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Stevens johnson syndrome (sjs) is a severe adverse drug reaction that can result in disability and mortality . Sjs is defined as having a widespread distribution throughout the whole body surface area with <10% extent of skin detachment and skin lesions . Some drugs, such as carbamazepine, have been reported to have a greater correlation to sjs . Although clozapine use has been mentioned as a risk factor for development of sjs, no report has clearly described the features of sjs as a reaction to clozapine use . Herein, we report the case of a patient presenting sjs after long - term clozapine treatment . He was hospitalized in a mental institute and received clozapine 200 mg / day for 2 years, without discomfort or drug side effects . He developed acute - onset mouth edema, multiple oral and ocular ulcers, oral and ocular mucosa swelling, and multiple erythematous skin rashes over his entire body and extremities with hypertension and high fever . Accumulated lymphocytes and macrophages in the epidermis and elevated tnf- might cause an immune reaction and apoptosis and result in the clinical presentation of sjs . Clozapine is believed to modulate the immunologic reaction, and therefore might induce sjs through immunomodulation . This case highlights the importance of considering the possibility of sjs resulting from the use of drugs for which there are no reports of such a severe complication . There have been many reports and articles discussing skin eruptions due to different classes of medication.14 these reactions result in different presentations, based on severity, from the simplest urticaria to the most severe reaction, stevens johnson syndrome (sjs).5 sjs is a rare but severe adverse drug reaction with a cutaneous presentation, and can progress rapidly and even result in disability and mortality.6,7 sjs is considered to be a spectrum disease, a group that consists of sjs, erythema multiforme, and toxic epidermal necrolysis.8 in a report by roujeau, sjs was defined as having a widespread distribution with <10% extent of skin detachment and skin lesions including macules, blisters, flat atypical targets, and mucosal lesions.8 it also consists of some nonspecific symptoms and signs, such as occasional high fever, arthralgia, and general weakness . When affected, patients enter the most severe phase rapidly and may develop severe complications, such as septic shock.9 mortality ranges widely according to different studies.5,10 the etiology of sjs is unclear and idiosyncratic . Some drugs, such as carbamazepine and phenytoin, have been reported to have a greater correlation to sjs than others.9 clozapine is one of the most widely used atypical antipsychotics in current clinical practice . Despite its excellent effect on refractory psychosis, it has numerous side effect profiles,11 which include leukocytosis, hypersalivation, sedation, pneumonia, electrolyte imbalance, seizure, agranulocytosis, and its most severe complication, neuroleptic malignant syndrome . Although clozapine has been mentioned as a risk factor for the development of sjs,12 there is no report clearly describing the features of sjs in patients with clozapine use . Herein, we report the case of a patient presenting sjs after long - term clozapine treatment . Mr a was a 54-year - old male who had been diagnosed as having chronic schizophrenia, paranoid type . The baseline laboratory data when hospitalized revealed no significant abnormality, and the routine chest x - ray and electrocardiography showed no significant finding . He had received clozapine 200 mg / day for 2 years without discomfort or drug side effects . Two weeks before the event of sjs, he developed acute gastroenteritis with nausea and vomiting, and recovered from it spontaneously . However, he developed an acute onset of mouth edema, oral and ocular multiple ulcers, injected oral and ocular mucosa, and multiple erythematous skin rashes over his entire body and extremities . When the event of sjs came, he only took clozapine 200 mg daily without any other concomitant drugs . His vital signs revealed hypertension, 153/101, with a high fever of 39.1c . Because of the emergent condition, he was soon referred to a general hospital for intensive care, where sjs was diagnosed . The laboratory data at that time revealed acute inflammation with elevated c - reactive protein (143.2 mg / l) and leukocytosis (1.4710/l). After supportive treatment, his critical condition subsided and he returned to our hospital 1 month later . The follow - up laboratory data 1 month later revealed gradually improved leukocytosis . His psychotic symptoms were well controlled under risperidone 3 mg / day, which was prescribed after this event subsided . To the best of our knowledge, this is the first report to describe in detail the whole course of sjs in a patient with long - term clozapine use . Our patient fulfilled most of the diagnostic criteria of sjs found in different reports.7,8 besides, according to the naranjo algorithm, the probability of adverse drug reactions by clozapine is highly probable, with a score of 6.13 he recovered smoothly under supportive treatment, and had no sequelae . Some researchers have focused on the relationship between hla - b*1502 and sjs with carbamazepine use.14 however, this finding could be replicated only in han chinese subjects receiving carbamazepine; in japanese and korean subjects receiving carbamazepine, different hla - b families are found to be related to sjs.15,16 at the present time, most published reports are focused on the relationship between hla - b families and antiepileptic drugs only.17,18 although some reports suggest that genetic screening techniques should be devised to screen for the risk of sjs before prescribing such drugs for patients with either epilepsy19,20 or mood disorder,21 another report has tried to investigate the implication of this pharmacogenetics technique in terms of other drugs, but this seems not implacable to be promoted to other drugs at the present time.22 however, the pathologic mechanism of sjs is still poorly established . In pathological reports, accumulated cd t lymphocytes and macrophages (eg, langerhans cells) in the epidermis23,24 and tnf- elevated by macrophages25 might cause a cytotoxic cellular immune reaction and apoptosis in the epidermis and result in the clinical presentation of sjs.9 clozapine actually is believed to elevate plasma tnf- levels.26,27 besides, in another report, clozapine revealed its ability to modulate the immunologic reaction, including that of il-1, il-8, monocyte chemotactic protein (mcp-1), and nf - kb1.28 therefore, clozapine might, at least partially, induce sjs through its potential ability of immunomodulation . This case highlights the importance of monitoring and considering the possibility of sjs as a reaction to drugs for which there are no previous reports of such severe complications.
Small bowel diverticuli are encountered most frequently in the duodenum, however complicated and symptomatic diverticuli occur most commonly in the jejunoileal region . Small bowel diverticuli are pulsion lesions acquired by herniation of mucosa and submucosa through a weak muscularis layer, hence forming a false diverticulum diagnosis of small bowel diverticuli is often made during diagnostic or therapeutic interventions for unrelated issues . While a small bowel barium study and enteroclysis are optimal diagnostic modalities for this disease, a computed tomography (ct) scan of the abdomen and pelvis is most ideal to identify the complications of small bowel diverticuli, such as formation of an abscess as was seen in our patient . A 25-year - old african american female with no significant past medical or surgical history presented with moderate to severe abdominal pain and fever (tmax 39.1c) of three days duration . She experienced a single episode of bilious vomiting, but denied any other obstructive symptoms . Vital signs were normal . On physical exam, her abdomen was soft but diffusely tender with rebound and guarding . Laboratory findings revealed leukocytosis (wbc 13,600/mm) and an elevated partial thromboplastin time (ptt 46). A pelvic ultrasound showed a small amount of free fluid in cul - de - sac, but no ovarian mass or uterine fibroids . A ct scan of the abdomen and pelvis (fig . 1) revealed a thickened jejunal wall with mild dilatation, several small adjacent enlarged mesenteric lymph nodes, and an air - fluid - containing structure measuring 1.9 2.9 cm in the left abdomen . The patient was taken to the operating room for an exploratory laparotomy . At exploration, diffuse serous fluid was encountered, and a segment of the proximal jejunum appeared erythematous and edematous . A large abscess cavity in the adjacent jejunal mesentery was also identified . Segmental resection of the involved jejunum and mesentery including the abscess cavity was performed followed by a side - to - side anastomosis . Post - operatively, the patient had an uneventful recovery and was discharged home on hospital day 7 . In a young female as the one described above, severe abdominal pain and tenderness with fever and leukocytosis, all of which suggest an acute abdomen, would draw a long list of differential diagnoses . The most common things would involve perforated appendicitis, ectopic pregnancy, pelvic inflammatory disease, tubo - ovarian abscess, or hemorrhagic or ruptured ovarian cysts . However, the ct scan image revealing an inflamed small bowel wall with an adjacent abscess was vital in determining the need for surgery . In retrospect, laparoscopy followed by segmental resection of the involved bowel and the abscess would have been preferable over open surgery . Small bowel diverticuli are encountered most frequently in the duodenum, but complications arise most often when they occur in the jejunoileal region . Small bowel diverticuli are false diverticuli, which means they involve only the mucosa, submucosa and serosa, excluding the muscularis layer . Both duodenal and jejunoileal diverticuli are acquired pulsion lesions that may clinically present in a vague and diverse manner, such as epigastric or periumbilical abdominal pain, satiety, bloating, constipation and diarrhea . Although colonic diverticular disease may be one of the extrarenal manifestations of autosomal dominant polycystic kidney disease (adpkd), only a single case of small bowel diverticuli in a chronically hemodialysed adpkd patient has been reported, and there is insufficient evidence to suggest an association between these two entities . Duodenal diverticuli are common and found in 2 - 5% of patients undergoing upper gastrointestinal series . Etiologically most duodenal diverticuli are thought to be acquired by herniation of the mucosa and submucosa through a wall defect caused by entrance of large vessels . Rarely duodenal diverticuli are intraluminal, which may be congenital resulting from incomplete intestinal lumen canalization . Clinically less than 10% of duodenal diverticuli are symptomatic with approximately 1% requiring definitive treatment . Rare complications of duodenal diverticuli include obstruction, perforation, bleeding and diverticulitis, and interestingly they are strongly associated with a high frequency of common bile duct stones . Jejunoileal diverticuli are found in 1 - 2% of the general population with most presenting during the sixth or seventh decade of life . Jejunoileal diverticuli are frequently associated with intestinal motility disorders such as progressive systemic sclerosis, visceral neuropathies or myopathies . Atrophy of the myopathic jejunal wall on one side and increased luminal pressure from the opposite leads to protrusion of small intestinal mucosa through defects in the lamina muscularis mucosae . Jejunoileal diverticuli are mostly asymptomatic, but malabsorption due to bacterial overgrowth within them may be a major clinical feature . They may also present in unusual ways, such as with a gastrointestinal hemorrhage due to a ruptured congenital arteriovenous malformation in the diverticular submucosal, perforation with abscess progressing into a jejuno- ileo- abdominal wall fistula formation, or episodes of bowel obstruction secondary to volvulus, stricture formation or an enterolith impaction . Jejunal diverticulitis occurs in about 2 - 6% of cases and has a mortality rate as high as 24% . Diagnosis of small bowel diverticuli is often incidental at endoscopic retrograde cholangiopancreatography, laparotomy, or double - balloon enteroscopy . Although endoscopy (capsule and double - balloon) may diagnose intraluminal abnormalities of the small bowel, its utility in emergency is limited . Barium follow - through and enteroclysis are the diagnostic modalities of choice for small bowel diverticuli . A ct scan of the abdomen and pelvis is most useful to identify abscess formation following diverticular perforation . Although non - specific, this finding may suggest jejunoileal diverticulitis amongst other differential diagnoses, namely neoplasms, focal crohn's disease, foreign body perforations, medication - induced ulcers and traumatic hematomas . In the absence of contraindications, diagnostic laparoscopy is a useful tool that may allow a thorough intra - abdominal surveillance to reach a precise diagnosis . Open or laparoscopic resection of the involved small bowel segment and primary anastomosis is indicated for jejunoileal diverticulitis, bowel perforation or bleeding [23, 24]. Approximately 15% of patients with jejunal diverticulosis may require small bowel resection for treatment of such complications . Unlike colonic diverticulitis, conservative management involving bowel rest and antibiotic administration is rarely successful . Macari et al . Have reported success in only one out of three patients with jejunal diverticulitis treated conservatively . Jejunoileal diverticulosis and its associated complications are rare . Its preoperative diagnosis is difficult, but should be considered in cases of unexplained abdominal symptoms and peritonitis . Given the high mortality rate of jejunal diverticulitis, a precise diagnosis followed by appropriate therapeutic intervention is essential . If suspected, diagnostic laparoscopy unless contraindicated seems to be an apt approach to confirm the diagnosis . Despite the successful outcome and uneventful recovery in our patient, we believe laparoscopic bowel resection may have been similarly beneficial in limiting the morbidity and recovery time associated with an open procedure . All the authors listed declare that there are no conflicts of interest and that they accepted no financial sponsorship in producing and presenting this article.
Data about the past 30-day use of cigarettes among us students in grades 6 to 12 were 9.2%, with 14.7% using 1 or more tobacco products . Adolescence is a critical time for prevention because 88% of initiation of tobacco use occurs before age 18 . Most young daily smokers become adult smokers, with half experiencing premature mortality from cigarette use . Proven strategies for preventing initiation and achieving cessation among adolescents are critical . Despite widespread knowledge about consequences of smoking, delivery of the advice barriers about addressing it have included insufficient clinician time and training, inadequate reimbursement, and lack of privacy because some parents wish to be present during interviews with patients . Perceived adolescents were unlikely to acknowledge smoking because of negative reactions from parents . In a national sample, two - thirds of adolescents at checkups the us preventive services task force described computer - delivered tobacco prevention programs during office visits may facilitate delivery of anticipatory guidance . If a new and innovative tool leveraged technology and minimized time when delivering preventive counseling about tobacco use, it could make a difference in helping adolescents to lead tobacco - free lives ., we compare 2 approaches for referring outpatients to a program delivered by the internet titled a smoking prevention interactive experience (aspire). Originally tested in a group randomized trial, aspire demonstrated a significant reduction in the initiation of tobacco use among participants presenting with the greatest number of risk factors for predicting uptake of smoking . E - mail link and involves e - mailing the aspire url via personal e - mail . The second tested approach is card referral, where clinicians handed a printed card with the url to participants . In both approaches, clinicians provide a message to avoid tobacco with instructions to work through the program at a location with access to high - speed internet . Secondary aims were to assess tobacco use, measure whether tobacco - related knowledge among participants was compromised, report evaluation of the program by participants, and describe whether refinements were needed to enhance future implementation . This randomized pilot study was conducted at a medical and dental clinic in houston, texas . The clinic is located in close proximity to the most ethnically diverse county in the united states . The protocol was approved by the institutional review board at md anderson cancer center (2014 - 0023). The recruitment goal was 200, determined by a sample size calculation set a priori . Written parental consent and adolescent assent were obtained for participants under age 18 . Eligible participants had e - mail, high - speed internet access, and the ability to read or speak english or spanish . Three participants were found to be duplicates, leaving a total sample of 197 . At enrollment participants were randomized via a computer program to 1 of the 2 approaches (ie, e - mail link or card referral). Consort - like diagram . * a smoking prevention interactive experience (aspire) evidence - based program delivered via the internet . Its electronic link is posted on the website of the national cancer institute s research - tested interventions . It combines interactivity and entertainment to engage users through animations, videos of high school students, and task - oriented activities . It is a self - administered, 4-hour activity with 5 modules that can be completed in several sessions . The other 4 modules are intended for smokers, with one of the modules describing benefits of quitting for smokers disinterested in quitting . Three modules provide practical strategies encountered during quitting that facilitate the likelihood of successful cessation . Because clinic outpatients would be completing activities unsupervised, the full - length program was modified to give participants the option of selecting the most relevant modules to work through . To receive compensation of a us $30 gift card, they were required to work through one 20- to 30-minute standalone module and complete pre- and post - tests . Those who did not access the program within 6 days of registering could have received up to 13 reminders (ie, 10 e - mails and 3 telephone calls). Knowledge questions were generated based on materials from the module selected by participants with multiple - choice responses . After post - tests, participants were asked whether they learned new facts from the program, whether it influenced decisions not to use tobacco, and whether they would recommend it to family and friends . Descriptive analyses and the proportion of participants accessing the program by referral approach were computed . The likelihood ratio test was used to determine the overall statistical and numerical differences between referral methods . The percentage scoring 70% between the 2 referral strategies was determined using logistic regression models . Results were summarized with odds ratios (ors) and 95% confidence intervals (cis). The statistical significance level was set at p <.05 . Post hoc power was calculated for sample size based on access rates by referral strategy . A 2-group test with a 0.050 2-sided test level would have 80% power to detect differences between a group 1 proportion, 1, of 0.535 and a group 2 proportion, 2, of 0.602 (or: 1.31) when each group had 857 participants . A 2-group test with 0.050 2-sided test level would have 15% power to detect the difference between a group 1 proportion, 1, of 0.535 and a group 2 proportion, 2, of 0.602 (or: 1.31) when the sample size in each group had 99 participants . This randomized pilot study was conducted at a medical and dental clinic in houston, texas . The clinic is located in close proximity to the most ethnically diverse county in the united states . The protocol was approved by the institutional review board at md anderson cancer center (2014 - 0023). The recruitment goal was 200, determined by a sample size calculation set a priori . Written parental consent and adolescent assent were obtained for participants under age 18 . Eligible participants had e - mail, high - speed internet access, and the ability to read or speak english or spanish . Three participants were found to be duplicates, leaving a total sample of 197 . At enrollment participants were randomized via a computer program to 1 of the 2 approaches (ie, e - mail link or card referral). Consort - like diagram . * a smoking prevention interactive experience (aspire) evidence - based program delivered via the internet . Participants were referred to aspire . Its electronic link is posted on the website of the national cancer institute s research - tested interventions . It combines interactivity and entertainment to engage users through animations, videos of high school students, and task - oriented activities . It is a self - administered, 4-hour activity with 5 modules that can be completed in several sessions . The other 4 modules are intended for smokers, with one of the modules describing benefits of quitting for smokers disinterested in quitting . Three modules provide practical strategies encountered during quitting that facilitate the likelihood of successful cessation . Because clinic outpatients would be completing activities unsupervised, the full - length program was modified to give participants the option of selecting the most relevant modules to work through . To receive compensation of a us $30 gift card, they were required to work through one 20- to 30-minute standalone module and complete pre- and post - tests . Those who did not access the program within 6 days of registering could have received up to 13 reminders (ie, 10 e - mails and 3 telephone calls). Knowledge questions were generated based on materials from the module selected by participants with multiple - choice responses . After post - tests, participants were asked whether they learned new facts from the program, whether it influenced decisions not to use tobacco, and whether they would recommend it to family and friends . Descriptive analyses and the proportion of participants accessing the program by referral approach were computed . The likelihood ratio test was used to determine the overall statistical and numerical differences between referral methods . The percentage scoring 70% between the 2 referral strategies was determined using logistic regression models . Results were summarized with odds ratios (ors) and 95% confidence intervals (cis). The statistical significance level was set at p <.05 . Post hoc power was calculated for sample size based on access rates by referral strategy . A 2-group test with a 0.050 2-sided test level would have 80% power to detect differences between a group 1 proportion, 1, of 0.535 and a group 2 proportion, 2, of 0.602 (or: 1.31) when each group had 857 participants . A 2-group test with 0.050 2-sided test level would have 15% power to detect the difference between a group 1 proportion, 1, of 0.535 and a group 2 proportion, 2, of 0.602 (or: 1.31) when the sample size in each group had 99 participants . Those completing pre - tests, accessing aspire but were not completing the post - tests after reminders were dropouts . Baseline information and tobacco use were available for 197, however, 85 (43%) of the 197 did not login to the program (ie, noncompleters; table 1 and figure 1). A total of 112 (57%) of the 197 connected to aspire: 47.3% in e - mail link and 52.6% in card referral . At baseline, for breakdown by race / ethnicity, 37.5% were hispanic / latino, 33.9% were asian, 17.8% were black, 4.5% white, and the remainder were others . No use of tobacco or nicotine products was reported by 98% (table 1). Among 82 completers, 94% (n = 77) selected the aspire module for committed nontobacco users . Those remaining (n = 5; 6%) selected the aspire module intended for smokers related to managing stress when trying to quit . Using baseline characteristics, participants were compared to noncompleters . After adjusting for age, gender, and ethnicity, the e - mail link group did not differ from card referral (adjusted or = 1.3, 95% ci = 0.8 - 2.4, p = .312; table 2). Non - hispanics were more likely to connect than hispanics (64% vs 48%, adjusted or = 2.1, 95% ci = 1.2 - 3.8, p = .012). We compared completers and dropouts on baseline sociodemographic characteristics and pre - test scores on knowledge . Using and 2-sample t tests, significant association for differences by age or gender was determined between completers and noncompleters . However, those who completed had higher scores on pre - tests compared to dropouts at post - test (p = .03). Fifty percent scored 70% on the pre - tests and maintained similar or higher scores on post - tests . Hence, those scoring 70% on knowledge either at pre- or post - test were compared between referral approaches . Seventy percent graduated in the e - mail link compared to 64% in the card referral . Adjusted logistic regression analysis indicated that the e - mail link group did not differ from card - referral group (adjusted or = 1.4, 95% ci = 0.5 - 3.5, p = .517). Age, ethnicity, and gender were not significantly associated with knowledge scores (data not shown). We provide results for the 94% who selected the module covering education and support for those committed to remain tobacco - free (table 3). At pre - test, 40% or more had compromised knowledge on questions 1, 4, 5, 7, and 9 . Mean percent correct responses were compared between pre- and post - tests with mcnemar test for related samples . Improvement at post - test was found for most items with statistical significance for item 4 . Participants rated the program favorably: up to 97% learned new facts, 95% indicated the program influenced them about abstinence, and 95% would recommend to family and friends (data not shown). Knowledge questions and mean pre- and post - test scores for those completing module about remaining tobacco - free . Using baseline characteristics, participants were compared to noncompleters . After adjusting for age, gender, and ethnicity, the e - mail link group did not differ from card referral (adjusted or = 1.3, 95% ci = 0.8 - 2.4, p = .312; table 2). Non - hispanics were more likely to connect than hispanics (64% vs 48%, adjusted or = 2.1, 95% ci = 1.2 - 3.8, p = .012). We compared completers and dropouts on baseline sociodemographic characteristics and pre - test scores on knowledge . Using and 2-sample t tests, significant association for differences by age or gender was determined between completers and noncompleters . However, those who completed had higher scores on pre - tests compared to dropouts at post - test (p = .03). Fifty percent scored 70% on the pre - tests and maintained similar or higher scores on post - tests . Hence, those scoring 70% on knowledge either at pre- or post - test were compared between referral approaches . Seventy percent graduated in the e - mail link compared to 64% in the card referral . Adjusted logistic regression analysis indicated that the e - mail link group did not differ from card - referral group (adjusted or = 1.4, 95% ci = 0.5 - 3.5, p = .517). Age, ethnicity, and gender were not significantly associated with knowledge scores (data not shown). We provide results for the 94% who selected the module covering education and support for those committed to remain tobacco - free (table 3). At pre - test, 40% or more had compromised knowledge on questions 1, 4, 5, 7, and 9 . Mean percent correct responses were compared between pre- and post - tests with mcnemar test for related samples . Improvement at post - test was found for most items with statistical significance for item 4 . Participants rated the program favorably: up to 97% learned new facts, 95% indicated the program influenced them about abstinence, and 95% would recommend to family and friends (data not shown). Knowledge questions and mean pre- and post - test scores for those completing module about remaining tobacco - free . The 2 tested referral approaches to aspire were equally effective, an encouraging finding supported by 57% of participants accessing aspire . With a majority accessing aspire, this can be considered an advancement in consistent delivery of tobacco prevention education to outpatients . Organizations interested in improving health - care quality are encouraged to use e - mail - based links and/or printed cards to refer patients to education about tobacco use . Unfortunately they may have joined because of social desirability, parental influence, and monetary compensation but reconsidered their participation afterward . After the study, when clinic providers were asked for their thoughts about noncompleters of the study, they suggested some may not trust researchers . A possibility suggested by providers about facilitating future program dissemination could be to involve case workers employed at the clinic with whom patients have professional, trusting relationships . It is possible participants were a very low - risk group for tobacco use but this is unknowable . Many were of mexican or pakistani origin, countries with norms about tobacco use more accepting than in the united states . Providers were asked after the study for their explanations about why so few participants reported tobacco use . The providers felt it was logical participants were uneager to reveal tobacco use when accompanied by parents because of desiring to avoid disapproval . Universally high agreement was indicated by study participants when asked if they learned new facts about tobacco, were influenced not to use tobacco, and would share the program with friends and family . Referral to antitobacco modules such as aspire by providers has potential for great promise, however, fine - tuning is needed . The physicians were appreciative of enhanced prevention activities made possible by research staff (ie, enrolling, tracking, placing follow - up reminders, and mailing compensation). One solution could be to use automated systems such as computerized telephone and e - mail messaging approved by the health insurance portability and accountability act . Counseling about nicotine and tobacco use prevention and cessation among adolescents is one of the most meaningful investments in population health that clinicians can implement . Additional work is needed to refine implementation and reduce barriers in order to efficiently link adolescents to an evidence - based program encouraging a tobacco - free lifestyle.
Given the relatively conservative cultural climate in india, those belonging to any sexual minority are subject to prejudices . The delhi high court revoked section 377 of the indian penal code which criminalized consensual acts of same - sex adults in private, and held that it violated the fundamental right of life and liberty and the right to equality as guaranteed in the constitution . However, this verdict was overturned by the supreme court of india in december 2013 . An anti - homosexual stance can trickle down into the attitudes of doctors, which could translate into an unconscious bias in treatment encounters with lesbian, gay, bisexual and transgender (lgbt) patients despite an adequate medical education . The diagnostic and statistical manual of mental disorders (dsm) has already recognized that it is not a disorder . Considerable evidence has shown that the lgbt population has unique physical and mental health care needs . Yet, they tend to avoid routine health care for the fear of stigmatization by the medical community . Medical curricula in canada and the united states were found to devote little time to impart formal training about lgbt related topics to in - the - making clinicians . Physicians across specialties agree that they lack the required skills to address issues related to their patients sexual orientation . The problem may be further compounded by physician homophobia, which impacts the doctor - patient relationship adversely and diminishes a patient's ability to disclose sensitive issues . Although there has been a substantial reduction in physician homophobia over the last 30 years in the developed world, it is still not completely done away with . A survey in austria revealed that medical students had more negative attitude towards homosexuals than non - medical students . Studies from australia, china and serbia have also found medical students tend to be prejudiced against the now increasingly visible sexual minority . It may be argued that homophobia is difficult to gauge because, as with discrimination, underreporting often occurs, yet the effort to find it out in medical professionals may prove to be worthwhile as it has implications in patient care and hence the present study assesses knowledge and attitudes among medical students and interns in an indian medical college . The study protocol was approved by the institutional ethics committee and written informed consent was taken from all participants . A cross sectional survey design with a convenience sampling . All undergraduate medical students and interns studying and working respectively in the institute to which the authors are affiliated were considered for inclusion in the study . The first year (second semester), second year (fourth semester) and third year (sixth semester) medical students were approached en masse during a lecture session after permission from the respective heads of department of anatomy, pharmacology and preventive and social medicine . The students were told that they would receive briefing about a new study, but the nature of the study was not told to them . Students and interns were then briefed about the types of sexual orientation, the unique health needs that minorities have, and how these need to be addressed in clinical practice . Anonymity was ensured by asking those who consented not to reveal their identity on the filled questionnaire . At least three of the authors were present for the entire duration of the survery, the students filled the forms at a distance from each other and discussion between students was not permitted . The filled forms were dropped by both the students and the interns in a sealed drop box . Age, gender, religion, marital status, sexual orientation (heterosexual, homosexual or bisexual), and year of medical schooling (first, second or third year medical student or intern). Participants were required to express their opinion on the validity of 32 statements as true, false, or do nt know . The score on this scale ranged from 0 - 32, where 32 represented the score with all correct answers . The instrument was found to have a cronbach's alpha of 0.724 in this study, but it was not validated prior to use . The questionnaire has been used previously by dunji - kosti et al . And was created by compilation of statements used in three previous studies . It contains 20 statements regarding homosexuals, their lifestyle, and their social position and is scored by the participants on a 5-grade likert type scale ranging from 1 (strongly agree) to 5 (strongly disagree). The score range on this scale was 20 - 100, with a higher score indicating a more negative attitude towards homosexuals . This instrument is also a compilation of items used in three previous studies, and was used by dunji - kosti et al . For their work . The instrument was found to have a cronbach's alpha of 0.810 in this study, but it was not validated prior to use . Descriptive statistics were used to describe the data, frequencies and percentages for categorical variables and mean values with standard deviations for continuous variables . Independent samples t test and one way anova were used to compare the knowledge and attitude questionnaire scores across different variables . Pearson's correlation was employed to find the correlation between age, knowledge and attitude questionnaire scores . Since only one participant identified himself / herself as homosexual and since only three participants reported being married, the variables sexual orientation and marital status were not analyzed separately . The study protocol was approved by the institutional ethics committee and written informed consent was taken from all participants . A cross sectional survey design with a convenience sampling . All undergraduate medical students and interns studying and working respectively in the institute to which the authors are affiliated were considered for inclusion in the study . The first year (second semester), second year (fourth semester) and third year (sixth semester) medical students were approached en masse during a lecture session after permission from the respective heads of department of anatomy, pharmacology and preventive and social medicine . The students were told that they would receive briefing about a new study, but the nature of the study was not told to them . Students and interns were then briefed about the types of sexual orientation, the unique health needs that minorities have, and how these need to be addressed in clinical practice . Anonymity was ensured by asking those who consented not to reveal their identity on the filled questionnaire . At least three of the authors were present for the entire duration of the survery, the students filled the forms at a distance from each other and discussion between students was not permitted . The filled forms were dropped by both the students and the interns in a sealed drop box . Age, gender, religion, marital status, sexual orientation (heterosexual, homosexual or bisexual), and year of medical schooling (first, second or third year medical student or intern). Participants were required to express their opinion on the validity of 32 statements as true, false, or do nt know . The score on this scale ranged from 0 - 32, where 32 represented the score with all correct answers . The instrument was found to have a cronbach's alpha of 0.724 in this study, but it was not validated prior to use . The questionnaire has been used previously by dunji - kosti et al . And was created by compilation of statements used in three previous studies . It contains 20 statements regarding homosexuals, their lifestyle, and their social position and is scored by the participants on a 5-grade likert type scale ranging from 1 (strongly agree) to 5 (strongly disagree). The score range on this scale was 20 - 100, with a higher score indicating a more negative attitude towards homosexuals . This instrument is also a compilation of items used in three previous studies, and was used by dunji - kosti et al . For their work . The instrument was found to have a cronbach's alpha of 0.810 in this study, but it was not validated prior to use . Age, gender, religion, marital status, sexual orientation (heterosexual, homosexual or bisexual), and year of medical schooling (first, second or third year medical student or intern). Participants were required to express their opinion on the validity of 32 statements as true, false, or do nt know . The score on this scale ranged from 0 - 32, where 32 represented the score with all correct answers . The instrument was found to have a cronbach's alpha of 0.724 in this study, but it was not validated prior to use . The questionnaire has been used previously by dunji - kosti et al . And was created by compilation of statements used in three previous studies . It contains 20 statements regarding homosexuals, their lifestyle, and their social position and is scored by the participants on a 5-grade likert type scale ranging from 1 (strongly agree) to 5 (strongly disagree). The score range on this scale was 20 - 100, with a higher score indicating a more negative attitude towards homosexuals . This instrument is also a compilation of items used in three previous studies, and was used by dunji - kosti et al . For their work . The instrument was found to have a cronbach's alpha of 0.810 in this study, but it was not validated prior to use . Descriptive statistics were used to describe the data, frequencies and percentages for categorical variables and mean values with standard deviations for continuous variables . Independent samples t test and one way anova were used to compare the knowledge and attitude questionnaire scores across different variables . Pearson's correlation was employed to find the correlation between age, knowledge and attitude questionnaire scores . Since only one participant identified himself / herself as homosexual and since only three participants reported being married, the variables sexual orientation and marital status were not analyzed separately . A total of 339 medical students and interns in all were approached and invited to participate, out of which 273 (80.5%) consented to participate and returned the filled questionnaires . Due to missing / invalid data, 29 (10.6%) forms were excluded so that the final sample for analysis consisted of 244 filled questionnaires [figure 1]. Distribution of sample across different professional years table 1 displays the socio - demographic characteristics of the study population . The mean age of the respondents was 20.04 (sd = 1.62) years, range 17 - 25 . The least participation in the study was from interns, who accounted for only 12.7% of the study population . Socio - demographic characteristics of the study sample (n = 244) * others included muslims, jains, christians, undergraduate students table 2 shows the association of gender, designation and religion with knowledge and attitude towards homosexuality . Females were found to have a more positive attitude towards homosexuals vis - - vis males, although their knowledge about homosexuality did not differ significantly from males . Interns and third year medical students scored better than first and second year students on knowledge (p = 0.046), but the difference did not reflect in their attitude towards homosexuals . Although when all the respondents were considered as a single group, those with higher knowledge had more favorable attitudes [table 3]. Likewise, non - hindus had greater knowledge about homosexuality as against hindus, but the difference in their attitude was not statistically significant . Association of gender, designation and religion with knowledge and attitude towards homosexuality (n = 244) * sex education and knowledge about homosexuality questionnaire, attitudes towards homosexuals questionnaire, undergraduate students correlation between age, knowledge and attitude towards homosexuality (n = 244) * sex education and knowledge about homosexuality questionnaire attitudes towards homosexuals questionnaire p<0.01 the score on sekhq [table 3] correlated positively with age (being older entailed greater knowledge), and inversely with score on ahq (those having higher knowledge had more positive attitudes). On stepwise multiple regression [table 4], knowledge about homosexuality and gender could significantly predict attitude towards homosexuals explaining 14.3% and 2.8% of the variance respectively, while all the variables taken together accounted for 16.3% of the variance . Apart from attitude, religion emerged as a significant predictor of knowledge about homosexuality explaining 3.2% of the variance . Predictors of knowledge and attitude towards homosexuality on linear regression (n = 244) * sex education and knowledge about homosexuality questionnaire, attitudes towards homosexuals questionnaire table 5 shows the percentage of correct responses on each item of the sekhq . On 28 of the 32 questions about knowledge related to homosexuality, less than 10 percent of the participants could correctly respond that there has not been an increase in homosexuality in the last 25 years . The highest correct response of about 72.5 percent was for the statement that homosexuals usually disclose their sexual identity to a friend before they tell a parent . The mean total score of the respondents on the sekhq was 10.59 (sd 4.19), range 1 - 21 . Response on sex education and knowledge about homosexuality questionnaire table 6 presents the mean scores obtained on each item of the ahq . Negative attitudes towards homosexuals were most reflected on statements like if gay men want to be treated like everyone else, then they need to stop making such a fuss about their sexuality / culture, in today's tough economic times, tax money should nt be used to support gay men's organizations, gay men have become far too confrontational in their demand for equal rights, and the mean total score of the respondents on the ahq was 56.52 (sd 8.63), range 25 - 85 . There is dearth of indian literature that has systematically investigated issues related to homosexuality, and this is the first such effort to study the knowledge and attitude of medical students towards the same in an indian set up . The findings suggest that overall the participants lacked adequate knowledge about homosexuality, although they endorsed a more or less neutral stance insofar as their attitude towards homosexuality is concerned . Western medical school curricula inadequately address and give very less time to the health and sexuality issues of lgbt people . In absence of any publications or formal reports, it is not exactly known how much importance medical curriculum gives to homosexuality and related health issues in india, although it may be apprehended that it may not be getting its due, which is reflected in the low knowledge level of students in this study . In fact, indian medical textbooks give misleading information on the subject, promoting a sense of bias against homosexuals . Knowledge about homosexuality emerging as the strongest predictor of a positive attitude towards homosexuals in this study only reiterates the previously proposed notion that enhancing knowledge maybe a possible tool to reduce the prejudice meted out to and stigmatization faced by the sexual minorities, especially during their encounters with medical professionals . To increase the knowledge and awareness of doctors about issues related to homosexuality, it becomes imperative to incorporate education about alternate sexuality in the medical teaching programs, not to champion the cause of homosexuality but to expose students to alternative views of sexuality, challenge their values and beliefs, and celebrate diversity . This in turn could enhance their professionalism and help them offer better health care to their lgbt patients with a minimal sense of discrimination creeping in, as health care providers who have negative attitudes toward same - sex behavior have been found to provide inadequate care for lgbt individuals . Lack of disclosure of sexual orientation to physicians significantly decreases the likelihood that appropriate health services are recommended to such patients . On the brighter side, previous research shows that practicing physicians and medical students alike have expressed the need to include such training at undergraduate and postgraduate levels . Greater clinical exposure to lgbt patients at the undergraduate level enhances knowledge of lgbt health care concerns and brings about a more positive attitude, further emphasizing the need . The association of american medical colleges has recommended that medical school curricula ensure that students master the knowledge, skills, and attitudes necessary to provide excellent, comprehensive care for lgbt patients by including comprehensive content addressing the specific healthcare needs of lgbt patients and training in communication skills with patients and colleagues regarding issues of sexual orientation and gender identity . Laying of such guidelines or recommendations, though highly desired has eluded the indian medical education scenario . It cannot be exactly explained why religion affects knowledge but not attitudes in this work . The wide gap in the number of hindu and non - hindu participants could be a probable reason . Females medical students tend to have a more tolerant attitude towards homosexuals, as also substantiated by this study . Although the regression models for both knowledge and attitude were highly significant (p <0.001), the relatively limited variance explained by the independent variables included in the models suggests the need to examine other factors that may influence medical students knowledge about homosexuality and attitudes towards homosexuals . Factors that may affect and shape attitudes could be cultural orientation and personal religious beliefs (and not just religious affiliation), family environment and effect of media amongst others . India, being a culturally and sexually conservative country, where respondents may feel less comfortable in expressing their views on sexuality related issues; such a response rate is still deemed to be decent . It may be apprehended that those who denied to participate had more negative attitudes towards homosexuals . Secondly, the questionnaires used in this study were not assessed for their psychometric properties other than internal consistency reliability which was found to be satisfactory . Thirdly, conducting this survey in a classroom may have introduced a bias - in such a setting, one may not get truthful answers about people's own sexual orientation, and more importantly, there may be an over representation of neutral opinions about homosexuality . Also, the fact that only 41.3% interns opted to participate when approached one to one may indicate that most other students would have done the same, had it not been for the en masse approach used . That the participants were told as a part of introduction to the study that lgbt patients may have special needs and that as doctors they would be expected to help out may be another potential source of bias causing over reporting of positive views . Lastly, the results of this work reflect the attitudes of medical students of a single medical teaching institute of india, and so cannot be extrapolated to the larger population of medical students of india . India, being a culturally and sexually conservative country, where respondents may feel less comfortable in expressing their views on sexuality related issues; such a response rate is still deemed to be decent . It may be apprehended that those who denied to participate had more negative attitudes towards homosexuals . Secondly, the questionnaires used in this study were not assessed for their psychometric properties other than internal consistency reliability which was found to be satisfactory . Thirdly, conducting this survey in a classroom may have introduced a bias - in such a setting, one may not get truthful answers about people's own sexual orientation, and more importantly, there may be an over representation of neutral opinions about homosexuality . Also, the fact that only 41.3% interns opted to participate when approached one to one may indicate that most other students would have done the same, had it not been for the en masse approach used . That the participants were told as a part of introduction to the study that lgbt patients may have special needs and that as doctors they would be expected to help out may be another potential source of bias causing over reporting of positive views . Lastly, the results of this work reflect the attitudes of medical students of a single medical teaching institute of india, and so cannot be extrapolated to the larger population of medical students of india . This study gives preliminary insight into the knowledge and attitude of indian medical students about homosexuality, although further research on a larger scale is needed across the country to have a more comprehensive impression about the stand of medical students on the issue, which could help to draft guidelines on inclusion of homosexual patients health needs in the indian medical curriculum . It has been rightly commented that medical treatment has more to do with doctors values and attitudes than with objective realities . The art of medicine depends on the ability to acknowledge and respect these differences and treat every patient as an individual . Medical students need to be trained to maintain a non - homophobic attitude and to be aware about how their own attitude affects clinical judgment . Enhancing knowledge of medical students by adequate incorporation of lgbt issues in the curriculum could help reduce practicing doctors prejudice faced by lgbt patients and improve the health care offered to such patients.
Edible plants (100 g) were minced into 5 mm fragments and extracts were obtained with methanol (200 ml) overnight at room temperature . The extracts were filtered and the residue was re - extracted under the same conditions as above . The combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . The specimen was dissolved in dmso (1.0 ml). Human umbilical vein endothelial cells (no . Jcrb0408, health science research resources bank, osaka, japan) were cultured at 37c in sfm cs - c medium (ds pharma biomedical, osaka, japan) supplemented with 1.5 g / ml fungizone under air - co2 (95:5) (referred to hereafter as cs - cf medium). For screening study purposes, huvecs were seeded in 60 mm culture dishes (1.5 10 cells). Then, huvecs were incubated with 1 g / l (normal glucose medium) or 4.5 g / l (high glucose medium) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed, one receiving 1) continuous 10 l meep, and the other receiving 2) continuous 100 l meep . Determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer's protocol (b - bridge international, inc ., supernatants cultured with each sample were added to elisa plates to measure the esrage content . Dna of huvecs was extracted from the homogenates using a dna extractor wb kit (wako pure chemical industries, osaka, japan) according to the protocol . The 8-ohdg level was determined by using the 8-ohdg check elisa kit (japan institute for the control of aging, shizuoka, japan) according to the manufacturer's protocol . The recovery rate of 8-ohdg level was expressed as% recovery of the normal glucose medium a450 nm (experimental a450nm / normal glucose medium a450 nm 100). Peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein (dcdhf) to 2,7-dichlorofluorescein (dcf) was estimated based on the method described by crow . Samples or control (dmso) were added to 100 mm pbs (ph 7.4) containing 100 m diethylenetriamine - n, n, n, n, n-pentaacetic acid (dojindo molecular technologies, inc ., japan), 5 m sodium peroxynitrite and 100 m dcdhf . The peroxynitrite scavenging activity was expressed as% inhibition of the control a500 nm ([control a500 nm experimental a500nm]/control a500 nm 100). Because meep were methanolic extracts, we measured their tpc . Tpc was measured with a modified version of the folin briefly, 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . We allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer (nihonbunko ltd ., the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . A statistical comparison between the groups was carried out using either anova or student's t - test . Edible plants (100 g) were minced into 5 mm fragments and extracts were obtained with methanol (200 ml) overnight at room temperature . The extracts were filtered and the residue was re - extracted under the same conditions as above . The combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . Jcrb0408, health science research resources bank, osaka, japan) were cultured at 37c in sfm cs - c medium (ds pharma biomedical, osaka, japan) supplemented with 1.5 g / ml fungizone under air - co2 (95:5) (referred to hereafter as cs - cf medium). For screening study purposes, huvecs were seeded in 60 mm culture dishes (1.5 10 cells). Then, huvecs were incubated with 1 g / l (normal glucose medium) or 4.5 g / l (high glucose medium) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed, one receiving 1) continuous 10 l meep, and the other receiving 2) continuous 100 l meep . Determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer's protocol (b - bridge international, inc ., supernatants cultured with each sample were added to elisa plates to measure the esrage content . Dna of huvecs was extracted from the homogenates using a dna extractor wb kit (wako pure chemical industries, osaka, japan) according to the protocol . The 8-ohdg level was determined by using the 8-ohdg check elisa kit (japan institute for the control of aging, shizuoka, japan) according to the manufacturer's protocol . The recovery rate of 8-ohdg level was expressed as% recovery of the normal glucose medium a450 nm (experimental a450nm / normal glucose medium a450 nm 100). Peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein (dcdhf) to 2,7-dichlorofluorescein (dcf) was estimated based on the method described by crow . Samples or control (dmso) were added to 100 mm pbs (ph 7.4) containing 100 m diethylenetriamine - n, n, n, n, n-pentaacetic acid (dojindo molecular technologies, inc ., japan), 5 m sodium peroxynitrite and 100 m dcdhf . The peroxynitrite scavenging activity was expressed as% inhibition of the control a500 nm ([control a500 nm experimental a500nm]/control a500 nm 100). Because meep were methanolic extracts, we measured their tpc . Tpc was measured with a modified version of the folin briefly, 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . We allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer (nihonbunko ltd ., the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . A statistical comparison between the groups was carried out using either anova or student's t - test . We examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . The result showed an increase in esrage production in the following gight samples (eggplant [solanum melongena], carrot peel [daucus carota subsp . Sativus], young sweet corn [zea mays saccharata], broad bean [vicia faba], japanese radish [raphanus sativus var . Longipinnatus] sprout, jew's marrow [corchorus olitorius] and cauliflower [brassica oleracea var . The results of the esrage production of these eight samples on huvecs are summarized in figure 1 . Figure 1a shows the effects of the eight samples (10 l) on esrage production in high glucose - induced huvecs . The esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . Genistein (10 m) increased esrage production by approximately 12%, but egcg (10 m) caused no increase . The results for plant samples suggest that white radish sprout, jew's marrow and cauliflower have esrage production potential on induced endothelial cells . In particular, white radish sprout, which had higher esrage production potential than genistein, significantly (p <0.01) increased esrage production . In addition, white radish sprout showed an esrage production potential approximately 2.4 times higher than the control (4.5 g / l of glucose only). On the other hand, in 100 l meep [figure 1b], more samples had a significant influence on esrage production . The results suggest that eggplant, carrot peel, young sweet corn, broad bean, japanese radish, and white radish sprout have esrage production potential on induced endothelial cells . However, different meep seem to have different effects on esrage production in the presence of high glucose . In 100 m concentration, genistein also showed a tendency to increase slightly, but the increase was not seen in egcg . In studies to date, no plant has yet been shown to increase esrage production . Therefore, the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . However, it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . Furthermore, lu et al . Indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation, down - regulating rage expression and up - regulating srage in the kidney . From the above - mentioned report, the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . However, although lee and lee show that 5 m egcg down - regulates the rage expression, this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . Effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products (esrage) production in human umbilical vein endothelial cells cultured in high glucose . The esrage productions of control (dimethyl sulfoxide) and samples in human umbilical vein endothelial cells cultured in high (4.5 two groups of experiments were formed, one receiving continuous 10 l methanolic extracts from edible plants (meep) (a) and the other receiving continuous 100 l meep (b) values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . Performed the study to compare concentration of srage and its ligands (en - rage and hmgb1) in type 1 (t1 dm) and t2 dm . Similarly, en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . They concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . Therefore, the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . On the other hand, some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . On other word, showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . In addition, liu et al . Showed that administration of euk134 (peroxynitrite scavenger) attenuated rage expression, and decreased cardiomyocyte apoptosis in diabetic mice . Therefore, we examined the correlation between esrage production, and the two indicators mentioned above (peroxynitrite and 8-ohdg levels) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . Showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . In our experiment, the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml (data not shown). The formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide (sin-1; peroxynitrite donor) was also inhibited by egcg . In our experiment, egcg (100 m) showed 8-ohdg recovery rates of approximately 90%, but genistein (100 m) caused 8-ohdg recovery rates of approximately 60% . The calculated 8-ohdg recovery rates for white radish sprout, broad bean, young sweet corn, japanese radish, eggplant, jew's marrow, cauliflower, and carrot peel were 61.9%, 47.7%, 68.9%, 55.5%, 43.8%, 62.0%, 60.2%, and 77.1%, respectively in comparison to the normal glucose medium [figure 2] indicating that the samples are potent inhibitors of 8-ohdg . In particular, carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein, significantly (p <0.05) decreased 8-ohdg level . In addition, carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . Figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . The results show that the 8-ohdg level does not correlate with esrage production (r = 0.086). The 8-ohdg recovery rates of control (normal glucose medium) and samples (high glucose medium) are indicated by unshaded and shaded columns, respectively . Values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls . The correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants figure 4 shows the calculated peroxynitrite scavenging activities of eight extracts . Because peroxynitrite has a short half - life, the measurement of the intracellular scavenging activity is difficult . Therefore, we performed the scavenging activity measurement of the sample in the test tube . Amongst the eight extracts, white radish sprout revealed the highest scavenging activity (44.8%) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . The peroxynitrite level significantly correlated with esrage production (r = -0.706, p <0.10). Wu et al . Showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . This was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . Values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the correlation between esrage production and tpc was analyzed [figure 6]. The correlation coefficient for esrage production was found to be greater than 0.6 (r = 0.660, p <0.10). A positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . The correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . However, the seven meep except for eggplant contain a lot of quercetin . Because there was a positive correlation between tpc and esrage [figure 6], it is very likely to be quercetin that had an effect . The effect of these seven plants may resemble the mechanism that zhang et al . Showed . Hyperoside, quercetin-3-o - galactoside, is a flavonoid isolated from many medicinal plants . In their studies, quiescent ecv304 (human endothelial - like) cells were treated in vitro with advanced glycation end products (ages) in the presence or absence of hyperoside . The results demonstrated that ages induced c - jun n - terminal kinases (jnk) activation and apoptosis in ecv304 cells . Furthermore, hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells, whereas knockdown of rage inhibited age - induced jnk activation . These results suggested that ages may promote jnk activation, leading to viability inhibition of ecv304 cells via the rage signaling pathway . Furthermore, from the above - mentioned report, the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . Our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes . We examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . The result showed an increase in esrage production in the following gight samples (eggplant [solanum melongena], carrot peel [daucus carota subsp . Sativus], young sweet corn [zea mays saccharata], broad bean [vicia faba], japanese radish [raphanus sativus var . Longipinnatus] sprout, jew's marrow [corchorus olitorius] and cauliflower [brassica oleracea var . The results of the esrage production of these eight samples on huvecs are summarized in figure 1 . Figure 1a shows the effects of the eight samples (10 l) on esrage production in high glucose - induced huvecs . The esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . Genistein (10 m) increased esrage production by approximately 12%, but egcg (10 m) caused no increase . The results for plant samples suggest that white radish sprout, jew's marrow and cauliflower have esrage production potential on induced endothelial cells . In particular, white radish sprout, which had higher esrage production potential than genistein, significantly (p <0.01) increased esrage production . In addition, white radish sprout showed an esrage production potential approximately 2.4 times higher than the control (4.5 g / l of glucose only). On the other hand, in 100 l meep [figure 1b], more samples had a significant influence on esrage production . The results suggest that eggplant, carrot peel, young sweet corn, broad bean, japanese radish, and white radish sprout have esrage production potential on induced endothelial cells . However, different meep seem to have different effects on esrage production in the presence of high glucose . In 100 m concentration, genistein also showed a tendency to increase slightly, but the increase was not seen in egcg . In studies to date, no plant has yet been shown to increase esrage production . Therefore, the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . However, it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . Furthermore, lu et al . Indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation, down - regulating rage expression and up - regulating srage in the kidney . From the above - mentioned report, the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . However, although lee and lee show that 5 m egcg down - regulates the rage expression, this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . Effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products (esrage) production in human umbilical vein endothelial cells cultured in high glucose . The esrage productions of control (dimethyl sulfoxide) and samples in human umbilical vein endothelial cells cultured in high (4.5 two groups of experiments were formed, one receiving continuous 10 l methanolic extracts from edible plants (meep) (a) and the other receiving continuous 100 l meep (b) values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . Performed the study to compare concentration of srage and its ligands (en - rage and hmgb1) in type 1 (t1 dm) and t2 dm . Similarly, en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . They concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . Therefore, the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . On the other hand, some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . On other word, showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . In addition, liu et al . Showed that administration of euk134 (peroxynitrite scavenger) attenuated rage expression, and decreased cardiomyocyte apoptosis in diabetic mice . Therefore, we examined the correlation between esrage production, and the two indicators mentioned above (peroxynitrite and 8-ohdg levels) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . Showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . In our experiment, the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml (data not shown). The formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide (sin-1; peroxynitrite donor) was also inhibited by egcg . In our experiment, egcg (100 m) showed 8-ohdg recovery rates of approximately 90%, but genistein (100 m) caused 8-ohdg recovery rates of approximately 60% . The calculated 8-ohdg recovery rates for white radish sprout, broad bean, young sweet corn, japanese radish, eggplant, jew's marrow, cauliflower, and carrot peel were 61.9%, 47.7%, 68.9%, 55.5%, 43.8%, 62.0%, 60.2%, and 77.1%, respectively in comparison to the normal glucose medium [figure 2] indicating that the samples are potent inhibitors of 8-ohdg . In particular, carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein, significantly (p <0.05) decreased 8-ohdg level . In addition, carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . Figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . The results show that the 8-ohdg level does not correlate with esrage production (r = 0.086). The 8-ohdg recovery rates of control (normal glucose medium) and samples (high glucose medium) are indicated by unshaded and shaded columns, respectively . Values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls . The correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants because peroxynitrite has a short half - life, the measurement of the intracellular scavenging activity is difficult . Therefore, we performed the scavenging activity measurement of the sample in the test tube . Amongst the eight extracts, white radish sprout revealed the highest scavenging activity (44.8%) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . The peroxynitrite level significantly correlated with esrage production (r = -0.706, p <0.10). Wu et al . Showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . This was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . Values are the mean standard deviation of three measurements . * * p <0.01, * p <0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the tpc of the eight meep significantly correlated with esrage production . The correlation coefficient for esrage production was found to be greater than 0.6 (r = 0.660, p <0.10). A positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . The correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . Because there was a positive correlation between tpc and esrage [figure 6], it is very likely to be quercetin that had an effect . The effect of these seven plants may resemble the mechanism that zhang et al . Showed . Hyperoside, quercetin-3-o - galactoside, is a flavonoid isolated from many medicinal plants . In their studies, quiescent ecv304 (human endothelial - like) cells were treated in vitro with advanced glycation end products (ages) in the presence or absence of hyperoside . The results demonstrated that ages induced c - jun n - terminal kinases (jnk) activation and apoptosis in ecv304 cells . Furthermore, hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells, whereas knockdown of rage inhibited age - induced jnk activation . These results suggested that ages may promote jnk activation, leading to viability inhibition of ecv304 cells via the rage signaling pathway . Furthermore, from the above - mentioned report, the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . Our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes.
They fully integrate into the middle ear and may be used to reconstruct the ossicular chain where there is insufficient autologous material . Tutoplast processed ossicular allografts (tutoplast ossicula auditus) consist of dehydrated human malleus or incus and provide a matrix for new bone formation through bone remodelling . This process involves osmotic destruction of tissue cells, followed by denaturation using sodium hydroxide and hydrogen peroxide to inactivate all pathogens, and finally dehydration and sterilization by gamma irradiation . The tutoplast process inactivates all living organisms and spores from donated tissue and achieves sterility assurance level of 10 . Each transplant can be tracked back to the original donor [1, 2]. Tutoplast ossicula auditus is licensed as a medical product in germany and fulfils european union and usa medical drug regulations . We present a series of 7 consecutive cases demonstrating excellent long - term hearing improvements in tympanoplasty using tutoplast processed malleus to reconstruct the middle ear following mastoidectomy . Seven consecutive patients with cholesteatoma aged 1169 years (four male, three female) underwent canal - wall - down mastoidectomy and tympanoplasty between may 2009 and january 2011 . All patients had canal - wall - down mastoidectomy for removal of cholesteatoma, followed by ype iii tympanoplasty including myringoplasty with tragal perichondrium in a single - stage procedure . This would entail the placement of a tutoplast processed malleus (tutoplast ossicula auditus, tutogen medical gmbh, neunkirchen, germany) onto the stapes if the patient's own ossicles were found to be either absent, eroded or unsuitable for an autograft . All patients had their ear dressed with 2 silastic sheets, one being placed in the mastoid cavity to facilitate epithelialisation of the cavity and the other to cover the tympanic membrane . Bismuth iodine paste gauze dressing was applied into the external auditory meatus for 2 - 3 weeks . Once sufficient healing was ascertained, patients were instructed in the valsalva manoeuvre and were encouraged to perform it 2030 times per day . Hearing assessment was by pure tone audiograms in accordance with the british society of audiology recommended procedure (2004). Values are given in decibel hearing level (db hl) for testing frequencies of 250, 500, 1000, 2000, 4000, and 8000 hertz (hz). Air - bone gaps were calculated in accordance with the american academy of otolaryngology - head and neck surgery (aao - hns) 1995 guideline . Average hearing levels are given in db hl and standard deviations are applied where appropriate . Where applicable, student's t - test (equal sample size, unequal variance) was performed and p values were given . Written consent, including the use of tutoplast ossicula auditus, was obtained . All investigations and procedures were performed according to best clinical practice and the medical principles of the declaration of helsinki . The national research ethics service of the united kingdom has confirmed that formal ethics approval procedure is not required (nres ref 04/26/31) as this is a retrospective study using a fully licensed product . There was partial destruction of the stapes suprastructure in one patient, with one crus being present, and, in this case, the prosthesis was placed on the preserved crus . The average preoperative hearing loss (air conduction) was 49.5 12.7 db (3673 db). The average preoperative air - bone gap was 32.7 6.6 db (2343 db) (table 1). Patients were followed up between 15 and 34 months after surgery, on average 25 6 months . All patients had a safe dry ear upon clinical examination and reported substantial improvement to their hearing . Postoperative hearing thresholds (air conduction) in the operated ear had improved to 29.1 9.7 db (p = 0.006). The air - bone gap had narrowed to 13.8 6.0 db after surgery (424 db) (p = 0.0001). Six patients (86%) had a postoperative air - bone gap of less than 20 db . The air - bone gap closure achieved was on average 18.9 8.5 db (429 db, table 1 and figure 1). Auditory ossicles are often eroded, making them insufficient for ossiculoplasty, and they can also harbour remnants of cholesteatoma matrix which can facilitate disease recurrence . Surgical options in these cases include the use of ossicular replacement prostheses or ossicular allografts . Apprehension in using such allografts over a fear of infection transmission has not made them widely known surgical options in recent years and many surgeons have no experience in using them . Since the inception of tutoplast processed human cadaveric allografts in the 1970s and, there have been no reported cases of graft rejection or disease transmission . Studies on allograft ossiculoplasty dating back to the 1970s demonstrate that allograft ossicles (notched incus homograft) achieve excellent integration and restoration of hearing . Tutoplast processed bone grafts achieve the highest mesenchymal stem cell adherence in vitro, hence making it an ideal environment for bone regeneration . A study on a postmortem temporal bone confirms minimal resorption of allograft ossicles and longevity of these grafts is excellent, as no osteoclastic bone resorption occurs . A series of 465 cases reported closure of air - bone gap to 15 db in 63% of cases and to 20 db in 73% of cases with partial ossicular replacement prostheses (porp). A series of 650 cases, also using plastipore porp, reported postoperative air - bone gaps of 20 db in 68%, although the average air - bone gap was 18 11 db after 12 months . Another group reported postoperative air - bone gap closure (20 db) in tympanoplasty following mastoidectomy for 46% and 33% for titanium and hydroxyapatite prostheses, respectively . Average postoperative air - bone gap was 26.5 db, with 23.8 db for titanium group and 29.8 db for hydroxyapatite group after 1 year . Closure of the air - bone gap fourteen years following mastoidectomy and tympanoplasty using plastipore porp was reported to be 60% in a group of 5 patients . Tympanoplasty with allogeneic ossicles can restore hearing to levels comparable to autograft, and hearing benefit is often favourable to prostheses . Early reports by wehrs report a graft take rate between 92 and 96% and a satisfactory hearing outcome between 77 and 89% . In a case series on using homologous or autologous incus interposition grafts, there was no significant difference in hearing gain between allografts and autografts . Postoperative air - bone gap was 19 db, with 66% of patients achieving an air - bone gap closure of 20 db or better after 15 months . Another study on malleus allograft ossiculoplasties reported air - bone gap closure of 20 db in 81% of cases one year postoperatively, but, in all cases, stapes suprastructure was missing and ossiculoplasty was performed as a secondary procedure, making these outcomes less straightforward to compare . Others report less favourable hearing outcomes compared to autografts or glass ionomer cement . In a study of 293 patients comparing different means of ossicular reconstruction, cholesteatoma removal was the primary cause for surgery in 62 cases (21%), with a mean postoperative air - bone gap of 15 8 db . Allograft ossicles were used in 39 out of 293 cases (13%), resulting in a postoperative air - bone gap of 13 9 db (mean air - bone gap closure 17 9 db). There is no distinct group undergoing canal - wall - down mastoidectomy for cholesteatoma using allograft ossicles in this study, which again makes this difficult to compare with other studies . The average closure of the air - bone gap is 18.9 db, and 86% of patients had a postoperative air - bone gap of 20 db or less . Our outcomes gained from a single - stage procedure exceed the air - bone gap closures reported for porp in some of the larger studies of tympanoplasty [7, 8]. They also compare favourably to results achieved in canal - wall - down mastoidectomy [9, 10, 13]. Moreover, the majority of operations in these studies were performed for chronic suppurative otitis media or were secondary procedures, and canal - wall - down mastoidectomy was either performed as a separate stage or not at all . In addition to potentially advantageous hearing benefit, allogeneic ossicular grafts integrate into the middle ear and rarely extrude, with historic failure rates between 4 and 8% and more recent extrusion rates of 0% . Extrusion rates between 4 - 5% [8, 10] and 7% have been reported in studies using prostheses . No extrusion was observed in our series but, due to its low group size, a conclusion on graft extrusion rates is not possible . We demonstrate that tutoplast processed malleus restores hearing in 7 patients undergoing canal - wall - down mastoidectomy and tympanoplasty for cholesteatoma.we recommend consideration of tutoplast processed malleus in cases of cholesteatoma where autologous material cannot be used and where a single - step operative procedure to eradicate cholesteatoma with concomitant reconstruction of the middle ear is desired.in this small study with a 25-month follow - up, no graft extrusion or other complications were observed, and we are encouraged to offer allograft ossicular reconstruction as an alternative to ossicular prostheses to our patients undergoing mastoid exploration for cholesteatoma who wish to have ossiculoplasty but who have insufficient autologous ossicles . We demonstrate that tutoplast processed malleus restores hearing in 7 patients undergoing canal - wall - down mastoidectomy and tympanoplasty for cholesteatoma . We recommend consideration of tutoplast processed malleus in cases of cholesteatoma where autologous material cannot be used and where a single - step operative procedure to eradicate cholesteatoma with concomitant reconstruction of the middle ear is desired . In this small study with a 25-month follow - up, no graft extrusion or other complications were observed, and we are encouraged to offer allograft ossicular reconstruction as an alternative to ossicular prostheses to our patients undergoing mastoid exploration for cholesteatoma who wish to have ossiculoplasty but who have insufficient autologous ossicles.
These microdeletions often occur between low copy repeats and are commonly due to liability to unequal crossing over . It was recently shown that the well known classical microdeletion syndromes such as digeorge and williams syndromes account for about 5% of all patients with mental retardation . Besides the detection of known microdeletion syndromes, microarray - based comparative genomic hybridization (array cgh) or molecular karyotyping recently led to the discovery of novel recurrent microdeletion syndromes such as 17q21.31 and 15q24 [12, 20, 21]. To date 14 patients have been reported who have interstitial deletions at 15q22-q24 [1, 3, 4, 7, 13, 20, 22], but only three of them are restricted to chromosomal band 15q24 [4, 20]. So far, only a few of these cases have been molecularly characterized in order to determine the size of the affected regions . Despite considerable phenotypic variability, patients with 15q24 microdeletion syndrome share common features including global developmental delay, hypotonia, and genital abnormalities in males . In order to further delineate the 15q24 genotype phenotype correlation, a novel patient with 15q24 microdeletion detected by array cgh the further course of pregnancy was uneventful and delivery by caesarean section was at 38 weeks of gestation after preterm hydrorrhoea . Physical examination at the age of 10 years revealed a height of 132.5 cm (p50), weight of 35.7 kg (p5075) and head circumference of 53.5 cm (p75). Our patient presented with developmental retardation, craniofacial dysmorphism, truncal obesity, and micropenis (fig . He showed lumbar hyperlordosis, mild genua valga, and joint hypermobility . At the age of 6 months coordination disturbances note truncal obesity, widely spaced, inverted nipples, as well as coarse face with high forehead, broad medial eyebrows, periorbital fullness, slight antimongoloid slant, deep set eyes, hypoplastic nostrils, long philtrum and full cheeks . C, d hands show mild brachydactyly, clinodactyly of fourth finger (left hand) and broad thumbs . E note hypoplasia of the distal phalanges, short mesophalanges, delayed carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb a, b patient at the age of 10 years . Note truncal obesity, widely spaced, inverted nipples, as well as coarse face with high forehead, broad medial eyebrows, periorbital fullness, slight antimongoloid slant, deep set eyes, hypoplastic nostrils, long philtrum and full cheeks . C, d hands show mild brachydactyly, clinodactyly of fourth finger (left hand) and broad thumbs . E note hypoplasia of the distal phalanges, short mesophalanges, delayed carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb speech development was retarded and a mild mental retardation (iq 65) was observed . Nasal speech and inguinal and umbilical hernias as well as unilateral retentio testis inguinalis were surgically corrected at age 4 years . Patient s hands show mild brachydactyly, clinodactyly of fourth finger (left hand), and broad thumbs (fig . Radiological investigations including cranial ct were normal except for hand radiographs disclosing hypoplasia of distal phalanges, short mesophalanges, delay of carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb (fig . Echocardiography as well as renal ultrasound and neurophysiological investigations (i.e. Nerve velocity, eeg, ecg) did not reveal any pathological findings . In repeated metabolic screenings triglycerides and he was only able to walk up to 100 m. clinically, a metabolic defect was suspected . However, examinations concerning mucopolysaccharidoses and cdg syndrome disclosed no abnormalities . All serum parameters including growth related factors (igf1, igfbp3) fsh (18 u / l) and testosterone (1.4 ng / ml) values were normal for the patient s age . This study was approved by the ethics committee of the charit, universittsmedizin berlin, and all individuals gave their informed consent prior to their inclusion in the study . The further course of pregnancy was uneventful and delivery by caesarean section was at 38 weeks of gestation after preterm hydrorrhoea . Physical examination at the age of 10 years revealed a height of 132.5 cm (p50), weight of 35.7 kg (p5075) and head circumference of 53.5 cm (p75). Our patient presented with developmental retardation, craniofacial dysmorphism, truncal obesity, and micropenis (fig . He showed lumbar hyperlordosis, mild genua valga, and joint hypermobility . At the age of 6 months coordination disturbances note truncal obesity, widely spaced, inverted nipples, as well as coarse face with high forehead, broad medial eyebrows, periorbital fullness, slight antimongoloid slant, deep set eyes, hypoplastic nostrils, long philtrum and full cheeks . C, d hands show mild brachydactyly, clinodactyly of fourth finger (left hand) and broad thumbs . E note hypoplasia of the distal phalanges, short mesophalanges, delayed carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb a, b patient at the age of 10 years . Note truncal obesity, widely spaced, inverted nipples, as well as coarse face with high forehead, broad medial eyebrows, periorbital fullness, slight antimongoloid slant, deep set eyes, hypoplastic nostrils, long philtrum and full cheeks . C, d hands show mild brachydactyly, clinodactyly of fourth finger (left hand) and broad thumbs . E note hypoplasia of the distal phalanges, short mesophalanges, delayed carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb speech development was retarded and a mild mental retardation (iq 65) was observed . Nasal speech and a hoarse voice were apparent . Prominent veins were evident on the trunk . Skin laxity was noted especially in the face and hands . Inguinal and umbilical hernias as well as unilateral retentio testis inguinalis were surgically corrected at age 4 years . Patient s hands show mild brachydactyly, clinodactyly of fourth finger (left hand), and broad thumbs (fig . Radiological investigations including cranial ct were normal except for hand radiographs disclosing hypoplasia of distal phalanges, short mesophalanges, delay of carpal ossification (corresponding to the state of a 3 year old child), irregular epiphyses of the second and fifth mesophalanges and of the distal phalanx of the thumb (fig . Echocardiography as well as renal ultrasound and neurophysiological investigations (i.e. Nerve velocity, eeg, ecg) did not reveal any pathological findings . In repeated metabolic screenings triglycerides and he was only able to walk up to 100 m. clinically, a metabolic defect was suspected . However, examinations concerning mucopolysaccharidoses and cdg syndrome disclosed no abnormalities . All serum parameters including growth related factors (igf1, igfbp3) fsh (18 u / l) and testosterone (1.4 ng / ml) values were normal for the patient s age . This study was approved by the ethics committee of the charit, universittsmedizin berlin, and all individuals gave their informed consent prior to their inclusion in the study . Karyotyping of gtg - banded chromosomes from lymphocytes at 450 bands resolution was performed according to standard procedures . Bac clones rp11414j4 (genomic position 72.772.9 mb) and rp1194p14 (genomic position 75.275.3 mb) were obtained from the rzpd (deutsches ressourcenzentrum fr genomforschung, berlin, germany). Bac dna was fluorescently labelled using nick translation and hybridized to metaphase spreads of the patient s and parents lymphocytes using standard procedures . Centromere probe, cep15 (d15z1; vysis, downers grove, il), labelled with spectrum green was used as control probe . Hybridization of the commercial probes for snrpn (prader - willi / angelman region) and tuple1 (digeorge / vcfs region) were performed on metaphase spreads of the patient as recommended by the manufacturer (vysis, downers grove, il). Array cgh was carried out using a submegabase whole human genome tiling path bac array consisting of the human 32k re - array set (http://bacpac.chori.org/phumanminset.htm) [14, 18], the 1 mb sanger clone set (wellcome trust sanger institute), and a set of 390 subtelomeric clones (generated in the course of the eu initiative costb19: molecular cytogenetics of solid tumours). Array cgh was performed as described previously and 33028 bacs were included in the analysis . The log2ratio of test to reference was calculated and plotted according to chromosomal position of the clones . Copy number gains and losses were determined by a conservative threshold of 0.3 and 0.3, respectively . Profile deviations consisting of three or more neighbouring bac clones were considered as genomic aberrations and were further evaluated by fish, unless they coincided with a published variant as listed in the database of genomic variants (http://projects.tcag.ca/variation/; version oct . 11, 2006). For visualizing the content of low copy repeats in the ratio plots (fig . 2a, b), each bac clone was classified into one out of seven categories and colour - coded as described previously . The red and green lines indicate the log2ratio thresholds 0.3 (loss) and 0.3 (gain), respectively . Note: the aberration close to the centromere on 15q11.2 constitutes a known cnv indicated by the turquoise colour of the spots . The microdeletion is flanked by low - copy repeats indicated by the turquoise colour of the spots . Horizontal bars represent dna copy number variants as listed in the database of genomic variants (dec . The red and green lines indicate the log2ratio thresholds 0.3 (loss) and 0.3 (gain), respectively . Note: the aberration close to the centromere on 15q11.2 constitutes a known cnv indicated by the turquoise colour of the spots . The microdeletion is flanked by low - copy repeats indicated by the turquoise colour of the spots . Horizontal bars represent dna copy number variants as listed in the database of genomic variants (dec . Karyotyping of gtg - banded chromosomes from lymphocytes at 450 bands resolution was performed according to standard procedures . Bac clones rp11414j4 (genomic position 72.772.9 mb) and rp1194p14 (genomic position 75.275.3 mb) were obtained from the rzpd (deutsches ressourcenzentrum fr genomforschung, berlin, germany). Bac dna was fluorescently labelled using nick translation and hybridized to metaphase spreads of the patient s and parents lymphocytes using standard procedures . Centromere probe, cep15 (d15z1; vysis, downers grove, il), labelled with spectrum green was used as control probe . Hybridization of the commercial probes for snrpn (prader - willi / angelman region) and tuple1 (digeorge / vcfs region) were performed on metaphase spreads of the patient as recommended by the manufacturer (vysis, downers grove, il). Array cgh was carried out using a submegabase whole human genome tiling path bac array consisting of the human 32k re - array set (http://bacpac.chori.org/phumanminset.htm) [14, 18], the 1 mb sanger clone set (wellcome trust sanger institute), and a set of 390 subtelomeric clones (generated in the course of the eu initiative costb19: molecular cytogenetics of solid tumours). Array cgh was performed as described previously and 33028 bacs were included in the analysis . The log2ratio of test to reference was calculated and plotted according to chromosomal position of the clones . Copy number gains and losses were determined by a conservative threshold of 0.3 and 0.3, respectively . Profile deviations consisting of three or more neighbouring bac clones were considered as genomic aberrations and were further evaluated by fish, unless they coincided with a published variant as listed in the database of genomic variants (http://projects.tcag.ca/variation/; version oct . 11, 2006). For visualizing the content of low copy repeats in the ratio plots (fig . 2a, b), each bac clone was classified into one out of seven categories and colour - coded as described previously . The red and green lines indicate the log2ratio thresholds 0.3 (loss) and 0.3 (gain), respectively . Note: the aberration close to the centromere on 15q11.2 constitutes a known cnv indicated by the turquoise colour of the spots . The microdeletion is flanked by low - copy repeats indicated by the turquoise colour of the spots . Horizontal bars represent dna copy number variants as listed in the database of genomic variants (dec . The red and green lines indicate the log2ratio thresholds 0.3 (loss) and 0.3 (gain), respectively . Note: the aberration close to the centromere on 15q11.2 constitutes a known cnv indicated by the turquoise colour of the spots . The microdeletion is flanked by low - copy repeats indicated by the turquoise colour of the spots . Horizontal bars represent dna copy number variants as listed in the database of genomic variants (dec . Standard karyotyping of g - banded chromosomes did not reveal any chromosomal abnormalities . According to the fish analyses there was no evidence for a deletion of the prader - willi syndrome region or a microdeletion 22q11.2 . Therefore, we performed array cgh analysis with the patient s genomic dna using a submegabase resolution whole genome tiling path array to detect microdeletions or duplications . A submicroscopic interstitial deletion on chromosome 15q24.1-q24.3 represented by 33 bac clones was detected (fig . 2). The size of the deletion is approximately 3.7 mb extending from 72.2 mb to 75.9 mb (ensembl release 43; feb . 2007). According to the array cgh data the two breakpoints are located between rp11247c02 and rp11672a20 (proximal breakpoint) and rp11758j16 and rp11745i11 (distal breakpoint). Both breakpoints were found to be enriched for low copy repeats, which, according to the segmental duplication database (http://humanparalogy.gs.washington.edu/), share a 49.9 kb segment with 95% sequence similarity (no .9417), and the proximal breakpoint coincided with published dna copy number variants . Given this high sequence similarity the deletion most likely emerged through nonallelic homologous recombination (nahr) of the two breakpoint flanking low copy repeats (lcrs), a mechanism associated with genomic rearrangements . The deleted region comprises 39 annotated genes (based on v39, june 2006, ensembl genome browser; (http://www.ensembl.org). In addition, a deletion on 15q11.2 was observed which represents a cnv listed in the database of genomic variants and was therefore considered as a benign variant without pathological relevance . For verification of array cgh data by fish we used a set of bac clones mapping to 15q24 . As expected from the array data (log2ratio <0.3) only one signal on the patient s metaphases was detected for bac clones rp11414j4 and rp1194p14 (data not shown). Fish on metaphase chromosomes of the patient s parents and his brother detected two signals on chromosome 15 for all probes investigated and karyotypes were normal (data not shown). Recently, sharp et al . Described a recurrent microdeletion of 15q24 in four cases ranging in size from 1.73.9 mb characterised by molecular cytogenetic techniques . The minimal deletion region was delineated to be 1.7 mb in size (72.1573.85 mb). The patient presented here constitutes an interstitial microdeletion at 15q24 which shares common proximal (bp1) and distal breakpoints (bp3) with cases imr349 and c45/06 of the publication by sharp et al . (fig . 3). Since all deletions occurred in the maternal lineage the possibility of an underlying imprinting effect was pointed out . A clinical comparison of our patient to these patients from the literature is shown in table 1 . The phenotype between patients with microdeletion 15q24 is variable; however, our patient shares all of the major features described for 15q24 microdeletion syndrome by sharp et al ., i.e. Unusual facial features (high frontal hairline, broad medial eyebrows, downslanted palpebral fissures, and long philtrum), developmental delay, digital abnormalities, genital abnormalities, and loose connective tissue (manifestation: joint laxity, inguinal hernia). However, our patient does not present with microcephaly (3/4), prenatal and postnatal growth deficiency (3/4), hearing problems (2/4), or bowel atresia (2/4). The critical region is located between the recurrent breakpoints bp1 and bp2table 1phenotypic comparison of patients with interstitial 15q24 deletion present casecushman et al . C45/06male, 10 yearsmale, 23 monthsfemale, 11 yearsmale, 14 yearsmale, 15 yearsmale, 33 yearsmale, 14 yearsshort stature+++hypotonia+++ndndnd+developmental delay / mental retardation+++++mild+microcephaly+++strabismus++nd++hypertelorismnd++++micrognathia+ndpalate anomaly++full lower lip++++ear anomaly+nd+++epicanthal folds+nd+high frontal hair line+nd++++broad medial eyebrows+nd++++long / smooth philtrum+nd++++downslanted palpebral fissures+ndnd+++herniainguinal and umbilicalnddiaphragmatic, inguinal and hiatalgenital anomalymicropenismicropenis, small scrotumndcoronal hypospadias with phimosismild hypospadiashypospadiasmusculoskeletal anomalyjoint laxity, clinodactyly of fourth finger (left hand), brachymeso - phalangy vclinodactyly of fifth fingers, distal tapering of fingersndjoint laxity, long slender fingers, proximally implanted thumbs, sandal gap, scoliosisjoint laxity, hypoplastic right thumb, contractures of fingers, pes cavus, camptodactyly of toesjoint laxity, small hands, distal brachy - dactyly, mild cutaneous syndactylyproximal implanted thumbs, scoliosis+ feature present, feature absent, nd feature not described schematic representation of 15q24 region . The three recurrent breakpoints as delineated by sharp et al . As well as the deletion sizes of the presented case and published cases are indicated . The critical region is located between the recurrent breakpoints bp1 and bp2 phenotypic comparison of patients with interstitial 15q24 deletion + feature present, feature absent, nd feature not described among the deleted genes are several coding for enzymes, e.g. Mannose phosphate isomerase (mpi), alpha - mannosidase 2c1 (man2c1), and alpha - subunit of electron transfer flavoprotein (etfa), as well as the cytochrome p450 side - chain cleavage enzyme (p450scc) which catalyzes the first step in steroidogenesis . Several of these enzymes are involved in the synthesis, export, and degradation of glycoproteins . Mutations in the mpi gene (mim 154550) cause congenital disorder of glycosylation type ib (cdg ib) [9, 19]. Man2c1 (mim 154580) is involved in the degradation of oligomannosides derived from dolichol intermediates, the degradation of newly synthesized glycoproteins, and in the processing of free oligosaccharides that are formed in the cytosol . Secretory carrier membrane proteins (scamp2, scamp5) are a family of post - golgi and golgi membrane proteins which have been implicated in vesicular trafficking . Scamps interact with nhe7 (na / h exchanger) and participate in the shuttling and retrieval of nhe7 from peripheral recycling endosomes to the trans - golgi network . Thus, we cannot exclude uncovering of a recessive condition which could result in a homozygous loss of alleles which leads to deficiency of one of the other enzymes in this region . The cholesterol side chain cleavage enzyme p450scc is responsible for the conversion of cholesterol to pregnenolone in mitochondria . So far, only one patient with a heterozygous mutation in p450scc has been described . This patient had a late - onset form of congenital lipoid adrenal hyperplasia without the characteristic enlargement of the adrenals . A complete absence of p450scc activity causes congenital adrenal insufficiency, complete 46,xy sex reversal, and severe adrenal failure [8, 10]. Congenital adrenal hyperplasia is a severe disorder with an impairment of adrenal and gonadal steroid synthesis . Although, we did not observe such a severe phenotype, and current steroid levels are normal in our patient . The haploinsufficiency of p450scc might contribute to the genital abnormalities in our patient and other affected male patients (microphallus with small scrotum, undescended testes, hypospadias) with deletions spanning the p450scc locus on 15q24.1 [4, 20]. In summary, the patient presented here constitutes an interstitial microdeletion at 15q24 and represents another case of the recently described 15q24 microdeletion syndrome . This is another example of the impact of array cgh as a diagnostic tool in clinical medicine.
The mental rotation task is a well - established paradigm to study the cognitive process of mentally rotating objects . Typically, observed reaction time (rt) profiles show an increase in rt for increased angle of rotation indicating that participants mentally rotate stimuli to the upward position (shepard and metzler 1971; parsons 1994; jeannerod and decety 1995). They used a task in which participants had to judge whether differently rotated 3d cube figures were identical or mirror reversed images of that figure (shepard and metzler 1971). It was found that rts increased linearly with increasing angle of rotation . Other mental rotation studies used different paradigms in which the participants had to judge object or body part laterality, referred to as a laterality judgment task, first introduced by sekiyama (1982). He found that rts for judging hand laterality also increased with increased angles of rotation . However, rt did not increase linearly but quadratic and was not symmetrical about 180 as is the case for 3d cube figures . This asymmetric quadratic rt profile was interpreted as evidencing kinesthetic influence of the laterality judgment of hands (sekiyama 1982). It was postulated that participants engage in an embodied mental process in which biomechanical constraints influence the duration of the mental rotation process (sekiyama 1982; parsons 1987; jeannerod 1994; decety 1996a; shenton et al . The influence of kinesthetic aspects or biomechanical constraints was further postulated by parsons (1994). He showed that the time needed to actually rotate the own hand to an identical position as the displayed rotated hand corresponds to the time needed for a hand laterality judgment (parsons 1987; parsons 1994). More specifically, he showed that hand rotations away of the mid - sagittal plane (i.e., lateral rotation) resulted in larger rts than hand rotation toward the mid - sagittal plane (i.e., medial rotation) for both overt as imagined movement . Thus, the rt profiles of imagined hand rotations were subject to the same biomechanical constraints experienced for overt movement . Laterality judgments of hands that are subject to biomechanical constraints make it conceivable that participants engage in embodied cognitive processing . This embodied processing is referred to as motor imagery (mi) (de lange et al ., rt differences between lateral- and medial - rotated stimuli can be used as excellent measure to test for engagement in mi . When engaged in mi, participants mentally plan and perform a movement from a first person perspective without overtly performing the movement and without sensory feedback (decety 1996a, b). The embodied nature of mi in a hand laterality judgment task was further evidenced by showing that rt profiles change as a consequence of changing the participant s posture (parsons 1994; sirigu and duhamel 2001; de lange et al . 2007; ionta et al . 2007; ionta and blanke 2009). Extending these findings, children at 57 years of age were also found to engage in mi in a hand laterality judgment task, possibly even more than adults (funk et al . 2005). Visual imagery (vi), on the other hand, encompasses simulating executing a movement from a third person perspective (e.g., steenbergen et al . 2007). Thus, vi is not subject to biomechanical constrains and as such not an embodied process . Studies using hand laterality judgment tasks have not unequivocally shown to induce an mi strategy . As an example, we showed that participants with congenital alterations of posture on one side of the body, i.e., participants with hemiparetic cerebral palsy, lacked the presumed influence of posture on laterality judgments (steenbergen et al ., it was hypothesized that these participants would show a different rt profile for laterality judgments of their affected versus their non - affected hand, but this was not evidenced by the data . In the study by lust et al . (2006), groups of adults, healthy children and children suffering from developmental disorder were tested on their engagement in mi during a hand laterality judgment task with only hand stimuli shown from the back . Their results showed that rts for both groups of children were subject to biomechanical constraints, as evident by the increased rt for difficult hand postures when compared to hand postures that are easier to adopt . However, the adult group showed no effect of biomechanical constraints and hence no engagement in mi (lust et al . We propose that the differences observed in literature on the effects of posture or, more general, biomechanical constraints on laterality judgments are due to the particulars of the stimulus set . The argument for this is following . (2006) and our recent study, the stimulus set was largely the same . That is, all displayed pictures of hands were presented from a back view perspective in different angles of rotation . In contrast, studies showing strong effects of posture on rt used stimulus sets in which hands were presented from different viewpoints . See table 1 for a short overview of literature on the hand laterality judgment task . Obviously, the use of these different perspectives from which the hand stimuli are presented yielded an increase in number of rotational axes and, as a consequence, an increase in overall task difficulty . Another factor that differed among studies and which may potentially cause the differential results is the number of rotational steps of the displayed stimuli, see table 1 . However, the number of rotational steps appears to have only a marginal influence because in the study by steenbergen et al . (2007) eighteen different rotational angles were used but no engagement in mi was found . Other studies, however, do show engagement in mi with as little as four angles of rotation (de lange et al . 2006).table 1studies on hand laterality judgment taskauthoryearviewsteps of rotationmi engagementionta and blanke2009hands and feet, back, palm, thumb, little finger6yesde lange et al.2008back, palm7yesionta et al.2007as ionta and blanke (2009)6yessteenbergen et al.2007back18nohelmich et al.2007back, palm8yesde lange et al.2006back, palm4yeslust et al.2006back8yes (children)no (adults)thayer and johnson2006back, palm6yessauner et al.2006back, palm8yesfunk et al.2005back, palm4yesshenton et al.2004back, palm6yesparsons1994back, palm, thumb, little finger, front finger, back palm12yesparsons1987back, palm, thumb, little finger, front finger, back palm12yessekiyama1982thumb, little finger, palm8yesnote studies on hand laterality judgment task and results on mi engagement . Both studies using only back view hand stimuli show no mi engagement (for adults). In contrast, studies using multiple viewpoints of hands within their stimulus set do show engagement in mi studies on hand laterality judgment task note studies on hand laterality judgment task and results on mi engagement . Both studies using only back view hand stimuli show no mi engagement (for adults). In contrast, studies using multiple viewpoints of hands within their stimulus set do show engagement in mi given such diverging results, we hypothesize that the use of more rotational axes within a stimulus set facilitates mi engagement . The argument for this is that simple tasks, in which only in - plane - rotated back view hand stimuli are used (i.e., one axis of rotation), may promote the use of an alternative strategy based on the combination of finger and thumb orientation to solve the task (lust et al . 2006). For more complex tasks, including for instance the use of both palm and back view hand stimuli, such a strategy will not suffice because one additional judgment on hand view is necessary next to judging hand laterality from the finger and thumb orientation . The inclusion of multiple rotational axes, and thus judgments, may therefore promote engagement in mi . That there is a neurophysiological difference between two - dimensional (2d) and three - dimensional 2007) showed that task difficulty enhanced by rotation dimensionality plays an important role in the selection of a motor strategy . They used three - dimensional cube figures, similar to shepard and metzler (1971). Participants were presented a set of these figures rotated in 2d (i.e., in - plane) or 3d (i.e., in - plane and in - depth). The results showed that the right superior parietal lobule was activated for 2d - rotated stimuli . In the case of 3d - rotated stimuli, the right dorsal premotor cortex (pmd) these findings indicate that 2d and 3d rotation of cube figures activate different brain regions and may therefore be (partly) different neuronal or cognitive processes . In the present study, we used three different conditions in which the number of axes of rotation increased from, respectively, only 1 axis to 3 axes of rotation . We expected an increasing difference in rts between lateral- and medial - rotated pictures of hands as a function of the number of axes of rotation . More specifically, for 1 axis of rotation, we expected to find no or marginal engagement in mi which will be evidenced by a marginal difference in rt between medial and lateral rotation, whereas increasing engagement in mi (i.e., increasing difference in rt between lateral- and medial - rotated hand pictures) is expected in the conditions with 2- and 3 axes of rotation . Twelve healthy right - handed participants were included in the present study (8 women, age 22.5 3.7 years, mean sd). The study was approved by the local ethics committee, and all participants gave written informed consent prior to the experiment, in accordance with the helsinki declaration . We used a custom made 3d hand model designed in a 3d image software package (autodesk maya 2009, usa). From this realistic model, we constructed all stimuli that were used in the experiments . All stimuli were displayed on a 19 lcd computer screen, at a distance of approximately 70 cm from the participants eyes, resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes, resulting in three different rotational directions, namely: in - plane, longitudinal (referred to as 1 . Three different sets of stimuli were used, namely: set-1 containing only in - plane - rotated stimuli, set-2 with both in - plane- and longitudinal - rotated stimuli, and set-3 containing in - plane-, longitudinal-, and in - depth - rotated stimuli . With 0 of in - plane angular disparity as the upright position with the fingers pointing upward, see fig . 1 . Left and right hands were mirror images of each other but otherwise identical.fig . 1shown are all used hand stimuli for set-1: upper row, left column; set-2: upper row; set-3: all stimuli . Angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1: upper row, left column; set-2: upper row; set-3: all stimuli . Angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1, we used back view left and right hand stimuli in six different angles of in - plane rotation (i.e., 0, 60, 120, 180, 240, and 300), resulting in 12 different stimuli, see upper left cell of fig . 1 . For set-2, we used both back and palm view (i.e., two longitudinal rotational steps) stimuli of hands . All other aspects were identical to set-1, resulting in 24 different stimuli, see upper row of fig . 1 . In set-3, our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations (i.e., 0, 60, 300) with 0 parallel to the vertical plane, resulting in 72 different stimuli, see fig . 1 . All stimuli were repeated three times resulting in 36 (12 * 3), 72 (24 * 3) and 216 (72 * 3) stimuli for set-1, -2, and -3, respectively . All three conditions were preceded by a test of 24 stimuli to familiarize the participants with the task . Stimuli were presented using custom - developed software in presentation (neurobehavioral systems, albany, usa). Participants had to respond by pressing the left button with their left hand for left - hand stimuli and the right button with their right hand for right - hand stimuli . Participants were instructed to judge the laterality of the hand as fast and as accurate as possible, without explicit instructions on how to solve the task . These seven blocks comprise: 1 block of set-1 stimuli, 3 blocks of set-2 stimuli, and 3 blocks of set-3 stimuli . All sets were presented blockwise and sequential, preventing mixing of blocks of the difficulty conditions over the experiment . Reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis (total loss 3.2% of trials); these upper and lower boundaries are based on similar studies using a hand laterality judgment task (sekiyama 1987; parsons 1994; ionta et al . Right hand and vice versa and amounted to a total 6.4% of all trials . For analyses purposes, the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation (dor) in order to measure engagement in mi . Medial - rotated stimuli consisted of right hand 240 and 300 and left hand 60 and 120 in - plane - rotated stimuli . Lateral - rotated stimuli consisted of right hand 60 and 120 and left hand 240 and 300 in - plane - rotated stimuli . First, we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . To this aim, we used an anova with the following design: 2 within - subject factors (set, angle), with 3 levels for set (set-1, -2, and -3) and 4 levels for angle (0, 60, 120, and 180). With 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . A significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation (shepard and metzler 1971). Then, we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design: 2 within - subject factors (set, dor), with 3 levels for set (set-1, -2 and -3) and 2 levels for dor (lateral rotation, medial rotation). A significant set by dor interaction would indicate that the influence of biomechanical constraints (as measured with dor) differs between sets . Ad hoc analysis was bonferroni corrected, and alpha level was set at p = 0.05 . Twelve healthy right - handed participants were included in the present study (8 women, age 22.5 3.7 years, mean sd). The study was approved by the local ethics committee, and all participants gave written informed consent prior to the experiment, in accordance with the helsinki declaration . We used a custom made 3d hand model designed in a 3d image software package (autodesk maya 2009, usa). From this realistic model, we constructed all stimuli that were used in the experiments . All stimuli were displayed on a 19 lcd computer screen, at a distance of approximately 70 cm from the participants eyes, resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes, resulting in three different rotational directions, namely: in - plane, longitudinal (referred to as 1 . Three different sets of stimuli were used, namely: set-1 containing only in - plane - rotated stimuli, set-2 with both in - plane- and longitudinal - rotated stimuli, and set-3 containing in - plane-, longitudinal-, and in - depth - rotated stimuli . With 0 of in - plane angular disparity as the upright position with the fingers pointing upward, see fig . 1 . Left and right hands were mirror images of each other but otherwise identical.fig . 1shown are all used hand stimuli for set-1: upper row, left column; set-2: upper row; set-3: all stimuli . Angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1: upper row, left column; set-2: upper row; set-3: all stimuli . Angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1, we used back view left and right hand stimuli in six different angles of in - plane rotation (i.e., 0, 60, 120, 180, 240, and 300), resulting in 12 different stimuli, see upper left cell of fig . 1 . For set-2, we used both back and palm view (i.e., two longitudinal rotational steps) stimuli of hands . All other aspects were identical to set-1, resulting in 24 different stimuli, see upper row of fig . 1 . In set-3, our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations (i.e., 0, 60, 300) with 0 parallel to the vertical plane, resulting in 72 different stimuli, see fig . 1 . All stimuli were repeated three times resulting in 36 (12 * 3), 72 (24 * 3) and 216 (72 * 3) stimuli for set-1, -2, and -3, respectively . All three conditions were preceded by a test of 24 stimuli to familiarize the participants with the task . Stimuli were presented using custom - developed software in presentation (neurobehavioral systems, albany, usa). Participants had to respond by pressing the left button with their left hand for left - hand stimuli and the right button with their right hand for right - hand stimuli . Participants were instructed to judge the laterality of the hand as fast and as accurate as possible, without explicit instructions on how to solve the task . These seven blocks comprise: 1 block of set-1 stimuli, 3 blocks of set-2 stimuli, and 3 blocks of set-3 stimuli . All sets were presented blockwise and sequential, preventing mixing of blocks of the difficulty conditions over the experiment . Reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis (total loss 3.2% of trials); these upper and lower boundaries are based on similar studies using a hand laterality judgment task (sekiyama 1987; parsons 1994; ionta et al . Right hand and vice versa and amounted to a total 6.4% of all trials . For analyses purposes, the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation (dor) in order to measure engagement in mi . Medial - rotated stimuli consisted of right hand 240 and 300 and left hand 60 and 120 in - plane - rotated stimuli . Lateral - rotated stimuli consisted of right hand 60 and 120 and left hand 240 and 300 in - plane - rotated stimuli . First, we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . To this aim, we used an anova with the following design: 2 within - subject factors (set, angle), with 3 levels for set (set-1, -2, and -3) and 4 levels for angle (0, 60, 120, and 180). With 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . A significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation (shepard and metzler 1971) then, we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design: 2 within - subject factors (set, dor), with 3 levels for set (set-1, -2 and -3) and 2 levels for dor (lateral rotation, medial rotation). A significant set by dor interaction would indicate that the influence of biomechanical constraints (as measured with dor) differs between sets . Ad hoc analysis was bonferroni corrected, and alpha level was set at p = 0.05 . The total number of incorrect responses (i.e., 6.4% of all trials) corresponds to former studies (de lange et al . 2006; ionta et al . The amount of erroneous responses differed significantly [f(2,20) = 4.692, p <0.05, = 0.319]. Within set-1, set-2, and set-3, the amount of errors was 3.3, 4.6, and 7.3% of the number of trials per set, respectively . The mental rotation analysis revealed a significant main effect for both set [f(2,22) = 43.380, p <0.001, = 0.798] and angle [f(3,33) = 38.772, p <0.001, = 0.779]. Additionally, a significant interaction of set by angle [f(6,66) = 7.716, p <0.001, = 0.412] was found . Ad hoc analysis revealed a significant increase in rt with increasing angle of rotation for all three sets . Set-1 [f(3,33) = 37.732, p <0.001, = 0.774], set-2 [f(3,33) = 20.941, p <0.001, = 0.656], and set-3 [f(3,33) = 28.789, p <0.001, = 0.724]. However, the mean rt for 0 rotated stimuli in set-3 was larger than the mean rt for 60 rotated stimuli . This larger rt for 0 was caused by the in - depth - rotated hand stimuli . Overall, rts increased significantly with increasing angle of rotation for all three sets, see fig . 2reaction times for all three sets, mirrored at 180 (i.e., 60 represents average rt for 60 and 300 rotated hand stimuli). Error - bars indicate standard error of the mean (sem) reaction times for all three sets, mirrored at 180 (i.e., 60 represents average rt for 60 and 300 rotated hand stimuli). Error - bars indicate standard error of the mean (sem) the analysis on the influence of biomechanical constraints showed a significant main effect for set [f(2,22) = 33.422, p <0.001, = 0.75] and dor [f(1,11) = 9.890, p <0.01, = 0.47]. The set effect was accounted for by the increase in rt for increasing number of axes of rotation, 848 ms, 1,123 ms, and 1,214 ms for set-1, set-2, and set-3, respectively . Set-1 differed significantly from both set-2 and set-3 (p <0.001 and p <0.001, respectively). Set-2 and set-3 did not differ significantly from each other (p = 0.168). The dor effect was accounted for by the increased rt for laterally rotated stimuli (1,117 ms) compared to medially rotated stimuli (1,006 ms). In order to support our hypothesis about increasing difference in rt between laterally and medially rotated stimuli, we expected a significant interaction between set and direction of rotation (dor). The anova resulted in a significant interaction of set by dor [f(2,22) = 8.196, p <0.01, = 0.43], which was accounted for by the increasing difference in rts between laterally and medially rotated stimuli over the stimulus sets, resulting in significant simple dor effects in set-2 [f(1,11) = 5.964, p <0.05, = 0.35] and set-3 [f(1,11) = 18.005, p <0.01, = 0.62]. Crucially, the simple dor effect of set-1 was not significant (p = 0.578), see fig . 3 . The simple dor effects differed significantly between set-1 and set-2 [f(1,11) = 6.276, p <0.05, = 0.363], and set-1 and set-3 [f(1,11) = 13.344, p <0.01, = 0.548] but not between set-2 and set-3 (p = 0.198).fig . Lateral rotation indicates rotations away from the mid - sagittal plane, and medial rotation indicates rotations toward the mid - sagittal plane . As can be seen, the significant interaction of set by dor (p <0.01) as represented by the differences in rts between lateral and medial rotation (i.e., dor) increases with increasing number of axes of rotation . * indicate significance at the p <0.05 level, * * indicate significance at the p <0.01 level . Error - bars indicate standard error of the mean (sem) reaction times for all 3 sets divided into lateral rotation and medial rotation . Lateral rotation indicates rotations away from the mid - sagittal plane, and medial rotation indicates rotations toward the mid - sagittal plane . As can be seen, the significant interaction of set by dor (p <0.01) as represented by the differences in rts between lateral and medial rotation (i.e., dor) increases with increasing number of axes of rotation . * indicate significance at the p <0.05 level, * * indicate significance at the p <0.01 level . Error - bars indicate standard error of the mean (sem) to control for the differences in mean rts between the sets and its possible influence on the obtained dor effects of the sets, we conducted an additional anova with the data being normalized into the range of [01]. The anova contained the following design: 2 within - subject factors (set and dor) with 3 levels for set (set-1, set-2 and set-3) and 2 levels for dor (lateral rotation, medial rotation). The results replicate the results with the non - normalized data except for an insignificant set effect (p = 0.074). The lack of significant set effect indicates that the normalization was effective as it reduced the differences in absolute value of the mean rts between the sets . As we only obtained a significant dor effect in the sets including palm view stimuli, one might argue that only the palm view stimuli accounted for the obtained dor effect in set-2 and set-3 . To test whether a dor effect is also present for in - plane - rotated back view stimuli (i.e., set-1 stimuli) within set-2 and set-3, we performed an anova identical to the anova testing the influence of biomechanical constraints, described in the data analysis section . However, we now only included rt values of the set-1 stimuli in all sets . This test enables us to see whether a dor effect is also present for back view stimuli in set-2 and set-3 and as a consequence enables us to examine whether only palm view or also back view stimuli induce the use of mi, depending on the context in which they are embedded . Additionally, this test shows whether participants used the same strategy for in - plane - rotated back view hand (i.e., set-1) stimuli for all three sets or not . The anova revealed a significant main effect of set [f(2,22) = 21.694, p <0.001, = 0.664] and dor [f(1,10) = 4.999, p <0.05, = 0.312] and an interaction of set by dor [f(2,22) = 3.429, p 0.05, = 0.238]. The interaction was accounted for by a lack of significant dor effect for set-1 stimuli in set-1 (p = 0.578) and set-2 (p = 0.237) but a significant dor effect for set-1 stimuli within set-3 [f(1,11) = 8.620, p <0.05, = 0.439]. In this study, we investigated the influence of multiple angles of rotation in the stimulus set in a hand laterality judgment task on participants engagement in mi . We hypothesized that an increase in the number of axes of rotation facilitates an mi strategy . The number of axes of rotation cumulated from 1 (i.e., in - plane) to 2 and 3 axes over 3 separate stimulus sets . According to the low error rates in all sets, which are well below chance level and the increasing rt in all sets for increasing angle of rotation (see fig . 2), we may assume that participants did indeed mentally rotate the stimuli and were able to solve the mental rotation task accurately (shepard and metzler 1971; sekiyama 1987; parsons 1994; helmich et al . We showed increased rt differences between laterally and medially rotated hand stimuli (e.g., direction of rotation (dor) effect) with increasing number of axes of rotation (see fig . Thus, there is an increasing influence of biomechanical constraints on rt with increasing number of axes of rotation, which exemplifies increased engagement in mi (parsons 1994; lust et al . (containing only in - plane - rotated back view stimuli), no effect of dor was found . In contrast, both set-2 (in - plane - rotated, back- and palm view stimuli) and set-3 (in - plane- and in - depth - rotated, back- and palm view stimuli) revealed a significant dor effect . The dor effects in both the set-2 and set-3 conditions were accounted for by a smaller rt for medial compared to lateral rotations, which is in correspondence with literature (parsons 1987; parsons 1994). Additionally, the difference in obtained dor effects between the sets was not accounted for by the differences in absolute mean rt between the sets as the analysis on the normalized data shows the same results . The process of mi is dependent on embodied cognitive processing and therefore affected by both the desired end - state and the current position of one s body (jeannerod 1994; de lange et al . The desired end - state is the presented hand posture . Because mi is subject to biomechanical constraints, rts for lateral rotations of hands are prolonged compared to medial rotations if the participant engages in mi (parsons 1987; parsons 1994; decety 1996a). However, one might argue that participants memorized the set-1 stimuli together with their laterality, leading to fast responses and no engagement in mi for set-1 . This explanation of the results is plausible because the set-1 stimuli are seen in all three sets, therefore facilitating the memorizing of the stimuli . However, if participants indeed memorized the set-1 stimuli, this would result in a lack of use of mental rotation at all . 2), which was evident from the increase in rt with increasing angle of rotation . Hence another possible explanation might be that the simple fact of adding palm view stimuli to a stimulus set results in the obtained difference in rt between medially and laterally rotated stimuli irrespective of the use of back view stimuli . The results indicate that we obtained this influence of biomechanical constraints only for the sets in which palm view stimuli were used . As a consequence, we sought to see whether the strategy used for the set-1 stimuli (i.e., only in - plane - rotated back view stimuli) changes when they are embedded within a set of stimuli rotated around other rotational axes . The results indicate that the context in which the stimuli (i.e., set-1 stimuli) are embedded is of influence on the strategy use, as the interaction of set by dor is significant . The changing effect of dor over the sets indicates that the particulars of a stimulus set (i.e., number of rotational axes) influences the strategy used to solve the mental rotation task for the complete set and that strategies are not linked to a particular type of stimuli . We expected an increase in rt from set-1 till set-3 because of the systematically adding of an extra rotational axis, thereby increasing the task difficulty . However, the general rt increase over all angles does not linearly increase from set-1 to set-3, as can be seen in both figs . 2 and 3 ., the set-1 stimuli were presented in a two - dimensional framework and the stimuli in both set-2 and set-3 were presented in a three - dimensional framework . It might be possible that, because of the dimensionality differences between the frameworks, the complexity increase is largest from set-1 to set-2, thereby introducing a larger overall increase in rt . Second, the non - linear increase may well be accounted for by the use of mi in both set-2 and set-3 and no mi in set-1 . This converges with findings of de lange and colleagues, who showed that mi is a much slower strategy than the use of a third person s perspective (i.e., vi) (de lange et al . 2005). These findings beg the question as to what is the underlying cause of the observed differences in engagement in mi between the sets? This question may be answered by examining the differences in dor effects and the particulars of the stimuli between the three stimulus sets . For the set-1 condition, it was sufficient for participants to judge the hand laterality by focusing on the combined finger and thumb orientation by, for example, determining whether an l-shape can be observed . If this is possible, then the presented hand must be a left hand or otherwise a right hand . This one possible strategy can be used for all angles of in - plane - rotated stimuli . However, when palm view stimuli are added, the use of that same strategy is not sufficient because, for an upright hand orientation, the same thumb orientation can lead to both left and right judgments due to the inverted view (i.e., thumb oriented to the left denotes a right back view hand or a left palm view hand). Therefore, an additional judgment needs to be incorporated into the strategy, namely a view judgment . It might be that the incorporation of multiple judgments into a strategy facilitates the use of the own body representation in order to judge the hand laterality . According to this suggested difference in strategy use, the inclusion of palm view stimuli within a stimulus set is crucial to induce mi engagement . It has already been shown that humans are able to choose between two strategies in order to solve a mental rotation task (kosslyn et al . Studies on the hand laterality judgment task with children also show that there are two different strategies . At the same time, these studies showed that the implicit use of these different strategies differs between adults and children (lust et al . Collectively, it is likely that participants in our study use two different strategies to solve the mental rotation task, which encompasses a visually based strategy for set-1 (as no influence of biomechanical constraints is evident) and a motor - guided strategy for set-2 and set-3 . In summary, this study shows that the number of axes of rotation of a stimulus set does critically influence the engagement in mi during a hand laterality judgment task . More specifically, combined use of palm and back view stimuli increases differences in rt between lateral and medial rotation compared to only presenting back view stimuli, implying a facilitated engagement in mi . Our results therefore show that participants do not automatically engage in mi in a hand laterality judgment task, but that engagement is critically dependent on the set of stimuli used . A simple set of stimuli might result in developing and using a strategy that lacks mi engagement, whereas a more difficult set of stimuli promotes engagement in mi . These results have implications for generalization of results of different studies on the hand laterality judgment task and may explain some of the differences observed in mi engagement between studies . Motor imagery is used for rehabilitation purposes by, for instance, patients with stroke (braun et al . 2006; sharma et al . 2006), patients with parkinson s disease and patients with complex regional pain syndrome (see dickstein and deutsch 2007 for a review). A major challenge in these studies is to engage participants into the use of motor imagery . Only then similar brain networks are active as in actual motor control, which enhances motor recovery . One way to establish this is by only including participants that are able to use motor imagery . Validated questionnaires such as the movement imagery questionnaire (miq) in addition, the use of a task that implicitly induces the use of a mental movement, such as the hand laterality judgment task, would probably facilitate the patient s ability to engage in a mental movement as this engagement does not depend on the conscious effort of the patient in imagining the movement . Finally, our results show that a hand laterality judgment task must be designed such that participants do not develop a strategy based on the visual characteristics of the stimuli.
Bullous lupus erythematosus (ble) is a rare subset of systemic lupus erythematosus (sle). There have been few studies addressing hypopigmented lesions in lupus erythematosus (le). In this study, we would like to introduce a rare case of sle, and show immunohistochemical and ultrastructural findings of the vitiliginous lesions following erosive erythema . A 57-year - old male patient had noticed itchy erythema on the frontal chest in may 2011, and thereafter experienced disease enlargement and partial crust formation . Since no clinical effect of 10 mg / day of oral prednisolone was observed, he was referred to our hospital on july 7, 2011 . In terms of the clinical findings at the first visit, symmetric red papules and centrifugal erythema, surrounded by an annular erosive crust, were distributed on the upper extremities and trunk in line with the seborrheic region . The center of the erythematous lesions in the skin tended to be notably resolved (fig . Although his skin disease was initially suspected to be a bullous disease, neither anti - desmoglein 1/3 nor anti - bp 180 antibodies were detected . Laboratory tests revealed cytopenia (wbc, 10,470/l; neu, 72.6%; lymph, 13.0%; mono, 14.0%; eosino, 0.1%; baso, 0.3%; rbc, 3.85 10/l; hb, 12.7 g / dl; ht, 38.5%; platelet, 180 10/l), ana (1/320, homogeneous + speckled pattern), positive anti - sm antibodies, anti - rnp, anti - cardiolipin antibodies, and low c3 and c4 levels . Metal patch and photo tests were negative, and no internal malignancy was detected by whole - body imaging . A biopsy of the erythematous lesion showed individual apoptotic keratinocytes and liquefaction with partial cleft formation on the epidermis, and lymphocytes predominantly infiltrated in the upper dermis and perivascular area (fig . The direct immunofluorescence findings of the uninvolved lesion were granular deposition of igg and igm in the basal membrane . These laboratory and histological findings led to a final diagnosis of ble type i. in an immunohistochemical analysis for immunocompetent cells, cd8 cells, instead of cd4 cells, and hla - dr - activating t cells were found to have densely infiltrated into the epidermis of the erythematous lesion . The cd68 macrophages densely infiltrated into the upper dermis, and there was a slight infiltration of il-17a cells and foxp3 cells (fig . 2a h). An increased dose of oral prednisolone was effective not only for spiking fever, cytopenia and hypocomplement level, but also crusted bullae and fused erythema (fig . Instead, annular incomplete hypopigmentation tracing the crusted lesions was noted 1 month later (fig . Our histological observations of the hypopigmented lesion showed a decrease, but not total absence, of melanosomes and melanocytes detected by masson - fontana (fig . In addition, an ultrastructural analysis of a hypopigmented lesion revealed attenuated melanin production, with the presence of immature melanosomes in the remaining melanocytes, whereas no disturbance of melanin transfer to adjacent keratinocytes was noted (fig . Ble is a rare variant of sle, with an incidence of less than 0.2 cases per million per year, and represents only 23% of the cases of autoimmune subepidermal bullous dermatoses . Clinically, ble is characterized by a disseminated vesiculobullous skin rash that may or may not be limited to areas exposed to light . Although it is considered to be an acquired bullous disease caused by anti - type vii collagen antibodies, the same as epidermolysis bullosa acquisita, the bullous lesions in ble usually remit without scar formation, which is common in cases of epidermolysis bullosa acquisita . Since antibodies such as anti - desmoglein 1/3 antibodies and anti - bp 180 antibodies were not detected, the present case of ble was diagnosed according to the diagnostic criteria . A histopathological examination revealed subepidermal blisters with neutrophilic microabscesses in the dermal papillae, perivascular inflammatory infiltration composed of lymphomononuclear cells, and in some cases, leukocytoclastic vasculitis . Our case also showed individual apoptotic keratinocytes, liquefaction with a partial cleft on the epidermis and lymphocytes predominantly infiltrating into the upper dermis and perivascular area . In our case franca and de souza reviewed the histological features of 12 patients with depigmented or hypopigmented lesions in 220 patients with cutaneous le, and the most common histological findings were summarized as: cellular infiltration (75%), hyperkeratosis (66.7%), thinning and flattening of the epidermis (58.3%) and lower number of melanocytes compared to normal skin . We observed similar histological findings in our case, including apoptotic keratinocytes, atrophy of the epidermis and superficial lymphocytic infiltration in the dermis and a decreased number of melanocytes . For a more detailed observation, we carried out the immunohistochemical and ultrastructural analyses for both the erythematous and subsequent hypopigmented lesions . For the immunohistochemical analysis, cd8 cells (instead of cd4 cells) and hla - dr - activating t cells were found to have densely infiltrated into the epidermis of the erythematous lesion . There was a decrease in the number of cd1a langerhans cells . While the cd68 macrophages densely infiltrated the upper dermis, there was a slight infiltration of il-17a cells and foxp3 cells . Liquefaction, degeneration and dense infiltration of cd8 t cells into the epidermis were detected, possibly resulting in hypopigmentation accompanied by le . Whereas the cd8 t cells were densely found in the affected lesions, the il-17a cells and foxp3 regulatory t cells were sparsely present moreover, we detected a large number of th17 cells infiltrating in the depigmented lesions compared to the uninvolved skin in nonsegmental vitiligo patients [6, 7]. With regard to ultrastructural findings, while melanosome production in melanocytes was attenuated, adjacent keratinocytes took over a significant number of mature melanosomes, indicating that there were decreases in both the melanocyte and melanosome number without impaired melanosome transfer to keratinocytes . Taking into consideration both these immune responses and the lupus condition, we considered the following process to be involved in the pathogenesis of the annular hypopigmentation; cytotoxic t cells penetrated into the epidermis followed by melanocyte destruction . Thereafter, pigment incontinentia followed by the recruitment of numerous cd68 melanophages in the upper dermis induced activation of melanin - specific antigen presentation, resulting in positive loop cellular immunity . The cd8 cytotoxic cell - related immune response was thus induced, and melanocyte were attacked, leading to basal membrane degeneration, which may have played a major role in the hypopigmentation associated with le . Early induction of systemic prednisolone treatment may have led to lesion improvement owing to the sufficient inhibition of this vicious disease cycle . We experienced a case of le associated with annular hypopigmentation . We suppose that a cd8 cytotoxic cell - related immune response in addition to basal membrane degeneration may play a major role in the development of hypopigmentation associated with le . Early administration of effective treatment may be important for restoration of the pigmentation . Although annular hypopigmentation is generally regarded as secondary hypopigmentation after local inflammation, annular hypopigmentation following ble also occurs in some patients . We believe that using a detailed morphological and hematological analysis in additional patient samples will help to elucidate the exact mechanism(s) underlying the development of hypopigmentation in patients with ble.
