayed igg, c3, and c4 in the peritoneal lavage fluid . Igg was measured by total protein stained sds - page (figure 2(a)), due to high background signal for elisa . Euglycemic rats showed a 2.5-fold increase in igg compared with diabetic rats at 2 hours after infection (p = 0.03). At 24 hours after infection, insulin - rescued rats showed a 2-fold increase in igg compared with diabetic rats (p = 0.001). These results suggest that hyperglycemia inhibits the influx of igg to the site of s. aureus infection and that insulin treatment of hyperglycemia reverses this effect . Complement c4, a critical component of classical and lectin complement pathway activation, was assayed by elisa (figure 2(b)). At 2 hours after infection, a trend towards increased c4 influx was noted for euglycemic rats compared with diabetic rats (p = 0.08). At 24 hours after infection, insulin - rescued rats demonstrated a 2-fold increase in c4 concentration compared with diabetic rats (p = 0.02). C3 influx into the peritoneum was assayed by elisa (figure 2(c)). At 2 hours after infection, c3 values were not significantly different, but at 24 hours after infection, insulin - rescued rats demonstrated a 1.7-fold increase compared with diabetic rats (p = 0.01). In order to confirm c3 elisa results and evaluate the c3 forms present in the peritoneal fluid, we performed western blot analyses of the peritoneal fluid . At 2 hours after infection, western blot analysis suggested increased c3-forms for euglycemic rats compared with diabetic rats and showed that this was predominantly unactivated c3 with some ic3b (figure 2(d)). Several of these samples were analyzed by western blot along with purified c3, c3b, and ic3b confirming the identity of c3b and ic3b (data not shown). At 24 hours after infection, western blot analysis showed increased c3-forms for insulin - rescued rats compared with diabetic rats (figure 2(e))., these results suggest that hyperglycemia inhibits the influx of critical complement components to the site of s. aureus infection, but insulin rescue can improve the influx of complement components compared with persistent hyperglycemia . In order to further evaluate hyperglycemic effects on s. aureus - initiated complement activation as well as anaphylatoxin generation, we measured c3a and c5a concentrations in the peritoneal lavage samples . Complement anaphylatoxins are direct indicators of complement activation and play vital roles in neutrophil chemotaxis and neutrophil activation, as well as increasing vascular permeability [28, 29]. C3a concentrations were measured using a quantitative western blot (figure 3(a)), due to high background levels with elisa . Peritoneal c3a levels increased over baseline measurements by 2 hours after infection, at which time c3a concentration in euglycemic rats was 2.5-fold greater than diabetic rats (p = 0.03). At 24 hours after infection, c3a concentrations increased to 2-fold which is greater for insulin - rescued rats compared with diabetic rats (p = 0.01). At 2 hours after infection c5a levels were not significantly different between the groups (figure 3(b)), but at 24 hours after infection, c5a concentrations increased to 2-fold greater for insulin - rescued rats compared with diabetic rats (p = 0.02). Together, these results show that hyperglycemia was associated with less anaphylatoxin generation in the rat peritoneum in response to s. aureus infection . The 24-hour data suggest that insulin rescue and reversal of hyperglycemia significantly increased complement activation and anaphylatoxin generation in response to s. aureus infection . In order to evaluate the effects of hyperglycemia on complement opsonization of s. aureus in the peritoneum, we recovered the bacteria from the peritoneal wash samples and assayed them by flow cytometry or stripped the bound complement opsonins and assayed them by elisa and western blot . Complement - mediated opsonization with c3b and ic3b is critical for efficient phagocytosis of s. aureus and survival of bacteremia [22, 23, 30]. Opsonization of s. aureus with c4 forms was measured by elisa and normalized for numbers of bacteria present (figure 4(a)). C4-fragment opsonization of s. aureus was increased by 4-fold at 2 hours for euglycemic rats compared with diabetic rats (p = 0.02). C4-opsonization was increased 15-fold for insulin - rescued rats at 24 hours after infection compared with diabetic rats (p = 0.05). These results suggest that classical or lectin complement pathway activation on the s. aureus surface was greatly decreased in hyperglycemic conditions . Flow cytometry analysis of efficiency of c3-opsonization of s. aureus recovered in the peritoneal wash samples was measured for area under the curve and normalized for 10,000 counts . At 2 hours after infection a 2-fold increase in c3-opsonization was noted for euglycemic rats compared with diabetic rats (p = 0.01), but diabetic rats compared with insulin - rescued rats at 24 hours after infection did not show a significant difference (figure 4(b)). In order to confirm flow cytometry c3-opsonization results, we also performed elisa measurements of c3-fragments stripped from the s. aureus surface and normalized for numbers of bacteria present . Consistent with the flow cytometry data, at 2 hours after infection, a trend towards increased c3-fragment opsonization was noted for euglycemic rats compared with diabetic rats (p = 0.06) (figure 4(c)). At 24 hours after infection, a small but statistically significant increase in c3-fragment opsonization was found for insulin - rescued rats compared with diabetic rats (p = 0.05). In order to evaluate the forms of c3 bound to s. aureus, we performed c3 western blot analysis on bacteria recovered at 2 hours after infection . No demonstrable c3b could be identified, but a mixture of opsonic ic3b and nonopsonic c3d was recovered from the surface of s. aureus from both euglycemic and diabetic rats (figure 4(d)). Together these results suggest that hyperglycemia inhibits complement opsonization of s. aureus early in infection and that insulin rescue may improve c4-mediated opsonization . Neutrophil migration into the peritoneum in response to complement activation and anaphylatoxin is well established [18, 19, 31]. In order to evaluate hyperglycemic effects on neutrophil migration in response to anaphylatoxin generation, we performed leukocyte counts on peritoneal wash fluids by microscopy of cytospin slides . After 2 hours of infection, nearly all of the leukocytes were neutrophils and both groups showed a considerable increase over baseline value with diabetic rats demonstrating a 2-fold increase in leukocytes compared with euglycemic rats (p <0.01) (figure 5(a)). The finding of increased neutrophils present for diabetic rats at 2 hours after infection was surprising given the increased levels of c3a present in the peritoneum of euglycemic rats . However, our previous s. aureus dose ranging experiments showed that an i.p . Dose of 10 cfu yielded fewer recovered bacteria and fewer recovered neutrophils compared with a dose of 10 cfu (figures 5(d) and 5(e)). Microscopic examination of the slides from rats dosed with 10 cfu demonstrated more cellular debris, suggesting that there may be an increased inflammatory response at 10 cfu resulting in more bacterial killing by neutrophils as well as increased neutrophil death . In order to evaluate whether there could be increased neutrophil death for the euglycemic rats, we measured myeloperoxidase (mpo) activity and free dna in the peritoneal lavage fluid using tmb and picogreen, respectively (figures 5(b) and 5(c)). At 2 hours after infection, myeloperoxidase activity was increased 1.7-fold for euglycemic rats compared with diabetic rats (p = 0.04) and free dna was increased by 1.6-fold for euglycemic rats compared with diabetic rats (p = 0.03). These results suggest that the increased numbers of neutrophils present in the peritoneum of diabetic rats at 2 hours after infection may in part be due to less neutrophil death from activation and degranulation . This is consistent with the expectation that neutrophils in the euglycemic rats will undergo increased phagocytosis and degranulation due to stimulation by c3a compared with diabetic rats . In order to evaluate the downstream effect of hyperglycemic inhibition of complement effectors against s. aureus we have previously shown in vitro that hyperglycemic inhibition of complement opsonization of s. aureus inhibits phagocytosis by euglycemic neutrophils . To evaluate the in vivo effects of hyperglycemia on neutrophil function in this rat model, neutrophil phagocytosis efficiency was assayed by fluorescence microscopy of acridine orange and crystal violet stained cytospin slides of peritoneal wash samples . At 2 hours after infection, euglycemic rats demonstrated a 1.6-fold increase in neutrophils phagocytosis of bacteria (figure 6(a)) compared with diabetic rats (p = 0.02). At 24 hours after infection there was no significant difference between groups . At 2 hours after infection, the number of bacteria phagocytized by 100 neutrophils (figure 6(b)) was increased 3-fold in euglycemic rats compared with diabetic rats (p = 0.02). There was a nonsignificant trend towards increased numbers of bacteria phagocytized at 24 hours after infection in insulin - rescued rats (p = 0.1). S. aureus survival in the rat peritoneum was assessed by colony counting of peritoneal wash samples . At 2 hours after infection, s. aureus survival was 3-fold higher in diabetic rats compared with hyperglycemic rats (p = 0.03) (figure 6(c)). At 24 hours after infection there was no statistically significant difference in s. aureus survival between diabetic and insulin - rescued rats . Taken together, these results suggest that hyperglycemic inhibition of complement effectors in s. aureus peritonitis may contribute to decreased phagocytosis efficiency and increased bacterial survival, at least early in infection . These experiments were designed to evaluate the effects of hyperglycemia on complement effectors against s. aureus in a rat model of peritoneal infection . Hyperglycemia adversely affected the early (i.e., 2-hour) influx of critical humoral immune components to the site of s. aureus infection . A likely explanation for this effect is hyperglycemia inhibiting s. aureus activation of complement components already present in the peritoneum at the time of inoculation . Previous investigators have shown that complement activation in the peritoneum greatly increases vascular permeability and leakage of plasma proteins via anaphylatoxin . Our data show there are low concentrations of igg, c4, and c3 present in peritoneal fluid in the absence of infection . Hyperglycemia inhibition of s. aureus - initiated complement activation at the time of inoculation, as suggested by our in vitro data, would decrease the amount of anaphylatoxins generated by the complement components already present in the peritoneal fluid . Uninhibited anaphylatoxin generation, as would be expected for the euglycemic animals, should increase histamine release and increase vascular permeability facilitating the extravascular transit of plasma components . Evaluation of these relatively short - lived anaphylatoxins minutes after infection could potentially be more revealing . Reversal of hyperglycemia by administering insulin improved the influx of humoral immune components to the site of infection at 24 hours after infection compared with animals that did not receive insulin . C3a and these results suggest that reversing hyperglycemic conditions may enhance anaphylatoxin generation and increase humoral immune component influx to the site of infection . Thus, these results suggest that hyperglycemia inhibits early humoral responses to s. aureus infection and that these effects can be reversed, at least partially, with insulin therapy . Opsonization of s. aureus in euglycemic rats was increased for both c4-fragments and c3-fragments at 2 hours after infection compared with diabetic rats . These findings demonstrate that a hyperglycemic environment inhibits s. aureus - initiated complement activation and opsonization of s. aureus, consistent with our prior in vitro findings . Decreased c4-fragment opsonization in hyperglycemic conditions suggests that the classical pathway, or lectin pathway, or both, may have been inhibited . It is also possible that high glucose environments may have a direct effect on the c4 molecule, as we demonstrated for the evolutionarily related c3 molecule . Alternatively, excess glucose may alter the surface of microbial pathogens inhibiting complement effectors, as has been shown for candida albicans [35, 36]. Phagocytosis of s. aureus by neutrophils was increased at 2 hours after infection for euglycemic rats compared with diabetic rats . This correlated inversely with s. aureus survival where diabetic rats showed increased numbers of live bacteria at 2 hours after infection compared with euglycemic rats . We have previously shown that hyperglycemic conditions inhibit complement - mediated opsonization of s. aureus resulting in decreased phagocytosis by euglycemic neutrophils, where neutrophil function was not affected by excess glucose . In these in vivo experiments, however, our prior in vitro findings and present demonstration of decreased complement opsonization of s. aureus in vivo suggest that hyperglycemic inhibition of complement responses to s. aureus likely contributed to decreased phagocytosis and increased bacterial survival for diabetic rats . In summary, these rat experiments show that hyperglycemia inhibited humoral effector recruitment, complement - mediated opsonization of s. aureus, and complement anaphylatoxin generation (figure 7). Treatment with insulin after infection improved some of these complement effectors compared with persistent hyperglycemia.
A 15-year old boy attempted suicide by taking a high dose of disulfiram (20 mg). Upon arrival at the hospital, he was alert and vital signs were stable . One month after, he suddenly developed an acute insult consisted of anxiety attack and subsequent retrocollis and forceful upward deviation of the eyes . During the attack, he could not close his eyes and speak, making only grunting noises . He could not move his eyes voluntarily at all, but horizontal oculocephalic reflexes could be elicited . The frequency, duration and intensity of the attacks increased gradually over a period of eight years, occurring more than 10 times a day and lasted up to 2 hours . He had no motor weakness or sensory deficits, but he needed a help to walk due to foot dystonia . T2-weighted brain mri studies showed high signal intensity lesions at the bilateral globus pallidus and left substantia nigra (figure 1). Levodopa treatment was not effective, but the frequency of the attacks decreased markedly after trihexyphenidyl hydrochloride (2.5 mg tid) treatment . Also, the foot dystonia improved partially and the patient could walk a short distance without assistance . Disulfiram is metabolized to cyanide disulfide (cs2) and produce lesions at the globus pallidus and substantia nigra pars reticulata in monkeys.7 in huans, pallidal or lenticular lesions after disulfiram intoxication have been reported.6,7 however, to our knowledge, lesion at the substantia nigra after disulfiram intoxication is rarely documented on brain mri studies . Ogc occurs frequently in association with neuroleptic treatment, postencephalitic parkinsonism, and focal brain lesions at the putamen or globus pallidus.1,2,4,5 these findings suggest that ogc can be caused by basal ganglia dysfunction, particularly of the dopaminergic system . In our patient, neuronal discharges from pathologically reorganized basal ganglia circuit to the midbrain ocular motor center might lead to tonic deviation of the eyes . We reported a patient who developed disulfiram induced parkinsonism, dystonia, and ogc, responding markedly to the anticholinergics treatment.
Lung cancer is one of the leading causes of cancer - related deaths in the world . Although it is well known that smoking is the primary risk factor for lung cancer, lung cancer develops in less than 20% of people who smoke throughout their life . Moreover, lung cancer is a multi - cellular and multistage process involving a number of genetic changes in oncogenes, suggesting that genetic factors may play an important role in its development . The glutathione s - transferases (gsts) are a gene superfamily of phase ii metabolic enzymes that detoxify free radicals, particularly in tobacco smoke, products of oxidative stress, and carcinogens such as benzopyrene and other polycyclic aromatic hydrocarbons . Phenotype assays have confirmed this lack of function by demonstrating a strong concordance between phenotype and genotype . Previous studies have suggested that individuals with null genotypes of gstm1 may be unable to eliminate electrophilic carcinogens efficiently and have a high risk of lung cancer . However, the results from previous reported studies in asians were inconclusive [333]. Therefore, we conducted a meta - analysis to explore the effect of gstm1 null genotype on lung cancer risk in asians . We searched the pubmed and embase to identify published case - control studies investigating the associations of gstm1 null genotype with risk of lung cancer in asians . Glutathione s - transferases or gstm1 and lung cancer, without restriction on language . Additional studies were identified by a manual search of references of original studies or review articles . The inclusion criteria were: (1) original papers containing independent data; (2) studies should provide the sample size, odds ratios (ors), and 95% confidence intervals (cis), as well as the genetic distribution or the information needed to infer the results; and (3) case - control or cohort studies . The major exclusion criteria for studies were: (1) overlapping data; (2) insufficiently useful data; and (3) case - only studies or family - based studies; (4) reviews, abstracts, or commentaries; (5) not relevant to lung cancer or gstm1; and (6) not conducted in asians . Two independent researchers extracted raw data according to the inclusion criteria . The following information was collected from each study using a data extraction form: the surname of the first author, year of publication, country of origin, sex of subjects, histology, smoking status, number of cases and controls, adjustment, and ors and the corresponding 95% confidence intervals (cis) of lung cancer risk . For the gstm1 gene, we estimated the risk effect of the null genotype on lung cancer compared with the non - null genotypes in the recessive model (null vs. heterozygous + wild type). The strength of the association between the gstm1 gene and lung cancer risk was measured by ors with 95% cis . The ors with corresponding 95% cis from individual studies were pooled using fixed or random effects models, according to the heterogeneity . When the p value for cochran s q statistic was less than 0.1, and a significant heterogeneity existed across the included studies, the random effects model (dersimonian and laird method) was used for meta - analysis, or the fixed - effects model (mantel - haenszel method) was used . Sensitivity analysis was further performed by excluding a single study to assess the impact of an individual study on the pooled estimate . Funnel plots and egger s regression test were used to assess the potential publication bias . Data analysis was performed using stata 12 (statacorp lp, college station, texas, usa). We searched the pubmed and embase to identify published case - control studies investigating the associations of gstm1 null genotype with risk of lung cancer in asians . Glutathione s - transferases or gstm1 and lung cancer, without restriction on language . Additional studies were identified by a manual search of references of original studies or review articles . The inclusion criteria were: (1) original papers containing independent data; (2) studies should provide the sample size, odds ratios (ors), and 95% confidence intervals (cis), as well as the genetic distribution or the information needed to infer the results; and (3) case - control or cohort studies . The major exclusion criteria for studies were: (1) overlapping data; (2) insufficiently useful data; and (3) case - only studies or family - based studies; (4) reviews, abstracts, or commentaries; (5) not relevant to lung cancer or gstm1; and (6) not conducted in asians . The following information was collected from each study using a data extraction form: the surname of the first author, year of publication, country of origin, sex of subjects, histology, smoking status, number of cases and controls, adjustment, and ors and the corresponding 95% confidence intervals (cis) of lung cancer risk . For the gstm1 gene, we estimated the risk effect of the null genotype on lung cancer compared with the non - null genotypes in the recessive model (null vs. heterozygous + wild type). The strength of the association between the gstm1 gene and lung cancer risk was measured by ors with 95% cis . The ors with corresponding 95% cis from individual studies were pooled using fixed or random effects models, according to the heterogeneity . When the p value for cochran s q statistic was less than 0.1, and a significant heterogeneity existed across the included studies, the random effects model (dersimonian and laird method) was used for meta - analysis, or the fixed - effects model (mantel - haenszel method) was used . Sensitivity analysis was further performed by excluding a single study to assess the impact of an individual study on the pooled estimate . Funnel plots and egger s regression test were used to assess the potential publication bias . Data analysis was performed using stata 12 (statacorp lp, college station, texas, usa). A total of 31 studies were retrieved based on the search criteria for lung cancer susceptibility related to the gstm1 polymorphism [333]. The evaluations of the association between gstm1 polymorphism and lung cancer risk are summarized in table 2 . The null genotype of gstm1 was associated with a significantly increased risk of lung cancer when compared with present genotype (or=1.43; 95% ci, 1.301.58; figure 2). The combination of adjusted ors for lung cancer was 1.38 (95% ci, 1.231.54). When stratified by sex, significantly elevated risks were observed in men (or=1.38; 95% ci, 1.061.78) and women (or=1.30; 95% ci, 1.031.64). In the subgroup analysis according to histology, significantly increased risks were observed in adenocarcinoma (or=1.27; 95% ci, 1.051.55) and squamous cell carcinoma (or=1.40; 95% ci, 1.101.78), but not in small - cell lung cancer (or=1.22; 95% ci, 0.811.83). Subgroup analysis based on the smoking status showed that increased risks were found in non - smokers (or=1.49; 95% ci, 1.251.79) and smokers (or=1.78; 95% ci, 1.432.23). As shown in figure 3, the results showed that the pooled ors tended to be stable . A single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors, and the corresponding pooled ors were not materially altered (figure 4). Funnel plot and egger s test were used to assess the publication bias of the literature . The shape of the funnel plot did not reveal any evidence of obvious asymmetry (figure 5). A total of 31 studies were retrieved based on the search criteria for lung cancer susceptibility related to the gstm1 polymorphism [333]. The evaluations of the association between gstm1 polymorphism and lung cancer risk are summarized in table 2 . The null genotype of gstm1 was associated with a significantly increased risk of lung cancer when compared with present genotype (or=1.43; 95% ci, 1.301.58; figure 2). The combination of adjusted ors for lung cancer was 1.38 (95% ci, 1.231.54). When stratified by sex, significantly elevated risks were observed in men (or=1.38; 95% ci, 1.061.78) and women (or=1.30; 95% ci, 1.031.64). In the subgroup analysis according to histology, significantly increased risks were observed in adenocarcinoma (or=1.27; 95% ci, 1.051.55) and squamous cell carcinoma (or=1.40; 95% ci, 1.101.78), but not in small - cell lung cancer (or=1.22; 95% ci, 0.811.83). Subgroup analysis based on the smoking status showed that increased risks were found in non - smokers (or=1.49; 95% ci, 1.251.79) and smokers (or=1.78; 95% ci, 1.432.23). As shown in figure 3, the results showed that the pooled ors tended to be stable . A single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors, and the corresponding pooled ors were not materially altered (figure 4). Funnel plot and egger s test were used to assess the publication bias of the literature . The shape of the funnel plot did not reveal any evidence of obvious asymmetry (figure 5). The present meta - analysis, including 5347 lung cancer cases and 6072 controls from 31 case - control studies, explored the association of gstm1 null genotype with lung cancer risk . We demonstrated that the null genotype of gstm1 was associated with a significantly increased lung cancer risk in asians . Furthermore, in the stratified analysis by sex, we found that both men and women with gstm1 null genotype had increased lung cancer risk . More studies are needed to assess the association between gstm1 null genotype and lung cancer risk in males and females . Cigarette smoking is a pro - inflammatory stimulus and an important risk factor for lung cancer . Our results showed significant associations between gstm1 polymorphism and lung cancer risk among smokers and non - smokers . We also found that patients with gstm1 null genotype had increased non small - cell lung cancer (adenocarcinoma and squamous cell carcinoma) risk . However, we failed to find a significant relationship between gstm1 null polymorphism and small - cell lung cancer risk . This result suggested that gstm1 null polymorphism may play an important role in the development of non - small - cell lung cancer . Gsts are biotransformation enzymes, and they are phase ii enzymes with both catalytic activities and non - catalytic functions . Previous studies have shown that individuals with the gstm1 null genotype have a decreased capacity to detoxify certain carcinogens . Thus, it is biologically plausible that the gstm1 null genotype may increase risk of lung cancer . First, the methodological issues for meta - analysis such as one - way sensitivity analysis and cumulative meta - analysis were well investigated . Results from one - way sensitivity analysis and cumulative meta - analysis suggested high stability and reliability of our results and significant heterogeneity was not observed in this meta - analysis . Moreover, funnel plots and egger s tests were used to find potential publication bias . Second, lack of the original data from the eligible studies limited the evaluation of the effects of the gene - gene and gene - environment interactions in the development of lung cancer . Third, only published studies were included in this meta - analysis; therefore, publication bias may have occurred even though the statistical test did not show it . In conclusion, this meta - analysis suggests that an increased risk of lung cancer was associated with the null polymorphism of gstm1 in asians.
It is caused by a complex interplay of motor, sensory, and cognitive impairments2 . These neurological deficits are the prime cause of reduced quality of life and social participation3 . Thus, gait and balance recovery is regarded as a chief goal in stroke rehabilitation4 . Until now, various exercise programs such as progressive exercise5, 6, muscle strength exercise7, rhythmic pattern exercise8, 9, and virtual training10 have been used to regain the balance, mobility, and endurance of stroke patients in clinical and research settings . Among these interventions, repetitive motor training can alter brain representation maps and is mainly and basically used for managing the motor function recovery in stroke patients11 . Skilled activity is necessary to drive brain changes that might lead to improvements in functional activities such as gait12 . Stationary cycling, which requires less balance capability, has been used for training patients with or without nervous system disorders who have difficulty in maintaining balance and independent gait13 . Cycling and walking share similar locomotor patterns of reciprocal flexion and extension movements and alternating muscle activation of antagonists14 . Cycling can improve functional mobility and acts as a pseudo walking task - oriented exercise2 . Besides improving muscle strength, cycling exercise also facilitates muscle control of the lower limbs, which may allow putting more weight on the affected leg while standing . For this reason, stationary cycling exercise has been used with various other interventions in the clinical environments15 . However, the pure effect of cycling exercise is uncertain in chronic stroke patients . Therefore, this study investigated the effects of stationary cycling exercise on the balance and gait abilities of chronic stroke patients . This study was designed as a randomized, double - blind, pretest - posttest controlled trial . In this study, all experimental procedures and contents were explained to each participant, who provided written informed consent thereafter . All of the experimental procedures were approved by the institutional review board of sahmyook university . The inclusion criteria were as follows: presence of hemiparesis secondary to stroke that had occurred in the past 6 months; ability to walk 10 m independently with or without an assistive device; ability to communicate and understand, with a mini - mental status examination score of more than 21 points; no visual disorders or visual field deficit; and no known musculoskeletal conditions that would affect the ability to walk safely . The 6 subjects who refused to participate in the present program or did not meet the inclusion criteria were excluded from the study . The subjects were randomly assigned to the following 2 groups by using a table of random sampling numbers: the experimental and the control group . Both the subjects and the therapist were blinded to group assignments of the patients . All of the participants were evaluated before training and at the end of the 4-week training period . The patients in the experimental group performed the cycling exercise 30 minutes a day, 5 times a week for 4 weeks . Both groups received traditional therapy for 30 minutes per session, 5 times a week for 4 weeks . Stationary bicycle training has been used in order to improve the balance and walking abilities of stroke patients . In this study, only the lower extremity part of a dual - extremity ergometer (super dynamic 3000, shingwang medical) was used . To perform the exercise, the patient mounted a stationary bicycle safely under supervision, and the therapist adjusted the position of the seat and tied the ankles and calf to the pedals . The therapist was fully aware of the order and method of the stationary bicycle training, and the patients performed the exercise after receiving instructions and familiarizing themselves with the ergometers, including test runs . The resistance was set at 1 of 4, which corresponded to 2530 w on the stationary bicycle . The patient was riding the bicycle at 5060 rpm without stopping, for 30 min, 5 times a week for 6 weeks . The subjects balancing skills were rated using the berg balance scale (bbs; range, 056), and the timed up - and - go (tug) test was used to evaluate dynamic balance abilities . The patients were asked to stand up, walk at a comfortable speed to a point marked 3 m away from their chair, turn around, walk back, and sit down in the chair . The cut - off tug test score that indicated normal versus below normal performance was 12 seconds . The intra - rater (r = 0.99) and inter - rater (r = 0.98) reliability values were high16 . The bbs and tug test scores were obtained by an experienced physical therapist blinded to the group assignment . Gait ability was measured by using the timed 10-m walking test (10mwt). For the 10mwt, 10 m was measured on the floor by using a tape measure, and the start and end points were marked with tape . In order to provide sufficient distance for acceleration and deceleration, intervals of 2 m were added before the start and after the end marks . The 10-m walking time was measured with a stopwatch for the period from the moment the subject s feet passed the starting line to the moment they crossed the finish line . The subjects practiced once, and all measurements were made 3 times, using the average value of the 3 measurements in the analysis . . For the 10mwt, the test - retest and inter - rater reliability have been reported to be 0.95 and 0.90, respectively; both of these values are very high17 . The statistical package for the social sciences (spss) ver . 18.0 was used for data analysis . Descriptive statistics was used to analyze the general characteristics of the subjects . In order to examine the effects of the intervention in each group, a paired t test was conducted . In order to investigate differences between the groups, the demographic characteristics of the participants are shown in the table 1table 1.general characteristics of the subjectsexperimental group(n = 16)control group(n = 16)gender (male / female)12/413/3age (years)65.2 6.461.7 6.1height (cm)165.0 7.9169.0 6.1weight (kg)69.5 10.466.8 10.0lesion side (right / left)7/910/6mmse (score)26.10 1.7425.80 2.12data are expressed as mean sd . Mmse: mini - mental state examination as shown in table 2table 2.changes in balance and gait abilitiesexperimental groupcontrol groupbbs scorepretest36.15 5.9837.06 5.61posttest37.90 5.65 * 37.44 5.62postpre1.75 1.520.40 0.88tug (sec)pretest25.11 5.4024.19 3.47posttest16.74 3.07 * 19.48 3.90postpre8.4 4.354.71 4.86 10mwt (sec)pretest44.75 18.4045.93 13.22posttest37.74 15.70 * 43.96 12.04postpre7.02 7.021.96 3.13bbs: berg balance scale; tug: timed up - and - go; 10mwt: 10-m walking test . Significant difference, paired t test: * p <0.05; significant difference, independent t test: p <0.05, the experimental group showed significant improvements in bbs, tug test, and 10mwt scores after the intervention (p <0.05), whereas the control group showed significant improvements in their tug test and 10mwt scores, but not in their bbs score (p <0.05). Moreover, the experimental group showed greater improvement than the control group in 3 outcome measurements (p <0.05). Bbs: berg balance scale; tug: timed up - and - go; 10mwt: 10-m walking test . Significant difference, paired t test: * p <0.05; significant difference, independent t test: p <0.05 the aim of this study was to test the effects of cycling exercise on the balance and gait abilities of chronic stroke patients . The results demonstrated that the stationary cycling exercise supplemented with conventional therapy led to better balance and gait abilities than the conventional therapy alone . First, the stationary cycling training was found to have a positive effect on dynamic balance as measured by using the tug test . The results are similar to those obtained in a previous study by kim et al ., who compared ergometer bicycle training with treadmill walking training in stroke patients and reported significant improvement in tug test scores in both groups, although the differences between the groups were not significant18 . This suggests that the effectiveness of cycling training in improving locomotor function is similar to the effectiveness of treadmill exercise in stroke patients . Preliminary evidence suggests that cycling training programs reduce musculoskeletal impairment after stroke . In terms of muscle strength, a study by kuo and zajak suggested that the muscles that may be particularly important for this purpose are the hamstrings, rectus femoris, gastrocnemius, and tibialis anterior . These were all activated during the cycling task, which requires reciprocal flexion and extension movements of the hip, knee, and ankle19 . It is interesting that lustosa et al . Reported a significant correlation between muscle strength and improvement in tug test score . Therefore, we assumed that in this study the mechanism of tug test score improvement in stroke patients was muscle strengthening20 . Repetitive bilateral training and treadmill walking with or without suspension have a positive effect on walking ability21 . Repetitive practice is known to be important for motor learning, as the repetitions enable the system to coordinate muscle synergies5 . Cycling and walking share similar locomotor patterns of repetitive reciprocal flexion and extension movements . Hence, stationary cycling exercise, which employs reciprocal movement of the lower limbs and requires coordination of corresponding muscles, effectively increased the gait ability . Cycling training stimulates motor regions in the central nervous system and activates the cerebral cortex which eventually improves motor learning and balance . Based on the results, stationary cycling training can be effective in rehabilitation of stroke patients with gait and dynamic balance deficits . This study has the following limitations that we plan to address in future studies, including the small sample size and relatively short intervention duration . Further large - scale, long - term controlled clinical studies are required to verify the clinical benefits of stationary cycling exercise training.
Grace data recorded postseismic increase of the gravity field by 6 galviscoelastic relaxation and afterslip were examined with the gravity datathe grace data constrain the biviscous rheology model for the asthenosphere grace data recorded postseismic increase of the gravity field by 6 gal viscoelastic relaxation and afterslip were examined with the gravity data the grace data constrain the biviscous rheology model for the asthenosphere the great (mw 9.1) 11 march 2011 tohoku - oki earthquake disturbed not only the ground and ocean surface but also the earth's gravity field permanently . It generated transient seismic and tsunami waves and produced large coseismic stress change that led to gradual stress redistribution and subsequent postearthquake crustal motions . The dense geodetic data such as gps, interferometric synthetic aperture radar, and acoustic seafloor measurements reported coseismic displacement (a few tens of meters of horizontal motion and a few meters of vertical motion) and rapid postseismic displacement that yielded 10% of the equivalent seismic moment of the main shock in 2 weeks [ozawa et al ., 2011; sato et al ., 2011 additionally, the gravitational effect of the earthquake has been measured through changes of instantaneous relative motions of two gravity recovery and climate experiment (grace) satellites coorbiting around 500 km altitude [han et al ., subsequently, the time series of monthly grace gravity field data have been processed by optimizing the signal over the earthquake region [matsuo and heki, 2011; cambiotti and sabadini, 2012, 2013; wang et al . Most of the large earthquakes are followed by seismic or aseismic sliding (afterslip) on the coseismic fault and its lateral extension, by viscoelastic flow as a process of gradual relaxation of coseismic stresses, and/or by deformation induced by pore fluid migration in response to coseismic stresses [e.g., cohen, 1999; wang et al . Inverted the postseismic gps displacement data over the 1.5 years after the 2011 tohoku - oki earthquake for afterslip and viscoelastic relaxation models, and concluded afterslip is a dominant process while viscoelastic relaxation is responsible for only 11% of the postseismic moment . However, they did not evaluate biviscous processes that may explain transient deformation during the first few months as well as afterslip . For example, after the 2004 sumatra - andaman earthquake, in addition to gps displacement data at teleseismic distances [pollitz et al ., 2006], the grace gravity data were useful for constraining postseismic processes consistent with biviscous relaxation [han et al ., 2008; panet et al ., 2010; hoechner et al ., 2011]. It is imperative to constrain the complex response of the earth to the 2011 tohoku - oki earthquake to understand the rheology of the crust and mantle and the cycle of strain accumulation and release, ultimately, in order to estimate the subsequent seismic hazards in the surrounding region [wang et al ., 2012a]. To this end, we analyzed spatially and temporally continuous grace gravity observations implying the broadscale (500 km) deformation of the surface and interior in response to regional - scale postearthquake stress / strain redistribution and evaluated two alternate postseismic processes of viscoelastic relaxation and afterslip . Additionally, we calculated the high - resolution gravimetric response to this event and used these results with other available geodetic data to further discriminate between alternate mechanisms . We analyzed a total of 11 years of grace gravity data from 2003 to 2013 using the rl05 level-2 (l2) monthly data products generated by center for space research, university of texas [tapley et al ., 2004]. The march 2011 data were not used since the earthquake occurred in the middle of that month . The spherical harmonic coefficients up to degree and order 40 were used to represent gravity variations at a spatial resolution of 500 km, which is a conservative estimate of the grace data resolution . We first determined an analytical model of annual and semiannual sinusoids and a linear trend in the grace data by fitting the model to the monthly grace time series from january 2003 to february 2011 (i.e., prior to the earthquake). We extended the estimated trend out to the end of 2013 and removed it from the entire set of grace data before the analysis . Figure 1a presents the grace gravity changes calculated by subtracting the mean gravity field over a preearthquake period (february 2008 to february 2011) from the mean field over a postearthquake period (april 2011 to december 2013). Therefore, it illustrates the coseismic deformation as well as the postseismic deformation averaged over a period from 11 march 2011 to december 2013 . Next, we computed the average coseismic gravity changes (figure 1b) using the seismic centroid moment tensor (cmt) solutions (global cmt and u.s . Geological survey w - phase moment tensor) and two seismic finite fault models, models ii and iii, from shao et al . . The finite fault models we used present the largest slip updip of the hypocenter close to the trench consistent with the recent analysis including tsunami and seafloor data presenting the rupture out to the trench [lay et al ., 2011; iinuma et al ., the dominant negative anomaly found in the seismic models indicates extensive crustal density decrease by volume extension under the influence of the ocean mass redistribution [han et al ., 2006, 2013; (a) the grace observed gravity change following the 2011 tohoku - oki earthquake computed by differencing the 3 year mean field before and after the earthquake . (b) the average synthetic gravity change caused by the coseismic deformation computed using four seismic models . (c) the postseismic grace gravity change computed by subtracting the seismic model prediction shown in figure 1b from the mean difference field shown in figure 1a . (d) the same as figure 1a but during the 3 years between 20082010 and 20052007 (no large earthquake in - between). Figure 1c elucidates the postseismic gravity change computed by removing the coseismic (elastic) contribution (figure 1b) from the grace data (figure 1a). It indicates that the magnitude of postseismic deformation is a fraction of the coseismic deformation . During the first 3 years after the main shock, on average, 6 gal of postseismic gravity change was estimated mostly around the epicenter . During a time period that does not include the perturbation due to a large earthquake (for example, the 20082010 mean field minus the 20052007 mean field), the mean field difference is within 2 gal (figure 1d)a measure of grace data noise and inherent gravity variations in this region . While the coseismic gravity change produced the positive anomaly offshore and the stronger negative anomaly landward, the postseismic gravity change yielded the positive anomaly prominently around the epicenter where the coseismic perturbation was small . To understand this, we further examine the temporal dependence of the postseismic gravity data and compare it with models of viscoelastic relaxation and afterslip . The temporal characteristics of postseismic gravity changes provide constraints on the physical processes and the rheological structure of the earth . In particular, pollitz [1997b] found that asthenospheric viscosity controls the temporal pattern of long - wavelength postseismic relaxation following a large megathrust event . Using the global normal mode formalism and viscous relaxation theory given in pollitz [1997b] and the fault geometry notation of kanamori and cipar [1974, figure 10] and kanamori and given [1981, figure 1], we express the gravitational potential change induced by earthquake as follows: 1where a point dislocation source is at the (north) pole and described with the moment tensor components, mrr, mr, mr, m m, and, m. the five independent and orthogonal basis functions of the earthquake geopotential change, gmrr, gmr, gmr, gm m, and gm, are excited by each moment tensor component with a physical dimension of m / s per nm . They are computed with the eigenfunctions of viscoelastic normal modes (pollitz [1997b], pollitz et al ., and han et al . They are dependent only on the earth's viscoelastic structure (i.e., precomputable regardless of seismic sources). The examples of the spatial pattern of each function for the elastic response (evaluated at t = 0) are shown in figure 2 (left). (left) the spatial patterns of the gravity changes to be excited by five independent moment tensor components of mrr, mr, mr, m m, and, m, respectively . The exact scale is dependent on the moment tensor (the negative to positive values are depicted with blue to red colors). The coordinates of the dislocation source are 38.5n, 142.6e (depicted as a red star), and its depth is 20 km . (right) the corresponding temporal variations of each component of the gravity change before and after the earthquake . The mean value of the synthetic gravity changes computed from various seismic models is shown in thick black solid line, and the 1 sigma variation of the models is depicted in thin black dashed line . Depicted are viscoelastic gravity changes computed from the maxwell asthenosphere model with the viscosity of 5 10 pa s (blue), 10 pa s (green), 5 10 pa s (red), and 10 pa s (cyan) and from the biviscous (burgers body) asthenosphere model with transient viscosity of 10 pa s and steady state viscosity of 10 pa s (purple). The temporal evolution of each gravity component, mrr, mr, mr, m m, and m, are simply obtained by the inner product of each basis function (e.g., gmrr) and the grace gravity data over unit sphere (see the supporting information for the explicit forms). The observed temporal changes from grace are presented in figure 2 (right). The gravity changes corresponding to the moment tensor components of mr and m can be resolved with the highest accuracy of 2030 10 nm (or mw = 8.28.3), while the other components have the accuracy of 80 10 nm (or mw = 8.6). The north - south sampling nature of the grace intersatellite tracking enables the best sensitivity in estimation of mr and m components, 34 times better than other components . This indicates the grace's sensitivity to earthquakes as small as mw = 8.2 in this region, depending on the focal mechanism . The average and standard deviation of four seismic models are shown as thick black solid and thin black dashed lines in figure 2, respectively . The first few months of grace data after the earthquake agree with the coseismic changes from seismic models; however, subsequently, there are substantial variations observed particularly in mrr and m m. there is no significant postseismic change discerned from other components . We used a spherically stratified viscoelastic earth model that includes a global ocean layer (3 km thick), an elastic lithosphere (60 km thick) overlying an asthenosphere extending to a depth of 220 km, and an upper (220670 km) and lower (6702891 km) mantle [pollitz et al ., 2006]. The elastic thickness of 63 km near the japan trench was estimated independently from the marine gravity and flexural analysis [levitt and sandwell, 1995]. The stratified density structure is consistent with preliminary reference earth model [dziewonski and anderson, 1981]. The upper and lower mantle was modeled with a maxwell rheology with the viscosity of 10 pa s and 10 pa s, respectively . We tested five different rheological models for the asthenosphere; (1) maxwell viscosity of 5 10 pa s, (2) 10 pa s, (3) 5 10 pa s, (4) 10 pa s, and finally, (5) transient (kelvin) viscosity of 10 pa s and steady state (maxwell) viscosity of 10 pa s with biviscous (burgers body) rheology [ivins and sammis, 1996]. The lithosphere overlying the asthenosphere is assumed to be elastic by assigning a very large viscosity of 10 pa s. we use the computer code visco1d [pollitz, 1997b] and its modification to compute the postseismic gravity response to a dislocation source for alternate spherical viscoelastic models [pollitz, 1997a; pollitz et al ., the pollitz [1997b] method is applicable to any deformation field represented as a viscoelastic normal mode sum on a laterally homogeneous spherical model, specifically, a weighted spherical harmonic sum of displacement - stress vectors . Although originally designed for the case of static gravity changes, it is also directly applicable to the case of postseismic gravity changes by employing equation (21) of pollitz [1997a] as the viscoelastic normal mode sum . Figure 2 presents the various synthetic viscoelastic gravity changes, depicted in solid colored curves . These viscoelastic simulations were computed with the cmt solution obtained from han et al . . The equivalent simulations with a finite fault model such as model ii of shao et al . Are shown in the figure s1 in the supporting information, and they are not different significantly at the spatial scale of consideration because of the concentrated coseismic slip of this earthquake . For the components mrr and m m, the model runs with the asthenospheric maxwell viscosity of 5 10 and 10 pa s (blue and green curves, respectively) predict rapid gravity change within a few months after the main shock . This may be reconciled with the short - term grace observations; however, the long - term changes with these viscosities are predicted to be overly large . The simulations with the asthenospheric maxwell viscosity of 5 10 and 10 pa s (red and cyan curves, respectively) do not reproduce the rapid change observed during the first few months but capture the cumulative long - term change . The rapid change followed by gradual variation in the grace gravity data are best reproduced with the biviscous relaxation model with the transient viscosity of 10 pa s and steady state viscosity of 10 pa s (purple). For the other components of mr, mr, and m that do not show discernible postseismic gravity change observation, any model with the steady state viscosity smaller than 5 overall, the biviscous asthenospheric model satisfactorily explains the postseismic gravity changes observed by grace, similar to the findings for the 2004 sumatra - andaman earthquake [pollitz et al ., 2006; han et al ., 2008; panet et al ., 2010; hoechner et al ., 2011] alternatively, the afterslip on the coseismic rupture surface and its downdip extension is also a viable candidate to explain postseismic deformation after large megathrust earthquakes . We use the finite fault models of afterslip to quantify the predicted gravity for comparison with the time series of grace gravity data . The (kinematic) afterslip model was constrained by gps measurements of postseismic displacement accumulated over various intervals (7, 20, 48, 107, 200, 289, 381, and 564 days) up to the 1.5 years after the main shock, assuming that the postseismic displacement is entirely due to afterslip [diao et al ., the maximum cumulative slip of 4 m over the period of 1.5 years and the equivalent seismic moment of 2.3 10 nm was found by elastic dislocation modeling of postseismic gps data [ozawa et al ., 2011; diao et al .,, we calculated the deformation associated with afterslip using dislocation theory [e.g., cohen, 1999]. Figure 3a presents the grace postseismic gravity data (same as figure 1c), and three different locations where the positive and negative (to a lesser extent) gravity changes are identified . The gravity change of the mrr component is responsible mostly for the positive gravity at p2, while the one of m m is for the negative gravity change at p1 and p3 . The grace gravity time series at each location are presented with the error estimates of 2 gal in figure 3b . The elastic gravity change was computed with model ii of shao et al ., and the viscoelastic gravity change was computed using the same seismic source model and the biviscous earth's model characterized with the transient viscosity of 10 pa s and steady state viscosity of 10 pa s for the asthenosphere . The time series of the afterslip gravity change from the finite fault model of diao et al (a) grace observation of postseismic gravity change (same as figure 1c). (b) time series of the grace gravity change at p1, p2, and p3 . An arbitrary bias of 17 and 17 gal was added to the ones of p1 and p3, respectively, for plotting clarity . The model prediction of coseismic slip, afterslip, and viscoelastic relaxation is shown, respectively, as black, blue, and red lines . (c) a snapshot of postseismic gravity change 1 year after the main shock from the viscoelastic relaxation model . (d) same as figure 3c but from the afterslip model . The snapshot of the synthetic gravity change 1 year after the main shock is depicted in figures 3c and 3d, respectively, for the viscoelastic relaxation and the afterslip models . Both postseismic processes produce similar gravity change in spatial and temporal pattern as well as the magnitude . The positive anomaly around the epicenter (p2) is a dominant feature consistently found in the grace measurements and in both viscoelastic relaxation and afterslip models . The secondary anomalies with 34 times smaller magnitude are the negative changes in the west and the east of the positive anomaly (p3 and p1, respectively). The afterslip model predicts a slightly larger anomaly in the west than the one in the east . So far, we examined the postseismic free - air gravity changes measured from grace and computed from viscoelastic relaxation and afterslip models at a spatial resolution of 500 km; it is difficult to discriminate these competing postseismic processes from the grace data alone for the period of a couple of years after the main shock . In order to further understand gravity change and deformation associated with these postseismic mechanisms, we computed the gravity effect of surface vertical deformation (uplift / subsidence of the ocean - seafloor interface) and interior deformation (bouguer gravity anomaly) separately [han et al ., 2006]. The gravity change of seafloor vertical motion by viscoelastic relaxation (figure 4a) is characterized by a dominant positive anomaly around the epicenter implying broadscale (500 km) seafloor uplift . The bouguer anomaly of viscoelastic relaxation (figure 4b), computed by subtracting the effect of seafloor vertical motion from the free - air anomaly, shows a similar pattern of gravity change but with a magnitude 23 times smaller . The bouguer gravity change is explained mostly by vertical deformation of the density interfaces of the upper and lower crust and of the crust and upper mantle (moho). It indicates that the broadscale uplift gradually occurs with negligible change in bulk volume (or density) as viscous asthenosphere and mantle yield to the coseismic stresses . (a, c, and e) gravity change due to seafloor vertical motion computed, respectively, from biviscous viscoelastic relaxation, afterslip, and coseismic slip . (b, d, and f) same as figures 4a, 4c, and 4e, respectively, but for the bouguer gravity . In comparison to the uplift by viscoelastic relaxation, the afterslip model predicts twice larger seafloor uplift around the epicenter as highlighted by the doubled magnitude of the positive gravity (figure 4c). The bouguer gravity of afterslip (figure 4d) is characterized by a negative anomaly orthogonal to the strike direction . This negative anomaly is primarily due to volume expansion or dilatation associated with the downdip afterslip within the lower crust and the upper mantle . We also computed the gravity changes of seafloor vertical motion and bouguer anomaly (figures 4e and 4f, respectively) from the coseismic slip model, model ii of shao et al . . The majority of the coseismic slip is estimated at the top 25 km of the crustal layer, while the afterslip is concentrated mostly at depths of 4050 km [ozawa et al . Deeper sources like the downdip afterslip yield smaller volume change due to larger rigidity and bulk modulus at the mantle depth, while shallower coseismic slip associated with smaller rigidity and bulk modulus at the crustal depth yields larger volume change . As a result, the deep afterslip produces the smaller bouguer gravity anomaly (figure 4d) relative to the gravity from seafloor vertical deformation (figure 4c) and thus results in the smaller negative change in the free - air gravity (figure 3d). On the other hand, the coseismic bouguer gravity anomaly (figure 4f) is as large as the coseismic vertical displacement (figure 4e), which reduces the positive contribution to the free - air gravity (figure 1b). The downdip afterslip also moves the peak gravity change landward (west), relative to the coseismic gravity change . We repeated the same calculations of synthetic gravity changes for the viscoelastic, afterslip, and coseismic deformation but at a resolution of 55 km by extending the spherical harmonic degree and order up to 360 and by using a finite fault model shao et al . We found that the high degree expansion of the viscoelastic simulation predicts local subsidence superimposed on the broadscale uplift across the japan trench . While the east coast of the island and the east of the trench undergo uplift, the area localized above the rupture zone undergoes subsidence, as implied by the positive and negative gravity change, respectively . This is the very characteristic pattern of surface vertical motion followed by thrust earthquakes when the elastic lithosphere overlying the viscoelastic asthenosphere is only partially ruptured [melosh, 1983; cohen, 1999]. Melosh found that the top boundary of the viscous asthenosphere experiences local extension beneath the fault if the fault stops within the lithosphere, which causes the local subsidence on top of regional uplift . However, if the fault cuts through the lithosphere, the boundary of the asthenosphere is subject to net shortening and the surface undergoes uplift . Sato et al ., burgmann et al ., and hu et al . Reported the postseismic subsidence of the seafloor above the rupture zone by a few decimeters within a couple of years after the main shock using the gps - acoustic positioning and seafloor pressure data, while the gps data implied postseismic uplift along the coast by a few centimeters [japan coast guard, 2012; japan coast guard and tohoku university, 2013]. Such subsidence may yield tens of gal gravity decrease (7 gal per 10 cm of seafloor subsidence; the bouguer correction with the density contrast between the crust and ocean). No seafloor measurement is available in the pacific plate, to the east of the trench, where viscoelastic uplift is predicted . The local subsidence from the viscoelastic model is diminished by the uplift in the periphery of the overthrust and underthrust blocks at a broadscale resolution, which is what the grace data observed . Does not produce subsidence, although the model is consistent with onland postseismic gps motions . Reported such inconsistency in the postseismic vertical motion from afterslip following the great 2006 and 2007 kuril earthquakes . Hu et al . Found that the poroelastic rebound may yield uplift above the rupture zone by a few decimeters in 2 years . Although all of these postseismic processes are likely present and compensate each other at times, we hypothesize the viscoelastic relaxation triggered by the partially ruptured elastic lithosphere is a main driver of the local subsidence above the rupture region . Grace observed postseismic gravity increase by 6 gal (4050% of the coseismic gravity change) within a couple of years after the 2011 tohoku - oki earthquake . The postseismic free - air gravity changes from viscoelastic relaxation and afterslip are similar at the scale of 500 km commensurate with the grace data resolution . However, the bouguer gravity responses are distinct; the bouguer anomaly of viscoelastic relaxation is positively correlated with the free - air anomaly, while the bouguer anomaly of afterslip is negatively correlated . The broadscale postseismic gravity observation and the local subsidence measurement above the rupture zone the viscosity is the most important factor to govern evolution of the long - wavelength stress field after the earthquake . Large - scale monitoring of the postseismic deformation (gravity as well as vertical displacement) will help characterize the rheological properties of the earth's interior that may lead to a better understanding of increased seismic hazard following great earthquakes.
Thais clavigera, a taupe shuttle - like conch, inhabits in the mesolittoral zone, on the rocks, and under the pebbles along the coasts from south to north in china and most coasts in japan . In recent years, it is usually applied as indicator of heavy metal pollution especially organotin in ocean [2, 3]. It has also been used as chinese medicine for over 1000 years, and all of its parts including the shell, flesh, and operculum of t. clavigera could be used as medicine, which are called liaoluo, hailuo, and hailuoyan, respectively . Since the flesh of t. clavigera tastes delicious, it has been often used as dishes by locals since ancient time . As there has been a certain amount of researches on nutrient and functional composition of some economic snails in ocean, the studies on these components in thais including t. clavigera are quite few . In previous studies, nutritional composition of wild t. clavigera in only a region (intertidal zone of dongji island in zhoushan, china) the results showed that it contains rich proteins, fatty acids, sterols, and mineral elements . And 17 amino acids were checked out in the species, which accounted for 60.17 g of the total dry weight; essential amino acids accounted for 35.57% of the total amino acids, which indicates that t. clavigera is high - quality protein source . As we all know, amino acids are basic structure units of biomacromolecules such as protein and enzyme . Moreover, the free amino acids associated with buthus martensii, cervus nippon temminck, and calculus bovis [79] have been paid considerable attention [1013]. As for the nucleosides and nucleobases, since more and more researches in ganoderma lucidum, cordyceps sinensis, and isatis indigotica have been carried out, they have also been proven to be important nutritional and functional compositions [1719]. For a nutrient and functional perspective, it is essential to determine the amino acids, nucleosides, and nucleobases in function foods for the quality control and potential useful value . However, the amino acids were usually determined by visible spectrophotometry after derivatization with ninhydrin in previous study, which has low sensitivity and complex pretreatment procedure . In the present study, we collected t. clavigera samples from 19 areas along the coasts in china . To determine these hydrophilic compounds in two categories, hydrophilic interaction ultra - performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (hilic - uplc - tq - ms / ms) was used for simultaneous identification and quantification of amino acids, nucleosides, and nucleobases in the extracts of t. clavigera . After content analysis, a pca method was further performed for comparing their content variation in different distribution regions . Shanghai, china), ammonium acetate (sinopharm chemical reagent co., ltd ., shanghai, china), formic acid (merck millipore, germany), and acetonitrile (tedia company inc ., united states) were of analytical grade . The deionized water (h2o) was purified by a superpurification system (eped technology development, nanjing, china). 41 standards including adenosine-5-monophosphate (1), inosine (2), guanosine (3), thymidine (4), 2-deoxyuridine (5), 2-deoxyinosine (6), cytidine-5-monophosphate (7), 2-deoxyadenosine-5-monophosphate (8), 2-deoxycytidine (9), 2-deoxyguanosine (10), thymine (11), adenine (12), cytidine (13), uracil (14), guanine (15), glycine (17), gaba (18), asparagine (27), glutamine (28), citrulline (30), hydroxyproline (32), taurine (35), and ornithine (37) were purchased from sigma (st . Louis, mo, usa). Standards including xanthine (16), leucine (19), isoleucine (20), phenylalanine (22), tryptophan (23), alanine (24), threonine (25), serine (26), glutamate (29), proline (31), tyrosine (34), valine (36), aspartate (38), lysine (39), histidine (40), and arginine (41) were obtained from the national institute for the control of pharmaceutical and biological products, beijing, china . A chemical standard of cysteine (33) was obtained from aladdin chemical, shanghai, china . A reference of methionine (21) was purchased from sinopharm chemical reagent, beijing, china . The samples of t. clavigera are collected from 19 different habitats of china in august 2014 . The t. clavigera were collected from the habitats and identified by professor ding shaoxiong (xiamen university, fujian province, china). The voucher specimens were deposited in nanjing university of chinese medicine, china . A mixed standard stock solution containing the reference compounds 141 dried to constant weight was prepared in methanol / water (9: 1, v / v). Working standard solutions for calibration curves were prepared by diluting the mixed standard stock solution with 10% methanol at different concentrations . After unfreezing at the temperature of 4c and removing the shell, the flesh of t. clavigera was freeze - dried by the vacuum freeze dry systems (labconco, united states), and then they were weighed and smashed, respectively . One gram of each dry sample was accurately weighed into 50 ml conical flask, and 40 ml distilled water was added . All of the mixtures were precisely weighed and placed into an ultrasonic bath (40 khz) for 60 min at room temperature and distilled water was added to compensate for the loss of water during extraction . The supernatants of extracting solution were separated after centrifugation (13000 r / min) for 10 minutes and then eliminated protein by adding acetonitrile to double volume and then stored at 4c and filtered through 0.22 m cellulose membrane filters prior to injection . A waters acquity uplc system (waters, milford, ma, usa) equipped with a quaternary pump solvent management system, the chromatographic separation was carried out on an acquity uplc beh amide column (2.1 mm 100 mm, 1.7 m). The mobile phase was composed of a (5 mmol / l ammonium formate, 5 mmol / l ammonium acetate, and 0.2% formic acid in aqueous solution) and b (1 mmol / l ammonium formate, 1 mmol / l ammonium acetate, and 0.2% formic acid in acetonitrile) with a gradient elution: 03 min, 10% a; 39 min, 1018% a; 915 min, 1820% a; 15 - 16 min, 2046% a; 1618 min, 46% a. the flow rate and column temperature were set at 0.4 ml / min 35c . The mass spectrometry analysis was performed on xevo triple quadrupole ms (waters corp ., milford, ma, usa) equipped with an electrospray ionization (esi) source operating in positive ionization mode . And the conditions of the esi source were set as follows: the desolvation gas flow rate was set to 1000 l / h, the desolvation temperature was set at a temperature of 350c, the cone gas flow rate was set at 20 l / h, the source temperature was set at 120c, and the cone voltage and collision energy were set depending upon the mrm for each compound . In this study, in order to obtain the best extraction efficiency, variable factors during the extraction including solvent (water, aqueous methanol of different concentrations), solvent volume (10, 20, 30, 40, 50, and 60 ml), extraction temperature (20, 40, 60, 80, and 100c), extraction methods (refluxing and ultrasonication), and extraction time (10, 20, 30, 40, 50, and 60 min) were optimized . During the optimization according to the peak area as evaluated criteria, it revealed that ultrasonic bath with the solvent of 50 ml water at room temperature for 60 min was the best condition . To achieve better results of analysis the separation was investigated on an acquity beh amide (100 mm 2.1 mm, 1.7 m) after comparison of two columns, an acquity beh c18 (100 mm 2.1 mm, 1.7 m), and an acquity beh amide (100 mm 2.1 mm, 1.7 m). Acetonitrile with better elution ability, separation selectivity, and peak shape compared to methanol was used as organic phase . In addition, the dissolution of ammonium acetate and ammonium formate in acetonitrile could improve the separation of amino acids, nucleosides, and nucleobases for hplc analysis . Meanwhile, formic acid was also used to inhibit solute ionization to improve the shape of peak . Consequently, acetonitrile mixed with 1 mmol / l ammonium formate, acetate, and 0.2% formic acid were chosen for organic phase, and deionized water mixed with 5 mmol / l ammonium formate, ammonium acetate, and 0.2% formic acid were chosen for aqueous phase . The flow rate and column temperature were both optimized, and they were set at 0.4 ml / min and 35c, respectively . The typical chromatograms of the 40 analytes are presented in figure 1 . As for ms / ms condition, all of the compounds were examined separately in direct infusion mode by full - scan ms method in both positive and negative ionization modes for better analysis . It was found that both higher sensitivity and clearer mass spectra were obtained in the positive ion mode compared to the negative ion mode . The proposed uplc method was validated by determining the linearity, lod, loq, precision, repeatability, stability, and recovery . And all the calibration curves exhibited good linear regressions (r> 0.9919) within the test ranges . The lod was determined at signal - to - noise (s / n) ratio of 3, and the loq was determined at s / n ratio of 10, each of which was 0.0030.112 g / ml and 0.0080.352 g / ml, respectively . The overall rsds of intraday precisions and interday precisions were <3.48% and <3.24%, respectively . The rsds of repeatability and stability assessed were <3.42% and <4.89%, respectively . The recoveries of the 41 compounds were in the range of 92.62%110.51% and the rsds were <4.10% . Moreover, it was proved to have no significant matrix effects in relatively complex functional food matrices within 24 h. as a result, the established method for simultaneous determination of 41 amino acids, nucleosides, and nucleobases in t. clavigera was accurate, sensitive, and repeatable . The amino acids, nucleosides, and nucleobases in t. clavigera collected from 19 different habitats in china were analyzed . The contents of the 41 compounds are listed in table 2, which indicate that there is a large difference among different samples . The results showed that the samples are rich in amino acids, nucleosides, and nucleobases and the total contents vary from 119.41 mg / g (sample 12) to 317.10 mg / g (sample 18). The results showed that t. clavigera collected from hainan was mostly higher in nutritional value compared to the other habitats . And the shape and appearance of the t. clavigera of hainan were different, which were much bigger and purer . All of these may be attributed to many factors such as much better marine environments surrounding hainan province . The 19 samples tested by the methods mentioned above mainly contain all the 16 nucleosides and nucleobases determined in the study . However, comparing to the amino acids, nucleosides and nucleobases were mostly of microgram magnitude, and total contents of them in different samples vary from 2.65 mg / g to 20.49 mg / g . The contents of t. clavigera collected from different sea areas were in the following order: hainan> shandong> liaoning> jiangsu> fujian> zhejiang . However, the quantities of all nucleosides and nucleobases we have determined in the test in one sample have no obvious differences . The samples collected from hainan showed great advantages, which resulted in the total contents of samples from hainan being almost ten times as much as the lowest one from other habitats . As for the specific constituents, the contents of inosine varied from 0.31 mg / g to 3.61 mg / g taking up 7.43% to 38.21% of total contents of nucleosides and nucleobases in the samples . As for the amino acids, the quantities of detected amino acids except gaba and cysteine were of milligram magnitude per gram of the samples . The total contents of amino acids in the samples were between 116.74 mg / g and 298.58 mg / g, and the contents of t. clavigera collected from different sea areas were sequenced as follows: hainan> fujian> jiangsu> shandong> liaoning> zhejiang . Among the amino acids, the contents of proteinogenic amino acids varied from 98.47 mg / g to 261.05 mg / g, and the contents of 8 essential amino acids between 38.79 mg / g and 92.72 mg / g were about 1/3 of the total contents of proteinogenic amino acids . Particularly, the content of lysine is far more than others, which are between 8.19 mg / g and 19.10 mg / g . In consideration of the good taste, flavor amino acids in t. clavigera were studied as well, and the results displayed that, among the 25 amino acids determined in the exam, the total contents of 22 flavor amino acids from 105.18 mg / g to 276.80 mg / g in t. clavigera take the most portion which are over 90% of the total contents . Due to the high contents of essential amino acids and flavor amino acids, t. clavigera is high in nutritious value with good taste . Besides the proteinogenic amino acids the total content of nonprotein amino acids in t. clavigera has the highest amount of taurine, which varied from 8.05 mg / g to 20.78 mg / g in the samples . And ornithine as nonprotein amino acid second only to taurine varied from 3.44 mg / g to 11.91 mg / g . To explore the 25 amino acids and 16 nucleosides and nucleobases in the samples from different habitats, principal component analysis (pca) was performed on the basis of the contents of the 41 compounds . According to the results of pca analysis, the first three principal components (pc1, pc2, and pc3) with the total proportion of 77.93% were extracted for further information mining . And all components of the three accounted for 60.68% (pc1), 10.95% (pc2), and 6.30% (pc3). The components loading matrix is shown in table 3 . According to the loadings, pc1 had good correlations with the most compounds especially those such as adenosine-5-monophosphate, adenine, xanthine, gaba, threonine, serine, hydroxyproline, and histidine . And pc2 had good correlations with analytes of thymidine, 2-deoxycytidine, guanine, and lysine whereas pc3 had good correlations with analytes of cytidine and aspartate . The sample scatter plot is shown in figure 3 where each sample is represented as a marker . It was noticeable that the samples were clearly clustered into three clusters including cluster a (the bohai sea and the yellow sea including samples 1, 2, 3, 4, 5, 6, 7, and 8 collected from liaoning, shandong, and jiangsu), cluster b (the east china sea including samples 9, 10, 11, 12, 13, 14, and 15 collected from zhejiang and fujian), and cluster c (the south china sea including samples 17, 18, and 19 collected from hainan). The regional distribution of t. clavigera is shown in figure 4 . From the plot, it turned out that there are significant differences between the t. clavigera samples collected from different geographical environment . The determination results of amino acids showed that the contents of lysine are between 8.19 mg / g and 19.10 mg / g accounting for 5.2212.11% in our study, which was more than that in dongji island (4.91%) by previous report . As we all know, in essential amino acids, lysine is relatively less in grain . As a consequence, the high contents of lysine in t. clavigera mean that people who eat grain as staple food or who eat it often can increase the intake of lysine and balance the essential amino acids in the body [20, 21]. We have recognized that the microbial catabolism of amino acids produces flavor compounds of importance for foods . Aspartate, glutamate, glycine, and alanine are present as amino acids with characteristic of umami . Among the 22 flavor amino acids determined in t. clavigera, their total contents were varied from 25.21% to 37.99%, which could fully explain the delicate flavour of t. clavigera . As functional food, it has to be mentioned that there are five nonprotein amino acids, which account for 7.9717.26% . Taurine relating to various biological processes including development of the central nervous system, membrane stabilization, and immunity appeared to be abundant in t. clavigera [2325]. It is the intermediate product of ureogenesis as well as precursor substance of amino acids such as citrulline and histidine metabolism . It has come to public attention in recent years due to its multifunctional health care function especially the protection of liver [26, 27]. Although there have researches about determination of amino acids in t. clavigera, there are few methods on nucleosides and nucleobases . However, according to the results, the nucleosides and nucleobases in t. clavigera are various, and the contents are balanced . Among the contents, inosine, which is widely distributed in animals, takes up the most of the total nucleosides and nucleobases contents . Meanwhile, inosine is reported to protect liver and inosinic acid is studied to be fresh aid related to the delicious taste . In this study, it is the first time that hilic - uplc - tq - ms / ms has been utilized for the simultaneous determination of these bioactive compounds in t. clavigera from 19 habitats as a sea snail . The results showed that, as traditional seafood, t. clavigera has excellent source of amino acids, nucleosides, and nucleobases with great nutritional and functional values . Importantly, inosine, lysine, glycine, and taurine in t. clavigera as top contents in each categories could be recognized as makers for establishing quality standards . And these research results also provided good data for establishing quality standard of t. clavigera even thais for the further development and utilization of the marine organism.
The search for new therapeutic approaches capable of preventing and treating musculoskeletal dysfunctions is progressively increasing in conjunction with current technological innovations . In this context, taping techniques have developed as a complement to the treatment of musculoskeletal dysfunctions, and has improved over time to provide therapeutic effects which do not hinder the functionality of a particular body segment . In 1973, dr . Kenzo kase developed an elastic tape with elastic properties similar to the skin, and named it kinesio tape1,2,3,4 . The kinesio taping method originated from the hypothesis that an external component could aid the functions of muscles and other tissues5, 6 . It is thin and elastic by design, and can stretch to 40% to 60% of its original length, which makes it very elastic compared to traditional taping materials, allowing complete range of motion5,6,7 . Various authors have described the benefits of kinesio taping as being dependent on the stretch of the tape and the form of placement on the skin, which elicits: positional stimulus and correction of muscle function; improvement of fascial tissue alignment; facilitation of bodily fluid circulation; repair of injured tissues; sensory stimulation assisting or limiting movement, thereby improving proprioception; edema control by guiding lymph toward lymph nodes; and correction of joint position2, 5,6,7,8,9,10,11 . According to kenzo kase, the stretch applied to the tape creates tension in the skin which improves communication with mechanoreceptors and increases the number of motor units recruited during a muscle contraction5 . Through these effects, the tape can improve muscle function by facilitating the contraction of inactive muscles . Therefore, the application of kinesio taping over the gripping musculature of the hand could possibly be used to complement therapeutic treatment of manual dysfunction once its influence has been assessed on healthy individuals . The human hand is clearly the most important and complex structure of the upper extremity due to its extensive mobility and the sensitive capabilities of its surrounding tissues, which allows gripping and feeling, its essential functions2, 12 due to the increasing utilization of kinesio tape in clinical settings, studies are necessary to confirm the purported benefits of the method and establish evidence - based standards for this technique . In light of the scarcity of research regarding kinesio taping, and the fundamental role of handgrip strength in activities of daily living, this study assessed the effects of kinesio taping on the handgrip strength of healthy women, as measured by handgrip dynamometry . This study recruited 75 healthy women volunteers, aged between 18 and 30 years, who were randomly subdivided into three groups (n=25): kinesio, kinesio without tension (kwt), and the control groups . The kinesio group received tape application at 25% to 35% of tension; the kwt group received tape application with no longitudinal stretch; while the control group did not receive any taping techniques . Subjects from both the kinesio and kwt groups were unaware of the tension utilized on their respective taping techniques to avoid possible influences on the results . The subjects of this study were physical therapy students of the university salgado de oliveira in brazil, who were invited to participate as volunteers through advertisements placed inside their classrooms . The inclusion criteria required a signed informed consent form and agreement to participate during all phases of the study . The exclusion criteria were: outside of the established age range; absence from the 24-hour or 48-hour follow - up assessment of handgrip strength; presence of limiting factors which could have influenced the results, such as cardiopulmonary, hormonal, or osteomyoarticular disorders; joint or bone deformities, congenital or acquired, in either of the upper extremities; central or peripheral neurological deficits; use of anabolic substances; injury or surgery to the upper extremities within the last six months; or consumption of alcoholic beverages or pharmaceutical substances 24 hours prior to the start of this study . This research was approved by the research ethics committee of the hospital of tropical diseases under protocol number 009/2011 . The instrument utilized to measure handgrip strength was a jamar dynamometer, which has been validated as a gold standard tool for this purpose by the american society of hand therapists (asht)11 . This instrument allows simple and quick readings of handgrip strength, which is measured in kilograms / force . Initially, the subjects sat on an adjustable chair which was adjusted so that subjects backs were straight, with their knees and hips in 90 of flexion with their feet on the floor . The shoulders were positioned in adduction next to the trunk with the elbows in 90 of flexion, the forearm and wrist in the neutral position, and the arm unsupported, while the examiner held the dynamometer for each reading, as recommended by the asht14 . The subjects performed the handgrip movement with maximum effort, only during exhalation and after a verbal cue given by the examiner: three measurements were made for each hand, alternating the test sides, starting with the right hand a rest interval of 60 seconds was provided between trials in order to avoid muscle fatigue during the assessment . Subjects were instructed to maintain maximum contraction for 5 seconds in each trial, since research has demonstrate that peak force is reached between 3 to 10 seconds of contraction13 . Subjects from the kinesio and kwt groups had the skin of their forearms cleaned with a cotton pad and 70% alcohol before the application of the respective kinesio taping techniques . A single researcher certified in the kinesio taping method conducted the taping, and pink kinesio tex gold tape was used . The technique performed for the kinesio group aims to influence muscle function by activating the flexor digitorum superficialis muscles . The tape was initially anchored with 2.5 to 5 cm of tape at the medial epicondyle, then 25% to 35% of tension was applied as it was wound over the target muscle toward the hand . The kwt group received the same tape application, but no tension was added as the tape was applied (0% of stretch). The control group only performed the handgrip strength assessment and did not receive any taping . The handgrip strength dynamometry was reassessed for all groups after 30 minutes, 24 hours, and 48 hours of taping . The subjects did not exercise their gripping muscles during the 48 hours of this study . The variables were normally distributed, and the statistical package for the social science software (version 15.0) was used . Initially, a descriptive analysis of the data was conducted in order to obtain the mean, standard deviations, minimums, and maximums of the mesured items . The anova test (complemented by the student s t - test) was used to verify and compare the effects of kinesio taping on handgrip strength after 30 minutes, 24 hours, and 48 hours . The initial sample size totaled 83 subjects, with an average age of 21.5 years (sd 2.60). Eight subjects did not participate in the study s entirety (did not return after 24 or 48 hours for reassessment of handgrip strength, or presented limiting factors which could have interfered with the results), and were excluded following the exclusion criteria, reducing the sample size to 75 healthy women . Regarding the behavior of handgrip strength values, it was observed that only the kinesio group presented an increase in the average values of each at the assessment times (table 1table 1.average strength of the right and left hands of the different groups (kg / f)time of assessmentkinesio groupaverage (sd)control groupaverage (sd)kinesio with notension average (sd)right handpre - taping (baseline)24.6 (3.5)25.0 (3.3)25.4 (2.9)30 minutes later26.5 (3.6)24.4 (3.3)25.2 (2.9)24 hours later27.1 (3.5)24.8 (3.1)25.2 (2.7)48 hours later26.9 (3.5)24.3 (3.0)25.6 (3.2)left handpre - taping (baseline)23.3 (3.8)24.3 (3.5)25.1 (2.6)30 minutes later25.9 (4.4)24.0 (3.2)24.6 (2.9)24 hours later26.2 (3.9)24.0 (2.9)24.8 (2.9)48 hours later26.2 (3.7)23.8 (3.3)25.2 (2.9) * statistically significant (p<0.05)). * statistically significant (p<0.05) it was also observed that a significant increase in strength values occurred in the right hand, compared to the initial values, after 24 hours and 48 hours of taping . For the left hand, an increase in strength was observed, compared to pre - taping, after 30 minutes 24 hours, and 48 hours of taping (p<0.05). The analysis of the data regarding the duration of taping and the different handgrip assessment times among the groups revealed there were no significant findings between the control and kwt groups . The right hand of the group that received kinesio taping showed significant differences in strength from the control group of taping, and the left hand showed a significant difference after 48 hours of taping . In comparison of the kinesio and kwt groups only the right hand showed a significant difference after 24 hours of taping (p<0.05). In the comparison of handgrip strength between the dominant and non - dominant hands, it was observed that the dominant hand demonstrated greater handgrip strength at all the assessment times, when considering the average dynamometry value, especially in the kinesio and control groups (table 1). Research regarding kinesio taping is still scarce in the scientific literature since the technique has only received international attention within the last ten years . Moreover, the available studies did not have large, homogeneous samples and were conducted with questionable methodologies . It should be noted that the present study is the first to utilize a large sample size in order assess the effects of kinesio taping on muscle strength . The primary objective of this study was to observe the influence of kinesio taping on handgrip strength by comparing the average handgrip strength as measured by dynamometry prior to tape application with its respective values after 30 minutes, 24 hours, and 48 hours of taping . The results demonstrate statistically significant differences after 24 hours and 48 hours of taping for the right hand, and at the three post - taping assessmentimes for the left hand . The results for the right hand after 30 minutes of taping revealed a p - value of 0.0575, which although the results was not significant, was very close to the level of significance adopted by this study . Similar findings were not observed in the kwt and control groups, thus confirming the hypothesis of this study, that kinesio taping can increase handgrip strength when applied with systematic standards for that purpose . According to kenzo kase, the kinesio taping method can improve the strength of muscles weakened by correcting muscle function with stimuli and reinforcement . Our present results are similar to those reported by vithoulka et al.15 in which kinesio taping increased eccentric muscle strength in healthy adults . In contrast to our present results, chang et al.16 evaluated the influence of kinesio taping on the maximum handgrip strength of 21 healthy students, all of them men and athletes . The technique employed was specifically for medial epicondylitis, and no significant changes in strength were observed after taping . However, a confounding factor in this study was that the tension applied to the placebo group, which was the same as that of the kinesio group (between 15% and 20%). Also, with the start of taping being more distal than the insertion point, the tension zone was the same, thus making the purpose of comparison between these two groups questionable . Another study investigated the immediate and delayed effects of two directions of kinesio taping on maximal isometric strength of the wrist and finger muscles of healthy adults . Inhibition and facilitation kt techniques were separately used to tape the dominant and non - dominant forearms of the participants, respectively . Maximal isometric strength of wrist extension, middle finger extension, and the grip of both hands were measured before taping, immediately after taping, and after 24 h of taping (with the tape in situ). Compared with the baseline, the average maximal isometric strength of middle finger extensors increased considerably after application of the facilitation kinesio tape . No significant time effect was observed on the middle finger extension strength on the dominant side or on the wrist extension and grip strength on either side . Divergences are also seen in the results presented by fu et al.16, in which kinesio taping was applied with 120% of longitudinal stretch over the quadriceps and hamstring muscles of 14 healthy athletes (7 men and 7 women), and assessed in the following manner: before tape application, immediately after taping, and 12 hours afterward . An isokinetic dynamometer was utilized in order to verify the muscle strength, and no significant increase was found in the muscle strength of these athletes . An issue with the method of this study is that the actual tension applied to the tape was 120%, which should only be utilized for taping procedures of a ligamentous injury . Such a high tension may hinder muscle contraction and decrease joint mobility, which could also lead to non - functional movement and muscle inactivation . Some authors have reported increases in electromyographic activity induced by the use of kinesio tape: e.g. Slupik et al.18, chen et al.19, hsu et al.8, and thelen, dauben and stoneman6 . In all of these studies,, there are consistency issues with the assessments of kinesio taping, as witnessed by the lack of a systematic approach in data collection, which did not permit a reliable assessment of initial benefits, the duration of benefits or the residual effects after kt removal . Some authors have evaluated the influence of kinesio taping immediately after its application8, 16, other have assessed it immediately afterward and 12 hours later17, as well as 24 hours and 72 hours later18, and only after 72 hours of taping15 . With regards to the stretch applied to the tape, some authors do not specify how much was utilized in their studies8, 19 . Other studies simply do not follow the standards recommended by the kinesio taping association . An example of this is related to the tension for muscle activation, which has been established to be between 25% and 35% . Nevertheless, some authors have opted to utilize 15% to 20% of tension16 and even 120%15 . Also a simple tape just placed without tension as performed for the control group in this study with no specific direction, will elicit tape stimulus and some effects should be apparent . Kinesio tape applied to the skin without tension will exert minimal tensions during body and skin movements . No tension is also used in the kinesio taping method for patients that are high sensitive to external stimulous . So it ca nt be called placebo, it is just a different degree of stimulus . When comparing the kinesio and control groups of the present study, the greatest handgrip strength values were observed in the kinesio group at 24 hours and 48 hours after application of the taping technique to the right hand, and after 48 hours for the left hand . These results indicate that handgrip strength increased and remained elevated in the right hand for 48 hours . For the left hand, considering that changes in handgrip strength were observed after 24 hours in the right hand, which lasted up to 48 hours, and that such changes were noted only after 48 hours of taping in the left hand, we believe that the sensitivity of the right hand is greater than that of the left hand . Therefore, the mechanoreceptor stimulus induced by the kinesio taping provided a faster response in the right hand than in its counterpart . The left hand possibly required greater tension and a longer period of time for it to receive sufficient stimulation to achieve the same results as for the right hand . With regards to the augmented handgrip strength observed for the right hand of the kinesio group in relation to the kwt group after 24 hours of taping, we assume that the tactile stimulus to the dermis and epidermis are present even when kt is applied without tension, since the skin maintains it normal mobility by stretching and recoiling in relation to the tape during upper extremity movements . Another result noted was that the dominant hand presented greater handgrip strength when considering the average values at each of the four assessment times, namely, before kinesio taping application, and 30 minutes, 24 hours, and 48 hours later . This finding was observed for all the groups, but with a higher percentage in the kinesio and control groups . Considering the present results, it is possible to conclude that an increase in handgrip strength occurred over pre - taping values at 30 minutes, 24 hours, and 48 hours after kinesio taping application . A statistically significant increase was observed in the kinesio group, when compared to control group after 24 and 48 hours of taping for the right hand, and after 48 hours of taping for the left hand . Between the kinesio and kwt groups, the dominant hand presented greater handgrip strength values in all assessments of all the groups.
Initiatives such as the surviving sepsis campaign, launched in 2002 as a collaborative initiative of the european society of intensive care medicine, the international sepsis forum, and the society of critical care medicine, aim to effectively reduce risk of death from severe sepsis and septic shock . Nonetheless, although substantial benefits raised from the implementation of this campaign have been obtained, much work remains if we are to realise the full potential promised by this strategy . Recently, new treatment approaches based on interventions for coagulation or inflammation have failed to improve survival in sepsis . A deeper understanding of the processes leading to sepsis is necessary before we can design an effective suite of interventions . Dysregulation of the immune response to infection is acknowledged to contribute to the pathogenesis of the disease . Critical illness itself, surgery and concomitant comorbidities such as diabetes, chronic renal failure or chronic obstructive pulmonary disease affect host responses to infection, which could in turn facilitate the development of sepsis or impair outcome once sepsis is established . Despite these precedents, the potential role of immunological monitoring in this disease has not been appropriately considered to the present moment . For years, two phases have been described in sepsis: an initial systemic inflammatory response syndrome followed by the negative feedback of a secondary compensatory anti - inflammatory response syndrome . In contrast to this long - held view, marchant and colleagues and our group have observed that production of the immunosuppressive cytokine il-10 occurs from the very first hours following the diagnosis of severe sepsis or septic shock, and that it is directly associated with the secretion of proinflammatory cytokines [7 - 9]. Levels of igg, igm and iga at diagnosis have been reported to correlate directly with survival . In turn, nonsurvivors have lower levels of c4 (a protein of the complement system) than survivors . Natural killer cell counts and function also seem to have an important role in this disease . Severe depletion of immune effector cells is a universal finding in all age groups during sepsis . Quantification of lymphocyte subsets and evaluation of their function could thus have diagnostic and prognostic value in sepsis . At the genomic level, repression of networks corresponding to major histocompatibility complex antigen presentation cumulative evidence supports the notion that the immunological situation of the patient is linked to the final outcome in sepsis . Immunological monitoring could thus contribute to the prevention or the treatment of sepsis in a personalised and timely manner . Interestingly, there is no currently available information on the potential role of proper immunological monitoring for the prevention of sepsis . Immunological monitoring could help to identify patients with immunological deficiencies (secondary to their disease process, treatment, and so forth) at higher risk for developing community acquired or nosocomial sepsis . Periodic monitoring of patients hospitalised in key services (that is, oncology, transplantation units, critical care units) could help to identify specific humoral or cellular immunity defects that could be addressed by implementation of prophylactic measures, such as administration of intravenous immunoglobulin (ivig) or proper cover with broad - spectrum antibiotics . Prompt implementation of an accurate treatment is key to the final outcome in patients with sepsis . Immunological monitoring could help to improve outcome in sepsis, by providing early detection of individuals at higher risk for developing complicated outcomes as well as relevant information for guiding treatment . For example, the treatment effect of ivig on mortality for patients with septic shock is currently controversial . Measuring endogenous levels of immunoglobulins could improve the render of ivig in sepsis, helping the clinician to better select those patients to be treated (those exhibiting marked hypogammaglobulinemia). Once more, there is a dramatic absence of information on the potential role of prior endogenous immunoglobulin quantification in the clinical assays evaluating ivig for the treatment of this disease . Immunological monitoring could help also to guide therapies with immunomodulatory drugs with an anti - inflammatory effect or, alternatively, with an immunostimulatory effect . Although not currently practiced, monitoring of the patient's immunological situation is feasible (at least from a quantitative point of view) in most hospitals . Quantification of immunoglobulins, complement proteins and t cells (cd4, cd8) in peripheral blood is an easily available routine test . Other tests of potential interest for immunological monitoring in sepsis include the quantification of hla - dr in the surface of blood monocytes or the evaluation of percentages of circulating cd4cd25t - regulatory cells in blood . Lastly, the use of genomic signatures (gene expression, mirna, dna methylation profiles) offers new opportunities to assess the immunological status of the patient . Although diffuse and limited, current available information supports the development of large comprehensive studies aimed to urgently evaluate immunological monitoring as a tool to prevent or treat sepsis, and thereby to diminish the morbidity and mortality associated with this severe condition . Hla: human leukocyte antigen; ivig: intravenous immunoglobulin; il: interleukin; mirna: microrna . Ra, jw and jfb - m provided the immunological insight and participated in writing the article . Et, da - o, im - l and pr provided the clinical insight and participated in writing the article . The authors want to thank dr david livermore (norwich medical school, university of east anglia) for his constructive comments on this letter.
Unfortunately this demand is more than the supply in terms of surgeons performing these surgeries . Robotic surgery fills the gap between having skills and not having one . A surgeon with limited skills this leads to added advantage both to the patient as well as the surgeon . Notwithstanding the cost, robot helps to duplicate the laparoscopic surgeries which otherwise would be beyond the reach of surgeon with limitation of skills . In april 2005, davinci robot was food and drug administration cleared for gynaecologic procedures based on preliminary evidence of safety and efficacy from their early experience with myomectomy and hysterectomy at the university of michigan . Robotics is best for single quadrant surgery and for fixed structures and hence is especially useful in gynaecological surgery . It has added advantages of 3d perception, wristed instrumentation, intuitive movements and dexterity . Since november 2009 until date, we have performed 80 robotic gynaecological cases in galaxy care laparoscopy institute, pune . We standardized the port positions as a 12 mm camera port was placed 2 cm above the umbilicus.an 8 mm robotic port on the either side was placed 10 cm lateral and 12 cm caudal to the camera port.the right sided robotic port was a mirror image of the left robotic port.two assistant 10 mm ports were placed pararectally at the level of the camera port . An 8 mm robotic port on the either side was placed 10 cm lateral and 12 cm caudal to the camera port . Two assistant 10 mm ports were placed pararectally at the level of the camera port . The robotic cart was docked from in between the legs a zero degree scope was used for the procedure . For all the surgeries, robotic docking time.console time surgical time.blood loss.complications.conversion to either lap or open robotic docking time . Conversion to either lap or open for the oncological surgeries, other parameters such as paracervical clearance, nodal yield and vaginal margins were also recorded . Of the total of 80 gynecological cases performed robotically in our institute, 29 were benign and 51 were malignant cases . Of the benign cases, 24 were hysterectomies for various complex benign pathologies like big fibroids, previous abdominal surgeries . 2 tubotuboplasties, 1 endometriotic cyst excision, 1 metroplasty, 1 rectovaginal fistula repair were also performed with good results at our institute . Of the total of 51 oncological cases performed robotically, 27 were robotic radical hysterectomies for cancer cervix, 10 total robotic hysterectomies with bilateral salpingo - opherectomy with ilioobturator node dissection for cancer endometrium, 2 were total robotic hysterectomy with bilateral salpingo- opherectomy and omentectomy for cancer ovary, 7 were exenterations, 5 were parametrectomy . Total robotic hysterectomy: we compared our outcomes with those of previously done standard studies as shown in the table 1 . Comparison of our data of total robotic hysterectomy with previously reported series our operative time, estimated blood loss was considerably lower when compared with other standard international studies . There was no conversion to open surgery; furthermore no major intraoperative or postoperative complications were noted . Robotic radical hysterectomy: we have compared the results of our radical hysterectomies with those of other standard reported cases in literature . As shown in table 2 our operative time, effective blood loss was less than that of others . Two cases of ureteric fistulas reported were during initial phase after acquiring the robot . As we got acquainted with these procedures comparison of our data of radical robotic hysterectomy with previously reported series table 3 below provides comparison of parameters between our laparoscopic cases with the robotic cases . Total robotic hysterectomy: we compared our outcomes with those of previously done standard studies as shown in the table 1 . Comparison of our data of total robotic hysterectomy with previously reported series our operative time, estimated blood loss was considerably lower when compared with other standard international studies . There was no conversion to open surgery; furthermore no major intraoperative or postoperative complications were noted . Robotic radical hysterectomy: we have compared the results of our radical hysterectomies with those of other standard reported cases in literature . As shown in table 2 our operative time, effective blood loss was less than that of others . Two cases of ureteric fistulas reported were during initial phase after acquiring the robot . As we got acquainted with these procedures comparison of our data of radical robotic hysterectomy with previously reported series table 3 below provides comparison of parameters between our laparoscopic cases with the robotic cases . The advantages of robotic assistance include enhanced dexterity, improved 3-d vision, and more intuitive instrument manipulation . It thus helps to bridge the skill gap for a laparoscopic surgeon helping to overcome skill limitations . This may make a complex surgical task more accessible to surgeons without much laparoscopic experience . Placement of robotic trocars has to be in such a way to prevent collision of robotic working arms and accessory ports . The accessory ports are used for retraction, for energy sources like ligasure or for applying clips . The preoperative planning helps in performing a smooth procedure as well as reducing the docking time . The docking time was previously 30 min which later reduced to 10 min because of standardization of ports, team gaining experience and getting acquainted with the system . In 1989, laparoscopy was first used to perform a hysterectomy . In 2002, the use of the davinci robot for hysterectomies was first reported . Payne and dauterive concluded that robotic hysterectomy was quicker and with less risk for abdominal conversion than standard laparoscopy . We have an extensive experience in laparoscopic hysterectomies with which we have started doing robotic hysterectomies . The patients with high body mass index and patients with narrow pelvis were preferred for robotic procedure . The time taken and blood loss was the same as in laparoscopy . Standardization of procedure and proper training of the technique was necessary to prevent complications and for safe outcome . The salient steps of our technique use of combined anaesthesia, ergonomic port positioning, use of myoma screw for traction, use of bipolar forceps medial to uterine stump, colpotomy at the level of uterosacral ligaments . Use of combined anaesthesia, ergonomic port positioning, use of myoma screw for traction, use of bipolar forceps medial to uterine stump, colpotomy at the level of uterosacral ligaments . All our patients had stage iv endometriosis and the rectum and ureter could be separated well due to the high magnification and the intuitive movements of the robot . Robotic - assisted laparoscopic surgery promises to provide advantages in the management of women with severe endometriosis secondary to 3-dimensional visualization, decreasing surgeon's fatigue and hand tremors and improving surgical precision . With the davinci system, the challenges of visibility with laparoscopy can be overcome as well as many other limitations of laparoscopy . Hence, the field of gynaecologic oncology has begun adopting the davinci system in performing oncological surgeries due to its shorter learning curve and ease of use . Studies, suggest that robotic radical hysterectomy (rrh) is preferable over laparoscopic radical hysterectomy (lrh) due to its decrease in blood loss, hospital stay, recovery time, and complications . However, rrh appears to be equivalent to lrh in the hands of experienced surgeons . In patients predicted to have a high chance for conversion to open from laparoscopy, davinci system may prevent conversion . The international federation of gynaecology and obstertrics staging requires lymphadenectomy for assignment of stage, and morbid obesity is usually cited as the most common limiting factor for completing a satisfactory lymphadenectomy . The multi - centre gynecologic oncology group lap-2 trial with 1,696 laparoscopic cases reported a 23% conversion rate and a mean operative time of 3.3 hrs, indicating difficulty with a significant proportion of cases . Reasons for not adopting laparoscopic surgery often cited by surgeons are prolonged operating times, surgeon fatigue, a difficult and prolonged learning curve, and lack of formal training in advanced laparoscopic technique . We have reported our experience of laparoscopic gynaec oncology procedures such as radical hysterectomy, anterior exenteration and total pelvic exenteration . The use of hybrid technique and using bipolar energy near the ureters have prevented any further fistulas in subsequent patients . The two larger series from boggess et al ., and lowe et al ., show that there were no transfusions, length of stay was one day, and total complications were less than that usually associated with either open or laparoscopic approaches . The results are comparable to those of laparoscopy, robot helps to duplicate many procedures with exceptional laparoscopic skills.