There is some evidence to suggest that working memory might be impaired in patients who show jtc compared to those who do not . A small study by ormrod et al69 found that visual working memory performance was affected in first - episode psychosis patients who showed a jtc response style . In addition, in sz patients with strong current delusions, working memory (but not premorbid iq) was worse in those who demonstrated jtc.70 as noted above, broome et al67 found that jtc in the prodrome was linked to working memory impairments . Nevertheless, the presence of a memory aid during the urn task does not affect jtc in patient groups.51,53 this would appear to undermine any suggestion that jtc stems from a relative inability to maintain the task items in memory . Working memory impairments might correlate with a tendency to jtc, but the relationship is unlikely to be causal . One explanation for the jtc response style observed in sz is that patients simply make decisions based on less evidence . This liberal acceptance account53,71 was founded on evidence that sz patients tend not to converge on one particular interpretation of a situation (eg, when asked to judge the plausibility of multiple interpretations of a picture). Healthy controls ruled out interpretations that patients continued to liberally entertain, giving higher ratings to a wide range of interpretations . Sz patients are proposed to more readily accept a response option, while healthy participants are more cautious in doing so . In situations where only two (mutually exclusive) options moritz et al71 increased the number of jars to four to provide additional ambiguity: this was found to abolish the jtc in patients . Although sz patients made a less systematic information search and were more likely to consider less valid information, they did not inspect fewer pieces of information compared to healthy controls and thus did not demonstrate jtc . Effects were found on confidence ratings however, with patients more likely to be overconfident, using extreme confidence ratings under inappropriate circumstances . This is consistent with work showing that sz patients are less confident of their correct answers and overconfident when they make errors, during word recall tasks.72,73 although there were no overall differences between patients and controls, symptomatology in the patient group was linked to information search . A correlation was observed between symptom severity scores (positive and negative syndrome scale [panss] positive, panss delusion) and the degree of information search, with higher - scoring participants tending to gather less pieces of information . Panss score did not predict use of extreme confidence ratings, but nevertheless this study would suggest that jtc can be largely abolished and can manifest as overconfidence only when information is presented in the right manner . Impulsiveness is unlikely to be a factor because patients with sz show similar reaction times as healthy controls51,53 and draw more beads when the task is made harder.51,55,74 motivational factors are also unlikely to play a part: the possibility that the patients overestimate the cost of gathering more information (possibly due to a greater need for closure) has been discounted because patients do not seem to experience a greater cost for gathering more information.75 an alternative explanation is that jtc manifests not through a lowered threshold for making a decision but through each piece of evidence being relatively overvalued . When asked to report belief estimates after each draw, it has been shown that patients make more drastic updates after each piece of evidence.46,56,77 speechley et al78 found that delusional patients, when asked to give likelihood ratings for each urn on each trial, gave higher ratings for whichever urn matched the current evidence, while ratings for the nonmatching urn did not differ from those of the control groups . The authors argue that this provides evidence of a reasoning bias characterized by hypersalience of evidence that matches a hypothesis, but with reasoning that appears intact for nonmatches . The literature is inconsistent regarding patients responses to nonmatches (disconfirmatory evidence). Because delusions are maintained in the face of contradictory evidence, it is unsurprising that patients tend to show a bias against disconfirmatory evidence.79 it has been argued that hypersalience could underlie this effect: hypersalience of evidence hypothesis matches may lead to an enhancement of weak matches, leading to difficulties in integrating disambiguating information.80 some studies point to hypersalience of disconfirmatory, as well as confirmatory, evidence: deluded patients show a tendency to overadjust when presented with potentially disconfirmatory evidence,53,56 although this effect might be linked to miscomprehension of the task,81 especially because such a tendency would contradict the evidence of reversal - learning deficits discussed in the previous section . These findings are consistent with an aberrant salience account of sz,82 whereby dysregulated dopamine transmission generates context - inappropriate salience attributions, potentially due to aberrant signaling in the ventral striatal dopaminergic pathway, which is thought to regulate stimulus response pairings.83 moore and sellen84 built a simple network model in which the gain, or signal - to - noise, parameter (which describes the likelihood of a node firing when presented with some input) was varied . The gain parameter was assumed to represent striatal dopaminergic activity, and increasing this parameter meant that the model successfully mimicked the jtc response style seen in research data from delusional patients . This might be overly simplistic however, because imaging studies have shown that the striatum is downregulated in arms85 and sz patients.86 nevertheless, it is important to note that evidence for hypersalience has been observed across various other cognitive biases present in sz8789 and as such represents a convincing account of jtc . As mentioned previously, another suggestion concerning the development of delusions in sz focuses on impairments in theory of mind.42 langdon et al77 compared 35 sz patients with a history of delusions to healthy controls, on a battery of tasks that included two versions of the urn task, three theory - of - mind tasks, and a questionnaire on attributional biases . A jtc response style was found in the patient group, as well as impairments on the theory of mind tasks and evidence of an externalizing attributional bias . Performance on the urn tasks correlated with that of the theory - of - mind tasks, while attributional bias scores did not correlate with other task measures . Delusion proneness (measured by the questionnaire) correlated with probabilistic reasoning and theory - of - mind measures, while externalizing bias did not; iq and memory ability were accounted for . Although it was draws - to - decision that correlated most robustly with delusion proneness, these data prompted the authors to suggest that a common underlying mechanism might operate in sz to drive probabilistic reasoning and theory of mind deficits . They speculate that this could be a difficulty in inhibiting sensory input reflecting the immediate perceived reality, thus making patients more likely to be influenced by current data when making decisions on probabilistic reasoning tasks and making it harder to maintain an abstract viewpoint as required by theory - of - mind tasks . However, it should be noted that a large meta - analysis found no evidence for a link between theory of mind deficits and positive symptoms; instead, deficits in theory of mind were correlated with negative symptoms, disorganization, and cognitive impairment.90 there is also some suggestion that poor emotion regulation might have a role to play . In one study, 90 healthy individuals with varying levels of psychosis vulnerability (assessed by the community assessment of psychic experiences) were recruited.74 half the sample received an anxiety induction procedure, the other half did not . Paranoid delusions and jtc were then assessed during the session . The anxiety induction procedure promoted delusions and jtc, and participants with higher psychosis vulnerability showed greater increases in paranoid delusional ideation . Furthermore, jtc appeared to mediate the association between anxiety and delusions, prompting the authors to suggest that paranoid delusions result from an interaction of anxiety and reasoning biases . In the glckner and moritz91 study, it was found that increasing stress (through time pressure and the addition of affective valence) led to diminished performance in patients . Thus, improved self - monitoring in terms of better emotion regulation, combined with metacognitive training targeted at reasoning biases, could be beneficial in reducing delusion formation in sz . On balance, the explanations best supported by evidence are those of liberal acceptance and hypersalience . At present, it is hard to say which is best supported by the evidence available: this is possibly because these two explanations are by no means mutually exclusive . One difficulty in differentiating these explanations is that the urn task offers limited information regarding learning and decision - making processes . In one of our studies,92 we investigated performance in patients on a sequence - learning task (sequences of four button presses were learnt using two buttons, feedback after each button press) and the typical urn task . Patients were able to learn the correct sequence, but learning was slower compared to that in healthy controls . Interestingly, learning from positive (but not negative) feedback in the sequence task correlated with draws - to - decision in the urn task: patients who showed a jtc response style were impaired in learning from positive feedback . Thus, these findings clearly favor a liberal acceptance account over hypersalience, but further work is needed to distinguish liberal acceptance and hypersalience, as well as to explore potential interactions with emotion regulation and other self - monitoring activities . In the next section, we discuss work aimed at elucidating the neural basis of jtc . Various studies have attempted to induce the jtc bias in healthy controls using pharmacological manipulations; others have used fmri . A drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . There is some evidence that the noncompetitive n - methyl - d - aspartate (nmda) receptor antagonist ketamine could serve as such a model . Because ketamine can be safely administered under clinical supervision and has relatively short - lived effects, the drug could represent a useful tool for studying processes underlying jtc . Ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly, ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . Prediction error is defined by the mismatch between expectations and experience, and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli (both internal and external), consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . Corlett et al105 found that ketamine could strengthen the memory trace of a previously conditioned stimulus when it was presented again, without reinforcement, offering tentative support for this hypothesis . However, another study failed to find a jtc response style during the urn task in healthy controls receiving ketamine . This suggests that although ketamine can induce delusions in healthy controls, delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . Interestingly, work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc, suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . L - dopa, had no effect on draws - to - decision, or confidence, whereas a dopamine antagonist (haloperidol) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise, dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum, attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . Thus, it would seem that jtc cannot be attributed solely to general dopaminergic overactivity, at least when induced acutely: the relationship might not be linear, or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . Alternatively, the disrupted interactions between several neurotransmitter systems might be critical . Fmri can potentially indicate the neural mechanisms contributing to probabilistic reasoning in the urn task . Studies in healthy controls implicate a distributed network of brain areas, including parietal cortex (typically around intraparietal sulcus), prefrontal cortex (typically dorsolateral), anterior insula, and striatum . These fmri studies use paradigms, including draw events (stimuli for which participants choose to gather more information) and urn events (in which participants decide they have enough evidence and so select an urn). Some of these studies compared urn task blocks (which collapse over draw and urn decision events) with blocks where participants performed a control task on the same stimuli . The most consistent finding across these block design studies are parietal responses near the intraparietal sulcus, which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals, compared to the responses in healthy controls.113 this conclusion is tentative, however, as this study failed to replicate the classic jtc behavioral effect in sz . These block - design studies are limited, as they cannot distinguish fmri responses to draw choice events (decisions to gather more information) from fmri responses associated with final choices of an urn . Jtc occurs when data gathering is discontinued in favor of choosing an urn, so brain areas contributing to urn choice events are likely to be involved in jtc . Studies that contrast fmri responses to urn choices versus draw choices yield similar results as the block - design contrasts . Urn choices, compared to draw choices, activate anterior insula, striatum,115,116 acc, and parietal cortex, including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here, this network of areas was associated with deciding on an option currently available (eg, a used car), compared to deciding to sample more possible options (eg, viewing more cars). Furl and averbeck116 found further roles for this parietal cortex area in the urn task . Parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . These latter findings link parietal responses to individual differences in information - gathering behavior . However, this between - participant variability was within a nonclinical sample, and it remains to be confirmed whether it also extends to clinical cases such as sz . Involvement of areas such as striatum, dorsolateral prefrontal cortex, and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking (as in jtc) is perhaps not surprising . For example, measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex, striatum, and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas, which contributes to the urn task and other decision - making tasks in healthy participants, may be compromised in sz patients who show jtc behavior . The striatum, for instance, signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior, as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum, responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics, compared to those in healthy individuals, when performing tasks requiring reward prediction.126 in sum, schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . Dysfunctional interactions between the dopaminergic striatum and cortical areas, such as intraparietal sulcus and dorsolateral prefrontal cortex, might explain jtc behavior in sz patients . More brain - imaging studies using sz patients and the urn task are required to test this hypothesis . A drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . There is some evidence that the noncompetitive n - methyl - d - aspartate (nmda) receptor antagonist ketamine could serve as such a model . Because ketamine can be safely administered under clinical supervision and has relatively short - lived effects, the drug could represent a useful tool for studying processes underlying jtc . Ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly, ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . Prediction error is defined by the mismatch between expectations and experience, and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli (both internal and external), consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . Corlett et al105 found that ketamine could strengthen the memory trace of a previously conditioned stimulus when it was presented again, without reinforcement, offering tentative support for this hypothesis . However, another study failed to find a jtc response style during the urn task in healthy controls receiving ketamine . This suggests that although ketamine can induce delusions in healthy controls, delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . Interestingly, work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc, suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . L - dopa, had no effect on draws - to - decision, or confidence, whereas a dopamine antagonist (haloperidol) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise, dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum, attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . Thus, it would seem that jtc cannot be attributed solely to general dopaminergic overactivity, at least when induced acutely: the relationship might not be linear, or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . Alternatively, the disrupted interactions between several neurotransmitter systems might be critical . Fmri can potentially indicate the neural mechanisms contributing to probabilistic reasoning in the urn task . Studies in healthy controls implicate a distributed network of brain areas, including parietal cortex (typically around intraparietal sulcus), prefrontal cortex (typically dorsolateral), anterior insula, and striatum . These fmri studies use paradigms, including draw events (stimuli for which participants choose to gather more information) and urn events (in which participants decide they have enough evidence and so select an urn). Some of these studies compared urn task blocks (which collapse over draw and urn decision events) with blocks where participants performed a control task on the same stimuli . The most consistent finding across these block design studies are parietal responses near the intraparietal sulcus, which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals, compared to the responses in healthy controls.113 this conclusion is tentative, however, as this study failed to replicate the classic jtc behavioral effect in sz . These block - design studies are limited, as they cannot distinguish fmri responses to draw choice events (decisions to gather more information) from fmri responses associated with final choices of an urn . Jtc occurs when data gathering is discontinued in favor of choosing an urn, so brain areas contributing to urn choice events are likely to be involved in jtc . Studies that contrast fmri responses to urn choices versus draw choices yield similar results as the block - design contrasts . Urn choices, compared to draw choices, activate anterior insula, striatum,115,116 acc, and parietal cortex, including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here, this network of areas was associated with deciding on an option currently available (eg, a used car), compared to deciding to sample more possible options (eg, viewing more cars). Furl and averbeck116 found further roles for this parietal cortex area in the urn task . Parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . However, this between - participant variability was within a nonclinical sample, and it remains to be confirmed whether it also extends to clinical cases such as sz . Involvement of areas such as striatum, dorsolateral prefrontal cortex, and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking (as in jtc) is perhaps not surprising . For example, measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex, striatum, and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas, which contributes to the urn task and other decision - making tasks in healthy participants, may be compromised in sz patients who show jtc behavior . The striatum, for instance, signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior, as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum, responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics, compared to those in healthy individuals, when performing tasks requiring reward prediction.126 in sum, schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . Dysfunctional interactions between the dopaminergic striatum and cortical areas, such as intraparietal sulcus and dorsolateral prefrontal cortex, might explain jtc behavior in sz patients . More brain - imaging studies using sz patients and the urn task are required to test this hypothesis . Some studies have suggested that the jtc bias could represent an important therapeutic target . Using an emotionally salient version of the urn task, menon et al64 found that within 2 weeks of initiating treatment with antipsychotics, patients demonstrated an increase in the number of trials - to - decision alongside attenuation of psychotic symptoms and delusions . Although these measures were not correlated, baseline performance on the task had some predictive power over the individuals who would show improvements in symptomology in response to medication: jtc performance at baseline could therefore be useful in guiding treatment . Moreover, treatment - related improvements in jtc can predict probability of regaining full employment, measured over a 6-month window . This was not the case for positive symptoms or neuropsychological performance, suggesting that jtc might act independently to influence real - world functioning.127 it is important to note that most studies have failed to show improvements in jtc on the standard urn task following antipsychotic treatment,64,128 although there is some evidence to dispute this.63,65 if jtc does indeed fluctuate with delusional symptoms, it would provide strong evidence of its importance as a treatment target: interventions that specifically target cognitive biases (known as metacognitive training programs) have already been shown to improve delusions and other positive symptoms.129,130 it is also important to note that most studies typically detect jtc in only approximately 50% of their patient samples . This heterogeneity needs to be explored further, to determine how it might relate to heterogeneity of symptomatology or whether it could be an issue of task sensitivity . Again, individual differences in jtc performance could be useful in determining the best course of treatment . Training programs that aim to ameliorate the jtc response style might prove to be an important adjunct to established therapies . In sum, jtc in sz seems to be a consistent finding and there is strong evidence linking jtc to delusion formation . Both liberal acceptance and hypersalience accounts of jtc are well supported by the literature, but attempts to replicate jtc in healthy controls using pharmacological manipulations have largely failed, undermining attempts to develop a neurobiological account of jtc . Fmri studies have implicated a network involved in making urn choices, which includes striatal, insula, parietal, and prefrontal areas; further patient work is required, particularly in the context of evidence suggesting that jtc could represent a valuable therapeutic target.
Development of skin lesions in the course of bullous pemphigoid (bp) results from destruction of basement membrane components . Complex structure of this membrane is responsible for maintaining the integrity of dermo - epidermal junction . Two glycoproteins of molecular mass 230 kd (bpag1) and 180 kd (bpag2) are the autoantigens in bullous pemphigoid . Structural studies revealed that extracellular fragment of bpag2 with cooh - terminal collagenous domain connects the basement membrane with epidermal hemidesmosomes . Nc16a fragment of bpag2, located within its extracellular fragment, is thought to be the most immunogenic part of the antigen . Binding of autoantibodies directed against these autoantigens, localized in the basement membrane of the epidermis, activates a series of immunological and enzymatic phenomena [1, 2]. Inflammatory infiltrates in the dermis, formed by neutrophils and eosinophils, and bound in vivo igg and c3 deposits along the basement membrane of the epidermis are observed . Ultrastructural studies confirmed also the presence of intensive inflammatory infiltrate at dermo - epidermal junction, as well as destruction of hemidesmosomes and components of extracellular matrix . Formation of the infiltrates is preceded by early accumulation of leukocytes, depending on activity of adhesion molecules, especially selectins and integrins . Schmidt et al . Observed that binding of autoantibodies with bpag2 leads to activation of keratinocytes, which release interleukin 6 and interleukin 8, as well as activation of c5 component of the complement . Mast cells and neutrophils activated by the same immunological reaction migrate through walls of blood vessels with help of e and l selectins and secrete specific proteases that can digest a series of basement membrane proteins . Matrix metalloproteinases, released by inflammatory cells and keratinocytes, are finally responsible for blister formation [5, 6]. Migration of immunocompetent cells from blood vessels into foci of inflammation has several phases . In each stage of this process different families of adhesion molecules are involved, responsible for rolling, adhesion, activation, binding, and diapedesis of leukocytes . During the first stage (leukocyte rolling), binding of e and l selectins with oligosaccharide ligands results in the so - called marginalization of the cells, for example, direct contact of leukocytes with the endothelium . Second stage, leukocyte activation, is characterized by increased affinity of integrin receptors on rolling leukocytes . Cytokines produced locally in foci of inflammation cause halting of rolling cells and their pushing through the epithelium [68]. Subfamilies of integrins, for example, 1 and 3, are involved in the third stage, termed strong adhesion . During the fourth stage diapedesis and migration in tissues they are transmembrane glycoproteins composed of extracellular, intramembrane, and cytoplasmatic functional domains . The 4 integrin is additionally involved in connecting the filaments of the cell to the basement membrane and forming hemidesmosomes . . Continued research concerning modulation of activity of selected selectins and integrins that are involved in processes taking place within the skin may contribute to development of new therapeutic methods for various dermatoses, especially bullous skin diseases . There are few literature data concerning variations of selected adhesion molecules serum levels or examinations on animal models in bullous pemphigoid . The aim of the study was to determine the localization and expression of e and l selectins and 1, 3, and 4 integrins in lesional skin of patients with bullous pemphigoid and in skin of healthy persons . The study included 21 patients with bp (15 women and 6 men, aged between 58 and 84 years, mean age 68.5) in active stage of the disease . All patients had skin lesions - blisters, vesicles, papules, or erythema . Control group consisted of 10 healthy persons (5 women and 5 men, ages between 19 and 49 years, mean age 42). Diagnosis of bullous pemphigoid was based on clinical picture as well as histological and immunological examinations . 11 patients had skin lesions in form of blisters, vesicles, and itching erythematous papules, the rest of patients presented with only erythema . In all patients, direct immunofluorescence test revealed bound in vivo igg / c3 linear deposits along basement membrane zone . In salt split test, the deposits were observed in epidermal part of the blister, or both in epidermal and dermal parts . By indirect immunofluorescence test circulating igg antibodies were found in 17 out of 21 patients, while elisa test (mbl, nagoya, japan) showed the presence of anti - nc16a autoantibodies in 19 cases . In histological examination, features consistent with diagnosis of bp were observed, such as neutrophilic, eosinophilic, and lymphocytic infiltrates, and in 11 cases subepidermal blisters . Biopsies for immunohistochemical examination were obtained from lesional skin in patients and from the nape region in healthy controls . Paraffin - embedded sections were used for routine h + e staining and for immunohistochemistry in dako envision detection system using immunoperoxidase method . The following primary monoclonal antibodies were used: cd29 (1 family), cd61 (gpiii) (3 family), cd104 (4 family), cd62e (e - selectin), and cd62l (l - selectin), (novocastra, united kingdom). For immunohistochemistry the paraffin - embedded sections were placed on adhesive plates and dried at 56c for 24 hours, later deparaffinated in a series of xylens and alcohols with decreasing concentrations (96%, 80%, 70%, 60%). Activity of endogenous peroxidase was then inhibited with 3% hydrogen peroxide solution in methanol for 5 minutes . In order to retrieve the antigenicity of tissues and allow them to react with antibodies, the following procedures were used for each of the tested antibodies: for cd62e the sections were heated in 0.001 m versenian buffer (edta) of 8.0 ph in water bath at 95 for 30 minutes; for cd62l the sections were boiled in 0.001 m versenian buffer (edta) of 8.0 ph in microwave oven at 700 w for 15 minutes; for cd29 the sections were heated 6 times in 0.01 m citrate buffer of 6.0 ph in microwave oven at the following power levels: 150 w (5 minutes), 350 w (5 minutes), 450 w (5 minutes), and 650 w (6 minutes); for cd61 and cd104 the sections were heated in dako target retrieval solution in water bath at 95 for 30 minutes . Cooled sections were then rinsed in 0.05 m tris buffer (tbs) at 7.6 ph for 30 minutes and incubated for 60 minutes at room temperature in damp chamber with, respectively, diluted antibodies cd62e (e - selectin) 1: 50, cd62l (l - selectin) 1: 50, cd29 1: 40, cd61 (gpiii) 1: 25, cd104 1: 50 . After incubation the sections were rinsed twice in tbs buffer and dako envision double - step visualization system was next applied in order to visualize the antigen - antibody reaction . The following semiquantitative scale was applied for evaluation of the intensity of immunohistochemical reaction of cd29, cd61, cd104, cd62e, and cd62l in skin biopsies:: no reaction, +: weak intensity in most cells, 2 +: moderate intensity, 3 +: strong intensity . Expression of selectins and integrins was assessed by two independent pathologists using olympus bx 41 microscope (japan). Results of semiquantitative evaluation of expression of studied integrins and selectins in immunohistochemical examination are presented in table 1 . Immunoexpression of integrin 1 (cd29) was detected in all basal keratinocytes . In skin samples from patients with bp, mean intensity of immunoexpression was weak (14/21) (see figure 1). Moderate expression of cd29 was observed in 4 of 21 patients . In skin samples obtained from healthy volunteers expression was very weak and detected only in single cells (see figure 2). Reaction with cd61 antibody was positive in skin samples from patients and was localized in basal keratinocytes and focally also in other layers of the epidermis (see figure 3). Mean intensity of immunoexpression of cd61 was weak (14/21). In control immunoexpression of integrin 4 (cd104) was detected in hemidesmosomes in bp samples as well as in control biopsies . In lesional skin biopsies from patients, expression was irregularly scattered along basement membrane (see figure 4), while in healthy skin it was regular and linear (see figure 5). Immunoexpression of e - selectin in skin samples from bp patients was detected on endothelial cells and neutrophils (see figure 6). Mean intensity of cd62e immunoexpression in patients was weak (17/21). In biopsies from healthy skin, only single endothelial cells showed very weak expression of cd62e . Immunoexpression of l - selectin was detected on limphocytes, macrophages, and neutrophils (see figure 7). In biopsies from healthy skin very weak expression was observed only on single endothelial cells . Pemphigoid is a subepidermal bullous dermatosis in which pathological processes result in disconnection between basal layer of the epidermis and the dermis, causing formation of tense blisters . It was proved that administration of rabbit anti - mbp180 antibodies to newborn mice causes production of pathological antibodies, accumulation of leukocytes, and formation of complement deposits along basement membrane that results in development of blisters [4, 9]. Scare literature data revealed the role of certain adhesive molecules in the pathogenesis of bp . Recent studies established biochemical properties of metalloproteinases and their tissue inhibitors and their high affinity to components of the basement membrane zone . These enzymes are produced by eosinophils and neutrophils attracted to the basement membrane by selectins and integrins . Integrins belong to particles that are very important for maintaining the dermo - epidermal junction as well as cell - to - cell connections . Lack of 1 integrin during fetal development in mice is a lethal mutation [8, 10, 11]. Genetic or autoimmune dysfunction of 64 integrin causes bullous lesions on the skin and mucous membranes in the course of such diseases as junctional type of bullous epidermolysis or cicatricans pemphigoid . Expression of various integrins on keratinocytes, firoblasts, endothelial cells, and migrating cells is also necessary for normal healing processes and for occurrence of the apoptosis phenomenon [12, 13]. Increased expression of integrins is associated with promotion of cell migration, production of metalloproteinases, and angiogenesis [14, 15]. Activation of various signal transduction pathways depends on the family of integrins, type of associated ecm protein, and stimulating factors . Integrin stimulating factors are cytokines, growth factors, chemokines, and other adhesion molecules, including cytokines themselves [1416]. Some literature date confirm the role of these cytokines, chemokines, and ecm enzymes in pathogenesis of bp [4, 5]. Integrins are heterodimers build from and chains connected noncovalently that pass through the cytoplasmatic membrane, joining extra and intracellular environments [7, 17, 18]. Linkage of an integrin with ligand initiates signaling cascade that modulates cell behavior and gene transcription . Integrins are main cellular receptors for binding with extracellular matrix components (e.g., plectin, fibronectin, vitronectin, collagen) through special short protein fragments and are involved in intercellular adhesion in epidermis [20, 21]. 1 subfamily integrins are involved mainly in the interactions between cells and connective tissue macromolecules (e.g., fibronectin, laminin, collagen); 2 are associated with cell - to - cell interactions, while 3 play a role in connections with ligands such as fibrinogen, vitronectin, thrombospondin, and von willebrand factor [7, 18]. 1 integrin subunit was detected in upper and lateral parts of cellular membrane of basal keratinocytes, while it was very seldom observed in its lower parts . This observation may suggest that this molecule is involved mainly in maintaining the intercellular connection . Molecules that belong to 1 subfamily of integrins may become expressed as late as 2 to 7 weeks after lymphocyte stimulation . In our study, expression of 1 integrin in all basal keratinocytes in lesional skin biopsies from pemphigoid patients was observed, in comparison with very weak signal from only single cells in the control group, revealing the important role of lymphocyte stimulation and production of cytokines in this disease . It binds with cd51 molecule, creating a receptor for vitronectin, and thus becomes expressed in the cells of many tissues . Moreno et al . Used monoclonal antibodies against this integrin, revealing its presence in epithelium of mouse's kidneys and testicles . Structural and functional analysis of 3 integrin fragments showed its strong adhesive potential and ability to influence the function of fibrinogen, explaining the expression of this integrin during the inflammatory process as well as its presence on other cells, including epithelial cells [4, 22]. In our study, expression of this integrin was observed on single epidermal cells . Positive signal was detected in lesional skin biopsies, comprising basal keratinocytes or focally cells from other layers of the epidermis . It seems that the inflammatory process and destruction of dermo - epidermal junction may be induced by expression of 3 on keratinocytes, but such phenomenon should be confirmed by other diagnostic methods . They are composed, among others, from 64 integrin, bp180, cd151, and plectin [21, 23]. Results of in vitro studies reveal that 64 integrin plays a key role in development of hemidesmosomes . This integrin is involved in connecting the intermediate filaments of a cell to the basement membrane and antibodies directed against this molecule inhibit both creation of hemidesmosomes and function of existing structures [23, 24]. Pemphigoid antigens bp180 and bp230 are linked not only to each other, but bp230 and 4 bind also with bp180 . Interaction of bp180 and 64 integrin subunit is necessary for stabilization of hemidesmosomes structure [25, 26]. It was demonstrated that sole interaction between bp180 and plectin is not sufficient for hemidesmosome formation when lacking 64 integrin . Complex interactions at the dermo - epidermal junction are also confirmed by other in vitro studies . Studies in patients with ophthalmologic type of cicatrisans pemhigoid underline the role of antibodies directed against 4 integrin . Serum form patients with cicatrisans pemphigoid recognized a protein of molecular mass 205 kda, for example, that of 4 integrin . Such protein was not recognized by sera from patients with bullous pemphigoid or pemhigus vulgaris [2830]. Revealed that serum from pemphigoid patients who had lesions on oral mucosa selectively binds human 64 integrin . In 48-hour culture with human oral mucosa obtained from the cheeks, antibodies may bind intracellulary with specific domains leading in consequence to development of skin lesions [14, 31]. Using immunoblot method revealed decrease of antibodies' reactivity against bp180 and 4 integrin that correlated with decreased activity of ocular pemphigoid resulting from introduced treatment . Rashid et al . Examined sera from 20 patients with mucous membrane pemphigoid in active stage of the disease and demonstrated that all studies sera bound with 6 integrin subunit in immunoprecipitation and immunoblot methods . Focal loss of expression of 64 integrin is associated with loss of adhesion to the basement membrane . Examined the role of 64 in hemidesmosome formation on an in vitro model of wound healing and revealed that antibodies against 4 subunit did not disturb migration of epithelial cells but they initiated disruption of formerly assemblied hemidesmosomes . New hemidesmoses were also no longer formed and antibodies against 64 integrin completely isolated epithelial cells from the area of healing . Our study revealed weak but linear and regular expression of 4 integrin in skin biopsies from healthy persons . Such expression proves that the presence of this molecule is constitutive in epidermal anchoring structures . However, in the course of immunologic delamination of the epidermis in bp, mediated by antibodies directed against basement membrane components and caused by a cascade of cytokine and enzymatic reactions, this expression was significantly changed . Irregular and focal expression of this integrin within the inflammatory infiltrates and blisters may reflect the destruction of the dermo - epidermal junction . Selectins belong to the first family of adhesion molecules that initiate diapedesis, for example, rolling of leukocytes . This family encompasses three transporting glycoproteins with similar structure: leukocyte (l), platelet (p), and endothelial (e) selectin . Selectins are involved in leukocyte recruitment to the inflammatory foci and in the initial stages of inflammation play a role in rolling of leukocytes on the endothelium of blood vessels . E - selectin is present in the stimulated endothelial cells . Production of this molecule is induced by a bacterial endotoxin, cytokines, thrombin, or tumor necrosis factor (tnf). Expression of e - selectin can be observed only in the foci of inflammation . In our studies, expression of this selectin was observed in walls of blood vessels, slightly weaker in neutrophils . It confirms the role of the e - selectin in inflammatory process and development of pathological changes in pemphigoid . In a preliminary study examined serum concentration of soluble e - selectin in pemphigoid and pemphigus vulgaris patients and revealed its significant increase in patients in comparison with the control group, as well as significant correlation with a number of skin lesions . In course of therapy, both severity of skin lesions and concentration of studied selectin decreased in parallel, which may prove the usefulness of e - selectin as a treatment efficacy marker . L - selectin is constitutively present on leukocyte surface and disappears after their stimulation . It has an important role in neutrophil recruitment to foci of inflammation and lymphocyte adhesion to endothelial cells of blood vessels in peripheral lymph nodes . The soluble form of this molecule is probably involved in regulation of adhesion processes [3, 18, 35]. Based on the fact that the inflammatory process is associated with leukocyte stimulation, lower expression of l - selectin in patients in comparison with the control group was expected in our study . Immunoexpression of l - selectin was however observed on lymphocytes, macrophages, and neutrophils . It may prove that not all inflammatory cells are simultaneously stimulated and some of them can still retain the l - selectin on the surface before its desquamation . Recent literature data as well as results of our studies confirm the role of selectins and integrins in pathogenesis of pemphigoid . The elucidation of the role of inflammatory cells, their soluble mediators, adhesion molecules, and signal transduction pathways in the pathogenesis of the bullous diseases may be helpful in the development of new targeted therapies . Extensive research has focused on modulating activity of adhesive molecules . Among the new therapeutic modalities, humanized monoclonal antibodies against adhesion molecules are in early phase of clinical trials.
Cutaneous crohn's was first described by parks et al ., in 1965, refers to extra intestinal mucocutaneous manifestations of crohn's disease in which there are noncaeseating, granulomatous mucocutaneous lesions preceding or manifesting after systemic manifestations of intestinal crohn's disease . Other dermatological conditions found to be associated with crohn's disease include pyoderma gangrenosum, erythema nodosum, erythema multiforme, epidermolysis bullosa acquisita, polyarteritis nodosa and vitiligo . Here, we report a case of anogenital crohn's and its rare association with vitiligo . The case we present here is about a 23-year - old female, presented with vitiligo patch on legs and back for 1 year and vulval swelling with anogenital and oral erosions for 9 months [figure 1]. There were multiple, painful, nonhealing erosions seen on anogenital area and oral cavity [figures 2 and 3]. She also had on- and off - history of loose stools for last 1 month . Investigations on admission had shown low hemoglobin 10 gm%, total count of 7100/cumm, differential count of n: 58, l: 34, m: 4, e: 4, b: 0, platelet count 4.89l acs / cumm . Erythrocyte sedimentation rate was 40 mm in 1 h, blood urea 10 mg / dl, serum creatinine 1 mg / dl, random blood sugar 80 mg / dl . Serum glutamic pyruvic transaminase 10 u / l, serum glutamate - oxaloacetate transaminase 54 u / l, serum bilirubin 0.8 mg / dl, prothrombin time 14 s, activated partial thromboplastin time 34 s, total protein 5.3 g, albumin: globulin 2.9:2.4 . Koh smear was taken from vaginal discharge, which was negative and report of gram - stain is not available . Ultrasonography (usg) of patient showed 17 cm bowel segment with 15 mm thickened wall and narrowing of the lumen with the diagnosis of inflammatory bowel disease . Multislice computed tomography (ct) scan of the abdomen with pelvis showed mild circumferential wall thickening with stratification and increased enhancement . Findings were in favor of crohn's with early sacroillitis . Colonoscopy showed dilated bowel segment with edema, erythema, telangiectasia, and few ulcers [figure 4]. Biopsy from anal wall showed acute as well as chronic inflammation with noncaseating granulomas suggestive of crohn's disease [figure 5]. Patient was treated with tablet mesalamine (800 mg) eight hourly and capsule doxycycline (100 mg) and neomycin cream on ulcers . Clinical photograph of vitiligo patches on legs clinical photograph of patient showing vulval edema clinical photograph showing anal erosions colonoscopy showing erosions in the terminal ileum histopathological picture crohn's disease, first described by sir crohn et al . In 1932, is a type of inflammatory bowel disease that may affect any part of the gastrointestinal tract from mouth to anus . It affects 1 in 400,000 - 600,000 people globally with male: female ratio of 1:1.2 . The age of onset of cutaneous crohn's is 10 - 69 years . In our case, the basic underlying etiology of crohn's is currently unknown, though various theories suggest an interaction between environmental, immunological and bacteriological factors in genetically susceptible individuals (nod2 gene). The lesions in cutaneous crohn's can be classified as: (1) specific lesions of cutaneous crohn's disease, (2) reactive dermatoses, (3) cutaneous manifestations secondary to malabsorption, and (4) cutaneous manifestations secondary to treatment . Cutaneous crohn's disease can also be divided clinically into two forms: genital (56%) and extra genital (44%). Approximately, two - third of children and half of adults with crohn's disease present with genital involvement . Anogenital signs and symptoms (in approximately 25% in illeal and 80% in colonic involvement) may appear before the other systemic manifestations and may be the first sign in the diagnosis of crohn's disease . Cutaneous manifestations associated with crohn's disease are erythema nodosum, pyoderma gangrenosum, epidermolysis bullosa acquisita, polyarteritis nodosa, and vitiligo . In our case, association of crohn's disease and vitiligo has been seen in other studies also . In the study by tanusin et al . Mcpoland and moss have reported a case of crohn's disease and vitiligo . In both studies, the differentials of chronic vulval edema with erosions are granulomatous vulvitis, sarcoidosis, and chronic lymphedema due to obstruction, tuberculosis, subcutaneous mycoses, hidradenitis suppurativa and langerhans cell histiocytosis . Clinically, presence of chronic erosions, swelling, and erythematous discoloration of genitals with gastrointestinal symptoms and on investigation typical findings in usg, ct scan and colonoscopy favors diagnosis of crohn's disease . Absence of lymphadenopathy, vegetating growth, scarring, and negative x - ray findings excludes other differential conditions . Although histological picture of cutaneous crohn's may be indistinguishable from other granulomatous diseases, but histopathology of skin along with colonoscopy findings together leads to diagnosis of crohn's disease . Many treatment modalities are available as systemic steroids, sulfasalazine, mesalamine (active moiety of sulfasalazine), oral metronidazole, hyperbaric oxygen, and antitumor necrosis factor- antibody (infliximab). Our patient was treated symptomatically with tablet mesalamine (800 mg) eight hourly, capsule doxycycline (100 mg) and topically neomycin cream and was referred to higher center for operative procedures.
First performed in late 1980s, deep brain stimulation (dbs) of the subthalamic nucleus (stn) and the internal globuspallidus (gpi) or ventral intermedius nucleus (vim) of the thalamus has developed and become a distinguished symptomatic treatment for parkinson's disease (pd). Especially dbs of the stn and gpi is an effective option to improve motor symptoms and manage long - term motor complications resulting from levodopa treatment, such as wearing - off phenomena and dyskinesias . Furthermore patients mobility, activities of daily living, emotional well - being and health - related quality of life which are impaired by motor symptoms and complications (damiano et al ., 2000; 2013), can be enhanced by dbs (volkmann et al ., 2001; deuschl et al ., 2006). The other aim is to give an overview of the clinical effects and side effects of dbs with a focus on neuropsychological aspects . Few of them already providing long - term data between 8 and 10 years of follow - up (fasano et al ., 2010; castrioto et al ., 2011) could demonstrate an improvement of levodopa - responsive motor symptoms and motor complications, a reduction of the levodopa equivalent dose and an increase in quality of life after dbs (table 1). These trials provide a high level of evidence namely: level i ii due to the prospective randomized nature of the trials (oxford centre for evidence - based medicine levels of evidence; march 2009). However, placebo controlled trials assessing prospectively quality of life are not available for dbs in pd . Prospective randomized controlled clinical trials of deep brain stimulation (dbs) in parkinson's disease (pd) an essential aspect influencing the outcome after dbs in pd is patient selection and timing of surgery . Main indications for dbs in pd patients are levodopa - induced motor fluctuations, dyskinesias and unmanageable tremor . Preoperative indicators for a good outcome are younger age and shorter disease duration, high levodopa - response, few axial motor symptoms, absence of dementia, stable psychiatric conditions and no or non - severe comorbidities (bronstein et al ., 2011). With the exception of non - levodopa - responsive tremor, the preoperative levodopa - response is one of the most important outcome predictors (charles et al ., 2002). Several studies have shown a positive correlation between preoperative levodopa - response and motor improvement (kleiner - fisman et al ., 2003; kim et al ., 2013). Regarding cognitive impairment and psychiatric disorders, dementia is one of the most common exclusion criteria (bronstein et al ., 2011). The management of mild cognitive impairment is still handled more heterogenously . Besides age and levodopa - equivalence dosage the axial subscore, i.e., speech, neck rigidity, posture, rising from chair, gait and postural instability, in the unified pd rating scale (updrs) is reported to be a predicting factor for executive dysfunction after dbs (daniels et al ., 2010). Prognostic factors for the development of depressive symptoms after surgery are preoperative persistently increased scores for depression and anxiety measured by the state - trait anxiety inventory (stai), beck depression inventory (bdi) and clinical global impression - improvement scale (cgi - i) (schneider et al ., 2010). Therefore depression and other unstable psychiatric conditions require a careful preoperative assessment, a stabilizing treatment and a close - meshed postoperative follow - up (bronstein et al ., 2011). Concerning time point selection, age, hoehn and yahr stage and disease duration are important criteria . There is no clear age cut off, but patients over 70 years have a higher incidence of relevant comorbidities and cognitive impairment resulting in an increased risk for peri- and postoperative complications (russmann et al ., 2004; lang et al ., 2006; ory - magne et al ., 2007). In the past dbs used to be performed after 11 to 13 years of disease duration in patients with advanced motor - complications (follett et al ., 2010; williams et al ., 2010 recent data show high efficacy of dbs concerning an improvement of motor symptoms and quality of life in patients with shorter disease duration and early motor complications (schpbach et al ., 2013). In this study patients with a mean age of 52 years, a mean pd duration of 7.5 years and motor fluctuations of any severity persisting for 1.7 years were treated with stn - dbs and medication or received best medical treatment . The quality of life - measured by the pd questionnaire (pdq-39) - improved by 26% in the stimulation group, whereas it worsened in the medication group . The updrs motor score improved by 53% in the stimulation group in comparison to 4% in the medication group . These results favor the application of stn - dbs in an earlier stage of pd . Although mechanisms of action of dbs are not completely known up to date, modulation of pathological local (beta) oscillations (khn et al ., 2008) and modulation of the basal ganglia - cortical network including the hyperdirect pathway seems to play an important role (gradinaru et al ., 2009). Stn - dbs was first performed in 1993 in an advanced pd patient leading to a reduction of motor fluctuations, dyskinesias and dopaminergic medication (benabid et al ., 1994). Since then, several studies have reported an improvement of levodopa responsive symptoms (limousin et al ., 1998; 2006), motor fluctuations and dyskinesia after stn - dbs (follett et al ., 2010; okun et al ., 2012). Stn - dbs is usually performed bilaterally, but unilateral stn - dbs can be effective in highly asymmetric tremor - dominant pd patients (kumar et al ., 1999). Currently, stn - dbs is the most frequently used surgical therapy in pd . However, concerning the improvement of major pd symptoms no significant difference in efficacy has been shown between the stn and the gpi (weaver et al ., 2012), concerning the reduction of dopaminergic medication doses the stn is favorable (follett et al . Furthermore, stn - stimulation requires lower electrical power and results in longer battery life - spans (volkmann et al ., 2001). Another advantage in comparison to stn seems to be a better outcome regarding depression scores (follett et al ., in contrast to stn- and gpi - dbs, stimulation of the vim has no effects on dyskinesia, motor fluctuations, rigidity and bradykinesia but a clear and immediate effect on tremor (benabid et al ., 1996; ondo et al ., 1998). Certainly, vim - dbs is a therapeutic option for patients with essential tremor and elderly patients with a unilateral tremor - dominant pd . There is evidence revealing that stimulation of the ppn might have positive effects on parkinsonian gait disorder, postural instability and freezing (pereira et al ., 2008). Other reported effects concern a modification of vigilance and quality of sleep (alessandro et al ., 2010). Stimulation of the substantia nigra pars reticulata, partially in combination with the stn, remains experimental . An assumed effect on axial motor symptoms could not yet be proved, but an improvement of freezing of gait has been observed (weiss et al ., 2013). Changes of neurocognitive function, behavior and mood after dbs in pd have been described in several studies with partially conflicting results . Recent data suggest that the most frequent cognitive side effect, verbal fluency deficits, may be caused by surgical implantation (okun et al ., 2012). In this study, 136 patients underwent dbs device implantation, whereof 101 patients received immediate stn - stimulation and 35 received stimulation after 3 months . Verbal fluency, measured by delis - kaplan executive function system, degraded similarly in both groups without further aggravation after 3 months . Witt et al . (2013) observed a decline in mattis dementia rating scale in 7 out of 31 patients with stn - dbs . In comparison to patients without cognitive impairment lead trajectories in these 7 patients harmed a significantly larger volume of the caudate nucleus . However, there is also evidence that stimulation itself has an effect on verbal fluency: low - frequency (10 hz) stn - dbs has been shown to improve verbal fluency in comparison to higher stimulation frequencies (130 hz) and no stimulation (wojtecki et al ., 2006). Also other factors of stimulation intensity, the localization of the electrode and respective volume of tissue activated impact verbal fluency (mikos et al ., 2011). Regarding speech performance the cognitive (executive - function) aspect has to be clearly distinguished from the voice / articulation / loudness aspect that can be ameliorated or deteriorated by dbs depending on factors such as preoperative speech impairment (tripoliti et al . Regarding depression, there are some clinical trials reporting an improvement after stn - dbs (witt et al ., 2008; one explanation for these results might be an increase in quality of life and reduction of motor symptoms and complications . Others revealed a beneficial effect of gpi - dbs on depression and a worsening effect of stn - dbs (odekerken et al ., 2013). The deteriorating effect of stn - dbs might be caused by a reduction of dopaminergic medication . Apart from that, disease progression has to be taken into account as well (houeto et al ., 2002; follett et al ., 2010). Furthermore, there are reports of a detrimental effect of stn - dbs on fatigue . Okun et al . (2012) described that stn - stimulation rather than the surgical procedure appears to be responsible for this side - effect . In comparison to pd patients with stn - stimulation, their control group received implantation without stimulation for three months . Impulse control disorders typically caused by dopamine agonists are expected to improve after stn - dbs, mainly due to reduction of dopaminergic medication (demetriades et al ., 2011). Nevertheless, there are studies reporting regression, new development or persistence of impulse control disorders (smeding et al ., 2007; most frequently, stereotactic magnetic resonance imaging (mri) is used for target identification and target coordinates are calculated relative to the stereotactic frame placed on the patient's head (dormont et al ., 2010). Further options apart from direct targeting are fusion of mri and computed tomography (liu et al ., 2001) and stereotactic ventriculography, which is still but rarely used by some teams (benabid et al ., 2009). Intraoperative neurophysiology consists of intraoperative microelectrode recording (mer) and test stimulation and is used to improve targeting accuracy . For mer multiple trajectories mer of the stn is characterized by typical activity patterns, proprioceptive responses to passive movements and asymmetrical spikes at high frequency in a bursting manner (benabid et al ., 2009). Some studies suggest a significantly better clinical outcome after microelectrode recording (mann et al ., 2009; however, local anesthesia allows a communication with the patient during the operation and thereby a more precise assessment of side effects and the effect on a variety of symptoms, i.e., rigidity, tremor, coordination and speech . In contrast, general anesthesia is a helpful option to reduce patient's stress and pain (lefaucheur et al ., 2008; benabid et al ., the final lead is implanted and subcutaneously connected to the implantable pulse generator (ipg). Typical sites for the ipg are infraclavicular area and lower abdomen . At present, there are rechargeable and non - rechargeable ipgs available . Depending on stimulation parameters higher stimulation amplitude and pulse width result in shorter battery life spans (ondo et al ., 2007) non - rechargeable ipgs need to be replaced after approximately 5 years by surgery and are favored for elderly patients and patients with few technical skills . The main aim of programming is reducing motor - symptoms and complications and simultaneously avoiding or minimizing side effects of stimulation . Stimulation devices of the first generation delivered electrical stimulation in a voltage - controlled mode whereas following devices predominantly use the constant - current mode or can be switched into this mode . In comparison to constant - current devices, where the stimulation field is kept stable in size, the stimulation field produced by constant - voltage devices is vulnerable to changing tissue impedances (lempka et al ., 2010; okun et al ., 2012). The most frequent programming parameters are monopolar stimulation, impulse duration 6090 s and frequency 130 hz (volkmann et al ., 2006). Considering individual symptoms, the amplitude is increased carefully with a simultaneous reduction of dopaminergic medication during several programming sessions . However, the increase of amplitude is limited by stimulation - related side effects such as gait disorder and disequilibrium, dysarthria, oculomotor dysfunction, paraesthesia and increased muscle tone . To decrease these side effects, the stimulation field can be minimized by bipolar stimulation, but the necessity of higher stimulation intensities has to be taken into account (volkmann et al ., 2006). Alternatively, a modification of the stimulation field is achieved by current steering with multiple stimulation sources . Current steering is possible with a new device and allows for an individual shaping of the stimulation field by shifting the current towards another contact (barbe et al ., 2014). Interleaving stimulation is another method to shape the stimulation field by using alternating stimulation of different programs on two electrode contacts . Amongst others, this can be advantageous in patients with motor symptoms that require different contacts or amplitudes for the best therapeutic effect (wojtecki et al ., 2011). Apart from altering the stimulation field, a current pilot study (custom - dbs) could show that a lower pulse width of 30 s results in a wider therapeutic spectrum with higher side effect thresholds (volkmann et al ., 2014). A further stimulation technique under development is directional dbs . In comparison to current devices with omnidirectional stimulation (using one or more contacts of a quadripolar or octopolareelectrode) presently available data are based on intraoperative measurements and need to be verified by chronic implantation (pollo et al ., 2014). During the last 25 years, dbs has developed and become an established therapy for pd . Nevertheless, there are ever - growing findings concerning the effectiveness of dbs and the pathomechanisms of side effects resulting in development of new devices and stimulation paradigms . The main aim is a further reduction of side effects and better adaption to individual courses of pd . Inter alia, further development of closed loop stimulation, i.e., adaptive and individual stimulation depending on recorded beta activity of the stn might be an important step for the future (rosin et al ., 2011; little et al ., 2012)
There is increasing evidence suggesting a link between phpt and risk of malignancy outside a hereditary pathway (multiple endocrine neoplasia syndrome). The establishment of this association is important in deciding on the management of patients as regards screening for malignancy in phpt and the decision to opt for surgery . Several studies indicate an increased risk of malignancy in phpt compared to the normal population which is a contributor to the risk of premature death . However, the tumor sites are different in different studies implying that the relationship between phpt and cancer is more general in character than specific . A retrospective study from the swedish cancer registry in 1988 involving 4,163 operated patients followed up for 22 years showed a significantly increased relative risk of developing gastrointestinal cancers, endocrine tumors (involving adrenals, thymus, pituitary and pancreas), kidney carcinoma and mammary carcinoma . Another retrospective study from sweden in 1990 involving 896 operated patients followed up for about 13 years showed the presence of 24 different tumor locations among the 72 patients who died from malignancy with a significant association only with pancreatic adenocarcinoma . In 2002, a record - linkage study among 2,425 patients of hyperparathyroidism using the danish national cancer registry showed a 25% increased risk of cancer with the risk being higher in women . Hematopoietic malignancies (particularly multiple myeloma) were significantly more in phpt while patients with unspecified hyperparathyroidism had significantly increased carcinoma of the urinary tract and thyroid gland . Patients with secondary hyperparathyroidism had an insignificant overall cancer risk suggesting that malignancies were not due to parathyroid hormone (pth) itself . A retrospective danish study in 2004 involving 1,578 phpt patients found a total of 77 cases of death from malignancy with significantly increased mortality from oral and oesophageal cancer, as well as hematological malignancy in men and colonic cancer in women . In a swedish prospective cohort study in 2006 involving 7,847 women, serum calcium levels showed inverse association with breast cancer risk in premenopausal women in a dose - response manner but a positive association in overweight peri / post - menopausal women . In 2007, a retrospective cohort study using the swedish cancer registry including 9,782 operated patients showed an increased overall risk of cancers in both genders with risk persisting beyond 15 years after parathyroidectomy . Breast cancer accounted for a quarter of cancer incidence in women and an increased risk of kidney, colonic and squamous cell cancer was found in both genders . The risk of endocrine and pancreas cancer was increased in the small subgroup who were operated before 40 years of age . A recent record linkage study from scotland involving 3,039 phpt patients showed increased incidence and mortality from cancers in both surgically and conservatively managed patients with the commonest cancers being those of the skin (non - melanoma), breast, colon and lung . However, it is pertinent to note that as the absolute number of organ - specific tumors in all the above mentioned studies is small, organ - specific malignancy data need to be interpreted with caution . Although there is a wide variability in tumor site involvement, multiple studies have conclusively shown an increased risk of malignancy in phpt which persisted even after surgery . The increased risk could be related to hypercalcemia or they could share common aetiologic factors, either genetic and/or environmental . Increased mitotic activity induced by hypercalcemia is a possible mechanism and experimental studies have also shown pth to have anti - apoptotic and tumor promoting effects . Both, calcium and pth have been found to potentiate the mitogenic activity in bone marrow and lymphocyte mitogenesis . However, the prolonged persistence of risk post surgery has diluted the possibility of biochemical derangements contributing to cancer risk and has shifted the focus to other possible mechanisms . Vitamin d induces apoptosis, cell - cycle arrest, and differentiation and inhibits invasiveness and angiogenesis, thereby producing an anti - tumor effect and vitamin d receptors (vdr) are expressed in several body cells . A genetic predisposition due to disturbances in vdr alleles may cause impaired regulation of parathyroid glands, as well as defective apoptosis and increased incidence of preneoplastic lesions . A number of studies have established an increased risk of mortality in phpt from cardiovascular disease, malignancy and renal disease, with risk probably persisting for several years post surgery and factors like preoperative serum calcium and pth level and post - operative parathyroid adenoma weight being able to predict the same . A retrospective swedish study involving 441 operated phpt patients in 1987 showed an increased mortality risk predominantly due to cardiovascular disease which decreased only 5 to 8 years after surgery . Immediate post - operative mortality rate (within 1 month of surgery) was 1.8% and were among patients with hypercalcemic crisis or among elderly persons (older than 70 years) with severe arteriosclerosis . A similar finding was seen in another swedish study published in 1990 in 896 operated patients in which malignancy and renal disease (uremia) were found to be additional contributors to increased mortality . In 1995, another swedish study in 713 patients operated for single parathyroid gland disease showed adenoma weight to be significantly related to mortality risk, as well as preoperative serum calcium although serum calcium did not have an independent predictive value . Since, it is impossible to estimate the exact duration of hyperparathyroidism before diagnosis, the size of parathyroid adenoma could be reflective of the same and could further strengthen the concept of adopting a liberal attitude towards early surgery . However, a study from mayo clinic published in 1997 involving 435 phpt patients (of whom only 126 were operated) put forward certain contrary suggestions . Overall mortality or mortality due to cardiovascular disease or cancer was not increased in this cohort although there was a non - significant trend for decreased survival in patients treated conservatively, especially after 10 years following initial calcium elevation . Patients in the highest quartile of serum calcium had worse survival compared to the three lower quartiles, although this increased mortality risk showed a non - significant trend when compared to age and gender matched controls . The contradictory findings in this north american study compared to those reported by scandanavian authors is in all probability due to the presence of less advanced disease in this cohort . The earlier swedish studies were corroborated by another study from the same country in 1998 involving 4461 operated patients which showed an increased mortality risk from cardiovascular disease even in mild or moderate phpt with significant risk reduction following surgery . A german study in 2000 involving 360 operated patients demonstrated an increased mortality from cardiovascular disease predominantly in women . In an interesting swedish study published in 2001, 172 patients with mild hypercalcemia (confirmed 2 years post initial diagnosis) who were followed up for 25 years showed an increased risk of dying from cardiovascular disease despite a significant decrease in serum calcium value with time and had little risk of developing clinically apparent renal damage . An elaborate danish study published in 2004, found a significantly increased risk of premature death from cardiovascular disease and cancer in 1,578 patients with a hospital diagnosis of phpt . Women treated conservatively had significantly increased mortality from ischemic heart disease and cerebrovascular disease and there was a trend towards improved survival with surgery . Men had significantly increased mortality from cerebrovascular disease but not ischemic heart disease and this risk was not influenced by surgery . It is today well - confirmed that symptomatic phpt patients suffer from increased mortality before and after treatment with parathyroidectomy although, more definitive and conclusive evidence for the same in asymptomatic patients is warranted . Phpt has been associated with hypertension, left ventricular hypertrophy (lvh), increased calcification of the valves and coronary arteries, abnormal endothelial vasodilatory response and increased arterial stiffness along with metabolic abnormalities like dyslipidemia, obesity and insulin resistance; all of which contribute to increased cardiovascular death and some of which regress following surgery . Lvh is a strong and independent predictor of cardiovascular mortality while valvular sclerosis increases risk of cardiac events . Following surgery, reversal of lvh is seen, particularly in normotensive patients and no further deterioration occurs in sclerosis of the aortic and mitral valves . Some studies have demonstrated an impaired endothelium - dependent vasodilatation in phpt while others have shown decreased vascular smooth muscle mediated vasodilatation to be responsible for vascular dysfunction . Stiffening of the arteries might cause an augmentation of the pressure in central arteries and thereby increasing afterload on the heart . Pth acts on adult cardiomyocytes by binding to the pth / pthrp receptor, thereby inducing a rise in the intracellular levels of calcium which activates protein kinase c and mediates hypertrophic, as well as metabolic effects on the cardiomyocyte . Pth also appears to have an effect on the energy utilization in heart cells with both in vitro and in vivo studies showing pth to significantly lower the content of creatinine phosphate, atp, adp, amp and decrease the mitochondrial oxygen consumption in the cardiomyocyte . Lvh in phpt develops irrespective of blood pressure, patients biochemical profile and disease symptoms implying that even patients with mild and asymptomatic phpt may have an increased risk of cardiovascular death . A reasonable and educated opinion is the fact that absolute serum calcium and pth levels and duration of disease are all culprits in the harmful effects of phpt . The contradictory results in clinical trials of phpt could be due to the fact that the duration of disease is an unknown variable . A large multicentric prospective randomized controlled trial is needed to settle the issue of surgical intervention in asymptomatic patients who do not otherwise fulfil the present day nih criteria . Parathyroid surgery in the present day is performed by specialists with an impeccable success rate and minimal complications which could justify and tilt the balance in favor of establishing a lower threshold for surgery . Even in patients otherwise managed conservatively, being vigilant about possible malignancy and prompt management of cardiovascular aberrations as far as practicable could significantly contribute to increased survival in this disorder.
The src family kinases (sfks) are a unique group of enzymes that have diverse functions in cell proliferation, survival, differentiation, adhesion, and migration . They not only play important regulatory roles in hematopoiesis, but also contribute to development of hematopoietic cancers . One historically prominent paradigm of sfk action is cell proliferation and contribution to cell transformation . Sfks are known to have functional importance in chronic myelogenous leukemia (cml) because of the interaction between sfks and the oncogenic fusion protein bcr - abl . Sfk members hck and lyn readily interact with bcr - abl via kinase - dependent and -independent mechanisms, and this interaction cannot be inhibited by the bcr - abl inhibitor, imatinib [2 - 4]. In addition, this interaction between sfks and bcr - abl appears to be crucial for the transforming activity of bcr - abl, which is particularly essential for bcr - abl - driven b cell leukemia . Lyn is the predominant active sfk expressed in acute myeloid leukemia (aml) cells . Blocking sfk activity according to recent studies using gene knock - out mice, sfks, most notably lyn, fgr, and hck, play a role in regulation of myelopoiesis in vivo . Mice lacking lyn manifest an increase in myeloid progenitors, enhancement of granulocyte colony - stimulating factor - stimulated granulopoiesis, and development of a myeloproliferative disorder leading to development of monocyte / macrophage tumors . Similar findings were reported in triple knock - out mice lacking lyn, fgr, and hck . Sfk activity and expression could also modulate all - trans - retinoic acid (atra) differentiation induction therapy . In a recent study reported by miranda et al ., the sfk inhibitor pp2 was found to potentiate atra - induced gene expression and enhanced the differentiation marker cd11b in myeloid nb4, hl-60, and primary acute promyelocytic leukemia (apl) cells and the synergic effects of pp2 were dose dependent . Arsenic trioxide (as2o3, ato) has considerable efficacy in treatment of previously untreated and relapsed apl . Although initial studies focused on the apl fusion protein, promyelocytic leukemia (pml)-retinoic acid receptor (rar), in mediating response to ato, subsequent investigations have revealed that ato acts on numerous intracellular targets . In this study, we investigated the question of whether sfk inhibitor pp2 has an equal synergistic effect on differentiation of nb4 cells when combined with atra or ato . In addition, we examined the changes in retinoic acid (ra)-induced gene expression in order to determine the possible mechanism for the different effect of pp2 on atra- or ato - induced differentiation of nb4 cells . Nb4 cells were grown in rpmi1640 medium (gibco, rockville, ny) supplemented with 10% heat - inactivated fetal bovine serum (gibco) and 1% penicillin / streptomycin (gibco) in a humidified 5% carbon dioxide and 95% air incubator at 37. atra was obtained from sigma, and 5 mm stock solution was prepared in ethanol and stored at -20. ato (as2o3), nitroblue tetrazolium (nbt), and phorbol 12-myristate 13-acetate (pma) were obtained from sigma (st . Louis, mo). The src family kinase inhibitor pp2 was obtained from sigma and 20 mm stock solution was prepared in dimethyl sulfoxide and stored at -20. antibodies against cathepsin d and intercellular adhesion molecule-1 (icam-1) were purchased from santa cruz biotechnology (santa cruz, ca). Secondary anti - rabbit and anti - mouse horseradish peroxidase conjugated antibodies were purchased from dako (glostrup, denmark). In order to induce differentiation, nb4 cells were treated with atra or ato in the absence or presence of pp2 for 72 hours . For analysis of cell surface antigens, cells were harvested and washed twice with washing buffer (phosphate buffered saline [pbs] containing 0.2% bovine serum albumin and 0.1% nan3); they were then incubated with anti - cd11b - pe antibody (bd biosciences, san jose, ca) for 30 minutes at 4 in the dark . The cells were analyzed for cd11b expression using flow cytometry (fc500, beckman coulter, fullerton, ca). Briefly, each cell suspension was mixed with an equal volume of a solution containing 1 mg / ml of nbt and 2.5 g / ml of pma and incubated at 37 for 30 minutes . The differentiated cells were identified by their intracellular blue formazan deposits . Using a light microscope, a minimum of 200 cells was counted, in order to determine the percentage of differentiated cells . The annexin - v fitc kit (biosource, camarillo, ca) was used for detection of apoptotic cells . Briefly, nb4 cells were treated with atra or ato in the absence or presence of pp2 for 72 hours . Cells were washed twice with pbs and stained in binding buffer containing annexin - v fitc and propidium iodide (pi) at room temperature for 15 minutes in the dark . The reaction was stopped by addition of 400 l of binding buffer and analyzed by flow cytometry (fc500) with a total of 10,000 events acquired for each sample . Cells were treated with atra or ato, in the absence or presence of pp2 for 72 hours, and were harvested and washed with pbs . Total cellular proteins were isolated by sonication using ice - cold pbs containing 1% nonidet p-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (sds), 2.0 g / ml aprotinin, and protease inhibitor cocktail (roche, mannheim, germany), and centrifuged at 12,000 g for removal of cellular debris . The protein concentrations in extracts were determined using the bicinchoninic acid colorimetric method (pierce, rockford, il). Equal amounts of proteins were separated by 10% sds - polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (bio - rad, hercules, ca). The membranes were then blocked with 5% nonfat dried milk (bio - rad) in ttbs (50 mm tris - hcl [ph 7.5], 150 mm nacl, and 0.1% [v / v] tween 20) for 1 hour at room temperature and then incubated with primary antibodies diluted to 1:1,000 - 1:3,000 in 5% nonfat dried milk / ttbs overnight at 4 with gentle shaking . The membranes were then washed with ttbs (315 minutes) and subsequently incubated with secondary anti - rabbit or anti - mouse horseradish peroxidase conjugated antibodies diluted to 1:5,000 in 5% nonfat dried milk / ttbs for 2 hours at room temperature . The membranes were then washed as described above and developed using the enhanced chemiluminescence detection system (amersham, buckinghamshire, uk). Nb4 cells were grown in rpmi1640 medium (gibco, rockville, ny) supplemented with 10% heat - inactivated fetal bovine serum (gibco) and 1% penicillin / streptomycin (gibco) in a humidified 5% carbon dioxide and 95% air incubator at 37. atra was obtained from sigma, and 5 mm stock solution was prepared in ethanol and stored at -20. ato (as2o3), nitroblue tetrazolium (nbt), and phorbol 12-myristate 13-acetate (pma) were obtained from sigma (st . Louis, mo). The src family kinase inhibitor pp2 was obtained from sigma and 20 mm stock solution was prepared in dimethyl sulfoxide and stored at -20. antibodies against cathepsin d and intercellular adhesion molecule-1 (icam-1) were purchased from santa cruz biotechnology (santa cruz, ca). Secondary anti - rabbit and anti - mouse horseradish peroxidase conjugated antibodies were purchased from dako (glostrup, denmark). In order to induce differentiation, nb4 cells were treated with atra or ato in the absence or presence of pp2 for 72 hours . For analysis of cell surface antigens, cells were harvested and washed twice with washing buffer (phosphate buffered saline [pbs] containing 0.2% bovine serum albumin and 0.1% nan3); they were then incubated with anti - cd11b - pe antibody (bd biosciences, san jose, ca) for 30 minutes at 4 in the dark . The cells were analyzed for cd11b expression using flow cytometry (fc500, beckman coulter, fullerton, ca). Briefly, each cell suspension was mixed with an equal volume of a solution containing 1 mg / ml of nbt and 2.5 g / ml of pma and incubated at 37 for 30 minutes . The differentiated cells were identified by their intracellular blue formazan deposits . Using a light microscope, a minimum of 200 cells was counted, in order to determine the percentage of differentiated cells . The annexin - v fitc kit (biosource, camarillo, ca) was used for detection of apoptotic cells . Briefly, nb4 cells were treated with atra or ato in the absence or presence of pp2 for 72 hours . Cells were washed twice with pbs and stained in binding buffer containing annexin - v fitc and propidium iodide (pi) at room temperature for 15 minutes in the dark . The reaction was stopped by addition of 400 l of binding buffer and analyzed by flow cytometry (fc500) with a total of 10,000 events acquired for each sample . Cells were treated with atra or ato, in the absence or presence of pp2 for 72 hours, and were harvested and washed with pbs . Total cellular proteins were isolated by sonication using ice - cold pbs containing 1% nonidet p-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (sds), 2.0 g / ml aprotinin, and protease inhibitor cocktail (roche, mannheim, germany), and centrifuged at 12,000 g for removal of cellular debris . The protein concentrations in extracts were determined using the bicinchoninic acid colorimetric method (pierce, rockford, il). Equal amounts of proteins were separated by 10% sds - polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (bio - rad, hercules, ca). The membranes were then blocked with 5% nonfat dried milk (bio - rad) in ttbs (50 mm tris - hcl [ph 7.5], 150 mm nacl, and 0.1% [v / v] tween 20) for 1 hour at room temperature and then incubated with primary antibodies diluted to 1:1,000 - 1:3,000 in 5% nonfat dried milk / ttbs overnight at 4 with gentle shaking . The membranes were then washed with ttbs (315 minutes) and subsequently incubated with secondary anti - rabbit or anti - mouse horseradish peroxidase conjugated antibodies diluted to 1:5,000 in 5% nonfat dried milk / ttbs for 2 hours at room temperature . The membranes were then washed as described above and developed using the enhanced chemiluminescence detection system (amersham, buckinghamshire, uk). To determine the effect of pp2 on atra- or ato - induced differentiation of nb4 cells, we examined cd11b expression by flow cytometry . Treatment of nb4 cells with 10 m of pp2 alone, 0.001 m of atra alone, or 0.5 m of ato alone for 72 hours resulted in only 11.23%, 13.64%, or 14.06% of cd11b - positive cells, respectively (fig . However, co - treatment with atra plus pp2 or ato plus pp2 resulted in significant enhancement of cd11b - positive cells (60.73% and 31.58%, respectively; p<0.05) (fig . The synergistic effect of pp2 combined with atra was more significant than that of pp2 combined with ato (p<0.05). Similar results were obtained in five independent experiments, and were confirmed with nbt staining of nb4 cells (fig . This means that treatment with pp2 resulted in significantly enhanced atra- or ato - induced differentiation of nb4 cells in vitro and that the synergistic effect of pp2 was higher in combination with atra than in combination with ato in nb4 cells in vitro . To determine the effect of pp2 on atra- or ato - induced apoptosis of nb4 cells, we evaluated apoptotic cell death after annexin v - fitc and pi staining using flow cytometric analysis . 3 shows that treatment of nb4 cells with atra or ato alone or in combination with pp2 for 72 hours did not induce apoptosis in nb4 cells . This means that the ability of pp2 to enhance atra- or ato - induced differentiation of nb4 cells was not due to its effect on apoptosis in nb4 cells in vitro . To determine the possible mechanism for the effect of pp2 on atra- or ato - induced differentiation of nb4 cells, we examined the changes in expression of proteins (icam-1 and cathepsin d) derived from rar target genes . 4, control nb4 cells that were not treated with any reagents expressed low levels of these proteins . Treatment with pp2 alone resulted in a modest induction of icam-1 and cathepsin d expression . Treatment with atra alone resulted in increased expression of icam-1, as well as a modest induction of cathepsin d expression . Treatment with ato alone resulted in increased expression of cathepsin d, whereas it had no effect on expression of icam-1 . This induction of expression by atra or ato was further enhanced when cells were treated with pp2 in combination with atra or with pp2 in combination with ato . In particular, icam-1 expression showed a significant increase in cells treated with pp2 in combination with atra, whereas cathepsin d expression was significantly increased in cells treated with pp2 in combination with ato . These findings suggest that pp2 has different effects on atra and ato - induced differentiation of nb4 cells . Pp2 promotes atra - induced differentiation of nb4 cells through icam-1 and ato - induced differentiation of nb4 cells through cathepsin d. to determine the effect of pp2 on atra- or ato - induced differentiation of nb4 cells, we examined cd11b expression by flow cytometry . Treatment of nb4 cells with 10 m of pp2 alone, 0.001 m of atra alone, or 0.5 m of ato alone for 72 hours resulted in only 11.23%, 13.64%, or 14.06% of cd11b - positive cells, respectively (fig . However, co - treatment with atra plus pp2 or ato plus pp2 resulted in significant enhancement of cd11b - positive cells (60.73% and 31.58%, respectively; p<0.05) (fig . The synergistic effect of pp2 combined with atra was more significant than that of pp2 combined with ato (p<0.05). Similar results were obtained in five independent experiments, and were confirmed with nbt staining of nb4 cells (fig . This means that treatment with pp2 resulted in significantly enhanced atra- or ato - induced differentiation of nb4 cells in vitro and that the synergistic effect of pp2 was higher in combination with atra than in combination with ato in nb4 cells in vitro . To determine the effect of pp2 on atra- or ato - induced apoptosis of nb4 cells, we evaluated apoptotic cell death after annexin v - fitc and pi staining using flow cytometric analysis . 3 shows that treatment of nb4 cells with atra or ato alone or in combination with pp2 for 72 hours did not induce apoptosis in nb4 cells . This means that the ability of pp2 to enhance atra- or ato - induced differentiation of nb4 cells was not due to its effect on apoptosis in nb4 cells in vitro . To determine the possible mechanism for the effect of pp2 on atra- or ato - induced differentiation of nb4 cells, we examined the changes in expression of proteins (icam-1 and cathepsin d) derived from rar target genes . 4, control nb4 cells that were not treated with any reagents expressed low levels of these proteins . Treatment with pp2 alone resulted in a modest induction of icam-1 and cathepsin d expression . Treatment with atra alone resulted in increased expression of icam-1, as well as a modest induction of cathepsin d expression . Treatment with ato alone resulted in increased expression of cathepsin d, whereas it had no effect on expression of icam-1 . This induction of expression by atra or ato was further enhanced when cells were treated with pp2 in combination with atra or with pp2 in combination with ato . In particular, icam-1 expression showed a significant increase in cells treated with pp2 in combination with atra, whereas cathepsin d expression was significantly increased in cells treated with pp2 in combination with ato . These findings suggest that pp2 has different effects on atra and ato - induced differentiation of nb4 cells . Pp2 promotes atra - induced differentiation of nb4 cells through icam-1 and ato - induced differentiation of nb4 cells through cathepsin d. apl, a subtype of aml, with distinctive biologic and clinical features, is now highly curable . Translocation of the pml gene on chromosome 15 adjacent to the rar gene on chromosome 17 produces a fusion protein that can be quantitatively monitored using polymerase chain reaction in order to document disease burden and to ultimately confirm molecular remission . The unique ability of atra to produce differentiation in apl blasts can reverse coagulopathy, which is the major cause of death during induction . Emerging evidence has implicated sfks as regulators of proliferation and survival of myeloid lineage cells and regulators of cytokine - induced myelopoiesis . Aml is a diverse malignancy characterized by defective myeloid differentiation and accumulation of proliferative blasts . Recent studies have demonstrated that sfks act to negatively regulate ra - induced gene expression and myeloid differentiation and suggest that the combination of sfk inhibition and ra treatment may be therapeutically beneficial in aml . In this study, we investigated the question of whether sfk inhibitor pp2 enhanced the differentiation of nb4 cells when combined with ato or atra . Ato, an effective drug for treatment of apl, was shown to exert dose - dependent dual effects on apl cells, i.e., triggering apoptosis and inducing partial differentiation . It has also been suggested that these effects were related to ato - induced modulation / degradation of pml - rar protein . We found that treatment with sfk inhibitor pp2 resulted in significantly enhanced atra- or ato - induced differentiation of nb4 cells and that the synergistic effect was significantly stronger when pp2 was combined with atra than when pp2 was combined with ato . To the best of our knowledge, this is the first evidence that sfk inhibitors can enhance ato - induced myeloid differentiation of apl cells, even though the synergistic effect between atra and pp2 was significantly stronger than that between ato and pp2 . Of particular importance, these synergistic differentiation - enhancing effects were not related to apoptosis when pp2 was combined with ato as well as when pp2 was combined with atra . These results suggested that, even in ato - induced apoptosis and differentiation of apl cells, the synergistic effect between ato and pp2 was also related to the presence of rar ., reported that sfks regulated rar - mediated gene transcription and that sfk inhibition potentiated ra - induced gene expression . In our study, ra - induced gene expression was different in both groups . Expression of icam-1 showed a significant increase in cells treated with pp2 in combination with atra, whereas cathepsin d expression was significantly increased in cells treated with pp2 in combination with ato . These results suggest that the mechanism of sfk inhibitor - induced synergistic effect of apl cell differentiation on atra or ato is associated with ra - induced gene expression and that the difference in apl differentiation between atra and ato is related to disparate activation of ra - induced genes . Dasatinib, a us food and drug administration (fda)- approved compound, was developed as an inhibitor of abl and sfks . Recent studies have demonstrated the effects of dasatinib in hematopoietic malignancies other than cml and have suggested additional therapeutic opportunities . Because some investigators have demonstrated that dasatinib promotes atra - induced differentiation of aml cells, fda - approved dasatinib in combination with atra may be beneficial in treatment of apl and non - apl aml . In 2004, shen et al . First published the outcomes of single agent atra, single agent ato, or the combination of both drugs . While complete remission rates exceeded 90% in all three arms, the decline in the level of pml - rar fusion protein was significantly higher with the use of the combination . Hematologic recovery was more rapid and relapse - free survival was improved at 18 months . For subsequent similar data, national comprehensive cancer network (nccn) guidelines indicate that atra plus ato is an alternative for patients who cannot tolerate anthracycline therapy . A recent trend in cancer treatment is moving toward the use of targeted therapy and we eagerly look forward to obtaining therapeutic benefit due to low toxicity of targeted therapy . Our data showed that sfk inhibitor enhanced apl cell differentiation when combined with either atra or ato with difference in activation of ra - induced genes . This suggests that a new combination of sfk inhibitor, pp2 plus atra and ato may be therapeutically beneficial in apl.
We identified key actors by applying a snowball method starting with key actors in our own networks . Because the netherlands is a relatively small country, this was a feasible method of reaching sufficient people . The interviews were conducted by two researchers who had not published any research on threatening communication (gjp and kees de jong) to minimize socially desirable answers based on familiarity with an interviewer's work . Specifically, we interviewed 18 intervention developers and two administrators at health promoting service providers, four scientists, three policymakers, three politicians, and three advertising professionals . Five participants worked at regional hsps (e.g., municipal health services); the remaining 14 worked at national hsps, each addressing one or several behavioral themes such as smoking, safe sex, exercise, substance use, and diet patterns . All four scientists worked at dutch universities, specifically in the fields of medicine, business communication, epidemiology, and risk communication . Of the policymakers, one worked at a dutch ministry; one at a national funding organization; one at a national hsp; and one at a regional hsp . Of the politicians, one was active in national politics as a member of parliament, and the other two in regional politics as municipal counselors . The advertising agency employees all worked at advertising agencies that had developed health promotion campaigns for national hsps . The interview protocol (see http://sciencerep.org/5) started with gathering information on the background of each participant . After that, questions addressed participants' experience with intervention development in general; what information participants gathered before making decisions about interventions; how certain choices were made; and what were particularly good or bad interventions in participants' opinion . Specifically, the interviews focused on interventions with behavior change as a goal in general (no specific interventions participants had developed). During these conversations, when threatening communication would come up, this issue would be probed in more depth . To ensure that threatening communication would be discussed in all interviews, a number of examples of interventions were included for the interviewee to judge, two of which used threatening communication (a picture of blackened lungs on a pack of cigarettes and a speeding intervention; see http://sciencerep.org/5 for these pictures). We chose this approach to let threatening communication come up in the interview naturally, which decreased the possibility of defensive reactions . We anticipated defensive reactions when intervention designers would be confronted directly with the question of why they use threatening communication . The interviews were recorded, transcribed, and imported into qsr nvivo, where they were coded by gjp . First, all interview fragments where threatening communication was discussed were coded in the same fear appeal category . Then these fragments were reread and coded with more specific categories (see results section). The second coding sweep yielded a number of systematically occurring beliefs, which were categorized in a tree structure with the following main categories: reasons for fear appeal use; reasons against fear appeal use; the process of intervention development; related beliefs (not otherwise categorized); and environmental constraints . Within some categories, subcategories were formed, such as within the process of intervention development category, the subcategories goals of intervention development and target group involvement . The beliefs identified in these categories and subcategories were then organized in a narrative in four sections, each describing largely coherent sets of beliefs . All participants knew a lot about how to develop effective interventions . In this paper, however, we focus on beliefs that were inconsistent with the scientific state of the art . That narrow focus may cause these results to paint a bleak picture, but this does not mean that the interviewed participants were ignorant or incompetent in terms of intervention development . Yet there is always room for improvement, and this paper aims to facilitate such improvement . Inducing fear was cited relatively rarely as a reason to use threatening communication . More often, the goal was to confront people with the negative consequences of a given risky behavior . The most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing, and attention is a limited resource . Given that target population members are exposed simultaneously to multiple stimuli, behavior change interventions need to compete for attention . Evoking strong emotions the need to break through and draw attention was often reported: of course, we don't have as much media budget as a commercial organization . You want to draw attention, you don't want to shock, i don't think that's the right word, but you do want to do something that reaches people . So you want to hit a certain emotion, and i think that we managed with our campaign . Because many people saw it . (intervention developer, 191110) of course, we don't have as much media budget as a commercial organization . You want to draw attention, you don't want to shock, i don't think that's the right word, but you do want to do something that reaches people . So you want to hit a certain emotion, and i think that we managed with our campaign . Because many people saw it . (intervention developer, 191110) emotion was also assumed to render interventions memorable, which, combined with the memory of the evoked emotion itself, was assumed to prompt desirable behavior: and why do you have to strike an emotion? Yes, again, people get to see so many communications in a day, that if you want to strike somebody at all, then especially that emotion is important . Because only by letting people really feel something, you can prompt people to act . So at the moment you, for example, strike someone, in the sense of, we have a collection campaign, and you manage to strike someone, then such a person will be willing to donate money in the collection box . Or maybe even extra this year . Oh, yes, i saw that commercial, that struck me personally . Then that works . (intervention developer, 191110) and why do you have to strike an emotion? Yes, again, people get to see so many communications in a day, that if you want to strike somebody at all, then especially that emotion is important . Because only by letting people really feel something, you can prompt people to act . So at the moment you, for example, strike someone, in the sense of, we have a collection campaign, and you manage to strike someone, then such a person will be willing to donate money in the collection box . Or maybe even extra this year . Oh, yes, i saw that commercial, that struck me personally . Then that works . (intervention developer, 191110) a second example of this reasoning was provided by a participant from the advertising world: (discussing the first prompting intervention, see http://sciencerep.org/5) what do you think? Is this a good intervention, or not? I think that in this case, you want to address a specific target group, and for that, i think it's good . I think that in that case you don't have to bother with a boring communication indeed, i think that if you want to address a certain target group, with a lot of sexual contacts, it's fine . Before you know it, you have it, so be wise . Becoming aware of easily contracting an sti and continuing your old behavior doesn't seem useful . (advertising professional, 51183) (discussing the first prompting intervention, see http://sciencerep.org/5) what do you think? I think that in this case, you want to address a specific target group, and for that, i think it's good . I think that in that case you don't have to bother with a boring communication indeed, i think that if you want to address a certain target group, with a lot of sexual contacts, it's fine . Before you know it, you have it, so be wise . Becoming aware of easily contracting an sti and continuing your old behavior doesn't seem useful . (advertising professional, 51183) another common belief was that when a communication manages to evoke emotions in target population members, this would cause reflection about the relevant behavior: for example, i spoke to a doctor, a doctor for young people from the municipal health centre, and he had a parent meeting at school, in group 7/8 [ages 1012], and there they showed a brain scan with pictures . So one picture of a child who had not consumed alcohol, and another scan of a child who had consumed alcohol . And showed what it does to your brains . Well, they showed that at the meeting, and then people could ask questions . And then i thought, i think that works, because people then start thinking . (politician, 191115) for example, i spoke to a doctor, a doctor for young people from the municipal health centre, and he had a parent meeting at school, in group 7/8 [ages 1012], and there they showed a brain scan with pictures . So one picture of a child who had not consumed alcohol, and another scan of a child who had consumed alcohol . And showed what it does to your brains . Well, they showed that at the meeting, and then people could ask questions . And then i thought, i think that works, because people then start thinking . (politician, 191115) this belief was based on the assumption that target population members act and reason rationally (i.e. Consistent with a subjective expected utility model; see e.g. Tversky & kahneman, 1981). A related and possibly derived belief was participants' presumption that their own common - sense reasoning about how communications would be received and processed further would resemble reality . For example, one scientist explained: i would, if i would read, like, it gives you lung cancer (scientist, 4119) i would, if i would read, like, it gives you lung cancer (scientist, 4119) also related, achieving awareness (or increasing risk perception to a certain threshold) was often considered a sufficient condition for behavior change: you try to increase this motivation to quit . So the more you make them aware of the fact that it's bad, the stronger your motivation becomes with respect to your addiction . So the more you make them aware of the fact that it's bad, the stronger your motivation becomes with respect to your addiction . (advertising professional, 22114) to participants who assumed that target population members who perform the undesirable behavior are ignorant as to the negative consequences, showing these negative consequences was considered necessary education . Similarly, target population members were generally assumed to underestimate the negative consequences of the behavior . Thus, participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . Intervention developers had often heard negative messages about threatening communication, but did not usually understand the dynamics at play . In general, it could be said that interviewees seemed to have some pieces of the puzzle, but lacked a coherent framework of the working mechanisms of threatening communication . This sometimes manifested as ambivalence regarding the effectiveness of threatening communication: yes, but risk communication does not work with this population? Yes, i think that this [pictures on cigarette packs] is a good method, but i also think that there's no other way than this [to change behavior]. (scientist, 4119) yes, but risk communication does not work with this population? Yes, i think that this [pictures on cigarette packs] is a good method, but i also think that there's no other way than this [to change behavior]. (scientist, 4119) when logical arguments against the use of threatening communication were acknowledged, this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand, often the nature of the behavior at hand (e.g., smoking is a difficult behavior). It was not uncommon for participants to express beliefs that in terms of the eppm were logically inconsistent . For example, participants could state that they thought that low susceptibility, rather than low severity, was the main cause of unhealthy behavior; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . One participant explained that they tried to solve the problem of low susceptibility in their target population by tailoring an intervention, enabling them to communicate that target population members belonged to a subpopulation that was at a relatively high risk . While this reasoning is partly in line with eppm, for example, one participant remarked: yes, this [picture of blackened lungs] is ok, because you can do something about it by quitting smoking, so, um, i mean, if you would say, (advertising professional, 22114) yes, this [picture of blackened lungs] is ok, because you can do something about it by quitting smoking, so, um, i mean, if you would say, breathing causes lung cancer [that would be a different story]. (advertising professional, 22114) related to this, participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high, but then often underestimated the required intensity of an intervention to enhance efficacy . In fact, this was sometimes reported as a necessary ingredient of interventions using threatening communication . Scientists were often cognizant of the eppm but, like other participants, did not always realize that the threatening stimulus in this model can be any stimulus . Some classes of intervention types were considered to not be fear - inducing (i.e., carry the risk of fear control responses), such as scenario messages or personalized risk assessments . Similarly, often participants indicated that they did not use threatening communication, while from their explanation it became clear that they did: [discussing a campaign against smoking that would focus on cancer] some people may call this a fear inducing message i don't call this fear inducing, because question one is, what is fear exactly and what is fear inducing? I consider it more of an awareness campaign . Because people often say, well, the risk is not so bad, and well, cancer it is more of a confronting campaign, where the health effects you can expect from smoking regarding cancer are made clear, visually . (policymaker, 26114) [discussing a campaign against smoking that would focus on cancer] some people may call this a fear inducing message i don't call this fear inducing, because question one is, what is fear exactly and what is fear inducing? I consider it more of an awareness campaign . Because people often say, well, the risk is not so bad, and well, cancer it is more of a confronting campaign, where the health effects you can expect from smoking regarding cancer are made clear, visually . In fact, few participants were aware of the conceptual distinction between personal determinants (psychological variables that can be influenced to eventually engender behavior change, such as risk perception or self - efficacy), behavior change methods or techniques (theoretical methods to influence a given determinant, such as fear appeals or modeling), and applications of these methods (an incarnation of a theoretical method, such as a picture of blackened lungs or a movie with a role model). One participant who was partially aware of this distinction explained: but then the step from those modifiable determinants to good methods to change that, kind of, that is often difficult . What are effective methods to achieve those goals and how do you utilize these? Often, the time and knowledge to do this thoroughly is lacking . (intervention developer, 22612) but then the step from those modifiable determinants to good methods to change that, kind of, that is often difficult . What are effective methods to achieve those goals and how do you utilize these? Often, the time and knowledge to do this thoroughly is lacking . (intervention developer, 22612) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication: there were no known good alternatives . Intervention developers often did not have an array of behavior change methods at their disposal, and sometimes they applied the same method in all interventions . Some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . Specifically, some participants indicated that it was important that an intervention originated from within the target population . However, these target population members generally advocated threatening communication approaches: yes, or, they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign ., last year, we had a study done into interventions targeting young immigrants, who were also like yes, we actually don't want these things addressed with humor, but actually it should just be, not too hard, but confronting, because bad things can happen if you have unsafe sex . (intervention developer, 21210) yes, or, they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign yes, no, it has to be more serious . For example, last year, we had a study done into interventions targeting young immigrants, who were also like yes, we actually don't want these things addressed with humor, but actually it should just be, not too hard, but confronting, because bad things can happen if you have unsafe sex . (intervention developer, 21210) because participants often assumed that target population members knew how they themselves could be influenced, this reliance on target population members' preferences for threatening communication could easily result in the development of threatening interventions . Among employees of advertising agencies and policymakers, the belief existed that there are no ways to predict in advance whether a campaign would be successful: and can you estimate a bit, when you've made many campaigns, whether campaigns work or not? Regardless of indicators like market share, on the basis of your experience? Yes, that is very hard to say . Because sometimes you're surprised by campaigns that turn out to work very well, while you think, this has to be great, but it doesn't work, or a customer gives it insufficient chance . That is, um, when you would be able to determine this in advance, you would have found the holy grail . (advertising professional, 9416) and can you estimate a bit, when you've made many campaigns, whether campaigns work or not? Regardless of indicators like market share, on the basis of your experience? Yes, that is very hard to say . Because sometimes you're surprised by campaigns that turn out to work very well, while you think, or campaigns of which you think, this has to be great, but it doesn't work, or a customer gives it insufficient chance . That is, um, when you would be able to determine this in advance, you would have found the holy grail . (advertising professional, 9416) intervention development was considered largely a trial - and - error endeavor, with the development process often being seen as a black box (in advertising, a creative process engaged in by the creatives). Yet, at the same time, in advertising, there were some basic guidelines on campaign development . For example, one interviewee explained that a commercial for a product such as soup should always visually show both the soup and the packaging . Another such heuristic was the merit of creativity or originality: you can do something very strange and very new, but if you do it a second or a third time, then it's already not surprising any more . I think that in the end clients choose to be creative within a given campaign, with a given idea . And (advertising professional, 9416) you can do something very strange and very new, but if you do it a second or a third time, then it's already not surprising any more . I think that in the end clients choose to be creative within a given campaign, with a given idea . And (advertising professional, 9416) among participants from advertising agencies, sometimes confrontation and humor were two options to choose from when trying to reach people . When confrontation was considered inappropriate, humor would be the alternative approach: well, i think that you choose humor at a time you actually want to point something out to people, but you don't want to pedantically wag your finger . And if you do it in a humorous way then it does come across differently to people than when you say, eh, you're not allowed to do this, or you have to do that . Yes . That you then mainly choose humor . So that it does come across, but that people don't think, (advertising professional, 22111) well, i think that you choose humor at a time you actually want to point something out to people, but you don't want to pedantically wag your finger . And if you do it in a humorous way then it does come across differently to people than when you say, eh, you're not allowed to do this, or you have to do that . Yes . That you then mainly choose humor . So that it does come across, but that people don't think, (advertising professional, 22111) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . At the same time, when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable (i.e., not in line with known goals or constraints), intervention developers would generally communicate this and demand adjustments . Participants generally conceded that evaluating whether an intervention had successfully induced behavior change was complicated . Understandably, besides the scientists, very few participants mentioned the possibility of examining such evaluations in a laboratory setting . Instead one such indicator was the degree to which an intervention was recognized or remembered, or the perceived impact as reported by introspection of target population members: so how do you want to verify whether goals you set for an intervention, whether those are met after some time? Well, for now that is mainly in narrative form, it's mainly about statements of people, to go start and maintain a dialogue with young people and teachers and other involved people, like, well, did it touch you, did it do something for you? (intervention developer, 18314) so how do you want to verify whether goals you set for an intervention, whether those are met after some time? Well, for now that is mainly in narrative form, it's mainly about statements of people, to go start and maintain a dialogue with young people and teachers and other involved people, like, well, did it touch you, did it do something for you? (intervention developer, 18314) some participants reported favoring pictures on packs of cigarettes, not to induce fear, but simply to make the packaging less attractive: i also don't believe that a picture in itself necessarily causes somebody to quit, if you say that i don't think you can deliver . Now the tobacco industry still tries, very glamorizing, to make it attractive, make it pretty . In america, they have special pink packages, to target women . In other countries they have packages with only five cigarettes, to make it more attractive for young people . (policymaker, 26114) i also don't believe that a picture in itself necessarily causes somebody to quit, if you say that i don't think you can deliver . . Now the tobacco industry still tries, very glamorizing, to make it attractive, make it pretty . In america, they have special pink packages, to target women . In other countries they have packages with only five cigarettes, to make it more attractive for young people . Participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem, they would often prioritize quick and clearly visible results over effective evidence - based interventions (note that this fits well with advertising agencies' drive for originality): and in practice people then start to do something, eh, because the municipality also has to show that something is happening, and politics also asks for it, and also asks for visible activities and results preferably . (intervention developer, 31317) and in practice people then start to do something, eh, because the municipality also has to show that something is happening, and politics also asks for it, and also asks for visible activities and results preferably . (intervention developer, 31317) in addition, intervention developers sometimes operated within intermediary organizations, such as schools, that were in favor of threatening approaches, and were sometimes hard to convince otherwise . When participants were not in favor of threatening communication, their reasons were not always based on the understanding of the inefficacy of threatening communications . Other reasons included that fear was not considered an intrinsic motivator (reflecting the implicit assumption that intrinsic motivation is superior to extrinsic motivation) and that their organization just did not believe in motivating through fear . One participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated, which might decrease donations or susceptibility to future messages . Finally, one additional overarching observation was that although most beliefs did not seem specific to one or a few categories of key actors, one clear pattern emerged . Participants who were closer to actual intervention development often had a generic idea that there was something wrong with inducing fear (though they rarely grasped the underlying dynamics). Participants who were further removed from the intervention development activities often did not have this basic heuristic belief . Inducing fear was cited relatively rarely as a reason to use threatening communication . More often, the goal was to confront people with the negative consequences of a given risky behavior . The most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing, and attention is a limited resource . Given that target population members are exposed simultaneously to multiple stimuli, behavior change interventions need to compete for attention . Evoking strong emotions the need to break through and draw attention was often reported: of course, we don't have as much media budget as a commercial organization . You want to draw attention, you don't want to shock, i don't think that's the right word, but you do want to do something that reaches people . So you want to hit a certain emotion, and i think that we managed with our campaign . Because many people saw it . (intervention developer, 191110) of course, we don't have as much media budget as a commercial organization . You want to draw attention, you don't want to shock, i don't think that's the right word, but you do want to do something that reaches people . So you want to hit a certain emotion, and i think that we managed with our campaign . Because many people saw it . (intervention developer, 191110) emotion was also assumed to render interventions memorable, which, combined with the memory of the evoked emotion itself, was assumed to prompt desirable behavior: and why do you have to strike an emotion? Yes, again, people get to see so many communications in a day, that if you want to strike somebody at all, then especially that emotion is important . Because only by letting people really feel something, you can prompt people to act . So at the moment you, for example, strike someone, in the sense of, we have a collection campaign, and you manage to strike someone, then such a person will be willing to donate money in the collection box . Or maybe even extra this year . Oh, yes, i saw that commercial, that struck me personally . Then that works . (intervention developer, 191110) and why do you have to strike an emotion? Yes, again, people get to see so many communications in a day, that if you want to strike somebody at all, then especially that emotion is important . Because only by letting people really feel something, you can prompt people to act . So at the moment you, for example, strike someone, in the sense of, we have a collection campaign, and you manage to strike someone, then such a person will be willing to donate money in the collection box . Or maybe even extra this year . Oh, yes, i saw that commercial, that struck me personally . Then that works . (intervention developer, 191110) a second example of this reasoning was provided by a participant from the advertising world: (discussing the first prompting intervention, see http://sciencerep.org/5) what do you think? I think that in this case, you want to address a specific target group, and for that, i think it's good . I think that in that case you don't have to bother with a boring communication indeed, i think that if you want to address a certain target group, with a lot of sexual contacts, it's fine . Before you know it, you have it, so be wise . Is this of course, you have behavior change and awareness . What do you think of it in those respects? Becoming aware of easily contracting an sti and continuing your old behavior doesn't seem useful . (advertising professional, 51183) (discussing the first prompting intervention, see http://sciencerep.org/5) what do you think? Is this a good intervention, or not? Do you think this would work, or not? I think that in this case, you want to address a specific target group, and for that, i think it's good . I think that in that case you don't have to bother with a boring communication indeed, i think that if you want to address a certain target group, with a lot of sexual contacts, it's fine . Before you know it, you have it, so be wise . Becoming aware of easily contracting an sti and continuing your old behavior doesn't seem useful . (advertising professional, 51183) another common belief was that when a communication manages to evoke emotions in target population members, this would cause reflection about the relevant behavior: for example, i spoke to a doctor, a doctor for young people from the municipal health centre, and he had a parent meeting at school, in group 7/8 [ages 1012], and there they showed a brain scan with pictures . So one picture of a child who had not consumed alcohol, and another scan of a child who had consumed alcohol . And showed what it does to your brains . Well, they showed that at the meeting, and then people could ask questions . And then i thought, i think that works, because people then start thinking . (politician, 191115) for example, i spoke to a doctor, a doctor for young people from the municipal health centre, and he had a parent meeting at school, in group 7/8 [ages 1012], and there they showed a brain scan with pictures . So one picture of a child who had not consumed alcohol, and another scan of a child who had consumed alcohol . And showed what it does to your brains . Well, they showed that at the meeting, and then people could ask questions . And then i thought, i think that works, because people then start thinking . (politician, 191115) this belief was based on the assumption that target population members act and reason rationally (i.e. Consistent with a subjective expected utility model; see e.g. Tversky & kahneman, 1981). A related and possibly derived belief was participants' presumption that their own common - sense reasoning about how communications would be received and processed further would resemble reality . For example, one scientist explained: i would, if i would read, like, it gives you lung cancer (scientist, 4119) i would, if i would read, like, it gives you lung cancer (scientist, 4119) also related, achieving awareness (or increasing risk perception to a certain threshold) was often considered a sufficient condition for behavior change: you try to increase this motivation to quit . So the more you make them aware of the fact that it's bad, the stronger your motivation becomes with respect to your addiction . So the more you make them aware of the fact that it's bad, the stronger your motivation becomes with respect to your addiction . (advertising professional, 22114) to participants who assumed that target population members who perform the undesirable behavior are ignorant as to the negative consequences, showing these negative consequences was considered necessary education . Similarly, target population members were generally assumed to underestimate the negative consequences of the behavior . Thus, participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . Intervention developers had often heard negative messages about threatening communication, but did not usually understand the dynamics at play . In general, it could be said that interviewees seemed to have some pieces of the puzzle, but lacked a coherent framework of the working mechanisms of threatening communication . This sometimes manifested as ambivalence regarding the effectiveness of threatening communication: yes, but risk communication does not work with this population? Yes, i think that this [pictures on cigarette packs] is a good method, but i also think that there's no other way than this [to change behavior]. (scientist, 4119) yes, but risk communication does not work with this population? Yes, i think that this [pictures on cigarette packs] is a good method, but i also think that there's no other way than this [to change behavior]. (scientist, 4119) when logical arguments against the use of threatening communication were acknowledged, this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand, often the nature of the behavior at hand (e.g., smoking is a difficult behavior). It was not uncommon for participants to express beliefs that in terms of the eppm were logically inconsistent . For example, participants could state that they thought that low susceptibility, rather than low severity, was the main cause of unhealthy behavior; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . One participant explained that they tried to solve the problem of low susceptibility in their target population by tailoring an intervention, enabling them to communicate that target population members belonged to a subpopulation that was at a relatively high risk . While this reasoning is partly in line with eppm, for example, one participant remarked: yes, this [picture of blackened lungs] is ok, because you can do something about it by quitting smoking, so, um, i mean, if you would say, breathing causes lung cancer (advertising professional, 22114) yes, this [picture of blackened lungs] is ok, because you can do something about it by quitting smoking, so, um, i mean, if you would say, breathing causes lung cancer [that would be a different story]. (advertising professional, 22114) related to this, participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high, but then often underestimated the required intensity of an intervention to enhance efficacy . In fact, this was sometimes reported as a necessary ingredient of interventions using threatening communication . Scientists were often cognizant of the eppm but, like other participants, did not always realize that the threatening stimulus in this model can be any stimulus . Some classes of intervention types were considered to not be fear - inducing (i.e., carry the risk of fear control responses), such as scenario messages or personalized risk assessments . Similarly, often participants indicated that they did not use threatening communication, while from their explanation it became clear that they did: [discussing a campaign against smoking that would focus on cancer] some people may call this a fear inducing message i don't call this fear inducing, because question one is, what is fear exactly and what is fear inducing? I consider it more of an awareness campaign . Because people often say, well, the risk is not so bad, and well, cancer, my grandfather lived to 92 with two packs of cigarettes a day . It is more of a confronting campaign, where the health effects you can expect from smoking regarding cancer are made clear, visually . (policymaker, 26114) [discussing a campaign against smoking that would focus on cancer] some people may call this a fear inducing message i don't call this fear inducing, because question one is, what is fear exactly and what is fear inducing? I consider it more of an awareness campaign . Because people often say, well, the risk is not so bad, and well, cancer it is more of a confronting campaign, where the health effects you can expect from smoking regarding cancer are made clear, visually . In fact, few participants were aware of the conceptual distinction between personal determinants (psychological variables that can be influenced to eventually engender behavior change, such as risk perception or self - efficacy), behavior change methods or techniques (theoretical methods to influence a given determinant, such as fear appeals or modeling), and applications of these methods (an incarnation of a theoretical method, such as a picture of blackened lungs or a movie with a role model). One participant who was partially aware of this distinction explained: but then the step from those modifiable determinants to good methods to change that, kind of, that is often difficult . What are effective methods to achieve those goals and how do you utilize these? Often, the time and knowledge to do this thoroughly is lacking . (intervention developer, 22612) but then the step from those modifiable determinants to good methods to change that, kind of, that is often difficult . What are effective methods to achieve those goals and how do you utilize these? Often, the time and knowledge to do this thoroughly is lacking . (intervention developer, 22612) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication: there were no known good alternatives . Intervention developers often did not have an array of behavior change methods at their disposal, and sometimes they applied the same method in all interventions . Some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . Specifically, some participants indicated that it was important that an intervention originated from within the target population . However, these target population members generally advocated threatening communication approaches: yes, or, they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign ., last year, we had a study done into interventions targeting young immigrants, who were also like yes, we actually don't want these things addressed with humor, but actually it should just be, not too hard, but confronting, because bad things can happen if you have unsafe sex . (intervention developer, 21210) yes, or, they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign ., last year, we had a study done into interventions targeting young immigrants, who were also like yes, we actually don't want these things addressed with humor, but actually it should just be, not too hard, but confronting, because bad things can happen if you have unsafe sex . (intervention developer, 21210) because participants often assumed that target population members knew how they themselves could be influenced, this reliance on target population members' preferences for threatening communication could easily result in the development of threatening interventions . Among employees of advertising agencies and policymakers, the belief existed that there are no ways to predict in advance whether a campaign would be successful: and can you estimate a bit, when you've made many campaigns, whether campaigns work or not? Regardless of indicators like market share, on the basis of your experience? Yes, that is very hard to say . Because sometimes you're surprised by campaigns that turn out to work very well, while you think, this has to be great, but it doesn't work, or a customer gives it insufficient chance . That is, um, when you would be able to determine this in advance, you would have found the holy grail . (advertising professional, 9416) and can you estimate a bit, when you've made many campaigns, whether campaigns work or not? Regardless of indicators like market share, on the basis of your experience? Yes, that is very hard to say . Because sometimes you're surprised by campaigns that turn out to work very well, while you think, or campaigns of which you think, this has to be great, but it doesn't work, or a customer gives it insufficient chance . That is, um, when you would be able to determine this in advance, you would have found the holy grail . (advertising professional, 9416) intervention development was considered largely a trial - and - error endeavor, with the development process often being seen as a black box (in advertising, a creative process engaged in by the creatives). Yet, at the same time, in advertising, there were some basic guidelines on campaign development . For example, one interviewee explained that a commercial for a product such as soup should always visually show both the soup and the packaging . Another such heuristic was the merit of creativity or originality: you can do something very strange and very new, but if you do it a second or a third time, then it's already not surprising any more . I think that in the end clients choose to be creative within a given campaign, with a given idea . And (advertising professional, 9416) you can do something very strange and very new, but if you do it a second or a third time, then it's already not surprising any more . I think that in the end clients choose to be creative within a given campaign, with a given idea . And (advertising professional, 9416) among participants from advertising agencies, sometimes confrontation and humor were two options to choose from when trying to reach people . When confrontation was considered inappropriate, humor would be the alternative approach: well, i think that you choose humor at a time you actually want to point something out to people, but you don't want to pedantically wag your finger . And if you do it in a humorous way then it does come across differently to people than when you say, eh, you're not allowed to do this, or you have to do that . Yes . That you then mainly choose humor . So that it does come across, but that people don't think, (advertising professional, 22111) well, i think that you choose humor at a time you actually want to point something out to people, but you don't want to pedantically wag your finger . And if you do it in a humorous way then it does come across differently to people than when you say, eh, you're not allowed to do this, or you have to do that . Yes . That you then mainly choose humor . So that it does come across, but that people don't think, (advertising professional, 22111) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . At the same time, when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable (i.e., not in line with known goals or constraints), intervention developers would generally communicate this and demand adjustments . Participants generally conceded that evaluating whether an intervention had successfully induced behavior change was complicated . Understandably, besides the scientists, very few participants mentioned the possibility of examining such evaluations in a laboratory setting . Instead one such indicator was the degree to which an intervention was recognized or remembered, or the perceived impact as reported by introspection of target population members: so how do you want to verify whether goals you set for an intervention, whether those are met after some time? Well, for now that is mainly in narrative form, it's mainly about statements of people, to go start and maintain a dialogue with young people and teachers and other involved people, like, well, did it touch you, did it do something for you? (intervention developer, 18314) so how do you want to verify whether goals you set for an intervention, whether those are met after some time? Well, for now that is mainly in narrative form, it's mainly about statements of people, to go start and maintain a dialogue with young people and teachers and other involved people, like, well, did it touch you, did it do something for you? Some participants reported favoring pictures on packs of cigarettes, not to induce fear, but simply to make the packaging less attractive: i also don't believe that a picture in itself necessarily causes somebody to quit, if you say that i don't think you can deliver . Now the tobacco industry still tries, very glamorizing, to make it attractive, make it pretty . In america, they have special pink packages, to target women . In other countries they have packages with only five cigarettes, to make it more attractive for young people . (policymaker, 26114) i also don't believe that a picture in itself necessarily causes somebody to quit, if you say that i don't think you can deliver . Now the tobacco industry still tries, very glamorizing, to make it attractive, make it pretty . In america, they have special pink packages, to target women . In other countries they have packages with only five cigarettes, to make it more attractive for young people . Participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem, they would often prioritize quick and clearly visible results over effective evidence - based interventions (note that this fits well with advertising agencies' drive for originality): and in practice people then start to do something, eh, because the municipality also has to show that something is happening, and politics also asks for it, and also asks for visible activities and results preferably . (intervention developer, 31317) and in practice people then start to do something, eh, because the municipality also has to show that something is happening, and politics also asks for it, and also asks for visible activities and results preferably . (intervention developer, 31317) in addition, intervention developers sometimes operated within intermediary organizations, such as schools, that were in favor of threatening approaches, and were sometimes hard to convince otherwise . When participants were not in favor of threatening communication, their reasons were not always based on the understanding of the inefficacy of threatening communications . Other reasons included that fear was not considered an intrinsic motivator (reflecting the implicit assumption that intrinsic motivation is superior to extrinsic motivation) and that their organization just did not believe in motivating through fear . One participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated, which might decrease donations or susceptibility to future messages . Finally, one additional overarching observation was that although most beliefs did not seem specific to one or a few categories of key actors, one clear pattern emerged . Participants who were closer to actual intervention development often had a generic idea that there was something wrong with inducing fear (though they rarely grasped the underlying dynamics). Participants who were further removed from the intervention development activities often did not have this basic heuristic belief . One of the main reasons for using threatening communication was to confront people with the consequences of a behavior . The first perceived goal of emotions was to draw attention to the intervention and prompt self - reflection, which would then lead to the desirable behavior, because target population members were assumed to act rationally on hypothesized (successful) increments in risk perceptions . This assumption of rationality also explained the presupposition that achieving awareness or raising risk perception would cause behavior change . This enhanced awareness was the second perceived goal emotions could serve: explicitly emotionally defined risk was often assumed to directly enhance awareness . This resulted in, for example, threatening interventions not being considered fear - inducing; underestimation of what is required to enhance self - efficacy; and overestimation of efficacy levels in the target population . Most participants did not know many behavior change methods, threatening communication sometimes being the best - known solution . Participants often believed the target population could help in identifying useful methods, and some relied on advertising agencies, which mainly advocated originality, confrontation, and humor . The complexity of the evaluation of behavior change interventions often led participants to adopt proxies for effectiveness, such as how well known an intervention was . Finally, working with external organizations sometimes facilitated the choice for threatening communications: funders or intermediary organizations such as schools sometimes preferred threatening communication, and politicians often desired quick and salient, rather than thoroughly researched, interventions . This last finding was already hypothesized decades ago (soames job, 1988), but has not been empirically investigated until now . It is clear that the decades of communication by behavior change scientists regarding the ineffectiveness of health threats has had results, as most intervention developers got the gist of it, albeit usually not more than the gist which still leaves many opportunities for threatening communications . This makes sense, as most intervention developers were not behavior change scientists, or even psychologists . This also explains a second erroneous assumption, namely that humans act rationally, being driven by something similar to expected utility theory (which is known to be incorrect; see kahneman & tversky, 1979). At the same time, the choice for threatening communication was also based on some characteristics of behavior change communications that were consistent with the scientific evidence: it is necessary to draw attention to an intervention, and the interventions do have to inspire self - reflection to a degree . However, people generally direct their attention away from threatening information (bar - haim, lamy, pergamin, bakermans - kranenburg, & van ijzendoorn, 2007), especially if they are not confident they can deflect the danger (kessels et al ., 2010; nielsen & shapiro, 2009). It became clear that intervention developers often did not know many behavior change methods, sometimes knowing no alternatives to threatening communication at all . Sometimes they relied on target population members to tell them which methods to use; but target population members often advocated fear (biener, ji, gilpin, & albers, 2004; goodall & appiah, 2008; lennon & rentfro, 2010). Some intervention developers also relied on advertising agency employees, who lacked knowledge on behavior change methods (and those advertising agency employees following the literature in their field may in fact be in favor of threatening communication; see for example latour, snipes, & bliss, 1996). At the same time, intervention developers would also correct advertising agencies when they noticed errors, although time constraints often restrained possibilities for adjustments . Integrating these various aspects of the decision - making process, some possible actions emerge that may be taken to decrease the use of threatening communication . First, of course, it is important to provide intervention developers with a toolbox of different behavior change methods . Such a toolbox is available, for example, in intervention development protocols such as intervention mapping (kok, bartholomew, parcel, gottlieb, & fernandez, 2014, this issue; see also bartholomew et al ., 2011) and the methodology being developed based on the behavior change techniques (bct) taxonomy (abraham & michie, 2008) and the behavior change wheel (michie et al ., 2011) second, it seems useful to educate intervention developers on two psychological knowledge elements: firstly, it is unwise to consider humans rational (kahneman & tversky, 1979); and secondly, threatening information averts attention (bar - haim et al ., 2007) and prompts defensive reactions (erceg - hurn & steed, 2011; kessels et al ., 2014, this issue), use of negative emotions is very dangerous, and one should exercise caution when labeling something as not fear - inducing . This is connected to the third point: intervention developers should be urged to apply the framework for ethical justification provided by brown and whiting (2014, this issue). Fourth, the use of determinant studies as a method of establishing intervention targets can be promoted . Comparison of the relevance of several cognitive determinants shows that often threat (i.e., severity and susceptibility) is not the most expedient intervention target (but note that when the target of the threat is somebody else, such as one's children, threat does seem effective). Sixth, and this is a very important point indeed: it would be beneficial if intervention developers were convinced of their expertise in this matter . Intervention developers need to be empowered to act as professionals when dealing with intermediary organizations, advertising agencies, and potentially even funders and politicians, who as well - meaning lay people are susceptible to the intuitive appeal of threatening communication . To this end, providing intervention developers with an overview of the evidence regarding the behavior change potential of threatening information can be helpful . Finally, alternative tools should be provided to attract attention, as this was one of the reasons for using threatening communication . Somewhat ironically, such tools may be found in the marketing and advertising literature (maughan, gutnikov, & stevens, 2007; pieters & wedel, 2012). A concrete recommendation is simply the opposite of the belief that threatening communication attracts attention: people look more at advertisements they like (maughan et al ., 2007). Note that in compiling guidelines for attracting attention, it is crucial to respect the parameters of effectiveness of behavior change methods (bartholomew et al ., 2011). An intervention implementing these elements may succeed in eradicating the widespread reliance on threatening interventions, paving the way to the use of more effective behavior change methods . In addition, the findings from the current study should be corroborated by quantitative studies, so that the pertinent beliefs of these groups of key actors can be mapped in terms of their relative importance.
As lifestyles become more complex, the skin is exposed to an ever - increasing spectrum of chemical and biological allergens . While the skin barrier is relatively impermeable to large molecules, contact allergens, because of their small size, easily penetrate the skin barrier and reach the living layers . One of the extended applications of patch testing is whenever the physician suspects past or a recent history of superimposed acd . When the existence of one or more positive reactions is found, relevance must then be determined . The most important question is whether the reaction is a manifestation of the presenting dermatitis or the expression of an acd that occurred previously . This prospective, observational study was conducted among 110 patients at the outpatient department of dermatology, manipal hospital, bangalore, between march 2009 and december 2010 . Subjects of any age with any form of psoriasis of more than six months duration who were unresponsive to conventional topical treatments were included . Pregnant women, patients with extensive psoriasis, current or recent dermatitis at patch test site and patients receiving oral prednisolone more than 20 mg / day or recent phototherapy were excluded from the study . Koh mount for fungal identification and skin biopsy were performed in some patients as part of diagnostic work up . All patients were patch tested with the indian standard series (iss) including parthenium . Patch test readings were taken on day 2 (48 hours) and day 4 (96 hours) and interpreted according to the international contact dermatitis research group criteria . The relevance of positive reactions was assessed and explained to patients as being related to the present problem or not and for cautioning against future exposure [figures 1 and 2]. The relevance of positive allergens was recorded as definite, probable, possible, past, or unknown . Current relevance was defined as present relevance (definite, probable, or possible). Relevance was considered definite if a use test with the item containing the suspected allergen was positive or a positive patch test to the object / product was observed . For use test, the patient was asked to use the suspected substance in the same way as when the dermatitis developed . For example, if a hand cream is suspected, it is applied over a small marked area (1 1 cm) on the hand for 1 week . If an eczematous skin reaction occurs during the test period, the test is considered positive . Relevance was considered probable if the substance identified by patch testing could be verified as present in the unknown skin contactants of the patient . Relevance was considered possible if the patient was exposed to circumstances in which skin contact with materials unknown to contain the allergen would likely occur . The current relevance was tabulated by adding the number of patients with the relevance coded as definite, probable, or possible and converting this to a percentage of patients with a positive test result for the allergen . Source of exposure for each relevant positive allergen was determined based on discussion with the patient . Use test was performed in 10 patients and patch test with patient's own product as is was done in 24 patients . Positive iss antigen reactions in those who had a positive use test or a positive as is were grouped as those reactions with a definite relevance . In those patients who had not undergone a use test/as is patch test, positive iss antigen reactions were grouped as those with a possible or probable relevance or past or unknown relevance . A taxi - driver with chronic, recalcitrant palmar psoriasis was patch tested a taxi - driver with chronic, recalcitrant palmar psoriasis was patch tested the statistical software namely spss 15.0, stata 8.0, medcalc 9.0.1, and systat 11.0 were used for the analysis of the data and microsoft word and excel have been used to generate graphs and tables . The age group ranged from 6 to 70 years with an average of 40 years . Most males were in the age group of 21 to 30 years and most females in 51 to 60 years . The duration of illness ranged from two months to 20 years with an average of four years . For most men, the duration of illness was one to two years and in case of women, it was more than two years . 20% of subjects had intense pruritus over legs, palms, and soles; 13.6% had burning sensation over palms and soles; 9.1% had lichenification over the legs; and 7.3% had hyperpigmentation over the legs . Palmoplantar psoriasis (32.7%) was the most commonly patch - tested type of psoriasis [table 1]. Diagnosis of palmoplantar psoriasis was made clinically by the presence of psoriasiform, scaly plaques with well - demarcated margins; presence of papules and plaques elsewhere in the body that showed a positive auspitz sign (this was observed in 28 (25.4%) patients); nail changes like pitting and subungual hyperkeratosis . In doubtful circumstances, tinea manuum and pedis and atopic dermatitis were ruled out on clinical grounds and by performing 10% koh mount and serum ige tests . Personal history of atopy was found in 12 patients (10.9%), family history of atopy in five patients (4.5%) who had a final diagnosis of psoriasis . Different types of psoriasis that were patch tested the commonly tested positive allergens were fragrance mix (13.6%), nickel sulphate (9.1%), parthenium (6.4%), and balsam of peru (5.5%) [table 2]. 47.3% of subjects showed a positive reaction on day 2 and 42.7% of the subjects showed positive reaction to one or more allergens on day 4 . 28.2% showed reaction to only one allergen; 8.2% to two allergens; 1.8% to three allergens, and 4.5% to more than three allergens . Number of patients tested for positive for each antigen in the current study, fragrance mix, ppd (paraphenylenediamine), paraben mix, lanolin, black rubber mix, para - tertiary - butyl formaldehyde resin, mercapto mix, chlorocresol, and cobalt chloride showed 100% current relevance [table 3], followed by balsam of peru (83.3%), nickel sulfate (80%), and parthenium (71.4%). Nitrofurazone had 100% past relevance followed by neomycin sulfate (50%) and gentamicin sulfate (50%). Under current relevance, ppd, paraben mix, chlorocresol, black rubber mix, neomycin sulfate, and gentamicin sulfate had 100% definite relevance [table 4], followed by nickel sulfate (75%) and fragrance mix (73.3%). Cobalt chloride had a high (100%) probable relevance and mercapto mix had a high (100%) possible relevance . Overall relevance of positive antigens present relevance of positive antigens allergens that were most commonly relevant were those that are found in different topical products like fragrances, topical medications, and preservatives [table 5]. These allergens are often found in cosmetic products or healthcare products and hence form the major sources of allergens in patients with psoriasis . Any chronic dermatosis such as psoriasis can be complicated by contact dermatitis due to an impaired cutaneous barrier . Pre - existing or concomitant constitutional and/or irritant contact dermatitis damages the skin, affecting its barrier function and producing increased opportunities for allergen absorption and secondary sensitization . The extent to which contact allergens play a role in the etiology of psoriasis has always been contemplated . It has been suggested that acd is uncommon in psoriasis. [16] some studies say that acd has been under - represented in patients with psoriasis . Other studies have observed positivity in 20 to 25% of patients with psoriasis. [391517] one study showed no difference in the frequency of allergic reactions between atopics, healthy persons, or psoriatics, whereas henseler and christophers calculated that acd was 3 times less frequent in patients with psoriasis compared with a control group . Another study by stinco et al . Revealed that the number of positive patch tests in patients with psoriasis were similar to that in normal population, nickel being the commonest allergen in both groups, which was in agreement with other studies . Nickel sulfate, coal tar, dithranol, and fragrance mix were the most common allergens in most studies. [38915182021] this study reveals that 47/110 (42.7%) of the subjects had a positive patch test to one or more allergens . Fragrance mix, nickel sulfate, parthenium, and balsam of peru were the most common sensitizers . Fragrance mix showed 100% current relevance and nickel sulfate showed 80% current relevance to the existing dermatitis . Relevance and avoidance of these allergens can be tricky when one considers the large variety of products each person uses on a daily basis and the choices available . It is possible that careful screening of skin products and avoidance of the selected antigens may alleviate chronic, recalcitrant psoriasis . Patients confirmed as having positive and relevant contact allergy have been shown to have a significant improvement in both perceived eczema severity and dermatology life quality index score two months after patch testing . Acd significantly affects quality of life (qol), especially when it affects the hands, face or is occupationally related . Outcomes in patients with acd were improved by early diagnosis and subjects enjoyed their best qol six to twelve months after patch testing . Patch testing and interpretation have been found to bring about greater improvement in the disease severity index and percentage disease activity than diagnosis without patch testing . Patch testing was found to be the most cost - effective in patients with a disease duration of two months to one year . To conclude, secondary contact dermatitis is common in patients with psoriasis and patch testing is necessary to determine the triggering or aggravating factors in these patients, to avoid sensitizers and to improve qol.
Tumors involving the sacrum are rare.1 the most common tumors requiring sacrectomy are primary sacral tumors and locally advanced rectal cancer infiltrating the sacrum.2 the optimal primary therapy for these latter cases includes en bloc resection of the tumor together with the structures adherent to the tumor.3 there is evidence that this approach improves overall survival and local control.4 when the coccyx or sacrum are involved, a total (all of s1), subtotal (at s1), or partial (at or below s3) sacrectomy has to be performed.5 sacrectomies are still a challenge for the surgeon, as all of them exhibit a high rate of complications, such as neurologic, urinary, infectious, and wound - healing complications.5 6 7 to achieve an abdominoperineal resection together with a partial sacrectomy, the most common approach has been the abdominosacral technique described by wanebo and marcove.8 this procedure implies an anterior approach for the abdominoperineal amputation and a posterior route to accomplish the sacral osteotomy and resection . This means a change of patient position during surgery and the additional morbidity related to a posterior approach . Anterior - only approaches are indirectly mentioned in literature within long series of rectal cancer surgery.9 we report a successful partial sacrectomy through a one - stage abdominoperineal approach without posterior exposure of the sacrum and with delivery of the specimen through the perineal incision . A 73-year - old woman with a history of surgically resected pt2n0 gallbladder cancer had been previously diagnosed with stage iiic rectal cancer . Preoperative computerized tomography (ct) scan and magnetic resonance imaging (mri) showed in proximity to uterus invasion of the mesorectal fat and the sacrum at s4 and s5, predominantly on the right side (fig . 1a, b, and c), with two left internal iliac adenopathies (of less than 1.2 cm) and one in the mesorectal fat (0.5 cm). (a) axial t1-wi magnetic resonance imaging (mri) showing invasion of the low sacrum . This image has been used as a scheme to demonstrate the transabdominal route, necessary to gain control of the internal iliac vessels, to dissect the superior and most of the anterior part of the tumor and to do the sacral osteotomy . The perineal route was utilized to dissect the inferior part of the tumor, the coccyx, and the inferior and posterior part of the sacrum . Under general anesthesia and continuous electromyographic control, a digital rectal exam showed the tumor was at 5 cm of the anal verge, infiltrating the posterior wall of the vagina and very adherent to the sacrum . The cut end was used as a handle to aid with the dissection of the posterior part of the rectum down to s4, as it was fixed to the sacrum . As the vagina was invaded by tumor, hysterectomy and posterior vaginectomy the right s3 root was sacrificed, as no electrical activity was shown . With a high - speed drill, a horizontal osteotomy was performed through the s3 vertebral body, lower to the left s3 foramina and at the level of the right one . Once in the canal, no thecal sac was encountered, and the remaining roots were transected . While this field was being developed, the perineal phase was being advanced . A perineal typical elliptical incision including the external sphincter muscle was made and monopolar cautery was used to cut the ischiorectal fascia . After coagulating the inferior hemorrhoidal vessels, the tip of the coccyx was exposed and the levator ani, transverse perinei, and rectourethralis muscles were divided . The muscular attachments of the gluteus maximus muscle were taken down with the monopolar coagulator to expose the lateral distal sacrum . Detachment of the muscles proceeded up to the level of the sacral osteotomy (fig . (a) surgical field through the transabdominal approach at the end of the resection . The arrow points to the gloved surgeon's finger coming through the perineal approach along the posterior part of the sacrum . The arrow points to one hand that was introduced through the transabdominal field and that is showing through the perineal incision after accompanying the specimen during the final resection . Finally, the lateral part of the sacrum was dissected by cutting the insertion of the pyramidalis muscle and the sacrotuberous and sacrospinalis ligaments . Once free, the whole specimen (including rectum, uterus, and the posterior part of the vagina) was removed en bloc through the perineal incision (fig . 2b and 2c). 3a, b, and c. the only postoperative complication was right dorsiflexion paresis that disappeared completely after 1 month . After 18 months of follow - up, the patient is completely ambulatory and has no local recurrence but has developed lung and liver disease . Note asymmetry of resection (lower on the left side to respect one s3 root, as the tumor was higher on the right side). (b, c) axial and sagittal images showing extent of resection . No stabilization was required . A 73-year - old woman with a history of surgically resected pt2n0 gallbladder cancer had been previously diagnosed with stage iiic rectal cancer . Preoperative computerized tomography (ct) scan and magnetic resonance imaging (mri) showed in proximity to uterus invasion of the mesorectal fat and the sacrum at s4 and s5, predominantly on the right side (fig . 1a, b, and c), with two left internal iliac adenopathies (of less than 1.2 cm) and one in the mesorectal fat (0.5 cm). (a) axial t1-wi magnetic resonance imaging (mri) showing invasion of the low sacrum . This image has been used as a scheme to demonstrate the transabdominal route, necessary to gain control of the internal iliac vessels, to dissect the superior and most of the anterior part of the tumor and to do the sacral osteotomy . The perineal route was utilized to dissect the inferior part of the tumor, the coccyx, and the inferior and posterior part of the sacrum . Under general anesthesia and continuous electromyographic control, the patient was placed in the lithotomy position . A digital rectal exam showed the tumor was at 5 cm of the anal verge, infiltrating the posterior wall of the vagina and very adherent to the sacrum . Together with digestive system surgeons, a midline laparotomy was performed . The cut end was used as a handle to aid with the dissection of the posterior part of the rectum down to s4, as it was fixed to the sacrum . As the vagina was invaded by tumor, hysterectomy and posterior vaginectomy the right s3 root was sacrificed, as no electrical activity was shown . With a high - speed drill, a horizontal osteotomy was performed through the s3 vertebral body, lower to the left s3 foramina and at the level of the right one . Once in the canal, no thecal sac was encountered, and the remaining roots were transected . A perineal typical elliptical incision including the external sphincter muscle was made and monopolar cautery was used to cut the ischiorectal fascia . After coagulating the inferior hemorrhoidal vessels, the tip of the coccyx was exposed and the levator ani, transverse perinei, and rectourethralis muscles were divided . The muscular attachments of the gluteus maximus muscle were taken down with the monopolar coagulator to expose the lateral distal sacrum . Detachment of the muscles proceeded up to the level of the sacral osteotomy (fig . (a) surgical field through the transabdominal approach at the end of the resection . The arrow points to the gloved surgeon's finger coming through the perineal approach along the posterior part of the sacrum . The finger can be seen through the osteotomy . The arrow points to one hand that was introduced through the transabdominal field and that is showing through the perineal incision after accompanying the specimen during the final resection . Finally, the lateral part of the sacrum was dissected by cutting the insertion of the pyramidalis muscle and the sacrotuberous and sacrospinalis ligaments . Once free, the whole specimen (including rectum, uterus, and the posterior part of the vagina) was removed en bloc through the perineal incision (fig . 2b and 2c). 3a, b, and c. the only postoperative complication was right dorsiflexion paresis that disappeared completely after 1 month . After 18 months of follow - up, the patient is completely ambulatory and has no local recurrence but has developed lung and liver disease . Note asymmetry of resection (lower on the left side to respect one s3 root, as the tumor was higher on the right side). Appropriate surgical management of locally advanced rectal cancer includes multivisceral resection,3 10 11 as there is a high probability of leaving residual disease at the local site because of tumor adherence or fixation . En bloc resection of adjacent organs or structures adherent to the tumor avoids separating the adhesions, in which tumor is found between 40 and 84% of the cases,3 and is the only way to achieve an r0 resection in locally advanced cases . This aggressive management provides better local control and survival.4 nevertheless, 5-year survival is more dismal and has been reported between 52 and 64%, mainly due to distant metastases.12 13 like in our case, the presence of lymph node involvement seems to be associated with a worse prognosis.13 whether the liver and lung metastases that appeared come from the rectal or the gallbladder disease is uncertain . In cases of locally advanced rectal cancer, the usual technique (wanebo technique) to perform a partial sacrectomy is a combined anterior and posterior approach . The anterior field is developed through a transabdominal exposure or both a transabdominal and perineal approach to carry out an abdominoperineal amputation . Curiously enough, sacral resection through the perineum is not the regular procedure for low sacrectomy . It is fairly easy to develop as no major vessels are in relation with the posterior wall of the sacrum up to s3 . Furthermore, it avoids changing the position of the patient, which is cumbersome in a lengthy procedure . It also avoids a posterior incision, which is usually subjected to a high risk of dehiscence and infection (25 to 46%).14 nevertheless, in our case, as an abdominoperineal amputation was done, the risk of infection due to proximity to the passage of feces is much lower . The other s3 root, which was severed, did not show any electrical activity in the intraoperative electromyographic control . Both the parasympathetic pelvic nerve, which supplies the detrusor fibers, and the somatic pudendal nerve, which innervates the external urethral sphincter, originate from the sacral cord at s2s4.15 some cases have been described in which the preservation of both s2 roots was enough to maintain urinary continence.6 this is why, together with the preservation of the bladder and ureters, a urinary diversion procedure such as a double - barreled wet colostomy was not planned from the beginning.16 17 notwithstanding, guo et al reported a strong association between s3 nerve root integrity and continence: more than 30% of their patients with unilateral s3 nerve root resection showed incontinence, and 75% of those with bilateral s3 nerve root severance were incontinent.14 in our case, the patient was rendered incontinent despite preservation of both s2 roots and one s3 . Similar complications have been described, and traction injury to the lumbosacral plexus or l5 nerve root have been invoked as causes of the deficit.18 this anterior - only approach was intended for a locally advanced rectal cancer in which an abdominoperineal resection was planned . Partial sacrectomies in primary tumors are one limitation of the applicability of this procedure, as in these cases the rectum and the anal region are to be preserved, and no perineal incision is usually undertaken.19 whether some form of sphincter - preserving technique can be done to access the anterior part of the sacrum through the perineal approach in case of a low sacral primary tumor remains speculative.20 high sacrectomies that require spinopelvic fixation are another limitation of this technique . The anterior part of the s1 level can be exposed through the transabdominal approach, and, through the perineal approach, dissection could be probably extended higher . Nevertheless, biomechanical studies show that sacral resections through s1 may damage more than a 70% of the sacroiliac joint and therefore cause a significant decrease of stiffness and both compressive and rotational instability.21 22 any sacrectomy above the s1s2 junction requires a spinopelvic fixation, whereas resections at or below s2 do not need further stabilization . As spinopelvic fixation is performed through a posterior approach, in high sacrectomies our perineal incision is rendered unnecessary . Body mass index definitively has to be taken into account when indicating this anterior - only approach and can be also considered as a relative limitation of this technique . There is some evidence in literature that obese patients encounter more wound - healing complications in perineal approaches done specifically for abdominoperineal resections.23 we believe that in cases of rectal cancer involving the low sacrum, in which a perineal amputation has to be done, the combination of a transabdominal and perineal route is a feasible approach that avoids changes of surgical positioning and the morbidity related to posterior incisions . The dissection of the posterior wall of the lower sacrum and the removal of the surgical specimen can be easily accomplished through the perineal route . This strategy should be considered when deciding on undertaking partial sacrectomy in locally advanced rectal cancer.
Tourette syndrome (ts) is associated with alterations in the development of brain networks that result in neural circuits with imbalanced excitatory and inhibitory influences . It is generally acknowledged that cortical - striatal - thalamic - cortical (cstc) circuits are dysfunctional in ts, with subsets of striatal neurons becoming active within inappropriate contexts, resulting in the disinhibition of thalamocortical circuits and the hyperexcitability of motor regions of the brain [47] that in turn lead to the occurrence of tics . Ts has been linked to alterations in inhibitory -aminobutyric acid (gaba) signaling [15, 16]. Postmortem examination has demonstrated that there are substantial decreases in the number of gaba interneurons found within the striatum of individuals with ts, and positron emission tomography imaging has revealed widespread reductions in gabaa receptor binding in ts . Finally, studies of cortical - spinal excitability (cse) in ts have demonstrated reduced intracortical gabaergic inhibition [47]. Together, these findings predict reduced phasic gabaergic inhibition in individuals with ts, which has most often been interpreted as a primary cause of the disorder contributing to the occurrence of tics . Ts often follows a developmental time course characterized by a reduction in the frequency and intensity of tics during adolescence . It has been proposed that individuals gain control over their tics through the development of compensatory mechanisms that lead to enhanced control over motor outputs based upon increased tonic inhibition [8, 10, 11, 1820]. Consistent with this proposal, it has been shown that the gain of transcranial magnetic stimulation (tms)-induced motor excitability (i.e., tms recruitment curves) (; see supplemental information available online) and the gain of motor excitability immediately prior to volitional movements are both significantly reduced in individuals with ts [7, 12, 13]. These findings have been interpreted as a secondary consequence of, or adaptation to, the disorder and have been associated with a reduction in clinical symptoms . Importantly, it has been demonstrated that both reduced inhibition (e.g., reduced short - interval cortical inhibition) and enhanced inhibition (e.g., reduced gain for tms - induced motor excitability) are observed in the same group of individuals with ts . The supplementary motor area (sma) is a likely focus for these control mechanisms . The sma is a major site for thalamocortical projections and has been linked previously to the volitional control of action and nonconscious, effector - specific control of motor outputs . Gaba concentrations within the sma are correlated with performance on behavioral tasks that index nonconscious control of motor outputs . Most importantly, cortical excitability within the sma is linked to the genesis of tics in ts . Thus, the hyperexcitability within primary motor cortex (m1) that is observed in ts is likely due to increased functional interaction between sma and m1: there is increased activity in the sma of individuals with ts that immediately precedes the occurrence of tics, and inhibitory repetitive tms (rtms) delivered to the sma has been shown to decrease tic frequency in individuals with ts [2628]. Here we offer a novel perspective on how this increased control over motor outputs can arise as a consequence of localized increases in tonic inhibition based upon increased levels of nonsynaptic, extracellular gaba concentration that operate to alter the gain of cortical spinal excitability (cse) locally . We used h magnetic resonance spectroscopy (mrs) at ultra - high field (7 t) to investigate in vivo concentrations of gaba within the primary and secondary (sma) motor areas of 15 adolescents (mean age 15.75 3.05 years) with a confirmed clinical diagnosis of ts and a control group of age- and gender - matched typically developing individuals . Mr spectroscopy data were collected from three 20 mm regions of interest (rois) located within the hand area of left primary sensorimotor cortex (m1), and bilaterally from within the sma and the primary visual cortex (v1). To aid localization of the hand area of m1 and sma, participants performed a brief bimanual, sequential finger - thumb opposition task (i.e., with both hands continuously tap each finger sequentially against the thumb until instructed to stop) while functional mr images were obtained (figure 1a). Anatomical (mp rage) images were analyzed to estimate, for each participant, the fraction of cerebral spinal fluid (csf), gray matter (gm), and white matter (wm) within each volume of interest (voi). The csf, gm, and wm fractions were compared separately between groups using independent - samples t tests . These analyses confirmed that there were no significant differences in the proportion of each tissue type between groups for each voi (maximum t(27) = 1.2, p> 0.1). Group differences in the magnitude of the fmri blood oxygen level - dependent (bold) signal change for the tap> rest behavioral contrast were examined for each of the m1 and sma vois using an independent - samples t test . The ts group had a significantly larger bold signal within the sma voi compared to controls (means: ts group = 1.88% 1.3%, control group = 1.1% 0.8%; t(27) = 2.1, . There was no significant between - group difference in fmri bold within the m1 voxel . The ts group had significantly increased absolute concentrations of gaba within the sma compared to the control group (means: ts group = 1.1 0.43 mol / g, control group = 0.75 0.28 mol / g; t(27) = 2.5, p <0.05). Gaba concentrations within the m1 and v1 rois did not differ significantly between groups (both p> 0.5). Following convention (e.g.,), we measured gaba concentrations as a ratio of n - acetylaspartate (naa) concentrations within each roi . Importantly, preliminary analyses confirmed that naa concentrations did not differ significantly between groups for any of the three rois . The analyses revealed that gaba / naa ratios were significantly increased relative to the matched control group within the sma (means: ts group = 0.14 0.04, control group = 0.11 0.04; t(27) = 2.2, p <0.05) but were not different from control group levels for the m1 or v1 rois (figure 1b). Our finding of selectively increased concentrations of gaba within the sma in individuals with ts (hereafter mrs - gaba) is consistent with the proposal that mrs - gaba primarily measures extracellular gaba concentrations that have been linked to alterations in levels of tonic inhibition (; see for review). A discussion of the cellular basis for tonic inhibition is beyond the remit of this paper (but for recent reviews see [3235]). It should be noted that mrs - gaba was measured at rest in the current study, and participants were instructed to remain still throughout . We cannot rule out that active suppression of tics contributed to the increased mrs - gaba in sma that we observed . An increase in mrs - gaba in the sma during tic suppression in ts would be consistent with previous reports that mrs - gaba concentrations within the sma of neurologically healthy individuals correlate with individual levels of performance on behavioral tasks that index control of motor outputs . We investigated the association between mrs - gaba and fmri bold within the sma and m1 voxels . Previous studies reported a negative correlation between mrs - gaba concentrations and fmri bold in the visual and motor cortices of healthy adults [29, 36, 37]. For the ts group, the analyses confirmed that the fmri bold signal change within the sma voxel was significantly negatively correlated with gaba / naa ratio (r = 0.65, p <0.01). The correlation between fmri bold response and gaba / naa within the m1 voi did not approach statistical significance (p> 0.1). The correlations between mrs - gaba and fmri bold in m1 and sma did not reach statistical significance for controls . Our finding that mrs - gaba concentrations within the sma are inversely associated with the fmri bold response is consistent with previous reports [29, 36, 37]. It is also consistent with the proposal that increases in mrs - gaba are linked to localized increases in tonic inhibition [30, 31], and that increased control over motor outputs in ts are brought about by reducing the gain of corticospinal excitability in cortical motor regions through increased tonic inhibition [7, 12, 13]. Individuals with ts exhibit significantly reduced gain for tms - induced cse and also preceding the execution of volitional movements [7, 12, 13]. Furthermore, gain in cse is inversely related to tic severity (; see supplemental information). These findings have been interpreted as evidence that the gain of cse is reduced in ts due to increased levels of tonic inhibition [7, 13]. To investigate the relationship between mrs - gaba and cse in ts, we examined the pearson correlation between mrs - gaba within sma and levels of cse within primary motor cortex (m1). Cse was measured using single - pulse tms delivered to the hand area of the left m1 region in the period immediately preceding (81%100% of the movement preparation period) volitional movements of the right hand in a subset of ts patients who had taken part in the current study and also in the study reported by draper et al . . This analysis revealed a significant negative correlation (r = 0.86, p <0.006) between mrs - gaba in the sma roi and cse measured within the left m1 (figure 2). Although statistically significant, this result should be interpreted with caution due to the relatively small sample size . Nevertheless, taken together with the finding for fmri bold reported above, it indicates that increases in mrs - gaba within the sma in the ts group are likely associated with decreases in motor excitability . If mrs primarily measures nonsynaptic, extracellular gaba concentrations that are linked to ambient levels of tonic inhibition [30, 31], then a key issue is to understand factors associated with mrs - gaba increases . One possibility is that mrs - gaba increases are triggered by neural projections arriving from other brain areas; another is that they are associated with tic severity scores . As noted previously, the sma is a major site for thalamocortical and cortical - cortical projections, and cortical excitability within the sma is strongly linked to the genesis of tics in ts . Previous studies have demonstrated altered wm microstructure (i.e., reduced fractional anisotropy [fa] values in ts relative to matched controls) within regions of the corpus callosum (cc) linking sensorimotor areas of cortex . Furthermore, these alterations in fa are positively correlated with tic severity [20, 38]. We used diffusion tensor imaging (dti) and tractography to test the hypothesis that increased projections to and from the sma, as measured by fa values in the region of the cc projecting to the sma, would be positively associated in ts with increased tic severity and increased mrs - gaba in the sma . We identified a 6 mm roi within the body of the cc from which fibers clearly connected to the sma within each hemisphere (figure 3a; see supplemental information for details). We then measured mean fa values within this cc roi for the ts group and correlated these with tic severity scores and mrs - gaba within the sma roi . The analyses revealed that fa values within the cc roi were positively correlated with motor tic severity scores (r = 0.87, p <0.001). These data confirm the previous finding that a reduction in cc projections to motor cortical areas is associated with reduced motor tic severity [20, 38]. More importantly, the analyses also revealed that fa within the cc roi was significantly positively correlated with mrs - gaba within the sma (r = 0.75, p <0.05). To investigate which of several key variables might contribute to mrs - gaba concentrations within the sma in the ts group, we carried out linear regression analyses for the following variables: participant age, current tic severity (yale global tic severity scale [ygtss] impairment, global, and motor scores), and wm microstructure (fa) in the region of the cc projecting to the sma . The analyses confirmed that participant age and nonmotor indices of tic severity (i.e., ygtss impairment and global scores) were not significant predictors of mrs - gaba within the sma (p> 0.05). By contrast, motor tic severity (ygtss motor score) (rsq = 0.54, adj - rsq = 0.46, f = 6.97, p <0.04) and callosal fa (rsq = 0.62, adj - rsq = 0.55, f = 9.7, p <0.025) were each significant predictors of mrs - gaba within the sma for the ts group . However, callosal fa is itself highly positively correlated with motor tic severity (r = 0.87, p <0.001). To determine the joint contribution of these factors, we entered them into a stepwise regression model . The analyses demonstrated that when motor tic severity is entered into the model first, callosal fa accounts for no additional variance and is no longer a significant predictor of mrs - gaba (t = 1.13, p> 0.1). By contrast, if callosal fa values are entered into the model first, motor tic severity accounts for no additional variance and is no longer a significant predictor of sma gaba (t = 0.42, p> 0.1). We used ultra - high - field (7 t) h mrs to investigate for the first time in vivo concentrations of gaba within primary and secondary motor areas of individuals with tourette syndrome (ts). We demonstrate that concentrations of gaba within the sma a brain area consistently linked with the cortical genesis of motor tics in ts are significantly elevated in individuals with ts relative to a control group of age - matched typically developing individuals . By contrast, gaba levels in primary motor cortex (m1) and in a control site within occipital cortex (v1) do not differ between groups . We investigated the relationship between elevated mrs - gaba observed for the ts group and measures of motor tic severity, motor cortical excitability, fmri bold response, and the structural connectivity of the sma region . We demonstrate that mrs - gaba within the sma is strongly negatively correlated with the fmri bold response in sma and also cortical excitability values within sensorimotor cortex, as measured by single - pulse tms delivered immediately prior to a volitional movement of the contralateral hand . Importantly, we report for the first time that mrs - gaba levels within the sma are strongly positively predicted by both motor tic severity and the fa values within a region of the cc that projects to the sma, and that these factors are themselves highly positively correlated . It has been argued that an important secondary consequence of ts is that enhanced control over volitional movements, and the suppression of tics, may arise as a result of increased tonic inhibition [713]. This proposal is consistent with the repeated finding that the gain in cortical excitability is reduced in ts ahead of volitional movements [7, 12, 13] and in response to increasing levels of tms stimulation . Based upon the findings of the current study, we propose that this increase in tonic inhibition may be due to localized increases in extracellular gaba within the sma . We believe that these findings are particularly important for understanding how localized adaptive changes in brain function may accompany neurodevelopmental disorders and play a key role in the control of behavioral symptoms.
In voltage - dependent sodium, potassium, and calcium channels, the voltage dependence of gating has usually been attributed to that intrinsic part of the protein molecule known as the voltage sensor (s4 region), although a number of very recent studies have reported some dependence on the presence and nature of the permeating cation (yellen, 1997). For example, lower than normal concentrations of external potassium substantially reduce the open probability of some k channels . By comparison, cloning and sequencing of the voltage - dependent clc family of cl channels has failed to reveal an s4 type of voltage sensor and experiments on the torpedo cl channel, clc-0, indicate that there is little or no contribution to gating from intrinsic protein charge movement (chen and miller, 1996). Indeed, for two family members, clc-0, and the skeletal muscle cl channel, clc-1, a number of experiments in which cl has been replaced by glucose or by other anions suggest that activation of these channels is mainly controlled by the presence and concentration of external cl (pusch et al . These authors submit that the permeant anion also serves as a ligand that regulates channel opening when it binds to a specific intra - pore site . For a constant external cl concentration, the availability of extracellularly derived cl at the regulatory site depends on the transmembrane electric field and this confers the experimentally observed voltage dependence upon gating, no intrinsic voltage sensing component of the channel protein being required . Similar experiments using methanesulfonate as the impermeant cl substitute along with the effect of a mutation in clc-1 (d136 g), meanwhile, led fahlke et al . (1995, 1996) to conclude that d136 is part of a voltage sensor that controls gating, this voltage sensor possibly being subject to electrostatic perturbation by anion binding within the channel . Mutations in clc-0 (ludewig et al ., 1997) and clc-1 (fahlke et al ., 1997c) at very different positions, however, cause the same inwardly rectifying gating as d136 g, making it unlikely that this residue serves as a voltage sensor . In their latest interpretation of gating, fahlke et al . (1997b) endorse the functional linkage between ion permeation and gating, but maintain their different viewpoint with respect to the involvement of a voltage sensor . They argue that the voltage responsiveness resulting from voltage - dependent conformational changes can be modulated by permeant ions, and hence be modified by the presence of foreign anions . Methanesulfonate, which has often been used as the preferred impermeant cl substitute because of its negligible permeability and apparent lack of interaction with cl in mole fraction substitution studies of membrane conductance in the rat diaphragm muscle (palade and barchi, 1977). Other anions (br, i, so4, and methylsulphate) tested by these authors, appeared to interact to varying degrees with cl, displaying nonlinear and anomalous mole fraction effects on membrane conductance when substituted for cl . Similar effects of foreign anion substitution on fish skeletal muscle had been demonstrated previously (hagiwara and takahashi, 1974). Early work on frog muscle with halide and no3 substitution defined the substantial specificity of skeletal muscle anion channels for cl over other anions (for review see bretag, 1987). Woodbury and miles (1973) tested an extensive range of foreign anions on frog muscle and found that they could be separated into two groups according to how they affected the ph dependence of membrane conductance . For cl itself and chloride - like anions, membrane conductance decreased at low ph, while, for benzoate - like anions, it increased . More recently, the effects of foreign anions on cl channels of tissues other than muscle have been analyzed at the single channel level (bormann et al ., 1987; halm and frizzell, 1992; linsdell et al ., 1997a; tabcharani et al ., 1997) and, based on the extensive kinetic information obtained, substantial models of multi - ion pores have been developed (bormann et al ., 1987; halm and frizzell, 1992; linsdell et al ., 1997b). The skeletal muscle cl channel, clc-1, has a conductance that is too small to allow observation of single channel currents (pusch et al . . Nevertheless, investigation of ensemble currents can provide valuable insight into the gating and permeation characteristics of this channel (steinmeyer et al . ; astill et al ., 1996; fahlke et al ., 1996; rychkov et al ., 1996; fahlke et al we have investigated the influence of foreign anions on current kinetics, apparent popen permeability, and conductance of clc-1 channels that allow a number of predictions to be made about the nature of the channel pore . Rat clc-1 (rclc-1) was expressed in sf9 (a spondoptera frugiperda insect cell line) cells as described in detail previously (astill et al ., 1996). Cultured sf9 cells were infected with baculovirus bvda6.3 containing rclc-1 cdna and incubated for 2830 h at 28c in air . After incubation, infected cells were seeded onto glass coverslips and maintained at room temperature until required . Whole - cell patch - clamp experiments were performed on sf9 cells between 28 and 34 h postinfection using an epc7 patch - clamp amplifier and associated standard equipment (list electronic, darmstadt, germany). The usual bath solution contained (mm): 170 nacl, 2 mgso4, 2 ca - gluconate, and 10 hepes, adjusted to ph 7.5 with naoh . Lower concentrations of cl in the bath solution were achieved by equimolar substitution of 5, 10, 25, 50, 75, 90, or 100% of external cl by a particular anion; for impermeant anions the highest concentration in the bath solution was 95% . Electrodes were made of borosilicate glass and had a resistance of 13 m when filled with a normal internal solution containing (mm): 40 kcl, 120 k - glutamate, 10 egta - na, and 10 hepes, adjusted to ph 7.2 with naoh . In some experiments, the concentration of nacl in the bath solution was raised to 340 mm, in which case the concentrations of kcl and k - glutamate in the internal solution were doubled . When hco3 was used to substitute for cl, bath hepes concentration was increased to 20 mm and the solution was used within 20 min . To reduce possible acidification of the internal medium due to co2 diffusion into the cell, 120 mm k - glutamate in the internal solution was replaced with equimolar tris - glutamate, a substitution that, of itself, had no effect on clc-1 channel properties . When necessary, pentobarbitone (0.5 mm), added to the bath solution, was used to block native anion channels in sf9 cells (birnir et al ., 1992). Data were collected, filtered at 3 or 10 khz, and analyzed on an ibm - compatible pc using pclamp v6.0 software (axon instruments, foster city, ca). Liquid junction potentials between the bath and electrode solutions were estimated by using jpcalc (barry, 1994) and corrected where specified . Experiments were conducted at a room temperature of 24 1c . To obtain permeability ratios, membrane potentials for at least seven different concentrations of each foreign anion were measured in current clamp mode . Shifts in potential (relative to control cl solution) were plotted against mole fraction of the anion and fitted with a goldman - hodgkin - katz equation of the form: 1\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}y=58.4\;log\;\;\{1/[(1-m_{f})+am_{f}]\},\end{equation}\end{document} where y is the shift of the membrane potential, mf (mole fraction) is the concentration of foreign anion, x, relative to the control cl concentration (mf = [x]/[x] + [cl]), and a = (px/ pcl) is the permeability ratio for this anion relative to cl . To determine relative conductances in the presence of external foreign anions, outward conductance was estimated as a chord conductance (approximating the slope conductance) from the outward current interpolated at a point 20 mv to the right of the reversal potential on the current voltage (i - v) plot . We constructed these i - v plots from steady state currents obtained at the end of 100-ms test voltage pulses . Tail currents for voltage steps to 100 mv after 150-ms test pulses in the range from 160 to + 100 mv were extrapolated back to the beginning of the pulse by fitting with two exponentials plus a constant (rychkov et al ., 1996). Instantaneous currents so obtained were used to produce apparent popen curves by fitting with a boltzmann distribution of the form: 2\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}x(v)=x_{o}+(1-x_{o})/\{1+\;exp[(v_{1/2}-v)/k]\},\end{equation}\end{document} where xo is an offset, v is the membrane potential, v1/2 is the potential at which x = (1 + xo)/2, and k is the slope factor . The slope factor k was used to calculate the apparent gating valence z, (z = 25.4/k). In these experiments, the bath solution contained (mm): 100 nax (x = cl, br, etc ., or 50 cl + 50 i), 2 mgcl2, 5 hepes, adjusted to ph 7.3 with naoh, and the pipette solution contained (mm): 100 nacl, 2 mgcl2, 5 egta, 5 hepes, adjusted to ph 7.3 with naoh . Inside - out patches containing hclc-1 were also produced from xenopus oocytes . In this case, the pipettes were filled with control bath solution containing (mm): 100 nacl, 2 mgcl2, 5 hepes, adjusted to ph 7.3 with naoh, and the patch was exposed to (mm): 100 nacl, 2 mgcl2, 5 egta, 5 hepes, adjusted to ph 7.3 with naoh . Where specified, various fractions of the 100 mm nacl were replaced by equimolar amounts of nabr or nai . Various foreign anion substitutions were first tested against the effects of nacl replacement by glucose . Our presumptions were that permeation through clc-1 would not be influenced by glucose, that glucose would not interact with any regulatory site or any process modulating clc-1, and that anion permeation through clc-1 could be satisfactorily monitored without interference from any other permeation pathways through the sf9 cell membrane . When nacl in the external solution was replaced by glucose, the slope conductance of clc-1, estimated near the equilibrium potential, showed saturation at physiological external cl concentrations, only becoming dependent on external cl at low concentrations (fig . Almost identical results were obtained when glutamate, gluconate, glucuronate, and diatrizoate were substituted for cl (fig . 1, relative permeabilities for these anions, obtained from the fit of the goldman - hodgkin - katz equation to shifts in membrane potential, were <1% and could not be considered different from zero . Substitution of cl by glutamate or glucose influenced current kinetics, increasing the rate of deactivation of inward currents, and shifted the apparent popen curve in the depolarizing direction, as we reported previously (rychkov et al ., 1996). Both of these were dependent on the extent to which cl had been replaced . When other anions in this group were substituted for cl, they also made current deactivation faster and shifted the apparent popen curve to more positive potentials to the same extent as glutamate (fig . Two much smaller anions, hco3 and bro3, although having a measurable permeability relative to cl (table i) had the same (fig . 2 c) or a similar (fig . 2 d) effect on current kinetics as the large impermeant organic anions from the previous group . They also shifted the apparent popen curves to more positive potentials without changing the gating charge (table i), but unlike glutamate and other anions from that group they appeared to interact with cl inside the channel to some extent, since the relationship between conductance and concentration was different from that for glucose (fig . Bicarbonate was less permeant than bro3 (table i) and its effects were closer to those of glutamate: at 95% concentration, hco3 shifted v1/2 by +60 mv, while bro3, at the same concentration, shifted v1/2 rather less, by +50 mv (compare with the shift of +70 mv for glutamate; table i). Bromate showed stronger interaction with cl inside the channel than hco3 since conductance of the channel in the outward direction in the presence of bro3 was smaller than in the presence of hco3 (figs . 1 and 2, c and d). At the same time, bro3 weakly blocked inward current, while hco3 did not show any block (compare the relative amplitudes of inward current in the presence of bro3 and hco3; fig . 2). Cyclamate and methanesulfonate were as impermeant as the other large organic anions, but they changed currents through clc-1 very differently from glutamate (fig . 3). Cyclamate (when substituting 95% of cl) blocked 35% of inward current, shifted v1/2 by + 28 mv (compared with +70 mv for glutamate; table i), and increased the slope coefficient, k, of the popen curve, thus reducing the apparent gating charge, z, to 0.63 . Methanesulfonate, at the same concentration, blocked up to 70% of inward current and substantially modified the kinetics of current deactivation . Furthermore, the apparent popen curve was shifted in the opposite direction, to more negative potentials, by 45 mv, and the apparent gating charge was decreased (fig . Bromide, no3, clo3, i, clo4, and scn fall into a separate category of foreign anions in regard to their action on clc-1 . With increasing amounts of br replacing cl in the external solution, the slope conductance, calculated for outward current near the reversal potential, decreased until just 15% of its initial value remained when 100% br substituted for cl (fig . Inward current amplitude (at 120 mv) was reduced by 50%, indicating that br blocks the clc-1 channel (fig . 5). The relative permeability for br, again obtained using the goldman - hodgkin - katz equation and data for different mole fractions of br, was 0.37 0.01 . There was no evidence of an anomalous mole - fraction effect on permeability for br . Kinetics of current deactivation were not substantially altered, nor was there any significant shift in the voltage dependence of the apparent popen, as indicated by v1/2 (table i). The main difference was an increased proportion of steady state current at negative potentials (compare figs . 2 and 5). 4) and, with relative permeabilities of 0.25, were somewhat less permeant than br . In addition, they blocked up to 80% of the inward current at 120 mv when substituting 100% of the cl (fig . Apparent popen in the presence of either of these anions at 50% concentration was shifted to more negative potentials without significant change in the slope of the curve, but if the concentration of no3 or clo3 was increased up to 100%, the slope factor of the apparent popen curve was increased (table i), implying a reduction in gating charge to 0.6 (normally 1.2). Time constants of the deactivating components of the inward current were not appreciably changed by these anions, but the relative proportion of steady state current at 120 mv increased from 7 to 50% (compare figs . 2 and 5). Although i was quite permeant, with a relative permeability of 0.22 0.02, conductance of i ions through clc-1 was negligibly small and not readily distinguishable from leakage . Inward current was blocked by 90% at 120 mv when all cl was replaced with i and current showed little deactivation at negative potentials (fig . Apparent popen curves obtained at lower mole fractions of i, where current deactivation at negative potentials was more obvious, showed that the voltage dependence of channel gating was shifted to more negative potentials by i and the apparent gating charge decreased (table i). Thiocyanate and clo4 at low concentrations (up to 10%) had effects very similar to i, they blocked inward current (by 90% in the case of 10% scn), reduced current deactivation at negative potentials, shifted v1/2 to more negative potentials, decreased the slope of apparent popen curves (table i), and shifted membrane potentials towards more positive values . When the concentration of scn (or clo4) was raised above 10%, inward current was blocked almost completely, but the amplitude of the outward current started to increase and reached its maximum when all cl in the external solution was replaced with scn (fig . 5), and scn was more permeant than cl since membrane potentials were shifted to values more negative than the cl equilibrium potential (fig . Endogenous volume - regulated cl channels in sf9 cells were more permeable to scn than to cl and, furthermore, control cells showed outwardly rectifying currents in the presence of scn qualitatively similar to the currents recorded under the same conditions from the cells expressing clc-1 (not shown). To make sure that the anomalous mole - fraction effect and outward rectification in solutions containing scn were not the result of increased activity of the native cl channels, concomitant with complete block of clc-1 channels, experiments with scn substitution for cl if currents recorded in the presence of scn were passing only through native channels, their amplitudes should not be much different from cell to cell and should not depend on the cl current amplitude through clc-1 . In reality, there was a high level of correlation between the amplitudes of outward currents, measured in cells expressing clc-1, bathed in control cl solution, and when scn replaced cl (fig . We can, therefore, have confidence that clc-1 is permeable to scn (and, likewise, to clo4) and that outward currents recorded in the presence of scn are mainly due to conductance through clc-1 . External substitution of br, no3, and i on hclc-1 expressed in xenopus oocytes had very similar effects to those described above for rclc-1, although block by br, i, and methanesulfonate may have been slightly weaker (table ii). 7), performed on outside - out patches containing hclc-1 at 140 mv to gain information about the mechanism of this block, show (table ii) that single channel current amplitude is reduced by foreign anion substitution in the same proportion as whole - cell current . As illustrated in figs . 1 and 8, conductance to outward currents is saturated at physiological cl concentrations with a kd of 6 mm . Since it was not possible to change internal cl concentration during whole - cell patch - clamp experiments in sf9 cells, results from separate whole - cell experiments were combined after normalizing in the following way . For different cells, the ratio between chord conductance at 120 mv and current at + 80 mv was found to be constant for external cl concentrations from 170 to 340 mm and a fixed internal cl concentration, presumably reflecting the ratio between single channel currents at 120 and + 80 mv . Similarly, ratios were calculated from results obtained using a variety of cl concentrations in the pipette, plotted against internal cl concentrations and fitted with a one - binding site hyperbola . The maximal ratio obtained from the fit was used to normalize the data points, making it possible to present the saturation curves for internal and external cl on the one graph (fig . 8). Similar to its external application, br applied internally had little effect on apparent popen curves (table iii). But unlike external br, which did not affect inward current kinetics, internal br slowed down current deactivation and appeared to have a stronger blocking action (table ii indicates 20% block of hclc-1 and 40% block of rclc-1 from the outside, while fig . These estimates of block from attenuation of inward currents are not strictly comparable as br applied from inside blocks inward current to a greater extent than outward current, whereas the opposite is true for block by externally applied br . In each case, the block is voltage dependent . 9) than br, just as occurs when they are applied from the outside . In 100 mm intracellular i (leaving 4 mm internal cl), inward currents through hclc-1 were almost completely blocked and it was impossible to extract parameters describing the voltage dependence . Partial replacement of cl by i led to a slight shift of v1/2 to more positive values and to a decrease in the apparent gating charge, z. the shift of v1/2 is opposite to the effect of external i, whereas both external and internal i led to a reduction in gating charge (table iii). In our receptor - operated model of gating in the clc-1 channel, open probability is controlled by a binding site for cl, or certain other anions, which is accessible only from outside and which must be correctly occupied for opening to occur (rychkov et al ., 1996). Consistent with this model, anions we have studied can be divided into three groups according to their ability to open the channel and to permeate it: impermeant anions that are unable to open the channel, anions that can open the channel but cannot permeate it, and anions that can both open the channel and permeate it . Large anions such as glutamate, gluconate, and glucuronate belong to the first group . All of these cause a shift of the deactivation curve to positive potentials similar to that caused by glucose, which provides evidence that the shift in voltage dependence of the channel is due to the absence of cl rather than to the presence of the foreign anion . Since glucose, presumably, does not interact with binding sites regulating the behavior of clc-1, any difference between those relative conductances obtained when glucose substitutes for nacl and those where a foreign anion substitutes for cl, reflects interaction between cl and that anion in the pore . Impermeant anions that do not interfere with the gating, such as glutamate and glucuronate, also do not change relative cl conductance . By contrast, hco3, which has just 3% of cl permeability, clearly reduces cl conductance (fig . 1) and shifts the apparent popen curve to less positive potentials than the impermeant glutamate . The slightly more permeant bro3 has more pronounced effects on channel conductance and gating . That gating is intrinsically linked to permeation and appears to be supported by the behavior of these anions . In the present work, however, we have found other anions, such as cyclamate and methanesulfonate, that can have a substantial effect on gating without being permeant . For example, cyclamate and bro3 have much the same effect on conductance and current kinetics and the amount of block of the inward current is also very similar, but while bro3 is permeant, cyclamate is impermeant and, in addition, it reduces the gating charge to 0.62 . These results suggest that cyclamate can bind (more readily than bicarbonate or bro3) to the regulatory binding site that opens the channel and that this site lies external to the selectivity filter, which cyclamate cannot pass . Thus, gating is not always coupled to permeation, probably because specificity of the pore selectivity filter differs from that of the site within the pore that regulates channel opening . It could be that when blocking anions, such as methanesulfonate, are bound to the latter site, they hinder cl passage through the pore . Alternatively, although less likely (see below), block could be due to binding at some second site (perhaps the selectivity filter), which must also be deep inside the pore, as indicated by diminished inward current block at negative potentials . In these ways, clc-1 appears to differ from clc-0, not because of any profound difference in their mechanisms of fast gating, but, probably, because the selectivity filter and regulatory binding site have very similar specificity in clc-0, which allows channel activation to be closely linked to permeation, whereas these specificities are different in clc-1 with the consequences that this entails . Small inorganic anions all showed potential - dependent block at low concentrations in the sequence: br <no3 <clo3 <i <clo4 <scn, which coincides with the ability to shift the voltage dependence of gating, to modify current kinetics, and also coincides with the lyotropic series . For br, no3, i, and methanesulfonate, we have good evidence from noise analysis that all reduction in current amplitude is due to a reduction in single channel current . In the lyotropic series, anions are arranged in order according to their ability to bind or adsorb to proteins, to potentiate the strength of a muscle twitch, and to unwind macromolecules . It is believed that, for binding to follow the lyotropic series, a site requires the combination of two molecular attributes: appropriate anion - attracting groups and neighboring hydrophobic groups (dani et al ., 1983). If conductance is limited by binding inside the pore, then, the stronger the binding, the smaller the conductance . This seems to be the case when scn concentration is low, where strong binding to only one of several intrapore binding sites is sufficient to block the channel . Possibly, as concentration is increased, all intrapore binding sites are occupied by scn, and electrostatic repulsion between these ions accelerates the rate - limiting exit steps . When all cl outside is replaced by scn, positive potentials applied from the inside reduce the probability of internal cl entering the channel, so the channel starts to rectify outward just as it also rectifies inward at strongly negative potentials when all scn is likely to be displaced from the channels . As is typical of the anomalous mole fraction effects seen in pores with multiple intrapore binding sites, the lowest conductance occurs when a mixture of ions is present in the channel . An anomalous mole - fraction effect for mixtures of cl and scn is not unique for clc-1 . Some other cl channels, such as the -aminobutyric acid (gaba) and glycine receptor channels (bormann et al ., 1987) and the cystic fibrosis transmembrane conductance regulator (tabcharani et al ., 1993), have similar properties . With a kd of 6 mm, outward cl conductance for outward currents through clc-1 is saturated at physiological external cl concentrations . Saturation is presumed to arise when the binding unbinding steps of permeation become rate limiting (rate of ion entry approaches the maximum rate for the unbinding steps). For inward currents, the kd is 33 mm, which suggests that there is a deeper energy well for cl ions passing through the channel from the external side than for those passing from the inside . This can be interpreted in terms of the rate limiting binding site being closer to the external side . When positive potential is applied from inside, this binding site is occupied by cl, but other intrapore sites will be free . Conductance is then very low due to strong binding of cl to this site . When cl is moving out under the influence of a positive potential on the outside, all binding sites in the channel will be occupied . Electrostatic repulsion between cl ions can then accelerate the rate - limiting exit step and increase conductance (hille, 1992). In this way, open channel rectification and the different kds on either side of the membrane may be explained . The effects of foreign anions applied to the inside are also different from their effects on the outside . From inside, br and i block inward current without affecting outward current, whereas, from outside, both currents are blocked . Also, from the inside, i shifts the apparent popen curve in the opposite direction, to more positive potentials . These results are all consistent with and extend the view of clc-1 as a channel with multiple internal binding sites having different characteristics and, especially, they support the proposal that there is at least one inner and one outer binding site inside the channel that are quite distinct from each other (see fahlke et al ., 1997b).> hco3 (f), while just for halides it was cl> br> i> f. this sequence corresponds to a cationic site of moderately strong field strength in the membrane (eisenman sequence 4; eisenman, 1965). However, not only electrostatic but also hydrophobic interactions are involved in anion permeation through clc-1, and so the permeability sequence need not be directly related to the electrostatic field strength of the relevant binding site . From blocking ability when applied externally, the binding sequence is scn> br> bro3> hco3 (f), which corresponds to the low field strength eisenman sequence 1 (eisenman, 1965). These results agree with early studies of foreign anion permeability (woodbury and miles, 1973) and of block of cl efflux (harris, 1958) by foreign anions in frog muscle where the same sequence difference occurred . This would indicate that the site accessible from outside, at which block occurs, is different from the site governing selectivity and because blocking ability corresponds to opening ability, that block probably occurs at the regulatory binding site . The effects of some of the anions studied in this work on rat clc-1 have been investigated in human clc-1 by fahlke et al . (1997a), who obtained a similar sequence for relative permeability: cl scn> br> (1997a) were somewhat higher than in our work, which can be explained by differences in internal cl concentration . In a multi - ion pore although for halides the differences were relatively small, methanesulfonate was found to be impermeant in rat clc-1, while in human clc-1, pmetsulf / pcl was 0.2 . With impermeant anion substitution, we have found that the sf9 cells cannot for long sustain membrane potentials as highly positive as +80 mv with 140 mm of cl inside and only 8 mm of cl remaining outside . As external cl was replaced with impermeant anion to achieve this condition, membrane potential increased to approximately the predicted + 80 mv, but soon dropped to less positive values due to increased leakage . We therefore generally used a lower concentration of cl (40 mm) in the internal solution . The difference in results might also be accounted for by cl accumulation near the external mouth of the channel, which could be higher at higher internal cl concentration . It is also possible that the inconsistencies could be due to a real difference between rat and human clc-1 . Some results, such as nondeactivating currents at negative potentials in the presence of i, are similar in both studies but explained differently . (1997b) sug - gest that inherently voltage - dependent conformational changes are modulated by i, which locks the channel in a nondeactivating state . While we do not dispute that i might lock the channel in the open state, evidence is lacking for the existence of conformational changes dependent upon an intrinsic voltage sensor in clc channels (see introduction). Our interpretation is, instead, based on a mechanism (pusch et al . 1995a; rychkov et al ., 1996) that is more akin to gating in receptor - operated channels . Hence, a binding site that has a higher affinity for i than for cl will maintain channel opening in the presence of i, achievable negative potentials being unable to displace i from the site . Estimates of the dimensions of a number of cation and anion channels have been made using a simple cylindrical pore model (hille, 1975; bormann et al ., 1987) relative permeabilities can be described by the equation: 3\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}p_{x}/p_{cl}=c / d(1-d / a)^{2},\end{equation}\end{document} where c is a constant, a is the pore diameter, and d is the stokes diameter for the particular ion . Although for the clc-1 channel this equation could not reliably be fitted to the experimental points, the relative permeability of anions included in the present study clearly decreased with increasing size of the anion (fig . Is still measurably permeant and methanesulfonate (5) is not, this might place an upper limit on the pore diameter . For comparison, stokes diameters of the permeant hydrophobic anions is significantly bigger, up to 6 for hexanoate . However, if those anions are approximated by a cylinder, their cylindrical diameter is 4.3 (halm and frizzell, 1992). Still, they are much more permeant than hco3 and their relative permeability increases along with the size of the hydrophobic part of the molecule, suggesting the likelihood of significant hydrophobic interactions with the wall of the pore (see woodbury and miles, 1973, for similar results on frog muscle). As well as having an anomalous relationship between permeability and size, these hydrophobic anions have complicated effects on clc-1 channel gating, which will be described in detail elsewhere . It may well be that the aromatic monocarboxylate blockers (bretag, 1987; astill et al . 1997) have their effect due to their ability to enter the pore because of their charge but interact very strongly with the pore wall . For several anion channels that were studied at the single channel level, multiple binding site models have been developed based on extensive kinetic data (bormann et al ., 1987; halm and frizzell, 1992; linsdell et al ., 1997b). It is apparent that very similar models of anion permeation could account for the characteristics of clc-1, but that a detailed quantitative description of such a model must await more information at the single channel level . In conclusion, we have shown (a) the multi - ion nature of clc-1 with demonstrable anomalous mole fraction effects of scn and clo4; (b) two of the anion binding sites, an inner and an outer site, are accessible from the cytoplasmic solution and from the external solution, respectively, and have different binding properties even for cl; (c) the outer binding site appears to have characteristics that make it the prime candidate for regulation of channel opening and for channel block by external anions, but it is not simultaneously the site that determines channel selectivity; (d) block of macroscopic currents by foreign anions can be accounted for entirely by a reduction in single channel conductance without any requirement for an effect on popen; (e) minimum pore diameter is 4.5; and (f) foreign anions can be clearly categorized into three different groups according to their interaction with clc-1 . Membrane conductance of sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace cl in the external solution . Relative conductance, gx + cl / gcl, is plotted against mole fraction, [x]/([x]+[cl]), of the foreign anion . Kinetics of whole - cell currents in sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace 95% of cl in the external solution . Currents were elicited by the standard activation protocol from a holding potential of 30 mv with a prepulse to + 40 mv followed by steps from 120 to + 80 mv in 20-mv increments . Since experiments were performed on different cells, currents have been normalized to the peak inward current in cl solution at 120 mv . Peak inward currents from cell to cell ranged between 5 and 10 na . Relative permeabilities for different foreign anions and their effect on the parameters of the popen curves voltages have been corrected for liquid junction potentials (see methods). The same number of experiments was performed with a particular anion for each different percent chloride replacement . 2) and 95% of external cl was replaced by (a) cyclamate or (b) methanesulfonate . Apparent popen curves (c) are plotted for control (), methanesulfonate () and cyclamate (). Membrane conductance of sf9 cells expressing rclc-1 when various permeant anions replace cl in the external solution . Relative conductance has been plotted against mole fraction of foreign anion as for fig . Kinetics of whole - cell currents in sf9 cells expressing rclc-1 when various permeant anions replace 100% of cl in the external solution . Amplitudes of outward current in cl solution and in scn solution were found to be closely correlated (a). Anomalous mole fraction effects were apparent for both permeability and conductance (b). Relative conductance () in this case was determined from the chord conductance measured at + 80 mv . Foreign anion block of macroscopic currents, i, from whole - cell studies and of single channel currents, i, estimated by noise analysis for ease of comparison, peak inward currents for a step to 140 mv in the presence of foreign anions have been normalized to the current in external chloride solution for rc1c-1 and hc1c-1 as appropriate . Currents, ix and ix, are inward currents carried by internal chloride in the presence of external anion x. these anions were substituted in equimolar amounts for 127.5 mm cl (75% of external cl substituted) in experiments on rclc-1 expressed in sf9 cells and for 100 mm cl (98% of external cl substituted) in experiments on hclc-1 expressed in xenopus oocytes . Nonstationary noise analysis performed on outside - out patches from xenopus oocytes expressing hclc-1 in the presence of cl, no3, i, or br . Variances (top) and means (middle) of current records (n = 150) are shown for standard voltage protocols incorporating a prepulse to + 60 mv followed by a 70-ms test pulse to 140 mv . (bottom) variance mean current plots are fitted with parabolas according to the equation: = 0 + ii i /n, where 0 is baseline noise variance, i is single channel current, i is mean current, and n is number of channels . Fitted values for the parabolas shown are: (a) for chloride, i = 0.26 pa; (b) for nitrate, i = 0.1 pa; (c) for 50% iodide/50% chloride, i = 0.077 pa; (d) for bromide, i = 0.21 pa . Relationship between cl conductance and concentration in rclc-1 . In this case, cl was replaced by glucose in the external solution but by glutamate in the internal solution . From fig . 1, it can be seen that the effects of glucose or glutamate replacement in the external solution are almost identical . Parameters of apparent popen curves from inside - out patches of xenopus oocytes after internal application of foreign anions kinetics of currents in inside - out patches from xenopus oocytes expressing hclc-1 when br and i are substituted for cl in the internal solution . A shows control currents in the patch to which 100% br was then applied (b), while c shows control currents in the patch to which 50% i was later applied (d). Voltage protocol: after a 50-ms prepulse to + 60 mv, the membrane potential was stepped for 150 ms from 140 to + 80 mv in 20-mv increments, followed by a step to 100 mv for 50 ms . Dependence of relative permeability of rclc-1 expressed in sf9 cells on the apparent ionic diameter of various anions . Apparent ionic diameters, d, were calculated from the einstein - stokes relation, d = 183.6/, where is the limiting conductance for the ion (robinson and stokes, 1959). Relative permeability for f was determined from the biionic potential with 160 mm of f in the internal solution and 170 mm of cl in the external solution.
Cardiovascular disease (cvd), including ischemic heart disease and stroke, is the most common cause of morbidity and mortality in the world . Established modifiable risk factors for ischemic heart disease include tobacco smoking, a diet high in saturated fatty acids, physical inactivity, high cholesterol, high blood pressure, and high body mass index . These are all risk factors associated with retinal microangiopathy found in epidemiological studies [3, 4] and in animal models . The eye offers noninvasive direct access to the microvasculature and, through high - quality imaging and software - assisted grading methods, the retinal vessel may be evaluated with high reproducibility [6, 7]. A number of studies have shown that changes in retinal vessel diameter predict risk of cardiovascular disease (cvd) [8, 9] and stroke mortality [10, 11], independently of traditional risk factors . Despite these data, there is an incomplete understanding of the relationship between retinal vessel diameter and cardiovascular events with inconsistent findings in the literature . Cardiovascular risk factors can lead to deaths that are not primarily classified as cardiovascular, such as chronic obstructive pulmonary disease, diabetes mellitus, dementia, or even cancer [13, 14]. It is relevant to study the role of microvasculature in noncardiovascular mortality, because of the established link between small vessels and diseases such as diabetes mellitus, dementia, and chronic obstructive pulmonary disease . It has been shown that decrease in risk factors was associated with the decrease in morbidity and mortality [15, 16]. The aim of this study is twofold: (1) to examine the associations between retinal vessel diameters and cardiovascular risk factors and (2) to assess the association between retinal vessel diameter and all - cause mortality . It is difficult to compare associations between retinal vessel diameter and mortality between studies because methods, study populations, length of follow - up, ethnicity, and mortality in the background population tend to differ across studies . This is the first study to examine such relationships in a danish population - based cohort . The inter99 study is a population - based nonpharmacological intervention study of cardiovascular and metabolic characteristics and lifestyle . The aim of the inter99 study was to assess the effect on incidence of ischemic heart disease (ihd) after screening and repeatedly group - based nonpharmacological intervention on lifestyle . An age- and sex - stratified random sample of 13,016 individuals from seven birth cohorts (years of birth 1939 - 40, 1944 - 45, 1949 - 50, 1954 - 55, 1959 - 60, 1964 - 65, and 1969 - 70) living in 11 municipalities of the south - western part of copenhagen county on december 2nd 1998 were invited to participate, of which 6906 turned up for the investigation . Of these, 122 were excluded due to either alcoholism or drug abuse or because of linguistic problems; thus 6784 subjects were included in the study . The study participants were drawn from the danish civil registration system in which a unique 10-digit number registers all inhabitants in denmark, making linkage across time and registers accurate . All participants went through a screening program at the research centre for prevention and health at glostrup university hospital, copenhagen, denmark . All had their blood pressure (bp) measured twice with a mercury sphygmomanometer after 5 min of rest in lying position . Height and weight were measured without shoes, and body mass index (bmi) was calculated (kg / m). Waist and hip circumference was measured in cm and waist / hip ratio was calculated . Fasting blood samples were drawn for assessment of total cholesterol, hdl cholesterol, and triglycerides . Vldl and ldl were calculated by friedewald's equation . An oral glucose tolerance test (ogtt) history of lifestyle, education and profession, chronic diseases, family history of chronic diseases, health care system contacts, and lifestyle consciousness was obtained from questionnaire . Information on age, sex, height, familial occurrence of acute ihd, previous ischemic disease, diabetes, systolic bp, cholesterol (incl . Hdl), weight, smoking, and diabetes was entered into the precard program to assess an individuals' 10-year risk of fatal and nonfatal ischemic heart disease . Each person was simulated in the computer program as 60 years old to be able to compare risk among different age groups . The precard program contains a new coronary risk score (the copenhagen risk score) for myocardial infarction . A subgroup of 1437 persons was chosen based on the following criteria: (1) an age- and sex - stratified control group was randomly polled to match the background population and (2) persons with a high risk of ihd, type 2 diabetes mellitus diagnosed by the oral glucose tolerance test, known diabetes mellitus, or impaired glucose tolerance . Individuals were considered as high risk if they either had an absolute risk in the upper quintile of the distribution (according to the precard classification) stratified according to age and sex or had one or more of the following risk factors: daily smokers (one or more grams of tobacco daily), systolic blood pressure of 160 or more (the lowest value of at least three measurements) or in antihypertensive treatment, total cholesterol of 7.5 mmol / l or more, body mass index of 30 or more, history of diabetes, or had diabetes or impaired glucose tolerance as evaluated from the ogtt . Persons without fundus photographs of acceptable quality (n = 62) were excluded, leaving 908 persons available for analysis . Excluded participants did not differ from the included (parameters from table 1 were tested). Only information from right eye was used, because large epidemiological studies have demonstrated that the correlation of computer - assisted measurements is high between the two eyes for crae and crve and that measurement from only one eye can adequately represent the retinal vessel diameters [20, 21]. If the retinal photographs from the right eye were not gradable, photographs from the left eye were used . Study participants were asked about present or past history of ophthalmic disorders, surgery, ocular medication, and family history of cataract and glaucoma . The ophthalmic examination included a determination of visual acuity, fundus photography (trc-50x camera; topcon corp . Tokyo, japan; with 1024 1024 pixel cv-1000 back - piece, angiovision 1000; medivision, yokneam illit, israel), and ophthalmoscopy . Image analysis was made of 60-degree digital grey - scale (red - free) fundus photographs centred on the optic disc or macula . A green filter for red - free photographs was used to enhance the sharpness and contrast of the blood column in the vessels . Retinal vessel diameters were measured using a danish custom - developed semiautomatic computer algorithm . According to international standards (vessel measurement system, ivan protocol, version 2, university of wisconsin), retinal vessel diameters were measured in 60 digital grey - scale (red - free) fundus photographs . We tested the ivan software against our own custom - developed software in twenty red - free fundus photographs . Absolute distances were calculated assuming a uniform vertical optic disc diameter of 1800 m . A standard grid containing three concentric circles the inner circle demarcated an average optic disc (circle was placed manually), the middle circle demarcated 0.5 disc diameters (dd) from the outer rim of the optic disc, and the outer circle demarcated 1.0 dd from the outer rim of the optic disc . The program identified the six largest arteries and the six largest veins and calculated the central retinal artery equivalent (crae) and the central retinal vein equivalent (crve) according to the formulas described by knudtson et al . . The grader then identified arteries and veins between the outer two circles, using red and blue lines, respectively, to delineate the borders of the blood vessels, which were defined, in this context, as the edges of the blood column the vessel wall being translucent . Vessel diameters were expressed as crae and crve and were measured in micrometres at the baseline examination . The participants' unique personal identification number was linked to the danish civil registration system, to the national patient registry, and to the danish register of causes of death for vital status and cause of death . Ihd was defined as either admission to hospital (inpatient or outpatient) or causes of death with icd-8 codes 410414 and icd-10 codes i20i25 or surgery codes 300.09304.99 and kfnc - kfnh (since 1996; bypass, recanalization, or reconstruction of coronary arteries). Stroke events were defined as admission to hospital (inpatient or outpatient) or causes of death with icd-8 codes 430434 + 436 and icd-10 codes i60i64 + i69 . The following codes covered noncardiovascular mortality: c00c97 (cancer), f00f03 (dementia), j15j19 (pneumonia), j30j98 (chronic respiratory disease), or k72 (acute and subacute hepatic failure) and the remaining codes (other). Participants were followed up from date of study entry until date of death, up till the 31st of december 2013 . Associations of retinal vessel diameters with the estimated 10-year absolute risk of ihd at 60 years (precard score) were analyzed using univariate linear regression analyses . Relations between retinal vessel diameters and individual risk factors for ihd were analyzed using multiple linear regression . First, each risk factor was modeled separately to analyze the relationship of each risk factor with crae and crve . When high colinearity was found for an independent variable in a regression model with several independent variables (variance inflation factor> 5), this variable was removed from the regression model . Step 1 was to include all the variables that were significant in unadjusted analyses (age, systolic blood pressure, total cholesterol, hdl cholesterol, smoking, diabetes, hba1c, fasting p - glucose, 2-hour ogtt p - glucose, bmi, and weight). Step 2 was to remove the variable with the highest p value and to run the model once more . Vessel diameters were chosen as dependent variables, and the estimated 10-year risk of ischemic heart disease (ihd) and individual risk factors for ihd were chosen as independent variables . Multivariate cox regression analyses were used to determine the association of retinal vessel diameters with all - cause mortality . Estimates were presented as hazard ratios (hrs) (95% confidence intervals) after adjustment for confounders (age, gender, blood pressure, and smoking). This means that the associations between crae and crve and cardiovascular risk factors were similar in the two ascertainment groups . Therefore, we presented the results of the total population of 908 persons . The level of statistical significance was set at p = 0.05 . Mean (sd) crae was 163.3 (15.8) m and crve was 251.0 (20.9) m (table 1). When we tested the ivan software against our danish custom - developed semiautomatic software in twenty red - free fundus photographs, we found an intraclass correlation coefficient (icc) of 0.8 (95% ci: 0.5 to 0.9) for crae and 0.9 (95% ci: 0.8 to 0.9) for crve . Furthermore, when we tested the ivan software against our danish custom - developed software, there were no statistically significant differences between measurements of crae (p = 0.448) and crve (p = 0.828). The intergrader reliability (icc) between 2 independent masked graders using the danish custom - developed software was 0.9 for arteries and 0.8 for veins, measured on 45 right eyes . The mean difference in vessel diameter among the two graders was 3.9 m (0.024%) for arteries and 6.2 m (0.025%) for veins . When adjusting for age no association between crae and ihd risk multiple linear regression analyses showed that smokers had wider crae than nonsmokers and that crae decreased with increasing age, increasing hdl cholesterol and increasing systolic blood pressure (table 3). When adjusting for age a significant association between wider crve and ihd risk was found, p <0.001 (table 2). Multiple linear regression analyses showed that smokers had wider crve than nonsmokers and that crve decreased with increasing hdl (table 3). Of the 61 participants who died, 12 persons died of cvd (8 from ihd and 5 from stroke) and 49 died from other causes . The mean (sd) age at the time of death was 58 (6) years for participants who died of cvd causes and 60 (7) years for participants who died from other causes . Noncardiovascular cause of death in 49 participants (n = 49) was due to cancer (n = 28), dementia (n = 4), chronic respiratory disease (n = 2), acute and subacute hepatic failure (n = 1), and/or unknown causes (n = 14). There was no significant difference in crae (p = 0.324) and crve (p = 0.954) between participants who died from cvd compared with other causes of death (data not tabulated). All - cause - mortality was not significantly associated with crae with a hr of 0.75 (95% ci: 0.3 to 1.5) (p = 0.431), in a model adjusted for age, gender, blood pressure, and smoking (table 4). Cardiovascular mortality was not significantly associated with crae with a hr of 0.57 (95% ci: 0.1 to 2.5) (p = 0.465), in a model adjusted for age, gender, blood pressure, and smoking (data not tabulated). When the third tertile of crve was compared to the first tertile of crve, all - cause mortality was significantly associated with higher crve with a hr of 2.02 (95% ci: 1.0 to 3.8, p = 0.033), in a model adjusted for age, gender, and blood pressure . When additionally adjusting for smoking it was no longer significant with a hr of 1.34 (95% ci: 0.6 to 2.6, p = 0.392) (table 4). Cardiovascular mortality was not significantly associated with crve with a hr of 1.06 (95% ci: 0.2 to 5.0) (p = 0.933), in a model adjusted for age, gender, blood pressure, and smoking (data not tabulated). In this large general population study of adult danes, we confirmed the classical associations between retinal vessel diameters and known cardiovascular risk factors . We did not find an association between retinal vessel diameters and all - cause mortality . Higher serum hdl cholesterol was independently associated with crae narrowing and crve narrowing in our study, which is consistent with findings from some [26, 27] but not all studies [7, 28]. However, clinical studies have not presented a consistent pattern of association between retinal vessel diameter and dyslipidemia . Some genetic mechanisms that raise plasma hdl cholesterol do not seem to lower risk of myocardial infarction . Thus, it is suggested that more may be learned from a closer inspection of those mechanisms . In our study mechanisms behind these associations remain unclear, but smoking - induced increase in nitrous oxide production, potassium channel activation, and possible tissue degeneration might explain the association with a wider crve [3134]. In our cohort we confirmed an association between narrowed crae and aging as well as between narrowed crae and high blood pressure [27, 35]. In our study, which used the precard program based on the copenhagen risk score, higher crve was associated with ihd risk, which is in accordance with findings from previous studies . Thus, the data from the study suggest a single fundus photograph cannot yet be used in the evaluation of an individual patient's risk of cardiovascular disease . It has been hypothesized that changes in the retinal vessel diameters might be markers for general health and not necessarily for cardiovascular health only . In 1978, in a study of 50-year - old men, svardsudd et al . Found that focal arteriolar narrowing was associated with increased 12-year all - cause and cardiovascular mortality rates after controlling for systolic blood pressure and other risk factors . An unexpected association with cancer and other noncardiovascular related mortality was also found . In february 2016, mutlu et al . Showed that narrower retinal arteriolar diameters and wider retinal venular diameters were associated with all - cause mortality . They followed 5674 persons from an adult dutch population during 25 years, where 3794 persons died, 1034 of those from cardiovascular causes . While narrower arterioles were associated with cardiovascular mortality, wider venules were equally associated with cardiovascular and noncardiovascular mortality . In our study, we compared if all - cause mortality risk was increased in patients who have the widest vs thinnest vessels . Larger crve explained the relationship between retinal vessel diameters and all - cause mortality, but this relationship was no longer significant after additional adjustment for smoking . Adjustment for smoking reduced the estimated difference between the highest tertile compared to the lowest tertile of retinal venular caliber by 34% . It is reasonable to think that the results represent a cofounding effect of smoking in the investigated association between retinal vascular diameters and all - cause mortality . Other factors explaining the lack of association may be the relatively short follow - up time and the low number of deaths . Diseases as chronic obstructive pulmonary disease, dementia, and even cancer, which are primarily classified as noncardiovascular diseases, may have common risk factors and a partly vascular pathogenesis . Still, most population - based cardiovascular studies are focused on large vessels such as aorta or carotid arteries, despite evidence that microcirculation is important for cardiovascular health as well . In our cohort, since studies of past smokers suggest that the impact of smoking on crve is reversible, it remains for our results to be proven in a larger study population . In several large population - based epidemiological studies, ivan software has demonstrated substantial reproducibility (intergrader correlation coefficient ranged from 0.6 to 0.9). The icc results in our study indicate that retinal vessel diameters calculated by the danish custom - developed semiautomatic software are comparable with retinal vessel diameters calculated by the ivan software and have a high interrate reliability . . Therefore, 20 pictures are significant for calculating these iccs . Thus, the danish custom - developed software provides a reliable research tool for objective assessment of structural vascular changes . The study was designed to optimize fundus vessel imaging by applying direct digital imaging in red - free illumination rather than digitization of colour slides as used in earlier studies [40, 41]. Limitations include the absence of correction for refraction and electrocardiographic synchronization of the fundus camera to the cardiac cycle which may influence the vessel calibers and the low number of deaths . Furthermore, calibration of the computer - assisted program is crucial when determining the true size of a fundus feature . Studies published in the 1990s showed that the true value of one standard vertical disk diameter was equivalent to 18001900 m; later it was standardized to 1800 m . This is now accepted as a reference for calibration to compensate for the effect of camera magnification on the vessel caliber measurement in the computer - assisted programs . This method potentially introduces bias, since individuals have different size optic discs, refraction, and axial length . Researchers within the field have accepted the magnification problem as bias, for which there is no good solution at the moment . In conclusion, we confirmed the classical associations between retinal vessel diameters and known cardiovascular risk factors, and we found that larger crve is associated with increased ihd risk . Furthermore, we did not find an association between retinal vessel diameters and all - cause mortality after adjusting for relevant confounders . However, given the heterogeneity of previous studies which differ in methods, populations, ethnicity, and length of follow - up, it remains for our results to be proven in a larger study population . Longer follow - up time may reveal associations that were not demonstrable at ages 3060 years, after 15 years of follow - up and with a high probability of survival.
Schistosomiasis mainly occurs in developing countries and it is the most important human helminth infection in terms of global mortality . This parasitic disease affects more than 200 million people worldwide causing more than 250,000 deaths per year . Furthermore, schistosomiasis causes up to 4.5 million daly (disability adjusted life year) losses annually . Current schistosomiasis control strategies are mainly based on chemotherapy but, in spite of decades of mass treatment, the number of infected people remains constant . Extensive endemic areas and constant reinfection of individuals together with poor sanitary conditions in developing countries make drug treatment alone inefficient . Many consider that the best long - term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment . A vaccine that induces even a partial reduction in worm burdens could considerably reduce pathology and limit parasite transmission . The advent of technologies that allowed large - scale studies of genes and proteins had a remarkable impact on the screening of new and potential vaccine candidates of schistosoma mansoni . Mass spectrometry- (ms-)based proteomics [710], transcriptome, and genome of s. mansoni offered a vast repertoire of potential targets for vaccine and drug therapies . Despite this possibility to generate information about dna and protein sequences, it remains an obstacle how to select them and which molecules would have the highest potential among thousands or hundreds of potential candidates . In this postgenomic scenario, bioinformatic technologies have emerged as important tools to mine transcriptomic, genomic, and proteomic databases . These new approaches have the potential to accelerate the identification of new generation of vaccine candidates that may induce greater protection than the previous schistosome antigens studied to date [13, 14]. Specific algorithms allow the identification of molecules containing transmembrane domains, signal peptides, signal anchors, and other posttranslational modifications that can be used as predictors of excretory - secretory products or components exposed to the surface of the s. mansoni tegument [9, 13]. Additionally, predicting the peptides that bind to mhc class ii molecules can effectively reduce the number of experiments required for identifying helper t - cell epitopes and play an important role in rational vaccine design . The tegument is a dynamic host - interactive surface involved in nutrition, immune evasion and modulation, excretion, osmoregulation, sensory reception, and signal transduction [16, 17]. Consequently, the tegument is considered an important source of parasite antigens for the development of a schistosome vaccine . Currently, the most promising schistosome vaccine candidates are located in the tegument, such as tsp-2 and sm29 . Throughout the next sections we will discuss and present the recent studies, approaches, and bioinformatics tools that have been used to search and validate new vaccine targets present in the tegument of s. mansoni . The sequencing of the s. mansoni transcriptome [11, 12] and the development of proteomic and microarray technologies have dramatically improved the possibilities for identifying novel vaccine candidates [21, 22]. In the search for an effective schistosome vaccine, several available bioinformatic tools can be helpful and a rational design of possible vaccines has replaced the trial - and - error approach . A first step for a rational vaccine design is the identification of target antigens . For schistosoma, a potential vaccine should include proteins that are preferentially surface exposed and/or secreted ones, expressed in the intramammalian host . The genes sequences can be obtain at the genbank database (http://www.ncbi.nlm.nih.gov/genbank/), and at the site of the whole schistosome genome sequencing project (http://www.schistodb.org). The proteins can be identified by proteomic analysis which has resulted in a remarkable understanding of the protein composition of the schistosome tegument [710, 24, 25]. If necessary, the target sequence can be translated using the translate tool which allows the translation of nucleotide (dna / rna) sequence to a protein sequence (http://expasy.org/tools/dna.html). Based on their amino acid sequences, topology prediction to confirm the presence of transmembrane helices can be performed using tmhmm server v. 2.0 (http://www.cbs.dtu.dk/services/tmhmm-2.0/) or sosui (http://bp.nuap.nagoya-u.ac.jp/sosui/) and subcellular localization can be performed using the wolfpsort (http://wolfpsort.org/aboutwolf_psort.html.en). The identification of domains within proteins can, therefore, provide insights into their function and some databases are able to identify known functionally important sequence motifs that may not be identified on the basis of sequence homology by itself . Such searches can be performed using different tools as pfam (http://pfam.janelia.org, http://pfam.sanger.ac.uk/search?tab=searchsequenceblock), interpro scan which integrates search in prosite, pfam, prints, and other family, and domain databases (http://www.ebi.ac.uk/tools/pfa/iprscan/) or scanprosite that scans a sequence against prosite or a pattern against the uniprot knowledgebase (swiss - prot and trembl) (http://expasy.org/tools/scanprosite/). The prediction of either hla - peptide binding or proteasomal processing of antigens can be predict for databases like syfpeithi (http://www.syfpeithi.de/scripts/mhcserver.dll/epitopeprediction.htm) epitope prediction algorithm, which comprises more than 7000 peptide known sequences binding class i and class ii mhc molecules or netchop (http://www.cbs.dtu.dk/services/netchop/) server that has been trained on human data only, and will therefore presumably have better performance for prediction of the cleavage sites of the human proteasome . However, since the proteasome structure is quite conserved, we believe that the server is able to produce reliable predictions for at least the other mammalian proteasomes . Primary structure analysis can be performed at protparam (http://expasy.org/tools/protparam.html) tool which allows the computation of various physical and chemical parameters for a given protein stored in swiss - prot or trembl or for a user entered sequence . Post - translational modification prediction as n - glycosylation, o - glycosylation and signal peptide can be analyzed using the netnglyc 1.0 (http://www.cbs.dtu.dk/services/netnglyc/), yinoyang (http://www.cbs.dtu.dk/services/yinoyang/) and signalp (http://www.cbs.dtu.dk/services/signalp/), respectively (figure 1). Schistosome membrane - bound antigens have currently become frequent targets of vaccines studies . Upon publication of the transcriptome data and its scrutiny for genes with functions that would indicate their surface exposure and likely interaction with the host immune system, a series of novel genes were offered as potential vaccine candidates based on their functional classification by gene ontology [11, 29]. The use of bioinformatic programs to screen and select potential vaccine targets from the available s. mansoni sequence databases, such as transcriptomes, genome, and proteomics is an important strategy for the rational design of vaccines, allowing the prediction of antigens in silico . These in silico analysis have lead to the selection of several schistosome vaccine candidates by different research groups and the protective effect of some tegument antigens that were already tested in vivo will be further discussed in this section and summarized in tables 1 and 2 . In the context of searching for new protective tegument proteins, in 2006 cardoso et al . Identified 34 proteins with membrane - bound protein motifs . At the same time other researchers across the world published the proteomic analysis of s. mansoni tegument [8, 9]. One of the first tegument proteins selected and studied, as a potential schistosome vaccine, was the schistosome membrane - bound protein sm29 that was identified through membrane - bound motif search using bioinformatic analysis [20, 51]. Sm29 is a membrane - bound protein with predicted n - glycosylation and o - glycosylation sites, with unknown function and no homolog outside the schistosoma genus [20, 21]. Recently, it was demonstrated that sm29 is gpi - anchored on the tegument surface of s. mansoni and shaved off under phosphatidylinositol - specific phospholipase c (piplc) treatment . Sm29 as recombinant vaccine induced a th1 type of immune response in rsm29-immunized mice, 50% reduction in liver granuloma, 60% reduction in intestinal eggs, and 51% reduction in adult worms . Another group of proteins that are exposed on the tegument surface, hence potential vaccine candidates, are the integral membrane proteins tetraspanins tsp-1 and tsp-2 . Tetraspanins cross the cell membrane four times and play multiple roles in mammalian cell signaling, such as interactions between immune effector cells and their ligands . Vaccination of mice with these recombinant proteins resulted in 57% reduction in worm burden and 34% reduction in liver eggs for rtsp-1 and 64% reduction in worm burden and 52% reduction in liver eggs for rtsp-2 . As mentioned before, the current most promising schistosome vaccine candidates are located in the tegument, such as tsp-2 and sm29 . The calcium - dependent, neutral cysteine - protease (calpain) was previously purified from s. mansoni and reported to be excreted / secreted and present on the tegument surface . The large subunit of calpain (sm - p80) was tested as recombinant vaccine which provided a 2939% reduction in worm burden in immunized mice challenged with s. mansoni . This antigen (sm - p80) was also evaluated in different protocols of immunization, such as dna vaccine with sm - p80 solely (39% of protection), combined with il-2 (57% of protection), or associated with il-12 (45% of protection). The authors attribute these results to the pivotal role of calpain on surface membrane biogenesis of schistosomes, as well as in the immune evasion process . Other recent publications of this group reported higher levels of protection by sm - p80 dna - based vaccines, 59% reduction in worm burden in mice, decrease in egg production by 84%, and a predominant th1 immune response . Mice prime - immunized with dna vaccine and boosted twice with rsm - p80 reduced up to 70% in worm burden and decreased egg production by up to 75% with an induction of a th1 and th17 cytokine profiles . Sm - p80 was also cloned in vr1020, an fda - approved vector for human use . Additionally, the protective efficacy of this vaccine formulation was tested in a murine model . A glycosylphosphatidylinositol (gpi - anchored) protein with unknown function was initially identified in the tegument [56, 57] and termed later 200 kda, sm200, or ecl [33, 58, 59]. Had suggested the use of this protein as immunogen to trigger the immune response against s. mansoni . Furthermore, the immune and protective responses induced by immunization with plasmid dna (ecl - encoded) complexed with protamine sulphate as adjuvant was evaluated . The protection elicited was 38.1% and the spectrum of the elicited immune response induced by the vaccine formulation without protamine were characterized by a high level of igg (igg1> igg2a). Sm200, like sm29, was released from the tegument surface by an enzymatic shaving confirming its tegument surface localization . Sm25, a major antigen on the tegument surface of s. mansoni, is a 25 kda n - glycosylated glycoprotein that was previously reported to be palmitoylated . Recently, it was demonstrated that sm25 is a gpi - anchored protein on the tegument surface . Differently from previous tegument surface antigens reported above that engendered substantial levels of protection as schistosomiasis vaccine candidates, the studies using rsm25 failed to protect mice against s. mansoni cercariae challenges, despite the induction of specific anti - sm25 igg . Some proteins present in the tegument of s. mansoni, regardless of being exposed on the surface, when used as vaccines have shown significant levels of protection in mice . The most relevant tegument proteins tested will be discussed below and are summarized in table 2 . Proteomic analysis of s. mansoni tegument [8, 9] provided the schistosome vaccinology field with promising candidates for vaccine design . Based on these new proteomic databases, it was selected a protein with unknown function that was termed smig because of the presence of an immunoglobulin domain . Smig tested as recombinant vaccine failed to induce worm burden reduction, but immunized mice had significant reduction of liver granuloma area and fibrosis content, showing a mixed th1/th2 type of immune response . Sm22.6 gene, encoding a tegument protein, was first identified in a s. mansoni lung - stage cdna library . Sm22.6, a specific schistosome lung - stage protein, was tested as dna and recombinant vaccine, but only the recombinant form provided reduction on adult worm burden (34%) and induced a mixed th1/th2 type of immune response . A membrane - associated protein localized on the s. mansoni adult worm, sm21.6, was also tested as recombinant vaccine . Sm21.6 showed 45% identity with sm22.6, both possess ef - hand antigen from the family of ef - hand containing parasite proteins with sequence similarity to dynein light chain . Bioinformatic analysis predicted sm22.6 as a soluble protein with neither signal peptide nor transmembrane domain, and confocal microscopy analysis revealed this protein as membrane - associated localized on the s. mansoni adult worm . Mouse immunization with rsm21.6 induced a mixed th1/th2 cytokine profile and no protection against infection, but vaccination with rsm21.6 reduced by 28% of liver granuloma numbers, 21% of granuloma area, and 34% of fibrosis . Further, a s. mansoni stomatin like protein-2 (smstolp-2) was demonstrated to be localized on the tegument and its recombinant form was also tested as a schistosomiasis vaccine . The function of stomatins is still unknown . In erythrocytes, it may link stomatin or other integral membrane proteins to the peripheral cytoskeleton, playing a role in the regulation of ion channel conductance or in the organization of sphingolipids and cholesterol - rich lipid rafts . Immunization with rsmstolp-2 engendered 32% reduction in adult worm burden and a th1 predominant immune response . Dif5 gene, a possible homologue of human cd59/ly6, predicted to be gpi - anchored on the s. mansoni tegument surface conferred 22% reduction in adult worm burden in mice immunized with the dif5 gene as dna vaccine . Sm21.7, a protein that was localized on the tegument of s. mansoni by immunofluorescence techniques, was tested as recombinant and dna vaccines . Similar to sm22.6, sm21.7 has a motif strongly homologous to the ef hand calcium binding domain; however, the change of the invariant glycine to glutamine in the calcium binding loop, makes this domain nonfunctional, as shown by the inability of sm21.7 to bind calcium . Sm21.7 was identified in cercariae, adults, and eggs whereas sm22.6 and sm20.8 were expressed in adults . Sm21.7 as recombinant and dna vaccines resulted in 4170% and 4153% reduction in the number of adult worms, respectively, following challenge by s. mansoni cercariae [37, 43]. Both forms of sm21.7 vaccine revealed a decrease in the number, size, and cellularity of the granuloma in the liver of the vaccinated in comparison with unvaccinated mice [37, 43]. Sm21.7 dna vaccine was also evaluated in association with the sm fimbrin gene as dna vaccine . This sm21.7/fimbrin vaccine showed 56% reduction in adult worm burden, 41% and 55% reduction in liver and intestine eggs, respectively . Sm fimbrin has homology to actin binding proteins, presents a calcium - binding site like in calmodulin molecules was localized on the tegument of adult worms and its recombinant form (solely) conferred 39.441.6% of protection in immunized mice . Sm23, an integral membrane protein detected with antibodies in all stages of the parasite host forms, engendered different levels of protection (from 18% to 44%) depending on the vaccine formulation [41, 42, 64]. Sm23 is a 23 kda integral membrane protein member of the tetraspanin family, possessing four hydrophobic putative transmembrane domains, some of them involved in signal transduction . Besides being an integral protein, sm23 is additionally linked to the membrane by a glycosylphosphatidylinositol anchor, but is not released from the surface after cleavage with piplc . This protein is expressed in all schistosome life stages examined and in several tissues, including the adult tegument . Sm20.8, another tegument protein, is a member of a family of soluble tegument antigens that contain ef - hand motifs, it interacts with dynein light chain and is recognized as antigenic targets in protective antisera . Antioxidant enzymes have been shown to be interesting targets eliciting high levels of protection against s. mansoni challenging . Moreover, mice immunized with cytosolic cu - zn superoxide dismutase and glutathione peroxidase as dna vaccines showed reductions in worm burden at levels 4170% and 85%, respectively, depending on the antigen and the vaccine formulation used in the studies . The association of cu - zn superoxide dismutase or glutathione peroxidase with a partial sequence of the structural protein filamin, as dna vaccines, resulted in protections ranging from 39% to 50% . Another schistosome antioxidant enzyme, glutathione s - transferase (sm28gst), when associated with il-18 as dna vaccine resulted in 28% reduction in egg laying and 23% reduction in worm burden in mice and up to 59% reduction in worm burden in rats immunized with a single dose of rsm28gst, using either aluminum hydroxide or bacillus calmette - gurin as adjuvants . Sm28gst was extensively explored as a potential antigen against schistosomiasis in different immunization protocols and vaccine formulations [6875]. These studies exploring sm28gst as vaccine candidate and its evaluation in human clinical trials are revised in capron et al . 2005 . A strong evidence of the potentiality of s. mansoni tegument - bound antigens as vaccine candidates was recently demonstrated using an extract of tegument proteins from lung - stage schistosomula named smteg . Smteg - immunized mice showed a th1 type of immune response associated with 48% reduction in worm burden, 65% reduction in liver eggs, 60% reduction in fecal eggs, and 41% reduction in liver granuloma . Even though funding for vaccine development against schistosomiasis has dropped significantly, there is a common understanding within the scientific community that long - term effective disease control will benefit from the combination of vaccination and chemotherapy, plus sanitation and public health control measures . The current vaccine candidates may prove not to be the most effective, but it is important to continue identifying new target antigens . Consequently, the characterization and better understanding of the s. mansoni tegument composition, coupled with new bioinformatic approaches and tools will definitely initiate a new era for the development of schistosome vaccines . A recent publication identified several tegument - associated proteins released through enzymatic shaving . This work presented a new repertoire of interesting and potential vaccine candidates to be explored, such as annexin, s. mansoni nucleotide pyrophosphatase / phosphodiesterase type 5 (smnpp-5), and an amino acid transporter, schistosome permease 1 (sprm1). Besides these surface exposed antigens, other potential tegument antigens to be further evaluated are the ones related to uptake or transport of nutrients, drugs, and other molecules across the tegument, such as aquaporins [82, 83]. Other candidates previously identified as tegument proteins, although not tested as vaccine, are the receptors such as the histamine receptors smgpr-1 and smgpr-2 . The major challenge to develop vaccines using defined and single antigens is finding molecules able to stimulate appropriate immune responses that can lead to resistance . However, a strategy that could accelerate the achievement of an effective antischistosomiasis vaccine would be the association of different recombinant antigens that previously resulted in partial protection or even the use of pools of antigens known as multivalent or multiepitope vaccines . This strategy was evaluated for some researchers using associations of s. mansoni synthetic peptides or even dna - based vaccines but their approach did not engender higher levels of protection when compared to the use of a single antigen [39, 86]. This demonstrates that no additive or synergistic effects were obtained from the different antigens selected in those studies but it could be related to the specific combination of antigens and/or the type of immune response resulted from this association and do not exclude the possibility of success using other parasite antigens . Taken together, all proteins discussed here, and many others identified by bioinformatic tools, could improve the search for an effective antischistosomiasis vaccine, and consequently, disease control.
As widely demonstrated, the plastic potential of the brain strongly depends on the preserved white matter connectivity sub - serving fundamental functional epicentres (duffau, 2014b). An extensive anatomical knowledge of the major white matter bundles is mandatory in order to tailor the surgical approach and to optimize the resection of various subcortical lesions . However, although white matter (wm) structures seem to share more common anatomical features across individuals at the deep white matter regions (dwm) (burgel et al ., 1997), only a few studies have analyzed the peripheral and more superficially located white matter (swm), which fills the space between the deep white matter and the cortex . For example, the swm is known to contain short cortical association fibres, but their locations, number, and trajectories are not sufficiently defined and for this reason their description has never become a crucial part of the neurosurgical training and preoperative planning . Only recently, with the advent of several new techniques such as mr diffusion tensor tractography (dtt) (oishi et al ., 2008), polarized light imaging (pli) (dammers et al ., 2012), optical coherence tomography (magnain et al ., 2014) new insights have been provided regarding the organization of the swm . The predominant intra - territorial connections within the superficial white matter are likely to be u - shaped association fibres, although neighbouring cortical regions within a territory may be linked at their border by intra - cortical fibres . Tractographic evidence and previous anatomical dissection studies suggest that territories are connected by multiple parallel long and intermediate association fibre pathways (catani et al ., 2005). The direction of these pathways into the subcortical regions should be always considered in order to optimize the surgical approach minimizing the destruction of several wm fibres . Despite its important role for research and educational applications unexpected artefacts can arise due to the discrete sampled, noisy and voxel - averaged tensor data . The degree of uncertainty in the estimation of the fibre orientation, which is typical of dti tractography algorithms, may increase variability across subjects in regions containing crossing fibres in which the fractional anisotropy decreases as the confidence intervals in fibre orientation (i.e., cone of uncertainty) especially close the cortical endpoints (jones et al ., 2003). A high level of anatomic knowledge and great care thus this cannot be considered the best tool for presurgical planning when minimal functional damage to the swm is warranted (duffau, 2014a). Even with some limitations, white matter dissection of previously formalin fixed brains still remains one of the most trustable techniques in order to demonstrate white matter connectivity and to acquire a comprehensive three - dimensional orientation during neurosurgical training (agrawal et al ., 2011). Our group has recently proposed an alternative method to the standard klingler's technique for cadaver specimen preparation, which seems to improve the three - dimensional orientation of the superficial and deep white matter fibres (latini et al ., 2015). The intra - carotidal perfusion process provides a very homogeneous fixation with many fine anatomical details preserved even at the grey / white matter junction . In a previous article we demonstrated that the cortical terminations of the major associative bundles could be identified until the pial layer with a very good accuracy . The main goal of this paper is to support a perspective renewed role of the white matter dissection for the acquisition of a three - dimensional orientation within the superficial white matter . A more systematic organization of the white matter terminations underneath the sulcal or gyral surface can be demonstrated with this technique as shown in figure 1 . While short intra - cortical u fibres interconnect territories that share the same sulcus, the adjacent gyri shares u fibres with a variable length and seem disposed in a constant disposition between the gyrus and the sulcus . In this organization, the gyral surface seems sub - served preferentially by a high number of long association fibres terminations, which present an oblique / vertical direction from the deep territories to the pial surface (figure 1a, b). As demonstrated by other techniques, a higher density of axonal terminations run underneath the gyral region, while the cortices that share the same sulcus present mostly very short u - fibres at least at the most superficial layer (nie et al ., 2012 high magnification views of the intra - cortical white matter terminations exposed during different stages of dissection and with different densities and numbers in different regions . (a) right hemisphere; the cortex of the superior parietal lobule (spl) has been split in order to expose the long and intermediate fibres running within the deep white matter layer at the level of posterior portion of corona radiata (cr) and ended perpendicular to the pial surface but underneath the gyral region and not at the sulcal level . (b) detail of deep white matter fibres and their distribution underneath the gyral surface (yellow dotted square). (c) right hemisphere, fibres of the superior longitudinal fasciculus (slf) complex connect the peri - sylvian regions . Some of them terminate at the level of the frontal operculum (fo) just above the insula (ins). The direction of the long associative fibres (slf) ended at the level of the gyral surface (black arrows), while the short u - fibres subserve close territories (red arrows). (d) right insular region with the same organization of gyral/ sulcal white matter architecture exposed . The black arrows indicate the intermediate fibres, which run along the extreme capsule, while the very short u - fibres are mainly disposed along the sulcal regions (red arrows). Pos: parieto - occipital sulcus; m1: primary motor cortex; sss: sagittal stratum of sachs . The full comprehension of this superficial white matter organization is important in order to minimize potential and avoidable damage to long or intermediate association fibre bundles during every step of a surgical approach . In this light, several studies report the impossibility to completely resect areas with lower potential of compensation by plasticity phenomena (ius et al ., 2011; sarubbo et al ., these areas include for instance the primary motor and somatosensory areas which are mainly uni - modal and probably organized serially, sub - served preferentially by projection fibre bundles (duffau, 2014b). The balance between extent of resection and functional outcome is still very crucial in this area . In a recent series 75% of patients operated with the use intra - operative monitoring because for pre / post central sulcus lesions had residual tumours (noell et al ., 2015). The rate of postoperative permanent deficits in rolandic area is reported between 4, 8 and 16% (keles et al ., 2004; carraba et al ., 2007; the absence of parallel alternative pathway may explain the impossibility to efficiently restore their function after damage . However, it is also well known that patients with wm lesion may improve, even if the recovery is rarely complete . Several mechanisms related to the integrity of the perilesional wm pathways (latent parallel networks, accessory pathways, additional relays, parallel long association pathways) have been hypothesized as supporting some level of functional compensation (duffau, 2009). Hence according to the new insights into the intracortical wm fibres, standard approaches to such eloquent areas may be carefully reanalysed starting from the first superficial layer in order to minimize avoidable damage to swm able to support a postoperative functional compensation . One of the limitations of the white matter dissection is that it is hard to merge the three - dimensional orientation acquired in the laboratory with the intraoperative experience because of the obvious differences between the prepared specimens and the living tissues . On the other hand vascular structures can be identified constantly during operations, guiding the strategy and the surgical approach to subcortical lesions . An advantage of our specimens is the use of the physiological intracranial vascular network for the fixation process, which preserves vascular landmarks at each stage of wm dissection . These fine and constant details allow correlation between vessels and white matter structures with the obvious advantage in three - dimensional orientation that can be employed during any standard neurosurgical procedure . Despite in clinical practice is the nature / location of the lesion that often dictates the surgical strategy; a transcortical approach is always the first step in many neurosurgical procedures . In these cases, the corticotomy is mostly based on the lesion's features, the vascular architecture of the region and the personal experience of the neurosurgeon . The possibility to observe with a different and more detailed anatomical view the wm architecture from the first cortical layer encouraged us to reconsider some of the standard trans - cortical approaches with the aim of minimizing the quantitative damage to the superficial white matter . Three examples of trans - cortical approaches in the post central gyrus are herein demonstrated, showing how the different layers of white matter fibres can be identified since the sub - pial surface is associated with almost constant vascular landmarks . In all the approaches a balance between the vascular damage and the quantitative damage to the white matter the classical subcortical vascular architecture described by yasargil (1987) was adopted as reference and confirmed during the dissection . A trans - gyral trans - cortical approach was the first to be tested (figure 3, yellow square, approach n1). The number of vascular structures encountered from the pial layer underneath the central area of the gyrus was abundant, with vessels located only approximately 2 mm apart . This organization forces the neurosurgeon to an inconvenient trajectory with a prolonged manipulation or retraction of long association fibres underneath the gyral grey / white matter junction . According to the data provided on the density of axonal terminations, even for small subcortical lesions this approach (according to figure 1) the junction between the long / intermediate (figure 1, black arrows) and short u - fibres (figure 1, red arrows). According to nie et al . (2012), a lesser density of white matter terminations was reported, which was confirmed by our dissection . Moreover, a single artery coming from the lateral wall of the sulcus is often encountered at a 90 angle from the sulcus leading to the deep wm underneath the gyral - sulcal transitional zone (figure 3 orange square, approach number 2). This trans - cortical artery often joins the deep arterial anastomosis at the level of the deep white matter and for this reason should be preserved (figure 2b, figure 3 orange square, approach number 2). Following this artery within the deep wm, the microsurgical dissection should be adapted to the longitudinal direction of the fibres of superior longitudinal fasciculus (slf) and arcuate fasciculus (af) (figure 2b). The minimal manipulation of vascular structures (basically only a single vessel) and the tangential trajectory in respect to the long and intermediate fibres may lead to a reduced damage to both vascular and wm structures in the grey / wm junction . (a) a cadaver specimens (right hemisphere) prepared with intracarotidal perfusion fixation process (latini et al ., 2015). The regions chosen on the post - central region for testing the trans - cortical approaches were labelled (1, 2 and 3). (b) advanced stage of dissection with the longitudinal fibres of the superior longitudinal fasciculus (slf) exposed together with deep arterial anastomosis (light blue patch). The post - central gyrus has been removed in order to study the trajectory of the underlying fibres and vessels . Cs: central sulcus; ins: insula; m1: primary motor cortex; ofc: orbito - frontal cortex; stg: superior temporal gyrus . Yellow square: the trans - gyral approach (number 1) is then exposed in three steps with the associated vascular anatomical structures encountered (see text for more details). Orange square: the para - sulcal approach (number 2) is shown in several steps with the associated vascular anatomical structures encountered . Red square: the trans - sulcal approach (number 3) has been performed with the reference for associated vascular anatomical structures encountered . (see text for more details). The third approach (trans - sulcal) (figure 3, red square, approach number 3) has already demonstrated a potential to lead to a safe surgery for the limited amount of vascular structures encountered (yasargil, 1987; mikuni et al ., 2006). Once the sulcal artery has been dissected in the subarachnoid space no other visible crucial vascular structures are present until the deep arterial and venous anastomosis (figure 2b) (latini et al ., 2015). According to previous data (nie et al ., 2012) and confirmed by our dissections, the sulcal areas present a lower density of associative fibres . However the commonly accepted cortical incision during trans - sulcal approach is generally in accordance with the sulcal direction . The dissection of the superficial white matter showed on the other hand that the direction of the u - fibres underneath the sulcal area is actually perpendicular to the sulcal direction . In case of eloquent areas like the somatosensory cortices, a more extended disconnection of the closer intra - cortical territories can lead to significant post - operative deficits . The incision of the sulcal cortex should rather follow the direction of the fibres as showed in figure 3 (red square, approach 3, red arrow). The fibres of superior longitudinal fasciculus (slf) and the horizontal segment of the af run in a longitudinal direction (figure 2b) and so even for a limited trans - sulcal approach this modified subcortical dissection may be suggested in order to minimize the wm damage and the disconnection between functional epicentres . For instance the vascular manipulation of the parasulcal (approach n2) or the sulcal artery (approach n3) as well as the related arterioles can lead to vascular / wm damage . Thus we cannot claim that one approach is safer than the other based on these anatomical results . Moreover the microvasculature of the subcortical grey and grey - white interface with short- to intermediate - length arterioles and arteries (that form a dual source of blood supply) is much more complex than the one visible during white matter dissection (moody et al ., many factors should be taken into consideration in order to possibly predict the functional outcome after standard neurosurgical procedures . Therefore, the main aim of this study is not to suggest the best transcortical approach for a certain region but to support the role of a more detailed wm comprehension during the neurosurgical training and presurgical planning . We believe that besides the superficial vascular architecture and the widely described major associative wm bundles, a more comprehensive knowledge of the superficial white matter should be mandatory nowadays in order to tailor cortical and subcortical resection with the least invasive possible surgical trajectories even at the most superficial layers . Our dissection technique showed how the main vascular landmarks could be constantly associated with each respective layer of white matter structures . A more widely use of this presurgical training can give to every neurosurgeon a more comprehensive orientation of the peripheral and more superficially located white matter (swm), which fills the space between the deep white matter and the cortex . These important insights into the possible surgical trajectories and subsequent quantitative damages of both vessels and white matter fibres (associative, projection, intermediate or short fibres) can help readapt even the most standard and widely accepted approach trough the brain cortex . A more detailed study of these fine anatomical details (with also new dti techniques, study of the microvascular architecture, study of micronetworks etc .) May become in the near future a fundamental part the neurosurgical training and the preoperative planning . The ideal goal would be to direct new attention to the architecture of this poorly understood region, minimizing avoidable damage to swm and improving the functional outcome in the daily neurosurgical practice.