Clinical decision - making should be founded on the highest level of evidence available . According to current hierarchies, randomized control trials (rcts) govern the top echelon due to the lowest possible influence of bias . As such, well - executed rcts are the gold standard for clinicians assessing therapeutic effectiveness and treatment options . Borawski et al ., performed the first formal evaluation of the levels of evidence in urological literature . Independent reviewers familiar with the level of evidence concept rated 600 studies using a standardized evaluation form adapted from the center of evidence based medicine . The studies were randomly selected from four major urology journals (the journal of urology, european urology, bju international, and urology) in the periods 2000 and 2005 . Overall, 60.3% of studies addressed questions of therapy / prevention, 11.5% addressed etiology / harm, 11.3% addressed prognosis, and 9.2% addressed diagnosis . Articles centered mainly on adult populations (86%) with oncology as the topic of choice (38.8%). Disturbingly, the levels of evidence provided by these studies were low: 5.3% level i, 10.3% level ii, 9.8% level iii, and 74.5% level iv . From 2000 to 2005, the authors conclude by suggesting that the majority of studies in urological literature cannot adequately guide clinical decision - making as a result of such low level of evidence . Several barriers to providing the highest level of evidence among surgical subspecialties have been previously identified, such as lack of surgeon patient equipoise about certain therapies, difficulty of standardizing quality of a given surgical procedure, and limited funding mechanisms . However, another looming possibility exists: is there paucity in statistical sense among urologists? In line with low levels of evidence, findings at scientific meetings do not see the light of full - text publication in many cases . Failure to publish is problematic for two main reasons: 1) clinicians looking to apply research findings lack the necessary detail in abstracts to critically appraise a given study for validity and impact; 2) it is wasteful of resources, unethical, and can lead to unnecessary replication of studies . Smith et al ., reviewed clinical research abstracts accepted for publication at 2002 and 2003 aua meetings . Literature search follow - up of published articles was performed in 2005 . Out of 1683 abstracts, the majority of abstracts from north america (62.5%), reported single institution efforts (68.2%) mainly in the domain of therapy / prevention (51.6%). Forty - four percent of these abstracts were published with a median follow - up of 27.8 months and 54.2% indicated formal statistical hypothesis testing . Kaplan - meier analysis showed less time to publication of abstracts that had statistical testing (912) compared to those that did not (771) (log - rank p = 0.009). Univariate analyses identified statistical hypothesis testing with time for publication along with other predictors as significant factors contributing to the difference in publication rates . This was confirmed in multivariable analysis, as reporting to statistical testing remained predictive (hr 1.2, 95% ci 1.11.4). The authors highlighted how 61% of studies are affected by nonpublication of research findings two years after presentation at the aua meeting due to a lack of statistics . Increasingly, statistical methodology has transitioned from the realm of statistical journals to medical research . With the advent and plethora of available statistical software, not using statistics is one weakness, but making errors in statistical testing and reporting of results can compromise the health of research animals, human subjects, and ultimate recipients of therapies . In research literature, scales and colleagues performed a systematic assessment of statistical usage in urology literature . Using a single issue (august 2004) of four leading urology journals (journal of urology, british journal of urology, urology, and european urology), two independent raters with formal statistics training reviewed the articles using a standardized evaluation form developed with an experienced biostatistician . Out of 97 articles that met eligibility criteria, cohort design comprised the majority of studies (44%). Of the 12.4% of studies that were randomized trials, 42% detailed clinically significant differences, 50% detailed power calculations, and 30% described method of randomization . Descriptive statistics were widely reported (94%) and articles mainly included simple statistical comparisons of two groups (77%). Distressingly, 71% of studies with statistical comparisons had at least one statistical error, including incorrect test (28%), faulty use of a parametric test (22%), and failure to adjust for multiple comparisons (65%). In addition, overfitting a regression model was a common problem (39%) in the 29% of studies that applied multivariable analysis . Such flawed application of statistics can potentially increase the likelihood of type i error and should be identified as a potential threat to validity of conclusions . Statistics is paramount to success for the urologist as a researcher and as a clinician in urology . The remainder of this review will focus on probing the underlying problem of statistical use among clinicians and offer solutions that can be applied to rectify this situation . To exercise evidence - based medicine (ebm), physicians need access to full - fledged research reports to critically evaluate study analysis and interpretation . However, surveys dating back to the 1980s identified physicians who had a poor grasp of statistical tests and interpretation of statistical results due to a lack of formal training in biostatistics. [1012] this problem is even more explosive today in light of increased complexity of statistical methods used in the literature . In response, graduate medical educators have increased training in biostatistics throughout the expanse of medical education . Medical schools have incorporated statistics courses and accreditation council for graduate medical education (agcme) guidelines since residency competency stipulates that residents must have a solid basic foundation in statistical methodology as it pertains to scientific research . While residency programs address this issue through ebm curricula and journal clubs,[1517] a few, if any, programs focus on selection and interpretation of statistical results . To broadly assess residents' knowledge and skills in ebm, windish et al ., conducted a seminal multiprogram assessment of 11 internal medicine residency programs in connecticut . By first reviewing research articles in six leading general medical journals between january and march 2005 on the basis of statistical methods used, the researchers developed a survey instrument of questions focused on identifying and interpreting results in the most frequently occurring statistical tests . Questions were multiple - choice, centered on a clinical vignette, and required no calculations . Attitudes and confidence questions were adapted from surveys on the assessment resource tools for improving statistical thinking website, rated on a 5-point likert scale . This instrument was validated and reformulated by pilot testing the questions on 5 internal medicine faculty with advanced training in biostatistics and 12 primary care internal medicine residents . In terms of respondent characteristics, out of 277 residents, 48% were female, 60.8% aged 2630 years with no advanced degrees (85.1%), and a modest distribution of years since medical school (35.0% <1 year, 26.8% 13 years, 30.1% 410 years). Of the foreign medical graduates in the population, 38.6% completed their medical school training outside the u.s ., 68.8% had previous coursework in biostatistics [69.5% of which were during medical school (15.9% college, 3.2% residency)]. Over 50% had previous training in epidemiology and ebm, and regularly read medical journals . Interestingly, the number of residents who could correctly identify and interpret statistical results was low . Approximately 25.6% could correctly identify chi - squared analysis, 13.0% could correctly identify cox proportional hazard regression, 11.9% could interpret a 95% ci and statistical significance, and only 10.5% could interpret kaplan - meier analysis results . Using a forward stepwise regression model, advanced degrees, successive years since medical school, and prior biostatistics training were all factors found to be independently associated with knowledge scores . In terms of attitudes and confidence, 95% of residents agreed that knowledge of statistics is essential to being an intelligent reader of literature and 77% indicated they would like to learn more statistics . While over 58% of residents reported using statistics in forming opinions or making clinical decisions, 75% indicated they did not fully understand the statistics reported in literature . Only 38% of residents felt confident assessing the appropriateness of statistical testing used and respondents with a higher confidence level in statistical knowledge fared better on the knowledge questions . While their report was confined to internal medicine residents, high internal consistency, good discriminative validity, and similarity in results among different residency programs lend credibility to the illustrated problem . A comprehensive review of biostatistics teaching indicates that 90% of medical schools taught biostatistics in preclinical years only with varying breadth and depth of education . Another pressing issue is that senior residents performed worse than junior residents, indicating a time correlation . Most likely, loss of knowledge over time, coupled with lack of adequate reinforcement could lead to loss of statistical competency . If clinicians cannot evaluate appropriate statistical tests and accurately interpret results, risks could be carried over to incorrect clinical decision - making . West and colleagues performed a similar study in 2005 on 301 medical students, internal medicine residents and faculty, about their attitudes toward biostatistics in medicine . According to their findings, 48.3% of those surveyed felt biostatistics is a difficult subject, 87.3% felt that understanding biostatistics would help their careers, and 17.6% felt their training in biostatistics was adequate for their needs . Furthermore, 23.3% of respondents could evaluate appropriateness of statistical methods used in a study, 88% felt knowledge of statistics is necessary for evaluating medical literature, and 48.5% felt that biostatistics is a necessary skill for clinicians not involved in research . In essence, the survey strongly indicated that clinicians were uncomfortable with biostatistics and even more dissatisfied with this cognizance . It is unclear why physicians are queasy regarding statistics even though they use statistics in their daily routine . Perhaps the finding that 20% of respondents felt their biostatistics coursework was taught effectively calls into question as to how clinicians are being educated about statistics in healthcare fields traditional teaching methods in schools employ a stepwise approach entailing formulae, data, and spoon - fed instructions . Bordering on a moral quandary is the question of whether expectations for the average urologist are too high . Would the urologist who is not a researcher be better suited to appraise practice guidelines, derived by experts with the necessary statistical knowledge, rather than interpret statistics? Urology is a highly competitive field that is constantly evolving and as such, expectations will continue to be shattered and stacked higher . The current consensus will most likely rest on the urologist having a strong statistical repertoire because research is an increasingly integral component of residency and fellowship programs, because guidelines can change given new information, and because treatment accountability ultimately rests with the physician's ability to evaluate evidence and make decisions . Most of the studies examining the use of statistics and knowledge of clinicians have thus far been centered in the u.s . In urology, only major journals have been examined leaving other international journals indexed in medline, such as brazilian journal of urology and indian journal of urology out of the loop . It is vital to assess how these journals and how urology practitioners in these regions fare in comparison to the current data through future investigations of this nature . Although errors in statistics and a lack of comprehensive understanding in methodology are common in the literature, modifications to current mindsets can still be made in the best interests of the patient . Curran - everett and benos have proposed guidelines for reporting statistics in journals published by the american physiological society . A set of 10 guidelines, ranging from advice to consult a biostatistician to interpretation based on confidence intervals and p - values, address reporting of statistics in the materials and methods, results, and discussion sections of a manuscript . A cursory look at additional references cited in the manuscript provides additional resources for urologists interested in looking at the framework of statistics and presentation issues . In addition, a commentary aimed at the publication of these guidelines by murray clayton provides an excellent critique of when to use the guidelines . Clayton argues that the algorithmic approach of guidelines may not always serve the practitioner or peer - reviewer well as situational cues dictate statistical testing and interpretation . As such the word is still out as to whether these guidelines truly represent the best practices in statistics . Focusing on urology, scales and colleagues have produced two publications that can serve as a starting point of quick statistical reference . First, they provided a series of non - technical explanations of basic statistical concepts encountered in urological literature . In terms of results, they discuss various outcome measures, how to summarize continuous data, how to summarize non - normal continuous and ordinal data, how to summarize unordered categorical data, how to interpret cis, how to interpret rrs, the difference between odds, or, and rr, how to interpret a km curve, and how to interpret multivariable analyses . In addition, the authors provide examples of common statistical flaws involving type i and ii errors, sample size calculations, multiple comparisons, and confounding variables to increase awareness of study limitations in light of statistical restrictions . By providing a statistical roadmap, the authors provide advice on choosing appropriate statistical tests as a brief introductory roundup for the practicing urologist . Scales and colleagues also provided a complementary companion primer on evidence - based clinical practice (ebcp) for urologists using examples from the literature . Principles of ebcp are discussed followed by a step - by - step approach to implementing ebcp . Sources of evidence are discussed along with methods to evaluate a study for therapeutic effectiveness . With appendices that summarize levels of evidence, electronic databases of primary evidence, and web addresses of online ebcp centers, this primer can provide urologists with the tools and questions that can aid in accumulating evidence and clinical decision - making . Faculty who are implementing biostatistics curricula can access these teaching resources . Without a doubt, teaching of statistics to medical students, residents, and fellows can be improved . Rather than sparse statistical exchanges during journal clubs, medical education should be expanded to make biostatistics less daunting and more meaningful to urologists in practice . More time should be allotted to biostatistics education in medical school in a clinical problem - based learning format . Rather than a one - shot infusion of statistics through an isolated course or a seminar, reinforced and integrated ideally, medical students will have exposure to statistics throughout their training . In residency, this can be complemented by recurring seminars from available biostatisticians or visiting faculty from nearby universities . These can be in the form of a retreat with a distribution of problem - sets at the end . Small - group work can be encouraged for a gathering and review of solutions a week later . Yet another option is online - educational courses offered by a variety of universities . For instance, harvard university extension school offers a semester - long course on introductory graduate biostatistics . Students can view streamed video lectures, post questions on an online discussion board, ask questions from professors and teaching assistants and receive feedback on homework and examinations as if they were partaking in a live course . While mailing outside the u.s . For graded assignments poses a time - lag problem, courses such as these provide an alternative if the means of quality education and expertise are lacking in the area . Such courses provide the welcome opportunity of immersing oneself in statistical software and learning the realities behind a particular formula . Ultimately, broader facilitation should be imparted at the departmental level to enable urologists to better answer research questions . Considering a hectic schedule of surgeries in the or and clinic presence, accessibility of literature for review, adequate data management infrastructure, availability of statistics know - how, and project supervision by faculty are the key factors that can dissuade even the most curious physician . Urology training programs need to be more trainee - centered to imbibe a statistical way of thinking to work around the areas of uncertainty . Statistical software that can transform raw data from a database into meaningful results using a core set of statistical tests should be freely available for use . Softwares such as stata, spss, sas, sigmaplot, r, jmp, and comprehensive meta analysis, to name a few, understandably require institutional licenses . Although these licenses are expensive, the investment is worthwhile because residents and fellows will get hands - on exposure to working with numbers . If such expenses are prohibitive, regional collaborations are encouraged to allow such software packages to be transitive in distribution . Departmental oversight of this nature can help ensure competency in fields of data management, statistical formula application, critical analysis, and study interpretation . Competencies should be expanded in medical school and residency to mandate a certain level of proficiency in order to progress from one training year to the next . In conjunction with better education of urologists, attitudes toward, and use of statistics medicine is evolving at a rapid pace with publications increasing to the rate that journals have a backlog of articles that see print six months after acceptance . At this pace, urologists need to be less intimidated by biostatistics . As important as the stethoscope, statistical sense is crucial to evaluate research findings and examining patient research . If not just for clinical decision - making, at least the physicians have a mechanism of expressing to patients why they are making a particular decision . The current problem of a low level of statistical evidence in urology literature coupled with a significant lag between abstract presentation and a full - text publication represent a lack of understanding of, and comfort with, statistics . This is reflected in errors in statistical usage that can be corrected by increased awareness of the problem and readiness to act by improving medical education of statistics.
Patients with graves orbitopathy have a higher probability of myasthenia gravis than the normal population.1 according to a large retrospective case series, myasthenia gravis was diagnosed in two of 150 patients with graves orbitopathy (1.3%).2 patients with graves orbitopathy commonly show eyelid retraction and/or hypotropia and/or esotropia, as well as exophthalmos, lagophthalmos, exposure keratopathy, and less frequently, compressive optic neuropathy.3 however, patients with myasthenia gravis often demonstrate ptosis and/or exotropia, but a consistent pattern of eye movement disturbances is usually not present.3 upper eyelid retraction in graves orbitopathy and myasthenia gravis (go - mg) is frequently camouflaged, and these patients may show a normal upper eyelid height . We herein report a patient with go - mg with left normal eyelid height, but in whom the upper eyelid retraction was disclosed after edrophonium chloride administration . A 46-year - old man with graves disease, well controlled by thiamazole 5 mg / day, was referred to our clinic to treat his graves orbitopathy . Although both eyes had exophthalmos (20 mm od, 19 mm os; normal range, <17 mm)4, he demonstrated a normal left eyelid height and right ptosis, ie, margin reflex distance-1, 3.5 mm os and 0.5 mm od, respectively (figure 1). His primary eye position was exotropic, dominantly on his right side, and the upward gaze of the right eye was severely restricted (figure 2). Coronal computed tomography of the orbits showed bilateral enlargement of the inferior rectus muscle, medial rectus muscle, superior oblique muscle, and superior rectus muscle - levator muscle complex (figure 3a). Axial computed tomography demonstrated bilateral medial and lateral recti muscle enlargement (figure 3b). Localization of these muscle involvements did not match the characteristics of the eye movement disorder (figure 3c). Because the aforementioned findings raised suspicion for the presence of both graves orbitopathy and myasthenia gravis, we performed an edrophonium chloride test.5 the margin reflex distance-1 had improved to 2.5 mm od and 8.0 mm os (figure 4), although the eye movement had not clearly improved . Some days later, the result of a serum acetylcholine receptor antibody assay was positive (1.8 nmol / l; normal, <0.2 nmol / l). Because the patient could completely close his left eye, we prescribed pyridostigmine bromide 180 mg / day,5 and the eyelid height stabilized the association between graves orbitopathy and myasthenia gravis has long been recognized.2,6 although it is not difficult to differentiate clinically between simple graves orbitopathy and myasthenia gravis, overlapping clinical features, such as upper eyelid height and/or eye movement disturbances occasionally cause diagnostic confusion, especially in patients with normal thyroid function . The most frequent sign of graves orbitopathy is upper eyelid retraction,7 which is caused by overaction of the mller s muscle because of excess catecholamines and cicatricial contraction of the levator muscles after the inflammatory process.7 for the overaction, topical guanethidine therapy is sometimes effective.8 for the contraction, steroid administration and botulinum toxin injection are indicated during the active phase, and surgical correction is necessary in the static phase.7 graves orbitopathy also shows eye movement disturbances, which have a restrictive myopathy pattern.3 the inferior and medial rectus muscles are frequently involved, and imaging discloses their hypertrophy when in the active phase . On the other hand, ptosis and diplopia are present in about 90% of patients with myasthenia gravis, and these ocular symptoms are an initial complaint in 20% of patients with myasthenia gravis.911 muscle hypertrophy does not exist on imaging in myasthenia gravis.12 no particular predilection of extraocular muscle involvement is shown in myasthenia gravis . However, solitary paresis usually occurs, in the form of an adduction deficit resulting in exotropia, which was the case in the present patient . Exotropia is more common in ocular myasthenia gravis.3 in spite of the obvious differences between graves orbitopathy and myasthenia gravis, each condition shares several features, potentially making it difficult to reach a correct diagnosis . Upper eyelid ptosis, typically suggestive of myasthenia gravis, may be seen with graves orbitopathy as a result of levator myopathy,13 apical compression,14,15 and pseudoptosis secondary to upper eyelid retraction in the fellow eye.16 however, if ptosis develops in a patient with graves orbitopathy, simultaneous myasthenia gravis should be considered,17 although ocular myasthenia gravis with orbital pain may imply orbital inflammation.18 in our patient, the right ptosis and exotropia suggested concomitant myasthenia gravis, which motivated us to perform an edrophonium chloride test . The left upper eyelid, appearing to have a normal height, was retracted to margin reflex distance-1 8.0 mm, and the right side, regarded as ptosis, moved to margin reflex distance-1 2.5 mm (figure 4). This examination demonstrated that the left upper eyelid originally had an upper eyelid retraction that was camouflaged by the ocular myasthenia gravis . Treatment of our patient was complicated because of laterality and strabismus induced by myasthenia gravis . When the eyelid height stabilizes after medication with improvement of strabismus, a sling surgery may be performed on the right upper eyelid . However, if strabismus persists, this strategy must be abandoned . When left upper eyelid retraction is conspicuous with lagophthalmos, left upper eyelid lengthening surgery may be performed.19 in conclusion, upper eyelid retraction in patients with go - mg may be camouflaged by a myasthenia effect . The upper eyelid height must be carefully monitored in patients with graves orbitopathy to detect the presence of concomitant myasthenia gravis.
At the duke - nus graduate medical school in singapore, a 1-year longitudinal family medicine clerkship was developed in 2008 to teach core precepts of primary and family care . This included a community - based component to enhance learning about chronic disease management and community-, patient- and family - centred care . This component involved students following hospitalized patients to the community with continuing clinic visits and home visits . Students participants were two consecutive classes of third - year medical students consisting of a total of 44 students from the duke - nus graduate medical school who rotated through a required 10-month - long longitudinal family medicine clerkship in 2009/2010 (21 students) and 2010/2011 (23 students). The third - year curriculum comprised a longitudinal family medicine clerkship and a research project . The research project occupied 4 of 5 weekdays and the clerkship took up 1 day each week . The family medicine clerkship is a 160-hour experience consisting of three modules: the knowledge foundation module (kfm), the continuing clinics module (ccm) and the patient centred care module (pccm). The clerkship is graded on an honours, high pass, pass or fail basis . The learning objectives of the pccm module are to (1) participate in the illness experience from the perspective of the patient and care givers, (2) engage in the management of chronic medical conditions and (3) gain exposure to care continuity between hospital / community and other health care providers . In the pccm, students identified two patients with chronic conditions whom they encountered in the hospital wards during the first month of the clerkship . The students were expected to keep in contact with their patients for the duration of their clerkship, make at least two home visits, accompany the patients on their clinic visits and visit them in hospital in the event of re - hospitalization . The students also received one overview of the module requirement by the clerkship coordinator, two face - to - face tutorials and a home visit demonstration by the faculty mentor . Students were required to reflect on their hospital and community encounters with their patients and to submit written narratives addressing all three components of the following: (1) the world of the patient and its impact on illness, (2) the impact of illness on the patient s world and (3) the patient s interactions with the health care system . The first narrative was after the first encounter in the hospital ward and was due in month 2 of the clerkship . The second narrative was after the home visit and was due in month 4 of the clerkship . A final end - of - module narrative served as a summary of their longitudinal experience . The written narratives without patient identifiers were submitted electronically to the faculty mentor and shared with their pccm group . The pccm accounted for 30% of the grade, and was graded based on completion of an adequate narrative and small group participation . A narrative was considered adequate if students addressed all three components of the above - mentioned guiding statements . The primary data source comprised student narratives submitted at the three stated time points . Each student submitted a total of six narratives (three narratives per patient followed) over 10 months . Student narratives were electronically submitted to a research administrator who de - identified the narratives by assigning a code number that permitted longitudinal linkage of narratives for each student and his / her patient . The first two faculty coders (rp and ffv) independently read through an initial sample of 60 narratives several times to identify themes . Content was analysed in part using the three original guiding statements: the world of the patient and its impact on illness, the impact of illness on the patient s world and the patient s interactions with the health care system and on drawing on the grounded theory analysis methodology (28). The first two coders met after their independent coding and constructed categories of themes using an iterative process of discussion, refining and revision of the coding schema, and consensus building . Two themes were identified for each narrative to permit equal weighting of narrative themes by student . A third coder (dl) was then added and trained in the coding schema . Each of the remaining narratives after the initial 60 was independently coded by at least two coders, with agreement reached by consensus discussion for each narrative . Where consensus could not be reached by the two primary coders, the third coder not assigned to the narrative would act as an adjudicator . Theme frequency was also determined for narratives from each of the three time points to examine temporal patterns across the longitudinal experience . Member checking (29), a common technique to support validity, was performed after the coding was completed by faculty . Two students who had completed the family medicine clerkship, who were also participants in the study, were asked to examine and code a random sample of 24 narratives already coded by faculty pairs . Participants were two consecutive classes of third - year medical students consisting of a total of 44 students from the duke - nus graduate medical school who rotated through a required 10-month - long longitudinal family medicine clerkship in 2009/2010 (21 students) and 2010/2011 (23 students). The third - year curriculum comprised a longitudinal family medicine clerkship and a research project . The research project occupied 4 of 5 weekdays and the clerkship took up 1 day each week . The family medicine clerkship is a 160-hour experience consisting of three modules: the knowledge foundation module (kfm), the continuing clinics module (ccm) and the patient centred care module (pccm). The clerkship is graded on an honours, high pass, pass or fail basis . The learning objectives of the pccm module are to (1) participate in the illness experience from the perspective of the patient and care givers, (2) engage in the management of chronic medical conditions and (3) gain exposure to care continuity between hospital / community and other health care providers . In the pccm, students identified two patients with chronic conditions whom they encountered in the hospital wards during the first month of the clerkship . The students were expected to keep in contact with their patients for the duration of their clerkship, make at least two home visits, accompany the patients on their clinic visits and visit them in hospital in the event of re - hospitalization . The students also received one overview of the module requirement by the clerkship coordinator, two face - to - face tutorials and a home visit demonstration by the faculty mentor . Students were required to reflect on their hospital and community encounters with their patients and to submit written narratives addressing all three components of the following: (1) the world of the patient and its impact on illness, (2) the impact of illness on the patient s world and (3) the patient s interactions with the health care system . The first narrative was after the first encounter in the hospital ward and was due in month 2 of the clerkship . The second narrative was after the home visit and was due in month 4 of the clerkship . A final end - of - module narrative served as a summary of their longitudinal experience . The written narratives without patient identifiers were submitted electronically to the faculty mentor and shared with their pccm group . The pccm accounted for 30% of the grade, and was graded based on completion of an adequate narrative and small group participation . A narrative was considered adequate if students addressed all three components of the above - mentioned guiding statements . The primary data source comprised student narratives submitted at the three stated time points . Each student submitted a total of six narratives (three narratives per patient followed) over 10 months . Student narratives were electronically submitted to a research administrator who de - identified the narratives by assigning a code number that permitted longitudinal linkage of narratives for each student and his / her patient . The first two faculty coders (rp and ffv) independently read through an initial sample of 60 narratives several times to identify themes . Content was analysed in part using the three original guiding statements: the world of the patient and its impact on illness, the impact of illness on the patient s world and the patient s interactions with the health care system and on drawing on the grounded theory analysis methodology (28). The first two coders met after their independent coding and constructed categories of themes using an iterative process of discussion, refining and revision of the coding schema, and consensus building . Two themes were identified for each narrative to permit equal weighting of narrative themes by student . A third coder (dl) each of the remaining narratives after the initial 60 was independently coded by at least two coders, with agreement reached by consensus discussion for each narrative . Where consensus could not be reached by the two primary coders, the third coder not assigned to the narrative would act as an adjudicator . Theme frequency was also determined for narratives from each of the three time points to examine temporal patterns across the longitudinal experience . Member checking (29), a common technique to support validity, was performed after the coding was completed by faculty . Two students who had completed the family medicine clerkship, who were also participants in the study, were asked to examine and code a random sample of 24 narratives already coded by faculty pairs . Mean age was 26 years (range 2334), gender distribution was one - third males and two - thirds females . Forty - three students were science majors prior to entering medical school and 1 student was humanity major . Data from the two classes are reported in aggregate because no difference in theme frequency or distribution was seen in the narratives from the two cohorts . A total of 88 patients were followed during the pccm module by the 44 students . Therefore, 88 narratives were submitted after the first encounter in the hospital (narrative 1) and 88 narratives were submitted after the home visit (narrative 2). However, due to patients being lost to follow - up, only 77 narratives were submitted at the end of the module (narrative 3). Each of two faculty coders (rp and ffv) independently identified two themes for each of the initial sample of 60 narratives . They then met and agreed on six unique themes (see table 1 for illustrative quotes). The two coders then returned to the 60 initial narratives and coded them according to the agreed upon schema . The two coders agreed on themes for 90% of the sample with agreement on the remaining 10% reached by consensus . Each of the remaining 193 narratives was then coded by two of the three coders . The coder pairs for each narrative were able to agree on 92% of themes, and a third coder was used for adjudication in 8% of the narratives . In total, 506 themes were derived from the 253 narratives (two themes per narrative). In other words, theme saturation was achieved with the first 60 narratives . Thematic content and illustrative quotes selected from 253 student reflective narratives, duke - nus graduate medical school 20092011 member checking confirmed findings of the six themes and student members agreed with the coding schema as being reflective of the narrative content for the 24 narratives assigned to them . A discussion of the narrative content for each theme is as follows . Under this theme, students wrote about understanding chronic disease management, disease progression and the importance of patient education and self - management . Student narratives also showed appreciation of the care continuity and team - based collaborative care by health care providers . Students recognized challenges to lifestyle and medication compliance and noted the use of alternative therapy in chronic diseases . For example, one student wrote: there are problems with his compliance to medications . I got the impression he was not very concerned about what medicines he is receiving and even less concerned about taking them regularly . When the pharmacist relayed information he appeared disinterested and mumbled occasionally later he told me that he prefers to take his traditional chinese medication for treatment of hypertension, and as his traditional practitioner told him not to mix the medications, he plans to stop taking his tablets i got the impression he was not very concerned about what medicines he is receiving and even less concerned about taking them regularly . When the pharmacist relayed information he appeared disinterested and mumbled occasionally later he told me that he prefers to take his traditional chinese medication for treatment of hypertension, and as his traditional practitioner told him not to mix the medications, he plans to stop taking his tablets students wrote about understanding the illness experience from the patient s perspective, the importance of relating to the patient as a person and attention to patient preference in illness management, as well as the importance of emotional and physical comfort and the role of the family in the patient s care . T at home and waiting with her at the clinic enabled me to see her out of the traditional medical student / patient relationship . Seeing other parts of her life enabled me to see her more completely as a human being rather than a patient seeing mdm . T at home and waiting with her at the clinic enabled me to see her out of the traditional medical student / patient relationship . Seeing other parts of her life enabled me to see her more completely as a human being rather than a patient students reported better understanding of the working of the health systems as their narratives progressed in time . This understanding often came from having to navigate the health system with the patients, for example attending clinics, filling prescriptions and visiting patients in hospital during readmissions . Students often reflected on the high cost of health care for the patients and their families and showed awareness of the availability of health care financing . For example, one student reflecting on his elderly patient wrote: our health care system doe not offer free health care for all although we discussed application for medifund (emergency funds for needy patients), mr l still feels he will not be able to cope with his medical bills and he is considering refusing treatment they are concerned about not having enough money to last while they are alive and worry immensely when they fall ill that they have not enough for their medical bills . Our health care system doe not offer free health care for all although we discussed application for medifund (emergency funds for needy patients), mr l still feels he will not be able to cope with his medical bills and he is considering refusing treatment they are concerned about not having enough money to last while they are alive and worry immensely when they fall ill that they have not enough for their medical bills . Students demonstrated a consolidation of their medical knowledge through their description of patients symptoms and illness progression . The risk of mrsa, c diff colitis has to be weighed against her risk of recurrence of osteomyelitis. The clinical question is when to stop her ciprofloxacin . The risk of mrsa, c diff colitis has to be weighed against her risk of recurrence of osteomyelitis. Students described their enhanced understanding of the role and availability of different community resources in managing patient s social and medical needs . They cited specific examples of such services that they learnt about, including home nursing care, meals on wheels and elder care services . I think it is a good community programme that allows patient to remain independent and reduce need for institutionalization . I think it is a good community programme that allows patient to remain independent and reduce need for institutionalization . This included feelings of inadequacy in meeting patients needs and confusion about their roles as health care learners versus friends to the patients . For example, one student reflected: while i am touched and feel privileged that mdm . H found it easy to share her problems with me, i realised how awkward it can be to suddenly be involved in someone else s personal issues . However, i realise that such an interaction is not uncommon in medical practice h found it easy to share her problems with me, i realised how awkward it can be to suddenly be involved in someone else s personal issues . However, i realise that such an interaction is not uncommon in medical practice of the 506 themes, the most frequent was chronic disease management (n=128/25%) followed by patient centred care (n=112/22%), health care systems (n=106/20.9%), biomedical issues (n=100/19.7%), community services (n=48/9.5%) and student s role conflict (n=12/2.3%). We noted a shift in the relative frequency of the different themes, as students moved from hospital to community with their patients (see fig . 2). The most frequent theme in narrative 1, after the hospital encounter was biomedical (44.3%) followed by health systems (18.2%) and patient centredness (12.5%). However, for narrative 2, after the first home visit, the dominant themes were chronic disease management (31.8%), patient centred care (23.3%) and health systems (21%). The most frequent themes in narrative 3, after 10 months of regular follow - up of the patient, were chronic disease management (35.1%), patient centredness (31.8%) and health systems (23.4%). Schema for data collection and flow of student narratives: duke - nus graduate medical school, family medicine clerkship 20092011 . Relative theme frequency, expressed as percentage, after analysis of the 253 narratives represented according to time frame: family medicine clerkship, duke - nus graduate medical school 20092011 . Under this theme, students wrote about understanding chronic disease management, disease progression and the importance of patient education and self - management . Student narratives also showed appreciation of the care continuity and team - based collaborative care by health care providers . Students recognized challenges to lifestyle and medication compliance and noted the use of alternative therapy in chronic diseases . For example, one student wrote: there are problems with his compliance to medications . I got the impression he was not very concerned about what medicines he is receiving and even less concerned about taking them regularly . When the pharmacist relayed information he appeared disinterested and mumbled occasionally later he told me that he prefers to take his traditional chinese medication for treatment of hypertension, and as his traditional practitioner told him not to mix the medications, he plans to stop taking his tablets i got the impression he was not very concerned about what medicines he is receiving and even less concerned about taking them regularly . When the pharmacist relayed information he appeared disinterested and mumbled occasionally later he told me that he prefers to take his traditional chinese medication for treatment of hypertension, and as his traditional practitioner told him not to mix the medications, he plans to stop taking his tablets students wrote about understanding the illness experience from the patient s perspective, the importance of relating to the patient as a person and attention to patient preference in illness management, as well as the importance of emotional and physical comfort and the role of the family in the patient s care . T at home and waiting with her at the clinic enabled me to see her out of the traditional medical student / patient relationship . Seeing other parts of her life enabled me to see her more completely as a human being rather than a patient seeing mdm . T at home and waiting with her at the clinic enabled me to see her out of the traditional medical student / patient relationship . Seeing other parts of her life enabled me to see her more completely as a human being rather than a patient students reported better understanding of the working of the health systems as their narratives progressed in time . This understanding often came from having to navigate the health system with the patients, for example attending clinics, filling prescriptions and visiting patients in hospital during readmissions . Students often reflected on the high cost of health care for the patients and their families and showed awareness of the availability of health care financing . For example, one student reflecting on his elderly patient wrote: our health care system doe not offer free health care for all although we discussed application for medifund (emergency funds for needy patients), mr l still feels he will not be able to cope with his medical bills and he is considering refusing treatment they are concerned about not having enough money to last while they are alive and worry immensely when they fall ill that they have not enough for their medical bills . Our health care system doe not offer free health care for all although we discussed application for medifund (emergency funds for needy patients), mr l still feels he will not be able to cope with his medical bills and he is considering refusing treatment they are concerned about not having enough money to last while they are alive and worry immensely when they fall ill that they have not enough for their medical bills . Students demonstrated a consolidation of their medical knowledge through their description of patients symptoms and illness progression . The risk of mrsa, c diff colitis has to be weighed against her risk of recurrence of osteomyelitis. The clinical question is when to stop her ciprofloxacin . The risk of mrsa, c diff colitis has to be weighed against her risk of recurrence of osteomyelitis. Students described their enhanced understanding of the role and availability of different community resources in managing patient s social and medical needs . They cited specific examples of such services that they learnt about, including home nursing care, meals on wheels and elder care services . I think it is a good community programme that allows patient to remain independent and reduce need for institutionalization . I think it is a good community programme that allows patient to remain independent and reduce need for institutionalization . This included feelings of inadequacy in meeting patients needs and confusion about their roles as health care learners versus friends to the patients . For example, one student reflected: while i am touched and feel privileged that mdm . H found it easy to share her problems with me, i realised how awkward it can be to suddenly be involved in someone else s personal issues . However, i realise that such an interaction is not uncommon in medical practice while i am touched and feel privileged that mdm . H found it easy to share her problems with me, i realised how awkward it can be to suddenly be involved in someone else s personal issues . However, i realise that such an interaction is not uncommon in medical practice of the 506 themes, the most frequent was chronic disease management (n=128/25%) followed by patient centred care (n=112/22%), health care systems (n=106/20.9%), we noted a shift in the relative frequency of the different themes, as students moved from hospital to community with their patients (see fig . 2). The most frequent theme in narrative 1, after the hospital encounter was biomedical (44.3%) followed by health systems (18.2%) and patient centredness (12.5%). However, for narrative 2, after the first home visit, the dominant themes were chronic disease management (31.8%), patient centred care (23.3%) and health systems (21%). The most frequent themes in narrative 3, after 10 months of regular follow - up of the patient, were chronic disease management (35.1%), patient centredness (31.8%) and health systems (23.4%). Schema for data collection and flow of student narratives: duke - nus graduate medical school, family medicine clerkship 20092011 . Relative theme frequency, expressed as percentage, after analysis of the 253 narratives represented according to time frame: family medicine clerkship, duke - nus graduate medical school 20092011 . We performed a qualitative analysis of student narratives written during a longitudinal experience following two patients from a hospital admission to their homes and communities over 10 months . The longitudinal community experience was associated with an increased understanding of chronic disease management, patient centredness, health care systems, community services and biomedical aspects of diseases . Our overall results demonstrated learning that coincided with the learning objectives of the pccm experience . Dominant themes changed from biomedical to psychosocial and community - based between the beginning and end of the experience . Several authors have advocated for longitudinal clerkships and patient care in the community (5, 6, 7, 12, 13, 3032), but reports about how learning in these clerkships differed from learning during block rotations are limited . Our study provides an insight into how long - term relationships with patients can alter student perspectives about the role of community, family and personal values in illness experience and disease progression . An unexpected finding from the narratives was that students reported conflicts in their roles as medical students; for example, in the blurring of the role between health care provider and friend . We speculate that longitudinal patient care, with its constant interaction with the patients and increasing patient demands, resulted in some students feeling overwhelmed and ill - prepared to respond to patient expectations . This is an aspect that the faculty will need to anticipate and address in longitudinal non - hospital - based experiences . Another unanticipated finding was that students often stated the facts of their encounters and told a story without writing reflectively . This could be attributed to our students being predominantly trained in the sciences rather than in the humanities . Reflective writing is a skill that may need to be taught, to better help students to learn from their clinical encounters (33, 34). Theme saturation was reached within 60 of 253 narratives, and the three coders achieved excellent consistency in theme identification . Other than the family medicine clerkship, the only other curriculum during the third year was the research project . As such, our results were likely reflective of the exposure to the longitudinal pccm experience rather than learning from other clinical settings . This is a unique aspect of our study since most third year medical school curricula comprise multiple clerkships and isolating the learning from a particular patient care experience would be a challenge in those settings . Thus we were not able to attribute all learning to the longitudinal construct of the pccm . We believe that our data remain robust because we achieved theme saturation within the numbers of narratives analysed . The narratives were based on three guiding questions that gave students a cue and structure . It is possible that the students may have had a more variation in the themes if the narratives were free flowing . Although two is a small number, the students were able to learn patient centredness, care continuity and chronic disease management from the longitudinal community experience . Previous authors have reported transformative learning from the illness narrative of longitudinal follow - up of even a single patient and family (24). The 2010 carnegie report advocated standardizing learning outcomes and individualizing the learning process, promoting multiple forms of integration, incorporating habits of inquiry and improvement, and focusing on the progressive formation of the physician s professional identity (6). Our study suggests that student participation and immersion in patients lives through a hospital - to - community longitudinal experience can combine those processes in a developmentally appropriate manner . The diverse perspectives of individual patient s experiences can be harnessed as a powerful, previously untapped or underutilized teaching tool . Future studies will examine how patients themselves experience the longitudinal care from students, whether the learning gained by students is durable, and if students apply their newfound knowledge, skills and attitudes to hospital and outpatient assessment and management of future patients in the form of richer and more meaningful biopsychosocial histories and patient - centred management plans . The authors report no conflicts of interest . The authors alone are responsible for the content and writing of the paper.
No author has any financial or proprietary interest in any material or method used in this study . One hundred and twenty patients (240 eyes), who were between the ages of 30 and 45 years (mean age = 37.3 sd 2.3 years) and were treated bilaterally with prk for myopia (mean = 4.5 sd 1.5 d) were enrolled in this prospective randomized study . All subjects provided written informed consent after the nature of the procedures had been explained in detail . We enrolled patients using the following inclusion criteria: mean spherical equivalent (se) within the range 3 and 6 d, bcva equal to or greater than 20/25 and corneal central thickness (cct) within the range of 540 microns and 550 microns . All subjects with ocular disease (which could cause a deficit in visual functionality), diabetic retinopathy, glaucoma, diseases affecting the anterior segment, macular degeneration, either vitreous or retinal disorders, retinal vasculopathies, anterior and/or posterior uveitis and systemic disorders (which could affect the ocular system and modify the corneal healing process) were excluded from this study . We also excluded patients with any evidence of lid disease, progressive or unstable myopia and keratoconus, a history of herpetic keratitis and previous intraocular and/or corneal surgery . All subjects included in the study were randomized into two treatment groups (group 1 and group 2). Sixty patients were included in the study group (group 1) and were treated postoperatively with standard topical antibiotics, corticosteroids, artificial tear therapy and topical b - fgf eye drops plus oral l - cysteine supplements . In addition, 60 subjects were included in the control group (group 2) and received standard topical antibiotics, corticosteroids and artificial tears as postoperative therapy plus b - fgf eye drops only . All patients enrolled were evaluated the day prior to surgery (visit 0) and received a complete ophthalmological examination, including bcva, biomicroscopy, tonometry (altair ultrasonic pachymeter; optikon 2000, rome, italy), corneal endothelial cell count (cellcheck xl; konan medical inc, irvine, ca usa), topography (cso cm02/cm - p02, cso, florence, italy) and funduscopy evaluation . All patients received corneal pachymetry to measure the central corneal thickness and corneal topography to map the curvature of the corneal surface . Tear function was assessed using shirmer's test without corneal anesthesia, and we also determined the tear film breakup time (but). Soft contact lens wearers were invited to discontinue use for a minimum of 3 days prior to surgery, while rigid gas - permeable contact lens wearers were required to suspend use for a minimum of 3 weeks prior to the ophthalmological examination performed the day before surgery . A single operator carried out the refractive procedure . Prk was performed using a schwind esiris excimer laser (schwind gmbh, germany) with an emission wavelength of 193 nm . This excimer laser uses a 0.8 mm flying spot, a gaussian bean with a repetition rate of 250 hz and a 250 hz infrared eye tracker, which monitors the pupil margin and centers the ablation on the entrance pupil center . All surgical procedures took place between 9 am and 1 pm preoperatively, patients were treated with oxibuprocaina hydrochloride 0.4% (novesina 0.6 ml 0.4%, novartis farma spa, italy) and the eye lashes and lids were treated with a povidone - iodine swab . The surgeon marked a 9.0-mm zone with an approved dye, centering it over the image of the pupil . Ethyl alcohol (90%) diluted in physiological saline solution (20%) was used to remove the central 9 mm of the corneal epithelium . This was followed by laser photoablation of both the bowman's layer and the anterior corneal stroma . After prk, a contact lens was applied and all patients received the standard topical medication recommended by the current guidelines, consisting of topical tobramycin (tobral 0.3%; alcon, tx, usa) and diclofenac eye drops (voltaren - ofta 0.1%; novartis, italy) every 6 h for the first week, followed by twice a day for 1 month . Artificial tears were applied every hour until corneal epithelial healing was completed (hyaluronate 0.2%; hyalistil, sifi, italy). Subjects included in group 1 also received topical medication with b - fgf at dose of 10 g per 10 l (recombinant b - fgf, prodotti gianni, italy) four times a day (every 6 h) for 7 days beginning on the day of surgery . These patients were also treated with oral l - cysteine supplements (l - cysteine; natural point s.r.l ., italy) at dose of 500 mg once a day for a period of 15 days beginning 7 days prior to prk . Beginning on the day after surgery, all subjects were evaluated daily until corneal healing appeared complete by biomicroscopy and corneal central thickness was increased according to pachimetry . We also recorded the rate of corneal re - epithelialization using daily slit - lamp evaluation . The follow - up period lasted 6 months, during which time all patients underwent complete ophthalmological examinations every 30 days, including bcva, slit - lamp biomicroscopic observation, applanation tonometry, pachimetry and retina examination . During the follow - up visits, we performed the student's t test for unpaired samples to evaluate differences in means between the two treatment groups . We also calculated the p value to define the statistical significance of the results, with p 0.05 considered statistically significant . There were no significant differences in preoperative variables between the two groups and the variables followed a gaussian distribution . However, the re - epithelialization rates differed between group 1 and group 2 . Complete corneal epithelial resurfacing was achieved starting at day 3 after prk in 57% of the eyes included in the study group (group 1) and in 44% of the eyes in the control group (group 2). At day four, 99% of the eyes included in group 1 showed complete healing, while just 89% of the eyes in group 2 achieved the same result . Furthermore, we observed complete re - epithelialization in all patients treated with topical b - fgf eye drops plus oral l - cysteine supplementation at day 5, while the patients in group 2, who were treated with topical b - fgf only, did not achieve complete resurfacing until day 6 or day 7 after the surgical procedure . Moreover, subjects treated with b - fgf plus l - cysteine demonstrated a re - epithelialization time of 3.3 days sd 0.3, whereas patients treated with b - fgf only required a longer time to achieve a complete re - epithelialization (4.8 days sd 0.5). The kaplan meier curves also demonstrated a statistically significant difference between the two groups (p <0.0001) from day 3 to day 5 postsurgery [fig . 1]. No statistically significant differences were observed regarding haze prevalence or bcva after prk between group 1 and group 2 . Kaplan - meyer curves for epithelial healing all subjects treated in both groups showed a bcva that ranged from 20/25 to 20/20, and no loss of lines of the bcva was detected in either group . No side - effects related to the treatments were reported during the study and none of the subjects were lost to follow - up during the 6-month follow - up period . Trace to mild corneal haze was observed in both groups, including in four eyes of the study group and six eyes of the control group, and statistical analysis did not show any significant differences between the two groups . Prompt corneal restoration after injury, both surgical and accidental, is required to maintain the optical properties of the eye . Furthermore, proper restoration of the epithelial layer is necessary for stromal recovery and to promote the stromal phase of wound healing . Altered epithelial repair induces excessive and disorganized stromal healing, with alterations to the corneal transparency and impairments in visual function . The degree and extent of keratocyte apoptosis also varies with the type of overlying epithelial injury, and can be influenced by the surgical technique and drugs . Therefore, rapid re - epithelialization of the cornea would likely promote wound healing of the underlying stroma with minimal cell apoptosis . Furthermore, the identification of factors that aid re - epithelialization after corneal epithelial injury and prevent complications related to delayed cicatrization, such as scar or haze formation, would optimize surgical outcomes . Additionally, more rapid epithelial healing has been shown to limit postoperative problems such as haze formation . Several growth factors, vitamins and amino acids have also been shown to have a primary and active role in corneal re - epithelialization after corneal injury . Growth factors are also known to play a primary role both in corneal wound healing and in remodeling of the ecm . The production of growth factors by corneal cells and their presence in tears is essential for the maintenance and renewal of normal tissue and the prevention of undesirable immune or angiogenic reactions . The wound healing response following excimer laser corneal photoablation has been extensively studied at the clinical, microscopic, ultrastructural and immunohistochemical levels, and these findings have provided considerable evidence supporting the close relationship between the structural and the biological wound healing responses of the cornea and the resulting optical clarity and stability of the intended refractive change . In addition, the efficacy of recombinant human b - fgf on epithelial healing has been demonstrated in rabbits after mechanical anterior keratectomy . In this study, we found that patients treated with both b - fgf eye drops and l - cysteine oral supplements benefited from more rapid corneal re - epithelialization, which indicates that these two compounds, which act at different subcellular sites, may deliver synergistic effects when used in combination . Moreover, this combination appeared to be safe on human eyes and more effective than topical b - fgf treatment alone . However, no significant differences in corneal haze formation or bcva were noted between the two groups . Further studies are needed to test this innovative treatment in patients with an expected delay in corneal epithelium healing and to expand the treatment's potential application to other corneal pathological diseases.
Psoroptid mites of the genus caparinia affect hedgehogs and a few other mammals causing skin disease . The mite passes all stages on the host and feeds on sloughed skin cells and epidermal debris, similar to chorioptes species . Among 5 species of the genus caparinia that have previously been classified, 2 species are known to infest hedgehogs; c. tripilis and c. erinacei . Among them, c. tripilis shows higher pathogenicity than c. erinacei and may burrow into the skin of the head, ears, flanks, and inner sides of the legs where they form clusters . It can cause irritation, dermatitis, and self - trauma due to pruritus which can lead to a secondary infection and the host may eventually die . First reported in england in 1889 by michael who found very active mites on the surface of hedgehogs and described " running up and down the spines of hedgehogs with great rapidity ", the mite was introduced to new zealand but received little attention until brokie examined 100 hedgehogs from wellington between 1954 and 1957 . In relatively recent years, c. tripilis has also been introduced to new mexico, united states, through breeding colonies of african hedgehogs for sale as pets . So far, the mite has not been reported in asia, possibly because the animal has neither been popular as pet nor has been considered as endangered species . Although there has been a great increase in the population of hedgehogs as pets, limited studies on the ectoparasitic diseases of hedgehogs are available . We report in this paper an outbreak of dermatitis in hedgehogs caused by c. tripilis with description of keys for the identification of the family psoroptidae and the genus caparinia . In february 2010, dermatitis characterized by scale and self - trauma due to pruritus was recognized in a group of 22 four - toed hedgehogs (a. albiventris wagner, 1841) from a local pet shop in gwangju, korea . Three of the hedgehogs were males and the rest were females with an average weight of 339.3 g. the mantle of the skin was flaking, and scales and crusts were present . Two of the severely affected hedgehogs died of self - trauma and secondary bacterial infections . For parasitological examinations, skin scraping samples from severely affected areas of the skin were collected and preserved in 70% methanol . For morphological comparison, adult male mites of otodectes cynotis and of chorioptes texanus were obtained from naturally - infested dogs and a holstein cow, respectively . The identification of the genus caparinia was based on the key provided by lawrence and description by fain . Body length and width of 5 mites per each developmental stage were measured (table 1; fig . Tarsal caruncles were bell - shaped on all legs of male while they were absent on legs iii and iv of females . There were 3 long setae on the third pair of legs in both sexes (fig . Adult males had a posterior end of the abdomen with a trilobate projection on each side, each lobe with a long seta (fig . Average length and width of adult males and females were 313.6240.5 m and 418.6287.1 m, respectively . Based on these morphological features, 4), and the quadrilateral space (fig . 3a, double - sided arrow) of the abdomen of males was wider than its length . A distinctive feature was that they had 3 long setae both on the third and fourth pairs of legs . Two pairs of long setae which were present at the posterior end of the body were well separated from the posterior margin (fig . Long humeral seta of the dorsal surface was inserted in a small distinct oval scutum (fig . Based on these features, the species of the mite was identified as c. tripilis . This study reports the first outbreak of c. tripilis infestation in a colony of african pygmy hedgehogs in korea . The genus caparinia belongs to the family psoroptidae and is closely related to chorioptes except for the presence of a caruncle on leg iv of female mites and to otodectes except for the presence of posterior lobate projections in male mites (fig . 3b). Caparinia spp . Infest hedgehogs and some other mammals, including the cape polecat, hyena and european fox leading to skin diseases . Since this is the first report of caparinic mite infestation in korea, identification keys for the family psoroptidae and the genus caparinia that have been modified from lawrence, fain, and michael are provided at the end of the discussion . The genus caparinia have been classified into 3 well - documented species; c. tripilis, c. erinacei, and c. ictonyctis . Two additional species, c. setifera (mgnin, 1880) and c. vulpis (mgnin, 1880), were mentioned by lawrence but his description was based only on females and were too brief to differentiate between these 2 species and c. tripilis . For this reason, the diagnostic key provided in this paper includes features of female mites of 5 species, while features of male mites of c. setifera and c. vulpis are not provided . The taxonomic status of c. tripilis and c. ictonyctis is based on the mophological characteristics of adult male mites which differ from c. erinacei by the number of lobes on each laminate projection on the posterior end of the body and the number of the setae attached on each lobe . Male mites of c. tripilis and c. ictonyctis have 3 paired posterior lobes, each equipped with a long seta, whereas c. erinacei has 2 paired lobes with a long seta on each lobe . The presence of posterior lobate projections is also an important feature when it is needed to be distinguished from the genus otodectes which does not have these lobate projections (fig . 3b). The posterior dorsal scutum is wider than its length in c. tripilis, whereas that in c. ictonyctis is longer than its width (fig . 4). Furthermore, while the 2 posterior projections of males enclose a quadrilateral space which is as long as its width in c. ictonyctis and wider than its length in c. tripilis (fig . Features of adult female mites of c. tripilis are very similar to those of both c. ictonyctis and c. erinacei . Legs iii and iv do not have a caruncle, which is a characteristic feature to distinguish from chorioptes spp . Female mites have 3 long setae at the end of the third and fourth pairs of legs whereas male mites have those on only leg iii . On the other hand, two pairs of long setae were present at the posterior end of the body of adult females (fig . 2i, black arrow). Furthermore, 1 of the distinctive features of c. tripilis females is that the insertion of posterior abdominal setae is well separated from the posterior margin . By contrast, both c. ictonyctis and c. erinacei had posterior abdominal setae inserted on the edge of the posterior margin . The life cycle of c. tripilis includes an egg, larva, protonymph, deutonymph (including pubescent female), and either an adult male or an adult female . Two bosses are present on the surface of an egg and are on the same side of the cleavage (fig . The adult males and pubescent females form an attachment pair which is more or less permanent up until the time of emergence of the adult females from the deutonymphal exuviae (fig . 2h). Except for the presence of a couple of copulatory tubercles at the posterior end of a pubescent female (female deutonymph, fig . 2f, arrow head), egg, larvae, protonymph, and deutonymph stages of caparinia do not show sexual dimorphism . Observations suggest that the complete life cycle encompass about 3 weeks . C. tripilis may burrow into the skin of hedgehogs, and symptoms may include pruritus, hair loss, spine loss, deformation of the ears, and scaly, encrusted skin lesions, leading to secondary infections . Severely infected animals become feeble, lose weight, scratch the affected skin, and may abandon their normal nocturnal behavior to become active in the daytime . C. erinacei, on the other hand, has low pathogenicity and does not form clusters on its hosts . A sarcoptic mange mite, notoedres muris, one of the most common pet breed of hedgehogs is the african pygmy hedgehog or 4-toed hedgehog (a. albiventris). It is smaller than the western european hedgehog, has a white abdomen, and is characterized by lacking the first toe of the hind leg . The animal had previously been classified in the genus erinaceus (macdonald, 1986) like western european hedgehogs, but recently it has been reclassified as part of the genus atelerix (wilson, 1993). Hedgehogs are becoming popular pet animals but relatively little research has been carried out on their ectoparasitic diseases . As previously stated, this is the first outbreak of c. tripilis in a colony of hedgehogs in korea . Some identification key aspects regarding the morphological features of the family psoroptidae and the genus caparinia are provided . The identification keys in this article are adapted from lawrence, fain, and michael . Pedicel of tarsal caruncles segmented and long--psoroptespedicle of tarsal caruncles not segmented, short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes pedicel of tarsal caruncles segmented and long--psoroptes pedicle of tarsal caruncles not segmented, short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3host: cape polecat -------------------------------------- c. ictonyctishost: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setiferalength and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3 host: cape polecat -------------------------------------- c. ictonyctis host: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setifera length and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis pedicel of tarsal caruncles segmented and long--psoroptespedicle of tarsal caruncles not segmented, short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes pedicel of tarsal caruncles segmented and long--psoroptes pedicle of tarsal caruncles not segmented, short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3host: cape polecat -------------------------------------- c. ictonyctishost: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setiferalength and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3 host: cape polecat -------------------------------------- c. ictonyctis host: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setifera length and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis pedicel of tarsal caruncles segmented and long--psoroptespedicle of tarsal caruncles not segmented, short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes pedicel of tarsal caruncles segmented and long--psoroptes pedicle of tarsal caruncles not segmented, short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections; tarsus iii in both sexes with 2 long setae ----------- otodectes posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis posterior end of abdomen with bilobate laminate projection on either side, each lobe with a long seta, host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections, each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long, quadrilateral space of abdomen is wider than it is long, side of the body are rounded, larger than c. ictonyctis, host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide, quadrilateral space of abdomen is as long as it is wide, side of the body are parallel, smaller than c. tripilis, host cape polecat ----------------------------------------------------------- c. ictonyctis abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3host: cape polecat -------------------------------------- c. ictonyctishost: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setiferalength and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin; humeral seta of dorsal surface not inserted in a scutum, or this scutum very indistinct ------------------ 3 host: cape polecat -------------------------------------- c. ictonyctis host: hedgehog ------------------------------------------- c. erinacei length and width of body 360280 m; posterior abdominal setae as long as body; host, hyena --------- c. setifera length and width of body 450400 m; posterior abdominal setae half as long as those of c. setifera; host, european fox -------------------------------------------------------- c. vulpis
Chronic unreduced anterior dislocations of the shoulder are not very common . Neurological and vascular complications may occur as a result of an acute anterior dislocation of the shoulder or after a while in chronic unreduced shoulder dislocation . We report a case of neglected bilateral anterior shoulder dislocation with bilateral displaced greater tuberosity fracture . To the best of our knowledge, only a handful cases have been reported in literature with bilateral anterior shoulder dislocation with bilateral fractures . Delayed diagnosis / reporting is a scenario which makes the list even slimmer and management all the more challenging . We report a case of a 35-year - old male who had bilateral anterior shoulder dislocation and bilateral greater tuberosity fracture post seizure and failed to report it for a period of 30 days . One side was managed conservatively with closed reduction and immobilization and the other side with open reduction . Shoulder dislocations should always be suspected post seizures and if found should be treated promptly . The most common bilateral shoulder dislocation is posterior resulting from seizure or convulsion due to epilepsy, electric shock or other reasons [2, 3]. Simultaneous bilateral anterior shoulder dislocation is usually of traumatic origin and occurs rarely [4, 5]. Isolated displaced greater tuberosity fractures are thought to occur in less than 2% of proximal humeral fractures, and are normally associated with anterior shoulder dislocations . The greater tuberosity fragment is pulled superiorly by the supraspinatus and posteriorly by infraspinatus and teres minor [8, 9]. Neglected anterior dislocations are less frequent than neglected posterior dislocations, because anterior shoulder dislocations are more familiar to the orthopaedic surgeon, and their radiological diagnosis is relatively easy . The purpose of this paper is to report a case of neglected bilateral anterior shoulder dislocation with bilateral fracture, its occurrence, diagnosis and management . A 35-year - old male presented to the casualty 30 days after his first episode of seizure with pain in both shoulders and difficulty in movements . Patient had not taken any consultations with any doctor post the episode of seizure . Past history failed to shed any light as to the cause of the seizure with no history indicating earlier episodes involving head trauma, substance abuse / withdrawl or any pre - existing neurological cause . On initial evaluation, the patient complained of decreased bilateral shoulder function and motion . On examination, normal shoulder contour was lost, shoulder movement was restricted especially abduction with arm in attitude of external rotation and pain was elicited with movement . X - rays showed bilateral anterior shoulder dislocations with displaced greater tuberosity fractures (fig . 1). Pre - operative x - ray both shoulders at the time of presentation . Left side closed reduction was done under general anesthesia followed by 3 weeks of immobilization and intermittent physiotherapy (fig.2). Closed reduction of the right shoulder had been attempted but the shoulder was locked and attempts proved futile . During second sitting, open reduction was performed . Even after complete release the gt was freed, repositioned and fixed with ethibond after repositioning, the joint was reduced, capsule repaired, joint was stable and was put in an immobilizer . After subscapularis was released, still there had been difficulty in reduction due to capsular adhesions . After releasing the capsule, adhesions were released and greater tuberosity was brought back into position fixed with ethibond and joint was reduced . Joint found stable and greater tuberosity was well reduced and in position under c arm . Patient was put in a shoulder immobilizer and continued for 3 weeks . For rehabilitation, gentle pendulum exercises were started post operatively, but the patient was non compliant and moved his shoulder joint inspite of instructions . Patient came for follow up after 3 weeks and x- ray showed partial displacement of greater tuberosity . However, for displaced greater tuberosity, we decided to continue rehabilitation and monitor progress . At 6 weeks follow up, patient had active assisted forward flexion of 110 degrees, active abduction of 60 degrees . At 12 weeks we report post - operative follow up of 3 weeks and 3 months wherein at the end of 3 weeks the side that underwent closed reduction had full abduction and the side that underwent open reduction had no wound complications, had started pendular movements and was under physiotherapist care for gradual mobilization (fig . 4, 5). At the end of 3 months, the side reduced closed had full abduction, no complaints of pain and the side reduced open had 150 - 160 degrees range of motion (fig . 6, 7, 8) with 3 month follow up x - ray also posted showing union of greater tuberosity (figs . 9, 3 weeks follow up x - ray . 3 weeks follow up clinical picture with left shoulder showing full abduction . 3 months follow up clinical picture with left shoulder showing full abduction and right shoulder showing nearly 150 -160 degrees abduction . Posterior shoulder dislocations usually occur following unbalanced muscle contractions (electric shock, epileptic seizure etc). The posterior dislocations are more common after seizure since the contraction of the relatively weak teres minor and infraspinatus and the posterior fibers of the deltoid are overcome by the more powerful subscapularis leading to internal rotation and posterior subluxation . Keeping the above in mind, it is difficult to explain why an episode of seizure would lead to bilateral anterior shoulder dislocation . Connor- read l et al suggested that anterior dislocation with seizure may occur not during the muscle contractions but from the trauma of the shoulders striking the floor, after the collapse . This could have been so in the case presented here as well but the patient did not report fall post seizure or could not remember but the presence of similar looking fracture dislocation patterns on both sides is perplexing . Even with trauma bilateral anterior shoulder fracture dislocation pattern is difficult as almost always one extremity takes the brunt of the impact . They have a unique mechanism of injury and were first described in 1902 in patients in whom excessive muscular contraction occurred as a result of camphor overdose by mynter . Associated fracture of the greater tuberosity occurs in 15% of the anterior dislocation cases and indicates an associated rotator cuff tear . This may cause long term instability and functional impairment if the fragment is not anatomically reduced . Thus, internal fixation after the reduction must be the ruled out in such cases . However with regards to the demands of the patient, cost constraints, closed reduction on one side and open reduction on the other without any implant aided fixation was done . Mri was not done on either side to evaluate rotator cuff but open reduction was done on one side due to block in closed reduction . Long head of biceps or subscapularis tendon, or as is more likely in our case a bony block . A displaced glenoid labrum, a bony fragment from the glenoid rim, greater tuberosity fragment or an impacted humeral head into inferior lip of glenoid are the mentioned osseous causes of an irreducible anterior shoulder dislocation [25 - 28]. In our case, both soft tissue interposition caused by tissue and capsular contractures as well as bony block due to adherent greater tuberosity seen while doing open reduction were the cause causing interference in reduction . We could find only 5 other reports of bilateral anterior fracture dislocation patterns involving the greater tuberosity which were chronic . Yadav reported a case (also caused by seizure) of a 56- years - old man, which was reported 6 weeks after injury but the patient was treated with benign neglect and, at 6-month follow - up, no union was seen at fracture site but the patient could perform some functional activities and had accepted possibility of future ailments like arthritis and periarthritis . Carew - mccoll described a case that had been caused by electrocution and treated with open reduction and no fixation . At final follow - up, the 78-year- old female patient had abduction 80 bilaterally and no external rotation . Thomas and graham treated a similar injury in a 65- year -old woman who had fallen from a bus but not been diagnosed until 8 months later . Salem reported the same injury in a 37- year - old man who had been electrocuted at work . Diagnosis was delayed 9 weeks, and he was treated with bilateral open reductions without fixation . Seth d. dodds published a report involving a 27-year - old man having similar injury post seizure 2 month late presentation treated bilaterally by open reduction internal fixation . Closed reduction of a neglected anterior shoulder dislocation can be performed only up to six weeks post injury . After this period the danger of an iatrogenic fracture or neurovascular damage rises too high and operative procedures shall be followed . Given the delay in presentation in our patient the possibility of avascular necrosis must also be considered and should be followed up at intervals to assess the same . Closed reduction without hampering the soft tissue attachments and blood vessels which would otherwise be in jeopardy in open surgery could be construed as naive as incidence of avn is more common in fracture dislocations involving surgical or anatomical humeral neck . Shoulder fracture dislocations best treated acutely . In old cases special attention needs to be given to patient expectations, functional motion, risk of avn and management given taking into account all relevant factors . Shoulder dislocation is extremely common after episodes of seizure and should be evaluated clinically as well as radiologically . Fractures of greater tuberosity may also be associated with it, hence any attempt at closed reduction should be made only after radiological evaluation with preparedness for open reduction if the need arises.
All patients gave written informed consent for the provision of blood for the purpose of this research . The protocol under which these samples were obtained was approved by the ethics committee of the university of tbingen, germany . Patient characteristics white blood cell counts were determined by routine testing at the central laboratory of our hospital . Flow cytometric analysis was performed with fresh whole blood specimens after staining with fluorochrome - labeled antibodies . The following antibodies were used: anti - human cd3-apc (1/100) clone ucht1, anti - human cd4-fitc (1/100) clone sk3, anti - human cd8-percp (1/100) clone sk1, anti - human cd19-fitc (1/100) clone hib19, anti - human cd27-pe (1/50) clone l128, anti - human cd38-apc (1/50) clone hit2, anti - human cd56-pe (1/50) clone b159, anti - human cd197(ccr7)-pe (1/50) clone 3d12, all from bd biosciences, and anti - human cd45ra - apc (1/50) clone hi100 from biolegend . As a reference absolute numbers of cd4 and cd8 t cells were calculated by multiplying lymphocyte numbers obtained from the white blood cell count with the frequency of cd4 and cd8 cells in flow cytometric analysis . Jcv copy numbers were determined by quantitative pcr at the institute for virology, heinrich heine university, dsseldorf, germany . Statistical analysis was performed by unpaired t test using spss 22 (ibm corp ., all patients gave written informed consent for the provision of blood for the purpose of this research . The protocol under which these samples were obtained was approved by the ethics committee of the university of tbingen, germany . White blood cell counts were determined by routine testing at the central laboratory of our hospital . Flow cytometric analysis was performed with fresh whole blood specimens after staining with fluorochrome - labeled antibodies . The following antibodies were used: anti - human cd3-apc (1/100) clone ucht1, anti - human cd4-fitc (1/100) clone sk3, anti - human cd8-percp (1/100) clone sk1, anti - human cd19-fitc (1/100) clone hib19, anti - human cd27-pe (1/50) clone l128, anti - human cd38-apc (1/50) clone hit2, anti - human cd56-pe (1/50) clone b159, anti - human cd197(ccr7)-pe (1/50) clone 3d12, all from bd biosciences, and anti - human cd45ra - apc (1/50) clone hi100 from biolegend . As a reference absolute numbers of cd4 and cd8 t cells were calculated by multiplying lymphocyte numbers obtained from the white blood cell count with the frequency of cd4 and cd8 cells in flow cytometric analysis . Jcv copy numbers were determined by quantitative pcr at the institute for virology, heinrich heine university, dsseldorf, germany . Statistical analysis was performed by unpaired t test using spss 22 (ibm corp ., armonk, ny). Three female and 2 male patients between 39 and 56 years of age were recruited at our institution at the initial presentation of pml between 2012 and 2015 (table). Two patients had hiv - associated pml, 2 patients were on treatment with fumaric acid (fumaderm) for psoriasis for more than 6 months, and one patient with relapsing - remitting multiple sclerosis was on natalizumab for more than 24 months . Pml outcome was related to the jcv copy numbers within the csf at the time of pml diagnosis . In one patient with negative jcv - pcr, an immune reconstitution inflammatory syndrome (iris) developed in both patients with hiv - pml following initiation of combined antiretroviral therapy and in the natalizumab - treated patient with multiple sclerosis following plasma exchange . Iris was characterized by the development of new neurologic symptoms and new contrast - enhancing lesions on cerebral mri . Two patients with less than 500 jcv copies per ml csf had only moderate residual symptoms and 2 patients who displayed> 10,000 copies of the jcv dna per ml of csf died of pml within 44 and 61 days, respectively . All patients had reduced lymphocyte numbers at the initial diagnosis of pml (table). At the initial diagnosis of pml, peripheral blood samples were obtained from all patients and cells were directly analyzed by flow cytometry after staining with the indicated fluorochrome - labeled antibodies (figure 1). As compared with healthy controls, the frequency of central memory cd4 t cells was highly significantly reduced (figure 2; p <0.00001), as was the frequency of naive cd4 t cells (p = 0.04). The proportion of effector memory cd4 t cells was increased (p = 0.01). In contrast, the frequencies of naive, central memory and effector memory cd8 t cells (figure 2), cd19 b lymphocytes, cd19cd38 plasma cells, cd19cd27 memory b cells, and cd56 natural killer cells (figure 3) were not significantly different from healthy controls . Expression of ccr7 and cd45ra was determined on live cd4 and cd8 cells, and expression of cd38 and cd27 was determined on live cd19 cells as indicated . Reduced frequency of central memory cd4 t cells (cd4tcm) (p <0.00001), naive cd4 t cells (p = 0.04), and effector memory cd4 t cells (cd4tem) (p = 0.01) in patients with pml . Healthy controls (hc) are depicted in the first column of each dotplot, patients with pml in the second . Frequency of cd19 b lymphocytes, cd19cd38 plasma cells, cd19cd27 memory b cells, and cd56 nk cells does not significantly differ from healthy controls (hc). Healthy controls are depicted in the first column of each dotplot, patients with pml in the second . Nk = natural killer; pml = progressive multifocal leukoencephalopathy . Longitudinal analysis of peripheral immune cells was performed during the course of pml in patients 1 and 2 . Patient 1 displayed a high jcv viral load within the csf (27,300 copies / ml csf) and died 61 days after initial pml diagnosis . He displayed highly reduced frequencies of naive cd4 t cells and central memory cd4 t cells and an increased proportion of effector memory cd4 t cells throughout the progressive course of the disease (figure 4). In patient 2, the reduced frequencies of naive cd4 t cells and central memory cd4 t cells returned almost to normal levels after initiation of combined antiretroviral therapy and during subsequent resolution of pml . Patient 1 neither clinically nor radiologically showed signs of iris whereas patient 2 already showed radiologic signs (edema, gadolinium enhancement) when diagnosed with pml . Frequency of naive, central memory (tcm), and effector memory (tem) cd4 t cells over the course of pml in patient 1 (open circles) and patient 2 (closed triangles). Gray areas indicate normal values (mean values 2 sds of healthy controls). Three female and 2 male patients between 39 and 56 years of age were recruited at our institution at the initial presentation of pml between 2012 and 2015 (table). Two patients had hiv - associated pml, 2 patients were on treatment with fumaric acid (fumaderm) for psoriasis for more than 6 months, and one patient with relapsing - remitting multiple sclerosis was on natalizumab for more than 24 months . Pml outcome was related to the jcv copy numbers within the csf at the time of pml diagnosis . In one patient with negative jcv - pcr, an immune reconstitution inflammatory syndrome (iris) developed in both patients with hiv - pml following initiation of combined antiretroviral therapy and in the natalizumab - treated patient with multiple sclerosis following plasma exchange . Iris was characterized by the development of new neurologic symptoms and new contrast - enhancing lesions on cerebral mri . Two patients with less than 500 jcv copies per ml csf had only moderate residual symptoms and 2 patients who displayed> 10,000 copies of the jcv dna per ml of csf died of pml within 44 and 61 days, respectively . All patients had reduced lymphocyte numbers at the initial diagnosis of pml (table). At the initial diagnosis of pml, peripheral blood samples were obtained from all patients and cells were directly analyzed by flow cytometry after staining with the indicated fluorochrome - labeled antibodies (figure 1). As compared with healthy controls, the frequency of central memory cd4 t cells was highly significantly reduced (figure 2; p <0.00001), as was the frequency of naive cd4 t cells (p = 0.04). The proportion of effector memory cd4 t cells was increased (p = 0.01). In contrast, the frequencies of naive, central memory and effector memory cd8 t cells (figure 2), cd19 b lymphocytes, cd19cd38 plasma cells, cd19cd27 memory b cells, and cd56 natural killer cells (figure 3) were not significantly different from healthy controls . Expression of ccr7 and cd45ra was determined on live cd4 and cd8 cells, and expression of cd38 and cd27 was determined on live cd19 cells as indicated . Nk = natural killer cells; nkt = natural killer t cells . Reduced frequency of central memory cd4 t cells (cd4tcm) (p <0.00001), naive cd4 t cells (p = 0.04), and effector memory cd4 t cells (cd4tem) (p = 0.01) in patients with pml . Healthy controls (hc) are depicted in the first column of each dotplot, patients with pml in the second . Frequency of cd19 b lymphocytes, cd19cd38 plasma cells, cd19cd27 memory b cells, and cd56 nk cells does not significantly differ from healthy controls (hc). Healthy controls are depicted in the first column of each dotplot, patients with pml in the second . Longitudinal analysis of peripheral immune cells was performed during the course of pml in patients 1 and 2 . Patient 1 displayed a high jcv viral load within the csf (27,300 copies / ml csf) and died 61 days after initial pml diagnosis . He displayed highly reduced frequencies of naive cd4 t cells and central memory cd4 t cells and an increased proportion of effector memory cd4 t cells throughout the progressive course of the disease (figure 4). In patient 2, the reduced frequencies of naive cd4 t cells and central memory cd4 t cells returned almost to normal levels after initiation of combined antiretroviral therapy and during subsequent resolution of pml . Patient 1 neither clinically nor radiologically showed signs of iris whereas patient 2 already showed radiologic signs (edema, gadolinium enhancement) when diagnosed with pml . Frequency of naive, central memory (tcm), and effector memory (tem) cd4 t cells over the course of pml in patient 1 (open circles) and patient 2 (closed triangles). Gray areas indicate normal values (mean values 2 sds of healthy controls). Herein, we provide data indicating that alterations in the composition of peripheral cd4 helper t cell subpopulations are associated with development of pml . Cd4 t cells have a pivotal role in the defense against viral infections as has been shown in numerous human diseases including hiv, hepatitis c virus, and jcv . The development of pml is known to be associated with low total numbers of cd4 t cells in patients with late - stage hiv infections and patients with idiopathic cd4 t cell lymphopenia . Our patients with pml did not display any significant alterations in the frequencies of naive, central memory and effector memory cd8 t cells, b lymphocyte populations, and natural killer cells . In contrast, the proportions of cd4 central memory and naive t cells were highly significantly decreased and the proportion of effector memory cd4 t cells was increased . Central memory t cells express the chemokine receptor crr7 and cd62l and recirculate through secondary lymphoid organs . Effector memory cd4 t cells do not express ccr7 and cd62l, molecules required for entry into lymph nodes, but express chemokine receptors such as ccr5, which direct their migration to the sites of inflammation . They are preferentially located within nonlymphoid tissues or remain within the blood and rapidly exhibit effector functions after a new antigen encounter . The fact that the proportion of cd4 effector memory t cells was high in the peripheral blood of patients with pml thus indicates that a large proportion of peripheral cd4 t cells is actively engaged in viral defense . This is consistent with the previously observed prominent infiltration of cd4 t cells into inflammatory cns lesions in pml and further supports the central role of cd4 rather than cd8 t cells in immune defense against jcv . Before the diagnosis of pml, 2 patients had been on immunomodulatory treatment with fumaric acid and one patient with natalizumab . These treatments are known to induce alterations in peripheral immune cells and may thus have influenced our results . Dimethyl fumarate has been shown to both reduce the frequency of lymphocytes in peripheral blood and to disproportionally reduce the frequency of cd8 t cells, while natalizumab increases peripheral lymphocyte counts and the proportion of peripheral b lymphocytes . The fact that the reduction of naive and central memory cd4 t cells was partially restored in patient 2 after resolution of pml and not in patient 1 who displayed a fatal course of pml may further indicate that the frequencies of these cells negatively correlate to pml disease activity . Collectively, these observations are in line with previous reports that found reduced frequencies of cd62l - expressing cd4 t cells in natalizumab - treated patients with relapsing - remitting multiple sclerosis who developed pml . In our cohort, the frequency of central memory cd4 t cells was reduced in patients with hiv - associated pml as well as in patients who developed pml under immunomodulatory treatment . This further supports the notion that the reduced frequency of central memory cd4 t cells represents a state of the immune system with reduced immunocompetence against jcv . Furthermore, these data indicate that additional in - depth analysis of the role of cd4 helper t cell populations during the course of pml could provide important insights into the immune conditions that predispose to this disease . Reconstitution of cd4 t cells in longitudinal analysis might be a predictor for clinical outcome . Obviously, this investigation is limited by the small number of patients, by the different conditions and medications under which pml developed, and the different courses of disease (i.e., with and without development of iris). If confirmed, the observed alterations in cd4 t cell subpopulations could provide new insights into the pathogenesis of pml and may ultimately represent a biomarker for pml disease activity or to detect patients at risk of pml development . This would be urgently needed as jcv infections of the nervous system including pml, jcv meningitis, and cerebellar granule cell neuronopathy become more clinically prominent . Study concept and design: f.b . Acquisition of data: e.d . Analysis and interpretation: e.d . E. dubois reports no disclosures . C. ruschil is on the scientific advisory board for novartis pharma gmbh . F. bischof served on the scientific advisory board for genzyme, novartis, and roche, received speaker honoraria and travel funding from biogen idec, genzyme, and novartis, and received research support from novartis.
Microcystic adnexal carcinoma (mac) is a rare malignant cutaneous neoplasm with pilar and eccrine gland differentiation . Mac was first described as a separate clinical entity by goldstein et al in 1982 . It is locally aggressive but rarely metastasizes, usually presenting as a slow growing asymptomatic lesion on the head and neck . Fewer than 300 cases have been reported worldwide, according to an analysis by yu et al . A 67-year - old man presented to a primary care skin cancer clinic in melbourne, australia for a routine six - month skin cancer examination . There was a long history of recreational sun exposure . Seven separate basal cell carcinomas had required excision from his forehead, nose, pinna, posterior neck, mid back calf in the last decade . Most recently a moderately differentiated squamous cell carcinoma had been excised from his right upper forehead some six months previous . A whole body skin examination was undertaken with the aid of a heine delta 20 non - polarizing dermatoscope (heine optotechnik, herrshing, germany). Digital clinical and dermatoscopic images were taken with a medicam 800 fotofinder non - polarizing camera (fotofinder systems gmbh, aichner, birnbach, germany), the dermatoscopy images being at 20 magnification . Examination confirmed fitzpatrick skin type 2 with severe actinic damage to the skin of his face, upper trunk and distal limbs with multiple solar lentigines and actinic keratoses . Significant actinic damage to the lower lip (actinic cheilitis) was apparent . During the examination the patient pointed out a white, scar - like lesion on his mid left cheek measuring 8 4 mm in diameter . It was non - pigmented and was composed of a clearly demarcated flat white plaque (figure 1). Dermatoscopically the lesion exhibited a dense white structureless area with fine linear branched blood vessels centrally . A notable feature was the white clods of variable diameter superiorly (figure 2). Eccrine syringoid carcinoma, a rare malignant cutaneous adnexal tumor, should also be included in the differential diagnosis . An excisional biopsy was performed using an elliptical excision, and the specimen was submitted for assessment by a specialist dermatopathologist . Examination of the histological sections revealed a deeply invasive dermal neoplasm composed superficially of keratin filled cysts with calcification, and in the underlying reticular dermis, of infiltrative aggregates of basaloid cells in slender strands and syringomatoid aggregates . The differential diagnosis was between a microcystic adnexal carcinoma and a fibrosing basal cell carcinoma with follicular differentiation . This staining pattern, although not entirely specific, was more in favour of microcystic adnexal carcinoma than fibrosing basal cell carcinoma . Ber - ep4 expression has been noted in 38% of microcystic adnexal carcinomas and 100% of basal cell carcinomas . However ck15 is expressed in 92% of microcystic adnexal carcinomas, whereas basal cell carcinomas are negative . Hence, in conjunction with the positive cea, the findings favoured a microcystic adnexal carcinoma (figures 3 to 11). A search of the literature has not discovered any previously published dermatoscopy images of a microcystic adenexal carcinoma . The two most notable dermatoscopic features of the lesion we present were the dense white structureless area centrally and the white clods of variable diameter peripherally . White clods of this pattern have also been observed in the more common adnexal skin tumor trichoepithelioma and may represent keratin retention cysts . Microcystic adenexal carcinoma is currently considered a rare tumor . However, such rarities will present more often as the world population increases in age and has increased access to modern medicine . The authors feel it is important to publish such dermatoscopic images as ours to as wide an audience as possible to aid clinical diagnosis in future.
Approximately 29 000 of those children are in sub - saharan africa where resources for pediatric cancer are very limited . The overall cure rate is 80% for children with cancer in high - income settings like the united states and europe . In low- and middle - income countries (lmics), where children are often diagnosed too late or one of the many barriers to survival of pediatric cancer in lmics is late presentation . The only services for children with cancer in botswana are offered at princess marina hospital (pmh), the main government referral hospital in the capital of gaborone . Since 2007, baylor college of medicine (bcm) and texas children s cancer and hematology centers (txch) have maintained a full - time pediatric hematologist - oncologist at pmh . To build local capacity, numerous pediatric cancer training programs have been offered at pmh through bcm / txch, including the development of a comprehensive pediatric hematology - oncology curriculum for a 5-day workshop at pmh to a multidisciplinary group of 30 health care workers in botswana . Whereas most training focused on the care of a child with cancer and was based at pmh, training that focused on timely recognition and referral by primary health care workers throughout the country was necessary . Therefore, a pediatric cancer awareness training program was developed to reach health care workers at hospitals throughout botswana . Botswana is similar in size to the state of texas, with a population of 2.2 million people . It has 25 government medical hospitals, including primary, district, and two of which are referral hospitals . There are also two faith - based mission and government partnership hospitals, and there is one psychiatric hospital . Hospitals are strategically located across the country, with district and referral hospitals in the population centers . Hospitals and clinics in rural botswana have long relied on outreach from pmh and gaborone for medical education and clinical assistance . The goal of the pediatric cancer recognition program was to visit at least 50% of the hospitals throughout botswana to educate health care workers on warning signs of pediatric cancer and to inform health care workers of pediatric cancer services available at pmh . Pediatric cancer recognition training workshops were organized with leadership at each hospital . The full - time bcm / txch pediatric hematologist - oncologist based at pmh developed and delivered the teaching . The teaching format was open - forum didactic sessions, with lectures using pictures of physical exam, radiographic, hematological, and/or pathological findings to illustrate presentations of pediatric cancers . The length of the workshops was determined by hospital leadership and varied from 2 hours to 1 day . All health care workers were welcome to attend with a focus on physicians and nurses who typically have the most patient contact and are the decision makers with respect to clinical care . All attendees of the training sessions were asked to complete a pretest evaluation assessing their knowledge of the burden of cancer in children, survivability of pediatric cancer in ideal settings, and the most common types of cancer in children . Additionally, attendees were asked about any training in pediatric oncology either during their preservice and/or clinical training years . Finally, attendees were asked to indicate their comfort with caring for children with cancer and their understanding of the referral process to pmh . Following the presentations, they were asked to complete the same questions regarding pediatric cancer, their comfort level with pediatric cancer patients, and their ability to refer a child with suspected cancer to pmh as well as an evaluation of the speaker and the presentations . Statistical analysis of the questionnaires included expressing responses as percentages, evaluating ordinal data as means, and analyses of respondents responses by medical specialty . Statistical analyses were done using stata, version 11 (statacorp lp, college station, tx). Pediatric cancer recognition training workshops were offered at 14 (53.8%) of the 26 government / mission hospitals in botswana, excluding pmh, over a 10-month period (figure 1). Of the 362 health care workers who attended a workshop, 279 (77.1%) completed the pretest, posttest, self - assessment, and evaluation . Nurses represented the majority of attendees who identified their profession, at 49.2% (178/362), whereas physicians represented 30.1% (109/362). Only 7.4% (20/272) of attendees completing the evaluations indicated that they had some formal training in pediatric oncology, including a fellowship, clinical rotation, and/or workshops . Most of those who had some formal training were physicians (at 23.5% [12/51]), whereas only 1.8% were nurses (2/114; p .01). When asked if their clinical training program included a pediatric oncology component, 26.8% (64/239) confirmed that their training program had either clinical or didactic pediatric cancer components, with the most common group being physicians (63.8%, 30/47) when compared with nurses (15.8%, 16/101; p .01; table 1). Pediatric cancer training by profession before the workshop, the majority of attendees were not familiar with the worldwide incidence of pediatric cancer (42.0%, 107/255, answered correctly) or the survival percentage of pediatric cancer in an ideal setting (43.5%, 113/260, answered correctly). When attendees were asked if they understood how to refer a child with suspected cancer to pmh, only 32.7% (84/257) answered positively (4 - 5 on a likert scale of 1 - 5 where 1 = strongly disagree, 3 = neutral, 5 = strongly agree) prior to the seminar . Physicians were most aware of the process (at 54.9% 28/51) compared with nurses (25.9%, 28/108) and others / unidentified (28.6%, 28/98; p .01). After the workshop, 79.5% indicated that they understood how to refer a child with suspected cancer to pmh (table 2). Attendees were asked to critically evaluate the workshop for clarity and content . On a 1 to 5 likert scale, with 5 denoting strongly agree, all criteria had a mean of 4.49 or greater (figure 2). Pre- and post - test responses by profession evaluation of workshop by health care workers (mean scores on a 1 - 5 likert scale where 1 = strongly disagree, 3 = neutral, 5 = strongly agree). In visiting 50% of government hospitals in botswana, 362 health care workers were trained on how to recognize and refer children with suspected cancer to pmh, the only hospital in the country with pediatric oncology services . Assessment of attendees found that the majority of health care workers in botswana had no prior training or clinical experience in pediatric oncology . Though treatment is available at pmh for children with cancer, recognition of suspected cancer and timely referral are essential to offer curative options for batswana children . Delayed presentation and diagnosis of pediatric cancer is a major issue in lmics . The reasons for delay of diagnosis are multifactorial, including the malignancy type, patient / family demographics, and multiple factors within health care systems in lmics . A south african study found that the median time of physician delay, the time from the child s presentation to the health care system until diagnosis of cancer, was 20 days, contrasted with patient delay, the time lapse between onset of symptoms until the first health care encounter, which was 5 days . A turkish study had similar results, with a median physician delay of 28 days versus 3 days for the patient / parent delay . This study also found that the physician delay was longer when the first patient contact was with a general practitioner instead of a pediatrician and even longer if the first contact physician was a nonpediatric specialist . Of the 109 workshop attendees in botswana who identified as physicians, only 10 (9.2%) identified as a pediatrician . Because of the late presentation of childhood cancers in south africa, the southern african children s cancer study group (saccsg) developed the saint siluan warning signs of childhood cancer . The warning signs were distributed throughout the johannesburg area, with a catchment area population of 6 million, over a 6-month period, through lectures (610 attendees) and posters . Public awareness was also included through a multimedia campaign . Compared with the 12 years that preceded the awareness campaign, new referrals and new cancer diagnoses increased in the 6 years following the program from a mean of 78.4/year to 109.3/year . In botswana, the pediatric hematology - oncology program at pmh has primarily relied on 1 bcm / txch pediatric hematologist - oncologist to serve the entire country, including a busy hematology service with the country s only comprehensive hemophilia program for adults and children . Because of the addition of a second physician through the baylor international pediatric aids initiative (bipai)/texas children s hospital global health corps and a txch project manager / care coordinator based at pmh, the full - time pediatric hematologist - oncologist was able to engage in nationwide outreach for the first time since the program s inception in 2007 . With limited time and resources, more than 50% of government hospitals were reached, with 362 health care workers attending the training . The long - term impact on referral patterns and new cancer diagnoses remains to be seen, but the short - term impact can be seen in the respondents increased awareness of basic pediatric cancer knowledge and the services available at pmh for children with cancer . Although this cancer recognition program met its goals, limitations of the program emerged that will inform future pediatric cancer education initiatives . Expenses for this training program included travel costs for the physician, educational materials, and catering at some sites . The overall cost of the program was $4054.02 or $11.20 per health care worker trained . With limited full - time staffing in botswana, it is not possible for the pmh oncologist to engage in prolonged educational initiatives while maintaining the quality of the clinical service . Future training models may involve intensively training representatives from hospitals and clinics throughout botswana to serve as a local resource for their colleagues and as a direct link to the pediatric hematology - oncology service at pmh . Txch is also currently developing a web - based didactic teaching model to include both physician and nurse education . With only 26.8% of attendees of the pediatric cancer recognition workshops indicating that their clinical training program had a pediatric oncology component, there is a tremendous need to incorporate pediatric cancer training into preservice training programs for physicians, nurses, and all health care workers in botswana . Engaging the government and other stakeholders to promote pediatric cancer modules in training programs will be paramount . There are many challenges to improving diagnosis and survival of pediatric cancer in lmics . Even where programs exist to treat children with cancer, such as the program at pmh, successful treatment is dependent on children being appropriately recognized and urgently referred to advanced care by health care workers in the primary care setting . Despite significant deficiencies in human resources, a pediatric cancer recognition program was presented at half of all government / mission hospitals throughout botswana . Sustained efforts are necessary to maximize the impact of such programs to lead to improved survival of pediatric cancer patients in lmics . Jss contributed to the conception and design; contributed to acquisition, analysis, and interpretation of data; drafted the manuscript; critically revised the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy . Ei contributed to the conception and design; critically revised the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy . Psm contributed to the conception and design; critically revised the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy.