Imaging evaluation of patients with cancer makes up a substantial portion of the workload in radiology departments . Discordant interpretations of computed tomography (ct) scans are common and have been reported in 3137% of patients, while multicenter trials have reported changes in patient management due to discordant readings in up to 23% of patients . In addition, since medical malpractice lawsuits filed against imaging specialists are related to missed diagnoses in 70% of cases, with cancer - related claims being the most frequent, improved accuracy in the interpretation of oncological ct studies could reduce legal challenges in radiology departments . In a study of the variables influencing the accuracy of interpretation of abdominal ct studies, loughrey et al . Found that the only contributing factor to reach statistical significance was the skill of the individual radiologist . Simultaneous supervision of interventional procedures, level of training of the assigned resident physician, and tumor type did not influence the error rate . Accuracy of interpretation of imaging studies in general is also improved when the interpreting physician has access to appropriate clinical findings, and when current studies are compared with previous examinations . Standardization of imaging protocols that use intravenous and oral contrast material may thus also improve diagnostic accuracy . Particular attention should be paid to known problem areas and pitfalls specific to the underlying disease . It is also important to carefully assess blind spots in patients with primary tumors and metastatic disease . Over the past 15 years, visualization of metabolic activity using the glucose analogue [f]fluorodeoxyglucose (fdg) in combination with the high anatomic resolution of ct in a single integrated positron emission tomography (pet)/ct examination has shown increasing importance in the diagnosis, staging and follow - up of a wide range of malignant diseases . However, the cost of pet / ct remains high and patient access is limited . Radiologists who are familiar with the appearance of lesions on pet / ct may come to better appreciate how these subtle lesions appear on ct, as many of the areas that are easily missed are fdg avid . If these ct scans were being read without the added experience from pet / ct, these findings would probably not have been picked up on ct . Familiarity with pet / ct may thus improve detection rates and avoid errors . In this article, we present regions of primary and metastatic disease that can be missed on multi - detector computed tomography (mdct). In our experience, these blind spots and overlooked regions are more easily detected on ct by a reader who is experienced in reading pet / ct . Breast cancer is the most common form of cancer and the second most common cause of cancer - related deaths in women . In 2009, approximately 190,000 new cases of breast cancer were diagnosed in the united states and over 40,000 women died of the disease; the incidence in europe is 94.3 per 100,000 women . While mammography is the most efficient way to screen for primary breast cancers, the ability to spot primary breast lesions and metastases on routine ct scans is important, as this may lead to a diagnosis before the patient would have otherwise come to medical attention, and improve the accuracy of staging for patients with a breast cancer diagnosis . Ct scans may demonstrate incidental findings that must be identified as either pathologic or benign . Reported a 30% rate of malignancy in patients referred to a breast clinic for assessment of incidental breast lesions detected on ct . Lesion spiculation and irregularity were strongly suggestive of malignancy, while calcification patterns did not aid in the diagnosis . Malignant lesions were significantly larger than benign lesions . Detecting breast malignancies on ct and differentiating them from normal breast parenchyma is challenging, and breast lesions can be missed . A side - by - side comparison of breast lesions on ct and fused pet / ct is presented in fig . 1 . (a) ct scan demonstrates a rounded soft tissue area seen in the lateral part of the right breast . (b) pet / ct demonstrates increased fdg uptake, compatible with breast cancer . A 55-year - old woman with breast cancer . (a) ct scan demonstrates a rounded soft tissue area seen in the lateral part of the right breast . (b) pet / ct demonstrates increased fdg uptake, compatible with breast cancer . The presence of distant metastasis in patients with breast cancer is a key prognostic factor . Ct is a useful modality for detecting metastases, with a sensitivity of 83% and a specificity of 85%, while pet has sensitivity and specificity of 87% and 83% respectively . Blind spots that are most often overlooked on ct are metastases to the bone and lymph nodes, especially internal mammary, retropectoral, mediastinal, and axillary lymph nodes . Thus, special attention should be paid to these areas when reviewing ct scans of a breast cancer patient . Pet / ct effectively demonstrates metastases in these regions (figs . 2 and 3). (a) ct reveals an enlarged rounded lymph node secondary to breast cancer spread between the pectoralis muscles . (b) the lymph node is visible as an area of increased fdg uptake on pet / ct . (a) ct reveals a subtle bone metastasis causing disruption of the posterior border of the l5 body . (b) the lesion is illustrated clearly on pet / ct due to its increased fdg uptake . A 53-year - old woman with breast cancer . (a) ct reveals an enlarged rounded lymph node secondary to breast cancer spread between the pectoralis muscles . (b) the lymph node is visible as an area of increased fdg uptake on pet / ct . (a) ct reveals a subtle bone metastasis causing disruption of the posterior border of the l5 body . (b) the lesion is illustrated clearly on pet / ct due to its increased fdg uptake . In europe and the united states, lung cancer is the second most frequently diagnosed primary malignancy and the leading cause of cancer - related mortality for both men and women . The incidence in europe in 2006 was 75.3 and 18.3 per 100,000 in men and women, respectively . In detecting primary lung cancer lesions on ct, white et al . Studied the characteristics of 15 primary lung cancers that were overlooked in 14 patients . Li et al . Reviewed the characteristics of 32 lung cancers missed on low - dose helical ct screening . The missed tumors were found in all lobes of the lungs, often in the central region, with some lesions overlapping with hilar structures . In many patients, there was underlying lung disease, and other more obvious lesions were identified while the lung cancer was missed . Small cell lung cancer is assumed to have metastasized at presentation, thus detection of metastases is less relevant to surgical decision making . However, determining whether non - small cell lung cancer has metastasized is central for treatment decisions and prognosis . In comparison with pet and ct individually, pet / ct was shown to improve the diagnostic accuracy of tumor, node, and distant metastasis (tnm) staging for non - small cell lung cancer . Reported overall tnm staging accuracy in 96% of patients with pet / ct, 70% with ct alone, and 74% with pet alone . Assessment of mediastinal lymph node involvement had an accuracy of 93%, 63%, and 89%, respectively, for pet / ct, ct alone, and pet alone . Lardinois et al . Found correct and unequivocal classification of tumor extent in 88% with pet / ct versus 58% with ct and 40% with pet; lymph nodes were accurately staged in 81% with pet / ct versus 59% and 49% with ct and pet, respectively . The heart or pericardium was involved in 1731% of lung cancer patients in autopsy studies . Tumor extension to the heart, pericardium, or great vessels may affect tnm classification and decisions on patient management . The differential diagnosis includes malignant pericardial effusion, inflammation, fibrosis due to radiation therapy, drug - induced pericarditis, infection, and other causes . A comparison of classic presentation of pericardial thickening associated with tumor extension on ct and pet / ct is found in fig . (b) pet / ct reveals increased fdg uptake, indicating that metastatic cancer is the cause of this thickening . A 59-year - old man with lung cancer . (b) pet / ct reveals increased fdg uptake, indicating that metastatic cancer is the cause of this thickening . Metastatic adenocarcinoma is the most common malignancy in the pleura, while mesothelioma is the more frequently diagnosed primary malignancy . Ct can be used for the identification of these pleural masses, but radiological findings can be subtle . There are several criteria that help differentiate between benign and malignant pleural disease on ct . These include circumferential or nodular pleural thickening, thickening greater than 1 mm, and mediastinal pleural involvement (fig . 5). (b, d) note increased uptake indicating malignancy on the pet / ct views . A 47-year - old man with lung cancer . (b, d) note increased uptake indicating malignancy on the pet / ct views . Once integrated pet / ct becomes more widely available, it may become the optimal tool for non - invasive staging of non - small cell lung cancer . However, ct currently plays a significant role in staging and restaging, since it is more accessible and less expensive than pet and pet / ct . As such, an appreciation for lesions that are the incidence of colorectal cancer in europe is 55.4 per 100,000 in men, 34.6 per 100,000 in women . In the united states, colorectal cancer is the third most common cancer in both men and women, with approximately 147,000 new cases diagnosed and 50,000 deaths in 2009 . With the exception of virtual colonoscopy, pet, ct, and pet / ct have limited roles in the primary diagnosis of colon cancer; however they have important roles in staging, assessing response to treatment, and detecting recurrence . In a series of 38 patients, kantorova et al . Reported ct and pet sensitivity for the detection of primary colorectal tumors at 49% and 95%, respectively, of lymph node involvement as 0% and 29%, and sensitivity for liver metastases as 67% and 78% . Ct sensitivity in the detection of liver metastases was 38% and specificity was 97%, versus sensitivity and specificity of 88% and 100% for pet . Selzner et al . Compared findings for contrast - enhanced ct and pet / ct in 76 patients with metastatic colorectal cancer . They reported 53% and 93% sensitivity of ct and pet / ct, respectively, for detection of local recurrence at the primary colorectal site . Ct and pet / ct had comparable sensitivity for detection of intrahepatic masses; however pet / ct was more successful in detecting intrahepatic recurrences following hepatectomy . Extrahepatic metastases in colorectal cancer patients were detected with 64% sensitivity by contract ct versus 89% sensitivity with pet / ct . Picking up local invasion (fig . 6a, b) as well as subtle metastases of colonic adenocarcinoma to lymph nodes, peritoneum, liver, lungs, skeleton (fig . 6c, d) and soft tissue (figs . 7, 8) is important, as their detection may upstage a diagnosis and alter patient management . Metastases to the skeletal muscle characteristically present as rim - enhancing lesions with central hypoattenuation . Pet / ct has good sensitivity for the detection of soft tissue metastases, which may have prognostic implications and provide easily accessible biopsy sites . (a, b) ct shows invasion of adjacent structures, which is depicted easily on pet / ct . (c, d) ct reveals a subtle skeletal mass with increased fdg uptake on pet / ct . (a, b) ct shows invasion of adjacent structures, which is depicted easily on pet / ct . (c, d) ct reveals a subtle skeletal mass with increased fdg uptake on pet / ct . Discovery of this metastasis led to restaging of the patient . A 45-year - old man with cancer of the colon . (b) the lesion is more clearly demarcated on pet / ct . A 53-year - old man with colon cancer . About 21,500 women in the united states are diagnosed with ovarian cancer each year, with about 14,600 annual deaths from the disease . Most women are diagnosed at a late stage, causing the overall 5-year survival rate to be only 55% . European incidence and mortality in 2008 were reported as 13.5 per 100,000 and 7.6 per 100,000, respectively . Detection of metastases is important for prognosis assessment and treatment planning, as well as for patient follow - up, since 50% of ovarian cancer patients will present with recurrence after first line surgery and chemotherapy . Sebastian et al . Found consistently higher sensitivity and accuracy of pet / ct compared with ct for detection of metastases from ovarian tumors in the body, chest, and abdomen . The most common route of ovarian metastasis is to the peritoneum, which leads to peritoneal carcinomatosis . Detection of carcinomatosis is important, as this may influence a decision to forego or delay surgery in favor of immediate systemic therapy in some patients . Coakley et al . Reported sensitivity for the detection of peritoneal implants on spiral ct as 8593% overall, although sensitivity remained low at 2550% for metastases 1 cm and smaller . Interobserver variability in detecting the smaller lesions was significant, showing the challenges inherent in accurate assessment of these patients with ct . The best ct criteria for identification of peritoneal metastasis is the presence of a nodular, plaque - like, or infiltrative soft tissue lesion in the peritoneal fat or on the peritoneal surface . The presence of ascites, parietal peritoneal thickening, or small bowel thickening may also indicate peritoneal metastases . Fig . 9 illustrates the presentation of a subtle peritoneal metastasis on ct and pet / ct . (b) the lesion is well illustrated on pet / ct . A 39-year - old woman with ovarian cancer . The incidence in the united states is rising, with 69,000 new cases and 9000 deaths in 2009 . Pet, pet / ct and ct imaging are not used for initial diagnosis, which is usually made clinically . Sentinel node biopsy remains the gold standard for assessment of regional spread in patients with stage i or ii disease . Patients with stage iii or iv melanoma are at high risk for metastasis, which may be detected with ct, pet, or pet / ct . In earlier studies, ct sensitivity and specificity was reported as 55% and 83%, respectively, compared with 94% and 83% for pet . In 2007, mottaghy et al . Found pet alone to be more accurate than ct in detecting skin lesions, malignant lymph nodes, and metastases to the abdomen, liver, and bone . However, as ct is more readily available and less expensive than pet and combined pet / ct, the ability to detect melanoma metastasis using this modality remains important . The liver, subcutaneous tissue, lymph nodes, and bone are regions where lesions can be especially subtle on ct scans . Lesions in these regions are illustrated clearly on pet / ct (figs . 10 and 11). (b, d) increased fdg uptake on pet directs the reader to these blind spots . (a) a tiny lytic lesion to the rib cage, consistent with melanoma metastasis, is seen on ct . (b, d) increased fdg uptake on pet directs the reader to these blind spots . (a) a tiny lytic lesion to the rib cage, consistent with melanoma metastasis, is seen on ct . Thrombosis is a significant cause of morbidity and mortality among oncology patients, especially patients with pancreatic and hepatocellular carcinoma . In one report, thrombosis was present in 6.8% of oncologic staging ct scans, and another study found a 9% prevalence of pulmonary embolism among inpatients with malignant disease . There is significantly higher fdg uptake in vessels containing thrombi, however care is required to distinguish between thrombotic and tumor emboli, and to rule out other potential causes of increased metabolic activity . A comparison of the ct and pet / ct appearance of deep vein thrombosis is shown in fig . (a) ct demonstrates a deep vein thrombosis in the right common femoral vein . (b) on pet / ct this lesion is seen as an area of increased fdg uptake, indicating its malignant nature . A 32-year - old woman with advanced - stage melanoma . (a) ct demonstrates a deep vein thrombosis in the right common femoral vein . (b) on pet / ct this lesion is seen as an area of increased fdg uptake, indicating its malignant nature . Experience in pet / ct reading may highlight blind spots when reading oncology ct . It is not our intent to underestimate the routine use of pet / ct in oncology patients, but rather to bring illustrative cases in which familiarity with the presentation of malignant lesions on pet / ct may improve the ability to detect subtle changes on mdct when it is used as the sole imaging tool for staging or follow - up of oncology patients . Based on experience with pet / ct, radiologists can be better prepared to identify subtle and readily overlooked lesions when only ct is available, thus avoiding errors and helping to improve patient management.
The utilization of radial (ra) over femoral arterial access (fa) for acute coronary intervention in stelevation myocardial infarction (stemi) is supported by a reduction in mortality, ischemic, and bleeding endpoints.1, 2, 3, 4, 5, 6, 7, 8 this has led various interventional societies to encourage its utilization over femoral access.9, 10, 11 although the basis of this evidence includes patients undergoing both primary and rescue percutaneous coronary intervention (pci), the majority of randomized data are confined to patients treated with primary pci (ppci).2, 7 ra in patients treated with a fibrinolytic pharmacoinvasive strategy (pi) has had limited exploration,12, 13, 14 as has any direct comparison with patients treated with ppci . Moreover, the application of a pi strategy creates at least 2 distinct patient subsets at the time of angiography, each with a different risk profile with respect to ischemic outcomes and major bleeding / accesssite complications.15 the first is the nonreperfused patient requiring urgent rescue pci who has received recent fibrinolysis with adjunctive antithrombotic and antiplatelet therapy sometimes associated with hemodynamic or electrical instability . The second is the stable reperfused stemi undergoing scheduled pci 6 to 24 hours later . Intuitively, a comparable efficacy and safety advantage as observed in ppci in favor of ra is expected in patients treated with a pi strategy, particularly in the subset undergoing rescue pci . The strategic reperfusion early after myocardial infarction (stream) trial,16 which randomized patients to a fibrinolytic pi (rescue and scheduled pci) versus ppci treatment strategy, provided a unique opportunity to address this issue in an early presenting, rapidly treated contemporary stemi cohort . Accordingly, within stream, we evaluated the relationship between arterial access site and the 30day primary composite (all cause death, shock, congestive heart failure [chf], and reinfarction) as well as major bleeding events in stemi according to the treatment strategy received . The stream trial protocol and primary results have been published previously.16, 17 the study protocol was approved by national regulatory authorities and by the local ethics committee at each study center . Acute stemi patients presenting within 3 hours of symptom onset and unable to undergo ppci within 1 hour of first medical contact were randomized to either fibrinolysis with a protocoldefined pi strategy or ppci . In the pi strategy, bolus weightbased tenecteplase (tnk), aspirin, clopidogrel, and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography (within 624 hours). After 21% of the ultimate population had been enrolled, the executive committee amended the protocol on august 24, 2009, to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage (ich) in that age group . The need for rescue pci was determined by site investigators according to <50% st segment resolution in the electrocardiogram (ecg) lead with the maximal stelevation 90 minutes after tnk bolus, hemodynamic instability, or refractory ventricular arrhythmias as mandated by the study protocol . Primary pci was conducted after expeditious transfer from the point of randomization, and early initiation of aspirin, clopidogrel, and antithrombotic therapy, including discretionary glycoprotein iib / iiia (gp 2b/3a) antagonists based on best standard practice . Ecgs were performed at baseline, 90 minutes posttnk, and 30 minutes postcatheterization in the pharmacoinvasive arm (including postpci in those undergoing pci) and baseline and 30 minutes postcatheterization or postpci (if performed) in the primary pci arm . Interpretation was performed at the canadian vigour center ecg core laboratory (edmonton, alberta, canada), and, for this study, the prespecified ecg metrics included: worst lead stelevation resolution; worst lead stelevation resolution 50%; and worst lead residual stelevation . Both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access, either ra or fa based upon local practice (table s1). Angiographic assessment detailing coronary anatomy, need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction (timi) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . The results of the current report are based on the perprotocol analysis according to the access site utilized to complete the angiographic procedure . The primary outcome of this study was a 30day composite of death, cardiogenic shock, chf, or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days, nonintracranial major bleeding or ich, stroke, and 1year allcause mortality . Detailed definitions for both the components of the primary composite and bleeding endpoints have been published previously.17 briefly, a stroke review independent panel adjudicated all strokes centrally . The global use of strategies to open occluded arteries (gusto) definition of bleeding20 was implemented in this trial . Major bleeding was classified as: (1) intracranial or (2) nonintracranial severe bleed (bleed that leads to hemodynamic compromise requiring intervention [blood or fluid replacement, inotropic support, ventricular assist device, or surgical repair] or lifethreatening or fatal bleed) or moderate bleed (bleeding requiring blood transfusion, but that does not lead to hemodynamic compromise requiring intervention). All bleeds, excluding intracranial bleeds, were investigator reported . Related major bleeding was defined as major bleeding that occurred less than 48 hours postpci . Categorical variables are summarized as percentages and as median (25th, 75th percentiles) for continuous variables . Baseline characteristics and concomitant treatment are reported according to access site (radial vs femoral) and study treatment received . Differences between groups were tested with the chisquare test (or fisher's exact test when count was <5 for at least 1 cell) for categorical variables and wilcoxon ranksum test for continuous variables, respectively . The association between access site and primary clinical outcome (composite of death, cardiogenic shock, chf, or reinfarction) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized, a propensity score for access site was created using a multivariable logistic regression model . Variables considered in the model were based on literature review, expert opinion, and univariate tests with p<0.10 . The covariates in the final model were forced in; that is, no conventional statistical selection techniques (eg, stepwise, forward, or backward) were used . Variables included in the final propensity score model were age, sex, weight, history of hypertension, history of diabetes mellitus, heart rate, systolic blood pressure, killip class, inferior myocardial infarction (mi), sum st elevation at baseline, q waves at baseline, time from symptom onset to randomization, and country of enrollment (table s2). The interaction between access site and study treatment (ie, pi vs ppci) on the 30day primary clinical composite outcome was also examined . A further prespecified similar subgroup analysis of access site within the pi (rescue vs scheduled) strategy was performed . To test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes, the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . Statistical analyses were performed using sas software (version 9.4; sas institute inc ., cary, nc). The stream trial protocol and primary results have been published previously.16, 17 the study protocol was approved by national regulatory authorities and by the local ethics committee at each study center . Acute stemi patients presenting within 3 hours of symptom onset and unable to undergo ppci within 1 hour of first medical contact were randomized to either fibrinolysis with a protocoldefined pi strategy or ppci . In the pi strategy, bolus weightbased tenecteplase (tnk), aspirin, clopidogrel, and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography (within 624 hours). After 21% of the ultimate population had been enrolled, the executive committee amended the protocol on august 24, 2009, to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage (ich) in that age group . The need for rescue pci was determined by site investigators according to <50% st segment resolution in the electrocardiogram (ecg) lead with the maximal stelevation 90 minutes after tnk bolus, hemodynamic instability, or refractory ventricular arrhythmias as mandated by the study protocol . Primary pci was conducted after expeditious transfer from the point of randomization, and early initiation of aspirin, clopidogrel, and antithrombotic therapy, including discretionary glycoprotein iib / iiia (gp 2b/3a) antagonists based on best standard practice . Ecgs were performed at baseline, 90 minutes posttnk, and 30 minutes postcatheterization in the pharmacoinvasive arm (including postpci in those undergoing pci) and baseline and 30 minutes postcatheterization or postpci (if performed) in the primary pci arm . Interpretation was performed at the canadian vigour center ecg core laboratory (edmonton, alberta, canada), and, for this study, the prespecified ecg metrics included: worst lead stelevation resolution; worst lead stelevation resolution 50%; and worst lead residual stelevation . Both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access, either ra or fa based upon local practice (table s1). Angiographic assessment detailing coronary anatomy, need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction (timi) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . The results of the current report are based on the perprotocol analysis according to the access site utilized to complete the angiographic procedure . The primary outcome of this study was a 30day composite of death, cardiogenic shock, chf, or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days, nonintracranial major bleeding or ich, stroke, and 1year allcause mortality . Detailed definitions for both the components of the primary composite and bleeding endpoints have been published previously.17 briefly, a stroke review independent panel adjudicated all strokes centrally . The global use of strategies to open occluded arteries (gusto) definition of bleeding20 was implemented in this trial . Major bleeding was classified as: (1) intracranial or (2) nonintracranial severe bleed (bleed that leads to hemodynamic compromise requiring intervention [blood or fluid replacement, inotropic support, ventricular assist device, or surgical repair] or lifethreatening or fatal bleed) or moderate bleed (bleeding requiring blood transfusion, but that does not lead to hemodynamic compromise requiring intervention). All bleeds, excluding intracranial bleeds, were investigator reported . Related major bleeding was defined as major bleeding that occurred less than 48 hours postpci . Categorical variables are summarized as percentages and as median (25th, 75th percentiles) for continuous variables . Baseline characteristics and concomitant treatment are reported according to access site (radial vs femoral) and study treatment received . Differences between groups were tested with the chisquare test (or fisher's exact test when count was <5 for at least 1 cell) for categorical variables and wilcoxon ranksum test for continuous variables, respectively . The association between access site and primary clinical outcome (composite of death, cardiogenic shock, chf, or reinfarction) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized, a propensity score for access site was created using a multivariable logistic regression model . Variables considered in the model were based on literature review, expert opinion, and univariate tests with p<0.10 . The covariates in the final model were forced in; that is, no conventional statistical selection techniques (eg, stepwise, forward, or backward) were used . Variables included in the final propensity score model were age, sex, weight, history of hypertension, history of diabetes mellitus, heart rate, systolic blood pressure, killip class, inferior myocardial infarction (mi), sum st elevation at baseline, q waves at baseline, time from symptom onset to randomization, and country of enrollment (table s2). The interaction between access site and study treatment (ie, pi vs ppci) on the 30day primary clinical composite outcome was also examined . A further prespecified similar subgroup analysis of access site within the pi (rescue vs scheduled) strategy was performed . To test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes, the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . Statistical analyses were performed using sas software (version 9.4; sas institute inc ., cary, nc). Figure 1 illustrates the 2 treatment group cohorts from the 1820 perprotocol treated patients enrolled in stream categorized by access site . As evident, there was comparable utilization of either access site within each treatment strategy (fa: pi 53.4% [n=478] and ppci 57.6% [n=533]). In addition, within the pi strategy, both access sites were comparably distributed in the rescue (fa: 52.8%; n=200) and scheduled pci (fa: 53.9%; n=278) subgroups . Fa indicates femoral; pi, pharmacoinvasive; ppci, primary percutaneous coronary intervention; ra, radial . Overall, stemi patients treated by fa were younger, had more past hypertension, lower systolic bp at presentation, lower timi risk score, and more stelevation on the baseline ecg compared to the ra group (table 1). Selected baseline patient characteristics according to access site and study treatment continuous variables presented as median (25th75th percentiles). Fa indicates femoral; mi, myocardial infarction; ppci, primary percutaneous coronary intervention; ra, radial; ste, stsegment elevation; timi, thrombolysis in myocardial infarction . Evaluated by ecg core laboratory at the canadian vigour centre . As described in table 2, patients treated by fa had shorter time from symptom onset to randomization in both treatment strategies . In addition, shorter times from symptom onset to femoral, compared to radial sheath, insertion were observed in the ppci, but not in the pi, strategy . Ischemic times, medications, angiographic findings, and posttreatment ecg according to access site and study treatment continuous variables presented as median (25th75th percentiles). Ecg indicates electrocardiogram; fa, femoral; gp, glycoprotein; iabp, intraaortic balloon pump; ppci, primary percutaneous coronary intervention; ra, radial; timi, thrombolysis in myocardial infarction; tnk, tenecteplase . Evaluated by ecg core laboratory at the canadian vigour center . A significantly higher utilization of gp2b/3a inhibitor use was noted in the ra, compared to fa, group (40.2% vs 24.1%; p<0.001), particularly in those undergoing ppci . Those patients treated with ppci strategy and ra had higher rates of postpci timi3 flow grade than fa patients . Evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts, as evidenced by higher rates of worst lead residual st elevation <1 mm and lesser rates in patients with 2 mm residual st elevation . Irrespective of treatment strategy, the unadjusted primary composite of 30day death, shock, chf, or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group (table 3). After adjustment, the benefit favoring ra persisted (adjusted or, 0.59; 95% ci, 0.440.78; p<0.001), as seen in figure 2 . Efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure; ppci, primary percutaneous coronary intervention . Fa indicates femoral; pi, pharmacoinvasive; ppci, primary percutaneous coronary intervention; ra, radial . Analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci (adjusted or, 0.63; 95% ci, 0.430.92) and pi cohorts (adjusted or, 0.57; 95% ci, 0.370.86; p [interaction]=0.730; figure 2). Within the pi group, a trend for ra advantage was evident in the highrisk rescue pci (13.4% vs 26.3%; adjusted or, 0.65; 95% ci, 0.391.07) subgroup with no significant difference in patients undergoing scheduled pci (5.5% vs 5.4%; adjusted or, 0.55; 95% ci, 0.241.26). However, no interaction was evident between rescue pci or scheduled pci as it relates to the advantage of ra after adjustment (p [interaction]=0.739). The observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome (ra: gp 2b/3a use vs not, 9.3% versus 8.7%; and fa: 20.2% vs 14.3%; p [interaction]=0.988); neither did the implementation of the amendment (halfdose lytic in patients 75 years) of the stream trial protocol (ra and fa: pre and postamendment, respectively, 9.0% vs 8.9% and 18.4% vs 15.2%; p [interaction]=0.920). The increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding (ra vs fa: adjusted hazard ratio, 0.56; 95% ci, 0.281.12; p [interaction]=0.087). Radial access was associated with an observed reduction in 30day mortality (2.4% vs 4.7%; p=0.009), cardiogenic shock (2.9% vs 6.4%; p=0.001), and heart failure (5.0% vs 8.4%; p=0.005; table 3). No significant differences in ischemic stroke or ich were noted in either vascular access site across the 2 treatment groups . At 1 year, no difference in allcause mortality was noted in either accesssite category across both study treatment groups . Overall, a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group (5.2% vs 6.0%; p=0.489; table 3). This was also evident within the 2 treatment strategies (pi and ppci: ra vs fa, 5.5% vs 7.8% [p=0.179] and 4.9% vs 4.3% [p=0.698], respectively); however, within the pi strategy, a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue, compared to the scheduled, subgroup was observed (rescue and scheduled: ra vs fa, 6.1% vs 11.6% [p=0.064] and 5.1% vs 5.1% [p=0.996]). Both major accesssite (ra vs fa, 2.8% vs 4.1%; p=0.163) and nonaccesssite (ra vs fa, 2.3% vs 1.9%; p=0.487) bleeding were similarly distributed in the overall study population . Major vascular access complication (pseudoaneurysm or arteriovenous fistula development) occurred in 0% in the ra and 1.4% in the fa group . Figure 1 illustrates the 2 treatment group cohorts from the 1820 perprotocol treated patients enrolled in stream categorized by access site . As evident, there was comparable utilization of either access site within each treatment strategy (fa: pi 53.4% [n=478] and ppci 57.6% [n=533]). In addition, within the pi strategy, both access sites were comparably distributed in the rescue (fa: 52.8%; n=200) and scheduled pci (fa: 53.9%; n=278) subgroups . Fa indicates femoral; pi, pharmacoinvasive; ppci, primary percutaneous coronary intervention; ra, radial . Overall, stemi patients treated by fa were younger, had more past hypertension, lower systolic bp at presentation, lower timi risk score, and more stelevation on the baseline ecg compared to the ra group (table 1). Selected baseline patient characteristics according to access site and study treatment continuous variables presented as median (25th75th percentiles). Fa indicates femoral; mi, myocardial infarction; ppci, primary percutaneous coronary intervention; ra, radial; ste, stsegment elevation; timi, thrombolysis in myocardial infarction . Evaluated by ecg core laboratory at the canadian vigour centre . As described in table 2, patients treated by fa had shorter time from symptom onset to randomization in both treatment strategies . In addition, shorter times from symptom onset to femoral, compared to radial sheath, insertion were observed in the ppci, but not in the pi, strategy . Ischemic times, medications, angiographic findings, and posttreatment ecg according to access site and study treatment continuous variables presented as median (25th75th percentiles). Ecg indicates electrocardiogram; fa, femoral; gp, glycoprotein; iabp, intraaortic balloon pump; ppci, primary percutaneous coronary intervention; ra, radial; timi, thrombolysis in myocardial infarction; tnk, tenecteplase . Evaluated by ecg core laboratory at the canadian vigour center . A significantly higher utilization of gp2b/3a inhibitor use was noted in the ra, compared to fa, group (40.2% vs 24.1%; p<0.001), particularly in those undergoing ppci . Those patients treated with ppci strategy and ra had higher rates of postpci timi3 flow grade than fa patients . Evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts, as evidenced by higher rates of worst lead residual st elevation <1 mm and lesser rates in patients with 2 mm residual st elevation . Irrespective of treatment strategy, the unadjusted primary composite of 30day death, shock, chf, or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group (table 3). After adjustment, the benefit favoring ra persisted (adjusted or, 0.59; 95% ci, 0.440.78; p<0.001), as seen in figure 2 . Efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure; ppci, primary percutaneous coronary intervention . Fa indicates femoral; pi, pharmacoinvasive; ppci, primary percutaneous coronary intervention; ra, radial . Analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci (adjusted or, 0.63; 95% ci, 0.430.92) and pi cohorts (adjusted or, 0.57; 95% ci, 0.370.86; p [interaction]=0.730; figure 2). Within the pi group, a trend for ra advantage was evident in the highrisk rescue pci (13.4% vs 26.3%; adjusted or, 0.65; 95% ci, 0.391.07) subgroup with no significant difference in patients undergoing scheduled pci (5.5% vs 5.4%; adjusted or, 0.55; 95% ci, 0.241.26). However, no interaction was evident between rescue pci or scheduled pci as it relates to the advantage of ra after adjustment (p [interaction]=0.739). The observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome (ra: gp 2b/3a use vs not, 9.3% versus 8.7%; and fa: 20.2% vs 14.3%; p [interaction]=0.988); neither did the implementation of the amendment (halfdose lytic in patients 75 years) of the stream trial protocol (ra and fa: pre and postamendment, respectively, 9.0% vs 8.9% and 18.4% vs 15.2%; p [interaction]=0.920). The increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding (ra vs fa: adjusted hazard ratio, 0.56; 95% ci, 0.281.12; p [interaction]=0.087). Radial access was associated with an observed reduction in 30day mortality (2.4% vs 4.7%; p=0.009), cardiogenic shock (2.9% vs 6.4%; p=0.001), and heart failure (5.0% vs 8.4%; p=0.005; table 3). No significant differences in ischemic stroke or ich were noted in either vascular access site across the 2 treatment groups . At 1 year, no difference in allcause mortality was noted in either accesssite category across both study treatment groups . Overall, a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group (5.2% vs 6.0%; p=0.489; table 3). This was also evident within the 2 treatment strategies (pi and ppci: ra vs fa, 5.5% vs 7.8% [p=0.179] and 4.9% vs 4.3% [p=0.698], respectively); however, within the pi strategy, a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue, compared to the scheduled, subgroup was observed (rescue and scheduled: ra vs fa, 6.1% vs 11.6% [p=0.064] and 5.1% vs 5.1% [p=0.996]). Both major accesssite (ra vs fa, 2.8% vs 4.1%; p=0.163) and nonaccesssite (ra vs fa, 2.3% vs 1.9%; p=0.487) bleeding were similarly distributed in the overall study population . Major vascular access complication (pseudoaneurysm or arteriovenous fistula development) occurred in 0% in the ra and 1.4% in the fa group . The results of this study indicate that in early presenting stemi (<3 hours from symptom onset), utilization of ra over fa is associated with a significant reduction in the composite of major adverse cardiovascular events regardless of the application of a fibrinolytic pi or ppci strategy . In addition, within the pi strategy, the prognostic advantage of ra applies particularly to the higher risk rescue pci cohort in whom a doubling of adverse outcomes appeared evident within the femoral access subgroup . Within the stream study at baseline, patients treated by fa were more hypotensive and had greater st elevation at presentation, reflective of adverse outcomes . Given this clinical profile, it is reasonable to suppose that this may have influenced the choice of fa and subsequently be reflected in worse outcomes . However, even after adjustment, the ra 30day composite clinical outcomes and mortality advantage persisted . The stream study enrolled patients presenting within 3 hours of symptom onset and thus represents a very early presenting stemi population as compared to other trials that randomized access site in stemi patients (1224 hours).2, 3, 7 hence, this study evaluated a distinct stemi cohort undergoing early cardiac catheterizations (except for scheduled pci cohort) in the presence of potent antiantithrombotic and fibrinolytic agents . It would therefore seem that lower major bleeding would be associated with the observed significant reduction in the primary composite and 30day mortality favoring ra across both treatment strategies . Although there was nominally less nonintracranial major accesssite and nonaccesssite bleeding in those pharmacoinvasivetreated patients undergoing ra, this was not statistically significant . Apart from lower major bleeding, the mechanism by which radial access might relate to allcause mortality currently remains unclear in the existing literature.13, 22 one plausible explanation for the observed prognostic difference favoring ra relates to selection bias introduced by operator experience and centerspecific differences . Greater radial interventional expertise in highvolume centers may have been associated with improved outcomes resulting from reduced vascular complications . To provide some context, it is noteworthy that 50% of the patients analyzed in the current study were treated with a fibrinolytic pi strategy, as compared to a much lower incidence in the radial versus femoral randomized investigation in stelevation acute coronary syndrome (rifle steacs; 7.6% failed fibrinolysis)2 and radial versus femoral access for coronary intervention (rival; 12%)7 trials . Hence, the current results not only support the effectiveness of ra within a pitreated stemi patient subset, but also allow comparison of outcomes between roughly equalsized groups undergoing different reperfusion strategies . Compared to randomized trials that enrolled postfibrinolytic patients,2, 7 a higher proportion of major overall and major accesssite bleeding was noted in the current study; for instance, radial versus femoral, respectively, in rival,7 non coronary artery bypass graft major bleeding (rival definition) was noted in 0.84% versus 0.91%, whereas in riflesteacs (timi definition) 1.8% versus 2.8% compared to 5.2% and 6.0% in our study . The reasons for this disparity in major bleeding is unclear, but could relate to: (1) investigator rather than central adjudication of major bleeding in the current study; (2) heterogeneity in definition of major bleeding; (3) differences in the proportion of the postfibrinolytic stemi population; and (4) variability in operator expertise and sitespecific vascular access protocols . Despite no difference in nonintracranial major bleeding between the 2 access sites in this study, our adjusted analysis of 30day mortality showed a persisting benefit associated with the radial approach . It is noteworthy that a recent study from the national cardiovascular data registry's cathpci registry on bleeding complications in fibrinolytictreated patients undergoing rescue pci reported a major bleeding rate of 6.9% in radial versus 12.0% femoral access patients (adjusted or, 0.67; 95% ci, 0.520.87; p=0.003).23 ra was employed in only 16% of these patients, and the authors highlighted the need for further data in this given that the choice of vascular access site was left to investigator discretion and absence of access to detailed procedural elements, we cannot exclude the impact of unmeasured confounders . Additionally, selection bias introduced by absence of information on center and operatorspecific interventional volumes cannot be excluded . Although we found no overall difference in vascular access bleeding, the trend toward more fa access bleeding in the pi patients undergoing rescue pci suggests less bleeding hazard in the presence of recent fibrinolytic treatment when radial access is employed . Stream excluded patients in cardiogenic shock and advanced kidney disease: hence, our findings do not apply to this population . Given that stream was an openlabel trial without central adjudication of bleeding endpoints, investigator bias may have played a role in the disparity in bleeding rates between this study and existing literature . However, to the best of our knowledge, it is the largest data set comparing a randomized, multicenter fibrinolytic pi strategy (rescue and scheduled pci) in a very earlytreated stemi population to ppci, demonstrating that the outcomes advantage with ra occurs in both the pi and ppci strategy . Irrespective of whether a ppci or pi reperfusion strategy is used, these results support the utilization of radial access as the preferred arterial access site in stemi . Funding for this trial was provided by boehringer ingelheim . Clinical trials identifier: nct00623623 . Welsh r discloses research funding from abbott vascular, alere, astrazeneca, bayer, boehringer ingelheim, canadian institute of health research, csl behring llc, edwards lifesciences, eli lilly, jansen, johnson & johnson, matrizyme pharma, pfizer, population health research institute, and university of alberta hospital foundation and personal funding from astrazeneca, bayer, and bristolmyers squibb / pfizer . Steg discloses a research grant (to inserm u1148) from sanofi and servier; has received speaking or consulting fees from amarin, astrazeneca, bayer, boehringer ingelheim, bristolmyers squibb, cslbehring, daiichisankyo, glaxosmithkline, janssen, lilly, merck, novartis, pfizer, regeneron, roche, sanofi, servier, and the medicines company; and owns stocks in aterovax . Dr armstrong's financial activities outside the submitted work are posted and routinely updated through http://www.vigour.ualberta.ca/en/about/conflictofinterest.aspx . Radial versus femoral access according to country of enrollment table s2 . Logistic regression model (propensity score model) of access site and baseline characteristics appendix s1.
Corneal blindness has been a global topic of interest in recent years [1, 2], and corneal infection remains a major cause of corneal blindness, especially in the developing world [3, 4]. With improved economic development and access to care, countries in the developing world are becoming better equipped to treat and cure infectious corneal ulcers . Nevertheless, in many cases, advanced infectious corneal ulcers may progress to corneal perforation, resulting in severe ocular morbidities and even loss of globe . Therapeutic penetrating keratoplasty (tpk) remains the most vital treatment strategy for perforated infectious corneal ulcers [811]. Successful outcomes after tpk for perforated infectious corneal ulcers have been reported in the literature derived from the developing world . However, in developed countries such as the united states where patient's access to healthcare resources and fortified antibiotics is greater, corneal ulcers apparently have a considerably lower incidence of perforation than in the developing world as evidenced by the sparse clinical data from the developed world [13, 14]. Furthermore, sterile corneal ulcers continue to be an important cause of corneal perforation in the united states and other developed nations [15, 16]. In this study, we compare the clinical course and long - term outcomes of infectious versus sterile perforated corneal ulcers after primary tpk in the united states . The srs institutional review board (iorg0007600/irb00009122) approved this retrospective, consecutive chart review that included all patients from august 2010 through august 2015 that received tpk for a perforated corneal ulcer at a single center in amarillo, tx . All research components adhered to the tenets of the declaration of helsinki and were carried out in accordance with accepted human research regulations and standards . The operative eyes of all patients that underwent primary tpk for a perforated corneal ulcer by a single surgeon (swr) during the aforementioned study interval were included . Patients without completion of at least 18 months of follow - up and patients that underwent corneal gluing prior to tpk were excluded from the analysis . The baseline demographic features and characteristics, preoperative diagnoses with existing ocular comorbidities, and postoperative outcomes were collected . The baseline characteristics included subject age, gender, ethnicity, laterality, lens status, history of contact lens wear, location of perforation, and preoperative best corrected visual acuity (bcva). The postoperative outcomes were collected over a 36-month follow - up period and included whether or not there was eradication of the underlying disease with initial tpk, the use and type of any adjunctive surgical measures to facilitate postoperative graft healing, the presence of graft clarity after initial tpk, whether or not reperforation occurred following the initial tpk, occurrence of regrafting after the initial tpk, the total number of corneal grafts received during the study interval, whether or not the patient received a boston type 1 keratoprosthesis, the postoperative bcva at 36 months, and whether or not the operative eye underwent enucleation / evisceration . The perforated ulcer was classified as central if the perforation was confined in its entirety within a 6 mm radius from the central apex of the cornea . The perforated ulcer was classified as peripheral if any portion of the perforation was located beyond a 6 mm radius from the central apex of the cornea . For perforated ulcers to be classified as infectious, the underlying pathogen must have been positively identified either by culture or else seen in the corneal button on pathology slides . Perforated ulcers classified as sterile all had negative culture results, clinical appearance without infiltrates or other findings that were suspicious for infectious agents, a clinically identifiable entity and known underlying pathology responsible for the ocular surface disease and corneal melting process that led to perforation, and corneal buttons that were negative for microorganisms when examined in the pathology lab . Various clinical findings including historical information, medications history, and corneal sensitivity testing were used to classify the underlying disease condition for the sterile perforated ulcers, but there was no consistent testing or method done uniformly for all of these patients . All tpk procedures were performed within 48 hours from the time in which the corneal perforation was diagnosed . A sixteen interrupted 10 - 0 nylon sutures' technique was used on all tpk surgeries . Graft size varied in diameter based upon the patient's existing anatomy and the location of the perforation but ranged between 7.5 and 10.0 mm . All donor corneal tissues had endothelial cell counts greater than 2,000 cells / mm according to preliminary eye bank testing . Adjunctive surgical measures done at the time of the tpk were discretionary according to the surgeon and sometimes included suturing or gluing of an amniotic membrane graft over the ocular surface and lateral suture tarsorrhaphy . The jmp 11 mathematical software package from the sas institute (cary, nc, usa) was used to execute the statistical analysis and calculate means with standard deviations . The outcome variables were not assumed to have a normal distribution, so one - way analysis of the variance (and likelihood ratios, when appropriate for nominal variables) was used to compare the baseline characteristics and postoperative outcomes among the infectious and sterile perforated ulcer groups . The mean age of the overall study population was of 58.2 (21.1) years with 60% male . The mean follow - up among both groups collectively was 38.6 (6.9) months (including 9 patients that deceased during the study interval). Table 1 classifies all corneal ulcers as sterile and infectious and details the underlying diagnosis and pathology that led to corneal perforation requiring tpk . For the thirteen bacterial perforated ulcers, four cultures were positive for staphylococcus aureus (two of them methicillin - resistant), two cultures were positive for haemophilus influenzae, two cultures were positive for streptococcus pneumoniae, two cultures were positive for pseudomonas aeruginosa, and one culture each was positive for citrobacter koseri, arthrobacter spp ., and a nonidentified atypical acid fast bacillus . For the seven fungal perforated ulcers, aspergillus spp ., and bipolaris spp ., and there were hyphae or other fungal elements identified on pathology specimen without positive culture for the remaining three cases . Table 2 compares the baseline features and preoperative characteristics among the sterile and infectious cohorts . The infectious group was more likely to have a history of contact lens wear (p = 0.003) and previous keratoplasty (p = 0.0138), while the sterile group was more likely to have a peripheral location of the perforation site (p = 0.0333). Patients presenting with sterile perforated ulcers were more likely to have recurrence of the underlying disease condition (p = 0.0321), have corneal reperforation (p = 0.0079), achieve worse bcva (p = 0.0130), develop nlp vision (p = 0.0053), and eventually require enucleation / evisceration (p = 0.0252) when compared to the infectious perforated ulcer group during the study interval . The sterile ulcer group was also more likely than the infectious group to receive adjunctive surgical measures in the immediate postoperative period (p <0.0001): 15 eyes received amniotic membrane grafting and 9 eyes received suture tarsorrhaphy in the sterile group, while just 1 eye received amniotic membrane grafting and 2 eyes received suture tarsorrhaphy in the infectious group . Subset analysis among the various sterile ulcer pathologies showed no significant difference in any of the different outcomes measured . There were 9 study subjects (20%) who deceased during the 36-month study follow - up: three patients due to end stage cancer, two patients due to complications from chronic autoimmune disease, two patients due to end stage renal disease and other complications from diabetes, and two patients due to chronic heart disease . If the patient in either perforated ulcer group resided in a nursing home (n = 11), then they were more likely to decease (p = 0.0020) and develop worse bcva (p = 0.0031) when compared the remainder of the study subjects . But nursing home residence did not correlate with either sterile or infectious perforated ulcer type (p = 0.1405). In addition, all phakic patients in both groups developed at least some degree of cataract progression during the follow - up interval, while three patients in the sterile group and two patients in the infectious group developed persistent increased intraocular pressure requiring topical medication . Primary tpk is considered the most definitive treatment option for large perforated corneal ulcers regardless of the underlying etiology [811], although recently there has been some interest in alternative techniques such as using autologous fibrin membrane combined with solid platelet - rich plasma, tenons patch grafting, grafting with processed pericardium combined with synthetic materials, and partial thickness lamellar grafting techniques . To our knowledge, this is the first case series to specifically compare long - term outcomes of primary tpk in the setting of sterile versus infectious perforated corneal ulcers in the developed world . Our data suggest that sterile corneal perforations may be more common than infectious corneal perforations in the united states . A recent study by yokogawa et al . Also reported a higher frequency of sterile perforated ulcers to infectious perforated ulcers in the developed world, but their study had too few cases in which primary tpk was performed to make a valid comparison to the results of our study . Furthermore, we observed that the patient population most likely to develop a perforated corneal ulcer in the united states often has substantial baseline risk factors with an immunocompromised state such as end stage cancer, end stage renal disease, poorly controlled diabetes, an advanced autoimmune disorder, existing corneal graft, or residence in a nursing home . Our results indicate that not only is there severe ocular morbidity for many patients presenting with a perforated corneal ulcer in the united states, but also that there is increased mortality (20% of our study subjects) due to other preexisting systemic comorbidities that can be associated with the ocular disease . For these reasons, a valid comparison cannot be made among studies from the developing world where tpk is frequently performed prior to occurrence of corneal perforation for infectious ulcers that typically occur in otherwise healthy patients . Amniotic membrane grafting, autologous serum topical therapy, and tarsorrhaphy have been used as adjunctive measures in the management of perforated corneal ulcers [2123]. In our study, most of the sterile perforated ulcer patients received aggressive adjunctive measures in combination with tpk but still had worse visual and anatomic outcomes compared to the infectious perforated ulcer patients . This highlights the importance of earlier detection and treatment of sterile corneal ulceration to prevent more advanced disease and perforation from occurring . Nursing home residents with corneal ulcers in particular are an extremely vulnerable group requiring more prompt identification, attention, and specialized care before corneal perforation develops . Peripheral ulcerative keratitis was the most common underlying cause of sterile ulcer perforation in this series . Patients with peripheral ulcerative keratitis often had other severe comorbidities due to their underlying rheumatologic disorder that likely contributed to delayed corneal healing after tpk . In our series, three out of the seven patients that developed an autoimmune - related perforation were, at minimum, treated with 40 mg of daily oral prednisone at the presentation of their ulcer whereas the other four of these seven patients presented with large perforation already existing . All seven of the patients were continued on minimum of oral prednisone 40 mg daily for 2 weeks after tpk and tapered off over a period of weeks and sometimes months . During the same time period that this study was conducted, there were 5 other patients that had small perforated autoimmune - related ulcers that were treated with glue and never had tpk . Disease - modifying biologic agents may assist in the management in autoimmune disease that develops peripheral ulcerative keratitis, although further studies are needed to determine their impact on corneal ulcer perforation prevention . Our study weaknesses include the retrospective data collection and the small number of study cases . In conclusion, patients in the united states undergoing tpk for a sterile perforated corneal ulcer are more likely to have the perforation in the peripheral cornea and recurrence of the underlying disease condition in the corneal graft, require adjunctive surgical measures in the immediate postoperative period, have reperforation after tpk, and have worse vision with loss of globe compared to patients undergoing tpk for an infectious perforated corneal ulcer.
Oral allergy syndrome (oas) is an allergic reaction in the oral cavity subsequent to the consumption of food, such as fruits, nuts, and vegetables, which occurs in adults who suffer from allergic rhinitis . It has been described under various names including pollen - food allergy syndrome, pollen - food syndrome, and pollen - associated food allergy syndrome . Oas in adults probably represents the most common allergic reaction caused by food; and more than 60% of all food allergies are actually cross - reactions between food and inhaled allergens . Unlike other food allergies, oas is a reaction limited to the oral mucosa, lips, tongue, and throat . The first description of the oas that associated a hypersensitivity to fruits and vegetables to birch pollinosis was written in 1942 by tuft and blumstein . First denominated it as oral allergy syndrome upon presenting a mainly oral clinical manifestation . Due to the increasing popularity of exotic fruits and vegetables in the diet, oas manifestations occur after the patient who is allergic to pollen consumes certain fruits, vegetables, or nuts . Oas belongs to the allergy type i group, that is, allergic reactions mediated by immunoglobulin e (ige). In susceptible patients, the immune system produces ige antibodies against the proteins of pollen which causes hay allergy . Pollen allergies are caused by repeated exposure to the pollen of some plants, which are usually pollinated by air and have such pollen quantities that inhalation of the pollen easily reaches the surface of the pulmonary alveoli . The oas patient is first sensitized by inhaling pollen that contains the antigens, and then after consuming food that contains cross - antigens (to the inhaled antigens) the symptoms characteristic of oas appear . Plant - derived proteins responsible for allergy include various families of pathogenesis - related proteins, protease and -amylase inhibitors, peroxidases, profilins, seed - storage proteins, thiol proteases, and lectins, whereas homologous animal proteins include muscle proteins, enzymes, and various serum proteins . Cross - reactivity between birch pollen and various fruits and vegetables is due to homology among various pathogenesis - related proteins, which are important in the defense against plant diseases . For example, mal d 1, the major apple allergen, is 63% homologous to the major birch pollen allergen, bet v 1 . Other birch pollen - related, pathogenesis - related proteins have been identified in hazelnut and celery (api g 1). Similarly, the birch pollen profilin, bet v 2, cross - reacts with profilins found in apple (mal d 2), celery (api g 2), and potato . The same immune system can trigger allergic symptoms in two different ways: in the presence of pollen it leads to rhinitis and in the presence of a particular food it leads to symptoms of food allergy . Different allergens vary in their stability, with differences in digestion survival, storage, high temperature, cold, and cooking or pasteurization survival . As important drivers of anaphylaxis, the lipid transporting proteins play an important role, since they cannot be easily denatured by digestion or cooking . Antibodies can react to linear amino acid sequences of the protein or a conformational epitope . Persons who respond to the linear sequence of the protein can tolerate neither raw nor cooked food, while those that respond to a conformational epitope can consume cooked food but not the raw food . Certain foods like peanuts are able to sensitize and elicit reactions after oral exposure and could trigger responses that generalize to related foods (legumes). In other groups of foods like apples, sensitization to homologous proteins encountered through respiratory exposure (e.g., birch pollen) may mediate reactions to cross - reacting proteins in the food . This distinction is based on clinical appearance, the predominantly affected group of patients (children or adults), and disease - eliciting food allergens . Primary (class 1) food allergy starts in early life and often represents the first manifestation of the atopic syndrome . The most common foods involved are cow's milk, hen's egg, legumes (peanuts and soybean), fish, shellfish, and wheat . The allergens contained in these foods not only elicit allergic reactions in the gastrointestinal tract but also often cause urticaria, atopic dermatitis, and bronchial obstruction . With a few exceptions, most children outgrow class 1 food allergy within the first 3 to 6 years of life . Secondary (class 2) food allergy describes allergic reactions to foods in mainly adolescent and adult individuals with established respiratory allergy, for example, to the pollen of birch, mugwort, or ragweed . This form of food allergy is believed to be a consequence of immunological cross - reactivity between respiratory allergens and structurally related proteins in the respective foods . Food allergens that induce oas rapidly dissolve in the oral cavity and are readily broken down by digestive enzymes . Preservatives in foods may also trigger the manifestation of the disease . Due to the structural similarity of individual protein molecules, a large number of allergens that exist in nature can be classified into groups as follows [1, 8] (table 1). Among the allergens in each group there is a possibility of cross - reactivity of ige antibodies, that is, antibodies binding to one of two or more allergens . The reaction may start with one type of food, and subsequently allergies to other food types can develop . Latex allergens can also sensitize patients to cross - react to the protein found in some foods . One of the most notable features of latex allergy is the patients' cross - reactivity to various fruits and vegetables, a condition often called latex - fruit syndrome . The first report of an allergic reaction to banana in a latex allergic patient was published in 1991 [9, 10]. Structurally similar proteins in many kinds of plants must be responsible for such extensive cross - reactivity . Commonly reported cross - reactive foods include banana, avocado, kiwi, chestnut, potato, and papaya, and numerous latex allergens cross - react with food and pollen proteins . Rarely oas is induced by the ingestion of other foods in subjects without pollen sensitization, for example, shellfish and pork [12, 13]. Grains of pollen are admixed to this raw material which retains their allergenic properties during the honey making process . More and coworkers reported that some patients presented with oas symptoms after eating foods cooked over mesquite wood and individuals were positive to skin prick test with mesquite pollen extract . They concluded that the transfer of allergens in foods cooked over mesquite wood might lead to symptoms in sensitized individuals . Some studies have confirmed that the oas is more common in female patients [16, 17]. Patients who show symptoms of oas may have a number of other allergic reactions that start very quickly, even minutes after consuming trigger food . Usually, it is manifested by itching and a burning sensation of the lips, mouth, ear, and throat or by the appearance of perioral erythema and generalized urticaria . The patient may develop swelling of the lips, tongue, and uvula, occasionally a sense of suffocation, and rarely anaphylaxis . In rare situations, oas may be manifested as difficulty in breathing, appearance of a rash, or hypotension (table 2). Have recorded symptoms after oral challenge with offending food and the reaction was classified in 4 grades of severity: (i) only oral mucosa symptoms; (ii) oral mucosa and gastrointestinal symptoms; (iii) oral mucosa and systemic symptoms, such as urticaria, angioedema, rhinoconjunctivitis, and asthma; and (iv) oral mucosa and life - threatening symptoms, such as laryngeal edema and shock . However, in most of the cases, oas presents with mild symptoms . Food allergy diagnostics are one of the most difficult tasks in allergology, especially when there is no clear connection between the development of the clinical features and the ingested food or when food allergy takes an atypical or chronic course . The diagnostic methods can be divided into two groups: clinical and laboratory . Among the group of the clinical methods clinical history, eating habits investigation, skin tests, and challenge tests are used for their high informative value . The specific ige antibodies assay is the most important among the laboratory methods . For a correct diagnosis, history of an allergy, whenever reported, should be recorded in the patient's medical history [1, 20]. Clinical history should contain details about the development of the clinical features, food eaten, symptoms, and period of time between the intake of the food and the onset of the signs, and sequence of the manifestations . In patients with allergies to airborne particles, the appearance of oral itching or tingling after eating fresh fruit or vegetables is enough to suspect oas . Skin testing for ige mediated reaction can be carried out using different methods: the prick method (prick test), the application of allergens via scratching the skin (scratch test), and rarely an intradermal test (application of allergens into the skin by a needle). Commercial extracts are used for prick tests, determining allergy to peanuts, hazelnuts, and peas . Prick tests are not carried out in areas of dermatitis or in areas where dermocorticosteroids or immunomodulating creams have been applied . The skin prick test is performed with commercial extracts of pollens and food on the forearm or the back, measuring the wheal after 15 minutes, and is considered positive if the diameter of the wheal is greater than 2 mm of the negative control sample . The commercially available fruit extracts used in allergy testing are not usually reliable indicators of allergy in patients with oral allergy syndrome, because the cross - reactive epitopes have been destroyed by the manufacturing process . Prick - plus - prick testing (prick the fruit and then prick the skin) with freshly prepared fruit extracts is more sensitive in detecting allergen specific ige antibody . If the history is positive and the prick test is negative, a provocation test with a fresh food should be conducted . An oral provocation test represents the safest confirmation of the presence of the disease . In doing so, the person first consumes a suspected food, and subsequently the onset of symptoms is recorded . To set up an accurate diagnosis, it would be necessary to keep a diary of food consumption as the basis for determination of which food tests to undertake . Good history can focus the testing on a specific type of food, and thus the doctor can act more rationally . For most ige mediated reactions, 8 10 gm of the dry food or 100 ml of wet food (double amount for meat / fish) at 10 15 min intervals is given over about 90 min followed by a larger, meal size portion of food a few hours later . The symptoms should be recorded and frequent assessments are to be made for symptoms affecting the skin, gastrointestinal tract, and/or respiratory tract . Blood tests are mostly performed as rist (radioimmunosorbent test) for the determination of total ige and rast (radioallergosorbent test) for the determination of specific ige antibodies to a particular allergen . Extensive research has led to the identification of principle allergens in cross - reactive food . Many allergenic components have been produced in a recombinant form maintaining their immunoreactivity and allergenic epitopes . These allergens are applied to a chip based microarray that uses small quantities of serum and provides ige antibody profiles to over 100 food and pollen allergens . However, most of the in vitro diagnostic tests are expensive and the cost factor limits its use . A multidisciplinary approach in patients with oas is necessary, which involves different professions (ear - nose - throat specialists, oral pathologists, allergologists, immunologists, dermatologists, pediatricians, gastroenterologists, and various other specialties). The oas should be managed according to the clinical presentation . Since many of the immunogenic proteins in fruits and/or vegetables are unstable (heat - labile), patients will tolerate food cooked and canned well and fresh or raw foods badly . It has been shown that cooking food can sometimes eliminate allergens in certain species like apples, while it is impossible to destroy allergens in celery and strawberries . For some types of food (e.g., nuts) that contain more than one allergen, heat treatment will destroy certain allergens, while some of them can cause a reaction even after that . Patients, their families, close relatives, and caregivers should be aware of risk situations and should be instructed in reading labels and how to avoid the relevant food allergens both inside and outside the home . Most patients with oas can be treated with a combination of allergen avoidance and pharmacotherapy . Oral antihistamines such as cetirizine 10 mg or intramuscular aqueous epinephrine at the dose of 0.01 ml / kg of 1: 1000 dilution can alleviate allergic symptoms by blocking specific immune pathways . The use of topical preparation of mast cell stabilizers like cromolyn sodium or antihistamines like levocetirizine prior to food intake has been effective in helping some patients with food allergy . Patients with a history of anaphylaxis should always carry a shot with a dose of epinephrine (such as epipen which contains epinephrine 0.3 mg in 0.3 ml) with them . In the event of a reaction, the patient is advised to stay calm, rinse their mouth with plain water, and rest . The patient can help himself / herself with hot (but not boiling) beverages that can inactivate residual allergens . This usually leads to withdrawal of the sensation of prickling, itching, and swelling, which stops within 30 minutes to an hour (before the antihistamine makes an effect) [1, 29]. When the patient is able to swallow a dose of antihistamines, they definitely need to be taken however, severe symptoms are rare in patients with oas [1, 29]. In patients with suspected oas, different reactions may appear at different times, such as sneezing attacks during scraping of fruits and vegetables, when particles can get into the air, or conjunctivitis if the patient touches his / her eyes after touching the fruit or vegetables . It is also recommended to avoid latex (rubber gloves) that can cause cross - allergic reactions to foods of plant origin . If the patient avoids areas of certain types of pollen, the syndrome usually relieves after two to three years . Desensitization to the pollen with immunotherapy is recommended in some cases and can sometimes help minimize cross - reactions . Subcutaneous specific immunotherapy (sit) has been tried and has significantly reduced oas symptoms associated with ingestion of the responsible fruit and vegetables . According to a study by asero, at least in some patients pollen sit can exert a long - lasting effect on pollen - associated food allergies (patients sensitized to birch pollen were still able to eat apples without any complaints as long as 30 months after the end of sit). A study by bergmann et al . Also suggested that pollen - specific sit can reduce oas triggered by pollen - associated foods in patients with pollen - induced rhinoconjunctivitis . Allergy or intolerance to the food we eat may be a problem routinely encountered . Though oas is mainly managed in allergy clinics, it is equally important for the oral physicians to be aware of the symptoms and clinical features of oas . It is equally important to record patient's history accurately regarding previous episodes of allergies . The dentists should pay attention especially to the individuals with a history of asthma, atopy, or any other allergic problems during the dental treatment procedures . Even though the symptoms of oas are mild in most of the cases, they can manifest life - threatening complications occasionally.
Superficial barrett s esophageal adenocarcinoma (s - bea) in barrett s esophagus is frequently found in the right wall of the esophagus 1 2 3 4 . Showed that more than half of s - beas were located at the 0 3 oclock position in the distal esophagus 1 . Also reported that, in barrett s maximal segments of 5 cm or less, around half of all high grade dysplasias and early adenocarcinomas were located at the 2 5 oclock position 2 . There is also a report indicating that the directional distribution of s - bea is not influenced by the distance of the lesion from the gastroesophageal junction (gej) 3 . Overall, the available evidence indicates the potential importance of surveillance in these quadrants for early detection of s - bea in patients with barrett s esophagus . On the other hand, it has been reported that esophageal mucosal breaks also mainly occur in the right anterior wall of the distal esophagus 4 5 6 . Edebo et al . Have reported that mucosal breaks in patients with grade a or b esophagitis occurred most frequently in the right wall of the distal esophagus 5 . Tongue - like short - segment barrett s esophagus (ssbe) was more frequent in the right anterior wall (in the 0 2 oclock position) than at other locations 4 7 . Using a ph catheter with eight sensors, ohara et al . Reported that patients with non - erosive reflux disease (nerd) and reflux esophagitis had radial asymmetric acid exposure that was predominant on the right wall of the distal esophagus 8 . So far, however, no published reports have examined the correlation between the location of s - bea and the direction of acid or non - acid reflux individually . In the present study, we investigated this correlation in individual patients with barrett s esophagus, hypothesizing that identification of the direction of acid or non - acid reflux in patients with barrett s esophagus might be useful for early detection of s - bea . In a preliminary study, we performed 24-h ph monitoring in five healthy subjects, one patient with nerd, and two patients with s - bea who were not receiving proton pump inhibitors (ppis). We defined acid reflux as ph <4.0 and non - acid reflux as ph> 8.0 . The catheter we employed (sme medizintechnik gmbh, germany) has four ph sensors arranged circumferentially at two different levels . This catheter has a blue line on ph sensors 1 (lower channel) and 5 (upper channel) located at the 6 oclock position in the lower esophagus (fig . 1). 8 are arranged counterclockwise at each level, and the upper channel is 5 cm distant from the lower channel (fig . 1). The catheter was inserted transnasally into the esophagus after taking calibrations at ph 4.0 and 7.0, based on the manufacturer s instructions, and the lower ph channel of the catheter was positioned 2 cm above the squamo - columnar junction (scj), close to the usual site of mucosal breaks in patients with low grade esophagitis and s - bea in patients with ssbe . Ph data from the eight sensors can be recorded simultaneously by connecting the catheter to four portable digital recorders (pocket monitor gmms-200ph; star medical). The catheter used in our preliminary study had four sensors arrayed circumferentially at each of two levels . In our main study, the ph catheter had eight sensors (white arrows) arrayed circumferentially at the same level as the catheter . This catheter had a blue line on channel 1 located at the 6 oclock position, and the eight sensors were arranged counterclockwise from that position (from ref . This preliminary examination was performed during daily activity, and ph monitoring was done on a normal diet . The possibility of horizontal rotation and vertical movement during ph monitoring was examined by endoscopy . However, no horizontal rotation or vertical movement was observed when individuals were standing, sitting or supine, or when head and swallowing movements were performed, and we excluded any results for which the data indicated that the catheter had fallen into the stomach . We measured the total percentage period when ph was <4.0 as an indicator of acid reflux . Channels 2, 3, 6, and 7 were located on the right anterior side . The numbers in table 1 indicate the total percentage period when ph was <4.0 . This percentage exceeded 4%, and was highest and second highest in nerd and s - bea patients, respectively . On the other hand, the * numbers show the distribution of high acid readings in each of the healthy subjects . These results indicated that acid exposure was located predominantly on the right side in nerd and s - bea patients, whereas in healthy subjects there was no radial variation of acid reflux (table 1). Nerd, non - erosive reflux disease; s - bea, superficial barrett s esophageal adenocarcinoma . Sensors 2, 3, 6, and 7 were located on the right anterior side . One nerd and two s - bea patients had acid exposure predominantly on the right side . In contrast, in healthy subjects, there were no radial variations in acid reflux . For this study, we retrospectively enrolled 37 s - bea patients with solitary lesions who were treated with endoscopic submucosal dissection (esd) or surgery between 2011 and 2014 at our hospital . However, the digital recorder failed in three patients and the catheter fell into the stomach in one patient, so the results of ph monitoring in these four patients were finally excluded, leaving 33 s - bea patients as the study subjects in whom all 33 lesions occupied no less than a third of the esophageal circumference . We histopathologically confirmed the diagnosis of s - bea in all cases according to the japanese classification of esophageal cancer 9 . Four of the patients (12.1%) were women, and the mean patient age was 63.4 years (range 46 twenty - eight patients (84.8%) had ssbe and the remaining five patients (15.2%) had long - segment barrett s esophagus (lsbe). The depth of invasion in 18 of the s - bea cases (54.5%) was the mucosa, whereas in the remaining 15 cases (45.5%) it was the submucosa . In ssbe cases, 23 lesions were distributed at the 0 3 oclock position, four at the 3 6 oclock position, and one at the 6 one lesion was distributed at the 0 3 oclock position, one at the 3 6 oclock position, and three at the 9 6 oclock, one at 6 9 oclock, and three at 9 0 oclock (fig . Ssbe, short - segment barrett s esophagus; lsbe, long - segment barrett s esophagus . Distribution of the direction of s - bea in (a) ssbe, (b) lsbe, and (c) all cases of barrett s esophagus . We performed 24-h ph monitoring before treatment of s - bea by esd or surgery . In this study, all of the patients had inevitably taken ppis because some had suffered prolonged esd beforehand . We defined acid reflux as ph <4.0 and non - acid reflux as ph> 8.0, even under ppi medication . The catheter we employed has eight ph sensors circumferentially arrayed at the same level as that in the device developed by shimane medical university and star medical (tokyo, japan) (8-channel ph catheter sa800). This catheter also had a blue line on sensor 1 located at the 6 oclock position in the lower esophagus, and sensors 1 8 were arranged in turn in a counterclockwise direction (fig . 1). It was inserted transnasally into the esophagus and positioned at the same level as the neoplasia in all 33 s - bea patients . Catheter insertion was performed in the afternoon, and in the evening the patients took liquid food . On the following day, esd or surgery was performed . Channel 1 of the catheter was positioned at 6 oclock in the lower esophagus, and this positioning was confirmed by endoscopic observation of the blue marker line (fig . 1). The conditions employed, such as daily activities and diet, were the same as those for our preliminary test . We measured the maximal total duration of acid and non - acid reflux (maximal total duration of acid reflux [mtd - a] or maximal total duration of non - acid reflux [mtd - na]) for 24 hours in all 33 s - bea patients . We then divided the esophageal locations of the catheter sensors into eight parts circumferentially . When the direction of mtd - a and mtd - na coincided with the location of the s - bea, the case was defined as a coincidental case and we calculated the rate of coincidence and the probability of the rate of coincidence this is a typical s - bea case located at the 0 3 oclock position in the distal esophagus . In this case, the direction of mtd - a and mtd - na was located at sensor 4 in the 0 3 oclock position . A s - bea case located at the 0 3 oclock position in the lower esophagus . Mtd - a and mtd - na were detected by sensor 4 in the 0 3 oclock position, and therefore this case was coincidental with acid and non - acid reflux . The study was conducted in accordance with the declaration of helsinki, and the study protocol was approved by the medical ethics committee of our hospital . The risks and benefits of this examination were explained beforehand, and written informed consent was obtained from all 33 patients . The study was conducted in accordance with the declaration of helsinki, and the study protocol was approved by the medical ethics committee of our hospital . The risks and benefits of this examination in 28 patients with ssbe, including four s - bea patients without acid reflux, the location of 19 s - beas (67.9%) corresponded to the direction of mtd - a (95% ci 0.48 16 of the s - beas (84.2%) were located at the 0 3 oclock position, two (10.5%) at 3 6 oclock, and one (5.3%) at 6 9 oclock in the lower esophagus . In all five patients with lsbe, the location of the s - beas (100.0%) corresponded to the direction of mdt - a (95% ci 0.48 1.00). Among these five coincidental cases, one s - bea (20.0%) was located at the 0 3 oclock position, one (20.0%) at the 3 6 oclock position, and the others (60.0%) at the 9 0 oclock position in the lower esophagus . Among the 33 s - bea cases, the mtd - a or mtd - na coincided in 24 (72.7%) (95% ci 0.54 0.87). On the other hand, among the 28 ssbe patients with non - acid reflux, including 3 s - bea patients without non - acid reflux, the location of 20 of the s - beas (71.4%) corresponded to the direction of mtd - na (95% ci 0.51 17 of the s - beas (89.5%) were located at 0 3 oclock, two (10.0%) were located at 3 6 oclock, and one (5.0%) was located at 6 9 oclock in the lower esophagus . Among the five non - acid reflux patients with lsbe, four of the s - beas (80.0%) corresponded to the direction of mtd - na (95% ci 0.28 0.99); three of these (60.0%) were located at 9 0 oclock, and the remaining one (20.0%) was located at 3 6 oclock . Among the 33 non - acid reflux patients with s - beas, the mtd - na coincided in 24 (72.7%) (95% ci 0.54 0.86). Overall, the rate of coincidence of either mtd - a or mtd - na was 30 /33 (90.9%) (95% ci 0.76 0.98) (table 3). Mtd - a, maximal total duration of acid; mtd - na, maximal total duration of non - acid; ssbe, short - segment barrett s esophagus; lsbe, long - segment barrett s esophagus . Fig . 4 illustrates a case of s - bea at the 9 10 oclock position in barrett s esophagus . Mtd - a was detected by sensors 5 7, in the 9 0 oclock position, and mtd - na was detected at sensors 6 8, in the 8 10 oclock position . Therefore, in this case, the positions of acid and non - acid reflux coincided . A s - bea case located at the 9 10 oclock position in mtd - a was detected by sensors 5 7 in the 9 0 oclock position, and mtd - na was detected by sensors 6 8 in the 8 mucosal breaks in patients with grade a or b esophagitis and tongue - like ssbe occur most frequently in the right wall of the distal esophagus 4 5 6 7 . S - bea is frequently found in the right wall of the esophagus 1 2 3 4 . However, no published studies have investigated the reason for this . In the ssbe patients we studied, the 0 3 oclock position in the esophagus accounted for the majority of s - beas: 23 cases . On the other hand, in lsbe patients, the 9 0 oclock position accounted for the majority of s - beas: three cases . The position of the latter three lesions coincided with the direction of mtd - a or mtd - na . Among the 33 s - bea patients as a whole, the direction of either mtd - a or mtd - na coincided in 30 (90.9%). The present study investigated whether mtd - a or mtd - na could be used as an indicator of the site of occurrence of s - bea . No obvious indicators of the site of s - bea occurrence have been reported so far, although a few studies have attempted to monitor esophageal acid exposure by ph detection in barrett s esophagus . Reported that patients with lsbe had significantly more frequent esophageal acid exposure than patients with ssbe . The duration of esophageal acid exposure seems to be an important factor determining the length of barrett s esophagus 10 . Menges et al . Suggested that there is a good correlation between the duration of esophageal exposure to acid and bile and the severity of pathological change in the esophagus 11 . However, no reports have attempted to define the duration of acid exposure as a risk factor for s - bea . Virchow described the presence of leukocytes in tumors, and hypothesized that the origin of cancer lay at the site of chronic inflammation . It has been reported that chronic infections are associated with 15 20% of malignant tumors 12 13 . In bea, therefore, we considered that mtd - a and mtd - na could be defined as indicators of s - bea occurrence . Recently, especially in japan, endoscopic treatments, including esd and endoscopic mucosal resection, have been shown to be safe, effective, and minimally invasive for s - bea in patients with barrett s esophagus 14 15 16 . In the present series, esd was selected for tumors without any submucosal invasion, whereas surgery was indicated for tumors showing obvious submucosal invasion . Patients with high grade intraepithelial neoplasia and intramucosal cancer have been shown to have only a minimal risk of lymph node metastasis, and therefore endoscopic therapy is generally regarded as curative . When cancer invades the submucosal layer, the risk of lymph node positivity rises to 20% 17 18 . Prevention of cancer death therefore requires early detection by endoscopy surveillance when the cancer is still curable at an early stage . Although no prospective and randomized controlled study has clarified the efficacy of surveillance endoscopy for prevention of cancer - related death in patients with barrett s esophagus, it is globally recommended by most gastroenterology societies 19 20 21 . Retrospective series have lent support to the opinion that endoscopic biopsy surveillance of barrett s esophagus facilitates detection of s - bea at an early and curable stage, thereby potentially reducing mortality due to esophageal adenocarcinoma 22 23 . A recent randomized, crossover trial has shown that narrow - band imaging (nbi) targeted biopsies can detect intestinal metaplasia at a rate similar to high definition white - light endoscopy examination with the seattle protocol, while requiring fewer biopsies . In addition, nbi targeted biopsies can detect more areas with dysplasia, and obviate the need for biopsies of those lesions showing regular surface patterns 24 . Therefore, if observation of the location and distribution of acid or non - acid reflux by ph monitoring could be combined with nbi target biopsy, detection and curability of s - bea would be maximized . First, almost all of the 33 s - bea patients were taking ppis during ph monitoring . In practice, however, we have found that, in patients not taking ppis, esd was prolonged and the esophagitis was worsened, making the borderline of s - bea less clear . Our purpose in this study was to reveal the prevailing distribution of acid or non - acid reflux . The most essential point was to evaluate the direction of mtd - a or mtd - na individually . The possibility of this occurring during ph monitoring was examined by endoscopy, but none was observed during standing, sitting or remaining supine, or during head movements and swallowing . In addition, the digital recorder failed in three patients and the catheter fell into the stomach in one patient, so the results of ph monitoring for these four patients had to be excluded . Third, the numbers of patients examined were small, especially those with lsbe . In order to investigate the correlation between the direction of mtd - a and mtd - na with the location of s - bea, more patients with lsbe moreover, it will be necessary to verify prospectively whether endoscopic surveillance of the direction of acid and non - acid reflux, by performing ph monitoring of barrett s esophagus with no neoplasia, would aid the early detection of s - bea . Here we found that the location of s - bea mostly corresponded to the direction of mtd - a or mtd - na . Currently, no method of endoscopic surveillance for barrett s esophagus has been devised . However, our present experience shows that it is possible to predict the location of s - bea using a ph catheter with eight sensors . Therefore, endoscopic observation of the prevailing distribution of acid or non - acid reflux by ph monitoring appears to have potential for early detection of s - bea.
Although spontaneous thrombosis of large and giant aneurysms is not uncommon, this situation in a small ruptured cerebral aneurysm is a rare event with incidence of 1 - 2% . Its possible pathophysiology with contributing factors has been well discussed in the literature.1)2)3)8)12)15)18) in addition, subsequent recanalization within the next few weeks has also been described . We report on a case of acute thrombosis of the aneurysm, which occurred immediately after coil extraction, and suggest the next way to prevent recanalization, which can lead to re - rupture of the aneurysm . A 54-year - old male experienced a sudden onset of headache which had developed 2 days ago . After the initial diagnosis of subarachnoid hemorrhage (sah) using brain computed tomography (ct) in another hospital, he was transferred to the emergency room where his glasgow coma scale score was e4v5m5 . Despite neck stiffness, there was no focal neurological sign . A brain ct scan taken on admission showed focal sah concentrated in the anterior portion of the basal cistern (fig . 1). Digital subtraction angiography (dsa) confirmed the presence of the anterior communicating artery (acoa) aneurysm and 3d rotational angiography (3dra) showed that the aneurysm had a very narrow neck, which measured 5.9 2.7 1.7 (width height neck diameter) (fig . As morphologic features of the aneurysm were adequate for coil embolization, we decided to perform an emergency endovascular coil embolization on the day of admission . Dsa was performed again to assure the aneurysm . After confirming that the result on dsa was identical to the previous one, a microcatheter was carefully guided over a microguidewire into the aneurysm . As the tip of the catheter was kept at the neck of the aneurysm, the first coil (target detachable coil 3 mm 8 cm; stryker neurovascular, fremont, ca, usa) placement was attempted very slowly . At the last moment of deploying the first coil, the tip of the microcatheter was seen to move back from the aneurysm and the projection of the coil loop outside the aneurysm was observed (fig . However, a subsequent angiogram showed near obliteration of the aneurysm without evidence of vasospasm . Because acute thrombosis facilitated by an exposed coil within the aneurysm was assumed, and, sooner or later, recanalization could occur, we decided to insert a smaller coil into the remaining stump of the neck . With the coil (hypersoft 3d complex coil 2 mm 4 cm; microvention, tustin, ca, usa), embolization was performed safely and post - embolization dsa showed complete occlusion of the aneurysm (fig . There was no occurrence of procedure - related complication during coil embolization and the patient recovered without neurologic deficit after the procedure . Follow - up dsa the next day showed persistent occlusion of the aneurysm (fig . The patient was discharged without neurologic deficit of the modified rankin scale score 0 . Magnetic resonance (mr) angiography at 3-month follow - up showed no evidence of residual neck or recurrence of recanalization . On 1 year follow - up dsa, despite change in the configuration of the coil, the aneurysm remained to be completely obliterated (fig . A 54-year - old male experienced a sudden onset of headache which had developed 2 days ago . After the initial diagnosis of subarachnoid hemorrhage (sah) using brain computed tomography (ct) in another hospital, he was transferred to the emergency room where his glasgow coma scale score was e4v5m5 . Despite neck stiffness, there was no focal neurological sign . A brain ct scan taken on admission showed focal sah concentrated in the anterior portion of the basal cistern (fig . 1). Digital subtraction angiography (dsa) confirmed the presence of the anterior communicating artery (acoa) aneurysm and 3d rotational angiography (3dra) showed that the aneurysm had a very narrow neck, which measured 5.9 2.7 1.7 (width height neck diameter) (fig . As morphologic features of the aneurysm were adequate for coil embolization, we decided to perform an emergency endovascular coil embolization on the day of admission . Dsa was performed again to assure the aneurysm . After confirming that the result on dsa was identical to the previous one, a microcatheter as the tip of the catheter was kept at the neck of the aneurysm, the first coil (target detachable coil 3 mm 8 cm; stryker neurovascular, fremont, ca, usa) placement was attempted very slowly . At the last moment of deploying the first coil, the tip of the microcatheter was seen to move back from the aneurysm and the projection of the coil loop outside the aneurysm was observed (fig . However, a subsequent angiogram showed near obliteration of the aneurysm without evidence of vasospasm . 3b). Because acute thrombosis facilitated by an exposed coil within the aneurysm was assumed, and, sooner or later, recanalization could occur, we decided to insert a smaller coil into the remaining stump of the neck . With the coil (hypersoft 3d complex coil 2 mm 4 cm; microvention, tustin, ca, usa), embolization was performed safely and post - embolization dsa showed complete occlusion of the aneurysm (fig . There was no occurrence of procedure - related complication during coil embolization and the patient recovered without neurologic deficit after the procedure . Follow - up dsa the next day showed persistent occlusion of the aneurysm (fig . The patient was discharged without neurologic deficit of the modified rankin scale score 0 . Magnetic resonance (mr) angiography at 3-month follow - up showed no evidence of residual neck or recurrence of recanalization . On 1 year follow - up dsa, despite change in the configuration of the coil, the aneurysm remained to be completely obliterated (fig . The reported incidence of acute thrombosis of the aneurysm after sah is 1 - 2%,7)9)17) and several possible mechanisms have been reported in the literature . In large or giant aneurysms, in particular, the incidence of spontaneous total thrombosis of the aneurysm has shown a marked increase, ranging from 13% to 20%,10) and the volume - to - orifice ratio of the aneurysm has been suggested as the most reliable mechanism of spontaneous thrombosis.1) however, in small aneurysms, other factors including anti - fibrinolytic agent, non - ionic contrast media, systemic hypotension, increased blood coagulability, increased platelet aggregation, vasospasm, and hemodynamics in the parent artery have also been suggested as an influential mechanism.3)6)8)15)18)20) in our patient, we supposed several factors that promoted thrombosis of the aneurysm . The most important and potent factor was a narrow neck of the aneurysm of 1.7 mm with a large volume - to - orifice ratio . Extremely narrow neck of the aneurysm was regarded as sufficient potential to induce thrombosis under special circumstances like the following . First, the endovascular procedure - including microcatheter placement on the neck of the aneurysm and aborted first coil - could interrupt the intrasaccular blood flow and trigger aneurysm thrombosis . Second, inadequate systemic heparinization might induce spontaneous thrombosis . According to the protocol of our institute, we generally induce systemic heparinization after successful placement of the first basket coil in treatment of a ruptured cerebral aneurysm . In this patient, systemic heparin was not administered during the procedure, and the endovascular procedure was performed under inadequate systemic heparinization . Third, induced hypotension during the procedure could be a potential factor in spontaneous thrombosis . From this experience, if the neck of a ruptured aneurysm was very narrow, a rapid and adequate endovascular procedure, sufficient heparinization and exclusion of excessive hypotension during the procedure should be considered in order to avoid spontaneous thrombosis of the ruptured aneurysm . Lee et al.13) pointed out that the mechanism of recanalization might be the result of liquefaction of the thrombus and subsequent intrathrombotic dissection by blood flow . Another opinion regarding recanalization following thrombosis of the aneurysm suggests that when the thrombosis was induced by endovascular treatment, recanalization of the thrombosed aneurysm might occur spontaneously.14) although the pathophysiology of spontaneous recanalization has not been fully elucidated, recanalization ensuing from complete thrombosis of aneurysms has been reported in many studies.4)5)11)12)16)19)21)22) in these studies, recanalization of acute thrombosis of the aneurysm was observed in the days and weeks after thrombosis . Therefore, follow - up angiogram and careful observation during the follow - up period are required for fear of recanalization resulting in rebleeding of the aneurysm . In our patient, coil embolization was performed using a small coil in the tiny stump of the remaining neck . We believed that this procedure might prevent spontaneous recanalization rather than inducing recanalization because the inserted coil would generate an additional thrombus as much as to block the narrow neck, thus resulting in obstruction of blood flow . Fortunately, a satisfactory result was obtained with complete occlusion of the aneurysm after 1-year follow - up . Thrombosis in a ruptured small aneurysm may be generated during coil insertion and the extraction process when the neck of the aneurysm is very narrow . Because recanalization may occur within the next few weeks, we believe that coil insertion on the remaining neck would provide a measure to prevent spontaneous recanalization.
It has been estimated that introduction will succeed only if fitness is greater than 80% . One of the prime targets for such modification is anopheles gambiae, one of the primary vectors in sub - saharan africa, where more than 90% of the world's malaria is transmitted . Whilst size of males females that mate with males that are two days old may be more likely to oviposit than if they mate with older males . Hence, younger males may be fitter and, for the introduction to succeed, released males will need to mate with wild females early in their life . In many areas of africa funestus is likely to have little long - term effect on malaria in many areas . Given their sympatry and the likelihood of a similar mating period, some aspects of the mating behaviour of an . Gambiae, like many other anophelines, mating is associated with swarming [5 - 9]. Funestus, however, is that of harper who observed swarming males on the threshold of a thatched dwelling 1 km from nyanza, lake victoria . Indeed the doyens of african medical entomologists, gilles and de meillon, in their extensive review state,' we have never found them (swarms) ourselves, even in areas of high density' . An . The greater the contrast the more likely will a swarm form and they can be induced to form over artificial markers . Although thought to be a eurygamic species (which requires large volumes of space to successfully mate) harper describes an . No marker was described and no mating was recorded despite the fact that estimated numbers in the swarm, which stretched almost a metre across, exceeded 500 . In a recent study of swarming behaviour of an .,, however, considered that swarm site and marker were unlikely to be specific to a particular species . They also threw doubt on the idea of' swarm arenas' proposed by charlwood and jones . Funestus were commonly observed, in a village in southern mozambique, in areas similar to those used by an . Funestus in these areas and to determine if any aspects of the observed behaviour differed from that recorded for the m form of an . The study took place on nineteen evenings between the 23march and 3may 2002, in the village of furvela, 650 km north of maputo and 7 km south of the town of morrumbene, in the province of inhambane, on the main maputo - beira highway (en1). The village lies circa 3 km from mangrove - bordered coast and is delineated by the furvela river a small stream within a 2 km wide valley . Most of the villagers live by subsistence farming, growing maize, manioc, peanuts and beans . Cashew nut trees and coconut palm are common . In the river valley sugar cane, dry rice and bananas are grown . Direct observation of swarms was undertaken in the manner described by charlwood & jones and charlwood et al ., . Swarming males and insects in copula were collected by sweep net, modified in the manner described by marchand . Artificial markers used were the same as those used in so tom . In order to determine whether size affected likelihood of a male swarming or mating the wing lengths of males collected resting, exiting houses, swarming and mating were measured . Collections of indoor resting insects were performed, with a torch and aspirator, for 10 minutes in buildings shown in figure 1 and exiting insects at dusk were monitored by placing a conical, double - size bednet over the gable - ends of houses within the study area . Map of four sites in which swarms of anopheles funestus were observed, furvela village, mozambique . Wing lengths, between the alula notch and the wing tip, excluding scales, of unselected samples of insects from all collections were measured using an ocular micrometer on a stereoscope to the nearest 0.03 mm . Wing lengths of the different groups were compared using anova . In order to determine if differences in flight sound between an . Gambiae, which might swarm together, differ (and therefore may act as potential barrier to prevent hybridization) the frequency distribution of individual insects, in free flight confined inside netting covered paper cups, was determined according to the method outlined in brogdon . Briefly, recordings of individual insects were made with a microphone (a sony ecm - d870p) either directly onto a portable computer or onto mini - disc and then transferred to computer and stored as wave files . The files were digitally sampled at 20,000 hz and then re - sampled at 5,000 hz and sonograms, plots of sound frequency versus time, prepared using computer software (spectrogram, public domain by richard horne). Temperature data was obtained for the two nearest weather stations (inhambane and vilanculos) available from the noaa website and the mean value used . There was, however, no obvious pattern as to the location of the swarms which could be at the edge or in the middle of the clearing (figure 1, figure 2). Swarms occurred 24 m off the ground, occupied a similar volume, and appeared to consist of a similar number of insects . At some sites two or more swarms occurred within a few metres of each other (figure 1). It was not possible to determine with any certainty, what the characteristics were that enabled males to maintain their position . At the site closest to the river valley (the putative breeding site) the swarm occurred in a gap in vegetation that was illuminated by the western sky (figure 1a). When this gap was artificially closed (by extending a sheet across it) males flew higher (so that they continued swarming in a gap). Two further swarms were discovered by looking for such gaps, and similar profiles occurred at other swarm sites, although in all other cases the' silhouette' was created at a much greater distance from the place where the swarm occurred . It was rarely possible to see the first male at the swarm site; rather, several insects started swarming at the same time . Numbers rapidly built up and within five minutes of the start had reached maximum size and density . On two occasions, dragonflies (libellula sp .) On one occasion all four males in a swarm were caught and eaten in a matter of seconds . Funestus were collected from a swarm of apparently larger insects that formed close to a higher and larger swarm from which 1 an . Funestus were collected swarming (a total of 601 males from 13 swarms) in sharp contrast to the an . Gambiae from so tom, swarming males avoided black or white horizontal markers and dispersed when these were introduced under the swarm . Despite there being a considerable number of male an . The number and time of pairs in copula was noted on 9 evenings of observation . The time at which the first pair was observed, relative to the time of the start of swarming, was inversely proportional to the total number observed throughout the swarming period (figure 3) limitation of resources meant that comparisons between sites on the same day were unavailable . 3.7 min after the start of swarming and had reached a maximum 4 min later . By the end of the observation period (when it became impossible to see the insects with the naked eye) numbers of mating pairs had almost declined to zero (figure 4). Relationship between the time of the first copula observed after the start of swarming and the total number of pairs subsequently counted . Number of pairs of anopheles funestus observed relative to the start of swarming, itself co - incident with sunset . All were newly emerged with undeveloped ovaries . Of the 71 unfed females dissected from resting collections 68 were virgins . The number of unfed females and males resting inside houses were positively associated (figure 5) but there was no association in the number of either of these categories with gravid and blood fed insects in resting collections . Relationship between the number of males and unfed (virgin) females collected resting inside houses, furvela village, mozambique . Overall both male and female wing size distributions were approximately normal and were not significantly different (figure 6). The wing length of males from resting collection, leaving houses, collected from swarms and in copula was similar (table 1) (figure 7a). The mean wing size of males differed according to collection site (one - way anova for 2 d.f ., p = 0.037). Among the females, those collected in light - traps (i.e. Host seeking) were smaller than the other categories (figure 7b). The wing size of females collected in copula were similar to that of newly emerged insects collected resting or exiting from houses . Wing length distribution of male and female anopheles funestus from furvela village, march - may 2002 . Winglength of male anopheles funestus collected at different phases of the activity cycle, furvela village, mozambique . During the period of study temperatures dropped from an average of 29c to 26c . Female wing sizes increased over this period but male size did not (figure 8). Mean male and female wing length of anopheles funestus and ambient temperature from furvela village, mozambique, by date of collection . Funestus were so common in furvela that it is strange that they have not been observed elsewhere . They differed from those recorded by harper in that they all occurred in very open spaces several metres off the ground rather than the threshold of houses a few centimetres off the floor . Anopheles funestus is now known to belong to a group of species, some of which are non - vectors and it is not certain which member of the group the author had observed . Gambiae seen in so tom . In both species they occurred at sunset 24 m above the ground in relatively open areas . Swarm size, dimensions and the estimated number of males per swarm appeared to be relatively constant between sites and species but the number of females entering swarms showed considerable variation . Swarm sites, each of which has a fixed capacity, may determine the dispersal of males . One might expect relatively more mating to take place in the swarms closest to the female emergence site . We were unable to determine the number of males in a swarm and hence unable to determine if male mating success rates were similar in the different swarms . The fact that male and newly emerged female ratios resting in houses were relatively constant implies that location of swarm has little influence on mating success . The lack of a relationship between either of these two groups and older females highlights the fact that entry routes, reasons for entry, sense used and time entered differs between the two categories . Males and virgin females use their eyes and enter houses at dawn usually via the darkest orifice (which usually means windows and doors). They are in search of a bloodmeal and use their sense of smell for direction . With a single exception funestus does not seem to be required since they are only likely to encounter con - specific females . On the one evening was obtained two distinct swarms, one of an apparently larger mosquito were seen prior to their being sampled . The swarms occurred circa 50 cm apart, with the larger insects swarming closer to the ground . It is not certain that the species were mixed . Given that they were caught swarming at least once during the study begs the question of why swarms of an . Were not more commonly observed or induced over markers in furvela as they were in so tom . One reason may be that the member of the complex observed in so tom was the m form of an . It would appear to be a different species from the other members of the complex and thus may behave differently . In particular all size groups were caught in copula, the caveat being that the numbers involved were small . The sampled houses were situated at varying distances from the river valley, the putative breeding site . . This may be due to the relatively large sample size or because size influenced male dispersal recently it has been shown that in small cages smaller female an . One of the difficulties with this interpretation is that a male can inseminate four or more females whilst females are largely monogamous . Given the equal sex ratio at emergence an average male will only meet a single female in his lifetime . If so it would seem unlikely that males would be too selective since there is probably another set of genes around that is not and should a further female come along then she can still be fertilized . Indeed the size distribution of females caught in copula was not significantly different from those emerging . As temperatures drop the duration of larval and pupal stages increase and the size of emerging insects increases . The duration of the larval stage in an . The effect of the drop in temperature on mean female size but not male size may be because the adult males collected had not been exposed as larvae, or particularly as pupae to such low temperatures as the females . Funestus avoided such markers (both light and dark) and dispersed if they were placed underneath naturally occurring swarms . The response to potential horizontal markers is likely to be sufficient to prevent mixed swarms from occurring and this is likely to be the factor responsible for isolation between an . Charlwood conducted the work in the field and helped prepare the manuscript thompson provided logistical support to the fieldwork madsen helped prepare the manuscript, was responsible for figures and provided statistical support jdc would like to thank marcia, elsa and luis for their help in swarm watching . This study was undertaken as part of the mozdan project and was supported by the danish bilharziasis laboratory.
In this debate, the author takes the position that ldl cholesterol is a major causative factor in the huge prevalence and incidence of arteriosclerotic disease that has plagued the western world and which is now growing rapidly in developing countries . In examining population data, the risk of clinical vascular disease is a continuous function of rising plasma cholesterol, which is attributable to a rise in ldl cholesterol . Those cultures estimated to have lifelong ldl cholesterol values at or below 100 mg / dl have previously experienced myocardial infarction and coronary death rates that are 70 to 90% below those in eastern europe where ldl has been 50 to 70% higher . As economic changes have brought about dietary changes, plasma cholesterol levels have risen, as have other major risk factors such as obesity and diabetes . Even in societies with low total cholesterol levels, preliminary evidence indicates that declining risk extends to values below 160 mg / dl (4.2 mmol / l), which is equivalent to an ldl level of approximately 100 mg / dl (2.6 mmol / l). Lowering plasma cholesterol by a variety of means - including diet, surgical bypass of the distal ileum, bile acid sequestrants, fibric acid derivatives and most recently statins - has led to a reduction in events both in patients with known clinical vascular disease as well in those without . It is highly probable that reducing the population distribution curve of ldl cholesterol values to a mean of around 100 mg / dl (total cholesterol of approximately 150 - 160 mg / dl) would cause a major change in the incidence of this problem . Furthermore, reductions well below 100 mg / dl, where feasible and cost effective, are predicted to give an additional margin of protection that could save millions of lives . The national cholesterol education program (ncep) in the us and the joint european commission have taken the position that health care providers should set the most aggressive treatment goals in those at highest risk . The ncep has chosen the ldl - c target of less than 100 mg / dl for all patients with clinically evident arteriosclerotic vascular disease . Furthermore, this organization is considering using other indicators of impending events, such as diabetes mellitus, reduced ankle / arm blood pressure ratios, and integrated risk analysis (' global risk'), to define additional populations that should be included in this ldl - c goal . The european recommendations suggest setting an ldl - c goal of 115 mg / dl (3.0 mmol / l), or less, in those patients with a global risk analysis that predicts a 20% probability of having a myocardial infarction (mi) or coronary artery death (cad) in the next 10 years . Accordingly, we will attempt to develop the argument for reducing the ldl cholesterol to values below 100 mg / dl (2.6 mmol / l) by sequentially focusing on defined populations ranked by increasing risk of suffering a major cardiovascular event . Those at highest risk are patients who have suffered a recent major spontaneous event such as a myocardial infarction or hospitalization for unstable angina . Recurrent hospitalization for cad or coronary death occurs in 12 to 20% of this group within 1 year . The recently reported myocardial ischemia reduction with aggressive cholesterol lowering trial (miracl) [17, 18] selected 3080 patients in hospital for non - q wave mi or unstable angina and randomly assigned them to receive atorvastatin (80 mg / d) or placebo for four months . Initially the ldl - c was only 124 mg / dl . By the end of the study, it had risen to 135 mg / dl in the control group but had been reduced to 72 mg / dl in those on the statin . Those treated with atorvastatin had experienced 16% fewer endpoints defined as' primary' (14.8% versus 17.4%). These included total death, nonfatal acute mi, cardiac arrest with resuscitation, and urgent hospitalization for angina (with objective evidence of ischemia). The hospitalizations for angina, when considered alone, were reduced by 26% (6.2% versus 8.4%). A second high - risk group consists of those symptomatic patients who are evaluated by coronary angiography and who are found to be suitable for treatment with percutaneous transluminal coronary angioplasty . The atorvastatin versus revascularization treatments (avert) study randomly assigned 341 such patients to receive either the angioplasty and usual medical care, or to forego the angioplasty and instead begin immediately on atorvastatin (80 mg / dl). After eighteen months, 21% of those receiving standard care had had a major vascular event compared to only 13% of those with more aggressive ldl - c reduction . The time to the first ischemic event after randomization was significantly longer in those given atorvastatin but no angioplasty . At the close of the trial, the mean ldl - c was 77 mg / dl in the atorvastatin treated group compared to 119 mg / dl with usual care . It is of note that over 70% of the latter group had received various lipid - lowering medications during the trial but at low doses, and that these were often started later in the study . Patients who have undergone coronary artery bypass surgery are at high risk of worsening disease in native arteries as well as new lesions in the vein grafts . The post - coronary artery bypass graft study (post - cabg) selected 1351 patients who had undergone this procedure one to eleven years earlier, had ldl - c levels of 130 to 175 mg / dl, and who had patent vein grafts . The cohort was randomly divided into two groups, one to be aggressively treated, and the other to have more modest ldl - c reduction . Diet and drug regimens containing lovastatin and cholestyramine were used to titrate the ldl - c to values of 95 to 97 mg / dl in an aggessively treated group . These were compared to a second randomly selected group in which the ldl - c was reduced to only 135 to 137 mg / dl, thus leaving them some 40 mg / dl higher for the duration of the study . After 4.3 years, the mean luminal diameter, the number of new lesions, and the percentage narrowing at major stenoses were all significantly better in the group with the greater reduction of ldl - c . Furthermore, the incidence of new revascularization procedures was reduced by 29% . The number of major clinical events was also reduced in the aggressively treated group after an additional three years of monitoring . Large long - term clinical trials using statins have achieved reductions in ldl - c of 26 to 35% with concomitant reductions in major vascular disease events of 24 to 37% . When considered separately, the cohorts with known chd and those without demonstrated a strong trend to lower event rates with lower group mean ldl - c during treatment with either drug (simvastatin) or placebo . In the 4s study, this high - risk group of coronary patients experienced a stepwise lower incidence of clinical events when ranked by tertiles of ldl - c while on treatment . The lowest tertile on simvastatin, with ldl - c of less than 104 mg / dl, had an incidence of mi and cad of 10.8% compared to rates of 13.3% in the middle (ldl - c 105 to 126 mg / dl) and 18.9% for the upper tertile (> 126 mg / dl). Other studies, particularly those using pravastatin such as cholesterol and recurrent events (care) and west of scotland coronary prevention study (woscops), have not reported similar findings . Those groups on treatment, who demonstrated a fall in ldl - c of more than approximately 25%, did not appear to enjoy any additional benefits . It should be kept in mind, however, that comparisons of subgroups within study cohorts are often distorted by various biases that are generated by the assumptions and selection criteria of the study and by the study procedures themselves . The only scientifically valid method to measure the benefits of further reduction in ldl - c is to perform a randomized and blinded comparison of groups treated to various ldl - c values . Fortunately, such studies are underway with interesting names such as treating to new targets (tnt), incremental decrease in endpoints through aggressive lipid lowering trial (ideal), study of the effectiveness of additional reductions in cholesterol and homocysteine (search), and heart protection . A definitive answer to whether leaving ldl - c just above 100 mg / dl is adequate or whether a significant further risk reduction can be achieved by lowering ldl - c by an additional 30 to 50%, will be available within the next five years . However, it should be recognized that these studies address the middle aged and older individuals who already have clinically evident disease . The extremely important question of the optimum ldl - c for children, young adults, and those without clinical disease will remain with us for some time . For the next decade it will be necessary to compromise our efforts in an attempt to use our current resources to give the greatest gain in controlling the clinical disease . The cost of screening, monitoring, and current drugs adds unwanted economic burdens to most societies . It is in this context that a rational argument can be made for more relaxed goals than those medically possible today . However, we should not be satisfied with the status quo since studies, such as pathobiological determinants of atherosclerosis in youth (pday), demonstrate that after 30 years of age the majority of adult patients in the usa already have the disease of arteriosclerosis and we are only delaying its clinical appearance . The result is a growth in the number of elderly patients whose lives are compromised by vascular disease . Gaining knowledge of the effects of lowering ldl - c to much lower levels (ie around 50 mg / dl) is extremely important and relevant to practical issues . Setting targets at this level will soon be feasible for most patients as new and more powerful statins appear and as new drugs with totally different mechanisms of action (eg reducing bile acid absorption, cholesterol absorption and or lipoprotein synthesis) are developed . Clearer concepts of the total societal costs, as well as the potential economic benefits of various preventive measures and treatments, must evolve so that the best policies can be developed to take advantage of thedemonstrated efficacy of such drugs . With a world already experiencing approximately 11,000,000 deaths annually from chd and stroke, the potential volume of usage will put major pressure on those attempting to reduce costs and improve efficiency of drug and health care delivery systems . Current trends strongly indicate that earlier treatment and more aggressive goals for ldl - c reduction will be targets for the future.
Glenohumeral (gh) joint contracture can occur after repair of rotator cuff tears, frozen shoulder, and rheumatoid arthritis1 . This can be caused by capsular contracture, tendon shortening, and scars2, 3 . Manual shoulder joint test is the end feel that the physical therapist feels during the resistance of the tissue against the force applied manually from the different directions of the articular surface and greatly depends on subjective evaluation4 . To test the stiffness and looseness of the articular capsule, one of the causes of limited shoulder movement, various orthopedic manual techniques are used5, 6 . According a study on manual therapy, traction can be used as a diagnostic tool to assess joint play or as treatment to relieve pain or improve joint mobility7 . In particular, the traction method according to the kaltenborn - evjenth concept is a safe and effective method to test the condition of the articular capsule in the field of orthopedic physical therapy8 . G1 represents the force necessary to remove the compressive forces acting on the joint . In grade 2 (g2), the slack is taken up from the tissue surrounding the joint, which are then tightened . Finally, in grade 3 (g3), additional force is applied and the soft tissues surrounding the joint are stretched; thereby, separation of the joint surfaces is achieved . Traction is preferably performed in the maximal loose - packed position (mlpp) of the joint (shoulder 55 abduction and 30 horizontal adduction in the transverse plane). In the mlpp, the traction is generally assumed to be most effective because the joint capsule is most relaxed10 . Dvorak and coworkers stated that up to 5 mm of separation of the joint surfaces is physiological during joint play testing11 . Gokeler et al . Reported that in the comparison of joint space width (jsw) between the humeral head and the glenoid fossa after applying a 14-kg load to the gh joint of the asymptomatic subjects during manual traction, jsw was widened by about 0.3 mm, but difference was not significant12 . Meanwhile, in the research by sato et al ., jsws were compared after applying traction to the point that the maximum resistance in the hip joint was detected in symptomatic subjects13 . The results of the research indicated that jsw was widened by about 0.9 mm, with a significant difference . Perhaps, the structural difference between the gh and the hip joints in both research studies might have affected the results . Therefore, when applying traction to the gh point, the exact degree of separation of the humeral head from the glenoid fossa is unknown . In addition, quantitative reports related to the application of traction are lacking, and traction remains dependent on the sensation felt by therapists in their fingertips . The aim of this study was to analyze the jsw of the humeral head and glenoid fossa during traction under 2 grade conditions (g2 and g3). The subjects of this study were individuals who did not have medical histories of shoulder injury and damage to the nervous and musculoskeletal systems, which may affect the range of shoulder movement, and have not performed shoulder muscle strength training during the recent 6 months prior to the study . The research subjects were selected from among 20 male employees who worked at h hospital, located in ulsan metropolitan city . Before starting the test, they were given enough explanation on the test and participated voluntarily in the test after submitting a signed consent form . This study was approved by the ethics commission of daegu university according to the declaration of helsinki . The general characteristics of the subject are shown in table 1table 1.general characteristics of subjects (n = 20)characteristicsmean sdage (years)31.72.9height (cm)174.63.7weight (kg)70.57.0bmi (kg / m)23.12.0bmi: body mass index . Traction was applied to the dominant side of the subjects shoulder . The dominant hand, defined as the hand used for eating and writing, was the right hand in all of the subjects . The subjects lied down on the radiography table, rotating the trunk 30 to the right so that the scapula at the shooting side touched on the table . The opposite shoulder was supported by using a triangular support angled at 30 so that the subject could lie down comfortably while maintaining the rotated position . To prevent scapular movement during traction, the tester positioned the subject s shoulder abduction to 50 after the subject s palms were positioned facing up . This position was used as the resting position and the mlpp of the shoulder joint . Taking the proximal humerus with both hands, the humerus was pulled toward the perpendicular direction against the glenoid fossa . The traction was performed by a physical therapist who completed educational courses on the keltenborn - evejenth concept and had at least 10 years of experience with joint mobilization . Immediately after applying traction all the radiographies were performed by the same radiologist, with anteroposterior projection (diagnostic radiography, listem, co., ltd ., japan). For random assignment of order by grade, the subjects were told to pick one card after placing cards with nos . 2 or 3 written on it in a box . Radiography was performed by applying the resting position, g3 on the first day, and g2 on the next day . For those who picked no . 2, radiography was performed by applying the resting position, g2 on the first day, and g3 on the next day . Radiography was performed at the same hour for the same person for 2 days . Other grades were applied in the same manner after 1 day so that the structure of the joint can return to its normal condition . In the computer that received the images taken by using the pacs program, data were calculated by using the method suggested by petersson and redlund - johnell14 . In this position, the projection of the joint surface of the humeral head forms a semicircle, the diameter of which is the line joining the two terminal points of the joint surface projection . One point was determined each at the superior and inferior edges of the glenoid fossa, making a total of three points . Each of the three points of the glenoid cavity was connected to the center point of the humeral head by placing the ruler . The radiologist was told how to conduct the measurement before the test and made to practice adequately how to measure by using the shoulder joint pictures obtained by other radiologists . In addition, information on the traction grade was withheld until all the measurements were finished . Prior to the analysis, data normality were tested by using the kolmogorov - smirnov . Icc(3.1) (icc, intraclass correlation coefficients) was used to derive the intra - rater reliability of the jsw measurements . To compare jsw according to gh traction grade, one - way repeated - measures analysis of variance (anova) was performed, and a least square difference was performed for post hoc analysis . The results of the statistical analyses are shown in table 2table 2.joint space width by the shoulder traction grade (mean sd)grouprestinggrade 2grade 3jsw (mm)4.00.34.50.44.70.5significant difference between resting and grade 2 traction (p <0.05). Significant difference between resting and grade 3 traction (p <0.05) jsw: joint space width . The results of the one - way repeated - measures anova to verify the changes in jsw according to grade indicated significant differences in jsw . Post hoc analysis showed that the jsw was significantly higher with g3 traction than with g2 traction and resting position . However, no significant difference in jsw was found between g2 and g3 . The intra - rater reliability for jsw measurements was good to excellent (icc3,1 = 0.880.98) significant difference between resting and grade 2 traction (p <0.05). Significant difference between resting and grade 3 traction (p <0.05) jsw: joint space width this study was performed to analyze the changes in jsw between the glenoid fossa and humeral head, while gh traction was applied according to grade . The jsw for the resting position was 4 mm . In the kaltenborn - evjenth concept, g1 traction is explained as the stage to remove the pressure force to the joint without separation of joint10 . Therefore, in this study, g1 traction was not applied in the resting position . In the g2 traction, jsw was 4.5 mm, increased significantly by 0.5 mm from that in the resting position . G2 traction refers to the moment that the slack of the articular capsule is released, and the collagen fibers within the tissue become tight by increasing the tension gradually . In addition, the jsw in the g3 traction was 4.7 mm, increased significantly by 0.7 mm from that in the resting position . In the g3 traction, greater force than the traction given in g2 was applied to the tissue, stretching the collagen fiber . In this moment, it does not cross the rupture point of the collagen fiber, which explains the reason why jsw in the g3 traction was increased by 0.2 mm, more than the 0.5 mm in the g2 traction . Al . Reported that jsw was increased significantly by 0.9 mm, from 3.86 to 3.95 mm, as a result of hip joint traction in the maximum relaxation position in the healthy adults13 . Applied a 14-kg force, which is equivalent to the gh traction, to a healthy person for repair of the scapula, and the jsw was increased by approximately 0.3 mm12 . The difference is due to the position of the subject during traction . In this study, traction was performed in the supine position; whereas in the research by goker et al ., it was performed in the sitting position . Considering that in the sitting point, the humerus is affected by gravity, the resistance of the tissue might be increased while in the sitting position . As the resistance of the tissue was small because most of the connective tissues are not affected by the gravity dvorak and coworkers stated that up to 5 mm of separation is normal in the mlpp during joint play testing but did not provide evidence to support their statement11 . When g3 traction was applied, only an increase of 0.6 mm was observed . Jsw was increased by 0.2 mm in g2 and g3, but without significant difference . This means that jsw was not increased greatly even when greater force was applied through the g3 traction to the joint capsule stretched tightly in the g2 traction . However, the therapists can adequately feel the difference in the end feel of the tissue during the g3 traction . The limitations of this study are as follows: first, as the number of the subjects was small, the study results are hard to generalize . Second, as this study was performed with healthy persons, we could not apply traction to the shoulder joint of patients with a frozen shoulder . Many physical therapists have used traction according to the kaltenborn - evjenth concept, but because they judge the grade subjectively, simply depending on the sensation of the therapist and the end feel of the joint, they could not know exactly how much space within the joint is separated, until recently . In conclusion, in this study, we found that although the g2 and g3 tractions during the gh traction in the healthy persons did not significantly differ, the increase in the jsw between the glenoid fossa and the humeral head was highest during the g3 traction . This is deemed to provide help for therapists in the safe and effective application of traction techniques in clinical cases.
Spinal subdural hematomas (ssdhs) have been reported to occur after minor trauma, lumbar puncture, spinal anesthesia,1 or spinal surgery, especially in the presence of intraoperative dural tears.2 nontraumatic (spontaneous) ssdhs are much more rare, with a recent review having identified 106 cases reported in the english literature.3 several predisposing factors have been associated with the occurrence of spontaneous ssdhs, such as coagulation abnormalities, anticoagulation therapy,4 platelet dysfunction,5 polycythemia vera,6 pregnancy,7 arterial wall abnormalities,8 and, more rarely, the presence of spinal arteriovenous malformations.9 most ssdhs occur at the lower thoracic region (the second most common location being the upper cervical spine) and typically extend from two to five segments.10 the vast majority of ssdhs are located anteriorly to the spinal cord, while most epidural spinal hematomas are located posteriorly to the spinal cord.11 this difference is related to the fact that the posterior longitudinal ligament closely adheres to the vertebral bodies, thus limiting anterior epidural collections . Interestingly, the vast majority of case reports on operated ssdhs identified no intermingling between the subdural blood and the cerebrospinal fluid (csf). Because the hematoma is confined to the extra - arachnoid space, some authors have suggested that what is commonly designated as an ssdh is, in fact, a dissection between the two inner layers of the spinal dura.12 this concept is supported by anatomic studies that demonstrated that under physiologic conditions, the spinal subdural space is only a capillary slit, which may occasionally extend into a genuine space under pathologic conditions such as during a subdural bleeding.10 13 percutaneous vertebroplasty is a therapeutic strategy that has gained increasing interest in the neurosurgical community for the treatment of refractory axial mechanical pain in patients with vertebral compression fractures . One of the main therapeutic mechanisms of cement augmentation has been proposed to be the improvement in the spinal stability provided by the injection of polymethyl methacrylate (pmma) into the fractured vertebral body.14 several complications of vertebroplasty have been reported in the literature, with the vast majority of them being related to cement extravasation to the epidural canal leading to spinal cord compression (some series report extravasation rates of up to 20% with approximately one - third of such patients being neurologically symptomatic and requiring surgical intervention)15 or related to cement migration through the epidural veins to the venous system leading to pulmonary embolism (reported to occur in 0.8 to 2.1% of the patients, with the vast majority of patients being asymptomatic).16 in this context, the severity grade of the fracture and a low viscosity of pmma cement have been identified as strong and independent risk factors for cement leakage.17 in this article, we report a patient who developed an acute ssdh following a percutaneous vertebroplasty without signs of major cement extravasation to the spinal canal . The authors perform a comprehensive literature review on the pathophysiology of ssdh and highlight the specific nuances of the presented case report to support a new hypothetical role of venous congestion as a possible etiologic factor involved in the pathophysiology of ssdh . Finally, directions for future experimental and clinical research for further investigation of such hypothesis are also delineated . A 49-year - old woman presented to the outpatient clinic with complaints of axial midthoracic pain and a history of a recent fall down the stairs . At that time, the computed tomography (ct) scan of the thoracic spine demonstrated a t8 compression fracture affecting mainly the midportion of the vertebral body, with preservation of the posterior cortex (fig . She was treated conservatively with a tlso brace and analgesics . However, at the 3-months follow - up, she still presented with episodes of severe, deep axial pain centered on her midthoracic spine . The pain was essentially mechanical in nature, with worsening of the symptoms with activity and partial improvement with bedrest . The 3-months follow - up x - ray demonstrated progression of the compression fracture with further loss of height especially in the anterior third of the vertebral body leading to worsening of the kyphotic deformity (fig . (a) initial sagittal computed tomography scan demonstrating a t8 compression fracture affecting mainly the midportion of the vertebral body, with preservation of the posterior cortex . (b) lateral plain x - ray of the thoracic spine performed at the 3-months follow - up after failed conservative treatment demonstrating progression of the fracture with further loss of height especially in the anterior third of the vertebral body, leading to worsening of the kyphotic deformity . After cannulation of the left t8 pedicle and the initial injection of pmma, a small posterior extravasation of cement to the epidural veins was observed (fig . After awaking, the patient presented diffuse numbness on the left side (both in the superior and inferior limbs) as well as diffuse weakness (strength 3/5) in the left leg . An immediate ct scan demonstrated only a very small posterior leakage of pmma toward the epidural space as well as into the adjacent costotransverse joint (fig . 2). However, it was possible to observe a hyperdense collection anterior to the spinal cord beginning one level above the site of the vertebroplasty and extending up to the cervical spine . The presence of pneumorrhachis at a point distant to the level of the vertebroplasty was also identified . (a) intraoperative fluoroscopy demonstrating a small extravasation of cement to the posterior epidural veins in the most superior region of the vertebral body . Immediate postprocedure (b) sagittal and (c) axial computed tomography scans demonstrating only a very small posterior leakage toward the spinal canal, as well as the presence of (d) a spinal subdural hematoma (black arrow) extending from one level above the vertebroplasty to the lower cervical spine . Note also the presence of pneumorrhachis (white arrow). Due to the acute motor deficit in the left lower limb, the patient was submitted to an immediate decompressive laminectomy at the level of the vertebroplasty and extending one level above and one level below to encompass those levels in which the cross - sectional area of the spinal canal was significantly compromised . No major compression of the thecal sac or epidural bleeding was identified during the surgical procedure . The postoperative magnetic resonance imaging (fig . 3) confirmed the presence of a collection with imaging characteristics of an ssdh, beginning one level above the vertebroplasty and extending up to the lower cervical spine, but without any residual signs of compression, as demonstrated by the presence of csf posterior to the spinal cord (fig . The subdural collection is located anterior to the spinal cord and extends up to the cervical spine . The acute spinal subdural hematoma is hyperdense in both t1- (a) and t2-weighted imaging (b). The axial images demonstrate no residual compression either at the laminectomy level (c) or in the levels above (d) as evidenced by the presence of cerebrospinal fluid posterior to the spinal cord . After physical therapy and rehabilitation the patient progressively recovered the motor function in the left leg . At the 3-months follow - up, she presented with almost complete recovery of her strength in the left leg (grade 4+/5 for hip flexion, hip extension, leg flexion and extension, and dorsiflexion and plantar flexion), although the sensory symptoms still persisted in the whole left side, both in the superior and inferior limbs, requiring treatment with gabapentin . The vertebral venous plexus is a complex, large - capacity, plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . Additionally, due to this plexus' valveless, bidirectional flow, it also provides a direct route for tumor or infection dissemination from the pelvic and lower lumbar region to the thoracolumbar spine . The fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover, the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal, intrathoracic, intracranial, and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries; special acknowledgments must be given to the works of batson (18941979), after whom this venous plexus is named, and breschet (17841845).20 the vertebral venous plexus has been classically divided into an internal (intradural) vertebral plexus (which possesses an anterior and a posterior component) and an external (or epidural) vertebral plexus . Recently, it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies, several small subsystems of plexiform veins work synergistically to accomplish their venous drainage (fig . 4). The basivertebral system, which is arranged horizontally in the middle of the vertebral body, forms a large - scale venous grid into which the vertical veins of the vertebral body flow from above and below . The subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins, which abruptly turn to run horizontally, parallel to the vertebral endplates, ultimately draining posteriorly into the epidural venous plexus.23 (a) schematic representation of the vertebral venous system, which can be divided into an internal vertebral plexus (composed of 1: anterior internal vertebral venous plexus; 2: posterior internal vertebral venous plexus), and an external (or epidural) vertebral plexus (composed of 4: posterior external vertebral venous plexus; 5: anterior external vertebral venous plexus; and 8: radicular vein) and the (6) radiculomedullary vein, which connects both . The drainage of the vertebral body is mainly performed by the (3) basivertebral plexus and the subarticular collecting plexus (not shown in this image). (b) a radiograph of a thin coronal section near the central area of a lumbar vertebra . Note the stellate arrangement of tributaries draining into the central vein of the basivertebral plexus . (c) radiograph of a thin sagittal section cut laterally near the vertebral pedicle . The horizontal subarticular collecting venous system of the vertebral body can be seen running parallel to the inferior vertebral endplate . This system drains by vertical stems through perforations in the vertebral endplates into the larger horizontal subarticular collecting vein system . (a) reproduced with permission from groen rjm, grobbelaar m, muller cjf, et al . Morphology of the human internal vertebral venous plexus: a cadaver study after latex injection in the 2125-week fetus . (b and c) reproduced with permission and copyright of the british editorial society of bone and joint surgery from crock hv, yoshizawa h, kame sk . J bone joint surg br 1973;55(3):528533.23 similar to its arterial counterparts, multiple radiculomedullary veins provide segmental drainage to the emerging spinal nerve roots . These radiculomedullary veins run in the same oblique course as the radiculomedullary arteries, but typically arise at different spinal levels . It has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system (the internal vertebral venous plexus) and the epidural space (the external vertebral venous plexus).24 although presenting a small caliber, such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . In fact, several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover, although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . Additionally, no previous histologic study has investigated the actual vulnerability of such apparently weak connection point between the epidural vertebral venous system and the internal venous plexus, as well as its response to congestive venous hypertension . Although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder, the exact pathophysiology of ssdh still remains obscure . Intracranial subdural hematomas have been commonly ascribed to the rupture of subdural bridging veins . However, unlike the intracranial subdural space, it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly, previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure (such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae (a place of lower resistance) in the vascular venous system between the internal and external vertebral venous plexus, which, when submitted to excessive pressures due to venous congestion, would possibly rupture, ultimately leading to extravasation of blood into the subdural space.35 36 alternatively, some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin, it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case, another factor suggesting an etiology involving venous congestion (related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . In the same way, the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally, the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas (the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis (presence of air in the subdural space) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure, ultimately leading to focal accumulation of air in the subdural space . Similarly, previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure (such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature (with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage), it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . Also, in the absence of radiologic evidence of spinal cord compression (such as in the reported case, in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value, especially in extensive lesions involving several levels as in the reported case.26 in the long term, it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh, ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the vertebral venous plexus is a complex, large - capacity, plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . Additionally, due to this plexus' valveless, bidirectional flow, it also provides a direct route for tumor or infection dissemination from the pelvic and lower lumbar region to the thoracolumbar spine . The fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover, the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal, intrathoracic, intracranial, and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries; special acknowledgments must be given to the works of batson (18941979), after whom this venous plexus is named, and breschet (17841845).20 the vertebral venous plexus has been classically divided into an internal (intradural) vertebral plexus (which possesses an anterior and a posterior component) and an external (or epidural) vertebral plexus . Recently, it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies, several small subsystems of plexiform veins work synergistically to accomplish their venous drainage (fig . 4). The basivertebral system, which is arranged horizontally in the middle of the vertebral body, forms a large - scale venous grid into which the vertical veins of the vertebral body flow from above and below . The subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins, which abruptly turn to run horizontally, parallel to the vertebral endplates, ultimately draining posteriorly into the epidural venous plexus.23 (a) schematic representation of the vertebral venous system, which can be divided into an internal vertebral plexus (composed of 1: anterior internal vertebral venous plexus; 2: posterior internal vertebral venous plexus), and an external (or epidural) vertebral plexus (composed of 4: posterior external vertebral venous plexus; 5: anterior external vertebral venous plexus; and 8: radicular vein) and the (6) radiculomedullary vein, which connects both . The drainage of the vertebral body is mainly performed by the (3) basivertebral plexus and the subarticular collecting plexus (not shown in this image). (b) a radiograph of a thin coronal section near the central area of a lumbar vertebra . Note the stellate arrangement of tributaries draining into the central vein of the basivertebral plexus . (c) radiograph of a thin sagittal section cut laterally near the vertebral pedicle . The horizontal subarticular collecting venous system of the vertebral body can be seen running parallel to the inferior vertebral endplate . This system drains by vertical stems through perforations in the vertebral endplates into the larger horizontal subarticular collecting vein system . (a) reproduced with permission from groen rjm, grobbelaar m, muller cjf, et al . Morphology of the human internal vertebral venous plexus: a cadaver study after latex injection in the 2125-week fetus . (b and c) reproduced with permission and copyright of the british editorial society of bone and joint surgery from crock hv, yoshizawa h, kame sk . J bone joint surg br 1973;55(3):528533.23 similar to its arterial counterparts, multiple radiculomedullary veins provide segmental drainage to the emerging spinal nerve roots . These radiculomedullary veins run in the same oblique course as the radiculomedullary arteries, but typically arise at different spinal levels . It has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system (the internal vertebral venous plexus) and the epidural space (the external vertebral venous plexus).24 although presenting a small caliber, such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . In fact, several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover, although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . Additionally, no previous histologic study has investigated the actual vulnerability of such apparently weak connection point between the epidural vertebral venous system and the internal venous plexus, as well as its response to congestive venous hypertension . Although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder, the exact pathophysiology of ssdh still remains obscure . Intracranial subdural hematomas have been commonly ascribed to the rupture of subdural bridging veins . However, unlike the intracranial subdural space, it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly, previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure (such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae (a place of lower resistance) in the vascular venous system between the internal and external vertebral venous plexus, which, when submitted to excessive pressures due to venous congestion, would possibly rupture, ultimately leading to extravasation of blood into the subdural space.35 36 alternatively, some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin, it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case, another factor suggesting an etiology involving venous congestion (related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . In the same way, the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally, the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas (the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis (presence of air in the subdural space) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure, ultimately leading to focal accumulation of air in the subdural space . Similarly, previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure (such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature (with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage), it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . Also, in the absence of radiologic evidence of spinal cord compression (such as in the reported case, in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value, especially in extensive lesions involving several levels as in the reported case.26 in the long term, it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh, ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the occurrence of such a complication in a level different from that of the intervention and in the absence of a major pmma extravasation into the spinal canal (as well as in the presence of pneumorrhachis) strongly suggests a venous congestive mechanism as the most plausible etiologic explanation . In this article, we propose a new pathophysiological scheme that may explain the development of ssdhs, at least in a subset of patients . This newly proposed etiologic pathway emphasizes the role of venous congestion leading to the rupture of the fragile radiculomedullary veins into the subdural space . Ultimately, this case report highlights the necessity of further anatomical and histologic studies of the radiculomedullary veins to better investigate their behavior in the face of congestive venous hypertension, as well as the supposed relationship between their possible rupture and the development of ssdhs.
The methionine cycle provides methyl units for a variety of reactions such as the methylation of proteins, dna, rna and lipids, allowing for the modulation of their biological functions (bauerle et al ., 2015, gut and verdin, 2013). S - adenosyl methionine (sam) is the primary methyl donor molecule utilized in cellular methylation reactions and is synthesized directly from the essential amino acid methionine (cantoni, 1952) (figure s1a). The function of methylation reactions is context and substrate dependent; for example, dna and histone methylation are epigenetic modifications that have important influences on chromatin structure and regulation of gene expression (cedar and bergman, 2009, gut and verdin, 2013). Previously, dna methylation was perceived as a relatively stable epigenetic modification with the propensity to encode heritable epigenetic information (dolinoy, 2007, dolinoy et al . However, recent work has highlighted the importance of dynamic control of dna methylation, for example, in embryogenesis (guo et al ., 2014, smith et al ., 2014), cardiomyocyte development (gilsbach et al ., 2014), and actively transcribed and regulatory regions of dna (kangaspeska et al ., 2008, mtivier et al ., 2008, schbeler, 2015). It is therefore increasingly appreciated that existing dna, as well as newly synthesized dna, can be dynamically methylated and demethylated (bhutani et al ., 2011,, 2012, feldmann et al ., 2013, kohli and zhang, 2013, yu et al ., 2012). Accordingly, it is important to define the cellular processes that control dynamic methylation of nucleic acids . As the fields of epigenetics and cellular metabolism particularly cancer cell metabolism have developed in recent years, so has the appreciation of the fundamental crosstalk between these processes (gut and verdin, 2013, hino et al ., 2013, previous studies have shown that histone modifications are responsive to metabolite levels; for example, glucose - derived acetyl - coa influences histone acetylation via atp - citrate lyase (wellen et al ., 2009). In gliomas, idh1 mutation is responsible for the generation of 2hg, which inhibits the dna hydroxylase tet and leads to altered methylation of histones and dna, so driving the phenotype of these tumors (christensen et al ., 2011, figueroa et al ., 2010, turcan et al ., 2012). Mouse es cells depend on threonine to maintain sam synthesis, with threonine starvation leading to decreased histone methylation and inhibited proliferation (shyh - chang et al ., 2013). These findings and others the importance of folate - mediated one - carbon metabolism for cancer cell proliferation has long been appreciated (locasale, 2013). Serine plays a key role in feeding one - carbon units to the tetrahydrofolate (thf) cycle and supports both nucleotide synthesis and nadph production (fan et al . Cancer cells have high demand for serine, which they meet by a combination of exogenous serine uptake and de novo synthesis from glucose (locasale and cantley, 2011, maddocks et al ., 2013, interestingly, the serine synthesis pathway enzymes can be epigenetically activated by histone h3 methyltransferase g9a to support cancer cell survival and proliferation (ding et al ., 2013). In addition to nucleotide production, one - carbon metabolism can also contribute to the methionine cycle by providing one - carbons, in the form of methyl groups, to recycle homocysteine to methionine (herbig et al ., 2002, lu and mato, 2012). Partitioning of one - carbon units between nucleotide synthesis and homocysteine remethylation can be controlled by cytoplasmic serine hydroxymethyltransferase (shmt1) (herbig et al ., 2002, with a decreased flux to thymidine synthesis associated with increased uracil incorporation into dna and enhanced cancer risk in some models (macfarlane et al ., 2011). By contrast, modulation of mthfd1 has been shown to lower sam levels but decrease uracil incorporation into dna, suggesting a switch toward nucleotide synthesis (macfarlane et al ., 2009). Nevertheless, this change in folate metabolism also increased tumorigenesis in some tumor models (macfarlane et al ., 2011). Dietary and genetic perturbation of folate mediated one - carbon metabolism have been shown to disrupt both the synthesis and methylation of dna (stover, 2004), and while these two pathways are intimately linked, how they are coordinated remains to be fully elucidated . Atp is the major energy carrier used in cellular metabolism (hardie et al ., 2012). The inter - conversion of amp, adp, and atp is a fundamental reaction in cellular biology allowing for the transfer of energy from energy yielding reactions (e.g., glycolysis) to energy consuming reactions (e.g., fatty acid synthesis)(hardie et al ., however, there is a clear but potentially underappreciated biochemical distinction between atp turnover via energetic reactions (i.e., regeneration of atp by re - phosphorylation of amp and adp) and atp synthesis (i.e., atp generated by de novo purine synthesis, a process that requires glycine and one - carbon units, which are both commonly derived from serine in cancer cells). Our previous studies highlighted the importance of serine in supporting purine synthesis, showing that glycine did not provide the one - carbon units necessary for the de novo synthesis of amp, adp, and atp in the cancer cells we studied (labuschagne et al . While purine synthesis provides vital nucleotides for nucleic acid synthesis, atp generated by this biosynthetic pathway has the potential to be used in other metabolic reactions . In this study, we analyze the transfer of c labeled carbon from methionine and serine directly onto dna or rna in cancer cells . Under methionine - fed conditions, serine did not contribute one - carbon units to recycle methionine, although serine did support dna and rna methylation under these conditions . Surprisingly, we show that this contribution of serine is through the de novo synthesis of atp, which is required to convert methionine to sam . Our data show that non - energetic metabolic stress can cause a dramatic decrease in total atp levels in rapidly proliferating cells, which can lead to changes in methyl group transfer without inducing activation of amp - activated protein kinase (ampk). We have previously seen that serine availability has a major influence on the growth and metabolism of colorectal cancer cells (labuschagne et al ., 2014, maddocks et al ., 2013), with a clear impact on one - carbon metabolism and purine synthesis . To analyze the contribution of serine - dependent one - carbon metabolism to the methionine cycle and methylation reactions (figure s1a) in these cells, we developed an assay to track one - carbons from extracellular nutrients, through the methionine cycle, into methylated substrates (figure 1a). We chose to focus on the nucleic acids dna and rna as the methyl acceptor substrates, as the most common forms of methylated dna and rna (methyl - cytidine and methyl - adenosine, respectively) are well characterized (fu et al . A number of assays have been developed to analyze relative quantities of modified and unmodified dna bases by mass spectrometry, including global dna methylation (kok et al ., 2007),, 2015, herbig et al ., 2002), and incorporation of deuterium - c - labeled methionine into dna (bachman et al ., 2014). Based on an acid hydrolysis technique (kok et al ., 2007), we developed our assay to use cn - serine or cn - methionine in cell culture medium for 3 hr . Using this method, we were able to track c - labeled serine and methionine into methyl groups on dna and rna . This method permitted relative quantification of the one - carbon contribution from extracellular serine and methionine onto dna and rna over a fixed time period (figure 1a). Using this method we found as expected that one - carbons from labeled methionine were readily used to methylate dna and rna . However, no significant contribution of one - carbons from serine was detected under the same conditions (figures 1b and 1c). As re - methylated methionine is derived from its downstream metabolite homocysteine (figure s1a), removal of exogenous methionine also deprives cells of the homocysteine they require for re - methylation (shlomi et al . Therefore we supplemented methionine - starved cells with exogenous homocysteine and vitamin b12 (co - factor for re - methylation reaction) to ensure these nutrients were not limiting . Surprisingly, although serine did not contribute one - carbons under these conditions, serine starvation clearly decreased the contribution of one - carbon units from methionine to dna and rna after 9 hr (figures 1b and 1c). Treatment with a low dose of the dna methyltransferase inhibitor azacytidine, which directly blocks dna methyl transfer, caused a similar decrease in the transfer of methyl groups to dna as serine starvation (figure 1d). After 30 hr of serine starvation, cells restored their ability to maintain the transfer of methionine - derived methyl groups to dna (figure 1e). Cancer cells adapt to serine starvation both by controlling serine utilization and by upregulating de novo serine synthesis, which allows recovery of serine levels (maddocks et al ., 2013), potentially explaining the recovery of methyl transfer by this later time point . Analysis of total dna and rna methylation showed that serine depletion did not significantly impact total levels of methylated dna or rna, whereas methionine starvation caused decreased methylation (figure s1b). This finding suggests a degree of coordination between serine availability and methyl group transfer, which is lacking for methionine . As expected, azacytidine caused a decrease in total dna methylation, but did not alter total rna methylation (figure s1b). As serine starvation decreases the proliferation rate of cancer cells, we tested whether serum starvation, which also decreases proliferation rate (figure s1c), had a similar effect . Serum starvation did not impede transfer of methyl groups to dna to the same extent as serine starvation (figure s1d), suggesting that the changes in methyl transfer that we detected are not simply a reflection of decreased proliferation . To understand why serine availability influenced the transfer of methyl groups to dna and rna, despite not directly contributing one - carbons for methylation, we analyzed metabolite levels in the methionine cycle by liquid chromatography mass spectrometry (lcms). Addition of cn - labeled methionine showed that serine - starved cells had increased levels of methionine but lower levels of downstream metabolites sam and sah (figures 2a and 2b), suggesting a decrease in the conversion of methionine into the methyl donor molecule sam . Given that conversion of methionine into sam is atp dependent, we hypothesized that methylation could be influenced by cellular atp levels . To test this, we assessed the impact of serine depletion on atp levels and compared this to glucose starvation as a positive control . Glucose is a major cellular energy source, and as expected, glucose depletion caused increased levels of amp and lower levels of atp (figure 3a). This corresponded with activation (by phosphorylation) of the cellular energy sensor ampk (figure 3b). By comparison, we noted that serine starvation alone decreased both atp and amp levels (figure 3a). This is a reflection of the requirement of serine - derived one - carbon units for de novo purine synthesis, and as reported previously (labuschagne et al ., 2014), this effect was further exacerbated by both removing serine and increasing glycine, which further inhibits nucleotide synthesis . In previous work, we found that pyruvate partially rescued cells from serine starvation and showed that acute depletion of exogenous pyruvate and serine resulted in activation of ampk (maddocks et al ., 2013). In the present study, cells were routinely grown without pyruvate, hence were adapted to its absence . In this context, the subsequent removal of serine led to a coordinated decrease in both amp and atp levels (figure 3a), without activation of ampk (figure 3b). While de novo atp synthesis may be slower in comparison to the rate of atp turnover (as in energy generating reactions), these results suggest that without significant de novo atp synthesis, cancer cells do not have an adequate pool of adenine nucleotides for biosynthetic reactions (such as sam synthesis) that require atp to contribute adenosine . In order to test whether de novo synthesis of atp could make a direct contribution to the methionine cycle, we analyzed sam cycle metabolites by lcms after feeding cells cn - serine . In support of the dna and rna methylation data (figures 1b and 1c), labeled serine did not contribute one - carbons to methionine re - methylation (i.e., the m+1 pool of methionine was at background levels, as seen in the control cells). However, after just 3 hr, both sam and sah were labeled by serine - derived carbon and nitrogen (figures 4 and s2a). Labeling was not seen in homocysteine, reflecting the fact that the atp - derived adenosine nucleotide is lost on conversion of sah to homocysteine (figure s2a). As a result, atp also contributes adenosine to the synthesis other metabolites (e.g., nad(h)). In comparison to sam, the contribution of labeled serine to nad(h) was smaller over 3 hr (figure s2a), with serine starvation having a relatively modest impact on total nad(h) levels over 624 hr . Excess glycine had a greater impact on nad(h), in line with its more dramatic effects on atp levels . These data suggest that sam levels may be more sensitive to variations in de novo atp synthesis than other metabolites . To test whether de novo nucleotide synthesis is required for incorporation of methyl units into dna, we cultured cells under conditions which inhibited de novo nucleotide synthesis (i.e., no serine / no serine + high glycine). Analysis of total atp, methionine, and sam pools (without using labeled metabolites) showed that these conditions caused a dramatic decrease in cellular atp levels concurrent with increased methionine, and lower sam levels (figures 5a and s3a). These metabolic changes translated to an increased methionine / sam ratio (figures 5b and s3b) and a decrease in the contribution of methyl units from the methionine cycle to dna (figure 5c). Exogenous formate directly provides one - carbons to the thf cycle and restores de novo nucleotide synthesis in the presence of excess glycine (labuschagne et al ., 2014). Consistent with the requirement for de novo atp to support methionine to sam conversion, the addition of formate restored transfer of methyl groups from methionine to dna (figure 5c). Wider analysis of nucleotide synthesis intermediates showed that serine starvation generally impeded the synthesis of purines (due to lack of one - carbon units, as previously reported; labuschagne et al ., 2014). By contrast, pyrimidine levels upstream of dtmp either showed little change or increased in response to serine starvation (figure s4). As the pyrimidine nucleotides upstream of dtmp do not require one - carbon units for de novo synthesis, they can be made in the absence of serine . Furthermore, the lack of purines will inhibit nucleic acid synthesis, allowing unused pyrimidines to accumulate . The synthesis of dtmp from dump requires serine - derived one - carbon, and accordingly, the levels of dtmp were also lower in serine - starved cells . As we were unable to detect the use of serine - derived one - carbons for methionine re - methylation after 9 hr (figures 1b and 1c), we considered that a longer period of methionine starvation could be necessary to promote detectable re - methylation . After 24 hr of methionine starvation (with supplementary vitamin b12 and homocysteine) with labeled serine for the final 3 hr, we were able to detect the transfer of serine - derived one - carbons to dna and rna (figure 6a). Supplementing cells with homocysteine and vitamin b12 provided varying degrees of proliferative rescue (figure 6b), most likely reflecting the efficiency with which these cells can recycle methionine from homocysteine . The small additional gain in rescue achieved by supplementary vitamin b12 suggests that background vitamin b12 levels are already present in the dialyzed serum in the protein bound form . Our data suggest that serine can contribute to the methionine cycle both by providing one - carbon units for re - methylation of methionine and by supporting de novo atp synthesis to allow the conversion of methionine to sam . To assess the full extent of the contribution of serine to nucleotide synthesis and the methionine cycle, we cultured cells with labeled serine for 24 hr, either in the presence of methionine or without methionine plus homocysteine and vitamin b12 . All three cell lines readily took up the exogenous serine, which was converted into glycine . One - carbons from serine and serine - derived glycine molecules entered de novo nucleotide synthesis so that almost the entire cellular pools of amp, adp, atp gmp, gdp, and gtp were labeled after 24 hr (figures 7a and s5). At this time point most nad(h) was also labeled, although the proportion of the pool labeled was less than that seen for atp and sam (figures 7a and s5). The major atp isotopomer contained four labeled carbons and one labeled nitrogen (m+5), indicating incorporation of serine - derived glycine (two carbons, one nitrogen) and two serine - derived one - carbon units (figures 7a and 7b). While total methionine levels were lower in the methionine - starved cells, an increase in the proportion of m+1 labeled methionine was observed . Hence the ratio of labeled to unlabeled methionine was dramatically increased in the methionine - starved cells . Labeling of sam generally mirrored atp labeling; however, an additional m+6 isotopomer was also observed in sam, which was increased during methionine starvation . This m+6 isotopomer is a result of sam labeling via re - methylated methionine (one carbon from serine) plus serine - labeled atp (four carbons and one nitrogen) (figures 7a and 7b). This labeling pattern indicates that serine can contribute simultaneously to two independent pathways that support the sam cycle . For technical clarity, we have provided examples of chromatogram peaks for adenine, methyl - adenine, cytosine, and methyl - cytosine with theoretical and measured masses (figure s6), as well as a breakdown of our data analysis and presentation for c methyl transfer (figures s7a s7c) and total rna / dna methylation analysis (figures s7d s7f). One of the most important cellular uses for serine is to provide one - carbon units to support the thf - cycle (tibbetts and appling, 2010). While the cleavage of glycine could also provide one - carbons, we showed previously that glycine cannot substitute for serine, and increasing glycine in cells starved of serine further depletes the one - carbon pool as cells convert the glycine to serine (labuschagne et al ., 2014). Serine - dependent one - carbon metabolism is necessary for purine synthesis, but it can also contribute to several other metabolic pathways . The biochemical capacity for transfer of one - carbons from the thf cycle to homocysteine re - methylation is well known (field et al . However, the activity of this pathway in cancer cells is poorly characterized . In the present study, we show that during methionine starvation, serine can provide one - carbon units to recycle homocysteine to methionine and so support the methionine cycle . Intriguingly, however, we also found that serine contributes to the sam cycle and subsequent dna and rna methylation, even in methionine - fed cells, although this was not a reflection of methionine recycling . Instead, we found that serine - dependent de novo atp synthesis is needed to support the conversion of methionine to sam and that limitation of this atp pool can impact the rate of sam generation and new methylation of dna and rna . The method we have used to track transfer of methyl units from c - labeled methionine and serine has potential utility to evaluate methylation dynamics of dna and rna in other contexts . There is a growing realization that methylation of dna and rna is more dynamic than previously appreciated . The ability to analyze these changes with a temporal dimension (e.g., by adapting the method to include multiple time points) may allow this technique to be used to achieve this goal . In addition, the analysis method we employed for total cytosine / adenine methylation offers a robust method for global methylation analysis . The advantages of this lcms method compared to other measures of global methylation (e.g., immuno - detection of 5 mc) are that it is direct and properly normalized for total cytosine / adenine content . However, the present method is not sensitive enough to detect small changes (e.g., less than 2%) in methylation or to provide information on where in the genome these changes may occur . Serine has been shown to play an important role in supporting the proliferation of cancer cells . Although not an essential amino acid, serine starvation promotes the induction of a program of metabolic adaptation including the de novo serine synthesis pathway, allowing cells to generate serine from glycolytic intermediates . In some cancers, amplification of the ssp enzymes renders the cells less dependent on exogenous serine . However, most cancer cells respond to serine starvation with an abrupt depletion of intracellular serine . Successful adaptation to serine starvation depends on the ability to partition the depleted serine pool into pathways important for cell survival (such as the generation of glutathione to control ros) while limiting flow of serine - derived one - carbon units into nucleotide synthesis . While this strategy is consistent with meeting the needs of cells that have undergone a transient cell - cycle arrest (in response to serine depletion) and so have lower need for nucleic acid synthesis, our data indicate that a broader view of the contribution of purines especially atp to cell growth and survival needs to be considered . Numerous studies demonstrate that cells monitor and sense energetic stress through ampk, which detects elevated amp / adp levels, signifying an increase in the amp - to - atp ratio (hardie et al ., 2012, xiao et al ., 2013). Ampk activation causes a host of changes that promote atp regeneration and inhibit atp consumption, so allowing the cell to balance atp demand with atp supply (hardie et al ., 2012, hochachka and mcclelland, 1997). The intracellular atp level is frequently considered to be a function of changes in atp turnover (i.e., the regeneration of atp by phosphorylation of amp / adp) (hardie et al ., 2012, hochachka and mcclelland, 1997, xiao et al ., 2013), and clearly, the changing ratios of these metabolites in relation to atp are signals for the activation of energetic stress responses like ampk (xiao et al ., 2013). Less explored, however, is the contribution of de novo atp synthesis (i.e., atp generated by de novo purine synthesis, a biosynthetic pathway that requires glucose and amino acids). Cancer cells show enhanced rates of de novo purine synthesis, which has been interpreted as contributing to increased demand for nucleic acid (primarily rna and dna) synthesis . However, atp generated by this pathway has the potential to be used in other metabolic reactions, including functions in energy transfer, as a phosphate donor, or as an adenosine donor as in sam and nad(h) synthesis . It is possible that these alternative functions for the de novo synthesized purines (both atp and gtp) could help explain why cancer cells devote so many resources to this pathway . Indeed, guanine nucleotides (gmp, gdp, and gtp) are critical for supporting the ras pathway and a range of other signaling and metabolic processes important in cancer (grewal et al ., 2011, ostrem et al ., 2013). Given that we observed almost complete labeling of the atp pool within 24 hr, our data also suggest that just as other pools of cellular components (such as dna and proteins) need to be replicated to support cell division, so do pools of free nucleotides such as atp and gtp . Since ampk is seemingly insensitive to concurrent decreases in amp and atp, it is tempting to speculate that other mechanisms exist to detect absolute cellular atp (and gtp) content . Given the dependence of the methionine cycle on atp and its ability to influence gene expression and protein activity by methylation, it is conceivable that the methionine cycle has a role in sensing absolute atp levels . In the present work, we observe the contribution of newly synthesized atp to the sam cycle and a requirement for serine in supporting transfer of methyl groups to dna and rna through atp synthesis . A wealth of literature supports the role of serine - derived one - carbon in methionine re - methylation through the synthesis of 5-methyl thf, and polymorphisms in one - carbon metabolism enzymes can influence chromatin methylation and ultimately disease risk (stover, 2011). However, we did not observe the expected serine - dependent re - methylation in cancer cell lines when methionine was present . We show that non - energetic metabolic stress can have a dramatic and rapid effect on total atp levels, causing an even greater decrease than glucose starvation . However, this drop in atp is not clearly detected by the classic energetic stress sensor ampk, likely because lower de novo atp synthesis is accompanied by lower levels of amp . We see that despite dramatic decreases in atp levels, cells survive and maintain proliferative potential . These are important observations given the widely held belief based on studies involving energetic stress that cells maintain constant atp levels at all costs and that atp concentration is universally homeostatic the term atp synthesis is very commonly used (pubmed returns over 600 papers with this term in the title) to describe the generation of atp by adding phosphate(s) to amp or adp . However, this form of atp generation can more accurately be referred to as atp turnover or regeneration, as the adenosine nucleotide is conserved during such reactions . As the present study demonstrates, true (i.e., de novo) atp synthesis achieved by the assembly of glucose and amino acids through multi - step de novo purine synthesis is also a major contributor to the functional atp pool in cancer cells . Hct116, rko, and sw480 cells were obtained from atcc and subsequently authenticated using the promega geneprint 10 system according to the manufacturer s instructions . Unless otherwise stated, cell culture media were purchased from gibco, product numbers are shown in parenthesis . Stock hct116 and rko cells were grown in mccoys 5a medium (26600) supplemented with 10% fbs and penicillin - streptomycin . Sw480 cells were grown in dmem (21969) supplemented with 2 mm l - glutamine 10% fbs . All stock cell culture and experiments were conducted in 37c, 5% co2 incubators . For experiments where nutrient levels were manipulated, cells received formulated medium containing mem vitamins (11120), dialysed fbs (hyclone, thermo scientific), penicillin - streptomycin, 17 mm d - glucose (sigma), sodium bicarbonate (sigma) lacking methionine, and serine and glycine (but containing all other amino acids), with varying concentrations of these nutrients added back as specified in each experiment . For re - methylation experiments (where cells were deprived of methionine) 0.8 mm homocysteine (sigma) and 1 m vitamin b12 (methylcobalamin, sigma) were added . Usa, via ck gas ltd, uk) were used at concentrations specified in each experiment . Cells were seeded in mccoys / dmem in 6-well plates and left for 1624 hr; initial seeding density was 2 to 8 10 cells per well and was calculated so that cells were approximately 80% confluent at the time of harvest . Cells were washed once with pbs and received formulated assay medium as described above under nutrient deprivation . For each experiment, cells were initially grown with unlabeled nutrients for various time periods (stated in each experiment) followed by a fixed period of 3 hr with labeled c3n1-serine or c5n1-methionine . The relative contribution of these labeled metabolites to dna / rna methylation over the 3 hr period was then assessed as follows: cells were removed from wells with trypsin, and each well was split into two cell pellets that were frozen on dry ice/80c . One pellet was used for dna extraction using qiaamp dna mini kit (qiagen, 51304) with rnase treatment, rna was extracted from the second pellet using rneasy mini kit (qiagen, 74104) with dnase treatment according to manufacturer s instructions . The protocol for dna / rna acid hydrolysis 1ug dna or 3ug rna was placed in a 1.5ml tube and dried at 40c under nitrogen gas . 100ul of formic acid (sigma) was added to the dry pellets and incubated at 130c for 3.5 hr . After cooling, the acid was dried off at 40c under nitrogen gas, dry pellets were re - suspended in 25ul lcms - grade water for 20 min at room temperature . 100ul of ice - cold lcms - grade methanol (62.5%) acetonitrile (37.5%) solution was added to each sample . Samples were vortexed and spun at 4c for 15 min, supernatant were transferred to lcms vials . Bases from hydrolyzed dna and rna were analyzed on a dionex ultimate 3000 lc system coupled to a q exactive mass spectrometer (thermo scientific). Chromatographic separation was achieved using a sequant zic - philic column (2.1 150 mm, 5 m) (merck) with elution buffers (a) and (b) consisting of 20 mm (nh4)2co3, 0.1% nh4oh in h2o) and acetonitrile, respectively . The lc system was programmed to maintain a flow rate of 200 l / min with the staring condition at 80% (a), which linearly decreased to 20% (a) over 10 min followed by washing and re - equilibration steps (20%80% [a]) over 7 min . Ionization of the analytes occurred in a heated electrospray ionization (hesi) probe fitted to the mass spectrometer that operated in negative ion mode over a mass range between 75 and 200 m / z at a resolution of 70,000 . Thermo lcquan software was used to identify and analyze the nucleotides . To quantify the contribution of labeled serine / methionine to dna / rna methylation the peak area for m+1,methyl - cytosine / m+1,methyl - adenine for quantification of total dna / rna methylation, only unlabeled metabolites were used, and the peak area for methyl - cytosine / methyl - adenine was divided by peak area for cytosine / adenine, respectively . Examples of chromatogram peaks and a graphical representation of the data analysis and presentation are included in figures s6 and s7 . Assays were performed as described previously (labuschagne et al ., 2014). Briefly, cells were plated in 6-well plates and cultured in mccoys 5a medium or dmem for 24 hr before replacing the medium with fresh medium containing labeled amino acids and incubated for the indicated times . Cells were washed with pbs followed by metabolite extraction with ice - cold extraction buffer consisting of methanol, acetonitrile, and h2o (50:30:20). Extracts were analyzed by lcms using a dionex ultimate 3000 lc system coupled to a q exactive mass spectrometer (thermo scientific). A sequant zic - philic column (2.1 150 mm, 5 m) (merck) was used to separate the metabolites using the same elution buffers ([a] and [b]) as described above . A gradient program starting at 80% (a) and linearly decreasing to 20% (a) over 17 min was used followed by washing and re - equilibration steps . The q exactive was operated in full scan mode over a mass range of 751,000 m / z at a resolution of 35,000 with polarity switching . Whole - cell protein lysates were prepared in ripa - buffer supplemented with complete protease inhibitors (roche), sodium orthovanadate, and sodium fluoride (both sigma). Lysates were separated using precast nupage gels (invitrogen, life technologies) and transferred to nitrocellulose membranes . Proteins were detected and quantified using a li - cor odyssey infrared scanner and software (li - cor biosciences). Primary antibodies used were as follows: anti - ampk - a (2532) and anti - phospho - ampk - a (2535) both from cell signaling technology . Secondary antibodies for the relevant species were irdye680lt and irdye800cw conjugated (li - cor biosciences). Hct116, rko (2 to 3 10 cells per well in mccoys medium) and sw480 (5 10 cell per well in dmem) were seeded in 24-well plates and allowed to adhere overnight . Adherent cells were washed with pbs and were fed formulated assay medium supplemented with the stated nutrients (see nutrient deprivation above). A separate time - zero media were changed every 24 hr, and plates were counted after 2 and 4 days . Relative cell number was calculated by comparison to cell number at time - zero . For counting cells were trypsinized, re - suspended in pbs - edta, and counted with a casy model tt cell counter (innovatis, roche applied science).
The reconstruction of defective and degenerative bones has been a significant challenge in oral and maxillofacial surgery [1, 2]. Autologous tissues derived from intra- or extra - oral sites are considered to be a gold - standard in terms of regenerative potential . However, the limited access and amount of bones as well as the pain and psychological fear that patients tend to suffer due to the additional surgical operation required have been raised as concerns . Bone grafts have been introduced as an alternative to the use of autologous bones, and they have been found to be useful for filling bony defects and expanding the quantity and quality of bones available for implantation [46]. When compared to allogenic or xenogenic materials, which may cause immune and/or disease problems, synthetic bone grafts are considered relatively safe for use in large quantities [3, 4]. Over the past few decades, several types of synthetic bone grafts have been proved by clinical trials and commercialized, and ceramics or glasses classified as bioactive materials comprise the major category [58]. Bioactive glasses obtained from melt - quenching or sol - gel process are known to form direct bonds with hard human tissues through a specific attachment mechanism, such as the formation of a bone mineral like carbonated hydroxyapatite layer that forms on the surface of the glasses under physiological conditions . One of the benefits of bioactive glasses is the ability to easily incorporate elements tuned to the required tissue responses . For example, the addition of magnesium to melt glass 45s5 has been found to improve bioactivity, while the addition of silver to bioactive glass was reported to elicit antimicrobial activity and the addition of strontium to sol - gel bioactive glass was shown to enhance bone cell responses . In this study, zinc was added to the sol - gel bioactive glass based on the previous reports demonstrating zinc roles in bone formation [1315]. As one of the trace elements found in natural bone, zinc has been studied in bone biology, particularly in osteoporotic patients [13, 14]. Furthermore, several recent studies have utilized zinc as an additive to medical materials, including zinc - substituted hydroxyapatite powders, and coatings, tricalcium phosphate powders, and bioactive glass granules [1520]. Small concentrations of zinc within the bioactive glasses have been shown to affect bone - associated cell responses such as proliferation and matrix synthesis in vitro [1820]. Stem cell - based tissue engineering is gaining considerable opportunities for the successful reconstruction of bone tissue . Mesenchymal stem cells (mscs) derived from adult bone marrow are one of the most popular cell sources available for the regenerative therapy, including osteogenesis . Compared to the stem cells of embryonic origin, relatively large quantities of mscs can be obtained without significant ethical concerns, and these cells can be applied clinically using autologous cells [21, 22]. In this study, as a first step towards bone tissue engineering using zinc - added bioactive glass, the effects of the addition of small concentrations (2 and 5% by mole) of zinc to the sol - gel bioactive glass on the growth and osteogenic potential of mscs were investigated . The sol - gel - derived bioactive glass was produced using precursors of tetraethyl orthosilicate and calcium nitrate tetrahydrate (from sigma - aldrich) in an ethanol - water solvent, with hydrochloric acid as a catalyst . To incorporate the zinc, calcium nitrate tetrahydrate was replaced with zinc nitrate hexahydrate (from sigma - aldrich) at 2 and 5 mol% to produce three different compositions of the bioactive glasses: 70sio2 - 30cao (0% zn), 70sio2 - 28cao-2zno (2% zn), and 70sio2 - 25cao-5zno (5% zn). The prepared sol was then left to age for 24 h and transformed into a gel, which was subsequently dried in an oven (60c) and then thermal - treated by heating to 650c at a ramping rate of 2c / min, where it was maintained for 3 h, after which the sol was furnace - cooled in the air . The calcined granules containing diameters ranging from 500 to 1000 micrometers were then gathered using metal sieves . For the cell test samples, the glass granules were sterilized and different quantities (3, 10, and 30) of the granules of each composition (0% zn, 2% zn, and 5% zn) were placed in the individual wells of 24 culture well plates . The surface morphology of the glass granules was observed by scanning electron microscopy (sem, hitachi). For observation of the bone bioactivity, the zinc - added bioactive glass granules were soaked in simulated body fluid (ionic concentration: 142.0 mm na, 5 mm k, 1.5 mm mg, 2.5 mm ca, 147.8 mm cl, 4.2 mm hco3, 1.0 mm hpo4, and 0.5 mm so4) for 7 days, after which the surface was examined by sem . The dissolution of ions (si, ca and zn) from the glasses was detected using inductively coupled plasma atomic emission spectroscopy (icp - aes). For the dissolution test, glass granules were incubated in an acellular culture medium (serum free) for up to 7 days, during which time the medium samples were analyzed . Mesenchymal stem cells (mscs) were isolated from the bone marrow of tibia and femora of adult rats (48 weeks, korea) using a method described in our previous study . The bone marrow mixture was then centrifuged, after which the supernatant was gathered and maintained on a culture flask containing growth medium (-minimal essential medium; mem, 2 mm glutamine, 100 u / ml penicillin, and 100 mg / ml streptomycin) supplemented with 10% fetal bovine serum (fbs). After cell adhesion for 1 day, the cells were washed with phosphate buffered saline solution (pbs) and cultured for up to 7 days until they reached near confluence . Glass granules (5001000 micrometers in size) with three different compositions (0, 2, and 5% zn) were contained within the individual wells of 24-well plates at three different quantities (3, 10, and 30). Thus, a total of ten sample groups were used, including a glass - free blank control . An aliquot of 100 l of the cell suspension prepared at a density of 5 10 cells / ml was then seeded into each well . The medium for the cell growth was supplemented with 50 g / ml sodium ascorbate, 10 m sodium -glycerol phosphate, and 10 m dexamethasone to induce osteoblastic differentiation . The samples were then incubated for 6 h, after which 0.1 ml of culture medium without cells was added to each well . Next, the samples were cultured for up to 21 days, with the medium being refreshed every 2 - 3 days . The cell growth level was measured at days 3 and 7 based on the mitochondrial nadh / nadph - dependant dehydrogenase activity, which was measured using a cell proliferation assay kit (celltiter 96 aqueous one solution, promega). Briefly, 100 l of mts regent was added to each sample and incubated for 3 h. the absorbance was then determined at 490 nm using an elisa plate reader . The morphology of cells at each culture time was determined by optical microscopy (motic). The cell growth on the glass granules was examined by sem after fixation of the samples with glutaraldehyde, dehydration in a graded series of ethanol, and treatment with hexamethyldisilazane solution . Sem was conducted at an accelerating voltage of 15 kv after gold coating the surface of the samples . The effect of the glass granules on the in vitro osteogenic differentiation of the mscs was assessed based on the alkaline phosphatase activity . Cells were seeded onto each sample at 5 10 cells / ml and then cultured for 7 and 14 days . After washing the samples with pbs the cells were gathered and added with cell lysis buffer (10% triton x-100, 1 m tris - hcl, 0.5 m nacl, and 0.5 m edta) containing protease inhibitor . Total protein content was measured using a commercial dc protein assay kit (biorad, hercules, usa). Bovine serum albumin (from sigma) was used as the reference curve for the protein assay . The quantity of each sample used for an enzymatic reaction was determined when normalized to the total protein content . The enzymatic activity of the alp produced was determined using an alp assay kit (procedure no . Three replicate samples were used for the assay (n = 3). To observe the osteogenic marker, bone sialoprotein (bsp), next, the samples were washed with pbs and incubated with the primary antibody at a concentration of 1: 500 (polyclonal rabbit antibone sialoprotein: bsp) at 4c, after which they were washed with pbs . Subsequently, the alexfluor 555 secondary antibody (molecular probe) was added to the samples at 1: 200 and incubated at ~25c in dark condition . After washing with pbs, the samples were mounted and visualized by fluorescence microscopy . For the mineralization study, the cells were cultured under the influence of the eluted products from glass granules that were present within the transwell insert . After culturing the cells, the samples were rinsed in pbs, fixed with 10% formaldehyde, and then stained with 1% alizarin red s (ars) solution (ph 4.1) for 30 min . After washing fully with distilled water the mineralization was quantified by detecting the amount of calcium present on the cellular constructs . After culturing, the cell layer (including extracellular matrix) collected from each specimen was homogenized in diluted hcl solution by vigorous shaking . The cell lysate was centrifuged and the supernatant was used to determine the calcium content . The calcium concentration in each sample was quantified with a regent solution using orthocresolphthalein complexone (ocpc) as a metallochromic indicator . Standard calcium solutions were prepared after suitable dilutions of 1 mg / ml cacl2 solution . An aliquot of the regent was added to each sample to induce ca - ocpc complexometric reaction, and the absorbance was read at 590 nm using a microplate reader . Data were presented as mean one standard deviation for three or five different sets of samples in each group . Statistical analysis was performed by the student t - test and significant level was considered at p <.05 . Figure 1 shows the typical morphology of the glass granules (2% zn) that were prepared by the sol - gel synthesis and subsequent heat treatment (figure 1(a)). Granules with sizes of approximately 500 to 1000 m were chosen for further biological tests . When the glass granules were immersed in a simulated body fluid and incubated for 3 days at 37c, calcium phosphate crystallites were found to precipitate on the surface of the glass granules, and after 7 days of incubation, the surface of the granules was fully covered by a thick mineral layer (representative image of 2% zn - added glass shown in figures 1(b) and 1(c)). Similar in vitro mineralization behavior in sbf was observed in the other compositions (data not shown here). For the biological tests, the glass granules were evaluated at three different concentrations (low (3), medium (10), and high (30)) contained within each well of a 24-well culture dish . The ionic elusion (si, ca, and zn) from glass samples cultured in an acellular medium was measured using icp - aes for up to 7 days (table 1). The results revealed that the addition of zinc to the glass led to a slight decrease in the dissolution of silicon and calcium . In addition, a trace amount of zinc was released from the zinc - added glass granules . The reduced release rate of the cations with time may be due to the depletion of cations with time and the resultant slow - down in further diffusion release process . Mesenchymal stem cells (mscs) extracted from rat bone marrow were cultured in the presence of the bioactive glass granules . Three different quantities (3, 10, and 30) of glass granules were placed in each well of a 24-well culture dish, and cellular responses such as growth and osteoblastic differentiation were investigated . Figure 2 shows the optical morphology of cells grown in culture wells for 7 days, including wells that contained the glass granules (0, 2, and 5% zn) at different concentrations (3, 10, and 30 granules), as well as a glass - free culture well as a control (blank). When low and medium concentrations (3 and 10) of the glass granules were present, the cells reached near confluence and had an elongated and well - spreading morphology similar to that of the control . However, when the high concentration (30) of the granules was present, the cell morphology became less elongated and more granular - like . The cell morphology on the contained glass granules was observed with sem (figure 3). After 14 and 21 days of culture, the cells were found to actively grow on the surface of glass granules . Furthermore, the cells had flattened bodies and many cytoskeletal extensions that were in intimate contact with the glass substrate . At day 21, the cells appeared to be aggregated (figure 3(e)), and mineral - like products were noticeable around / below the cellular construct (figures 3(e) and 3(f)). Figure 4 summarizes the cell viability for periods of up to 7 days in the presence of bioactive glass granules with different compositions and concentrations, as measured by an mts method . When cultured in the presence of low and medium concentrations of granules for 3 and 7 days, the cell growth was equal to or higher than that of the control . However, a slight decrease in cell growth was observed with a high quantity (30) of the glass (2% and 5% zn - added), and even significant decrease was noticed in the 0% zn - added glass . In particular, zinc - containing glasses induced significantly higher cell growth at day 3 when they were contained at low (3) and medium (10) quantity . After prolonged culture periods (7 days), the cell viability affected by the glass granules became similar to that of the blank control, regardless of the composition of the glass . The alkaline phosphatase (alp) activity of the mscs cultured in the presence of various glasses was evaluated (figure 5). Two sets of experiments were conducted using different quantities of glasses (10 and 30 granules). At day 14, the addition of medium quantity (10) of glass granules significantly increased the alp level, and this improvement increased as the zinc content within the glass increased (figure 5(a)). A similar trend was observed in samples containing a high quantity of glass granules (30 granules) (figure 5(b)). The expression of the osteogenic marker bone sialoprotein (bsp) produced by the cells in the presence of bioactive glass granules was observed by immunofluorescence staining . As shown in figure 6, the red - colored spots representing the expressed bsp were obvious (2% zn shown as a representative sample), particularly near the bioactive glass granules . The immunofluorescence stain was more obvious in the samples that contained the glass granules than in the control . When compared to the blank control, the addition of glass granules significantly enhanced the expression of bsp . Moreover, the stimulation of bsp was greater in response to a higher concentration of glass granules . The mineralization behavior of the mscs was investigated by alizarin red s (ars) staining . Specifically, 30 glass granules for each composition (0, 2, and 5% zn) were placed in a transwell insert, and the mscs were then cultured under the influence of the ionic eluants from the glass granules . This was conducted to eliminate the possible interference between ca within the glass and ars dye . As shown in figure 8(a), all the glass - containing groups showed the cellular constructs stained dark - red in response to the influence of the glass eluants, confirming significant level of cellular mineralization . The mineralized level was assessed by detection of calcium quantity of the cellular constructs . As presented in figure 8(b), the mineralization was significantly higher on all the glass - containing samples than on the blank control, and furthermore, the 2% zn - added glass sample showed significantly higher level of mineralization than the pure bioactive glass . The use of bioactive inorganics is considered a promising tool in the reconstruction of oral and maxillofacial defects [1, 2]. Among the various compositions evaluated, bioactive glasses have been widely studied due to their excellent bioactivity and ability to direct - bonding to bone, which is primarily associated with the bone mineral - like phase created on their surface [8, 9]. Moreover, the composition of bioactive glasses is believed to be easily controlled by the introduction of biologically relevant elements, such as silicon, magnesium, strontium, silver, and zinc [1012]. Zinc is considered a key element present in bone and has been shown to regulate osteoblastic function and bone formation . Osteoporotic patients have often been found to have a lack of zinc; therefore, it is considered to be useful for the treatment of osteoporosis [13, 14]. Additionally, several recent studies have been conducted to utilize zinc within the composition of biomaterials . Those studies have shown that zinc played an effective role in the stimulation of bone cell functions when it was present in calcium phosphate compounds such as hydroxyapatite and tricalcium phosphate [1518]. In the present study, zinc was added to the composition of bioactive glass during the sol - gel synthesis, and the effects of zinc addition (2 and 5%) on the responses of rat bone marrow mscs were evaluated . Mscs are considered a potential cell source for application in regenerative medicine, including the treatment of bone disorders [2123]. For stem cell - based bone tissue engineering, the material substrate should guide the initial anchorage of cells and their multiplication and further stimulate the differentiation into an osteogenic lineage . In order to utilize the zinc - bioactive glass as a scaffold for the mscs, this study was carried out as a first step to gain insight into the behavior of mscs in response to the glasses that contain zinc . Different quantities of granules (3, 10, and 30) with variable glass compositions (0, 2, and 5% zn - added) were used to evaluate the mscs responses . For all compositions, the initial cell adhesion and growth were shown to be favorable in the presence of small quantities of glass granules (figures 2 and 4). Although some downregulation of cell viability was noticed initially, particularly in the case of the high concentration of 0% zn - added glass, the cell growth level became almost equal to that of the blank control after a prolonged culture time (7 days). Conversely, a small quantity of glass granules led to a significant increase in cell viability, suggesting that the existence of glasses and their eluted products should favor the proliferative potential of mscs . Based on the ionic release data (table 1), the addition of zinc to the glass led to a slight decrease in the elution of ions such as si and ca . Thus, the initial high release of ions from the 0% zn - added glass might be one of the possible reasons for the decreased cell viability . Although the initial ionic release was high, the release rate decreased with time, with cells eventually reaching their normal proliferation level after a prolonged culture period (over 7 days). The images of the cells grown directly on the glass particles at day 14 (figure 3) confirmed the viable cellular status, that is, the presence of extensive cytoskeletal processes and the formation of collagenous fibrillar extracellular matrix by the proliferative cells . The effects of the zinc - added bioactive glass on the osteogenic differentiation of the mscs were evaluated based on the alkaline phosphatase (alp) activity of cells . Alp is a marker of osteoblastic cells that is evident at relatively early stage of osteogenic differentiation of progenitor or stem cells . In the present study, alp was significantly upregulated when grown in the presence of bioactive glass granules, particularly with larger quantity of granules and at later culture times (over 14 days) (figure 5). Moreover, the increase appeared to be greater in the presence of zinc - added glasses . As another marker for osteogenic differentiation, bone sialo - protein (bsp) was evident in the presence of glass, particularly near the glass granules (figures 6 and 7). These results indicate that mscs are triggered to undergo osteogenic development by the eluants of the bioactive glass particles . The ions released from the bioactive glasses further stimulated cellular mineralization, as confirmed by the ars staining and calcium quantification of samples (figure 8). The released calcium ion should participate directly in the mineralization of extracellular matrices (ecms), which would be secreted by the differentiated cells . Although the mscs differentiate down to an osteogenic lineage within the total culture medium used herein, the presence of bioactive glasses should alter the secretion of proteins, and thus the ecm components . As noticed, the mineralization behavior under the influence of the glasses was noteworthy with respect to that of glass - free control which was conditioned only with the osteogenic medium . These results indicate that the differentiated cells and the synthesized ecm must be properly conditioned to induce mineralization under the influence of the bioactive glasses eluants . As to the effects of zinc, the results demonstrated that the mscs expressed alp and bsp at levels largely similar to those of zinc - free bioactive glass, and sometimes at even higher levels depending on the zinc concentration and the quantity of glass used . The different levels of ions released from the zinc - added bioactive glasses should affect the differentiation responses of stem cells and further cellular mineralization, and the zinc ions released may participate in the regulation of cellular functions to some extent . Although no significant difference in bsp levels was noticed, the upregulations of alp and mineralization by the zinc - added glass support the possible role of zinc in the differentiation of mscs to the osteoblastic pathway . Taken together this study may provide some useful information regarding the effects of zinc addition to bioactive glasses on the mscs growth and their stimulation into osteogenic differentiation . Accordingly, we are currently developing zinc - added bioactive glasses as 3d scaffolds to use them in bone tissue engineering with the mscs . The cell growth and osteogenic differentiation of mscs with respect to zinc - added bioactive glasses (0, 2, and 5%) in granular form were investigated . The presence of a small quantity of glass enhanced the initial cell growth, whereas a downregulation was noticed in response to a large quantity of zinc - free glass . The osteogenic development, as determined based on the alkaline phosphatase (alp) activity and bone - sialo protein (bsp) secretion, was significantly enhanced by the bioactive glass granules . Moreover, the zinc - added glass induced cells to differentiate to a level similar to or even greater than that of zinc - free glass . The cellular mineralization was also significantly stimulated by the zinc addition to the bioactive glass . Taken together, zinc - added bioactive glasses may be useful in bone regeneration and tissue engineering with adult stem cells because they preserve the active growth of mscs and stimulate further osteogenic differentiation.
It is done to prevent the incompatible red cell transfusions which may result in immune - mediated hemolytic transfusion reaction . It ensures that transfused cells have an acceptable survival rate as well as there is no significant destruction of recipient's own red blood cells . Conventional tube technique (ctt) although this technique is believed to be the gold standard, it has got its own limitations . The end - points of the reaction are unstable; reading and grading require a high level of expertise leading to interobserver variation . In the last few decades, there has been rapid technological advancement in blood banking . In 1976, low - ionic - strength solution (liss) based additives and tube liss indirect antiglobulin test (tube liss - iat) was introduced which significantly increased sensitivity for antibody detection in a shorter duration of time . In 1990's, the lack of washing phase in mct decreases the potential for false weak or negative reactions and makes it ideal for automation . However, the incidence of false positives is more with mct when compared to conventional tube methods . With the aim to improve the efficiency, different laboratories select methods tailored to meet their needs . There are conflicting data in the literature about the relative sensitivities of various techniques being used for the serological crossmatch and in detection of clinically significant antibodies . This present study has been designed to compare the efficacy of three crossmatch techniques (ctt, liss - iat, and mct) used in the blood bank serology laboratory . This was a prospective study which was conducted in a tertiary care hospital from january 2011 to september 2012 after approval by the institutional ethics committee . During the study period, we received request for cross match of 150 samples from patients who had received two or more transfusions on two different occasions (with at least 72 h between two transfusions). Detailed transfusion history, any relevant medical, surgical, and obstetric history was recorded . The grading system of ctt reactions was followed according to the american association of blood banking [table 1]. Grading of agglutination reaction in conventional tube technique standard procedure for the preparation of liss was followed as per dghs technical manual . To prepare 1 l of liss, 18 g of glycine was dissolved in 500 ml of distilled water . Phosphate buffer (0.15 m) ph 6.7 was prepared by adding 0.15 m nah2po4.2h2o (23.4 a volume of 20 ml of phosphate buffer (0.15 m) ph 6.7 was added to the above - prepared glycine solution . 1.79 g of nacl dissolved in 100 ml of distilled water was added to this solution . The standard procedure for performing liss - iat was followed as per dghs technical manual . The diamed - id microtyping gel system (eu patent 0305337) was used for cross matching . Depending on the intensity of the reaction, erythrocytes penetrate the gel to varying degrees and reactions are graded as either 4 + (reaction is represented by a solid band of agglutinated red cells at the top of the gel column)/3 + (reaction is represented by a predominant amount of agglutinated red cells toward the top of the gel column with a few agglutinates staggered below the thicker band)/2 + (reaction is characterized by red cell agglutinates dispersed throughout the gel column with few agglutinates at the bottom of the microtube)/1 + (reaction is characterized by red cell agglutinates predominantly observed in the lower half of the gel column with red cells also at the bottom)/weak + (few agglutinates remaining in the gel area just above the red cell pellet at the bottom of the microtube) or negative (red cells forming a well - delineated pellet in the bottom of the microtube). Mixed field reaction is recognized as a layer of red cell agglutinates at the top of the gel accompanied by pellet of unagglutinated cells at the bottom of the microtube . Samples showing positive result by any of the technique were further evaluated using antibody screen (id - diacell i - ii - iii, diamed gmbh 1785 cressier, switzerland) and identification (id - diacell panel, diamed gmbh 1785 cressier, switzerland) by gel technique (id - micro typing system, diamed ag 1785 cressier, switzerland) on igg + c3d liss / coombs cards (diamed gmbh 1785 cressier, switzerland). The strength of agglutination reaction observed with three techniques was compared to determine the efficacy of these techniques . The statistical analysis was carried out using statistical package for social sciences (spss version 15.0 for windows inc ., chicago, il, usa). For age, we calculated mean and standard deviation . Proportions were compared using chi - square or fisher's exact test whichever was applicable . To see the agreement between two methods, kappa test of agreement was applied . All statistical tests were two - sided and performed at a significance level of = 0.05 . The grading system of ctt reactions was followed according to the american association of blood banking [table 1]. Grading of agglutination reaction in conventional tube technique standard procedure for the preparation of liss was followed as per dghs technical manual . To prepare 1 l of liss, 18 g of glycine was dissolved in 500 ml of distilled water . Phosphate buffer (0.15 m) ph 6.7 was prepared by adding 0.15 m nah2po4.2h2o (23.4 a volume of 20 ml of phosphate buffer (0.15 m) ph 6.7 was added to the above - prepared glycine solution . 1.79 g of nacl dissolved in 100 ml of distilled water was added to this solution . The liss prepared was stored at 4c . The standard procedure for performing liss - iat the grading system of ctt reactions was followed according to the american association of blood banking [table 1]. Standard procedure for the preparation of liss was followed as per dghs technical manual . To prepare 1 l of liss, 18 g of glycine was dissolved in 500 ml of distilled water . Phosphate buffer (0.15 m) ph 6.7 was prepared by adding 0.15 m nah2po4.2h2o (23.4 a volume of 20 ml of phosphate buffer (0.15 m) ph 6.7 was added to the above - prepared glycine solution . 1.79 g of nacl dissolved in 100 ml of distilled water was added to this solution . The standard procedure for performing liss - iat was followed as per dghs technical manual . The diamed - id microtyping gel system (eu patent 0305337) was used for cross matching . Depending on the intensity of the reaction, erythrocytes penetrate the gel to varying degrees and reactions are graded as either 4 + (reaction is represented by a solid band of agglutinated red cells at the top of the gel column)/3 + (reaction is represented by a predominant amount of agglutinated red cells toward the top of the gel column with a few agglutinates staggered below the thicker band)/2 + (reaction is characterized by red cell agglutinates dispersed throughout the gel column with few agglutinates at the bottom of the microtube)/1 + (reaction is characterized by red cell agglutinates predominantly observed in the lower half of the gel column with red cells also at the bottom)/weak + (few agglutinates remaining in the gel area just above the red cell pellet at the bottom of the microtube) or negative (red cells forming a well - delineated pellet in the bottom of the microtube). Mixed field reaction is recognized as a layer of red cell agglutinates at the top of the gel accompanied by pellet of unagglutinated cells at the bottom of the microtube . Samples showing positive result by any of the technique were further evaluated using antibody screen (id - diacell i - ii - iii, diamed gmbh 1785 cressier, switzerland) and identification (id - diacell panel, diamed gmbh 1785 cressier, switzerland) by gel technique (id - micro typing system, diamed ag 1785 cressier, switzerland) on igg + c3d liss / coombs cards (diamed gmbh 1785 cressier, switzerland). The strength of agglutination reaction observed with three techniques was compared to determine the efficacy of these techniques . The statistical analysis was carried out using statistical package for social sciences (spss version 15.0 for windows inc ., chicago, il, usa). For age, we calculated mean and standard deviation . Proportions were compared using chi - square or fisher's exact test whichever was applicable . To see the agreement between two methods, kappa test of agreement was applied . All statistical tests were two - sided and performed at a significance level of = 0.05 . This prospective study included 150 patients out of which 86 patients were male and 64 were female . Study group included multitransfused patients, i.e., thalassemic children, patients with chronic renal failure, radiotherapy patients, patients admitted to icu and miscellaneous group [figure 1]. Antibody specificity did not show any statistically significant association with sex and age of the patient . Out of 16 cases, 14 were clinically significant (anti - c = 5, anti - k = 4, anti - e = 2, anti - s = 2, anti - jk = 1) and 2 nonclinically significant antibody cases (anti - le). Mct detected all the 14 clinically significant antibody cases and no case of nonclinically significant antibody . Tube liss - iat detected 14 antibody cases including 2 cases of non - clinically significant antibody but failed to detect 1 case of anti - c and the only case of anti - jka . Ctt detected only 10 antibody cases including 2 cases of nonclinically significant antibody and but failed to detect 3 cases of anti - c, 1 case of anti - k, 1 case of anti - e, and the only case of anti - jk [table 2]. Crossmatch in transfusion medicine is a complex testing which is performed before blood transfusion for the detection of red cell antibodies . Its main aim is to detect maximum number of clinically significant antibodies, keeping clinically insignificant antibodies to minimum in a timely manner . Red cell antibodies play an important role in perinatal immunohematology and are important factors contributing to the risks of immune - mediated hemolytic transfusion reactions . Some of the hemolytic transfusion reactions may have serious consequences including hemoglobinemia, disseminated intravascular coagulation, and death . The techniques commonly employed to detect these antibodies in blood banks include ctt, liss - iat, mct, polyethylene glycol tube test, solid phase red cell adherence assay, etc . Our study compared three techniques (ctt, liss - iat, and mct) in terms of strength of agglutination reaction observed with the specific antibody [table 2]. In our study, mct was found to be most sensitive in the detection of clinically relevant antibodies of rh, kell, kidd, and anti - s specificity as compared to liss - iat and ctt . Mct detected all the 14 clinically significant antibodies; tube liss - iat detected 12 of them and ctt detected only 8 antibodies . Similar set of antibodies have been identified in various studies conducted in the past in multitransfused patients [table 3]. On extensive review of literature, no study comparing three techniques was found . However, the results of our study are similar to few other studies where two techniques have been compared [table 4]. Few studies have contradictory findings in detection of kell antibody . In our study, ctt was less sensitive than tube liss - iat and mct in detecting anti - k while studies by various authors have shown ctt to be more sensitive in detecting kell antibody when compared to liss - iat and mct . This may be due to intense hydrophilic nature of kell antigen due to which anti - kell binds less efficiently in low - ionic solutions when compared to normal saline . Our study has shown mct to be more sensitive in detection of anti - jk and anti - s when compared with tube liss - iat while study by phillips et al . Has shown opposite results, which was attributed to unfamiliarity with the technique by the concerned personnel . In our study, two cases of le were identified which were detected in immediate spin phase by ctt and liss - iat . Our result is contradictory to the studies conducted in the past which have shown gel technique to be more sensitive in detecting cold - reactive antibodies . It may be due to incubation of patient's serum at 37c with donor cells in mct without immediate spin which might affect the reactivity of le at that temperature . Antibodies identified in multitransfused patients comparison of different crossmatch techniques different methods have their own merits and demerits when compared to each other . The sensitivity and specificity of properly performed ctt crossmatch have been estimated to be 100% with anti - human globulin phase included in the procedure . Over - vigorous agitation to dislodge the cell button can cause false - negative results . The end - points of the reaction are unstable; reading and grading require a high level of expertise ., ctt also requires prolonged incubation phase which can delay the release of blood in emergency situations . Liss medium increases the rate and amount of alloantibody uptake and decreases the incubation time to approximately 15 min, thus preventing the unnecessary delay in releasing blood in emergency situations . However, it also increases the uptake of gamma globulins and complement which leads to increased incidence of false positive reactions . The reactivity of certain antibodies, especially kell antibodies is known to decrease in liss medium . Liss - iat is still the most frequently used test tube method for identification of alloantibodies . The lack of washing phase does not cause elution of antibodies and thus contributes to the improved sensitivity of the test which makes it ideal for automation . The reactions are stable for several days and can be photocopied or photographed for future reference . Although gel technology is known to be more sensitive in detection of clinically significant antibodies; it also shows increased detection rates for benign cold - reacting antibodies which can cause undue hindrance in releasing blood in emergency situations . In addition, the incidence of false positives was more with mct in certain studies when compared to conventional tube methods . The results of the previous studies comparing the sensitivity of different crossmatch techniques have been inconsistent . Certain studies found ctt to be a better option while certain studies concluded liss - iat and mct to be a better substitute for detecting clinically significant antibodies . The present study compared the efficacy of three techniques (ctt, liss - iat, mct) in terms of strength of agglutination reaction observed with the specific antibody and found mct to be most efficacious when compared to ctt and tube liss - iat in detecting clinically significant red cell antibodies; although mct missed 2 cases of le antibody, which were detected by ctt and liss - iat . The present study also found the rate of alloimmunization in multitransfused patients which came out to be 10.6% . This suggests that this group of patients is at constant risk of alloimmunization and needs antigen - matched blood by appropriate crossmatch technique which can detect clinically significant antibodies in these patients . The old saying still holds true that no one method will detect all antibodies of clinical relevance . The answer to the question which of these systems be employed in blood banks is influenced by the cost of the procedure, technical skills of the concerned personnel, sensitivity and specificity of the method as well as the possibility of automation.
This study adhered to the helsinki declaration and the good clinical practice (gcp) guidance and was approved by the research ethics committee of the hospital clnico san carlos (madrid, spain). This single - center, prospective, single - blind phase iv study included adults of either sex, who were scheduled for ocular surgery after the treatment . Exclusion criteria included any kind of ocular or systemic disease, past or present, and any ocular and/or systemic treatments / antibiotics within the last 6 months . The study lasted 5 days and the patients were examined at day 0 (d0), day 3 (d3), and day 5 (d5). The 5-day treatment was applied to only 1 eye that is, the eye selected for surgery; the other eye served as control with no treatment applied . A small towel was heated in a microwave on full power for 10 to 15 sec (which rises towel temperature to approximately 40c) which was repeated every 2 min . It was applied to the closed eye area with sufficient pressure to ensure that the towel remained in contact with the eyelids inward downward and the lower lid inward upward, to mobilize the debris adhered to the eyelashes and meibum, and finally they use a sterile eyelid wipe and slide it across the eyelid from the inside to the outside, to clean the upper and lower lids . The blephaclean (laboratoires tha, clermont - ferrant, france) sterile wipes contain a solution of hyaluronic acid, capryloyl glycine, iris florentine, and centella asiatica . Capryloyl glycine and iris florentine extracts are natural emollients show antibacterial activity and present a sebum - regulator effect and centella asiatica acts repairing dermal tissue, increasing collagen production, and activating blood circulation . They were used twice - a - day for 5 days in the selected eye . Visual acuity was measured at d0 and d5, and a slit - lamp examination was performed in each visit, to ensure there was no adverse reaction on the treated tissues . Microbiota samples were taken from the surface of the lower lid and the inferior conjunctival sac fundus of both eyes at d0, d3, and d5 using sterile swabs moistened with saline solution without administering topical anesthesia . The nontreated eye was used as a control and microbiota samples were obtained in the same way . Samples were cultured in specific media on blood and chocolate agar plates and incubated aerobically for 48 hr at 37c . Sterile cotton swabs (materlab sl, madrid, toledo, spain) were used wet with 50 l of saline solution . The volume of saline was controlled and applied by a pipette . Initially a sample from the eyelid was taken and spread onto 1 semipart of the dish and, by the same procedure, another cotton swab was taken from the conjunctival sac of the conjunctiva and seeded onto the other semipart of the dish . Dishes were divided in two semiparts; the left side corresponding to the eyelid culture and the right side to the conjunctiva . Grown colonies were photographed with a high - resolution digital camera, and the total area of microbial growth on the plates was analyzed by imagej software . The percentage of the area occupied by the colonies was determined as follows: (1) the original image of the plate, shown on the left side of figures 1 and 2, is a full rgb tiff, which was converted to 8-bit grayscale . The upper plates belong to the right eye and the lower plates to the left eye . (2) a mask was created by marking out the region where the colonies may grow . The mask is a semicircle covering half the plate, corresponding to the region in which the colonies from the lid or the colonies from the conjunctiva were grown . (3) the selected region is transformed to 1-bit image by selecting an adequate threshold . The contrast between the colonies and the background (culture medium) was very good and a sharp contour of the colonies was achieved . (4) the ratio of the area occupied by the colonies and the total available growing region was obtained as the ratio of the number of pixels set to 0 and the number of pixels set to 1 . Dishes are divided in two semiparts: the left sides correspond to the eyelid culture and the right sides to the conjunctiva, the upper plates belong to the right eye and the lower ones to the left . Once the photographs of the dishes were taken, microorganisms were identified by gram staining, catalase, coagulase, dnase, and mannitol fermentation tests . Differences in microbiota numbers between d0, d3, and d5 were assessed using the student t test, assuming a significance level of 0.05 . Microbiota samples were taken from the surface of the lower lid and the inferior conjunctival sac fundus of both eyes at d0, d3, and d5 using sterile swabs moistened with saline solution without administering topical anesthesia . The nontreated eye was used as a control and microbiota samples were obtained in the same way . Samples were cultured in specific media on blood and chocolate agar plates and incubated aerobically for 48 hr at 37c . Sterile cotton swabs (materlab sl, madrid, toledo, spain) were used wet with 50 l of saline solution . The volume of saline was controlled and applied by a pipette . Initially a sample from the eyelid was taken and spread onto 1 semipart of the dish and, by the same procedure, another cotton swab was taken from the conjunctival sac of the conjunctiva and seeded onto the other semipart of the dish . Dishes were divided in two semiparts; the left side corresponding to the eyelid culture and the right side to the conjunctiva . Grown colonies were photographed with a high - resolution digital camera, and the total area of microbial growth on the plates was analyzed by imagej software . The percentage of the area occupied by the colonies was determined as follows: (1) the original image of the plate, shown on the left side of figures 1 and 2, is a full rgb tiff, which was converted to 8-bit grayscale . The upper plates belong to the right eye and the lower plates to the left eye . (2) a mask was created by marking out the region where the colonies may grow . The mask is a semicircle covering half the plate, corresponding to the region in which the colonies from the lid or the colonies from the conjunctiva were grown . (3) the selected region is transformed to 1-bit image by selecting an adequate threshold . The contrast between the colonies and the background (culture medium) was very good and a sharp contour of the colonies was achieved . (4) the ratio of the area occupied by the colonies and the total available growing region was obtained as the ratio of the number of pixels set to 0 and the number of pixels set to 1 . Dishes are divided in two semiparts: the left sides correspond to the eyelid culture and the right sides to the conjunctiva, the upper plates belong to the right eye and the lower ones to the left . Once the photographs of the dishes were taken, microorganisms were identified by gram staining, catalase, coagulase, dnase, and mannitol fermentation tests . Differences in microbiota numbers between d0, d3, and d5 were assessed using the student t test, assuming a significance level of 0.05 . The study included 45 consecutive patients (17 men, 28 women) with a mean age of 53.86.3 years (range 4573 years). Visual acuity in the treated eye was in a range between 1.0 and 0.54 logmar and remained stable during the treatment, and the slit - lamp examination showed no adverse reaction on the treated tissues . At d0, the eyelid showed the presence of s. epidermidis and corynebacterium spp (94.7% and 32.98% of the plates, respectively) as the most frequent microorganisms . In a smaller percentage, s. aureus, micrococcus spp, and bacillus spp (6.38%, 8.51%, and 1.06%, respectively) were isolated . From the conjunctival swabs, s. epidermidis and corynebacterium spp (54.26% and 38.29%) were identified as well as s. aureus and micrococcus spp (5.32% and 1.06%). Microbiota profiles for eyelid and conjunctiva before treatment the microbial area of growth on the plates was measured according to the method described in the methods section . A photograph of a typical petri dish, along with the mask used for measuring the area occupied by the colonies is shown in figure 1 . The colonies' area analysis yielded a higher microbial load from the eyelids (17.9%) compared with the conjunctiva (1.4%) in most of the samples, as it is seen in figure 2 . Percent reduction in microbial load on the eyelid in the treated eyes was 58% at d3 and 63% at d5 63% (p=0.001) (fig . There was a reduction, although nonsignificant, of 72% and 69% at d3 and d5, respectively (fig . 4) in the numbers of microbes on the conjunctiva . Table 2 and figures 1 and 2 show the microbiota growing areas from eyelid and conjunctiva in all groups . Eyelid microbiota throughout the study (d0, d3, and d5) for the treated eye (te) and the nontreated eye (nte). Conjunctiva microbiota throughout the study (d0, d3, and d5) for the treated eye (tc) and the nontreated eye (ntc). Mean values of microbiota growing areas from eyelid and conjunctiva for days 0, 3, and 5 the eyelid showed the presence of s. epidermidis and corynebacterium spp (94.7% and 32.98% of the plates, respectively) as the most frequent microorganisms . In a smaller percentage, s. aureus, micrococcus spp, and bacillus spp (6.38%, 8.51%, and 1.06%, respectively) were isolated . From the conjunctival swabs, s. epidermidis and corynebacterium spp (54.26% and 38.29%) were identified as well as s. aureus and micrococcus spp (5.32% and 1.06%). The microbial area of growth on the plates was measured according to the method described in the methods section . A photograph of a typical petri dish, along with the mask used for measuring the area occupied by the colonies is shown in figure 1 . The colonies' area analysis yielded a higher microbial load from the eyelids (17.9%) compared with the conjunctiva (1.4%) in most of the samples, as it is seen in figure 2 . Percent reduction in microbial load on the eyelid in the treated eyes was 58% at d3 and 63% at d5 63% (p=0.001) (fig . There was a reduction, although nonsignificant, of 72% and 69% at d3 and d5, respectively (fig . 4) in the numbers of microbes on the conjunctiva . Table 2 and figures 1 and 2 show the microbiota growing areas from eyelid and conjunctiva in all groups . Eyelid microbiota throughout the study (d0, d3, and d5) for the treated eye (te) and the nontreated eye (nte). Bars represent meansd . Differences between te and nte were statistically significant (p<0.0001). Conjunctiva microbiota throughout the study (d0, d3, and d5) for the treated eye (tc) and the nontreated eye (ntc). Mean values of microbiota growing areas from eyelid and conjunctiva for days 0, 3, and 5 in addition to the classical procedures such as cataract surgery, the number of intraocular interventions is increasing because of the development of new therapies . Endophthalmitis caused by microorganisms present on the ocular surface may occur as a consequence of these treatments . The results obtained in this study demonstrated a significant reduction in the microbiota present on the lids and conjunctiva after 3 days of rubbing the lids with the wipes, demonstrating the importance of lid hygiene before ocular surgery or an intraocular procedure, as a complementary prophylactic approach . The percentages of microorganisms existing on the eyelids and conjunctiva in the study were similar to that described previously with the most commonly isolated bacteria being coagulase - negative staphylococci and, more specifically, s. epidermidis followed by corynebacterium spp between 20% and 80% of swabs from the conjunctiva and between 30% and 100% of swabs from the lids showed growth of the bacteria s. epidermidis . Carron et al . Found the most common bacteria were coagulase - negative staphylococci (66.7%), followed by corynebacterium spp (11.5%) from the conjunctiva, and li et al . Described a prevalence of 62.9% and 32.4% for s. epidermidis and corynebacterium spp, respectively, from the conjunctiva . Detected between 90.4% and 94.4% of staphylococcus species from the conjunctiva, of which 71% were s. epidermidis . The rate of isolation of the same bacteria in the study by hfling - lima et al . Ranged between 50% and 80% for the conjunctiva and between 76% and 100% for the eyelids . The microbial isolation of the conjunctiva was reduced on presurgical prophylaxis of wiping the eye after briefly heating the eye . Presented a similar study analyzing the use of palpebral cleansing solution with capryloyl glycine over the conjunctiva microbiota . A clinical reduction was seen from the third day of applying the cleansing solution that lasted until day 5 . In the study of hueso abancens, the maximum reduction obtained after 5 days of treatment was approximately 10% and the main bacteria was staphylococcus spp coagulase - negative . The smaller reduction found by hueso abancens et al . Than this study could be due to the hygiene protocol used to clean the lids . In the study by hueso abancens et al ., the patients were not told to apply heat and massage the lids before using the cleansing solution . In addition, many studies have been conducted on the reduction of eyelid and conjunctiva microbiota after the application of different antibiotics on their own or in combination with pvi . Hfling - lima et al . Examined the efficacy of topical lomefloxacin 0.3% and tobramicin 0.3% 3 days before cataract surgery in reducing biota, and showed statistical reductions in conjunctiva of 66.7% and in lid biota of 3.9% with lomefloxacin, and of 75% and 34.5%, respectively, with tobramicin . Carron et al . Described a 4.3% reduction in conjunctiva biota after the administration of ciprofloxacin 0.3% 1 day before cataract surgery that increased to 60.9% after the application of pvi . The use of pvi before cataract surgery has become a standard of care and a mandatory step in reducing ocular surface microbiota . Showed a 91% reduction in the number of colonies of the conjunctiva after the application of pvi . Reported a 38% reduction after the application of pvi 3 min before surgery and a 34% reduction with the topical application of moxifloxacin 0.5% 2 hr before surgery and pvi 3 min before surgery . The percentage reductions in the microbiota from this study are comparable in some ways to those obtained after the application of topical antibiotics . Eyelid hygiene did not sterilize the tissue, it just decreased the number of bacteria present in the external tissue of the eye without modifying the composition of saprophytic biota on both eyelids and conjunctiva . The reason for these reduced values is probably due to the hygiene protocol that patients followed of cleaning their eyelids after the application of heat and the massage . Although microbiota is present in eyelids and conjunctiva, the percentage of area covered by colonies shows an important microbial load from the eyelids, which highlights the need to maintain preoperative and postoperative asepsis of both eyelid and conjunctiva . Results obtained from bacteria cultures can indicate the type of antibiotic prophylaxis to use for minimizing ocular postoperative infections: broad - spectrum antibiotics or, preferably, antibiotics with specific activity against gram - positive bacteria . Eyelid hygiene cannot replace general preoperative procedures focused on preventing contamination, such as the use of pvi solutions for eyelids and conjunctiva or antibiotics . Nevertheless, eyelid hygiene can be used as a complementary prophylactic approach to prevent endophthalmitis . The use of cleansing eyelid wipes may help in the case of undiagnosed blepharitis before cataract surgery or other kinds of ocular surgery, as they could also reduce the risk of postoperative endophthalmitis . Further studies are needed regarding the convenience of the medical regimen of use 3 and 5 days before surgery; the time the microbiota takes to recover itself after the use of the eyelid wipes; and what occurs if the patient uses the wipes more than 5 days . Another aspect worthy of study is the usefulness of applying the eyelid wipes as a postoperative prophylaxis or the medical regimen of use . This study demonstrated that the application of heat and a massage to the eyelids before the use of the eyelid wipe improves the reduction in the microbiota present in the ocular surface in 5 days, and eyelid hygiene can be use as a complementary prophylactic approach to prevent ocular infections.
It has been reported that the unicompartmental knee arthroplasty has good long - term outcomes for western and japanese patients . Alternatively, several reports have described reoperations after unicompartmental knee arthroplasty because of post - operative knee pain and sometimes it is difficult to diagnose the cause of pain . We treated a patient with anteromedial knee pain caused by intra - articular scar tissue that contained residual cement fragments on the anterior surface of a femoral implant following oxford unicompartmental knee arthroplasty . After arthroscopic resection of the scar tissue and removal of the 3 mm residual cement covered with the scar tissue, the patient s post - operative symptoms were considerably alleviated . This is the first report describing a case of painful intra - articular scar tissue following unicompartmental knee arthroplasty . It has been reported that the oxford unicompartmental knee arthroplasty (oxford uka, biomet, warsaw) has good long - term outcomes for western and japanese patients [1, 2] and lesser frictional polyethylene wear . The clinical outcomes have been good, and out of 350 patients, at present, only one patient has required conversion to total knee arthroplasty (tka). Alternatively, several reports have described reoperations after oxford uka because of post - operative knee pain and it has been reported that reoperations, including conversion to tka, has been required for various reasons such as lateral compartment arthritis, loose implants, bearing dislocation, tibial fracture, lateral meniscus injury, osteonecrosis, and synovitis [4, 5, 6]. Diagnosis of the causes leading to the requirement of conversion to tka requires the careful evaluation of clinical and image findings, therefore, post - operative unexplained pain persists in 5 - 8% [4, 5, 7] of patients and diagnosis can be difficult . Furthermore, some reports indicate that the formation of intra - articular soft tissue can cause pain following tka such as patella clunk or soft tissue impingement [8, 9]. Takahashi et al . Found that eight patients had abnormal soft tissue formation in the patellofemoral joint following tka, and indicated that all patients improved after arthroscopic surgery . However, to the best of our knowledge, there are no reports of soft tissue related pain following uka . Therefore, we describe a case of painful intra - articular scar tissue formation caused by residual cement fragment following uka, which showed considerable improvement after arthroscopic resection . Post - operative radiographs of his left knee showed that the femoral and tibial implants were placed properly, and there were no loose cement fragments . Post - operative course was good, and the patient was discharged at 3 weeks walking alone . At that time, pain developed on the anterior knee surface of the left knee, and at two and a half years after surgery; the pain became localized around the medial margin of the patella of the left knee . The symptoms led to suspicion of soft - tissue - mediated pain, and a local anesthesia injection temporarily achieved complete pain relief . However, the patient s symptoms subsequently persisted, and an additional surgery was scheduled . Pre - operative range of motion of the left knee was 0 - 140, and there was no marked swelling of that joint . There was pain and tenderness at the medial margin of the patella, pain when standing from a bending position, and pain during twisting movement . The radiolucent line was verified beneath the tibial components in both knees 1 year after surgery on plain radiographs, and we diagnosed it as physiological radiolucency because it was no more than 2 mm thick and was surrounded by sclerotic margin . The surgical findings showed that scar tissue extending from the intercondylar area to the posterior part of the infrapatella fat pad covered the anterior surface of the femoral implant to the anterior groove of the implant, and we were able to confirm that the scar tissue was in contact with the femoral implant (fig . While surgically removing the scar tissue, a 3 mm diameter cement fragment was identified in the scar tissue and this was removed along with all of the scar tissue (fig . The symptom disappeared immediately, and at present, 2 years after the additional surgery, there has been no recurrence of symptoms and the pain has disappeared . Physiological radiolucent line was more than 2 mm thick and was surrounded by sclerotic margin; however, this did not expand over time . There is no movement of the bone fragments on the medial aspect of the tibia . Left: 1 year following surgery, right: 2 and a half years following surgery . (a) scar tissue extending from the intercondylar area to the posterior part of the infra - fat pad covered the anterior surface of the femoral implant and was in contact with the implant . (b) while removing the scar tissue a 3 mm loose cement fragment within scar tissue was identified and removed . The patient was informed that his data would be submitted for publication, and he gave his consent for the same . We describe a patient requiring removal of painful intra - articular scar tissue caused by retained cement fragment, because of anteromedial pain following uka . We assumed that the cause of pain might be intra - articular soft tissue because pain was localized from the anterior surface of the knee to the medial patella . Furthermore, local anesthesia injection to the area of pain temporarily yielded complete pain relief and showed considerable improvement after arthroscopic resection . The fact that patient had been completely asymptomatic for several months after primary uka and additional arthroscopic surgical findings implied that the new soft tissue had come into contact with the femoral implant causing irritation . Through the same mechanism as medial patellar plica, scar tissue causes a fibrotic reaction as a result of irritation, which is thought to have caused the pain in the current case and scar resection considerably relieved the pain . During the last 10 years, painful soft tissue formation occurred in 0.3% (one of 350) patients undergoing the procedure, and this is a relatively rare phenomenon . As mentioned above, to the best of our knowledge, there are no reports of new painful intra - articular soft tissue formation following uka . The rare painful scar tissue formation, in this case, lucas et al . Claimed that foreign bodies are one cause of new intra - articular soft tissue formation in tka and the fact that the symptoms in our patient only developed on one side, despite surgery on both knees, led us to assume that retained cement was involved in the scar tissue formation . However, this theory was an assumption, because we could not have histological findings of resected soft tissue . Many reports have described complications due to loose cement fragments following uka [12, 13, 14]. Complication from such fragments in uka was first reported by berger et al ., after which howe et al . Reported four cases of pain and swelling from 2 to 14 months following minimally invasive uka, and jung et al . Reported a patient who developed severe pain and hemarthrosis 6 months following surgery however, in our patient, the cement fragment was not loose but covered with the scar tissue, which differs from previous clinical symptoms caused by the direct contact of a loose cement fragment . Depending on the diagnosis of the cause of post - uka pain, it may be improved with arthroscopic surgery . In addition, post - operative mri is not a useful diagnostic tool because of halation generated by implants following uka . Arthroscopic surgery was performed in our patient because intra - articular soft tissue formation was speculated to be the cause of pain . Pandit et al . Reported seven cases of arthroscopic surgery following oxford uka for meniscus damage in four patients, osteonecrosis of the lateral femoral condyle in one patient, and synovitis in two patients, and found that symptoms improved in all patients except for the patient with necrosis . Similarly, bergeson et al . Reported seven cases of arthroscopic surgery following oxford uka and indicated that the underlying causes were retained cement, lateral meniscal damage, or synovitis . This shows that in a small number of cases, treatment by arthroscopy without conversion to tka will alleviate symptoms . In our patient in addition, conversion to tka should be avoided with the physiological radiolucent line proposed by goodfellow et al . [10, 15] often observed just below the tibia implant on post - operative plain radiograph . It can be mistakenly interpreted as looseness, resulting in revision surgery in some patients . The accurate diagnosis of pain following uka is also important to avoid such unnecessary revision procedure . We have reported on a case of painful intra - articular scar tissue formation following uka, which improved considerably after arthroscopic scar tissue resection for the first time . This report indicated that scar tissue formation could be one of the causes of knee pain following uka and arthroscopic surgery could be a treatment option for knee pain following uka.
In the may issue of genome medicine, belmont and mcguire make the case for a' uniform electronic health record' (ehr) that includes both genotype and phenotype information . By uniform they mean a single data standard across different ehr databases and user interfaces, rather than a single database or a single user interface (this has been confirmed by personal communication with the authors). It is certainly true that a clearer picture of a patient's health is possible when their genotype data are combined with phenotype data . The quantity and quality of these data are improving, along with the analytical tools that allow us to interpret them . Patients, clinicians and researchers can all benefit from a better understanding of these data, and belmont and mcguire's article describes efforts in europe and the usa to unify the datasets . However, other parties that would benefit from better understanding include public health officials, government bureaucrats, insurance companies and employers . And in some cases, there are conflicts of interest; for example, an insurance company could use genetic information to raise premiums or deny cover, whereas a patient might use the same information to seek increased cover when they learn of the risk for future diseases . There are ways to solve the conflicts of interest that can arise from the use and availability of patient data . First, as belmont and mcguire describe, efforts such as the personal genome project allow patients to opt in to fully disclose their genetic information for the benefit of researchers . Patientslikeme.com has an openness policy alongside their privacy policy so that participants can agree to share all their data, and tens of thousands of people from around the world have already agreed to do so . The value to researchers is currently limited because the data are self - submitted rather than independently verified, but the proof that patients are willing to share their personal information is there . The principle must still stand, however, that data sharing begins with and is controlled by the patient . This favors a single personal health record (phr) as a database rather than a single electronic health record . Phrs are records owned and controlled by the patient, as opposed to ehrs, which are owned and controlled by health care practitioners . Useful data standards for phr and ehr communication should be expanded to fit the genomic vision that belmont and mcguire outline . In particular, the continuity of care record (ccr) data format is the digital equivalent of a referral letter from one clinician to another about a patient . It is supported by phr providers such as google health and microsoft healthvault; pharmacies such as walgreens and cvs; and providers such as minuteclinic . The department of health and human services at the national cancer institute unveiled a standard earlier this year for family history . De - identification is a better term than anonymization because the latter implies a binary process, which is misleading, while the former accurately conveys a spectrum . We know that de - identification algorithms are already in use when the public interest demands phenotype sharing but patient consent is not possible or practicable . Examples include notifiable disease surveillance, public health planning and large - scale research . In these cases, looking after the patient's privacy requires measures that ensure they cannot be identified through illicit use of those data . Illicit patient re - identification has three sources of risk: the research team, all other people who have access to these data and finally the inherent readability of the data itself . Building a single system to be accessed by hundreds or thousands of researchers across tens or hundreds of projects such systems can therefore never be adequately private . What might work, when public interest demands but consent is not possible, are schemes that separately copy just the minimum of phenotype and genotype data from various health management systems for a specific group of vetted researchers working within a highly protective legal context . Any change in project purpose would necessitate a re - assessment of the prevailing risks . A system in which highly vetted organizations were permitted to collect and link minimal data from all its various sources would be ideal . In addition, the architecture for a single ehr or phr is not a simple one . It is desirable and correct to view all the relevant data at the time of making a clinical decision or coming to a research conclusion . However, that does not mean all the data should be viewable . For the person viewing the data, their storage in a single place but for the people whose data are viewed, such a data warehouse is ripe for abuse . Citizens have expressed their distrust of such systems on many occasions, and security experts have repeatedly pointed out the risks of data warehouses . Federated architectures, where data are spread across multiple sites and queried as needed, have been deployed and are made easier by new approaches, such as service - oriented architecture . And knowing how much protection to put in place is made easier by couching privacy concerns in terms of the risk of illicit patient re - identification . All of the above discussion is not to say that a single ehr is a bad idea . Belmont and mcguire make a good case for the need to unify data in the service of laudable aims, including providing good patient care and advancing medical research . However, just because something can be done does not mean that it should be done, and in health care it is patients who should decide what should be done . They will be the most affected by privacy breaches, so they must be the ones who decide which of the benefits to take advantage of . The good news is that mature technologies exist that do put patients in control . As professionals we need to earn their trust by using these technologies when we ask for data sharing that makes our jobs easier . Ma is the ceo of patients know best, a company that makes and sells personal health record software . Rn is the ceo of sapior, a company that makes and sells de - identification software for the private sharing of health data . Ma wrote the sections on personal health records and rn wrote those on de - identification.
It usually appears several years after abdominal surgery or trauma, and causes substantial problems with the interpretation of clinical and morphological findings . There are few case - reports published and all of them show tumors in the surgical site of the previous surgery . A 32-year - old man was referred to our hospital complaining of a painful mass in the left hypochondrium, postprandial distension and a weight loss of about 14 kg in the previous three months . Ultrasonography (usg) of the abdomen showed a 2 cm 2 cm hypoechoic lesion in contact with the abdominal wall (fig . 1). Computerized tomography (ct) of the abdomen showed a heterogeneous mass in the great omentum below the stomach and above the transverse colon (fig . Laparoscopic exploration revealed an omental mass firmly attached to the abdominal wall . A great deal of purulent fluid spread during the procedure . Due to the difficult exploration of the abdominal cavity, the procedure was converted to hand assisted laparoscopy by means of a wound retractor (alexis, applied medical, rancho santa margarita, ca, usa), enlarging 4 cm the incision of the 5 mm trocar on the left hypochondrium . We find an omental tumor involving the stomach and the transverse colon . Inside the mass there were purulent material and non - absorbable polypropylene sutures (figs . 3 and 4). Debridement of the wall abscess was performed and a piece of the wall was sent for frozen histology examination which proved intra - operative, negative for neoplasic cells . An intra - operative fistulography revealed that the abscess cavity was not communicated with the bowel lumen . This tumor was first described by schloffer in 1809 . He evaluated four patients with chronic inflammatory tumor of the abdominal wall following inguinal hernioplasties . Bacteriologic study reveals low virulent germ, generally staphylococcus, and a thick wall of fibrous tissue . The volume varies from a small tumor to giant masses of 2 or 3 kg with a central abscess . Histological examination reveals abundant fibrous granulation tissue and lymphocyte infiltration caused by foreign body reaction . These tumors occur postoperatively after an undetermined free period, which can range from months to years . In abdominal surgery it has been described after appendectomy, hernioplasty, hysterectomy, oophorectomy, gastrectomy, cholecystectomy, biliary surgery or colonic resections . Abdominal wall, stomach, colon, omentum, testis, and bladder can be invaded . The tumor - like symptoms predominate over inflammatory symptoms, and general status is not affected . The sonographic appearance of suture granulomas is characterized by hyper echoic double lines (rail - like lines) or single lines within a hypoechoic lesion . It was suggested that st should also be taken into consideration as differential diagnosis in patients with a history of past operations and cumulative operative wounds of the abdomen revealed by fdg - pet . Accumulation images in fdg (ring - shaped appearance) should alert surgeons of the possibility of diagnosing foreign body granuloma, in addition to patient's history, us and ct findings . Some authors suggest that complete tumor resection should always be performed, and others just simply drain the tumor . In our case, we only performed abscess drainage and removal of non - absorbable suture material after histological confirmation of benignity . The interesting thing about this case is the appearance of the tumor after a one - year interval, in a place far away from surgical site . The patient relates he had had a large left hernia, surely with omentum inside . When a tumor appears after surgery, st should always be suspected . In many laparotomies performed on daily basis using non - absorbable suture material, we suggest that a mini - invasive approach to these tumors should always be performed . Complete resection is not required, if we have a negative frozen histologic examination . A simple drainage and removal of suture material, without major resections, solves the problem of these patients . The authors have no financial and personal relationships with other people or organizations that could inappropriately inuence (bias) this submission . Written informed consent was obtained from the patient for publication of this case report and its accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Carlos m. canullan, nicolas f. baglietto and roberto f. klappenbach: performed the operation . Juan alvarez rodriguez, luis t. chiappetta porras: study design and interpretation.key learning pointsschloffer tumor is an inflammatory pseudotumor.it usually appears several years after abdominal surgery.we suggest a mini - invasive approach, a simple drainage and removal of suture material, without major resections . Schloffer tumor is an inflammatory pseudotumor.it usually appears several years after abdominal surgery.we suggest a mini - invasive approach, a simple drainage and removal of suture material, without major resections . We suggest a mini - invasive approach, a simple drainage and removal of suture material, without major resections.
Brain ct is still the technique of choice for evaluation of head trauma7,12,13,16,17,22) and it is an important part of the diagnostic armamentarium in a head injury as neuro - cranial imaging2,21,23,24). It is readily obtainable in most medical centers and is the cornerstone for rapid diagnosis5). The availability of ct, its rapidity of scanning, reconstructive ability and compatibility with medical resuscitation devices underlie its use in acute head injury imaging5,10,17,22). Contrast from ct scans, mri findings typically demonstrate the lesions from the onset of injury, but many facilities cannot perform mri on an emergent basis . In addition, mri examination can take up to an hour to perform, and patients may require sedation to minimize motion artifacts . Moreover, national insurance in korea usually does not cover for mri if brain ct scan does not show abnormal lesions in head injury patients . Thus, in such cases, doctors have usually skip the components of a further study, such as brain mri . However, in a few cases, mri can show abnormal findings, such as cerebral contusion even in such a case12). It is important to find out whether there is an intracranial injury or not which is not shown in a brain ct due to legal issues, medical insurance, and neuropsychiatric dysfunction . Few studies have been taken in case of mild head injury to decide whether only a ct scan is sufficient to evaluate it or not and when further study such as mri is needed . The purpose of this study was to clarify the debate through a study of a large number of patients with negative brain ct after head trauma . This study was designed to determine; 1) the incidence of abnormal brain mri findings even in the negative brain ct scan, 2) specific cases which mri is needed in mild head trauma patients with normal intracranial finding of brain ct scan, and 3) which predictable risk factors including patients' characteristics [age, gender, past medical history, associate symptoms, coagulopathy, loss of consciousness, initial glasgow coma scale (gcs) score, symptom duration], injury mechanism, and lesion location are highly related with positive brain mri finding even if negative intracranial finding of brain ct scan . From january 1, 2009 to february 28, 2011, we prospectively registered cases of mild head injury having negative brain ct scan, who visited the neurosurgery department of our hospital . During a 2-year period, we prospectively evaluated brain ct and brain mri of 180 patients with mild head injury . Mild head injury was defined as gcs score of 13 to 14 or gcs score of 157), with at least one of the following risk factors: history of loss of consciousness, short - term memory deficit, amnesia for the traumatic event, post - traumatic seizure with blunt injury to the head, such as traffic accident, pedestrian trauma, fall down injury, slipped down injury or sports injury11,16,19,24). Persons whose continuous observation were possible during hospitalization and underwent both brain ct and mri, were included in this study . Patient with a gcs score <13, contraindications for ct, or concurrent intracranial injuries in head ct at presentation were excluded . After the original clinical examinations, all patients underwent standard ct scan of the head according to the judgment of the treating physician . All brain ct scans, 64-mdct (somatom sensation, siemens medical solution, mnchen, germany), were performed within 8 hours of presentation to the hospital and non - contrast axial whole brain ct scans were obtained with 4.8 mm of slices thickness . Also, brain and bone windows were obtained from all patients . All the including patients underwent early brain mri [4.672.65 days after injury (meansd)] within 7 days after first brain ct scan for confirm whether they have intracranial injuries or not . We performed a mri protocol on a 3.0-t scanner (achieva tx, philips medical system, amsterdam, netherlands) with axial and sagittal t1-weighted sequence, axial t2-weighted sequences, t2 flair, and axial gre sequence using a 16-in diameter coil . Saturation recovery images were obtained with a 500-msec repetition time, using averages of four signals, a matrix size of 340191, a slice thickness of 5 mm, and multislice data acquisition . All studies were interpreted by two neurosurgeons and one neuroradiologist, who had a certificate for added qualification in neuroradiology . According to brain ct and brain mri findings 1a - e) with negative brain ct and negative brain mri, group ii (fig . Patients with intracranial abnormalities of initial brain ct scan after mild head injury or incidental findings of cerebral infarction, brain tumor, or cerebral aneurysm were excluded .' Negative' ct scan and brain mri imaging were defined as having no traumatic intracranial lesion, except scalp swelling or simple linear skull fracture . Simple linear skull fracture if it was not combined with intracranial lesion was considered to be' negative' brain ct scan or brain mri imaging . Besides,' positive' brain mri imaging were considered to show intracranial abnormal finding on brain mri imaging, such as traumatic intracranial lesion (depressed skull fractures, focal or diffuse contusion, parenchymal hematoma, epidural hematoma, subdural hematoma, subarachnoid hemorrhage). Patients were classified by interpretation according to presence or absence of abnormal brain mri finding . Two neurosurgeons and one neuroradiologist validated the results of interpretation of the images both brain ct scan and brain mri double blindly . An independent staff who was unaware of the agreement between two sets of readings was analyzed with the use of cohen's kappa test and the statistical package for the social sciences version 12.0 software (ibm spss statistics, ibm corporation, new york, usa). There was intra - observer agreement between the two interpretations for 20 imaging study as follows: (kappa: 1.000) for ct, [kappa: 0.773 (p<0.001), confidence interval (ci): 0.527 - 0.907] for mri . To determine the reproducibility of the ct scan and mri data, 20 patients were reviewed and examined by a second physician at the time of the initial evaluation . There was inter - observer agreement between the two sets of evaluations for 20 patients as follows: (kappa: 1.000) for ct, [kappa: 0.773 (p<0.001), ci: 0.527 - 0.907] for mri . Data from the electric charts were tabulated into patient's characteristics and injury characteristics categories . The principal investigators reviewed total medical records in order to determine reliability and validity of the data collection method . These data included in age, gender, past medical history, coagulopathy, main symptom, loss of consciousness, initial gcs score, symptom duration, injury type, mechanism of injury, and trauma type . According to the injury type, simple injury was defined as head trauma only, and multiple injury was defined as head trauma with other tissue injury . Coagulopathy was defined as history of bleeding or clotting disorder or current treatment with aspirin, clopidogrel or warfarin17,18). Low initial gcs score was defined as gcs score of 13 to 14 . In table 1, the chi - square test in order to validate the significance of gender, main symptom, the loss of consciousness, and symptom duration . The fisher's exact test was used in order to validate the significance of past medical history, coagulopathy, and initial gcs score . Fisher's exact test for injury type and chi - square test for injury mechanism, and abnormal extracranial lesion were used for significance testing . A 53-year - old woman visited our hospital because of headache after pedestrian traffic accident . She had past medical history of hypertension and not taken any other medications . At the time of admission, gcs score was 14 points, and she had loss of consciousness . She showed normal level of coagulation factor on routine hematology examination . Except for scalp laceration, external wound of the other head parts was not observed . Initial brain ct scan which was taken at other institution did not show abnormal lesion, except scalp swelling . Thus, we performed brain mri which showed hemorrhagic contusion at left frontal lobe (fig . After we managed with medication and closed observation, she was discharged without any neurologic deficit 1 week later . From january 1, 2009 to february 28, 2011, we prospectively registered cases of mild head injury having negative brain ct scan, who visited the neurosurgery department of our hospital . During a 2-year period, we prospectively evaluated brain ct and brain mri of 180 patients with mild head injury . Mild head injury was defined as gcs score of 13 to 14 or gcs score of 157), with at least one of the following risk factors: history of loss of consciousness, short - term memory deficit, amnesia for the traumatic event, post - traumatic seizure with blunt injury to the head, such as traffic accident, pedestrian trauma, fall down injury, slipped down injury or sports injury11,16,19,24). Persons whose continuous observation were possible during hospitalization and underwent both brain ct and mri, were included in this study . Patient with a gcs score <13, contraindications for ct, or concurrent intracranial injuries in head ct at presentation were excluded . After the original clinical examinations, all patients underwent standard ct scan of the head according to the judgment of the treating physician . All brain ct scans, 64-mdct (somatom sensation, siemens medical solution, mnchen, germany), were performed within 8 hours of presentation to the hospital and non - contrast axial whole brain ct scans were obtained with 4.8 mm of slices thickness . Also, brain and bone windows were obtained from all patients . All the including patients underwent early brain mri [4.672.65 days after injury (meansd)] within 7 days after first brain ct scan for confirm whether they have intracranial injuries or not . We performed a mri protocol on a 3.0-t scanner (achieva tx, philips medical system, amsterdam, netherlands) with axial and sagittal t1-weighted sequence, axial t2-weighted sequences, t2 flair, and axial gre sequence using a 16-in diameter coil . Saturation recovery images were obtained with a 500-msec repetition time, using averages of four signals, a matrix size of 340191, a slice thickness of 5 mm, and multislice data acquisition . All studies were interpreted by two neurosurgeons and one neuroradiologist, who had a certificate for added qualification in neuroradiology . According to brain ct and brain mri findings, two groups were classified as follows; group i (fig . 1a - e) with negative brain ct and negative brain mri, group ii (fig . Patients with intracranial abnormalities of initial brain ct scan after mild head injury or incidental findings of cerebral infarction, brain tumor, or cerebral aneurysm were excluded .' Negative' ct scan and brain mri imaging were defined as having no traumatic intracranial lesion, except scalp swelling or simple linear skull fracture . Simple linear skull fracture if it was not combined with intracranial lesion was considered to be' negative' brain ct scan or brain mri imaging . Besides,' positive' brain mri imaging were considered to show intracranial abnormal finding on brain mri imaging, such as traumatic intracranial lesion (depressed skull fractures, focal or diffuse contusion, parenchymal hematoma, epidural hematoma, subdural hematoma, subarachnoid hemorrhage). Patients were classified by interpretation according to presence or absence of abnormal brain mri finding . Two neurosurgeons and one neuroradiologist validated the results of interpretation of the images both brain ct scan and brain mri double blindly . An independent staff who was unaware of the agreement between two sets of readings was analyzed with the use of cohen's kappa test and the statistical package for the social sciences version 12.0 software (ibm spss statistics, ibm corporation, new york, usa). There was intra - observer agreement between the two interpretations for 20 imaging study as follows: (kappa: 1.000) for ct, [kappa: 0.773 (p<0.001), confidence interval (ci): 0.527 - 0.907] for mri . To determine the reproducibility of the ct scan and mri data, 20 patients were reviewed and examined by a second physician at the time of the initial evaluation . There was inter - observer agreement between the two sets of evaluations for 20 patients as follows: (kappa: 1.000) for ct, [kappa: 0.773 (p<0.001), ci: 0.527 - 0.907] for mri . Data from the electric charts were tabulated into patient's characteristics and injury characteristics categories . The principal investigators reviewed total medical records in order to determine reliability and validity of the data collection method . These data included in age, gender, past medical history, coagulopathy, main symptom, loss of consciousness, initial gcs score, symptom duration, injury type, mechanism of injury, and trauma type . According to the injury type, simple injury was defined as head trauma only, and multiple injury was defined as head trauma with other tissue injury . Coagulopathy was defined as history of bleeding or clotting disorder or current treatment with aspirin, clopidogrel or warfarin17,18). Low initial gcs score was defined as gcs score of 13 to 14 . In table 1, the chi - square test in order to validate the significance of gender, main symptom, the loss of consciousness, and symptom duration . The fisher's exact test was used in order to validate the significance of past medical history, coagulopathy, and initial gcs score . Fisher's exact test for injury type and chi - square test for injury mechanism, and abnormal extracranial lesion were used for significance testing . A 53-year - old woman visited our hospital because of headache after pedestrian traffic accident . She had past medical history of hypertension and not taken any other medications . At the time of admission, gcs score was 14 points, and she had loss of consciousness . She showed normal level of coagulation factor on routine hematology examination . Except for scalp laceration, external wound of the other head parts was not observed . Initial brain ct scan which was taken at other institution did not show abnormal lesion, except scalp swelling . Thus, we performed brain mri which showed hemorrhagic contusion at left frontal lobe (fig . After we managed with medication and closed observation, she was discharged without any neurologic deficit 1 week later . Eighteen patients (10.0%) of 180 after mild head injury had intracranial injury, even though they had not intracranial abnormal brain ct finding . The mean age of the 180 patients was 48.9 years (range, 13 to 88), and 51% were male . The mean ages for group i and ii were 48.2 years and 54.6 years respectively . In group i, female (n=83) was more common than male (n=79) and male (n=12) was more common than female (n=6) in group ii . However, there was no statistical significance between the two groups in the age (p=0.083) and gender (p=0.233) (table 1). A total of 71 patients had past medical history, including 42 patients (23.3%) with hypertension, 14 patients (7.8%) with diabetes, 7 patients (3.9%) with liver disease, and 8 patients (4.4%) with others . Thirty patients (16.7%) had dizziness, 13 patients (7.2%) had blurred vision, 8 patients (4.4%) had insomnia, and 36 patients had others . Ten patients (55.6%) of the group ii patients and 40 patients (24.7%) of the group i patients had loss of consciousness . Compared with group i, group ii has a higher proportion than group i with the loss of consciousness, which is statistically significant (p=0.006). Group ii showed higher percentage of low initial gcs score than those of group i (28% vs. 6%). Patients presenting initial low gcs score was statistically significant to have intracranial lesions (p=0.009). Most of them, symptoms of 102 cases sustained less than 2 weeks (56.7%). As for symptoms lasting more than two weeks of the entire inter - group analysis, there were 63 cases (38.9%) of group i compared with that (83.8%) of group ii which was significantly different (p=0.0003). Other injury types, except 115 cases without external damage, were as follows: scalp laceration (n=10) and scalp abrasion (n=38). The most common cause of head injury was 55 cases (30.6%) of simple rear - end motor vehicle crashes . Injury mechanisms were standing fall [35 (19.4%) cases], pedestrian versus vehicle [19 (10.6%) cases], motorcycle crash, and fall from height [9 (5.0%) cases] were in row . Large impact trauma, such as fall from height or pedestrian versus vehicle, was relatively frequent than simple rear - end motor vehicle crash, which refers less impact trauma such as in group ii . Simple injury was more common than multiple injury (158 cases vs. 22 cases), but multiple injury in group ii was significantly frequent than group i cases (27.8% vs. 10.5%) (p=0.049). These findings included subdural hematoma [5 (27.8%) cases], single focal contusion [5 (27.8%) cases], diffuse cerebral contusion [5 (27.8%) cases], subarachnoid hemorrhage [2 (11.1%) cases], and epidural hematoma [1 (5.6%) case] (table 3). The most common location of abnormal brain contusion after mild head injury was a temporal base (n=8), and second location was a frontal pole (n=5). In addition, our study showed the other location as follows: falx cerebri (n=2), basal ganglia (n=1), sylvian fissure (n=1), and tentorium (n=1) (fig . Eighteen patients (10.0%) of 180 after mild head injury had intracranial injury, even though they had not intracranial abnormal brain ct finding . The mean age of the 180 patients was 48.9 years (range, 13 to 88), and 51% were male . The mean ages for group i and ii were 48.2 years and 54.6 years respectively . In group i, female (n=83) was more common than male (n=79) and male (n=12) was more common than female (n=6) in group ii . However, there was no statistical significance between the two groups in the age (p=0.083) and gender (p=0.233) (table 1). A total of 71 patients had past medical history, including 42 patients (23.3%) with hypertension, 14 patients (7.8%) with diabetes, 7 patients (3.9%) with liver disease, and 8 patients (4.4%) with others . Thirty patients (16.7%) had dizziness, 13 patients (7.2%) had blurred vision, 8 patients (4.4%) had insomnia, and 36 patients had others . Ten patients (55.6%) of the group ii patients and 40 patients (24.7%) of the group i patients had loss of consciousness . Compared with group i, group ii has a higher proportion than group i with the loss of consciousness, which is statistically significant (p=0.006). Group ii showed higher percentage of low initial gcs score than those of group i (28% vs. 6%). Patients presenting initial low gcs score was statistically significant to have intracranial lesions (p=0.009). Most of them, symptoms of 102 cases sustained less than 2 weeks (56.7%). As for symptoms lasting more than two weeks of the entire inter - group analysis, there were 63 cases (38.9%) of group i compared with that (83.8%) of group ii which was significantly different (p=0.0003). Other injury types, except 115 cases without external damage, were as follows: scalp laceration (n=10) and scalp abrasion (n=38). The most common cause of head injury was 55 cases (30.6%) of simple rear - end motor vehicle crashes . Injury mechanisms were standing fall [35 (19.4%) cases], pedestrian versus vehicle [19 (10.6%) cases], motorcycle crash, and fall from height [9 (5.0%) cases] were in row . Large impact trauma, such as fall from height or pedestrian versus vehicle, was relatively frequent than simple rear - end motor vehicle crash, which refers less impact trauma such as in group ii . Simple injury was more common than multiple injury (158 cases vs. 22 cases), but multiple injury in group ii was significantly frequent than group i cases (27.8% vs. 10.5%) (p=0.049). These findings included subdural hematoma [5 (27.8%) cases], single focal contusion [5 (27.8%) cases], diffuse cerebral contusion [5 (27.8%) cases], subarachnoid hemorrhage [2 (11.1%) cases], and epidural hematoma [1 (5.6%) case] (table 3). The most common location of abnormal brain contusion after mild head injury was a temporal base (n=8), and second location was a frontal pole (n=5). In addition, our study showed the other location as follows: falx cerebri (n=2), basal ganglia (n=1), sylvian fissure (n=1), and tentorium (n=1) (fig . Approximately two thirds of patients with head trauma in the united states are classified as having mild head injury5,11). Mild head injury include patients with scores of 13 to 15 on the gcs, indicating little or no impairment in the consciousness4,5,12,13,15 - 17,19,20,23). Thus, it is important to exclude whether there is brain contusion or not in such a case . In early 1990s, several retrospective studies of patients with minor head injury reported substantial proportions with intracranial lesions on ct (17 to 20%)5). Head ct images obtained immediately after the traumatic event often show no evidence of brain swelling or edema, but practically normal3,6). Approximately less than 10% of patients with mild head injury in the united states had positive findings on a brain ct scanning, implying that greater than 90% had normal ct findings5,15). In another study of patients with a score of 15 on the gcs, the rate of intracranial lesions on brain ct was similar (6 to 9%)2). Ct is considered as the first choice in the assessment of the patients with acute head injury15,22). A ct scan is probably recommended for all patients because one in five will have an acute lesion detectable by the scan with head injury . It can be performed quickly; newer ct scanners can complete a scan within 5 minutes . Ct scan findings help identify abnormalities that may need acute intervention and can be performed in the presence of life support equipment . Thus, it is easily accessible in most hospitals and a good screening tool to triage mild head injury so as to ascertain who should be safely discharged home or admitted22). Ct is the preferred tool for skull lesions and the sensitivity of ct for significantly higher than mri for evaluation of fracture . In addition, advantageously poor quality ct images, due to motion blurring, are easily repeated . Compared many advantage of ct scan, it has several weak points in mild head injury . The detection of superficial contusions using ct scans is hampered by artifacts from adjacent bone . Imaging findings in brain contusions tend to vary because of the stages of evolution common to these lesions . Initially, ct findings can be normal or minimally abnormal because the partial volumes between the dense microhemorrhages and the hypodense edema can render contusions isoattenuating relative of the surrounding brain8). When patient, whose initial brain ct was normal, complains of continuous symptoms after discharge, brain mri may show an abnormal intracranial pathology . This is reason why mri is the choice for full assessment of brain lesions after head injury . Mri is more sensitive and accurate than ct for detecting contusions because of its multiplanar capability and greater sensitivity for edema8,9). Mri has clear advantage over ct in the evaluation of lesions seen in minor traumatic brain injury like nonhemorrhagic cortical contusions, and follow - up parenchymal changes8). Also, the sensitivity of mri is significantly higher than ct in detecting diffuse axonal injury, brain stem lesions, non - hemorrhagic contusion or subacute subdural bleed and sinus invasion8,22). Streak and beam hardening artifacts particularly degrade the imaging of lesions close to the cerebral convexities22). On these regards, mri with superior soft tissue contrast and multi - planarity is more sensitive for chronic traumatic head injury, subtle abnormality and has strong correlations with long term neuropsychological outcome22). Mri findings typically demonstrate the lesions from the onset of injury, but many facilities cannot perform mri on an emergent basis . Estimation of lesion volume based on mri was frequently greater than with that of ct . In addition, mri examination can take up to an hour to perform, and patients may require sedation to minimize motion artifacts . Longer scanning time, poor sensitivity to skull fractures and sah as well as the inability to monitor patients in the mri fields are its drawbacks22). But, in mild head injury, false negative brain ct scan possibly include insensitive to concussion, diffuse axonal injury, early cerebral edema and has poor resolution in imaging of posterior cranial fossa22). Mri outperforms ct in visualization of small intraparenchymal abnormalities as small contusions and small foci of traumatic axonal injury14). If ct cannot demonstrate pathology, mri is warranted to adequately be explained to account for clinical state . Yuh et al.25) reported that 27% of mild traumatic brain injury patients with normal admission head ct had abnormal early brain mri which was performed 1239 days after injury . They had shown that a subset of mild traumatic brain injury patients have significant alterations in neuropsychiatric functioning within weeks to months of injury, and approximately 15% have measurable deficits at 1 year . Our study has shown that 10 percent of patients after mild head injury had positive findings on brain ct scanning and had no required neurosurgical intervention15). As mri becomes more widely available, it may have a greater role in the evaluation of patients with mild head injury . Diffusion tensor imaging is more sensitive to white matter injury than conventional mri and ct . Recently, the use of diffusion tensor imaging of mild traumatic brain injury14) is especially important in cases where the patient experiences chronic postconcussive symptoms despite unrevealing conventional imaging . Mri cannot always be performed in all patients with mild head injury due to high prices . If so, when would mri be really indicated in the patients with negative brain ct scan in mild head injury? The use of clinical findings as predictors of intracranial lesions in patients with mild head injury has been evaluated in several studies1,4). Our study had meaningful understandable results about two views, such as 1) patients characteristics and 2) injury mechanism to find out predictable risk factors of intracranial pathology in group ii . Head trauma at the age of 60, coagulopathy, history of neurosurgical procedure or epilepsy, and drug or alcohol consumption were widely known risk factors for intracranial pathology on a brain ct scan after mild head injury1,11,16,21,24). In our study, group ii of higher proportion of patients with loss of consciousness, initial low gcs score, and symptoms duration showed statistically significant risk factors . Thus, careful investigation on these factors especially in mild head trauma patients should be executed . When head trauma patients admit to the hospital, investigator often overlooks the information about the injury mechanism . Our study provides that this information is important to predict intracranial pathology in patients with negative brain ct scan after mild head injury . Such as falls from height, falls from standing, pedestrian traffic accident, rather than simple rear - end motor vehicle accident, demonstrated more significantly common in group ii patients . In the trauma type, group ii showed having a high percentage of linear skull fracture rather than external lesions such as scalp laceration or abrasion . These results suggest that they may cause to exert more direct blow to make an occurrence of the intracranial pathology in patients with negative brain ct scan after mild head injury than simple accident . In addition, long duration of symptoms (> 2 weeks) was significantly higher percentage in group ii than group i. accordingly, the authors recommend that doctors should be alert in cases of multiple injury and complaining of more than 2 weeks . As above results, even the negative brain ct, brain mri should be recommended to perform especially in limited cases of accompanying those risk factors . Although our study consisted of 180 consecutive trauma patients, some limitations exist . During time interval between brain ct and brain mri, if trauma progresses, new intracranial pathology which was absent in the initial brain ct can be visible in brain mri at a later point in time . Although this hypothesis occur probably quite low because this study is confined in mild head injury patients with negative initial brain ct scan, this is a weak point of our study, and further study is needed in the future . Our study shows that a few patients (10%) with mild head injury, even in negative brain ct scan have intracranial pathology on brain mri . It was more commonly associated with multiple injury than simple one and when the patients complain sustained symptoms more than 2 weeks, with loc, and with initial low gcs score after mild head injury . Thus, physicians including neurosurgeons should be alert and do not hesitate to perform brain mri to carefully watch the regions of temporal base, frontal pole, or falx cerebri in cases of mild head injury accompanying these risk factors.