Data on 1,276 cases of human stec infection reported in ontario from january 1996 to december 1998 were obtained from the reportable disease information system (rdis) of the ontario ministry of health and long term care . Infection with stec is notifiable in ontario, and more than 95% of reported cases are due to e. coli o157:h7 (3). Cases were excluded if they were identified as part of a communitywide outbreak resulting from a single source, such as contaminated water supply . Consolidated census subdivision (ccs) identifiers were added to the database via a software package that links ccs to appropriate postal codes (postal code conversion file; statistics canada). The human population distribution of ontario was obtained from georef 1996 census (statistics canada), which is based on data collected from all households in the province . Ccs areas, which are coded in square kilometers, were also extracted from the georef database . Livestock distribution and land use data were collected from the 1996 census of agriculture (statistics canada), and area units were converted from hectares to square kilometers . Information on soil development and drainage characteristics for ontario were obtained from the canadian soil information system (cansis) website (http://res.agr.ca/cansis/). All data were aggregated to the ccs because this was the most detailed level at which agricultural data were available . Ccs data from the northern portion of the province were excluded to avoid a potential bias, since this area was sparsely populated and had little agricultural activity . Incidence rates were determined over a 3-year period, from 1996 to 1998, and were expressed as the number of stec cases per 100,000 population per year in ontario ccs . Arcview version 3.1 (esri, redlands, ca, usa) was used to create chloropleth maps based on disease rates and ldi measures . For the purposes of mapping, the method of nested means, as adapted by michel and colleagues (9), was used to classify incidence rates . Quintile breaks in the data were used to catergorize continuous ldi measures (for example, the lower 20% quintile for the ratio of beef cattle to human population is 0.000 to 0.049). Arcview was also used to calculate ccs centroid locations (latitude and longitude) to allow calculation of autocorrelation measures . Spacestat version 1.9 (regional research institute, west virginia university, morgantown, wv, usa) was used to calculate the euclidean distance between ccs centroids, so that an inverse square distance matrix could be produced . This matrix was used in the calculation of moran s i and gi statistics for the 435 townships in the study area . Soil landscape coverage version 2.2 and hydrological data version 2.2 were downloaded and imported into arcview . Attributes containing information on the dominant soil type and drainage characteristics of the soil were mapped . Arcview s geoprocessing extension was used to clip overlay analysis that would remove areas normally covered with bodies of water . The spatial analyst extension of arcview was used to perform a cross - tab query to obtain soil type and drainage characteristic in each ccs . Soil typing within each ccs was based on the predominent type of soil; if two or more soil or drainage types occupied equal areas within a township, then the variable was set to a null value . Soil development and drainage characteristics were based on the canadian system of soil classification (10). For this study, an ldi was defined as a measure of livestock farming intensity that captures information on the number of animals or the amount of their fecal waste relative to various agricultural and environmental factors within a given geographic area . Ldis were created by combining a series of variables that were considered a priori to be potentially spatially associated with human stec infection, based on a comprehensive analysis of possible sources and pathways of infection . We grouped these variables into dimensions and components (table 1). Dimensions included variables related to number of animals, area of manure application, land uses, and human population within a given ccs . Within the dimensions were components that provided further refinement . For example, components within the dimension animal included numbers of various animal species within a given ccs, while components within manure consisted of specific manure characteristics and methods of application (table 1). (table 2) to form the ldi . Within each frame, all possible combinations of the relevant components were used . For example, frame 3 (human population / manure) was used to create four separate ldi consisting of the ratios of total human population in a ccs to the area having manure applied either 1) by solid spreader, 2) by irrigation, 3) as liquid on the soil surface, or 4) as liquid by injection into the soil . Each generated ldi was examined for biological and logical plausibility, and those considered inappropriate were discarded . Data manipulation, merging of data sets, and statistical analyses were conducted by using the statistical analysis system for personal computers, version 6.12 (sas institute inc ., univariate associations between each indicator and the incidence of human stec infection were examined by using poisson regression analysis . The glimmix macro in sas was used, and census division was entered as a repeated effect to induce a correlation structure in an attempt to control for the spatial effects inherent in the data . Initially, ldi were grouped along common components in their numerator (e.g., all ldi with dairy cattle in the numerator were combined into a single group). Ldi within each group were then entered into a separate multivariate model, and non - significant ldi were removed by backward elimination until a minimum of one variable remained (stage 1 models). Variables thus identified were then offered to a second series of multivariate models (stage 2 models), each of which was subjected to a backward elimination procedure . Variables offered to stage 2 models consisted of all combinations of one variable from each of the models developed in stage 1 . Moran s i and gi statistics were calculated for stec incidence rates, which provided a measure of overall and local spatial autocorrelation . For all statistical analyses, data on 1,276 cases of human stec infection reported in ontario from january 1996 to december 1998 were obtained from the reportable disease information system (rdis) of the ontario ministry of health and long term care . Infection with stec is notifiable in ontario, and more than 95% of reported cases are due to e. coli o157:h7 (3). Cases were excluded if they were identified as part of a communitywide outbreak resulting from a single source, such as contaminated water supply . Consolidated census subdivision (ccs) identifiers were added to the database via a software package that links ccs to appropriate postal codes (postal code conversion file; statistics canada). The human population distribution of ontario was obtained from georef 1996 census (statistics canada), which is based on data collected from all households in the province . Ccs areas, which are coded in square kilometers, were also extracted from the georef database . Livestock distribution and land use data were collected from the 1996 census of agriculture (statistics canada), and area units were converted from hectares to square kilometers . Information on soil development and drainage characteristics for ontario were obtained from the canadian soil information system (cansis) website (http://res.agr.ca/cansis/). All data were aggregated to the ccs because this was the most detailed level at which agricultural data were available . Ccs data from the northern portion of the province were excluded to avoid a potential bias, since this area was sparsely populated and had little agricultural activity . Incidence rates were determined over a 3-year period, from 1996 to 1998, and were expressed as the number of stec cases per 100,000 population per year in ontario ccs . Arcview version 3.1 (esri, redlands, ca, usa) was used to create chloropleth maps based on disease rates and ldi measures . For the purposes of mapping, the method of nested means, as adapted by michel and colleagues (9), was used to classify incidence rates . Quintile breaks in the data were used to catergorize continuous ldi measures (for example, the lower 20% quintile for the ratio of beef cattle to human population is 0.000 to 0.049). Arcview was also used to calculate ccs centroid locations (latitude and longitude) to allow calculation of autocorrelation measures . Spacestat version 1.9 (regional research institute, west virginia university, morgantown, wv, usa) was used to calculate the euclidean distance between ccs centroids, so that an inverse square distance matrix could be produced . This matrix was used in the calculation of moran s i and gi statistics for the 435 townships in the study area . Soil landscape coverage version 2.2 and hydrological data version 2.2 were downloaded and imported into arcview . Attributes containing information on the dominant soil type and drainage characteristics of the soil were mapped . Arcview s geoprocessing extension was used to clip overlay analysis that would remove areas normally covered with bodies of water . The spatial analyst extension of arcview was used to perform a cross - tab query to obtain soil type and drainage characteristic in each ccs . Soil typing within each ccs was based on the predominent type of soil; if two or more soil or drainage types occupied equal areas within a township, then the variable was set to a null value . Soil development and drainage characteristics were based on the canadian system of soil classification (10). For this study, an ldi was defined as a measure of livestock farming intensity that captures information on the number of animals or the amount of their fecal waste relative to various agricultural and environmental factors within a given geographic area . Ldis were created by combining a series of variables that were considered a priori to be potentially spatially associated with human stec infection, based on a comprehensive analysis of possible sources and pathways of infection . We grouped these variables into dimensions and components (table 1). Dimensions included variables related to number of animals, area of manure application, land uses, and human population within a given ccs . Within the dimensions were components that provided further refinement . For example, components within the dimension animal included numbers of various animal species within a given ccs, while components within manure consisted of specific manure characteristics and methods of application (table 1). Dimensions were combined mathematically according to equations denoted as frames (table 2) to form the ldi . Within each frame, all possible combinations of the relevant components were used . For example, frame 3 (human population / manure) was used to create four separate ldi consisting of the ratios of total human population in a ccs to the area having manure applied either 1) by solid spreader, 2) by irrigation, 3) as liquid on the soil surface, or 4) as liquid by injection into the soil . Each generated ldi was examined for biological and logical plausibility, and those considered inappropriate were discarded . Data manipulation, merging of data sets, and statistical analyses were conducted by using the statistical analysis system for personal computers, version 6.12 (sas institute inc ., univariate associations between each indicator and the incidence of human stec infection were examined by using poisson regression analysis . The glimmix macro in sas was used, and census division was entered as a repeated effect to induce a correlation structure in an attempt to control for the spatial effects inherent in the data . Initially, ldi were grouped along common components in their numerator (e.g., all ldi with dairy cattle in the numerator were combined into a single group). Ldi within each group were then entered into a separate multivariate model, and non - significant ldi were removed by backward elimination until a minimum of one variable remained (stage 1 models). Variables thus identified were then offered to a second series of multivariate models (stage 2 models), each of which was subjected to a backward elimination procedure . Variables offered to stage 2 models consisted of all combinations of one variable from each of the models developed in stage 1 . Moran s i and gi statistics were calculated for stec incidence rates, which provided a measure of overall and local spatial autocorrelation . For all statistical analyses, a significance level of 5% was used (p=0.05). Geographic distribution of the yearly incidence of human stec infection in ontario between 1996 and 1998 is shown in figure 1 . According to the nested means techniques, stec incidence rates were classified as very low incidence (0.00 to 0.95 per 100,000) in 204 ccs areas, low (0.96 to 4.54 per 100,000) in 95, average (4.55 to 5.38 per 100,000) in 15, high (5.39 to 15.01 per 100,000) in 78, and very high (15.02 to 77.52 per 100,000) in 41 . Ccs where the incidence of stec infection was classified as high or very high were located primarily in the northwestern portion of southern ontario, with smaller numbers in eastern ontario . Yearly incidence of shiga toxin - producing escherichia coli infection (per 100,000 population), southern ontario, 1996 - 1998 . Moran s i calculation for the incidence of stec infection indicated a significantly positive autocorrelation (p=0.012). Gi statistics for the ccs areas with the 10 highest and 10 lowest incidence rates were also statistically significant (p<0.003). A total of 8,316 ldi were generated, of which all but 80 were eliminated on the basis of biological plausibility . Of these 80 variables, of these 33, 9 (27.3%) were based on the number of beef cattle, the total number of cattle per ccs, and measures of manure application; 4 (12.1%) were based on the number of standardized animal units per ccs; and 1 (3.0%) was based on both the number of dairy cattle and chickens per ccs . The number of sheep or goats, soil type, or drainage characteristics were not significantly associated with the incidence of human stec infection in the univariate analysis . The 10 ldi having the highest r values in univariate analyses are shown in table 3 . All of these ldi were based on either the number of cattle, beef cattle, or animal units per ccs . Multivariate modeling resulted in the creation of 16 unique stage 2 models with r values ranging from 0.0932 to 0.266 . The multivariate model having the highest r value, shown in table 4, consisted of humbcow (the ratio of the number of beef cattle to the human population in a ccs), pigfarm (the total number of swine per km of farm land in a ccs), smanccs (the proportion of land in a ccs in which manure is applied by a solid spreader), and lsmancrp (the proportion of cropland in a ccs in which liquid manure is applied to the soil surface . Humbcow, smanccs, and lsmancrp were all positively (and independently) associated with the incidence of human stec infection, whereas pigfarm was negatively associated . The results of our analyses are consistent with the findings of michel et al (3), who demonstrated a higher incidence of human stec infection in rural areas of ontario, as opposed to urban areas, and a spatial association between the incidence of human stec infection and cattle density . These findings are also consistent with other reports in the literature, including outbreaks of stec infection related to consumption of unpasteurized milk (2) and water from shallow wells, direct contact with cattle (5), and an association between endemic stec infection and exposure to agricultural environments (2,11). To our knowledge, this is the first time the application of manure to land has been identified as a potential risk factor for endemic human stec infection . Runoff from agricultural land that has been treated with manure has the potential to contaminate local surface water and wells that supply water for human consumption (12). A relationship between agricultural activities, such as manure spreading, animal density, and elevated fecal bacterial counts in local streams . Was demonstrated in 1989 by meals (13). An outbreak of stec infection in new york state was associated with contaminated well water used in the preparation of beverages and ice at a county fair (6). It was thought that the well in question became contaminated with manure - laden water as a result of recent heavy rains . More recently, contamination of a municipal water supply with e. coli o157:h7 and campylobacter spp . In walkerton, ontario, canada, resulted in the largest documented outbreak of gastroenteritis caused by multiple pathogens . Strong evidence suggests that contamination of walkerton s water supply was due to manure runoff from a nearby farm that entered a shallow well supplying the municipal water system (14). The density of swine within a ccs was negatively associated with the incidence of human stec infection . This apparent protective effect may simply be the result of a relative absence of cattle in areas where swine are intensively farmed . Although swine commonly harbor stec within their intestinal tract, they are not considered to be important reservoirs of e. coli o157:h7 (15). Past studies have identified sheep and goats as important reservoirs for stec (15,16), but these animals were not identified as important predictors of human stec infection in our study . One explanation may be the relatively low numbers of these animals compared with other livestock types . The approach enabled us to examine a large pool of potential covariates from which appropriate indicators could be assessed and used to evaluate the association between livestock intensity and incidence of human stec infection . The chosen indicators were biologically plausible and allowed for identification of a previously unreported risk factor . By using a systematic construction, we identified ldi that were more strongly associated with the incidence of stec infection than has been reported previously (3). When modeled at the same geographic scale, the r value for the best model from our investigation (i.e., the ratio of beef cattle to human population as a measure of cattle density) was 0.27 compared with 0.14 for the total cattle density model used in michel s report (3). These differences in r values may be the result of our selecting beef cattle for the ldi, rather than total number of cattle . It is worth noting that this difference in association is not necessarily evident from maps (compare figures 2 and 3), because both indicators suggest roughly similar distributions of cattle density, with the greatest concentration in ccs located in south - central and eastern ontario . Ratio of beef cattle to human population (number of animals per person), southern ontario, 1996). Caution should be exercised when interpreting our study results, however, because not all potential confounding variables (e.g., age or gender of the infected humans) were included in the analysis . Also, systematic errors arising from differential reporting rates may have biased the relationship between the incidence of human stec infection and the risk factors studied . Since several ldi were investigated, some associations we observed may have arisen from chance alone . Through linkage of existing data sources, spatial analytic techniques provide a means of identifying populations at high risk and potential risk factors for stec infection . The approach outlined in this study provides a rational, practical, and powerful tool for public health . As spatial analysis becomes more widely used in epidemiology, we anticipate that the development of such approaches will take on increasing importance.
The causes are multifactorial, with extramedullary pulmonary hematopoiesis being described as a rare etiological factor (2). This occurs in conditions such as myeloproliferative disorders, hemoglobinopathies and marrow infiltrating diseases . In chronic myeloproliferative disorders, extramedullary hematopoiesis in the lungs may be suspected on computed tomography (ct) chest scans in the presence of diffuse ground - glass opacities or interstitial thickening (3). We report the case of a 66-year - old man in whom chest ct scan did not reveal any lung parenchymal abnormality . A pulmonary extramedullary hematopoiesis causing pulmonary hypertension and severe tricuspid regurgitation was diagnosed on technetium-99 m (tc-99 m) sulfur colloid bone marrow scan and single - photon emission computed tomography (spect)/ct . A 66-year - old man with a known primary myelofibrosis (diagnosed in december 2008 at an outside institution) presented to our hospital with complaints of shortness of breath and abdominal bloating in march 2012 . He had multiple hospital admissions for the worsening of its dyspnea over the previous two years . On clinical examination, he was dyspneic with an elevated jugular venous pressure and bipedal pitting edema . The total white cell count was markedly raised (41.2 10/l) with a low hemoglobin level (5.3 g / dl). A ct pulmonary angiogram did not show any pulmonary embolism; however, the right atrium and the main pulmonary arteries were dilated, suggesting a pulmonary hypertension with reflux of contrast into the inferior vena cava and hepatic veins (fig . 1e). The two - dimensional echocardiography confirmed a severe tricuspid regurgitation with marked right atrial dilatation and moderately impaired right - ventricular systolic function . A tc-99 m sulfur colloid bone marrow scan with spect / ct imaging was performed (symbia, siemens medical solutions, erlangen, germany) with a background history of primary myelofibrosis to exclude pulmonary hematopoiesis as the cause of pulmonary hypertension . The planar gamma imaging showed a hepatosplenomegaly with increased tracer accumulation in the spleen, compatible with an increased hematopoietic activity resulting from an underlying myelofibrosis (fig . A moderate degree of tracer activity in a diffuse pattern was noted in both thoracic regions (fig . The spect / ct imaging confirmed the location of an increased thoracic tracer activity in the lungs (fig . A lung biopsy was not carried out in view of the increased risk of pulmonary hemorrhage in the presence of pulmonary hypertension . A hydroxyurea treatment was started for the patient to control the markedly raised total leukocyte count and massive splenomegaly . Concurrently, he was referred to a short course radiation - therapy to the lungs with view to achieve control of the pulmonary hematopoiesis . The usual sites of extramedullary hematopoiesis are the liver, spleen and intrathoracic paraspinal regions (3). Lungs and pleura are rare sites of extramedullary hematopoiesis with less than forty cases of pulmonary extramedullary hematopoiesis described in the current literature (4). A causal association between pulmonary hematopoiesis leading to dyspnea and pulmonary hypertension has been postulated in few case reports and a limited number of cohort studies (1, 2, 5). (1) listed hematopoietic infiltration of the pulmonary parenchyma as one of the probable causes of pulmonary hypertension in patients with myelofibrosis . (6) described the case of a 49-year - old man in whom dyspnea due to pulmonary hematopoiesis was a presenting feature of myelofibrosis . In most of case reports describing pulmonary extramedullary hematopoiesis, the diagnosis was suspected on the chest ct scan due to abnormalities such as ground - glass opacities (4) or interstitial septal thickening (6). Ground - glass opacities per se are non - specific ct findings with more common differentials of congestive cardiac failure and lung infection in patients presenting with dyspnea . In the setting of myelofibrosis, additional possibilities include pulmonary extramedullary hematopoiesis, thromboembolism or chemotherapy - induced lung toxicity (7). In our case, an increased lung uptake of tc-99 m sulfur colloid is not specific to an extramedullary pulmonary hematopoiesis . It can occur after liver and bone marrow transplantation, hepatic veno - occlusive disease, liver cirrhosis, liver metastases or hepatic chemotherapy toxicity (8). A possible mechanism is the stimulation of reticulo - endothelial elements in the lung interstitium or an induction of pulmonary macrophages . The localization of increased tracer activity in the lungs was sufficiently demonstrated on spect / ct imaging . In addition, spect / ct imaging also aided in confirming absence of tracer activity within the pleural spaces, thus excluding any pleural site of extramedullary hematopoiesis . We believe that spect / ct imaging, if available, should be included in the imaging protocol of suspected pulmonary hematopoiesis, as it can localize tracer activity in the lungs or pleura, which is not possible on a two - dimensional planar imaging . A histopathological confirmation of pulmonary hematopoiesis could not be obtained as a lung biopsy was not performed in our patient . However, pulmonary hematopoiesis was considered the most likely diagnosis in the absence of other complicating factors which could have resulted in an increased lung uptake of tc-99 m sulfur colloid . In summary, extramedullary pulmonary hematopoiesis in myelofibrosis leading to pulmonary hypertension and tricuspid regurgitation is a rare condition . This case report highlights the utility of tc-99 m sulfur colloid bone marrow scan in cases of unexplained pulmonary hypertension with underlying myeloproliferative disorders . We believe that an inclusion of spect / ct imaging helps to confirm and characterize the pulmonary involvement.
Antimicrobial agents are usually incorporated into hygiene products for the treatment and prevention of plaque and gingivitis . Dental caries is a public oral health problem and an infectious - contagious disease that implies an imbalance of normal molecular interactions between the tooth surface / subsurface and the adjacent bacterial biofilm thus, antimicrobial mouth rinses that augment daily home care may provide an efficient income of remove or controlling bacterial plaque to limit gingivitis and periodontitis . Here is a continuous need of new antimicrobial components due to rapid appearance of multiple drug - resistance bacteria . The genus juglans (family juglandaceae) comprises several species and is widely dispersed throughout the world . Many parts of green walnuts such as shells, kernel and seed, bark, and leaves are used in the pharmaceutical and beauty industry [6, 7]. Juglans regia l. bark is used in some countries as a toothbrush and as a dye for coloring the lips for makeup purpose . Walnut (juglans regia l.) bark has been claimed to own anti - inflammatory, blood purify, anticancer, depurative, diuretic, and laxative activities . Juglans regia stem bark contains chemical constituents, namely, -sitosterol, ascorbic acid5, juglone, folic acid, gallic acid, regiolone, and quercetin-3--l - arabinoside [10, 11]. Antifungal, antibacterial, and antioxidant activities of this plant have been described [1216]. Its extract of juglans regia bark showed a broad spectrum antimicrobial activity in a dose - dependent manner . It inhibited the growth of several pathogenic microorganisms such as gram - positive bacteria (staphylococcus aureus and streptococcus mutans), gram - negative bacteria (escherichia coli and pseudomonas aeruginosa), and pathogenic yeast (candida albicans). The extract has either synergistic or additive act when tested with a broad variety of antibacterial drugs . Rahul tested the stem bark extracts of j. regia l. for antimicrobial activity against the microbes present in the saliva specimens of patients suffering from dental caries . Acetone extract was found to be more effective of the extracts as antimicrobial against the oral microflora . Since many of currently used natural products are either detrimental or ineffective, natural folk medicines with antimicrobial effects have been under investigation during the few past decades . Growing emphasis of plant studies in the field of dentistry is due to the antibiotic - resistant bacteria, side effects of chemical antibiotics, and their high cost in developing countries . Juglans regia bark species have been analyzed chemically adequately in several studies, and in agreement among most of them, with respect to the essential oil composition, the major components are phenolic compounds, terpenoids, alkaloids, flavonoids, and steroids . It is reported that leaves from j. regia l. contain monoterpenes and sesquiterpenes, and the bark contains ketones like juglone, regiolone, sterol, and flavonoid . Juglans regia bark is a medicinal plant used in iranian folk medicine as antimicrobial medicine [19, 20]. The aim of the present study was to evaluate the antimicrobial activities of ethanolic and aqueous extracts of juglans regia bark against four species of oral bacteria . The plant material (stem bark) of the species was collected from local market from kordestan, iran, and confirmed at department of botany, school of agriculture, shahid chamran university, ahvaz, iran . Juglans regia bark powdered (100 g) was extracted by 500 ml of ethanol (for preparing the ethanolic extract) and 500 cc of water (for preparing the aqueous one) consecutively using soxhlet extractor with not exceeding the boiling point of the solvent . The extracts were filtered by whatman filter paper and then concentrated in vacuum at 40c by means of a rotary evaporator . The bacteria strains were used for antibiogram pattern including streptococcus mutans ptcc1683, streptococcus salivarius ptcc1448, streptococcus sanguis ptcc1449, and staphylococcus aureus ptcc: 1112, were provided by the iranian microbial type culture collection . The strains were inoculated in blood agar and incubated at 37c at least for 24 h, until emergent adequate colonies . The bacteria strains were touching to 4 - 5 colonies raised from pure microorganism culture and inoculated at the concentration in order to attain the mc . Farland no: 0.5 density and then streptococci species and staphylococcus aureus incubated in muller - hinton and blood agar, respectively . Extracts were diluted in water, and thus 1 gr of each extract inoculated in 1 ml distil water and diluted it to obtain different concentrations as 1000, 500, 250, 125, and 62.5 mg / ml for aqueous and ethanolic extracts . The blank discs (padtan teb co, iran) were inoculated with 20 l of every concentration extracts and placed on the muller - hinton and blood agar were cultivated with bacterial strains . Negative controls used the same solvents to dissolve the extracts, and tetracycline (30 g) and erythromycin (15 g) were used as positive references . Antimicrobial pattern was evaluated by measuring the zone of inhibition against the test bacteria based on millimeters . The mic values were read as the antibacterial concentration at the point where dense colonial growth intersected the disc [22, 23]. Analysis of variance (anova) was used to determine the significance (p 0.05) of the data obtained in all tests . The inhibition zones due to aqueous extract, negative control (water), and positive control (tetracycline 30 g, erythromycine 15 g) are showed in table 1 . Aqueous extract had significantly antibacterial effect against staphylococcus aureus, s. salivarius (figure 2), and s. sanguis (figure 4) compared to control and was significant (p <0.0001), but it did not show effect on s. mutans when compared with erythromycin . Also the results were shown that ethanolic extracts were significant (p <0.0001) with inhibitory effect on the growth of four tested bacteria, in contrast to negative control (table 2). The mic was evaluated for the antimicrobial activity of ethanolic and aqueous extracts of juglans regia on bacteria and the results were shown in table 3 . According to the obtained mic values, ethanol extract of juglans regia bark had the lowest mic of 1.25 microg / ml on s. sanguis (figure 3). Ethanol extract mic value for s. salivarius (figure 1) and aqueous extract mic value for s. sanguis and s. salivarius were similar, 2.50 microg / ml . Antibacterial activity of the aqueous extract on staphylococcus aureus was the least sensitivity with a highest determined mic of 125 microg / ml, while mic value for the ethanolic extract was 2.00 microg / ml . Aqueous extract did not have any inhibitory effect on s. mutans in terms of antimicrobial activity; however, for the ethanol extract it was 5.00 microg / ml . The most important cause of gingival inflammation and dental caries is bacterial plaque . Some of people keep away from chemical mouth rinse because of the presence of alcohol, artificial preservation, or artificial color in mouth rinses the present study showed that juglans regia are potential antimicrobial agents and can be used in oral hygiene products . According to our finding, we indicated that the high concentrations of ethanolic and aqueous extracts have had antimicrobial effects against s. sanguis, s. mutans, s. salivarius, and staphylococcus aureus with significant difference in contrast to control . We indicated that ethanolic extract exhibited zones of inhibition against all the tested samples, whereas aqueous extract is active with comparatively smaller zones of inhibition (tables 1 and 2). Reported that the acetone extract of j. regia l was found to be more effective of the extracts as antimicrobial against the oral microflora . . Showed that walnut leaves could be used as an easily available source of natural compounds to inhibit the growth of different gram - positive bacteria responsible for dental plaques and oral hygiene problems . Recently, darmani et al . Reported the growth inhibition of various cariogenic bacteria (streptococcus mutans, streptococcus salivarius, lactobacillus casei, and actinomyces viscosus) by walnut aqueous extract . The data of this study clearly indicated that ethanolic and aqueous extracts of juglans regia bark significantly inhibited the growth of the tested oral bacteria, and those reports are compatible with our finding . The antibacterial property of the plant material may be due to the presence of phenolic compounds, terpenoid, alkaloids, flavonoids, and steroids . This study has confirmed the antimicrobial potentials of this kind of iranian plant, thus supporting its folklore application as a preventive remedy for various microbial diseases (caries and periodontal disease) in the oral cavity in iran . It provides the basis for the present rapidly increasing interest for the use of natural antioxidants and antimicrobials . Further studies are required to find these effects in order to replace synthetic medications with natural remedies . We concluded that iranian bark of juglone, regiolane, has the antibacterial effects against the important oral bacteria, and ethanolic extract was of higher effectivity against tested bacteria than aqueous extract.
Dendritic cells (dcs) are non - lymphoid antigen - presenting cells distributed widely throughout the body and are distinguishable from macrophages by their lack of both phagocytic activity and capacity to act as effectors cells . Morphologically, they present a complex of dendritic cytoplasmic projections, one or more lobulated nucleoli, clear cytoplasm with scattered organelles and lack of both phagocytic activity and capacity to act as effectors cells . T - cell associated dcs include the interdigitating dendritic cells (idcs), indeterminate cells, langerhans cells, connective tissue dendritic cells and veiled cells (dendritic leukocytes)9,22,23 . Fdcs are located in b - cell dependent areas of the lymphoid follicles of secondary lymphoid organs . Functionally, they have an ability to bind and retain antigens through linking complement and immune complexes for a long time, and are involved in b - cell proliferation, selection and differentiation18,21 . Fdcs can be stained through the complement receptors, c3d (cd21) and c3b (cd35), and by the low affinity to ige receptor (cd23). Also, fdcs are immunopositive to cell cycle markers, fdcs - associated antigen (dcr-1; ki - m4; cna.42; dr53), intermediated filament, adhesion molecules, cytokine receptors and to calcium - bindings proteins (calmodulin; caldesmon; annexin ii; annexin vi and s100 protein -subunit). The immunophenotype, as well as ultrastructural features, of fdcs are variable depending on their distribution in the zones of lymphoid follicles . Idcs are located in the t - cell areas of lymph node, spleen, and thymus, and are related to the function of presenting antigens to t - cells . In contrast to langerhans cells, idcs are immunonegative to cd1a5,20 . Due to the relationship between fdcs / b - cell and idcs / t - cell, the microenvironment of the neoplastic or reactive transformation of b- and t - cell produce alterations in expression of dendritic cells types . Also, a comprehensive investigation on this subject might provide valuable information about diagnosis of lymphomas1,3,7,12,14,17,24 . The goal of this study was to evaluate the presence and distribution of fdcs and idcs in oral lymphomas . The experimental protocol was approved by the committee of bioethics in research of the dental school of the university of so paulo, so paulo, sp, brazil (number 150/00). Routinely processed paraffin sections from 50 oral lymphomas were selected from the files of the oral pathology service at the university of sao paulo, brazil . Oral lymphomas were classified according to the world health organization classification/200110 and colomo, et al.4 by morphology, immunophenotype, epstein barr virus detection and igh gene rearrangement . Representative samples were diffuse large b - cell lymphoma (dlbcl, n=17); plasmablastic lymphoma of the oral mucosa type (pblomt, n=11); burkitt lymphoma (bl, n=15); extranodal marginal zone b - cell lymphoma of mucosa- associated lymphoid tissue (malt lymphoma, n=2); mantle cell lymphoma (mcl, n=1); extranodal nk / t - cell lymphoma, nasal type (etcl, n=3), and peripheral t - cell lymphoma, unspecified (ptcl, n=1). All cases were primary of the oral cavity, since physical exam did not demonstrate signals of disease in other regions of the body . Fdcs were characterized with the antibodies anti - cd21, anti - cd35, and anti - caldesmon . Since caldesmon is also expressed in the vessel walls, consecutive sections were also stained with anti - cd34 and both stain were submitted to quantification with the use of a computerized system (imagelab software, lido, fousp, brazil). Five fields of each section were selected in a light microscope (laborlux; leitz, wetzlar, germany) at 100x magnification . Images were transferred to a computer monitor with area of 640 x 480 pixels, and the quantification was performed by subtraction of images . The values of the caldesmon+/fdcs were obtained through the total of caldesmon+/ immunoexpression minus cd34+/immunoexpression and expressed in m . Since langerhans cells also express s100 protein, antibody anti - cd1a was also used in consecutive sections to exclude the langerhans cells . S100+/idcs were counted in the light microscope (laborlux; leitz, 400x magnification). Ten fields were selected for each case using an integration reticule and values expressed in mm . The statistical analysis was considered only in the dlbcl, pblomt and bl, because the sample of these cases was sufficient to statistical analysis . This analysis was performed with mann - whitney test and significance statistical was evaluated at 0.05 level . A universal automatic system of staining (dako auto staining, dako corporation, carpinteria, ca, usa) was used for immunohistochemistry reactions with the strepatavidin - biotin standard protocol in 3-m - thick sections . The primary antibodies used were: cd21 (dako, clone 1f8, 1:50, incubation for 40 min at 37c), cd35 (dako, clone ber - mac - cdr, 1:20, incubation for 40 min at 37c), caldesmon (novocastra, clone td107, 1:50, incubation for 30 min at 37c), cd1a (serotec, clone 010, incubation for 30 min at 37c), s100 protein (dako, clone z0311, incubation for 30 min at 37c), and cd34 (novocastra, clone qbend/10, 1:50, incubation for 30 min at 37c). The sections were submitted to antigen retrieval . Sections for the reactions with cd21 and cd35 were immersed in target retrieval solution ph 6.0 (dako, s1700) heated to 95c for 30 min . To caldesmon, cd1a, and cd34, louis, mo, usa) buffer 0.01 m, ph 6.0 and heated to 95c for 30 min . Routinely processed paraffin sections from 50 oral lymphomas were selected from the files of the oral pathology service at the university of sao paulo, brazil . Oral lymphomas were classified according to the world health organization classification/200110 and colomo, et al.4 by morphology, immunophenotype, epstein barr virus detection and igh gene rearrangement . Representative samples were diffuse large b - cell lymphoma (dlbcl, n=17); plasmablastic lymphoma of the oral mucosa type (pblomt, n=11); burkitt lymphoma (bl, n=15); extranodal marginal zone b - cell lymphoma of mucosa- associated lymphoid tissue (malt lymphoma, n=2); mantle cell lymphoma (mcl, n=1); extranodal nk / t - cell lymphoma, nasal type (etcl, n=3), and peripheral t - cell lymphoma, unspecified (ptcl, n=1). All cases were primary of the oral cavity, since physical exam did not demonstrate signals of disease in other regions of the body . Fdcs were characterized with the antibodies anti - cd21, anti - cd35, and anti - caldesmon . Since caldesmon is also expressed in the vessel walls, consecutive sections were also stained with anti - cd34 and both stain were submitted to quantification with the use of a computerized system (imagelab software, lido, fousp, brazil). Five fields of each section were selected in a light microscope (laborlux; leitz, wetzlar, germany) at 100x magnification . Images were transferred to a computer monitor with area of 640 x 480 pixels, and the quantification was performed by subtraction of images . The values of the caldesmon+/fdcs were obtained through the total of caldesmon+/ immunoexpression minus cd34+/immunoexpression and expressed in m . Since langerhans cells also express s100 protein, antibody anti - cd1a was also used in consecutive sections to exclude the langerhans cells . S100+/idcs were counted in the light microscope (laborlux; leitz, 400x magnification). Ten fields were selected for each case using an integration reticule and values expressed in mm . The statistical analysis was considered only in the dlbcl, pblomt and bl, because the sample of these cases was sufficient to statistical analysis . This analysis was performed with mann - whitney test and significance statistical was evaluated at 0.05 level . A universal automatic system of staining (dako auto staining, dako corporation, carpinteria, ca, usa) was used for immunohistochemistry reactions with the strepatavidin - biotin standard protocol in 3-m - thick sections . The primary antibodies used were: cd21 (dako, clone 1f8, 1:50, incubation for 40 min at 37c), cd35 (dako, clone ber - mac - cdr, 1:20, incubation for 40 min at 37c), caldesmon (novocastra, clone td107, 1:50, incubation for 30 min at 37c), cd1a (serotec, clone 010, incubation for 30 min at 37c), s100 protein (dako, clone z0311, incubation for 30 min at 37c), and cd34 (novocastra, clone qbend/10, 1:50, incubation for 30 min at 37c). The sections were submitted to antigen retrieval . Sections for the reactions with cd21 and cd35 were immersed in target retrieval solution ph 6.0 (dako, s1700) heated to 95c for 30 min . To caldesmon, cd1a, and cd34, louis, mo, usa) buffer 0.01 m, ph 6.0 and heated to 95c for 30 min . Cd35 positively stained fdcs in four cases of oral lymphomas: (a) two cases of dlbcl, (b) one malt lymphoma, and (c) on the case of mcl . In the dlbcl, immunoexpression was observed in pseudofollicular proliferation centers14,17 . Malt lymphoma presented fdcs as a dense and confluent meshwork corresponding to colonized follicles (figures 1c and 1d). Mcl presented the distribution of fdcs as a loosely arranged, ill - defined and expanded meshwork (figures 1e and 1f). Caldesmon+/fdcs were present in all oral lymphomas and appeared as a dense and confluent meshwork (lacy pattern) among the neoplastic lymphoid cells (figure 1 g). In cases immunopositive to cd21 and cd35, caldesmon+/ fdcs were coincident with cd21 and cd35 stain . S100+/idcs were large cells, disclosing a round nucleus and irregular cytoplasm (figure 1h). Mean values and standard deviation of the quantification of caldesmon+/fdcs and s100+/idcs are presented in table 1 . There is not statistical difference of the caldesmon+/fdcs and s100+/idcs quantification between bl and pblomt, and dlbcl and pblomt, respectively . More frequently fdcs are evaluated in reactive follicles and follicular lymphomas because of microenvironmental similarity between reactive follicles and follicular lymphomas1,3,7,15,24 . Oral lymphomas are almost exclusively b - cell neoplasm, presenting a diffuse pattern of growth10 . Therefore, the finding of rare cd21+/cd35+/ fdcs is expected . Using antibodies anti - cd21 and anti- cd35, fdcs were visualized in two cases of dlbcl as a sparse and disrupted meshwork in neoplastic pseudofollicular proliferation centers, as demonstrated by maeda, et al.14 and mori, et al.17 that evaluated fdcs in b- cell neoplasm with a diffuse pattern . Actually, the role of fdcs is to present antigens to b - cell9,21 . In the process of the antigen - presenting, the activation of complement receptors c3d (detected by cd21) and c3b (detected by cd35) takes place in the fdcs19 . Therefore, the detection of fdcs by cd21 and cd35 depends on their activation, which occurs in reactive follicles, neoplastic pseudofollicular proliferation centers or in neoplastic follicles of follicular lymphomas14,17,19 . Morphologically, malt lymphomas present reactive follicles10 . In the present study, cd21 and cd35 identified fdcs in the case of malt lymphoma that exhibited reactive follicles . Bagdi, et al.1 also verified that stain for cd21, cd23 and cd35 revealed dense fdcs meshwork in reactive follicles of seven cases of primary salivary gland or gastric malt lymphomas . In the present study, so, in malt lymphoma the immunolocalization of the fdcs by cd21 and cd35 is also dependent on the microenvironment organized by neoplastic or non - neoplastic lymphoid cells in the reactive follicles . Mcl typically present a loosely arranged, ill - defined and expanded meshwork of fdcs at the periphery of the neoplasm in nodular or diffuse pattern as demonstrated in the studies of bagdi, et al.1 and mori, et al.17 . This finding is important since this pattern of distribution of fdcs resembles that of the mantle zone of non - neoplastic lymphoid follicles . Also, the immunohistochemistry of fdcs in mcl is helpful to diagnosis of this disease1,17 . In t - cell and nk - cell neoplasm, the cd21+/cd35+/ fdcs are not frequently identified, as in our study1,15,24 . However, angioimmunoblastic t - cell lymphoma presented a expanded meshwork of cd21+/cd23+/cd35+/fdcs . It has been suggested that these cells may be not real fdcs, but rather fibroblastic reticular cells showing overexpression of cd21, cd23 and cd351013 . Tsunoda, et al.24 used caldesmon to evaluate neoplastic follicles of follicular lymphoma and verified a difference in fdcs immunoexpression in follicular lymphomas grade i, ii and iii . Caldesmon stained fdcs in all the studied oral lymphomas, and fdcs were visualized as a lacy pattern meshwork among neoplastic lymphocytes . Thus, in the b - cell neoplasms (dlbcl, pblomt, bl, malt lymphoma and mcl) evaluated, caldesmon+/fdcs might represent nonactive fdcs, since active fdcs are immunoexpressed in follicular microenvironment1,9,21 . In the t - cell and nk - cell neoplasms (etcl and ptcl) evaluated, caldesmon+/fdcs also may represent non - active fdcs or fibroblastic reticular cells with an overexpression of caldesmon . Tsunoda, et al.24 suggested that caldesmon on actin filaments, and extracellular matrix adhesion receptors on fdcs may be the main way whereby fdcs twine around extracellular fibers, contributing to the formation and maintenance of the meshwork structure . In accordance to tsunoda, et al.24, may be that in oral b - cell, t - cell and nk - cell neoplasms caldesmon is also the major way of the formation and maintenance of the meshwork structure of fdcs or fibroblastic reticular cells overexpressing caldesmon . Idcs were identified by the immunoexpression of s100 protein8,9,22 . In order to exclude the possibility of langerhans cells, reactions to cd1a thus, it can be asserted that in the present study the cells positive to s100 protein were idcs, as shinzato, et al.20 and fonseca, et al.5 showed . As idcs are known as antigen - presenting cells to t - cells, they probably represent active cells in oral lymphomas, since a few non - neoplastic t - cells are always present in all b - cell neoplasm . Another possibility is that idcs represent a casual feature in lymphomas due to a favorable microenvironment as also seen is non - neoplastic lymphoid tissue9,19 . Thus, this study represents the first one to perform a quantitative analysis of the dcs in lymphomas . Caldesmon and s100 protein were quantitatively analyzed because the immunoexpression was consistently present in all cases . It was observed that bl presented a lower statistically significant number of caldesmon+/fdcs and s100+/idcs than dlbcl . This finding may be related to the fact that bl is a specific entity and presents a high proliferation rate, shown by the high mitotic counting, spontaneous cell death, and high counting (close to a 100%) of ki-67 positive cells10 . Also, pblomt represent a subgroup of dlbcl that is more frequent in hiv - patients4,10 . Meug - moraw, et al.16 evaluated reactive bone marrow biopsies and observed that hiv - patients did not present differences in fdcs immunoexpression . Therefore, cell proliferation in oral lymphomas may be related to the appropriated microenvironment to development of dcs . The microenvironment determined by neoplastic lymphoid cells in oral lymphomas is responsible by the development and expression of dendritic cells types.
A 70-year - old female with oculocutaneous albinism, presented with proptosis of the right eye for 15 days and defective vision of both eyes since birth, which got worsened in the right eye for the past 15 days . Ophthalmological examination revealed visual acuity of hm and 20/400 in re and le, respectively . Eccentric proptosis of right eye was seen (27 mm hertel in re and 15 mm hertel in le) with fullness in the superotemporal quadrant of the right orbit [fig . 1]. Anterior segment of both eyes showed iris transillumination defects, a normal pupillary reaction and nuclear sclerosis . Proptosis of right eye in oculocutaneous albinism ultrasound of right eye showed low to moderate echogenic mass in the superotemporal quadrant of the orbit . 2] computed tomography (ct) and magnetic resonance imaging (mri) of orbit showed a well - defined lobulated, elongated mass of mixed intensity, measuring 7.1 3.6 2.8 cm with well - enhancing solid and non - enhancing cystic areas in the superior part of the right orbit . Superiorly there was erosion of roof of the orbit with intracranial and extra dural extension . There was erosion of medial wall of the orbit with extension into the posterior ethmoidal sinus [figs . 3 and 4]. Imaging features were in favor of orbital neurofibroma, so radiologist suggested fine needle aspiration cytology or biopsy to confirm the diagnosis . However, they could not guarantee a total extirpation of the intracranial part of the plexiform neurofibroma . B - scan shows orbital mass indenting the eye ball ct orbit shows cystic mass with peripheral enhancement extending into cavernous sinus with eroding lesser wing of sphenoid bone mri orbit shows mass with cystic space extending into cavernous sinus histopathological study reveals plexiform neurofibroma h and e, 10) neurofibromas of the orbit are rare and account for 0.6 - 2.4% of all orbital tumors . Plexiform neurofibromas, the most common orbital subtype, occur exclusively in neurofibromatosis type 1 . Diffuse neurofibromas are usually the dermal variants; they rarely affect the orbit and are clinically indistinguishable from the plexiform subtype . Histologically, diffuse neurofibromas show greater cellularity, less collagen deposition, and lack the cellular peri - neural sheathing characteristic of plexiform neurofibromas . Both the plexiform and diffuse subtypes lack clear margination and tend to be highly vascular . They are relatively well circumscribed and much less vascular . Though this patient was diagnosed to have plexiform neurofibroma of the orbit, other clinical manifestations of neurofibromatosis 1 were absent . Isolated plexiform neurofibromas without the systemic features of neurofibromatosis 1 have been reported at other anatomical sites; however, their location in orbit is relatively rare . Similar cases of orbital plexiform neurofibromas without the systemic features of neurofibromatosis 1 have been reported in literature . Oculocutaneous albinism is associated with syndromes such as prader willi syndrome, angelmann syndrome, hermansky pudlack syndrome and chediak - higashi syndrome . A very rare case has been reported showing the genetic associations between partial albinism and neurofibromatosis in two daughters of a family along with axenfield's defect, congenital deafness and peroneal muscular dystrophy . However, isolated orbital plexiform neurofibroma in association with albinism has not been reported so far . This rare case is reported for the coincidental presentation of oculocutaneous albinism and isolated orbital plexiform neurofibroma without any systemic features of neurofibromatosis 1 in the same patient and probably the first case to be reported.
Retinal injuries due to military and industrial lasers occur in less than 15 individuals yearly, world wide, despite the increasing use of lasers in the health care, military, and educational sectors, and in commercial laboratories.1 in the military, lasers are used as range finders, target designators and for long distance communications.123456 even in ophthalmology the use of lasers has increased significantly . The increase in the use of laser devices has resulted in a concomitant increase in ocular exposure to laser radiation . Accidental momentary laser exposure can be annoying and distracting.78910 prolonged viewing of the beam for more than 10 s especially at close range, can cause retinal damage.10 recently unregulated lasers have been imported in the middle east and can be easily acquired by the public here, we present three cases of military personnel with unilateral visual loss and retinal lesions following alleged exposure to laser pointers . None of the individuals were aware that the bright blue - green light projected into their eyes was from a laser pointer and was harmful . Two young soldiers (cases 1 and 2) aged 27 and 28 years respectively, serving in the oman army, projected penlight like devices emanating bright blue - green light into each others eyes (left eye for case number 1 and right eye for case number 2) for about 510 s. they competing with each other to determine who could bear the light longer while celebrating the success of a local football game . Both individuals experienced some after images, severe photophobia and headache followed by blurred vision the next day . Case 1 presented to the emergency clinic 1-day after laser exposure complaining of poor vision in the left eye . On examination, the best corrected visual acuity was 6/6 with a refraction of 0.25 d sphere in the right eye and 1/60 with a refraction of 0.500.25 90 in the left eye with no improvement on pin - hole testing . Anterior segments and intraocular pressures were normal bilaterally amsler grid testing was normal for the right eye and there was central scotoma in the left eye . On dilated fundus evaluation, the cup - to - disc ratios (cdr) were 0.40.5 bilaterally, the retinal vessels and fovea and fovea, in the right were normal . Optical coherence tomography of the macula in the right eye was normal, in the left eye there was a shadow effect due to the subhyaloid haemorrhage [figure 2]. The patient declined yittrium aluminium garnet (yag) laser hyaloidotomy for the left eye . At 1-month follow up subhyaloid hemorrhage had completely resolved with a dull foveal reflex on fundus evaluation and no improvement in vision in the left eye [figure 3]. Optical coherence tomography of macula in the left eye showed irregularity of the retinal layers [figure 4]. (b) fundus photograph os showing subhyaloid hemorrhage at the posterior pole with horizontal level (a) optical coherence tomography od - normal macula . (b) optical coherence tomography os - showing shadowing of macula by subhyaloid hemorrhage, horizontal level and sup macula seen, ilm irregular fundus photograph os - showing dull foveal reflex after 1-month optical coherence tomography os showing irregular retinal microstructure (a) fundus fluorescein angiography od showing normal dye transit (b) fundus fluorescein angiography os showing - normal dye transit case 2 presented a week after laser exposure complaining of poor vision in the right eye . Best corrected visual acuity was 1/60 with a refraction of plano 0.25 85 in the right eye with no improvement with pinhole and 6/5 with a refraction of 0.50 0.25 60 in the left eye . Amsler grid testing indicated a central scotoma in the right eye and the left eye was normal . There was premacular subhyaloid hemorrhage in the right eye approximately 1 disc diameter in size covering the foveal area horizontally [figure 6]. Optical coherence tomography indicated a shadow effect due to the subhyaloid hemorrhage in the right eye and the left eye was unremarkable [figure 7]. Fundus fluorescein angiograms indicated blocked fluorescence corresponding to the hemorrhage at the fovea in the right eye and was unremarkable in the left eye [figure 8]. Hyaloidotomy with yag laser was unsuccessful in the right eye as the blood covering the fovea was organised [figure 6c]. (c) fundus photo graph od - organised blood clot at posterior pole following yittrium aluminium garnet lysis (a) optical coherence tomography macula od - shadowing due to subhyaloid haemorrhage . (b) optical coherence tomography macula os - normal (a) fundus fluorescein angiography od - showing blocked fluorescence due to subhyaloid hemorrhage at posterior pole . (b) fundus fluorescein angiography os - normal dye transit an unlabelled laser pointer was recovered from case number 1 and case number 2 [figure 9]. Unlabelled laser pointer recovered from cases 1 and 2 case 3 was another soldier who was 28 years old and presented approximately 89 months after presentation of cases 1 and 2 with a similar history . However, he reported to the clinic immediately after exposure . On examination, best corrected visual acuity was 6/5 with a refraction of 0.25 d sphere in the right eye and 1/60 with a refraction of 0.25 d sphere in the left eye with no improvement on pinhole testing . Amsler grid testing indicated normal results in the right eye and a central scotoma in the left eye . On fundus evaluation the cdrs were 0.3 bilaterally, the retinal vessels and fovea in the right eye were normal . There was a well circumscribed round hole at the fovea in the left eye [figure 10]. On optical coherence tomography, the macula was normal in the right eye, and there was a full thickness macular hole with cystoid changes at the edges of the hole and increased reflectivity at the base in the left eye [figure 11]. Fluorescein angiography of the right eye was unremarkable and there was a window defect in the left eye corresponding to the full thickness macular hole [figure 12]. (a) fundus photograph od - showing normal fundus (b) fundus photograph os - showing full thickness macular hole (a) optical coherencetomoraphy od - macula normal (b) optical coherence tomoraphy os showing fill thickness hole, cystoid changes at the edges, with increase reflectivity at the base of the hole (a) fundus fluorescein angiography od - normal dye transit . (b) fundus fluorescein angiography os - showing hyperfluorescence at the site of full thickness macular hole two young soldiers (cases 1 and 2) aged 27 and 28 years respectively, serving in the oman army, projected penlight like devices emanating bright blue - green light into each others eyes (left eye for case number 1 and right eye for case number 2) for about 510 s. they competing with each other to determine who could bear the light longer while celebrating the success of a local football game . Both individuals experienced some after images, severe photophobia and headache followed by blurred vision the next day . Case 1 presented to the emergency clinic 1-day after laser exposure complaining of poor vision in the left eye . On examination, the best corrected visual acuity was 6/6 with a refraction of 0.25 d sphere in the right eye and 1/60 with a refraction of 0.500.25 90 in the left eye with no improvement on pin - hole testing . Anterior segments and intraocular pressures were normal bilaterally amsler grid testing was normal for the right eye and there was central scotoma in the left eye . On dilated fundus evaluation, the cup - to - disc ratios (cdr) were 0.40.5 bilaterally, the retinal vessels and fovea and fovea, in the right were normal . Optical coherence tomography of the macula in the right eye was normal, in the left eye there was a shadow effect due to the subhyaloid haemorrhage [figure 2]. The patient declined yittrium aluminium garnet (yag) laser hyaloidotomy for the left eye . At 1-month follow up subhyaloid hemorrhage had completely resolved with a dull foveal reflex on fundus evaluation and no improvement in vision in the left eye [figure 3]. Optical coherence tomography of macula in the left eye showed irregularity of the retinal layers [figure 4]. (b) fundus photograph os showing subhyaloid hemorrhage at the posterior pole with horizontal level (a) optical coherence tomography od - normal macula . (b) optical coherence tomography os - showing shadowing of macula by subhyaloid hemorrhage, horizontal level and sup macula seen, ilm irregular fundus photograph os - showing dull foveal reflex after 1-month optical coherence tomography os showing irregular retinal microstructure (a) fundus fluorescein angiography od showing normal dye transit (b) fundus fluorescein angiography os showing - normal dye transit case 2 presented a week after laser exposure complaining of poor vision in the right eye . Best corrected visual acuity was 1/60 with a refraction of plano 0.25 85 in the right eye with no improvement with pinhole and 6/5 with a refraction of 0.50 0.25 60 in the left eye . Amsler grid testing indicated a central scotoma in the right eye and the left eye was normal . There was premacular subhyaloid hemorrhage in the right eye approximately 1 disc diameter in size covering the foveal area horizontally [figure 6]. Optical coherence tomography indicated a shadow effect due to the subhyaloid hemorrhage in the right eye and the left eye was unremarkable [figure 7]. Fundus fluorescein angiograms indicated blocked fluorescence corresponding to the hemorrhage at the fovea in the right eye and was unremarkable in the left eye [figure 8]. Hyaloidotomy with yag laser was unsuccessful in the right eye as the blood covering the fovea was organised [figure 6c]. (c) fundus photo graph od - organised blood clot at posterior pole following yittrium aluminium garnet lysis (a) optical coherence tomography macula od - shadowing due to subhyaloid haemorrhage . (b) optical coherence tomography macula os - normal (a) fundus fluorescein angiography od - showing blocked fluorescence due to subhyaloid hemorrhage at posterior pole . (b) fundus fluorescein angiography os - normal dye transit an unlabelled laser pointer was recovered from case number 1 and case number 2 [figure 9]. Unlabelled laser pointer recovered from cases 1 and 2 case 3 was another soldier who was 28 years old and presented approximately 89 months after presentation of cases 1 and 2 with a similar history . However, he reported to the clinic immediately after exposure . On examination, best corrected visual acuity was 6/5 with a refraction of 0.25 d sphere in the right eye and 1/60 with a refraction of 0.25 d sphere in the left eye with no improvement on pinhole testing . Amsler grid testing indicated normal results in the right eye and a central scotoma in the left eye . On fundus evaluation the cdrs were 0.3 bilaterally, the retinal vessels and fovea in the right eye were normal . There was a well circumscribed round hole at the fovea in the left eye [figure 10]. On optical coherence tomography, the macula was normal in the right eye, and there was a full thickness macular hole with cystoid changes at the edges of the hole and increased reflectivity at the base in the left eye [figure 11]. Fluorescein angiography of the right eye was unremarkable and there was a window defect in the left eye corresponding to the full thickness macular hole [figure 12]. (a) fundus photograph od - showing normal fundus (b) fundus photograph os - showing full thickness macular hole (a) optical coherencetomoraphy od - macula normal (b) optical coherence tomoraphy os showing fill thickness hole, cystoid changes at the edges, with increase reflectivity at the base of the hole (a) fundus fluorescein angiography od - normal dye transit . (b) fundus fluorescein angiography os - showing hyperfluorescence at the site of full thickness macular hole laser pointers are simple, handheld battery operated devices, used for teaching and training purposes . Laser pointers are comprised of a diode emitting laser light, with an energy output between 1 and 5 mw.12345 initially commercially available laser pointers were red lasers with a wavelength of 670 nm . However, other wavelengths such as green, blue, yellow and violet lasers are also available and have become popular because of their advantages . For example, green lasers have brighter beams visible both in daylight and night, allowing star gazing and pointing far off objects, blue and violet lasers light up to different colors depending upon where they are projected, yellow lasers dazzle like gold and are used as a laser guide star for use with astronomical adaptive optics.1234567 as the use of laser pointers becomes more popular, physicians need to understand which lasers can cause eye injury.12345678 lasers are divided into four classes based on their output (class 1 and 1 m, class 2 and 2 m, class 3a, 3b, 3r and class 4).123 class 1 lasers, (energy <0.4 mw) are the safest and cannot cause damage even if viewed for long periods of time . Visible laser pointers (400700 nm) operating at <1 mw and 15 mw power are class 2 and class 3a lasers respectively . Class 3b and 3r lasers generate between 5 and 500 mw of power; class 4 lasers generate more than 500 mw of power . Class 2 lasers cause damage to retina if the beam is viewed for more than 10 s at close range . Class 3 lasers, especially 3r and 3b are hazardous and prohibited in many countries but are easily available online and are popular with teenagers.7 class 4 lasers are the most powerful lasers, and are used in military and occupational settings, such as laser shows . They are capable of producing extensive ocular damage.45678910 the output of laser pointers available to the general public is limited and varies by country.678910 as per the united states food and drug administration code of federal regulations, demonstration laser products like pointers must comply with applicable requirements for class i, iia, ii, or iiia devices.12345678910 transmission and absorption of optical radiation by ocular media depends upon the wavelength of the incident ultraviolet (uv) radiation, visible light or infrared radiation.56 for lasers, wavelength, spot size, pulse duration and irradiance determine the magnitude and extent of thermal damage in tissues exposed to the laser beam . Potential harmful effects to the eye, occur due to photomechanical, thermal or chemical injuries, or a combination of these effects.34567 these effects are applied in a controlled manner for the treatment of eye diseases.12345 multiple ocular symptoms such as pain, redness, irritation, corneal signs and retinal injury have been reported in patients exposed to laser pointers.12345678910 scotoma, photophobia, metamorphopsia, chromatopsia or decreased visual acuity can occur hours after exposure.12345678910 the energy from the pointers at the ocular surface is insufficient to cause any appreciable harmful effect.456 however, amplification of irradiance caused by the ocular media to approximately 104 times makes the retina the most susceptible tissue in the body to laser pointer injury.789 redness or surface irritation in a patient with laser pointer exposure is likely due to secondary rubbing of the eye . Pain following exposure to lasers may be due to corneal injury caused by eye rubbing following exposure.12345 anterior segment injuries are rare as the cornea and crystalline lens absorbs most of the uv and infrared energies . Natural responses such as the blink response, squinting, pupillary constriction, and aversion from the uncomfortably bright light protects the retina from accidental injury.123 most often, retinal injuries are subtle with no objective findings making diagnosis of laser induced damage difficult . In such cases diagnosis is facilitated by a set of six questions formulated by mainster et al.1 visual prognosis is excellent if retinal findings are minor or spare the fovea.123456 severity of visual loss, depends upon the distance of the laser impact site from the centre of the fovea, the extent of the chorioretinal disruption and amount of chorioretinal bleed.12345 rapid tissue expansion or distortion caused by extremely high irradiances during short exposure can result in retinal, subhyaloid, subretinal and or choroidal hemorrhages that cause temporary visual deficit if involving the central fundus . These deficits were present in cases number 1 and 2 in our series.3456 permanent visual deficit occurs if there is an underlying damage to the retinal structure.5678 for example case number 1 had persistent poor vision due to alteration of retinal structure noted on optical coherence tomography . In case number 2, allen et al.2 reported full thickness foveal hole in a 20-year - old man due to accidental exposure from a hand held nd: yag laser range finder device . Presence of strong reflections from the choroid underlying the hole on optical coherence tomogram, lead the authors to study the injury pattern by experimentally producing laser induced macular holes in non human primate models . They noted that this specific type of laser induced trauma required a minimum total intraocular energy of about 13 mj.234 laser induced macular holes were similar to idiopathic macular holes clinically and on angiography, but differed on optical coherence tomography, in that the former had increased reflectivity at the base due to scarring of the underlying choroid, similar to case 3 in our series.1 retinal lesions following laser injury generally heal on their own, without any specific therapy . Systemic corticosteroids have been used, with little conclusive evidence indicating faster recovery.8910 hossein et al.9 noted clinical and objective improvement of laser induced maculopathy on spectral - domain optical coherence tomography in a patient who was treated with high dose systemic steroids . The patient was exposed to a class 3r laser for <1 s. spectral domain optical coherence tomography disclosed a hyper - reflective band in the foveal region . However, residual disruption of the outer retinal layer at the fovea remained unchanged.9 nd: yag laser hyaloidotomy is a safe and effective procedure, achieving rapid resolution of premacular subhyaloid haemorrhage with restoration of visual function while preventing the need for vitreoretinal surgery.8 in our case series, treatment was declined by case number 1 and ineffective in case number 2 . Kasaoka et al.10 used animal models and reported that the rpe cells initiate a post - injury process in response to pathologic states and transform from a stationary epithelial state to a spindle - shaped, migratory, proliferative mesenchymal state, leading to the transretinal membrane formation associated with the development of proliferative vitreoretinopathy.10 this rpe transdifferentiation and its migration across the retinal surfaces is mediated by a tyrosinase receptor called c - met . Control of this activity may be a future therapeutic target to minimize retinal damage following laser injury.910 retinal injuries due to laser pointers or devices have legal, financial and medical consequences . Most accidents are prevented by natural reflexive protective mechanisms.456 a lack of information on the types of lasers and the hazards, mis - information or lack of information to consumers, by laser device manufacturers, easy availability of hazardous lasers that resemble safe lasers are a few of the factors that can lead to careless use.1234567 strict legislation, prohibiting the manufacture, use or possession of hazardous laser pointers, public education and education of the military sector about the hazards of lasers is mandatory, especially due to increased use of lasers for military applications.12345678910
We are also grateful to darby proctor for useful comments on a previous version of the manuscript . The present study was approved by the ethics committee of azabu university (japan) (no . 1303042). This research was supported by the jsps research fellowships for foreign researchers (no . P10311) and the mext grant - in - aid for scientific research (no . 26380981) to t. r., the mext grant - in - aid for challenging exploratory research (no . 23650132) to t. h., and the grant - in - aid for scientific research on innovative areas (no.
Recent evidence has shown that fat accumulation, especially in the abdominal cavity, causes dysregulation of adipokines, including increase in leptin, tumor necrosis factor- (tnf-), interleukin-6, and monocyte chemotactic protein-1, and decrease in adiponectin, leading to the development of various metabolic disorders and atherosclerotic cardiovascular diseases [1, 2]. Adiponectin, a member of the c1q / tnf - related protein (ctrp) family, is one of the most extensively studied adipokines that possesses insulin - sensitizing, anti - inflammatory and antiatherogenic effects [35]. To date, 15 additional ctrp family members have been identified that are related to adiponectin in sequence and structural organization [6, 7]. Of all the ctrps, ctrp9 has the highest amino acid identity to adiponectin in its globular c1q domain [6, 8]. Ctrp9 is predominantly expressed in the adipose tissue and plays protective roles in diet - induced obesity, glucose intolerance, and insulin resistance in mice [8, 9]. Several basic studies have shown the beneficial effects of ctrp9 on the cardiovascular system [1014]. Ctrp9 was shown to induce endothelium - dependent vasorelaxation, attenuate neointimal formation after vascular injury, and protect against cardiac injury [10, 11], and adverse cardiac remodeling after acute infarction in mice . Consistent with those experimental studies, serum ctrp9 levels were found to be inversely related to obesity, insulin resistance, and dyslipidemia in a community - based population and in patients with coronary artery disease . In contrast, serum ctrp9 levels were positively associated with bmi in morbidly obese subjects requiring bariatric surgery and with obesity, insulin resistance, and arterial stiffness in subjects with type 2 diabetes (t2d). No study is currently available on the association between ctrp9 and morphological evidence of atherosclerosis in human subjects . Moreover, the association of ctrp9 with atherosclerosis remains to be fully investigated because patients with renal dysfunction or chronic kidney disease (ckd), who have a high risk of cardiovascular mortality, were not included in the preceding studies [15, 16, 18]. Therefore, in this study, we measured the plasma levels of ctrp9 in subjects with t2d representing a wide range of renal function and investigated the clinical association of plasma ctrp9 level with the intima - media thickness (imt) of the carotid artery separately in subjects with ckd and in those without ckd . We consecutively enrolled 419 subjects with t2d (245 men and 174 women) who were admitted to the diabetes center of the osaka city university hospital for the purpose of glycemic control, education, and/or evaluation of diabetic complications between january 2009 and july 2014 . Subjects with type 1 diabetes and other types of diabetes were excluded from the present study . In our analyses, the estimated glomerular filtration rate (egfr) was calculated by using the japanese egfr equation, and the subjects were divided into the ckd (egfr <60 ml / min/1.73 m) or the non - ckd (egfr 60 ml / min/1.73 m) group for analyses . This study was done in accordance with the declaration of helsinki (1975, as revised in 2013). The study protocol was approved by the ethics committee of our institution (number 164). All study participants provided written informed consent . Blood pressure (bp) was determined by using the conventional cuff method with an automatic sphygmomanometer after the subjects had rested for at least 15 min . Blood was drawn after an overnight fast, and biochemical parameters were analyzed by means of a standard laboratory method in the central clinical laboratory of the osaka city university hospital (certification #15 - 0240 by the japanese associations of medical technologists). Glycated hemoglobin a1c (hba1c) was assessed as the national glycohemoglobin standardization program equivalent value (%), which was expressed by adding 0.4 point to the hba1c (jds;%) measured by standard laboratory methods and the previous japanese standard materials . Immunoreactive insulin levels were measured for subjects not receiving insulin therapy (n = 243) by electrochemiluminescence immunoassay (cobas 8000(502/602), roche diagnostics) in the central clinical laboratory . Homeostasis model assessment of insulin resistance (homa - r) was calculated according to the following formula: fasting insulin (u / ml) fasting glucose (mg / dl)/405 [24, 25]. Plasma levels of ctrp9 (ser877hu, uscn life science, houston, tx, usa) and total adiponectin (commodity code #410614, otsuka, tokyo, japan) were measured by using an enzyme - linked immunosorbent assay following the manufacturer's instructions . The minimum detectable levels of ctrp9 and adiponectin were 1.29 ng / ml and 23.4 pg / ml, respectively . The intra- and interassay coefficients of variation of ctrp9 were <10% and <12%, respectively, whereas those of adiponectin were <10% and <10%, respectively . Ultrasonography of the common carotid artery (cca) was performed by using a phase - locked echo - tracking system, which was equipped with a high - resolution real - time 13 mhz linear scanner (prosound ssd 6500 and f75; hitachi aloka medical, ltd ., the imt value was determined by using a measurement software (intimascope; media cross co. ltd, tokyo, japan), as described elsewhere . In brief, images were obtained 20 mm proximal to the origin of the bulb at the far wall of both ccas . The average value of 416 points in this region and the largest value, including plaque lesions, in the cca were measured separately . The mean - imt of the right and left cca (mean - imt) and the greatest imt among the left and right cca (max - imt) were used as markers of atherosclerotic changes . Data are expressed as number (%) or median (interquartile range) as appropriate . For comparisons between the non - ckd and ckd groups, -test or wilcoxon rank - sum test multiple regression analyses were performed to explore the factors associated with carotid imt after adjustment for age, sex, body mass index (bmi), systolic bp, egfr, hba1c level, triglyceride level, high - density lipoprotein (hdl) cholesterol level, low - density lipoprotein (ldl) cholesterol level, treatment with statins, treatment with angiotensin ii receptor blockers or angiotensin - converting enzyme inhibitors (arbs / aceis), smoking status, ctrp9 level, and adiponectin level . A p value of <0.05 was considered significant . Statistical analyses were performed by using the jmp 10 software (sas institute inc ., cary, nc, usa). The clinical characteristics of the total study population, as well as of the subjects with and without ckd, are shown in table 1 . The median age, duration of diabetes, and bmi of the subjects were 65 years, 11 years, and 25.0 kg / m, respectively . One hundred and sixty - one subjects (38.4%) were categorized into the ckd group and the remaining 258 (61.6%) were categorized into the non - ckd group . The median egfr was 42.7 and 76.7 ml / min/1.73 m for the ckd and the non - ckd group, respectively . As expected, subjects with ckd were older and had a longer duration of diabetes than those without ckd . The systolic bp and serum triglyceride levels were higher, and the hba1c, serum hdl - cholesterol, and ldl - cholesterol levels were lower in subjects with ckd than in those without ckd . The prevalence of subjects treated with oral hypoglycemic agents such as sulfonylureas, biguanides, and thiazolidinediones was lower, whereas that of subjects treated with insulin was higher, in the ckd group than in the non - ckd group . The ckd group had a higher prevalence of subjects treated with arbs / aceis and statins for hypertension and dyslipidemia, respectively, than the non - ckd group . The median plasma ctrp9 and adiponectin levels for the total population were 17.4 g / ml (range, 0.0684.3 g / ml) and 6.2 g / ml (range, 0.8546.4 g / ml), respectively . Of note, both plasma ctrp9 and adiponectin levels were inversely correlated with egfr (figure 1, supplementary tables 1 and 2 in supplementary material, available online at http://dx.doi.org/10.1155/2016/8624313), and, accordingly, the plasma levels of ctrp9 and adiponectin were markedly higher in the ckd group than in the non - ckd group (table 1). No significant correlation between plasma ctrp9 and adiponectin levels was found in either the total population, the non - ckd group, or the ckd group (supplementary table 1). In univariate analyses, the plasma ctrp9 levels were positively correlated with age, systolic bp, serum creatinine, and triglycerides and negatively correlated with egfr, hba1c, and hdl - cholesterol (supplementary table 1). On the other hand, the plasma adiponectin levels were positively correlated with age, diabetes duration, systolic bp, serum creatinine, and hdl - cholesterol and negatively correlated with bmi, egfr, immunoreactive insulin, homa - r, and serum triglyceride levels (supplementary table 2). The median of max - imt and mean - imt was 1.08 mm (range, 0.502.86) and 0.76 mm (range, 0.341.57), respectively . Subjects with ckd had higher max - imt and mean - imt than those without ckd (table 1). We then examined the association of plasma ctrp9 levels with carotid imt by using univariate analyses in the non - ckd and the ckd groups, separately . In the non - ckd group, plasma ctrp9 levels were positively correlated with max - imt and mean - imt (figures 2(a) and 2(b)). In contrast, neither max - imt nor mean - imt was significantly correlated with plasma ctrp9 levels in the ckd group (figures 2(c) and 2(d)). On the other hand, plasma adiponectin levels were positively correlated with mean - imt (= 0.147, p = 0.019), but not with max - imt (= 0.036, p = 0.563), in the non - ckd group . Neither max - imt (= 0.039, p = 0.627) nor mean - imt (= 0.090, p = 0.259) was significantly correlated with plasma adiponectin levels in the ckd group . Finally, we performed multiple regression analyses to identify an independent association between plasma ctrp9 levels and carotid imt after adjusting for bmi, plasma adiponectin level, and other potential confounders including age, sex, systolic bp, egfr, hba1c, serum triglyceride level, hdl - cholesterol level, ldl - cholesterol level, smoking status, and presence of treatment with statins and arbs / aceis in the non - ckd or the ckd groups, separately . In the non - ckd group, plasma ctrp9 level was found to be an independent determinant of max - imt (= 0.128, p = 0.037) and mean - imt (= 0.124, p = 0.028) (table 2). Notably, among variables other than ctrp9, the independent determinants were only age for max - imt and only age and ldl - cholesterol level for mean - imt in the non - ckd group . On the other hand, no significant association was observed between plasma ctrp9 level and carotid imt in the ckd group (table 2). In the present study, we measured plasma ctrp9 levels in patients with t2d representing a wide range of renal functions and investigated the impact of plasma ctrp9 level on carotid imt separately in subjects with or without ckd . Importantly, the association was independent of obesity, plasma adiponectin levels, and other traditional cardiovascular risk factors . The results also revealed that plasma ctrp9 levels were elevated in subjects with ckd compared with those without ckd; however, plasma ctrp9 levels were not significantly associated with carotid imt in the ckd group . This study clearly demonstrated that plasma ctrp9 levels were independently and positively associated with carotid imt in diabetic subjects without ckd . Previous experimental studies in mice consistently demonstrated the beneficial effects of ctrp9 on vascular endothelial function, vascular smooth muscle cell proliferation, and the profile of inflammatory cytokines in macrophages [28, 29]. Several studies have investigated the association of ctrp9 with cardiovascular risk factors in human subjects [15, 16, 18], whereas only two reports have shown the association of ctrp9 with atherosclerosis or cardiovascular disease . In a study in which 35% of subjects had diabetes, the ctrp9 level in serum and epicardial adipose tissue negatively predicted the presence of coronary artery disease . In contrast, a study exclusively of diabetic subjects showed that serum ctrp9 levels were independently and positively associated with arterial stiffness . The present study showed a positive relationship between ctrp9 and carotid imt, suggesting that the impact of ctrp9 on atherosclerosis or cardiovascular disease differs depending on the characteristics of study population, at least with regard to diabetic status . Unlike a prior study on subjects with t2d that evaluated subclinical atherosclerosis according to brachial - ankle pulse wave velocity, we were able to demonstrate in diabetic subjects a close relationship between plasma ctrp9 level and carotid imt, the most established surrogate marker for cardiovascular diseases . Moreover, our subjects were older (age, 65 versus 58 years) and had poorer glycemic control (hba1c, 8.6% versus 7.0%) and lower renal function (egfr, 76.7 versus 94 ml / min/1.73 m) than those in a prior study, even in the non - ckd group . Therefore, this study further suggests a significant impact of ctrp9 on atherosclerosis in patients with t2d who are exposed to a relatively high risk of cardiovascular diseases . Importantly, the positive relationship between plasma ctrp9 levels and carotid imt was independent of obesity, plasma adiponectin levels, and other traditional cardiovascular risk factors in the non - ckd group . Considering the inhibitory effect of ctrp9 on vascular smooth muscle cell growth and neointimal formation in mice, a detrimental impact of ctrp9 on carotid imt in this study could be explained by a compensatory response of ctrp9 to the conditions predisposing to the development of atherosclerosis in subjects with t2d . Upregulation of ctrp9 expression in the adipose tissue was observed in 8-week - old ob / ob mice relative to age - matched controls . The protein expression of ctrp9 in cardiac tissue was also increased at 4 weeks in mice that received a high - fat diet and decreased thereafter . A prior study showing an independent and positive association of serum ctrp9 level with arterial stiffness in human subjects with t2d also indicated a role of ctrp9 as a compensatory factor in the pathogenesis of arterial stiffening . Our research group previously reported that plasma adiponectin levels were inversely associated with carotid arterial stiffness in nondiabetic but not in diabetic subjects . No antiatherogenic effect of adiponectin with carotid imt taking these results together, a loss of antiatherogenic effect of adiponectin in diabetic condition could be one of the explanations for the compensatory response of ctrp9 to advanced atherosclerosis in patients with t2d . This study also suggests a possibility of a direct pathophysiological link between ctrp9 and atherogenesis in patients with t2d; however, this needs to be confirmed with future studies . In this study, previous studies investigating ctrp9 levels in the circulation of human subjects excluded those with renal dysfunction [15, 16] or targeted t2d subjects with normal renal function . This study is the first to demonstrate elevated plasma ctrp9 levels in human subjects with renal dysfunction compared with those without renal dysfunction . The plasma levels of adiponectin, which belongs to the c1q family and has high homology to ctrp9, were also elevated in subjects with ckd and inversely correlated with egfr in the present study . The plasma levels of the other isoform of ctrp, ctrp3, were also associated with egfr in human subjects including those with t2d . Although the elevated adiponectin level in patients with ckd is reported to play a protective role against atherosclerosis and cardiovascular diseases [35, 36], it is currently unknown whether the elevated ctrp9 levels in subjects with ckd play a role or are merely due to reduced renal excretion . In the present study, the plasma ctrp9 levels were associated with systolic hypertension, renal dysfunction, and metabolic dysregulation (e.g., obesity, high triglyceride level, and low hdl - cholesterol level) in the ckd group, whereas no parameter other than age and systolic bp was correlated with ctrp9 levels in the non - ckd group (supplementary table 1). These observations may indicate an association of ctrp9 with obesity - related metabolic dysregulation in diabetic subjects with ckd; however, its functional significance needs to be confirmed in further studies . In contrast to diabetic subjects without ckd, those with ckd exhibited no significant association between plasma ctrp9 levels and carotid imt . It is well recognized that individuals with ckd are at a higher risk for cardiovascular mortality than those with preserved renal function . In patients with ckd, not only a clustering of several traditional risk factors, but also nontraditional risk factors such as anemia, hyperphosphatemia, hyperhomocysteinemia, and inflammation play a role in cardiovascular damage . Indeed, in the multivariate analyses (table 2), the explanatory factors including traditional cardiovascular risk factors along with plasma ctrp9 and adiponectin levels did not significantly explain max - imt (r = 0.088, p = 0.515) or only minimally explained mean - imt (r = 0.160, p = 0.032) in the ckd group . Therefore, it is possible to speculate that nontraditional factors related to ckd, although not evaluated in this study, attenuated the relationship between ctrp9 and carotid imt in subjects with ckd . The concentrations of circulating ctrp9 in this study population were found to be different from those in the prior studies [15, 17, 18], even with the same assay kits . However, it needs to be mentioned that the ctrp9 levels were nearly one hundred times different among the prior studies (mean 4.69 ng / ml, 115.3 ng / ml, and 415 ng / ml). Moreover, ctrp9 concentrations obtained by the other assay kits were quite different between the assays (96 pg / ml and 5.0 ng / ml). Based on these facts first, we examined only ctrp9 based on its high similarity to adiponectin and the abundance in experimental evidence of its vascular effects [1014]. Since ctrp1 and ctrp3 [34, 40] were also indicated to be linked to atherosclerosis, the other members of the ctrp family need to be examined in the future . Second, because this was a cross - sectional study, a causal relationship between ctrp9 and carotid atherosclerosis could not be confirmed . Third, our subjects were receiving antihypertensive drugs and statins, which could have affected arterial thickness and its related risk factors . To minimize the effects of these drugs, we adjusted for the presence of these treatments in the multivariate analyses . Fourth, no healthy controls were included in this study, and we could not confirm how diabetic status affects plasma ctrp9 level or its association with carotid imt . Finally, because our subjects with t2d were hospitalized in a university hospital and were largely without adequate glycemic control, the current results cannot be generalized to the entire population of patients with t2d . This study clearly shows that plasma ctrp9 levels are independently and positively associated with carotid imt in diabetic patients without ckd, but not in those with ckd . Our data indicate an important link between ctrp9 and carotid arterial wall thickness, a powerful predictor of cardiovascular diseases, in patients with t2d . This study further proposes that plasma ctrp9 level is a potential biomarker of atherosclerosis in t2d patients without renal complications . Further longitudinal studies are required to clarify whether plasma ctrp9 levels are predictive of the progression of atherosclerosis in patients with t2d who are at a high risk of cardiovascular diseases.