Reduction of elevated intraocular pressure is the only established modifiable risk factor shown to reduce the risk of glaucoma - associated optic neuropathy.1,2 topical beta - adrenergic antagonists and carbonic anhydrase inhibitors are well accepted medical treatments for reducing production of aqueous humor.3,4 newer prostaglandin analogs reduce intraocular pressure by promoting uveoscleral aqueous outflow.46 however, the ocular hypertension treatment study showed that almost 40% of patients will require a combination of two or more medications to achieve a 20% reduction in intraocular pressure.1 complex drug regimens requiring the use of numerous bottles can reduce the therapeutic effect, and use of multiple bottles has also been shown to be a barrier to compliance.79 coadministration of drops may introduce a washout effect if not adequately spaced in time.10 similarly, eye drop tolerability will affect compliance and treatment outcomes.7 multiple - drop therapies have been formulated to address some of these compliance issues . Azarga (alcon laboratories inc, fort worth, tx) is a fixed combination of brinzolamide 1% + timolol 0.5%, and is effective in reducing intraocular pressure.11 in comparison, a fixed combination of brinzolamide 1% + timolol 0.5% is noninferior to a fixed combination of dorzolamide 2% + timolol 0.5% and achieved lower mean intraocular pressures at nine of 12 study visits.12 tolerability comparisons of these two fixed - combination eye drops seem to show a reduction in eye irritation using the brinzolamide formulation, but an increase in blurred vision.1215 in this study, we directly switched patients requiring multiple - drug treatments from a fixed combination of dorzolamide - timolol to brinzolamide - timolol and investigated the impact of this change on tolerability and compliance . This study was performed at the glaucoma unit in the royal hallamshire hospital, sheffield, uk . Patients were switched from a fixed preparation of dorzolamide - timolol to brinzolamide - timolol . Following the switch, patients were contacted by telephone and a number of questions were asked over a period of 426 weeks after changing eye drops . One independent questioner read a preformed questionnaire to patients over the telephone (figure 1). Telephone consultations were initiated by confirming the patient details and that correct antiglaucoma treatment was concurrent . A primary yes / no question was asked to confirm if a specific side effect had been experienced . If a side effect was noted for either drug, a comparison of the two types of eye drop was made by asking patients to assign a numerical value (1 to 9) comparing the new (brinzolamide - timolol) and old (dorzolamide - timolol) drops . Thirty - one consecutive patients whose medication had been altered were contacted and questionnaires were successfully completed . Thirty - one consecutive patients (12 males and 19 females aged 4189 years) successfully completed the questionnaire . The data were analyzed for significant differences from the assumed mean of 5 using the t - test if the sample set was> 20 . When the sample set was <20 the wilcoxon signed - rank test was performed . A test for proportions was used to compare the numbers of patients experiencing a specific side effect . The population varied in the percentages experiencing side effects, as shown in table 1 . Comparison of the percentages of side effects indicated less stinging but more blurring for brinzolamide - timolol compared with dorzolamide - timolol eye drops (table 1). There were no differences in the proportions of patients who experienced altered taste or redness following instillation of the eye drops . A comparison of the severity and chronicity of the side effects of the two types of fixed - combination eye drops is shown in table 2 . The fixed combination of brinzolamide - timolol produced significantly less stinging and for a shorter amount of time than the dorzolamide - timolol eye drops; it also produced less eye redness and for a significantly shorter amount of time . However, the fixed combination of brinzolamide - timolol produced more blurring, although the length of time this was present was similar to that with dorzolamide - timolol . No differences between the two eye drops were found for taste, overall impression, and likelihood of compliance . Previous studies have shown the fixed - combination brinzolamide - timolol eye drop to be significantly more effective than its individual components and noninferior to fixed - combination dorzolamide - timolol eye drops in lowering intraocular pressure.11,12 we suggest that although this crossover study was unidirectional and of limited size, it does demonstrate a different side effect profile for the two types of fixed - combination eye drops . Stinging was significantly less and lasted for a shorter period of time with the brinzolamide - timolol eye drops, which is consistent with previous findings.1215 apart from any intrinsic differences in the two molecules which may alter the stinging profile of the two drugs, the ph of the eye drops containing brinzolamide is relatively more neutral than that of dorzolamide (ph 7.5 and ph 5.6, respectively). Furthermore, eye drops containing dorzolamide use sodium citrate as a buffer whereas none is present in the eye drops containing brinzolamide . All these differences seem to cause the brinzolamide - timolol eye drop to sting less . Redness can be a problematic side effect of any antiglaucoma medication, and accordingly reduces compliance, but may also be a surrogate of general irritation and stinging . We found that fixed - combination eye drops containing brinzolamide or dorzolamide show a slight reduction in severity of redness, but of significantly shorter duration . Given that this represents another reduction in side effects, it should have the effect of increasing tolerability.7,9 blurring of vision is a known side effect of brinzolamide,11 but studies have shown conflicting results when compared with dorzolamide . Stewart et al did not find any difference between the two drops.17 however, silver found a higher incidence of visual blurring for brinzolamide than with dorzolamide, as did manni et al and mundorf et al.12,13,16 this increase in blurring of vision with the eye drop containing brinzolamide was also observed in our study, but does not seem to last any longer than the blurring caused by the eye drop containing dorzolamide . The increase in visual blurring is probably due to the viscosity of the eye drop, with the brinzolamide - timolol combination eye drop being much thicker than the dorzolamide - timolol eye drop . Indeed, seven of the 31 patients we interviewed stated that they found the brinzolamide - timolol eye drop difficult to apply due to its thickness . Also, one patient switched back to the dorzolamide preparation because of this application problem, even though she preferred the side effect profile of the brinzolamide eye drop . Unfortunately absolute levels of blurring and the finite time for which the blurring persisted were not assessed in this study . Thus, no assumptions can be made regarding how blurring affects general daily activities using either type of eye drop . It could be that blurring persists long term and causes a significant reduction in ability to perform daily tasks, or it could be very transient and cause few problems . An answer to this question would help to make a more meaningful comparison of the side effect profiles of the two combination preparations . Initial studies by silver showed a higher incidence of altered taste sensation with brinzolamide than with dorzolamide,16 although subsequent studies, including our study, showed no difference.12,17,18 there was no statistically significant difference between the overall comparison and likelihood of compliance with the fixed - combination brinzolamide - timolol and dorzolamide - timolol eye drops in this study . Previous studies have found a patient preference for the drop containing brinzolamide over the one containing dorzolamide.13,15 however, this and other studies show a reduction in stinging and redness at the cost of increased blurred vision . It is possible that the exchange of one side effect for another leaves the eye drops being equivalent overall . Interestingly, jampel et al found that patients would pay more for an eye drop with reduced blurring but would not pay more for a reduction in any other side effect.19 this observation would suggest a preference for the dorzolamide combination overall, but this does not appear to be the case in this study . Our study confirms the finding of other researchers pertaining to the side effect profiles of fixed combinations of brinzolamide - timolol and dorzolamide - timolol, ie, a reduction in stinging and redness but an increase in blurred vision . The advantage of one eye drop over the other then becomes patient - specific, depending on which side effect they find more tolerable . We suggest that both types of eye drop are acceptable and interchangeable choices for treating patients with glaucoma, but compliance may be an issue as a result of the specific side effects of one drop or the other.
More than a century ago, the first scientific report was published about fracture fixation using plates . The pioneers in plating fractures were the belgian surgeon albin lambotte (18661955) and the scottish surgeon sir william arbuthnot lane (18561938), who introduced internal plate fixation for fractures . During the 1950s, open reduction and plate fixation for fractures were standardized by the founders of the swiss association for the study of internal fixation . The ultimate goal of fracture treatment and internal fixation, as noted in the first edition of the arbeitsgemeinschaft fr osteosynthesefragen, is to restore the function of the injured limb . Plate osteosynthesis is capable of providing anatomical reduction and stable fixation, as well as allowing early functional mobilisation and usually leading to better clinical outcomes . Since the introduction of plate fixation for fractures, several plates and screws have been developed, all with their own advantages and disadvantages . Material dysfunction was first described by lane in 1895, who described corrosion, and lambotte in 1909, who characterized the insufficient strength of the metal used . In 1926, both alloys and stainless steel were introduced in fracture surgery, and from that moment on the quality of the implant material improved over the years . In order to achieve greater fracture stability, it was thought that thicker and heavier plates should be used for internal fixation . Although these heavy plates resulted in a strong and rigid fixation, they had the disadvantage of a compromised blood supply to the fractured bone, often resulting in bone necrosis and ultimately delayed union or non - union . In the search for a better plate, the dynamic compression plate was developed and eventually evolved into the limited - contact dynamic compression plate . A new type of plate was introduced with up to 50% less cortical contact than the dynamic compression plate, thereby preserving the periosteal blood supply . With the search for and development of new materials and techniques for fracture fixation, less invasive procedures have become increasingly popular over the last few decades [46]. In order to accomplish reduction and ultimately a stable fixation of the fracture, large incisions have often been used for adequate exposure . The dissection and accompanying devitalization of the soft tissue and bone create a less favourable environment for bone healing and increase the risk of infection . The benefits of less invasive procedures are obvious in smaller surgical wounds, where they result in less postoperative pain, improved postoperative function, the preservation of blood supply, and fewer surgical site infections [46]. Due to an increasing demand for minimally invasive procedures, these systems were designed to be inserted though small, strategically placed incisions . Following this development, minimally invasive plate osteosynthesis (mipo) was introduced for fracture fixation . With the mipo technique, procedures could be performed with smaller incisions and thus with less soft tissue damage and a better preserved blood supply . Mipo has so far only been described in diaphyseal and metaphyseal fractures of the femur, tibia, and the humerus . Blunt thoracic trauma often leads to isolated or multiple fractured ribs, especially in elderly patients with osteoporotic bones . In the united states alone, over 450,000 patients with fractured ribs present to the emergency department each year . It has been reported that the presence of multiple fractured ribs is associated with a mortality rate of up to 30% and a major morbidity rate of up to 70% . Fractured ribs can lead to respiratory problems, especially when fractured in more than one place, as in flail chest . Generally, patients with fractured ribs suffer from pain, resulting in less efficient breathing, impaired ventilation, and ultimately the development of pneumonia . Studies have shown that up to 30% of patients with isolated or multiple fractured ribs develop pneumonia . This potentially lethal complication could lead to a prolonged hospital stay, the need for mechanically supported ventilation with all its associated complications (e.g., pneumothorax and ventilation - acquired pneumonia), and ultimately an increase in health care costs . However, even without pneumonia, the presence of fractured ribs is associated with the need for mechanical ventilation, especially in patients with flail chest . Flail chest is defined as three or more consecutive, ipsilateral, doubly broken ribs . These patients often show impaired ventilation due to the paradoxical movements of fractured ribs during normal breathing . Although the majority of patients with fractured ribs can be treated conservatively, the surgical fixation of fractured ribs has become increasingly popular . The ultimate goal of surgical rib fixation is to reduce pain, optimise oxygenation and ventilation, and ultimately avoid the need for mechanical ventilation . However, an increasing amount of evidence suggests that surgical rib fixation improves the outcome of patients with flail chest . Recent studies have shown that operative stabilization in flail chest patients is associated with fewer days on mechanical ventilator support and a lower incidence of pneumonia . Relative indications for surgical rib fixation, such as delayed or non - union of fractured ribs, severely displaced fractured ribs, and excessive pain despite maximal analgesia, are still the subject of debate, and future studies must show whether patients with these conditions benefit from surgical rib fixation . Many surgical approaches for rib fixation have been described in the older literature, but most of these techniques are now obscure . In the early years of the twentieth century, the severe clinical consequences of flail chest were already recognized, with a reported mortality of up to 80%, mostly due to ventilatory problems . This high mortality rate prompted many surgeons to develop techniques that could fix the fractured ribs, with the ultimate goal of improving ventilation and reducing mortality . In 1926, the first report of rib fixation in a patient with flail chest was described . A bullet forceps on traction was used to percutaneously apply external support to the chest wall . Some examples include the cape town limpet, analogous to a regular sink plunger that was applied to the chest wall, resulting in expansion of the thorax, and the introduction of a hook from a clothes hanger into the sternum, which was connected to a weight, resulting in traction on the chest wall . The rationale of all these old - fashioned techniques was to restore the biodynamic characteristics of the chest wall and to reduce pain . The first open surgical open reduction and fixation of fractured ribs was described in 1967 . Two k - wires were used to fix the fractured ribs during thoracotomy . Following this report, many intramedullary techniques have been described, as well as suturing and bridging techniques with which the fractured ribs were surrounded or connected by metal wires and sutures . Since 1980, plate osteosynthesis has become more popular, probably due to the high complication rate and unsatisfactory results of the aforementioned techniques . Several specially designed plates and screws have been described, but only in small studies with a short period of follow - up . Over the past decades, the quality of the plates and screws has improved, ultimately leading the current use of anatomic and angularly stable titanium plates that easily fit onto the fractured ribs . As described above, mipo has become increasingly popular in orthopaedic surgery in patients who have experienced trauma, especially in patients with fractures of the long bones . So far, no case series or technical reports have described the role of mipo in surgical rib fixation . Since a solitary rib could be seen as a long bone, similar to the tibia, humerus, and femur, although much more fragile, mipo may be a promising alternative technique to the current maximally invasive approaches . Remarkably, in 1975, paris et al . Reported a less invasive technique for rib fixation in a handful of patients, but this technique has never been described in further detail . Currently, large incisions with considerable surgical insult are most commonly used to stabilise rib fractures . Therefore, we describe this technique in more detail below . In order to successfully apply the mipo principles to rib fixation, it is crucial to position the patient correctly and to determine the correct location of the incision . Fractured ribs are difficult to palpate, and preoperative imaging techniques should therefore be used to determine the localisation of the fracture and, consequently, the location of the incision . In the preoperative setting, we think that a computed tomography (ct) scan is essential . The literature shows that plain chest x - rays underestimate the amount of fractured ribs, as well as the severity of the fractures [2022]. In our opinion, plain chest x - rays should only be used to obtain general information about the number and severity of the fractured ribs . If there is any doubt about the diagnosis or if there is any clinical suspicion of multiple fractured ribs, a ct scan should be performed . With the ct scan, an exact localisation of the fractured ribs can be performed and the severity of the fractures can be assessed . Three - dimensionally (3d) reconstructed pictures of the rib cage are a helpful tool for obtaining a global overview of the fractured ribs (fig . 1). Caution, however, is warranted, as the 3d technique tends to smoothen out the fracture lines of the ribs, with the ultimate risk of missing certain fractures . Therefore, we still depend on the conventional transverse slices of the ct scan for planning the operation . Subsequently, all fractures are drawn in a schematic diagram, creating a roadmap for the operation (fig ., a plan can be made to identify which ribs require correction and which can be left without surgical treatment . The next step is to determine the location of the incision(s). This can be done by using four anatomical landmarks, which can always be found regardless of the phenotype of the patient: (1) jugular notch of the sternum, (2) xiphoidal point of the sternum, (3) mid - axillary line, and (4) scapular point . The first three anatomical landmarks are always in exactly the same position, while the location of the scapular point is position - dependent . When using the scapular point as an anatomical landmark, it is essential to know whether the patient was positioned during the preoperative ct scan with the arm elevated or with the arm parallel along the torso . In the majority of cases, the location of the fractured ribs and, thus, the location of the incision can easily be determined using the aforementioned anatomical landmarks . If the surgeon needs an even more accurate localisation, ultrasound imaging can be used . The literature indicates that ultrasound in experienced hands is a reliable imaging tool for determining the location of the fractured ribs . The day before the operation, we always perform an ultrasound to obtain an even more exact localisation . During the ultrasound, the radiologist positions the patient exactly as he or she will be placed during the operation . This last step is essential, as one can imagine that the location of the marked skin in relation to the fractured ribs will vary depending on the position of the patient . 3 shows a patient with the preoperative ultrasound localisation of four fractured ribs on the anterior chest wall . When initiating the operation, additional tools and techniques facilitate the application of the mipo principles . The alexis wound retractor is a frequently used instrument in thoracoscopic- and laparoscopic - assisted procedures . However, no scientific reports have yet described the use of the alexis wound retractor in approaches to the chest wall and surgical rib fixation . When starting the mipo procedure, the skin is incised at the predetermined location, followed by division of the subcutaneous tissue . Depending on the location of the fractures, muscle fibres are divided in a muscle - sparing manner until the chest wall is reached . Subsequently, a cavity is created between the chest wall and the overlying soft tissue, enabling the placement of the alexis wound retractor . This retractor is available in several sizes, but we mainly use the retractor with a diameter of 4 cm . After placement of the inner ring between the chest wall and the soft tissue, the retractor is tensioned by rolling itself on the outer ring, creating an outward retracting force and forming a window through which the procedure can be performed . Another advantage of the alexis wound retractor is that the rubber seal between the inner and outer ring has a haemostatic feature, creating a dry operating window . Analogously to the mipo technique used in long bone fractures, additional tools are needed to facilitate surgery . These additional tools are available in a separate mipo matrix kit (depuy - synthes, west chester, pa, usa) (fig . This kit, together with the 90-degree drill and screwdriver, enables the surgeon to stabilize almost every fracture type . In the next paragraphs the 90-degree drill and screwdriver enable the surgeon to drill through a small incision in a perpendicular fashion to the plate and rib, ensuring the correct placement of the locking screws . The drill has a knob at the end, making it possible to manually drill a hole . However, if power drilling is preferred, the rear knob can be substituted with a maxilla facial power drill or the pendrive (depuy - synthes). The 90-degree drill device also functions as a 90-degree screwdriver if the drill bit is substituted with a screwdriver bit . The challenge with using the 90-degree drill device is to drill a hole perpendicular to the rib exactly in the middle of a well - positioned plate . It is important to realize that an off - angle placement with a maximum of only five degrees will be tolerated by the plate and screw . In order to facilitate this perpendicular drilling, two drill guides with four different approach angles one of these tools is a trocar set with a soft tissue clamp (fig . An appropriate plate is pre - bent by the surgeon and positioned on the rib ., a trocar is placed in a strategic manner enabling the placement of multiple screws . Next, through the trocar, a drill guide is placed in the designated hole and screwed in the thread of the plate . Additionally, a soft - tissue clamp is placed underneath the tissue envelope on the same trocar . When lifted and closed, the clamp will tension the soft tissue, creating a window through which the plate can be visualised and approached . Subsequently, the rib is drilled with the appropriate drill length, the drill guide is removed, and an appropriate - length screw is placed . The mipo kit also provides a threaded reduction tool (trt), which is extrapolated from the less invasive stabilisation system used for long bone fractures . This trt consists of a threaded drill with a nut at the end . With this tool, after drilling, the nut at the end of the trt should be turned down, pushing the trocar with the plate onto the rib (fig . This results in a temporarily fixed plate, enabling the surgeon to drill and place all other screws into the plate . Finally, the trt is removed and a screw can be placed, using the hole that was pre - drilled . This clamp enables the perpendicular fixation of a plate onto a rib through a small incision (fig . One should, however, realise that the clamp has a limited range corresponding to the thickness of the rib . The introduction of mipo in rib fixation has led to new difficulties that are not likely to occur in conventional surgery . As addressed in the previous paragraphs, the location of the incision is crucial, since a certain number of rib fractures must be fixed through the same small incision . Furthermore, working through small incisions makes it challenging for the surgeon to determine the screw length . In the pre - operative plan, the ct images are crucial for determining the rib thickness and, thus, the length of the screws . A helpful trick in regard to drilling and determining the screw length is to pay attention to the behaviour of the drill penetrating the first and second cortex and the remaining length of the drill in relation to the trocar . A surgeon experienced with this technique can use it to determine whether the chosen drill length was adequate . It may sometimes be necessary to adjust the length of the drill and screws, as an estimation made on the basis of the ct scan may not always prove accurate . This is much more demanding than doing so through a large incision and requires some experience . It is essential not to rush the procedure, since an inaccurate contour or incorrect placement of the plate will result in insufficient osteosynthesis . Especially in surgeons who are not familiar with mipo as mentioned before, no scientific reports have so far described the use of mipo for rib fixation . This reflects the fact that mipo for rib fixation is a new and developing technique . Comparable with mipo techniques for the long bone surgery, it is expected that in the near future, new tips and tricks will become available, resulting in improved patient care . The unique feature of a fractured rib compared to a fractured long bone is the pleural cavity underneath the rib . In theory, it may be possible to fix the fractured rib through a thoracoscopic approach, but no adequate tools or implants yet exist that would make this possible . Due to the continually increasing interest in surgical rib fixation, new developments and approaches are expected in the near future . In order to successfully apply the mipo principles to rib fixation, it is crucial to position the patient correctly and to determine the correct location of the incision . Fractured ribs are difficult to palpate, and preoperative imaging techniques should therefore be used to determine the localisation of the fracture and, consequently, the location of the incision . In the preoperative setting, we think that a computed tomography (ct) scan is essential . The literature shows that plain chest x - rays underestimate the amount of fractured ribs, as well as the severity of the fractures [2022]. In our opinion, plain chest x - rays should only be used to obtain general information about the number and severity of the fractured ribs . If there is any doubt about the diagnosis or if there is any clinical suspicion of multiple fractured ribs, a ct scan should be performed . With the ct scan, an exact localisation of the fractured ribs can be performed and the severity of the fractures can be assessed . Three - dimensionally (3d) reconstructed pictures of the rib cage are a helpful tool for obtaining a global overview of the fractured ribs (fig . 1). Caution, however, is warranted, as the 3d technique tends to smoothen out the fracture lines of the ribs, with the ultimate risk of missing certain fractures . Therefore, we still depend on the conventional transverse slices of the ct scan for planning the operation . Subsequently, all fractures are drawn in a schematic diagram, creating a roadmap for the operation (fig ., a plan can be made to identify which ribs require correction and which can be left without surgical treatment . The next step is to determine the location of the incision(s). This can be done by using four anatomical landmarks, which can always be found regardless of the phenotype of the patient: (1) jugular notch of the sternum, (2) xiphoidal point of the sternum, (3) mid - axillary line, and (4) scapular point . The first three anatomical landmarks are always in exactly the same position, while the location of the scapular point is position - dependent . When using the scapular point as an anatomical landmark, it is essential to know whether the patient was positioned during the preoperative ct scan with the arm elevated or with the arm parallel along the torso . In the majority of cases, the location of the fractured ribs and, thus, the location of the incision can easily be determined using the aforementioned anatomical landmarks . If the surgeon needs an even more accurate localisation, ultrasound imaging can be used . The literature indicates that ultrasound in experienced hands is a reliable imaging tool for determining the location of the fractured ribs . The day before the operation, we always perform an ultrasound to obtain an even more exact localisation . During the ultrasound, the radiologist positions the patient exactly as he or she will be placed during the operation . This last step is essential, as one can imagine that the location of the marked skin in relation to the fractured ribs will vary depending on the position of the patient . 3 shows a patient with the preoperative ultrasound localisation of four fractured ribs on the anterior chest wall . When initiating the operation, additional tools and techniques facilitate the application of the mipo principles . The alexis wound retractor is a frequently used instrument in thoracoscopic- and laparoscopic - assisted procedures . However, no scientific reports have yet described the use of the alexis wound retractor in approaches to the chest wall and surgical rib fixation . When starting the mipo procedure, the skin is incised at the predetermined location, followed by division of the subcutaneous tissue . Depending on the location of the fractures, muscle fibres are divided in a muscle - sparing manner until the chest wall is reached . Subsequently, a cavity is created between the chest wall and the overlying soft tissue, enabling the placement of the alexis wound retractor . This retractor is available in several sizes, but we mainly use the retractor with a diameter of 4 cm . After placement of the inner ring between the chest wall and the soft tissue, the retractor is tensioned by rolling itself on the outer ring, creating an outward retracting force and forming a window through which the procedure can be performed . Another advantage of the alexis wound retractor is that the rubber seal between the inner and outer ring has a haemostatic feature, creating a dry operating window . Analogously to the mipo technique used in long bone fractures, additional tools are needed to facilitate surgery . These additional tools are available in a separate mipo matrix kit (depuy - synthes, west chester, pa, usa) (fig . This kit, together with the 90-degree drill and screwdriver, enables the surgeon to stabilize almost every fracture type . In the next paragraphs the 90-degree drill and screwdriver enable the surgeon to drill through a small incision in a perpendicular fashion to the plate and rib, ensuring the correct placement of the locking screws . The drill has a knob at the end, making it possible to manually drill a hole . However, if power drilling is preferred, the rear knob can be substituted with a maxilla facial power drill or the pendrive (depuy - synthes). The 90-degree drill device also functions as a 90-degree screwdriver if the drill bit is substituted with a screwdriver bit . The challenge with using the 90-degree drill device is to drill a hole perpendicular to the rib exactly in the middle of a well - positioned plate . It is important to realize that an off - angle placement with a maximum of only five degrees will be tolerated by the plate and screw . In order to facilitate this perpendicular drilling, two drill guides with four different approach angles one of these tools is a trocar set with a soft tissue clamp (fig . An appropriate plate is pre - bent by the surgeon and positioned on the rib ., a trocar is placed in a strategic manner enabling the placement of multiple screws . Next, through the trocar, a drill guide is placed in the designated hole and screwed in the thread of the plate . Additionally, a soft - tissue clamp is placed underneath the tissue envelope on the same trocar . When lifted and closed, the clamp will tension the soft tissue, creating a window through which the plate can be visualised and approached . Subsequently, the rib is drilled with the appropriate drill length, the drill guide is removed, and an appropriate - length screw is placed . The mipo kit also provides a threaded reduction tool (trt), which is extrapolated from the less invasive stabilisation system used for long bone fractures . This trt consists of a threaded drill with a nut at the end . With this tool, it is possible to temporarily fix the plate to a rib . After drilling, the nut at the end of the trt should be turned down, pushing the trocar with the plate onto the rib (fig . This results in a temporarily fixed plate, enabling the surgeon to drill and place all other screws into the plate . Finally, the trt is removed and a screw can be placed, using the hole that was pre - drilled . This clamp enables the perpendicular fixation of a plate onto a rib through a small incision (fig . One should, however, realise that the clamp has a limited range corresponding to the thickness of the rib . The introduction of mipo in rib fixation has led to new difficulties that are not likely to occur in conventional surgery . As addressed in the previous paragraphs, the location of the incision is crucial, since a certain number of rib fractures must be fixed through the same small incision . Furthermore, working through small incisions makes it challenging for the surgeon to determine the screw length . In the pre - operative plan, the ct images are crucial for determining the rib thickness and, thus, the length of the screws . A helpful trick in regard to drilling and determining the screw length is to pay attention to the behaviour of the drill penetrating the first and second cortex and the remaining length of the drill in relation to the trocar . A surgeon experienced with this technique it may sometimes be necessary to adjust the length of the drill and screws, as an estimation made on the basis of the ct scan may not always prove accurate . This is much more demanding than doing so through a large incision and requires some experience . It is essential not to rush the procedure, since an inaccurate contour or incorrect placement of the plate will result in insufficient osteosynthesis . Especially in surgeons who are not familiar with mipo as mentioned before, no scientific reports have so far described the use of mipo for rib fixation . This reflects the fact that mipo for rib fixation is a new and developing technique . Comparable with mipo techniques for the long bone surgery, it is expected that in the near future, new tips and tricks will become available, resulting in improved patient care . The unique feature of a fractured rib compared to a fractured long bone is the pleural cavity underneath the rib . In theory, it may be possible to fix the fractured rib through a thoracoscopic approach, but no adequate tools or implants yet exist that would make this possible . Due to the continually increasing interest in surgical rib fixation, new developments and approaches are expected in the near future.
It significantly reduces the risk of death (by over 60% compared to dialysis), doubles the expected survival time and greatly improves quality of life . Improvements in surgical techniques and upgrades in pharmacotherapy protocols, such as the reduction of steroid dosage and the introduction of multi - drug immunosuppression, have significantly decreased the incidence of surgical / urological complications during the last decades . However, urological complications in renal transplant recipients are still common and their occurrence is associated with significant morbidity, impairment of graft function and, in some cases, graft loss and even recipient death . Urological complications can be divided into early (occurring during first 90 days after the procedure) and late (occurring after 90 days post - procedure). We aimed to identify retrospectively late urological complications in renal transplant recipients at a single center and analyze the treatment modalities and their outcome . Between january 2008 and december 2014, a total of 58 patients after ktx were treated in the department of urology as a result of post - transplant urological complications that occurred during follow - up (1922 months) at the transplant outpatient department . A total of 460 kidneys were transplanted between 2008 and 2014 in the department of vascular surgery of wrocaw university hospital . All kidneys were harvested in the classic manner as per accepted standards concerning the removal of organs of the abdominal cavity . All ureters were anastomosed by the lich - gregoire procedure . In the lich - gregoire technique, the bladder mucosa is reached via a single cystotomy, and the distal ureter is sutured to the mucosa with an absorbable monofilament 5 - 0 suture . Routinely, in every recipient a double j (dj) stent was inserted into the anastomosed ureter during transplantation and was removed after 6 weeks in the majority of cases . Patients received calcineurin inhibitors (cyclosporine or tacrolimus), mycophenolate mofetil and steroids as immunosuppressive therapy . In high - risk patients, the role of the urology department is centered on patient qualification for transplantation, assistance during the procedure in complicated cases and complication management . Urological complications were diagnosed according to clinical symptoms and kidney function markers, and the use of ultrasound, ct scan, renal scintigraphy and antegrade or retrograde pyeloureterography . All urological complications were categorized according to the clavien - dindo classification . The choice of treatment modality depended on the severity of symptoms and complication type . Between january 2008 and december 2014, of the total of 22 patients after ktx underwent various oncological procedures and 5 were treated due to benign prostatic hyperplasia (bph) in our department of urology . Fifty - eight patients (19 women, 39 men) were treated as a result of post - transplant urological complications . The mean age in the group of recipients who had experienced urologic complications was similar to those without complications (46.1 vs. 47.8 years). Thirty - eight patients (14 females, 24 males) were admitted with ureteral stenosis (clavien grade iii). Diagnosis was confirmed mainly by ultrasound, and in some cases by percutaneous transplant nephrostomy followed by antegrade ureterogram, revealing pyelocaliectasis or ureteropyelocaliectasis . Thirty - five patients had stenosis located at the ureterovesical junction (92.2%). Initially, in 10 patients percutaneous nephrostomy was performed and in 15 patients a double j catheter was reinserted to decompress the collecting system and to ensure the patency of the kidney prior to further treatment . A total of nine open operations were carried out: in one patient because of a failed endoscopic approach and recurrent stenosis; in a patient with ureteropelvic junction obstruction, a hynes - anderson operation was performed; in a case of stenosis in the central ureter, resection of short stenosis and end - to - end anastomosis was carried out; one instance of ureter dissection from massive adhesions; in 2 patients with ureterovesical junction stenosis, reimplantation of the ureter, and 2 y - v plasties were performed . In all patients a double - j catheter was left in place after the procedure for 6 weeks . Patients mean kidney function, estimated by gfr, improved from 30 to 48 ml / min after urological treatment . Ten patients (3 females, 7 males) presented with symptomatic lymphocoele (clavien grade iii). Nine patients were successfully treated with ultrasonography - guided percutaneous drainage performed in the operating theatre . In 6 patients, the procedure was performed only once, whereas three patients required 23 drainages, and one, with recurrent lymphocoele (more than 3 drainages), required open surgical drainage with marsupialization . Five patients (1 female, 4 males) were admitted with stones in the ureters of the transplanted kidneys (clavien grade iii). One patient had 3 consecutive extracorporeal shock wave lithotripsies (eswl) performed and expulsed the fragments of stone quickly . The second unerwent 2 eswl sessions, but these proved unsuccessful; ursl with holmium laser was subsequently performed . Five patients (1 female, 4 males) with urethral strictures were treated in our department . One patient required open urethroplasty because of long - standing stenosis resulting from iatrogenic injury during catheterization . There was no vesicoureteral reflux or ureteral necrosis requiring surgical intervention, no graft loss or death related to urological complication and treatment (table 1). Urological complications in kidney transplant patients ktx kidney transplantation; usrl ureterorenoscopic lithotripsy; eswl- extracorporeal shock wave lithotripsy; the first successful renal transplantation was carried out on 23 december 1954 by dr joseph murray, a plastic surgeon, and dr hartwell harrison, an urologist . For years, urologists were the primary surgeons to perform renal transplantations . At present, the surgical role of the urologist in renal transplantation has become less important because of, among others, reduced training in vascular surgery for specializing urologists . However, due to specific problems related to the genitourinary tract, urological input in renal transplantation is still vital and the urologist s familiarity with operations on the genitourinary tract is frequently invaluable . Moreover, in many centres urologists perform laparoscopic living donor nephrectomy (ldn). In our urology department, we also perform laparoscopic ldn procedures without postoperative complications . Finally, urological evaluation and intervention is often necessary in patients after ktx to save the transplanted kidney and patient . Complications such as urine leakage, ureteral stenosis, lymphocoele, lithiasis, urethral stricture and vesicoureteral reflux are reported to occur in between 2.5% and 30% of all recipients, depending on the criteria . The majority of urological complications were managed by endourological approach (82.5%) instead of open surgery . Since 2002, routine insertion of a ureteral stent resulted in a substantial reduction of urinary leakage or fistula . Ureteral stenting, despite lowering the frequency of urinary leaks and early obstruction due to anastomotic edema, can cause urinary tract infections or urethral injury with bleeding . In our centre, the ureteral stent was generally maintained for 6 weeks (but now reduced to 34 weeks) and then removed during cystoscopy . Yet, some authors advocate the early removal of the stent at the end of 2 weeks after renal transplantation to decrease the rate of urinary tract infections [7, 8]. Ureteral stenosis, with urine flow obstruction, was the most frequently observed complication, having occurred in 38 patients . In the literature, ureteral stenosis has been reported as the major long - term urological complication, at a rate of 38% [9, 10], but accounted for about 50% of all urological complications . Almost all of our patients (92%) had stenosis located at the ureterovesical junction as a consequence of development of fibrosis at anastomosis site . Previous reports indicate that anastomotic stenosis occurred in 6095% of patients with stenosis . In our department, some authors have described an average time to ureteral stenosis of 5.4 months, but the 10-year risk post - transplantation is estimated to be 9% . Our group observed increased frequency of ureteral stenosis in male patients whose donors were men . It is believed that ureteral strictures and their subsequent obstructions are caused by surgical errors in anastomosis, ureter ischemia, immunological factors and acute or chronic rejection episodes, infections, and immunosuppressive drugs . It has been proven that dissection of periureteral connective tissue and excessive manipulation of the so - called golden triangle (the site confined by the ureter, kidney and renal artery) should be avoided . Necrosis of the distal ureter was reported in up to 70% of patients with damage to this site . It has been shown that ureteral spatulation of more than 10 mm is effective in decreasing this complication . Another proven risk factor of late ureteral stenosis is polyomavirus bk (bkv) infection . The therapy of ureteral obstruction must be introduced as early as possible to avoid loss of graft function . A wide range of therapeutic methods is available, depending on stricture location and aetiology . In our department, of the 38 patients with ureteral obstruction, 25 were initially treated with nephrostomy or ureteral stent insertion to restore renal function . Twenty - nine patients with anastomotic stricture underwent single endoscopic ureter orifice incision and double - j stent placement to good effect . The remaining 9 patients (24%) underwent open surgical procedures, including a hynes - anderson operation, resection of central ureter stenosis and end - to - end anastomosis, reimplantation of a ureter and 2 y - v plasties . In patients with multiple or long ureteral stenosis, ureteropyelostomy or ureteroureterostomy can be employed and in cases with an unusable ureter, the boari flap procedure may be considered [12, 14, 15] in the majority of the patients, kidney function significantly improved following urological treatment . The first - line treatment in every patient with symptomatic lymphocoele was ultrasonography - guided percutaneous drainage without sclerotic agent administration, performed in the operation theatre . Percutaneous drainage and fenestration (both open and laparoscopic) are commonly applied in the management of lymphocoele . It has been shown that laparoscopic fenestration is a safe treatment for symptomatic lymphocoele and is associated with the lowest risk of lymphocoele recurrence . Yet, it seems that small and benign lymphocoeles should be treated with percutaneous drainage [16, 17]. It is believed to result from accumulating lymph, originating from lymphatic vessels surrounding the iliac vessels damaged during the operation or from lymph vessels of the transplanted kidney itself . Cautious and limited dissection, with careful ligation of damaged lymphatic vessels, is recommended to decrease the incidence of this complication . It is also important to run proper post - operative drainage . In our department, transplant urolithiasis occurred 6 years after transplantion . Though the incidence of urinary stones following renal transplantation is low (0.171.8%), it is not negligible . Urolithiasis is a dangerous complication due to the risk of obstruction, sepsis, and potential loss of allograft function [1821]. Factors predisposing to stone formation include amongst others hyperparathyroidism, recurrent urinary tract infection, hypercalciuria, and hypocitraturia [20, 22, 23] treatment options for urolithiasis in renal transplant recipients include all methods used in the general population . However, because of altered anatomy and the patient s immunocompromised condition, therapy can be difficult and may be associated with a higher risk of complications . In our department, 4 patients were successfully treated with ursl and 1 patient with 3 consecutive repeated eswl procedures . Other authors reported less effective treatment of transplant calculi with ursl . On the other hand, del pizzo et al . Reported a 100% success rate using endoscopic removal of ureteral calculi . In our study, no complications were observed with the above - mentioned procedures . In addition to the above, 5 patients with urethral strictures were treated in our department . The successful urethrotomy with optic urethrotome was performed in 4 patients, with the remaining patient requiring open urethroplasty because of long - standing stenosis, resulting from iatrogenic injury during catheterization . Urethral strictures following ktx are rare, yet serious complications, possibly leading to hydronephrosis and graft function deterioration . Urethrography is very useful in revealing the precise location and length of the urethral stenosis . Urethral strictures may be associated with iatrogenic urethral injury, prolonged catheterization time and urinary tract infection . The urological treatment was successful in all patients: renal transplant function improved, with neither graft loss nor patient death . Over the last decade, the ratio of endoscopic to open surgical procedures in the treatment of urological complications has more than doubled, with about 76% of complications treated endoscopically . In our previously published paper concerning the period 19831999 ureteral stenosis, with urine flow obstruction, was the most frequently observed complication, having occurred in 38 patients . In the literature, ureteral stenosis has been reported as the major long - term urological complication, at a rate of 38% [9, 10], but accounted for about 50% of all urological complications . Almost all of our patients (92%) had stenosis located at the ureterovesical junction as a consequence of development of fibrosis at anastomosis site . Previous reports indicate that anastomotic stenosis occurred in 6095% of patients with stenosis . In our department, some authors have described an average time to ureteral stenosis of 5.4 months, but the 10-year risk post - transplantation is estimated to be 9% . Our group observed increased frequency of ureteral stenosis in male patients whose donors were men . It is believed that ureteral strictures and their subsequent obstructions are caused by surgical errors in anastomosis, ureter ischemia, immunological factors and acute or chronic rejection episodes, infections, and immunosuppressive drugs . It has been proven that dissection of periureteral connective tissue and excessive manipulation of the so - called golden triangle (the site confined by the ureter, kidney and renal artery) should be avoided . Necrosis of the distal ureter was reported in up to 70% of patients with damage to this site . It has been shown that ureteral spatulation of more than 10 mm is effective in decreasing this complication . Another proven risk factor of late ureteral stenosis is polyomavirus bk (bkv) infection . The therapy of ureteral obstruction must be introduced as early as possible to avoid loss of graft function . A wide range of therapeutic methods is available, depending on stricture location and aetiology . In our department, of the 38 patients with ureteral obstruction, 25 were initially treated with nephrostomy or ureteral stent insertion to restore renal function . Twenty - nine patients with anastomotic stricture underwent single endoscopic ureter orifice incision and double - j stent placement to good effect . The remaining 9 patients (24%) underwent open surgical procedures, including a hynes - anderson operation, resection of central ureter stenosis and end - to - end anastomosis, reimplantation of a ureter and 2 y - v plasties . In patients with multiple or long ureteral stenosis, ureteropyelostomy or ureteroureterostomy can be employed and in cases with an unusable ureter, the boari flap procedure may be considered [12, 14, 15] in the majority of the patients, kidney function significantly improved following urological treatment . Lymphocoele occurred in 10 renal transplant recipients . The first - line treatment in every patient with symptomatic lymphocoele was ultrasonography - guided percutaneous drainage without sclerotic agent administration, performed in the operation theatre . Percutaneous drainage and fenestration (both open and laparoscopic) are commonly applied in the management of lymphocoele . It has been shown that laparoscopic fenestration is a safe treatment for symptomatic lymphocoele and is associated with the lowest risk of lymphocoele recurrence . Yet, it seems that small and benign lymphocoeles should be treated with percutaneous drainage [16, 17]. It is believed to result from accumulating lymph, originating from lymphatic vessels surrounding the iliac vessels damaged during the operation or from lymph vessels of the transplanted kidney itself . Cautious and limited dissection, with careful ligation of damaged lymphatic vessels, is recommended to decrease the incidence of this complication . Transplant urolithiasis occurred 6 years after transplantion . Though the incidence of urinary stones following renal transplantation is low (0.171.8%), it is not negligible . Urolithiasis is a dangerous complication due to the risk of obstruction, sepsis, and potential loss of allograft function [1821]. Factors predisposing to stone formation include amongst others hyperparathyroidism, recurrent urinary tract infection, hypercalciuria, and hypocitraturia [20, 22, 23] treatment options for urolithiasis in renal transplant recipients include all methods used in the general population . However, because of altered anatomy and the patient s immunocompromised condition, therapy can be difficult and may be associated with a higher risk of complications . In our department, 4 patients were successfully treated with ursl and 1 patient with 3 consecutive repeated eswl procedures . Other authors reported less effective treatment of transplant calculi with ursl . On the other hand, del pizzo et al . Reported a 100% success rate using endoscopic removal of ureteral calculi . In our study, no complications were observed with the above - mentioned procedures . In addition to the above, 5 patients with urethral strictures were treated in our department . The successful urethrotomy with optic urethrotome was performed in 4 patients, with the remaining patient requiring open urethroplasty because of long - standing stenosis, resulting from iatrogenic injury during catheterization . Urethral strictures following ktx are rare, yet serious complications, possibly leading to hydronephrosis and graft function deterioration . Urethrography is very useful in revealing the precise location and length of the urethral stenosis . Urethral strictures may be associated with iatrogenic urethral injury, prolonged catheterization time and urinary tract infection . The urological treatment was successful in all patients: renal transplant function improved, with neither graft loss nor patient death . Over the last decade, the ratio of endoscopic to open surgical procedures in the treatment of urological complications has more than doubled, with about 76% of complications treated endoscopically . In our previously published paper concerning the period 19831999 the improvement of transplant surgery techniques has subsequently decreased the incidence of severe urological complications in renal transplant recipients.
A 30-year - old male was admitted to our hospital due to a midline anterior neck mass . On the enhanced ct images, the mass was located at the infrahyoid midline neck and it showed the imaging features of a thyroglossal duct cyst . The enhanced oblique coronal multiplanar reconstruction ct images revealed a variation of the aortic arch as an aberrant right subclavian artery that was distal to the left subclavian artery (fig . The right vertebral artery had its origin from the right common carotid artery at the inferior border of the right thyroid gland, and it had an aberrant entrance to the c5 transverse foramen (fig . The prevertebral segment of the right vertebral artery was located in the retro - thyroid area and very close to the thyroid gland (fig . A 67-year - old female was admitted to our hospital for an operation for thyroid cancer . The enhanced ct images revealed an aberrant right subclavian artery distal to the left subclavian artery (fig . In addition, the left vertebral artery originated from the aortic arch between the left common carotid artery and the left subclavian artery . The right vertebral artery had an origin from the right common carotid artery and also an aberrant entrance to the c5 transverse foramen (fig . The prevertebral segment of the right vertebral artery was located in the retro - thyroid area and close to the thyroid gland (fig . A 30-year - old male was admitted to our hospital due to a midline anterior neck mass . On the enhanced ct images, the mass was located at the infrahyoid midline neck and it showed the imaging features of a thyroglossal duct cyst . The enhanced oblique coronal multiplanar reconstruction ct images revealed a variation of the aortic arch as an aberrant right subclavian artery that was distal to the left subclavian artery (fig . The right vertebral artery had its origin from the right common carotid artery at the inferior border of the right thyroid gland, and it had an aberrant entrance to the c5 transverse foramen (fig . The prevertebral segment of the right vertebral artery was located in the retro - thyroid area and very close to the thyroid gland (fig . A 67-year - old female was admitted to our hospital for an operation for thyroid cancer . The enhanced ct images revealed an aberrant right subclavian artery distal to the left subclavian artery (fig . In addition, the left vertebral artery originated from the aortic arch between the left common carotid artery and the left subclavian artery . The right vertebral artery had an origin from the right common carotid artery and also an aberrant entrance to the c5 transverse foramen (fig . The prevertebral segment of the right vertebral artery was located in the retro - thyroid area and close to the thyroid gland (fig . The embryologic mechanism of arsca with a right va - cc has been explained in the several studies (2, 4, 8). The normal vertebral artery (va) builds up due to the process of longitudinal anastomosis and obliteration of the horizontal parts of the cervical intersegment artery . Normally, the first to the sixth cervical intersegment arteries (cias) develop into the va and the seventh cia makes the subclavian artery (sca). If longitudinal anastomosis of the right cia stops between the 6th and 7th cia, and the right side of the dorsal aorta is obliterated proximal to the 7th cia, then the right side subclavian artery (sca) originates from the left side aorta distal to the left sca and the right va originates from the right common carotid artery (fig . Aberrant right subclavian artery has been reported with the incidence of less than 1% (8). A combination of these two variations is rare, but the true incidence of right va - cc with underlying arsca is not known . Fifteen cases of arsca were confirmed by enhanced ct or ct angiography in our hospital during the recent three years . Among these cases, only the two cases we present herein showed the right va - cc variation . Also, only these two cases had an aberrant level of the entrance of the va into the transverse foramen of the cervical spine . Based on our results, the right va - cc variation is not likely to frequently occur with an underlying arsca . There have been many reports about the variation of the arsca with a right va - cc, but only three reports remarked about the aberrant entrance of the va into the transverse foramen of the cervical spine (3, 7, 10). The vertebral artery usually enters into the transverse foramen of the 6th cervical spine (the c6 entrance). Those three reports revealed different entrance levels as c2, c3 and c4 . In our two cases, there have been no reports describing the anatomical course of this type of va - cc . In our cases, this va - cc had a close spatial relation with the thyroid gland . It was located in the retro - thyroid area and very close to the thyroid gland . During its course up to the transverse foramen this anatomical characteristic of the va - cc bears watching during anterior cervical spine surgery, thyroid surgery or other interventions . If this va - cc were overlocked, it may be pulled with the longus colli muscle, or it may become lacerated during the anterior crevical spine surgery . During thyroidectomy, the inferior thyroid artery is usually ligated . As mentioned by one autopsy result (7), the inferior thyroid artery may be near to the right va - cc, so meticulous care may be needed to avoid an inadvertent injury to the va - cc during thyroidectomy . During thyroid aspiration, the needle occasionally penetrates the posterior surface of the thyroid gland and it reaches the vertebral body . If the va - cc is near to the thyroid gland, then there is a possibility to puncture the va during thyroid aspiration . Therefore, knowledge of this aberrant course of the va may be helpful to avoid injury of the va when performing these procedures.
A 49-year - old woman was admitted with sign of acute onset tonic clonic grand mal epilepsy and seizure . She had a throbbing frontal headache resistant to analgesics and intractable vomiting and blurred vision since three months ago . Also, she had a positive medical history of craniotomy for removing brain tumor when she was 30 years old . The complaint of the patient 19 years ago was also epilepsy, which was relieved by operation and anticonvulsant therapy (carbamazepin, 200 mg, tds) for 11 years . Only the report of first ct scan was accessible, which said: a right frontal neopalastic tumofaction with severe peri lesion edema . A 56 cm round radio dense mass lesion in anterior interhemispheric arising from the falx was seen . At the site of previous craniotomy a surgical defect in the right temporal bone and adjacent right frontal lobe encephalomalacia was seen (figure 1). A 56 cm round radio dense mass lesion in anterior interhemispher after a bicoronal incision at the previous site, a craniotomy was done by removing the right frontal bone . After opening the dura matter, sub frontal and interhemispheric dissection anteriorly revealed the mass originating from falx cerebri which extended laterally in to the right frontal lobe and was tough, fibrous and beige in color . The capsule of the mass was opened and yellowish cotton wool and creamy soft tissue was removed without complication . After operation anti convulsant therapy continued and the patient was discharged with good condition and without any symptoms . Her headache and seizure were resolved . Gross examination of the received specimen showed fragments of gray - white firm tissue measuring 213 cm, totally (figure 2). The gross view of intracranial foreign body microscopic examination of the several h&e stained slides showed hyalininized tissue containing several foreign body granulomas (figure 3). Microscopic view of the intracranial foreign body polarizing microscope revealed refractile material gauze fibers (figure 4). A 56 cm round radio dense mass lesion in anterior interhemispheric arising from the falx was seen . Punctuate hyper dense area due to calcium deposition was also noted . At the site of previous craniotomy a surgical defect in the right temporal bone and adjacent right frontal lobe encephalomalacia was seen (figure 1). A 56 cm round radio dense mass lesion in anterior interhemispher after a bicoronal incision at the previous site, a craniotomy was done by removing the right frontal bone . After opening the dura matter, sub frontal and interhemispheric dissection anteriorly revealed the mass originating from falx cerebri which extended laterally in to the right frontal lobe and was tough, fibrous and beige in color . The capsule of the mass was opened and yellowish cotton wool and creamy soft tissue was removed without complication . After operation anti convulsant therapy continued and the patient was discharged with good condition and without any symptoms . Her headache and seizure were resolved . Gross examination of the received specimen showed fragments of gray - white firm tissue measuring 213 cm, totally (figure 2). The gross view of intracranial foreign body microscopic examination of the several h&e stained slides showed hyalininized tissue containing several foreign body granulomas (figure 3). Microscopic view of the intracranial foreign body polarizing microscope revealed refractile material gauze fibers (figure 4). A 56 cm round radio dense mass lesion in anterior interhemispheric arising from the falx was seen . Punctuate hyper dense area due to calcium deposition was also noted . At the site of previous craniotomy a surgical defect in the right temporal bone and adjacent right frontal lobe encephalomalacia was seen (figure 1). A 56 cm round radio dense mass lesion in anterior interhemispher after a bicoronal incision at the previous site, a craniotomy was done by removing the right frontal bone . After opening the dura matter, sub frontal and interhemispheric dissection anteriorly revealed the mass originating from falx cerebri which extended laterally in to the right frontal lobe and was tough, fibrous and beige in color . The capsule of the mass was opened and yellowish cotton wool and creamy soft tissue was removed without complication . After operation anti convulsant therapy continued and the patient was discharged with good condition and without any symptoms . Her headache and seizure were resolved . Gross examination of the received specimen showed fragments of gray - white firm tissue measuring 213 cm, totally (figure 2). The gross view of intracranial foreign body microscopic examination of the several h&e stained slides showed hyalininized tissue containing several foreign body granulomas (figure 3). Microscopic view of the intracranial foreign body polarizing microscope revealed refractile material gauze fibers (figure 4). Clinical symptoms caused by foreign body granulomas can be noticed from months to many years after surgical procedure . Suture material is the most common etiology of granulomas in noncentral nervous system surgery; and various symptoms depending on deposited location can be seen . They can simulate neoplasm clinically, radiologically, and even grossly.56 two cases reported by leong et al, illustrate the difficulty of detecting an unsuspected foreign body in mri . When the signal characteristics and morphology may appear conflicting or uncertain on mri, consideration can be given to the use of ct, which is more sensitive to the presence of hemorrhage, gas, calcium or manmade material . While a foreign body may be overlooked or difficult to demonstrate on mri, addition of ct, ultrasound or plain x - ray may provide a useful adjunct to diagnosis.7 searching published papers from 1965 to 2001 with keywords foreign body granuloma and brain, 20 such reports were found . Cotton materials used commonly for homeostasis during various operations may cause a foreign body granulomatous reaction that may resemble recurrent or progressive neoplasm or abscess . When suspecting a central nervous system mass lesion as primary or recurrent, a second look laparatomy should be planned to determine the true nature . The final treatment decisions depend on the nature of the mass lesion . In case of tumor recurrence, surgical resection and additional oncologic consultation should be recommended; if the resection yields a foreign body reaction, potentially harmful therapy can be withheld or postponed.9 although rare, differential diagnosis of foreign body granuloma is stressed to pay attention not to leave cotton pledgets during operations.10 hs carried out the experiment, analyses, interpretation, and presentation and wrote the paper . Tf planned the project and assisted in analyses, interpretation, and presentation and edited the manuscript.
Hydatidosis is an endemic parasitic disease in mediterranean countries, north africa, turkey, the middle east, australia, new zealand, south america, baltic areas, the philippines, northern china, and the indian subcontinent . However, physicians and surgeons worldwide may encounter the disease sporadically because of increased travel and immigration . It is caused by the parasite, echinococcus granulosus, which is a cestode that lives in the small intestine of dogs and other canines . Eggs are eliminated in the feces and when ingested, liberate their larvae in the duodenum of an intermediate host . The intermediate host can be sheep/ goat (pastoral hydatidosis) or reindeer / moose / caribou (sylvan hydatidosis). The larvae cross the intestinal wall and via the portal system reach the hepatic sinusoids where they develop into cysts . Some larvae are not filtered in the liver, but remain in the blood to reach the next station, the lungs . In addition, some may pass through the pulmonary circulation and travel to other organs . Larva transported in the mesenteric lymphatics are carried to the cisterna chili, the thoracic duct, and into the general circulation, ending up in a variety of distant sites . The most common site of occurrence of hydatid cysts in humans is the liver (50% to 93%). Left untreated, the cyst grows and follows one of several courses: forms fistulas into adjacent organs or the biliary system, ruptures into the peritoneal cavity causing seeding of multiple daughter cysts throughout the peritoneal cavity, developing daughter cysts within or rarely dying de novo . Older cysts have an increased risk of exogenous daughter cyst formation, which is an important factor for recurrence of disease after surgery . Medical therapy consists of albendazole alone or in combination with praziquantel by which stabilization of the disease has been reported . A variety of surgical operations have been advocated from complete resection (eg, total pericystectomy or hepatectomy) to minimally invasive procedures (eg, percutaneous aspiration of cysts). The world health organization (who) recommends percutaneous aspiration, irrigation and re - aspiration (the pair approach). However, fear of anaphylactic shock resulting from spill - age of hydatid fluid during treatment by the minimally invasive method may be discouraging a wider adoption of this technique . This study evaluates the efficacy of a new trocar - cannula system, the palanivelu hydatid system (phs), specifically designed to prevent spillage of hydatid fluid for laparoscopic management of hydatid cysts . In all patients, the diagnosis of echinococcal cyst was based on history, physical examination, ultrasound (us), and computed tomography (ct) scan . All patients were treated with albendazole 10 mg / kg / day for at least 2 weeks preoperatively and continued postoperatively for 4 weeks . The exclusion criteria were no informed consent; serious coagulation abnormalities; known allergy to local anesthetics or albendazole; deep - seated cysts; posteriorly located cysts (segments i, vii and viii); cysts less than 3 cm in diameter; pregnancy, or women who refused contraception for the time of albendazole treatment; and cysts characterized by a heterogeneous complex mass (gharbi type 4) or a calcified wall (gharbi type 5). The palanivelu hydatid system (phs) consists of a trocar and cannula along with 5-mm and 3-mm reducers . Its tip is pyramidal shaped with each facet of the pyramid bearing a fenestration to enable any fluid leaking on its insertion to be sucked into its hollow body by the cannula placed within . Its long shaft also bears 2 fenestrations opposite each other at a distance of 17 cm from the tip . Its outer nozzle is designed so that the suction tube can be fit onto it in an airtight manner (figure 1). After introducing the camera port through the umbilicus following creation of pneumoperitoneum, the hydatid cyst is identified on the surface of the liver . Then, the phs trocar with cannula is introduced into the peritoneal cavity directly over the hydatid cyst . Once inside the peritoneal cavity, the trocar is removed and the cannula alone is advanced till suction is applied through the side channel to maintain the contact between the cyst and the cannula opening . Thereafter, the trocar with a 5-mm suction nozzle inside connected to another suction machine is introduced into the cannula and, by steady pressure, is pushed into the cyst along with the cannula . Any fluid spillage on puncture of the cyst wall is immediately suctioned either into the body of the hollow trocar through its fenestrated tip and then into the suction cannula or into the outer cannula and thence, into the suction side - channel . Once the phs enters into the hydatid cyst, the trocar is removed and the cavity is irrigated through the main channel while continuous suction is simultaneously maintained all the time . In this way, fragments of laminated membrane, daughter cysts, and debris are easily removed . Once the retaining fluid is clear, co2 is insufflated at low pressure (3 mm hg to 4 mm hg), and the telescope is introduced into the cavity through the cannula to visualize the interior for any overt cyst - biliary communication . In the absence of overt cyst - biliary communication (verified by the absence of bile staining in the suctioned fluid and nonvisualization of the opening within the cyst cavity), 0.5% cetrimide is instilled into the cyst cavity as a scolicidal agent . In case of overt signs of cyst - biliary communication, use of a scolicidal agent is avoided and after marsupialization, the opening is sutured closed with 3 0 vicryl . Postoperatively, the cyst size is monitored by us at 2 weeks and 1, 3, 6, 12, 18, and 24 months . The primary end points were defined as complete cyst collapse by us at the end of the procedure, disappearance of cyst cavity or at least 50% reduction in cyst size at follow - up imaging, and disappearance of complications, such as pain, cystobiliary fistulas, vascular or biliary compression, and infection . The secondary end points of the study were recurrence of cyst cavity to> 50% of its initial size, vascular or biliary compression, fistulas, pain, and infection within 2 years after surgery, death, withdrawal from the study, or loss to follow - up . The palanivelu hydatid system (phs) consists of a trocar and cannula along with 5-mm and 3-mm reducers . Its tip is pyramidal shaped with each facet of the pyramid bearing a fenestration to enable any fluid leaking on its insertion to be sucked into its hollow body by the cannula placed within . Its long shaft also bears 2 fenestrations opposite each other at a distance of 17 cm from the tip . Its outer nozzle is designed so that the suction tube can be fit onto it in an airtight manner (figure 1). After introducing the camera port through the umbilicus following creation of pneumoperitoneum, the hydatid cyst is identified on the surface of the liver . Then, the phs trocar with cannula is introduced into the peritoneal cavity directly over the hydatid cyst . Once inside the peritoneal cavity, the trocar is removed and the cannula alone is advanced till its tip is in total contact with the hydatid cyst surface . Suction is applied through the side channel to maintain the contact between the cyst and the cannula opening . Thereafter, the trocar with a 5-mm suction nozzle inside connected to another suction machine is introduced into the cannula and, by steady pressure, is pushed into the cyst along with the cannula . Any fluid spillage on puncture of the cyst wall is immediately suctioned either into the body of the hollow trocar through its fenestrated tip and then into the suction cannula or into the outer cannula and thence, into the suction side - channel . Once the phs enters into the hydatid cyst, the trocar is removed and the cavity is irrigated through the main channel while continuous suction is simultaneously maintained all the time . In this way, fragments of laminated membrane, daughter cysts, and debris are easily removed . Once the retaining fluid is clear, co2 is insufflated at low pressure (3 mm hg to 4 mm hg), and the telescope is introduced into the cavity through the cannula to visualize the interior for any overt cyst - biliary communication . In the absence of overt cyst - biliary communication (verified by the absence of bile staining in the suctioned fluid and nonvisualization of the opening within the cyst cavity), 0.5% cetrimide is instilled into the cyst cavity as a scolicidal agent . After 10 minutes, the scolicidal agent is suctioned and the cyst is marsupialized . In case of overt signs of cyst - biliary communication, use of a scolicidal agent is avoided and after marsupialization, the opening is sutured closed with 3 0 vicryl . Postoperatively, the cyst size is monitored by us at 2 weeks and 1, 3, 6, 12, 18, and 24 months . The primary end points were defined as complete cyst collapse by us at the end of the procedure, disappearance of cyst cavity or at least 50% reduction in cyst size at follow - up imaging, and disappearance of complications, such as pain, cystobiliary fistulas, vascular or biliary compression, and infection . The secondary end points of the study were recurrence of cyst cavity to> 50% of its initial size, vascular or biliary compression, fistulas, pain, and infection within 2 years after surgery, death, withdrawal from the study, or loss to follow - up . Age and sex of 66 patients operated on with the palanivelu hydatid system the majority of patients (51.5%) presented with pain . Symptoms leading to discovery of hydatid disease most of the patients (n=60, 90.9%) had only a single cyst, while 6 patients (9.1%) had 2 cysts . The right lobe of the liver was more commonly involved (n=36, 54.5%) than was the left lobe (n=26, 39.4%). The remaining, 9 patients (13.6%) presented with cyst - biliary communication, one (1.5%) was a case of secondarily infected cyst and 2 cases (3%) were recurrent cysts . Out of the recurrent cysts, one had recurred after a laparoscopic attempt at removal elsewhere, while the other case was following open hydatid surgery (table 3). In 55 patients (83.3%), simply evacuation of the hydatid cyst by phs was done . In 9 cases (13.7%), this was followed by left lobectomy, as the cysts were large, occupying almost the entire left lobe of the liver . Transcystic fenestration of underlying cyst was carried out in 2 (3%) of the 6 patients with multiple cysts while the other 4 were dealt with by separate insertions of phs into individual cysts . Omentoplasty was performed by inserting a plug of omentum into the remnant cavity and suturing it intracorporeally with the edge of the cavity with vicryl 2 0 interrupted sutures . The average duration of the surgery was 52 minutes (range, 36 to 94). Postoperatively, 2 patients (3%) had infection, and 9 patients (13.7%) had a minor biliary leak that stopped draining by 5 days to 7 days (table 5). Types of complications classification by clavien et al.27 grade i: alterations from the ideal postoperative course, nonlife - threatening and with no lasting disability . Complications of this grade necessitate only bedside procedures and do not significantly extend hospital stay . Grade iii: complications resulting in residual long - term disability, including organ resection or persistence of life - threatening conditions . Grade iv: complications leading to patient death . Of the 66 patients, regular follow - up has been maintained in 52 patients with an average follow - up period of 5.8 years . Physicians worldwide should be aware of the presentation and management of hydatid disease, which is endemic to certain regions, as they may encounter it sporadically due to increased travel and migration . Though the common mode of infection is the unhygienic practice of consuming unwashed or improperly washed infected raw fruits and vegetables, direct contact with infected dogs is also another means of contracting the disease, especially in children . The youngest patient in the study was 14 years old though the average age of 38.6 years was in keeping with the average age of presentation in endemic areas . However in nonendemic areas, all the age groups are usually equally affected with the average age of presentation being older . Males were predominantly affected in the study though other studies have reported equal infestation in both sexes or predominant female infestation . Abdominal pain was the most common mode of presentation (51.5% of cases) in this study, which has also been reported by other authors . Liver hydatidosis is one of the most common causes of acute abdomen in endemic regions . Echinococcal infestation should be suspected in patients who present with an abdominal mass, pain, fever, jaundice, or anaphylaxis . However, in nonendemic countries, most of the cases are asymptomatic and are detected fortuitously . The most common pathology was a single cyst in the right lobe of the liver . Ultrasonography and ct are both effective imaging modalities for the detection of liver hydatid disease . Us is particularly useful for the detection of cystic membranes, septa, and hydatid sand, while ct best demonstrates cyst wall calcification and cyst infection . Certain features in us, ct, or magnetic resonance imaging (mri) may warn of biliary communication or impending cyst rupture . Ultrasonographic appearances have also formed the basis of classification of liver hydatid cysts by various authorities like gharbi, who, and milicevic . Serologic tests have a sensitivity of 65% to 90% but are not routinely performed in our institution . Several nonsurgical options have been explored . In endemic countries with scarce surgical resources, a percutaneous approach of aspiration, saremi described a percutaneous approach in which a special cutting instrument is used to fragment and evacuate daughter cysts and laminated membrane while the cavity is continuously irrigated with scolecidals . Percutaneous evacuation of cyst content (pevac) using a large bore catheter has been advocated by schipper et al . Drug therapy in the form of oral albendazole is given for specific conditions in liver hydatid, viz . Widely disseminated hydatid disease, localized disease in poor surgical risk patients, ruptured cysts, and patients in whom significant intraoperative spillage has occurred . A variety of surgical procedures are done for hydatid cysts of the liver, which are tailored to suit each individual case . These include marsupialization, closed total cystectomy, partial pericystectomy, partial pericystectomy with capitonnage, modified capitonnage, partial pericystectomy with omentoplasty, and typical and atypical liver resections . The first report of laparoscopic treatment of hydatid cyst of the liver was published in 1994 followed soon thereafter by the first report of anaphylactic shock complicating laparoscopic treatment of hydatid cysts of the liver . In fact, an exaggerated fear of anaphylaxis seemed to discourage surgeons from more widely adopting minimal access techniques for the treatment of hydatid cysts . However, gradually reports started appearing in the world literature detailing laparoscopic management of liver hydatid disease . The indications, contraindications, advantages, and disadvantages of this technique have been elucidated in our study, we have performed various procedures laparoscopically, viz, evacuation and marsupialization, transcystic fenestration, and left lobectomy . We have used the palanivelu hydatid system (phs), a specially designed trocar to obtain a totally contamination - free management of liver hydatid disease . Various laparoscopic techniques described are total pericystectomy, puncture and aspiration of contents followed by marsupialization, unroofing and drainage, unroofing and omentoplasty, and omentoplasty using helical fasteners . One of the problems faced in laparoscopic treatment of liver hydatid cysts is the difficulty in evacuating the particulate contents of the cyst, the daughter cysts, and laminated membrane . Bickel et al initially advocated the use of a large transparent beveled cannula . Later on, they modified the technique somewhat by creating a continuous vacuum inside the cannula while its tip was firmly adhered to the cyst wall . Saglam described a perforator - grinder - aspirator apparatus designed specifically for the evacuation of hydatid cysts . Kayaalp directly inserted a laparoscopic trocar into the hydatid cyst but reported greater success for anterior and unilocular cysts than for posterior and multi - locular cysts . Zengin et al used another perforator and aspirator called the per - fore - aspirator . Of all these, the isolated hypobaric technique described by bickel et al is the only one that has attempted to deal with the problem of spillage . Phs not only prevents any spillage of hydatid fluid but also assists complete evacuation of the cyst content and allows intracystic magnified visualization for cyst - biliary communication . Indications for performing procedures postoperatively, 2 of our patients had infections (clavien type i complication), while 9 of the patients had minor biliary leakage (clavien type ii complication). Complications seen in open surgery include pleural effusions, infections, biliary fistulae, subdiaphragmatic collection, liver abcesses . Thus, with laparoscopic management, the severity of complications decreases as compared with that in open surgery . With an average follow - up of 5.8 years various reports in the literature reveal a recurrence rate varying from 0.9% to 22% for open surgery . We have found phs highly effective and safe for the laparoscopic management of hydatid disease . Due to the freedom from spillage of hydatid fluid we recommend phs for management of hepatic hydatid disease . We have found its efficacy to be optimum for preventing spillage, evacuating contents of hydatid cysts, performing transcystic fenestration, and for dealing with cyst - biliary communications . The only limitation of this system is related to the anatomical relation of the cyst . At present, we do not recommend this technique for posteriorly located cysts, small cysts, and cysts deep within the hepatic parenchyma.
The diversity of microbes within a given body habitat can be defined as the number and abundance distribution of distinct types of organisms, which has been linked to several human diseases: low diversity in the gut to obesity and inflammatory bowel disease, for example, and high diversity in the vagina to bacterial vaginosis . For this large study involving microbiome samples collected from healthy volunteers at two distinct geographic locations in the united states, we have defined the microbial communities at each body habitat, encountering 8199% of predicted genera and saturating the range of overall community configurations (fig . Oral and stool communities were especially diverse in terms of community membership, expanding prior observations, and vaginal sites harbored particularly simple communities (fig . This study established that these patterns of alpha diversity (within samples) differed markedly from comparisons between samples from the same habitat among subjects (beta diversity, fig . For example, the saliva had among the highest median alpha diversities of operational taxonomic units (otus, roughly species level classification, see http://hmpdacc.org/hmqcp), but one of the lowest beta diversities - so although each individual s saliva was ecologically rich, members of the population shared similar organisms . Conversely, the antecubital fossae (skin) had the highest beta diversity but were intermediate in alpha diversity . The vagina had the lowest alpha diversity, with quite low beta diversity at the genus level but very high among otus due to the presence of distinct lactobacillus spp . The primary patterns of variation in community structure followed the major body habitat groups (oral, skin, gut, and vaginal), defining as a result the complete range of population - wide between - subject variation in human microbiome habitats (fig . Within - subject variation over time was consistently lower than between - subject variation, both in organismal composition and in metabolic function (fig . The uniqueness of each individual s microbial community thus appear to be stable over time (relative to the population as a whole), which may be another feature of the human microbiome specifically associated with health . No taxa were observed to be universally present among all body habitats and individuals at the sequencing depth employed here, unlike several pathways (fig . 2 and supp . 2, see below), although several clades demonstrated broad prevalence and relatively abundant carriage patterns . Instead, as suggested by individually focused studies, each body habitat in almost every subject was characterized by one or a few signature taxa making up the plurality of the community (fig . Signature clades at the genus level formed on average anywhere from 17% to 84% of their respective body habitats, completely absent in some communities (0% at this level of detection) and representing the entire population (100%) in others . Strikingly, less dominant taxa were also highly personalized, both among individuals and body habitats; in the oral cavity, for example, most habitats are dominated by streptococcus, but these are followed in abundance by haemophilus in the buccal mucosa, actinomyces in the supragingival plaque, and prevotella in the immediately adjacent (but low oxygen) subgingival plaque . Additional taxonomic detail of the human microbiome was provided by identifying unique marker sequences in metagenomic data (fig . 3b). These two profiles were typically in close agreement (supp . Fig . 3), with the former in some cases offering more specific information on members of signature genera differentially present within habitats (e.g. Vaginal prevotella amnii and gut p. copri) or among individuals (e.g. Vaginal lactobacillus spp .) C pathogens above 0.1% abundance (aside from s. aureus and e. coli) from the healthy microbiome, but the near - ubiquity and broad distribution of opportunistic pathogens as defined by patric . Canonical pathogens including vibrio cholerae, mycobacterium avium, campylobacter jejuni, and salmonella enterica were not detected at this level of sensitivity . Helicobacter pylori was found in only two gut samples, both at <0.01%, and e. coli was present at> 0.1% abundance in 15% of stool microbiomes (> 0% abundance in 61%). Similar species level observations were obtained for a small subset of stool samples with 454 pyrosequencing metagenomics data using phylotu . Pathogens were detected in the healthy microbiome (at> 1% prevalence of> 0.1% abundance, supp . Table 2), all opportunistic and, strikingly, typically prevalent both among hosts and habitats . The latter is in contrast to many of the most abundant signature taxa, which were usually more habitat - specific and variable among hosts (fig . This overall absence of particularly detrimental microbes supports the hypothesis that even given this cohort s high diversity, the microbiota tend to occupy a range of configurations in health distinct from many of the disease perturbations studied to date . Inter - individual variation in the microbiome proved to be specific, functionally relevant, and personalized . One example of this is illustrated by the streptococcus spp . Of the oral cavity . The genus dominates the oropharynx, with different species abundant within each sampled body habitat (see http://hmpdacc.org/hmsmcp) and, even at the species level, striking differences in carriage within each habitat among individuals (fig . As the ratio of pan- to core - genomes is high in many human - associated microbes, this variation in abundance could be due to selective pressures acting on pathways differentially present among streptococcus species or strains (fig ., we observed extensive strain - level genomic variation within microbial species in this population, enriched for host - specific structural variants around genomic islands (fig . Gene losses associated with these events were common, for example differentially eliminating s. mitis carriage of the v - type atpase or choline binding proteins cbp6 and cbp12 among subsets of the host population (fig . 4d). These losses were easily observable by comparison to reference isolate genomes, and these initial findings suggest that microbial strain- and host - specific gene gains and polymorphisms may be similarly ubiquitous . Other examples of functionally relevant inter - individual variation at the species and strain levels occurred throughout the microbiome . In the gut, bacteroides fragilis has been shown to prime t cell responses in animal models via the capsular polysaccharides psa, and in the hmp stool samples this taxon was carried at a level of at least 0.1% in 16% of samples (over 1% abundance in 3%). B. thetaiotaomicron has been studied for its effect on host gastrointestinal metabolism and was likewise common at 46% prevalence . On the skin, staphylococcus aureus, of particular interest as the cause of methicillin - resistant s. aureus (mrsa) infections, had 29% nasal and 4% skin carriage rates, roughly as expected . Close phylogenetic relatives such as s. epidermidis (itself considered commensal) were, in contrast, universal on the skin and present in 93% of nares samples, and at the opposite extreme pseudomonas aeruginosa (a representative gram negative skin pathogen) was completely absent from both body habitats (0% at this level of detection). These and the data above suggest that the carriage pattern of some species in the human microbiome may be analogous to genetic traits, where recessive alleles of modest risk are maintained in a population . In the case of the human microbiome, high - risk pathogens remain absent, while species that pose a modest degree of risk also appear to be stably maintained in this ecological niche . Finally, microorganisms within and among body habitats exhibited relationships suggestive of driving physical factors such as oxygen, moisture and ph, host immunological factors, and microbial interactions such as mutualism or competition (supp . 4). Both overall community similarity and microbial co - occurrence and co - exclusion across the human microbiome grouped the 18 body habitats together into four clusters corresponding to the five target body areas (supp . There was little distinction among different vaginal sites, with lactobacillus spp . Dominating all three and correlating in abundance . However, lactobacillus varied inversely with the actinobacteria and bacteroidetes (see supp . 23), as also observed in the cohort of ravel et al . Gut microbiota relationships primarily comprised inverse associations with the bacteroides, which ranged from dominant in some subjects to a minority in others who carried a greater diversity of firmicutes . A similar progression was evident in the skin communities, dominated by one of staphylococcus (phylum firmicutes), propionibacterium, or corynebacterium (both phylum actinobacteria), with a continuum of oral organisms (e.g. Streptococcus) appearing in nares communities (supp . These observations suggest that microbial community structure in these individuals may sometimes occupy discrete configurations and under other circumstances vary continuously, a topic addressed in more detail by several hmp investigations . An individual s location within such configurations is indicative of current microbial carriage (including pathogens) and of the community s ability to resist future pathogen acquisition or dysbiosis; it may thus prove to be associated with disease susceptibility or other phenotypic characteristics . As the first study to include both marker gene and metagenomic data across body habitats from a large human population, we additionally assessed the ecology of microbial metabolic and functional pathways in these communities . We reconstructed the relative abundances of pathways in community metagenomes, which were much more constant and evenly diverse than were organismal abundances (fig . We were likewise able to determine for the first time that taxonomic and functional alpha diversity across microbial communities significantly correlate (spearman of inverse simpson s r=0.60, p=3.6e-67, n=661), the latter within a more proscribed range of community configurations (supp . The most abundant of these core pathways include the ribosome and translational machinery, nucleotide charging and atp synthesis, and glycolysis, and reflect the basics of host - associated microbial life . Also in contrast to taxa, few pathways were highly variable among subjects within any body habitat; exceptions included the sec (orally, sd . 0.0033) secretion systems, indicating a high degree of host - microbe and microbe - microbe interactions in the healthy human microbiota . This high variability was particularly present in the oral cavity, for phosphate, mono- and di - saccharide, and amino acid transport in the mucosa, as well as lps biosynthesis and spermidine / putrescine synthesis and transport on the plaque and tongue (http://hmpdacc.org/hmmrc). The stability and high metagenomic abundance of this housekeeping core contrasts with the greater variability and lower abundance of niche - specific functionality in rare but consistently present pathways, e.g. Spermidine biosynthesis, methionine degradation, and hydrogen sulfide production, all examples highly prevalent in gastrointestinal body sites (nonzero in> 92% of samples) but at very low abundance (median rel . Long tail of low - abundance genes and pathways also likely encodes much of the uncharacterized biomolecular function and metabolism of these metagenomes, the expression levels of which remain to be explored in future metatranscriptomic studies . Protein families showed diversity and prevalence trends similar to those of full pathways, ranging from maxima of only ~16,000 unique families per community in the vagina to almost 400,000 in the oral cavity (fig . B, http://hmpdacc.org/hmgi). A striking fraction of these families were indeed functionally uncharacterized, including those detected by read mapping, with a minimum in the oral cavity (mean 58% sd . Likewise, many genes annotated from assemblies could not be assigned a metabolic function, with a minimum in the vagina (mean 78% sd . The latter range did not differ substantially by body habitat and is in close agreement with previous comprehensive gene catalogs of the gut metagenome . Taken together with the microbial variation observed above throughout the human microbiome, functional variation among individuals might indicate pathways of particular importance in maintaining community structure in the face of personalized immune, environmental, or dietary exposures among these subjects . Determining the functions of uncharacterized core and variable protein families will be especially essential in understanding the microbiota s role in health and disease . We finally examined relationships associating both clades and metabolism in the microbiota with host properties such as age, gender, bmi, and other available clinical metadata (fig . 5; supp . Table 3). Using a sparse multivariate model, 960 microbial, enzymatic, or pathway abundances were significantly associated with one or more of 15 subject phenotype and sample metadata features . A wide variety of taxa, gene families, and metabolic pathways were differentially distributed with subject ethnicity at every body habitat (fig . 5a), representing the phenotype with the greatest number 266 at fdr q<0.2) of total associations with the microbiome . Vaginal ph has also been observed to correlate with microbiome composition, and we detected in this population both the expected reduction in lactobacillus at high ph and a corresponding increase in metabolic diversity (fig . Not previously observed, subject age was most associated with a collection of highly differential metagenomically encoded pathways on the skin (fig . 5c), as well as shifts in skin clades including retroauricular firmicutes (p=1.0e-4, q=0.033). The examples of associations with ethnicity and vaginal ph are among the strongest associations with the microbiome, however, and most correlates (e.g. With subject bmi, fig . 5d) are more representatively modest . This lower degree of correlation held for most available biometrics (gender, temperature, blood pressure, etc . ), with even the most significant associations possessing generally low effect sizes and considerable unexplained variance . We conclude that most variation in the human microbiome is not well - explained by these phenotypic metadata, and other potentially important factors such as short- and long - term diet, daily cycles, founder effects such as mode of delivery, and host genetics should be considered in future analyses . This extensive sampling of the human microbiome across many subjects and body habitats provides an initial characterization of the normal microbiota of healthy adults in a western population . The large sample size and consistent sampling of many sites from the same individuals allows for the first time an understanding of the relationships among microbes, and between the microbiome and clinical parameters, that underpin the basis for individual variation--variation that may ultimately be critical for understanding microbiome - based disorders . Clinical studies of the microbiome will be able to leverage the resulting extensive catalogs of taxa, pathways, and genes, although they must also still include carefully matched internal controls . The uniqueness of each individual s microbiome even in this reference population argues for future studies to consider prospective within - subjects designs where possible . The hmp s unique combination of organismal and functional data across body habitats, encompassing both 16s and metagenomic profiling, together with detailed characterization of each subject, has allowed us and subsequent studies to move beyond the observation of variability in the human microbiome to ask how and why these microbial communities vary so extensively . Many details remain for further work to fill in, building on this reference study . Do epidemiological patterns of transmission of beneficial or harmless microbes mirror patterns of transmission of pathogens? Which co - occurrences among microbes reflect shared response to the environment, as opposed to competitive or mutualistic interactions? How large a role does host immunity or genetics play in shaping patterns of diversity, and how do the patterns observed in this north american population compare to those around the world? Future studies building on the gene and organism catalogs established by the human microbiome project, including increasingly detailed investigations of metatranscriptomes and metaproteomes, will help to unravel these open questions and allow us to more fully understand the links between the human microbiome, health, and disease . Microbiome samples were collected from up to 18 body sites at one or two time points from 242 individuals clinically screened for absence of disease . Samples were subjected to 16s rrna gene pyrosequencing (454 life sciences), and a subset were shotgun sequenced for metagenomics using the illumina gaiix platform . 16s data processing and diversity estimates were performed using qiime, and metagenomic data were taxonomically profiled using metaphlan, metabolically profiled by humann, and assembled for gene annotation and clustering into a unique catalog . Potential pathogens were identified using the patric database, isolate reference genome annotations drawn from kegg, and reference genome mapping performed by bwa to a reduced set of genomes to which short reads could be matched . Microbial associations were assessed by similarity measures accounting for compositionality, and phenotypic association testing was performed in r. all data and additional protocol details are available at http://hmpdacc.org.
Thyroid gland is rarely affected by tuberculosis, even in countries where tuberculosis is common . Children constitute very small proportion of these cases and according to terzidis et al . And razmpa et al . We present a case of 11-year - old female child of thyroid tuberculosis who presented with a solitary thyroid nodule for 2 months and was diagnosed on cytological examination . The present case is about an 11 years female child presented with a history of swelling in the thyroid region for preceding 2 months . On physical examination, thyroid gland was enlarged measuring 4.0 2.5 cm, firm in consistency and moving with deglutition . Computed tomography (ct) scan neck showed large bilateral lobes of thyroid right measuring 21 13 mm and left 16 18 mm showing heterogeneous tissue density with evidence of necrosis, bilateral cervical lymphadenopathy . X - ray chest revealed non - homogenous opacity in the lower zone of lung on the right side . Enlargement of hilar and mediastinal lymph nodes, right side minimal pleural effusion and pleural thickening were found on ct scan of the thoracic cavity . Ultrasound of the abdomen revealed enlarged lymph nodes in periportal, portal, peripancreatic para - aortic regions . Hemoglobin was 10% g and erythrocyte sedimentation rate was 40 mm in 1 h. peripheral smear of blood revealed microcytic hypochromic anemia . T3, t4 and thyroid stimulating hormone were within the normal limits and anti - thyroid peroxidase antibody was negative . On fine - needle aspiration from the thyroid swelling, thick cheesy fluid was aspirated . The fluid was centrifuged and smears of the sediment showed caseating necrosis, few epithelioid cell granulomas and occasional benign follicular epithelial cells [figures 1 and 2]. Based on caseating necrosis, epithelioid cell granulomas and demonstration of afb, diagnosis of thyroid tuberculosis was made . With treatment with antitubercular drugs, benign follicular cells and background of caseating necrosis (giemsa stain, 400) epithelioid cell granuloma with caseating necrotic background (giemsa stain, 400) the estimation of prevalence of this condition is difficult . However, reported prevalence rate varied from 0.1% to 1% . Rarity of tuberculosis in thyroid has been attributed to bactericidal property of colloid, high vascularity and excess of iodine, enhanced activity of phagocytes in hyperthyroidism and possible antitubercular roles of thyroid hormone . Involvement of thyroid by tuberculosis can occur by hemetogenous and lymphatic routes or by direct extension from cervical lymphadenitis . Earlier the criteria described for diagnosis of thyroid tuberculosis were finding of afb within thyroid, a necrotic or abscessed gland and presence of tuberculous focus outside the thyroid . Since it is not always possible to demonstrate afb, recently it is stated that multiple coalesced and caseating epithelioid cell granulomas along with giant cells are considered to be diagnostic of thyroid tuberculosis . In our case, the diagnosis of tuberculosis of thyroid was confirmed by demonstration of tubercular bacili in the specimen . Other extra thyroid lesions were also found in the present case which is similar to findings noted in many previous reports . Although pathological assessment of these extra thyroid tissues was not done in the present case to avoid invasive investigations, however, regression of these lesions with anti - tubercular drugs lends evidence to the possible tuberculous etiology in these lesions as well . One of the peculiarities of our case is that involvement of large number abdominal lymph nodes (periportal, portal, peripancreatic para - aortic regions) besides cervical, mediastinal lymph nodes and pulmonary lesion . To the best of our knowledge, no case of thyroid tuberculosis was reported in the past wherein such widespread extra thyroid involvement was observed . Usually the tubercular involvement of lymph nodes in such patients was limited to one or two lymph node regions with or without pulmonary tuberculosis . Khan et al . In their study have reported four cases of thyroid tuberculosis, among them one case had cervical and axillary lymphadenopathy, another case had tuberculosis of cervical lymph nodes and third one had pulmonary tuberculosis and remaining one case did not reveal any involvement of extra thyroid tissue . Likewise in the case reported by oklah and al - kaisi concomitant pulmonary tuberculosis and tubercular mediastinal lymphadenitis was found and mondal and patra in a study of 18 cases of thyroid tuberculosis found three cases had associated tubercular cervical lymphadenitis and four cases had pulmonary tuberculosis . Other atypicality is the younger age of our case, only very few cases were reported in this age group in the past, the most of previously reported patients were adults with the median age around 40s and . There are case reports that suggest the possibility of primary thyroid tuberculosis in which there is no known focus of tuberculosis outside thyroid and such condition is even more uncommon . Like in current case, thyroid function test is generally found to be normal in these patients, but cases of thyrotoxicosis and hypothyroidism have also been reported . Being a rare entity, tuberculosis as the cause of thyroid swelling is unlikely to be suspected clinically . However, like in the present case, fine - needle aspiration has also been found to be diagnostic in cases of thyroid tuberculosis thus preventing the unnecessary surgery . Differential diagnosis of granulomatous inflammation in thyroid includes subacute thyroiditis, goitrous autoimmune thyroiditis, tuberculosis and sarcoidosis . It may be concluded that in cases of thyroid swellings tubercular thyroiditis should be considered as one of the differential diagnosis.
Current understanding see psoriatic arthritis (psa) as a seronegative inflammatory disease of the joints, enthuses and periarticular connective tissue, associated with any clinical type of psoriasis . Psa has been included in the group of hla - b27-associated spondyloarthropathies as it shares many clinical features with them . Psa is more common in white persons than in persons of other races; it typically develops in persons aged 35 - 55 years with equal sex distribution . Recent data indicates that the incidence of psa in the general population has been rising over the past 30 years in both men and women with an annual incidence rate of 7.2/100.000 . Similarly the psa prevalence in patients with psoriasis has varied widely during the past decade with estimated rates of 6% and 19% in 2009, and of 11% in 2005 . Reasons for this increase are not clear: it may be related to a true change in incidence or a greater overall awareness of the diagnosis by physicians . The occurrence of psa is linked to the complex interplay of genes, environment, and immune system . A genetic predisposition is commonly accepted and the role of genetic factors is evident when considering the strong heritability of psa . In addition to the genetic influences, environmental factors and immunological mechanisms (in which cd8 + t cell and t - cell - derived cytokines play crucial roles) are thought to be prominent in the development, amplification, and perpetuation of the disease . Psa is characterized by a wide variety of articular, periarticular, and extra - articular features, along with comorbidities that may complicate the disease course . Regarding diagnosis of enthesitis ultrasonography (us) allows detailed assessment of both tendinous and bony - side subclinical involvement . Early us signs of enthesitis include hypoechoic swelling of the tendon insertion, increase of blood flow detectable with power doppler, and bursal enlargement [figure 1]. Considering articular involvement, psa can be classified in axial disease (with or without peripheral arthritis) and peripheral disease, which include five patterns: asymmetrical olygoarthritis, symmetrical polyarthritis, distal interphalangeal arthropathy, and arthritis mutilans . Extra - articular features comprise involvement of the skin, the nails [figure 2], and the eye (iridocyclitis, conjunctivitis, and other anterior uveitis in most cases), followed by less common involvement such as inflammation of the aortic valve, iga nephropathy, or amyloidosis . Psoriasis vulgaris [figure 3] is the main form of psoriasis associated with psa but pustular psoriasis, flexular psoriasis, and guttate lesions have also been recognized . Nail changes include onycholysis, hyperkeratosis, transverse ridging, nail pitting [figure 4], oil drop discoloration, and splinters; all these features refer to the definition of psoriatic onychopathy [figure 5]. It is often reported that when the nail organ is involved in nail psoriasis, a secondary affection of the joint may develop and vice versa; this is because the nail is functionally integrated with enthesis associated with the distal phalanx. [1012] moreover, when skin and joint disease begin simultaneously, nail involvement is frequently present at the onset, and severe deforming arthritis of the hands and feet is frequently associated with extensive nail involvement . Us imaging of the nail in a patient with psa: (a) loss of the normal tri - laminar appearance of the nail plate, which appears with an irregular shape; (b) nail bed is clearly thickened and power doppler us reveals a marked signal indicative of an increase of blood flow at the nail bed level an ultrasound image of a healthy nail psoriatic plaque (a), related us findings of lesional skin (b) and perilesional healthy skin (c) showing increased neoangiogenesis in the lesional skin nail pitting and transverse ridging in a patient with psoriasis clinical presentations of psoriatic onychopathy in two patients (a, b) with psa the natural course of psa is characterized by flares and remissions . The following factors influence the severity of the disease and are related to a more aggressive course: onychopathy, specific clinical subsets (e.g., arthritis mutilans, symmetric polyarthritis), involvement of five or more joints at onset, female sex, family history of arthritis, and hla markers (especially hla - b39 and hla - b27 in the presence of hla - dr7). Moreover, the use of medication before the first clinical visit, evidence of radiologic damage, and elevated erythrocyte sedimentation rate have been related to significantly increased mortality . Finally, research over recent years has highlighted that moderate to severe psa and psoriasis are linked to cardiovascular disease, the metabolic syndrome, and increased mortality. [1416] between 6% and 42% of patients suffering from psoriasis will develop psa, with over two - thirds developing psoriasis usually 10 years before articular involvement . Otherwise, approximately 15% of patients develop articular manifestations, especially arthritis, before the onset of psoriatic skin lesions . Juvenile psa generally develops in children aged 9 - 10, with a female predominance, and it accounts for 8 - 20% of childhood arthritis . In 52% of cases, arthritis precedes psoriasis onset, and usually children have a higher frequency of simultaneous onset of psoriasis and arthritis than adults . Genetic factors have long been recognized to play an important role in psa . Identifying genes underlying disease susceptibility involves a series of complex investigations beginning with familial aggregation studies followed by segregation analysis, linkage analysis, association analysis and functional studies to identify and characterize genes . Moreover, compared with most other rheumatic diseases, heredity plays a particularly strong role in the development of psa . The genetic factors underlying susceptibility to psa are closely intertwined with that of cutaneous psoriasis since almost all patients diagnosed with psa have either personal or family history of psoriasis . The immune system is a tightly regulated network of cells and cytokines, and genetic factors that lead to immune alterations may likely tip the balance toward inflammation in the skin and synovium of psoriasis and psa, respectively . Thus, psa has a strong genetic component involving loci within or outside the major histocompatibility complex (mhc) region . Polymorphisms in the genes coded in the hla region on chromosome 6p have been shown to be associated with psa . This dense region codes for a number of genes important in the immune response, including hla and non - hla alleles . Class i antigens (hla - b13, hla - b57, hla - b39, hla - cw6, hlacw7) have consistently shown a positive association with psoriasis and psa . While hla - b13, -b16, and its splits -b38 and -b39, b17, and cw6 are associated with psoriasis, with or without arthritis, b27 and b7 are specifically associated with psa . Associations with class i alleles are stronger with hla - b than hla - c alleles . The association of hla - c with psa was found to be due to association with early onset psoriasis, since no association was found in patients with psa and late onset psoriasis . Hla antigens may also identify patients with a particular pattern of psa: hla - b27 with spinal involvement, and b38 and b39 with peripheral polyarthritis . Hla - b39 alone, hla - b27 in the presence of hla - dr7, and hla - dqw3 in the absence of hla - dr7, each conferred an increased risk for disease progression . The rheumatoid arthritis (ra) shared epitope was found to be associated with radiological erosions among patients with psa . Recently, patients with psa carrying both hla - cw6 and hla - drb107 alleles were found to have a less severe course of arthritis, as measured by the number of damaged and involved joints . Non - hla genes within the mhc region have also been proposed to be associated with psa . These include mhc class i chain - related antigen a (mica) and tumor necrosis factor alfa (tnf-) gene . Mica is one of the most polymorphic non - hla genes in the human genome . It lies 47 kb centromeric to the hla - b locus, within the heart of the mhc complex . Its protein product is expressed in a wide variety of epithelial cells, the skin and the synovium and interacts with the natural killer - cell receptor (kir) nkg2d to activate the immune response . Mica has two main forms of polymorphism: a gct triplet repeat in the transmembrane region and a large number of single nucleotide polymorphisms (snps) in the part of the gene that encodes the extracellular binding domains . Some studies have found an association between psa and the mica - a9 (mica allele which has nine gct repeats), but relatively small numbers of patients have been studied considering the complexity of the condition . In a spanish population, the trinucleotide repeat polymorphism mica - a9 corresponding to the mica-002 allele was associated with psa (but not psoriasis), independent of hla cw0602 . Similar associations have been shown with jewish, croatian, and british patients. [2933] another high - priority candidate is the tnf- gene, which is located 250 kb centromeric from the hla - b locus . Tnf- is a key inflammatory cytokine in psoriasis and psa and is found in high levels in the serum, synovial fluid, and synovial membrane of patients with psa . Associations have been reported between the tnf-308 polymorphism in the promoter region of the tnf- gene and psa . The tnf308 polymorphism has been associated with the presence of joint erosions, progression of joint damage, and early age of the onset of psa . Patients show marked clinical response to treatment with biological and it would be interesting to investigate whether these polymorphisms can be related to responsiveness to treatment with anti - tnf agents . It would appear that any association with the tnf genes and psa is more linked with disease severity and clinical expression than overall disease susceptibility . Association studies in psa have identified a number of genes outside chromosome 6p including interleukin-1 (il-1) gene cluster (chromosome 2q), killer - cell immunoglobulin - like receptors (kirs) genes (chromosome 19q), and il-23r (chromosome 1p). Subsequently, a study revealed two regions contributing independently to risk of psa: a region spanned by markers rs3783547, rs3783543, and rs17561 in il-1a, and a region near the end of il-1b . Kirs are one of two groups of receptors on natural killer (nk) cells . There is a high degree of polymorphism and complexity in the genes that encode the kirs . A particular kir haplotype encodes a distinct set of receptors for an individual's nk cells . Each nk cell has a combination of inhibitory and activating receptors that interact with certain hla alleles to influence the immune response . In addition, mica is also a ligand for an nk receptor (nk2gd). Polymorphism within this gene may also influence susceptibility to psa through altered interactions with nk cells . Interestingly, the association between kir2ds1 and psa was only found if the hla ligand for the corresponding inhibitory receptor (kir2dl1) was absent . There was a statistically significant difference between psa and psoriasis alone at three loci; hla - c and il-23r were more strongly associated with psoriasis alone, and il-12b with psa . However, an adequately genome - wide association study on psa has not yet been done . Thus, a number of susceptibility loci for psa have been described, but many others remain to be identified . These discoveries can help us to understand pathogenesis, identify drug targets, and predict the disease course or the response to pharmacotherapy . The goal of genetic screening is to identify subjects for preventive or early treatment or extended surveillance prior to the onset of symptoms . Therefore, the sensitivity of the test should be high and similarly, high specificity is desired to increase the efficacy of the screening and minimize the number of subjects who will be treated unnecessarily . Current psa screening techniques identify symptomatic patients after the onset of inflammatory arthritis . Identifying psa in an earlier or a pre - clinical stage will allow treatment to be initiated at a time when intervention has a greater likelihood of succeeding . Early screening combined with tailored treatment will help preventing disease progression and irreversible slow joint destruction . The need for early screening and medical intervention for psa is underscored by the fact that psa becomes more severe when left not properly treated or untreated, leaving patients with significant joint damage, functional impairment, and reduced quality of life . Therefore, a genetic screening test has been recently developed in order to identify patients at a high risk for developing psa prior to the onset of arthritic symptoms . The genetic screening test is most appropriate for individuals with psoriasis who have not yet developed psa . It is also useful to assess the risk for individuals who have a family history of psoriasis or psa . A genetic sample is collected using a cheek swab, and the sample is mailed for analysis at a certified laboratory . The genetic screening test for psa provides information on the presence of a specific variation (a triple repeat polymorphism) on the mica immune response gene located on chromosome 6p, a variant called mica - a9 . Thus, the test shows whether a person has the high or low risk variant of the mica gene . Patients with mica - a9 variant have a higher risk for psa; patients without this variant have a lower risk . The clinical validity of genetic screening test has been demonstrated using standard statistical methods . The authors pooled data from two independent published studies (on jewish and spanish populations) that demonstrated statistical significant association between the mica variants measured by this genetic test and psa . The data reported by the studies are presented in table 1 . Based on these data, they calculated sensitivity (the probability that a person with psa will test positive) = tp/(tp+fn) = 59%specificity (the probability that a person without psa will test negative) = tn/(tn+fp) = 71%positive predictive value = tp/(tp+fp) = 60%negative predictive value = tn/(tn+fn) = 70% sensitivity (the probability that a person with psa will test positive) = tp/(tp+fn) = 59% specificity (the probability that a person without psa will test negative) = tn/(tn+fp) = 71% positive predictive value = tp/(tp+fp) = 60% negative predictive value = tn/(tn+fn) = 70% association between psa and the mica variants measured by psoriasisdx genetic test using this genetic test, a physician can conclude that a patient who tests positive for the mica - a9 variant has approximately 60% chance of developing psa; thus, the patient will likely benefit from early treatment . Similarly, a physician can conclude that a patient that tests negative has approximately 70% chance of not developing psa; thus, the patient may be able to avoid costly treatment . However, due to the limitations of the test to identify all the psoriasis patients who will ultimately develop psa, the genetic screening test for psa should be used as an adjunct to currently used diagnostic criteria to determine the need for medical therapy . Unlike traditional medical diagnostics, genetic tests may have no immediate clinical benefit, but may have great utility for the patients and the society as well as the social, economic, and health impact on the individual . Assessed clinical parameters as predictors of the number of exposures of narrow - band ultraviolet b (nb - uvb) needed to clear psoriasis and of the duration of remission . The influence of the fok1, apa1, bsm1, taq1, and rs4516035 polymorphisms of the vitamin d receptor (vdr) gene on treatment response was also evaluated . Vitamin d3 is a potent anti - proliferative and pro - differentiation factor for keratinocytes and modulates immunological processes such as t - cell activation, cytokine secretion, and dendritic cell maturation . Vitamin d3 exerts the majority of its effects by binding to the vitamin d receptor (vdr) which belongs to the nuclear hormone receptor superfamily of receptors . There are several polymorphisms of the gene encoding the vdr which may alter its activity . In this study, the authors investigated whether polymorphisms which modify the effects of nb - uvb - induced vitamin d may have an influence on treatment response . Authors used nb - uvb to treat 119 patients with chronic plaque psoriasis until clearance was achieved and then monitored the patients for up to 1 year or until relapse occurred . In all, 105 of the patients completed the course of phototherapy . The median number of exposures to clear psoriasis was 26 and the median remission duration was 16 weeks . The taq1 polymorphism of the vdr gene was shown to be predictive of remission duration . Patients homozygous for the c allele, which is associated with the decreased activity of the vdr, had a shorter remission duration than those heterozygous for the allele and those homozygous for the t allele . As yet, routine testing for vdr polymorphisms has been considered too expensive for use in clinical practice . This is the first prospective study to demonstrate that both clinical and genetic parameters may predict therapy response and remission duration in patients with psoriasis treated with nb - uvb . While nb - uvb was shown to be a very effective treatment in the majority of patients, a subset of patients was identified who demonstrated an inefficient treatment response . As nb - uvb treatment is time consuming, expensive, and potentially carcinogenic, the ability to predict patients who will clear quickly with prolonged remission would be useful in terms of patient care and health economics . Systemic treatments for moderate - severe psoriasis and psa cannot only be potentially dangerous and time consuming for the patient but also very expensive for the national health systems . The ability to predict which subset of patients will develop the most severe forms of the disease (i.e., psa) or will respond to well - established (i.e., uvb irradiation) or other systemic treatments is now required by physicians and patients associations . Genetic tests today seem to represent a reliable investigation procedure which could rapidly and safely improve the diagnostic ability of the dermatologist and contribute to the early and correct treatment of the different subsets of psoriatic patients, especially regarding the identification of patients with psa . These genetic tests, if properly adopted, could highly reduce the potentially dangerous side effects of systemic treatments of psoriatic patients, allow physicians to start in the earliest phase the correct treatment for the different subsets of patients, and eventually drastically reduce the cost of the optimal treatment of psoriatic patients.