The diagnosis of alzheimer's disease (ad) is currently based primarily on clinical symptoms . Whereas the sensitivity of the clinical diagnosis for possible and probable alzheimer dementia according to national institute of neurological and communicative disorders and stroke and the alzheimer's disease and related disorders association (nincds - adrda) criteria is over 80% the term mild cognitive impairment (mci) was introduced for subjects who complain about verifiable cognitive disturbances but who show a preserved general cognitive functioning and no impairment in the activities of daily living . These patients can be further subdivided into those with an impaired memory function (amnestic mci) and those whose memory is preserved but who show disturbances of language, executive function, or visual - spatial skills (nonamnestic mci). If only one of the above - mentioned cognitive domains is impaired, patients are called single - domain mci; if two or more domains are affected, they are referred to as multidomain mci . Although the term mci is solely descriptive and allows no conclusion on the aetiology, the classification allows some prediction of the course of the disease . For amnestic mci patients, an accurate early diagnosis in mci patients or even a predictive diagnosis in individuals without cognitive disturbances is still virtually impossible . As there is evidence that pathological biochemical changes start many years before the occurrence of functional symptoms, identification of biological markers in individuals with early - stage dementia is the most promising way to facilitate a predictive diagnosis [46]. Improving the early and predictive diagnosis of ad is of paramount importance if, in the future, preventive and disease - modifying therapies become available . In this regard, enormous efforts are under way . Although most therapies failed to show efficacy in phase iii trials, there are still some promising approaches like a lowering compounds, inhibitors of inflammation, inhibitors of tau phosphorylation and aggregation, and compounds interfering with cholesterol metabolism under investigation . Although the brain has some limited regenerative capacity, neurons are still difficult to replace [8, 9]. Therefore, it is clear that maximal benefit for the patients can be expected when the treatment can be initiated as early as possible in the course of the disease . Furthermore, biologically valid and clinically accurate biomarkers may serve in the development of novel therapeutic strategies and may provide important information in clinical trials of therapies . Well - documented biomarkers for ad in cerebrospinal fluid (csf) include alterations in a 1 - 42, total - tau, and phospho - tau . Importantly, these particular changes are detectable in early dementia stages as well as in individuals with mild cognitive impairment (mci) who are at high risk of conversion to ad . When analyzed in well - characterized clinical samples, the measurement of a 1 - 42, tau, and phospho - tau in cerebrospinal fluid generally allows the diagnosis of ad and even the prediction of the conversion from mci to ad with a specificity and sensitivity of about 85% . However, some report a lower sensitivity of below 50% for single biomarkers when these biomarkers are measured as part of a routine diagnostic test in a memory clinic . This drop in sensitivity can be explained by the fact that in clinical practice the reference cohort is not a group of cognitively healthy individuals but consists of patients with other neurodegenerative and neurologic diseases who may also have slightly elevated total - tau, phospho - tau, or a 1 - 42 levels . The application of these markers in the differential diagnostic of neurodegenerative diseases therefore proves to be particularly problematic . Consequently, there is a need for additional and more sensitive csf biomarkers for the early and differential diagnosis of alzheimer's disease . There is the additional problem of lumbar puncture to obtain csf, since although the rate of complications during and after lumbar puncture is below 24% and restricted to mild to moderate postlumbar puncture headache [1518], it must still be seen as invasive method for which special precautions must be taken . Consequently, there is a pressing need for new biomarkers in more easily accessible body - fluids such as peripheral blood . Clinical proteomics is a fast developing field dedicated to the search for new biomarkers applicable to support the clinical diagnosis . At present, a number of potential new biomarker - candidates for ad have been reported from proteomic studies [20, 21]; unfortunately, however, the published data is often contradictory and in many cases, a solid reassessment by other methods and with independent samples is required . Taking this into account, the eu - project clinical proteomics for neurodegenerative diseases (cneupro) is not only dedicated to the detection of potential new biomarker candidates for neurodegenerative diseases in csf and blood, but also to the implementation of in - depth reassessments and validation studies . Finally, promising biomarker candidates will be studied for their suitability as routine test analytes by prototype assays . Cneupro (http://www.cneupro.eu/) is a specific targeted research project (strep) within the sixth framework program of the european commission . It started in april 2007 and is coordinated by jens wiltfang, university of duisburg - essen . For the general aims of cneupro, (see box 1). The consortium consists of 14 academic partners (university of duisburg - essen, centre hospitalier universitaire de montpellier, sahlgrenska academy at the university of gothenburg, vu university medical center, university of ulm, university of newcastle upon tyne, university of aveiro, university of szeged, university of perugia, ruhr - university bochum, heinrich heine university of duesseldorf, university of eastern finland kuopio, institut de la sant et de la recherche mdicale, university of erlangen) as well as four small to medium enterprises . (matrix advanced solutions germany gmbh, microdiscovery gmbh, protagen, biogenes gmbh). Cneupro integrates almost all different levels of biomarker research: the primary phase involves the comprehensive clinical characterization of patients and standardized sample - acquisition and handling by specialized geriatric psychiatrists and neurologists . These samples are subsequently used in the search for candidate biomarkers, their biochemical identification by mass spectrometry, and their reassessment in a second, independent set of high quality samples . Finally, the identified biomarkers will be integrated into novel prototype assays (figure 1). The research within cneurpo concentrates on individuals diagnosed with mci at baseline who subsequently either developed ad, other dementias, or who did not progress to dementia . As the samples had been taken at baseline, clinical information obtained during follow - up allows the identification of predictive biomarker candidates retrospectively . In addition, clinical samples from patients with early ad at baseline or other dementias in the early stages are also included in the analysis . In the search for new biomarker candidates in csf or blood, hypothesis - free proteomic approaches such as urea - based gel electrophoresis, multidimensional liquid chromatography, combined with two - dimensional differential gel electrophoresis (2d - dige), several mass spectrometric methods (e.g., seldi - tof, maldi - tof, nanolc - maldi - tof / tof, nanolc - esi, nanolcqftms), and array - based methods are conducted . Additionally, specific and potentially interesting molecules are studied in detail in the sense of hypothesis - driven approaches . Where applicable, published information in terms of the biological function or a possible role of selected candidates in the pathophysiology of ad will also be considered . The selected candidates will be reassessed with a further independent high quality clinical sample of age- and sex - matched patients and controls and with assays allowing for intermediate sample throughput and quantitative comparisons . For those biomarker candidates that can be successfully validated, cneupro will devise novel poly- and monoclonal antibodies . Finally the biomarkers will be integrated into novel elisa - type assays and, if appropriate, in multiplex - assays . An essential prerequisite for a successful multicenter biomarker - discovery study is the standardization of the clinical diagnostics, the preanalytical sample handling procedures, and the measurements of the known biomarkers total - tau, phospho - tau, and a 1 - 42 in csf . To this end, two neurochemical dementia diagnosis reference centers in hungary and portugal are currently being established, and european standard operating procedures for clinical diagnostics and preanalytical sample handling have been defined . In hungary, 42 dementia centers are responsible for the diagnosis and treatment of demented patients . Before 2009, the csf analysis of a 1 - 42, total - tau, and phospho - tau to support dementia diagnostics was not possible for these centers . As one of the aims of cneupro, the first reference center for neurochemical dementia diagnosis in hungary was launched in szeged . With the support of the cneupro consortium, state - of - the - art diagnostic and methodological standards have been implemented, and the center takes part in an ongoing quality control program organized by kaj blennow from sahlgrenska university hospital, mlndal, sweden . During its first twelve months of operation, the neurochemical dementia diagnosis reference center in szeged has received a total of 54 csf samples from 14 different dementia centers in hungary . This neurochemical dementia diagnosis center will now try to provide its service to further dementia centers in hungary and to start collecting samples for scientific purposes . Due to substantial intercenter variations, the reported accuracy of csf biomarkers is considerably lower in multicenter studies than in single center surveys [2224]. To this end, a multicenter study, supported by cneupro, provides guidance on how to establish, validate, and audit csf tau cutoff values using an unbiased, two - stage multicentre strategy . Furthermore, a hands - on workshop was organized by members of the cneupro consortium (paper submitted to the same issue of ijad). The aim of the workshop was to assess the differences in assay procedures as potential sources of error . During this workshop, 14 groups simultaneously performed the a 1 - 42, total - tau, and phospho - tau assays according to the guidelines of the manufacturer . At least 23 items in assay procedures were identified that varied between the laboratories, including procedures for washing, pipetting, incubation, finishing, and sample handling . Thus, even if centers use the same assays for a 1 - 42, total - tau, and phospho - tau measurement on a regular basis, they do not uniformly adhere to the procedures recommended by the manufacturer . The results of the workshop stress the importance of standardization of assay protocols . To facilitate biomarker research on a multicenter level, standard operating procedures for the clinical diagnosis and the preanalytical sample handling have been defined by the cneupro consortium (boxes 2 and 3). The standard operating procedures for sample acquisition, handling, and storage defined by cneupro meet the quality standards required for proteomic studies in csf and are in agreement with the recently published guidelines for csf collection and biobanking from the bioms - eu network . In the last decade, the levels of a peptides and tau proteins in csf have gained increasing importance in supporting the clinical diagnosis of ad [10, 33]. As no single marker alone allows for a diagnosis with the desired accuracy, several combinations of csf - biomarkers (a x-42, a x-40, total - tau, phospho - tau) have been proposed . For these markers, a diagnostic accuracy of up to 94% has been achieved in single center studies . Within cneupro, welge et al . Reported a sensitivity and specificity of 88% in the discrimination of ad subjects from other dementias and from elderly depressed individuals with cognitive complaints, by combining the measurement of a 1 - 40, a 1 - 38, and phospho - tau . With the use of maldi - tof mass - spectrometry for the study of csf samples from ad patients, an oxidized form of a 1 - 40 quantification by sds - page / western immunoblot revealed elevated a 1 - 40 levels in patients with ad as compared to probable vascular dementia and controls . Taken together, these pilot studies suggest that besides a 1 - 42, additional variants of a peptides may turn out to be specifically altered in ad patients . Although combinations of these csf biomarkers were reported to have a high predictive value in single - center studies, their application in multicenter - studies is hampered by relatively high intercenter variations . In an associated multicenter study, including 750 patients with mci who were followed for at least two years, the conversion to ad could be predicted with a sensitivity of 83% and a specificity of 72% by the ratio of a 1 - 42/phospho - tau and total - tau . As this is probably due to its high potential to form aggregates and to stick to test tubes, alternative markers related to app processing have been investigated within cneupro . In an associated multicenter study, sapp and sapp, two proteins secreted in the csf after the - or -secretase cleavage of app, were assessed in 188 patients with mci or mild to moderate ad . In previous studies, sapp and sapp were found to be unchanged [36, 37] or decreased [3840] in the csf of ad patients . Within cneupro, sapp and sapp levels in csf of mci and ad patients with elevated total - tau and reduced a 1 - 42 csf concentrations were compared to those from patients without a respective csf biomarker profile . Both were found to be higher in the csf from patients with an ad - indicative biomarker profile . Taken together, these results suggest that sapp and sapp may be indicators of altered app expression and/or metabolism . Reports on their value as candidate biomarkers are however so far contradictory . In a different study which was supported by cneupro, six novel n - terminal app - fragments with molecular masses of approximately 12 kda and starting at amino acid 18 of the app sequence were detected in csf by mass spectrometry . In a subsequent small pilot study, six of six ad patients and five of five controls additionally, immuno - ms analysis of csf has led to the detection of eleven novel app fragments, which begin n - terminally to the -secretase cleavage site, and end one amino acid before the proposed -secretase cleavage site (app / a peptides). Interestingly, seven of the twelve app / a peptides were significantly upregulated in ad . One of several kinases that have been suggested to be involved in the abnormal hyperphosphorylation of tau is the map - kinase erk1/2 . In a methodological pilot study, erk 1/2 and its doubly phosphorylated, activated form have been detected in a small number of csf samples from patients with ad, mci, and frontotemporal lobar degeneration (ftld). To evaluate the usefulness of erk 1/2 as a potential novel csf biomarker, erk1/2 levels in csf are currently being studied in a total of 110 csf samples from partners within the consortium with a chemiluminescent 96 well assay format . In accordance with a previous report, research within cneupro found glial fibrillary acidic protein (gfap), a marker for astrogliosis, to be increased in csf of ad and sporadic creutzfeldt - jacob disease (scjd) patients . Csf samples of 18 ad patients, 22 scjd cases, and 18 from nondemented controls were analyzed with the use of a commercially available elisa . In ad, a remarkable elevation in csf gfap levels with no overlap to controls was observed . Although a significant increase in gfap could be observed in cjd as well, this was not as pronounced as in ad . Consequently gfap might have some additive value as part of a biomarker supported diagnosis, although it lacks specificity for ad . Chronic inflammation associated with oxidative and nitrosative stress is another aspect which is considered to be important in the pathophysiology of ad . The most common protein markers of oxidative and nitrosative stress are protein - bound carbonyls and 3-nitrotyrosine . An increased oxidation of certain proteins and an increased concentration of 3-nitrotyrosine have been reported in tissue and csf [5052] of ad patients, but there is also contradictory data indicating no difference between ad and controls . In a study conducted by members of the cneupro consortium, where the concentrations of 3-nitrotyrosine and total protein carbonylation were measured, no change was found in csf of ad patients . Yet, slightly reduced levels of protein carbonyls were detected in apoe-4 carriers as compared to apoe-4 noncarriers . These results suggest that the concentrations of total protein carbonyls and 3-nitrotyrosine are at this stage not suitable to monitor the chronic inflammatory processes related to ad . In addition to promoting the early and predictive diagnosis of ad, cneupro is also dedicated to search for new biomarkers to support the diagnosis of other neurodegenerative diseases such as scjd, ftld, vascular dementia (vad), dementia with lewy bodies (dlb), parkinson's disease (pd), and parkinson's disease dementia (pdd). Two - dimensional differential gel electrophoresis (2d - dige) followed by maldi - tof mass - spectrometry indicated that csf from patients with scjd differed from csf from patients with other neurological deficits on the basis of several protein spots . Among these, several previously identified surrogate markers of scjd such as 14 - 3 - 3 protein, neuron - specific enolase, and lactate dehydrogenase were identified . Additionally, an unidentified protein of 85 kda was found to be significantly increased in scjd patients . In a separate cneupro investigation, seldi - tof mass spectrometry was applied in the analysis of csf from 32 scjd patients, 32 controls, and 31 patients with other dementias . Ubiquitin, an 8.6 kda protein involved in protein degradation, was found to be elevated in the csf of scjd cases ., the accuracy of a biomarker - based classification of the samples could be significantly improved by including ubiquitin in addition to tau, and 14 - 3 - 3 protein . This finding is in accordance with several previous reports where ubiquitin was also found to be elevated in the csf of scjd patients . As there is also evidence for altered levels of csf ubiquitin in ad [5759] and vascular dementia, it seems that this observation is related to neurodegenerative processes in general and not to a specific disease . Yet, in the steinacker study csf ubiquitin levels in scjd were higher than those in other dementias . Therefore, ubiquitin may still be a good biomarker for scjd if, as with tau protein, disease - specific cut - off values are applied . S100b, another astroglial marker, may also be useful to support the diagnosis of scjd . Within cneupro, s100b was measured in 54 csf samples from patients with scjd, ad, and control patients with the use of a commercial elisa . Supporting previous findings [62, 63], s100b was shown to be highly elevated in scjd with no overlap to the other groups . Others have found elevated s100b in familial cjd cases, but also in csf and serum of ad patients . These findings suggest that more attention might be paid to the use of astroglial markers in supporting the differential diagnosis of dementias . With respect to ftld, cneupro found elevated mean levels of the tar dna - binding protein 43 (tdp-43) and reduced a 1 - 42 levels [67, 68]. In line with the reported increased gene expression of tdp-43 in brain tissues, elevated 45 kda tdp-43 levels were found in the csf of 12 patients with ftld as compared to 13 nondemented controls by western - immunoblot . In the same sample, the assessment of different a peptide species, sapp and sapp, by electrochemiluminescence - based multiplex assays indicated no significant difference for sapp and sapp between the groups . However, reduced a 1 - 42 levels were found in ftld . These findings are supported by several earlier studies which found csf - levels of a 1 - 42 in ftld to be lower than in nondemented controls and higher than in ad [7073]. However, there are also contradictory publications, regarding levels of a species which did not find reduced csf a 1 - 42 concentrations in ftld [74, 75]. Although tdp-43 and fragments of app processing are currently not suitable as biomarkers because of a large overlap between the different diagnostic groups, these findings may still reflect aspects relevant for understanding the pathophysiology of these disorders . In an associated study focussed on the biomarker supported differential diagnosis of ad, pd, pdd, and dlb, csf a 1 - 42, total - tau, and phospho - tau were measured in the csf of a total of 80 patients . Although some significant differences in the average biomarker measurements were found between the groups, only ad patients could be effectively differentiated from patients with other dementias by phospho - tau . For a 1 - 42, total - tau, and phospho - tau, a large overlap between the other neurodegenerative diseases was observed . Interestingly, only in dlb were a 1 - 42 and total - tau found to correlate with the duration and the severity of dementia . Consequently, more and better biological markers are needed to support the differential diagnosis of these dementias . In addition to a previous report linking a reduced activity of -glucocerebrosidase to pd, a reduced activity of -glucocerebrosidase was specifically found in dlb within cneupro . In csf from nondemented controls, patients with ad or ftld, in contrast, the activity of -mannosidase, another lysosomal hydrolase, was found to be significantly reduced in all investigated neurodegenerative diseases as compared to controls . In order to support the hypothesis that csf -glucocerebrosidase activity might be a novel csf biomarker of synucleinopathies, the data need to be confirmed in larger studies . Several recent studies aimed at identifying ad biomarkers in blood were specifically targeted at determination of a peptides in blood plasma or serum . Within a cneupro associated substudy of the german kompetenznetz demenzen (http://www.kompetenznetz-demenzen.de/), a 1 - 40 and a 1 - 42 were assessed in blood plasma from 257 individuals with multiplexing technology on the luminex platform . A statistically significant decrease of the a 1 - 42/1 - 40 ratio was found in the plasma of the patients with early ad and mci of ad type whose clinical diagnoses were backed up by corresponding findings in the csf . Moreover, the cneupro associated french three - city study found that a reduction of the ratios a 1 - 42/a 1 - 40 as well as a x-42/a x-40 was associated with an increased risk of developing dementia within the next two years . In contrast, several other published studies have not reported significant differences in a peptide concentrations in blood plasma between ad patients and controls [8284]. In summary, there is no definitive conclusion as to whether plasma a reflects the changing level of central amyloid . Due to the substantial interindividual variations and a large overlap between the diagnostic groups, measuring the individual concentrations of a peptides in plasma is not suitable to support the clinical diagnosis of different dementia disorders . However, there is preliminary evidence that specific forms of a peptides in plasma prove to be helpful in the differential diagnosis of ad and other dementias . In a retrospective pilot study which was supported by cneupro, vascular dementia could be differentiated with a sensitivity and specificity of> 80% from other dementias and depressive controls by the ratio of a 1 - 38/a 1 - 40 . Currently, highly sensitive assays for the detection of a peptides in blood and csf are available for a x-38, a x-40, and a x-42 . For a detailed analysis of additional variants of a peptides in blood plasma, a highly sensitive two - dimensional gel separation method was established within cneupro . Using this method, at least 30 different a peptides were observed . Semiquantitative analysis revealed that the peptides a 1 - 40 and a 1 - 42 accounted for less than 60% of all a peptides that were detected by the specific antibody that was used in this study . At least 10% of the detected a peptides appear to be n - terminally truncated . One possible source of these n - terminally truncated a peptides detected in human plasma is mononuclear phagocytes . Cultures of human mononuclear phagocytes were shown to secrete complex a peptide patterns characterized by a high proportion of n - terminally truncated variants . Furthermore, the secretion of a peptides from human mononuclear phagocytes was differentially regulated in response to cell culture conditions and was elevated in cell cultures of mononuclear phagocytes from ad patients as compared to controls . Additional work is under way to evaluate several n- and c - terminally truncated a peptides in plasma as potential biomarkers for ad . The identification of valid biomarkers in blood is highly desirable because they have the advantage of being easily accessible . The search for potential biomarker candidates in plasma or serum is complicated by the presence of a number of highly abundant proteins . These proteins which are believed to have only small diagnostic potential make up about 90% of the whole plasma proteome . As a first step towards biomarker discovery in serum, it was shown that the depletion of 12 high abundant serum proteins by immuno affinity chromatography columns resulted in an increased number of detected peaks by subsequent analysis with seldi - tof mass spectrometry . In contrast, csf proteomics for biomarker discovery in neurodegenerative diseases is particularly attractive because of the proximity of csf to the brain . Again, the removal of highly abundant proteins resulted in an improved detection of low abundant csf proteins including brain - derived proteins . Additional separation procedures were introduced to account for the large dynamic range of the expression levels and to simplify the analysis of proteolytically generated peptides by mass spectrometry . For a comparative analysis of individual clinical samples and for a relatively in - depth search for potential novel biomarkers, reproducibility is an absolute requirement . Therefore, different multiaffinity depletion methods followed by gel - nanolc - ms / ms and spectral counting have been evaluated for the in - depth, label - free quantitative analysis of csf . Depletion in spin - filter format, coupled to gel - lc - ms / ms, provided a robust method that yielded ~800 csf proteins per analyzed sample, with acceptable reproducibility of protein identification (71%74% in technical replicates) and quantification (17%18% cv on spectral counts). To control for reproducibility, this proteomics approach was subsequently applied in both laboratories to the independent analysis of two separate cohorts of 20 individual csf samples each . In both cohorts the patients were clinically diagnosed, and csf was taken according to the cneupro standard operating procedures . Both discovery sets of samples included csf samples from five control subjects, from five subjects with mild cognitive impairment without conversion to ad, from five patients with mild cognitive impairment with conversion to ad within the follow - up of 2 years, and five patients with ad . Both datasets contained ~1100 identified proteins with a total of ~1600 unique csf proteins in the common dataset and an overlap of ~500 between the two laboratories . The biostatistical analysis is currently on - going to select the most promising candidates for a reassessment by targeted mass spectrometry and antibody - based methods in a larger set of samples . Within the first two years, cneupro confirmed sapp, various a peptide variants, gfap, s100b, and ubiquitin as biomarker candidates known from previous studies . Additionally, further app fragments were discovered and tdp-43 as well as -glucocerebrosidase and erk 1/2 were proposed as potential novel candidate biomarkers for the early and differential diagnosis of neurodegenerative diseases (table 1). Because of the high complexity of the blood proteome and probably because of its distance from brain pathology, novel biomarkers in serum or plasma are still elusive . To promote biomarkers in support of the clinical diagnosis of neuropsychiatric disorders in europe, cneupro devised european standard operating procedures for preanalytical sample handling and established a neurochemical dementia diagnosis reference center in hungary . Cneupro has now started to select the most promising biomarker candidates from two proteomic studies within cneupro and to reassess the most promising biomarker candidates with larger sample size and independent methods to finally integrate them into novel prototype assays . To increase the accuracy of a biomarker - based diagnosis, biomarkers in body - fluids have been combined with other biological markers such as structural and functional neuroimaging and neuropsychological testing . Whether the new biomarker assays which will be developed within cneupro will be useful in such a multimodal diagnostic workup remains to be elucidated.
In bacteria, the number of characterized small, noncoding rnas (srnas) that have intrinsic functions as regulators has steadily increased in recent years . Many srnas act by posttranscriptionally regulating mrnas via basepairing interactions (1). In escherichia coli, all of the srnas that act by basepairing affect either the stability or translation of the mrna target . In most cases, the mrna targets of srna regulation are trans - encoded, and the srna: mrna basepairing interactions are interrupted by gaps in the pairing . Further, most srnas in this class bind to the rna chaperone hfq, which has been shown to facilitate the interaction between many srnas and their targets (2). Here, we describe a program, targetrna, which can effectively predict mrna targets of basepairing srnas . While a number of approaches have been described for identifying targets of microrna genes in eukaryotes (37), there have been relatively few computational tools developed for characterizing targets of srna regulators in bacteria . (8) describe one such tool, which has been applied successfully in the genome of listeria monocytogenes, though the program is not available as a webserver . Vogel and wagner (9) offer a comprehensive review of approaches, both computational and experimental, for identifying targets of bacterial srnas . The program we describe here, targetrna, is accessible via a webserver that has been operating and publicly available since 2005, processing approximately 1000 sequence submissions per month . The webserver consists of a 16 cpu parallel computing cluster, and targetrnas underlying search algorithm has been designed for parallel computation, in order to increase performance and to process efficiently a large number of sequence submissions . The predictive performance of targetrna has been validated experimentally in e. coli using both northern blot and microarray experiments (10). Additionally, predictions from targetrna have been validated in other organisms, such as in vibrio cholerae (11) and in neisseria meningitidis (12), or are consistent with experimentally validated interactions, such as in salmonella (13). Targetrna takes, as input, a genomic sequence that may correspond to a srna gene . Targetrna then searches each annotated message in the user - specified genome for mrnas that evince statistically significant basepair - binding potential to the input srna sequence . Basepair - binding potential of a srna with each mrna is determined using one of two user - selected hybridization scoring methods . A detailed description of the hybridization scoring methods has been described previously (10). In summary, waterman dynamic program (14), except that instead of assessing homology potential, basepairing potential is assessed . The stacked basepair model of hybridization scoring is based on stacking and destabilizing energies of interacting sequences . The stacked basepair model calculates the minimum free energy of hybridization for two rna sequences, without allowing intramolecular basepairings . This model for hybridization scoring closely follows that developed and used for the rnahybrid algorithm (7). The stacked basepair model is computationally more intensive than the individual basepair model and, as a result, increases the time required for calculating basepair binding potential by a factor of approximately five for most sequence inputs . Once targetrna determines hybridization scores for the srna sequence with each message in a genome, the statistical significance of each potential srna: mrna interaction is assessed . Determination of statistical significance is similar to that described for the rnahybrid algorithm (7). Ten thousand random rna sequences are generated such that the nucleotides in the random sequences are drawn from the distribution of nucleotides in the actual mrna search space . The hybridization score is computed for each of these random sequences and the srna sequence . The resulting distribution of ten thousand hybridization scores is used to estimate a p - value for an srna: mrna hybridization score by determining the probability of observing a score, by chance, equal to or less than the given srna: mrna hybridization score . After computing the statistical significance of basepair binding between a srna sequence and each message in a genome, targetrna outputs a ranked list of mrnas in a genome whose basepair - binding potential with the srna sequence meets a significance threshold (the default is a p - value 0.01). Messages with statistically significant basepair - binding potential are considered candidate targets of srna regulation . Figure 1 illustrates example output from targetrna when searching for mrna targets of the srna gene spot42 in e. coli (15). As shown in figure 1, targetrna outputs, for each candidate mrna target identified, an annotation of the mrna, a visual representation of its predicted basepair binding with the srna, and a p - value corresponding to the significance of the hybridization score of the predicted srna: mrna interaction . To facilitate further investigation of candidate message targets, each identified target is linked to the entrez gene database (16) from the national center for biotechnology information . The time required for targetrna to process an input sequence and generate results depends on the length of the sequence and the parameter selection but typically takes only a few seconds . Output from targetrna is available via both a formatted web page and a text file . Figure 1.the figure illustrates example output from using the targetrna webserver with default parameter settings to search for message targets of the srna spot42 in escherichia coli . In the middle of the figure, the six message targets predicted by targetrna are summarized . In the bottom of the figure, the predicted interaction between spot42 and one of the six predicted targets, galk, is illustrated . The predicted interaction between spot42 and galk consists of 41 nucleotides (from nucleotide 21 to 61) in the 109 nucleotide srna spot42, and 39 nucleotides (from 19 nucleotides upstream of the galk start codon to 20 nucleotides downstream in the galk coding sequence) in the galk message . The figure illustrates example output from using the targetrna webserver with default parameter settings to search for message targets of the srna spot42 in escherichia coli . In the middle of the figure, the six message targets predicted by targetrna are summarized . In the bottom of the figure, the predicted interaction between spot42 and one of the six predicted targets, galk, is illustrated . The predicted interaction between spot42 and galk consists of 41 nucleotides (from nucleotide 21 to 61) in the 109 nucleotide srna spot42, and 39 nucleotides (from 19 nucleotides upstream of the galk start codon to 20 nucleotides downstream in the galk coding sequence) in the galk message . The targetrna webserver provides a number of advanced search options that allow users more flexible control over the target search space beyond that provided by the default parameter settings . Targetrna provides the option of automatically identifying and removing the region of the srna sequence corresponding to the terminator stem - loop since, in many cases, the terminator stem - loop does not participate in the srna: mrna interaction . Users have the option of focusing their search around the 5 utr or 3 utr of messages, specifying the number of nucleotides to include upstream and downstream of the messages start codon or stop codon . Specifying regions around the 5 utr to be searched may be advantageous since many documented target interactions occur in particular regions, such as around the ribosome - binding sites, of messages . A seed, which corresponds to a minimum required length for at least one stretch of consecutive basepaired nucleotides in the srna: mrna interaction, can also be specified . The seed is meant to reflect, biologically, the initial interaction between srna and mrna, which has been shown in some cases to be a stretch of unpaired nucleotides in a loop of the srna that first basepairs with the target message . While targetrna searches each annotated message in a genome by default, users have the option of searching an individual message in order to explore more carefully a particular interaction predicted by targetrna . When searching for targets of a srna in a given organism, targetrna also offers the option of calculating the hybridization scores of orthologous targets with orthologous srnas in other organisms . Since many srna genes are conserved across related species, the program can thus suggest whether it is likely that the targets and hybridization interactions are conserved . The various user - adjustable program parameters have the benefit of allowing a user to explore the tradeoff between sensitivity and specificity when assessing results of the targetrna program . For instance, removing the srna terminator sequence, focusing searches for targets to regions around translation start sites of messages, and setting a seed threshold above about five nucleotides can eliminate many false positive predictions . However, too much stringency with these parameters may result in a lack of identification of true targets . For example, some srna: mrna interactions include the terminator sequence of the srna, such as the oxys: fhla interaction (17,18) in e. coli, and some srnas interact with messages somewhat distant from the message's start of translation, such as dsra (19) and rpra (20) when interacting with rpos in e. coli . Thus, the default parameter settings provide only a starting point for message target investigation . Default parameter settings were determined by optimizing performance of targetrna on srna: mrna interactions in e. coli reported prior to 2005 . Considering the recent growth in the number of srna: mrna interactions reported in the literature across a range of bacterial species, we have revisited the issue of whether different default parameter settings would improve the performance of targetrna . When accounting for a broad set of srna: mrna interactions in different species, we did not find a set of parameters that led to significant improvement, over the current default parameter settings, in successfully identifying targets of srna regulation . A recent addition to the targetrna webserver is a companion program to targetrna called rnatarget, which provides reverse searching capabilities . Whereas targetrna takes as input the sequence of a candidate srna region and searches the genome for possible message targets, rnatarget takes as input the sequence of a candidate message target of an unknown srna regulator and searches the genome for regions containing possible srna regulators of the target sequence . Rnatarget searches all intergenic sequences greater than 50 nucleotides in length in the genome for regions that evince significant basepair - binding potential to the input candidate target sequence . Rnatarget outputs a ranked list of intergenic regions in the genome whose basepair - binding potential with the target sequence meets a significance threshold (the default is a p - value 0.01). Intergenic regions with statistically significant basepair - binding potential are considered as candidate regions corresponding to a srna regulator . Figure 2 illustrates example output from rnatarget when searching intergenic regions for candidate srna regulators of the mrna target galk in e. coli . It has been reported previously that the srna spot42, which resides in the intergenic region of the genome between genes pola and yiha, interacts with and regulates galk (21). As shown in figure 2, rnatarget outputs, for each intergenic region identified, the flanking protein - coding genes, a visual representation of the predicted basepair binding between the two genomic sequences, and a p - value corresponding to the significance of the hybridization score of the predicted mrna: srna interaction . It is worth noting that rnatarget searches only for genomic regions demonstrating basepair - binding potential to the target sequence, it does not attempt to identify other properties of genomic sequences suggestive of srna genes, such as transcription initiation or termination sequences . Several other computational programs are available for predicting srna genes in a genome based on various sources of evidence, including transcription signals and comparative genomics information (22). Figure 2.the figure illustrates example output from using the rnatarget program with default parameter settings to search for intergenic regions in e. coli that evince basepair binding potential with a region of the galk message around its ribosome binding site . (b) details of the predicted interaction between galk and one of the intergenic regions, between genes pola and yiha, is shown . The srna spot42, which resides in this intergenic region, is known to interact with and regulate galk (21). The predicted interaction between the intergenic region and galk consists of 41 nucleotides (from nucleotide 167 to 207) in the 380 nucleotide pola yiha intergenic region, and 39 nucleotides (from 19 nucleotides upstream of the galk start codon to 20 nucleotides downstream in the galk coding sequence) in the galk message . The figure illustrates example output from using the rnatarget program with default parameter settings to search for intergenic regions in e. coli that evince basepair binding potential with a region of the galk message around its ribosome binding site . (b) details of the predicted interaction between galk and one of the intergenic regions, between genes pola and yiha, is shown . The srna spot42, which resides in this intergenic region, is known to interact with and regulate galk (21). The predicted interaction between the intergenic region and galk consists of 41 nucleotides (from nucleotide 167 to 207) in the 380 nucleotide pola yiha intergenic region, and 39 nucleotides (from 19 nucleotides upstream of the galk start codon to 20 nucleotides downstream in the galk coding sequence) in the galk message . Targetrna is a freely available webserver that predicts message targets of srna action in bacteria . Assessment of targetrna's performance suggests that the predictive performance of targetrna varies between srnas . For some srna regulators where one or more targets have been reported in the literature, targetrna successfully identifies the majority of targets, whereas for other srna regulators with one or more targets, targetrna identifies few, if any, targets (10). Targetrna operates under the assumption that the basepair - binding potential of two genomic sequences, corresponding to an srna and a mrna target, can serve as a predictor, albeit imperfect, for the interaction of the two rnas . Conserved basepair - binding potential of two genomic sequences across different genomes can provide further evidence for srna: mrna interaction . However, other factors that may contribute to srna: mrna interactions, such as rna secondary structure or the role of hfq, are not modeled by the program . As more examples of srna: mrna interactions are reported across a range of bacteria, we will gain a better understanding of the various rna properties and components within the cell that contribute to the interactions, and computational methods that aid in investigation of these interactions can be evolved appropriately to incorporate the new insights.
The prevalence of aggression is 8 - 20% in three- to six - year - old children . Biological and genetic factors, environmental learning, cognitive processing and personal stimuli or motives are some important causes of aggression . Treatment of aggressive behavior at an early age is very important, as aggression in early years of life sets the ground for many problems in personal and interpersonal areas of the lives of aggressive children . For instance, it can lead to a weak self - concept, being rejected by peers, poor academic performance and can serve as a predictor of delinquency, depression, academic failure, substance abuse, more inappropriate and aggressive reaction to social issues, and choosing more aggressive solutions for solving problems later in life . On the other hand, childhood exposure to aggression may also influence life - long health through biological mechanisms . Also, the economic costs imposed on the community due to youth aggression, bullying and violence are extremely high . Yet, social - learning theorists contend that environmental factors are responsible for learning aggressive behaviors and continuing them . Therefore, the children who learn aggressive behaviors are able to avoid such behaviors by means of reinforcement behavior therapy . Reinforcement behavior therapy is defined as the employment of reinforcement and extinction in order to increase the occurrence of desirable behaviors and decrease the incidence of behaviors that interfere with intended behavior . (2011) compared the effectiveness of two methods, namely reinforcement and reward behavioral therapy and ellis s cognitive therapy, in the degree of aggression among 89 parentless children . The reinforcement and reward behavioral therapy method led to a significant reduction in aggression mean score before and after applying the procedure, whereas ellis s cognitive therapy technique did not show any significant impact on lowering aggression . The study of khazaie and colleagues did suggest the effectiveness of behavioral therapy in aggression; however, it was carried out by a psychotherapist . In many countries, factors, such as insufficient number of professional psychotherapists or expenses of the procedure, therefore, training kindergarten and day - care centers teachers can be an appropriate alternative for offering proper interventions under the supervision of a therapist, so that a greater number of preschool children can benefit from such procedures . School environment interventions that impact on a range of health risk behaviors, including aggression, are likely to be one of the most efficient ways of modifying population - level risk . Schools approaches to discipline, behavior management and aggression prevention vary widely and are rarely evidence - based, and that further resources and research are urgently needed to combat aggressive behaviors . Although a large number of studies have so far been conducted on the effectiveness of complimentary therapies in reducing children s aggression, most of them have fundamental limitations . Some such limitations include a greater focus on aggressive boys than girls, more emphasis on overt forms of aggression in comparison to more subtle forms like relational aggression . Another treatment alternative is family therapy which, although very effective in children s behavioral problems, is quite costly and time - consuming . There is, therefore, a pressing need to determine which interventions are effective in addressing aggression in preschoolers, and if the intervention can apply effectively by kindergarten teachers to scale up such interventions across local and national school networks especially in developing countries . Since children spend a great part of their active hours in kindergarten in close contact with their teachers, training the teachers can make a significant contribution to the elimination of aggressive behaviors . Moreover, given the fact that each teacher is in charge of several children, providing teachers with trainings will result in saving the time and cost . In addition, the existing studies have addressed almost all forms of aggression except for impulsive anger . Therefore, the present study has focused on all forms of aggression and aims to identify the effect of reinforcement behavior therapy by kindergarten teachers on all types of aggressive behavior among kindergarten and preschool children . It was a cluster randomized control trial research with a pretest / post - test design and control group . The study compared two groups of aggressive children, the intervention group receiving reinforcement behavior therapy and routine care by kindergarten teachers and the control group only receiving routine care . Mohr is located in iran, in the southernmost part of fars province, 360 km away from shiraz . Currently, there are 8 urban and 27 rural kindergartens and daycare centers in this city and about 1000 children attend these centers . The research population consisted of all three- to six - year - old kindergarten and preschool children in mohr . The inclusion criteria of the study were parental consent, children s age range of 3 - 6 years, and obtaining a minimum aggression score of 117.48 for girls and 125.77 for boys (according to preschoolers aggression questionnaire constructed by shahram vahedi et al . On the other hand, the children or families with emotional crisis during the 2 months of intervention 14 out of the 35 existing kindergartens were selected through cluster random sampling method, as recommended by a demographer . Then, all the 14 kindergartens were randomly allocated into either the intervention (n=7) or the control group (n=7) through block randomization by the researcher s assistant . The preschool aggression scale was distributed among the teachers to complete it for all 3 - 6 year old children in 14 kindergartens . Using a study entitled prevalence of behavioral problems in 3 to 6 year old children in hamadan city and based on effect size=0.95, power of 0.8, and =0.05, a 54 subject sample size (27 subjects in each group) was determined for the present study . Considering the probable loss in the sample, more than 117.48 for girls were randomly selected by simple randomization procedure by a table of random numbers from the list of aggressive children and their parents were asked to complete the written informed consent forms . The teachers of the kindergartens (no=14) in the intervention group received 4 educational sessions based on positive reinforcement behavior therapy, but the teachers of kindergartens in the control group did not receive any interventions during the research period . Each teacher in the intervention and control groups had about 15 children in his / her class . During the study, consort flow diagram of study participants in this study, the data were collected using preschoolers aggression questionnaire . This questionnaire aimed to evaluate various types of aggression, including verbal aggression, physical aggression, relational aggression, and impulsive anger . The reliability and validity of preschool aggression scale was studied by shahram vahedi et al . (2007) in a research on evaluation of aggression in preschool children of urmia, iran . The cronbach s alpha was estimated to be 0.98 for the whole scale, 0.93 for verbal aggression, 0.92 for physical aggression, 0.94 for relational aggression, and 0.88 for impulsive anger . A factor analysis of this scale using principal component analysis with varimax rotation resulted in four elements of verbal aggression, physical aggression, relational aggression, and impulsive anger, which was indicative of the construct validity of the scale . Therefore, this aggression measurement scale can be used as a valid and reliable instrument in educational and clinical settings . In this 43-item questionnaire, the first 14 questions are related to verbal aggression and the next 13 are about physical aggression; also, there are 9 questions on relational aggression, and the last 7 items deal with impulsive anger . This questionnaire was filled out by the teacher using 5 options (0=never, 1=rarely, 2=once a month, 3=once a week, and 4=often). The scores of verbal aggression, physical aggression, relational aggression, and impulsive anger were between 0 and 56, 0 and 52, 0 and 36, and 0 and 28, respectively . The children whose aggression scores were two standard deviations above the mean (117.47 for girls and four training sessions, each lasting for two hours, were planned for the teachers in the intervention group who were ready to take part in the study by an interventionist who was unaware of the aim of the study and was professional in behavior therapy . In the first session, the teachers were given some information about mental, psychological, and physical characteristics of the preschool children, and gained an understanding of how to deal with them properly . In the second session, the teachers learned about aggression in children, its types, causes, importance of early treatment of aggressive children, and general treatment techniques and methods . The third session intended to make teachers familiar with behavioral therapy and its various techniques, with a focus on reinforcement behavior therapy . The fourth and final session was entitled practical work on reinforcement and reward behavior therapy, and aimed to demonstrate how this technique works in practice, how to write down the details, and how to prepare an anecdotal record . The teachers were also given a written and practical test on proper practice of the technique . Then, the teachers were asked to practice what they had already learned during their four sessions of training for aggressive children in the intervention group for 8 weeks . They were also asked to complete anecdotal record for every aggressive event in children, so that the researcher could follow up any changes in the aggressive children as well as the way the teachers implemented the procedure . In these forms, the teachers were asked to describe their reactions towards the children s aggressive behaviors in details and free from any judgment . Then, in his weekly visits to the kindergartens, the researcher would review these forms and discuss them together with teachers . Afterwards, if necessary, the teacher s reactions were modified and the required instructions were given . The control group, however, received no interventions throughout the course of research . Yet, the demographic information questionnaire and the preschoolers aggression questionnaire were filled out by the teachers . After this project was carried out for two months, the teachers were asked to complete the same preschoolers aggression questionnaire for the children in both intervention and control groups for the second time and the results were evaluated by the researcher . The outcome measures in this study included the children s scores in the preschoolers aggression . The study data were collected by kindergarten teachers before and after the eighth week of the intervention . The teachers completed a demographic information questionnaire and the preschoolers aggression questionnaire . In this study, the data were analyzed using statistical package for the social sciences (version 15, spss inc, chicago, il). To evaluate the homogeneity of the participants characteristics in the intervention and control groups, chi - square smirnov test, paired t test and student s t - test were used for statistical analysis, as well . Besides, p<0.05 was considered as statistically significant . The study was conducted in accordance with the human subjects protection principles (declaration of helsinki). Ethics committee approval was obtained from the research ethics committee of shiraz university of medical sciences . A written informed consent was obtained from the teachers and family of children for participation in the study . It provided the subjects with some information about the study, such as the purpose, e procedure, possibility of sharing the study results after completion, and promised anonymity in the event of publication of the study results . In addition, the subjects were assured that participation / non - participation would not affect their received care . The participants were allowed to withdraw from the research at any stage of the process if they or their parents were unwilling to continue their cooperation . Moreover, after completion of the study, a handbook was prepared and given to the teachers and parents of the control group children . Mohr is located in iran, in the southernmost part of fars province, 360 km away from shiraz . Currently, there are 8 urban and 27 rural kindergartens and daycare centers in this city and about 1000 children attend these centers . The research population consisted of all three- to six - year - old kindergarten and preschool children in mohr . The inclusion criteria of the study were parental consent, children s age range of 3 - 6 years, and obtaining a minimum aggression score of 117.48 for girls and 125.77 for boys (according to preschoolers aggression questionnaire constructed by shahram vahedi et al . On the other hand, the children or families with emotional crisis during the 2 months of intervention 14 out of the 35 existing kindergartens were selected through cluster random sampling method, as recommended by a demographer . Then, all the 14 kindergartens were randomly allocated into either the intervention (n=7) or the control group (n=7) through block randomization by the researcher s assistant . The preschool aggression scale was distributed among the teachers to complete it for all 3 - 6 year old children in 14 kindergartens . Using a study entitled prevalence of behavioral problems in 3 to 6 year old children in hamadan city and based on effect size=0.95, power of 0.8, and =0.05, a 54 subject sample size (27 subjects in each group) was determined for the present study . Considering the probable loss in the sample, more than 117.48 for girls were randomly selected by simple randomization procedure by a table of random numbers from the list of aggressive children and their parents were asked to complete the written informed consent forms . The teachers of the kindergartens (no=14) in the intervention group received 4 educational sessions based on positive reinforcement behavior therapy, but the teachers of kindergartens in the control group did not receive any interventions during the research period . Each teacher in the intervention and control groups had about 15 children in his / her class . During the study, this questionnaire aimed to evaluate various types of aggression, including verbal aggression, physical aggression, relational aggression, and impulsive anger . The reliability and validity of preschool aggression scale was studied by shahram vahedi et al . (2007) in a research on evaluation of aggression in preschool children of urmia, iran . The cronbach s alpha was estimated to be 0.98 for the whole scale, 0.93 for verbal aggression, 0.92 for physical aggression, 0.94 for relational aggression, and 0.88 for impulsive anger . A factor analysis of this scale using principal component analysis with varimax rotation resulted in four elements of verbal aggression, physical aggression, relational aggression, and impulsive anger, which was indicative of the construct validity of the scale . Therefore, this aggression measurement scale can be used as a valid and reliable instrument in educational and clinical settings . In this 43-item questionnaire, the first 14 questions are related to verbal aggression and the next 13 are about physical aggression; also, there are 9 questions on relational aggression, and the last 7 items deal with impulsive anger . This questionnaire was filled out by the teacher using 5 options (0=never, 1=rarely, 2=once a month, 3=once a week, and 4=often). The scores of verbal aggression, physical aggression, relational aggression, and impulsive anger were between 0 and 56, 0 and 52, 0 and 36, and 0 and 28, respectively . The children whose aggression scores were two standard deviations above the mean (117.47 for girls and 125.77 for boys) were diagnosed as aggressive . Four training sessions, each lasting for two hours, were planned for the teachers in the intervention group who were ready to take part in the study by an interventionist who was unaware of the aim of the study and was professional in behavior therapy . In the first session, the teachers were given some information about mental, psychological, and physical characteristics of the preschool children, and gained an understanding of how to deal with them properly . In the second session, the teachers learned about aggression in children, its types, causes, importance of early treatment of aggressive children, and general treatment techniques and methods . The third session intended to make teachers familiar with behavioral therapy and its various techniques, with a focus on reinforcement behavior therapy . The fourth and final session was entitled practical work on reinforcement and reward behavior therapy, and aimed to demonstrate how this technique works in practice, how to write down the details, and how to prepare an anecdotal record . The teachers were also given a written and practical test on proper practice of the technique . Then, the teachers were asked to practice what they had already learned during their four sessions of training for aggressive children in the intervention group for 8 weeks . They were also asked to complete anecdotal record for every aggressive event in children, so that the researcher could follow up any changes in the aggressive children as well as the way the teachers implemented the procedure . In these forms, the teachers were asked to describe their reactions towards the children s aggressive behaviors in details and free from any judgment . Then, in his weekly visits to the kindergartens, the researcher would review these forms and discuss them together with teachers . Afterwards, if necessary, the teacher s reactions were modified and the required instructions were given . The control group, however, received no interventions throughout the course of research . Yet, the demographic information questionnaire and the preschoolers aggression questionnaire were filled out by the teachers . After this project was carried out for two months, the teachers were asked to complete the same preschoolers aggression questionnaire for the children in both intervention and control groups for the second time and the results were evaluated by the researcher . The outcome measures in this study included the children s scores in the preschoolers aggression . The study data were collected by kindergarten teachers before and after the eighth week of the intervention . The teachers completed a demographic information questionnaire and the preschoolers aggression questionnaire . In this study, the data were analyzed using statistical package for the social sciences (version 15, spss inc, chicago, il). To evaluate the homogeneity of the participants characteristics in the intervention and control groups, chi - square, fisher exact test and student s t test were applied . Smirnov test, paired t test and student s t - test were used for statistical analysis, as well . Besides, p<0.05 was considered as statistically significant . The study was conducted in accordance with the human subjects protection principles (declaration of helsinki). Ethics committee approval was obtained from the research ethics committee of shiraz university of medical sciences . A written informed consent was obtained from the teachers and family of children for participation in the study . It provided the subjects with some information about the study, such as the purpose, e procedure, possibility of sharing the study results after completion, and promised anonymity in the event of publication of the study results . In addition, the subjects were assured that participation / non - participation would not affect their received care . The participants were allowed to withdraw from the research at any stage of the process if they or their parents were unwilling to continue their cooperation . Moreover, after completion of the study, a handbook was prepared and given to the teachers and parents of the control group children . The two groups were compared in terms of the children s age, both parents age, both parents level of education, both parents occupation, and family income; they were similar regarding all the features, except for father s age . The means (sd) of aggression score in boys and girls were compared and shown in table 2 . All of the teachers were female between 20 - 35 years old and most of them had a bs degree (n=20, 72%); 28% of them had high school diploma level of education (n=8). Comparison of the demographic characteristics in the control and intervention groups meansd scores of aggression among boys and girls in both groups moreover, no significant difference was found between the two groups regarding the total aggression score (p=0.55) as well as the scores of the four subscales prior to the intervention (table 4) the mean score of aggression after the intervention showed a statistically significant difference between the two groups in this regard (p=0.01) (table 3). Moreover, a statistically significant change was observed in the intervention group s mean score of aggression after the intervention (p<0.02); also, such a difference was found in the control group (p<0.023), but the results showed that the mean score of aggression in the control group was increased in the post - test compared to the pre - test . Furthermore, the results revealed a decrease in the mean scores of verbal aggression, physical aggression, relational aggression, and impulsive anger in the intervention group after the intervention . Yet, the difference between the intervention and control groups was statistically significant only for physical and verbal aggression subscales (p=0.02, p<0.01) (table 4). Comparison of the frequency distribution of the participants in the intervention and control groups based on demographic characteristics x = chi - square; f = fisher exact test comparison of the changes in the mean scores of various types of aggression in the preschoolers in the intervention and control groups before and after the intervention the present study aimed to investigate the effect of training kindergarten teachers regarding reinforcement behavior therapy on reducing different forms of aggression in preschool children . The study findings indicated that using reinforcement behavior therapy by kindergarten teachers resulted in a significant decrease in the total aggression, verbal and physical aggression, scores in the intervention group compared to the control group . It did alleviate the children s covert aggression, including relational aggression and impulsive anger, as well, but this change was not statistically significant . This finding supported the results of a study that indicated the effectiveness of reinforcement behavior therapy on abatement of childhood aggression . Since children spend a long time at kindergartens in close contact with their teachers, it is possible to reform, or even eliminate, many behaviors that are possibly shaped at home . In this regard, a meta - analysis was performed by smeet et al, (2014), on twenty - five studies to identify predictors of treatment response regarding cbt . Furthermore, the treatment setting and duration did not seem to influence the treatment effect, which shows the need for development of more cost - effective and less - invasive interventions . There are limited studies that show the effect of behavior therapy by kindergarten teachers but to support the effectiveness of behavior therapy on the children s aggression, both verbal and physical, several studies have been conducted; their results are all consistent with the present research . Verbally and physically aggressive children can be detected more easily . Since preventing this form of aggression parents are more sensitive and try to rectify the child s behavior and even seek help and advice from teachers . The children who display relational aggression are less responsive to psychological treatments because of the nature of relational aggression compared to other types and the deeper mechanisms involved in it . Relationally, aggressive children were popular among their peers, causing short - term treatments not to have noticeable effects . The place these children gain among their peers because of their behavior makes them more resistant to medium - term psychological treatments . The results of the present study demonstrated a decrease in impulsive anger after the intervention; however, this reduction was not statistically significant . This can be due to the fact that impulsive anger is a covert form of aggression and, consequently, it is sometimes not considered as aggression at all . (2012) showed that group therapy using parent - child relationship also did not have any significant effects on covert aggression . Utilizing a primary, secondary and tertiary intervention model from the public health perspective can help the organization to address violence in each of these three domains . In the absence of school nurses in kindergartens, the teachers are the best choice for working with aggressive children . In this regard, the psychiatric mental health nurses in their community based approach can arrange educational programs for teachers to facilitate and improve their ability in providing psychiatric interventions such as reinforcement behavior therapy . Short duration of treatment, small sample size, all female teachers, potential contamination across schools and the fact that evaluation of children s aggression at home is not possible were some major limitations of the present study . Filling out the questionnaire by teachers can affect the result . In conclusion, the results of this research emphasized the effectiveness of reinforcement behavior therapy in reducing physical and verbal aggression in preschool children . Nevertheless, it was shown that this technique was not sufficient to alleviate relational aggression and impulsive anger . This reminds us of the necessity for continuation of the treatment or designing another type of intervention which is more appropriate for these types of behavioral problems in children . In this study, therefore, using other psychological interventions in aggressive children is suggested to be used in future studies . Studying the effect of positive reinforcement therapy on aggression and its sub - scales for more than 8 weeks is also recommended . Moreover, qualitative studies are recommended to be performed on aggression in preschoolers in future . To improve evidence - based nursing, further studies on the impact of this intervention
In october 2003, a man with a history of noninjection multiple - drug abuse (including methamphetamine) was admitted to a detention center (regional jail). His pre - incarceration history included unprotected sex with men in the community, most recently in april 2003; however, results of multiple serologic assays for hiv performed in the community had been negative, most recently in june 2003 and december 2002 . He had an unremarkable past medical history . On december 31, 2003, this patient came to the correctional facility's medical clinic with perianal and rectal discomfort; perianal condylomata were present . He reported that during december he had consensual, unprotected, receptive anal intercourse with 2 male inmates at the correctional facility; both of these inmates had chronic hiv infection . One, who was not receiving antiretroviral treatment, had a plasma hiv rna level of 53,000 copies / ml; the second, who was receiving antiretroviral treatment, had a plasma hiv rna level of 92 copies / ml . On january 6, enzyme immunoassay (eia) testing of the index patient for hiv was negative . On january 9, the patient came to the medical clinic with fever, sore throat, myalgia, headache, vertigo, nausea, and vomiting . Posterior pharyngeal erythema was present, as well as tender, minimally enlarged, anterior cervical lymphadenopathy . Laboratory testing showed plasma hiv rna of 436,000 copies / ml; cd4 + t - lymphocyte count of 616 cells/l (28%); serum alkaline phosphatase 183 iu / l; and negative serologic test results for hepatitis a, b, and c viruses . He denied participating in tattooing or injection drug use . On january 13, he reported vertigo and urinary retention; his temperature was 39.4c, and he was ataxic ., physical examination showed bilateral horizontal nystagmus and perianal ulcerations; plasma hiv rna was> 750,000 copies / ml . On january 15, a second eia for hiv was negative; however, a western blot of that serum sample showed an equivocal p24 band . On january 16, he reported difficulty defecating and urinating . Physical examination showed a scattered macular exanthem of discrete erythematous macules on the trunk and extremities with involvement of the palms; oral mucositis; a tender prostate; and a friable, mildly inflamed anal mucosa with some ulcerations and excoriations . Swab cultures of the rectum for herpesvirus, chlamydia, and neisseria gonorrhoeae were negative . A cd4 + t - lymphocyte count drawn on january 15 showed 338 cells/l (26%); plasma hiv rna level was 234,000 copies / ml . His exanthem had become maculopapular, diffuse, and pruritic . Antiretroviral therapy with efavirenz, zidovudine, and lamivudine was initiated at the correctional facility to treat his primary hiv infection . A genotype of his pretreatment hiv isolate collected on january 14 showed sensitivity to all antiretroviral agents . On january 21, he complained of paresthesias involving the tips of his fingers . On january 22, he was able to urinate spontaneously . On january 30, he reported a poor appetite; his weight had decreased 1.4 kg since january 16; physical examination showed a resolving, salmon - colored macular exanthem across the trunk and upper and lower extremities and resolution of his oral and anal lesions . On february 4, his plasma hiv rna was 2,463 copies / ml, and his cd4 + t - lymphocyte count was 1,575 cells/l . An eia for hiv was positive, and a western blot was positive with bands present for p24, gp40, p55, and gpl60 . On february 9, he reported that his appetite had recovered and his paresthesias had resolved; his weight had increased 0.5 kg since january 30 . On march 19, his plasma hiv rna was <50 copies / ml, and his cd4 + t - lymphocyte count was 1,056 cells/l . On april 1, the court ordered that he be released, and he moved to another state . (4) reported concerns that a resurgence of hiv / aids may be imminent, fueled in part by increasing indicators of high - risk behavior in the gay and bisexual population . (5) regarding men who have sex with men, use of methamphetamine, and transmission of hiv underscores these concerns . The high prevalence of hiv infection in overcrowded and understaffed correctional facilities further accentuates these concerns and poses a public health challenge . On december 31, 2002, 2.0% of state prison inmates were positive for hiv (1); among interviewed jail inmates, 1.3% disclosed they were hiv positive . Estimates of the proportion of inmates who indulge in homosexual intercourse while in prison range from 2% to 65%, and most of this sexual contact is likely unsafe because few correctional facilities address the issue of intraprison sex or distribute condoms (2). (6) proposed that the paucity of evidence for transmission of hiv infection within correctional facilities is probably accounted for by the difficulties in determining the time of hiv seroconversion in relation to the period of incarceration, rather than by the rarity of the event . Krebs and simmons (2) used surveillance data from a 22-year period (january 1, 1978january 1, 2000) to identify inmates who contracted hiv while incarcerated in the florida state prison system . They reported that a minimum of 33 inmates contracted hiv while in prison, compared to 238 who contracted hiv after leaving prison; inmates were more likely to have contracted hiv in prison by having sex with other men than through injection drug use . Additional reports of hiv transmission in correctional facilities have been published from illinois (8 hiv seroconversions) (7), nevada (2 seroconversions) (8), maryland (2 seroconversions) (9), australia (1 seroconversion) (10), and scotland (11). (11) determined that 13 inmates had acquired hiv infection by sharing needles during their incarceration . Acute retroviral syndrome and primary hiv infection may be frequently unsuspected by the evaluating clinician because the signs and symptoms are relatively nonspecific . However, within correctional facilities, the diagnosis of primary hiv infection should be considered in the differential diagnosis of any inmate with an acute febrile illness associated with pharyngitis and mucocutaneous lesions . Our report is limited in that virus was not sequenced to document transmission between inmates . Early diagnosis of primary hiv infection can lead to successful antiretroviral intervention (12) and prevention of secondary transmission . Whether antiretroviral treatment of acute hiv infection results in long - term virologic, immunologic, or clinical benefit is unknown . In october 2005, the us department of health and human services clinical practices panel noted that antiretroviral treatment of acute hiv infection is optional . If the clinician and patient elect to treat acute hiv infection with antiretroviral therapy, treatment should be implemented with the goal of suppressing plasma hiv rna to below detectable levels; resistance testing at baseline will likely optimize virologic response (13). We urge correctional facilities to address the issue of unprotected sex among inmates and the associated transmission of sexually transmitted diseases within institutions (14). In 2001, wolfe et al . (14) reported that from 1991 to 1999,> 5 outbreaks of syphilis occurred in alabama prisons; multiple concurrent sex networks involving 4, 7, and 10 inmates were identified in the 1999 outbreak . Wolfe et al . Recommended that condom distribution should be used to control sexually transmitted disease in correctional facilities . Nevertheless, in 2006, <1% of us correctional facilities provide inmates with condoms . Reasons for not providing condoms include the conflict with policies forbidding sexual intercourse (or sodomy) and the potential for condoms to be used as weapons or to smuggle contraband (15). In contrast, condoms are available to inmates in all canadian federal prisons and some provincial prisons; few problems related to condom distribution have been reported from those systems (15). Wolfe et al . Proposed that providing condoms to prisoners may yield additional public health advantages beyond the prison walls if exposure to and experience with condoms in this setting translate into increased use after release . Correctional staff and inmates should be educated about the consequences of unprotected sex and the signs and symptoms of acute retroviral syndrome . Because many correctional systems contract for medical care, and because staff turnover rates are high, annual education should be implemented . Education for staff who screen sick inmates is critical (14), and all inmates should have access to hiv counseling and testing.
A 51-year - old pakistani man presented to the emergency department with a painfully swollen right lower limb . His medical history revealed insulin - dependent diabetes mellitus and a recent hospitalization in pakistan with a crush trauma of the right foot . Apparently the patient had left the hospital without specific treatment and had flown directly to belgium . On admission, his blood pressure was 130/88 mmhg, heart rate 105 beats / minute, respiratory rate 26 breaths / minute, tympanic temperature 35.5c, and oxygen saturation 98% . Relevant laboratory results were as follows: c - reactive protein 348 mg / l (normal <5), white blood cell count 28.4 10 cells / mm with 88.5% neutrophils and> 10% band forms, urea 276 mg / dl, and creatinine 2.39 mg / dl . An arterial blood gas without supplemental oxygen showed ph 7.49, partial pressure of carbon dioxide in the blood 26 mmhg, partial pressure of oxygen in the blood 98 mmhg, base excess 3 meq / l, and a lactate level of 2.4 mmol / l . Physical examination was unremarkable except for extensive right foot necrosis with fetid purulent discharge and lymphadenitis . Right lower leg amputation was performed and the patient was transferred postoperatively to the intensive care unit (icu). Antibiotic treatment at that time consisted of ciprofloxacin (400 mg every 8 hours [q8h]) and clindamycin (600 mg q8h). Wound cultures grew methicillin - resistant staphylococcus aureus, -hemolytic streptococcus, bacteroides fragilis, morganella morganii, and new delhi metallo--lactamase-1 escherichia coli . The latter was susceptible only to colistin (minimum inhibitory concentration [mic] 0.125 g / ml) and tigecycline (mic 0.5 g / ml). Therapy was changed to meropenem (1 g q8h), vancomycin (continuous infusion over 24 hours, target plateau concentration 2530 g / ml), and colistin (3 million units [miu] q8h). Due to insufficiently controlled infection of the amputation stump, sepsis persisted and on day 4 after admission the right upper limb was amputated . Tigecyclin (100 mg loading, followed by 50 mg q12h) was added to the antibiotic regimen . Antibiotic treatment was continued, and intensive local wound control with daily local debridement was provided . On day 15, surgical site infection and sepsis recurred, necessitating broad wound surgical debridement under general anesthesia . Antibiotics were left unchanged except for a dose reduction of meropenem (2 g q12h) because of a slight postoperative plasma creatinine increase (figure 1). In the late afternoon of day 18, lumbar puncture yielded crystal - clear cerebrospinal fluid with normal cellular, protein, and glucose content . The patient experienced increasing difficulty breathing and suddenly developed apnea, necessitating urgent endotracheal intubation . Although highly probable, the diagnosis of apnea upon colistin intoxication cannot be definitively confirmed . Continuous venovenous hemofiltration (cvvh) was set up with an an69 st (surface treated) membrane known as a very adsorptive filter (gambro, lund, sweden). Cvvh was done exclusively in a hemofiltration mode at a dose of 35 ml / kg / h (gambro - prismafex, lund, sweden). Blood flow was set at 200 ml / min, and predilution and postdilution were, respectively, 65% and 35% . The surface area was 1.5 m. colistin concentrations in plasma and ultrafiltrate were determined simultaneously before the start of cvvh and at 1, 3, 5, 7, 11, and 15 hours thereafter . The initial plasma colistin concentration of 8.06 g / ml (peak level) fell within 12 hours to below 1 g / ml (figure 2, dotted line). The maximal colistin concentration (0.46 g / ml) in the ultrafiltrate was attained only after 3 hours (figure 2, plain line). The patient was completely anuric at this time, and thus colistin could be eliminated only by cvvh . It is administered parenterally as the prodrug colistimethate sodium, a fraction of which is hydrolyzed in vivo to colistin . Colistin causes rapid bacterial killing in a concentration - dependent manner . Following intravenous administration, the drug is mainly renally excreted, with 40% of the dose recovered in the urine within 8 hours . Colistin use has always been hampered by the occurrence of renal toxicity and, to a lesser extent, neurological adverse effects . New and less toxic antibiotics with a comparable or broader antibacterial spectrum progressively supplanted colistin during the 1970s . However, the mounting prevalence worldwide of infections due to multidrug - resistant gram - negative bacilli has renewed interest in this antimicrobial but has also revived the discussion about its toxicity.1 the interaction of colistin with neurons that have a high lipid content has been associated with neurotoxicity, including peripheral, oral, and facial paresthesias, vertigo, visual disturbances, hallucinations, mental confusion, ataxia, and seizures . The most dreaded neurotoxic event, however, is neuromuscular blockade presenting either as a myasthenia - like syndrome or as respiratory muscle paralysis producing apnea . Potential triggers of neurotoxicity are hypoxia, impaired renal function, and concomitant medication (muscle relaxants, narcotics, sedatives, anesthetic drugs, and corticosteroids).2 the incidence of colistin - associated neurotoxicity reported in the literature before 1975 was approximately 7%, with paresthesias constituting the main event . Only sporadic cases of apnea were reported, typically in patients receiving colistin intramuscularly, suffering either acute or chronic renal failure, or treated with medications known to potentially induce respiratory muscle weakness.2 more recent studies all retrospective in design did not observe a clear association between colistin treatment and neurotoxic events . However, three patients already had neurological symptoms before colistin was started, and in the one remaining patient polyneuropathic symptoms subsided, although colistin was continued for 11 more days . Two patients had either concomitant neurotoxic medication (gabapentin, baclofen, and tizanidine) or disorders (multifocal acute encephalopathy) that might have contributed to their neurological distress one patient with respiratory muscle weakness had received the equivalent of 13 miu of colistin base per day for 19 days whilst experiencing a doubling of plasma creatinine levels . In a cohort of 115 patients, cheng et al5 identified four cases of potential colistin - induced neurotoxicity, including three patients with focal seizures and one patient with altered mental status . These patients had normal kidney function, but details about concomitant treatment or comorbidities were not given . Of note, no clinically significant neurotoxicity was observed during colistin treatment in a large group of patients with underlying neurological disorders admitted to a neurosurgical icu.6 except for one (not so well documented) case of diffuse muscular weakness in an icu patient that spontaneously resolved 1 month after discharge, neuromuscular toxicity has never been described in prospective studies evaluating colistin treatment.2 diagnosis of neurotoxicity is mostly made on clinical grounds, making it difficult to discriminate eventual colistin - induced neurotoxicity from the more frequently observed critical illness polymyoneuropathy in icu patients . In only one study, electrophysiological measurement was performed in a limited number of patients who had received colistin for at least 7 days . Among these patients, 50% exhibited typical features consistent with critical illness polymyoneuropathy, but none had evidence of neuromuscular junction blockade.7 the sudden occurrence of respiratory muscle weakness and apnea in our patient was unexpected . He received intensive respiratory and muscle reinforcement physiotherapy daily and never complained of any neuromuscular discomfort . Carbapenems can produce neurotoxicity.8 however, carbapenem - induced neuromuscular blockade has never been described . Meropenem has the lowest neurotoxic potential of all carbapenems, and its dose was appropriately adapted to renal function . Product information (colistineb, forest laboratories, kent, uk) recommends a dose of 12 miu q8h in adults weighing more than 60 kg, which is, indeed, lower than the prescribed dose in our patient . However, during the last 2 years of a 7-year cohort study evaluating 258 patients of whom 86% were hospitalized in the icu, colistin dose was standardized to 9 miu / day.9 no significant neurotoxicity was observed even when treatment was given for more than 4 weeks.4 sparse pharmacokinetic and pharmacodynamic data in critically ill patients demonstrated maximum mean steady state concentrations of colistin base between 2.3 and 3.9 g / ml after administration of 3 miu q8h.10,11 the colistin base concentration measured at occurrence of neuromuscular blockade in our patient, though largely exceeding the mic value for the culprit escherichia coli, would still be considered suboptimal for treatment of pseudomonas aeruginosa and acinetobacter baumannii strains currently reported as sensitive (mic 2 g / ml). Finally, 70% of patients with cystic fibrosis receiving the equivalent of 13.518.5 mg / kg / day of colistimethate sodium for up to 35 days (compared with our patient receiving 9 mg / kg / day for 17 days) experienced paresthesias, headache, and lower limb weakness but never developed neuromuscular blockade.12 the late deterioration of renal function might have elicited neurotoxicity . However, this is difficult to anticipate . Plasma levels of colistin are not routinely measured and are not correlated with creatinine clearance values.10 also, a relationship between a given plasma level and the appearance of severe neurotoxic events has never been described . Product information suggests maintaining the amount of each colistin dose without extending the dosing interval when creatinine clearance is maintained between 20 and 50 ml / min . Calculated creatinine clearance during treatment in our patient never decreased below 40 ml / min . Colistin - induced neurological symptoms are mostly not evident and usually do not necessitate treatment modification . If more serious neurotoxicity is suspected, the antibiotic should be withdrawn . Colistin is also eliminated by intermittent or, as shown in our patient, continuous hemofiltration using the cvvh modality . Intermittent hemodialysis (ihd) rapidly removed normal colistin levels of around 2 g / ml.13 however, no data exist on the ability of ihd to efficiently remove potentially severe toxic levels of colistin exceeding 8 g / ml . Cvvh was used in our patient because of hemodynamic instability and to avoid any rebound effect of colistin as might occur with ihd . Indeed, this calculation was impossible, as an important fraction was eliminated by hemoadsorption . Moreover, by doing an indirect measure of the area under the curve (figure 2, dotted line), hemoadsorption was responsible for probably more than 80% of the elimination until membrane saturation was reached . Still, colistin elimination from plasma was as effective as in the patients reported by marchand et al.13 our case, however, is very different from the one presented by li et al.14 these authors used cvvh at a dose lower than 35 ml / kg / h (ie, 27 ml / kg / h with the associate dialysis), as well as a classical an-69 membrane (gambro) with less adsorptive properties than our an69 st membrane . The maximal concentration of colistin in the effluent was reached only after 3 hours of cvvh in our patient . If convection was the sole way of eliminating colistin, then concentrations in the effluent should completely mirror plasma concentrations . Colistin removal by convection alone would indeed have been greater, as initial plasma levels were high, thereby producing higher colistin levels in the effluent . As this was not observed during the first hours of cvvh in our patient, colistin must be largely adsorbed in the membrane . With saturation occurring after 3 hours adsorption is still an important mechanism of removal in cvvh.15 a careful look at the graph accompanying the case reported by li et al14 shows a similar pattern of removal, though less pronounced, as the used membrane was less adsorptive . To our knowledge, our case is the first description of colistin adsorption while employing cvvh with a highly adsorptive membrane . This finding may offer important perspectives with regard to urgent treatment of life - threatening colistin toxicity in hemodynamically unstable critically ill patients . According to the reports of li et al14 and our findings, adsorption is the main mechanism of removal of colistin and might be responsible for 80% of the whole removal . In conclusion, a case of colistin - associated neuromuscular blockade presenting as sudden severe respiratory muscle weakness and apnea is described . The rising incidence of multidrug - resistant gram - negative infections in the critically ill will probably produce an increase in colistin prescriptions . Moreover, recent clinical and pharmacological experience supports the use of a higher than currently prescribed colistin dose . Therefore, it is of utmost importance that clinicians remain aware of this rare but life - threatening complication when prescribing colistin . Cvvh at a dose of 35 ml / kg / h and using a highly adsorptive membrane might be an excellent tool for rapid and adequate elimination of colistin, avoiding any rebound effect.
Staphylococcus aureus is part of the microbiome of roughly 30% of people who show no clinical symptoms . It is an opportunistic human pathogen that can cause a wide variety of diseases and can involve any organ system in the human body . Diseases caused by s. aureus may include mild skin infections such as folliculitis and impetigo to fatal conditions such as pneumonia, osteomyelitis, and endocarditis . Treatment of s. aureus infections has become problematic as it has developed numerous mechanisms to become resistant to almost all known antibiotics [3, 4]. It was previously reported that exposure of s. aureus to oxacillin and other cell wall - active antibiotics increases the expression of msra1 and msrb both at the transcriptional and at the protein level [5, 6]. Pathogenic bacterial species are exposed to a variety of extremely potent reactive oxygen species (ros) by the host phagocytic cells during the course of phagocytosis that are damaging to all cellular macromolecules . Ros can cause damage to proteins by the oxidation of sulfhydryl groups, reduction of disulfides, oxidative adduction of amino acid residues close to metal - binding sites, and peptide fragmentation . In particular, ros oxidize the sulfur atom of protein - bound methionine residues resulting in methionine sulfoxide (meto) and loss of protein function . Msra and msrb proteins reduce s- and r - epimers of methionine sulfoxides (meto), respectively . In s. aureus, genes encoding msra1 and msrb are the first and second genes of a four - gene cluster that are cotranscribed . A mutation in the msra1 gene increased the susceptibility of s. aureus to oxidative stress [6, 9]. More recently, it was shown that the msra1 protein was critical for s. aureus in establishing an infection in mice . Interestingly, the msra1-deficient s. aureus was shown to possess an elevated level of msrb giving rise to the speculation of autoregulation of the msra1/msrb locus . Additionally, sigma factor b (sigb) is an alternative sigma factor that is involved in regulating the expression of stress response genes in s. aureus . Thus, it seems plausible that sigb may have a role in the regulation of the msra1/msrb locus . Findings of this study provide evidence that the msra1/msrb locus is negatively regulated by the products of this locus and sigb . S. aureus cultures were grown aerobically at 37c in tryptic soy broth (tsb) in a shaking incubator (220 rpm) or on tryptic soy agar (tsa) by incubation for 2448 h. overnight cultures of s. aureus reporter strains were prepared in the presence of erythromycin at 10 g ml . Oligonucleotide primers used in this study were obtained from eurofins and are shown in table 2 . Construction of msra1/msrb promoter - lacz reporter strain has been previously described . In this construct, a 1.3 kb dna fragment starting 44 nucleotides downstream and going upstream of the msra1 gene cloned in front of a promoterless lacz gene in the vector paz106 was integrated in the chromosome of s. aureus strain rn450 [5, 6]. The msra1/msrb promoter - lacz reporter was transduced into various strains of s. aureus using a phage 80 transduction procedure . Strains used in this study were verified by pcr . To determine if the msra1/msrb locus is autoregulated, the expression of lacz from the msra1/msrb promoter - lacz fusion was investigated in msra1-, msrb-, and msra1-msrb - deficient strains of s. aureus strains sh1000 and col . Therefore, the strength of the msra1/msrb promoter was also assessed in a sigb mutant . Overnight cultures of these strains were diluted (1: 100) and grown at 37c with shaking . These cultures were grown to od600 = 0.5 that was considered time 0 and the levels of -galactosidase activity in these cultures were measured at different time points (0, 90, 180, 270, and 360 min) as an indicator of the strength of the msra1/msrb promoter . Previous studies [5, 6, 9, 10, 13] have shown that, in the presence of oxacillin, there is an increased production of msra1 and msrb in s. aureus . To further investigate the regulation of the msra1/msrb locus and to see if it can be magnified in the presence of oxacillin, overnight cultures of wild - type and the derivative msra1-msrb mutant of s. aureus strain col were diluted (1: 100) in fresh tsb and grown to od600 of 0.5 . 10.0 ml of the culture was split into two 15 ml tubes . To one of the cultures both cultures with and without oxacillin were allowed to grow for an additional 2 h at 37c with shaking . The od600 of the culture was determined as a measure of cell density and cells were subsequently collected by centrifugation . For precise optical density readings, cultures were diluted appropriately to bring density into measurable range . The cell pellet was used to measure -galactosidase activity as described previously using o - nitrophenyl--d - galactopyranoside (onpg) as the substrate [5, 6, 13]. Qrt - pcr assays were used to verify induced expression of the genes of the msra1/msrb locus under oxacillin stress and to validate the lacz reporter expression data in sigb mutants . Cultures of s. aureus strain col were grown to od600 = 0.3 and divided into two tubes . One tube was stressed with oxacillin at a concentration of 1.0 mg ml for 2 h. total rna was extracted from these oxacillin stressed and control cultures as described previously . For the validation of lacz data, the wild - type and sigb mutant strains of s. aureus were allowed to grow for 90 min and 6 h after reaching the od600 = 0.5 and total rna from these cultures were extracted . Cdna from dnase treated 0.5 g of total rna was synthesized in a 20 l reverse transcription reaction containing random hexamers and superscript iii reverse transcriptase (invitrogen). All real - time pcr reactions were carried out with bio - rad icycler (iq5 system). The transcript level of msra1 was quantified using primers p13 and p14, that of msrb was quantified using p15 and p16, and that of the gene encoding the iia(pts) was quantified using primers p17 and p18 . Transcript levels of genes were normalized to dna gyrase mrna using primers p19 and p20 based on a previous report [15, 16]. All results are reported as the mean se of at least three independent experiments . Data were analyzed with student's t - test using r studio for windows (version 0.98.1103, 3.1.3). Previously created msra1, msrb, msra1-msrb, and sigb knockout mutants of s. aureus strain sh1000 [6, 9, 10] were transduced in the methicillin - resistant s. aureus strain col . These mutants and the presence of meca gene in these strains were verified by pcr (see supplemental figures s1-s2 in supplementary material available online at http://dx.doi.org/10.1155/2015/617925). The msra1/msrb promoter - lacz fusion was subsequently integrated into the chromosome of these mutant strains using a bacteriophage transduction procedure . Overall, five msra1/msrb promoter - lacz reporter strains were created in methicillin - resistant as well as methicillin - sensitive s. aureus backgrounds . Proper integration of the msra1/msrb promoter - lacz fusion was also confirmed by pcr (supplemental figure s3). Previously, we reported higher msrb levels in msra1-deficient s. aureus cells [9, 10]. This led to the speculation that the msra1/msrb locus may in part be regulated by the products of this locus . To investigate this possibility, the level of -galactosidase was measured in msra1-, msrb-, and msra1-msrb - deficient strains of s. aureus . -galactosidase activity levels were higher in these strains compared to the activity level in the wild - type s. aureus strain sh1000 (figure 1). The msra1/msrb promoter - lacz reporter was also studied in the methicillin - resistant strain col . Overall, the expression of lacz was lower in methicillin - resistant s. aureus compared to the methicillin - sensitive s. aureus (figures 1(b) and 2(b)). In addition, -galactosidase activity comparison revealed that only the msra1-msrb double mutant strains had higher activity levels compared to the wild - type col at the various time points (figure 2(b)). In the individual msra1 or msrb mutant strains, a significant increase in -galactosidase activity was not observed compared to wild - type s. aureus col (figure 2(b)). Measurement of -galactosidase activity demonstrated that there was increased expression of lacz from the msra1/msrb promoter when s. aureus was deficient of sigb in strain sh1000 (figure 3(b)). However, in s. aureus col, no such increase in the expression of lacz was observed from the msra1/msrb promoter under sigb - deficient conditions compared to the wild - type strain (figure 4(b)). In qrt - pcr assays, a relatively higher level of msra1 transcripts was observed in sigb mutant of s. aureus strain sh1000 compared to the wild - type strain (table 3). However, this increase in msra1 gene expression was not evident in the sigb mutant of s. aureus strain col (table 3) supporting the findings of the msra1/msrb promoter - lacz data in sigb mutant strains . Previous studies have shown that the msra1/msrb locus is induced by the cell wall - active antibiotics, oxacillin, vancomycin, and d - cycloserine, in a methicillin - sensitive s. aureus strain . In a later study, while the msra1/msrb locus remained inducible in the presence of d - cycloserine and vancomycin, no induction of this locus was noted in the presence of oxacillin, when similar experiments were carried out in a methicillin - resistant s. aureus strain col . However, in our experiments, a significantly increased -galactosidase activity clearly indicates a significant induction of msra1/msrb locus in the presence of oxacillin, even in a methicillin - resistant s. aureus (figure 5). We also investigated the expression of the downstream genes of msra1 locus in qrt - pcr assays . We determined that the oxacillin stress dramatically induced the expression of msra1, msrb, and the gene encoding iia(pts) (table 4). The expression level of the fourth gene of this locus was not investigated due to its very small size . This finding further supports our previous observation of cotranscription of the four genes of the msra1/msrb locus [5, 6]. While studying the regulation of the msra1/msrb locus in a methicillin - resistant s. aureus strain col, oxacillin was added during the growth of the msra1/msrb promoter - lacz reporter to investigate any magnification of the regulation . In these studies, while an increased lacz expression was observed in wild - type s. aureus strain col after oxacillin treatment, a more dramatic increase in the lacz expression in response to oxacillin was seen in msra1-msrb - deficient col (figure 6). Cell wall - active antibiotics have been used extensively for the treatment of infections caused by bacterial pathogens . S. aureus is a major human pathogen and is resistant to most commonly available antibiotics . Interestingly, cell wall - active antibiotics cause induction of a locus in s. aureus that leads to elevated synthesis of two methionine sulfoxide reductases (msra1 and msrb) [5, 6]. These enzymes reduce methionine sulfoxide and play important roles in maintaining protein integrity and function particularly under oxidative stress . These two proteins have also been shown to have roles in the virulence of bacterial pathogens [1923]. Msr - deficient bacterial mutants show a reduction in the ability to adhere to eukaryotic cells and are thus less likely to establish an inflection [21, 22, 24, 25]. It is speculated that the lack of the msr enzymes compromises the integrity of the bacterial surface proteins responsible for adherence to eukaryotic cells . Reduced msr activity decreases bacterial survival inside the phagocytic cells . In addition to increased levels of msra1 and msrb specifically in response to cell wall - active antibiotics, these proteins in s. aureus have been shown to play roles in the survival of bacterial cells under oxidative stress as well as in mice [6, 10]. We previously demonstrated that when the msra1 gene is deleted in s. aureus, there is an increase in msrb synthesis suggesting a possible role in the regulation of this locus . Findings of this study suggest that, in a methicillin - sensitive s. aureus strain sh1000, msra1 and msrb individually can downregulate the msra1/msrb locus . However, in methicillin - resistant s. aureus strain col, msra1 and msrb both are needed to downregulate the expression of the msra1/msrb locus . It is speculated that the msra1/msrb locus, to some extent, is differentially regulated between methicillin - resistant and methicillin - sensitive s. aureus strains . It is not uncommon to observe a differential gene expression pattern between different s. aureus strains . It has been demonstrated that the growth of methicillin - resistant s. aureus is slower than that of methicillin - sensitive s. aureus in the lag phase but not during the exponential phase and that the alterations in virulence between these two strains may at least partially be due to the growth rate differences . Deletion of a gene encoding nitric oxide synthase (nos) in a methicillin - resistant s. aureus reduced virulence as seen by decreased bacterial survival and smaller abscess formation . However, nos was shown to have a limited role in a methicillin - sensitive s. aureus . Expression of genes encoding staphylococcal superantigen - like (ssl) proteins also varies between s. aureus strains [29, 30]. Significant differences were also noted between the protein profiles of the methicillin - resistant and methicillin - sensitive s. aureus strains exposed to triton x-100 . It is well established that the msra1/msrb locus is selectively induced in the presence of cell wall - active antibiotics . These antibiotics interfere with the bacterial cell wall synthesis and, as a result, the cells become fragile and susceptible to lysis . Expression of msra1/msrb locus is not induced by antibiotics that target other bacterial metabolic pathways . In a previous report, it was shown that the msra1/msrb locus was not induced by the presence of oxacillin but was induced by the presence of d - cycloserine and vancomycin in a methicillin - resistant s. aureus . However, data from our study provide clear evidence that oxacillin does in fact induce the msra1/msrb locus in a methicillin - resistant background of s. aureus . The previous report did not observe any induction because the bacterialcells were not exposed to a high enough concentration to impose antibiotic stress in a methicillin - resistant s. aureus strain . Furthermore, we explored the induction of msra1/msrb genes in msra1-msrb double mutant in methicillin - resistant strain col . An increase in induction of the msra1/msrb locus was further magnified in msra1-msrb double mutant exposed to oxacillin compared to the wild - type s. aureus col in response to oxacillin . This further confirms the notion of downregulation of the msra1/msrb locus by msra1 and msrb and this is more likely an indirect effect . This speculation of an indirect regulation is based on the fact that, after conducting a protein domain search (http://prosite.expasy.org/), no specific dna - binding domain was observed in msra1 and msrb proteins . It is possible that the msra1 and msrb enzymes are critical in maintaining the integrity of a cytoplasmic transcriptional regulator that is involved in the regulation of expression of this locus . In recent years, regulation of msra and msrb has been studied extensively across multiple species; however, none have shown that msra or msrb directly or indirectly regulates its own expression . It has been demonstrated that rynb regulates the synthesis of escherichia coli msrb but not msra by binding to the 5 untranslated region of msrb mrna and interfering with its binding to the ribosome . Nitric oxide, which is induced in ulva fasciata upon exposure to light, upregulates the expression of msr genes in the intertidal macroalga . In saccharomyces cerevisiae, calcium phospholipid binding protein (cpbp) interacts with the msra promoter and enhances its expression . In bacillus subtilis, a transcriptional regulator, spx, however, in s. aureus spx mutant, teicoplanin exposure resulted in no significant induction of this locus, whereas, in the spx mutant strain complemented with the wild - type spx gene, msra1/msrb induction in response to teicoplanin exposure was restored . Additionally, in the spx mutant, basal msra1 mrna was significantly lower than spx complemented strain . Sigb is the alternative sigma factor in s. aureus that plays a role in the regulation of expression of stress responsive genes in s. aureus . In addition, sigb is also associated with the regulation of expression of the virulence genes in s. aureus . In a previous report, the level of expression of msra1/msrb locus was investigated between rn450 (sigb) and sh1000 (sigb). It was shown that, in s. aureus strain sh1000, msra1/msrb expression was 30% more induced than in s. aureus strain rn450 in the presence of oxacillin . In contrast, our study shows that sigb in fact downregulates the expression of msra1/msrb locus in s. aureus in the methicillin - sensitive s. aureus strain sh1000 and plays no role in the regulation of this locus in methicillin - resistant strain col . In summary, this study provides evidence that the expression of the msra1/msrb locus is enhanced when s. aureus is deficient in msra1, msrb, or both in a methicillin - sensitive s. aureus . However, in methicillin - resistant s. aureus, increased expression of the msra1/msrb locus was apparent only when the bacterial cells were deficient in both msra1 and msrb . In addition, sigb also in part downregulates the expression of this locus in methicillin - sensitive s. aureus but not in methicillin - resistant s. aureus.
Methadone (mtd), as a synthetic opioid with long elimination half - life, is the preferred drug for the treatment of opioid dependence and control of pain in iran . This drug is available in oral liquid formulation and tablets, has analgesic effects, and can be used in the management of severe chronic pain . Patients who refer after acute overdose are generally those who obtain multiple doses of take home methadone from methadone maintenance therapy (mmt) clinics [18]. In iran, it is a colorless liquid usually kept in water bottles at home and accidental acute poisoning with it is extremely common especially in children . Suicidal attempt with mtd syrup has also become so popular within the recent years . Delayed or recurrent respiratory depression, ventricular dysrhythmias, acute lung injury, and death are the major complications of mtd overdose . Respiratory arrest is the main cause of mtd - related deaths and usually develops 12 hours after the ingestion of the drug . Additionally, torsade de pointes (tdp)the arrhythmia induced by qt interval prolongation may contribute to death [11, 12]. Overdose may cause renal failure as a result of rhabdomyolysis and myoglobinuria in prolonged immobilization or coma . Some studies reported a 10-fold increase in mtd toxicity - related fatalities between 2000 and 2003 which could probably be because of its arbitrary use and use in pain clinics [13, 14]. It should be noted that many of mtd intoxication deaths are preventable with cautious prescription, good monitoring, and attention to the signs and symptoms of toxicity and the drug interactions . It is important to have a broad knowledge about the different epidemiological aspects of mtd usage and overdose . This study aimed to describe the demographic data of mtd - overdosed patients and compare mtd - intoxicated survivors and nonsurvivors to investigate the possible prognostic factors in this toxicity . In this retrospective, cross - sectional study, medical data of all mtd - poisoned patients older than 12 years who had been admitted to toxicology ward of loghman hakim hospital between 2007 and 2012 were evaluated . The diagnosis of mtd poisoning was based on history of its ingestion taken from the patients and/or their relatives, the presence of a minimum of two signs of opioid toxicity including central nervous system depression, respiratory depression, and miosis in physical examination, and the positive urine drug screen test for mtd (urine was checked twice, once at emergency department [ed] and the second time, 8 hours later at ward). All the available information including the demographic data, past medical history, signs and symptoms on presentation, laboratory findings, complications, and outcome was recorded . The cases with insufficient data, those with background diseases (i.e., ischemic heart disease, chronic renal failure, and advanced liver disorders), concomitant head trauma, and multidrug toxicity, and those on mtd treatment in mmt program and/or overdosed on other opioids were excluded from the study . Data analysis was performed by statistical package for social sciences (spss) version 16 . A p value less than 0.05 was considered to be statistically significant . Finally, the patients were divided into two groups, those with acute toxicity after single ingestion of a dose of mtd for the first time (suicide attempt or accidental) and those with acute on chronic toxicity poisoning that were on daily doses of mtd and had ingested it more than the usual dose (suicide attempt or recreational overdose). The study was performed according to the helsinki declaration and approved by our local ethics committee . A total of 456 mtd - poisoned patients had been admitted to toxicology ward of loghman - hakim hospital between 2007 and 2012 . Of them, 322 patients older than 12 years of age (mean age: 36.0 15.8 years) were included . The male / female ratio was 2.97 and 241 cases (74.8%) were male . Methadone syrup had been used by 129 patients (41.4%) while others had overdosed on mtd tablets . The mean ingested dose of mtd was 85.91 82.61 mg (range; 5 to 500 mg). Mean time elapsed between mtd ingestion and admission was 9.41 9.87 hours (range; 1 to 72 hours). In the first visit at ed, focused on cardinal signs of toxicity apnea, respiratory depression (respiratory rate [rr] <10/min), and miosis were detected in 23 (7.1%), 117 (36.3%), and 177 (60.78%) patients, respectively . Almost 32.5% of the cases were fully conscious on presentation while 59.1% were confused and sleepy, and 3.8% were in comatose status . Based on the mode of toxicity, mtd poisoning had intentionally happened (suicidal attempt, illicit use, or recreational use) in 270 patients (84.1%). Tracheal intubation had been performed in 79 (24.5%) patients at ed or during hospitalization due to respiratory compromise . The most common complication related to mtd poisoning was renal failure in 16 patients, in eight of whom, concomitant clinical and laboratory signs of rhabdomyolysis were present . Electrocardiographic (ecg) findings of 151 patients on admission were available and interpreted by a single cardiologist . The mean pr, ors, and corrected qt intervals (qtc) were 150.3 32.8 (range; 80 to 280) msec, 92.2 21.6 (range; 40 to 150) msec, and 446.3 50.7 (range; 289 to 584) msec, respectively . A total of 198 patients (61.5%) had intentionally or accidentally ingested mtd for the first time and 12 (6%) of them had died . Acute on chronic poisoning had happened in 123 patients (38.2%) who were users of daily doses of mtd . Of them, 92 (28.6%) were on mmt program and had ingested a higher dose of mtd to suicide or for recreational use and 31 (9.6%) had ingested it without prescription ., it was not clear if the overdose was due to the first - time use or he was an abuser . Methadone is a synthesized opioid with excellent oral bioavailability of 70% to 90% and a long plasma half - life (15 to 52 h). With many drug interactions, tablets or syrup of mtd are broadly prescribed for treatment of opioid dependency in iran, and therefore, mtd - related death has increased in this country within the recent decades . Profound hypoxia due to respiratory depression or arrest can lead to acute lung injury or acute respiratory distress syndrome (ards), brain injury, and renal or liver impairment . Some mtd - intoxicated patients need endotracheal intubation and artificial ventilation because of central hypoventilation and inability to protect airways . Dangerous tdp dysrhythmia is another uncommon cause of death in these patients . In our study, the mortality rate was significantly less in those who had ingested mtd for the first time (p = 0.04). This means that acute toxicity is more dangerous in the patients who are on mmt program . Reviewing the literature shows different but increasing rates of mtd - related fatalities in different parts of the world . For instance, in western australia, vermont, ontario, and zurich, a total of 84 (19931999), 76 (20012006), 54 (2004), and 146 (19982007) deaths have been reported due to mtd overdose [1518]. Our study is one of the first reports on mtd - related deaths from the capital of iran . The results showed that mean age of nonsurvivors was more than survivors (46.2.21.2 versus 35.0 14.9 years, resp . ; p = 0.01). It can be concluded that mtd poisoning can contribute to more complications in older ages and surely needs more medical attention in them . The mean age of our patients was similar to a norwegian study (36 15 versus 36 10 years, resp . ). According to our results also, against our expectation, the mean ingested dose was lower in nonsurvivors whose possible cause is their delayed hospital presentation . On the other hand, factors including older ages, acute on chronic toxicity, severe loss of consciousness, and decreased mean arterial pressures on admission were significantly associated with poorer outcomes . It should be noted that, in our cases, respiratory rate was significantly more in nonsurvivors . Since acute lung injury, ards, and pneumonia [table 4] may occur and accompany a higher respiratory rate, it can be suggested that tachypnea can be a predictor for poor prognosis in mtd poisoning . Although no statistically significant difference was detected in the level of creatinine phosphokinase (cpk) between the survivors and nonsurvivors, cpk was less in those who survived (1691 3457 versus 16058 31749). Acute renal failure was a poor prognostic factor for mtd toxicity - related deaths since eight dead cases had acute renal failure due to rhabdomyolysis and acute tubular necrosis . The frequency of endotracheal intubation, duration of hospitalization, lactate dehydrogenase (ldh), blood urea nitrogen (bun), creatinine (cr), aspartate transaminase (ast), and alanine transaminase (alt) were significantly higher in the nonsurvivors, while mean platelet count was significantly lower in this group . Arterial ph and hco3 were lower and pco2 was higher in nonsurvivors . Although the differences were not significant, these findings are due to central respiratory depression and profound hypoxia in severe mtd toxicity . Prolongation of pr (> 200 msec), qrs widening (> 120 msec), and prolongation of qtc interval (> 450 msec) were seen in 3.3%, 7.9%, and 45% of the patients, respectively . Previous studies had defined a qtc interval greater than 450 msec as a risk factor for cardiac dysrhythmia (such as tdp), syncope, and cardiac sudden death [10, 12, 19]. Qtc interval was reported to be 472.72 18.5 msec in iranian causalities receiving mmt and prolonged in 25% of the 100 studied patients . Mean dose of mtd had a significant relationship with the mean qtc interval (p = 0.009). There are different reports about the relationship between the mean dose of mtd and qtc interval; some show significant and others nonsignificant relationships [20, 21]. According to the literature, sudden death due to cardiac arrhythmia and especially qtc interval can occur in a wide range of doses of mtd, even in daily doses of less than 30 mg / day . Higher numbers of acute renal failure, rhabdomyolysis, aspiration pneumonia, hepatic failure, sepsis, and disseminated intravascular coagulation in nonsurviving patients suggest that these patients have serious complications which can lead to death . In a study performed in london in 2004, reduced respiratory rate, aspiration, pulmonary edema, bronchopneumonia, and heart and renal problems were reported as factors causing death in mtd abusers . Delayed hospital presentation, severe loss of consciousness, acute toxicity in patients who were on daily dose of mtd (acute on chronic toxicity), need for early endotracheal intubation, tachypnea (as a sign of acute lung injury or aspiration pneumonitis), acute renal failure, and rhabdomyolysis can be assumed as early poor prognostic factors for mtd - related death.
Laparoscopic cholecystectomy with three or four ports is the standard operation for gallbladder diseases worldwide . The established use of 5 or 10 mm instruments and ports leads to small skin incisions in the upper abdominal wall . However, these scars are still visible and might be a potential risk factor for incisional hernia or adhesions . Furthermore, new innovative methods such as notes (natural orifice transluminal endoscopic surgery) show promising first results regarding the technical feasibility and the possibility of scarless and painless operation [14]. At the present time, it becomes more important for younger patients to undergo surgery with none or at least very small scars . Notes surgery might be the solution but is in case of transvaginal access to the abdominal cavity limited to female patients, and the procedure is not as easy to learn . In addition, this technique requires special instruments, which do not exist in a regular department of surgery . But due to the discussion about notes, another approach for the treatment of both genders is getting more attention from the public . The transumbilical access, described in the literature amongst others as laparoscopic single - site surgery (less) [5, 6]. For this technique, a 15 to 20 mm incision is made direct through the umbilicus, which is defined as a natural embryonic scare, and, therefore, the procedure is also called e - notes (embryonic natural orifice transumbilical endoscopic surgery). Beside the positive cosmetic effect of transumbilical incision, less incisional this report describes our experience with single - incision cholecystectomies in 220 patients as standard procedure using a commercial available single - incision and conventional straight instruments . As far as we know, this is the largest series about single - incision surgery as standard procedure . Between september 2008 and march 2010, 220 laparoscopic single - incision cholecystectomies were performed at the vivantes klinikum am urban, berlin, germany by three experienced surgeons in standard technique . Single - incision operation using two 5 mm and one 10 mm trocar was performed in 24 patients (11%) between october 2008 and january 2009 . Patients with symptomatic acute and chronic gallbladder disease were included in the observation, and all operations were performed consecutively . Exclusion criteria for single - incision surgery were patients with gallbladder perforation, diagnosed in ultrasound or ct scan, with peritonitis or severe critical illness . All patients were hospitalized for at least two days and were completely informed about the single - incision technique . Both arms are rolled out and the monitor is placed on the opposite site of the surgeons near the right shoulder . The operation begins with a longitudinal incision direct through the umbilicus between both umbilical edges (approximately 1.5 to 2 cm). For a clear and safe closure of the linea alba at the end of the operation, the umbilicus has to be disconnected from the ground with a scissor . After good exposure of the linea alba with small hooks, the fascia has to cut with a scissor at a length of 15 to 20 mm . After dissolving of eventually existing adhesions with fingers, a langenbeck`s hook has to retract the inferior part of the incision and the single - port (triport, olympus, germany) can be safely brought into the abdominal cavity with the triport injector introducer . After establishing the pneumoperitoneum up to 14 mm hg with co, a 30 5 or 10 mm laparoscope was used for initial inspection of the abdominal cavity at a 1520 reverse trendelenburg, right - side - up position . We used as instruments conventional straight 5 mm graspers and a 5 mm hook electrocautery device . Because of the gel valves, a continuous grease of the instruments (instellagel, farco - pharma gmbh, germany) is important for a safe, nonfitful handling . A special technique for a better movement inside the abdominal cavity is cross - handedness of the instruments . That means to undercross the instrument in the left hand under the instrument in the right hand . For a right hander the right hand holds the gallbladder with a grasper, and the hilum was prepared with a hook electrocautery device to expose the cystic duct and cystic artery . Both structures were clipped with a 5 mm endoscopic clip applier (ligamax m / l, ethicon endo - surgery, oh, usa) and divided with scissors . After dissection the gallbladder from the fossa, the bladder was removed through the triport system without an endobag (figure 1). The fascial incision was closed with a nonabsorbable 0 suture (prolene, ethicon, germany). Finally, skin closure was done with an absorbable 4/0 suture (monocryl, ethicon, germany). Comparison of the umbilicus before (figure 2(a)) transumbilical incision and at the end of the operation (figure 2(b)) demonstrates that the incision is only at the ground of the umbilicus . The mean age of 142 females (65%) and 78 males (35%) was 47 years (range: 1589), and an elective surgery was planned for 154 patients (70%). The average american society of anesthesiologists (asa) classification was 2 (range: 13) and the body mass index (bmi) 28 (range: 1549). Mean hospital stay was 4 days (range: 220) and 103 patients (47%) had secondary diagnoses . Thirty - seven patients (17%) presented preoperative bile duct stones and of all received an endoscopic retrograde cholangiography (erc). Twenty - three patients (10%) had clinical signs of biliary pancreatitis in medical history . Preoperatively measured laboratory values were in a regular range for leucocytes, c - reactive protein (crp), aspartate aminotransferase (ast), alkaline phosphatase (ap), and bilirubin . All patients received the same prophylactic antibiotic treatment of 2 g cefotaxim and 0.5 g metronidazol as single - shot dosage . Mean operation time was 62 (range: 26174) minutes, and 31 patients (14%) had an incidential umbilical hernia . Twenty - two patients (10%) had previously undergone abdominal operation, and 54 patients (24%) showed peritoneal adhesions . Three patients (2%) required conversion to a three - port technique because of a very large gallbladder with deep positioned hilus . An exact identification of the structures in the hilum via single port was not possible . One patient had a severe bleeding out of the cystic artery after the first clip was accidentally removed . A wound drainage was positioned at the end of operation in five cases (3%). One patient developed a 2 cm necrosis of the bile duct after mirizzi's syndrome, two days after the initial operation . Of 220 patients, 202 (92%) had gallbladder stones and 150 patients (68%) multiple stones . Signs of gallbladder inflammation was diagnosed in 218 patients (99%), and 57 (26%) patients had an acute cholecystitis . The classification of inflammation grade was in 74 patients (34%) light, 101 patients (46%) moderate and 45 patients (20%) severe . A gallbladder hydrops was noted in 53 patients (24%), and ten patients (4.5%) developed a shrunked gallbladder . We could demonstrate in one of the largest series that single - incision cholecystectomy is feasible and safe as standard technique for elective and acute gallbladder disease . Most patients in our collective were satisfied with an almost scarless procedure and less pain after operation . Previously published studies about multiport technique which included more than 1000 patients showed similar results compared to our study group [911]. The conversion rate to an open procedure was in former studies between 2% and 7% and in our population only 1% . Major complications in multiport surgery such as bile duct or vessel injury were noted in 0.9% to 5.8% of all patients . Although we had a comparative high complication rate of 5%, major complications like bile duct injury and necrosis happened only in two patients (1%), which is within the international standard . While single - incision surgery is getting more and more popular, patient numbers of previously published reports are still low [1219]. A review of eight studies from 2009 about single - incision cholecystectomy shows only two studies with 100 patients [14, 17], and the largest series of single - incision surgery, published by lee et al . One of the most important points in the discussion about notes or single - incision surgery is the extended operation time, because of a more complicated access to the abdominal cavity and the difficult handling of the instruments . The mean operation time in eight studies about less surgery including 365 patients was 80 minutes (range: 5194) [1219]. But it is mentionable that studies with a higher case number like rivas et al . And one reason might be that the learning curve for this technique is certainly longer as for the three- or four - port technique . Rivas et al . Compared in their study including 100 patients the first 50 patients with the second 50 ones . Age and bmi were almost equal, but the operation time was considerable reduced from 73 to 45 minutes . Our mean operation time was 64 minutes, and it is still close to the regular time for conventional laparoscopic cholecystectomy . However, we had a high percentage of patients with acute cholecystitis compared to other studies with 5% and 9% [14, 15]. A partition of our 220 cases into patients with acute and chronic cholecystitis showed a considerably different operation time . Fifty - seven patients with acute cholecystitis required a mean operation time of 80 minutes (range: 34174) and in contrast patients with chronic cholecystitis or no inflammation only 57 minutes (range: 28159). In addition, we could not indicate a reduction of operation time between the first 50% of operations and the second 50% like rivas et al . We performed the operation in the first 110 patients in a median time of 61 minutes and the second 110 patients in a median time of 65 minutes . It seems that the learning curve has an important impact but is for an experienced laparoscopic surgeon not as important in large series . But one reason for the more extended time in later operations might be the different view on the indication for single - port surgery after a major experience . The first 30 patients had not undergone previously abdominal surgery and, therefore, the operation easier to perform with a reduced operation time . Another important point of criticism about single - incision surgery is the conversion rate to multiple ports . Several studies reported about a conversion rate between 0% and 5% and are similar to our results with 2% [1316, 18, 19]. Only one study of lee et al . Had a high conversion rate of 13.5% because of technical difficulties . Conversion rate to an open procedure was 1% in our study group and is described in the literature with 0% to 2% [1316, 18, 19]. We had to convert to an open procedure because of an acute bleeding from the cystic artery without an identifiable vessel in the hepatoduodenal ligament . A blind closure of the vessel with 5 mm clips or bipolar thermocoagulation could have injured structures in the ligament . The second patient had an unknown cholecystoduodenal fistula, which could not be closed in laparoscopic technique . Considering these results, the conversion rate in single - incision surgery is even to multiport standard . A view on the complication rates after single - site surgery in the literature shows a percentage between 0% and 5,4% [1219]. Four studies reported about no complications in their study population [13, 14, 18, 19]. In our study, eight patients (5%) developed postoperative complication, and six of these patients (3.5%) had to undergo reoperation . Except romanelli et al ., who had one case of postoperative hernia, other reports did not mention a reoperation . An analysis of our six patients showed that one of two patients with an incisional hernia had an incidential umbilical hernia and might have used a mesh for optimal wound closure . Two patients developed a wound infection, and a wound debridement had to be performed in both cases . In one patient, the gallbladder was opened for extracting the stone and that might be the reason for infection . If the use of an endobag is more safely for preventing wound infection is questionable . We did not use one endobag in our series and had only an infection rate of 1% . These infections would have healed secondary, but because of a good cosmetic result, we decided to reoperate the patient . These hernias could be safely repaired within the standard closure of the fascia using a nonabsorbable suture . In conclusion, we could demonstrate for the first time that laparoscopic single - incision cholecystectomy as standard procedure is feasible and safe compared to conventional multiport technique . Beside scarless operation, one major advantage in comparison to notes is the treatment option for both genders and the use of conventional instruments . Results of long - term followup have to answer the theoretical increased risk of incisional hernia.
Enterohemorrhagic escherichia (e.) coli strains are a subset of the shiga toxin - producing e. coli (stec) that cause diseases in humans and pose a threat to public health worldwide . Many environmental and food sources have caused e. coli o157 or e. coli o157:h7 infections, but they are primarily attributed to consumption of food of animal origin, especially cattle, or to direct or indirect contact with cattle or other farm animals . Human infection by e. coli o157:h7 has been reported in over 30 countries, and cattle appear to be the chief source of infection . Cattle and other ruminants have been established as major natural reservoirs of e. coli o157 and play a significant role in the epidemiology of human infections . Specifically, between 1% and 35.8% of cattle in the united kingdom, and the united states were estimated to be contaminated with e. coli o157 . E. coli o157 and e. coli o157:h7 are present in the intestines of cattle as a component of the native microbiota and they can contaminate both the meat and the slaughterhouse environment . As a result, contamination of both carcasses and the environment by e. coli o157 and o157:h7 from the intestinal contents of cattle during slaughter is one of the most significant risk factors in transmission to humans . Therefore, feces and hide removal are considered to be the main sources of e. coli o157 and e. coli o157:h7 contamination of carcasses during slaughter . Contamination of carcass meat with e. coli o157 and e. coli o157:h7 can occur during dressing, primarily during the skinning, but also during the evisceration phase . Once the e. coli is transferred to the carcass surface, handling and trimming operations can spread the pathogen to the beef trimmings . The pathogenicity of e. coli o157 and e. coli o157:h7, including stec, is associated with several virulence factors . The main factor contributing to their pathogenicity is their capacity to produce two potent phage - encoded cytotoxins called shiga - toxins (namely, stx1 and stx2). Shiga toxins cause diseases such as hemorrhagic colitis and hemolytic uremic syndrome through cytopathic effects on the vascular endothelial cells of the kidneys, intestines, central nervous system and other organs . In addition to the production of toxins, another virulence - associated factor expressed by stec is a protein called intimin, which is encoded by the eae gene and responsible for the intimate attachment of stec to the intestinal epithelial cells . The role of other virulence genes through the production of enzymes such as enterohemolysin, an extracellular serine protease, and a catalase / peroxidase in causing infection appears to be minor . According to studies reported from various parts of the world, cattle carcasses carry a potential risk of the presence of e. coli o157 and e. coli o157:h7 through fecal contamination . There have been many studies conducted to determine e. coli o157 and e. coli o157:h7 in only carcass or rectal samples at abattoirs worldwide, including turkey . However, to the best of our knowledge, there has been no attempt to detect e. coli o157 and e. coli o157:h7 in both rectal and carcass samples from the same animal . Therefore, this study was conducted to investigate the presence of both e. coli o157 and e. coli o157:h7 and to detect the presence of the stx1 and stx2 genes in isolates from both cattle carcasses and their rectal samples obtained from two commercial abattoirs located in the samsun province of turkey . In this study, a total of 200 swab samples obtained from 100 slaughtered cattle were tested to investigate the presence of e. coli o157 and e. coli o157:h7, as well as to detect the presence of stx1 and stx2 genes in the isolates . Swab samples were taken from 100 cattle carcasses and their rectal contents at two commercial abattoirs located in samsun province, turkey between december 2007 and march 2008 . Samples were collected immediately after removal of the hide (dressing step) from the carcass . Each carcass surface sample consisted of two pooled neck and rump subsamples . An area of 200 cm of the neck and rump regions of the carcass was swabbed (100 cm - 10 10 cm - of each region) using two different sterile cotton swabs . For isolation, the swabs were placed in a modified tryptone soy broth (mtsb - oxoid - cm 989; basingstoke, england) supplemented with novobiocine (20 mg / l, n1628; sigma, usa) in 10 ml tubes and then incubated at 41.5 for 24 h . A total of 100 rectal samples were collected and processed in the same manner as the carcass samples . Immunomagnetic separation (ims) was conducted using immunomagnetic beads coated with an anti - e . The 50 l ims bead complex finally recovered was spread onto tellurite (2.5 mg / l) - cefixime (0.05 mg / l) - sorbitol macconkey (tc - smac) agar (oxoid - cm 813, supl.sr 172 e; basingstoke, england) and incubated at 37 for 24 h. up to five colonies exhibiting typical presumptive positive characteristics of e. coli o157 colonies were identified, subcultured onto yeast extract - tripticase soy agar (oxoid - cm 131-l21; basingstoke, uk), incubated for 24 h at 37 and subjected to the confirmatory tests described below: the pinpoint indol test was conducted, after which indol test positive colonies were streaked onto 4-methylumbelliferly--d - glucuronide sorbitol macconkey (mug - smac) agar (oxoid - br 071 e; basingstoke, england) and incubated overnight at 37. colonies displaying no fluorescence when illuminated with 366 nm uv light were assumed to be exhibiting typical characteristics of e. coli o157 . The cellobiose test was subsequently conducted in purple broth base (difco-0227 - 01 - 6; difco, usa). Only colonies that tested negative to the cellobiose fermenting test were selected for further processing . All sorbitol non - fermenting, indole - positive, mug - negative, cellobiose - negative colonies were cultured on tsa and incubated at 37 for 24 h. the isolates were then examined by latex agglutination with e. coli o157 (#210753; denka - sheiken, japan) and o157:h7 antisera (#211057; denka - sheiken, japan). Colonies exhibiting a positive precipitation reaction with the o157 antiserum were identified as e. coli o157, while colonies showing a positive precipitation reaction with the h7 antiserum were identified as e. coli o157:h7 . Multiplex pcr was conducted to detect the presence of the shiga - toxin genes (stx1 and stx2) in e. coli o157 and e. coli o157:h7 isolates were trated according to a modified version by using multiplex pcr assays . Briefly, each positive colony was inoculated on tsa and incubated for 24 h at 37. two colonies were then selected and suspended separately in 500 l of sterile distilled water in microcentrifuge tubes, after which they were incubated at 95 for 10 min in a water bath (memert, germany). The tubes were then centrifuged (hettich - universal-320r unit) at 9,503 g for 10 min, after which the supernatant containing the dna to be used as the template dna was transferred into dnase / rnase - free microcentrifuge tubes . The extracted dna samples were stored at -20 until use . To detect the stx1 and stx2 genes, 10 l of extracted dna was used as a template in a reaction mixture with a final volume of 50 l that contained 200 mm of each deoxynucleoside triphosphate (dntp), 250 nm stx1 primer, 250 nm stx2 primers, 1u of taq dna polymerase in 1 pcr buffer and 2 mm of mgcl2 . The amplification of dna (mj mini; biorad, usa) was conducted as follows using a thermocycler (mj mini - ptc-1148; biorad, usa): initial denaturation at 94 for 10 min, 35 cycles of denaturation at 94 for 1 min, annealing at 60 for 1 min, extension at 72 for 1 min, and final extension at 72 for 7 min . For gel electrophoresis, the 20-l amplicon mixtures were supplemented with 4-l of loading dye and loaded onto a 2.0% agarose gel containing ethidium bromide (gene choice). Electrophoresis (power pac - basic; bio - rad, usa) was then conducted at 90 v for 1.5 h. a 100~1,000 bp molecular weight marker was used to identify the amplified products, which were visualized by uv illumination (wiseuv - wuv - l50; daihan scientific, korea). The nucleotide sequences and predicted product sizes of the primers are shown in table 1 . The pinpoint indol test was conducted, after which indol test positive colonies were streaked onto 4-methylumbelliferly--d - glucuronide sorbitol macconkey (mug - smac) agar (oxoid - br 071 e; basingstoke, england) and incubated overnight at 37. colonies displaying no fluorescence when illuminated with 366 nm uv light were assumed to be exhibiting typical characteristics of e. coli o157 . The cellobiose test was subsequently conducted in purple broth base (difco-0227 - 01 - 6; difco, usa). Only colonies that tested negative to the cellobiose fermenting test were selected for further processing . All sorbitol non - fermenting, indole - positive, mug - negative, cellobiose - negative colonies were cultured on tsa and incubated at 37 for 24 h. the isolates were then examined by latex agglutination with e. coli o157 (#210753; denka - sheiken, japan) and o157:h7 antisera (#211057; denka - sheiken, japan). Colonies exhibiting a positive precipitation reaction with the o157 antiserum were identified as e. coli o157, while colonies showing a positive precipitation reaction with the h7 antiserum were identified as e. coli o157:h7 . Multiplex pcr was conducted to detect the presence of the shiga - toxin genes (stx1 and stx2) in e. coli o157 and e. coli o157:h7 isolates were trated according to a modified version by using multiplex pcr assays . Briefly, each positive colony was inoculated on tsa and incubated for 24 h at 37. two colonies were then selected and suspended separately in 500 l of sterile distilled water in microcentrifuge tubes, after which they were incubated at 95 for 10 min in a water bath (memert, germany). The tubes were then centrifuged (hettich - universal-320r unit) at 9,503 g for 10 min, after which the supernatant containing the dna to be used as the template dna was transferred into dnase / rnase - free microcentrifuge tubes . The extracted dna samples were stored at -20 until use . To detect the stx1 and stx2 genes, 10 l of extracted dna was used as a template in a reaction mixture with a final volume of 50 l that contained 200 mm of each deoxynucleoside triphosphate (dntp), 250 nm stx1 primer, 250 nm stx2 primers, 1u of taq dna polymerase in 1 pcr buffer and 2 mm of mgcl2 . The amplification of dna (mj mini; biorad, usa) was conducted as follows using a thermocycler (mj mini - ptc-1148; biorad, usa): initial denaturation at 94 for 10 min, 35 cycles of denaturation at 94 for 1 min, annealing at 60 for 1 min, extension at 72 for 1 min, and final extension at 72 for 7 min . For gel electrophoresis, the 20-l amplicon mixtures were supplemented with 4-l of loading dye and loaded onto a 2.0% agarose gel containing ethidium bromide (gene choice). Electrophoresis (power pac - basic; bio - rad, usa) was then conducted at 90 v for 1.5 h. a 100~1,000 bp molecular weight marker was used to identify the amplified products, which were visualized by uv illumination (wiseuv - wuv - l50; daihan scientific, korea). The nucleotide sequences and predicted product sizes of the primers are shown in table 1 . In the present study, e. coli o157 or o157:h7 was detected in 52 of 200 samples tested (49 e. coli o157 and 3 e. coli o157:h7). The e. coli o157 strain was isolated from 24 carcasses and 25 rectal samples, while the e. coli o157:h7 strain was isolated from two carcasses and one rectal sample . Of the 49 samples that contained e. coli o157, 32 were from the rectal and carcass samples of the same animal, while the other 17 isolates were all from different cattle . Two of the e. coli o157:h7 isolates were obtained from the carcass and rectal swabs of the same animal, while the remaining isolate was obtained from a carcass sample (table 2). The results of multiplex pcr for the detection of stx1 and stx2 genes are shown in fig . While both the stx1 and stx2 genes were detected in 35 e. coli o157 isolates from 17 carcass and 18 rectal samples, they were detected in only one e. coli o157:h7 isolate from a carcass sample . The stx2 gene alone was detected in only two e. coli o157 isolates and the stx1 gene was not detected alone in any isolates . Neither of these genes was detected in the 12 e. coli o157 and two e. coli o157:h7 isolates from carcass and rectal samples (figs . 1 and 2). In turkey and other parts of the world, although there have been many studies conducted to determine the presence of e. coli o157 and e. coli o157:h7 in either carcass or rectal samples at abattoirs, the authors of the present study are unaware of any attempts to detect e. coli o157 and e. coli o157:h7 in both the rectal and carcass samples of the same animal . Therefore, the present study was conducted to investigate the contamination of carcasses and their rectal contents with e. coli o157 and e. coli o157:h7 at two abattoirs in samsun province of turkey because the majority of food - borne e. coli o157 and e. coli o157:h7 infections in humans occur after the consumption of contaminated beef and cattle products . Isolation rates of e. coli o157 and e. coli o157:h7 from bovine carcasses and feces ranging from low (0.39%) to high (17.0%) have been reported for mexico, ireland, belgium, england, france, poland, germany, the united states, turkey and other countries . E. coli o157 or e. coli o157: h7 isolates obtained from cattle carcasses or feces have also been found to contain at least one of the stx1, stx2, eaea, hyla and flich7 genes . In contrast to other countries, there has been only one study of carcasses reported in turkey . The results of that study revealed that 3.9% and 2.4% of the bovine carcasses were contaminated by e. coli o157 and e. coli o157:h7, respectively . There have been a few studies of cattle feces conducted in turkey, the results of which revealed that e. coli o157 or e. coli o157:h7 were present in 1.28% and 13.6% of the samples, respectively . It has also been reported that at least one virulence gene (stx1, stx2 or eae genes) was detected in the e. coli o157 or e. coli o157:h7 isolates from feces samples . In the present study, the e. coli o157 strain was isolated from 24 carcasses (24%) and 25 rectal samples (25%), while the e. coli o157:h7 strain was isolated from two carcasses (2%) and one rectal sample (1%). These values for e. coli o157 were higher than those of previously conducted studies . However, previously conducted studies show a wide range of isolation ratios for e. coli o157 and e. coli o157:h7 . This variation may be due, at least in part, to the sensitivity of the method, diverse geographical origins of cattle, numbers of cattle, study design, number of herds and cattle, sex and age of cattle, season, abattoir conditions and treatment with antimicrobial substances during the process . It has also been reported that the prevalence of e. coli o157 and e. coli o157:h7 varies with the seasons, generally increasing in the warm months of march - september in the northern hemisphere . Another important factor influencing the identification of individual strains of e. coli is the isolation method . Indeed, the detection of e. coli o157:h7 from cattle fecal samples is known to be very difficult due to their low concentration . One of the more sensitive methods is the ims technique, which is why enrichment / ims procedures were employed in the present study . Another study found that the ims technique was superior to the classic culture technique for salmonella isolation (unpublished data). In the present study, swab samples were collected from the rectum of the cattle instead of direct feces samples, because the recto - anal junction mucosa has been identified as the primary site of e. coli o157:h7 colonization in cattle . In attempting to manage e. coli o157 and e. coli o157:h7 contamination in abattoirs e. coli o157 and e. coli o157:h7 have been reported to spread easily onto carcass surfaces from the hide or during evisceration . The results of the present study support that contention, with many rectal and carcass samples of the same animal being positive . Epidemiological studies in cattle indicate that the horizontal transmission of e. coli o157 or e. coli o157:h7 occurs in groups of animals, and that contaminated water may facilitate its spread and persistence within herds . Therefore, control of the spread of e. coli o157 and e. coli o157:h7 at the farm level becomes very important . Moreover, cross contamination may occur during the slaughter of cattle and other processes at abattoirs . The results of the present study indicate that meat from cattle poses a risk to human health in turkey because of potential e. coli o157 and e. coli o157:h7 contamination . To minimize the risk to public health, implementation of the haccp system in abattoirs
Benign colorectal strictures can develop after diverticulitis, ischemic colitis, radiation colitis, or colonic resection . Recently, colorectal endoscopic submucosal dissection has become another possible culprit for the development of this benign stricture, which usually occurs after resection of a lateral spreading tumor, occupying 75% of the lumen . Anastomotic strictures after surgery, occurring in 5% to 20% of cases after low anterior resection, could be a serious condition that may require endoscopic or surgical treatment . Despite this serious condition, most strictures could be managed successfully with several treatment modalities, such as direct digital dilation, transanal surgical treatment, endoscopic balloon dilation, and stent insertion . However, other varieties of endoscopic or surgical techniques are required in refractory strictures, especially after failure of the first endoscopic management . Data on refractory cases in colorectal benign strictures are limited and this will be discussed . Endoscopic balloon dilation has been used as a first treatment modality for the treatment of benign colorectal strictures . However, varying results have been reported with regard to the success rate of this procedure.1,2 despite its simplicity and immediate efficacy in up to 80% of cases, this technique requires several treatment sessions and is associated with a significant rate of recurrent benign stenosis . Predictors of a successful outcome include: a relatively narrow stenosis (<10 mm), a short segment stricture (<4 cm), and anastomotic strictures . Poor predictors include: numerous strictures, complete obstruction, associated fistulas within the stricture, active inflammation around the stricture, recent surgery, a tight angulation, and malignancy.3 balloons usually exert a radial vector force against the strictured tissue, and these often require sequential dilation using a larger balloon over two to three endoscopic sessions in order to achieve long - term success . However, this may not be determined until the results of the first dilation are known . Immediate relief of symptoms has been reported in 77% of patients and long - term relief in 44% of patients.4 fifty - five patients with crohn symptomatic strictures were studied . The long - term success of endoscopic balloon dilation was reported to depend on the type of strictures, their location, and their length . Failure of endoscopic treatment was observed in long - segment strictures in the terminal ileum.5 in one study, 55 patients with ileocolonic strictures secondary to crohn disease underwent 78 balloon dilations . Dilatation resulted in complete relief of obstructive symptoms in 34 patients after one or repetitive dilatations . . However, four of these patients showed improvement with administration of intravenous fluid and antibiotics.6 balloon dilatation is more useful in patients with anastomotic strictures . In one study, endoscopic dilatation in 94 patients with postoperative anastomotic stenosis was successful in 59% of patients who underwent resection for cancer and in 88% who underwent resection for a benign condition . High success and low risk rates make endoscopic balloon dilatation the treatment of choice to avoid high risk of reoperation in patient with benign anstomoticstrictures.2 placement of a self - expandable metal stent (sems) has been suggested as one of therapeutic modalities for the relief of benign colorectal strictures.7 unlike uncovered stents, fully covered sems has several advantages in the management of benign strictures . These fully covered sems have limited local tissue reaction; thus, they are used in benign conditions such as colonic strictures, fistulas, perforation, and leaks in the digestive tract.8,9 permanent insertion of sems is also associated with a significant and unacceptable rate of complications, such as new stricture formation and perforation . Only a few studies have reported on the usefulness of fully covered sems in patients with benign colorectal strictures . One recent study, which included 43 patients with symptomatic strictures, reported on the effectiveness of fully covered sems in the management of benign colorectal strictures . The efficacy of the stent, technical success, stent retrieval, safety, and recurrence of symptoms were evaluated during the follow - up . The authors insisted that fully covered sems is safe and effective for treatment of symptomatic benign strictures, despite a high rate of spontaneous migration.10 although this study could not define the predictive risk factors for clinical success or recurrence, refractory patients who underwent several balloon dilation sessions were successfully managed . Conduct of future studies will be needed in order to define the best treatment option, such as balloon dilation vs. early fully covered sems insertion . Biodegradable stents have recently been developed for use in the management of refractory benign esophageal strictures . The poly - l - lactic based stent and polydiosanone based stent were mainly used for refractory benign esophageal strictures . In particular, a polydiosanone stent is a semicrystalline, biodegradable polymer, which degrades by random hydrolysis and at low ph . The prolonged dilatory effect before stent absorption and the progressive stent degradation could represent a favorable solution for patients with benign strictures refractory to standard dilation therapy compared with self - expandable metal and plastic stents for esophageal strictures.11,12 currently, use of biodegradable stents in the management of colorectal refractory benign strictures, same as benign esophageal strictures, has been attempted . Treatment of 10 patients with postsurgical colorectal strictures (n=7) and fistula (n=3) with biodegradable polydiosanone stents was an effective alternative in short to medium terms . The fistulas were successfully closed in all patients . Among the six patients who received stents for strictures, symptoms resolved in five; in the remaining patients, the stent migrated shortly after the endoscopy . If biodegradable stents are to be used for the treatment of strictures and fistulas, the proximity of the stricture should be close to the anus because of the inflexibility, and the need to fix stents in the colon was important.13 in another study, 11 patients with postsurgical benign strictures located within 20 cm from the anal verge, refractory to mechanical or pneumatic dilation (at least three sessions) were included . The overall success rate of the biodegradable stent was 45%.14 treatment using biodegradable stents in the management of refractory anastomotic colorectal stricture is very safe . They concluded that, instead of a nondedicated biodegradable stent which carried a high migration risk, dedicated stents with a large diameter and antimigration findings could improve the outcome of patients with refractory benign colorectal stricture . Endoscopic balloon dilation has been used as a first treatment modality for the treatment of benign colorectal strictures . However, varying results have been reported with regard to the success rate of this procedure.1,2 despite its simplicity and immediate efficacy in up to 80% of cases, this technique requires several treatment sessions and is associated with a significant rate of recurrent benign stenosis . Predictors of a successful outcome include: a relatively narrow stenosis (<10 mm), a short segment stricture (<4 cm), and anastomotic strictures . Poor predictors include: numerous strictures, complete obstruction, associated fistulas within the stricture, active inflammation around the stricture, recent surgery, a tight angulation, and malignancy.3 balloons usually exert a radial vector force against the strictured tissue, and these often require sequential dilation using a larger balloon over two to three endoscopic sessions in order to achieve long - term success . However, this may not be determined until the results of the first dilation are known . Immediate relief of symptoms has been reported in 77% of patients and long - term relief in 44% of patients.4 fifty - five patients with crohn symptomatic strictures were studied . The long - term success of endoscopic balloon dilation was reported to depend on the type of strictures, their location, and their length . Failure of endoscopic treatment was observed in long - segment strictures in the terminal ileum.5 in one study, 55 patients with ileocolonic strictures secondary to crohn disease underwent 78 balloon dilations . Dilatation resulted in complete relief of obstructive symptoms in 34 patients after one or repetitive dilatations . . However, four of these patients showed improvement with administration of intravenous fluid and antibiotics.6 balloon dilatation is more useful in patients with anastomotic strictures . In one study, endoscopic dilatation in 94 patients with postoperative anastomotic stenosis was successful in 59% of patients who underwent resection for cancer and in 88% who underwent resection for a benign condition . High success and low risk rates make endoscopic balloon dilatation the treatment of choice to avoid high risk of reoperation in patient with benign anstomoticstrictures.2 placement of a self - expandable metal stent (sems) has been suggested as one of therapeutic modalities for the relief of benign colorectal strictures.7 unlike uncovered stents, fully covered sems has several advantages in the management of benign strictures . These fully covered sems have limited local tissue reaction; thus, they are used in benign conditions such as colonic strictures, fistulas, perforation, and leaks in the digestive tract.8,9 permanent insertion of sems is also associated with a significant and unacceptable rate of complications, such as new stricture formation and perforation . Only a few studies have reported on the usefulness of fully covered sems in patients with benign colorectal strictures . One recent study, which included 43 patients with symptomatic strictures, reported on the effectiveness of fully covered sems in the management of benign colorectal strictures . The efficacy of the stent, technical success, stent retrieval, safety, and recurrence of symptoms were evaluated during the follow - up . The authors insisted that fully covered sems is safe and effective for treatment of symptomatic benign strictures, despite a high rate of spontaneous migration.10 although this study could not define the predictive risk factors for clinical success or recurrence, refractory patients who underwent several balloon dilation sessions were successfully managed . Conduct of future studies will be needed in order to define the best treatment option, such as balloon dilation vs. early fully covered sems insertion . Biodegradable stents have recently been developed for use in the management of refractory benign esophageal strictures . The poly - l - lactic based stent and polydiosanone based stent were mainly used for refractory benign esophageal strictures . In particular, a polydiosanone stent is a semicrystalline, biodegradable polymer, which degrades by random hydrolysis and at low ph . The prolonged dilatory effect before stent absorption and the progressive stent degradation could represent a favorable solution for patients with benign strictures refractory to standard dilation therapy compared with self - expandable metal and plastic stents for esophageal strictures.11,12 currently, use of biodegradable stents in the management of colorectal refractory benign strictures, same as benign esophageal strictures, has been attempted . Treatment of 10 patients with postsurgical colorectal strictures (n=7) and fistula (n=3) with biodegradable polydiosanone stents was an effective alternative in short to medium terms . The fistulas were successfully closed in all patients . Among the six patients who received stents for strictures, symptoms resolved in five; in the remaining patients, the stent migrated shortly after the endoscopy . If biodegradable stents are to be used for the treatment of strictures and fistulas, the proximity of the stricture should be close to the anus because of the inflexibility, and the need to fix stents in the colon was important.13 in another study, 11 patients with postsurgical benign strictures located within 20 cm from the anal verge, refractory to mechanical or pneumatic dilation (at least three sessions) were included . The overall success rate of the biodegradable stent was 45%.14 treatment using biodegradable stents in the management of refractory anastomotic colorectal stricture is very safe . They concluded that, instead of a nondedicated biodegradable stent which carried a high migration risk, dedicated stents with a large diameter and antimigration findings could improve the outcome of patients with refractory benign colorectal stricture . Most benign colorectal strictures could be managed with several sessions of balloon dilation or fully covered sems insertion . Several studies have reported on the use of combined endoscopic or a novel method other than these techniques . Variable therapeutic options continue to evolve and have taken much of their lead from the treatment of refractory benign esophageal strictures . A combined technique of endoscopic electroincision using the tip of a polypectomy snare or papillotome and balloon dilatation was used in 36 patients with benign colorectal anastomotic strictures . Recurrence of the stricture was found in only five of these patients at 1-year follow - up, and all were treated successfully by further balloon dilatation.15 the other combined modality was used at the completely obstructed anastomosis stricture . A puncture catheter (polyethylene catheter) with a needle and a flexible metallic sheath at the distal end could penetrate the central obstructed anastomosis stricture under endoscopic and fluoroscopic control . The guide - wire was passed through the catheter and pneumatic dilatation was then performed . More expandable dilatation was performed repeatedly for successful web destruction.16 another study also reported that endoscopic reanastomosis in a sigmoid cancer patient with a completely strictured colorectal anastomosis was performed successfully using a transrectal puncture needle and wire guided balloon dilatation.17 a novel hybrid technique using transanal endoscopic microsurgery (tem) and balloon dilation for the treatment of a benign complete colorectal anastomotic stricture has been reported.18 the stricture was incised with a vessel - sealing device using a 20-cm tem rectoscope . Tem - assisted balloon strictureplasty could be a minimally invasive solution for bridging the gap between radical surgery and conservative treatment . In refractory benign strictures involved around the rectum, electrocautery can be performed via flexible sigmoidoscopy using a urologic resectoscope19 or by tem with balloon dilatation . Laser ablation has also been reported for the treatment of strictures.20 one study reported the use of tem combined with a nd: yag laser for resection of an anastomotic stricture.21 dilation of the stricture using tem can be achieved at the same time as resection . Novel endoscopic techniques are emerging for the management of refractory benign colorectal strictures; these would be of great help in the management of patients who suffered from strictures . Endoscopic dilation of colonic strictures offers many advantages over surgical management, including preservation of intestinal length . The procedure for balloon dilatation is quick, simple, and can be easily controlled by endoscopists . Repeat dilations are often necessary, and the number of repeated dilations seems to vary between two and three or more . If a trial of several balloon dilatations fails, the patient will need an alternative modality . It is safe and effective for the treatment of refractory benign colonic strictures, despite a high rate of spontaneous migration . In particular, the biodegradable stent is very safe and does not need to be retrieved because it will be degraded naturally with tissue reaction . Combined investigational modality can also be considered if only one modality like balloon or sems cannot be performed due to a poor situation (e.g., numerous strictures). Wire guided balloon dilation after use of a puncture needle, eletroincision, and tem using balloon dilatation and combined techniques could be attempted . These different treatments suggest that a single method is not adequate for all refractory benign colorectal strictures . What is important is that the etiology and pathogenesis behind colorectal stricture should be analyzed, given the success rate of a nonoperative intervention method . Finally, surgical treatment is reserved for patients who fail all endoscopic remediation or those are not candidates for endoscopic treatment (fig.
Though fetal endocardial fibroelastosis (efe) has been reported in the past three decades, the classical echocardiographic features of efe following maternal organophosphorus ingestion at 20 weeks of gestation is reported for the first time in the literature . These signs completely regressed within a period or 14 weeks and a normal healthy full - term baby was delivered . This report is unique due to the fact that fetal echocardiographic features consistent with the diagnosis of endocardial fibrosis following maternal organophosphorus poisoning are not an indication for termination of pregnancy, which is contradictory to the common practice . A 23-year - old primigravida at 20 weeks of gestation was hospitalized, following suicidal attempt by ingestion of organophosphorus pesticide (carbofuran). She was treated with antidote, corticosteroid steroid, and supportive measures . Following recovery after 2 weeks, she was referred for a targeted scan for anomalies at 22 weeks of gestation . Both ventricular and interventricular septal endocardial surfaces showed diffuse hyperechoic thickening [figure 1]. This pearly white lining was extending along the crux and atrioventricular valves [figure 3]. The proximal outflow tracts, mainly up to the semilunar valves, were also brighter with normal crossing, dimensions, and flow [figure 5]. Pulsed doppler study (cursor directed through the left ventricle along the junction of the anterior leaflet of the mitral valve and left ventricular outflow tract) showed e / a ratios of mitral and tricuspid valves below the 2.5 percentile (0.41 and 0.45, respectively) for this period of gestation . The e / a ratio of the atrioventricular valves is calculated by dividing the peak velocity of e wave (due to early diastolic filling of the ventricle) by the peak velocity of the a wave (due to late diastolic filling of the ventricle from atrial contraction). Slightly oblique short axis view of the fetal heart at 22 weeks of gestation shows the pearly white endocardium of ventricles (rv: right ventricle, lv: left ventricle) and interventricular septum (ivs) magnified view of the interventricular septum shows the hyperechogenicity of the endocardial surfaces (apx: cardiac apex, rv: right ventricle, lv: left ventricle) the 4-chamber view shows the hyperechoic endocardium extending along the crux and the mitral leaflets . The hyperechoic mitral leaflets are opening into the left ventricle (lv: left ventricle, rv: right ventricle, la: left atrium, ra: right atrium) magnified view of the mitral leaflets shows the rolled tips within the left ventricle (lv: left ventricle, rv: right ventricle, la: left atrium, ra: right atrium) the outflow tracts show normal calibre and flow (apx: cardiac apex, rv: right ventricle, lv: left ventricle, ao: aorta, pa: pulmonary artery) the myocardial performance index (mpi), otherwise known as tei index (ti), for the left ventricle was more (0.71) than the normal value expected at this gestational age (average value at this age is 6). There were no significant doppler spectral changes of the inferior vena cava (such as reversal of a wave in the inferior vena cava or hepatic veins) or umbilical vein (biphasic rather than triphasic spectral pattern and umbilical venous pulsations) to suggest impending cardiac failure . Reassessment at 26 weeks of gestation showed non - progressive echocardiographic abnormalities and mild mitral incompetence as before . Follow - up echocardiography at 36 weeks of gestation showed normal fetal cardiac chambers and outflow tracts . Carbofuran (2, 3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) is a very toxic anticholine esterase carbamate widely used as a pesticide all over the world . The oral ld50 (median lethal dose) of this chemical in animals ranges from 8 to 19 mg / kg and for humans it is 11 mg / kg . The fatal concentration in the human blood is 0.32 - 11.6 g / ml . This mother had consumed diluted carbofuran of which some quantity was removed later by gastric lavage . The exact maternal serum concentration of carbofuran at the time of admission was not known . However, she had all the symptoms and signs of moderate degree organophosphorus poisoning and took 2 weeks for complete recovery . This fetus at 22 weeks of gestational age had all the classical echocardiographic features of endocardial fibroelastosis (the term was first coined by weinbergand himmelfarb). The efe is diagnosed by the echocardiographic features such as hyperechoic endocardial thickening extending along the ventricular walls and atrioventricular valves . These progressive changes lead to decreased cardiac function which is best assessed by the mpi (or ti). The left ventricular tei index was more in this fetus due to the increased isovolumetric relaxation time (irt) (> 2 sd above the mean for this age) and the shorter ejection time (et). This fetus had the typical diagnostic features of endocardial fibroelastosis which gradually subsided within a period of 14 weeks . This is likely to be due to the capability of the myocardium to recover from the temporary myocardial toxicity induced by the pesticide . The fetus in this case was exposed to acute maternal carbofuran poisoning at 20 weeks of gestation . Since fetal period is characterized by rapid cellular multiplication and organization, the endocardium is also affected by the toxin . It is suggested that any severe mechanical or toxin - induced stress to the cardiac tissue can stimulate the proliferation and transformation of fibroblasts of the endocardial lining to deposit more collagen and elastin . A study on the effect of organophosphorus compounds on fetal growth and preterm delivery did not highlight any cardiac sequelae . A recent follow - up of newborns exposed to maternal organophosphorus poisons during pregnancy has revealed growth disturbances, metabolic disorders, and cns morbidity, and no cardiac sequelae . This is likely to support the explanation that the cardiac tissue can withstand the damage more efficiently in the fetal period and the changes may be transient . Traditionally, the endocardial fibroelastosis has been classified as primary and secondary . In the primary type, there is no structural cardiac abnormality, whereas in the secondary type, a cardiac abnormality is demonstrable . The general agreement is that any severe myocardial stress is the basic pathology leading to endocardial fibroelastosis . The case illustrated here shows that efe may result from myocardial toxicity induced by organophosphorus poisoning, which may completely regress without further complications and is not a definite indication for the termination of pregnancy . The use of intensive maternal corticosteroid administration must have contributed to the complete regression of efe.
The majority of tooth extraction procedures are performed using a handpiece accompanied by a thin bur to remove both the tooth and the bone . Several articles have reported on the risk of percutaneous injuries to the surgeon caused by the bur, and injuries to the hand are the most commonly reported123 . In many cases, when the surgeons use the elevator to dislocate the third molar, they must stand beside the patient . Thus, due to the proximity of the handpiece to the surgeon's leg, the bur can accidently penetrate the lower limb, leading to an external injury . This report presents one such case and considers the precautions needed to avoid similar accidents . A 24-year - old male patient with no systemic medical history visited the department of oral and maxillofacial surgery of wonkwang university daejeon dental hospital (daejeon, korea) because of discomfort in his mandibular third molar . The tooth was extracted by creating an envelope flap and exposing the tooth and the bone, and a fissure bur connected to a handpiece was used to remove the bone of patient . After performing luxation using the elevator to dislocate the tooth, the surgeon, who was in a standing position, turned his body to pick up another instrument . At that moment, because the location of handpiece in a fixed upright position at the front of the instrument tray, the fissure bur broke and penetrated the surgeon's right femoral region . The handpiece was immediately removed, but the head of the bur was not found . Once the surgeon finished extracting the patient's tooth, the injured area was disinfected with potadine ointment (povidone iodine 450 g / bottle . ; samil, seoul, korea). Radiographs of the right femoral region were then obtained at various angles to determine the location of the head of the bur, which was found in the upper medial area of the right vastus lateralis muscle. (fig . 1) under local anesthesia, a 1-cm incision was made to verify the radiographic findings, and the bur was removed. (fig . 2) subsequently, the area was dressed with potadine ointment and sutured using 5 - 0 nylon sutures . To prevent tetanus and bacterial infection, intramuscular hyper - tet injection (green cross, yongin, korea) was administered, and antibiotics and analgesics were also administered for 3 days . Daily wound dressing was also performed for 10 days, and the treatment regimen was concluded with stitch removal . Approximately 1 month after the incident, no postoperative complications had been observed . Occupational injuries caused by sharp instruments such as needles, burs, blades and surgical wires occur frequently among dentists . Previous studies have described the danger of percutaneous injuries to the hands and fingers of both the patient and the surgeon due to the tools used during dental treatment . However, percutaneous injuries caused by burs have rarely been reported . Cleveland et al.2 reported in 1995 the percentage of percutaneous injuries associated with dental tools: endodontic files 1%, utility knives 5%, burs 2%, explorers 2%, needles 1%, pliers 1%, excavators 1%, and other instruments 1% . Also in 2007, a target study conducted with dental healthcare personnel reported that the percentage of percutaneous injuries associated with needles is 46%, burs 10%, scalpels 7%, scalers 5%, elevators 5%, explorers 4%, wires 3%, and other instruments 20%4 . Even though instrument breakage in surgical procedures involving the bur is common5, carlton et al.6 noted that puncture wounds occurred frequently in patients during oral maxillofacial surgery, but the proportion of such wounds occurring in the surgeon is low . To the best of our knowledge, this article is the first to report a case in which the surgeon suffered a percutaneous injury in the femoral area due to bur breakage . Complications from cutaneous penetration of burs include infections through direct contact with patient blood, including viral transmission (e.g., human immunodeficiency virus, hepatitis b and c viruses) and tetanus . A puncture wound from contaminated instruments carries the same health risks as a human bite7 . In this case, the injury was not serious, as it did not result in a severe soft tissue laceration, and the bur did not damage the bone or move to other parts of the body . We should have performed serological tests immediately, as blood - borne pathogens can lead to cross - infection . However, the patient didn't have a relevant medical history, so we thought that serological tests were not necessary . If a similar incident resembling our report occurs in the future, blood tests should be considered . The main treatment method in cases involving penetration of foreign substances is to completely remove the debris and to provide prophylaxis against potential infections with antibiotic treatment8, as well as through tetanus immunoglobulin shots9 . Similar to the case described in this report, numerous dentists experience light abrasions from the scaler tip because of the relatively close location of the dentist's chair to the patient . Although changing the design of the handpiece delivery system to that of the european dental unit (continental dental unit; adec inc ., bristol, in, usa) could help prevent such injuries, several studies have reported no difference in the incidence of percutaneous injuries associated with these systems10 . In addition, the main reason for bur breakage is the reuse and repeated disinfection of the instrument5 . Therefore, it is important to avoid overuse of the equipment . The most important factor, however, is to focus on prevention and care . Through this article, we hope to have provided guidance for oral and maxillofacial surgeons regarding the procedures and precautions necessary to avoid (or care for) accidental penetration of the femoral region with a piece of equipment, which can occur during extraction of the third molar.
The developmental origin of coronary hearth diseases proposes that undernutrition in utero permanently changes body functions and metabolism leading to an increased risk of coronary artery diseases (cad) in adult life 1 . The association between low birth weight (bw) and risk factors for cad, hypertension and type 2 diabetes reported in various longitudinal studies 2 - 5 is in line with the developmental theory . Some studies, however, suggest that birth weight may not be a major risk factor for development of hypertension and cardiovascular disease 6,7 . Gender differences in the association between bw and risk factors for cad have been reported in some studies 8 - 10, but not in others 11 . Wilkins and murphy 12 have suggested that gender - specific genes affecting insulin sensitivity are responsible for the gender difference in birth weight: females would be genetically less responsive to the trophic effect of insulin resulting in a lower birth weight . Hormonal differences between sexes influence intrauterine development and may interact with genetic and environmental factors resulting in a different pattern of susceptibility in adult life to coronary artery diseases in the present study we have considered the effect of gender and diabetes on the relationship between bw and cad . 226 subjects admitted consecutively for treatment of cad to valmontone hospital in rome, italy were studied . The sample may be representative of subjects with non fatal cad living in the area around the hospital . 395 consecutive newborn infants from the white population of rome studied between 1968 and 1972 were considered as controls . Among subjects with cad only 127 were found to have reliable information on birth weight . Informed consent was obtained by the patients to participate in the study that was approved by the ethical committee of valmontone hospital . Three way contingency tables were analyzed by a log linear model according to sokal and rohlf 13 . Given three variables a, b and c by this analysis is possible to study the effect of the categories of a variable (i.e. C) on the association between the other two variables (i.e. A and b). Chi square of independence, t test for difference between means and logistic regression were carried out by using the spss package 14 . Logistic regression is similar to discriminant analysis: it is useful to study the effect of categorical variables to separate classes of a dependent variable . Table 2 shows the distribution of bw in relation to gender . Among cad cases the distribution of bw classes was found to depend on gender (p=0.009), while in healthy newborns no statistical significant association was observed (p=0.541). Three way contingency table analysis shows a statistically significant interaction confirming that the pattern of association bw - gender observed in cad cases is different from that observed in healthy newborns . In females with cad there is a tendency toward a low bw, while in males with cad there is a tendency toward a high bw . Table 3 shows the mean birth weight in relation to gender . Among cad cases bw is higher in males than in females (p=0.001) while no statistically significant difference is observed among healthy newborns (p=0.688). In table 4 we examined the effect of diabetes on the association between bw and gender among cad cases . A highly significant association was observed in non - diabetics (p=0.001) while no significant association was observed in diabetic subjects (p=0.567). In non - diabetics there is a marked decrease of females with high bw and a marked increase of males with high bw . A three way contingency table analysis shows a highly significant interaction (p=0.01) suggesting that the pattern of association bw - gender in non - diabetics is different from that observed in diabetics . A logistic regression analysis with cad as dependent and birth weight and gender as independent has shown a highly significant effect of birth weight (p<0.01) and of the interaction between birth weight and gender (p<0.01) (data not shown). Correlation analysis between birth weight and systolic and diastolic blood pressure is shown in table 5 . Among females there is a positive correlation between birth weight and both systolic and diastolic blood pressure, while in males no correlation is observed . The negative correlation between bw and risk of cad present in females might appear in favour of the hypothesis that gender specific genes affecting insulin sensitivity present in females are responsible for low birth weight and risk of cad in adult life 12 . However the fact that the negative relation between bw and risk of cad is not present in females with diabetes argues against this hypothesis . Although the present study broadens the field of possible etiological elements of cad, various limitations are in order . The sample size is relatively modest and the pattern of associations should be re - examined in larger samples . The differences between males and females observed in our population could be due to differences in clinical severity leading to different rates of mortality between sexes . Indeed our cases include non fatal cad only and we have no information on fatal cases . Cad birth weight should be compared with birth weight of adults of the same age without cad . Birth weight of newborns refers to the period 1968 - 1972 while birth weight of cad patients refers to a period about twenty years before . However no appreciable difference in birth weight has been registered in italian population during that period . At present it is unclear which exposure in early life might be responsible for the association between fetal growth and cad in adult life . Moreover some observations indicate that nourishment before birth is not crucial to adult health 15 . The developmental origin theory of cad is generally presented in opposition to the current orthodoxy that cad results from unhealthy life styles with a contribution of genetic factors . The two theories are compatible and many factors may contribute to susceptibility to cad . Moreover section a depicts a true correlation while section b shows that genetic factors influencing bw may independently influence the risk of cad leading to a spurious correlation between bw and cad . It is becoming increasingly evident that the maternal - fetal relationship has an important role not only in intrauterine survival and development, but also in the well being of offspring during extra uterine life . Additionally, the data presented here suggest that the developmental origin theory of cad deserves increased attention in future investigations . Several studies point to a significant immune component in the pathogenesis of atherosclerosis that shows many feature of a chronic inflammatory disease 16 - 18 . Moreover it has been recently reported that a genetic polymorphism of ptpn22 19, known to be associated with autoimmunity, is associated with atherosclerosis 20 . Maternal and fetal genetic factors interact during intrauterine life in a manner that influences fetal development and the immunological orientation of the fetus . As a result, these interactions could modify the susceptibility of the individual to those diseases having an immunological component . Recent studies suggest that microchimerism is a common condition mainly due to the fractures of the placental barrier that allow the passage of cells in both directions during pregnancy . Microchimerism could contribute to immune attacks but may be also beneficial in special conditions 21,21 . Thus, the developmental origin hypothesis of cad also reflects an immunological facet that was not included in the original formulation . However, this association depends on gender: the risk of cad is correlated negatively to bw in females but positively in males . In the presence of diabetes this pattern of correlation disappears while it is much more evident in subjects without diabetes.
For millions of years, daily salt (sodium chloride) intake in man was about 1 g . Then recently, about 10,000 years ago, salt intake increased by about ten - fold because of the practice of using salt as a food preservative . It allowed former nomads to settle, grow grain and preserve foodstuffs over long periods . Over the last few millennia, humans got used to the taste of salt and enjoyed the benefits of non - perishable food . Genetically, humans are well - equipped with mechanisms that retain even tiny amounts of salt, a prerequisite for survival at those times when salt was scarce and intake was low . In keeping with this background, humans have less efficient excretory mechanisms when challenged with large salt loads, and the limiting factor is the rate of renal salt excretion . If salt intake exceeds the kidneys ability for salt excretion, then salt is deposited in the body, which, in synergy with aldosterone, affects heart, blood vessels and kidneys . It is estimated that at least 30% of the world's population develop hypertension (elevated blood pressure) when exposed to a high salt diet . In the past, salt sensitivity was thought to be the result of kidney malfunction, i.e. On the imbalance between salt input and salt output . However, recent observations suggest that the vascular system may also play an important role in this imbalance . More than 20 years ago, it was shown that the vascular endothelium expresses sodium channels similar to those in renal epithelia . Some years later, it was demonstrated that sodium channel function in endothelium was regulated, much as it is in the kidney, by the mineralocorticoid hormone aldosterone . The fact that the vascular system is also a potential target for aldosterone led to a paradigm shift in so far as the attention was no longer directed solely to the kidney but also to the vascular system [13 - 20]. Currently, there are far more data on the pathophysiology of aldosterone affecting vascular function than on the normal vascular physiology of this steroid hormone [21 - 33]. Sodium and aldosterone synergistically act on the endothelium . At cellular level, small changes in plasma sodium concentration can have a large impact on endothelial function as long as aldosterone (or aldosterone receptor function) is available . Even a 5% increase in plasma sodium concentration mechanically stiffens endothelial cells by about 25%, leading to cellular dysfunction (decreased nitric oxide release / increased vascular smooth muscle tone). A major component of this high sodium sensitivity is the sodium channel in the endothelial plasma membrane, which is identical to the epithelial sodium channel cloned from renal tissue . This channel allows sodium to enter the endothelial cells and, by yet unknown mechanisms, turn off endothelial nitric oxide synthase activity . Do these in vitro experiments translate into the in vivo setting and explain how plasma sodium as such affects blood pressure? This question is not easy to answer since changes in plasma sodium are usually accompanied by changes in osmolality, which may mask any direct action of sodium . However, a recent study properly corrected for any changes in osmolality, shows that there is indeed a marked alteration in blood pressure observable when plasma sodium is manipulated . Similarly, blood pressure in dialysis patients is known to decrease when sodium concentration in the dialysate is lowered . Furthermore, small but significant changes in plasma sodium, paralleled by concomitant changes in blood pressure, are known to occur in humans during acute or chronic salt intake . Finally, there is experimental evidence that the brain may be involved in the sodium - triggered increase of blood pressure . Blaustein and colleagues postulated an interesting hypothesis, namely that high sodium in the cerebrospinal fluid triggers the secretion of endogenous ouabain in the hypothalamus and suprarenal glands . Endogenous ouabain acts in the brain, increasing sympathetic nerve activity, but also acts on blood vessels . Both endogenous ouabain actions lead to vasoconstriction and increase in blood pressure . For obvious reasons, this more complex mechanism, involving different organs, most recently, the endothelial surface layer facing the blood stream has become a focus of interest [46 - 54]. This soft layer, termed endothelial glycocalyx, is a negatively charged biopolymer known to preferentially bind sodium . It has been calculated that about 700 mg of sodium can be transiently bound to the endothelial glycocalyx in the human body, which is about the amount contained in a single meal . Interestingly, the sodium buffering capacity of the endothelial glycocalyx is severely damaged by excessive sodium intake over time, leading to a significant reduction of the negatively charged heparan sulphate residues in the endothelial glycocalyx . These observations led to a new concept of vascular sodium permeability, namely that two (more or less) permeable barriers determine the rate of sodium elimination after a salty meal . One barrier is the endothelial plasma membrane, with a variable sodium permeability depending on the abundance of epithelial sodium channels . The other barrier, located on the surface of the endothelium, is the endothelial glycocalyx that transiently buffers ingested sodium and, thus, controls access to the epithelial sodium channels (figure 1). Sodium channel activity and glycocalyx function are inversely related to each other . A plasma sodium concentration in the high - physiological range (> 140 mm) reduces the negatively charged heparan sulphate residues of the endothelial glycocalyx, increasing the amount of sodium reaching the endothelial sodium channels, which in turn makes the channels more active . The overall effect is that the barrier against sodium entry fails under these conditions and the endothelium becomes more permeable to sodium (figure 2). As indicated above, evidence for a salt - sensitive endothelial glycocalyx and its relationship to endothelial epithelial sodium channels is based on in vitro experiments and, at best, on ex vivo studies in human tissue (e.g. Human umbilical veins). To our knowledge, there are no direct studies in humans . However, if aldosterone is viewed as a hormone that facilitates sodium retention and epithelial sodium channel expression in the vascular endothelium, then a link between salt and glycocalyx, albeit indirect, becomes visible . Clinical research often describes potentially important phenomena in the human without a mechanistic model . In this case, a mechanistic model based on in vitro experiments is available first, and it will be up to clinical research to test it in the human . This state of vascular function is associated with low daily sodium intake, and/or low aldosterone and/or favorable genetics . This state of vascular function is associated with high daily sodium intake, and/or high aldosterone and/or unfavorable genetics . After a salty meal, the translocation of sodium from the blood into the interstitium is delayed by the significant buffering capacity of the endothelial glycocalyx . Sodium will reversibly bind to / dissociate from the endothelial glycocalyx binding sites and, thus, can be readily excreted via the kidneys . Excessive sodium intake over time will damage the endothelial glycocalyx and lead to a decrease in its sodium buffering capacity because of the loss of negatively charged heparan sulphate residues . Following that, sodium gains direct access to the " unprotected " epithelial sodium channels . Thus, in addition to the paracellular transport route (i.e. Sodium transport between endothelial cells along its chemical gradient), sodium uses the transcellular pathway for entering the large extracellular space (about 30% of body weight). There, sodium is bound reversibly to the extracellular matrix [59 - 63]. After the ingested sodium has spread throughout the body, the plasma sodium concentration decreases . Now, sodium starts diffusing back into the vascular bed (along its chemical gradient directed from interstitium to blood) and will finally be excreted by the kidneys (figure 2). This detour of sodium " through the whole organism delays renal sodium excretion significantly . In the meantime, a sodium load from the next salty meal may arrive and so on, leading over time to sodium accumulation in the organism . Vascular salt sensitivity can be defined as the ratio of " endothelial sodium channel activity over endothelial glycocalyx sodium buffer capacity " . It should be noted that vascular salt sensitivity is not thought to be exclusively congenital [8,64 - 66] but could most likely be influenced (among other yet - unknown factors) by the amount of ingested salt and by endogenous aldosterone . As salt sensitivity correlates positively with sodium channel activity in the endothelium, the blockade of these channels by amiloride analogues should help in identifying individuals with a high sensitivity to salt . The test is described in general terms in figure 3 (see figure legend and, for more details, see). The four cylinders symbolize the vascular system . After an acute na load (5 g nacl orally), with and without addition of an epithelial sodium channel blocker (two separate sessions), blood pressure low vascular sensitivity is indicated by a ~ zero, high vascular sensitivity is indicated by a> zero . The endothelial glycocalyx appears to be a key structure for regulating body sodium . In vitro studies show that the quality of this biopolymer layer is determined by the abundance of its electrical negative charges . Loss of these surface charges renders the endothelial surface vulnerable to unwanted intruders, among them excessive sodium . We look forward to clinical research that will hopefully confirm these results in vivo in humans and substantiate the mechanism behind the protective effect of a low salt diet on the cardiovascular system.
It is freely available to any one who allows adequate exposure of skin to the ultraviolet radiation present in the sunrays . However, vitamin d deficiency is not uncommon even in the sunny land of india . The clinical syndrome vitamin d deficiency consists of osteomalacia in adults and rickets in children . Other manifestations of vitamin d deficiency include nonspecific backache, joint pains, and generalized body ache . With the availability of a method to accurately measure 25-hydroxyvitamin d [25(oh) d], a state of hypovitaminosis d without overt signs and symptoms of vitamin d deficiency has become apparent [1, 2]. The amount of vitamin d provided by sun exposure and diet is difficult to determine as the duration and intensity of sunlight exposure depends on age, clothing, and sunscreens . The insufficient intake of vitamin d has been implicated as one of the factors in the development of bone disease [46]. Various elements contribute to this insufficient vitamin d intake such as skin pigmentation, which presumably interferes with ultraviolet ray transmission through epidermis, genetic factors [8, 9], social customs such as avoidance of sun exposure, consumption of chapattis (flattened rounded wheat bread) that is high in phytates which bind calcium in the gut and interfere with calcium absorption, lack of sun exposure and malnutrition to name a few important contributing factors . This is exemplified by the high prevalence of vitamin d deficiency in house bound and elderly patients [1113]. While osteomalacia and rickets have been studied extensively, there is little information on vitamin d deficiency without obvious bone disease . The aims and objectives of this study were (a) to study the clinical symptoms and signs of vitamin d deficiency and their correlation with 25-hydroxyvitamin d [25(oh) d] levels, (b) to study the bone profile of patients of hypovitaminosis d, and (c) to observe the response to treatment with vitamin d on these individuals . The patients were seen between 1996 and 2001 at the rheumatology services of the hinduja hospital and medical research centre, mumbai, india . A proforma was prepared to record relevant details (appendix a - proforma: profile of vitamin d deficiency). This included details regarding symptom and signs, dietary history, duration and severity of illness, associated illnesses, sun exposure, renal disease, gastrointestinal tract disease and intake of anticonvulsants such as diphenylhydantoin sodium . The treatments given to these patients were recorded and their responses to treatment were noted . For the details not available in the medical chart, an attempt was made to contact the patients on telephone or in person to get complete information . Of these 38% (n = 27) were in the age group of 3140 years . Vitamin d deficiency in this population, while symptomatic, was without any of florid signs (63% showed no tenderness as shown in table 3). The bone profile of patients with vitamin d deficiency is shown in table 4 . The radiological data showing various vitamin d associated changes in the study's patients are shown in table 6 . Taking the normal range of vitamin d, 25(oh) d in our laboratory as 9.941.5 ng / ml, we classified our patients into 3 grades of vitamin d deficiency . The grades were mild 25-hydroxyvitamin d [25(oh) d] levels6 to 9.9 ng / ml, moderate 25-hydroxyvitamin d [25(oh) d] levels2 to 5.9 ng / ml, and severe 25-hydroxyvitamin d [25(oh) d] levels <2 ng / ml . The distribution of severity of vitamin d amongst these patients is shown in table 7 using these parameters . Vitamin d deficiency is variably defined as a 25(oh) d level less than 1015 g / l [16, 17]. The commonness of vitamin d deficiency in an otherwise healthy population was an eye - opener as well as a teaser . At the hinduja hospital and medical research center, mumbai, india, the awareness of vitamin d deficiency started with appreciation of vitamin d deficiency as a complicating factor in rheumatic diseases . A case is briefly described to make the point . A 59year - old male patient with ankylosing spondylitis of more than 30 years duration was admitted with a recurrence of left - sided hemiparesis and a recent increase in joint pains . He had diabetes for the past 10 years which was controlled on oral hypoglycemic agents . A year before his admission, he had developed left - sided hemiparesis from which he had recovered completely . The increase in joint pains started six months before he was admitted but he reported no swelling or stiffness at that time . There was no evidence of synovitis of peripheral joints, but he had severe diffuse bony tenderness, especially of his spine and ribs . Investigations revealed the following statistics: hemoglobin: 12.2 gm / dl, white blood cell count: 16,700/mm, platelets: 2.3 lac / mm, esr: 28 mm / h, serum calcium: 8.3 mg / dl (normal range: 8 to 10.4 mg / dl), phosphorus 2.9 mg / dl (normal range: 2.54.6 mg / dl), alkaline phosphatase: 590 iu / l (normal range: 40120 iu / l), 25(oh) vitamin d: 4.3 ng / ml (normal range: 9.941.5 ng / ml) and parathormone 333 pg / ml (normal range: 1272 pg / ml). A routine urine test showed trace proteins, 24 hours urinary proteins: 792 mg (0165 mg) and creatinine 1 mg / dl (normal range: 0.6 to 1.01 mg / dl). He was diagnosed with vitamin d deficiency - related osteomalacia with secondary hyperparathyroidism being made and he was initiated on a vitamin d treatment regimen (alpha d3 and vitamin d 60 000 units), along with one gram supplemental calcium . Within one week his pains started to decrease and were significantly more manageable in three weeks time and he was discharged . At this man's medical case was followed by the publication of a small series of letters to the editor in the journal of association of physicians of india . Soon patients with a variety of nonspecific musculoskeletal complaints were tested for vitamin d status . To our surprise thus of the samples tested for vitamin d levels in our hospital laboratory, 80% had low serum 25-hydroxyvitamin d [25(oh) d] levels, that is, <9.9 ng / ml . It therefore became imperative that we study the significance of low vitamin d levels in adults for clinical, therapeutic, and other aspects . Thus the case selection was based upon the presence of low vitamin d levels in individuals attending the clinic, for various nonspecific musculoskeletal symptoms . The present study has its inherent limitations but was thought necessary to understand the phenomenon of low vitamin d levels . All the study patients had musculoskeletal complaints and were suspected to suffer from rheumatoid arthritis, ankylosing spondylitis, or vertebral disc disease . Although it is not possible to judge the exact proportion of such patients, vitamin d deficiency is an important differential diagnosis of patients with such complaints . In rheumatology parlance, fibromyalgia and hypothyroidism closely resemble this symptomatology, and hence myalgia and nonspecific pain may be due to vitamin d deficiency . Muscle weakness led to inability to get up from squatting position and waddling gait in some patients . Vitamin d deficiency is an important consideration in a patient with proximal muscle weakness, (and may additionally complicate a case of polymyositis - dermatomyositis). Vitamin d deficiency in this population was without florid signs (63% showed no tenderness). This is important, as without bony tenderness (which would suggest osteomalacia) and without proper awareness on the part of physicians, vitamin d deficiency can easily be overlooked . When we tried to correlate the vitamin d levels with the symptoms (as shown in table 7), we came to the conclusion that although patients with low vitamin d levels present many nonspecific symptoms like backache, body ache, leg pain, and thigh pain, not all are osteomalacic . How does one explain the absence of clinical osteomalacia in individuals with very low vitamin d levels? Is it likely that even at low vitamin d levels, there is enough 1, 25 (oh)2 d3 to maintain homeostasis? Although this current study does not possess the funding to answer this question, it remains an interesting line of inquiry . There may be (over) active conversion of 25 (oh) d to 1, 25 (oh) 2 d3 to maintain bone mass . A parallel can be drawn with iron deficiency anemia; until body iron stores are exhausted iron deficiency does not manifest as anemia . An attempt was made to see if the severity of vitamin d deficiency correlated with the number of symptoms recorded (as shown in table 7). (unfortunately, the severity of individual symptoms cannot be analyzed as this was not recorded using a visual analogue scale .) Still there does not seem to be any such correlation given that only one patient had more than four out of the seven symptoms analyzed . We compared the records of 12 patients who were diagnosed as osteomalacic and found that 50% of the patients had vitamin d levels that measured at levels below 5 ng / ml . It is apparent that individuals with vitamin d deficiency may not suffer from clinical osteomalacia but can have nonspecific musculoskeletal symptoms accompanied with morbidity . It is likely that some may go on to develop frank nutritional osteomalacia unless treated . This is because, compared with the commonness of vitamin d deficiency, clinical osteomalacia is not a common disorder . It is difficult to guess the prevalence of vitamin d deficiency in the community, but it does seem logical to assume that osteomalacia (as well as rickets) are only the tip of the iceberg . The common etiological factors of vitamin d deficiency are lack of exposure to sun rays (solar ultraviolet - b exposure to as much of body surface as possible produces vitamin d) and decreased intake of foods rich in vitamin d. a specific history was not recorded in all patients studied, but a majority of these patients were vegetarians, with poor intake of milk or milk products and little outdoor activity . None of the patients belonged to a low socioeconomic group and hence likely had access to plenty of food . Thus paradoxically, vitamin d deficiency may be a disease of the rich! The case involves a 50-year - old gentleman, a businessman and an epileptic on diphenylhydantoin sodium living in a house with drawn curtains, and traveling in a car with tainted glass . When seen by the physician, he was nearly crippled by back pain with frank evidence of osteomalacia . The pain was treated as spondylosis, and he was even suspected to be suffering from tuberculosis!. Vitamin d, calcium, and regular sun exposure brought him back to normalcy . When we checked the response to vitamin d and calcium therapy, we found that out of 20 patients placed on calcium and/or vitamin d, 13 patients showed significant clinical improvement . Also followup information was not provided for 51 of these patients, and so it is impossible to speculate about their response to treatment . It should be noted that the cause for nonresponse to treatment in some patients may be due to factors other than vitamin d deficiency . Additionally, the symptoms these patients report, symptoms that indicate a lack of vitamin d, may also be caused by other incidental factors . For example a recent controlled study found that diffuse musculoskeletal pain may not be associated with low vitamin d and that this pain does not respond to treatment with vitamin d . As already stated vitamin d deficiency can complicate a rheumatic disease and make diagnosis difficult [18, 2023]. G, a 46-year - old female patient suffering from rheumatoid arthritis for more than 15 years had tried a few disease modifying agents at the initial stages of the disease without beneficial effects . Eight years prior to being seen, she had undergone a left hip replacement . For five of those years, she was bed ridden with deformities and complained of severe, debilitating, unbearable aches and pains . She could not even turn in bed, move her legs, or lift her arms due to the pain . Investigations revealed undetectable levels of vitamin d, low serum calcium, phosphorus, and increased alkaline phosphatase . A diagnosis of concomitant osteomalacia was made, and she was placed on three weekly bolus of 60 000 units of oral vitamin d followed by a daily supplement of 1.5 grams of calcium . Over a period of 1 month, her bone pains reduced significantly . She was able to lift her arms, eat with her own hands, and sit without support . The case was an eye - opener . In every case with pains out of proportion to clinical findings, it would be wise to check for vitamin d deficiency symptoms . Since this was a retrospective study where the study participants had to recall their dietary history, type of indoor and outdoor activities performed, and history of sun exposure, a recall bias is evident . Furthermore the data on assessment of muscle weakness were incomplete and were not evaluated by another independent rater . This small retrospective study shows that vitamin d deficiency is not uncommon but frank osteomalacia is uncommon . A subclinical vitamin d state exists which is characterized by nonspecific musculoskeletal symptoms . This state may complicate a rheumatic disorder, and it is important to consider this diagnosis as the results of treatment are most gratifying . Three conclusions can be drawn from this study: (a) vitamin d deficiency runs across the board; importantly it affects individuals in the prime of their life and affects the quality of life and the productivity of those suffering from it; (b) the preponderance of female patients compared to male patients suffering from vitamin d deficiencies possibly reflects social factors like the avoidance of milk and milk products and staying indoors; (c) a large chunk of vitamin d deficiency is subclinical . If estimation of vitamin d is not possible, a trail of oral vitamin d supplements and calcium is indicated to be beneficial . There is a new and emerging body of literature showing relationship between vitamin d deficiencies and respiratory infections, along with obstetrical conditions such as pre - eclampsia.
Chronic lung disease (cld) is still one of the major causes of morbidity and mortality in very low birth weight infants (vlbwi). From the early 1980s, dexamethasone has been administered to ventilator - dependent infants with cld, as several reports that dexamethasone improves the lung function and enables earlier extubation were published (1, 2). In the 1990s, postnatal dexamethasone was administered to preterm infants almost as a routine to reduce the time spent on the ventilator and to prevent cld (3). However, it has been known that although postnatal dexamethasone reduces the length of assisted ventilation and the incidence of cld both at the 28th day of life and at the 36th postmenstrual week, it does not reduce the length of hospital stay and mortality ultimately (4 - 9). Moreover, postnatal dexamethasone has been reported to be able to produce not only short - term adverse reactions such as gastrointestinal bleeding, intestinal perforation, sepsis, meningitis, and hyperglycemia, but also long - term adverse reactions such as adrenal suppression and growth failure (10 - 15). Even more remarkably, recently, there has been a series of reports that the use of postnatal dexamethasone is associated with developmental delay and cerebral palsy (16 - 19). Therefore, nowadays there is a trend not to use postnatal dexamethasone routinely simply for the prevention of cld in many neonatal intensive care units (nicu) (20 - 22). Similarly, in our nicu, considering the potential harm of postnatal dexamethasone to the developing brain, we have been reducing the use of postnatal dexamethasone gradually . We have attempted to confine postnatal dexamethasone therapy to infants who were still ventilator - dependent beyond the second week of life and showed ongoing deterioration in ventilator settings and radiological finding for more than 3 to 7 consecutive days . Moreover, in case of postnatal dexamethasone therapy, we have tried to defer it after the second week of life, and use it in a low dose . However, there have been persistent concerns about the increased risk for the cld due to the decreased use of postnatal dexamethasone (23, 24). Nevertheless, there are few studies looking at the change in the incidence of cld according to the decreased use of postnatal dexamethasone . Therefore, we did this study to delineate the change in the incidence of cld according to the gradual reduction of postnatal dexamethasone use in vlbwi in our nicu . Five hundred fifty nine vlbwi weighing less than 1,500 g at birth who were admitted to nicu unit at samsung medical center between november 1994 and december 2002 were enrolled . To see the trends over time, we divided the study period arbitrary by a two - year interval into four eras: era 1, november 1994-december 1996; era ii, january 1997-december 1998; era iii, january 1999-december 2000; era iv, january 2001-december 2002 . We reviewed the medical records of subject infants with a focus on following variables: demographic variables including birth weight, gestational age, and sex; clinical characteristics including chorioamnionitis, respiratory distress syndrome (rds), patent ductus arteriosus (pda), retinopathy of prematurity (rop) requiring laser or cryotherapy, high grade intraventricular hemorrhage (ivh grade iii, volpe's grading system, 25), periventricular leukomalacia (pvl), the extent of initial physiological weight loss, the length of synchronized intermittent mandatory ventilation (simv), the length of nasal continuous positive airway pressure (ncpap), the length of supplemental oxygen (fractional concentration of oxygen, fio20.3), and mortality; variables associated with postnatal dexamethasone therapy and cld including the use of antenatal steroid, the use of postnatal dexamethasone, the day when dexamethasone therapy was begun, the dose of dexamethasone used, and the incidence of cld at the 28th day of life and at the 36th postmenstrual week . Gestational age was based on obstetrical record and survival was defined as when a vlbwi left the hospital alive fulfilling the indication of discharge in our nicu: weighing over 1,800 g and gaining weight steadily about 20 - 30 g / day; ability to maintain temperature in an open crib; no need for any parenteral drugs or fluids . Chorioamnionitis was defined as histological chorioamnionitis or umbilical cord vasculitis of grade 2 or greater according to the grading system suggested by salafia et al . The diagnosis of rds required the presence of respiratory distress, increased oxygen requirement (fio20.4) and radiological and laboratory findings consistent with rds in the absence of the evidence of other causes of respiratory distress . Cld was defined by the need for supplemental oxygen (fio20.3) at the 28th day of life or at the 36th postmenstrual week with consistent radiological finding . In our nicu, high - dose regimen was as follows: starting at 0.5 mg / kg / day divided every 12 hr for 48 hr, then halving the dose every 48 hr for the next 5 days completing a total of 7 day - regimen . Low - dose regimen was the same as high - dose regimen except that the starting dose was 0.2 mg / kg / day . All categorical variables were designated as percent (%), and continuous variables were designated as meanstandard deviation . For this purpose, we used score test for trend (jonckheere - terpstra test for continuous variables, and linear by linear association for categorical variables). There were no significant difference in gestational age and sex by the four study eras (table 1). However, birth weight showed a significant trend to decrease as the time elapses from era i to era iv (p<0.01). There were no significant differences in the incidence of rds, pda, rop requiring laser and cryotherapy, and ivh (grade iii) by the four study eras except the incidence of pvl that revealed a significant trend to decrease as the time passes (p<0.05). There were no significant differences in the length of hospital stay by the study eras . However, the length of simv and supplemental oxygen, and mortality showed a significant trend to decrease as the time elapses (p<0.001, p<0.001, p<0.01, respectively), while the extent of initial physiological weight loss and the length of ncpap revealed a significant trend to increase as the time passes (p<0.001). The use of antenatal steroid showed a significant trend to increase as the time elapses from era i to era iv (p<0.001). However, the use of postnatal dexamethasone in vlbwi has significantly decreased as the time passes (p<0.001) (table 2). Especially, the use of high - dose regimen has markedly decreased (p<0.01), while the use of low - dose regimen did not reveal a significant trend across the study eras . The incidence of cld at the 28th day of life and that at the 36th postmenstrual week have all decreased significantly as the time elapses with the latter more evident (p<0.05, p<0.001, respectively). There were no significant difference in gestational age and sex by the four study eras (table 1). However, birth weight showed a significant trend to decrease as the time elapses from era i to era iv (p<0.01). There were no significant differences in the incidence of rds, pda, rop requiring laser and cryotherapy, and ivh (grade iii) by the four study eras except the incidence of pvl that revealed a significant trend to decrease as the time passes (p<0.05). There were no significant differences in the length of hospital stay by the study eras . However, the length of simv and supplemental oxygen, and mortality showed a significant trend to decrease as the time elapses (p<0.001, p<0.001, p<0.01, respectively), while the extent of initial physiological weight loss and the length of ncpap revealed a significant trend to increase as the time passes (p<0.001). The use of antenatal steroid showed a significant trend to increase as the time elapses from era i to era iv (p<0.001). However, the use of postnatal dexamethasone in vlbwi has significantly decreased as the time passes (p<0.001) (table 2). Especially, the use of high - dose regimen has markedly decreased (p<0.01), while the use of low - dose regimen did not reveal a significant trend across the study eras . The incidence of cld at the 28th day of life and that at the 36th postmenstrual week have all decreased significantly as the time elapses with the latter more evident (p<0.05, p<0.001, respectively). Around the year of 1999, a number of investigators reported that postnatal dexamethasone therapy for the prevention or treatment of cld might produce adverse neurodevelopmental outcome (11, 16 - 19, 27, 28). Recognizing this potential neurodevelopmental risk of postnatal dexamethasone, we have tried to reduce the use of postnatal dexamethasone for the prevention or treatment of cld in our nicu . The results of present study indicate that in our nicu, the use of postnatal dexamethasone, especially high - dose regimen has remarkably decreased as the time elapses from era i to era iv . Moreover, a large majority of the studies that reported the neurodevelopmental risk of postnatal dexamethasone dealt with the early treatment regimen that dexamethsone is given to vlbwi within the first two weeks of life, particularly within the first four days of life . Therefore, we have also tried to delay postnatal dexamethasone therapy after the second week of life . Actually, our results demonstrated a trend that the time when dexamethasone therapy was begun has been deferred from 147 postnatal day in era i to 2914 postnatal day in era iv . Our results also show that the incidence of cld has not increased, instead it has decreased despite the reduced use of postnatal dexamethasone during the same period . Many factors might be responsible for this decrease in the incidence of cld . In the present study, we observed the trends in the factors that have been previously known to be associated with the development of cld over time during the study period . Birth weight, gestational age, and the incidence of pathologically proven chorioamnionitis, rds, and pda have not changed significantly during the study period . However, the extent of initial physiological weight loss, the length of simv and ncpap showed a significant change over time during the study period . The extent of initial physiological weight loss has increased as the time elapses from era i to era iv . This change in the extent of initial physiological weight loss might reflect the alteration in fluid management strategy in our nicu . We applied fluid restriction therapy (less than 80 ml / kg / day during the first week of life) since 1999 . There have been several reports that excessive fluid administration in the early postnatal period is associated with the development of cld (29, 30). Therefore, it is possible that the increased extent of initial physiological weight loss might have contributed to the decreased incidence of cld during the study period . However, the effect of fluid restriction therapy on the prevention of cld remains to be further evaluated by randomized case - control study . The decrease in the length of simv and the increase in the length of ncpap are related to each other, because we have tried to wean the infants from simv to ncpap as soon as possible since the latter half of the study period . Early weaning from simv to ncpap is one of the strategies to reduce the ventilator - induced lung injury, and has also been applied in many other nicus (20 - 22). Its preventive effect on cld is supported by several studies (31 - 33). In our study, the decrease in the incidence of cld might be partly attributable to this weaning strategy . The incidence of cld has significantly decreased not only at the 36th postmenstrual week but also at the 28th day of life . Considering that in the latter half of the study period, the era iii and iv, the mean day when dexamethasone therapy was begun was around end of the 4th week, the decrease in the incidence of cld at the 28th day of life in our cohort is not thought to be the effect of postnatal dexamethasone . In contrast to the postnatal dexamethasone, the use of antenatal steroid to the mothers of vlbwi has increased across the four study eras . Antenatal steroid is known to facilitate the fetal lung maturation resulting in the improvement of the pulmonary function in the postnatal period (34), and is being widely used routinely when preterm labor is anticipated unless the case is contraindicated . Reported that the vlbwi whose mothers had been given antenatal betamethasone developed pvl on cranial ultrasonography 2 times less frequently than the vlbwi whose mothers had not, while the vlbwi whose mothers had been given antenatal dexamethasone developed pvl 1.5 times more frequently than the vlbwi whose mothers had not (35). Shankaran et al . Reported that there were no differences in the neurodevelopmental prognosis of vlbwi according to the use of antenatal steroid (36). All the more, they asserted that antenatal steroid decreased the incidence of high - grade (grade iii) ivh . Similarly, leflore et al . Reported that antenatal steroid did not affect the incidence of pvl and the scores of bayley scales of infant development (37). Our results also revealed that the incidence of pvl has, at least, not increased across the four study eras, while the use of antenatal steroid has markedly increased during the same period . However, according to the study of jobe et al . 's, antenatal steroid can cause symmetrical intrauterine growth retardation (38, 39). From the above contradicting results, at present, the neurodevelopmental safety of antenatal steroid may not be guaranteed yet, and it requires further investigations . Aside from the probable neurodevelopmental risk of antenatal steroid, the inverse relationship between the trends in the frequency of antenatal steroid use and the incidence of cld across the study eras suggests a possibility that the decrease in the incidence of cld might be due to the increased use of antenatal steroid considering its beneficial effect to the lung function . However, that possibility might be low, because the incidence of rds that is thought to be the ultimate outcome of the beneficial effect of antenatal steroid did not show a significant change over time during the same study period . Moreover, in our recent epidemiologic study to assess the risk factors for cld in out nicu, rds was not a significant risk factor for cld (40). Our results demonstrated that although the use of postnatal dexamethasone has been reduced, the incidence of cld has not increased . This favorable outcome is thought to be attributable to the introduction of other measures for the reduction of the lung injury such as early weaning from simv to ncpap and fluid restriction therapy in the early postnatal period . Therefore, as long as the possibility that postnatal dexamethasone produces neurodevelopmental adverse effect remains, it may be desirable to refrain from the routine use of postnatal dexamethasone for the prevention of cld and restrict postnatal dexamethsone therapy to the ventilator - dependent vlbwi who aggravates relentlessly clinically and radiologically beyond the second week of life.