Gingival recession is defined as apical migration of the junctional epitheliun, with exposure of root surfaces . Patients may complain of hypersensitivity of the teeth and poor esthetics, and the area may retain dental plaque, which can later cause root caries . Gingival recession may be localized or generalized and can be associated with one or more tooth surfaces . Periodontal treatment aims to protect and maintain the patient's oral health over his lifetime for adequate function as well as esthetic appearance . Over the last few decades, many different approaches for the treatment for gingival recession have been reported in the literature without a consistent consensus . The use of free autogenous grafts and pedicle grafts including advanced flaps and rotational flaps have been advocated . Combination grafts with either autogenous grafts or allograft and with guided tissue regeneration (gtr) membranes have been reported for root coverage . The double lateral sliding bridge flap technique was initially proposed by marggraf, to cover gingival recession in multiple teeth with or without adequate attached gingiva . Barrier techniques, using expanded poly - tetra - fluro - ethylene, polygalactin, polylactic acid and collagen are employed to exclude epithelium and the gingival corium from the root as they interfere with regeneration . According to a hypothesis formulated by melcher, certain cell populations residing in the periodontium have the potential to create new cementum, alveolar bone, and periodontal ligament, the use of gtr for root coverage has been extensively reported in the literature with considerable success . A recent innovation is the preparation and use of platelet - rich fibrin (prf), which is a concentrated suspension of the growth factors found in platelets . The platelet concentrate contains platelet - derived growth factor and transforming growth factor that modulate and up - regulate one growth factors function in the presence of second or third growth factor . This specific feature influenced the decision to use prf as the test material of choice in this case report . In the case described in this article, platelet rich derivative (prf membrane) was combined with the double lateral sliding bridge flap technique for root coverage of multiple teeth . A collagen membrane (healiguide) a 38-year - old male patient reported to the department of periodontology with the chief complaint of hypersensitivity in lower front tooth region for 4 months . Clinical examination revealed miller's class iii recession in mandibular anterior teeth measuring 2 mm each in mandibular central incisors, 1 mm in left lateral incisor, 3 mm in right lateral incisor and 4 mm each in mandibular canines [figure 1]. Preparation of the patient was carried out, which included scaling and root planning of the entire dentition and oral hygiene instructions . Blood of the patient was drawn in 10 ml test tubes without an anticoagulant and centrifuged immediately . Blood was centrifuged using a table top centrifuge (remy laboratories) for 12 min at 2700 rpm . The resultant product consisted of the following three layers: the top most layer consisting of acellular platelet poor plasmaprf clot in the middlered blood cells at the bottom [figure 2]. Figure 2preparation of platelet rich fibrin the top most layer consisting of acellular platelet poor plasma prf clot in the middle red blood cells at the bottom [figure 2]. Figure 2preparation of platelet rich fibrin preparation of platelet rich fibrin prf can be obtained in the form of a membrane by squeezing out the fluids in the fibrin clot [figure 3]. Platelet rich fibrin membrane the prf membrane was placed over gtr membrane (healiguide) [figure 4]. Placement of platelet - rich fibrin membrane over guided tissue regeneration membrane it included the bridge flap technique given by marggraf and later modified by romanos . Under local anesthesia, an arch shaped or semilunar incision was given in the vestibule at a distance, which was twice the amount of gingival recession plus 2 mm (2 * gingival recession + 2 mm). A split thickness flap was then elevated in the apicocoronal direction [figure 5] and it was connected with the first incision so that the two flaps communicated with each other . The patency of the reflection was checked with the help of a periosteal elevator or a periodontal probe [figure 6]. Prf in a collagen membrane carrier was placed over the sites with denuded root surfaces [figure 7]. The entire flap was then coronally positioned to cover the membrane and sling sutures were placed [figure 8]. A non - eugenol periodontal dressing (coe - pack, gc) was given over the surgical site . Patency of flap evaluated placement of platelet - rich fibrin membrane using the guided tissue regeneration membrane as carrier the patient was prescribed antibiotics (amoxicillin, 500 mg thrice daily) and analgesics (ibuprofen, 400 mg thrice daily) for 5 days and 0.12% chlorhexidine digluconate mouth rinse for 4 weeks . The patient was advised to follow routine post - operative periodontal mucogingival instructions, with minor modifications . The patient was recalled after 8 weeks, and the complete root coverage was observed [figure 9]. Preparation of the patient was carried out, which included scaling and root planning of the entire dentition and oral hygiene instructions . Blood of the patient was drawn in 10 ml test tubes without an anticoagulant and centrifuged immediately . Blood was centrifuged using a table top centrifuge (remy laboratories) for 12 min at 2700 rpm . The resultant product consisted of the following three layers: the top most layer consisting of acellular platelet poor plasmaprf clot in the middlered blood cells at the bottom [figure 2]. Figure 2preparation of platelet rich fibrin the top most layer consisting of acellular platelet poor plasma prf clot in the middle red blood cells at the bottom [figure 2]. Figure 2preparation of platelet rich fibrin preparation of platelet rich fibrin prf can be obtained in the form of a membrane by squeezing out the fluids in the fibrin clot [figure 3]. Platelet rich fibrin membrane the prf membrane was placed over gtr membrane (healiguide) [figure 4]. It included the bridge flap technique given by marggraf and later modified by romanos . Under local anesthesia, an arch shaped or semilunar incision was given in the vestibule at a distance, which was twice the amount of gingival recession plus 2 mm (2 * gingival recession + 2 mm). A split thickness flap was then elevated in the apicocoronal direction [figure 5] and it was connected with the first incision so that the two flaps communicated with each other . The patency of the reflection was checked with the help of a periosteal elevator or a periodontal probe [figure 6]. Prf in a collagen membrane carrier was placed over the sites with denuded root surfaces [figure 7]. The entire flap was then coronally positioned to cover the membrane and sling sutures were placed [figure 8]. A non - eugenol periodontal dressing (coe - pack, gc) was given over the surgical site . Patency of flap evaluated placement of platelet - rich fibrin membrane using the guided tissue regeneration membrane as carrier the patient was prescribed antibiotics (amoxicillin, 500 mg thrice daily) and analgesics (ibuprofen, 400 mg thrice daily) for 5 days and 0.12% chlorhexidine digluconate mouth rinse for 4 weeks . The patient was advised to follow routine post - operative periodontal mucogingival instructions, with minor modifications . The patient was recalled after 8 weeks, and the complete root coverage was observed [figure 9]. New materials and techniques are being developed these days to predictably satisfy the patient - centered esthetic demands . Root coverage can be performed to alleviate a patient's concerns regarding unsatisfactory esthetics and root hypersensitivity . Gingival recession provides a nidus for microbial plaque and calculus accumulation and can be difficult to maintain with normal oral hygiene measures . In addition, there is the potential for root caries to develop on the denuded root surfaces . Connective tissue grafts (ctgs) are currently considered the gold standard for root coverage since they are highly predictable procedures for treating recession defects . However, a common concern of patients is that ctgs require an additional surgical site and produce added morbidity . Harvesting a palatal or other intraoral donor site causes additional discomfort to the patient and increases chair time for the surgeon . As per the literature search, this is the first case report where double lateral sliding bridge flap was used along with prf in a collagen membrane carrier for root coverage of multiple teeth . The flap covers the denuded root surface of multiple teeth and is supplied by plasmatic circulation from capillaries in adjacent gingiva, allowing it to survive . In this case, miller defined complete root coverage in clinical terms as location of soft - tissue margin at the cej, presence of clinical attachment to the root, a sulcus depth of 2 mm or less and absence of bleeding on probing . The use of platelet concentrate gel in a collagen membrane carrier has been documented to be beneficial . Systematic reviews conducted by oates et al . And roccuzzo et al . Found that root coverage with gtr membrane showed significant results . A recent innovation in dentistry has been the preparation and use of prf, a concentrated suspension of the growth factors found in platelets . Placement of the prf membrane in recession defects can be used to restore the functional properties of the labial gingiva of the maxillary and mandibular teeth by repairing gingival defects and re - establishing the continuity and integrity of the zone of keratinized gingiva . Aleksic et al . Concluded that the use of prf and subepithelial ctg was equally effective in the treatment of gingival recession . They also concluded that the utilization of the prf resulted in a decreased post - operative discomfort and advanced tissue healing . Jankovic et al . Conducted a randomized controlled trial and concluded that the use of prf membrane in gingival recession treatment provided acceptable clinical results, followed by enhanced wound healing and decreased subjective patient discomfort compared to ctg treated gingival recessions and found no difference between the procedures in gingival recession therapy . Martinez - zapata et al . Conducted a systematic review and concluded that autologous plasma rich in platelets improved gingival recession . Soft - tissue maintenance is the primary line of defense in protecting the tissue from bacterial infection . In this report, double lateral sliding bridge flap along with prf in a collagen membrane carrier was found to have satisfactory results . It also appears that neither the quantity of gingival recession nor the qualities of the supporting tissues are prerequisites for the success of this technique . The main advantage of this technique is that it is a one - step procedure, which showed complete root coverage as well as increased the zone of keratinized gingiva . The use of prf and barrier materials in clinical practice has shown beneficial outcomes and holds promise for further procedures in the future.
In alzahra university hospital of isfahan, iran, the a 42-year - old homemaker woman, who used to suffer from rheumatoid arthritis since 2004 was admitted . During this time, rheumatoid factor and anti - cyclic citrollinated peptide (anti - ccp) always had been negative . In this period, she had wrists, shoulders, knees, and ankles involvement, and never been improved completely . The patient was under the treatment of oral methotrexate (10 mg per week), sulfasalazine (1500 mg / day), diclofenac (50 mg / day), and low dose prednisolone (7.5 mg / day). Dose escalation did not improve disease activity score of 28 joints (das28> 5.1) and laboratory parameters . She was admitted to the hospital due to refractory joints pain, tenderness, leukocytosis (12 10/ul), and elevated esr (65 mm / h) and crp (42 mg / l). In addition, she received steroid pulses (iv methylprednisolone sodium succinate 500 milligram) during each admission . She was the candidate for biologic therapy, but she could not afford it . Due to this problem after four weeks, she felt better, and ten - derness decreased, but swelling continued . Esr (35 mm / h) and crp (22 mg / l) improved slightly; but during this period, blood pressure and serum creatinine increased . Unfortunately, by tapering the drug dosage, the clinical and laboratory signs recurred . In respect of anti - inflammatory effect of pamidronate, after signing the inform consent by the patient, we decided to start single infusion of 60 mg drug for her . (in a 3-month period, patient received three infusions of 60 mg pamidronate). After the second infusion, all the clinical and laboratory indices of the patient resolved completely . Disease activity index decreased to less than 2.6 (das28 <2.6) (table 1). Das28 and changes of its components during the pamidronate infusion courses (first case) by ameliorating of the joint tenderness and swelling, we discontinued cyclosporine and tapered other drugs gradually . After three months of the last drug injection, the disease was under control by 7.5 mg of oral methotrexate per week and 1 gram of oral sulfasalazine per day . In alzahra university hospital of isfahan, a 23-year - old homemaker suffered from rheumatoid arthritis since 2007, was addmitted . In the disease course, she had right wrist, elbows, shoulders, jaw, knees, and ankles involvement . In the laboratory examinations, she had a negative rheumatoid factor and anti - ccp, elevated esr (87 mm / h) and crp (45 mg / l), and leukocytosis (13.4 10/ul). In the disease course, she was under the treatment of oral methotrexate (10 mg / week), sulfasalazine (2000 mg / day), naproxen (500 mg / day), and prednisolone (7.5 mg / day). This patient, as the previous case, could not afford biologic drugs . In this regrad, we started oral cyclosporine a (3mg / kg) due to refractoriness to conventional dmards . After three weeks, patient felt better and disease activity index, esr, crp and leukocytosis improved . However, after the dose reduction in each time, clinical and laboratory signs of disease relapsed . In this respect, after signing the inform consent by the patient, we decided to start intravenous pamidronate 60 mg per month until three months . After four weeks, laboratory and clinical signs of the patient decreased and after eight weeks, the entire patient's joint tenderness improved . Das28 and changes of its components during the pamidronate infusion courses (second case) even so, we gradually started to tapered drugs . After 3 months of the last infusion, the patient only received one - gram oral sulfasalazine per day and other drugs were discontinued . Rheumatoid arthritis is a disabling disease that in some cases is refractory to conventional dmards . Rheumatoid factor is positive in most of the cases, but some cases are seronegative and resistant.3 biologic drugs most commonly are used for refractory cases to conventional dmards,6 but they are expensive . Pamidronate is a cheap interavenous bisphosphonate that has some anti - inflammatory properties by increasing apoptosis of monocytes.2 we reported two cases of severe seronegative ra that responded to this drug . By this report, we can schedule a more extensive research in this subject to identify the useful property of pamidronate . Ms and pm carried out the design and coordinated the study, participated in data collection and prepared the manuscript.
Porcine eyes: fresh enucleated porcine eyes were purchased from a local abattoir in osaka, osaka meat and organs co., ltd . The eyes with the surrounding eyelids and conjunctiva were immediately enucleated from slaughtered pigs, and the eyelids were closed to minimize corneal damage . The eyes were placed in a cool box kept at 4c with moist conditions of saline (otsuka normal saline, otsuka, tokyo, japan) and transported to the laboratory at osaka prefecture university within an hour by motorcycle . Each porcine eye was rinsed with saline, and 1% (w / v) sodium fluorescein (sfl) solution prepared with saline and sfl powder (nacalai tesque, kyoto, japan) was applied to the porcine cornea in order to select porcine eyes with an intact cornea . The selected porcine eyes were gently rinsed with saline again, the eyelids were closed, and then, the eyes were again stored in the moist cool box (4c) until the experiment, within approximately one hour . Artificial tears: four types of artificial tears were used in the experiments: 1) saline, 2) 0.1%, 0.5% or 1% (v / v) sodium hyaluronate (sh) solution prepared with saline and sh (artz dispo 25 mg, seikagaku corporation, tokyo, japan), 3) 0.5%, 1% or 5% (v / v) castor oil (co) solution made with saline and co (sioe pharmaceutical co., ltd ., amagasaki, japan) and 4) a mixture solution of 0.5% (v / v) sh and 1% (v / v) co. each artificial tear was prepared before each experiment and stored in a sterilized eye dropper at room temperature (2022c). All eye droppers were shaken well just before application of the artificial tears to the porcine eyes . Experimental procedures: an in vitro porcine dry eye model was designed by modification of the procedures of choy et al . And fujihara et al . . The eyelids of each porcine eye with an intact cornea were held open, excess saline was removed from the conjunctival sac with swabs, and then, the eyes were securely positioned on a plastic cap with the corneal surface up in the chamber kept at 2022c and 4050% humidity without air flow in the room, with environmental conditions similar to those made with air - conditioners and dehumidifiers . In the first experiment on the in vitro porcine short - term dry eye model, the eyes treated without any artificial tears were desiccated for up to 360 min in the chamber described above . On the other hand, in the second experiment for evaluation of artificial tears including sh, co and a mixture solution containing 0.5% sh and 1% co, eyes were treated with 2 drops of each artificial tear (50 l / drop) of saline, 0.1%, 0.5% or 1% sh solution, 0.5%, 1% or 5% co solution or the mixture solution, blinked manually a couple of times by closing and opening the eyelids, held open again and then desiccated in the chamber for 60, 90 or 180 min . The control eyes were treated continuously with saline applied in the same chamber for the same desiccation time . In both experiments, all surrounding tissues of the eyes were removed after desiccation, and the corneas of the desiccated and control eyes were stained by dipping in 1% (w / v) methylene blue (mb) solution prepared with sterilized distilled water (dw) and mb powder (nacalai tesque) or 1% (w / v) lissamine green (lg) solution, made with sterilized dw and lg powder (nacalai tesque) for 1 min . Mb and lg were used for staining dead and membrane - damaged cells of the cornea, respectively [6, 12, 22]. The stained eyes were washed well with saline to remove staining solutions, and the cornea stained with mb or lg was removed with surgical knives and scissors from the limbus of each eye . The corneas stained with mb were immediately photographed and placed in 2 ml of acetone / saturated sodium sulfate solution (volume ratio of 7:3) for 16 hr at room temperature (22c) to extract mb . The photographed images were used for the evaluation of corneal staining scores indicating corneal integrity based on the extent of mb staining . The mb staining score was divided in 4 categories as follows: 1, less than 1/4 of the corneal area stained; 2, 1/41/2 of the corneal area stained; 3, 1/23/4 of the corneal area stained; and 4, more than 3/4 of the corneal area stained . The absorbance of mb - extracted solutions was measured at 660 nm with a spectrophotometer (smart spec 3000, bio - rad, hercules, ca, u.s.a . ). The density of lg - stained corneas was measured using an image analyzer (fpd-100s, fuji film, tokyo, japan). Histopathological examination: the harvested corneas from the desiccated and control eyes were fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned at 34 m, stained with hematoxylin and eosin (he) after deparaffinization and then examined microscopically . Statistical analysis: values of the corneal staining scores of mb, the absorbance of mb extracted from the cornea and the corneal staining density of lg are shown as the mean standard deviation (sd), and their averages and sds of continuous saline - treated control eyes were calculated from all of the control ocular data upon placement in the desiccated chamber for 60, 90 and 180 min, because the control data obtained from each time were similar . Data of artificial tears treated eyes were compared to the control values of ones treated continuously with saline, which were the group for which the eyes had similar conditions of normal eyes in the live animals . Comparison of values obtained in this experiment was carried out by using non - repeated measurement one - way analysis of variance (anova) and then scheffe s test or the kruskal - wallis h test, followed by the mann - whitney u - test with bonferroni correction (statcel 2nd ed . ; oms publishing co., tokyo, japan). A p - value less than 0.05 in vitro porcine short - term dry eye model: corneal damage induced by desiccation was evaluated by the staining of non - viable cells, which were detectable using mb, and membrane - damaged cells, which were observable using lg . The corneal staining scores of mb, absorbance values of mb extracted from the corneas and values of the corneal staining density of lg increased significantly with increasing desiccation time (table 1table 1.kinetics of the degenerative changes of the cornea upon desiccationdesiccation time(minutes)corneal staining scoreof methylene bluecorneal absorbance at660 nm of methylene bluecorneal staining densityof lissamine green01.0 00.017 0.0090.208 0.019602.4 0.60.056 0.0330.268 0.0081803.8 0.50.206 0.0270.470 0.0163604.0 00.272 0.0530.612 0.048different superscript letters indicate statistically significant differences between groups (p<0.05) (n=5).). In addition, a significant linear correlation was found between the staining score of mb and its absorbance (r=0.97, p<0.01); the regression equation between these 2 variables was y=0.07 x 0.06 (fig . 1.relationship between staining score of methylene blue (mb) and absorbance values of mb in the in vitro porcine short - term dry eye model . Mild cytoplasmic vacuolations were found in some basal cells on the control cornea treated continuously with saline when compared with the cornea harvested immediately from the slaughtered animals (fig . 2a and 2bfig . 2.histopathologies of the desiccated cornea in the in vitro porcine short - term dry eye model . When compared with the cornea obtained immediately from the slaughtered animals (a), mild cytoplasmic vacuolations could be found in some basal cells of the cornea treated continuously with saline (b). The cornea desiccated for 180 min (c) had marked swollen basal cells with cytoplasmic vacuolations and nuclear swelling and wing cells with vacuole in the cytoplasm, and there were corneal basal and wing cells with mild to moderate cytoplasmic vacuolations on the cornea desiccated for 60 min (d). Meanwhile, histopathological alterations with cytoplasmic vacuolations were minimal on the corneal basal and wing cells in eyes desiccated for 60 min and treated with 1% sh (e) or a mixture solution containing 0.5% sh and 1% co (f), when compared with saline non - treated eyes for 60 min of desiccation (d). 400 . He staining . ), whereas conspicuous alterations could be observed in the histopathology of cornea desiccated for 180 min . The cornea desiccated for 180 min had not only marked swollen basal cells with cytoplasmic vacuolation and nuclear swelling, but also wing cells with vacuole in the cytoplasm (fig . Different superscript letters indicate statistically significant differences between groups (p<0.05) (n=5). Relationship between staining score of methylene blue (mb) and absorbance values of mb in the in vitro porcine short - term dry eye model . Histopathologies of the desiccated cornea in the in vitro porcine short - term dry eye model . When compared with the cornea obtained immediately from the slaughtered animals (a), mild cytoplasmic vacuolations could be found in some basal cells of the cornea treated continuously with saline (b). The cornea desiccated for 180 min (c) had marked swollen basal cells with cytoplasmic vacuolations and nuclear swelling and wing cells with vacuole in the cytoplasm, and there were corneal basal and wing cells with mild to moderate cytoplasmic vacuolations on the cornea desiccated for 60 min (d). Meanwhile, histopathological alterations with cytoplasmic vacuolations were minimal on the corneal basal and wing cells in eyes desiccated for 60 min and treated with 1% sh (e) or a mixture solution containing 0.5% sh and 1% co (f), when compared with saline non - treated eyes for 60 min of desiccation (d). Corneal - protective effects of artificial tears on the porcine short - term dry eye model: table 2table 2.corneal-protective effects of artificial tears of sodium hyaluronate, alone castor oil alone, and a mixture containing sodium hyaluronate and castor oildesiccationtime (minutes)controlshcomixture1%0.5%0.1%5%1%0.5%0.5%sh+1%cocorneal staining score of methylene blue0 (cts)1.0 060(3.3 0.6)1.1 0.3 * 2.0 0.52.7 0.52.2 0.73.2 0.73.3 0.51.0 0 * 90(3.7 0.6)1.4 0.5 * 2.3 0.53.0 02.9 0.33.7 0.53.9 0.31.8 0.4180(4.0 0)2.0 0.72.9 0.33.8 0.43.6 0.53.9 0.34.0 02.1 0.3corneal absorbance at 660 nm of methylene blue0 (cts)0.048 0.00860(0.163 0.021)0.051 0.020 * 0.096 0.0450.113 0.0750.090 0.0110.114 0.0210.175 0.0610.058 0.006 * 90(0.182 0.017)0.074 0.034 * 0.114 0.0690.132 0.0890.091 0.0370.115 0.0430.176 0.0700.076 0.007180(0.205 0.031)0.162 0.0740.126 0.0620.151 0.0340.147 0.0470.134 0.0160.196 0.0770.113 0.013corneal staining density of lissamine green0 (cts)0.219 0.01560(0.340 0.046)0.232 0.010 * 0.262 0.0230.307 0.0230.293 0.0500.345 0.0210.340 0.0730.221 0.026 * 90(0.393 0.040)0.267 0.0360.301 0.0290.330 0.0250.328 0.0580.380 0.0240.364 0.0710.252 0.021180(0.430 0.030)0.317 0.0230.326 0.0410.352 0.0340.361 0.0790.426 0.0190.413 0.0760.289 0.018cts: continuous treatment with saline, sh: sodium hyaluronate, co: castor oil . A superscript asterisk indicates no significant differences between control (cts; 0 min of desiccation time) and each experimental group . N=89 (values with parentheses were calculated from the data of 3 eyes treated with saline). Shows the results of mb and lg staining in eyes treated with saline, sh solutions, co solutions and a mixture solution containing 0.5% sh and 1% co on the porcine short - term dry eye model . All control eyes with continuous treatment of saline (cts) for 60, 90 and 180 min showed similar results of mb and lg staining, and their minimal corneal damage was demonstrated by their staining, as shown in table 2, when compared with the ocular group indicated as 0 min of desiccation time in table 1 of the in vitro porcine short - term dry eye model, which was the group for which the eyes were stained with the dyes at the earliest time after enucleation . There were no significant differences between eyes treated continuously with saline (control) and those desiccated for 60 and 90 min and treated with 1% sh in terms of the staining scores and absorbance values of mb . No significant difference of the staining density of lg was only observed between control eyes and 1% sh - treated ones with 60 min of desiccation . Histopathological changes with cytoplasmic vacuolation were minimal on the corneal basal and wing cells in eyes desiccated for 60 min with 1% sh treatment, when compared with saline non - treated eyes for 60 min of desiccation (fig . Significant differences were found in the staining scores of mb and absorbance values of mb between control and 1% sh - treated eyes for 180 min of desiccation and in the values of the staining density of lg between control and 1% sh - treated eyes for 90 and 180 min of desiccation (table 2). There were no significant differences between control eyes and those desiccated for 60 min with the treatment of the mixture solution in terms of the staining scores of mb, absorbance values of mb and values of the staining density of lg (table 2), indicating that corneal protection similar to that for 1% sh - administered eyes for 60 min of desiccation was obtained in the eyes treated with the mixture solution containing 0.5% sh and 1% co. as shown in fig . 2e and 2f, the cornea desiccated for 60 min and treated with the mixture solution had minimal histopathological changes on the basal and wing cells, which were similar to the changes of 1% sh - treated eyes with 60 min of desiccation . Unfortunately, significant differences were observed in the results of mb and lg staining between the control and eyes treated with the mixture solution for 90 and 180 min desiccation time . There were significant differences in the staining results of mb and lg between the control and eyes treated with solution of 0.5% sh, 0.1% sh, 5% co, 1% co and 0.5% co for all desiccation times (table 2). Cts: continuous treatment with saline, sh: sodium hyaluronate, co: castor oil . A superscript asterisk indicates no significant differences between control (cts; 0 min of desiccation time) and each experimental group . N=89 (values with parentheses were calculated from the data of 3 eyes treated with saline). An in vitro porcine short - term dry eye model was established in this study . Three different staining dyes, sfl, mb and lg, were used in this model . Mb and lg were selected for the detection of non - viable dead cells and membrane - damaged cells of the corneal epithelium, respectively [6, 12, 22]. Sfl was employed to select eyes with an intact cornea without corneal defects, because the dye did not adhere to an intact epithelial surface . If mb or lg was used for ocular selection and the dye was left on the corneal surface, overestimated results might arise due to the remaining dye used at the time of the selection . However, sfl only stains the disrupted part of the corneal epithelium lightly, if there is little intercellular space on the cornea . The stained parts of sfl would be overlaid with dark dye of mb or lb, resulting in no influence on the evaluation system with mb and lg in the dry eye model . The stainabilities of mb and lg significantly increased with increasing desiccation time in the dry eye model (table 1), and there was a linear correlation between the staining score of mb, as a qualitative evaluation, and the absorbance of mb, as a quantitative analysis, using a spectrophotometer (fig . 1). These results are similar to the findings observed in an in vivo rabbit short - term dry eye model reported previously . The histopathological alterations observed in the desiccated porcine cornea were very similar to the alterations found in a mouse model of dry eye and experimental canine kcs [5, 8]. Moreover, both mb and lg staining was related to the degree of histopathological change in this study (table 1 and fig . All these findings suggest that the porcine short - term dry eye model can reflect the corneal changes upon desiccation in live animals and is available for evaluating the potencies for corneal protection of newly developed artificial tear substitutes by in vitro studies . The dry eye model should have the following advantages: 1) not only preventing exposure to unexpected adverse effects and/or inefficacies of the newly developed artificial tears, but also reducing the number of experimental animals in studies dealing with assessment of potential therapeutic agents, 2) being able to collect a large amount of data on novel artificial tears in a short period and 3) rapidly assessing the therapeutic effects of new artificial tears . On the other hand, the dry eye model cannot evaluate phenomena exhibited by live animals, such as blinking and corneal wound healing [2, 17, 19], and cannot evaluate long - term and adverse reactions of newly developed artificial tears, because it uses enucleated eyes, which exhibit postmortem changes . Therefore, the porcine short - term dry eye model established in this study is useful for screening in the selection of potential agents before experiments with live animals . There are plenty of artificial tear substitutes for lacrimomimetic therapy in animals with an absence or a reduction of lacrimal secretions, and they generally contain one or more ingredients that can replace deficiencies of tear components [9, 11]. It is well known that sh has mucinomimetic properties with viscosity and wettability and is a useful corneal protectant in animals with kcs [9, 11, 15, 18, 20, 23]. Judging from our results, 1% sh may be available as an artificial tear . However, the clinical usefulness of 1% sh would be low, because it is an expensive agent and may have a disadvantage of provoking an uncomfortable sensation when the drug is applied to the eyes, due to its high viscosity [11, 12, 18]. The concentrations of commercially available ophthalmic sh solutions are 0.1% and 0.3% in japan and 0.15% and 0.4% in other countries [9, 11, 18, 23]. However, it is still difficult to provide infrequent lacrimomimetic therapy, fewer than 6 times per day, to animals with tear film abnormalities in spite of the presence of good ophthalmic sh solutions [9, 11, 18]. This is not surprising, because sufficient corneal - protective effects could not be found in eyes treated with 0.5% sh, which was a greater concentration than that in commercially available ophthalmic sh solutions, in the porcine short - term dry eye model (table 2). Natural tear film consists of 3 components: lipid, an aqueous component and mucin, and the layer containing lipid inhibits the evaporation of the aqueous component from tear film [12, 14, 16, 18, 20]. Hence, insufficient corneal protection should be attributable to a lack of an effect inhibiting the evaporation of water from sh . A surface coating layer could be added to inhibit such evaporation, thereby improving sh ophthalmic solution to achieve infrequent lacrimomimetic therapy in animals . Lipid products or oils are generally used as ingredients of ophthalmic ointments [9, 11], although the solution type of artificial tear containing lipid products, such as co, was shown to be helpful for treating patients with tear film abnormalities or dry eye model animals [10, 14, 16]. In addition, co was shown to inhibit the evaporation of water from the corneal surface [14, 16]. Therefore, combined prescription of saline, sh and co, which is similar to natural tear film containing lipid, an aqueous component, and mucin [11, 18, 20], was established and assessed in the experiment . The saline - based tear substitute containing 0.5% sh and 1% co showed corneal - protective effects equal to those with 1% sh (table 2), indicating that this combination of an aqueous component (saline), mucinomimetics (sh) and lipid (co) is an effective prescription for an artificial tear substitute for treating cases with tear film abnormalities in animals . However, the maximum time of corneal protection was only 60 min for both 1% sh and the mixture solution containing 0.5% sh and 1% co in the porcine short - term dry eye model, when compared with the group having continuous treatment of saline (table 2). This corneal - protective time would still be too short for providing infrequent applications of artificial tears in animals with kcs . The prescription of our artificial tear substitute presented here should be improved to achieve infrequent lacrimomimetic therapy of fewer than 6 times per day in cases with tear film abnormalities . In order to determine the appropriate prescription for long - acting artificial tear substitutes, more detailed prescriptions should be studied or different types of sh and/or lipid products should be used in the future . In summary, the saline - based tear substitute containing 0.5% sh and 1% co had protective effects against corneal desiccation similar to those of 1% sh for 60 min . The combined prescription of saline, 0.5% sh and 1% co would be useful as an artificial tear substitute for treating animals with tear film abnormalities, such as kcs.
Vitiligo, an idiopathic skin depigmentation disorder, is caused by the destruction of melanocytes from the basal layer of the epidermis as a result of which irregularly shaped milky white patches appear on the lesional skin (shajil et al ., 2006). Vitiligo affects approximately 1% of the world population including both men and women unbiasedly from all ethnicities (tang et al ., 2013). Although, it is not a life threatening disorder but this could be psychologically devastating leading to mild embarrassment to a severe loss of self - confidence for the affected person especially the dark skinned people because of the presence of white patches on the skin (ongenae et al ., 2006). Melanocytes are solely responsible for the melanin pigment production by the process named melanogenesis which imparts color to the skin . Loss of melanocyte results in lack of melanin and thus, milky white patches appear at the lesion site . The exact pathophysiology of vitiligo is hard to pin down but various theories based on autoimmunity (kemp et al ., 2001,, neural factors (lerner, 1959) and genetic defects (shajil et al ., 2006) have been proposed to explain the reason of melanocyte destruction . Various researchers are focusing their efforts to explicate the mechanism of skin pigmentation and its regulation with the purpose of better management of pigmentary disorder . More than 150 genes are identified that affect pigmentation in skin, hair and eye along with multiple transcription factors, signaling factors and other biological factors involved in its regulatory pathway known to interfere with pigmentation process (bennett and lamoreux, 2003). Candidate gene association studies have reported more than 33 genes involved in generalized vitiligo (spritz, 2011). Liver x receptor (lxr) is ligand (such as oxysterol, high concentration of d - glucose) activated nuclear transcription factor which regulate the expression of target genes involved in various physiological processes . Lxr exists in two isoforms: lxr- and lxr-. Lxr- is highly expressed in several metabolically active tissues including liver, adipose tissue, macrophages, and intestine, whereas lxr- is ubiquitously expressed in most tissues (steffensen and gustafsson, 2004, jamroz - wisniewska et al . Recently, lxr has also been shown to express in skin tissue such as sebaceous gland, hair follicle, epidermal keratinocyte and fibroblast and was found to be linked with various skin disorder pathogeneses like psoriasis and acne vulgaris (russell et al ., 2007). Melanocytes express lxr but the functions of lxr in these cells are still elusive (kumar et al ., 2010). Kumar et al . Observed high expression of lxr- at both transcriptional and translational levels in the melanocytes obtained from perilesional skin compared to the normal skin of vitiligo patient (kumar et al ., 2010). Various researchers have reported the effect of activated lxr on melanogenesis (kumar et al . Further showed that lxr- agonist 22(r)-hydroxycholesterol treatment significantly downregulates the cell adhesion molecule, suggesting the existence of melanocytorrhagy in which melanocytes with defective adhesion system induces detachment from the basement membrane in perilesional vitiligo skin followed by melanocyte apoptosis (kumar and parsad, 2012). Lei et al . Demonstrated that matrix metalloproteinase 2 (mmp 2) plays an important role in melanoblast (melanocyte precursor) migration from the outer root sheath of hair follicle into clinically depigmented epidermis and crucial for the repigmentation of vitiliginous skin (lei et al ., 2002). Reported that mmp-2 and mmp-9 were downregulated in vitiligo patient and furthermore, it was shown that lxr- gene knock - down significantly increased the expression of mmps (kumar et al ., 2011). Recently, lee et al . Reported that the activated lxr inhibits the melanogenesis through the accelerated activation of extracellular signal - regulated kinase (erk) mediated microphthalmia - associated transcription factor (mitf) degradation (lee et al ., 2013). Thus, upregulated expression of lxr- in perilesional skin melanocytes significantly decreases the adhesion, proliferation and matrix metalloproteinases and increases apoptosis . Based on all these studies, lxrs appear to play a key role in vitiligo pathogenesis and considered as a potential therapeutic target for vitiligo (kumar et al ., 2012). Variation in gene expression which is considered as an intrinsic factor in a disease may be associated with both genetic factor (mutational changes, single nucleotide polymorphism, epigenetic change) and non - genetic factors (level of agonists e.g. Oxysterol, drugs such as statins, fibrates, thazolidinedione derivatives, level of antagonists e.g. Oxysterol sulfonates). We selected two lxr- single nucleotide polymorphisms (snps; rs11039155 and rs2279238) to investigate their contribution to the risk of vitiligo . These snps have been reported to be associated with many metabolic indicators and conditions, including circulating ldl and hdl - cholesterol concentrations (robitaille et al ., 2007; sabatti et al ., 2009), type 2 diabetes mellitus (dahlman et al ., 2009, ketterer et al ., 2011) and obesity (dahlman et al ., 2006). To the best of our knowledge, this is the first study to assess the association of lxr- polymorphisms with vitiligo disorder . Clinical history of vitiligo and any other autoimmune disease in patients was taken by clinical interview . A total of 75 (31 females/44 males) healthy individuals with no history of vitiligo or apparent autoimmune disease were included as control; they matched to patients with regard to age, sex and geographical distribution . Rajendra prasad government medical college and jaypee university of information technology, solan, himachal pradesh . The importance of the study was explained to all participants and written consent was obtained from patients and controls . Peripheral blood (2 ml) genomic dna was extracted from peripheral blood using a standard salting out procedure described earlier (miller et al ., 1988). Genotyping of lxr- 6 g> a and + 1257 c> t snp was done using the polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) method as described earlier (legry et al ., 2008, fukae et al ., 2011). Briefly, the dna fragments containing these snps were amplified in 25 l reaction mixture containing 12.5 l pcr master mix (promega, madison, us), 0.2 m of each primer and 40 ng of dna template . A (forward 5-gtg aga gga tca ctt gag c-3 and reverse 5-cag acc gca ggc tcc acg c-3; amplifies 366 bp fragment), and for + 1257c> t (forward 5-ctt tct gag cct cac ttt cc-3 and reverse 5-cgc agc tca gaa cat tgt ag-3; amplifies 377 bp fragment). The pcr amplification conditions were: 3 min initial denaturation at 94 c, followed by 35 cycles of denaturation at 94 c for 30 s, annealing for 6 g> a for 30 s at 62 c & for + 1257c> t for 45 s at 60 c and 30 s of extension at 72 c followed by final extension at 72 c for 5 min . For rflp analysis, pcr products were digested with 1 u of respective restriction enzyme (hpy188iii for 6 g> a and fnu4hi for + 1257c> t) for 4 h at 37 c . Weinberg equilibrium (hwe) was tested to determine if the population fulfilled the hwe at the variant locus . The observed genotype distribution of control was assessed for deviation from hwe using a chi square test (http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html). Odds ratios and 95% confidence intervals were calculated to assess the risk associated with alleles and genotype . The significance of the odds ratio was determined by z test (p <0.05 was considered statistically significant). Clinical history of vitiligo and any other autoimmune disease in patients was taken by clinical interview . A total of 75 (31 females/44 males) healthy individuals with no history of vitiligo or apparent autoimmune disease were included as control; they matched to patients with regard to age, sex and geographical distribution . Rajendra prasad government medical college and jaypee university of information technology, solan, himachal pradesh . The importance of the study was explained to all participants and written consent was obtained from patients and controls . Peripheral blood (2 ml) was collected from the patients and healthy subjects in na2edta coated tubes . Genomic dna was extracted from peripheral blood using a standard salting out procedure described earlier (miller et al ., 1988). Genotyping of lxr- 6 g> a and + 1257 c> t snp was done using the polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) method as described earlier (legry et al ., 2008, fukae et al ., 2011). Briefly, the dna fragments containing these snps were amplified in 25 l reaction mixture containing 12.5 l pcr master mix (promega, madison, us), 0.2 m of each primer and 40 ng of dna template . The primer pairs used were, for 6 g> a (forward 5-gtg aga gga tca ctt gag c-3 and reverse 5-cag acc gca ggc tcc acg c-3; amplifies 366 bp fragment), and for + 1257c> t (forward 5-ctt tct gag cct cac ttt cc-3 and reverse 5-cgc agc tca gaa cat tgt ag-3; amplifies 377 bp fragment). The pcr amplification conditions were: 3 min initial denaturation at 94 c, followed by 35 cycles of denaturation at 94 c for 30 s, annealing for 6 g> a for 30 s at 62 c & for + 1257c> t for 45 s at 60 c and 30 s of extension at 72 c followed by final extension at 72 c for 5 min . For rflp analysis, pcr products were digested with 1 u of respective restriction enzyme (hpy188iii for 6 g> a and fnu4hi for + 1257c> t) for 4 h at 37 c . Allelic and genotypic frequencies were calculated using microsoft excel software (microsoft corporation). Hardy weinberg equilibrium (hwe) was tested to determine if the population fulfilled the hwe at the variant locus . The observed genotype distribution of control was assessed for deviation from hwe using a chi square test (http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html). Odds ratios and 95% confidence intervals were calculated to assess the risk associated with alleles and genotype . The significance of the odds ratio was determined by z test (p <0.05 was considered statistically significant). The average ages of patients and controls were 27.38 14.06 yr and 23.05 7.08 yr, respectively . The average age at onset of disease was 18.09 10.44 yr . The majority of patients (75%) had less than 25% body coverage of the depigmented patches . Patient and control subjects were genotyped applying pcr - rflp assay for 6 g> a and + 1257c> t in lxr- gene . The genotypes and allele frequencies of lxr- polymorphisms (rs11039155 and rs2279238) in patients and controls are summarized in table 2 . The genotype distribution for both the snp showed no deviation from hardy weinberg equilibrium in control population (p> 0.05). Genotyping of the snps in the lxr- gene revealed that the allele a of rs11039155 was found in 13% of controls and 24% of patients; the allele t of rs2279238 was found in 47% of controls and 60% of patients . Both of the alleles 6a and + 1257 t were found to increase the risk of vitiligo (rs11039155: odds ratio (or) = 1.99; 95% confidence interval (ci) = 1.073.71; rs2279238: or = 1.70; 95% ci = 1.062.73) implicating that these snps could be a prognostic marker for the disease . Dominant and co - dominant models of rs11039155 (table 3) demonstrate that the presence of a allele in the individual's genotype predisposes that person to the vitiligo . Recessive and homozygous models of rs2279238 demonstrate that allele t is associated with the vitiligo susceptibility . Vitiligo is a multifactorial complex genetic disorder leading to skin depigmentation (shajil et al ., 2006). It has been reported that variations in the genes could affect their stability, expression or regulation which might account for disease susceptibility in the patients . Identification of these potential vitiligo susceptibility variants would lead to better understanding of disease pathogenesis and targeted disease treatment . Recent reports have shown that liver x receptor, an inducible transcription factor, plays an important role in vitiligo pathogenesis . Genes involved in regulation of melanocytes are the target genes of lxrs (kumar et al ., 2011, kumar and parsad, 2012, kumar et al ., 2012). Although the exact role of lxr- in development of vitiligo is not known, a number of reports have demonstrated higher expression of lxr- in the melanocytes obtained from perilesional skin of the vitiligo patient which speculates its role in vitiligo pathogenesis (kumar et al ., 2010),(kumar et al ., 2012, lee et al ., 2013). In the present study, we have investigated vitiligo patients for genetic variation in the lxr- gene . T) and rs11039155 (6 g> a) in lxr- gene to assess their involvement in the susceptibility to vitiligo in north indian population . Various studies have reported association of these snps with high levels of circulating ldl and hdl - cholesterol (robitaille et al ., 2007, legry et al ., 2008, sabatti et al ., 2009), type 2 diabetes mellitus (dahlman et al ., 2009, ketterer et al ., 2011) and obesity (dahlman et al ., 2006). To the best of our knowledge, this is the first report showing association of lxr- variants with the risk of vitiligo . Our data suggested that 6a and + 1257 t alleles were significantly associated with the risk of vitiligo . The 6 g> a polymorphism is located six base pairs upstream from the atg site and is part of kozak sequence could affect translation efficiency of a gene . This sequence in the lxr- gene (gaagagatgt) is different from the consensus kozak sequence (gacaccatgg). Moreover, polymorphism / change of g at 6 position to a may alter translation efficiency of this gene (kozak, 1987, legry et al ., 2008). Second snp, + 1257c> t is located at an exon splicing enhancer (ese) of lxr- where splicing factor srp55 binds . This polymorphic marker can affect mrna splicing, stability and translation efficiency (due to codon usage) (price et al ., 2011). However, in - depth molecular studies are required to elucidate the functional roles of the rs11039155 and rs2279238 for an understanding of its role in vitiligo which could guide the development of new ligands for the treatment of vitiligo . Furthermore, these snps could be in linkage disequilibrium with another snps affecting lxr- expression . Secondly, lack of knowledge about the functional influence of the identified polymorphisms with vitiligo remains to be determined . In conclusion, variant 6 a and + 1257 t alleles of lxr- were significantly associated with the risk of vitiligo in north indian population and underscore the importance of genetic factors in vitiligo pathogenesis . However, studies with large number of samples as well as from other populations are needed to further validate these findings . These data provide support for additional prospective studies since lxr- is an inducible transcription factor . Further studies elucidating the role of other functional snps of lxr- and other related genes involved in related biological pathways that are involved in the etiology of vitiligo would be useful . In addition to this, studies elucidating the role of these snps in the treatment response would be helpful for the better management of this disease.
The results of all leptospirosis mats for dogs from january 2002 through december 2004 were obtained electronically from antech diagnostic veterinary laboratory (los angeles, ca, usa). The 7 leptospira serovars included in the mats were canicola, grippotyphosa, icterohaemorrhagiae, hardjo, pomona, autumnalis, and bratislava . Mat results for each serovar were reported as the highest dilution of serum (1:100, 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, or> 1:12,800) at which> 50% agglutination of organisms occurred when compared with a control suspension . Calculation of seropositivity was performed separately by using cutoff titers of> 400,> 800, or> 1,600 . The percentage of seropositive test results for each serovar was calculated both as the number of positive test results divided by the total number of tests performed and by the total number of positive test results with 95% confidence limits . Proportions for categoric variables were compared by using the test for independence . A rank from 1 to 9 was assigned based on the serum dilution results . If 2 serovars had equivalent titers on a mat for a dog, both serovars received the same rank score appropriate for that dilution . All calculations were performed by using sas version 9.1.3 statistical software (sas, cary, nc, usa), and a p value> 0.05 was considered significant . Tests that used paired sera from the same dog or tests repeated on the same dog at a different time could not be identified because patient identifiers were not included in the database . Therefore, a few individual dogs could have contributed> 1 test to the dataset, but this possibility was considered uncommon . During the study, 23,005 serum samples were submitted for a leptospirosis mat, and 23,000 tests were performed for each of 5 serovars, namely canicola, grippotyphosa, icterohaemorrhagiae, hardjo, and pomona (table). Laboratory testing for serovars autumnalis and bratislava was initiated in 2003, and 11,600 tests were performed for each of these 2 serovars . The percentage of mats that were positive significantly increased from 2002 to 2004 by using cutoff titers> 400 (p<0.002),> 800 (p<0.0001), or> 1,600 (p<0.0001). At these 3 cutoff titers, the percentage of positive mat results was greatest for serovars autumnalis (9.1%, 6.5%, and 4.7%, respectively) and grippotyphosa (6.4%, 4.9%, and 4.0%, respectively). The proportion of positive mat results attributable to serovars canicola or icterohaemorrhagiae declined as the cutoff titer increased, while it generally increased for serovars autumnalis, bratislava, grippotyphosa, and pomona (figure 1). No consistent or distinct geographic pattern for positive mat results was observed in the study (figure 2), but seropositivity was greater in the midwest, south - central, and northwest regions of the united states . Percentage of positive microscopic agglutination tests by leptospira serovar, using 3 different cutoff titers for 23,005 canine sera from 20022004 . Serovars canicola and icterohaemorrhagiae have been used in canine bacterins for leptospirosis during the study period . Canine leptospirosis microscopic agglutination test results shown as the percentage positive and standard error, by state and year from 20022004 . A test was considered positive if the titer for any serovar was> 400 for autumnalis, bratislava, canicola, grippotyphosa, icterohaemorrhagiae, pomona, or hardjo serovars . Moderately strong positive correlation in seropositivity (r, 0.590.72) was present between serovars autumnalis, pomona, grippotyphosa, and bratislava, with the strongest correlation between serovars autumnalis and pomona . In contrast, weak positive correlation (r = 0.36) was found between serovars canicola and icterohaemorrhagiae, and each of these serovars was weakly correlated (r, 0.200.33) with serovars autumnalis, pomona, grippotyphosa, and bratislava . Serovar hardjo was excluded from correlation analysis because of the small number of positive test results . Positive leptospirosis mat results in dogs may indicate natural infection due to direct or indirect contact with wildlife maintenance hosts or recent vaccination (2). However, titers> 800 from vaccination are considered unlikely as postvaccinal titers wane rapidly (10) and most leptospiral bacterins available for dogs are bivalent for canicola and icterohaemorrhagiae, 2 serovars with low seropositivity in this study . Although the health and vaccination status of dogs from which sera were submitted was unknown, veterinarians most likely submitted samples for leptospirosis testing when they suspected leptospirosis based on clinical signs including vomiting, fever, lethargy, and anorexia . The most common serovar associated with a positive mat result was autumnalis, a serovar not currently included in licensed canine bacterins . Reactivity to this serovar in the mat has been considered a possible paradoxical cross - reaction with serovar pomona (11); a strong positive correlation in titers for these 2 serovars was found in this study . The autumnalis serovar has been isolated from raccoons in the southern united states (12), and seropositivity in dogs may represent natural infection from this source . The mat is not serovar - specific, but the 7 serovars evaluated in this study belong to different serogroups (13). Serovar grippotyphosa, the second most common positive serovar in this study, has also been associated with human leptospirosis outbreaks in the 1990s (8). The finding of a moderately high correlation in serologic reactivity between serovars autumnalis, pomona, grippotyphosa, and bratislava suggests that cross - protection to autumnalis could be induced by current bacterins that lack this antigen . Canine vaccines are now available with serovars grippotyphosa and pomona as well as the traditional serovars canicola and icterohaemorrhagiae . This vaccine may confer some immunity to serovar autumnalis, since some protein antigens are highly conserved among several pathogenic serovars (14). Limitations of the present study included the inability to determine if multiple tests had been performed for individual dogs, lack of data on clinical signs, and unknown vaccination status of the dogs . The geographic distribution of serologic reactivity during the study, however, indicates broad dispersion of leptospira pathogens that pose a risk to both domestic animals and humans . Dogs in suburban or rural environments have been shown to be at increased risk of leptospirosis (15), presumably because of greater likelihood of contact with wildlife habitats . Veterinary practitioners and public health officials need to be aware of the potential change in the ecologic environment and circulating endemic strains for this zoonotic organism.
The increased life expectancy of the population has led to increased demand for rehabilitation using osseointegrated implants and reconstructive procedures . To receive an implant, a site must have adequate alveolar bone volume . When there is an inadequate alveolar ridge for this purpose, certain techniques may be used to promote an increase in bone tissue, such as autogenous bone grafts and guided bone regeneration . Additionally, in association with the population s aging, there has been an increase in the prevalence of diseases that may interfere with the process of osseointegration, such as osteoporosis . This disease affects millions of people around the world and is characterized by decreased bone mass and structural deterioration, leading to an increased risk for fractures . Because the interference of osteoporosis with bone repair is highly debated, it is important to investigate the mechanisms by which this disease can jeopardize the osteointegration process . Several studies have aimed to describe this relationship, which observed that estrogen deficiency has a negative impact on the bone healing process during osteointegration after titanium implants were placed in ovx rats tibias, leading to less contact area between the implant surface and the bone, and showed that the bone healing of drill - hole defects in mice with ovx - induced osteoporosis exhibited impaired angiogenesis in the early stage, impaired osteogenesis in the middle stage, and impaired remodeling in the late stage, which resulted in compromised mechanical properties in the end . Despite the negative effects demonstrated by some studies mentioned above, other studies have presented contradictory, and showed that ovariectomy did not seriously affect bone healing after the placement of implants in cortical bone areas, but it reduced the bone contact ratio and the bone in the cancellous bone area, and that the systemic effects of estrogen deficiency are not crucial for fracture healing . There is a lack of information about the influence of estrogen deficiency on the healing process and about the markers for bone formation after onlay autogenous bone graft placement on the mandibula . About markers for bone formation, langille and solursh (1990) showed that culture of mesenchyme mandibular cells initiate expression of type i and ii collagen at early periods, followed by type x, which coincided with the onset of mineralization . After that, they loste cartilage markers and began to express bone sialoprotein ii (bsp ii), osteocalcin, and type i collagen . In addition, then, the cells formed significant mineral, detected by von kossa staining . According to nagata, et al . (1991), the initial distribution of bsp may modify bone formation and mineralization . Both osteopontin and bone sialoprotein are located ahead of the mineralization front, being necessary for the initiation of the process . Osteocalcin and osteonectin are not present in areas of initial crystal formation, but are seen in the entirely mineralized matrix . The three bone - forming markers were chosen based on their structural and mineral - inducing properties, in addition to these proteins capacity to alter recruitment, attachment, differentiation, and activity of bone cells . Therefore, the aim of this study was to evaluate the immunohistochemical expression of bone marker proteins in the bone repair of onlay autogenous grafts in ovariectomized rats . Additionally, the study aimed to observe whether the presence of an expanded polytetrafluoroethylene (e - ptfe) membrane has any influence on wound healing in this systemic condition . The present study was conducted in accordance with the ethical principles for animal experimentation adopted by the brazilian school of animal experimentation (cobea) and approved by the research ethics committee at the college of dentistry of so jos dos campos univ . Eighty 3-month - old, adult female wistar rats that weighed approximately 300 grams were enrolled in the present study . During the entire period of the study, the animals were housed in groups of six in plastic cages, and food and water were given ad libitum to all the animals . Prior to the beginning of the experimental procedures, the animals were allowed to acclimatize to the laboratory environment for 5 days . The animals were randomly divided into 2 groups: group ovx was subjected to an ovariectomy procedure, and group sham was subjected to sham surgery . Each group was divided into the following two subgroups: placement of an autogenous bone graft (bg) and an autogenous bone graft associated with an e - ptfe membrane (bgm) (wl gore, newark, delaware, usa). The animals were anesthetized using a 2% xylazine solution (rompum, bayer, so paulo, sp, brazil) and ketamine (dopalen, agribands, paulnia, sp, brazil) at a ratio of 1:1 (0.3 ml/100 g of body weight). In the ovx group (40 animals), two small incisions (~10 mm) were made beginning after the last rib, one on each side of the back of the animal . Afterward, the ovaries were held up and ligatures were made to avoid bleeding . Then, the ovaries were completely excised . In the sham group (40 animals), after the incisions, the bilateral ovaries were held up and then returned to their original position without being excised . The incisions were sutured using a resorbable material for the muscle layer (poligalatin 910 vycril 4.0 ethicon johnson & johnson, so jos dos campos, sp, brazil) and silk sutures for the skin (4.0 ethicon, johnson & johnson, so jos dos campos, sp, brazil) after 30 days, a new surgical procedure for the placement of autogenous bone grafts was performed as described by jardini, et al . The calvarium was used as the donor area, and the angle of the mandible was the recipient area . The graft was initially removed using a trephine drill with an external diameter of 4.1 mm (neodent, so paulo, sp, brazil) and then punctured in the center using a carbide bur (kg sorensen, cotia, sp, brazil) at low rotation and cooled with saline . Three perforations were made at the angle of the mandible using the carbide bur under cooling conditions, which enabled the stable attachment of the bone block to the recipient bed using a 5.0 green braided polyester suture (ethicon, johnson & johnson, so jos dos campos, sp, brazil). Afterward, the e - ptfe membrane was adapted, covering the bone graft in the ovx+bgm and sham+bgm . These procedures allowed close contact between the graft and the surface of the mandibular bone (figure 1). Figure 1surgical procedure: a) the calvarium was used as the donor area to graft removed; b) angle of the mandible was the recipient area; c) recipient bed; d) perforation was made at the angle of the mandible, which enabled the stable attachment of the bone block; e) bone block in position; f) e - ptfe membrane was adapted, covering the bone graft in the ovx+bgm and sham+bgm after the graft procedure, the muscle layer was sutured using a 5.0 absorbable polyglactin 910 suture (ethicon, johnson & johnson, so jos dos campos, sp, brazil), followed by suturing of the skin using a 4.0 silk suture (ethicon, johnson & johnson, so jos after the surgery, a single dose of antibiotics (1 mg / kg) was administered intramuscularly to all animals (pentabiotic, fort dodge, so paulo, sp, brazil). Euthanasia was performed using an overdose of anesthetics immediately after surgery (0 hour) and 7, 21, 45, and 60 days after the surgical procedure (figure 2). Figure 2periods of euthanasia the specimens were demineralized using a 10% edta solution at ph 7.8 in a microwave oven (pelco 3441, ted pella, california, usa). The blocks were sliced at a thickness of 3 m and subjected to immunohistochemistry for osteocalcin (occ) (fl-110:sc-30044; santa cruz biotechnology, paso robles, ca, usa), bone sialoprotein (bsp), and osteonectin (onc). The bsp (lf-87) and onc (lf-23) antibodies were kindly donated by dr . Larry w. fisher at the national institute of dental and craniofacial research at the national institutes of health (bethesda, antigen retrieval was performed using citrate (ph 6.0) in a microwave oven followed by the blocking of endogenous peroxidase using a solution of 50% methyl alcohol and hydrogen peroxide (20-volume solution) (1:1). The samples were incubated in bovine serum albumin (bsa) for 1 hour inside a moist chamber to block nonspecific antigens . Samples were then incubated with the primary antibodies (bsp, 1:150, 1 hour, room temperature; onc, 1:400, 1 hour, room temperature; occ, 1:400, 4c, overnight) followed by incubation with a secondary antibody (universal lsab tm kit / hrp, rb / mo / goat dako, carpinteria, ca, usa) for 30 minutes . A final incubation was performed using the tertiary complex streptavidin peroxidase (universal lsab tm kit / hrp, rb / mo / goat dako, carpinteria, ca, usa) for an additional 30 minutes . The reaction was visualized using diaminobenzidine (dab dako, carpinteria, ca, usa). Counterstaining was performed using mayer s hematoxylin, and the specimens were mounted in permount . As a positive control for bsp and onc was used on rat bone tissue repair area . Microscopic analysis was conducted using an axiophot 2 light microscope (carl zeiss, oberkochen, germany) coupled with an axiocam mrc 5 digital camera (carl zeiss, oberkochen, germany), which transmitted images to the axiovision release 4.7.2 computer software . The intensity of the immunohistochemical staining of predetermined structures and cells was classified for all periods, and antibodies were categorized as mild (+), moderate (+ +), or intense (+ + +). The present study was conducted in accordance with the ethical principles for animal experimentation adopted by the brazilian school of animal experimentation (cobea) and approved by the research ethics committee at the college of dentistry of so jos dos campos univ . Eighty 3-month - old, adult female wistar rats that weighed approximately 300 grams were enrolled in the present study . During the entire period of the study, the animals were housed in groups of six in plastic cages, and food and water were given ad libitum to all the animals . Prior to the beginning of the experimental procedures, the animals were allowed to acclimatize to the laboratory environment for 5 days . The animals were randomly divided into 2 groups: group ovx was subjected to an ovariectomy procedure, and group sham was subjected to sham surgery . Each group was divided into the following two subgroups: placement of an autogenous bone graft (bg) and an autogenous bone graft associated with an e - ptfe membrane (bgm) (wl gore, newark, delaware, usa). The animals were anesthetized using a 2% xylazine solution (rompum, bayer, so paulo, sp, brazil) and ketamine (dopalen, agribands, paulnia, sp, brazil) at a ratio of 1:1 (0.3 ml/100 g of body weight). In the ovx group (40 animals), two small incisions (~10 mm) were made beginning after the last rib, one on each side of the back of the animal . Then, the ovaries were completely excised . In the sham group (40 animals), after the incisions, the bilateral ovaries were held up and then returned to their original position without being excised . The incisions were sutured using a resorbable material for the muscle layer (poligalatin 910 vycril 4.0 ethicon johnson & johnson, so jos dos campos, sp, brazil) and silk sutures for the skin (4.0 ethicon, johnson & johnson, so jos dos campos, sp, brazil) after 30 days, a new surgical procedure for the placement of autogenous bone grafts was performed as described by jardini, et al . The calvarium was used as the donor area, and the angle of the mandible was the recipient area . The graft was initially removed using a trephine drill with an external diameter of 4.1 mm (neodent, so paulo, sp, brazil) and then punctured in the center using a carbide bur (kg sorensen, cotia, sp, brazil) at low rotation and cooled with saline . Three perforations were made at the angle of the mandible using the carbide bur under cooling conditions, which enabled the stable attachment of the bone block to the recipient bed using a 5.0 green braided polyester suture (ethicon, johnson & johnson, so jos dos campos, sp, brazil). Afterward, the e - ptfe membrane was adapted, covering the bone graft in the ovx+bgm and sham+bgm . These procedures allowed close contact between the graft and the surface of the mandibular bone (figure 1). Figure 1surgical procedure: a) the calvarium was used as the donor area to graft removed; b) angle of the mandible was the recipient area; c) recipient bed; d) perforation was made at the angle of the mandible, which enabled the stable attachment of the bone block; e) bone block in position; f) e - ptfe membrane was adapted, covering the bone graft in the ovx+bgm and sham+bgm after the graft procedure, the muscle layer was sutured using a 5.0 absorbable polyglactin 910 suture (ethicon, johnson & johnson, so jos dos campos, sp, brazil), followed by suturing of the skin using a 4.0 silk suture (ethicon, johnson & johnson, so jos dos campos, sp, brazil). After the surgery, a single dose of antibiotics (1 mg / kg) was administered intramuscularly to all animals (pentabiotic, fort dodge, so paulo, sp, brazil). Euthanasia was performed using an overdose of anesthetics immediately after surgery (0 hour) and 7, 21, 45, and 60 days after the surgical procedure (figure 2). The specimens were demineralized using a 10% edta solution at ph 7.8 in a microwave oven (pelco 3441, ted pella, california, usa). The blocks were sliced at a thickness of 3 m and subjected to immunohistochemistry for osteocalcin (occ) (fl-110:sc-30044; santa cruz biotechnology, paso robles, ca, usa), bone sialoprotein (bsp), and osteonectin (onc). The bsp (lf-87) and onc (lf-23) antibodies were kindly donated by dr . Larry w. fisher at the national institute of dental and craniofacial research at the national institutes of health (bethesda, md, usa). Antigen retrieval was performed using citrate (ph 6.0) in a microwave oven followed by the blocking of endogenous peroxidase using a solution of 50% methyl alcohol and hydrogen peroxide (20-volume solution) (1:1). The samples were incubated in bovine serum albumin (bsa) for 1 hour inside a moist chamber to block nonspecific antigens . Samples were then incubated with the primary antibodies (bsp, 1:150, 1 hour, room temperature; onc, 1:400, 1 hour, room temperature; occ, 1:400, 4c, overnight) followed by incubation with a secondary antibody (universal lsab tm kit / hrp, rb / mo / goat dako, carpinteria, ca, usa) for 30 minutes . A final incubation was performed using the tertiary complex streptavidin peroxidase (universal lsab tm kit / hrp, rb / mo / goat dako, carpinteria, ca, usa) for an additional 30 minutes . The reaction was visualized using diaminobenzidine (dab dako, carpinteria, ca, usa). Counterstaining was performed using mayer s hematoxylin, and the specimens were mounted in permount . As a positive control for bsp and onc was used on rat bone tissue repair area . Microscopic analysis was conducted using an axiophot 2 light microscope (carl zeiss, oberkochen, germany) coupled with an axiocam mrc 5 digital camera (carl zeiss, oberkochen, germany), which transmitted images to the axiovision release 4.7.2 computer software . The intensity of the immunohistochemical staining of predetermined structures and cells was classified for all periods, and antibodies were categorized as mild (+), moderate (+ +), or intense (+ + +). The ovx+bg and ovx+bgm groups presented greater marrow spaces and connective tissue compared to the sham groups, showing the influence of the absence of estrogen on bone repair (figures 3, 4, and 5). Structures labeled and staining intensity figure 4osteonectin (onc). Structures labeled and staining intensity figure 5osteocalcin (occ). Structures labeled and staining intensity within the same period, the 4 groups exhibited similar characteristics . At 0 hours, the bone matrix from the recipient bed and graft showed mild positivity in all groups for all periods . The ovx+bg and ovx+bmg groups showed moderate staining of the reversal lines of the recipient bed . At 7 and 21 days, newly bone tissue formed on the surface of the recipient bed exhibited intense staining . The osteoblasts present at the periphery of the recipient bed and around the immature bone trabecular showed intense staining as well as large osteocytes, which were interspersed in the immature bone trabecular . At day 45, the newly formed bone tissue, both at the bed - graft interface and at the periphery of the graft, presented mild to moderate staining in all groups . Large osteoblasts and osteocytes in this area were moderately stained . At day 60, the newly formed bone tissue in the bed - graft interface and around the graft showed diffuse staining that ranged from mild to moderate with a predominance of mildly positive areas . Osteocytes in this area sometimes accompanied the staining, and sometimes these samples showed mild to moderate staining only in the lacuna . Figure 6aa) bone sialoprotein (bsp) -day 0 group ovx - bgm: slight expression in recipient bed (rb) matrix and graft (g); b) day 0 group ovx - bgm: moderate marking in reverse lines (); c) day 7 group sham - bg: intense staining of newly formed bone (); d) day 7 group sham - bg: intense staining of newly formed bone (), osteoblasts () and osteocytes; e) day 21 . Group sham - bgm: intense staining of newly formed bone (); f) day 21 group sham - bgm: osteocytes () and osteoblasts () showing intense staining figure 6ba) bone sialoprotein (bsp) day 45 group sham - bgm: mild to moderate marking in newly formed bone () and the middle part of the graft; b) day 45 group ovx - bgm: osteoblasts () and osteocytes () showing moderate staining; c) day 60 group sham bgm: osteocytes () moderately marked on the middle portion of the graft; d) group ovx - bgm: osteoblasts () showing mild to moderate staining . G: graft; rb: recipient bed at 0 hours, the immunohistochemical staining was similar in all four groups . Only osteocytes located in the median region of the graft were stained, which varied from mild to moderate intensity . At day 7, the ovx+bg and sham+bg groups presented a large amount of intensely stained granulation tissue interposed between the graft and the recipient bed . The ovx+bgm and sham+bgm groups also showed this same staining pattern, though the amount of granulation tissue in this area was smaller . The granulation tissue surrounding the graft was mildly stained in the ovx+bg and sham+bg groups, while this tissue presented moderate to intense staining in the ovx+bgm and sham+bgm groups . Osteoblasts showed intense staining, and osteocytes in the immature bone trabecular exhibited varied staining that was sometimes mild and sometimes intense . The connective tissue present at the bed - graft interface presented intense staining in all four groups . The ovx+bg and sham+bg groups showed mild to moderate staining in the connective tissue surrounding the graft, while the ovx+bgm and sham+bgm groups showed intense staining . The osteoblasts showed moderate to intense staining, whereas the largest osteocytes present in the immature bone trabeculae showed more intense staining than those found in other areas . At day 45, the connective tissue interposed between the recipient bed and the graft showed intense staining in all four groups . The ovx+bg and sham+bg groups showed mild to moderate staining of the connective tissue surrounding the graft, whereas in the ovx+bgm and sham+bgm groups, the staining was intense . The majority of the osteocytes had no staining, even the largest ones . At day 60, when present, the connective tissue at the bed - graft interface showed mild to moderate staining in all four groups . The connective tissue surrounding the graft exhibited mild staining in the bg groups and moderate staining in the bg+m groups . The osteoblasts showed mild to moderate staining, while the osteocytes were not stained (figure 7a and figure 7b). Figure 7aa) osteonectin (onc) day 0 group ovx - bgm: no labeling of both the receptor bone matrix bed (l) and graft (e). At the bottom, intense staining was observed in skeletal striated muscle (); b) day 0 group ovx - bgm: osteocytes () present in the mid portion of the graft exhibit mild to moderate markup; c) day 7 group ovx - bg: connective tissue () exhibited varied staining that was sometimes intense and sometimes mild; d) day 7 group sham - bgm: osteoblasts () and osteocytes () showing intense staining; e) day 21 group sham - bg: connective tissue present at the surround bed - graft presented intense staining; f) day 21 group ovx - bg: osteoblasts () on the bone surface are moderately marked figure 7ba) osteonectin (onc) day 45 group ovx - bgm: in connective tissue surrounding the graft, the staining was intense; b) day 45 group sham - bgm: connective tissue surrounding newly bone () tissue showed intense staining; c) day 60 group sham - bg: granulation tissue in graft - bed () interface lightweight markup; d) day 60 group ovx - bgm group: osteoblasts () with mild cytoplasmic, osteocytes () the bone matrix of the recipient bed and the graft showed discrete and diffuse staining in all evaluated groups for all periods . At 0 hours, the osteocyte lacunae present at the midline region of the graft were moderately stained, while the osteocytes of the receptor showed more discreet marking . At day 7, the newly formed bone tissue showed no staining . The ovx+bg and sham+bg groups exhibited large amounts of intensely stained granulation tissue interposed between the recipient bed and the graft . The ovx+bgm and sham+bgm groups also showed this staining pattern, though the amount of granulation tissue in this area was smaller . The granulation tissue surrounding the graft exhibited mild to intense staining in the ovx+bg and sham+bg groups and intense staining in the ovx+bgm and sham+bgm groups . The osteoblasts showed mild to moderate staining, and the large osteocytes exhibited mild staining in all four groups . At day 21, the connective tissue present at the bed - graft interface showed intense staining . In the ovx+bgm and sham+bgm groups, the connective tissue surrounding the graft showed intense staining, whereas, in the ovx+bg and sham+bg groups, the staining was either negative or mild . In all groups, the osteoblasts had mild to moderate staining, and the osteocytes present in the immature bone trabecular, particularly the larger ones, showed mild staining in the sham+bg and sham+bgm groups, whereas, in the ovx+bg and ovx+bgm groups, the staining was negative . At day 45, the newly formed bone tissue had diffuse and discrete staining at this point, similar to that observed at the initial time point . At day 60, the newly formed bone tissue in the recipient bed - graft interface exhibited mild and diffuse staining, as observed at the initial time point . When present, connective tissues from the bed - graft interface showed intense staining in the ovx+bg and sham+bg groups and moderate staining in the ovx+bgm group . The connective tissue surrounding the graft showed mild or no staining in the g groups, whereas the staining in the bgm groups varied between moderate and intense . The osteoblasts exhibited intense staining in all regions where they were presented, like large osteocytes of the newly formed matrix (figure 8a and figure 8b). Figure 8aa) osteocalcin (occ) day 0 group ovx - bg: bone matrix with lightweight markup; b) day 0 group ovx - bg: moderately marked gaps of osteocytes () are seen in the center of graft; c) day 7 group ovx - bgm: connective tissue exhibited varied staining that was sometimes intense (); d) day 7 group ovz - bgm: osteoblasts in newly bone () stained less intense than connective tissue (); e) day 21 ovx - bg: connective tissue in the bed - graft interface showed intense staining; f) detail of connective tissue in day 21 ovx - bg specimen figure 8ba) osteocalcin (occ) day 45 group ovx - bgm: newly formed bone tissue showed diffuse and discrete staining; b) day 45 group ovx - bgm: in detail, newly formed bone tissue () had diffuse and discrete staining osteocytes (); c) day 60 group sham - bgm: moderate staining in the connective tissues from the bed - graft interface; d) day 60 group sham - bg: detail of bone matrix showing discrete expression of occ within the same period, the 4 groups exhibited similar characteristics . At 0 hours, the bone matrix from the recipient bed and graft showed mild positivity in all groups for all periods . The ovx+bg and ovx+bmg groups showed moderate staining of the reversal lines of the recipient bed . At 7 and 21 days, newly bone tissue formed on the surface of the recipient bed exhibited intense staining . The osteoblasts present at the periphery of the recipient bed and around the immature bone trabecular showed intense staining as well as large osteocytes, which were interspersed in the immature bone trabecular . At day 45, the newly formed bone tissue, both at the bed - graft interface and at the periphery of the graft, presented mild to moderate staining in all groups . Large osteoblasts and osteocytes in this area were moderately stained . At day 60, the newly formed bone tissue in the bed - graft interface and around the graft showed diffuse staining that ranged from mild to moderate with a predominance of mildly positive areas . Osteocytes in this area sometimes accompanied the staining, and sometimes these samples showed mild to moderate staining only in the lacuna . Figure 6aa) bone sialoprotein (bsp) -day 0 group ovx - bgm: slight expression in recipient bed (rb) matrix and graft (g); b) day 0 group ovx - bgm: moderate marking in reverse lines (); c) day 7 group sham - bg: intense staining of newly formed bone (); d) day 7 group sham - bg: intense staining of newly formed bone (), osteoblasts () and osteocytes; group sham - bgm: intense staining of newly formed bone (); f) day 21 group sham - bgm: osteocytes () and osteoblasts () showing intense staining figure 6ba) bone sialoprotein (bsp) day 45 group sham - bgm: mild to moderate marking in newly formed bone () and the middle part of the graft; b) day 45 group ovx - bgm: osteoblasts () and osteocytes () showing moderate staining; c) day 60 group sham bgm: osteocytes () moderately marked on the middle portion of the graft; d) group ovx - bgm: osteoblasts () showing mild to moderate staining . Only osteocytes located in the median region of the graft were stained, which varied from mild to moderate intensity . At day 7, the ovx+bg and sham+bg groups presented a large amount of intensely stained granulation tissue interposed between the graft and the recipient bed . The ovx+bgm and sham+bgm groups also showed this same staining pattern, though the amount of granulation tissue in this area was smaller . The granulation tissue surrounding the graft was mildly stained in the ovx+bg and sham+bg groups, while this tissue presented moderate to intense staining in the ovx+bgm and sham+bgm groups . Osteoblasts showed intense staining, and osteocytes in the immature bone trabecular exhibited varied staining that was sometimes mild and sometimes intense . The most intense staining was observed in larger osteocytes . At day 21, the connective tissue present at the bed - graft interface presented intense staining in all four groups . The ovx+bg and sham+bg groups showed mild to moderate staining in the connective tissue surrounding the graft, while the ovx+bgm and sham+bgm groups showed intense staining . The osteoblasts showed moderate to intense staining, whereas the largest osteocytes present in the immature bone trabeculae showed more intense staining than those found in other areas . At day 45, the connective tissue interposed between the recipient bed and the graft showed intense staining in all four groups . The ovx+bg and sham+bg groups showed mild to moderate staining of the connective tissue surrounding the graft, whereas in the ovx+bgm and sham+bgm groups, the staining was intense . The majority of the osteocytes had no staining, even the largest ones . At day 60, when present, the connective tissue at the bed - graft interface showed mild to moderate staining in all four groups . The connective tissue surrounding the graft exhibited mild staining in the bg groups and moderate staining in the bg+m groups . The osteoblasts showed mild to moderate staining, while the osteocytes were not stained (figure 7a and figure 7b). Figure 7aa) osteonectin (onc) day 0 group ovx - bgm: no labeling of both the receptor bone matrix bed (l) and graft (e). At the bottom, intense staining was observed in skeletal striated muscle (); b) day 0 group ovx - bgm: osteocytes () present in the mid portion of the graft exhibit mild to moderate markup; c) day 7 group ovx - bg: connective tissue () exhibited varied staining that was sometimes intense and sometimes mild; d) day 7 group sham - bgm: osteoblasts () and osteocytes () showing intense staining; e) day 21 group sham - bg: connective tissue present at the surround bed - graft presented intense staining; f) day 21 group ovx - bg: osteoblasts () on the bone surface are moderately marked figure 7ba) osteonectin (onc) day 45 group ovx - bgm: in connective tissue surrounding the graft, the staining was intense; b) day 45 group sham - bgm: connective tissue surrounding newly bone () tissue showed intense staining; c) day 60 group sham - bg: granulation tissue in graft - bed () interface lightweight markup; d) day 60 group ovx - bgm group: osteoblasts () with mild cytoplasmic, osteocytes () the bone matrix of the recipient bed and the graft showed discrete and diffuse staining in all evaluated groups for all periods . At 0 hours, the osteocyte lacunae present at the midline region of the graft were moderately stained, while the osteocytes of the receptor showed more discreet marking . At day 7, the newly formed bone tissue showed no staining . The ovx+bg and sham+bg groups exhibited large amounts of intensely stained granulation tissue interposed between the recipient bed and the graft . The ovx+bgm and sham+bgm groups also showed this staining pattern, though the amount of granulation tissue in this area was smaller . The granulation tissue surrounding the graft exhibited mild to intense staining in the ovx+bg and sham+bg groups and intense staining in the ovx+bgm and sham+bgm groups . The osteoblasts showed mild to moderate staining, and the large osteocytes exhibited mild staining in all four groups . At day 21, the connective tissue present at the bed - graft interface showed intense staining . In the ovx+bgm and sham+bgm groups, the connective tissue surrounding the graft showed intense staining, whereas, in the ovx+bg and sham+bg groups, the staining was either negative or mild . In all groups, the osteoblasts had mild to moderate staining, and the osteocytes present in the immature bone trabecular, particularly the larger ones, showed mild staining in the sham+bg and sham+bgm groups, whereas, in the ovx+bg and ovx+bgm groups, the staining was negative . At day 45, the newly formed bone tissue had diffuse and discrete staining at this point, similar to that observed at the initial time point . At day 60, the newly formed bone tissue in the recipient bed - graft interface exhibited mild and diffuse staining, as observed at the initial time point . When present, connective tissues from the bed - graft interface showed intense staining in the ovx+bg and sham+bg groups and moderate staining in the ovx+bgm group . The connective tissue surrounding the graft showed mild or no staining in the g groups, whereas the staining in the bgm groups varied between moderate and intense . The osteoblasts exhibited intense staining in all regions where they were presented, like large osteocytes of the newly formed matrix (figure 8a and figure 8b). Figure 8aa) osteocalcin (occ) day 0 group ovx - bg: bone matrix with lightweight markup; b) day 0 group ovx - bg: moderately marked gaps of osteocytes () are seen in the center of graft; c) day 7 group ovx - bgm: connective tissue exhibited varied staining that was sometimes intense (); d) day 7 group ovz - bgm: osteoblasts in newly bone () stained less intense than connective tissue (); e) day 21 ovx - bg: connective tissue in the bed - graft interface showed intense staining; f) detail of connective tissue in day 21 ovx - bg specimen figure 8ba) osteocalcin (occ) day 45 group ovx - bgm: newly formed bone tissue showed diffuse and discrete staining; b) day 45 group ovx - bgm: in detail, newly formed bone tissue () had diffuse and discrete staining osteocytes (); c) day 60 group sham - bgm: moderate staining in the connective tissues from the bed - graft interface; d) day 60 group sham - bg: detail of bone matrix showing discrete expression of occ the aim of the present study was to evaluate the expression of immunohistochemical markers of bone formation during the repair process of autogenous bone grafts that were both covered and not covered by an e - ptfe membrane in estrogen - deficient female rats . The results of the present study demonstrate that estrogen deficiency may not alter the expression of bone markers during the repair of onlay blocks placed on rat mandibles . However, the use of the e - ptfe may enhance the expression of the bone markers regardless of the presence of the systemic condition . The most intense staining of bsp could be observed at 7 and 21 days after surgery . The results obtained in the present study show a more intense staining of bsp in newly formed bone, whereas mature bone was weakly stained at all time periods . Based on analyses of alveolar bone formation in rats, (1993) analyzed the mandibular alveolar bone of swine fetuses and observed intense bsp staining in newly formed bone . Ivanovski et al . (2000) observed intense bsp staining of the newly formed bone in experiments on guided bone regeneration in dogs . In the present study, osteoblasts and osteocytes exhibited greater positivity for bsp on days 7 and 21, which gradually decreased until the final time point, when staining was mild and restricted to the lacunae . (1995) observed intense bsp staining of osteoblasts in the mandibles of newborn rats, whereas the osteocytes were not stained . (2000) observed intense staining of osteoblasts and osteocytes associated with newly formed bone during periodontal regeneration in dogs . (2002) observed weak bsp staining in osteoblasts present in the mandibles of fetal rats . Bsp was the only marker that revealed reversal lines, and staining was slightly more pronounced in the ovx groups on days 7 and 21 . Osteocalcin (occ) showed staining of the newly formed bone matrix on day 45 and at 60 days, revealing characteristics of the mature bone . (2000), who observed little or no staining by occ staining of the newly formed bone in experiments on guided bone regeneration in dogs; furthermore, ishigaki, et al . (2002) observed weak positivity for occ in the newly formed bone in the mandibles of rat fetuses . (2010) after analyzing alveolar bone repair in ovariectomized rats . In that study, rats that were not subjected to ovariectomy showed intense staining of the newly formed bone matrix by occ on days 14 and 21, while the ovariectomized group showed a more discrete expression for this marker . In this study, osteoblasts showed mild to moderate staining between 7 and 45 days, with intense staining observed at the last two time points . Osteocytes from newly formed bone presented mild positivity of their lacunae . At 21 days, staining was intense in the sham groups, while, in the ovx groups, it was moderate . (2000) observed intense occ staining of osteoblasts and osteocytes associated with newly formed bone during periodontal regeneration in dogs after 30 days, while ishigaki, et al . (2002) observed weak occ staining in osteoblasts in the mandibles of fetal rats . Osteoblasts showed intense positivity for onc between 7 and 45 days . On days 7 and 21, the onc positivity in osteocytes from immature bone trabeculae varied from mild to intense . The most intense staining was observed in larger osteocytes . On day 45, most osteocytes were no longer stained . (2002), who observed moderate to intense onc staining in osteoblasts present in the mandibles of fetal rats . Ovariectomy is widely recognized for its ability to induce osteopenia, as demonstrated in several classic studies . Although several clinical studies conducted on women with osteoporosis and in animal models have demonstrated a delay in bone repair, the results of the present study show that ovariectomy - induced osteoporosis did not influence autogenous bone graft repair . (2007) observed that estrogen deficiency was not a crucial factor in delaying bone repair . In the present study, (2002) found greater bone formation, maturation, and smaller amounts of bone loss when onlay grafts were covered by e - ptfe membranes compared to grafts without the membrane . (2002) demonstrated that the use of a membrane coating autogenous grafts accelerated the migration of osteogenic cells, the formation of new bone, and the mineralization process . (2005) evaluated the use of a e - ptfe membrane in specimens of autogenous grafts in rats and observed greater bone loss during the healing period in the group that did not receive the membrane . A similar experimental model developed by nascimento, et al . (2009) observed that grafts not covered by the e - ptfe membrane suffered significant reabsorption . One possible explanation for the present results is that estrogen deficiency may not negatively influence the expression of markers for bone formation since this condition is characterized by the increase in bone resorption and may not have any influence on bone formation . In contrast, the presence of a e - ptfe promoted an increase in intensity staining in the present study . One explanation for this may be the favorable environment for bone formation created by the presence of the barrier . According to the principle of guided bone regeneration there is a correlation between angiogenesis and bone regeneration . However, osteoporotic / osteopenic conditions may provide new challenges since they are a major public health threat for a large number of people around the world . Thus, it becomes necessary to recognize the impact of low bone mineral in the dental set . The results of the present study showed that the estrogen deficiency may not influence the expression of markers of bone formation . In contrast, the presence of an e - ptfe membrane created a favorable environment for bone formation . However, caution must be exercised because these results derive from an animal model, and other studies in humans and future research involving the bone forming markers of bone resorption could be useful for better understanding of the influence of estrogen deficiency on bone healing . Within the limits of the present study, we conclude that bone metabolism during the process of bone repair was more intense between days 7 and 21 . The expression of bone forming markers may not be altered by estrogen deficiency, but the presence of an e - ptfe membrane may have a beneficial effect.
It is effective in the detection and removal of adenomatous polyps,1,2 and is being increasingly and widely used.3 accordingly, biopsy and final confirmative diagnosis of endoscopic procedures, including polypectomy, mucosal resection, and submucosal dissection, are growing practices in the field of surgical pathology.4 histopathologic diagnosis of colorectal lesions plays a crucial role in patient management;5,6 therefore, accurate pathologic examination of colorectal lesions is of paramount importance . The current standard guideline for the colonoscopic management of polyps is to retrieve all resected tissue for pathologic assessment.7 recently, the american society for gastrointestinal endoscopy introduced the " resect - and - discard " strategy for diminutive colorectal polyps.8 the 2014 guideline of the european society of gastrointestinal endoscopy suggests that virtual and conventional chromoendoscopy can be used, under strictly controlled conditions, for the real - time optical diagnosis of diminutive colorectal polyps as a replacement to histopathologic diagnosis.9 these documents were developed from evidence - based methods and are expected to offer substantial cost savings; however, they have a number of limitations.10 these guidelines should incorporate a multisociety - based consensus, most important the pathologist's perspective, concerning diminutive and small colorectal polyps . Recent progress in advanced endoscopic imaging and electronic chromoendoscopy has allowed the real - time endoscopic estimation of the histology of polyps, and its main application is in the differentiation of adenomas from hyperplastic polyps (hps).10 pathologically, colorectal polyps may arise from mucosal glands, the lamina propria, or connective tissue, and can encompass a wide range of histogenetic origins . They can be neoplastic, hamartomatous, inflammatory, or of various reactive conditions (table 1).11,12 most of these are adenomas and hps; however, other polypoid lesions of the mucosa and submucosa are readily detected at the time of colonoscopy . From the practical point of view, the endoscopic diagnosis of all of these polyps can be either adenoma or nonadenoma, and this can be enough to make a diagnostic decision; however, with this policy, we cannot reliably assess the infrequent but sometimes significant polyps, and therefore this may distort the occurrence rate of a variety of pathologic lesions described in the literature . The classification and diagnosis of colorectal epithelial polyps became more challenging with the introduction of a third category, serrated polyps (sps). Sps have emerged as precursor lesions in cpg island methylation phenotype (cimp) colorectal carcinogenesis, known as a serrated neoplasia pathway, and account for 20% of all colorectal carcinomas.13,14 sps include lesions with heterogeneous morphological and molecular features . This heterogeneous group comprises hps, sessile serrated adenomas (ssas)/polyps, mixed polyps, and traditional serrated adenomas . Hps were initially considered nonneoplastic lesions; however, subsequent identification of clonal genetic aberrations including braf mutation, kras mutation, and cimp indicated that they are, in fact, neoplastic lesions (table 2).11 sps have a " saw - tooth " appearance on histology, as a result of crypt epithelial cell accumulation and luminal budding, secondary to inhibition of apoptosis . The classification of sps is complicated by their morphologic subtypes and overlapping features, such as the absence or presence of varying degrees of dysplasia.15 the histologic differentiation of ssas from hps is predominantly architecture dependent, and a morphologic continuum exists between the two categories . The clinical significance and histologic criteria of these polyps, as with the search for reliable diagnostic biomarkers, are currently under investigation.16,17,18 therefore, it is more important to create standardized diagnostic criteria and to understand the behavior of sps than discard most of the small - sized sps without verification . Sps are one example highlighting the importance of clinicopathologic correlation and communication between clinicians and pathologists . The size of the colorectal polyp (especially the adenoma) is important in terms of its relation to the likelihood of malignant transformation, and to the risk of synchronous and metachronous adenomas and carcinomas.11 it is one of the major factors determining the risk groups of adenomas according to the 10-mm criteria.5,6 small (<10 mm) polyps were further divided into diminutive and small polyps according to 5-mm dimensions.11 the resect - and - discard strategy is based on data showing a very low prevalence (<2%) of advanced histology in diminutive polyps.19 to adopt this size - based management strategy, accurate polyp size measurement is of paramount importance . However, endoscopic measurement of polyp size has been found to be inconsistent, and a substantial number of endoscopists overestimate or underestimate polyp size when the estimation is done visually or depending on the modality used for the measurement.20 use of the postfixation measurement provided by pathologists would be a preferable alternative; however, tissue fixation may cause shrinkage or enlargement of the polyps.21 another problem is that the malignant risk of a lesion in relation to its size is part of a continuum.21 does something specifically occur at the 5-mm threshold? Is it reasonable to manage a 5-mm polyp and a 5.1-mm polyp differently without considering the measurement error? There has been, and will continue to be, a great deal of controversy in determining the standardized method of polyp size measurement, and this could be one of the problems that complicate the development of an official and legal standard . Surgical pathology is used in many branches of medicine because it provides a critical and definitive diagnosis . In the past, the pathologic diagnosis of cancer is primarily done on the basis of surgical resection in the case of advanced cancers . Recent medical advances have broadened the diagnostic technologies and management options for cancer, the most effective of which are prevention, early detection, and complete cure . Nowadays, endoscopic resection of precancerous lesions and early cancer has increased explosively; the same is true for the increase in the pathologic diagnosis for these lesions.4 this brought about the need for a more elaborate tissue diagnosis and an additional consensus for the diagnostic terminology and histopathologic grading of precancerous lesions in almost every human organs, including colorectal adenoma.22 pathologists should be clearly aware of these facts and constantly strive to develop more applicable consensus criteria, as well as increase the interobserver agreement through consensus meetings, multicenter studies, and communicating with clinicians constantly, both informally and through interdepartmental conferences.4,23 in terms of the public health system, pathology reports are essential as they provide supporting confirmatory information about the performance of the medical procedure done on the patient, assign disease codes for national and private health insurance registration, and provide data for disease statistics, especially cancer.24,25 pathologic examination, diagnosis, and storage of all resected human tissue can provide legal authentication of patient management . Even a small human tissue such as a diminutive colorectal polyp harbors a vast amount of molecular information that may be crucial to medicine in the future although it seems trivial at the present time . Another important aspect of pathologic examination is the storage of human tissue for ongoing translational research . Pathologists can integrate the information from both the traditional morphologic examination and the newer techniques that are increasingly applicable to routinely processed tissue specimens.26 the discard policy of diminutive colorectal polyps may interfere with next - generation research that might be more beneficial in the field of medicine . The major potential advantage of the resect - and - discard strategy for diminutive polyps is the reduction in the costs of histopathologic examination.8,27 this may be the reality in the united states; however, such benefit cannot be estimated in other countries with a different medical reimbursement system . The classification of pathology services by the national health insurance system in korea consists of 13 categories, and polypectomy specimens are coded as c5916 or c5917 on the basis of the number of resection or paraffin blocks (table 3).28 following the definition, resected polyps (from a single organ) that are six or fewer in number are considered a single unit of code c5916 and seven or more polyps (unlimitedly) are considered a single unit of code c5917, without any overlap . It is considerably different from the anatomic pathology coding by the american medical association, which defines individual specimen as a unit of code and then the actual fee is determined by multiplying the number of polyps and the corresponding price of the code.29 in a simulation model conducted in the united states, the resect - and - discard policy resulted in a substantial economic benefit according to the feasibility rate of in vivo differentiation of diminutive polyps and the cost of the pathologic examination.27 in another model, the overall net cost saving per patient was estimated to be us $174.30 although there has been no cost - effectiveness analysis of the resect - and - discard strategy in korea, the net cost saving per patient can be roughly estimated to be us $30 to 35 . It is less likely that forgoing the pathologic examination of diminutive polyps has much economic benefit in korea . Instead, the resect - and - discard strategy may bring about increasing medical cost due to the incorrect determination of the surveillance intervals . Like many other paradigms of human activity, medical technologies and strategies are continuously and rapidly evolving . Recently, advanced colonoscopic imaging has been used to assess colorectal polyp histology, with a high prediction rate, particularly when done by an expert endoscopist . However in fact, the highly confident endoscopic estimation of polyp type is a novel valuable tool that can provide a symbiosis between gastroenterologists and pathologists to allow them to make a more evident diagnosis and management of patients with colorectal polyp . In the pathologist's point of view, the power of microscopic analysis and the amount of information that can be obtained even from a diminutive colorectal polyp represent a real acquisition, and there is no available technique that provides so much information in terms of data quality, quantity, and cost.
We present a case of an 82-year - old female, an immigrant from brazil, with past medical history of atrial fibrillation, colon cancer, and uterine cancer status post abdominal surgeries on remission who presented to our emergency room with severe abdominal discomfort, nausea, and 78 episodes of bilious vomiting without hematemesis . She did not have any constipation or diarrhea, and her last bowel movement was a day prior to presentation . She was saturating 93% in room air and her temperature was 97.8f . Physical examination revealed a diffusely tender abdomen with rebound but no guarding or rigidity was present . She had a hemoglobin level of 13 g / dl, a white blood cell count of 10,000 per microliter, and a platelet count of 80,000 per microliter . She had a lactate level of 1.8 mmol / l, and her lipase level was 29 abdominal x - ray showed centrally dilated small bowel with large amount of stool within the rectum . Computed tomography (ct) scan of the abdomen showed circumferential intramural air within the proximal mid stomach (figs . 1 and 2) and intrahepatic portal venous air within the left lobe of the liver (figs . 3 and 4) consistent with emphysematous gastritis with no evidence of small bowel obstruction . Ct abdomen showing intramural air (yellow arrow) within the proximal mid stomach consistent with emphysematous gastritis . Ct abdomen / coronal view showing intramural air (yellow arrow) within the proximal mid stomach consistent with emphysematous gastritis . Ct abdomen / coronal view showing portal venous air (yellow arrow) within the left lobe of the liver . Ct abdomen showing portal venous air (yellow arrow) within the left lobe of the liver . She was treated with intravenous hydration, intravenous vancomycin, cefepime, and metronidazole and kept npo (nil per os). Two conditions are included in the differential diagnosis of intramural gas in stomach: emphysematous gastritis and gastric emphysema (1). Emphysematous gastritis, first described in 1889, is a rare form of phlegmonous gastritis caused by gas - forming organisms invading the stomach wall through a mucosal defect . Recent systematic review suggested a mortality rate of 55% for emphysematous gastritis and 29% for gastric emphysema (4). The most common predisposing factors for emphysematous gastritis include ingestion of caustic substances, alcohol abuse, abdominal surgery, diabetes, immunosuppression, and phytobezoars (2, 3). Abundant blood supply, effective mucosal barrier, and an acidic ph make the stomach a very uncommon site for this finding (5)., clostridium spp ., pseudomonas aeruginosa, staphylococcus aureus (2, 3), sarcina spp . Previous reports have suggested a high mortality rate in cases with the finding of intramural stomach gas in association with hepatic portal venous gas (8, 9). Gastric emphysema is a more benign condition in which air usually reaches within the wall from outside (1). The predisposing factors for gastric emphysema are mucosal disruption due to procedures like endoscopy (10) or nasogastric tube placement (11), increased intraluminal pressure secondary to pyloric or enteral stenosis (12, 13), gastric paresis and forceful vomiting, and chest wall injuries . It is important to differentiate emphysematous gastritis from gastric emphysema mostly because of the difference in management and prognosis . Emphysematous gastritis usually presents in a sick, hemodynamically unstable patient and has increased mortality (15). It might also have an increased association of radiological finding of gas in the portal venous system in addition to the intramural stomach gas (5). In contrast, patients with gastric emphysema generally present with milder symptoms of dyspepsia and epigastric discomfort or may be asymptomatic . They are usually managed with conservative management and observation (15). A cystic mottled pattern is usually associated with emphysematous gastritis, while linear lucency along the greater curvature of the stomach is present in gastric emphysema . Radiologists use terminologies like linear, curvilinear, or cystic to describe non - gangrenous changes, whereas bubbly signifies gangrenous gastritis (14, 15). Our patient had prior history of multiple cancers and advanced alzheimer's disease and was bed bound . We concluded that she developed emphysematous gastritis secondary to her immunosuppressed status and possible mucosal tears in the stomach from multiple bouts of vomiting . Emphysematous gastritis was diagnosed based on the characteristic radiographic signs and a good response to antibiotics . The radiological finding of intramural stomach gas and portal venous air predicted a poor prognosis . However, she had a stable hospital course and resolution with medical management, likely due to early diagnosis and initiation of appropriate treatment . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
Four hc and three oligoarticular jia patients were included who all had active disease at the time of sampling . Sf was collected upon therapeutic joint aspiration and pb was drawn at the same moment . Sfmcs and pbmcs were isolated using ficoll isopaque density gradient centrifugation and were frozen down in fcs containing 10% dmso . Cd4cd45ro cells (ranging between 600.0001.200.000 cells per sample) were isolated from frozen hc pbmcs and jia sfmcs using flow cytometry, activated with human t - activator cd3/cd28 dynabeads (1 cell: 3 beads) and cultured for 16 h in the presence of 300 nm jq1(+) or jq1(). Subsequently, cells were treated for 4 h with phorbol 12-myristate 13-acetate (100 ng / ml) and ionomycin (1 g / ml). Total rna was extracted using the rnaeasy kit (qiagen), according to the manufacturer's instructions . The quality of the rna was assessed based on the rna profile generated by the bioanalyzer using the picochip . Removal of rrna and globin - encoding mrna, rna fragmentation, cdna generation, adapter ligation and pcr amplification was done using the truseq stranded total rna with ribo - zero globin sample preparation kit (illumina) and 75 bp single - end sequencing was performed using an illumina nextseq 500 platform through the utrecht dna sequencing facility . Read quality assessment, sample demultiplexing, and adapter trimming was performed by utrecht dna sequencing facility using basespace (illumina) software . Reads with quality score of q> 30 were selected for downstream analysis . Before starting downstream analysis, fastqc (babraham bioinformatics) 1a and b). As a human reference genome, hg19 was used, and reads were mapped to this reference sequence using tophat v.2.0.9 with bowtie 2.1.0 as index (tophat2 -p 8 library - type fr - firststrand g hg19.gtf). For each sample 91% of the reads were successfully mapped to the genome, indicating sufficient coverage . Rseqc was used to calculate how mapped reads were distributed over dna regions, such as coding sequences and utrs, and to assess uniform read coverage of genes (fig . Transcriptomes of each individual sample were generated using cufflinks v2.2.1 with hg19 gene annotation as a guide, . Quartile normalization was performed (library - norm quartile) to improve the detection of differential expression of less abundant transcripts . Reads mapping to rrna and trna were masked (-m option) from the quantification . Cuffnorm was used to generate quartile normalized count and fpkm tables . Using an fdr 0.05, 914 transcripts were identified that are significantly differentially expressed between hc and jia patients . Among these transcripts, 637 were upregulated in jia patients compared to hc, while 237 were downregulated in jia patients . Jq1(+) and jq1() treatments of jia patient - derived memory / effector t (tmem / eff) cells resulted in 633 differential transcripts, based on fdr 0.05, of which 338 were downregulated upon jq1(+) treatment and 295 upregulated . Principle component analysis (pca) on the complete transcriptome of hc and jia patient - derived t cells, either with and without jq1(+) treatment, demonstrated that four different groups can be distinguished, illustrating that hc and jia cluster separately and that jq1 affects gene expression in such way that treated samples cluster separately from untreated samples (fig . In addition, we performed pca on the genes significantly expressed between all different groups, which demonstrated that all replicates cluster within the same group (fig . Initial gene ontology term analysis revealed that the group of significantly genes upregulated in jia compared to hc as well as the genes downregulated upon jq1(+) treatment in jia, correlated with a defense response . Additional gene set enrichment analysis (gsea, broad institute) for genes differentially expressed between hc and jia showed that the genes positively correlated with this comparison, i.e. Genes that are upregulated in jia, are enriched for the same gene subsets as the genes that are negatively correlated with the comparison jq1() versus jq1(+) treatment of jia patient - derived t cells, i.e. Genes that are downregulated by jq1 (fig . This again illustrates that genes affected by jq1(+) strongly overlap with the genes that are upregulated in jia . In addition to functionally grouped network analysis, gsea also shows that genes associated with cytokines / cytokine receptor interactions are enriched within this subset of genes . To further validate our rna - sequencing results, we selected some inflammation - associated (activation) markers that were downregulated upon jq1(+) treatment and measured gene expression by qpcr . This resulted in the confirmation of downregulation of several cd markers on the rna level, of which cd38, cd152, and cd195 also showed downregulation on the protein level, measured by flow cytometry (fig . Four hc and three oligoarticular jia patients were included who all had active disease at the time of sampling . Sf was collected upon therapeutic joint aspiration and pb was drawn at the same moment . Sfmcs and pbmcs were isolated using ficoll isopaque density gradient centrifugation and were frozen down in fcs containing 10% dmso . Cd4cd45ro cells (ranging between 600.0001.200.000 cells per sample) were isolated from frozen hc pbmcs and jia sfmcs using flow cytometry, activated with human t - activator cd3/cd28 dynabeads (1 cell: 3 beads) and cultured for 16 h in the presence of 300 nm jq1(+) or jq1(). Subsequently, cells were treated for 4 h with phorbol 12-myristate 13-acetate (100 ng / ml) and ionomycin (1 g / ml). Total rna was extracted using the rnaeasy kit (qiagen), according to the manufacturer's instructions . The quality of the rna was assessed based on the rna profile generated by the bioanalyzer using the picochip . Removal of rrna and globin - encoding mrna, rna fragmentation, cdna generation, adapter ligation and pcr amplification was done using the truseq stranded total rna with ribo - zero globin sample preparation kit (illumina) and 75 bp single - end sequencing was performed using an illumina nextseq 500 platform through the utrecht dna sequencing facility . Read quality assessment, sample demultiplexing, and adapter trimming was performed by utrecht dna sequencing facility using basespace (illumina) software . Reads with quality score of q> 30 were selected for downstream analysis . Before starting downstream analysis, fastqc (babraham bioinformatics) 1a and b). As a human reference genome, hg19 was used, and reads were mapped to this reference sequence using tophat v.2.0.9 with bowtie 2.1.0 as index (tophat2 -p 8 library - type fr - firststrand g hg19.gtf). For each sample 91% of the reads were successfully mapped to the genome, indicating sufficient coverage . Rseqc was used to calculate how mapped reads were distributed over dna regions, such as coding sequences and utrs, and to assess uniform read coverage of genes (fig . Transcriptomes of each individual sample were generated using cufflinks v2.2.1 with hg19 gene annotation as a guide, . Quartile normalization was performed (library - norm quartile) to improve the detection of differential expression of less abundant transcripts . Reads mapping to rrna and trna were masked (-m option) from the quantification . Cuffnorm was used to generate quartile normalized count and fpkm tables . Using an fdr 0.05, 914 transcripts were identified that are significantly differentially expressed between hc and jia patients . Among these transcripts, 637 were upregulated in jia patients compared to hc, while 237 were downregulated in jia patients . Jq1(+) and jq1() treatments of jia patient - derived memory / effector t (tmem / eff) cells resulted in 633 differential transcripts, based on fdr 0.05, of which 338 were downregulated upon jq1(+) treatment and 295 upregulated . Principle component analysis (pca) on the complete transcriptome of hc and jia patient - derived t cells, either with and without jq1(+) treatment, demonstrated that four different groups can be distinguished, illustrating that hc and jia cluster separately and that jq1 affects gene expression in such way that treated samples cluster separately from untreated samples (fig . In addition, we performed pca on the genes significantly expressed between all different groups, which demonstrated that all replicates cluster within the same group (fig . Initial gene ontology term analysis revealed that the group of significantly genes upregulated in jia compared to hc as well as the genes downregulated upon jq1(+) treatment in jia, correlated with a defense response . Additional gene set enrichment analysis (gsea, broad institute) for genes differentially expressed between hc and jia showed that the genes positively correlated with this comparison, i.e. Genes that are upregulated in jia, are enriched for the same gene subsets as the genes that are negatively correlated with the comparison jq1() versus jq1(+) treatment of jia patient - derived t cells, i.e. Genes that are downregulated by jq1 (fig . 2c). This again illustrates that genes affected by jq1(+) strongly overlap with the genes that are upregulated in jia . In addition to functionally grouped network analysis, gsea also shows that genes associated with cytokines / cytokine receptor interactions are enriched within this subset of genes . To further validate our rna - sequencing results, we selected some inflammation - associated (activation) markers that were downregulated upon jq1(+) treatment and measured gene expression by qpcr . This resulted in the confirmation of downregulation of several cd markers on the rna level, of which cd38, cd152, and cd195 also showed downregulation on the protein level, measured by flow cytometry (fig . In order to increase insight into molecular mechanisms playing a role in autoimmune diseases, we performed genome - wide transcriptome profiling of autoinflammatory - site derived tmem / eff cells obtained from jia patients . We observed that the majority of genes differentially expressed between jia patients and hc were upregulated in jia patient - derived t cells, strongly suggesting that these genes might contribute to the disease . Furthermore, we treated patient - derived t cells with the bet - inhibitor jq1 and observed a preferential inhibition of the genes that were upregulated in jia patients, i.e. The disease - associated genes . Rna - sequencing results were confirmed by qpcr and both pca and gsea showed that jq1 can dramatically alter the cellular gene expression profile . In conclusion, our data suggests that bet - inhibition might be a novel, powerful therapeutic strategy in the treatment of autoimmune diseases.
Retroperitoneal sarcomas are rare tumors accounting for only 1%2% of all solid malignancies . Of all sarcomas, only 10%20% of sarcomas are retroperitoneal sarcomas, and the overall incidence is 0.3%0.4% per 100000 of the population . The peak incidence is in the 5th decade of life, although they can occur in any age group . The most common types of retroperitoneal soft tissue sarcomas in adults vary from study to study . However, in most studies, the most frequently encountered cell types are liposarcomas, leiomyosarcomas and malignant fibrous histiocytomas (mfh). Recently, the frequent diagnosis of mfh in the retroperitoneum has been - disputed . With the use of immunohistochemistry, many of these fibrous tumors have now been shown to represent other sarcoma types such as leiomyosarcomas or dedifferentiated liposarcomas [3, 4]. For this reason, it is anticipated that the number of these neoplasms that will be considered as mfh will be dramatically reduced in the future . Patients with sarcomas present late, because these tumors arise in the large potential spaces of the retroperitoneum and can grow very large without producing symptoms [5, 6]. Moreover, when symptoms do occur, they are nonspecific, such as abdominal pain and fullness, and are easily dismissed as being caused by other less serious processes . Imaging is important in the diagnostic workup of these patients, being required not only for tumor detection, staging, and operative planning, but also for guiding percutaneous or surgical biopsy of these tumors . As other neoplastic processes, such as lymphoma and metastatic disease, which are treated differently, may mimic retroperitoneal sarcomas, tissue diagnosis is of paramount importance . Therefore, image - guided and surgical biopsies have a relatively greater role to play in the diagnosis of retroperitoneal sarcomas than is the case for sarcomas elsewhere in the body [811]. Once the diagnosis is made, the surgical team needs to determine if the retroperitoneal sarcoma can be resected . Therefore one of the first determinations to be made is whether the tumor is localized, its local extent, and also if there is evidence of intra- or extra - abdominal metastatic spread of tumor . The location and size of the tumor, its relationship to adjacent organs, presence or absence of local extension, relationship to and/or involvement of major vascular structures, as well as the presence of normal anatomic variants and anomalies of major abdominal arteries and veins, are all crucial pieces of information that need to be provided . Since resection of one kidney is not uncommon, any radiographic evidence of unilateral renal dysfunction involving the kidney that is not adjacent to the tumor should be relayed to the surgical team . While it may be unavoidable that the patient will be left with a single poorly functioning kidney, the surgeon must be provided with all relevant information prior to attempted tumor resection . In evaluating preoperative imaging studies, the radiologist should be cognizant of two facts: (a) up to 75% of retroperitoneal sarcoma resections involve concomitant resection of at least one adjoining intra - abdominal visceral organ (commonly large or small bowel or kidney); (b) the most common types of vascular involvement precluding resection are involvement of the proximal superior mesenteric vessels or involvement of bilateral renal vessels . Accordingly, since these tumors tend to invade organs with which they are contiguous, such contiguity must be mentioned even in the absence of imaging evidence of gross tumor invasion of these organs . Also, the mesenteric and renal vessels must be carefully examined and their relationship to a mid or upper retroperitoneal tumor described . Accurate staging is important as it facilitates determination of appropriate surgery, establishes prognosis, and provides a guide for adjunctive therapy . The american joint committee staging system (tables 1 and 2) of extremity soft tissue sarcomas, which is based on the tnm classification, is most commonly used for most retroperitoneal soft tissue sarcomas, although it is better suited for extremity sarcomas . This staging system takes into consideration histological grade, tumor size and depth relative to the superficial muscular fascia, presence or absence of lymph node involvement, and the presence or absence of distant metastases (table 2). Nearly all retroperitoneal sarcomas are large and> 5 cm and are deep to the superficial fascia . Therefore localized retroperitoneal sarcomas are nearly always classified as stage iib (large, low - grade, and deep) or stage iii (large, high - grade and deep) neoplasms, with the distinction between these two stages being made only on the basis of histologic grade . Imaging cannot be reliably used to predict the cell types of most sarcomas (fig . 1(a), (b)), with rare exceptions being liposarcoma and intracaval leiomyosarcomas . The presence of macroscopic fat enables one to make the diagnosis of a liposarcoma (fig . 3). Some of these tumors may be composed almost entirely of soft tissue and fluid components . In these instances, a tumor within the lumen of the inferior vena cava with expansion of its lumen and enhancing tumor thrombus is pathognomonic of an intracaval leiomyosarcoma (fig . Fortunately, it has been shown that in the vast majority of sarcomas, cell type has no impact on treatment and long - term survival . The major factors that affect survival are the tumor grade and resectability [10, 11]. Patients who have had a successful complete resection and also have low - grade tumors have the best survival rates . The exception is a liposarcoma, and, in general, if a liposarcoma contains mostly fat and very little soft tissue, it is likely to be a low - grade tumor (fig . 5(a), (b)). However, the converse is not true . A liposarcoma containing a large amount of soft tissue and with little or no macroscopic fat, may be a low-, intermediate- or high - grade tumor . Calcification or ossification within a liposarcoma has been shown to be a poor prognostic feature, often indicating dedifferentiation . Ct is the most commonly used modality for identification, localization, and staging of retroperitoneal sarcomas [5, 8, 1820]. The use of magnetic resonance imaging is generally reserved for selected problem solving; such as to address questions regarding vascular invasion, and evaluate problematic indeterminate liver lesions . More recently pet - fdg imaging has been used in an effort to assess the tumor grade as well as to evaluate patients for tumor recurrence (fig . Chemotherapy and radiotherapy without surgical debulking have rarely been beneficial, when used alone or in combination . Pre-, intra- or post - operative radiotherapy has, however, been of benefit in some patients, but, in most instances, does not improve patient prognosis [5, 2427]. As these tumors are locally invasive, extensive and aggressive local resection of the tumor and any adjacent organs should be performed at the time of presentation . Resection of the tumor en - bloc with adjacent adrenals, kidneys, or segments of small bowel, or colon is often required [28, 29]. In a study of 28 patients with liposarcomas, adjacent organ resection was carried out in more than half the cases, with partial or total resection of the kidneys in 60%, colon in 50% and adrenal glands in 35% . Despite complete resections, 5- and 10-year survival rates are poor, being 51% and 36% respectively [30, 31]. Most tumor recurrences occur within 2 years of initial surgical resection [7, 32]. Since subsequent prognosis in these patients is affected by the ability to completely resect the local recurrences, early detection of tumor recurrence is important . When re - resections are performed early, they are successful in up to 90% of the patients [30, 31]. Unfortunately, many recurrences are diagnosed late in the course of the disease, leading to incomplete resection, which then leads to re - recurrence in about 50% of patients . As most recurrences are local, a careful scrutiny of the surgical bed for subtle changes on follow - up imaging should be made [2832]. Clinical follow is usually up not helpful as up to 50% of patients are asymptomatic, and if symptoms are present, they are usually nonspecific . Soft tissue attenuation recurrences may not be easily distinguished from post - operative scarring / fibrosis in the surgical bed . Detection of local recurrence in liposarcomas is especially difficulty as, when small, recurrent liposarcomas can be difficult to distinguish from normal retroperitoneal fat on imaging (fig . Frequently, closer scrutiny may show that the fat in a recurrent liposarcoma is of slightly higher ct attenuation when compared to normal retroperitoneal fat . Also, at times, recurrent liposarcomas can have different imaging characteristics than that of the primary tumor . In one ct study of fat - containing liposarcomas, four of the eight recurrent tumors did not contain any visible fat . As recurrent tumors are best treated with repeat surgical resection, all tumor - bearing sites should be identified to enable optimal and complete re - resection [32, 33]. Regional metastases are also frequent and a thorough search of the draining nodes, peritoneal surfaces, and liver should be made prior to evaluation for distant metastases . Follow - up imaging is usually performed with ct or mri with the frequency of follow - up being often dictated by the completeness of the tumor resection, tumor type and grade . One suggested follow up scheme is to obtain imaging at regular intervals (i.e. Ct or mri every 34 months for 2 years, then every 46 months for 35 years, and every 12 months thereafter . Follow up for greater than 5 years is recommended as although most sarcomas (whether high - grade or low - grade) recur within 2 years, marked delay in appearance of recurrent disease is not unusual . Contrast - enhanced axial ct shows two large retroperitoneal heterogenous neoplasms with areas of low density due to degeneration or necrosis . These proved to be malignant: a peripheral nerve sheath tumor (a) and retroperitoneal leiomyosarcoma (b), respectively . Contrast - enhanced axial ct shows large right retroperitoneal liposarcoma (arrow) composed predominantly of fat but also has areas of soft tissue density and calcific components . Contrast - enhanced axial ct shows left - sided predominantly soft - tissue density abdominal tumor (arrow) which proved to be a high - grade pleomorphic liposarcoma . Large inferior vena caval leiomyosarcoma (arrow) seen on (a) contrast - enhanced ct and (b) coronal contrast - enhanced gradient echo mr image (arrow). Note intraluminal tumor extension on both images . Well - differentiated low - grade and high - grade liposarcomas . (a) contrast - enhanced axial ct shows well encapsulated fatty mass (arrow) with no septations or soft tissue component . (b) contrast - enhanced axial ct shows a predominantly soft tissue pelvic mass (arrow) which proved to be a high - grade liposarcoma . (a) coronal pet - fdg image shows peritoneal metastatic nodule (arrow). (b) axial contrast - enhanced ct shows this metastatic nodule (arrow), which was not prospectively identified as it was thought to be part of the left lobe of the liver (a) contrast - enhanced axial ct shows right retroperitoneal mass (arrow) which as a liposarcoma . (b) on a 6-month post - resection follow - up contrast - enhanced axial ct, there is suggestion of recurrence (arrow). (c) at 12-month follow - up contrast - enhanced axial ct, the mass (arrow) has shown interval growth and is more obvious.
Cardiac calcified amorphous tumors (cats) are extremely rare cardiac masses which can arise in all four cardiac chambers.1)2) while several causes of cardiac cats have been suggested, the true etiology is not still clear . Cardiac cats are usually benign, but sometimes cause diverse symptoms due to obstruction or embolization.1)3) we recently encountered a patient with a cardiac cat causing multiple, calcific, pulmonary emboli and right - sided heart failure . A cardiac cat has not been reported previously in korea . A 33-year - old man sought evaluation in our outpatient clinic for progressive pretibial pitting edema and shortness of breath on exertion . There was no personal or family history of thromboembolic, hematologic, inflammatory, or malignant diseases . The physical examination revealed mild icteric sclera, a palpable liver, a distended abdomen, and jugular vein distention . A chest x - ray showed a dense calcification within the cardiac silhouette from the left lateral view . An electrocardiogram revealed incomplete right bundle branch block, right atrial enlargement, and right ventricular hypertrophy . Echocardiography demonstrated a diffuse calcified mass affecting the tricuspid chordal apparatus and the free wall of the right ventricle (rv), resulting in severe tricuspid regurgitation and markedly increased rv systolic pressure (70 mmhg) (fig . 1). On laboratory analysis, the serum parathyroid hormone of 50.07 pg / ml (normal, 15 - 65 pg / ml), calcium of 8.1 mg / dl (normal, 8.0 - 10.5 mg / dl), creatinine of 1.0 mg / dl (normal, 0.5 - 1.4 mg / dl), and glucose of 100 mg / dl (normal, 70 - 110 mg / dl) levels were within normal limits . A chest computed tomography (ct) showed multiple pulmonary thromboemboli, possibly calcific, which were noted on non - contrast ct imaging (fig . 2). Fluoroscopic imaging showed an irregular - shaped calcified mass in the rv which changed in shape and size during the cardiac cycle (fig . 3). Cardiac magnetic resonance imaging (mri) demonstrated a tubular calcified mass, which was separated from the right ventricular myocardium, extending from just below the tricuspid valve annulus to the right ventricular outflow tract, suggesting a cat or calcific fibroma (fig . An endomyocardial biopsy was not performed due to the risk of right ventricular rupture or prolapse . Heart - lung transplantation was deferred until the pulmonary arterial pressure improved and empirical anticoagulation was administered . A cardiac cat was first reported in 1997.4) cardiac cats can arise in all four chambers of the heart,1)2) although the proportion in each chamber is not known . Although mural thrombi,4)5) chest trauma,1) and increased parathyroid hormone and calcium phosphate product levels in hemodialysis patients6) have been described as the causes for cardiac cats, the pathogenesis of these lesions is still unknown . In the present case histologically, a cardiac cat is characterized by nodular calcium deposits over a matrix of fibrin and/or amorphous fibrin - like material, hyalinization, inflammatory cells, and degenerated hematologic elements.7) clinical tests usually show cardiac cats to be benign, although they may cause obstruction or embolism,4) and cases can evolve fatally.5) an endomyocardial biopsy was not performed because of the risk of right ventricular rupture and further calcific embolization.8) surgical removal of the tumor may be indicated if embolism has occurred or seems likely . Complete surgical resection should be pursued if possible, considering its recurrence.5) heart transplantation may be considered if not possible . We chose heart - lung transplantation in the present patient who had multiple calcified emboli and severe right ventricular dysfunction . During the differential diagnosis, cardiac neoplasias, especially myxomas and fibromas, are considerations, particularly if they are calcified, as are conditions involving infection or thrombosis.9) due to the lack of histology, calcified atrial myxoma, calcified thrombi or other cardiac neoplasms should be also considered as a differential diagnosis of calcific mass of rv . Echocardiography, and ct and mri provide important information on the size and shape, attachment site, and pattern of movement of the calcified tumor . Myxomas usually have a short, broad - based attachment and are pedunculated, although calcification may develop in approximately 10% of myxomas . Ct scans can detect intracardiac masses and define the extracardiac extension.10) ct scans can also detect even minute amounts of calcium, which facilitated detection of the calcific pulmonary embolism in the present case . We recommend utilizing a multimodality imaging approach to accurately characterize intracardiac masses and their complications.