Acute lung injury (ali) and the acute respiratory distress syndrome (ards) represent a spectrum of diseases characterized by the rapid onset of pulmonary infiltrates and progressive hypoxemia in the absence of significant left ventricular dysfunction . Within the early phases of ali, the role of mechanical ventilation and its influence on patient outcomes has been an area of specific interest . It is now widely acknowledged that the use of excessive tidal volumes in patients with underlying ali can further perpetuate lung dysfunction while limiting injury through the use of lower tidal volumes has been the only therapeutic maneuver shown to improve survival . Furthermore, the proinflammatory response associated with the mechanical stress of ventilation, known as biotrauma, represents one of the key mechanisms by which mechanical ventilation may be critical in determining patient outcomes . Prior studies have demonstrated that the injured lung serves as the primary origin of proinflammatory mediators which may decompartmentalize into the systemic circulation [68]. Recent studies from our laboratory have shown that these lung - derived mediators are capable of eliciting the expression of surface adhesion molecules in liver endothelial cells both directly and in a tidal volume - dependent fashion [9, 10]. From a clinical perspective, it has been demonstrated that patients ventilated with low - tidal volumes had a reduction in plasma proinflammatory mediator levels compared to those patients ventilated by conventional strategies and, notably these levels correlated with a reduction in multiple organ failure . Although such evidence implicates the lung as the primary source of mediators leading to systemic inflammation, the specific mechanisms that serve to perpetuate and propagate the ensuing proinflammatory signaling cascade remain uncharacterized . For example, it remains unknown, whether the marked rise in plasma cytokines can be attributed entirely from a spillover phenomenon of a mechanically ventilated, injured lung to the systemic circulation or whether a primary inflammatory signal generated by the lung may be secondarily amplified by downstream peripheral organs . Therefore, characterization of the discrete signaling processes which drive persistent increases in systemic inflammatory mediators and the localization of their specific cellular origins may be critical in the development of effective therapeutic agents aimed at mitigating the inflammatory response resulting from mechanical ventilation . One of the intracellular signaling pathways most widely recognized for its importance in inflammation is the nuclear factor kappa b (nf-b) signaling pathway . It has been well established that many receptors activate the nf-b pathway, the most extensively studied of which are the interleukin (il), tumor necrosis factor (tnf), and toll - like receptor families . Canonical activation of the nf-b pathway involves phosphorylation of p65 (rela), and its translocation to the nucleus leading to a number of proinflammatory responses including the upregulation of adhesion molecules (on both endothelial cells and leukocytes) and transcriptional regulation of a wide array of cytokines and chemokines . Although activation of the nf-b pathway may be involved in the resolution of inflammation, particularly through its alternative pathway, we describe studies involving the acute phase of inflammation wherein the proinflammatory actions of nf-b activation predominate . In the current study, it was hypothesized that inflammatory mediators generated by the lung in response to mechanical ventilation are secondarily amplified by downstream organs in a herein, we demonstrate that lung - derived mediators are definitively upregulated by liver tissues in both in vitro and in vivo models of mechanical ventilation - induced inflammation . Further studies examining specific intracellular pathways responsible for mediator amplification demonstrate that activation of the inflammation relevant nf-b signaling pathway in liver endothelial cells is in part responsible for these observations . In order to obtain inflammatory mediators generated and released specifically from the lung into the systemic circulation, the isolated perfused mouse lung (ipml) model was employed . Lungs were mechanically ventilated using the ex vivo impl setup and lung perfusate was obtained after a completion of the ventilation protocol . Subsequently, mouse liver endothelial cells were exposed to lung perfusate to determine whether subsequent increases in inflammatory mediators were observed and the signaling processes that may be involved such as the inflammation - associated nf-b pathway . Furthermore the physiological relevance of these ex vivo and in vitro studies was validated using an in vivo model of mechanical ventilation to observe similar findings in intact whole liver tissues . Male mice were used for experiments (charles river, saint - constant, canada). All procedures were approved by the animal use subcommittee at the university of western ontario in agreement with the guidelines of the canadian council of animal care . All animals were acclimatized a minimum of 72 hours prior to use in the experiments and had free access to water and standard chow, lab diet rodent diet 5001 (pmi nutrition international, st louis, mo). A model of ventilation - induced inflammation was employed as previously described to obtain lung - derived mediators [8, 10]. Using this technique, male 129x1/svj mice weighing between 25 and 30 grams briefly, the pulmonary artery was initially isolated, cannulated, and secured using 4 - 0 silk . A second cannula was then inserted into the left ventricle and single pass of perfusate (rpmi 1640 lacking phenol red, + 2% low endotoxin grade bovine serum albumin; sigma, st louis, mo) was utilized to clear the lung of the remaining blood . Subsequently, a continuous reperfusion of the pulmonary circulation was performed using approximately 10 ml of perfusate . This perfusate was used to replace the blood within the pulmonary vascular compartment, while bovine serum albumin was included to maintain the integrity of the pulmonary vessels . Animals were mechanically ventilated with room air for a period of 2 h with a tidal volume (vt) of 12.5 ml / kg, respiratory rate of 30 breaths / min, positive end expiratory pressure (peep) of 3 cm h2o while using 5% co2 to maintain the ph of the bicarbonate - buffered rpmi . At the completion of the ventilation protocol, lung perfusate was collected and immediately stored at 80c . Lung perfusate was pooled and the levels of inflammatory cytokines in lung perfusate were determined using a millipore milliplex kit according to the manufacturer's protocol (millipore, billerica, ma) for ten inflammation relevant analytes using a multiplex assay . Samples were analyzed using the luminex xmap detection system on the luminex (linco research, st charles, mo) as per manufacturer's instructions . New non - circulated perfusate media (control perfusate) were used as a blank control in the elisa as well as a baseline or negative control in subsequent in vitro cell culture experiments . Steven alexander (louisiana state health sciences center, shreveport, la, usa). Mlec were cultured in (minimal essential media) mem d - valine (promocell, heidelberg, germany) supplemented with 10% fetal bovine serum (fbs), 2 mm l - glutamine (invitrogen, burlington, on), mem nonessential amino acids (invitrogen), mem vitamin mix (invitrogen), and 1% penicillin / streptomycin (invitrogen). Mlecs were seeded in a 24-well plate (6 10 cells per well) 2 days prior to the experiment . The confluent mlec monolayers were challenged for 8 h in a cell culture incubator with 0.25 ml of: (a) control uncirculated perfusate, (b) uncirculated perfusate containing cytomix using equal concentrations of tnf-, il-1 and interferon (if)- (10 ng / ml), (which has been used to simulate inflammatory conditions in cell culture), or (c) lung perfusate . The obtained conditioned media were analyzed with the millipore milliplex kit and luminex xmap detection system as described above . Mlec were plated two days prior to experimentation in 6-well (western blot) or 24-well (elisa) plates at 1.5 10 or 6 10 cells per well, respectively . Following stimulation, cells were washed three times with cold phosphate buffered saline (pbs) and lysed in a buffer containing 0.5% sodium dodecyl sulfate (sds), 1 mm ethylenediaminetetraacetic acid (edta), 50 mm tris ph 7.5 plus 1: 100 protease inhibitor cocktail (sigma, st . Cell lysates were subsequently boiled and subjected to western blot analysis using an anti - phospho - p65 antibody (cell signaling, beverley, ma, usa) and anti - gapdh (cell signaling, danvers, ma) as previously described . For the detection of phospho - p65 by elisa, cells were lysed and processed according to the manufacturers instructions using the pathscan phospho - p65(ser536) elisa kit (cell signaling). Elisa results were normalized to the total protein content per well as determined by the micro bicinchoninic acid (bca) technique (thermo scientific, nepean, on). 1.5 10 mlecs were placed on 35 mm dishes 2 days prior to exposure to 0.8 ml of the indicated perfusates (with or without nf-b inhibitors) for 4 hours at 37c . Total rna was extracted from the cells using the rneasy plus mini kit (qiagen, toronto, on, canada). 1 g of total rna was reverse - transcribed using superscript iii reverse transcriptase (invitrogen) following the manufacturer's protocol . Qpcr was performed as described previously with the exception that the cq values were determined by linear regression in cfx manager v2.1 (biorad, mississauga, on). Cq data was exported into qbaseplus (biogazelle, zwijnaarde, belgium) for quantification of expression and statistical analysis . The gene - specific pcr efficiencies were determined using the qpcr package v1.36 in the data were fitted to a 5-parameter logistic curve using the smoothing option to determine reaction efficiencies using the cy0 method . The control perfusate samples were used as the calibrator in each reaction for cultured cells, unventilated control livers were arbitrarily set to 1 for graphing after analysis . The target gene expression was normalized to the -actin, gapdh, and 18s rna in all samples . Primer sequences were obtained from rtprimerdb: -actin i d: 168, il-6: 3269, tnf-: 3747, cxcl-2: 1068, or cxcl-1, gapdh: fwd 5-caacgaccccttcattgacctc-3 and rev 5-ccaatgtgtccgtcgtggat-3, 18s (a kind gift from dr . Aaron cox, western university, london, on, canada): fwd 5-acgatgccgactggcgatgc-3 and rev 5-cccactcctggtggtgccct-3. For experiments involving nf-b pathway inhibition, cells were preincubated with 15 m imd-0354 (tocris bioscience, minneapolis, mn) or 20 m caffeic acid phenethyl ester (cape) (tocris) for 20 minutes, prior to exposure with lung perfusate that also contained the same concentration of the indicated inhibitor . A short preincubation period was used to ensure the nf-b pathway would not be activated immediately upon exposure to the inflammatory mediators in the perfusate . C57bl/6 mice weighing between 20 and 30 grams were initially anesthetized with ketamine (100 mg / kg) and xylazine (5 mg / kg) and subsequently the left jugular vein and left carotid artery were exposed and cannulated with pe10 tubing which was secured in place with 5 - 0 silk . The arterial line was used to collect arterial blood samples (60 l each time) for blood gas measurements (abl 700, radiometer, copenhagen, denmark), monitor hemodynamics, and deliver fluids (sterile 0.9% nacl and 100 iu heparin / l) using an infusion pump at a rate of 0.5 ml/100 g / h . The venous line was used to deliver additional ketamine / xylazine anesthetic as needed and to deliver additional fluid (0.5 ml/100 g / h) continuously . Ketamine / xylazine was administered through the venous line to maintain a consistent level of anesthesia and avoid additional unnecessary animal handling . The trachea was exposed and a 14-gauge endotracheal tube was secured with 3 - 0 surgical silk . Animals were subsequently connected to the harvard mini - vent volume - cycled mechanical ventilator (harvard instruments, saint - laurent, canada) with the following parameters: vt = 10 ml / kg, peep = 3 cm h2o, respiratory rate = 150 breaths / min (bpm), and fio2 = 1.0 . After 15 minutes of ventilation, animals were assessed for initial inclusion criteria, which consisted of a ratio of arterial partial pressure of oxygen to fractional percentage of inspired oxygen (pao2: fio2) of> 400 mmhg . Every fifteen minutes, for the subsequent 240 minutes measurements were taken of peak inspiratory pressure (pip) blood pressure (bp), heart rate (hr) and recorded while temperature was constantly measured with a rectal probe attached to an omega engineering, hh-25tc thermocouple . After 4 h of ventilation, the animals were euthanized with an intravenous overdose of sodium pentobarbital (110 mg / kg). Liver samples were subsequently excised and snap frozen for later rna extraction using trizol reagent (invitrogen) as per the manufacturer's protocol . Groups were analyzed by one - way analysis of variance (anova) (cell culture samples) or student's t - test (livers) using graphpad prism v4.03 (graphpad software inc, la jolla, ca), except qpcr statistics were performed using qbaseplus's internal statistical analysis by one - way anova (cell culture samples) or student's t - test (livers). In order to elicit ventilation - induced lung inflammation in mice and obtain lung - specific mediators in a perfused solution, we ventilated euthanized mice on the ipml apparatus . Analysis of the inflammatory cytokine concentrations in lung - derived perfusate collected at the completion of the mechanical ventilation protocol is shown in table 1 . The concentrations of lung - specific mediators from ventilated mice were comparable to previous observations made by our group using this protocol . Mlecs were exposed to control uncirculated perfusate, lung perfusate, or uncirculated perfusate plus cytomix for 8 h. mlecs exposed to lung perfusate expressed significantly greater concentrations of granulocyte colony stimulating factor (g - csf), il-6, chemokine (c - x - c motif) ligand 1 (cxcl-1), cxcl-2, and monocyte chemoattractant protein 1 (mcp-1) measured within the conditioned media compared to mlecs exposed to control perfusate and compared to concentrations in the perfusate before incubation on mlecs . Four of the analytes included in the assay demonstrated no significant change in concentrations after 8 h of incubation with lung perfusate (if-, il-1, il-10, and tnf-) as compared to the baseline concentrations, while eotaxin decreased significantly from baseline (data not shown). Incubation of mlec with cytomix (10 ng / ml) demonstrated significant increases in mcp-1, granulocyte macrophage colony stimulating factor (gm - csf), tnf-, il-1, and eotaxin, while the remaining analytes demonstrated no significant change from control . Based on the observation that incubation with lung perfusate elicited the production of further inflammatory mediators, we investigated the role of the inflammation - relevant nf-b signaling pathway in this process . Incubation of mlec with lung perfusate resulted in a significant increase in nf-b - subunit p65 phosphorylation compared to cells incubated with control perfusate media (figure 2). Figures 2(a) and 2(b) depict a representative western blot and quantification of phospho - p65 from mlec stimulated with either lung perfusate or tnf- as a positive control . Similarly, in independent experiments, activation of nf-b was also confirmed employing an elisa approach to detect phospho - p65 (ser536) (figure 2(c)) with cytomix used as a positive control . P65 phosphorylation, detected by elisa, was significantly increased in lung perfusate and cytomix exposed cells compared to control perfusate alone . Based on the above observations, two structurally different nf-b inhibitors, imd-0354 (imd) and caffeic acid phenethyl ester (cape), were employed . These compounds have previously been determined to interfere with nf-b activation at two different points along the nf-b signaling cascade [19, 20]. Initial experiments were performed to determine the effective and minimally cytotoxic concentrations of both inhibitors . The obtained results indicated that imd and cape were effective in suppressing nf-b activation at 15 and 20 m, respectively (data not shown). Treating mlec with either imd or cape significantly mitigated the production of proinflammatory mediators released by mlec after incubation with lung perfusate as shown in figure 3 . To confirm that these changes occurred at the level of gene transcription, selected mediators were chosen for qpcr analysis in mlecs exposed to lung perfusate (figure 4). Gene transcription of il-6, cxcl-1 and cxcl-2 were significantly reduced by treating mlec with either imd or cape prior to exposure to lung perfusate, whereas neither inhibitor had a significant effect on the gene expression of tnf-, although there was a trend of reduced tnf- expression . Physiological parameters for animals undergoing 2 hours of mechanical ventilation are shown in figure 5 . Over the course of mechanical ventilation, there was a decrease in pao2 at both 120 and 240 minutes of mechanical ventilation compared to the baseline pao2; however, this decrease was not statistically significant . In contrast, the pip, also shown in figure 5, increased over the course of ventilation and was significantly increased at 60 minutes and thereafter compared to the baseline (time 0) pip . Additionally, blood pressure and partial pressure of co2 did not vary significantly from the baseline (data not shown). The lack of a significant change in the majority of these parameters suggested that a significant degree of lung dysfunction was not elicited by this ventilation protocol . Qpcr demonstrated a significant increase in cxcl-1, cxcl-2, il-6, and tnf- gene transcription in livers of mechanically ventilated animals compared to non - ventilated controls, a phenomenon consistent with our observations made in vitro . The results of the current study present a novel finding of an nf-b - dependent mechanism of proinflammatory cytokine amplification by liver endothelial cells secondary to mechanical ventilation . Previous studies have consistently demonstrated that the nf-b signaling cascade represents a key regulatory process controlling the transcription of many proinflammatory mediators as it is estimated that over 400 activators of this inflammatory pathway have been identified including physical stress, oxidant stress, and proinflammatory cytokines . Thus, while it may not be unexpected that lung perfusate obtained from ventilated mice that is rich in multiple proinflammatory cytokines is capable of activating the nf-b pathway in liver endothelial cells, we highlight unique aspects which we believe are relevant in the context of systemic inflammation subsequent to the initiation of mechanical ventilation . Firstly, through the use of the impl model, we show that specific mediators originating from a lung generated in response to mechanical ventilation are capable of inducing nf-b signaling in endothelial cells of a peripheral organ . The impl model allows the pulmonary circulation to be isolated from the systemic circulation, thereby facilitating the collection of mediators generated directly by the lung as a result of mechanical (ventilation) stress . Although other aspects of the ipml model may have contributed to the inflammatory mediators in perfusate, such as surgery and lack of blood, current literature suggests that the vast majority of these mediators are induced by the cell stretch due to ventilation [2527]. From a clinical standpoint, although the absolute rises in serum cytokines have been directly correlated with outcomes in the setting of ards, the specific origin of these mediators has been incompletely characterized . Therefore, based on the results of this study we speculate that although the injured lung serves as the primary origin of the systemic inflammatory response, the signal is promptly propagated by peripheral organs in a maladaptive feed - forward mechanism of systemic inflammation . Secondly, while this lung - derived perfusate contains elevated levels of multiple inflammatory mediators, equivalent or greater concentrations of cytomix (tnf-, il-1, if-) failed to elicit an equal magnitude of responses . These findings would suggest that the effects observed in our model may be an aggregate effect of multiple mediators present in lung perfusate samples which are generated specifically through the effects of mechanical ventilation . Furthermore, the downstream increase in inflammatory mediators originating from liver cells was not simply a global, nonspecific effect . Rather, although liver endothelial cells are capable of producing a wide spectrum on inflammatory mediators, the rise in mediators appeared to be restricted to a significant increase in 5 out of 10 analytes measured including g - csf, il-6, cxcl-1, cxcl-2, and mcp-1 . Notably, tnf- was not significantly elevated in the cell culture model, although tnf- gene transcription was significantly up - regulated in lung perfusate treated cells . This may be related to the known properties of the tnf- gene which is rapidly transcribed upon stimulation, but has subsequent translation tightly controlled . Although some mediators were not significantly increased upon exposure to the mlec cultures (if-, il-1, il-10, and tnf-), this is not to suggest these mediators are not important or do not contribute to inflammation . These findings not only underscore the complexity of the systemic inflammatory response secondary to mechanical ventilation, but also may explain why previous therapeutic interventions targeting isolated cytokines have not resulted in improvement in patient outcomes . Using the in vivo model of ventilation - induced inflammation highlights several interesting observations . Although the use of mechanical ventilation is obligatory in the setting of ali and ards to maintain host survival, the in vivo model adopted in the current study employed the use of mechanical ventilation alone to study its downstream effects on systemic inflammation . Despite the absence of marked changes in host physiology (oxygenation), significant proinflammatory changes were noted in liver tissues suggesting that systemic manifestations of mechanical ventilation may not only occur in the absence of physiological lung dysfunction but that pre - existing lung injury may not be an obligatory requirement for potentially deleterious systemic manifestations . Clinical studies in patients with ards have consistently demonstrated that stepwise increases in inflammatory cytokines in patients with ards have been correlated with greater adverse outcomes [31, 32]. For example, ranieri et al . Showed that patients exposed to protective modalities of mv had lower pulmonary and systemic inflammation compared to patients on conventional ventilation strategies . Furthermore, other studies have also demonstrated that patients ventilated with lower tidal volumes had a lower plasma level of il-6, as well as soluble tnf- and il-1 receptor antagonists compared to those ventilated with conventional strategies, thereby providing evidence that mechanical ventilation independently leads to systemic inflammation . The current study adds to the growing body of evidence that injudicious use of mechanical ventilation can contribute adversely toward a maladaptive systemic inflammatory response by peripheral organs, and furthermore, may provide insight into potential mechanism by which therapeutic approaches, such as low tidal volume mechanical ventilation, have been successful in improving patient outcomes . Our data would suggest that the adoption of either a primary or complementary strategy of mitigating peripheral organ responses early in the course of ards through the blockade of maladaptive pathways such as nf-b signaling in peripheral organs may be an effective approach to consider . Alternately, strategies aimed at minimizing the translocation of lung - derived mediators into the systemic circulation may represent a more proximal upstream approach; however, the specific mechanisms responsible for the release of these mediators remains as yet undetermined . Although we describe a potential mechanism whereby inflammatory signals originating the in the lung are subsequently amplified by cells of a downstream organ, we recognize that our model does have inherent limitations . Firstly, we chose to utilize liver endothelial cells as the cell type of interest due to the immediate proximity and exposure of this cell layer to lung - derived mediators which may circulate in vivo . Therefore, our findings are limited to this specific cell type and we have not accounted for the contribution of other tissue specific cells within the liver such as hepatocytes or kuppfer cells, for example . The contribution of other cell types from liver and other organs may account for why we did not observe significant increases in several mediators previously shown to be important in patient outcomes (e.g., il-1, tnf-). Nonetheless, the use of whole liver tissues employed in the in vivo model of mechanical ventilation indicates that increases in il-6, for example, may be expressed throughout the liver and not restricted to any one cell type . Secondly, our investigation focused primarily on proinflammatory effect, the contribution of anti - inflammatory mediators in this process may also be important to evaluate in future studies . Thirdly, it remains unknown whether similar links exist between the lung and other downstream organs such as the kidneys, heart or brain and whether an amplification of inflammatory mediators from theses other systemic organs contribute to a greater or lesser extent toward systemic inflammation . Whether the nf-b signaling cascade represents a common pathway of proinflammatory signaling within each organ or future studies to determine the generalizability of our findings beyond a single downstream organ are therefore warranted . In the current study, we demonstrate that inflammatory mediators generated by the lung in response to mechanical ventilation decompartmentalize to the systemic circulation in a murine model of ventilator - induced inflammation . Subsequently, we show that the levels of these inflammatory mediators are significantly amplified upon exposure to liver endothelial cells thereby resulting in a maladaptive upregulation of the systemic inflammatory response . The results of in vitro experiments illustrating this phenomenon are further confirmed in an in vivo model of ventilation induced inflammation whereby a significant increase in transcriptional activity in these mediators is observed in the liver . Ultimately, we show that the propagation of the systemic inflammatory response by the liver occurs through an nf-b - dependent mechanism and that inhibition of this signaling pathway can, in part, mitigate these responses . The significance of these findings will require further studies to determine whether blockade of the nf-b pathway in peripheral organ tissues would provide a rational means of therapeutic intervention.
A 68-year - old male presented complaining of sudden and profound loss of central vision in his left eye following blunt eye trauma . He reported seeing a large, dense, central scotoma that began fading 1 h after the injury . He was emmetropic, phakic, and had no significant ophthalmic history . On examination, 24-h postinjury, best - corrected visual acuity (bcva) in the left eye oct of the left macula (spectralis, heidelberg engineering, 5-line raster acquisition protocol) revealed abnormal hyper - reflectivity and nodularity of the photoreceptor os layer . Interestingly, the patient was found to have prominent abnormalities on infrared imaging using the infrared reflectance protocol of the spectralis oct [fig . 1]. Infrared imaging of the left fundus revealed diffuse infrared hypo - reflectance dotted with spots of very low pixel value (dark), creating a stippled appearance . Point - to - point correlation between the infrared and oct images using the spectralis software (heidelberg engineering, germany) demonstrated that the infrared dark spots correlated with foci of thickened os seen on 5-line raster oct . No pathology of the superficial retina, retinal pigment epithelium or choroid was detected using red - free reflectance, fundus auto - fluorescence, and enhanced - depth imaging (edi), respectively . 30 posttrauma, and at 8 months posttrauma, bcva in the left eye was 20/25 and the retina appeared normal on examination and when imaged with infrared reflectance, 5-line raster oct, fundus auto - fluorescence, and edi [fig . 2]. Left fundus images at day 1 postblunt trauma to the left eye . (b) infrared confocal scanning laser ophthalmoscope fundus image demonstrating stippled hypo - reflectance at the macula . (c) optical coherence tomography of the macula demonstrating abnormal hyper - reflectivity of the outer segments (between arrowheads). (b) infrared confocal scanning laser ophthalmoscope fundus image reveals resolution of the infrared hypo - reflectance in this case, there is a clear temporal relationship between eye trauma, transient vision loss, transient oct changes consistent with cr, and transient infrared hypo - reflectance . Cases of cr detectable on oct, but not fundus examination have been described, confirming that some cases of cr are subclinical . To the best of the authors knowledge the pathology of cr has been studied histologically and on oct, and comprises fragmentation of the photoreceptor os without injury to the inner retina . As demonstrated by our case, this manifests on 5-line raster oct as abnormal hyper - reflectivity of the os layer . This oct feature is subtle and is easily overlooked if the study is not carefully scrutinized . Our case implicates infrared imaging as a diagnostic adjunct for detecting subclinical cr . Since focal dark spots on infrared imaging correlated with points of nodular os thickening in our case, we infer that the infrared hypo - reflectance in cr is caused by increased absorption of infrared light by an abnormal os layer . With a wavelength of 820 nm, the infrared mode of the spectralis oct penetrates deeper into the retina than modalities using visible wavelengths of light . It is, therefore, useful for detecting outer retinal pathology, such as that of cr . The spectralis uses a confocal scanning laser ophthalmoscope (cslo) and records infrared reflectance without barrier filters . Compared with conventional recording techniques, cslo improves image resolution by using a higher emission of light energy and by reducing the capture of scattered light . Further studies are required to determine whether infrared hypo - reflectance is a consistent feature in all cases of cr, and whether this imaging modality has greater sensitivity for detecting subclinical cr than 5-line raster oct . Nevertheless, this case suggests that infrared imaging may have a role as a diagnostic adjunct for detecting subclinical cr . We recommend that clinicians consider performing infrared imaging of the retina when managing a patient who presents with unexplained vision loss after blunt trauma and a normal - appearing fundus.
Personal identification is the act of establishing the identity of a personby linking him / her to the stream of data in the information systems . It beholds a major portion in forensics and usually relies on the comparison of the known features to the unknown specimen . Identification of an individual is vital for the family not only from an emotional standpoint but is also a medicolegal requirement . In living individuals, identification plays a pivotal role in cases such as property disputes, insurance claims, issuance of passports, and various other licenses, whereas after death, it becomes important to identify the deceased as in case of a murdered victim, or file closure of a person missing for a prolonged period to facilitate the rituals of body disposition following death and permission of remarriage . So, the society's duty to preserve human rights and dignity beyond life begins with identity . Identification of the living individual is less cumbersome since simple methods such as direct visual recognition by a family member, friend, or acquaintance is possible that is done by the identification of unique physical characteristics such as scars, birthmarks and tattoos . This method of personal recognition can be applied to identify the decedent as well . However, standardized techniques such as antemortem and postmortem comparisons of fingerprints, palm prints, and footprints act as reliable tools for positive identification . Nevertheless, all of these methods depend on the preservation of soft tissue components of the body in question, and cannot be applied when the surface topography is unrecognizable or featurelessas in decomposed, burnt, mutilated, skeletonized, and fragmented states . In such circumstances, identification is solely dependent on the skeletal framework of the decedent, and the process starts with the determination of age and sex . Sex determination reduces the search of individuals by 50% and is 98% accurate when the whole skeleton is available . But finding an intact skeleton is not always likely . In such circumstances, pelvic bone possesses highest sex discriminant accuracy of 95% . The skull is the second best alternative and is 90% accurate to determine the sex of the decedent . However, in explosions, warfare and mass disasters, human remains are often obtained in fragmented states . In such circumstances, only strong bones that greatly resist fracture are likely to be recovered intact . The base of the skull is one such bone and establishing sex discriminant value of the skull base has attracted attention . It is located inferior to the sagittal suture and surrounded by the basilar, squamous, and lateral parts of the occipital bone . Situated in the deepest part of the posterior cranial fossa and covered by a large volume of soft tissue, it is an ideal structure for sex determination . Sexual dimorphism in fm dimensions is population - specific and highly influenced by environmental, socioeconomic, and genetic factors . This digital radiographic study aims at evaluating the accuracy of fm dimensions in sex determination of south indian adults . The results obtained by the study would help in personal identification of the given population . The present study was conducted on 150 south indian subjects comprising 75 males and 75 females . The subjects chosen were in the age group of 2565 years, randomly selected from patients of dayananda sagar college of dental sciences and hospital, bengaluru, karnataka, india . Hence, in order to prevent the age factor influencing the results, subjects under 25 years were not included in the study . Furthermore, patients beyond 65 years are unlikely to be in need of submentovertex (smv) radiographs . The radiographic procedure was explained to all the subjects and informed written consents were obtained . Sirona (new york, u.s.a .) Orthophos xg 5 machine was prepared for the submentovertex radiographic procedure by positioning it to c2 program, which is specifically designed for anteroposterior projections . A charge - coupled device (ccd) image receptor was placed in its slot and the unit was approximated to the patient's head position [figure 1]. Exposure parameters were personalized as per the manufacturer's recommendations during the radiographic exposure and the image was displayed on the digital display monitor . Patient positioned for digital submentovertex radiograph three qualified oral radiologists with an experience of 510 years evaluated the fm dimensions in the resultant image under standardized viewing conditions . During interpretation, the observers were permitted only to modify the contrast and brightness of the image (if and when required) just to facilitate the visualization of the fm boundaries . Each observer evaluated all the 150 images twice within a minimum period of 10 days . The following measurements of the fm were recorded in millimeters [figure 2]: digital submentovertex image with analysis of the foramen magnum dimensions longitudinal diameter / length of fm (ld): maximum anteroposterior dimension measured from the highest point on the anterior border up to the inferior - most pointtransverse diameter / width of fm (td): the maximum mesiodistal dimension measured from the point of highest convexity on the medial border up to the point of highest convexity on the distalmost margincircumference of fm (c): perimeter obtained by tracing the entire border of fmarea (a) of fm: calculated by substituting ld and td in radinsky's formularadinsky's formula: area = ld tdshape of fm: subjective assignment of one of the seven shapes to fm morphology according to the classification of chethan et al . Of fm shapes [figure 3]. Longitudinal diameter / length of fm (ld): maximum anteroposterior dimension measured from the highest point on the anterior border up to the inferior - most point transverse diameter / width of fm (td): the maximum mesiodistal dimension measured from the point of highest convexity on the medial border up to the point of highest convexity on the distalmost margin circumference of fm (c): perimeter obtained by tracing the entire border of fm area (a) of fm: calculated by substituting ld and td in radinsky's formula radinsky's formula: area = ld td shape of fm: subjective assignment of one of the seven shapes to fm morphology according to the classification of chethan et al . Of fm shapes all the recordings were tabulated and subjected to the statistical analysis using statistical package for the social sciences (spss) software version 16.0 (manufacturer- ibm spss statistics, formerly known as spss inc . ). Data comparison was done by applying student's t - test to find out the statistical significance of the obtained results . The mean and standard deviation for all the four measurements were obtained to derive the fm dimensions in the south indian population . Table 1 shows the mean values with the standard deviation for longitudinal diameter, transverse diameter, circumference, and area for 75 males and 75 females as assessed by all the three observers independently . The values for all the four parameters were higher in males as compared to females, highlighting sexual dimorphism in fm dimensions . To substantiate this, p value was derived by applying student's t - test and it was seen that p value was <0.05 for longitudinal diameter, transverse diameter, circumference, and area [table 2]. This suggests that the difference in the mean values between males and females in all the four measurements were statistically significant . Sexual dimorphism in the foramen magnum dimensions foramen magnum measurements in males and females as assessed by all the three observers to evaluate intra - observer agreement, r value (degree of correlation) was calculated for all the four fm dimensions as measured by the observers on the first day and second day of observation [table 3]. R value showed a strong degree of correlation for ld, td, and c, and a moderate degree of correlation for a, suggesting good intra - observer agreement . (r) of the foramen magnum dimensions based on the first day and the second day of observation to evaluate interobserver agreement, r value (degree of correlation) was calculated for all the four fm dimensions as measured by all the three observers . The resultant r value suggested a strong inter - observer agreement [table 4]. Inter - observer correlation . (r) of the foramen magnum dimensions to assess the accuracy of digital smv radiograph, at first, a formula was derived using discriminant function analysis: gender = [(-0.275 ld) + (0.163 td) + (0.461 c) + (0.677 a)] 104.89 by applying our data to the derived equation, canonical variables were derived for all the four fm dimensions . An accuracy of 67.3% was obtained when a total of 150 subjects were considered [table 5]. Also, an attempt was made to assess, which among the four parameters was most efficient in sex determination . It was seen that maximum accuracy was obtained for c and the least for ld . Thus, c was the best indicator for sex determination followed by a, td and ld . Accuracy of foramen magnum dimensions in sex determination morphology of the fm: the shapes assigned by all three observers for a total of 150 images, as determined twice, made it a total of 900 shapes . Using the classification given by chethan et al ., it was observed that egg shape was the most common shape and hexagon was the least common shape [figure 4]. Similar results were observed in the fm of males and females [figure 5]. Pie chart depicting the distribution of the foramen magnum shapes in 150 subjects bar diagram representing the distribution of the foramen magnum shapes in males and females positive identification of the deceased is a very crucial aspect of forensic science and sex determination beholds a major role in this regard . Sex determination helps to channelize the investigation by deducing the search to half the population, thus conserving both resources and the time required for identification . When the entire skeleton is present, sex determination is possible with 100% accuracy . However, in many instances human remains are likely to be obtained in fragmented states . In such situations, the pelvis has demonstrated maximum accuracy in sex determination followed by the pelvis if available with cranium, pelvis with long bones, and long bones or skull in isolation . The base of the skull, precisely the occipital bone is often recovered intact, even in cases of severe trauma due to its well - protected anatomical position and large amount of overlying soft tissue . But this sexual dimorphism is population - specific, as demonstrated by studies on the populations of iraq, turkey, brazil, poland, and nigeria; this is also seen in india in diverse geographical locations such as uttar pradesh, gujarat, chandigarh, and madhya pradesh . The dimensions of fm are also influenced by genetic, environmental, and social factors . Many authors have demonstrated sexual dimorphism in the south indian population as well but they have utilized dry skulls to derive their results . This limits the study sample . Moreover, archiving skulls of a known age and sex for the study is an added drawback . To overcome this, we performed the study with the help of archived digital smv radiographs and perhaps this is the first of its kind . It is unresponsive to secondary sexual changes, with no influence of musculature on its size and shape, making it considerably stable beyond adolescence . The fm measurements obtained in our study clearly showed statistically significant differences between the genders (p value <0.05) with all values significantly greater in males than in females . Babu found the mean ld values to be 35.68 mm in males and 32.57 mm in females; kanchan et al . The mean values of td as reported in these studies were 28.91 mm in males and 28.19 mm in females (babu r); 27.36 mm in males and 26.74 mm in females (kanchan et al . ). Our obtained values using digital smv radiograph are in accordance with those obtained on direct measurements of the skull as seen in aforementioned studies [table 1]. This justifies that smv can effectively replace dry skulls in the measurement of fm dimensions . A (area) of fm was calculated by using radinsky's formula . In a review of the literature, we observed that researchers had used two formulae to calculate the area: texeira formula and radinsky's formula . Among the indian studies, babu used both the formulae in their study and have opined that the value for a obtained by radinsky's formula is a better evaluator of sex . Based on this, we chose radinsky's formula to calculate a. the results showed statistically significant gender difference and are comparable to those obtained by kanchan et al . (744.33 mm in males and 706.93 mm in females) and babu r (811.67 mm in males and 722.66 mm in females). This is an expected result because the aforementioned studies were also conducted among subjects of karnataka who were representative of the south indian population . We presume that this is the only indian study to have assessed the c of fm . Hence, we compared the derived mean c values (males: 106.15 mm; females: 99.95 mm) with the next only available study conducted on the iraqi population by uthman et al . Where the mean c values was found to be 99.3 mm in males and 92.6 mm in females . Discriminant function analysis demonstrated that c of fm was 66% accurate and was the best predictor of sex among the four parameters, capable of differentiating males with 66.7% accuracy and females with 65.3% accuracy [table 5]. The next best predictor of sex was the a of fm (64.7%) followed by td (62.7%); ld (60.7%) formed the lowest predictor . The reliability of td, ld and a in sex determination has been demonstrated with variable results by different authors, with one parameter being better over the other . The greater accuracy of c in our study brings to light that the c of fm is the most important parameter and must always be evaluated during the morphometric analysis of fm for sex determination . We categorized the fm morphology of all the 150 subjects according to the shapes classified in the study on south indian subjects by chethan et al . We found egg shape to be the most common and hexagon to be the least common [figures 4 and 5]. Though we considered the classification of chethan et al . To categorize the shapes, the incidence of occurrence in their study showed slight variations from ours, with round being the commonest shape followed by egg and tetragonal shape . But the incidence rate of round shape in their study (22.6%) is similar to the incidence rate in our study . By this, we opine that in the south indian population, the fm commonly exhibits a round or egg shape . A high degree of inter - observer correlation [table 4] implies that fm dimensions are minimally affected by subjective variations and hence, are highly reproducible in establishing sexual dimorphism . Strong degrees of correlation for ld, td, and c and a moderate degree of correlation for a achieved by comparing the values obtained on the 2 days of observations by each observer suggest moderate to strong intra - observer association . This strengthens the hypothesis that digital smv radiograph can effectively be used to establish sexual dimorphism in fm . By applying discriminant function analysis, it was seen fm dimensions evaluated using digital smv radiograph were 67.3% accurate in differentiating sex and 65.3% and 69.3% accurate in determining males and females, respectively [table 5]. Our results are similar to the accuracy rate achieved by measuring fm dimensions on dry skulls by suazo et al . Thus, we opine that digital smv radiograph can be an excellent alternative to direct morphometry in evaluating sexual dimorphism using fm dimensions . However, the accuracy achieved by using smv radiographs is lower than the accuracy (81%) achieved in the study by uysal et al . On computed tomography (ct) images . In ct, though a contrast between the anatomical structures is enhanced, the greatest disadvantage is its high radiation dose to the patients (20 sv), which is 477 times higher than for the digital smv radiograph (0.6 sv). Achieving an accuracy of 67.3% at a reduced radiation exposure, digital smv the results of our study demonstrate that all the dimensions (ld, td, c and a) are higher in males than in females, with the values being similar to the studies performed earlier by direct morphometry or ct . Strong intra- and inter - observer agreements between the values of fm dimensions emphasize that digital smv radiograph is efficient for measuring fm dimensions and can be a good alternative to dry skulls and ct for sex determination . Our study elucidates its morphometric data and variations in the morphology with emphasis on its application in the identification of unknown individuals . We believe that data obtained from our study will be useful to forensic investigators, anthropologists, clinical anatomists and the neurosurgeons . Fm dimensions are population - specific; therefore, values derived from the respective population must be considered during evaluation of fm of unidentified skull remains . The c and a of the fm in south indian adults are useful indicators of sex . Yet considering the limited accuracy rate achieved by the study, the application of fm in sex determination should be restricted to cases where only a fragment of the skull base is brought for examination and should not be used in a situation where the complete cranium is present; wherein other reliable skull parameters can be used . To the best of our knowledge, this is the first study to make use of digital smv radiograph to establish sexual dimorphism in fm . Achieving a fair accuracy rate, we emphasize that digital smv radiograph can be a promising alternative to ct, offering reduced radiation exposure to the patient at an affordable price . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
For drugs that operate in a narrow therapeutic range, it can be desirable or even necessary to use pharmacodynamic measures to adjust the dose to achieve an appropriate level of response . For example, anticoagulants such as warfarin act by reducing the ability of the blood to form clots . Thus, pharmacodynamic measurement of blood clotting ability is used to adjust the dose within a specified range.12 establishing a dose adjustment algorithm requires quite a few decisions . What endpoint will be used to make dose adjustment decisions? What is the target exposure range to maintain this endpoint within? What should be the starting dose? Will monitoring and adaptive dosing continue indefinitely, or only during an initial period after treatment initiation? Simulations of these scenarios can be quite helpful in understanding the impact of these decisions . In this tutorial for illustration purposes, we have chosen a system where the pk is described by a twocompartment model, and the pd effect is modeled with an indirect response13 (figure 1). In order to maintain efficacy while avoiding adverse side effects, it has been determined that inhibition of the target should be in the range of 4060% . A twocompartment pharmacokinetic model with an indirect response pharmacodynamic model . First, we will demonstrate how to set up and run a simulation using rxode . The rxode package can be installed from github at https://github.com/hallowkm/rxode (see the supplementary material for installation instructions). Rxode is made available as open source under the gnu general public license version 2 or later . Figure 2 gives an overview of the workflow for performing a simple simulation in rxode with the model described above . C2 = centr / v2;c3 = peri / v3;d / dt(depot) = kadepot;d / dt(centr) = kadepot clc2 kout(1c2/(ec50+c2))eff; d / dt(depot) = kadepot; d / dt(centr) = kadepot qc2 + qc3; d / dt(peri) = qc2 qc3; d / dt(eff) = kin kout(1c2/(ec50+c2))eff names of parameters in the vector must be a superset of parameters in the ode model, and the order of parameters within the vector is not important . The number of ics must equal exactly the number of odes in the model, and the order must be the same as the order in which the odes are listed in the model . C(ka = 0.3, cl = 7, v2 = 40, q = 10, v3 = 300, kin = 0.2, kout = 0.2, ec50 = 8)inits <c(0, 0, 0, 1) #define parameters and initial conditions params <c(ka = 0.3, cl = 7, v2 = 40, q = 10, v3 = 300, kin = 0.2, kout = 0.2, ec50 = 8) inits <c(0, 0, 0, 1) the creation of an eventtable () object provides an extremely efficient and flexible way to specify dosing and sampling . The generated eventtable object has functions that allow easy addition of dosing and sampling events . The add.dosing () function allows specification of the dose amount, number of doses, dosing interval, compartment to dose into, rate (if an infusion), and dosing start time . More complex dosing schedules can be simulated by applying the add.dosing () function multiple times . The add.sampling () function allows specification of the time points to be included in the simulation output . The add.dosing () function provides an efficient method to specify a variety of dosing schedules calling the rxode () function, the model is translated into c code, compiled, and dynamically loaded into the running r process . A simulation can then be performed by calling the r model object's function run (), with the specified parameter vector, initial conditions vector, and event table as inputs . Rxode(model = ode, modname = mod1) #run simulationx <mod1$run(params, ev, inits) mod1 <rxode(model = ode, modname = mod1) x <mod1$run(params, ev, inits) all state variables as well as other variables computed in the model are returned in the output matrix, at the times specified in the eventtable . Thus, the simulation results are readily available for performing calculations and generating plots in r using any of the existing r packages (lattice, ggplot, etc). The user can also choose to specify the absolute and/or relative tolerance, as well as the type of solver to be used: x <m1$run(theta, ev, inits, stiff = f, atol = 1e8, rtol = 1e6) x <m1$run(theta, ev, inits, stiff = f, atol = 1e8, rtol = 1e6) rxode uses the lsoda and a rungekutta integrators for stiff and nonstiff equations, respectively . The lsoda (livermore solver for ordinary differential equations) fortran package is an automatic method switching for stiff and nonstiff problems throughout the integration interval . For purely nonstiff systems, rxode uses dop853, an explicit rungekutta method of order 8(5,3). A matrix of parameter values can be generated, where each row represents one set of parameter values . R sampling functions such as rnorm and mvrnorm can be utilized to build this matrix, but decisions on the level(s) of uncertainty to include are left to the discretion of the modeler, since this depends on the specific questions a given simulation is designed to address . The following code generates parameters for 100 subjects with correlated interindividual variability on cl and v2 . Mvrnorm(n = nsub, rep(0,2), sigma) #sample from covariance matrixcl <40exp(mv[,2])params.all <cbind(ka=0.3, cl = cl, v2=v2, q=10, v3=300, kin=0.2, kout=0.2, ec50=8) nsub matrix(c(0.09,0.08,0.08,0.25),2,2) #iiv covariance matrix mv <mvrnorm(n = nsub, rep(0,2), sigma) #sample from covariance matrix params.all <cbind(ka=0.3, cl = cl, v2=v2, q=10, v3=300, kin=0.2, kout=0.2, ec50=8) once this parameter matrix is generated, each subproblem can be simulated by looping through the parameter matrix, using each row as an input for the simulation, and collecting the output of each simulation in an output matrix . Res <null #loop through each row of parameter values and simulationfor (i in 1:nsub) {params <params.all[i,]x <mod1$solve(params, ev, inits = inits)#store results for effect compartmentres <cbind(res, x [, " eff"])}the same result can be achieved more efficiently with the following code: res <apply(theta.all, 1, function(theta) mod$run(theta, ev, inits) [, eff]) #loop through each row of parameter values and simulation params <params.all[i,] x <mod1$solve(params, ev, inits = inits) #store results for effect compartment res <cbind(res, x [, " eff "]) the same result can be achieved more efficiently with the following code: res <apply(theta.all, 1, function(theta) mod$run(theta, ev, inits) [, eff]) simulation results can be then be directly analyzed and visualized using any of the statistical and graphics tools available within r. figure 3 shows the results of the above simulation when the drug is given qd for 2 days . Simulation of effect for 2 days of qd dosing, correlated interindividual variability on cl and v2 . Since simulations are conducted in r, rxode simulation results can easily be summarized and visualized graphically . To return to the problem of simulating adaptive dosing, there are many factors that must be considered in a designing an adaptive dosing scheme . Utilizing rxode within the r environment, simulations of the impact of various factors can be quickly performed and evaluated . In order to simulate an adaptive dosing scenario, we will first simulate the following decision rule: the drug is to be dosed once daily, and trough pd effect levels will be measured 24 hours after each dose . If the measured pd effect is less than 40%, the dose will be doubled . If the measured pd effect is greater than 60%, the dose will be cut in half . If the pd effect is between 40 and 60%, no change will be made . The following r code is used to perform this simulation over 25 days . In brief, parameters governing the simulation are defined, including the decision rule limits and dose adjustments, number of days, starting dose, and sampling frequency . Then treatment is simulated one day at a time, and after each day the simulated trough level at the end of that day is used to determine the dose level for the next simulated day . This is repeated for the number of days specified, and the results are stored in a matrix . Results contained in this matrix can then be plotted using any available r plotting tools . Effect.limits = c(0, 0.4, 0.6, 9) #decision rule limitsdose.multipliers = c(0.5, 1, 2) #decision rule effectsndays <25; unit.dose <10000; start.dose <1; sampling.frequency <1 #sample every day #simulate each day . At the end of each day, test the effect level, and adjust the dose level according to the decision rulefor (i in seq(1, ndays, by = sampling.frequency)) {if (i==1) {#initialize on first dayinits <c(0, 0, 0, 1)last.multiplier <start.dosethis.multiplier <1} else {#use end of previous day as initial conditionsfor next day, compare trough effect with#decision rule limits and determine dose multiplier accordinglyinits <x[dim(x), vars]wh <cut(inits[eff], effect.limits)this.multiplier <dose.multipliers[wh]}this.multiplier <this.multiplierlast.multiplier #adjust doselast.multiplier <this.multiplier #store new dose#specify dosing and samplingev <eventtable()ev$add.dosing(dose <mod1$run(params, ev, inits) #run simulation #compile outputstime.total <length(time))x <cbind(x, time.total, doses);res <rbind(res, x)} effect.limits = c(0, 0.4, 0.6, 9) #decision rule limits dose.multipliers = c(0.5, 1, 2) #decision rule effects ndays <25; unit.dose <10000; start.dose <1; sampling.frequency <1 #sample every day #simulate each day . At the end of each day, test the effect level, and adjust the dose level according to the decision rule for (i in seq(1, ndays, by = sampling.frequency)) {if (i==1) {#initialize on first day inits <c(0, 0, 0, 1) last.multiplier <start.dose} else {#use end of previous day as initial conditions for next day, compare trough effect with #decision rule limits and determine dose multiplier accordingly inits <x[dim(x), vars] wh <cut(inits[eff], effect.limits) this.multiplier <dose.multipliers[wh] this.multiplier <this.multiplierlast.multiplier #adjust dose last.multiplier <this.multiplier #store new dose #specify dosing and sampling ev$add.dosing(dose = this.multiplierunit.dose, dosing.interval = 24, nbr.doses = sampling.frequency) ev$add.sampling(0:(24sampling.frequency)) x <mod1$run(params, ev, inits) #run simulation time.total <ev$get.eventtable()[,time]+(i1)*(24) doses <rep(last.multiplier, length(time)) x <cbind(x, time.total, doses); figure 4 shows the results of this simulation . After 10 days, a steady state dose that is 25% of the starting dose is reached, which maintains the trough levels within the desired range . However, during the first 5 days the level of inhibition is well beyond the desired target . The red dashed line shows the dose multiplier over time (actual dose is the dose multiplier times the starting dose). A stable dose that maintains the trough effect within the target range is reached after 10 days . For instance, figure 5 shows the simulation results if both trough and peak levels are controlled within the 4060% range (i.e., biomarker measurements are taken at tmax of 12 hours and at trough, and dose is adjusted daily). This full script for performing this simulation can be found in the appendix, but it requires only the addition of the following line to the script above: simulated adaptive dosing to maintaining both trough and peak effect within the target range of 4060% . The red dashed line shows the dose multiplier over time (actual dose is the dose multiplier times the starting dose). A pattern emerges, indicating that dosing 12.5% of the starting dose, but then giving a double dose every 5 days, would achieve this goal . #if effect at 12 hours is less than 0.4, cut dose in halfif (x[13, eff] <effect.limits) {this.multiplier <dose.multipliers} #if effect at 12 hours is less than 0.4, cut dose in half if (x[13, eff] <effect.limits) {this.multiplier <dose.multipliers in this case, a stable dose is not reached . Instead, a more complex pattern emerges, suggesting that dosing 12.5% of the starting dose, but then giving a double dose every 5 days, could achieve the desired result . It may also be of interest to explore the impact of pk and pd variability within the population on the resulting dose trajectory and final dose . We can simulate variability, as described above, by specifying a matrix of parameter sets, rather than a fixed set of parameters, and looping through this parameter matrix, performing the adaptive simulation as described above for each set of parameters . This is easily done by adapting the simulation of a single subject to contain an outer forloop that cycles through the parameter matrix . Again, we assume correlated interindividual variability on cl and v2, and simulate 100 subjects for 25 days . Trough levels for all subjects are controlled within the specified range by the end of the time period . 62% of subjects end at 25% of the starting dose, 35% require only 12.5%, and a few subjects (2.5%) require a higher dose (50% of starting dose). It took at least 20 days to reach a final dose in all subjects (compared to 10 days for the typical subject in the previous simulation). Simulation of adaptive dosing regimen in 1,000 subjects, with correlated interindividual variability on cl and v2 . (a) the adaptive dosing regimen successfully controls effect levels within the desired range after 5 days . (b) the dose trajectory and final dose varies among the population, and most subjects end on either 25% or 12.5% of the starting dose . The simulations performed thus far are static, in that fixed parameter inputs are defined, simulations are performed, and plots are generated . Often, when the results of such simulations are reviewed, further questions arise what happens if the range limits for our decision rule are relaxed or tightened? What happens if we change the starting dose? What happens if we sample every 2nd or 3rd day, rather than every day? These new questions usually require the modeler to go off and perform new simulations, and the team must reconvene days or weeks later to discuss new results . This introduces significant lag time, especially since several iterations may be required to reach a final set of simulations . Although it may be possible to compile a massive document containing all the permutations of scenarios of interest a priori, sifting through this document with team members in real time can be a daunting task, and is not advisable, as it can lead to confusion and/or loss of attention of key stakeholders . A better alternative is to encase the simulation procedure in an interactive application in which a wide range of scenarios can be evaluated in real time, as they arise . A major advantage of performing simulations within the r environment is the ability to take advantage of r's shiny package for developing interactive web applications . These web applications can be made available to the team online, and used in meetings to explore simulation scenarios in real time . This is extremely advantageous in facilitating modeler team interactions, because it can greatly reduce the number of iterations, separate meetings, and associated lag time . Shiny apps are easy to write, requiring no web development skills and very limited programming skills . Genshinyapp.template(appdir = shinyexample, verbose = true)library(shiny) #load the shiny packagerunapp(shinyexample) #run the example app genshinyapp.template(appdir = shinyexample, verbose = true) library(shiny) #load the shiny package runapp(shinyexample) #run the example app the function genshinyapp.template () generates a folder that contains the r shiny ui.r and server.r files for the template app, as well as an rda file containing the saved example model, parameters, and initial conditions . To incorporate a different r model, the user needs can save the new model to an rda file and load this new file from the server.ui file . #save the model, parameters, init values, etc . In the file rx_shiny_data.rda to be loaded by the server.rsave(mod1, params, inits, stiff = true, atol = 1e8, rtol = 1e6, file = rx_shiny_data.rda) #save the model, parameters, init values, etc . In the file rx_shiny_data.rda to be loaded by the server.r save(mod1, params, inits, stiff = true, atol = 1e8, rtol = 1e6, file = rx_shiny_data.rda) to tailor the app, the only two files that need to be altered by the user are the ui.r and server.r files . The ui.r file can be edited to add widgets for obtaining user input and to control the layout of the user interface . The server.r file can be edited to control how inputs are used and how outputs are displayed . There is excellent documentation on developing shiny app interfaces available online8 as well as a previous tutorial on this topic in this journal.14 figure 7 shows the shiny app interface for exploring different factors in an adaptive dosing regimen, including the starting dose, the number of days of simulation, the frequency at which the dose is adjusted, the lower and upper limits of the range for the decision rule, and whether trough, peak, or both levels are controlled by the decision rule . It displays the calculated% time above and below the decision rule limits, and it plots the effect and dose as a function of time . As the user moves the sliders or checks the check boxes, the outputs are adjusted interactively . The ui.r, server.r, rxode.run.r files and the compiled c model are available in the supplementary material . Rxode provides a function for generating interactive shiny apps, which can then be customized . This app allows users to vary the dose, number of days, sampling frequency, and decision rules, and to view simulation results in real time . Thus, starting within a baseline scenario, many different scenarios and combinations of scenarios can be quickly explored . While the modeler will likely facilitate evaluation of scenarios, these apps require no technical expertise or experience with r, and thus the apps are accessible to nontechnical partners . Also, importantly, shiny apps can be easily deployed online and do not require local installation or r. this is in contrast to other software such as berkeley madonna . While berkeley madonna can facilitate interactive model exploration, it also requires purchase of a software license, local installation of the software, and some user familiarity with the software and modeling . Rxode is an r package that provides tools for the efficient simulation of complex dosing regimens via pk / pd models described by odes . It provides great flexibility and speed in performing simulations with variability and uncertainty . As part of the r environment, rxode outputs can be combined with a multitude of r facilities to create advanced static and interactive visualization displays for effective communications with clinical team members and other consumers . Furthermore, unlike many other simulation tools, simulation and preparation of graphics can be conducted completely within a single, freely available, and opensource software . No licenses are required, and it does not require linking with any external software . Although currently focused on efficient simulations, rxode can also be used for parameter estimation through the many existing statistical estimation algorithms in r, including nonlinear mixed effects models,14 stochastic approximation expectationmaximization (saem),16 and bayesian methods using gibbs sampling, e.g., jags.17 future work includes developing functionality to aid users in linking rxode models with these estimation algorithms in a more efficient manner . Supplementary material is linked to the online version of the article at wileyonlinelibrary.com.cpt click here for additional data file.
Development of atherosclerosis and at the same time the peripheral arterial disease of lower limbs is multi gradual process resulting as a response on various forms of damages of endothelium (1). Persons with peripheral arterial disease (pad) are at increased risk for all - cause mortality, cardiovascular mortality, and mortality from coronary artery disease (2). High level of the total cholesterol can increase further development of arteriosclerosis (3). The most of epidemiological studies discovered that increased overall cholesterol and low hdl cholesterol independently, are associated with increased risk of arterial disease of lower limbs (4). Aim of the research was to determine health effects of programmed physical activities on blood fats in peripheral arterial disease of lower limbs or in examinees on medication therapy and examinees who simultaneously with medication therapy performed physical activities . Primary aim was to improve quality of life in patients with peripheral arteriosclerosis, and to compare the change in total amount of blood lipids before and after the conducted program . We hypnotized that physical activity can reduce total cholesterol and triglycerides level in patients with peripheral arteriosclerosis . The programmed physical activity is efficient in improvement of symptoms and increase in exercise capacity (5 - 8). Optimal sample size was determined using g power software . To obtain 80% power at p <0.05 minimal predicted number of patients was 52 in calculated . Overall research was carried out at the clinical for vascular diseases ccus . For cg age between 40 and 55; hospitalization due to other disease; complications of existing disease; self initiative interruption of therapy . Age range 40 - 55 years; hospitalization due to other disease; complications of existing disease; self - initiative interruption of the therapy; noncompliance with activities of the programmed physical activities . Inclusion criteria meet 100 examinees randomly divided into two equal groups: control (n=50) (mean sd: age 48.6 3.82) and test (n=50) (mean sd: 47.56 3.62). The examinees of tg were given a detailed instruction for daily program of physical activities . By analyzing the gender structure of examinees it has been established that the majority of examinees in both groups were males, 66% in control and 62% in test group, while 34% examinees in the control group and 38% examinees in the test group were females (table 1). Patients characteristics in our invsetigation doppler sonography of pedal arteries was used for diagnostic procedure and depending from results examinees were included or excluded into the research and were classified in one of two comparative groups . Except ultrasound findings instruments used in the research were: color doppler sonography of arteriae tibialis posterior (atp) performed on ultrasound device vivid 5 m high - frequency probe of 12 mhz, linear 4 cm . Physiological values are calculated when psv is determined as 30 - 50 cm / s . As a pathological flow clinical data: laboratory data lipid status was determined by enzymatic photometric method on biochemical analyzer hitachi 912, and interpreted as increased or decreased out of reference value limits (total cholesterol: 3.5 - 6.5 mmol / l, triglycerides: 0.5 - 2.1 mm / l) reference values of the clinical center laboratory, sarajevo university programmed activity for tg was consisted of: warm up from 5 to 10 minutes followed by walking on treadmill or fitness, aerobic with resistance . Intensity adjustment of the loading level was in order to cause the claudication symptoms in period of 3 to 5 minutes . Exercise intensity adjustment was increased until the appearance of illness claudicating, followed by rest period in standing or sitting position until recovery . Duration of exercises: initial training lasted approximately 35 minutes and each next session approximately 5 minutes longer until maximum of 50 minutes were reached . Ethical committee publicly approved research according to procedure of the clinical center of the sarajevo university and under principles proscribed by declaration of helsinki . Data analysis was conducted using statistical software program spss (v22, ibm corp . ). Shapiro wilk s (normality of data distribution) and leven s tests for homogeneity of variance for all data were calculated . Chi square (x- test) model 2x2 test was used for determining the differences between percentage structure of gender and smoking habits among and between groups . Changes in cholesterol and triglycerides were assessed over the experimental period for subjects of tg and cg using two factor (group x time) analysis of variance (anova). Data are presented as mean sd (standard deviation). Significance level was set at p <0.05 . Programmed activity for tg was consisted of: warm up from 5 to 10 minutes followed by walking on treadmill or fitness, aerobic with resistance . Intensity adjustment of the loading level was in order to cause the claudication symptoms in period of 3 to 5 minutes . Exercise intensity adjustment was increased until the appearance of illness claudicating, followed by rest period in standing or sitting position until recovery . Duration of exercises: initial training lasted approximately 35 minutes and each next session approximately 5 minutes longer until maximum of 50 minutes were reached . Ethical committee publicly approved research according to procedure of the clinical center of the sarajevo university and under principles proscribed by declaration of helsinki . Data analysis was conducted using statistical software program spss (v22, ibm corp . ). Shapiro wilk s (normality of data distribution) and leven s tests for homogeneity of variance for all data were calculated . Chi square (x- test) model 2x2 test was used for determining the differences between percentage structure of gender and smoking habits among and between groups . Changes in cholesterol and triglycerides were assessed over the experimental period for subjects of tg and cg using two factor (group x time) analysis of variance (anova). The shapiro wilk s test showed that data have normal distribution, and results of leven s test reviled that there is no violation in homogeneity of variance level . Analysis of the examinees age within gropus in relation to the gender structure established that there is no statistically significant difference in age between male and female within tg (f=0.044, p=0.835) and cg (f=1.232, p=0.272). Differences in age between males and females of tg compared to cg were insignificant as well (f=0.445; p=0.506). Hi square test determined that there is no statistically significant difference in gender structure of examinees between tg and cg with male examinees prevail, (=0.172; p=0.418). At baseline patients between groups were similar in bmi value (f=1.126, p=0.291). After treatment of physical activity bmi in tg decreased significantly (f=19.694, p=0.000) compared to cg . At baseline, anamnestic data reviled that 56% of examinees in cg and 54% of examinees in tg consumed cigarettes which was insignificant difference (=0,040; p=0,500) (table 2). At the end point of study, percentage share of smokers in tg and cg findings for blood cholesterol and triglycerides were obtained at the beginning of research for each examinee (table 3). There was no statistically significant difference in the values of cholesterol and triglycerides between tg and cg at the beginning of research (f=0.459; p=0.500) of cholesterol and triglycerides was within reference values . Mmol / l that was statistically significant difference between cg and tg (f=21.614; p=0.000). Analysis of mean values of triglycerides at the end of research did not determined statistically significant difference between cg and tg (f=2.91, p = 0.104). Average values of cholesterol and triglycerides in relation to tg at the beginning and at the end of research planned physical activities as measures rehabilitation medicine are used in treatment of affected with peripheral arterial disease of a lower limbs due to its affects to: metabolism of cholesterol and triglycerides, inflammatory changes and edema, blood flow through the muscle, development of collaterals, muscle pump and causes which disturb its work, drainage of lymph liquid . In this way they contribute to stabilization, change or elimination of functional deficit caused by arteriosclerotic disease, so they present an aspect of functional treatment of the disease (10, 11). When patient achieve a possibility of quality walk the intensity of exercise should be intensified by increased walking speed in order to provide permanent stimulus to retain claudication of pain during the exercise (training is satisfactory only when symptoms appear (12). At the beginning of the study, statistically significant difference were established at the end of study by analyzing mean values of cholesterol in both cg and tg . Statistically significant change of the mean value of the cholesterol was established by analyzing cholesterol and triglycerides values before and after the treatment in examinees of the cg and tg . It is common known fact that lowering smoking habit or stop consuming cigarettes can decrease total cholesterol and triglycerides levels . Claudication symptoms appear early among smokers and after quit smoking become equal like in non - smokers (13, 14). Smoking of cigarettes depending from dosage level represent greater risk factor for peripheral arterial disease then for coroner disease . By increasing of the level and intensity of physical exercise the appearance of cardiac symptoms as well as stenocardia, arrhythmia, great number of patients with symptomatic peripheral arterial disease present atypical symptoms (17). Functional diseases of peripheral arteries are conditions where the peripheral arterial circulation is disturbed and not caused by primary pathological - anatomical changes of the arterial wall but by disturbances of vascular tonus and flow in a vasospasm . Typically, atherosclerosis in people is being developed during the period of several years, usually in several decades . Enlarging of atherosclerotic plaques is probably not going on by linear progression but discontinuously, with period of relative stagnancy interrupted by period of quick evolution . Quite period, arteriosclerosis may become clinically manifested (18). Even within the given vascular bed the atherosclerosis shows tendency of focal localization, typically in predisposed regions . Mechanisms which condition the interruption of continuity in anatomic distribution of atherosclerosis remain unexplained so far . Risk factors of peripheral arterial disease are similar to those important in etiology of coronary arterial disease: smoking, dyslipidemia, diabetes mellitus and hypertension . However, for certain peripheral arterial positions the proofs connecting these factors with development of disease are limited . In addition, specific risk factors may be more significant for development of disease on certain areas (21,22). The most of epidemiological studies have discovered that increased overall cholesterol and low hdl cholesterol independently are connected with increased risk for arterial disease of lower extremities . Improvements in claudication following exercise rehabilitation in patients are dependent on improvements in peripheral circulation and walking economy (23). Improving exercise tolerance through supervised exercise training is an important part of the medical treatment of peripheral arterial disease and intermittent claudication (23). At the beginning of research the values of cholesterol and triglycerides are within reference values . Statistically significant difference between the control and test group was established by analyzing of average values of cholesterol at the end of research . Statistically significant change of the average value of the cholesterol was established by analyzing of the cholesterol and triglycerides values before and after the treatment in examinees of the control and test group . Adequate physical treatment of patients with peripheral vascular disease in our patient proved as very successful . Results indicate statistically significant changes of average values of cholesterol and triglycerides after the treatment of patients . There are obvious limitations of physical therapy such as diseases of muscle and joints, neurological diseases . Cardiac and/or pulmonary diseases may present limitation and decrease the capacity in achievement of adequate level of training required for maintenance of positive results . Concerning the practical aspects, such as difficulties in attendance in the session or negligence of continuous training, the actual results in clinical practice often are not so good as in those in studies.
Fat embolism and fat embolism syndrome (fes) are rare but life threatening complications associated with fracture and surgical manipulation of skeletal elements . None of the proposed diagnostic criteria have good diagnostic value as fes may present atypically such as, diffuse alveolar haemorrhage . A 25-year - old man was referred to us three days post road traffic accident . He was found to have open fracture shaft of left femur and fracture condyle of left humerus . There was no associated history of loss of consciousness, vomiting, convulsions or any bleed . Intra medullary nailing was done for fracture femur under general anesthesia in a peripheral hospital . Intra - operative and immediate post - operative period were uneventful . Within next 18 hours, he was found to be drowsy, restless, hypotensive, and there were signs of pulmonary hemorrhage in the form of blood stained endotracheal secretion with hypoxia . Initial resuscitation was done and he was started on noradrenalin infusion in view of hypotension . Initial laboratory parameters showed: hemoglobin 8.4 gm / dl, total leucocyte count 11300/cc, platelet count 80000/cc . Renal functions were deranged with serum urea 38 mg / dl and serum creatinine 1.2 mg / dl . His liver function tests showed serum billirubin 2.1 mg / dl (direct 1.0 mg / dl), sgot / sgpt 235 and 114 initial arterial blood gas analysis showed, ph 7.37, paco2 63.5 mm hg, pao2 70.8, sao2 93% with fio2 of 1 . Other investigations include serum d - dimer 4.3 (normal 0.0630.245 mg / dl), anti - ccp antibodies 0.43 (normal <5 ru / ml), ana (immunofluroscence) negative at 1:100 dilutions, pr-3anca (elisa) 1.5 iu / ml (normal <6 u / ml). Bronchoalveolar lavage fluid showed numerous debris laden alveolar macrophages and pearls stain was suggestive of intracellular positivity . There was evidence of subcutaneous emphysema with x - ray chest showing signs of pneumomediastenum . Initial high resolution computed tomography (hrct) chest scan showed confluencing air space opacity with ground glass densities and interlobar septal thickening involving bilateral lung fields suggestive of acute respiratory distress syndrome (ards) [figure 1]. He was electively ventilated for next three days, intercostal drain was placed in view of pneumomediastenum . Repeat hrct chest on day 6 revealed patchy ill defined air space opacity in bilateral lower lobes with ground glassing in bilateral upper and middle lobes . His neurological status gradually improved over next 5 days and was put on t piece on 12 day, which he tolerated well . Computer tomography of chest showing bilateral alveolar hemorrhage ct of chest showing recovering acute respiratory distress syndrome and pnemothoarax, pneumomediastenum with intercostal chest tube insitu fat embolism is defined as a blockage of blood vessels by intravascular fat globules ranging from 1040 m in diameter . Fat embolism syndrome (fes) comprises a defined set of clinical pattern and is a serious consequence of fat embolism . Though the major cause of fes is skeletal fracture associated with trauma, small percentage (5%) of cases do have atraumatic etiology . These atraumatic causes include bone marrow transplantation, pancreatitis, sickle cell disease, burns, prolonged high - dose corticosteroid therapy, diabetes mellitus, hepatic trauma, liposuction, lipectomy, external cardiac compression, gas gangrene, decompression sickness, and lipid infusions etc . Perioperative incidence of fes is between 3.5 to 5% in surgeries involving early fixation of fractures . Age is considered to be a determining factor in the development of fes: young men with fractures are at increased risk . The fat enters torn venules which are kept patent in the haversian canals and makes way into circulation . Fat globule ranging from 710 m in diameter has been documented to traverse the pulmonary vasculature . Systemic embolisation has been documented due to a patent foramen ovale which may be present in 25% of individuals . Even larger size fat globule can traverse the foramen if severe pulmonary hypertension is precipitated by fat embolism . Pulmonary hypertension can cause a pressure difference between the right and left atria which leads to embolisation of larger fat globule . Fes is caused by perivascular hemorrhage and edema following the accumulation of fat in the pulmonary, cerebral or dermal microvasculature and local liberation of free fatty acids (ffas). It may be delayed as much as 2 weeks after the insult . In our case, the patient developed respiratory distress and encephalopathy within 18 hours of surgical manipulation of the fracture . Classically, it presents with asymptomatic interval followed by pulmonary, neurologic, and skin manifestations . The initial symptoms are caused by mechanical occlusion of blood vessels with fat globules that are too large to pass through the capillaries . Unlike other embolic events, the vascular occlusion in fat embolism is often temporary or incomplete since fat globules do not completely obstruct capillary blood flow . The late presentation is due to hydrolysis of the fat to more irritating ffas which then migrate to other organs via the systemic circulation . The most common system involved is respiratory (95%) followed by the central nervous system . The incidence has been reported to be 50% to 96% of the affected patients and most of them require ventilation . Petechial rash in conjunctiva, oral mucosa, and skin folds in axilla and neck occurs in up to 60% of cases . The described mechanism for development of petechiae is embolization of small dermal capillaries leading to extravasation of erythrocytes . We noted oblivious petechiae in conjunctiva in both eyes of the patient [figure 3]. Conjunctival hemorrhage a number of minor features of fat embolism syndrome may be present and these appear to result from the release of toxic mediators secondary either to the initial injury or to dysfunctional lipid metabolism . These include tachycardia, myocardial depression, ecg changes indicative of right heart strain, soft fluffy retinal exudates with macular edema, scotomata (purtschers retinopathy), coagulation abnormalities (which mimics disseminated intravascular coagulation). The described pathogenesis is an inflammatory response caused by lipoprotein lipase which is activated by catecholamine surge caused by stress . It acts on the fat deposited in the pulmonary or systemic capillary network liberating high concentrations of toxic ffas locally . It causes platelet aggregation, a mild disseminated intravascular coagulation, and disruption of the pulmonary and cerebral capillary walls . Pulmonary histology usually reveals intra - alveolar hemorrhage, fat within pulmonary capillaries and oedema . Fes is a clinical diagnosis and none of the evaluated laboratory parameters found to be sensitive or specific enough to diagnose fes . Various definitive criteria (include clinical and laboratory parameters) have been described, which include gurds (four major and three minor criteria), lindeques criteria (four criteria) and schonfelds criteria (14 out of 15 points) [chart 1]. [2023] diagnostic criteria for fat embolism syndrome investigations are usually performed to support the clinical diagnosis or to monitor therapy which include: hematology and biochemistry: an unexplained anemia (70% of patients) and thrombocytopenia (platelet count <1,50,000/cumm in up to 50% of patients). Hypocalcaemia (due to binding of free fatty acids to calcium) and elevated serum lipase have also been reported . Hypofibrinogenemia, raised erythrocyte sedimentation rate (esr), and prolongation of prothrombin time may be present . Isolated case report suggests microemboli detected by trans cranial doppler (tcd) and magnetic resonance imaging (mri) can add in diagnosing fes. [62628] corticosteroids act as anti - inflammatory agents and reduce the perivascular hemorrhage and edema . Heparin is thought to have potential for activating lipoprotein lipase thereby increases the clearance of lipemic serum . On the other hand, there is a potential risk of increase in free fatty acid level there - by possible enhancement of inflammation . Bloody bronchoalveolar lavage specimens (with numerous erythrocytes and siderophages) majority of cases of pulmonary hemorrhge have an autoimmune cause and mainstay of the treatment include steroids and immunosuppression . As in our case the cause is nonimmune, we used high dose steroids (methyl prednisolone started at 60 mg twice daily and tapered over 2 weeks with improvement of clinical state). Fes is a rare but devastating complication in patients presenting with traumatic fracture of long bones or procedure related and involving manipulation of the same . The diagnosis of fes is clinical, though use of diagnostic criteria may be helpful . Though none of the available studies have clearly supported the role of steroids, we found good result with early use of steroid in our case.
Primary mesenchymal tumors of the intestinal tract are a heterogeneous group of tumors with a wide clinical spectrum ranging from benign incidentally detected nodules to frank malignant tumors . Traditionally the majority of these tumors were thought to be derived from smooth muscle cells . It has now been realized that the majority of these tumors are not smooth muscle or nerve sheath tumors, but represent a distinct clinico - pathological entity termed as gastrointestinal stromal tumor (gist). It is currently believed that gist is a specific mesenchymal neoplasm and the term is used to refer to those mesenchymal neoplasms of the gastrointestinal tract (git) which express cd117, a c - kit proto - oncogene protein, and show gain of function mutation of c - kit gene that encodes a growth factor receptor with tyrosine kinase activity . The introduction of a new targeted treatment for gist in the form of imatinib mesylate, a receptor tyrosine kinase inhibitor, has further validated this entity . Gist can occur at all levels of git and may also arise in extra - gi locations, principally mesentery, omentum and retroperitoneum and occasionally in pancreas . Approximately 50 - 60% of gists arise in the stomach, 20 - 30% in small bowel, 10% in large bowel and 5% in esophagus . About 30% of gist are malignant and liver is the most common site for metastasis. [710] the criteria for differentiation of benign from malignant gist remain controversial . Many parameters have been proposed, tumor size and proliferative activity have been found to be the most important prognostic indicators . Gist reported outside the git t as apparent primary tumors are designated as extra - gastrointestinal stromal tumor (egist). [1214] the frequency of egist is only 5 - 7% . Because of overlapping morphology, gists are cytologically difficult to distinguish from other gastrointestinal mesenchymal neoplasms including smooth muscle tumors and nerve sheath tumors. [1519] although many investigators have described various features used in the cytologic diagnosis of gist, few have reported their findings in patients with malignant gist . The present study is the largest series discussing the cytology of gists and egist from different anatomic sites including extremely rare sites like subcutaneous nodules, fluid cytology samples and metastasis from a primary pancreatic egist . In the present study we studied the cytomorphological features in 33 gist / egist from 27 patients . All patients of histologically and immunohistochemically confirmed gist and egist who underwent cytological examination over a period of ten years (jan 2000-july 2010) were retrieved from the records of the department of pathology . 33 specimens from 27 patients including 26 gist (13 primary, 12 metastatic, one recurrent) and seven egist (5 primary, 1 metastatic, 1 recurrent) were identified . These included guided (28) and unguided (2) fine - needle aspirates, imprint smear (1) and fluid cytology (2). Fine needle aspiration cytology (fnac) was performed using 22 gauge needle attached to 10 ml disposable syringe under ultrasound guidance of intra - abdominal tumors . Fna material was smeared on glass slides and slides fixed in 95% alcohol were stained with papanicolaou and hematoxylin and eosin stain while air dried smears were stained with may - grnwald - giemsa stain (mgg). Histological categorization of gist of different sites into various risk groups was done as suggested recently by miettinen et al . Cytology was reviewed in all cases with emphasis on the following cytological features: overall cellularity, smear pattern (cohesion vs. dispersed cells), palisading, crush artefact, prominent vascular pattern, spindle versus epithelioid cell morphology, nuclear grooves and inclusions, nuclear pleomorphism, presence of nucleoli, round or blunt - ended oval or wavy nuclei, multinucleation / bizarre cells / or giant cells, perinuclear vacuoles, cytoplasmic quality, mitoses and necrosis . The patients included 20 males and 7 females with male: female ratio of 2.67:1 . The mean age was 50.6 years (range: 26 - 76, median: 52 years). There were 18 primary tumours (5 gastric, 5 duodenum, 1 ileum, 1 ileocecal, 1 rectum, 4 intra - abdominal / retroperitoneal tumours, 1 mesentery), 9 hepatic metastases (5 gastric, 2 duodenum, 1 jejunum, 1 small bowel primary tumours), 1 ascitic fluid metastases (gastric primary), 1 pleural fluid metastases (jejunum primary), 1 subcutaneous nodule metastases (gastric primary), 1 lump at base of penis (rectum primary) and 2 recurrences (1 ileum, 1 intra - abdominal / retroperitoneal). The smears were variably cellular . Malignant and metastatic lesions were commonly highly cellular . In patients with predominantly spindle cells on cytology (n = 18) smears showed cells often arranged in cohesive to loose three dimensional clusters and singly scattered or dispersed cells [figure 1a and b]. The tumor cells often formed fascicles with parallel, side - by - side arrangements of nuclei . In these fascicles tumor cells the stroma of the cohesive sheets present between the nuclei was loosely fibrillary and stained pink to magenta on mgg [figure 1d]. Skenoid fibres were noted in a smear from liver metastasis of a patient with primary jejunal gist . Spindle cells with high cellularity, closely packed to loose clusters and dyscohesive cells (mgg, 100); (a) spindle cells displaying elongated to wavy nuclei with blunt to tapered ends (mgg, 200); (b) fascicles with parallel, side - by - side arrangements of nuclei with scant cytoplasm . Nuclear palisading is also observed (mgg, 100); (c) abundant extracellular stromal material (mgg, 200); (d) (inset: spindle cells with bipolar cytoplasmic processes (mgg, 400) focal to diffuse epithelioid cell morphology admixed with spindle cells were observed in twelve samples and 3 tumors had only epithelioid cell type [figure 2a]. Plasmacytoid appearance was noted in eight samples [figure 2c] few demonstrated rossetting and acinar structure arrangement reminiscent of adenocarcinoma [figure 2d]. The nucleoli were indistinct in low grade tumors and prominent or multiple nucleoli were seen in high grade tumors irrelevant of cell type . The presence of marked cytologic atypia with presence of bizarre and giant cells was identified in 6 tumors . Occasional tumors displayed bubbly appearance due to multiple cytoplasmic vacuoles . In 9 cases mitotic figures were observed . The pleural and ascitic fluid cytology smears exhibited loosely formed aggregates with epithelioid cell morphology . Smear showing groups of epithelioid tumors cells with round nuclei (mgg, 200); (a) multinucleation (mgg, 400); (b) plasmacytoid tumor cells displaying eccentric round nuclei with smooth nuclear membrane and fine chromatin (mgg, 400); (c) acinar arrangement of tumor cells mimicking epithelial tumor (mgg, 400); (d) (inset: mitotic figure in a mixed gist (mgg, 400) histology sections of all primary gist and egist were reviewed . The majority (n = 18, 66.67%) of tumors were classified as spindle cell type, while 3 (11.12%) were classified as epithelioid type and 6 (22.23%) as mixed cell type . The cellularity of the majority of tumors was subjectively assessed as moderate (n = 17). Categorization of gist of different sites into various risk groups was performed as suggested recently by miettinen et al . There was only one low grade tumor, 4 tumors with moderate risk, and 22 tumors were categorized as high risk . Immunohistochemically all tumors were positive for cd117 with strong intensity of positivity in the majority of tumors . The cytological studies on gist in literature are not comparable as many of the earlier published cytologic studies have combined gist into a group of neoplasms encompassing leiomyoma, schwannoma, leiomyosarcoma, and an epithelioid leiomyoblastoma. [1517] however recently, few authors have established fnac as a reliable method for diagnosing gist and egist before surgical procedure [table 1]. [1830] we observed several morphologic features suggestive of high grade gist and egist . In recurrent, metastatic and malignant gist however, it was difficult to find mitoses in the cytologic smears because most of the tumor cells occurred in closely packed cohesive thick tissue fragments . Li et al ., found that mitoses in the resected malignant gists were seldom seen in fnac smear . We also found that malignant gists sometimes had no significant pleomorphism in the cytologic smears . Nuclear inclusions were more commonly seen in malignant and metastatic tumors irrespective of cell type in our cases . Dirty or necrotic background again was least reliable indicator of malignancy as it was seen in two samples only . We agree with earlier descriptions that fna findings alone cannot reliably assess behavior of gist . Prediction of behavior by cytological features is likely to result in either underestimation or overestimation of malignant potential. [1825] summary of various series of gist and egist cytology cases described in the literature the differential diagnosis between gists and gastrointestinal leiomyomas is difficult owing to their overlapping clinical and cytologic features . Leiomyoma shows varying cellularity and are composed of bland spindle cells with abundant cytoplasm often having a fibrillary appearance . Leiomyosarcomas shows three - dimensional, tightly cohesive, sharply marginated syncytia of spindle cells, often with nuclear crush artefact . Benign and malignant nerve sheath tumors show fibrillar cytoplasm and wavy nuclei, similar to a subset of and characteristic features of nerve sheath differentiation, such as nuclear palisading, may be focal . Epithelioid gists may cause significant diagnostic confusion with carcinomas, neuroendocrine tumors, and melanoma, particularly when metastatic and even hepatocellular carcinoma . The regular round nuclei, with finely granular chromatin seen in many of cases raised the possibility of neuroendocrine tumor . Key features of melanoma are loose aggregates or isolated cells, cellular pleomorphism, enlarged nuclei with macronucleoli, binucleation, multinucleation and intracytoplasmic melanin . Intranuclear inclusions, intracytoplasmic bile pigment and no bile duct epithelium are important distinguishing features . The separation of metastatic gist from other metastasis is important since these may respond to imatinib . To conclude, firstly cytology is a useful method for preoperative diagnosis and follow - up of gists and egist . Gist show a broad morphologic spectrum on cytology and gist should be considered as a differential diagnosis of tumors with spindle or epithelioid morphology . In an appropriate clinical and radiologic setting the presence of closely packed spindle or oval cells forming fascicles with parallel side - by - side arrangements of nuclei suggests gist . The diagnosis of gist should also be considered in aspirates of the gastrointestinal tract, liver, mesentery, or abdominal wall mass lesions when epithelioid cells are the predominant cell type . The presence of cellular dyscohesion, nuclear pleomorphism, necrosis and mitosis suggest malignant behavior, however absence of these features on cytology is not diagnostic of low risk benign behavior.
Participants were autoantibody - positive relatives of t1d patients from the dpt-1 (n = 670) and tnnhs (n = 991) cohorts . Dpt-1 participants were all islet cell autoantibody (ica) positive, whereas tnnhs participants were all positive for gada, ia-2a, miaa, and/or ica . Both studies were approved by institutional review boards at all participating sites, and written informed consent or assent as appropriate were obtained in both studies . Two - hour oral glucose tolerance tests (ogtts) were performed at baseline and at 6-month intervals in both cohorts . Oral glucose (1.75 g / kg; maximum, 75 g of carbohydrate) was administered after fasting samples were obtained; glucose and c - peptide samples were then obtained every 30 min . Plasma glucose was measured by the glucose oxidase method . In dpt-1, c - peptide was measured by radioimmunoassay, whereas in the tnnhs, the tosoh assay was used . Values from the two assays were similar in split samples (r = 0.961; tosoh = 0.96 * rai + 0.1; n = 564). Undetectable fasting c - peptide values (<0.2 ng / ml) were assigned a value of 0.1 ng / ml . The methodology for performing autoantibody measurements in the tnnhs has been reported previously (12). The autoantibodies obtained in the tnnhs include ica, gada, miaa, ia-2a, and znt8a . The dptrs (7), developed from the dpt-1 cohort, is based on a proportional hazards model that includes the glucose sum of 30-, 60-, 90-, and 120-min values divided by 100, the c - peptide sum of 30-, 60-, 90-, and 120-min values divided by 10, log fasting c - peptide, log bmi, and age . There is a curvilinear relation between risk and the dptrs . As dptrs values increase above 6.50, the risk estimates increase more steeply (8). Dysglycemia was defined as any of the following on the baseline ogtt: a fasting glucose value between 110 and 125 mg / dl (impaired fasting glucose); a 30-, 60-, and/or 90-min value 200 mg / dl with a 2-h value <140 mg / dl (indeterminate); or a 2-h value between 140 and 199 mg / dl (impaired glucose tolerance [igt]). Participants were informed if they had a dysglycemic ogtt; however, no treatment was recommended . Analyses included the following: a comparison of the cumulative incidence of t1d between normoglycemic individuals with dptrs values> 7.00 and those with dysglycemia; a comparison of the cumulative incidence of t1d between dptrs values <7.00 or> 7.00 among those with dysglycemia and among those with two or more autoantibodies; a comparison of the cumulative incidence of t1d between the tnnhs and dpt-1 among those with dptrs values> 7.00 and among those with dysglycemia; and a comparison of the reliability between dptrs values> 7.00 and dysglycemia . The 7.00 threshold had previously been shown to identify high - risk individuals in the overall dpt-1 and tnnhs cohorts (8). The p values are two - sided . A p value <0.05 was considered to be statistically significant . Participants were autoantibody - positive relatives of t1d patients from the dpt-1 (n = 670) and tnnhs (n = 991) cohorts . Dpt-1 participants were all islet cell autoantibody (ica) positive, whereas tnnhs participants were all positive for gada, ia-2a, miaa, and/or ica . Both studies were approved by institutional review boards at all participating sites, and written informed consent or assent as appropriate were obtained in both studies . Two - hour oral glucose tolerance tests (ogtts) were performed at baseline and at 6-month intervals in both cohorts . Oral glucose (1.75 g / kg; maximum, 75 g of carbohydrate) was administered after fasting samples were obtained; glucose and c - peptide samples were then obtained every 30 min . Plasma glucose was measured by the glucose oxidase method . In dpt-1, c - peptide was measured by radioimmunoassay, whereas in the tnnhs, the tosoh assay was used . Values from the two assays were similar in split samples (r = 0.961; tosoh = 0.96 * rai + 0.1; n = 564). Undetectable fasting c - peptide values (<0.2 ng / ml) were assigned a value of 0.1 ng / ml . The methodology for performing autoantibody measurements in the tnnhs has been reported previously (12). The autoantibodies obtained in the tnnhs include ica, gada, miaa, ia-2a, and znt8a . The dptrs (7), developed from the dpt-1 cohort, is based on a proportional hazards model that includes the glucose sum of 30-, 60-, 90-, and 120-min values divided by 100, the c - peptide sum of 30-, 60-, 90-, and 120-min values divided by 10, log fasting c - peptide, log bmi, and age . There is a curvilinear relation between risk and the dptrs . As dptrs values increase above 6.50, the risk estimates increase more steeply (8). Dysglycemia was defined as any of the following on the baseline ogtt: a fasting glucose value between 110 and 125 mg / dl (impaired fasting glucose); a 30-, 60-, and/or 90-min value 200 mg / dl with a 2-h value <140 mg / dl (indeterminate); or a 2-h value between 140 and 199 mg / dl (impaired glucose tolerance [igt]). Participants were informed if they had a dysglycemic ogtt; however, no treatment was recommended . Analyses included the following: a comparison of the cumulative incidence of t1d between normoglycemic individuals with dptrs values> 7.00 and those with dysglycemia; a comparison of the cumulative incidence of t1d between dptrs values <7.00 or> 7.00 among those with dysglycemia and among those with two or more autoantibodies; a comparison of the cumulative incidence of t1d between the tnnhs and dpt-1 among those with dptrs values> 7.00 and among those with dysglycemia; and a comparison of the reliability between dptrs values> 7.00 and dysglycemia . The 7.00 threshold had previously been shown to identify high - risk individuals in the overall dpt-1 and tnnhs cohorts (8). The p values are two - sided . A p value <0.05 was considered to be statistically significant . The 991 tnnhs participants were significantly older than the 670 dpt-1 participants (mean age sd: 18.5 13.3 years [median 13.0 years] vs. 13.8 9.6 years [median 11.1 years]; p <0.001). In the tnnhs, the median follow - up of those diagnosed and those not diagnosed was 1.4 and 2.0 years, respectively . Whereas 125 had igt alone, 29 had an indeterminate ogtt alone, and 7 had impaired fasting glucose alone . Figure 1 shows that a dptrs threshold> 7.00 identified tnnhs participants with normoglycemia at baseline who were at substantial risk for t1d . The cumulative incidence of tnnhs participants with normoglycemia and dptrs values> 7.00 was comparable to the cumulative incidence of those with dysglycemia . The 3-year risk estimates were 0.38 for those with dptrs values> 7.00 and 0.33 for those with dysglycemia . Tnnhs participants with normoglycemia and dptrs values> 7.00 were much younger than those with dysglycemia (8.1 4.9 years for dptrs> 7.00 vs. 19.6 14.3 years for dysglycemia; p <0.001). Shown are cumulative incidence curves for t1d of tnnhs participants with normoglycemia (ngt) and the cumulative incidence curves are comparable . N.s ., not significant . When those in the tnnhs with dysglycemia were dichotomized according to dptrs values> 7.00 or <7.00 (fig . 2), there was a marked difference between the cumulative incidence curves (p <0.001). The 3-year risk estimate was 0.46 for those> 7.00, whereas the 3-year risk estimate was only 0.16 for those <7.00 . The 3-year risk for those with normoglycemia and dptrs values <7.00 was 0.08 . Shown are cumulative incidence curves of tnnhs participants with dysglycemia after they are dichotomized according to dptrs values <7.00 or> 7.00 . There is a large difference in the cumulative incidence (3-year estimates: 0.16 for <7.00 and 0.46 for> 7.00). We also assessed the differences in risk according to the 7.00 dptrs threshold among individuals with two or more autoantibodies in the tnnhs . There was again a marked difference in risk (p <0.001 for difference in cumulative incidence curves). Individuals with dptrs values> 7.00 (n = 87) had a 3-year risk estimate of 0.55, whereas those with dptrs values <7.00 (n = 209) had a 3-year risk estimate of 0.16 . The degree of consistency in estimating risk between dpt-1 and the tnnhs is shown for those with dptrs values> 7.00 in fig . Whereas there was no significant difference in the cumulative incidence of t1d between dpt-1 and tnnhs participants for those with dptrs values> 7.00, the cumulative incidence for those with dysglycemia was much higher in dpt-1 than in the tnnhs (p <0.001). Shown are cumulative incidence curves of tnnhs and dpt-1 participants who either had dptrs values> 7.00 (a) or dysglycemia (b). Among those with dptrs values> 7.00, the cumulative incidence was similar between the tnnhs and dpt-1, whereas among those with dysglycemia, the cumulative incidence was markedly lower in the tnnhs than in dpt-1 . Reliability was also compared between dysglycemia and the 7.00 dptrs threshold . Of those who had dysglycemia at baseline in the tnnhs of the 77, 41 (53%) had dptrs values <7.00 at baseline . Of those with dptrs values> 7.00 at baseline, a smaller proportion, 42 of 177 (24%), reverted to having values <7.00 at the next visit . Of the 42, 22 (52%) had normoglycemia at baseline . Of those with dptrs values> 7.00 who reverted to dptrs values <7.00, the glucose sum declined significantly (p <0.001). There was a decline in the c - peptide sum that was of borderline significance (p = 0.05). We have performed proportional hazards regressions to examine associations of t1d with dysglycemia and with the dptrs as single variables . Each was highly predictive of t1d when included alone (p <0.001 for both). However, when both were included in a model, while t1d and the dptrs were still strongly associated (p <0.001), there was no longer a significant association between t1d and dysglycemia (p = 0.783). Risk estimates are indicated at 2, 3, and 4 years of follow - up for those above certain dptrs thresholds at baseline in the tnnhs . If the dptrs threshold of 7.00 was used instead of dysglycemia for the selection of prevention trial participants, more would have been diagnosed (71 vs. 62) with a smaller number entered (191 vs. 221). Other thresholds could be selected according to the desired number of participants and their level of risk . For example, if the 6.75 threshold was chosen in place of dysglycemia for the selection of participants, appreciably greater numbers would have been entered (253 vs. 221) and diagnosed (85 vs. 62) even though the risk was still comparable . Risk estimates of t1d for dysglycemia and dptrs thresholds at baseline in the tnnhs (n = 991) we examined the occurrence of t1d for those above dptrs thresholds when dysglycemia was absent and for those below dptrs thresholds when dysglycemia was present (supplementary table 1). Whereas 22 of 64 (39%) were diagnosed of those normoglycemic with a dptrs value> 7.00, only 13 of 94 (13%) were diagnosed of those dysglycemic with a dptrs value <7.00 . The findings indicate that a reliance upon dysglycemia as a demarcation of risk in autoantibody - positive populations could result in a less - than - optimal classification of risk for prevention trials . The risk of certain individuals with normoglycemia could actually be higher than some with dysglycemia . The findings also show that the risk implications of dysglycemia can vary markedly according to the particular population that is studied . In addition, the presence of dysglycemia can be inconsistent when ogtts are repeated in individuals . It is evident that the dptrs can improve the accuracy of risk classification when it is used in conjunction with dysglycemia for prediction . However, the data in table 1 suggest that it might be advantageous to use dptrs thresholds in place of dysglycemia for prevention trials . In addition to improving accuracy, dptrs thresholds provide a choice of target populations with different risks . If greater risk homogeneity is desired for a trial, bounded categories could be used . Risk estimates of specific dptrs categories have previously been reported (8); these can be used for reference . The dptrs provides selection from a risk continuum, whereas dysglycemia only offers a dichotomous selection . The presence of multiple autoantibodies has been used as an indicator of higher risk for t1d . However, those with dptrs values <7.00 were at much lower risk than those with dptrs values> 7.00 . A recent article (12) examined prediction by an autoantibody risk score in tnnhs participants that takes both positivity and level into account . In that article, the area under the receiver operating characteristic curve was higher for the dptrs than the autoantibody risk score . Also, when the autoantibody risk score and the dptrs were both included in a regression model, the dptrs was still highly predictive . Risk can vary substantially for a given number of autoantibodies, depending upon which specific autoantibodies are positive (13). It is likely that risk prediction will be improved further with the refinement of autoantibody prediction (14,15) and the integration of autoantibody and metabolic information . However, it is relatively weak in comparison with other predictors, both overall (7) and among those with normoglycemia (6). Moreover, the performance of intravenous glucose tolerance testing is cumbersome for participants and labor intensive for staff . Individuals with normoglycemia identified as being at substantial risk for t1d were much younger than those with dysglycemia . This finding suggests that the t1d risk of children can be particularly underestimated when dysglycemia is used as an indicator of risk and highlights the importance of the consideration of age in assessing the risk of t1d . The dptrs is useful for identifying normoglycemic individuals at high risk in large part because it includes age, which is inversely related to the risk for t1d (7,8). The lack of consistency in the cumulative incidence curves between the tnnhs and dpt-1 among those with dysglycemia shows that a dependence upon glucose indices alone for risk estimation can be misleading . The greater consistency of dptrs values> 7.00 is possibly attributable to the consideration of other factors associated with risk . The frequent reversion from the dysglycemic state to the normoglycemic state in the tnnhs is consistent with previous findings in dpt-1 in which there were frequent fluctuations between states of glycemia during the progression to t1d (20). The lower reversion rate from dptrs values> 7.00 to <7.00 suggests that dptrs thresholds are more reliable indicators of risk than dysglycemia . Thus, the findings are not necessarily generalizable to other populations, such as those at genetic risk for t1d . However, since dpt-1 participants were selected on the basis of autoantibodies that differ from those in the tnnhs, the findings show that the dptrs has general utility for improving the risk classification of autoantibody - positive populations . Although no treatment was recommended, it is possible that some could have undertaken treatment on their own . Dysglycemia has been shown to be a frequent precursor to t1d (4,21), and an understanding of its development in the pathogenesis of diabetes is of importance . However, data now suggest that the prediction accuracy of t1d can be improved well beyond the predictive information provided by glycemia status . The findings indicate that the dptrs can refine prediction and ultimately improve the accuracy of t1d risk classification.
A 3.09 kg, 33-day - old boy with pierre robin syndrome was admitted to our hospital for a glossopexy . The patient was born at 39 weeks by c - section and was diagnosed with the syndrome during pregnancy . After birth, the patient showed a micrognathia, retrognathia, and a large tongue . The patient also showed moderate chest retraction with coarse breathing sounds presenting difficulty in maintaining patent upper airway . He was kept in a prone / lateral position under 5 l / min of o2 hood at nicu until the operation . In the operating room intraoperative monitoring devices such as an ecg, an automatic blood pressure cuff, and a pulse oximeter were applied to the patient . The initial oxygen saturation was 98% in room air and other vital signs (bp: 96/27 mmhg, pr: 161/min, rr: 42/min) were in normal ranges . A mask was applied before induction and the manual ventilation was well maintained with patient's spontaneous ventilation . Premedication with 0.004 mg / kg of glycopyrrolate was given intravenously and anesthesia was induced with gradual increment of sevoflurane concentrations from 2% to 4% with oxygen by mask while also maintaining spontaneous respiration . Brief direct laryngoscopic examination with a curved mcintosh blade (#1) was performed and only the hypopharynx was seen . Classical reusable laryngeal mask airway (lma) #1, in which two vertically placed bars were removed, was inserted and effective ventilation was assured by chest expansion, auscultation, and etco2 monitoring . While pausing the ventilation, ultrathin fiberoptic bronchoscope (fob) (olympus enf type xp, 1.8 mm od, length: 530 mm, tokyo, japan) was passed through the lumen of the lma and the vocal cords were well visualized . Under the visualization of the vocal cords by the fob, we passed the cook airway exchange catheter (caec) (1.6 mm i d, 2.7 mm od) (cook medical incorporated, bloomington, in, usa) through the lma into the vocal cords . However, passing the caec into the vocal cords under the guidance of the fob repeatedly failed because the caec frequently flexed backwards toward the esophagus . At that time, we recognized that the length of an uncuffed i d 3.0 mm endotracheal tube (length: 16 cm) was longer than that of lma#1 (length: 11 cm). Therefore, through the lma, the tracheal tube was mounted on the fob and introduced into the trachea . Successful ventilation was verified by manual ventilation . To prepare for any accidental removal of the tracheal tube while removing the lma, we inserted the caec into the lumen of the tracheal tube . After placing the caec in the midtrachea, lma was carefully removed while tightly holding both the caec and tracheal tube simultaneously . Despite this, the endotracheal tube was displaced out of the vocal cords while removing the lma . By holding the caec firmly inside the trachea, the displaced endotracheal tube after intubation, endotracheal tube position was verified again by auscultation of equal bilateral breathing sounds and etco2 . During the procedure, anesthesia was maintained with intermittent sevoflurane administration in oxygen (o2: 1 l / min, air: 1 l / min) through the lma to prevent awakening and desaturation of the patient . The maximum time allowed for the procedure was until the spo2 decreased lower than 95% . Atracurium 1.5 mg was given intravenously for muscle relaxation and mechanical ventilation was maintained during the operation . The extubation was carried out when the patient was in a completely alert state and the spontaneous ventilation was well maintained without any chest retraction following the extubation . The patient was then transferred to nicu in the supine position under 5 l / min of o2 mask . Patients with pierre robin syndrome may create difficulties during intubation and airway control is a major concern for anesthesiologists . Ventilation using a face - mask in the presence of a markedly recessed mandible may be difficult and patient's supine position frequently leads to a total obstruction of the airway . Visualization of the larynx is almost impossible once the patient is anesthetized . In a recent survey conducted amongst pediatric anesthetists in canada, 73% stated that they first attempted a direct laryngoscopy and, in the case of a failure, 51% would choose laryngeal mask airway - guided fiberoptic intubation . When fob is chosen to be used for intubation, most anesthesiologists generally tend to use lma as a conduit for the bronchoscope, indicating that this technique is now firmly established in an approach to the difficult pediatric airway . Although this approach seems to be very successful, one problem is that the endotracheal tube can be easily withdrawn from the trachea during the removal of the lma . In order to prevent this, selim et al therefore, it was possible to keep the intubated tube patent inside the trachea while lma was being removed, but instability of an elongated tube has still been a problem . In addition to this, some authors used a long guide wire inserted into the suction port of the fiberscope, which was then placed inside the trachea under direct vision . Having the guidewire left in the trachea, the fiberscope and lma were withdrawn, and the tracheal tube was railroaded over the guidewire . A disadvantage of this method was that the guidewire can be easily bent and flipped out of the trachea . Osses et al . Introduced a method of using an adult intubating stylet attached to the end of the tracheal tube so that lma would be removed while pushing the stylet inward . As an alternative approach, we used the caec under the guidance of the fiberscope to ease the insertion of the endotracheal tube into the trachea . However, when this catheter was passed down through the lma, it frequently emerged from the posterior aspect of the mask aperture of the lma and resulted in an esophageal intubation even though vocal cords were well - visualized with the ultrathin fob . Despite many trials using a caec, it was very difficult to approach the vocal cords with the tip of the catheter . Brimacombe and berry have reported that the success rate of blind placement of the caec via the lma in adults was only 30% despite good lma positioning . Therefore, this indicates that, even when the lma is perfectly positioned, blind tracheal intubation cannot always be performed successfully . In children, perfect positioning of the lma was observed in 44%, 29%, and 49% of all cases, which also indicates that there may have been some impediment to blind passage of the endotracheal tube, a bougie or introducer when this technique is attempted . To correctly place the caec into the trachea, after the guidewire passed through the suction channel of the fob, a stiffening device such as the caec or ureteral dilator railroaded over the guide wire through the lma might be helpful in placing the caec or dilator correctly in the trachea . In addition to this, we suggest another method for successful placement of the caec . As shown in fig . 1, a fishing line is passed through the lumen of a caec and form a guide - loop like the one of arndt endobronchial blocker (cook medical incorporated, bloominton, in, usa) at the distal end of the caec and a tight knot is made at the proximal end of the catheter, making sure that it does not come loose . Then, the fob and lma were removed while keeping the catheter in midtrachea and the endotracheal tube is mounted on the caec, followed by railroading over the caec . In a pediatric patient, weighing 7.5 kg, thomas and parry also used a caec through which a fob was inserted and the tip of the fob was extended from the distal aperture of the caec . Following introduction of caec with the enclosed fiberscope into the trachea, the fob was removed leaving the caec within the trachea . Although this is an interesting approach, considering that they used an olympus lf - p fiberscope (proximal od 2.2 mm and distal od 1.8 mm) and a caec (2.3 mm i d for use with tracheal tubes of i d 4 mm or larger), it cannot be used in a smaller pediatric patient because the olympus enf type xp or lf - p fiberscope cannot pass through the lumen of the caec (1.6 mm i d for use with tracheal tubes of 3 mm or larger) which we used . Another problem with this approach is that the caec has to be cut to an adequate length to ensure that the fob tip extends from the distal aperture of the caec . In our case, an uncuffed 3.0 mm i d endotracheal tube (length: 16 cm) was longer than that of lma#1 (length: 11 cm). However, in pediatric difficult airway cases in which the endotracheal tube length is similar to or shorter than the lma, the proximal end of the tracheal tube tends to disappear into the lma once the tracheal tube has passed through the vocal cords . In this situation, it might be helpful to insert a caec first through the lma using the suggested methods by walker, thomas and parry's methods, or our method in order to facilitate easy insertion of the caec into the trachea . In summary first, it can provide a more stable passage for the endotracheal tube since the diameter of the caec which we used (1.6 mm i d) is larger than the guide wire previously used to pass through the suction port . Second, it is much safer to attempt repeated intubation in the case of accidental tube displacement out of the trachea during removal of the lma.
Frontalis sling surgery is the treatment option for myopathic blepharoptosis with poor levator muscle action . Nontuberculous mycobacterial infections in the periocular region are rare and are usually caused by organisms belonging to runyon group iv including mycobacterium chelonae and mycobacterium fortuitum . Chang et al . Reported six cases of nontuberculous mycobacterial infection and found an association with nasolacrimal duct obstruction, implantation of foreign body, history of recent surgery, and immunosuppression . Mauriello found implantation of a foreign body in 5 out 13 patients of nontuberculous mycobacterial infections of the periocular region . Treatment of nontuberculous mycobacterial infections can be difficult because of their multi - drug resistance . Treatment usually involves surgical debridement with removal of infected foreign body and a prolonged course of antibiotics like amikacin, clarithromycin, ciprofloxacin, and doxycycline . We report a rare case of nontuberculous mycobacterial infection of the silicone rod after frontalis sling surgery . A 65-year - old apparently healthy male presented with complaints of pain and swelling of both eyelids of 2 months duration . He had undergone cataract and frontalis sling surgery for myopathic blepharoptosis in both eyes 2 years prior to presentation . The best corrected visual acuity for distance was 20/400 in right eye and 20/25 in the left eye . Examination of face showed multiple, erythematous nodules involving both upper eyelids and the brow . All nodules were localized to the scars from the previous ptosis surgery with a few showing signs of suppuration (fig . The posterior segment showed hypertensive retinopathy in both eyes and a scar at the macula in the right eye . Systemic workup was unremarkable and included a complete blood picture, chest x - ray and serological tests for hiv . The patient was clinically diagnosed to have chronic progressive external ophthalmoplegia status post bilateral frontalis sling and cataract surgery with multiple abscesses in forehead and upper eyelids . The patient underwent an incision and drainage of the abscesses in the brow and the purulent material was sent for microbiological examination . The smear showed the presence of acid fast bacilli on 20% acid fast staining (fig . 1b) and culture was significant for staphylococcus sp . And m. fortuitum (fig . The patient received intravenous amikacin (1 g / day, single dose) and ciprofloxacin ointment locally . Poor response to treatment necessitated exploration with explantation of the silicone rod sling in the right eye 10 days later . A repeat microbiological evaluation showed acid fast bacilli and significant growth of m. fortuitum on culture . As the isolate showed a similar antibiotic sensitivity pattern the patient was continued on intravenous amikacin (1 g / day). However, 11 days later, the patient suffered from another episode of pain and swelling in the left eye . A repeat exploration of the brow with drainage and silicone rod explantation was performed in the left eye . The patient showed a slow but definite improvement and received intravenous amikacin for 6 weeks . Five months after commencement of therapy the patient was asymptomatic and the left upper eyelid position was maintained (fig . 1a an apparently healthy 62-year - old man presented with painful, erythymatous nodules of bilateral brow and upper lids two years after frontalis sling surgery for severe myopathic ptosis . B microscopic picture of the purulent material showing long, slender acid fast bacilli along with polymorphonuclear cells (ziehl neelsen stain, total magnification 1,000). C sheep blood chocolate agar inoculated with purulent material along with silicone tube (left eye brow after 11 days of treatment) shows confluent growth of cream colored, opaque, medium size colonies of staphylococcus aureus and tiny, semi - opaque colonies of mycobacterium fortuitum (incubation: 4 days, 5% co2, 37 c). D five months after sensitivity - based antibiotic treatment and explantation of bilateral silicone rods, the patient showed resolution of symptoms . The left upper eyelid maintained an elevated position after silicone rod explantation a an apparently healthy 62-year - old man presented with painful, erythymatous nodules of bilateral brow and upper lids two years after frontalis sling surgery for severe myopathic ptosis . B microscopic picture of the purulent material showing long, slender acid fast bacilli along with polymorphonuclear cells (ziehl neelsen stain, total magnification 1,000). C sheep blood chocolate agar inoculated with purulent material along with silicone tube (left eye brow after 11 days of treatment) shows confluent growth of cream colored, opaque, medium size colonies of staphylococcus aureus and tiny, semi - opaque colonies of mycobacterium fortuitum (incubation: 4 days, 5% co2, 37 c). D five months after sensitivity - based antibiotic treatment and explantation of bilateral silicone rods, the patient showed resolution of symptoms . Several authors have reported about nontuberculous mycobacterial infections after periocular surgery [3, 4]. The median time interval between prior surgery and onset of infection in these reports was 6 weeks (range 0.511 months) [3, 4]. In our patient, multiple erythematous nodules with suppuration along the tract of the silicone rod more than 2 years after surgery led to the suspicion and eventual diagnosis of nontuberculous mycobacteria . Some aspects in our patient need to be highlighted . First is that a bilateral infection by nontuberculous mycobacteria involving the silicone rod after frontalis sling surgery has not been reported earlier . The lodgement of nontuberculous mycobacteria in the brow region in our case may be related to the persistent irritation from the silicone rod and its predeliction for fat [4, 5]. Sequestration of nontuberculous mycobacteria with fat allows its growth without detection by normal immunosurveillance [4, 5]. In our patient, sequestration of nontuberculous mycobacteria in the brow fat may have been responsible for delayed and bilateral infection involving the silicone slings . Further, fitzgerald et al . Found nontuberculous mycobacteria in 82% of fat globules in their series of 71 cases . Complete resolution in our case occurred only after silicone slings were removed from both eyes . Finally, removal of the silicone sling did not result in a recurrence of blepharoptosis . This phenomenon is postulated to be due to scarring and fibrosis along the tract of the silicone rod . Nontuberculous mycobacterial may be responsible for bilateral delayed infection after frontalis surgery with silicone slings in an immunocompetent adult . Surgical removal of infected slings and prolonged course of antibiotics are required for complete resolution . The authors do not have any financial interest or any conflicting relationship in any of the issues or products referred to in the manuscript . This article is distributed under the terms of the creative commons attribution license which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.
A 52-year - old woman with granulocytic sarcoma affecting the anterior cranial fossa and frontal sinus was commenced on ade (cytarabine, daunorubicin and etoposide) and mylotarg (gemtuzumab) through a peripherally inserted central catheter (picc) line as part of the aml 17 trial (http://aml17.cardiff.ac.uk/aml17/default.aspx). She was known to have a beta - lactam allergy manifest as an erythematous rash . On day 9 of chemotherapy, her fever resolved after 48 hours and meropenem was stopped after 7 days . On day 18 of chemotherapy she remained febrile over the next three days and caspofungin was started in view of high - resolution computed topography (hrct) chest findings compatible with possible fungal infection and a positive initial serum galactomannan assay (although the repeat specimen was negative). Meropenem was therefore replaced with ciprofloxacin as it was thought to be responsible for the rash . All cultures including multiple blood cultures and picc line site swabs were culture negative . On day 31 of chemotherapy she was still febrile and blood cultures from peripheral veins and the picc line taken on day 30 yielded gram - positive cocci in pairs and chains after 24 and 11 hours' culture respectively . The isolate was identified as leuconostoc lactis using the bd phoenix identification (becton, dickinson and company, usa) and api rapid i d 32 strep systems (biomrieux, france). Mean inhibitory concentrations (mics) determined by e - test (ab biodisk, sweden) were: penicillin 0.5 mg / l, vancomycin> 256 mg / l, teicoplanin 256 mg / l, ciprofloxacin 1.0 mg / l, tigecycline 0.064 mg / l, linezolid 1.5 mg / l and daptomycin 0.064 mg / l . On day 32 of chemotherapy, ciprofloxacin and teicoplanin the next day therapy was changed to tigecycline (50 mg iv q12h) because of concerns over myelotoxicity (anaemia and thrombocytopenia) associated with linezolid . Daptomycin was unavailable in the hospital formulary and ampicillin was not used due to concerns over beta - lactam allergy and a borderline penicillin e - test mic . The patient continued to have low - grade fevers over the next four days, despite a negative transthoracic echocardiogram . On the fourth day of treatment, however, her fever resolved and she was discharged from hospital after completing 8 days of tigecycline, to which she had no adverse effects . Leuconostoc spp . Are catalase - negative, gram - positive, facultatively anaerobic coccobacilli . They are environmental organisms often found on plants, dairy products, vegetables, wine and occasionally in human vaginal and stool samples . Although an uncommon human pathogen, cases of bacteremia, endocarditis, pneumonia, meningitis, osteomyelitis, peritonitis, brain and liver abscesses have been described . And other gram - positive antimicrobial - resistant organisms are increasingly recognised as important pathogens in neutropenic patients probably due, in addition to immunosuppression, to the use of indwelling intravascular devices, antibiotic prophylaxis and evolution of chemotherapeutic agents . Infection with leuconostoc may cause fever, intravenous catheter - related sepsis, bacteremia, abdominal pain, gastroenteritis, colitis or meningitis in this group of patients . Other reported risk factors for infection removal of intravenous catheters alone has been shown to be curative in some patients without the need for antimicrobial therapy . Are intrinsically resistant to glycopeptides, owing to the production of peptidoglycan precursors ending in d - ala - d - lac, but are usually susceptible to penicillin, ampicillin, aminoglycosides, clindamycin, minocycline and macrolides . In addition, linezolid and daptomycin have been used successfully to treat leuconostoc bacteremia, although linezolid mics of leuconostoc spp . Although the organism has been shown to be resistant to cefoxitin, it is susceptible to cefotaxime in vitro . Tigecycline, a glycylcycline, is a broad spectrum synthetic derivative of minocycline which has a broad spectrum of activity against various gram - positive and gram - negative bacteria including multidrug - resistant strains, anaerobic bacteria and atypical organisms . It has proven to be useful in the treatment of hospital - acquired infections caused by vancomycin - intermediate and vancomycin - resistant enterococci (vre), meticillin - resistant staphylococcus aureus (mrsa), extended - spectrum -lactamase (esbl)-producing enterobacteriaceae, multidrug - resistant acinetobacter baumanii and penicillin - resistant streptococcus pneumoniae . In the uk, its licensed indications are complicated intra - abdominal and complicated skin and soft tissue infection . It is also licensed for the treatment of community - acquired pneumonia in the us . Although tigecycline has been demonstrated to be a safe and effective second - line option in microbiologically documented infections in neutropenic patients, there have been no trials to determine whether tigecycline is effective in neutropenic bacteremia and there are also theoretical concerns surrounding low serum concentrations (due to a large volume of distribution) and its mostly bacteriostatic activity . It is for this reason that tigecycline is not generally recommended for primary bacteremia but it is used for secondary bacteremia associated with complicated skin and soft tissue infections, intra - abdominal infections and community - acquired pneumonia . Despite these concerns, here we report the first successful use of tigecycline in the treatment of leuconostoc bacteremia in a neutropenic patient.
Utilizing wearable technology to obtain body metrics is a trending phenomenon (3, 5). The ease of obtaining individual measures makes wearable technology an attractive option, however, there is very little literature supporting the notion that these apparatuses can be used for field research . Hexoskin wearable technology shirt (hxs) is designed to measure several physiological variables including heart rate (hr), respiratory rate (rr), total energy expenditure (ee), and total steps (sc). In a laboratory - based investigation, the validity of this technology was compared with standard laboratory equipment at intensities up to 80% of the estimated mhr . Minimal variability was reported and consistency was accepted (4). While there is evidence the hxs may be valid and reliable in a controlled laboratory setting, its application in an outdoor environment is largely unknown . Therefore, the purpose of this study was to utilize hxs technology to obtain data in various trail hiking situations . We used this opportunity as a means to pilot test the hexoskin for collecting data in a real - life, outdoor setting . Ten individuals (male n = 4, female n = 6) volunteered to participate (age = 2410 years, height = 1663 cm, mass = 6518 kg). Prior to involvement in the study, participants provided informed consent that was approved by the institutional review board (southern utah university protocol #13 - 092014). The protocol was a modification of a previous investigation completed by our research group (2). On the first day, volunteers completed two easy (class i, yosemite decimal system (yds)) 1.82 km (1.13 mile) trail hikes at a self - preferred pace with a 15-minute rest period between trials . Altitude was measured at 5,446 feet above sea level (4400 heat stress tracker, kestrel, boothwyn, pa). Body metrics provided by the hxs (hexoskin smart shirt, montreal, canada) were hr, mhr, ee, rr, mrr, sc and cadence (ca). The hxs collects data through a data collection device (dcd) that connects by a plug to the shirt itself . The hxs dcd was connected when the subject began the easy trail hike and was disconnected when they reached the finish point . Systolic blood pressure (sbp), diastolic blood pressure (dbp) and ratings of perceived exertion (rpe) was also taken . Sbp and dbp were measured with an automatic blood pressure device (omron, bp742, kyoto, japan). Spb, dbp, and rpe was taken at the very beginning (directly before hxs activation) and immediately at the finish for both easy trail hikes (directly after the hxs was disconnected) (1). On the second day, participants completed a strenuous (class i, yosemite decimal system (yds)) 1.82 km (1.13 mile) uphill hike (17.6% grade). Initial elevation was 5,757 feet above sea level, and rose to 6,443 feet at the summit . Hxs, spb, dbp, and rpe measurements were taken at the beginning and end of both stages of the strenuous trail hike in a similar manner as the easy trail hikes . The dependent variables of average hr, maximal hr, estimated calories, average breathing rate, maximal breathing rate, steps, cadence and rpe were analyzed between trail type (initial easy trail, strenuous uphill, strenuous downhill) using one - way repeated measures analysis of variance (anova) (spss, ver . Intraclass correlation coefficients (icc) for each of the previously listed dependent variables as well as sbp, dbp, and rpe were determined using the reliability analysis: intraclass correlation coefficient option (two - way mixed model, absolute agreement type) in spss . Pearson product moment correlation coefficients (r) were determined for each trail condition for relationships between rpe and the dependent variables of sbp, hr, and mhr; and between cadence and rr in spss using the bivariate correlation option and significance was accepted at p0.05 . Ten individuals (male n = 4, female n = 6) volunteered to participate (age = 2410 years, height = 1663 cm, mass = 6518 kg). Prior to involvement in the study, participants provided informed consent that was approved by the institutional review board (southern utah university protocol #13 - 092014). The protocol was a modification of a previous investigation completed by our research group (2). On the first day, volunteers completed two easy (class i, yosemite decimal system (yds)) 1.82 km (1.13 mile) trail hikes at a self - preferred pace with a 15-minute rest period between trials . Altitude was measured at 5,446 feet above sea level (4400 heat stress tracker, kestrel, boothwyn, pa). Body metrics provided by the hxs (hexoskin smart shirt, montreal, canada) were hr, mhr, ee, rr, mrr, sc and cadence (ca). The hxs collects data through a data collection device (dcd) that connects by a plug to the shirt itself . The hxs dcd was connected when the subject began the easy trail hike and was disconnected when they reached the finish point . Systolic blood pressure (sbp), diastolic blood pressure (dbp) and ratings of perceived exertion (rpe) was also taken . Sbp and dbp were measured with an automatic blood pressure device (omron, bp742, kyoto, japan). Spb, dbp, and rpe was taken at the very beginning (directly before hxs activation) and immediately at the finish for both easy trail hikes (directly after the hxs was disconnected) (1). On the second day, participants completed a strenuous (class i, yosemite decimal system (yds)) 1.82 km (1.13 mile) uphill hike (17.6% grade). Initial elevation was 5,757 feet above sea level, and rose to 6,443 feet at the summit . Hxs, spb, dbp, and rpe measurements were taken at the beginning and end of both stages of the strenuous trail hike in a similar manner as the easy trail hikes . The dependent variables of average hr, maximal hr, estimated calories, average breathing rate, maximal breathing rate, steps, cadence and rpe were analyzed between trail type (initial easy trail, strenuous uphill, strenuous downhill) using one - way repeated measures analysis of variance (anova) (spss, ver . Intraclass correlation coefficients (icc) for each of the previously listed dependent variables as well as sbp, dbp, and rpe were determined using the reliability analysis: intraclass correlation coefficient option (two - way mixed model, absolute agreement type) in spss . Pearson product moment correlation coefficients (r) were determined for each trail condition for relationships between rpe and the dependent variables of sbp, hr, and mhr; and between cadence and rr in spss using the bivariate correlation option and significance was accepted at p0.05 . Preferred hiking speed uphill was significantly slower (4.540.64 kmh) than the easy trail (5.840.45 kmh, p<0.001) as well as on the downhill portion of the strenuous trail (5.630.71 kmh, p<0.001). No difference was observed between the hiking pace on the easy trail or the downhill portion of the strenuous trail (p=0.80). Conversely, ratings of perceived exertion were significantly greater during the uphill portion of the strenuous trail (13.72.4) compared to both the easy trail (9.91.3, p<0.001) and the downhill portion (10.42.5, p<0.001). There was no difference in rpe between the easy trail or the downhill portion of the strenuous trail (p = 0.40). The uphill trail elicited significantly elevated hr (p=0.04, see figure 1) and ee compared to the other hiking conditions (p=0.02, see figure 2). The downhill portion of the strenuous trail produced significantly increased sc compared to the easy trail only (p=0.01, see figure 3). No differences were observed for any other condition (p>0.05). Additionally, downhill ca was significantly greater when compared to the strenuous uphill portion (p=0.01, see figure 4), but no differences were observed for any other condition (p>0.05). No significant differences for trail type were noted for mhr (up = 16822 beatsmin, easy = 16222 beatsmin, down = 14720 beatsmin; p=0.38), rr (up = 3817 breathsmin, easy = 347 breathsmin, down = 3914 breathsmin; p=0.45) or mrr (up = 5417 breathsmin, easy = 6425 breathsmin, down = 6420 breathsmin; p=0.31). Significant icc was observed for dbp (r = 0.80, p = 0.02), rr (r = 0.98, p = 0.01), sc (r = 0.97, p = 0.01), ca (r = 0.97, p = 0.01) and rpe (r = 0.94, p = 0.01). The icc for sbp (r = 0.65, p = 0.07), hr (r = 0.73, p = 0.14), mhr (r = 0.65, p = 0.91), ee (r = 0.53, p = 0.25), and maximal rr (r = 0.68, p = 0.09) were not significant . Ratings of perceived exertion were not significantly correlated with sbp, average hr, or mhr during any of the hiking stages (see table 1). Furthermore, there was no significant correlation between rr and ca in any of the hiking stages (easy trail r = 0.19, p = 0.49; strenuous uphill r = 0.52, p = 0.16; strenuous downhill r = 0.25, p = 0.49). The primary purpose of this investigation was to pilot test the hxs while obtaining physiological measurements in an outdoor trail hiking setting . We hypothesized this technology would allow us to record measures that provided face validity . While measurements of hr and ee demonstrated expected values, it was not the case for mhr, rr, or mrr . Additionally, while hxs measurements of rr, sc, and ca were found to be reliable, the measurements of hr, mhr, ee and mrr returned nonsignificant intraclass correlation coefficients . Based on the physiological responses that we reported in our previous investigation (2), we expected to observe a general increase during strenuous uphill hiking when compared with both the easy - rated trail and downhill portion of the strenuous trail . While we did observe this phenomenon for hr and ee (see figures 1 and 2), it was not consistent for mhr, rr, or mrr . The similar response in these variables to the different trail conditions may be due to the subjects self - selecting a slower pace for the strenuous uphill hike . Evidence for this is suggested by the lower cadence for the uphill hike (fig . 4) coupled with a significantly greater rpe . Additionally, while not significant, there was a trend for rpe obtained during the uphill strenuous portion of the hike to be correlated with maximal heart rate (p=0.051). We have also observed that the hxs occasionally returned spurious values which could account for the results obtained . This should be taken into account for investigators wishing to utilize hxs in the field . While we acknowledge that a great number of subjects are necessary to determine reliability measures for the hxs, the poor iccs in the current investigation are a concern this is another factor that should be taken into consideration for researchers using this technology to obtain physiological measures in an outdoor field setting . (4) will be necessary to confirm that the hxs technology is valid and reliable in both laboratory and field - based settings . The results of this study indicate that hxs technology may be utilized to provide select physiological data in an applied setting ., hr did not register in 5 out of 10 subjects on the easy trail, and 8 out of 10 participants during the strenuous hike . Due to the nature of field testing, we were not cognizant of this fact until we attempted to download the data at a later time . Additionally, estimated ee values for the hexoskin appears to be linked to hr intensity . While further testing is necessary to determine the validity of this algorithm, the returned ee will not be accurate in cases where hr does not register on the hxs device . This study demonstrated there may be issues concerning the hxs s ability to measure and record data in a real - life setting . This product should first be validated against established laboratory and field standards in order to confirm the manufacturer s claims that the hxs is indeed a useful tool for physical training, sleep, and personal daily activities . In conclusion, we recommend that validity and reliability be established before hxs are utilized for research purposes in a field - based environment.
Extraskeletal soft tissue chondroma is a very rare, slow - progressing, benign tumour . Chondromas are tumour - like masses formed by proliferation of chondrocytes in a mature hyaline matrix . A 22-year - male came with chief complaints of asymptomatic swelling on the palate since birth . He had symptoms of cleft palate, i.e., nasal regurgitation and poor speech . On examination, there was a complete cleft of secondary palate and the cleft was filled by non - tender, globular swelling of size 3 sq . There was a linear band of soft tissue extending from the anterior part of the swelling to the upper lip mucosal surface after passing through the diastasis of the upper central incisors [figure 1]. Pre - operative photograph showing palatal swelling completely obliterating the cleft with linear band extending to the lip on computed tomography scan, a 30 mm 28 mm size, well - defined, globular soft tissue mass was seen in the region of the hard palate, which showed fusion defect anteriorly . Plain computed tomography coronal section of the swelling differential diagnosis of dermoid cyst and chondroma were kept . Intraoperatively, the tumour was seen attached by a small pedicle to the nasal mucosa of the cleft margin on the right side . The tumour was excised along with the linear band of tissue extending to the mucosal surface of the upper lip [figure 3]. Histopathological examination revealed hypercellular lobules of cartilage composed of evenly spaced chondrocytes of uniform size and separated by collagen and adipose tissue . There was no recurrence after 2 years of follow - up [figure 5]. Histopathological appearance of the chondroma two - year follow - up photograph showing repaired palate having adequate length with no recurrence soft tissue chondromas are benign cartilage - forming tumours that are usually found in close proximity to the tendon or joint capsules . Only five cases of soft tissue chondroma of palate have been reported in the literature. [36] it has never been reported in a patient of cleft palate . One theory is that these lesions are from residual embryonal tissue (embryonic remnant theory). The other theory is that these lesions are from metaplasia of pluripotential mesenchymal cells (metaplastic theory). In our case, because the swelling was present since birth, this goes in favour of embryonic remnant theory . Even after a meticulous review of the literature, we could not establish the cause and effect relationship between chondroma and cleft palate . In fact, we feel that the embryonic changes in the palate during the process of cleft might lead to entrapment of mesodermal tissue (mesoderm giving rise to cartilage / bone). Clinically, these tumors present as slow - growing, firm masses not attached to the underlying bone and are, occasionally, painful . Radiologically, soft tissue chondroma show well - demarcated, extraskeletal, soft tissue masses.
Fragile x related genes are members of a small gene family whose founding member is the fragile x mental retardation 1 gene (fmr1). Inactivation of fmr1 causes fragile x syndrome, the most common cause of inherited mental retardation (1,2). The other members of this family, fxr1 and fxr2, are autosomal and have not been associated so far with any human disease (24). Animal models have been generated for fmr1 deficiency, recapitulating the phenotype of fragile x syndrome (5,6). Fxr2 null mice are viable and show some behavioral phenotypes, such as hyperactivity, similar to those observed in fmr1 knockout mice (7). Fxr1 null mice die shortly after birth most likely because of heart and/or respiratory failure due to alterations in muscle development (8). In xenopus, complete or partial inactivation of xfxr1 expression has dramatic muscle - specific effects (9). In vertebrates, members of the fxr protein family are structurally very similar and share a high degree of sequence homology in clustered regions corresponding to functional domains (24). Like fmrp, fxr1p contains several rna binding domains: two kh domains and one rgg box . It also contains a nuclear localization signal (nls), a nuclear export signal (nes) and a protein protein interaction domain (2,10). They also share the same gene structure, derived from their common ancestor in drosophila melanogaster (11). Fxr proteins are able to bind rna (3,4), but binding specificity has been studied in detail only for fmrp . Indeed, even if a few hundreds of different rnas have been proposed to be putative targets of fmrp, only two structures are specifically bound by this protein, the g - quartet and the kissing complex (1214) and one sequence, a poly(u) stretch (15). Fxr1p has been reported to bind au rich element (are) and, through the interaction with this element, to regulate the expression of the proinflammatory cytokine tumor necrosis factor (tnf) in macrophages (16). In the cytoplasm the three fxr proteins are associated with polyribosomes (17), while they share only two interacting proteins, cyfip2 and msp58, with fmrp (2,18,19). The fxr1 primary transcript is alternatively spliced, with the possibility to generate upto 15 isoforms (20), see also . Of notice, some of these isoforms are differentially expressed in various tissues (21). Up to date, the ability of full - length fxr1p and fxr2p to bind a g - quartet rna structure in a specific manner has not been reported . We analyzed here the rna binding properties of the three most abundantly expressed fxr1p isoforms and show that they have different affinities for the g - quartet rna structure . Since all protein members of the fxr family are able to heterodimerize with fmrp, they are believed to act together (4). In the present study we determined that, when complexed to fmrp, fxr1p isoforms can modulate its affinity for g - quartet rna and also the dynamics of this complex . Our data demonstrate that fxr1p has a synergistic molecular function with fmrp rather than a redundant role . Glutathione s - transferase (gst)-fmrp produced in the baculovirus system was purified as described previously (22). Pet21a / fmrp (iso1) vector was described previously (23). To construct pgex-4t-1/fmrp, iso1 cdna was excised from ptl1/fmrp iso1 and subcloned into the ecori / noti sites of pgex-4t-1 (amersham). To construct pet21a / fxr1p, isoe, isod and isoa isoforms were amplified by pcr using the primers (eurogentec): ecori forward-5-ggcgaattcatggcggacgtgacggtg-3; xhoi reverse-5-gccctcgagttatgaaacaccattcaggac -3, the pcr consisted of 1 cycle at 94c for 4 min, 30 cycles of three steps each, 94c for 30 s followed by 60c for 30 s and 68c for 2 min using the pfx polymerase (invitrogen). Pcr fragments were purified, digested and cloned into the ecori / xhoi sites of pet21a (novagen). The sequences of the cdnas corresponding to the different fxr1p isoforms were verified by sequencing . Briefly, an increasing amount of recombinant his - fxr1p (1, 2 or 4 m) was mixed with 4 m of gst - fmrp . Pull down assays were carried out in the following buffer: [50 mm tris hcl (ph 7.4) at 4c, 1 mm mgcl2, 1 mm edta, 150 mm kcl, 1 mm dtt], as described (22). After washing with the same buffer, the proteins bound to the beads and their interactors were eluted using 30 mm glutathione and separated by electrophoresis on 8% sds polyacrylamide gels . Fmrp was visualized by immunoblot using the 1c3 monoclonal antibody (24), fxr1p was revealed by the 3fx monoclonal antibody (21). The different rna fragments used in this study, n19 [rna sequence derived from fmr1 cdna and containing a g - quartet forming structure (13)] and n8 [rna sequence not containing g - quartet structures and corresponding to the 3-untranslated region (3-utr) of pp2ac (25)], were cloned in ptl1 plasmid . For filter binding assay, ptl1 plasmids linearized with psti were in vitro transcripbed with t7 rna polymerase (promega) (13). For binding experiments, n19 was labeled co - transcriptionally by incorporation of [-p]atp . Labeled rnas were purified on a 1% low - melting agarose gel (ambion)., 5 fmol) were renatured for 10 min at 40c in 4 l of binding buffer [50 mm tris hcl (ph 7.4) at 4c, 1 mm mgcl2, 1 mm edta, 150 mm kcl, 1 mm dtt] in the presence of 8 u rnasin (invitrogen), 0, 1 g of escherichia coli total trna and 0.01% bsa . After incubation, binding solutions were passed through mf - membrane filters (0.45 ha, millipore) and washed with 2 ml binding buffer . Competition experiments to determine the relative binding strength of the different proteins to g - quartet rna were carried out using labeled n19 rna incubated with 1 pmol of protein in the presence of increasing concentrations of unlabeled competitors ., 5 fmol of labeled n19 were incubated with 1 pmol of the appropriate protein in the binding buffer between 10 and 300 min on ice . For dissociation rate determination, 5 fmol of labeled n19 were incubated with 1 pmol of the appropriate protein in the binding buffer for 10 min on ice, 10 m of competitor rna (n19 or n8) were then added to the mixture and incubated between 10 and 300 min . Each binding curve is the result of at least three independent experiments performed with three replicates for each binding point . All data obtained for the different experiments of rna binding, calculating the standard deviation for each binding point, are shown in supplementary data . Glutathione s - transferase (gst)-fmrp produced in the baculovirus system was purified as described previously (22). Pet21a / fmrp (iso1) vector was described previously (23). To construct pgex-4t-1/fmrp, iso1 cdna was excised from ptl1/fmrp iso1 and subcloned into the ecori / noti sites of pgex-4t-1 (amersham). To construct pet21a / fxr1p, isoe, isod and isoa isoforms were amplified by pcr using the primers (eurogentec): ecori forward-5-ggcgaattcatggcggacgtgacggtg-3; xhoi reverse-5-gccctcgagttatgaaacaccattcaggac -3, the pcr consisted of 1 cycle at 94c for 4 min, 30 cycles of three steps each, 94c for 30 s followed by 60c for 30 s and 68c for 2 min using the pfx polymerase (invitrogen). Pcr fragments were purified, digested and cloned into the ecori / xhoi sites of pet21a (novagen). The sequences of the cdnas corresponding to the different fxr1p isoforms were verified by sequencing . Briefly, an increasing amount of recombinant his - fxr1p (1, 2 or 4 m) was mixed with 4 m of gst - fmrp . Pull down assays were carried out in the following buffer: [50 mm tris hcl (ph 7.4) at 4c, 1 mm mgcl2, 1 mm edta, 150 mm kcl, 1 mm dtt], as described (22). After washing with the same buffer, the proteins bound to the beads and their interactors were eluted using 30 mm glutathione and separated by electrophoresis on 8% sds fmrp was visualized by immunoblot using the 1c3 monoclonal antibody (24), fxr1p was revealed by the 3fx monoclonal antibody (21). The different rna fragments used in this study, n19 [rna sequence derived from fmr1 cdna and containing a g - quartet forming structure (13)] and n8 [rna sequence not containing g - quartet structures and corresponding to the 3-untranslated region (3-utr) of pp2ac (25)], were cloned in ptl1 plasmid . For filter binding assay, ptl1 plasmids linearized with psti were in vitro transcripbed with t7 rna polymerase (promega) (13). For binding experiments, n19 was labeled co - transcriptionally by incorporation of [-p]atp . Labeled rnas were purified on a 1% low - melting agarose gel (ambion)., 5 fmol) were renatured for 10 min at 40c in 4 l of binding buffer [50 mm tris hcl (ph 7.4) at 4c, 1 mm mgcl2, 1 mm edta, 150 mm kcl, 1 mm dtt] in the presence of 8 u rnasin (invitrogen), 0, 1 g of escherichia coli total trna and 0.01% bsa . After incubation, binding solutions were passed through mf - membrane filters (0.45 ha, millipore) and washed with 2 ml binding buffer . Competition experiments to determine the relative binding strength of the different proteins to g - quartet rna were carried out using labeled n19 rna incubated with 1 pmol of protein in the presence of increasing concentrations of unlabeled competitors ., 5 fmol of labeled n19 were incubated with 1 pmol of the appropriate protein in the binding buffer between 10 and 300 min on ice . For dissociation rate determination, 5 fmol of labeled n19 were incubated with 1 pmol of the appropriate protein in the binding buffer for 10 min on ice, 10 m of competitor rna (n19 or n8) were then added to the mixture and incubated between 10 and 300 min . Each binding curve is the result of at least three independent experiments performed with three replicates for each binding point . All data obtained for the different experiments of rna binding, calculating the standard deviation for each binding point, are shown in supplementary data . Our first aim was to assess whether fxr1p is able to bind g - quartet rna structure, which is considered to be a frequent structure recognized by fmrp and present in many of its mrna targets (12,13,26). Due to extensive alternative splicing of fxr1 mrna, at least seven isoforms of fxr1p are differentially expressed in various tissues (20). We decided to study the rna binding properties of three fxr1p isoforms: isod and isoa (figure 1), the two isoforms most highly expressed in brain (3), and isoe (figure 1) that is a fxr1p isoform highly expressed during myogenesis and in adult cardiac and skeletal muscle (21). The fxr1p - isod and isoa isoforms both lack exon 12 and 15 and only differ in their c - terminus due to the choice of a different splicing acceptor site in the mrna of the fxr1p - isoa isoform, resulting in a frameshift that induces an early stop codon (figure 1a). On the other side, it is interesting to underline that the only differences between fxr1p - isod and isoe isoforms are the insertion of 28 amino acid encoded by exon 12 and the presence of 27 amino acid encoded by exon 15 (20,21) (figure 1a). This 27 amino acid stretch is strongly recognized as a putative rna binding motif by two different predictive programs available online [(27) and (28)], whereas the sequence of exon 12 does not apparently display such properties . The presence or absence of exon 15 raises then the possibility that the 3 isoforms share different rna binding abilities . The fxr1p isoforms . (a) schematic representation of the c - terminal region of the three fxr1p isoforms analyzed: isoe 84 kda, isod 78 kda, isoa 70 kda . In the upper part of the figure a (+) under each amino acid indicates the predicted ability of the sequence to bind rna accordingly to the algorithm described by terribilini and coworkers (28). Equal amounts of hist - fmrp, his - fxr1p - isoe, isod, isoa and gst - msp58 were loaded on a 10% sds page gel and revealed by coomassie blue staining . As the tissue distribution of fxr1p isoforms had not been investigated completely, we performed rt pcr on various rna samples extracted from cell lines and tissues . Fxr1p containing exon 15 rna was detected at very low level in brain, and in particular in the cerebellum, cortex and hippocampus, as well as in the neuroblastoma cell line ng108, together with fxr1p isoforms lacking exon 15 (data not shown). To investigate the g - quartet binding properties of the three fxr1p isoforms, we generated in a bacterial system recombinant fxr1p isoforms: isoe, isod and isoa (figure 1a) (3,21), fmrp iso1 (29) and as a control msp58, a recently described g - quartet binding protein (19), tagged with his or gst (figure 1b). In a filter binding assay, recombinant fmrp protein produced in bacteria displays the same affinity for rna containing a g - quartet structure as recombinant fmrp produced in an insect cell system (data not shown), confirming that the system of production does not change fmrp affinity for g - quartet rna, in agreement with studies by darnell and colleagues (30). Also it has been shown that fmrp acts as a nucleic acids chaperone in low - salt binding conditions (31) and is also able to bind rna non - specifically, raising the possibility of introduction of a bias in the assesment of its binding affinities, as already suggested (13,32). Considering the high level of homology that exists between the fxr proteins (4), we reasoned that fxr1p could also display the same properties of aspecific binding to rna . As a result, to assess the rna binding properties of fxr1p isoforms, we used the rigorous and sensitive rna competition assays, which alleviate the contribution of aspecific binding (13,25). Using the previously described filter binding assay (30), we observed that fxr1p - isod and isoa isoforms do not bind specifically g - quartet rna structure since the amount of bound g - quartet radiolabeled probe is not competed by either the unlabeled g - quartet rna [n19, corresponding to the portion of the fmr1 transcript containing the g - quartet structure (13)] or another rna not containing g - quartet structures and not binding fmrp [n8, corresponding to the 3-utr of the pp2ac transcript (25)] (figure 2b). Indeed, at the equilibrium state, the dissociation constant (kd) is around 5 m for fxr1p - isod isoform and 0.8 m for fxr1p - isoa . Conversely, fxr1p - isoe binds g - quartet rna but with a lower affinity compared to fmrp or msp58 (figure 2a). As little as 1 nm of competitor rna is able to displace 50% of fmrp from g - quartet labeled probe, whereas 10 nm are necessary for fxr1p - isoe (figure 2a) when we used as competitor the n8 probe, that does not bind fmrp (25), no binding was observed for all proteins analyzed here (figure 2b). To confirm that fxr1p - isoe interaction with g - quartet rna is specific for the structure, we performed the binding assay either in the presence of k or in the presence of na . Indeed, fxr1p - isoe, like fmrp and msp58 (13,19) is unable to bind g - quartet containing fmr1 rna in the presence of na, a cation destabilizing the g - quartet structure (figure 2c). This finding suggests that the effect observed is not due to the recognition of a specific rna sequence, but to the g - quartet structure localized in the assayed rnas (13). In addition, we repeated the same analysis by competing the binding of p - labeled n19 probe with the g - quartet forming rna structures obtained from the 5-utr of pp2ac (that contains four g - quartet forming structures) (25) and obtained the same results (data not shown) that are described in figure 2a using n19 rna competition . (a) filter binding assay using fmrp, fxr1p - isoe, isod, isoa and msp58 . The rna probe used is p - labeled n19 rna, and competition was performed using the same unlabeled rna . (b) the same experiment was repeated using as competitor the n8 rna sequence, not containaing any g - quartet forming structure . (c) filter binding assay was repeated with an increasing amount of fmrp and isoe in the presence of na or k. (d) gst - pull down was performed as described in materials and methods . On the right part of (d), proteins used in gst - pull down assay were revealed by immunoblot . Lane 1: 4 m gst - fmrp complexed with 1 m his - fxr1p, lane 2: 4 m gst - fmrp complexed with 2 m his - fxr1p, lane 3: 4 m gst - fmrp complexed with 4 m his - fxr1p . On the left part of (d), proteins used in gst - pull down experiment were revealed by coomassie stained gel . (e) competition assay to determine the kd at the equilibrium state binding fmrp, the heterodimers fmrp / isoe, fmrp / isod, fmrp / isoa and the complex fmrp / msp58, with the p - labeled n19 probe and competed with unlabeled n19 . (f) the same experiment described in (e) was repeated using the n8 rna as unlabeled competitor . Since fxr1p isoforms display different g - quartet binding specificity, we asked whether they would affect differently fmrp binding affinity to g - quartet . First, we verified the amounts of fxr1p and fmrp that integrate the heterodimer complex . To this purpose, we performed gst - pull down experiments by mixing 4 m of gst - fmrp with increasing amounts (1, 2, 4 m) of his - fxr1p - isoe, isod or isoa (in figure 2d, the results are shown only for interaction between fmrp and fxr1p - isoe). The beads were then treated with glutathione and the eluted proteins were revealed by immunoblot using the two monoclonal antibodies 1c3 (24) and 3fx (21) recognizing fmrp and fxr1p, respectively . As shown in figure 2d, the ratio of released fxr1p and fmrp is around 1:1 when mixed in stochiometric amounts . This result shows that when the two proteins are mixed in vitro their association is dose - dependent and, on the other side, also shows that our results are not due to an unbalanced ratio of the two interacting proteins in the fxr heterodimers . Subsequently, we tested the ability of the fxr1p - isoe / fmrp heterodimer to bind g - quartet rna . Indeed, the fxr1p - isoe / fmrp complex binds g - quartet rna with a comparable affinity as the fmrp homodimer at different concentrations of competitor rna (figure 2e). Surprisingly, fxr1p - isod and isoa inhibit fmrp binding to g - quartet rna when these form a heterodimer with the latter protein (figure 2e). As a control, msp58 protein, that binds g - quartet rna in a specific manner (19), was used (figure 2e), and its binding to fmrp leads to the same results as when fmrp is complexed to fxr1p - isoe . When we used the n8 probe as a negative control, no displacement of the equilibrium was observed, as shown in figure 2f . In view of these results, we decided to better dissect the dynamics of fxr1p / g - quartet rna and fxr1p / fmrp / g - quartet rna interactions . We evaluated the velocity of interaction of the two fxr proteins with g - quartet rna . For this purpose 1 pmol of each protein was mixed with 5 fmol of labeled n19 rna . At different time points (10, 30, 60, 120, 240 or 300 min), the assay was stopped and the amount of radioactivity bound by the proteins evaluated by the filter binding assay . The time necessary for fmrp / fxr1p - isoe heterodimer to bind the half amount of total bound rna ligand was estimated to be 9.93 min, which is lower than the time employed by fmrp or fxr1p homodimers (33.65 and 19.04 min, respectively) to bind the same amount of rna probe (figure 3a and b). This result indicates that the kon for the heterodimer is higher than the kon for both homodimers . Conversely, the presence of msp58 complexed with fmrp did not influence its binding to g - quartet rna . (a) each protein was mixed with p - labeled n19 rna probe for a time lapse of 10, 30, 60, 120, 180, 240 and 300 min and then each reaction was filtered and the amount of retained radioactivity evaluated . (b) the same experiment described in (a) was repeated with the complex fmrp / msp58 and fmrp / isoe as indicated in the figure . We then investigated the kinetics of fxr1p - isoe and fmrp dissociation from the g - quartet rna structure, when both bind to it as homodimers or as heterodimers . Fmrp was mixed with p - labeled n19 rna and the complex was allowed to form for 10 min on ice . Then 1 m of cold rna [n19 or n8, as a negative control (25)] was added, the reaction was stopped after 10, 30, 60, 120, 240 or 300 min and the amounts of retained radioactivity evaluated by filter binding assay . In this experiment, it is interesting to observe (figure 4a) that the fxr1p - isoe homodimer releases 50% of total bound rna after only 15 min . As expected from its higher affinity, the fmrp homodimer releases the same amount of bound rna after a longer lapse of time, around 45 min . The binding of recombinant proteins to g - quartet containing rna was competed using the n8 probe . Indeed, only 2025% of the binding is competed after more than 4 h of incubation . Dissociation rate of fmrp, isoe, msp58 and the two complexes fmrp / isoe and fmrp / msp58 . (a) each protein was mixed with p - labeled n19 rna probe for 10 min on ice and then an equal amount of unlabeled n19 rna was added as competitor to each reaction, which was then filtered after a precise time lapse of 10, 30, 60, 120, 180, 240 and 300 min . (b) the same experiment described in (a) was repeated using unlabeled n8 rna as competitor . (c) the same experiment described in (a) was performed using the protein complexes fmrp / isoe and fmrp / msp58 . (d) the same experiment described in (c) was performed using the cold n8 rna as competitor . Finally, the heterodimer fxr1p - isoe / fmrp and the complex msp58/fmrp were analyzed . The effect of the heterodimer was dramatic: 50% of the labeled rna was released after 5 min and 65% of labeled rna was released after 10 min (figure 4c), while the interaction with msp58 does not change significantly the dynamics of the interaction between the g - quartet rna and fmrp . In figure 4d the negative controls are shown, confirming the specificity of the action described in figure 4c . This data shows that formation of the fxr1p - isoe / fmrp heterodimer increases the dynamics of protein fmrp is a component of multimolecular complexes involved in different steps of mrna metabolism (2,12). A growing list of proteins interacting with fmrp has been described, most of them being rna binding proteins (19,33). In addition, several hundreds of mrnas have been described as putative targets of fmrp (12,26), however the functional significance of most of the multiple interactions established by fmrp is still elusive (12,34). A widely accepted hypothesis proposes that fmrp can transport mrna in mrnps shuttling between structures where rna translation is repressed and polyribosomes (2). In the absence of fmrp, the equilibrium in mrnp normally containing fmrp is perturbed, resulting in the deregulation of the expression and localization of a subset of its target mrnas (12,26). Based on these considerations, we reasoned that rna binding proteins belonging to the same mrnp complex, fmrp and its interacting proteins may enter in contact with the same mrnas and decided to test the ability of fxr1p to bind the same mrna targets and to influence its affinity for them . Up to date, the functions of fxr1p (or fxr2p) were inferred to be, by homology and analogy, similar to that of fmrp (1). Here it is surprising that among the three isoforms analyzed only one, the isoe is able to bind a g - quartet rna forming structure, present in a large amount of putative target rnas of fmrp (26). However, it has been reported that, even though a peptide corresponding to the rgg box of fmrp binds specifically g - quartet forming rna (30), the corresponding peptide of the rgg box of fxr1p does not (35), strongly suggesting that the rgg box of fxr1p is not sufficient per se to bind the g - quartet structure . The only difference between the two isoforms isoe and isod are two short sequences of 28 (exon 12) and 27 amino acid (exon 15) (cf . Only this latter amino acids stretch appears to have putative rna binding properties and is encountered solely in the isoe isoform able to bind specifically the g - quartet structure . This 27 amino acid stretch encoded by fxr1p exon 15 being in close proximity to the rgg box of fxr1p encoded by sequences of exon 14 (figure 1a), it may contribute to the binding to g - quartet mrna structures together with the rgg box . Alternatively, the presence of this additional sequence in the fxr1p - isoe, as compared to the other shorter isoforms, may alter the structure of the c - terminal portion of fxr1p, thereby allowing the binding . In a similar way, a different affinity for rna was also shown for different fmrp isoforms . Indeed, iso18, a minor isoform of fmrp lacking a small portion of exon 17 (29), is still able to bind g - quartet rna (36), like iso1 and iso7 (13,30). In addition, iso 18 is able to bind the 3-utr of fmr1 mrna (36), that, conversely, is not bound by iso1 and iso7 (13,30). In adult muscle, where both fmrp and fxr2p are absent, the fxr1p isoforms encountered both contain exon 15 sequences and correspond to a doublet of 82 (isog) and 84 kda (isoe) (20,21,37). Their ability to bind g - quartet rna structure suggests that, in this tissue, specific rnas might be recognized by fxr1p via the interaction with g - quartet forming structure . Since fxr1p absence has a strong impact during muscle development (8,9), its rna binding capacities are critical per se, independently from fmrp's fonction . Indeed, a recent knock - down analysis for xfxr1 produced a list of putative fxr1p target rnas . Interestingly, several of their human homologues harbor a putative g - quartet structure [(9), and our unpublished data]. In addition, very little is known concerning the precise function of fxr1p in muscle, and it is possible that muscle - specific fxr1p interacting proteins might modulate its affinity for rna . Our analysis to dissect the binding capacities to g - quartet rna fxr1p and its heterodimer with we observed a dramatic effect of the fmrp / fxr1p heterodimer on the dynamics of complex formation with g - quartet rna . This effect was not observed when fmrp and its other partner msp58 were mixed together, suggesting that msp58 can probably compete for the same binding site as fmrp . In addition, the interaction of fmrp with fxr1p - isoa or isod strongly reduced fmrp specificity for g - quartet rna . These different behaviors of the two fmrp - interacting proteins illustrate the complexity of the functions and interactions that take place in fmrp - containing mrnps and in different tissues . Fxr1p - isod and isoa are the fxr1p isoforms with the highest expression in brain, suggesting that in neurons fmrp interacts mostly with these two isoforms that might regulate negatively its action . Since in brain and cerebellum fxr1p - isoe mrna is expressed at a low level as revealed by rt pcr (our unpublished data), probably only a very small portion of fmrp may be regulated by fxr1p - isoe . Conversely, fmrp and fxr1p - isoe and isog isoforms are co - expressed in myoblasts and in myotubes, suggesting a particular regulation of g - quartet containing target mrnas during muscle differentiation but not in adult muscle, where fmrp is not expressed anymore (21). The present study highlights the functional differences between fxr1p isoforms and therefore emphasizes the importance of the extensive tissue - specific alternative splicing undergone by fxr1 mrna . In view of these results, it is clear that in each mrnp the ratio between fmrp and fxr1p different isoforms becomes important to precisely regulate fmrp function . The modulation of the affinity and/or of the dynamics observed for the fxr1p / fmrp heterodimer may reflect a regulation of the exchange of mrnas between mrnps or trafficking granules and polyribosomes . The interaction domain of the two fxr proteins is localized in the n - terminal region of both proteins . This domain mediates the interaction between fmrp and several other proteins (fxr2p, cyfip1, cyfip2, nufip and 82-fip) (10,33). On the other hand, despite the high level of homology, the n - terminal region of fxr1p seems to interact only with cyfip2 and fxr2p (18). Cyfip2, together with cyfip1, that only interacts with fmrp, belongs to a small family of proteins linking fmrp to the rac pathway (18,33,38). We have previously proposed that the cyfip proteins might modulate the ability of the fxr family members to homo and/or heterodimerize (18). Fxr1p and fxr2p are believed to have distincts but overlapping function in conjunction with fmrp, with the possibility to partially compensate for its absence . Our results show here a completely different function for two different fxr1p isoforms, which modulate the action of fmrp . This data reveals how a full understanding of fmrp function may be achieved through the deciphering of the global action of fmrp - containing mrnp complexes.
Breast oncologic surgery is now widely focussed in conservative treatment with tissue sparing in order to obtain satisfying cosmetic results besides an adequate surgical resection [13]. Obviously, resection borders are becoming considerably closer to the neoplastic lesions; hence, tumor - free margins are hardly achieved in nonpalpable lesions, such as small lesions, lesions with ill - defined profile, and microcalcifications areas . A large number of studies proved that the condition of the resection margins represents a significant risk factor of recurrences in women who underwent breast conservative surgery, together with tumor size and tumor grading (see recent reviews from singletary and huston) [4, 5]. It was claimed that margin status represents an independent risk factor in distant metastasis development and overall survival . The worth of close or involved margins in predicting the presence of neoplastic residual in the area next to the surgical bed is still debated: a recent paper reported that 21% of a series of re - excised tumors with negative margins contained residual tumors . Unfortunately, some inconsistencies in reported data depend on the fact that a large consensus about the definition of free surgical resection margins has still to be reached . In recent years, the role of the intraoperative evaluation of surgical specimen in resection margin control was assessed, but the literature is still limited and dissimilar . A group of recent papers on series of conservative resections for breast malignancies proved that the intraoperative examination of resection specimens is useful in decreasing the rate of second procedures, employing gross examination techniques, specimen radiogram, or a combination of them . Another paper proved the efficacy of gross margin assessment combined with radiography in skin sparing mastectomies in reducing excision rate in breast conserving surgery for carcinoma in situ . Assuming that the re - excision procedures are useful tools in obtaining an adequate removal of the neoplastic lesions, thus reducing the risk of persistence of neoplastic foci in residual parenchyma, we believe it could be of interest to make an attempt at quantifying the amount of tumoral mass that lies beyond a margin that is considered close according to the ordinary criteria of intraoperative evaluation (mammography of the operative specimen, clinical examination of surgical bed specimen, and frozen sections examination). The literature about this topic is still somewhat limited and is mainly based on delayed excisions: two recent works [14, 15] analyzed 23 and 26 delayed re - excisions after positive surgical margins at definitive histology of primary surgery . The rate of re - excision with residual tumors was, respectively, 48% and 65%, and one of these studies stated that none of the examined risk factors was statistically related with the occurrence of residual tumor in re - excision specimen . The present survey is based on the examination of histological material from a consecutive series of conservative resections that comprehended re - excision procedures, both in the context of the same session and in delayed surgical treatments . The main goal of this study is the assessment of the extension and of the morphological characteristics of the tumoral residuals in re - excision specimens to define both the role of the re - excisions (and mainly of the limited re - excisions) as a tool to obtain the complete removal of neoplastic lesions in conservative breast surgery and the potential employment of other therapeutic options in obtaining an adequate removal of the target area . As a consequent achievement, we planned to weigh up some of the issues that could be related with the occurrence of tumoral residuals, as the invasive or preinvasive nature of the lesion, the method of evaluation of the first resection adequacy, and the time occurring between the first resection and the re - excision . For the present study, we selected 51 patients consecutively treated in our institution with a preliminary conservative approach, subsequently extended with further (intraoperative or delayed) procedures . Since resection specimen radiogram is considered the most reliable technique of margin status evaluation in our structure, all the first resection specimens were conveyed to the radiology department for intraoperative evaluation, and most of the re - excisions were based on the radiologist's advice . Considering the main lesions reported in final histology for each patient, 28 infiltrating carcinomas (55%, 4 multifocal), 19 in situ carcinomas (37%), and 4 (8%) benign lesions were resected . The main histological characteristics of infiltrating and in situ carcinomas are resumed in table 1 . 112 surgical specimens from 54 procedures (first resections and subsequent re - excisions) were evaluated in this study . The presence of the target lesion in the radiograms was first ascertained; the margins were judged close to the target lesion when the lesion was eccentrically placed in the same mammograms . Space - oriented specimens were examined for the final histology . In the initial resections, the surgical margins were marked with one or two different colour inks . In the re - excision specimens consecutive sections of the area of the neoplasm closest to the surgical margins were obtained in large specimens while the whole tissue was processed in smaller specimens or, in the cases in which the lesion's shape was not clearly identifiable, on macroscopic examination . Resection margin was considered close when the distance from the lesions was equal to or smaller than 2 mm . We divided the re - excisions in accordance with the time of re - excision and with the method of evaluation that defined the surgical procedure . 38 patients (74.5%) were re - excised intraoperatively: in 25, re - excision was supported by the radiological report of close margins after specimen mammogram and, in 13, direct re - excision was supported by clinical evidence of incomplete excisions during surgery; 3 patients of this group had delayed re - excisions performed after definitive histological report of close margins on first resection specimen . The remaining 13 patients (25.5%) had no intraoperative extension, since the ordinary method of immediate evaluation of first resection provided suggestions for further procedures, and were extended subsequently (with 13 related specimens) because of evidence of close margin on histology . The histological definitive diagnosis of the re - excisions specimens (considering the main lesion if the re - excisions consisted of more than one specimen) is reported in figure 1, matched with the main lesion reported at final histology for each case . The overall occurrence of residual neoplastic lesions in re - excisions selected for this study was 19 out of 54 (35%). In the series of the intraoperative procedures concerning patient with neoplastic lesions, 5 (13%) were effective in removing the target lesion, 9 (24%) harboured residual neoplastic lesions, and 20 (52%) were reported as normal breast parenchyma or contained benign lesions at definitive histology . On the other hand, 4 intraoperative re - excisions (11%) were related to cases with definitive histological diagnosis of benign lesions . Among the 16 delayed re - excisions, 10 (62%) were effective in eradicating residual tumor . The rate of residual lesions was compared in the two groups of intraoperative re - excisions (9/29 re - excisions, exclusive of the 5 re - excisions containing the target lesion and the 4 resections from patients with final diagnosis of benign lesion) and delayed re - excisions (10/16 re - excisions). The difference was statistically significant (p = .03) even considering the re - excision in the patients with final diagnosis of invasive carcinomas (28 re - excisions, 17 intraoperative, and 11 delayed; p = .01). Considering the morphological features of the 9 intraoperative re - excisions which were effective in removing neoplastic residual, in 8 cases the residual lesions were scattered foci of in situ carcinomas: 4 re - excisions performed in cases with definitive diagnosis of in situ carcinoma contained residual foci of the lesion, 4 re - excisions performed in cases with main diagnosis of invasive carcinoma contained residual foci of peritumoral in situ in 3 cases, and a focus of microinvasion (2 mm) in an area of in situ carcinoma in another case . In 3 of the 8 excisions, the in situ carcinomas were poorly differentiated sec holland . Another re - excision harboured residual foci of a lobular carcinoma with invasive features . In 7 cases (77%), the residual lesion was close to the new resection margin . In order to assess the role of invasive features in the efficacy of re - excision, we evaluated the rate of residual lesions in re - excisions in the two groups of patients with final diagnosis of invasive carcinoma (13/28) and with final diagnosis of in situ carcinomas (6/17). We excluded re - excision from patients in which the re - excision led to the removal of the target lesion (5 re - excisions). No statistical difference in the distribution of the neoplastic residual lesions between the two categories could be stated, both considering (p = .45) and ruling out (p = .45) the peritumoral in situ as residual lesion in resection for invasive lesions . Similar results were obtained considering only the intraoperative re - excisions (29 cases, p = .87 and p = .16, resp . ). The role of the technique of intraoperative evaluation of the first resection in removing residual neoplastic lesion was assessed in the group of the 29 intraoperative re - excisions, using the same criteria of exclusion described in the preceding sections . We matched the 20 radiology guided re - excisions and the 9 surgeon guided re - excisions . No statistical difference in the rate of neoplastic lesions was ascertained in the two groups (p = .10, p = .27, ruling out peritumoral in situ). Recent advances in breast surgery showed the efficacy of the conservative approach in surgical resection of breast carcinomas . One of the main requirements in conservative resection is to obtain a complete removal of neoplastic lesions, usually verified with tumor - free resection margins at final histology . The goal of tumor - free margins is reached in recent surgical practice with intraoperative resection specimen examination, mostly with mammography and with specimen frozen section microscopy . In other cases, the purpose is obtained with delayed re - excision or radicalizations ensuing from pathological evidence of close margins at first resection . The main purpose of this study is the evaluation of the efficacy of the immediate re - excision, performed after intraoperative evaluation of first re - excision specimen, in removing neoplastic residual . We believe it could be of interest to weigh the value of intraoperative re - excisions in obtaining the complete removal of the neoplastic mass, compared with other therapeutic options . A preliminary statement is that a significant part of (13%) of the re - excisions leads to the resection of the target lesions, supporting the usefulness of this practice in breast oncologic surgery . Considering the efficacy of intraoperative re - excisions in clearing residual parts of the main lesion, the histological definitive examination detected residual neoplasm in 24% of the cases . Interestingly, the rate of successful re - excision in this study is somewhat lower than the rate of occurrence in other studies available in the literature (see introduction) [9, 1417]. This discrepancy could be explained with different settlement of close margins or a dissimilar evaluation of specimens at mammography . If we look at the morphology of tumoral residuals in limited intraoperative re - excisions that were confirmed as neoplastic lesions at definitive histology, mutifocal, ill - defined lesions (8 re - excisions with in situ foci, with one case of microinvasion, and one re - excision with lobular invasive carcinoma foci) were detected, and in the 77% of cases the re - excisions were ineffective in completing the removal of the neoplasm, as the residual lesions were close to the new resection margin . Our data show that invasive or preinvasive nature of target lesion does not affect the rate of neoplastic lesions in re - excisions: it could be expected that the oncological adequacy of conservative surgery is dependent on lesion profile and on detection capability at radiological and clinical examination, more than on evidences of invasion . Postsurgical radiotherapy could be a good option in obtaining the local control of the residual lesion that we detected in our revision . A study on predictive factors of residual - positive re - excisions performed on 115 delayed resections achieved the same statements . Another study stated that recurrences in non - reexcised lumpectomies with negative and positive margins are not statistically different after radio chemotherapy . According to our data, the efficacy of the intraoperative re - excisions (grouping together radiogram and clinical guided re - excisions) is lower than the efficacy of delayed surgery after histological examination of first resection specimen, both considering all the re - excisions with cases with final diagnosis of neoplastic lesion (excluding those which harboured the target lesion; p = .004) and limiting the analysis to cases with final diagnosis of invasive lesions . Placing these results in the background of the debate about the real implication of the finding of close margins, apart from the evaluation methods, our findings could not confirm the hypothesis of a higher rate of tumoral residual in immediate versus delayed re - excision for the absence of repair processes; on the contrary, delayed re - excisions after histological evaluation of the first specimen seem to be more effective in removing tumoral tissue . Considering the evaluation method of the first resection adequacy, we could not ascertain any statistical difference between the efficacy of radiological evidence guided re - excisions and the surgeon's choice dependent re - excisions in completing tumoral excision . Probably, a larger number of cases are needed for a more accurate evaluation of these two techniques, but these results show that surgeon's evaluation of surgical bed has a pivotal role in removing neoplastic residual that are not evident in radiology . Concluding, this analysis suggests that intraoperative re - excisions are mandatory when the intraoperative examination doesn't confirm the presence of the target lesions in the first resection specimen, but is more questionable when the lesion is judged close to the resection margin . The lesions that were re - excised in the same surgical session were mainly ill - defined areas with foci of in situ carcinomas, and these findings suggest the employment of other therapeutic options, such as radiotherapy . Considering the adequacy of the resections, it must be underlined that the practice of conservative breast surgery must now face the recent theory of the sick lobe which asserts that conservative breast surgery must obtain the complete resection of the whole lobe involved in neoplastic disease . Following this theory, the resection of ill - defined neoplastic lesions such as in situ lesions, lobular invasive, and multifocal invasive carcinomas should be firstly planned in preoperative phase, with an accurate definition of the lesion's profile and with a careful evaluation of radiograms and other instrumental data (ultrasound, mr), integrated with cytological and microhistological presurgical sampling . On the other hand, the higher efficacy of the delayed re - excisions points out that the examination of the specimen with definitive histology is a more suitable procedure for establishing the morphological and biological characteristics of the lesion excised with the first excision and for planning further re - excisions.
Over the last few decades, a dramatic worldwide increase in infection rates by multidrug - resistant (mdr) pathogens has occurred, which is acknowledged as a public health crisis . Management of infections caused by these pathogens is often difficult due to the scarcity of available active drugs . The last report of the european antimicrobial resistance surveillance system (earss) network, which includes 30 european countries, describes a general european - wide increase in antimicrobial resistance for the gram - negative pathogens under surveillance (escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa). High proportions of antimicrobial - resistant p. aeruginosa have been reported by many european countries . In a study performed in 2000 in spain, 41% of acinetobacter baumannii indeed, the rate of carbapenem resistance has increased dramatically over the last decade, especially in the critical care setting . An ominous emerging threat is the appearance of gram - negative microorganisms harboring new beta - lactamases that confer high - level resistance to all available classes of beta - lactam antibiotics . Concerning gram - positive bacteria, methicillin - resistant staphylococcus aureus (mrsa) and enterococcus spp . The incidence of mrsa infections seems to have remained stable over recent years, although this pathogen causes severe infections . The issue of increasing incidence of mdr is clearly more complex in intensive care units (icus), where selection pressure and emergence of resistance, as well as the risk of patient - to - patient transmission, are highest . The spanish annual april - to - june icu national nosocomial infection surveillance study (estudio nacional de vigilancia de infeccin nosocomial, [envin]) confirms that multi - drug resistance is an unresolved problem in spanish intensive care, with worrisome rates of gram - negative mdr pathogens . In addition, mdr microorganisms often do not cause true infection, but only colonization, constituting a hidden reservoir for the spread of these pathogens . The prognosis of patients who develop nosocomial infection in the icu is poor, especially if an mdr pathogen is involved . Mortality rates and economic burden are significantly higher in infections caused by mdr pathogens, than in those caused by susceptible organisms . Moreover, even the acquisition of an mdr pathogen, without concomitant infection, is associated with an increased risk of death, length of hospitalization, and cost . The spanish society of intensive care medicine and coronary care units (semicyuc) and the spanish society of intensive care nursing (seeiuc) have recently completed their role as technical lead for two programs aimed at reducing icu - acquired infections, namely catheter - related bloodstream infections [zero bacteremia] and ventilator - associated pneumonia (vap) [zero vap]. Both projects were developed within a framework of zero tolerance . Zero bacteremia and zero vap were both promoted by the spanish ministry of health, more than 200 icus participated, and the programs consisted of the implementation of evidence - based infection prevention bundles for catheter - related bloodstream infection and vap . Highly successful results for both initiatives confirmed that these practices could be systematically implemented across spain, could reduce the rates of these infections and could contribute to diminish antimicrobial use in the participating icus . With the experience gained in the two previous projects, a new project named zero resistance was developed by the semicyuc with the support of the spanish ministry of health . This project uses the same structure created for zero bacteremia and zero vap, which is based on coordination at national, regional and local levels . A scientific expert committee (sec) for the development and implementation of this program was appointed as follows: semicyuc nominated nine intensivists chosen for their expertise in the field of prevention and management of infections in the critical care setting and seeiuc designated an intensive care nurse with experience in infection control . A microbiologist, an epidemiologist, an infectious diseases specialist, and two technicians from the ministry of health with broad knowledge in the field were also incorporated . The members of the sec reviewed the available evidence in pubmed indexed papers, including observational studies, clinical trials, guidelines, systematic reviews and meta - analyses . The following databases were searched: medline, embase, the cochrane library, and centre for reviews and dissemination, including the national health service economic evaluation database and the health technology assessment database . The implementation of bundles of effective measures, compared to individual interventions, has been proposed to reduce the incidence of catheter - related bloodstream infections or vap . With this concept in mind, the sec developed a bundle of 10 recommendations that was discussed and approved after review and analysis of the existing scientific literature . Admittedly, the evidence supporting some of the chosen recommendations is weak, but all were deemed to reach at least the level of expert recommendation. No grading system was used to support the strength and quality of recommendations . Criteria for defining mdr pathogens vary from institution to institution and are also not uniform in the published literature, although the most highly resistant strains are readily recognizable . Based on the pathogens considered most problematic in spanish icus, zero resistance collects information on episodes of infection and colonization of the pathogens listed in table 1 . Finally, because acquiring an infection may be the result of errors in patient - care, all three programs were designed to reduce and prevent these by incorporating an integral patient safety program .table 1 definitions of multidrug - resistant bacteria monitored in the zero resistance program microorganism resistance marker gram - positive staphylococcus aureus methicillin (mrsa) enterococcus spp.vancomycin (vre)gram - negative enterobacteriaceae 3rd generation cephalosporins (particularly esbl - producing)carbapenems (particularly carbapenemase - producing) pseudomonas aeruginosa 3 antibiotic classes, including carbapenems, cephalosporins, piperacillin - tazobactam, flouroquinolones, aminoglycosides and colistin acinetobacter baumannii carbapenems imipenem, meropenem or doripenem; ceftazidime or cefepime; ciprofloxacin or levofloxacin; gentamicin, tobramycin or amikacin . Esbl: extended spectrum beta - lactamase; mrsa: methicillin - resistant staphylococcus aureus; vre: vancomycin - resistant enterococcus . Definitions of multidrug - resistant bacteria monitored in the zero resistance program imipenem, meropenem or doripenem; ceftazidime or cefepime; ciprofloxacin or levofloxacin; gentamicin, tobramycin or amikacin . Esbl: extended spectrum beta - lactamase; mrsa: methicillin - resistant staphylococcus aureus; vre: vancomycin - resistant enterococcus . The main objective of the zero resistance project is reduction in the cumulative incidence of patients with icu - acquired mdr infections by 20% . Secondary objectives are to study the epidemiology of mdr infections in spanish icus, to be able to distinguish imported from icu - acquired cases, to promote and strengthen safety assurance in participating units, and to create a network of icus implementing safe, and evidence - based practices . The primary aim of the bundle recommendations is reduction of the three most influential factors contributing to the development and transmission of mdr, namely: 1) adequate prescription of antibiotics; 2) early detection and prevention of cross - colonization of mdr; and 3) elimination of reservoirs .first recommendation: in each icu, at least one intensivist will be designated as responsible for the use of antimicrobials . He / she should have extensive experience in infection control and in the treatment of severe infections . Analysis of antimicrobial use should include: review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .second recommendation: empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s) based on patient risk factors and/or knowledge of local ecology . Otherwise, narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens (carbapenems, colistin, tigecycline, glycopeptides, daptomycin, linezolid) should be started if needed . In all cases, samples for culture of the potential sources of infection should be obtained before starting antibiotic therapy.rationale: early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . However, delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection . More recently, a quasi - experimental, before - after observational cohort study concluded that, after adjusting for confounders, aggressive antimicrobial therapy was an independent predictor of mortality . In the aggressive period, antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . In the second period (conservative strategy), antimicrobial treatment was started only after objective findings confirmed the infection .the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking . However, it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal . In addition, the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology . Therefore, it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . Obviously, efforts to reduce the delay of microbiological results (use of rapid diagnostic techniques, direct contact with the microbiologist) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed.third recommendation: in each unit, at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens.rationale: success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals . Nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care.a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient . The participation of nurses is of extraordinary importance for the success of infection control programs in intensive care . In fact, most procedures performed to reduce the risk of nosocomial infection (vascular catheter care, artificial airway care, mouth hygiene, etc .) Are part of the nurse s daily tasks.programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders . This model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc . The zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens.fourth recommendation: it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay . These samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital.rationale: guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units, e. g., icus . The implementation of contact precautions in patients colonized or infected with mdr is widely accepted . In contrast, the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed . Initial screening is specially recommended for mrsa, although the same principles and practices apply to gram - negative mdr organisms, which actually now constitute the main threat.active surveillance programs are time and resource - consuming . The type and number of samples are selected according to local resources and epidemiology and should include at least nasal, rectal and oropharyngeal swabs (bronchial aspirates in intubated patients). In addition, other samples may be necessary to control potential reservoirs (infections, skin ulcers, etc. ).concerning surveillance cultures, two approaches are acceptable: all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist (see fifth recommendation).fifth recommendation: at admission to the icu, a checklist of risk factors (table 2) must be completed to identify patients at high risk of mdr pathogen carriage . Patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2 checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria risk factor 1 . Hospital admissionlasting> 5 days, during last 3 monthsyes no 2 . Institutionalized (prison, healthcare and social centers, geriatric centers, etc. )yes no 3 . Known colonization or infection with mdr pathogensyes no 4 . Antibiotic therapy 7 days in previous month (particularly 3rd and 4th generation cephalosporins, flouroquinolones and carbapenems)yes no 5 . Comorbidities associated with high incidence of colonization or infection with mdr pathogens: cystic fibrosis, bronchiectasis, chronic skin ulcers, etc.yes no rationale: several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified: prior antibiotic use, the presence of invasive devices and certain underlying diseases are the most frequently reported . Patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america (ats / idsa) criteria are: current hospitalization of 5 days or more, prior antibiotic therapy, prior hospitalization, residence in a nursing home or extended - care facility, home infusion therapy within 30 days, chronic dialysis within 30 days, home wound care, family member with an mdr pathogen, and immunosuppression . However, in a prospective evaluation, although these criteria had an excellent negative predictive value (96%), they had a very low positive predictive value (18%) for infection or colonization with an mdr pathogen at icu admission . In a case control study, immunosuppression was not independently associated with mdr bacteria in the icu .in other studies, risk factors for specific pathogens, like mrsa or a. baumannii, have been identified in an attempt to establish control measures that limit spread . This approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection.with this information, the sec generated a checklist (table 2) for detection of patients at high risk of carrying mdr pathogens . If one or more of these risk factors is present, screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms . The prospective validation of this checklist is one of the pending tasks of this program.sixth recommendation: compliance with preventive measures including those based on transmission mechanisms should be routinely measured.rationale: contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms . Briefly, contact precautions (by staff and visitors) consist of: hand hygiene and donning of gown and gloves immediately prior to room entry, and disposal of gown and gloves inside the patient s room, followed by hand hygiene immediately prior to leaving the room.adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study . Correct practice includes: (1) use of a contact precautions sign for every patient colonized / infected by mdr pathogens; (2) availability of contact precautions equipment at patient room entry; (3) barrier disposal containers inside patient room; and (4) monitoring of adherence to the contact precautions protocol by staff / visitors . If there are no closed rooms, precautions must be tightened.to achieve the desired results, all staff members should watch compliance with preventive measures . Concerning this issue, the sec of zero resistance considers that nurses have a special responsibility in implementing effective prevention . Therefore, the rest of the hospital staff and visitors must follow their recommendations.seventh recommendation: all units should develop a cleaning protocol for rooms of patients with mdr pathogens.rationale: many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas . Nevertheless, substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [37 - 39]. Cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee . This protocol should include fixed structures (floors and walls) as well as the bed (including main structure, rails and mattress). Cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology, frequency of cleaning and disinfectant products . Because different cleaning products are approved in each hospital, if deemed necessary, controls will be established to ensure mdr eradication .eighth recommendation: a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated.rationale: any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . Therefore, the first action is to remove all expendable materials, leaving work surfaces as free as possible . Equipment should be filed and information on the following aspects provided: staff responsible for cleaning, cleaning schedule and cleaning methodology (disinfection, sterilization). Each healthcare worker is responsible for cleaning and disinfection of equipment for personal use (stethoscopes, flashlights).ninth recommendation: to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens.rationale: several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [40 - 43]. A systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage, and possibly bloodstream infections, with gram - positive mdr pathogens (mrsa and vancomycin - resistant enterococci [vre]), whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak .in a recent randomized multicenter trial carried out in 13 icus, the effect of different infection control strategies on acquisition of mdr pathogens was assessed . Improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition, particularly of mrsa . Interestingly, in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing, screening and isolation of carriers did not reduce acquisition rates of mdr pathogens . More recently, a multicenter, open, crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .chlorhexidine solutions must contain 0.16 grams of chlorhexidine (digluconate) per liter (dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water). Contraindications for chlorhexidine use and adverse reactions should be taken into account . Because chlorhexidine is a cationic molecule, its activity can be reduced by natural soaps, various inorganic anions, non - ionic surfactants, and hand creams containing anionic emulsifying agents . Daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection.tenth recommendation: if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods.rationale: studies of outbreaks based on the phenotypic characteristics of microorganisms (antigenic properties, metabolic or antibiotic resistance) are limited and do not provide conclusive differences or similarities between them . Therefore, molecular typing methods, to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism, should be performed . This will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .the zero resistance program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory (national center for microbiology, institute of health carlos iii;), where the microbiological test will be performed free of charge . First recommendation: in each icu, at least one intensivist will be designated as responsible for the use of antimicrobials . He / she should have extensive experience in infection control and in the treatment of severe infections . Analysis of antimicrobial use should include: review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression . Review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration), evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation . Rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development . Prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression . Second recommendation: empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s) based on patient risk factors and/or knowledge of local ecology . Otherwise, narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens (carbapenems, colistin, tigecycline, glycopeptides, daptomycin, linezolid) should be started if needed . In all cases, samples for culture of the potential sources of infection rationale: early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . However, delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection . More recently, a quasi - experimental, before - after observational cohort study concluded that, after adjusting for confounders, aggressive antimicrobial therapy was an independent predictor of mortality . In the aggressive period, antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . In the second period (conservative strategy), the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking . However, it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal . In addition, the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology . Therefore, it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . Obviously, efforts to reduce the delay of microbiological results (use of rapid diagnostic techniques, direct contact with the microbiologist) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed . Third recommendation: in each unit, at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens . Rationale: success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals . Nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care . A multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient . The participation of nurses is of extraordinary importance for the success of infection control programs in intensive care . In fact, most procedures performed to reduce the risk of nosocomial infection (vascular catheter care, artificial airway care, mouth hygiene, etc .) Are part of the nurse s daily tasks . Programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders . This model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc . The zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens . Fourth recommendation: it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay . These samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital . Rationale: guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units, e. g., icus . The implementation of contact precautions in patients colonized or infected with mdr is widely accepted . In contrast, the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed . Initial screening is specially recommended for mrsa, although the same principles and practices apply to gram - negative mdr organisms, which actually now constitute the main threat . The type and number of samples are selected according to local resources and epidemiology and should include at least nasal, rectal and oropharyngeal swabs (bronchial aspirates in intubated patients). In addition, other samples may be necessary to control potential reservoirs (infections, skin ulcers, etc . ). Concerning surveillance cultures, two approaches are acceptable: all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist (see fifth recommendation). Checklist of risk factors (table 2) must be completed to identify patients at high risk of mdr pathogen carriage . Patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2 checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria risk factor 1 . Hospital admissionlasting> 5 days, during last 3 monthsyes no 2 . Institutionalized (prison, healthcare and social centers, geriatric centers, etc. )yes no 3 . Antibiotic therapy 7 days in previous month (particularly 3rd and 4th generation cephalosporins, flouroquinolones and carbapenems)yes no 5 . Comorbidities associated with high incidence of colonization or infection with mdr pathogens: cystic fibrosis, bronchiectasis, chronic skin ulcers, etc.yes no checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria rationale: several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified: prior antibiotic use, the presence of invasive devices and certain underlying diseases are the most frequently reported . Patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america (ats / idsa) criteria are: current hospitalization of 5 days or more, prior antibiotic therapy, prior hospitalization, residence in a nursing home or extended - care facility, home infusion therapy within 30 days, chronic dialysis within 30 days, home wound care, family member with an mdr pathogen, and immunosuppression . However, in a prospective evaluation, although these criteria had an excellent negative predictive value (96%), they had a very low positive predictive value (18%) for infection or colonization with an mdr pathogen at icu admission . In a case control study, immunosuppression was not independently associated with mdr bacteria in the icu . In other studies, risk factors for specific pathogens, like mrsa or a. baumannii, have been identified in an attempt to establish control measures that limit spread . This approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection . With this information, the sec generated a checklist (table 2) for detection of patients at high risk of carrying mdr pathogens . If one or more of these risk factors is present, screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms . The prospective validation of this checklist is one of the pending tasks of this program . Sixth recommendation: compliance with preventive measures including those based on transmission mechanisms should be routinely measured . Rationale: contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms . Adherence to these practices must be continuously reinforced and monitored . Briefly, contact precautions (by staff and visitors) consist of: hand hygiene and donning of gown and gloves immediately prior to room entry, and disposal of gown and gloves inside the patient s room, followed by hand hygiene immediately prior to leaving the room . Adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study . Correct practice includes: (1) use of a contact precautions sign for every patient colonized / infected by mdr pathogens; (2) availability of contact precautions equipment at patient room entry; (3) barrier disposal containers inside patient room; and (4) monitoring of adherence to the contact precautions protocol by staff / visitors . If there are no closed rooms, precautions must be tightened . To achieve the desired results, all staff members should watch compliance with preventive measures . Concerning this issue, the sec of zero resistance seventh recommendation: all units should develop a cleaning protocol for rooms of patients with mdr pathogens . Rationale: many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas . Nevertheless, substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [37 - 39]. Cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee . This protocol should include fixed structures (floors and walls) as well as the bed (including main structure, rails and mattress). Cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology, frequency of cleaning and disinfectant products . Because different cleaning products are approved in each hospital, eighth recommendation: a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated . Rationale: any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . Therefore, the first action is to remove all expendable materials, leaving work surfaces as free as possible . Equipment should be filed and information on the following aspects provided: staff responsible for cleaning, cleaning schedule and cleaning methodology (disinfection, sterilization). Each healthcare worker is responsible for cleaning and disinfection of equipment for personal use (stethoscopes, flashlights). Ninth recommendation: to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens . Rationale: several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [40 - 43]. A systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage, and possibly bloodstream infections, with gram - positive mdr pathogens (mrsa and vancomycin - resistant enterococci [vre]), whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak . In a recent randomized multicenter trial carried out in 13 icus, improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition, particularly of mrsa . Interestingly, in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing, screening and isolation of carriers did not reduce acquisition rates of mdr pathogens . More recently, a multicenter, open, crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia . Chlorhexidine solutions must contain 0.16 grams of chlorhexidine (digluconate) per liter (dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water). Contraindications for chlorhexidine use and adverse reactions should be taken into account . Because chlorhexidine is a cationic molecule, its activity can be reduced by natural soaps, various inorganic anions, non - ionic surfactants, and hand creams containing anionic emulsifying agents . Daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection . Tenth recommendation: if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods . Rationale: studies of outbreaks based on the phenotypic characteristics of microorganisms (antigenic properties, metabolic or antibiotic resistance) are limited and do not provide conclusive differences or similarities between them . Therefore, molecular typing methods, to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism, should be performed . This will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed . Program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory (national center for microbiology, institute of health carlos iii;), where the microbiological test will be performed free of charge . The main objective of the zero resistance project is reduction in the cumulative incidence of patients with icu - acquired mdr infections by 20% . Secondary objectives are to study the epidemiology of mdr infections in spanish icus, to be able to distinguish imported from icu - acquired cases, to promote and strengthen safety assurance in participating units, and to create a network of icus implementing safe, and evidence - based practices . The primary aim of the bundle recommendations is reduction of the three most influential factors contributing to the development and transmission of mdr, namely: 1) adequate prescription of antibiotics; 2) early detection and prevention of cross - colonization of mdr; and 3) elimination of reservoirs .first recommendation: in each icu, at least one intensivist will be designated as responsible for the use of antimicrobials . He / she should have extensive experience in infection control and in the treatment of severe infections . Analysis of antimicrobial use should include: review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .second recommendation: empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s) based on patient risk factors and/or knowledge of local ecology . Otherwise, narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens (carbapenems, colistin, tigecycline, glycopeptides, daptomycin, linezolid) should be started if needed . In all cases, samples for culture of the potential sources of infection should be obtained before starting antibiotic therapy.rationale: early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . However, delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection . More recently, a quasi - experimental, before - after observational cohort study concluded that, after adjusting for confounders, aggressive antimicrobial therapy was an independent predictor of mortality . In the aggressive period, antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . In the second period (conservative strategy), antimicrobial treatment was started only after objective findings confirmed the infection .the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking . However, it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal . In addition, the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology . Therefore, it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . Obviously, efforts to reduce the delay of microbiological results (use of rapid diagnostic techniques, direct contact with the microbiologist) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed.third recommendation: in each unit, at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens.rationale: success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals . Nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care.a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient . The participation of nurses is of extraordinary importance for the success of infection control programs in intensive care . In fact, most procedures performed to reduce the risk of nosocomial infection (vascular catheter care, artificial airway care, mouth hygiene, etc .) Are part of the nurse s daily tasks.programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders . This model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc . The zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens.fourth recommendation: it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay . These samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital.rationale: guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units, e. g., icus . The implementation of contact precautions in patients colonized or infected with mdr is widely accepted . In contrast, the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed . Initial screening is specially recommended for mrsa, although the same principles and practices apply to gram - negative mdr organisms, which actually now constitute the main threat.active surveillance programs are time and resource - consuming . The type and number of samples are selected according to local resources and epidemiology and should include at least nasal, rectal and oropharyngeal swabs (bronchial aspirates in intubated patients). In addition, other samples may be necessary to control potential reservoirs (infections, skin ulcers, etc. ).concerning surveillance cultures, two approaches are acceptable: all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist (see fifth recommendation).fifth recommendation: at admission to the icu, a checklist of risk factors (table 2) must be completed to identify patients at high risk of mdr pathogen carriage . Patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2 checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria risk factor 1 . Hospital admissionlasting> 5 days, during last 3 monthsyes no 2 . Institutionalized (prison, healthcare and social centers, geriatric centers, etc. )yes no 3 . Antibiotic therapy 7 days in previous month (particularly 3rd and 4th generation cephalosporins, flouroquinolones and carbapenems)yes no 5 . Comorbidities associated with high incidence of colonization or infection with mdr pathogens: cystic fibrosis, bronchiectasis, chronic skin ulcers, etc.yes no rationale: several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified: prior antibiotic use, the presence of invasive devices and certain underlying diseases are the most frequently reported . Patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america (ats / idsa) criteria are: current hospitalization of 5 days or more, prior antibiotic therapy, prior hospitalization, residence in a nursing home or extended - care facility, home infusion therapy within 30 days, chronic dialysis within 30 days, home wound care, family member with an mdr pathogen, and immunosuppression . However, in a prospective evaluation, although these criteria had an excellent negative predictive value (96%), they had a very low positive predictive value (18%) for infection or colonization with an mdr pathogen at icu admission . In a case control study, immunosuppression was not independently associated with mdr bacteria in the icu .in other studies, risk factors for specific pathogens, like mrsa or a. baumannii, have been identified in an attempt to establish control measures that limit spread . This approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection.with this information, the sec generated a checklist (table 2) for detection of patients at high risk of carrying mdr pathogens . If one or more of these risk factors is present, screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms . The prospective validation of this checklist is one of the pending tasks of this program.sixth recommendation: compliance with preventive measures including those based on transmission mechanisms should be routinely measured.rationale: contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms . Briefly, contact precautions (by staff and visitors) consist of: hand hygiene and donning of gown and gloves immediately prior to room entry, and disposal of gown and gloves inside the patient s room, followed by hand hygiene immediately prior to leaving the room.adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study . Correct practice includes: (1) use of a contact precautions sign for every patient colonized / infected by mdr pathogens; (2) availability of contact precautions equipment at patient room entry; (3) barrier disposal containers inside patient room; and (4) monitoring of adherence to the contact precautions protocol by staff / visitors . If there are no closed rooms, precautions must be tightened.to achieve the desired results, all staff members should watch compliance with preventive measures . Concerning this issue, the sec of zero resistance therefore, the rest of the hospital staff and visitors must follow their recommendations.seventh recommendation: all units should develop a cleaning protocol for rooms of patients with mdr pathogens.rationale: many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas . Nevertheless, substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [37 - 39]. Cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee . This protocol should include fixed structures (floors and walls) as well as the bed (including main structure, rails and mattress). Cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology, frequency of cleaning and disinfectant products . Because different cleaning products are approved in each hospital, if deemed necessary, controls will be established to ensure mdr eradication .eighth recommendation: a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated.rationale: any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . Therefore, the first action is to remove all expendable materials, leaving work surfaces as free as possible . Equipment should be filed and information on the following aspects provided: staff responsible for cleaning, cleaning schedule and cleaning methodology (disinfection, sterilization). Each healthcare worker is responsible for cleaning and disinfection of equipment for personal use (stethoscopes, flashlights).ninth recommendation: to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens.rationale: several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [40 - 43]. A systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage, and possibly bloodstream infections, with gram - positive mdr pathogens (mrsa and vancomycin - resistant enterococci [vre]), whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak .in a recent randomized multicenter trial carried out in 13 icus, the effect of different infection control strategies on acquisition of mdr pathogens was assessed . Improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition, particularly of mrsa . Interestingly, in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing, screening and isolation of carriers did not reduce acquisition rates of mdr pathogens . More recently, a multicenter, open, crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .chlorhexidine solutions must contain 0.16 grams of chlorhexidine (digluconate) per liter (dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water). Contraindications for chlorhexidine use and adverse reactions should be taken into account . Because chlorhexidine is a cationic molecule, its activity can be reduced by natural soaps, various inorganic anions, non - ionic surfactants, and hand creams containing anionic emulsifying agents . Daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection.tenth recommendation: if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods.rationale: studies of outbreaks based on the phenotypic characteristics of microorganisms (antigenic properties, metabolic or antibiotic resistance) are limited and do not provide conclusive differences or similarities between them . Therefore, molecular typing methods, to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism, should be performed . This will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .the zero resistance program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory (national center for microbiology, institute of health carlos iii;), where the microbiological test will be performed free of charge . First recommendation: in each icu, at least one intensivist will be designated as responsible for the use of antimicrobials . He / she should have extensive experience in infection control and in the treatment of severe infections . Analysis of antimicrobial use should include: review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression . Review of the indication for antimicrobials, evaluation of the appropriateness of the antimicrobial and the correct administration (dosing, intervals and duration), evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation . Rationale: antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge . Moreover, many pathophysiological changes associated with severe acute illness or sepsis, like capillary leak, third spacing, increased volume of distribution, and impaired renal and/or liver function, affect antimicrobial pharmacokinetics / pharmacodynamics . Therefore, it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality . This implies choosing optimal empirical antibiotics, appropriate mode of administration, and correct dosage . Administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations, which are associated with treatment failure and resistance development . Prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . However, as soon as microbiological information is available, empiric therapy should be adapted, if appropriate, by either reduction in number and/or narrowing of antimicrobial spectrum . In fact, de - escalation of empirical therapy is performed in less than 50% of patients . Recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression . Second recommendation: empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s) based on patient risk factors and/or knowledge of local ecology . Otherwise, narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens (carbapenems, colistin, tigecycline, glycopeptides, daptomycin, linezolid) should be started if needed . In all cases, samples for culture of the potential sources of infection rationale: early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . However, delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection . More recently, a quasi - experimental, before - after observational cohort study concluded that, after adjusting for confounders, aggressive antimicrobial therapy was an independent predictor of mortality . In the aggressive period, antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . In the second period (conservative strategy), the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking . However, it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal . In addition, the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology . Therefore, it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . Obviously, efforts to reduce the delay of microbiological results (use of rapid diagnostic techniques, direct contact with the microbiologist) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed . Third recommendation: in each unit, at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens . Rationale: success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals . Nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care . A multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient . The participation of nurses is of extraordinary importance for the success of infection control programs in intensive care . In fact, most procedures performed to reduce the risk of nosocomial infection (vascular catheter care, artificial airway care, mouth hygiene, etc .) Are part of the nurse s daily tasks . Programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders . This model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc . Zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens . Fourth recommendation: it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay . These samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital . Rationale: guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units, e. g., icus . The implementation of contact precautions in patients colonized or infected with mdr is widely accepted . In contrast, the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed . Initial screening is specially recommended for mrsa, although the same principles and practices apply to gram - negative mdr organisms, which actually now constitute the main threat . The type and number of samples are selected according to local resources and epidemiology and should include at least nasal, rectal and oropharyngeal swabs (bronchial aspirates in intubated patients). In addition, other samples may be necessary to control potential reservoirs (infections, skin ulcers, etc . ). Concerning surveillance cultures, two approaches are acceptable: all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist (see fifth recommendation). Checklist of risk factors (table 2) must be completed to identify patients at high risk of mdr pathogen carriage . Patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2 checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria risk factor 1 . Hospital admissionlasting> 5 days, during last 3 monthsyes no 2 . Institutionalized (prison, healthcare and social centers, geriatric centers, etc. )yes no 3 . Known colonization or infection with mdr pathogensyes no 4 . Antibiotic therapy 7 days in previous month (particularly 3rd and 4th generation cephalosporins, flouroquinolones and carbapenems)yes no 5 . Comorbidities associated with high incidence of colonization or infection with mdr pathogens: cystic fibrosis, bronchiectasis, chronic skin ulcers, etc.yes no checklist of risk factors for carriage of multidrug - resistant (mdr) bacteria rationale: several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified: prior antibiotic use, the presence of invasive devices and certain underlying diseases are the most frequently reported . Patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america (ats / idsa) criteria are: current hospitalization of 5 days or more, prior antibiotic therapy, prior hospitalization, residence in a nursing home or extended - care facility, home infusion therapy within 30 days, chronic dialysis within 30 days, home wound care, family member with an mdr pathogen, and immunosuppression . However, in a prospective evaluation, although these criteria had an excellent negative predictive value (96%), they had a very low positive predictive value (18%) for infection or colonization with an mdr pathogen at icu admission . In a case control study, immunosuppression was not independently associated with mdr bacteria in the icu . In other studies, risk factors for specific pathogens, like mrsa or a. baumannii, have been identified in an attempt to establish control measures that limit spread . This approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection . With this information, the sec generated a checklist (table 2) for detection of patients at high risk of carrying mdr pathogens . If one or more of these risk factors is present, screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms . The prospective validation of this checklist is one of the pending tasks of this program . Sixth recommendation: compliance with preventive measures including those based on transmission mechanisms should be routinely measured . Rationale: contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms . Briefly, contact precautions (by staff and visitors) consist of: hand hygiene and donning of gown and gloves immediately prior to room entry, and disposal of gown and gloves inside the patient s room, followed by hand hygiene immediately prior to leaving the room . Adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study . Correct practice includes: (1) use of a contact precautions sign for every patient colonized / infected by mdr pathogens; (2) availability of contact precautions equipment at patient room entry; (3) barrier disposal containers inside patient room; and (4) monitoring of adherence to the contact precautions protocol by staff / visitors . If there are no closed rooms, precautions must be tightened . To achieve the desired results, all staff members should watch compliance with preventive measures . Concerning this issue, the sec of zero resistance seventh recommendation: all units should develop a cleaning protocol for rooms of patients with mdr pathogens . Rationale: many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas . Nevertheless, substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [37 - 39]. Cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee . This protocol should include fixed structures (floors and walls) as well as the bed (including main structure, rails and mattress). Cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology, frequency of cleaning and disinfectant products . Because different cleaning products are approved in each hospital, eighth recommendation: a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated . Rationale: any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . Therefore, the first action is to remove all expendable materials, leaving work surfaces as free as possible . Equipment should be filed and information on the following aspects provided: staff responsible for cleaning, cleaning schedule and cleaning methodology (disinfection, sterilization). Each healthcare worker is responsible for cleaning and disinfection of equipment for personal use (stethoscopes, flashlights). Ninth recommendation: to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens . Rationale: several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [40 - 43]. A systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage, and possibly bloodstream infections, with gram - positive mdr pathogens (mrsa and vancomycin - resistant enterococci [vre]), whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak . In a recent randomized multicenter trial carried out in 13 icus, improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition, particularly of mrsa . Interestingly, in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing, screening and isolation of carriers did not reduce acquisition rates of mdr pathogens . More recently, a multicenter, open, crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia . Chlorhexidine solutions must contain 0.16 grams of chlorhexidine (digluconate) per liter (dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water). Contraindications for chlorhexidine use and adverse reactions should be taken into account . Because chlorhexidine is a cationic molecule, its activity can be reduced by natural soaps, various inorganic anions, non - ionic surfactants, and hand creams containing anionic emulsifying agents . Daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection . Tenth recommendation: if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods . Rationale: studies of outbreaks based on the phenotypic characteristics of microorganisms (antigenic properties, metabolic or antibiotic resistance) are limited and do not provide conclusive differences or similarities between them . Therefore, molecular typing methods, to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism, should be performed . This will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed . Program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory (national center for microbiology, institute of health carlos iii;), where the microbiological test will be performed free of charge . Active implementation of this type of program is clearly necessary in order to achieve the desired results . The agency for quality assurance of the spanish ministry of health will promote implementation in collaboration with the 17 regional healthcare authorities through dissemination, coordination and follow - up . Every autonomous region will create a coordinating team led by an intensivist, responsible for contacting hospital management . The hospital management will notify their local infection and patient quality assurance committees and nominate a local coordinating team consisting of at least an intensivist and an intensive care nurse . It is recommended that the local teams keep track of the number of healthcare workers, physicians, nurses and nurse aides that complete the web - based training modules and report their local educational indices to the regional coordinator . The impact of zero resistance, as in all quality programs, must be measured using quality indicators that can be broken down into structure, procedure and outcome indicators . Obviously, outcome measures are of greater interest since they reflect all aspects of care and are the ultimate objectives of the intervention . The proposed indicators are explained in detail in the program, but each local team should decide which indicators to monitor depending on the information systems and efforts necessary to obtain these measurements . Zero resistance program are committed to entering data required for calculation of the relevant indices in the web - based envin - helics registry . Data are recorded through a specific adaptation of the envin - helics web page . Summary descriptive statistics are available on - line for every individual unit, which can directly access its data on a daily basis . Bacterial resistance to antibiotics is growing day by day, particularly in hospitals, with a significant impact on mortality and morbidity . The lack of new antibiotics, especially for gram - negative mdr pathogens, aggravates this serious problem as noted by numerous agencies and professional societies . Antibiotics are often incorrectly prescribed: inadequate antibiotics or incorrect dosage for a particular infection, administration of antibiotics for non - bacterial infections, and excessively long treatment courses are all frequent . Zero resistance is a project developed by the semicyuc with the technical support of the spanish ministry of health, with the main objective of reducing the cumulative incidence of patients with icu - acquired mdr by 20% . This project contains a bundle of 10 recommendations aimed at improving prescription of antibiotics, detection and prevention of cross - colonization of mdr pathogens, and elimination of reservoirs . This initiative includes an integral patient safety program and educational modules to facilitate its implementation . Adherence to the project and its results will be evaluated through a series of indicators.
A 36 year old, para 2 female with a history of chronic right adnexal pain was admitted for a laparoscopic right salpingo - oophorectomy . The patient experienced worsening right ovarian pain which did not improve despite nonsteroidal anti - inflammatory medications . An ultrasound revealed a 4 cm complex ovarian mass which did not change in size with serial ultrasounds . Previous surgeries included an abdominal hysterectomy for a fibroid uterus, laparoscopic left salpingooophorectomy and lysis of adhesions for pelvic pain, an appendectomy, two previous cesarean sections, a bilateral tubal ligation and an inguinal herniorrhapy as a teenager . The patient underwent general endotracheal anesthesia and was prepped and draped in the usual manner after being placed in the dorsolithotomy position . After making a small incision in the umbilicus, a veress needle with its 2 mm trocar was carefully inserted into the abdomen with the panniculus elevated . The saline drop test revealed patency, and the veress needle was attached to the co2 for the pneumoperitoneum . The co2 was immediately stopped, the veress needle removed and a 2 mm microlaparoscope (minisite gold; u.s . Surgical corp ., norwalk, ct) inserted into the trocar, which revealed bowel lumen with stool . A second 2 mm trocar was placed in the right lower quadrant, and the microlaparoscope revealed a perforation into the anti - mesenteric border of the ileum . Was inserted under direct visualization lateral to the right rectus muscle at the level of the umbilicus . The 2 mm umbilical trocar was replaced with a new one of the same diameter . At the site of the small bowel entry, after irrigating the pelvic and abdominal cavities with 3 liters of normal saline with no further fecal spillage or bleeding, the decision was made to not suture the site of entry into the small bowel . Postoperatively, the patient had a nasogastric tube inserted, was placed on famotidine (pepcid) 20 mg every 12 hours and was given antibiotics consisting of ampicillin, gentamicin and clindamycin intravenously . On the evening of surgery, the patient developed a fever of 101.2 degrees fahrenheit . On postoperative day one, she had a single temperature spike of 100.9 after being afebrile . On postoperative day three, she had a small bowel movement, was advanced to a regular diet and discharged home in the afternoon on oral cephalexin . At her one week postoperative visit she remained afebrile, tolerating her diet and with no complaints . One month after surgery she was symptom free and her chronic right adnexal pain was gone . Most of the current data on laparoscopic complications is based on case reports and small series . Open laparoscopy was developed to reduce the risk of blind entry into the peritoneal cavity . However, injury to the bowel has been reported to occur at the same rate with this technique . Recent advances in microlaparoscopy have allowed us to perform both diagnostic and operative procedures through 2 mm trocars . In our patient, a microlaparoscope inserted through the entry site confirmed the complication . Had we performed traditional laparoscopy using a 10 mm trocar, the site of bowel injury would have required suturing, possibly a laparotomy . In cases where the site of perforation is free of adhesions and in need of repair, the bowel can be sutured externally by withdrawing the bowel through a 12 mm trocar . Our minimally invasive, conservative approach to this complication was possible because no further fecal material leaked through the site of perforation and the diameter of the injury was small . We felt that suturing in this case was unnecessary because the margins were regular, hemostatic and self - sealing . The stool that was forced through this site probably was a result of the increased intraluminal pressure in the bowel from the co2 insufflation . After copious irrigation with normal saline and no further spillage, we felt that the patient's morbidity would be greater with suturing of the bowel if it produced bleeding or further spillage . Except for an initial fever, the patient responded well to conservative management with bowel decompression, antibiotic therapy and close observation . Conditions requiring a laparotomy following a bowel perforation include any sign of peritonitis, persistent fever, ileus or prolonged vomiting . We believe that some small bowel perforations from 2 mm instrumentation can be managed conservatively without suturing provided that the site of injury is not actively leaking stool nor bleeding.
Primary malignant tumors of the spine are rare.123 the treatment of metastatic spinal tumors has evolved significantly with greater understanding of molecular biology of the tumors and advances made in the diagnostic and therapeutic modalities.456 high grade metastatic spinal tumors are invasive and without timely treatment, patients can succumb to progression of their disease.7 despite being an invasive and morbid surgery, current evidence suggests that only total enbloc spondylectomy (tes) with free tumor margins in selected cases has better survival rates.89 with this background, the authors describe their results of tes for solitary high grade metastatic spinal tumors with 18 months followup . Five patients underwent tes for solitary metastatic vertebral lesion between november 2012 and january 2014 . All patients underwent complete preoperative investigations, computed tomography (ct) scan, magnetic resonance imaging (mri) scan, and positron emission tomography (pet) scan to know any other metastatic lesions in the body . After thorough investigations, it was confirmed that each patient had high grade, solitary, metastatic vertebral body tumor . All patients had presented to us with spinal instability, unrelenting severe spinal pain and one patient with severe progressive radiculopathy . Visual analog scale (vas) all patients had eccentrically located metastatic lesion, in a single vertebral body (confined to operable zones as per wbb classification). All patients underwent preoperative digital subtraction angiography (dsa) guided arterial embolization of major feeding vessel to the tumor, 24 h prior to surgery . This not only helped in decreasing the tumor vascularity but also helped surgeons with clear bloodless operative field during surgery . The patient was placed in prone position, a midline incision extending three levels cephalad and three levels caudal was made . Skin, fasciae, and paraspinal muscles were elevated bilaterally, up to tips of transverse processes, on both sides . Pedicle screws were inserted two levels proximally and two levels caudally on both sides of proposed tes vertebra . Using kerrison punches (or osteotomes some times), bilateral pars osteotomy and posterior soft tissue release were performed . The posterior elements consisting of lamina, spinous process, and inferior articular process were removed in toto (first piece). The pedicle was osteotomied inside out at its junction with the vertebral body on the uninvolved side . Thus, the transverse process, superior articular process, and pedicle were removed in toto (second piece). Now, the proposed vertebral body undergoing tes is bluntly dissected to free the paravertebral soft tissue attachments (starting from side to front and cephalad disc to caudal, bilaterally). This involved combination of blunt dissection with finger tips, mono / bipolar cautery, and isolation with ligation of segmental arteries using vascular clips . The major vessel pair in front was separated from vertebral body using finger tip dissection . A large size sponge placed skirting the front and sides of the vertebral body, separating it from major vessels . The spinal cord / thecal sac / exiting roots were isolated throughout and secured bilaterally . Now, the rod was placed on the involved side, connecting the proximal and distal screws . Once the vertebral body is loose enough for removal, the connecting rod is exchanged to uninvolved side . Multiple attempts to extract the vertebral body in to were tried, releasing the loose tags hindering the extraction during each attempt . Extreme care is also taken not to use any metal extraction devices during these attempts, so as to avoid fracture at tumor - bone junction and subsequent tumor spillage . With these cares in place, after vertebral body extraction, the sponge behind the major vessels was removed and the tumor bed was inspected for any active bleed or any suspicious looking tissue . All three in toto pieces were reassembled to re - structure the vertebral body and were examined for any cortical violations / missing bony pieces . The interbody reconstruction was done using appropriate sized metal cage, filled with polymethyl methacrylate (pmma) cement . Inserting these large sized cages is also a challenge, which requires multiple attempts of negotiation and repositioning . With cage in proper place, the screw rod assembly is tightened and held under compression over the cage . Following surgery, all the patients were put on appropriate chemotherapy by oncologists and radiotherapy was started once the surgical wound was fully healed . All patients were ambulated by 3 postoperative day and were discharged from the hospital in 5 to 7 days . All patients had eccentrically located metastatic lesion, in a single vertebral body (confined to operable zones as per wbb classification). All patients underwent preoperative digital subtraction angiography (dsa) guided arterial embolization of major feeding vessel to the tumor, 24 h prior to surgery . This not only helped in decreasing the tumor vascularity but also helped surgeons with clear bloodless operative field during surgery . The patient was placed in prone position, a midline incision extending three levels cephalad and three levels caudal was made . Skin, fasciae, and paraspinal muscles were elevated bilaterally, up to tips of transverse processes, on both sides . Pedicle screws were inserted two levels proximally and two levels caudally on both sides of proposed tes vertebra . Using kerrison punches (or osteotomes some times), bilateral pars osteotomy and posterior soft tissue release were performed . The posterior elements consisting of lamina, spinous process, and inferior articular process were removed in toto (first piece). The pedicle was osteotomied inside out at its junction with the vertebral body on the uninvolved side . Thus, the transverse process, superior articular process, and pedicle were removed in toto (second piece). Now, the proposed vertebral body undergoing tes is bluntly dissected to free the paravertebral soft tissue attachments (starting from side to front and cephalad disc to caudal, bilaterally). This involved combination of blunt dissection with finger tips, mono / bipolar cautery, and isolation with ligation of segmental arteries using vascular clips . The major vessel pair in front was separated from vertebral body using finger tip dissection . A large size sponge placed skirting the front and sides of the vertebral body, separating it from major vessels . The spinal cord / thecal sac / exiting roots were isolated throughout and secured bilaterally . Now, the rod was placed on the involved side, connecting the proximal and distal screws . Once the vertebral body is loose enough for removal, the connecting rod is exchanged to uninvolved side . Multiple attempts to extract the vertebral body in to were tried, releasing the loose tags hindering the extraction during each attempt . Extreme care is also taken not to use any metal extraction devices during these attempts, so as to avoid fracture at tumor - bone junction and subsequent tumor spillage . With these cares in place, after vertebral body extraction, the sponge behind the major vessels was removed and the tumor bed was inspected for any active bleed or any suspicious looking tissue . All three in toto pieces were reassembled to re - structure the vertebral body and were examined for any cortical violations / missing bony pieces . The interbody reconstruction was done using appropriate sized metal cage, filled with polymethyl methacrylate (pmma) cement . Inserting these large sized cages is also a challenge, which requires multiple attempts of negotiation and repositioning . With cage in proper place, the screw rod assembly is tightened and held under compression over the cage . Following surgery, all the patients were put on appropriate chemotherapy by oncologists and radiotherapy was started once the surgical wound was fully healed . All patients were ambulated by 3 postoperative day and were discharged from the hospital in 5 to 7 days . There were four females and one male with an average age of 46.8 years (range 3961 years). All these patients presented to us with progressive, severe midback pain with typical history of rest pain and nocturnal aggravations (average vas of 9.4/10, range 9/1010/10), although one patient presented with severe upper limb radiculopathy and ipsilateral motor weakness . All these patients had solitary, high grade, metastatic spinal tumors which were histopathologically proven . On follow up mri scans, these lesions showed pure vertebral body lesions and soft tissue effacement around the spinal cord / thecal sac . On further ct and pet scan screening, it was confirmed that the existing active spinal metastasis was the only skeletal lesion in the body [with average maximum standardized uptake value (suv max) of 8.4, range (611)]. There were three patients of symptomatic, metastatic lesion to l3vertebra, followed by metastatic lesion to t1 vertebra in one patient and to l2 vertebral body was seen in another patient [table 1]. Clinical details of patients and surgical outcome individually, the lesions were nonsmall cell lung carcinoma with l2 vertebral body metastasis, nonkeratinizing squamous cell carcinoma with moderate degree of differentiation with l3 vertebral metastasis, poorly differentiated adenocarcinoma of unknown primary with metastasis to t1 vertebral body, hurthle cell adenoma with metastasis to l3 vertebral body and follicular carcinoma of thyroid with unknown primary with metastasis to l3 vertebral body . The patient with l2 vertebral body metastatic lesion was a known case of non small cell lung carcinoma . The patient had previously undergone pneumonectomy and had received six cycles of chemotherapy with gemcitabine, carboplatin, and avastin for the same . Upon investigation for progressive, severe mid back pain, with typical history of rest pain and nocturnal aggravations, the patient showed persistent l2 vertebral body lesion with soft tissue shadow effacing the thecal sac, on mri scan . Based on ct scan and pet scans, it was further confirmed that there was no lesion in the lung and l2 vertebral body lesion was the only active metastatic lesion (suvmax = 8.9) in the body . Hence, tes of l2 vertebra was performed from all posterior approach, l1 to l3 interbody reconstruction with mesh cage filled with pmma cement, two levels cephalad and two level caudal pedicle screw rod stabilization was done [figure 1a g follow up, the patient was pain free with no tumor recurrence on pet scan and roentgenograph showed implants in good position [figure 2a and b]. (a and b) t2w mid sagittal and axial view mri scan showing panvertebral body pathologic lesion . (c and d) anteroposterior and lateral x - rays of lumbosacral spine showing panvertebral sclerotic lesion (e) peroperative photograph showing en bloc l2 posterior vertebral removal and osteotomy of un - involved left pedicle (f) peroperative photograph showing circumferential separation of soft tissues bluntly and en bloc extraction was done (g) post - extraction re - assembly showing three complete, separate en bloc elements (i), (ii) and (iii) (h and i) peroperative photographs showing intervertebral reconstruction by titanium metal cage filled with pmma cement and spinal stabilization (a and b) x - ray anteroposterior and lateral views of dorsolumbar spine at 20 month followup showing no recurrence of the lesion, and implants in situ the patient of non keratinizing squamous cell carcinoma with metastasis to l3 vertebral body was treated previously with chemo and radiotherapy, 10 years back for uterine carcinoma . Patient presented with severe, progressive low back pain since 6 months with worsening left lower limb radiculopathy since 2 months, an expansile lesion involving l3 vertebra, with mild extension into left pedicle causing lateral recess stenosis and some posterior marrow retropulsion effacing the anterior thecal sac was seen on mri scan . Pet scan showed abnormal, intense degree of fdg uptake involving l3 vertebral body (suv max = 11.4) and there was no other fdg concentrating lesion anywhere in the body . Ct guided biopsy of the l3 vertebral body lesion, followed by histopathological examination confirmed metastatic nonkeratinizing squamous cell carcinoma with moderate degree of differentiation . Hence tes of l3 vertebra was performed from all posterior approach with interbody reconstruction using mesh cage filled with pmma cement, and posterior stabilization was performed by two level cephalad and two level caudal pedicle screw rod stabilization . At 18 months the patient with poorly differentiated adenocarcinoma of unknown primary (based on ct guided biopsy of the skeletal lesion) with symptomatic metastasis to t1 and left humerus was initially treated with chemotherapy and surgery for humeral lesion . The skeletal lesion resolved following surgery and six cycles of chemotherapy with gemcyte and taxol (suvmax = 2.1) and 5 cycles (20gy) of radiotherapy to both lesions, but t1 vertebral body lesion showed very mild improvement, with progressive weakness in both upper limbs (right side 3/5, left side 2/5). Hence, tes of t1 vertebra was done from all posterior approach, interbody reconstruction with 11 mm polyetheretherketone (peek) cage filled with pmma cement was done and posterior stabilization from c5 to t3 was performed [figure 3a f]. The patient experienced three episodes of hypotension and intermittent tachycardia intraoperative, which improved with fluid challenge and 98% oxygen therapy . The patient did not have any recurrence at 18 months followup with significant improvement in symptoms . Sagittal (a) and axial (b) ct scans; sagittal (c) and axial (d) t2w mri scans showing high grade metastatic tumor involving t1 vertebral body (e) x - ray anteroposterior view of cervicodorsal junction immediate post tes showing implant and cage in situ (f) x - ray lateral view of cervicodorsal spine at 18 months followup showing no recurrence and implant in situ patient with asymptomatic, hurthle cell adenoma with metastasis to l3 vertebral body was under regular follow up after total thyroidectomy, performed 5 years back . This patient presented with severe progressive low back pain with bilateral lower limb radiculopathy since 5 months . The patient also complained of difficulty in standing, walking with tingling, and numbness in both legs . New mri scan showed solitary, expansile, l3 vertebral body lesion with partial collapse and posterior bony retropulsion compressing thecal sac . A ct guided biopsy of the lesion was reported as metastatic hurthle cell adenoma with unknown primary . Hence, tes of l3 vertebral body was done from all posterior approach with interbody reconstruction using mesh cage filled with pmma cement, and posterior stabilization was performed by two level cephalad and two level caudal pedicle screw rod stabilization system [figure 4a, b (i iv) and c e]. The patient was administered 8 fractions of radiotherapy in the postoperative period (24 gy). The patient did not have tumor recurrence at 16 months followup with significant improvement in symptoms [figure 4f]. (a) sagittal and axial t2w mri pictures showing l3 vertebral body (hurthle cell adenoma of thyroid) metastasis (b) post tes pictures of specimen showing parts of vertebral body removed [b (i, ii, iii and iv)] (c) intraoperative picture of surgical field showing implants in situ on one side (d) intraoperative picture showing interbody reconstruction using pmma impregnated mesh cage and bilateral pedicle screw rods (e) fluoroscopic view anteroposterior and lateral pictures showing surgical reconstruction (f) anteroposterior and lateral x - ray of lumbosacral spine showing implants in situ and no tumor recurrence after 16 months of followup a post thyroidectomy patient under regular follow up (with mri scan and pet scan) for her l3 vertebral body metastasis presented to us with severe progressive low back pain with rest pain and nocturnal aggravations . New mri and pet scans showed highly active l3 vertebral body soft tissue lesion and there was no other metastatic lesion in the body . Ct guided biopsy of the lesion was diagnosed as high grade follicular carcinoma of thyroid with unknown primary . Tes of the l3 vertebral body was done from all posterior approach with interbody reconstruction using mesh cage filled with pmma cement, and posterior stabilization was performed by two level cephalad and two level caudal pedicle screw rod stabilization . The patient did not have any recurrence at of 16 months followup with significant improvement in symptoms . The average preoperative vas score of 9.4 (range 910) improved to 2 (range 14) at last follow up . There was one mortality in the immediate postoperative period due to septicemic shock (this patient was not part of the study). One patient suffered intra hepatic inferior vena cava thrombosis in the postoperative period, which was treated with anticoagulants . The same patient had to receive six units of fresh frozen plasma and vitamin k injections (with anticoagulants stopped) for deranged coagulation profile during antithrombotic treatment . One patient developed hematuria during chemotherapy in the postoperative period, which subsided following completion of chemotherapy course . There was one mortality in the immediate postoperative period due to septicemic shock (this patient was not part of the study). One patient suffered intra hepatic inferior vena cava thrombosis in the postoperative period, which was treated with anticoagulants . The same patient had to receive six units of fresh frozen plasma and vitamin k injections (with anticoagulants stopped) for deranged coagulation profile during antithrombotic treatment . One patient developed hematuria during chemotherapy in the postoperative period, which subsided following completion of chemotherapy course . Late presentation of spinal tumors (primary as well as metastatic) makes a surgical treatment challenging, often leading to choose palliative treatment . Tes wherein, the entire tumor within the vertebra with a cuff of healthy tissue around it, is removed as one specimen and has proven to be beneficial (due to its lower recurrence rates) in extending the long term survival rates and functional outcome for patients, especially with solitary metastatic disease of the spine.5101112 traditionally, tes for spinal tumors has been considered as morbid procedure.131415 however, with more understanding of surgical techniques and team approach with two spine surgeons trained in spine onco - surgeries, operating from either side as in this series, helps to reduce the complication rates . The average duration of surgery and blood loss was low to comparable, as compared to other existing series of tes procedures.161718 the team approach works in favor of reducing the surgical duration, complications, and thereby morbidity associated with tes surgery . The use of metal cages has been adequately supported in literature as it is believed to offer excellent day 1 stability to the spine.1920 although stacked carbon fiber reinforced polymer (cfrp) cages with biocompatibility and modulus of elasticity similar to that of cortical bone are ideal for vertebral body replacement following tes,21 issues associated with cost constraints and availability limit us from their usage . The fears of donor site morbidity with autograft / disease transmission with allograft usage lead us to use of pmma cement, which is known to resist compressive forces almost immediately once it solidifies . The irradiation of pmma cement during adjuvant radiotherapy causes no objective changes in its shear strength, compressibility or durability.222324 this was also due to the authors belief that relying on bony fusion may not be reliable in patients that will be subjected to irradiation and chemotherapy . The present series shows favorable short term results of tes for solitary, metastatic, high grade vertebral body tumors by a team approach.
Asthma severity and asthma control are often based on the presence and frequency of asthma symptoms, in particular respiratory symptoms such as exercise - induced asthma, limitation of physical activity, and frequency of night asthma symptoms . The revised version of the gina guidelines from 2006 recommends that disease classification can be changed from severity based on symptoms and airflow limitations to success of disease management . This is mainly based on frequency of symptoms and continues to include limitation of physical activity and night symptoms suggestive of level of airway inflammation and increasing need of inhaled corticosteroid (ics). Perception of respiratory symptoms may vary between individuals some might feel minor changes more than others . Physical activity is an important release of asthma symptoms, revealing exercise - induced bronchoconstriction [3, 4], which is widely believed to be related to either airway inflammation or smooth muscle dysfunction [58]. The primary treatment strategy in airway inflammation should be inhaled steroid; whereas smooth muscle dysfunction should be treated primarily with inhaled beta - agonist nighttime awakenings are probably related to increased airway inflammation [10, 11]; however, treatment strategies including lower dose of ics combined with long - acting beta2-agonist have been found to induce a more stable disease than a higher dose of ics alone, which is contradictory to increased inflammatory control . Eia and night asthma might arise from airway inflammation, but other factors, such as smooth muscle dysfunction or differences in the patient's perception / tolerance of symptoms, might play a role . The aim of the present study was to examine in a large population sample whether ahr is of importance for the perception of eia, that is, positive response to a questionnaire and nighttime symptoms . The hypothesis is that symptoms of eia in a general population are not usable for the diagnosis of astma . All asthmatic subjects (n = 793) participating in two large asthma studies in our research unit were pooled in the present analysis [13, 14]; inclusion and exclusion criteria and the examinations performed were the same in all studies . Subjects were aged 1444 years (to minimize entry of patients with copd) and all lived in copenhagen, denmark . Exclusion criteria were respiratory illness other than asthma, for example, rhinitis as single disease, sarcoidosis, and cardiac illness . Patients were excluded if they had withdrawn their consent after completing the questionnaires or if they had left the clinic without completing the clinical examination or diagnostic procedures . All participants received information in both oral and written form and gave their consent in writing before enrolment . Subjects were asked about respiratory and allergic symptoms (within the preceding four weeks and at any time (ever asthma)), use of medication, hospital referrals, and gp or specialist visits . The questions asked about asthma at the interview were adapted from studies by the american thoracic society, division of lung disease of the national heart, lung and blood institute . Asthma was defined as asthma symptoms and signs of reversible airway disease, that is, either airway hyperresponsiveness (ahr) to inhaled methacholine with a pd20 4 mol, peak expiratory flow (pef) variability 20%, or at least a 15% increase in forced expiratory volume in one second (fev1) after administration of a bronchodilator (minimum 300 ml). Asthma severity was classified according to the gina guidelines (1) mild intermittent: symptoms <once a week, nighttime symptoms <twice a month, fev1> 80% predicted . (2) mild persistent: symptoms> once a week but <once a day, nighttime symptoms> twice a month, fev1> 80% predicted . (3) moderate persistent: daily symptoms, nighttime symptoms> once a week or fev1 6080% predicted . (4) severe: continuous daytime symptoms, frequent nighttime symptoms or fev1 <60% predicted . Further, nighttime awakenings 0 (never), nighttime awakenings (nta) 3 per month (mild), nta 3 - 4 per month (mild persistent), nta> 4 per months and fewer than 2 - 3 per week (moderate), and nta> 4 per week (severe). The severity of asthma (mild, mild persistent, moderate, and severe) was classified according to the 2004 gina guidelines, based on symptom frequency only, and asthma control was assessed according to the definition in the 2006 gina guidelines . Details about education, socioeconomic aspects, and lifetime consumption of tobacco in pack years (tobacco consumption [g / day]/20 duration of smoking [years]) were collected . Never smokers were participants who have never smoked, whereas smokers included daily smokers and smokers with a more dispersed consumption . Height and weight were measured and body mass index (bmi) was calculated as (weight [kg])/(height [m]). Spirometry was performed on a 7-l dry wedge spirometer (vitalograph) in accordance with the ers and the percentage of predicted normal values of fev1 (fev1%pred) and fvc (fvc%pred), and the fev1/fvc ratio was calculated . Airway responsiveness to inhaled methacholine was measured in accordance with the method of yan et al . In all patients with fev1 the dose resulting in a 20% fall in fev1 (pd20) was calculated, and the ratio dose response (rdr) was calculated as the decline in fev1 from inhaled saline divided by the highest dose of methacholine administered . A constant of four was added to all dose - response ratios to eliminate negative and zero values . Measurement of fev1 was repeated 15 minutes after administration of 2 mg terbutaline in those with fev1 <70% or 15 minutes after the last inhalation of methacholine in those with either symptoms or a significant decrease in fev1 (i.e., 20%). Lastly, all subjects underwent an allergen skin prick test with ten inhalant allergens in accordance with the eaaci guidelines, and blood eosinophils were counted (10/l). All data were entered in the database by one person and 10% of all patient data were then proofread . When this was done, quality control was conducted by an experienced researcher on all outliers within the entire number of variables . Incidence rates were calculated for the entire group and differences were tested by chi - squared analysis . Further, differences in mean (sd) values between participants with and without respiratory symptoms were tested by parametric analysis (student's t - test). All included variables were examined in a univariate analysis with exercise - induced asthma or nighttime awakenings due to asthma as the dependent variable . In the event of a significant relationship, variables were entered in a linear regression analysis with backward elimination of all nonsignificant parameters after which a final regression analysis was performed . Lastly, an odds ratio analysis was applied in the case of dichotomy variables . A p - value <.05 was considered significant . All 793 asthmatic subjects in the present survey were included based on respiratory symptoms and a positive test to methacholine provocation, inhaled beta - agonist or day - to - day variation in pef (table 1). The frequency of rhinitis was 71% among those with certain asthma, 62% had positive skin prick test, and 55% were never smokers . The frequency of exercise - induced asthma was as follows: never symptoms 9%; 54% 2 times per week; 22% 26 times per week; 7% daily exercise symptoms; 8% reported eia more than once a day . The night awakening due to asthma was 69%, 12%, 7%, 6%, and 6%, respectively . Of the entire group, 29 asthmatic subjects (3.1%) reported both severe eia and nighttime awakenings, and 55% reported neither symptom (p <.001). A univariate analysis including severity of eia (table 2) and nighttime awakenings (table 3) showed a significant association between airway inflammation, indicated by responsiveness to inhaled methacholine (logrdr), and blood eosinophil count (10/l), or obstruction and severity of symptoms . The association between airway responsiveness and eia symptoms (f = 2.5, p <.05, and rho = 0.1, p = .01) was less close than that between airway responsiveness and nighttime awakenings due to asthma (f = 3.5, p <.01 and rho = 0.14, p <.001). Further, no significant association was found between rhinitis and severe eia (15.4% versus 15.1%, ns) and nighttime awakenings (11.9% versus 10.9%, ns); atopic diseases was seldom seen in those with severe eia (10.0% versus 14.3%, p = .08) or among those with severe night symptoms (12.4% versus 21.0%, p <.01). Experience of severe eia was frequently found among female participants (21.0% versus 7.3%, p <.01), and nighttime awakenings were found equally frequently among those who had severe eia (12.9% versus 8.5%, resp ., ns). Lastly, those with severe eia also had many nighttime awakenings (56.8% versus 11.4%, p <.001); those with many nighttime awakenings also experienced many symptoms of eia (24.0% versus 4.8%, p <.001). Concerning eia, by including all variables in a multivariate analysis, logrdr was eliminated; whereas fev1%pred (.16, p <.001), smoking (.098, p <.05), atopy (.16, p <.001), sex (.18, p <.001), and asthma treatment (.17, p <.01) were found to be of significant consequence for development of eia . Women reported persistent exercise symptoms more frequently than did men (21% versus 7%, p <asthmatic subjects with persistent exercise symptoms had lower lung function than those without symptoms (88% versus 94%, p <.001), and treatment with inhaled steroid was more frequently used among those with persistent exercise symptoms (41% versus 17%, p <.001). Including all variables in a multivariate analysis concerning nighttime awakenings showed that logrdr was eliminated as well; whereas frequency of shortness of breath during daytime (0.263, p <.001), coughing (0.243, p <.001), and eia (0.102, p = .066) were of significant consequence . Furthermore, a higher level of eosinophils was associated with a higher level of nighttime awakenings (0.155, p <.011). These findings showed that among those with frequent night symptoms, 64% experienced daily coughing, 31% had daily dyspnoea, and the eosinophil count increased from 0.22 10/l (no night symptoms) to 0.35 10/l (frequent night symptoms), (normal cell count 0 - 0.4 10/l). The relative risk of having nighttime awakenings due to asthma was more than twofold higher among those with eia symptoms than among those without eia symptoms . In addition, the odds ratio (ci95%) was 2.77 (2.03.8) (p <.001) of having nighttime awakenings due to asthma among those with eia symptoms . Although many different surrogate variables representing airway inflammation such as bronchial responsiveness, low level of lung function, signs of airway obstruction (low fev1/fvc ratio), positive skin prick test, high eosinophilic cell count, and many daily asthma symptoms were included in the analysis of this large uniform cohort of persons with asthma, the study showed great diversity . Eia and nighttime awakenings due to asthma symptoms are symptoms of equal importance in the gina guidelines when evaluating the severity of asthma . According to the present guidelines, severity of asthma is based equally on the frequency of respiratory symptoms, such as exercise - induced asthma symptoms, and nighttime awakenings due to asthma symptoms . Less controlled disease is usually an indicator of increased airway inflammation, and the guidelines therefore suggest initiation of inhaled steroid . From a theoretical viewpoint, exercise - induced bronchoconstriction could occur only in the event of airway inflammation, and the more severe the eia the more severe the airway inflammation that could probably be demonstrated [35, 11, 22]. Symptoms of eia in elite athletes are thus not to be used as a predictor for asthma [23, 24] as those symptoms are not related to either airway hyperresponsiveness to inhaled agents or to indirect provocation . It is thus unknown whether eia symptoms are closely associated with airway responsiveness and airway inflammation in a general population of asthmatic subjects who have demonstrated certain airway variability, because the majority of studies have been performed in groups with selected asthmatic subjects who report eia . However, the present study showed that factors important for the presence of eia symptoms and nighttime awakenings were outside the battery of inflammatory variables . These present findings indicate a close univariate association between eia symptoms and ahr, which is in agreement with earlier findings of a close association between degree of eia and ahr to inhaled agents [25, 26]. However, in the final analysis many inflammatory variables were statistically eliminated, whereas eia symptoms continued to show a significant association with low level of lung function and positive skin prick test, which could suggest an association between markers of airway inflammation and eia in asthmatic subjects with a history of eia symptoms . One of which, the most important predictor of airway inflammation in the present study, may be having low level of lung function, as low level of lung function could be due to inflammatory swelling of the airway mucosa [2729]. The asthmatic women frequently reported persistent exercise - induced symptoms; whereas nighttime awakenings in asthmatic participants in this large population were without sex association when interview by a specialist using a questionnaire - based interview with focus on the issue, as suggested by gina guidelines . It indicated that women may have a lower tolerance to eia or perhaps a different perception of eia compared with that of men . An explanation of women's lower tolerance to eia could be a symptom of low fitness, and not astma or airway inflammation . Women probably perform fewer physical activities than do men, especially among the oldest in this group; consequently, when they exercise they develop more symptoms . Another explanation could be the level of lung function, because although both women and men who reported severe eia generally had low level lung function, the women had a significantly larger reduction in fev1 percentage predicted than did the men (data not shown), indicating that low ventilatory capacity could also be a satisfactory explanation of the present results of the eia symptoms . Other studies indicate that asthmatic persons who are smokers have impaired effect of their asthma treatment, and compared with the asthmatic subjects who were non - smokers, the asthmatic smokers showed low level of lung function, less improvement in lung function, bhr, and symptoms [30, 31]. These findings support our finding of more eia symptoms among those asthmatic subjects who smoked, although it is unexplained whether it is due to the smoking history or the low level of lung function, but it seems not to be inflammation . Those asthmatic subjects with severe nighttime awakenings seem to have more severe illness than those with eia symptoms . Those with night symptoms had a high eosinophilic count; whereas the perception of exercise - induced symptoms had a cell count which was significantly lower . Those with nighttime awakenings due to asthma had a more severe degree of airway responsiveness than did those with exercise symptoms; moreover, those who had severe night awakening also had frequently signs of other asthma symptoms, such as dyspnoea and cough during daytime, all of which indicate that those with nighttime awakenings due to asthma might have severe airway inflammation and need treatment with inhaled steroid . These findings indicate that symptoms of eia may be less related to inflammation, whereas nighttime awakenings are more closely related to airway inflammation . The higher eosinophilic count found among those with many night awakenings is of significant importance as the prognosis of asthmatic subjects with high level of eosinophilic cell count; many respiratory symptoms and severe ahr have been shown to be serious with a more severe and more variable asthmatic disease . Nighttime awakenings due to asthma compared with eia symptoms without objective measurements are probably more likely to be based on airway inflammation, which indicates that these asthmatic subjects with nighttime awakenings, but not symptoms of eia, would probably benefit from increased treatment with inhaled steroid [3234]. However, we found a significantly increased risk of having nighttime awakenings among those with exercise - induced asthma symptoms . The risk was almost threefold increased among those with many eia symptoms and nighttime symptoms, indicating some association between the two different questions of asthma symptoms, and that although the nighttime awakenings seem to be more closely associated with airway inflammation, the presence of many exercise symptoms also has some relation to the presence of airway inflammation, but the majority of symptoms might be due to low level of fitness . Based on these findings and earlier findings in elite athletes [23, 24] the gina guidelines would most likely benefit from reducing the importance of eia symptoms as an initiator of increased treatment strategy of inhaled steroid without having an exercise test showing eib . These findings also emphasise the importance of objective measurement of asthma, including objective measurement of eia symptoms, before including eia as important for the level of asthma severity and the level of control . It could be speculated, however, whether some of these symptoms are based on a smooth airway dysfunction rather than inflammation or low level of fitness, indicating need of bronchodilator and not steroid, as all participants had proven asthma . In the present study a substantial number of asthmatic subjects were either untreated or undertreated; this has been reported in earlier publications . These findings of undertreatment in the present population of asthmatic subjects highlight the importance of the present findings . The subjects were not contaminated with inhaled steroid, and even those who reported serious night awakening due to asthma were not receiving treatment according to the guidelines . In conclusion, in this large group of asthmatic subjects we found that eia symptoms and night symptoms were associated with ahr in a univariat analysis, but other factors seem of importance for the perception of eia and night symptoms . Symptoms of eia in a general population are, like eia symptoms in elite athletes, not usable for the diagnosis of astma . Night asthma is more closely associated with airway inflammation, which verifies what we had expected.
Folic acid has emerged as a promising ligand for the selective delivery of imaging and therapeutic agents to target cells, such as cancer cells and activated macrophages, in inflammatory sites (1). As a selective targeting ligand, folic acid has several advantages such as 1) high affinity to its target, folate receptors (frs), even after conjugation with diagnostic / therapeutic agents, 2) ease of conjugation with a variety of imaging and therapeutic agents, and 3) very low or undetectable expression of its receptors on normal cells, despite its high expression on cancer cells and activated macrophages (1). Folic acid primarily enters non - pathogenic, normal cells through the reduced folate carrier to effect its functions (2), but folic acid linked with conjugating agents only enters cells through the fr (23). Fr is a cell surface glycosylphosphatidylinositol (gpi)-anchored glycoprotein, and there are three isoforms in humans: fr-, -, and - (45). Fr- is overexpressed on many types of cancer cells, including ovary, lung, breast, kidney, brain, endometrium, and colon cancer, whereas fr- is overexpressed on activated macrophages which are implicated in inflammatory pathologies, including rheumatoid arthritis, psoriasis, crohn's disease, and systemic lupus erythematosus (6). For this reason, frs have been regarded as promising molecular targets for both diagnostics and therapeutic treatment of a variety of human diseases . In this review, we will introduce the characteristics and structures of frs and discuss their expression profiles on normal, as well as pathogenic, cells . Furthermore, we will give an overview of recent studies describing strategies to target fr- selectively expressed on activated macrophages for the diagnosis and therapy of human inflammatory diseases . Folic acid, also known as folate and vitamin b9, is essential for cells to generate dna, rna, and metabolic amino acids that are required for their proliferation and division (78). Because eukaryotic cells are incapable of synthesizing folic acid, it is delivered into cells through either the reduced folate carrier, which is present in all cell types, or the fr, which is expressed in limited cells (9). Although folic acid is transported into the cells through either system, folate conjugates designed for diagnostics and therapeutics (fig . The fr is a cell surface glycoprotein of molecular weight in the range of 38~45 kda with a high affinity for folic acid (kd<10~10), and is attached to the plasma membrane by a glycosylphosphatidylinositol (gpi) anchor (45). Human fr (hfr) is encoded by a family of hfr genes, and three isoforms, hfr-, -, and -, have been reported to date (4). The gene which is predicted to encode hfr- was also found from genome data mining; however hfr--expressing cells and tissues have not yet been identified (10). Hfr- and - are membrane - associated proteins, whereas hfr- is a secreted protein because it does not have the signal peptide for the gpi anchor at its c - terminus (1112). Hfrs share 68~79% amino acid sequence identity and have n - glycosylation sites that are critical for their proper folding (1213). Hfr- and - transport folic acid into cells via receptor - mediated endocytosis . Although all hfrs have been reported to have high binding affinity with folic acid, relative affinities of hfr- and - for folate conjugates are significantly different, in the range of 2~100 fold (14). Studies of chimeric hfr constructs showed that amino acid sequences, such as leu49 in hfr-, and ala49, val104, and glu166 in hfr-, are critical for the differential ligand specificities of hfr- and - (1516). Frs are expressed on the cells of different tissues depending on the types of fr isoforms . Fr- is expressed in the epithelial cells of normal tissues, such as type i and ii alveolar cells in the lungs, choroid plexus, ovary, fallopian tube, uterus, epididymis, submandibular salivary and bronchial glands, placental trophoblasts, and the basolateral membrane of retinal pigment epithelial cells (51718). Because the fr- expressed in these normal tissues is distributed on the luminal surfaces of the tissues, it is protected from fr - targeted folate conjugates administered intravenously . However, fr- is expressed in proximal kidney tubules, which are exposed to folate conjugates filtered from the blood stream . Interestingly, fr- is overexpressed on many malignant tumor cells of epithelial origin, including lung, ovarian, cervical, endometrial, brain, and breast cancers (45), and significant correlation has been observed between the fr- expression level and the grade of the tumor (1920). Although many previous studies have focused on the expression of fr- on the cells of neoplastic tissues, recent studies have shown that fr- is expressed on different types of cells . Fr- is a marker protein in normal hematopoiesis of myelomonocytic lineage cells (5), and is expressed in neutrophils, cd34 hematopoietic progenitor cells, placenta, spleen, and thymus (212223). Expression of fr- is also found in pathogenic cells, such as acute myelogenous leukemia (aml) cells and chronic myelogenous leukemia (cml) cells (2124). Interestingly, fr- is highly expressed on activated, but not normal and resting macrophages, which are implicated in the pathogenesis of human inflammatory diseases, such as rheumatoid arthritis, psoriasis, crohn's disease, systemic lupus erythematosus, atherosclerosis, diabetes, ulcerative colitis, osteoarthritis, glomerulonephritis, and sarcoidosis (6). The expression of fr-, indeed, was confirmed in synovial macrophages obtained from patients suffering from rheumatoid arthritis and in a mouse rheumatoid arthritis model (2526). Fr- is expressed in normal and malignant hematopoietic cells in the spleen, bone marrow, and thymus as well as ovarian, cervical, and uterine cancer cells (1113). Although fr- mrna was detected in lymphoid leukemia cells, a secreted form of fr- protein has not been detected in the serum of patients with lymphoid leukemia (4). Because activated macrophages, which are regarded as key players in the pathogenesis of inflammatory diseases, express a high level of fr- on their surface, folate - conjugated imaging agents have been designed and synthesized to detect pathogenic activated macrophages in the lesions of inflammatory diseases by selectively targeting fr-. Folic acid has been linked to a variety of dyes or radiopharmaceuticals, such as fitc, texas red, alexa fluor, oregon green, technetium (tc), gallium (ga), and indium (in) (272829), and these folate imaging agents have been used for the detection of fr - positive cancer cells and pathogenic activated macrophages . Because these folate - conjugated imaging agents successfully imaged cancer cells in vitro, in vivo, and even clinically, they were further used for the imaging of activated macrophages in the lesions of inflammatory diseases for their diagnosis . Imaging studies of inflammatory diseases were initially conducted on rheumatoid arthritis to examine whether folate - conjugated imaging agents could selectively detect inflamed arthritic joints . First, arthritic dogs were treated with a folate radiotracer, tc - ec20 (fig . 1c), to image expected sites of inflammation, and dramatic uptake of tc - ec20 into arthritic joints was observed, whereas normal control dogs displayed background levels of radioactivity (30). Subsequent imaging studies were conducted in adjuvant - induced arthritic rats, and tc - ec20 was used for imaging inflamed arthritic tissues . The resulting images revealed accumulation of tc - ec20 in inflamed joints, livers, and spleens of arthritic rats, but not in those of healthy control rats (26). Importantly, treatment of the arthritic rats with excess free folic acid completely blocked the uptake of tc - ec20 into their joints and organs, strongly indicating that uptake of tc - ec20 is dominantly fr - mediated (26). Depletion of macrophages from arthritic rats with liposomal clodronate treatment also blocked the uptake of tc - ec20, and the binding of folate - conjugated agents was observed only in macrophages in the total cell populations obtained from the livers of atherosclerotic mice (31). An autoradiography study revealed that tc - ec20 was much more accumulated in the atherosclerotic lesions of apoe-/- mice fed on a high fat diet than their counterparts fed on a normal diet (31). Folate - conjugated fluorescent dyes and radioactive agents specifically targeted asthmatic lung macrophages in a murine asthma model, whereas little uptake by macrophages presented in healthy lung tissue (32). These observations reveal that only fr - expressing macrophages are selective targets for folate - conjugated imaging agents in animal models of human inflammatory diseases . Fr - targeted imaging using folate radiopharmaceuticals was also conducted in human patients suffering from both cancer and inflammatory disease . A patent with ovarian cancer was administered folate - diethylenetriaminepentaacetic acid (dtpa)-in to detect cancer tissues, and uptake was observed within the ovarian cancer tissues (33). Uptake of folate - dtpa - in and tc - ec20 was also detected in inflamed knee joints of a patient suffering from osteoarthritis and rheumatoid arthritis, whereas no uptake was observed in healthy knees (13435). These fr - targeted in vivo imaging studies of macrophage - mediated inflammatory diseases in animal models and human patients are summarized in table i, and all data strongly support the idea that folate - conjugated imaging agents can selectively target fr - positive activated macrophages in inflamed tissues . Furthermore, this fr - targeted imaging of activated macrophages could also be applied to other macrophage - mediated inflammatory diseases . Based on successful fr - targeted imaging of cancers and activated macrophages in inflamed tissues using folate - conjugated imaging agents, folate - drug conjugates were designed and used to treat cancers and inflammatory diseases . Initially, conjugation of conventional chemotherapeutic agents to folic acid was regarded as a good strategy for fr - targeted therapy of human diseases . However, many chemotherapeutic agents are hydrophobic, and hydrophobic chemotherapeutic conjugates exert considerably less target cell specificity than watersoluble ones, therefore, hydrophilic linkers are required to connect folic acid and its chemotherapeutic cargo (36). In addition, it is critical to conjugate the therapeutic agents to folic acid without decreasing the affinity of folic acid for its folate receptor . Most importantly, designing linkers that release free drug inside the cells after fr - mediated endocytosis is a major challenge limiting the therapeutic efficacy of folate - drug conjugates (36). With these guidelines, it is clear that an alternative strategy must be developed to improve the potential of fr - targeted therapy of diseases . Consequently, a new strategy named fr - targeted immunotherapy, which minimizes the limitations mentioned above, was developed to treat human diseases (fig . A highly immunogenic, low molecular weight hapten linked to folate beautifully decorates fr positive cancer cells or activated macrophages, leading to rapid elimination of these targeted cells by macrophages, nk cells, and complement components in the body's immune system through antibody - dependent cellular cytotoxicity (adcc) and complement - dependent cellular cytotoxicity (cdc) (fig . 2). Given the therapeutic efficacy of fr - targeted immunotherapy against cancers (37383940), immunotherapy using folate - hapten conjugates was also applied for the targeting of activated macrophages to treat several animal models of inflammatory diseases . Adjuvant- or collagen - induced arthritic rodents, previously immunized with a hapten (fitc), were treated with fr - targeted immunotherapy, resulting in the alleviation of arthritic symptoms by eliminating fr - positive activated macrophages in the inflamed lesions of the rodents (41). Fr - targeted immunotherapy also showed therapeutic effects on systemic lupus erythematosus in a murine disease model by alleviating disease symptoms and extending the life span of treated animals (42). Various folate - hapten conjugates were further used for fr - targeted immunotherapy of inflammatory diseases . Initial results with a folate - fitc conjugate showed a good therapeutic effect in animal models of rheumatoid arthritis and systemic lupus erythematosus (4142). Folate conjugates of dinitrophenyl (dnp) and trinitrophenyl (tnp) were used for fr - targeted immunotherapy of rheumatoid arthritis in a rodent animal model, and these folate - hapten conjugates exerted therapeutic effects (43). Fr - targeted recombinant immunotoxin was also used for fr - targeted immunotherapy of atherosclerosis . Administration of atherosclerotic apoe-/- mice with recombinant immunotoxin targeted fr - positive activated macrophages and alleviated the symptoms of atherosclerosis in the animal model (44). One concern that could be raised is whether fr - targeted immunotherapy eliminates the macrophage population in our body during treatment, resulting in significant loss of the body's immunity against pathogen infection . However, the conjugates used for the studies of fr - targeted immunotherapy mentioned above were well - tolerated by all animals, and they did not show any symptoms of pathogen infection or side effects during the immunotherapy . This strongly suggests that the possibility of the destruction of the body's immune system during fr - targeted immunotherapy is negligible, and fr - targeted immunotherapy effectively removes only fr - positive activated macrophages that play a critical role in the pathogenesis of inflammatory diseases . Given that therapeutic fr - targeted immunotherapy was successful for the treatment of macrophage - mediated inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and atherosclerosis (table i), this strategy is promising and could be further applied for the treatment of other types of macrophage - mediated inflammatory diseases . Inflammation is essential for host defense against pathogen invasion; however, uncontrolled or repeated chronic inflammation leads to pathogenic conditions and diseases such as inflammatory diseases and cancers (4546474849). Macrophages are critical immune cells to initiate inflammatory responses (50), and increasing numbers of studies have successfully proven that activated macrophages, which express high levels of fr-, are found in the inflamed tissues of a variety of inflammatory diseases and are actively involved in the pathogenesis of these diseases . This strongly suggests that selective targeting of frs using folate conjugated imaging or therapeutic agents could be a good strategy for the diagnosis, as well as therapeutic treatment, of macrophage - mediated inflammatory diseases . In spite of these successful studies proving the utility of frs as diagnostic and therapeutic targets for inflammatory diseases, moreover, few therapeutic drugs developed based on the strategy of selective fr targeting have been reported so far for these diseases, which raises the necessity of developing new potential drugs targeting frs with strong efficacies and minimal toxicities . In conclusion, selective targeting of frs, especially fr- on activated macrophages, could be a promising strategy for the diagnosis and treatment of macrophage - mediated inflammatory diseases, and there will be a high demand for the development of fr - targeted efficacious and safe therapeutics.
Fulvestrant (faslodex; astrazeneca) is a novel steroidal estrogen receptor (er) antagonist lacking agonist effects . By covalent binding to the er, receptors are rapidly downregulated, resulting in a decrease of cellular er levels and complete abrogation of estrogen - sensitive gene transcription . The efficacy of fulvestrant in patients with tamoxifen - resistant disease [2, 3] and in patients progressing on aromatase inhibitors has been confirmed, thus giving the opportunity to extend the overall period during which hormonal agents may be used in women with endocrine - responsive advanced breast cancer . Two recent clinical trials are focusing on fulvestrant high - dose regimens (500 mg monthly) to optimize fulvestrant treatment . The neoadjuvant newest trial showed significantly greater biological activity benefit with high - dose fulvestrant compared to the approved dose . The randomized, double - blind confirm (comparison of faslodex in recurrent or metastatic breast cancer) trial presented recently has shown a small but statistically significant increase in time to progression for fulvestrant 500 mg compared with fulvestrant 250 mg, without increase of toxicity . We report a case achieving long - term complete remission under fulvestrant introduced after no or poor response to prior endocrine therapies, in whom the most efficacious dosing regimen was below the approved dose . In january 1993, a 47-year - old, premenopausal woman was diagnosed with a left breast cancer (pt1c(m)pn0m0) after bilateral subcutaneous mastectomy for relapsing fibroadenomas . The tumor was er - positive (90%), pgr - positive (70%) as evaluated by immunohistochemistry and had a low - level her2-gene amplification ratio of 2.22 (positive> 2.20) as assessed in 2007 . Axillary dissection was carried out followed by radiotherapy (50 gy total dose), but no adjuvant endocrine therapy was given . In january 1998, distant recurrence was diagnosed in soft tissue (er - negative, pgr - positive by histological assessment), in bone, pleura and lung (lymphangiosis carcinomatosa). Anastrozole was started as 1st - line endocrine treatment in a clinical trial, but discontinued after 3 months due to progression in pleura / lung and in the contralateral breast . 1). Six cycles of chemotherapy with paclitaxel (175 mg / m) and doxorubicin (60 mg / m), every 21 days (eortc 10961), were given, followed by monthly pamidronate for 8 months . Partial remission for 10 months was achieved with normalized chest x - ray and ca 15 - 3 . In june 1999 she received 9 months of tamoxifen as 2nd - line endocrine treatment, which resulted in stable disease for 6 months prior to progression (ca 15 - 3 re - rising). Exemestane was started as 3rd - line endocrine treatment, but discontinued 14 days later due to severe allergic exanthema . In march 2001, cytological assessment of a further progression in the contralateral breast revealed an er - positive, pgr - negative tumor and fulvestrant (250 mg intramuscular, monthly) was initiated as 4th - line endocrine treatment . As the drug had not yet been licensed in switzerland at that time, fulvestrant was initially provided free of charge through a named patient program (supported by astrazeneca). Twelve months later complete remission was achieved (normal cervical / thoracic / abdominal computed tomography scan, disappearance of previous tracer uptake in bone scan). However, the patient experienced painful oral and vaginal dryness with loss of libido, ulcerative stomato - pharyngitis and an extensive thrombophlebitis of the left arm requiring temporary treatment interruptions . Symptomatic treatment was given in different combinations, but mucosal dryness and inflammation became an ongoing problem diminishing her quality of life . Therefore, in february 2004, we decided to continue fulvestrant at half dose (125 mg monthly). Mucosal symptoms improved; however, when fulvestrant was once administered in full dose by error, painful oral and vaginal mucositis reoccurred . In june 2006, complete remission was reconfirmed and fulvestrant was tentatively stopped 63 months after the 1st injection . However, complete remission was lost shortly thereafter (ca 15 - 3 re - rising, small suspicious pleura effusion), but re - achieved 10 months after renewal of fulvestrant (125 mg monthly) (fig ., we finally decided to determine the patient's fulvestrant plasma concentrations under the 125-mg dose by the high - performance liquid chromatography (hplc) method in the standard lab for fulvestrant plasma sample analyses . The maximum concentration detected was even below the predicted level (table 1). At follow - up in september 2008 thereafter, ca 15 - 3 ranged between upper normal limit (unl) and 1.3 unl, but so far progression of disease has not been confirmed on restaging . At the last follow - up in march 2010, the patient was doing well . As observed in several clinical trials and case studies, some patients experience prolonged duration of response with fulvestrant treatment for endocrine - responsive advanced breast cancer [3, 8, 9]. To our knowledge, a 9-years response to 4th - line endocrine treatment with fulvestrant has not been reported in the literature so far . This case is remarkable in several aspects: first, achievement of long - lasting complete remission (last reconfirmed 90 months after treatment start) is very rare, especially after no or poor response to prior anastrozole / tamoxifen, obviously both not predictive for the efficacy of fulvestrant in this patient . Second, reducing the dose of fulvestrant due to unanticipated intensity of mucosal toxicity maintained complete remission in this case . We think it is important to alert oncologists to the fact that the prior response to an aromatase inhibitor does not appear to be predictive for the benefit of fulvestrant, an observation also made in a phase ii trial investigating fulvestrant in patients with primary or acquired aromatase inhibitor - resistance . Abram et al . Presented a case with endocrine - responsive advanced breast cancer, which also appeared to respond better to fulvestrant than to prior endocrine therapies . They assumed that the intramuscular administration by a health care professional may have resulted in improved compliance . A belgian study found a greater activity of fulvestrant in tumors expressing both er and pgr . Other investigators found fulvestrant activity not being reduced in pgr - negative tumors . In our case, hormone receptor status was er - positive / pgr - negative when repeated prior to start of fulvestrant treatment, though the primary tumor was er - and pgr - positive . Pgr expression within the tumor is known to be patchy and a sampling error might have led to the er - positive, pgr - negative result in the immunocytochemical analysis of the fine needle aspiration material . Tumors for which fulvestrant therapy might be particularly appropriate are those er - positive tumors with her2 overexpression assuming ligand - independent er activation seems to be a key feature . However, available data are conflicting . In our case, her2 amplification with a ratio of 2.22 was low and unlikely to contribute to the superiority of fulvestrant compared to anastrozole and tamoxifen by the above - mentioned mechanism . Several large trials have shown that fulvestrant is well tolerated [2, 3, 4, 14]. Our patient experienced unusually severe mucosal toxicity together with complete remission under the approved 250-mg dose, but clearly less toxicity while maintaining complete remission under a 125-mg dose . The pharmacokinetic profile excluded faster absorption or a variation in drug metabolism which theoretically could have contributed to the observed toxicity and efficacy (table 1). Her fulvestrant plasma concentrations were below the observed mean for a 250-mg dose and, importantly, even below the predicted mean for a 125-mg dose . We therefore hypothesize an extraordinary sensitivity, but not a metabolic phenomenon as a key factor in the observations made in this case . This observation is of particular interest in the ongoing fulvestrant dosing debate: our case might serve as a basis for future strategies investigating individual dosing as a promising approach for optimizing fulvestrant treatment.
The lateral process of the talus is prone to fracture either as an isolated event or in conjunction with other ankle or talar injuries . Lateral process of the talus fractures are typically caused by axial loading with elements of dorsiflexion and eversion or inversion, and have been identified to have a high prevalence in snowboarders as a result of the particular stresses put on the foot and ankle in the boot - binding complex . Hawkins divided the fractures into 3 groups: a nonarticular avulsion, a single large fragment involving both the talofibular articulation and the subtalar joint, and a comminuted fracture fragment . Displacement of the fragment markedly increases the chance of nonunion or malunions with subsequent degenerative changes of the subtalar joint and pain in the sinus tarsi . Stress radiography has been recommended to quantify subtalar and tibiotalar instability . In langer et al's study, he reveals that there is a general acceptance defining ankle and subtalar joint stability . Using lateral, anteroposterior (ap), and 30-degree brodn view, it has been accepted that a 3-mm increase in anterior tibiotalar translation, 3-degree increase in tibiotalar tilt, a 5-mm increase in medial talocalcaneal motion, and> 5-degree increase in talocalcaneal tilt define instability of the ankle and subtalar joints, respectively . Despite appropriate treatment, whether conservative, open reduction internal fixation, or fragment excision, the size of the fragment and degree of displacement appear to be the critical factors in determining treatment . Less than 1 cm and undisplaced fragments are managed conservatively . For comminuted fragments, most authors recommend primary surgical excision to avoid development of arthritic changes in the subtalar joint . Displaced (> 2 mm) and> 1 cm single fragments offer a range of treatment options, from closed reduction to cast immobilization, open reduction internal fixation, and fragment excision . The objective of this study was to biomechanically assess the effect of a simulated large lateral talar process excision (up to 10 cm) on ankle and subtalar joint stability and ankle pressures . Seven fresh - frozen cadaveric lower legs (2 left, 5 right) were thawed for 24 h before experimental preparation . Radiographic evaluation was used to exclude specimens with bony deformities, prior trauma, or arthritis . Ranges of motion of at least 10-degree dorsiflexion and 20-degree plantarflexion in the ankle joint and at least 5-degree eversion and inversion in the subtalar joint were verified using a goniometer . A minimum of 6 specimens was required in this study as a sample size to achieve statistical power of 0.8 at a level of significance 0.05 . This calculation was based on data published by langer et al for the general acceptance that a 3-degree increase in tibiotalar tilt defines instability of the ankle joint . In addition, an expected 2-degree standard deviation of the tibiotalar tilt within the sample was considered . A vertical approach to the anterior and posterior ankle was performed . Transverse anterior and posterior arthrotomies were performed to allow access for insertion of a pressure sensor into the ankle joint . For radiographic analysis, 3 parallel 8-cm kirschner wires (k - wires) were laterally placed in the anterior distal tibia, anterior talar dome, and calcaneus (just below the subtalar joint). Each specimen was then fixed with molded polymethylmethacrylate (pmma; beracryl, suter kunststoff ag, jegenstorf, switzerland). The proximal 5 cm of the tibia and fibula were fixed in a pmma block . Plantarly, a steinman pin was inserted transversely into the plantar aspect of the calcaneus and was used to augment the fixation of the foot into the pmma molding . The hindfoot was held in the pmma molding in 5 degree of valgus to simulate normal plantegrade position . A custom - made seesaw rig was designed to simulate inversion / eversion stress loading forces and allow for radiographic and pressure analysis . The rig secures the proximal leg while allowing the distal pmma base of the foot to rotate on a platform (figure 1). Because the seesaw rig only allows inversion / eversion motion, the foot was oriented in 10-degree external rotation in relation to the sagittal plane to allow additional plantar and dorsiflexion, therefore simulating hind foot supination and pronation, respectively . (a) (top - left): fresh - frozen cadaver specimen placed in the seesaw test rig setup . The configuration allows for full radiographic assesment with inversion and eversion forces to the specimen . (b) (top - right): specimen is fixed proximally with pmma block, and plantarly in its pmma form to the sliding pivoting platform . K - wires are shown in the tibia, talus, and calcaneus, which serve as radiographic markers for biomechanical anaysis . (c) (bottom - left) and (d) (bottom - right): specimen in seesaw test rig with everted and inverted stress load of 10 kg with hanging weights . Pmma = polymethylmethacrylate . For ankle joint pressure and force measurements, pressure sensors (model #5033, tekscan inc, south boston, usa) were used . The calibration resulted in an approximate saturation pressure of 16 bars and the total matrix area was 1025 mm (46 32 sensels, 38.4 mm 26.7 mm) resulting in a spatial resolution of 0.696 mm per sensel . Pressure and force measurements of the tibiotalar joint were obtained before and after loading the ankle with an axial force while the foot was placed horizontally in the custom seesaw rig with the sliding elements of the rig in a locked position (figure 2). For ankle joint pressure and force measurements, static axial compression was increased from a fixed preload of 2 to 30 kg, according to half body weight . Maximum load was held for 6 s. load and contact pressure distribution was captured at 30 hz . The location and shift of the center of force were determined from the pressure sensor measurements . For data recording, a sensor software package (i - scan, tekscan inc, south boston, usa) was used . Subsequent data evaluation was performed using custom - made software based on matlab (mathworks inc ., test setup showing a specimen placed in the custom seesaw rig for ankle joint pressure and force measurements . Pressure sensors were inserted into the ankle joint in an anterior to posterior direction, being fixed with thumbtacks . Static axial compression was increased from a fixed 2-kg preload to 30 kg, according to half body weight . After analysis of the pressure sensor data, the following parameters were analyzed: peak force (n), center of force movement (mm), contact area (mm), and contact area pressure (bar). Peak force was defined as the maximum pressure at the highest loaded area (2 2 sensel) at 30-kg load . Center of force movement was defined as the length vector between initial and final center of force positions at 2- and 30-kg loading, respectively . Contact area was defined as final total area of the sensor covered by the tibiotalar joint surfaces at 30-kg load . Contact area pressure was defined as the average pressure of the final contact area of the sensor at 30-kg load . Tibiotalar pressure measurements were repeated after each consecutive fragment excision . For excision of the lateral process of the talus, we used a modified ollier approach, centered distal and anterior to the tip of the fibula . Initially, in the pre - excision group, soft tissue dissection was performed, but no osteotomy was carried out . Careful attention was paid to ensure that the lateral ligamentous complex was not compromised during the surgical dissection . A 1-cm osteotome (depuy synthes, west chester, usa) was used to osteotomize the lateral process of the talus . In the 5-cm excision step, 3 cuts were made relative to the apex of the lateral talar process and subtalar joint (figure 3). The apex of the lateral talar process was determined to be the most inferior point on the talus just above the talocalcaneal articulation and the most lateral aspect of the talus . Cuts were made superior and parallel, medial and perpendicular, and posterior and perpendicular . For final fragment excision of 10 cm, additional cuts were made medially, superiorly, and posteriorly next to the previous bony defect to simulate the next size fracture fragment . Lateral process of the talus after excision of a 5-cm fragment, using a modified ollier approach . Three cuts were made relative to the apex of the lateral talar process and subtalar joint . Ap radiographs were taken to assess tibiotalar tilt (tt) and subtalar joint tilt (stjt). Twenty - five - degree brodn views were taken to evaluate the talocalcaneal tilt (tct). The radiographs were taken in neutral hindfoot alignment, forced inversion and forced eversion with a 10-kg weight, which was attached to the rotating rig platform . The cortical surfaces of the tibia and talus were used for measurement of the tibiotalar tilt angle changes from neutral alignment to inversion and eversion respectively, as demonstrated on the ap radiograph (figure 4 a, b, c). The k - wires that were inserted into the talus and calcaneus were used as reference markers for indirect measurements of the subtalar joint angle changes as demonstrated on the ap radiograph (figure 4 d, e, f). The talocalcaneal tilt was calculated using a 25-degree brodn view measuring the angulated difference created by lines drawn along the cortical surfaces of the talus and calcaneus (figure 4 g, h, i). The data was analyzed to calculate the mean angle change from neutral alignment to forced inversion and eversion with respect to tt, stjt, and tct . Pre - excision radiographs were analyzed versus 5-cm fragment excision and 10-cm fragment excision of the lateral talar process . Measurements were taken of the angle between lines drawn through cortical surfaces or k - wire markers . (a) non - stress (neutral hindfoot alignment) ap view showing measurement lines of tt; (b) inversion stress ap view showing measurement lines of tt with 10-kg load; (c) eversion stress ap view showing measurement lines of tt with 10-kg load; (d) non - stress ap view showing measurement lines of stjt; (e) inversion stress ap view showing measurement lines of stjt with 10-kg load; (f) eversion stress ap view showing measurement lines of stjt with 10-kg load . (g) non - stress brodn view showing measurement lines of tct; (h) inversion stress brden view showing measurement lines of tct with 10-kg load; (i) eversion stress brodn view showing measurement lines of tct with 10-kg load . Ap = anteroposterior, stjt = subtalar joint tilt, tct = talocalcaneal tilt, tt = tibiotalar tilt . Statistical analysis was done using spss statistical software (ibm spss, chicago, il, usa). After having analyzed all parameters of interest for normal distribution applying the shapiro - wilk test, we used paired - sample t tests to compare pre - excision values to 5-cm excision and 10-cm excision values . P values were adjusted according to the bonferroni correction . Level of significance was set to p = .05 for all statistical tests . One specimen was excluded from the study due to fracture of the distal fibula during forced inversion . Table 1 illustrates the mean standard deviation angle changes from neutral hindfoot to forced inversion with 10-kg load in the pre - excision (control), 5-cm excision (post1), and 10-cm excision (post2) phases . Average tt angle increased from 2.32 1.40 (control) to 3.22 1.90 degree (post1) and 5.30 2.04 degree (post2). Evaluation of stjt revealed an average angle of 10.88 3.41 degree for the control group, 11.85 4.26 degree in post1 and 11.43 6.90 degree in post2 . A continuous increase of the average tct angle was observed between the excision states with an initial angle of 1.65 1.18 degree (control), further increasing to 2.50 1.64 degree (post1) and finally reaching 4.10 2.45 degree (post2). Angle changes in terms of mean value standard deviation, representing the tibiotalar tilt, subtalar joint tilt and talocalcaneal joint tilt of 6 specimens from neutral hindfoot alignment to forced inversion with 10-kg load in pre - excision, 5 cm excision and 10 cm excision phases table 2 illustrates the angle changes from neutral hindfoot to forced eversion with 10-kg load in the pre - excision, 5-cm excision, and 10-cm excision phases . A drop in the average tt angle from 0.92 0.64 (control) to 0.77 1.31 degree (post1) was observed while remaining at the same level in the post2 phase with 0.77 0.46 degree . Furthermore, the average stjt angle ascended steadily from 3.33 3.88 (control) to 3.72 4.37 degree (post1) and reached finally 3.80 2.84 degree in post2 . However, the average tct angle dropped initially from 0.58 0.53 (control) to 0.52 0.55 degree (post1) and then leveled up again to 0.58 0.33 degree in post2 . Angle changes in terms of mean value standard deviation, representing the tibiotalar tilt, subtalar joint tilt and talocalcaneal joint tilt of 6 specimens from neutral hindfoot alignment to forced eversion with 10-kg load in pre - excision, 5-cm excision and 10-cm excision phases table 3 illustrates the peak force, center of force movement, contact area, and contact area pressure of the tibiotalar joint after a 30-kg axial load application . Peak force declined from 3.92 0.29 (control) to 3.66 0.23 n (post1), but overreached the initial level after post2 revealing 4.16 0.45 n. thereby, center of force movement inclined steadily from 2.24 0.50 (control) to 2.38 1.21 mm (post1), measuring ultimately 3.32 0.96 mm (post2). The contact area initially rose from 347.67 80.63 (control) to 417.67 112.32 mm (post1), and settled down to 404.50 111.09 mm (post2). Similarly, the contact area pressure revealed initially 4.37 0.82 bar (control), which increased to 4.85 0.77 bar in post1 and finally dropped to 4.74 0.89 bar in post2 . Peak force, center of force movement, contact area and contact area pressure in terms of mean value standard deviation, as defined from the pressure measurements in the ankle joint of 6 specimens under axial load in pre - excision, 5-cm and 10-cm excision phases with respect to forced inversion, the only statistical significance was seen in tt angle change from neutral hindfoot alignment to forced inversion between pre - excision and 10-cm excision (p = .04, table 1). There were no significant differences in the stjt angle changes from neutral hindfoot alignment to forced inversion in both 5-cm and 10-cm excisions in comparison with control group . A trend was observed in the tct angle changes when inversion was forced between control and post1 (p = .08), respectively between control and post2 (p = .09). In view of forced eversion, there were no significant differences in the tt, stjt, and tct angle changes in both 5-cm and 10-cm excisions as compared with pre - excision (table 2). Referring to the ankle joint pressure and force measurements, there was no significant difference in each of the analyzed parameters: peak force, center of force movement, contact area and contact area pressure in both 5 and 10-cm excisions as compared with control group (table 3). Our biomechanical study demonstrates that excision of large 5 and 10-cm fragments of the lateral process of the talus does not produce significant instability in the subtalar joint . With respect to the ankle joint, a 10-cm fragment excision alone did produce a statistically significant tibiotalar tilt in inversion (p = .04) in comparison with the pre - excision phase . Regarding the compression forces at the ankle joint level, following the excision, no significant changes in peak force, center of force movement, contact area, and contact area pressure similar results have been shown supporting subtalar stability for smaller 1-cm fragments in previous studies, as they demonstrated that instability did not occur until 100% of the footprint of the lateral talocalcaneal ligament (ltcl) origin and approximately 15% of the anterior talofibular ligament (atfl) and posterior talofibular ligament (ptfl) footprints were reduced . As the ltcl is the only of these ligaments to cross the subtalar joint, it can be expected that additional resection of the atfl and the ptfl would not result in any further subtalar instability, given that the interosseous ligament remains intact . Based on several anatomical and also biomechanical studies, it has been shown that the talocalcaneal interosseous ligament is the greatest contributor to subtalar joint instability . By resecting a large fragment of the lateral process clinically, only a resection of a larger fragment in case of an additional subtalar dislocation would lead to subtalar instability . Not a single report in the literature has described ever the combination of a subtalar joint dislocation together with an isolated fracture of the lateral process of the talus . Another reason that the subtalar joint remained stable is the fact that the calcaneofibular ligament remained intact . It has been previously shown that this ligament contributes significantly to the stability of the subtalar joint . The stability was not maintained when evaluating the ankle joint; we measured a significant increase in the tibiotalar angle during inversion stress when resecting 10 cm but not 5 cm . By increasing the resection area, several studies have identified the atfl as the most stabilizing ligament with the foot in neutral position . It can be inferred that if the atfl and the ptfl are removed as part of the fracture and the ankle joint becomes unstable under an inversion stress, these ligaments must also have an varus stabilizing effect . We believe that the remaining stability under valgus stress at the level of the ankle joint can be explained by the fact that the integrity of the talar dome is not compromised during resection of the lateral talar process fragment, and the medial deltoid ligamentous complex is substantial . We chose to excise 5 and 10 cm to test the largest possible fragment that one could take without causing a talar body fracture extending into the talar dome . In a clinical setting in which one has a large comminuted fracture with the lateral process of the talus, we propose that excision with careful soft tissue dissection would not lead to ankle or subtalar instability and moreover, that forces acting on the ankle would not significantly be changed . We are unable to comment on the forces seen at the subtalar joint with this study design, and a large lateral talar process excision may contribute to joint pressure change, which could predispose to subtalar arthorsis . Of course, in the clinical setting, large fragment excision in the lower extremity needs more clinically relevant studies before application in common practice . One major limitation of the current study is that we could not measure the forces in the subtalar joint . This is mainly due to the fact that extensive medial dissection would have to be performed to insert the sensor, which would likely lead to ankle and subtalar instability . However, it could be extrapolated that, due to the axial configuration of the tibia, talus, calcaneus, and ground reaction force, pressure stability in the ankle joint implies subtalar joint stability as well . Contributing to this deduction is the fact that our test setup with the pmma foot form locks the forefoot to the hindfoot only allowing ankle and subtalar joint motion . It could have potentially happened that the approach has compromised surrounding tissue stabilizing both the ankle and subtalar joint . An anterior drawer pre - test with resected lateral talar process, independent of the size of the fragment, resulted in a complete anterior dislocation of the ankle joint at 10-kg load because the majority of the stabilizing fibers of the talus against an anterior shift (atfl and lctl) were dissected together with the resected fragment . During the complete dislocation, the remaining intact fibers of the atfl and ltcl were completely ruptured and we were not able to use these specimens for any further tests . We therefore elected to discontinue the anterior drawer test . Measuring angles on stress radiographs to investigate ankle and however, ankle and subtalar motion is a complex 3-dimensional process and taking a stress radiograph in a single plane under a uniform stress might be insufficient . Finally, we strongly acknowledge that our ex - vivo investigations were with limitations inherent to all cadaveric studies, with limited number of fresh - frozen human cadaveric lower legs and variability between the specimens that cannot truly mimic the in - vivo situation . The cadaveric limbs were embedded at the level of the mid tibia and tendon actuators were not incorporated in the study design . We attempted to account for any affect this might have had by performing the intact measurements after the specimens had been embedded, to mimic the study conditions . We were able to show that an excision of up to 10 cm of the lateral talar process does not cause a significant instability at the level of the subtalar joint but might be a destabilizing factor at the ankle joint under inversion stress . Further studies are required to determine the optimal treatment of ligament reconstruction and/or refixation of the detached ligaments following resection of a large lateral talar process fragment and its implication on late ankle osteoarthritis.

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