One hundred and fifty million people are infected by hepatitis c virus (hcv) worldwide, and chronic hepatitis c is now the leading indication for liver transplantation in the us . Transmitted principally by blood, hcv is passed by transfusion of inadequately screened blood and blood products, and by intravenous drug use; sexual and vertical transmission also occur, but at substantially lower rates than in other blood - borne viral infections . Unsterile dental equipment, accidental needle punctures in medical facilities, and tattooing are also linked to transmission . Following acquisition of infection, 70 to 85% of patients will develop persistent viremia, usually for the duration of their lives . Not all of these will develop liver failure, however - infection is often indolent for long periods . Nonetheless, two decades after infection about 20% of hcv - infected subjects will have developed end - stage liver disease . Hepatitis c is now a more common cause of cirrhosis than alcoholism . Though chronic hepatitis is the major result of hcv infection, there are other manifestations of the disease that stem from chronic inflammation and associated immune cell stimulation with cytokine release . These include arthritis, antibody - mediated thrombocytopenia, itching, porphyria cutanea tarda, dermatitis, glomerulonephritis and cryoglobulinemia . Hcv is a small (50 nm) single - stranded rna member of the flaviviridae family . The rna has one open reading frame, preceded by a ribosome - binding site within a utr (untranslated region). The open reading frame encodes a 3,011 amino acid protein that is cleaved into several different proteins by proteases of viral and cellular origins . The amino acid sequences include nucleocapsid, envelope, protease, helicase, transmembrane, and rna polymerase proteins . The hcv rna polymerase is highly error prone, and the sequences of hcv genomes display enormous amounts of variation . They differ from one another at nearly one - third of their nucleotide positions, but in practice are defined by mutations in the 5'utr . These six genotypes are stable, heritable variants that differ in their geographic distribution . Superimposed on this already substantial variation is the fact that large numbers of mutations occur in any given individual's hcv isolate over time - that is, as in hiv, each new infection gives rise to a cloud of sequence variants descended from the original infection; this cloud is sometimes referred to as a' quasispecies' . This extraordinary sequence diversity renders the virus very resistant to standard immune responses and inhibits the production of a useful preventive vaccine . The two coat proteins of the virus are heavily glycosylated and recognize receptors on the hepatocyte . Though not entirely proven, these receptors are thought to be cd81, claudin-1 and occludin [3 - 5]. Virions replicate at an enormous rate in hepatocytes, from which they are released into the blood (they may invade monocytes and b cells as well, but this is uncertain). The titer of virus in the blood may therefore provide a fairly accurate index of the hepatic load of virus . Recently a relatively well - conserved sequence of the non - coding region has been identified, permitting the development of an inexpensive and highly accurate diagnostic polymerase chain reaction (pcr)-based blood test for the virus and its titer . This protein activates the janus kinase / signal transducers and activators of transcription (jak / stat) signaling pathway to induce the transcription of interleukins and caspases that kill viral loaded cells, but the toxicity of the thrice - weekly treatment was considerable and the original remission rate was only 10 to 15% . Particularly poor results were seen in those infected with viral genotype 1, which accounts for 65% of us cases of hepatitis c - including the bulk of caucasian and the vast majority of american black patients . The addition of ribavirin brought the remission rate up to 20 to 25%, and a change to weekly pegylated interferon alfa further increased the response rate . But the treatment remains difficult, with side - effects that resemble the cytokine - releasing consequences of cancer chemotherapy, and the hemolytic anemia induced by ribavirin weakens patients still further . American patients with genotype 1 continue to respond less well than those with genotypes 2 and 3, but nonetheless a significant subset of genotype 1 carriers do benefit from treatment . Given the toxicities of interferon / ribavirin, it would be very useful to be able to predict which patients might fall into the responder subclass . Connected to this, a startling advance has recently been made by ge, thomas and their co - workers, who have shown that a polymorphism in close proximity to the il28b gene that encodes interferon lambda 3 predicts both spontaneous clearance of hcv and response to interferon and ribavirin treatment in genotype 1 hepatitis c infection . Clearance and response to therapy are not affected by the polymorphism in those with genotype 2 and 3 infections . The reasons for these disparities are not at all evident, since the genotypes are not associated with any known protein differences . Finally, the treatment of hepatitis c - induced chronic liver failure with liver transplantation illustrates the dilemma posed by the growth of technology in medical care . Indeed, all such liver transplants become re - infected by persistent virus, and in some patients the march to secondary cirrhosis is accelerated, necessitating a second transplant . Ironically, alcoholic cirrhotics are routinely denied transplants in the current donor - liver shortage because it is thought that their bibulous habits cannot be broken . Yet patients with hepatitis c receive transplants routinely, even though their livers are doomed to be re - infected and cirrhosis induced at a far faster rate than that caused by alcohol . Since the natural progression of the disease takes two or more decades, and since most patients come to transplant in their 50 s or 60 s, most do not require a second transplant but the costs of these decisions are massive . Hcv: hepatitis c virus; jak: janus kinase; pcr: polymerase chain reaction; stat: signal transducers and activators of transcription; utr: untranslated region . The authors are grateful to maureen jonas md and chinwe ukomadu md for their valuable help in the preparation of this discussion, and particularly express their appreciation to don ganem md for his careful review of the manuscript and his important suggestions.
Diffuse alveolar haemorrhage (dah) is a rare and life threatening condition characterized by hemoptysis, dyspnea, and alveolar infiltrates on chest radiograph and various degrees of anemia [1 - 3]. It may occur either as a primary disease of the lungs or a secondary condition due to cardiac, systemic vascular, collagen, or renal diseases . Pulmonary hemosiderosis is a very rare entity, possibly of the immunologic mechanism, causing a defect in the basement membrane of the pulmonary capillary, or of toxic origin . Idiopathic pulmonary hemosiderosis (iph) is a diagnosis made by the exclusion of other causes . It should be confirmed by the presence of many hemosiderinladen macrophages in bronchoalveolar lavage fluid obtained by bronchofiberoscopy . Iph is in some cases associated with celiac disease, so patients with idiopathic pulmonary hemosiderosis should routinely be tested for gluten intolerance . The estimated incidence of iph in children is 0.24 - 1.23 cases per million, with a mortality rate as high as 50% . Only about 500 cases of this disease have been described in medical literature, including 16 cases reported in polish medical journals . The rarity of this disease and the variable clinical course results in many diagnostic as well as therapeutic problems and pitfalls . The aim of this paper is to present our observations relating to the diagnosis and treatment of iph in a 9-year - old girl . A 9-year - old, previously healthy girl, at the age of 5 developed weakness, fatigue, a chronic cough, transient dyspnea, recurrent respiratory tract infections and anemia . Physical examination at that time revealed profound skin and mucous membrane pallor and tachycardia with a silent systolic murmur . Laboratory investigations showed anemia with hemoglobin (hb) level of 5.8 g / dl, hematocrit value (hct) - 0.22, red blood cells (rbc) - 3.4 t / l, microcytosis, hypochromia and poikilocytosis with anulocytosis . The values of mean corpuscular haemoglobin (mch) mean corpuscular volume (mcv), mean cell haemoglobin concentration (mchc), levels of serum iron and transferritin were decreased, and the reticulocyte rate was elevated (9%). The patient received a red cell transfusion followed by prolonged oral iron supplementation . Despite of a routine clinical work - up, purpura, laryngeal disorders, esophagitis and bleeding from the gastrointestinal tract, and congenital heart disease, were excluded . Due to hemoptysis, the patient was referred to the institute of tuberculosis and lung diseases in rabka, where bronchofiberoscopy was performed . This showed increased vascularization of the bronchial mucosa, most prominent in the middle lobar bronchus (figure 1). There was no active bleeding; however, the bronchoscopic picture was typical of diffuse alveolar hemorrhage . Microscopic examination of the bronchoalveolar lavage fluid revealed the presence of many hemosiderin - laden macrophages . Other causes of secondary pulmonary hemosiderosis, including glomerular and hemolytic syndromes, hemorrhagic purpura, and cardiac, vascular, rheumatoid, and immunologic disorders were excluded . The diagnosis of iph was made and therapy with glucocorticoids was initiated with partial and transient response . In order to exclude celiac disease serum markers of this condition were also investigated . However, antigliadin iga, igg and antiendomysial antibodies iggema were positive, thus a gluten - free diet was introduced . Glucocorticoids were tapered off and substituted with azathioprine, given at the dose of 5 mg / kg / day . Bronchofiberoscopy showing increased vascularization of bronchial mucosa . At present, the girl remains on a gluten - free diet and oral azathioprine . Episodes of alveolar hemorrhage occur at least every 2 - 3 months, most of them being triggered by viral infections of the upper respiratory tract . They are manifested by a cough, hemoptysis and dyspnea associated with rapid falls in the hemoglobin level and erythrocyte count . Chest x - ray taken at relapse is usually nonspecific, demonstrating bilateral reticular and nodular opacities (figure 2). Up to now these episodes have responded to intravenous administration of methylprednisolone in a dose of 20 mg / kg / day for 5 - 7 days . Even if better controlled, the disease is still active, thus the prognosis remains at least doubtful . Moreover, it is suspected that a substantial proportion of this age group is undiagnosed childhood - onset cases . It is probably due to the fact that iron deficiency anemia may be the first and the only manifestation of iph, preceding other symptoms and signs by several months . Iron deficiency anemia is the most common haematologic disorder seen in childhood . In infants and toddlers it results from poor dietary intake of iron and does not require any additional work - up . However, every child older than 24 months presenting with iron deficiency anemia should be suspected and evaluated for chronic blood loss . Our patient, when seen for the first time, presented with deep microcytic and hypochromic anemia and unspecific symptoms and signs from the respiratory tract, which subsided spontaneously after several days . Hemoptysis, which occurred several months later, drew our attention to a pulmonary hemorrhage . Pulmonary hemorrhage and hemoptysis they may be a manifestation of cystic fibrosis or congenital heart disease, but these causes, as well as purpura, laryngeal disorders, oesophagitis and bleeding from the gastrointestinal tract were excluded in our patient . As discussed by godfrey in his review, other causes of hemoptysis in children, including diffuse alveolar hemorrhage, are far less common . Gomez - roman classified diffuse alveolar hemorrhagic syndromes into 3 large groups: (i) those which generally involve pulmonary capillarities and are associated with the presence of antineutrophil cytoplasmic antibodies; (ii) syndromes caused by immune deposits, which can be detected by immunofluorescence; and (iii) a large group that includes drug reaction, infections, and idiopathic disease . Immune mediated syndromes may be associated with renal involvement; these so called pulmonary renal syndromes were extensively discussed by bruselle . They are usually manifested by nephritic syndrome and the presence of serum ancas; however, this was not the case in our patient . Susarla and fan proposed a revised classification of diffuse alveolar haemorrhage in children to include that condition with and without pulmonary capillarities . They also suggested that pulmonary capillaritis, an immune mediated form of dah, is more commonly found in adults than children . The precise and final diagnosis of entities manifested by dah can be made on the basis of specimens obtained by either transbronchial or open lung biopsy; however, this investigation was not done in the case of our patient . Instead, bronchoalveolar lavage was performed, showing the presence of many haemosiderinladen macrophages . As stated by ioachimescu et al, this finding is suggestive of iph, having a higher diagnostic yield than sputum examination . Several hypotheses have been proposed: genetic, autoimmune, environmental, metabolic, and allergic, but none of them have been proven . There might be a genetic predisposition, since familial clustering and a high incidence of iph in consanguinous marriages have been reported . Immunohistochemical examination of lung tissue did not support an immunological pathogenesis of iph; however, it is of interest that a proportion of patients with iph subsequently develop some form of autoimmune disease . Data from the center of disease control did not prove the role of exposure to insecticides or fungal toxins in the development of iph . There are reports indicating a link between iph and celiac disease and remission of iph after instituting a gluten - free diet [18 - 20]. Glucocorticoids control the acute phase of iph; however, their effect on the chronic phase is unclear . Moreover, prolonged glucocorticoid therapy in children results in several side effects with a potential impact on growth and development . This could also be observed in our patient, who subsequently developed cushing's syndrome . Prednisone was substituted with azathioprine, which is a' second line' immunosuppressant agent recommended in iph . The prognosis for patients with iph seems to improve over time . While two decades ago the mean survival was 3 years from diagnosis, our knowledge about iph is still very poor, and there is an urgent need for large registries and prospective studies on iph . However, due to the rarity of this condition the performance of such studies seems almost impossible.
Evidence continues to show that poor children have higher mortality than their wealthier peers, especially in low- and middle - income countries, and there are increasing calls for pro - poor programming . With few exceptions, studies demonstrating higher mortality among poor children are based on cross - sectional designs that provide little guidance about which interventions and program approaches are most effective in reducing these inequities . More evidence is needed to help ministries of health and their partners develop programs that will redress socioeconomic inequities in maternal and child health programs . The lives saved tool (list), as described earlier in this volume, supports program decision making by estimating the lives that can be saved by increasing coverage for proven maternal and child health interventions, alone or in combination, for user - defined populations and time frames . Our aim in this study was to determine whether list produces valid estimates for wealth subgroups within a population, allowing users to compare alternative program scenarios based on the extent to which they would differentially prevent child deaths among the poorest populations . Our original design for this study was to identify large population - based household surveys that collected data on deaths by cause, intervention coverage and household wealth at two points in time (say, 2000 and 2005) in a single setting . We would then have used list to model changes in the distributions of deaths by cause from 2000 to 2005 using the baseline (2000) cause - of - death distribution and changes in intervention coverage between 2000 and 2005 as inputs . We reviewed all demographic and health survey (dhs) data sets and were not able to identify a single country with two surveys that included measurement of under-5 deaths by cause . We therefore revised the design to allow us to pose the research question using a single data set: how well can list predict under-5 mortality and child deaths by cause using input data on intervention coverage from a single wealth quintile, using national measured results as baseline? In two countries, bangladesh and pakistan, the dhs survey included data on intervention coverage and household wealth, and was accompanied by a verbal autopsy study in which trained surveyors visited households reporting a child death to determine the cause of death . The verbal autopsy data were not yet available in pakistan; in bangladesh the 2004 bangladesh demographic and health survey (2004 bdhs) met these criteria and the investigators who conducted the verbal autopsy agreed to work with us on this analysis . We used the wealth index as defined in the 2004 bdhs, which uses standard procedures, and principal components analysis to categorize the population into one of five equal - sized groups from the lowest (poorest) to the highest (wealthiest). We reanalysed the 2004 bdhs data to obtain coverage estimates by wealth quintile for all interventions for which data were available (table 1). Table 1 intervention coverage from 2004 bdhs datainterventioncoverage indicatorantenatal interventions case management of pregnancypercentage of pregnant women with at least four antenatal care visits * syphilis detection and treatmentpercentage of pregnant women with at least four antenatal care visits * tetanus toxoid vaccinationpercentage of pregnant women who received two or more doses of tetanus toxoid during pregnancy or ever multiple micronutrient supplementationpercentage of women who bought or received iron supplementation during pregnancychildbirth care interventions antenatal corticosteroids for preterm labourpercentage of infants born in a facility * antibiotics for prevention of premature rupture of membranespercentage of infants born in a facility * labour monitoring, skilled delivery and access to emergency obstetric carepercentage of infants born in a facility * newborn resuscitationpercentage of infants born in a facility * clean delivery kitpercentage of infants delivered with a skilled attendant, among those delivering at homepostnatal preventive interventions infant postnatal carepercentage of infants delivered at home with a postnatal health contact / visit within six weeks of birth water connection in the homepercentage of households with water piped into home or yard improved water sourcepercentage of households with access to either piped water or a tubewell improved excreta disposalpercentage of homes with access to an improved latrine or flush toilet vitamin a supplementationpercentage of children aged 059 months receiving at least one dose of vitamin a in the past six monthsvaccinations measles vaccinepercentage of infants aged 1223 months having received one dose of measles - containing vaccine diphtheria pertussis tetanus (dpt) vaccinepercentage of infants aged 1223 months having received three doses of dpt vaccinepostnatal curative interventions case management of serious neonatal illnesspercentage of children delivering in a facility * oral rehydration salt (ors) for diarrhoeapercentage of children with diarrhoea given ors case management of pneumoniamedical care sought among children with fever / cough in previous 2 weeks antibiotics for dysenterymedical care sought among children with fever / cough in previous 2 weeks vitamin a for measles treatmentpercentage of children aged 059 months receiving at least one dose of vitamin a in the past 6 months*a standard fraction built into list . The exact formula used for each indicator can be found in the list manual at http://software.futuresgroup.com/spectrum/csmanual.pdf, page 53 (accessed on february 7, 2010). The exact formula used for each indicator can be found in the list manual at http://software.futuresgroup.com/spectrum/csmanual.pdf, page 53 (accessed on february 7, 2010). Data on the cause of child death were obtained from the verbal autopsy study conducted in association with the 2004 bdhs . When a child death in the previous 5 years was identified in the 2004 bdhs the primary cause of death was assigned using a hierarchical process, in which diagnoses that are more specific are given greater priority than less certain diagnoses . We grouped the deaths by age (neonatal = age 28 days; post - neonatal = age> 28 days) and cause to facilitate analysis . Neonatal deaths included those assigned causes of sepsis pneumonia, asphyxia, prematurity, diarrhoea, tetanus, congenital anomalies, and other . Post - neonatal deaths included those assigned causes of pneumonia, diarrhoea, measles, and injury / other . Deaths assigned to the dual - cause category of diarrhoea and acute respiratory infection (1.8%) were considered as diarrhoea deaths . Post - neonatal deaths assigned to the triple - cause category of measles and diarrhoea or acute respiratory infection (0.6%) were considered as measles deaths . The resulting data set included the full set of child deaths by cause in each of the five wealth quintiles . Neonatal and under-5 mortality rates were calculated for the 5 years preceding the survey for the total sample and by wealth quintiles using dhs methods . Typically, dhs uses women s birth history data to estimate directly childhood mortality using a synthetic cohort life table approach . Probability of death is calculated for small age segments of children up to 59 months of age and then combined into under-5 mortality using life table approach . We estimated standard errors using the jack - knife method and computed 95% confidence intervals (cis). We describe here how 2004 bdhs data were used to meet the requirements for list data inputs for the lowest and highest quintiles . National bdhs data were used to calculate estimates by quintile for neonatal, infant and under-5 mortality rates, stunting (height for age z - score <2) percent by age, and the percent breastfed exclusively, predominantly, partially, and not at all . The percent intrauterine growth retardation (iugr) was derived using data from unicef report on low birth weight and the prediction formula developed by de onis and colleagues . Table 1 shows the interventions for which coverage data from the 2004 bdhs were used in the list scenarios . Coverage estimates for several nutritional interventions included in list were not available in the 2004 bdhs . Coverage values for balanced energy supplementation and complementary feeding education and supplementation were imputed to generate corresponding modeled stunting rates that closely approximate the observed rates reported in the bdhs . However, there was a discrepancy in the observed and modeled stunting rates among children under one month because we assumed, at the national level, the same stunting rate for children under 1 month and those aged 15 months . Prevalence of iugr was estimated based on surveys in india and pakistan showing rates of iugr as 20% lower and 20% higher than the national mean in the lowest and highest quintiles, respectively ., the version of list used for this analysis calls for coverage of face - to - face counselling as an intervention to increase exclusive breastfeeding to 6 months, for which data were not available in the 2004 bdhs . We therefore used the prevalence of exclusive breastfeeding among children up to 6 months of age as reported in the survey as a basis for imputing coverage rates for the counselling intervention . We used list to model mortality rates in the lowest and highest wealth quintiles by assuming changes in coverage from the national level to the levels measured in each of the wealth subgroups . To do this, we assumed a 5-year time period to allow interventions to achieve their full effect by using 2004 as the baseline year and modelling the results that would occur in mortality given the measured coverage levels for each wealth quintile . We assumed that the entire change in coverage between the national estimate and the estimates for the lowest and highest quintiles occurred in the first year . We used list to produce predictions of mortality levels for two age groups (neonatal and post - neonatal) and two wealth quintiles (lowest and highest). The bdhs 2004 estimated that 47.3% of all under-5 deaths in the data set occurred in the neonatal period, compared to 55.7% of deaths based on the verbal autopsy data set . We investigated this by comparing the child deaths in the 2004 bdhs and verbal autopsy data sets by matching each death on cluster, household number, mother line number and birth year, and found that among 587 deaths in the verbal autopsy dataset, only 474 matched children in the 2004 bdhs data . We performed the analyses using both the full and limited data sets and the results were similar; here we report results from analyses using the full verbal autopsy dataset (n = 587). We describe here how 2004 bdhs data were used to meet the requirements for list data inputs for the lowest and highest quintiles . National bdhs data were used to calculate estimates by quintile for neonatal, infant and under-5 mortality rates, stunting (height for age z - score <2) percent by age, and the percent breastfed exclusively, predominantly, partially, and not at all . The percent intrauterine growth retardation (iugr) was derived using data from unicef report on low birth weight and the prediction formula developed by de onis and colleagues . Table 1 shows the interventions for which coverage data from the 2004 bdhs were used in the list scenarios . Coverage estimates for several nutritional interventions included in list were not available in the 2004 bdhs . Coverage values for balanced energy supplementation and complementary feeding education and supplementation were imputed to generate corresponding modeled stunting rates that closely approximate the observed rates reported in the bdhs . However, there was a discrepancy in the observed and modeled stunting rates among children under one month because we assumed, at the national level, the same stunting rate for children under 1 month and those aged 15 months . Prevalence of iugr was estimated based on surveys in india and pakistan showing rates of iugr as 20% lower and 20% higher than the national mean in the lowest and highest quintiles, respectively ., the version of list used for this analysis calls for coverage of face - to - face counselling as an intervention to increase exclusive breastfeeding to 6 months, for which data were not available in the 2004 bdhs . We therefore used the prevalence of exclusive breastfeeding among children up to 6 months of age as reported in the survey as a basis for imputing coverage rates for the counselling intervention . We used list to model mortality rates in the lowest and highest wealth quintiles by assuming changes in coverage from the national level to the levels measured in each of the wealth subgroups . To do this, we assumed a 5-year time period to allow interventions to achieve their full effect by using 2004 as the baseline year and modelling the results that would occur in mortality given the measured coverage levels for each wealth quintile . We assumed that the entire change in coverage between the national estimate and the estimates for the lowest and highest quintiles occurred in the first year . We used list to produce predictions of mortality levels for two age groups (neonatal and post - neonatal) and two wealth quintiles (lowest and highest). The bdhs 2004 estimated that 47.3% of all under-5 deaths in the data set occurred in the neonatal period, compared to 55.7% of deaths based on the verbal autopsy data set . We investigated this by comparing the child deaths in the 2004 bdhs and verbal autopsy data sets by matching each death on cluster, household number, mother line number and birth year, and found that among 587 deaths in the verbal autopsy dataset, only 474 matched children in the 2004 bdhs data . We performed the analyses using both the full and limited data sets and the results were similar; here we report results from analyses using the full verbal autopsy dataset (n = 587). Table 2 shows coverage levels, national and by wealth quintile, for the interventions included in the list analyses . These results, which have been reported elsewhere,, show the expected overall trend towards higher coverage as household wealth increases, but levels of inequality vary by intervention . There are relatively small differences between the lowest and highest quintiles in coverage for improved water source, vaccinations, vitamin a supplementation, and oral rehydration salt solution and much larger differences in coverage for antenatal care, facility - based births, infant post - natal care, and water connection in the home with coverage rates in the wealthiest quintile being more than seven times higher than coverage in the poorest quintile . Inequalities were moderate for iron supplementation, case management of pneumonia and improved excreta disposal . Table 2 reported coverage for list interventions, 2004 bdhsindicatorsnationalwealth quintilesratio highest / poorestlowestsecondmiddlefourthhighestantenatal care15.93.97.112.018.244.911.5tetanus toxoid immunization63.756.061.363.569.770.91.3iron supplementation50.031.644.648.157.476.12.4facility - based birth9.92.33.36.312.331.913.9skilled birth attendance13.43.44.510.517.439.611.6infant postnatal care18.66.89.214.321.347.97.0water connection in the home6.20.20.52.35.128.5142.5improved water source97.896.997.497.898.499.01.0improved excreta disposal56.224.346.061.277.989.43.7vitamin a supplementation71.969.172.469.772.777.51.1measles vaccination75.759.579.676.380.690.51.5dpt vaccination81.070.780.982.484.991.01.3case management of pneumonia16.68.211.216.121.133.84.1ors for diarrhoea67.956.062.069.486.477.31.4 reported coverage for list interventions, 2004 bdhs figure 1 shows the results of using these coverage levels to model mortality rates for the neonatal and post - neonatal age groups, assuming a change in coverage from national level to the level observed in lowest and highest wealth quintiles . In all four comparisons the modelled estimates rates fell within the 95% cis of the measured mortality . Figure 1 measured neonatal (nn) and under-5 mortality rates (u5mr), 95% cis and modelled rates for lowest (q1) and highest (q5) wealth quintiles measured neonatal (nn) and under-5 mortality rates (u5mr), 95% cis and modelled rates for lowest (q1) and highest (q5) wealth quintiles table 3 shows the causes of deaths in the neonatal period for the poorest and richest quintiles as measured and as modelled by list . Agreement between measured and modelled results for the lowest quintile was good, with no single cause showing a difference of greater than 6 percentage points and all estimates falling within the cis of the measured estimate . Agreement for the wealthiest quintile was less good, with the modelled estimate of deaths due to asphyxia falling 17.1 percentage points below the measured estimate, and the modelled estimates for deaths due to prematurity and other causes falling 6.3 and 8.6 percentage points above the measured estimates, respectively . Table 3 neonatal deaths in 2004 bdhs verbal autopsy data by cause, observed and modelled using listcausesnationallowest wealth quintilehighest wealth quintileobserved (95% ci)modelleddifferenceobserved (95% ci)modelleddifferencediarrhoea1.81.3 (0.03.9)1.90.65.4 (0.012.9)1.93.5sepsis pneumonia43.650.0 (38.461.6)44.85.237.8 (24.251.4)39.61.8asphyxia21.117.1 (7.726.5)21.34.237.8 (23.052.7)20.717.1prematurity11.09.5 (2.916.0)10.81.34.7 (0.09.9)11.06.3tetanus4.05.4 (0.110.7)4.31.12.6 (0.08.0)3.91.3congenital anomalies5.14.1 (0.08.5)4.60.53.7 (0.09.9)6.32.6other13.512.6 (4.121.1)12.40.38.0 (1.414.5)16.68.6total100.0100.0100.0100.0100.0n33810451 neonatal deaths in 2004 bdhs verbal autopsy data by cause, observed and modelled using list table 4 shows similar results for the post - neonatal period . Again the agreement between modelled and measured results was good for the poorest quintile and less good for the wealthiest quintile, with list predicting 17.1 percentage points more pneumonia deaths than measured and 14.5 percentage points fewer diarrhoea deaths . Table 4 post - neonatal deaths in 2004 bdhs verbal autopsy data by cause, observed and modelled using listcausesnationallowest wealth quintilehighest wealth quintileobserved (95% ci)modelleddifferenceobserved (95% ci)modelleddifferencediarrhoea13.313.4 (5.021.7)16.63.224.2 (6.442.1)9.714.5pneumonia63.266.4 (55.976.8)62.53.944.7 (27.861.6)61.817.1measles1.31.6 (0.03.8)1.60.010.01.11.1injury / other22.118.7 (9.927.5)19.30.631.1 (14.547.6)27.43.7total100.0100.0100.0100.0100.0n2709233no cases in the numerator . Post - neonatal deaths in 2004 bdhs verbal autopsy data by cause, observed and modelled using list no cases in the numerator . This first effort to assess how well list predicts mortality within wealth quintiles found promising results, with modelled estimates falling within the 95% cis of measured mortality for both neonatal and post - neonatal deaths in the lowest and highest quintiles . These results suggest that list can capture the impact of coverage inequities on neonatal and under-5 mortality rates in this context . Limitations in the results reflect the quality of the verbal autopsy and coverage data used . Small sample size for the verbal autopsy data resulted in lower precision for the estimates of proportions of deaths by cause . Similarly, estimates of all cause mortality for the lowest and highest quintiles suffered from small sample size in each quintile group . Furthermore, coverage estimates for some indicators for example improved water source were not sensitive enough to discriminate across quintiles . List performed well in predicting the cause - of - death profile for these two age groups for the poorest quintile of the population, but less well for the richest quintile . These findings merit further investigation through a closer examination of the categorization of deaths in the original verbal autopsy results and in this analysis . Cause of death was assigned through a hierarchical process, which is sensitive to the order of assignment . The lower predictive ability for deaths among the rich is not surprising, because one might expect list to perform less well as mortality levels drop and other interventions not taken into account in list (e.g. Hospital - based care) become more important . Of the sixty - eight countries that accounted for 97% of maternal and child deaths worldwide in 2006 identified by the countdown to 2015, bangladesh is among those with the lowest rate of skilled deliveries . Only 20% of deliveries in bangladesh are attended by a skilled professional compared with a median of 32% among the 68 countdown countries . Large socioeconomic inequities in access to delivery care may explain why list underestimated the percentage of deaths due to asphyxia in the wealthiest quintile . The more recent bdhs, in 2007, reported 51% of deliveries attended by a medically trained professional among the wealthiest quintile compared to only 5% among the lowest quintile, and 43% vs 4% for health facility deliveries . Effects of contextual factors on the prediction of neonatal and post - neonatal cause - of - death profiles using list can only be accurately assessed when cause - of - death data become readily available for several countries with different contextual characteristics . Further validation of list predictions of cause - specific deaths can be done only in settings where recent data on the causes of child deaths are available . This is one reason why verbal autopsies should be considered for inclusion in future dhs surveys . Sufficient sample sizes would be needed in these surveys to allow stable estimates of deaths by cause at national level and among specific subpopulations such as wealth subgroups . To further illustrate the possible programmatic applications of list, we used the data reported here to assess the number of child lives that could be saved if coverage for these proven interventions was increased among households in the poorest quintile of the population to the mean coverage levels reported for the national population . The results showed that in bangladesh, 8000 child deaths (10% of child deaths in the poorest 20% of the population) could be averted, or 3% of all deaths nationally, through this strategy . Increasing coverage levels in the poorest quintile to that of the wealthiest quintile would avert> 20 000 child deaths each year, or almost 10% of all child deaths in the country . List holds promise as a useful tool for those planning maternal and child health programs in low - income countries with high levels of socioeconomic inequity . Program planners can consider alternative scenarios and use list to assess the extent to which they are pro - poor and will contribute to redressing inequities in mortality by wealth status . This work was supported in part by a grant to the us fund for unicef from the bill & melinda gates foundation (grant 43386) to promote evidence - based decision making in designing maternal, neonatal and child health interventions in low- and middle - income countries, and a grant to the institute for international programs at the johns hopkins bloomberg school of public health from the canadian international development agency (grant 7052335) for real - time monitoring of child mortality . Key messages list performed well in predicting inequities in under-5 mortality (overall and by cause of death) for the poorest and least - poor population quintiles in bangladesh.list can contribute to the design and evaluation of programs that are pro - poor, but only if large - scale surveys in low - income countries measure intervention coverage, cause of death and household assets using adequate sample sizes.list produces accurate estimates for poor populations that are unable to access tertiary care; children in wealthier households are more likely to receive advanced biomedical interventions that are not included in list . List performed well in predicting inequities in under-5 mortality (overall and by cause of death) for the poorest and least - poor population quintiles in bangladesh.list can contribute to the design and evaluation of programs that are pro - poor, but only if large - scale surveys in low - income countries measure intervention coverage, cause of death and household assets using adequate sample sizes.list produces accurate estimates for poor populations that are unable to access tertiary care; children in wealthier households are more likely to receive advanced biomedical interventions that are not included in list . List performed well in predicting inequities in under-5 mortality (overall and by cause of death) for the poorest and least - poor population quintiles in bangladesh . List can contribute to the design and evaluation of programs that are pro - poor, but only if large - scale surveys in low - income countries measure intervention coverage, cause of death and household assets using adequate sample sizes . List produces accurate estimates for poor populations that are unable to access tertiary care; children in wealthier households are more likely to receive advanced biomedical interventions that are not included in list.
In their article in the present issue of critical care, wierdsma and colleagues validated a novel and feasible method to measure the degree to which enteral nutrition (en) is absorbed by the gastrointestinal tract . The accuracy of simply weighing daily faecal production to identify gastrointestinal dysfunction was validated against three reference methods . The contribution of protein and fat to the faecal nutritional losses were estimated by labour - intensive chemical analyses . The authors identified a daily faecal weight above> 350 g as a reliable indicator for gastrointestinal malabsorption . Of course, these results need to be confirmed in a larger study population, including the most critically ill and those with known gastrointestinal problems . The present study is of methodological and conceptual importance to nutritional research and clinical nutrition management . First, the validation of this new assessment technique has been done in a very accurate and complete way and thereby provides a new reliable tool . Secondly, these results focus on a rarely addressed problem in the critically ill: is the en administered to a patient truly absorbed? In 9 out of the 48 stable patients in this trial, the nutrition was only partially absorbed . This 19% represents a high incidence of gastrointestinal dysfunction since patients with known gastrointestinal problems were not included . Previous studies assessed and treated diarrhoea in critically ill patients, measuring the frequency, liquidity and volume of stools but not the proportion of en energy, proteins and fat lost by the patient . Whether studying the effect of nutritional interventions or managing nutrition in clinical practice, we will have to take into account these data on gastrointestinal energy losses . The discrepancy between prescribed and effectively infused en doses in critically ill patients is relevant . Interruptions of en for procedures, emesis, high gastric residual volumes (grvs), diarrhoea and feeding tube replacement are important culprits . En lost in grvs being discarded after grv assessment can be measured using a refractometer . An alternative is to reinject grvs after measuring or to refrain from measuring grvs, since evidence supporting this practice is limited . Briefly, we should be aware that the amount of en effectively taken up by the patient is the prescribed volume minus the volume not administered to the patients, minus the en discarded with grv minus the en lost in faeces . Indirect calorimetry, however, does not measure energy needs but energy consumption, and indirect calorimetry is less reliable in patients on renal replacement therapy, aggressive ventilation and other conditions often present in the critically ill . A recent randomised trial showed more morbidity in the icu, but an unexplained reduction in hospital mortality in the per - protocol analysis, when feeding was guided by indirect calorimetry . Recent trials found no benefit in early en administration up to the calculated target as compared with low en intake . Finally, what should be done if en uptake is insufficient? Probiotics or fibre - rich en reduced the volume or frequency of diarrhoea in critically ill patients in some of the few randomised controlled trials on this topic . Absorption of en by dysfunctional gut could be facilitated by the absence of proteins or even peptides in, respectively, semi - elemental en or elemental en; however, trials in critically ill patients failed to provide convincing evidence . The best controlled method to effectively complete insufficient en would be the intravenous administration of nutrition: parenteral nutrition . Supplementing insufficient en with parenteral nutrition during the first week of critical illness, however, resulted in more infections and delayed recovery in a large randomised controlled trial . In conclusion, the optimal en dose during different stages of critical illness is not yet known . Stool weight measurements could improve our knowledge of true en absorption, however, and could thus lead to more accurate research and clinical practice . Ku leuven received an institutional partial (<30%), unconditional and nonrestrictive research grant for the epanic trial from baxter healthcare (maurepas, france). Mpc receives funding from the research foundation flanders, belgium (fwo doctoral fellowship), and dm from the clinical research fund of the university hospitals leuven, belgium (postdoctoral fellowship).
Apoptosis is a distinct form of cell death characterized by a series of typical morphological events, such as shrinkage of the cell, fragmentation into membrane - bound apoptotic bodies and rapid phagocytosis into the neighboring cells without induction of an inflammatory response . Cardiomyocyte apoptosis is an important event after acute myocardial infarction (ami) and may be responsible for a significant portion of myocyte death during the acute ischemic stage . In the initial 1 - 7 days of myocardial infarction (mi), apoptotic myocyte cell death precedes cell necrosis and is the major determinant of infarct size . A quantitative study reported that apoptotic and necrotic myocyte cell death were both independent contributing variables of infarct size, but apoptosis accounted for 86% of the total loss of myocytes and necrosis for only 14% . The loss of cardiac myocytes is one of the mechanisms involved in mi - related heart failure; inhibition of cardiomyocyte apoptosis following mi may therefore improve left ventricular (lv) remodeling and cardiac function . Toll - like receptor 4 (tlr4), a class of pattern recognition receptors, has recently emerged as a key player in inflammation and innate immunity, including innate immune responses, antigen presentation and more importantly cytokine gene expressions . Conventional tlr4 signaling recognizes the ligands, activates nuclear factor-b (nf-b) pathway and is sufficient to trigger the inflammatory response . One main function of tlr4 in the non - immune system is to regulate apoptosis . Tlr4-nf-b pathways were markedly activated in failing and ischemic myocardium . Conversely tlr4 deficiency led to improved survival after mi mediated by attenuated apoptosis and lv remodeling . Carvedilol is a non - selective 1 - and -receptor blocker initially used in the treatment of hypertension . In addition to its antihypertensive property, carvedilol has been shown to significantly reduce morbidity and mortality in heart failure and post - ami patients . First reported that the protective effects of carvedilol on the ischemic myocardium involved an inhibition of apoptosis of cardiomyocytes in an experimental model of ischemia / reperfusion . Schwarz et al . Have further demonstrated that the antiapoptotic effects of carvedilol are independent of its -adrenoceptor blocking effects . However, whether the tlr4 signaling pathway is involved in the antiapoptotic effect of carvedilol has never been examined . In the present study, we hypothesized that the beneficial effects of carvedilol on ami - induced apoptosis could be related to the down - regulation of tlr4-mediated signaling activity . Rat mi model was generated by ligating the left anterior descending (lad) coronary artery according to a previously described method . Briefly, after being anesthetized by intraperitoneal injection of ethyl carbamate (1.0 g / kg), all animals underwent endotracheal intubation . Mechanical ventilation was provided with room air at 60 - 70 breath / min using a rodent respirator (taimeng company, chengdu, china). Left thoracotomy was performed to expose the heart at the fifth intercostal space; lad was ligated with a 5 - 0 silk suture . Ischemia was confirmed by the elevation of st segment in the electrocardiogram and cardiac cyanosis . Following these surgical procedures, rats were allowed to stabilize for 15 min and then the thoracic cavity was closed . The sham - operated rats underwent the same operative procedure, but the suture was loosely tied to avoid coronary artery occlusion . A total of 48 rats were randomized to the following groups before surgery: sham - operated group (n = 8), mi group (n = 10), 2 mg / kg carvedilol - treatment group (n = 10), 10 mg / kg carvedilol - treatment group (n = 10), 30 mg / kg carvedilol - treatment group (n = 10). Sham and mi groups were given vehicle and carvedilol groups received different dose carvedilol, by direct gastric gavage for 7 days . On the 4 day of drug or vehicle administration, forty rats (except sham group) were rendered mi by ligation of lad . All animal experiments were performed with permission from the medical ethics committee at anhui medical university and followed the protocol outlined in the guide for the care and use of laboratory animals published by the us national institutes of health (publication no . The heart was excised and placed on ice and the myocardium was flushed with ice - cold krebs buffer . One segment from the mid - ventricle was fixed in cold 10% formalin solution and embedded in paraffin for in situ tdt - utp nick - end labeling (tunel) and immunohistochemistry . Briefly, myocardial tissue sections (4 m) were incubated with proteinase k for 5 min at 37c and then washed with tris - buffered saline (tbs). Endogenous peroxidase was inactivated by treatment with 0.3% h2o2 for 5 min at room temperature and sections were incubated with the labeling buffer containing tdt, mn+, biotinylated - deoxyuridine 5-triphosphate at 37c for 70 min . Finally, the specimens were counter - stained with hematoxylin, washed with tbs and the signals were visualized . The number of apoptotic cardiomyocytes and their percentage of total cardiomyocytes were counted with the use of a microscope . Cardiomyocytes from at least three randomly selected sections per animal were evaluated immunohistochemically for these variables . The number of tunel - positive cells was calculated as cells per area of heart tissue at 400-fold magnification . The percentage of tunel - positive cells was calculated as a percentage of total cells viewed in five randomly selected fields for each group . The hearts fixed in 10% phosphate - buffered formaldehyde were routinely processed and paraffin - embedded . Tissue sections (4 m) mounted on poly - l - lysine - coated glass slides were deparaffinized with xylene . After washing with phosphate - buffered saline (pbs) solution, the sections were treated with 0.3% h2o2/methanol and heated for 5 min in 10 mmol / l citrate buffer at 95c . The normal goat serum - blocking solution was added the sections incubated at room temperature for 30 min and the extra liquid removed . The primary antibodies against tlr4 (1:150, santa cruz), nf-b p50 (1:200, santa cruz), bax (1:100, santa cruz), bcl-2 (1:300, santa cruz) were then added and the slides incubated over night at 4c . After washing with pbs solution, the sections were incubated with the secondary antibody (goat anti - mouse immunoglobulin g (zymed laboratories) 37c for 30 min . Streptomycete antibiotin - peroxidase solution was added and then freshly prepared dab solution for coloration . The optical density was evaluated with computer - assisted image analysis (image - pro plus 6.0, media cybernetics, silver springs, md, usa). All data are expressed as mean standard deviation (sd) statistical analysis was performed with the statistical package for the social sciences 13.0 . Rat mi model was generated by ligating the left anterior descending (lad) coronary artery according to a previously described method . Briefly, after being anesthetized by intraperitoneal injection of ethyl carbamate (1.0 g / kg), all animals underwent endotracheal intubation . Mechanical ventilation was provided with room air at 60 - 70 breath / min using a rodent respirator (taimeng company, chengdu, china). Left thoracotomy was performed to expose the heart at the fifth intercostal space; lad was ligated with a 5 - 0 silk suture . Ischemia was confirmed by the elevation of st segment in the electrocardiogram and cardiac cyanosis . Following these surgical procedures, rats were allowed to stabilize for 15 min and then the thoracic cavity was closed . The sham - operated rats underwent the same operative procedure, but the suture was loosely tied to avoid coronary artery occlusion . A total of 48 rats were randomized to the following groups before surgery: sham - operated group (n = 8), mi group (n = 10), 2 mg / kg carvedilol - treatment group (n = 10), 10 mg / kg carvedilol - treatment group (n = 10), 30 mg / kg carvedilol - treatment group (n = 10). Sham and mi groups were given vehicle and carvedilol groups received different dose carvedilol, by direct gastric gavage for 7 days . On the 4 day of drug or vehicle administration, forty rats (except sham group) were rendered mi by ligation of lad . All animal experiments were performed with permission from the medical ethics committee at anhui medical university and followed the protocol outlined in the guide for the care and use of laboratory animals published by the us national institutes of health (publication no . The heart was excised and placed on ice and the myocardium was flushed with ice - cold krebs buffer . One segment from the mid - ventricle was fixed in cold 10% formalin solution and embedded in paraffin for in situ tdt - utp nick - end labeling (tunel) and immunohistochemistry . Briefly, myocardial tissue sections (4 m) were incubated with proteinase k for 5 min at 37c and then washed with tris - buffered saline (tbs). Endogenous peroxidase was inactivated by treatment with 0.3% h2o2 for 5 min at room temperature and sections were incubated with the labeling buffer containing tdt, mn+, biotinylated - deoxyuridine 5-triphosphate at 37c for 70 min . Finally, the specimens were counter - stained with hematoxylin, washed with tbs and the signals were visualized . The number of apoptotic cardiomyocytes and their percentage of total cardiomyocytes were counted with the use of a microscope . Cardiomyocytes from at least three randomly selected sections per animal were evaluated immunohistochemically for these variables . The number of tunel - positive cells was calculated as cells per area of heart tissue at 400-fold magnification . The percentage of tunel - positive cells was calculated as a percentage of total cells viewed in five randomly selected fields for each group . The hearts fixed in 10% phosphate - buffered formaldehyde were routinely processed and paraffin - embedded . Tissue sections (4 m) mounted on poly - l - lysine - coated glass slides were deparaffinized with xylene . After washing with phosphate - buffered saline (pbs) solution, the sections were treated with 0.3% h2o2/methanol and heated for 5 min in 10 mmol / l citrate buffer at 95c . The normal goat serum - blocking solution was added the sections incubated at room temperature for 30 min and the extra liquid removed . The primary antibodies against tlr4 (1:150, santa cruz), nf-b p50 (1:200, santa cruz), bax (1:100, santa cruz), bcl-2 (1:300, santa cruz) were then added and the slides incubated over night at 4c . After washing with pbs solution, the sections were incubated with the secondary antibody (goat anti - mouse immunoglobulin g (zymed laboratories) 37c for 30 min . Streptomycete antibiotin - peroxidase solution was added and then freshly prepared dab solution for coloration . The optical density was evaluated with computer - assisted image analysis (image - pro plus 6.0, media cybernetics, silver springs, md, usa). All data are expressed as mean standard deviation (sd) statistical analysis was performed with the statistical package for the social sciences 13.0 . The occurrence of apoptosis was indicated by the cardiomyocyte nuclei with deoxyribonucleic acid fragmentation detected by tunel . Tunel - positive myocytes were barely detectable in sham [figure 1a and f], but significant increase in mi [32.50 4.5%, figure 1b and f, p <0.05 vs. sham]. In contrast to mi, carvedilol - treatment resulted in a significant reduction in the number of tunel - positive myocyte nuclei [figure 1c - f] (p <0.05 vs. mi group). The maximal inhibition ratio was 36% (seen in car 30 mg / kg group). Apoptotic cells in the infarcted area assessed by immunostaining of tdt - utp nick - end labeling - positive cells (brown) (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham, p <0.05 versus mi immunoreactivity of bax and bcl-2 was yellow - brown reactive product located in the cytoplasm of the cardiomyocytes . As shown in figure 2, there was limited expression of bax in sham group [figure 2a and f]. The expression of bax was significantly increased in mi group [figure 2b and f] (p <0.05 vs. sham group). Carvedilol - treatment [figure 2c - f] attenuated the increase (p <0.05). Figure 3 shows that the expression of bcl-2 was increased in mi and the three car groups (p <0.05 vs. sham), although the difference between mi and carvedilol - treatment groups was not statistically significant . The ratio of bax to bcl-2, a better apoptotic index than the two proteins considered separately, was increased in mi group (1.57) compared with sham group (0.89), but normalized by carvedilol treatment (1.11 for 2 mg / kg, 1.00 for 10 mg / kg and 0.88 for 30 mg / kg). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham, p <0.05 versus mi, p <0.05 versus car 2 mg / kg immunohistochemical staining of bcl-2 in myocardial sections (400). (a) sham; (b) myocardial infarction; (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; * p <0.05 versus sham tlr4 positive expression, which manifested as a pervasive brown - yellow color in the myocardial cells, was found in the myocardial tissue sampled from all the five groups . There was a low - level of expression of tlr4 protein in sham group [figure 4a and f]. The expression of tlr4 protein in mi group [figure 4b and f] was significantly higher (p <0.05 for all mi groups vs. sham). Carvedilol - treatment 2, 10 and 30 mg / kg, [figure 4c - f] consistently decreased the excessive expression of tlr4 protein induced by mi (p <0.05 versus . Effect on the expression of toll - like receptor 4 in different groups measured by immunohistochemistry (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham; p <0.05 versus mi the relationship between tlr4 and apoptosis was further analyzed . Changes to the level of tlr4 were closely correlated to the extent of mi - induced cardiomyocytes apoptosis as well as the ratio of bax to bcl-2 as the dose of carvedilol varied [figure 5]. (a) apoptotic cardiomyocytes; (b) bax / bcl-2 ratio the expression of nf-b p50 3 days after mi was measured by immunohistochemistry [figure 6]. Four days after mi, marked increase of p50 was observed, mostly in the nuclear staining of the infarcted region . Carvedilol treatment inhibited nf - kb subunit p50 expression induced by mi (p <0.05), especially in car 30 mg / kg group (p <0.05 vs. car 2 mg / kg). Effect on the expression of nuclear factor-b p50 in different groups examined by immunohistochemistry (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham, p <0.05 versus mi, p <0.05 versus car 2 mg / kg the occurrence of apoptosis was indicated by the cardiomyocyte nuclei with deoxyribonucleic acid fragmentation detected by tunel . Tunel - positive myocytes were barely detectable in sham [figure 1a and f], but significant increase in mi [32.50 4.5%, figure 1b and f, p <0.05 vs. sham]. In contrast to mi, carvedilol - treatment resulted in a significant reduction in the number of tunel - positive myocyte nuclei [figure 1c - f] (p <0.05 vs. mi group). The maximal inhibition ratio was 36% (seen in car 30 mg / kg group). Apoptotic cells in the infarcted area assessed by immunostaining of tdt - utp nick - end labeling - positive cells (brown) (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * immunoreactivity of bax and bcl-2 was yellow - brown reactive product located in the cytoplasm of the cardiomyocytes . As shown in figure 2, there was limited expression of bax in sham group [figure 2a and f]. The expression of bax was significantly increased in mi group [figure 2b and f] (p <0.05 vs. sham group). Carvedilol - treatment [figure 2c - f] attenuated the increase (p <0.05). Figure 3 shows that the expression of bcl-2 was increased in mi and the three car groups (p <0.05 vs. sham), although the difference between mi and carvedilol - treatment groups was not statistically significant . The ratio of bax to bcl-2, a better apoptotic index than the two proteins considered separately, was increased in mi group (1.57) compared with sham group (0.89), but normalized by carvedilol treatment (1.11 for 2 mg / kg, 1.00 for 10 mg / kg and 0.88 for 30 mg / kg). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham, p <0.05 versus mi, p <0.05 versus car 2 mg / kg immunohistochemical staining of bcl-2 in myocardial sections (400). (a) sham; (b) myocardial infarction; (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . Tlr4 positive expression, which manifested as a pervasive brown - yellow color in the myocardial cells, was found in the myocardial tissue sampled from all the five groups . There was a low - level of expression of tlr4 protein in sham group [figure 4a and f]. The expression of tlr4 protein in mi group [figure 4b and f] was significantly higher (p <0.05 for all mi groups vs. sham). Carvedilol - treatment 2, 10 and 30 mg / kg, [figure 4c - f] consistently decreased the excessive expression of tlr4 protein induced by mi (p <0.05 versus . Effect on the expression of toll - like receptor 4 in different groups measured by immunohistochemistry (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; (f) data of quantitative analysis are expressed as mean standard deviation . * p <0.05 versus sham; p <0.05 versus mi the relationship between tlr4 and apoptosis was further analyzed . Changes to the level of tlr4 were closely correlated to the extent of mi - induced cardiomyocytes apoptosis as well as the ratio of bax to bcl-2 as the dose of carvedilol varied [figure 5]. The expression of nf-b p50 3 days after mi was measured by immunohistochemistry [figure 6]. Four days after mi, marked increase of p50 was observed, mostly in the nuclear staining of the infarcted region . Carvedilol treatment inhibited nf - kb subunit p50 expression induced by mi (p <0.05), especially in car 30 mg / kg group (p <0.05 vs. car 2 mg / kg). Effect on the expression of nuclear factor-b p50 in different groups examined by immunohistochemistry (400). (a) sham; (b) myocardial infarction (mi); (c) car 2 mg / kg; (d) car 10 mg / kg; (e) car 30 mg / kg; * p <0.05 versus sham, p <0.05 versus mi, p <0.05 versus car 2 mg / kg in the present study, we have investigated the protective effect of carvedilol on cardiomyocyte apoptosis and the possible mechanisms in a rat model of mi closely mimicking human anatomy physiology . The main results of the present work are: (1) short - term administration of carvedilol significantly inhibited cardiomyocyte apoptosis in infarcted region 3 days after mi . (2) in parallel to the effect on apoptosis, carvedilol treatment alleviated the over - expression of tlr4 and nf-b proteins induced by mi . Occlusion of a major coronary artery in the rat is a well - characterized animal model of acute mi and its chronic sequelae, such as chronic heart failure . Cardiomyocyte apoptosis occurs in mi and importantly contributes to its pathological progression . In this model, apoptotic cardiomyocytes were seen in the central ischemic areas in the acute phase of infarction . In the chronic stage of mi as much as 54% of the tunel - positive cardiomyocytes appeared in the noninfarcted tissue; this was correlated with the degree of ventricular enlargement and remodeling, resulting in chronic heart failure . A recent clinical study suggests that carvedilol may be superior to other -adrenoceptor blockers in the improvement of heart failure and one possible mechanism underlying this particular beneficial effect could be regulating apoptosis . In animal experiments, carvedilol inhibited necrosis and apoptosis of ischemic myocardial cells, leading to improved post - mi remodeling . Bax and bcl-2, the two main members of the apoptosis family, critically influence the permeability of the mitochondrial membrane and regulate apoptosis . Bax is a pore - forming cytoplasmic protein and in response to an enhanced oxidative stress, translocates to the outer mitochondrial membrane, alters its permeability and induces cytochrome c loss from the intermembrane space of the mitochondria into the cytosol . The anti - apoptotic bcl-2, on the other hand, acts on the outer mitochondrial membrane and stabilizes the membrane permeability, thus preserving mitochondrial integrity and suppressing the cytochrome c release . The ratio of bax to bcl-2 may therefore better predict the apoptotic fate of the cell . The present study showed that the administration with carvedilol decreased tunel - positive cardiomyocytes and inhibited the increase of bax: bcl-2 ratio induced by mi . This suggests that the short - term administration of carvedilol can significantly suppress the apoptosis and affect its regulatory proteins in the acute stage of mi . The clinical implication is that carvedilol may act early to bring a beneficial effect to the cardiac function following mi . In recent years tlr4, the first toll receptor identified in mammal, manifests itself in all cell types and plays an important role in regulating the inflammatory reaction . Recent studies have suggested that tlr4 expression both at mrna and protein levels is increased in mi . Tlr4-mediated pathways played a key role in triggering the post - infarction inflammatory response by activating the nf-b system and in tlr4 knockout mice, the activation of nf-b induced in mi was markedly inhibited . An important function of tlr4 in the non - immune system is anti - apoptosis . In contrast to wild type - mi mice, the infarcted area of knock - out -mi mice displayed a significantly decreased content of tunel - positive apoptotic cells induced by mi (approximately 73%). More recently, tlr4 blocking antibody was also found to inhibit the apoptosis of isolated cardiac myocytes . In the present study, the expression of tlr4 and activity of nf-b in the infarction region short - term administration of carvedilol significantly inhibited the expression of tlr4 and the activity of nf-b . Furthermore, changes to mi - induced tlr4 expression largely mirrored the effects on apoptotic parameters with varying carvedilol doses . These data suggest that the anti - apoptotic effect of carvedilol may be associated with an inhibition of the excessive expression of tlr4-nf-b pathway . The results suggest that the short - term administration of carvedilol significantly reduces cardiomyocyte apoptosis in the infarcted area probably via an inhibition of the excessive expression of tlr4 and nf-b induced by mi.
Drug - induced hypersensitivity syndrome / drug reaction with eosinophilia and systemic symptoms (dihs / dress) is a severe drug - induced adverse reaction that in most cases occurs 3 to 6 weeks after drug administration [14]. The diagnosis can be confirmed if 5 out of the following 6 criteria are fulfilled: 1 . 4 . Leucocytosis (> 1010/l), atypical lymphocytosis, and eosinophilia; 5 . Hepatitis (alt> 100 u / l); and 6 . Because of the complexity and variability of dihs / dress, another diagnostic scoring system has been proposed by the regiscar study group . Numerous systemic complications may occur, including pneumonitis, pancreatitis, renal failure, encephalitis, thyroid disease, spleen rupture, eosinophilic colitis or esophagitis, coronary artery thrombosis, and myocarditis . The most common drugs associated with dihs / dress are: antiepileptic drugs (carbamazepine, phenytoin, phenobarbital, lamotrigine, levetiracetam), sulfonamide or its derivates (sulfamethoxazole, sulfadiazine, sulfasalazine, dapsone), allopurinol, minocycline, mexiletine, nevirapine, abacavir, and others [14]. The dihs / dress has several important clinical features that cannot be explained only by drug antigen - driven oligoclonal t cell expansion . These include paradoxical worsening of clinical symptoms, frequent flare - ups and a stepwise development of multiorgan failures when the causative drugs are withdrawn . The pathophysiology of dihs / dress resembles that of graft versus host diseases (gvhd) and immune reconstitution syndrome (irs). Herpesviruses, including herpesvirus-6 (hhv-6), herpesvirus-7 (hhv-7), epstein barr virus (ebv) and cytomegalovirus (cmv), can be reactivated during dihs / dress in the same sequential manner as in gvhd [611]. For example, diabetes type i, autoimmune thyroiditis, systemic sclerosis, and systemic lupus erythematosus (sle) have been described . The standard treatment of dihs / dress is with moderate- or high - dose corticosteroids [13]. Indeed, sometimes corticosteroid treatment is not effective, and tapering the dose may be associated with disease flaring . Adjunctive treatment with acetylcysteine, intravenous immunoglobulins, plasmapheresis, and cyclosporine have been tried with varying results . We report on a woman with sulfasalazine - induced dihs / dress treated using an unprecedented combination of n - acetylcysteine, prednisone, and valganciclovir . A 38-year - old woman was admitted to hospital on september 2010 because of high fever and a generalized erythematous rash . She had taken a course of acetaminophen 500 mg 3 times daily (a total of 19 tablets). She had 4 children, of whom 1 suffered from diabetes type i since the age of 2 years . Six years earlier she underwent a thyroidectomy for unknown reason and since then had been on l - thyroxine 125 g daily . Four weeks prior to this admission she was prescribed sulfasalazine 500 mg tds because of arthralgias . On the morning of the hospital admission she had discontinued sulfasalazine on her own . The month prior to hospital admission the patient was fasting for cultural reasons (muslim ramadan). On physical examination she had a fever 40c, a non - itchy generalized erythematous rash and mild facial edema . There were signs of dehydration, and body weight loss of 3 kg over 1 month . Laboratory analysis showed: white blood cells 410/l, neutrophils 75%, hemoglobin 10.8 g / l, mcv 63, ast 541 iu / l, alt 389 iu / l, ldh 1,703 iu / l, pt inr 1.21, ptt ratio 1.41, gamma - globulin 16.5% (8.73 g / l), crp 80 mg / l, urinary ketones 300 mg / l, albumin 26 acetaminophen blood level 10 hours after the last drug assumption was 3 g / ml . The prolonged fasting for ramadan was considered a risk factor for acetaminophen hepatotoxicity at therapeutic doses . Therefore intravenous n - acetylcysteine was immediately administered (10 g the first 2 hours, followed by 2.5 g every 6 hours for the next 3 days), followed by temporary hypotension (from 130/75 to 80/50 mmhg). Amoxi - clavulanate 2 g tds was given for treatment of the lung consolidation (as above). On day + 2 of admission, significant improvement was observed, with resolution of malaise, abdominal pain and erythematous rash . Nevertheless, she continued to have persistent fever, elevated ldh (figure 1), enlarging lymphadenopathies, liver and spleen, lymphocytosis (from 0.7 to 8.9510/l with 1.6210/l atypical lymphocytes), high cd4 + cell count (5.83210/l), leucocytosis (from 4 to 16.3710/l), eosinophilia (from 0.2 to 1.5410/l) for 1 week (figure 1), which was strongly suggestive of sulfasalazine - related dihs / dress syndrome . She has started taking this drug 4 weeks previously . On day + 7, treatment with 75 mg prednisone daily was commenced and amoxi - clavulanate was discontinued . She was advised to avoid taking sulfasalazine and other sulphonamides, including sulfamethoxazole . Microbiological analysis for hav - igm, hbs - ag, hbv - dna, hcv - ab, legionella - ag and streptococcus pneumoniae - ag in urine were all negative . Herpesvirus screening was performed and tested positive for cmv- ebv-, and hhv-6-igg (1:40); however, she tested negative for cmv-, ebv-, and hhv-6-dna (day + 8). Hhv-6-dna was detected in blood and serum samples on day + 12 from admission time . Four days later, because of general malaise, the dose was halved to 450 mg twice daily . The patient s clinical status was unremarkable and all laboratory values were within normal range . Usually sulfasalazine is split in the colon by bacterial azo - reduction into 5-asa and sulfapyridine . Sulfapyridine is almost completely absorbed in the colon and metabolized by hydroxylation, glucuronidation, and acetylation . Various oxidizing cytochrome p450 (cyp) enzymes, including cyp2c9, cyp2e1, and cyp3a4, can oxidize aryl amines to reactive metabolites and sulfapyridine will be converted into the unstable sulfapyridine hydroxylamine intermediate, which auto - oxidizes to nitroso - sulfapyridine . The results of the patch tests and lymphocyte transformation tests indicate that drug - specific t cells are the driving force behind dihs / dress . Sulfamethoxazole (smx) is commonly used to investigate the chemical and cellular basis of drug hypersensitivity . The smx metabolite nitroso - smx binds covalently to cysteine residues on cellular protein; this binding above a certain threshold may cause direct toxicity and provides an antigenic signal to nitroso - smx specific t cells . Dendritic cells, as antigen - presenting cells, play an important role in immunity, differentiating between tolerogenic and immunogenic responses . Dendritic cell maturation and costimulatory signals are required for t cell immunogenicity together with the antigen . There are a series of exogenous and endogenous signals that mature dendritic cells, leading to expression of costimulatory molecules . Exogenous signals include conserved microbial products that interact with pathogen - recognition receptors, such as toll - like receptors (tlrs). This may be of relevance to the increased risk of drug hypersensitivity reactions associated with viral infections such as ebv, human herpes 6, and hiv . Endogenous signals derive from necrotic cell death, apoptotic cell death or oxidative stress . In an experiment, antigen - presenting cells were incubated with smx in the presence of bacterial endotoxins, flu viral proteins, cytokines, inflammatory molecules, oxidants, and hyperthermia . Such conditions, also referred to as danger conditions, significantly increased the formation of smx - protein adducts . Soluble antioxidants were shown to prevent smx - hydroxyamine auto - oxidation and to reduce nitroso - smx back to smx - hydroxylamine, limiting nitroso - smx protein binding and t cell stimulatory capacity of nitroso - smx protein adducts . Therefore it was suggested that acute and chronic infections treated with smx require antioxidant supplementation . Activation of antigen - presenting cells through cd40 signalling is known to precipitate animal models of autoimmune diseases . Several autoimmune diseases have been reported to occur at intervals of months to years after clinical resolution of dihs / dress (see below). It was also demonstrated that cd4cd25foxp3 regulatory t (treg) cells are expanded at the acute stage of dihs / dress . This expansion may reflect an attempt to limit collateral tissue damage induced by activation of effector t cells while allowing latent herpesviruses to reactivate . Nitroso - smx responsive t cell clones from patients with smx hypersensitivity reaction displayed in vitro reactivity toward nitroso metabolites of sulfadiazine and sulfapyridine . T cell receptor cross - reactivity with nitroso sulfonamides displaying different side chains was thus demonstrated, and shows the clear potential for hypersensitivity reaction to develop different drug structures within the same chemical class through metabolite formation and targeting of identical binding sites on protein . The above described patient was therefore advised to avoid the entire class of sulfonamides and its derivates . In 1997 a rise of hhv-6 antibody titre was described for the first time in a patient with dihs / dress and a fulminant hemophagocytic syndrome . In addition, increased hhv-6 dna in the serum was detected by quantitative pcr and by in situ hybridization in the skin of patients with dihs / dress . It was also shown that other herpesviruses, hhv-7, ebv, and cmv, could reactivate in a severe drug - induced multiorgan reaction in the same sequential order as in gvhd . Hhv-6 reactivation was found in 62 of 100 patients with dihs / dress and was associated with flaring and severity of the syndrome . Dna was detected in patients serum from day 10 to day 27 after clinical onset, but not earlier . First, we do not know if during asymptomatic hhv-6 infection the virus does not replicate, if it replicates at a low level, or reactivates temporarily in localized compartments like the salivary gland or lymphatic tissue . In 1 patient with gvhd, hhv-6 reactivation in saliva was observed 10 days before onset of rash, and salivary hhv-6 dna became negative during the rash, followed by detection of hhv-6 dna in the blood . An ongoing virus replication or virus reactivation could add to the danger conditions that lead to dendritic cell maturation and expression of costimulatory molecules, thus favoring the immunogenicity of the drug protein adducts (see above). This would increase the rate of allergic drug reactions, as seen in infectious mononucleosis and hiv infection . Second, hypo - immunoglobulinemia, and low b cell and cd56 cell counts observed in the initial phase of some patients with dihs / drees may reflect immune depression caused by drug administration and/or may be a consequence of excessive regulatory t cell expansion (see below). Drug - discontinuation may reconstitute immunity, and the paradoxical worsening of symptoms after drug discontinuation could be interpreted as immune reconstitution syndrome (irs). Third, t cell activation and proliferation (induced by the drug metabolite protein adducts) may reactivate hhv-6 and other herpesviruses, which may be responsible for late organ complications such as nephritis, encephalitis, pneumonitis, myocarditis . Corticosteroid treatment may favor herpesvirus reactivation, and the clinical flare - ups observed when the corticosteroid dose is tapered too fast may reflect an irs . Currently, anti - cytomegalovirus drugs such as valganciclovir, cidofovir, and foscarnet are used to treat hhv-6 infections because in vitro studies show that they also have activity against hhv-6 . Several autoimmune diseases (type i diabetes mellitus, thyroiditis, systemic sclerosis - like manifestations, and sle) have been reported to occur at intervals of several months to years after clinical resolution of dihs / dress . A dramatic expansion of t regulatory (treg) cells has recently been found in the acute stage of dihs / dress . Treg cells are cd4cd25foxp3 t cells specialized in suppressing the activation of the immune system and thereby maintaining immune system homeostasis and tolerance to self antigens . The suppressive capacity of treg cells became defective after clinical resolution of dihs / dress . One possible explanation for treg dysfunction is that treg cells might be exhausted owing to repeated and excessive activation of effector t cells by drug protein adducts and/or reactivated herpesviruses . The functional defect of treg cells could be responsible for the development of autoimmune disease that can occur months to years after the resolution of dihs / dress . Also human herpesvirus binding tlr2 present on treg cells has been shown to temporarily abrogate their suppressive phenotype . Foxp3 expression, a key regulator of regulatory t cell function, was found to decrease following tlr2 stimulation of treg cells . Unfortunately we missed the opportunity to measure the quote of the cd4cd25foxp3 t cells among the excessively increased cd4 t cells of the described patient (figure 1). The patient s increased number of cd4 compared to cd8 t cells and her mild skin lesions might reflect the excessive expansion of treg cells, since treg cells are thought to alleviate the skin lesion severity of dihs / dress . However, excessive treg cells are thought to allow latent herpesviruses to reactivate in an uncontrolled fashion . Hypogammaglobulinemia observed at the onset of dihs / dress may be related to the expansion of treg cells, because treg cells have been shown to be capable of inducing b cell death . . Increased formation of the reactive intermediate metabolite n - acetyl - p - benzo - quinone - imine (napqi) and depletion of cellular glutathione have been proposed as the major reasons for the hepatotoxicity of acetaminophen overdose . In rare circumstances, even cyp2e1, among other cyp enzymes, was found to be the main activator of acetaminophen to napqi . In rats the repeated administration of subtoxic doses of acetaminophen has been shown to induce hepatic cyp enzymes (cyp2e1, cyp3a, cyp1a). Cyp2e1 is capable of carrying out the oxidation of acetone, a product of fatty acid oxidation, in a pathway that leads to the production of glucose, termed the propane diol pathway of gluconeogenesis . This explains the marked induction of cyp2e1 seen in fasted rats, because fasting results in an increase in acetone . Acetone has been shown to be an inducer of cyp2e1 by substrate stabilization of the enzyme . Cyp2e1 induction by repeated administration of acetaminophen and by acetone resulting from food restriction may have: 1 . Increased oxidative stress due to increased napqi formation; 2 . Increased oxidation of sulfapyridine to its hydroxylamine metabolite, and 3 . Depleted cellular glutathione stores and thus decreased the capacity to neutralize the reactive metabolites and thus favoring their covalent binding to cellular proteins (figure 2). Acetaminophen poisoning, either intentional or unintentional, has become the most common cause of acute liver failure . When given within the first 24 hours after ingestion, n - acetylcysteine can effectively prevent or minimize liver damage caused by acetaminophen, even after massive overdoses . Additionally, it has been shown that intravenous n - acetylcysteine improves transplant - free survival in early stage of non - acetaminophen acute liver failure [2931]. N - acetylcysteine, a synthetic precursor of glutathione, is used to treat patients with acetaminophen overdose for up to 48 hours after ingestion . It is well established that early treatment with n - acetylcysteine can improve the scavenging of the reactive metabolite n - acetyl - p - benzoquinone imine . Recent studies in mice showed that delayed treatment with high n - acetylcysteine doses can protect against acetaminophen overdose by 2 further mechanisms by scavenging of reactive oxygen and peroxynitrite due to cysteine supply for glutathione synthesis, and by providing excess amino acids (amino acids not used for glutathione synthesis) as substrates for the krebs cycle, supporting the mitochondrial energy metabolism . Furthermore, in animal studies n - acetylcysteine attenuates cerebral complications of non - acetaminophen - induced acute liver failure, and has nephroprotective properties in cyclosporine-, cisplatin-, and isophosphamide - induced nephrotoxicity, and in ischemia / reperfusion kidney injury . The defective detoxification of reactive intermediates of the drugs metabolized by cyp450 has been considered the triggering factor of the dress syndrome since its first description . It had been shown that smx - hydroxylamine and not smx caused dose - dependent toxicity to lymphocytes of normal volunteers in vitro, and that toxicity was decreased by co - incubating the lymphocytes with n - acetylcysteine . In another experiment, smx - hydroxylamine produced concentration - dependent toxicity in hiv - infected t - lymphoblasts toxicity that was significantly greater than the toxicity seen among non - infected t - lymphoblasts . Incubation with smx - hydroxylamine produced a concentration - dependent decline in glutathione content in both infected and non - infected t - lymphoblasts . Co - incubation with glutathione or n - acetylcysteine reduced the toxicity of smx - hydroxylamine in hiv - infected cells . In anecdotal case reports of sulphasalazine - related life - threatening adverse effects, n - acetylcysteine had been used and was suggested to be beneficial . In other cases of dihs / dress its beneficial effect it seems obvious that the potential beneficial effect of n - acetylcysteine administration is eliminated if it is given too late or if its use is not combined with other treatments . However, n - acetylcysteine as treatment of dihs / dress has not gained wide acceptance, and n - acetylcysteine is not mentioned in reviews on treatment of this syndrome [13]. Usually sulfasalazine is split in the colon by bacterial azo - reduction into 5-asa and sulfapyridine . Sulfapyridine is almost completely absorbed in the colon and metabolized by hydroxylation, glucuronidation, and acetylation . Various oxidizing cytochrome p450 (cyp) enzymes, including cyp2c9, cyp2e1, and cyp3a4, can oxidize aryl amines to reactive metabolites and sulfapyridine will be converted into the unstable sulfapyridine hydroxylamine intermediate, which auto - oxidizes to nitroso - sulfapyridine . The results of the patch tests and lymphocyte transformation tests indicate that drug - specific t cells are the driving force behind dihs / dress . Sulfamethoxazole (smx) is commonly used to investigate the chemical and cellular basis of drug hypersensitivity . The smx metabolite nitroso - smx binds covalently to cysteine residues on cellular protein; this binding above a certain threshold may cause direct toxicity and provides an antigenic signal to nitroso - smx specific t cells . Dendritic cells, as antigen - presenting cells, play an important role in immunity, differentiating between tolerogenic and immunogenic responses . Dendritic cell maturation and costimulatory signals are required for t cell immunogenicity together with the antigen . There are a series of exogenous and endogenous signals that mature dendritic cells, leading to expression of costimulatory molecules . Exogenous signals include conserved microbial products that interact with pathogen - recognition receptors, such as toll - like receptors (tlrs). This may be of relevance to the increased risk of drug hypersensitivity reactions associated with viral infections such as ebv, human herpes 6, and hiv . Endogenous signals derive from necrotic cell death, apoptotic cell death or oxidative stress . In an experiment, antigen - presenting cells were incubated with smx in the presence of bacterial endotoxins, flu viral proteins, cytokines, inflammatory molecules, oxidants, and hyperthermia . Such conditions, also referred to as danger conditions, significantly increased the formation of smx - protein adducts . Soluble antioxidants were shown to prevent smx - hydroxyamine auto - oxidation and to reduce nitroso - smx back to smx - hydroxylamine, limiting nitroso - smx protein binding and t cell stimulatory capacity of nitroso - smx protein adducts . Therefore it was suggested that acute and chronic infections treated with smx require antioxidant supplementation . Activation of antigen - presenting cells through cd40 signalling is known to precipitate animal models of autoimmune diseases . Several autoimmune diseases have been reported to occur at intervals of months to years after clinical resolution of dihs / dress (see below). It was also demonstrated that cd4cd25foxp3 regulatory t (treg) cells are expanded at the acute stage of dihs / dress . This expansion may reflect an attempt to limit collateral tissue damage induced by activation of effector t cells while allowing latent herpesviruses to reactivate . Nitroso - smx responsive t cell clones from patients with smx hypersensitivity reaction displayed in vitro reactivity toward nitroso metabolites of sulfadiazine and sulfapyridine . T cell receptor cross - reactivity with nitroso sulfonamides displaying different side chains was thus demonstrated, and shows the clear potential for hypersensitivity reaction to develop different drug structures within the same chemical class through metabolite formation and targeting of identical binding sites on protein . The above described patient was therefore advised to avoid the entire class of sulfonamides and its derivates . In 1997 a rise of hhv-6 antibody titre was described for the first time in a patient with dihs / dress and a fulminant hemophagocytic syndrome . In addition, increased hhv-6 dna in the serum was detected by quantitative pcr and by in situ hybridization in the skin of patients with dihs / dress . It was also shown that other herpesviruses, hhv-7, ebv, and cmv, could reactivate in a severe drug - induced multiorgan reaction in the same sequential order as in gvhd . Hhv-6 reactivation was found in 62 of 100 patients with dihs / dress and was associated with flaring and severity of the syndrome . Dna was detected in patients serum from day 10 to day 27 after clinical onset, but not earlier . First, we do not know if during asymptomatic hhv-6 infection the virus does not replicate, if it replicates at a low level, or reactivates temporarily in localized compartments like the salivary gland or lymphatic tissue . In 1 patient with gvhd, hhv-6 reactivation in saliva was observed 10 days before onset of rash, and salivary hhv-6 dna became negative during the rash, followed by detection of hhv-6 dna in the blood . An ongoing virus replication or virus reactivation could add to the danger conditions that lead to dendritic cell maturation and expression of costimulatory molecules, thus favoring the immunogenicity of the drug protein adducts (see above). This would increase the rate of allergic drug reactions, as seen in infectious mononucleosis and hiv infection . Second, hypo - immunoglobulinemia, and low b cell and cd56 cell counts observed in the initial phase of some patients with dihs / drees may reflect immune depression caused by drug administration and/or may be a consequence of excessive regulatory t cell expansion (see below). Drug - discontinuation may reconstitute immunity, and the paradoxical worsening of symptoms after drug discontinuation could be interpreted as immune reconstitution syndrome (irs). Third, t cell activation and proliferation (induced by the drug metabolite protein adducts) may reactivate hhv-6 and other herpesviruses, which may be responsible for late organ complications such as nephritis, encephalitis, pneumonitis, myocarditis . Corticosteroid treatment may favor herpesvirus reactivation, and the clinical flare - ups observed when the corticosteroid dose is tapered too fast may reflect an irs . Currently, anti - cytomegalovirus drugs such as valganciclovir, cidofovir, and foscarnet are used to treat hhv-6 infections because in vitro studies show that they also have activity against hhv-6 . Several autoimmune diseases (type i diabetes mellitus, thyroiditis, systemic sclerosis - like manifestations, and sle) have been reported to occur at intervals of several months to years after clinical resolution of dihs / dress . A dramatic expansion of t regulatory (treg) cells has recently been found in the acute stage of dihs / dress . Treg cells are cd4cd25foxp3 t cells specialized in suppressing the activation of the immune system and thereby maintaining immune system homeostasis and tolerance to self antigens . The suppressive capacity of treg cells became defective after clinical resolution of dihs / dress . One possible explanation for treg dysfunction is that treg cells might be exhausted owing to repeated and excessive activation of effector t cells by drug protein adducts and/or reactivated herpesviruses . The functional defect of treg cells could be responsible for the development of autoimmune disease that can occur months to years after the resolution of dihs / dress . Also human herpesvirus binding tlr2 present on treg cells has been shown to temporarily abrogate their suppressive phenotype . Foxp3 expression, a key regulator of regulatory t cell function, was found to decrease following tlr2 stimulation of treg cells . Unfortunately we missed the opportunity to measure the quote of the cd4cd25foxp3 t cells among the excessively increased cd4 t cells of the described patient (figure 1). The patient s increased number of cd4 compared to cd8 t cells and her mild skin lesions might reflect the excessive expansion of treg cells, since treg cells are thought to alleviate the skin lesion severity of dihs / dress . However, excessive treg cells are thought to allow latent herpesviruses to reactivate in an uncontrolled fashion . Hypogammaglobulinemia observed at the onset of dihs / dress may be related to the expansion of treg cells, because treg cells have been shown to be capable of inducing b cell death . Acetaminophen - induced toxicity is a clinically important model of drug - induced liver injury . Increased formation of the reactive intermediate metabolite n - acetyl - p - benzo - quinone - imine (napqi) and depletion of cellular glutathione have been proposed as the major reasons for the hepatotoxicity of acetaminophen overdose . In rare circumstances, even cyp2e1, among other cyp enzymes, was found to be the main activator of acetaminophen to napqi . In rats the repeated administration of subtoxic doses of acetaminophen has been shown to induce hepatic cyp enzymes (cyp2e1, cyp3a, cyp1a). Cyp2e1 is capable of carrying out the oxidation of acetone, a product of fatty acid oxidation, in a pathway that leads to the production of glucose, termed the propane diol pathway of gluconeogenesis . This explains the marked induction of cyp2e1 seen in fasted rats, because fasting results in an increase in acetone . Acetone has been shown to be an inducer of cyp2e1 by substrate stabilization of the enzyme . Cyp2e1 induction by repeated administration of acetaminophen and by acetone resulting from food restriction may have: 1 . Increased oxidative stress due to increased napqi formation; 2 . Increased oxidation of sulfapyridine to its hydroxylamine metabolite, and 3 . Depleted cellular glutathione stores and thus decreased the capacity to neutralize the reactive metabolites and thus favoring their covalent binding to cellular proteins (figure 2). Acetaminophen poisoning, either intentional or unintentional, has become the most common cause of acute liver failure . When given within the first 24 hours after ingestion, n - acetylcysteine can effectively prevent or minimize liver damage caused by acetaminophen, even after massive overdoses . Additionally, it has been shown that intravenous n - acetylcysteine improves transplant - free survival in early stage of non - acetaminophen acute liver failure [2931]. N - acetylcysteine, a synthetic precursor of glutathione, is used to treat patients with acetaminophen overdose for up to 48 hours after ingestion . It is well established that early treatment with n - acetylcysteine can improve the scavenging of the reactive metabolite n - acetyl - p - benzoquinone imine . Recent studies in mice showed that delayed treatment with high n - acetylcysteine doses can protect against acetaminophen overdose by 2 further mechanisms by scavenging of reactive oxygen and peroxynitrite due to cysteine supply for glutathione synthesis, and by providing excess amino acids (amino acids not used for glutathione synthesis) as substrates for the krebs cycle, supporting the mitochondrial energy metabolism . Furthermore, in animal studies n - acetylcysteine attenuates cerebral complications of non - acetaminophen - induced acute liver failure, and has nephroprotective properties in cyclosporine-, cisplatin-, and isophosphamide - induced nephrotoxicity, and in ischemia / reperfusion kidney injury . The defective detoxification of reactive intermediates of the drugs metabolized by cyp450 has been considered the triggering factor of the dress syndrome since its first description . It had been shown that smx - hydroxylamine and not smx caused dose - dependent toxicity to lymphocytes of normal volunteers in vitro, and that toxicity was decreased by co - incubating the lymphocytes with n - acetylcysteine . In another experiment, smx - hydroxylamine produced concentration - dependent toxicity in hiv - infected t - lymphoblasts toxicity that was significantly greater than the toxicity seen among non - infected t - lymphoblasts . Incubation with smx - hydroxylamine produced a concentration - dependent decline in glutathione content in both infected and non - infected t - lymphoblasts . Co - incubation with glutathione or n - acetylcysteine reduced the toxicity of smx - hydroxylamine in hiv - infected cells . In anecdotal case reports of sulphasalazine - related life - threatening adverse effects, n - acetylcysteine had been used and was suggested to be beneficial . In other cases of dihs / dress its beneficial effect was not confirmed . It seems obvious that the potential beneficial effect of n - acetylcysteine administration is eliminated if it is given too late or if its use is not combined with other treatments . However, n - acetylcysteine as treatment of dihs / dress has not gained wide acceptance, and n - acetylcysteine is not mentioned in reviews on treatment of this syndrome [13]. Its use has recently been suggested in combination with intravenous immunoglobulin administration . To the best of our knowledge no treatment of dihs / dress with the combination of n - acetylcysteine, corticosteroid and valganciclovir has been previously reported . We do not know if the favorable course of the dihs / dress in the reported patient was a consequence of treatment . Nevertheless, the knowledge of the sequential pathomechanisms describes above provides a rational basis for such a combined treatment . The immediate administration of n - acetylcysteine, which was associated with significant clinical improvement, might have neutralized the drug - derived reactive metabolites, which are responsible for protein adduct formation and specific t cell stimulation, and repleted the glutathione stores that counterbalance oxidative stress . Valganciclovir might have prevented hhv-6-associated pathology, including immune reconstitution syndrome (figure 2).
The 6-minute walk test (6mwt) was developed to measure cardiorespiratory function and endurance by assessing the maximum distance that a subject is able to walk in 6 minutes (6-minute walking distance, 6mwd)1 . The american thoracic society (ats) has proposed guidelines for safe and accurate performance of the 6mwt, which has demonstrated high accuracy and reproducibility in the evaluation of endurance in patients with cardiorespiratory disorders1 . Today, it is used for evaluation of physical endurance in the clinical field of physical therapy2,3,4 . The 6mwt has been reported to be useful for determining the ambulatory capacity of patients with duchenne muscular dystrophy (dmd)5, 6 and for evaluating the natural progression of the disease7, 8 . It has also been used in the evaluation of therapeutic efficacy in patients with neuromuscular diseases such as pompe disease, myotonic dystrophy, and mucopolysaccharidosis9,10,11,12,13,14,15 . The conventional 6mwt (c6mwt) can be used for patients 12 years of age, especially in patients with dmd16, and dmd is often associated with autism or mental retardation17, 18; it is therefore difficult for these patients to follow instructions and accurately perform the 6mwt . The ats guidelines for the 6mwt simply state the following: the object of this test is to walk as far as possible for 6 minutes . However, the ats guidelines do not provide specific instructions for patients with dmd in regard to stopping or running during the test5, 19, 20 . Therefore, this study attempted to develop a periodic sound - based 6mwt (ps6mwt) that is appropriate for assessing physical endurance in patients with dmd . A longer walking distance covered in 6 minutes controlling the stride length is generally difficult, but cadence can be easily adjusted by walking to match a sound . Therefore, a longer 6-minute walking distance might be achieved by adjusting the cadence to as fast as possible . The threshold limit for the cadence may differ among patients, and it is uncertain whether the walking distance at the fastest cadence would be the maximal distance, without controlling the stride length . Therefore, the best periodic sound (bps) was determined as the periodic sound when the subjects walked the longest distance in 1 minute . The purpose of this study was to verify whether the ps6mwt can evaluate physical endurance in patients with dmd more precisely than the c6mwt . This study confirmed the efficacy and safety of the protocol in healthy adult subjects and patients with dmd . Twenty randomly selected healthy males aged 2026 years were recruited from the school of health sciences, faculty of medicine, shinshu university, and 6 ambulant patients with dmd, aged 58 years, were recruited from shinshu university hospital . The physical characteristics of the healthy subjects are presented in table 1table 1.physical characteristics of the healthy adult subjectstotalgroup agroup bgroup a vs group bn201010age (years)22.2 3.121.9 3.822.5 2.1height (cm)171.1 3.9169.9 4.2172.3 3.2body weight (kg)66.2 11.362.5 4.269.8 14.5bmi (kg / m)22.6 3.621.6 1.123.5 4.8bmi: body mass index . Unpaired student s t - test ., and the clinical profiles of the 6 patients with dmd are presented in table 2table 2.physical characteristics of the patients with duchenne muscular dystrophypatient no.123456gendermalemalemalemalemalemaleage (years)657548weight (kg)171726161923bmi (kg / m)14.015.120.415.216.620.1gene mutationdmd del . Exons 25 - 55dmd nonsense mutation in exon 21dmd nonsense mutation in exon 48dmd nonsense mutation in exon 18dmd del . Exons 17 - 19dmd nonsense mutation in exon 44serum ck (u / l)31,35024,03021,63014,79018,97013,611walk alone (months)151418281721developmental disorderbiqbiqbiq, asd - n / abiq, asddrug / duration (months)prednisolone / 4prednisolone / 2910 m running (sec)3.94.42.84.95.34.2rising from the floor (sec)5.74.21.64.04.84.7nsaa (score)272833252024bmi: body mass index; dmd: duchenne muscular dystrophy; del . : deletion; n / a: not applicable; ck: creatine kinase; biq: borderline iq; asd: autistic spectrum disorder; nsaa: north star ambulatory assessment . This study was approved by the institutional ethics committee of the shinshu university school of medicine, japan (approval numbers: healthy subjects, 2,761; patients with dmd, 2,340). The aim and method of this study were explained to all subjects and/or parents, and consent was obtained based on the declaration of helsinki . : deletion; n / a: not applicable; ck: creatine kinase; biq: borderline iq; asd: autistic spectrum disorder; nsaa: north star ambulatory assessment all tasks were administered by 2 examiners, and one of the examiners followed the patients down the hall . The healthy subjects initially walked for 1 minute to a 110 steps / minute sound rate of a metronome, and the distance was measured . The sound interval was then gradually shortened to yield 120 steps / minute, 130 steps / minute, and up to 180 steps / minute, and each walking distance was measured for 1 minute . Patients with dmd initially walked for 1 minute to the sound rate obtained by rounding off and subtracting 20 steps / minute from the average value of the cadence in the c6mwt . The sound interval was then gradually shortened by 10 steps / minute, and each walking distance was measured for 1 minute . The rest time between the tests was 1 minute, and the measurements were continued until the walking distance was noted to show no increase . The bps was determined as the periodic sound when the subjects demonstrated the maximum walking distance; this method was named the period shortening walk test (pswt). 1.protocol of the pswt and allocation of subjects(a) the flowchart shows the procedure used to decide the best periodic sound (bps) in the pswt . S means the sound rate of the metronome in terms of the number of steps in one minute . S1, s of the first time; si, s of the second or subsequent times . The protocol is completed when the distance (di) at si is shorter than the distance (di-1) at si-1 . The bps was determined based on the si when the subject walked the longest distance in 1 minute . (b) healthy adults were allocated randomly into two groups (group a and b), and all patients with dmd were allocated into the group a. pswt: period shortening walk test; dmd: duchenne muscular dystrophy; ps6mwt: periodic sound - based 6-minute walk test; c6mwt: conventional 6-minute walk test; bps: best periodic sound . Protocol of the pswt and allocation of subjects (a) the flowchart shows the procedure used to decide the best periodic sound (bps) in the pswt . S means the sound rate of the metronome in terms of the number of steps in one minute . S1, s of the first time; si, s of the second or subsequent times . The protocol is completed when the distance (di) at si is shorter than the distance (di-1) at si-1 . The bps was determined based on the si when the subject walked the longest distance in 1 minute . (b) healthy adults were allocated randomly into two groups (group a and b), and all patients with dmd were allocated into the group a. pswt: period shortening walk test; dmd: duchenne muscular dystrophy; ps6mwt: periodic sound - based 6-minute walk test; c6mwt: conventional 6-minute walk test; bps: best periodic sound in the 6mwt, the subjects walked around 2 cones, which marked the path and were 25 m apart, in the counterclockwise direction . The total walking distance was calculated based on the number of round trips and a ruler set up between the cones . The distance per minute was measured based on the patient position at the end of the minute . The instructions provided during the c6mwt were according to the ats guidelines1 . To obtain the bps for the pswt, the initial instructions were as follows: first, walk as far as possible for 1 minute to this sound . Repeat walking in time to the sound for 1 minute until you are asked to stop . If you cannot continue walking in time to the sound, you can reduce the length of your stride . For the ps6mwt, the instructions were as follows: please begin the 6mwt with your bps, which is x steps / minute (x steps refers to the subject s bps). Keep step with this sound, and walk as far as possible for 6 minutes, but never run ten healthy adult subjects were randomly allocated to each of the 2 groups (group a or b). There were no differences in age, height, body weight, and body mass index (bmi) between the 2 groups (table 1). The task order in group a was (1) c6mwt, (2) pswt, and (3) ps6mwt; in group b, the order was (1) pswt, (2) ps6mwt, and (3) c6mwt . The interval between tasks was 2030 minutes for healthy subjects . In patients with dmd, the task order was (1) c6mwt, (2) pswt, and (3) ps6mwt . Further, in each task, a monitor (rcx5, polar electro, finland) was used to measure heart rate at 1 hz, the number of steps (steps / min) was measured, and a 3-axis accelerometer (jd mate, kissei comtec, matsumoto, japan) was used to measure energy expenditure (ee) at 1 hz; these measurements were performed continuously . Before and after tasks, systolic and diastolic blood pressure (sbp and dbp, respectively) were measured with a hemodynamometer (h55, terumo, tokyo, japan), and the oxygen saturation (spo2) was measured with a pulse oximeter (bo-650, japan precision instruments, shibukawa, japan). The degree of fatigue was also assessed with the borg cr10 scale, but only in healthy subjects21, 22 . The sound was generated by a metronome (me-110, yamaha corporation, hamamatsu, japan). The variance of normality of the data was tested by the shapiro - wilk test . Comparison between the 2 groups was performed using the unpaired student t - test or mann - whitney u - test for non - repeated measures and using the paired student t - test or wilcoxon signed - rank test for repeated measures . In the comparison among 3 or more - sample designs, one - way analysis of variance (anova) or friedman test was used in the case of 1 factor, and two - way anova was used in 2 factors . In multiple comparisons, as a post hoc test, bonferroni correction or the wilcoxon signed - rank test was used, as appropriate . Correlation between the 2 groups was examined using the pearson product - moment correlation coefficient . All analyses were conducted using the pasw statistics software (version 18.0, spss, inc . In healthy adult subjects, the sbp, maximum heart rate (hrmax), and cr10 score showed significant interaction effects between the evaluated points (pre vs. post) and tests (c6mwt vs. ps6mwt) (table 3table 3.changes in clinical parameters between before (pre) and after (post) the c6mwt and ps6mwt in the healthy adult subjectsc6mwtps6mwtpre vs. postc6mwt vs. ps6mwtinteractionprepostprepostsbp (mmhg)122.7 9.8135.0 13.5122.9 13.0146.8 13.4*******dbp (mmhg)74.6 9.179.1 9.376.2 11.183.2 8.7spo2 (%) 98.0 0.697.9 0.398.3 0.598.0 0.6hrmax (bpm)79.7 10.9133.5 25.178.4 14.6161.9 24.3******cr100.2 0.43.1 1.40.2 0.35.8 1.4*****ee (kcal / kg / min)0.105 0.0170.130 0.020distance (m)665.1 73.8791.3 61.3number of steps(steps / min)120.1 17.4147.7 10.8**p<0.001 . Anova (no normality). Measured before the c6mwt or ps6mwt while sitting on a chair . Measured during the c6mwt or ps6mwt . Paired student s t - test c6mwt: conventional 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; cr10: borg cr10 scale; ee: energy expenditure). The ee and number of steps were significantly different between the c6mwt and ps6mwt . A significant difference in the 6mwd was obtained between the c6mwt (665.1 73.8 m) and ps6mwt (791.3 61.3 m) (p <0.001) (table 3). The hrmax for the ps6mwt was significantly higher than that for the c6mwt (p <0.001), and the hrmax for the pswt was significantly higher than that for the c6mwt (p <0.01) and lower than that for the ps6mwt (p <0.001). After the pswt were significantly higher than those before the pswt (p <0.001) (table 4table 4.changes in clinical parameters between before (pre) and after (post) the pswt in the healthy adult subjectsprepostpre vs. post sbp (mmhg)120.3 12.0141.4 13.2dbp (mmhg)72.9 9.583.3 10.6spo2 (%) 97.9 0.598.2 0.6hrmax (bpm)75.9 14.1148.2 24.4cr100.1 0.24.9 1.9***p <0.001 . Paired student s t - test . Pswt: period shortening walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; cr10: borg cr10 scaletable 5.changes in clinical parameters between before (pre) and after (post) the c6mwt and ps6mwt in the patients with duchenne muscular dystrophyc6mwtps6mwtpre vs. postc6mwt vs. ps6mwtinteractionprepostprepostsbp (mmhg)101.7 14.498.6 15.784.4 15.497.1 14.3dbp (mmhg)61.3 8.064.3 14.255.7 10.256.5 5.8spo2 (%) 98.5 0.598.8 0.498.7 0.598.3 0.5hrmax (bpm)98.6 11.5144.5 11.096.7 12.3149.7 10.3ee (kcal / kg / min)0.100 0.0130.116 0.020distance (m)386.2 33.4427.4 32.5number of steps(steps / min)143.2 10.0151.0 10.0p<0.01; * * p<0.001 . Anova (no normality). Measured before the c6mwt or ps6mwt while sitting on a chair . Measured during the c6mwt or ps6mwt . Anova . C6mwt: conventional 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; ee: energy expenditure). The 1-minute walking distance in the ps6mwt was significantly longer than that in the c6mwt (p <0.001). The distance covered in the first minute was significantly longer than that in the second (p <0.001) and third minutes (p the statistical analyses were performed using two - way anova followed by the bonferroni post hoc test . C6mwt: 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test . * * p <0.01; * * * p <0.001). Among the walking distances for each sound interval in the pswt, the 1-minute distance at free speed (no sound) was significantly shorter than that with the bps (fig . All subjects (n = 20) walked at their usual speed with no sound (free) and then walked with 20, 10, 0 (bps), and + 10 steps / minute compared with the bps . (b) the number of steps was measured for each of the abovementioned conditions . The statistical analyses of the values were performed using one - way anova, followed by the bonferroni post hoc test . * p <0.05; * * p <0.01; * * * p <0.001). The distance with the bps was significantly longer than those of the sound rates with 20, 10, and + 10 steps / min compared with the bps . The number of steps at each sound interval in the pswt was increased until it reached + 10 steps / minute compared with the bps (fig . The results also indicated that the 1-min walking distance with the bps in the pswt was significantly correlated with the 6mwd in the ps6mwt (r = 0.738, p <0.001) but not with that in the c6mwt (fig . 4.correlations of the c6mwt and ps6mwt distances with the pswt distance in healthy adults . (a) the c6mwt distance was not significantly correlated with the 1-minute walking distance with the bps in the pswt . (b) the ps6mwt distance was positively correlated with the 1-minute walking distance with the bps in the pswt (r = 0.738, p <0.001). C6mwt: 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; pswt: period shortening walk test; bps: best periodic sound). * * * p<0.001 . Anova . Anova (no normality). Measured before the c6mwt or ps6mwt while sitting on a chair . Measured during the c6mwt or ps6mwt . Paired student s t - test c6mwt: conventional 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; cr10: borg cr10 scale; ee: energy expenditure * * * p <0.001 . Paired student s t - test . Pswt: period shortening walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; cr10: borg cr10 scale * * p<0.01; * * * p<0.001 . Anova (no normality). Measured before the c6mwt or ps6mwt while sitting on a chair . Measured during the c6mwt or ps6mwt . Anova c6mwt: conventional 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate; ee: energy expenditure changes in the c6mwt and ps6mwt distances in healthy adults . The statistical analyses were performed using two - way anova followed by the bonferroni post hoc test . C6mwt: 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test . * * p <0.01; * * * p <0.001 changes in the pswt distance and number of steps in healthy adults . All subjects (n = 20) walked at their usual speed with no sound (free) and then walked with 20, 10, 0 (bps), and + 10 steps / minute compared with the bps . (b) the number of steps was measured for each of the abovementioned conditions . The statistical analyses of the values were performed using one - way anova, followed by the bonferroni post hoc test . Pswt: period shortening walk test; bps: best periodic sound . * p <0.05; * * p <0.01; * * * p <0.001 correlations of the c6mwt and ps6mwt distances with the pswt distance in healthy adults . (a) the c6mwt distance was not significantly correlated with the 1-minute walking distance with the bps in the pswt . (b) the ps6mwt distance was positively correlated with the 1-minute walking distance with the bps in the pswt (r = 0.738, p <0.001). C6mwt: 6-minute walk test; ps6mwt: periodic sound - based 6-minute walk test; pswt: period shortening walk test; bps: best periodic sound in the results of the patients with dmd, the 6mwd in the ps6mwt (427.4 32.5 m) was significantly longer than that in the c6mwt (386.2 33.4 m) (p <0.01). The hrmax after the pswt was significantly increased compared with that before the pswt (p <0.01) (table 6table 6.changes in clinical parameters between before (pre) and after (post) the pswt in the patients with duchenne muscular dystrophyprepostpre vs. postsbp (mmhg)101.0 14.599.3 11.3dbp (mmhg)62.9 16.662.1 13.7spo2 (%) 98.2 0.798.5 0.8hrmax (bpm)111.0 8.0149.8 10.9***p<0.01 . Paired student s t - test . Pswt: period shortening walking test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate). The 1-min walking distance in the ps6mwt was significantly longer than that in the c6mwt (p <0.001). The results showed a significant correlation between the 1-min walking distance with the bps and the 6mwd in the ps6mwt (r = 0.884, p <0.05), but not between the 1-min walking distance with the bps and the 6mwd in the c6mwt . * p<0.01 . Pswt: period shortening walking test; sbp: systolic blood pressure; dbp: diastolic blood pressure; spo2: oxygen saturation; hrmax: maximum heart rate among the healthy controls, it was found that the 6mwd in the ps6mwt was significantly longer than that in the c6mwt . The 6mwd in the 6mwt in healthy elderly subjects has been reported to be influenced by age, height, weight, and gender16, 23 . Previous studies reported 6mwd values for the 6mwt in healthy adults of 670.1 m24 and 654.7 m25; the walking distances were comparable to the data for the c6mwt in the present study (table 3). The ps6mwt was also safe to administer, with no subjects falling or stopping exercise . The sbp, hrmax, and cr10 score showed significant interactions between the evaluated points and tests, suggesting that the physical load in the ps6mwt was greater than that in the c6mwt (table 3). Therefore, the ps6mwt could provide a more accurate evaluation of ambulatory potential compared with the c6mwt . The results also indicated a highly positive correlation between the 1-min walking distance with the bps in the pswt and the 6mwd in the ps6mwt . Because hrmax was significantly lower during the pswt than during the ps6mwt, the pswt could be conducted at a lower physical load and may be available for evaluation of physical endurance . The results showed that the 6mwd in the ps6mwt was significantly longer than that in the c6mwt (table 5). It has been reported that a decrease of motivation or concentration can affect the 6mwt performance in children with dmd20 . Several studies have reported that some patients were unable or unwilling to complete the 6mwt, even with permitted rest periods20, 26, 27 . The 2-minute walk test (2mwt) has been recommended for healthy adults, healthy children, and cardiac surgery patients26,27,28 . A previous study suggested that the 6mwd and 2-minute walking distance were highly correlated in the 6mwt29 . However, if the subjects were instructed to complete the test in just 2 minutes, the walking distance on the 2mwt might be further prolonged . A better method may be to stop at 2 minutes during the 6mwt, but this cannot be done repeatedly because of the difference in motivation of the subjects regarding the tests . It is necessary to confirm if the 2mwt is indicative of physical endurance in patients with dmd . Moreover, the results also indicated a significant correlation between the 1-minute walking distance with the bps and the 6mwd in the ps6mwt . The pswt is expected to be a better indicator of ambulatory potential in an evaluation of physical endurance compared with the c6mwt in patients with dmd . The results revealed that the patients did not show any adverse changes including the spo2, and none of the patients dropped out or wanted to stop the task halfway; therefore, it was consider that this experiment was conducted in a safe manner . In this study, a 6mwt based on a regular metronome sound was developed, and the bps, that is, the sound used when the subject walked the longest distance in 1 minute, was determined . The ps6mwt was administered to healthy young adults and ambulant patients with dmd, and the 6mwd was compared between the ps6mwt and c6mwt . All subjects showed a significantly longer 6mwd in the ps6mwt than in the c6mwt, and the 1-minute walking distance with the bps was significantly correlated with the ps6mwt distance . Both the ps6mwt and pswt may be useful in the evaluation of physical endurance.
The central paradigm of the microelectronics industry is the method of planar processing: the sequential deposition and patterning of thin films on the surface of a wafer of substrate material . Very often, the substrate is a single crystal, and the films need to be epitaxial, that is to say in crystal register with the underlying substrate . With semiconductor materials, this is typically achieved either using molecular beam epitaxy (mbe) in a laboratory setting or metalorganic vapor phase epitaxy (movpe) in manufacturing . Whilst the epitaxial growth of metals by mbe is possible, they are easily deposited by sputtering, and this is the most common method for the deposition of thin magnetic films in both research and industrial settings . Whilst this method is commonly associated with the growth of polycrystalline films, epitaxial growth on a single crystal substrate these generally include a raised substrate temperature (at least for the initial layers), a slow deposition rate, and a low vacuum chamber base pressure . This approach has been used to prepare giant magnetoresistance multilayer materials, for instance . In our own laboratory, we have used epitaxial sputtering to prepare a variety of magnetic materials on single crystal substrates . It has been possible to grow cofe alloy epilayers on gaas(001), for instance, by selecting the lattice - matched co70fe30 composition . This material is a solid solution, where the co and fe atoms randomly populate the bcc lattice sites . We have also grown chemically ordered magnetic alloys, where the different atomic species are required to take up particular lattice sites . The growth protocol we shall describe here was initially developed for the growth of l10-ordered fepd and fept alloys, which are of interest since they possess a very high magnetocrystalline anisotropy . We have studied the relationship between ballistic and diffusive spin - polarized transport and the anomalous hall effect in these materials, which are of comparable quality to layers grown by mbe . Here fe and rh will form alloys at any composition, however a b2-ordered compound is the equilibrium state for stoichiometries in the near - equiatomic range 49 - 53% atomic fe . This so - called -phase is an antiferromagnet (af) that exhibits a first - order phase transition on heating, becoming an '-phase ferromagnet (fm) around tt = 350400k . This metamagnetic transition between the two different but both fully ordered magnetic states (type ii af and fm) is accompanied by an isotropic 1% volume expansion in the b2 lattice, a large entropy release, a large drop in the resistivity, and a large increase in the carrier concentration . Neutron diffraction and more recently xmcd measurements indicate that part of the 3.3 b magnetic moment centered on the fe in the af phase is transferred to the rh in the fm phase, with fe~2.2 b and rh ~0.6 b . The curie temperature for the fm' phase is ~670 k, comparable to the curie temperature of alloys with x>0.53 . The metamagnetic transition temperature tt is highly sensitive to the composition in ferh1-, and is suppressed by ~8 k / t of applied magnetic field . This rich array of physical behavior depends critically on achieving the proper b2-ordered structure and so permits a wide variety of measurement techniques to be deployed to detect proper chemical ordering in a specimen, making it a convenient example to demonstrate a method of growing high - quality ordered alloy epilayers . In this protocol, thin films of the ordered ferh alloy are made with dc - magnetron sputtering on mgo (001) substrates . The samples are grown in a magnetic field of about 200 oe provided by a permanent magnet array, which is used to set an in - plane magnetic anisotropy . The target diameter is 50 mm and the distance between the target and substrates is about 10 cm . To grow ferh, the heaters are bulbs positioned 2 cm above the substrates and surrounded by a metal cylinder to keep the heated volume small . The maximum temperature possible in this system is ~1,050 k. this system is capable of holding 24 different substrates; however, we typically grow fewer than 10 when making epitaxial samples due to time constraints . The details presented here for this sample preparation protocol are known to work well in our vacuum system . As there are many equivalent vacuum systems that differ in their details, the requirements for the quantitative parameters such as temperature, time, etc . May well take different optimal values in other systems . In the detailed protocol below, it is assumed that the reader is familiar with the basics of good vacuum practice, such as the use of gloves to handle all components that will enter the vacuum chamber [see, for instance, reference 26]. This section describes the preparation of the sputter deposition chamber and the single crystal mgo substrates . Rinse the (001) mgo substrates in isopropanol and mount them in the substrate holders . Mount the ferh target in the magnetron gun and reassemble the gun . For a sample with an equiatomic composition, we have found that a target with fe47rh53 is most suitable, yielding the clearest magnetostructural phase transition . Once the vacuum is better than 1 x 10 torr, heat the substrates to 870 k. monitor the vacuum level and heating rate to ensure that the pressure does not rise above this level . One hour before commencing growth, begin to flow liquid nitrogen through the meissner trap . The pressure in the chamber should rise to the low mtorr range . Before the growth, pre - sputter the ferh target for 1,200 sec at 30 w. this section describes the deposition of the ferh layer by dc - magnetron sputtering . Adjust the set point of the mass flow controller to give a chamber pressure of 4 x 10 torr . Apply power to the magnetron to yield an overall deposition rate of 0.4 / sec . In chambers equipped with a quartz crystal monitor, if the chamber does not have a quartz crystal monitor, post - growth thickness measurements can be helpful, as well as a high level of reproducibility between runs . Open the shutter and deposit ferh on the heated substrate for a length of time suitable to give the desired thickness . For instance, a 500 sec deposition will yield a sample 20 nm thick . Shut off power to the magnetron . Increase the sample temperature to 970 k. hold the samples at this temperature for one hour . The effects of varying this anneal temperature can be found in de vries et al . Shut off heater power and cool the samples to room temperature . In this system, this takes at least three hours . Deposit any capping layer required, using steps similar to steps 2.1 - 2.7 . Depositing the capping layer at a temperature below ~370 k is essential to prevent interdiffusion into the ferh layer . This section provides an overview of the basic characterization steps carried out on the majority of our ferh samples . As there are many possible equivalent methods for making these measurements, the nature of the descriptions here are less detailed and prescriptive, and rather concentrate on the essential features of any such measurements . Mount the sample in the diffractometer and align it in with the detector angle 2 1. if available, should also be aligned . Run a standard -2 scan with running from 0 until the noise floor of the instrument is reached, typically once 6 for a good quality sample . Clear kiessig (thin - film interference) fringes should be visible, from which the epilayer thickness can be determined . Perform a high - angle -ray diffraction scan to determine the degree of chemical order . This can be carried out with the sample still mounted in the diffractometer from step 3.1 . The mgo substrate peak should be found (at 2 = 42.9 if cu k radiation is used) and the sample aligned again in . Again, run a -2, covering at least the range 12.5<<62.5 (again assuming cu k radiation) so that both the ferh (001) and (002) peaks, as well as the substrate peak, are captured . Perform a measurement of the temperature dependence of the sample resistivity to determine the transition temperature . Make electrical contacts to the sample such that a standard 4-point measurement can be made to avoid contact resistance problems . If a dc method is being used, make measurements for forward and reverse current directions and the resistances averaged in order to null off any thermal e.m.f . Then place the sample on a temperature controlled hot stage (in this set - up the stage is in a small turbo - pumped high vacuum chamber to be sure of avoiding any oxidation), and measure the resistance as a function of temperature on both heating and cooling sweeps so that any hysteresis in the first order magnetostructural phase transition can be determined . This section describes the preparation of the sputter deposition chamber and the single crystal mgo substrates . Rinse the (001) mgo substrates in isopropanol and mount them in the substrate holders . Mount the ferh target in the magnetron gun and reassemble the gun . For a sample with an equiatomic composition, we have found that a target with fe47rh53 is most suitable, yielding the clearest magnetostructural phase transition . Once the vacuum is better than 1 x 10 torr, heat the substrates to 870 k. monitor the vacuum level and heating rate to ensure that the pressure does not rise above this level . One hour before commencing growth, begin to flow liquid nitrogen through the meissner trap . The pressure in the chamber should rise to the low mtorr range . Before the growth, pre - adjust the set point of the mass flow controller to give a chamber pressure of 4 x 10 torr . Apply power to the magnetron to yield an overall deposition rate of 0.4 / sec . In chambers equipped with a quartz crystal monitor, if the chamber does not have a quartz crystal monitor, post - growth thickness measurements can be helpful, as well as a high level of reproducibility between runs . Open the shutter and deposit ferh on the heated substrate for a length of time suitable to give the desired thickness . For instance, a 500 sec deposition will yield a sample 20 nm thick . Shut off power to the magnetron . Increase the sample temperature to 970 k. hold the samples at this temperature for one hour . The effects of varying this anneal temperature can be found in de vries et al . Shut off heater power and cool the samples to room temperature . In this system, this takes at least three hours . Depositing the capping layer at a temperature below ~370 k is essential to prevent interdiffusion into the ferh layer . This section provides an overview of the basic characterization steps carried out on the majority of our ferh samples . As there are many possible equivalent methods for making these measurements, the nature of the descriptions here are less detailed and prescriptive, and rather concentrate on the essential features of any such measurements . Mount the sample in the diffractometer and align it in with the detector angle 2 1. if available, should also be aligned . Run a standard -2 scan with running from 0 until the noise floor of the instrument is reached, typically once 6 for a good quality sample . Clear kiessig (thin - film interference) fringes should be visible, from which the epilayer thickness can be determined . Perform a high - angle -ray diffraction scan to determine the degree of chemical order . This can be carried out with the sample still mounted in the diffractometer from step 3.1 . The mgo substrate peak should be found (at 2 = 42.9 if cu k radiation is used) and the sample aligned again in . Again, run a -2, covering at least the range 12.5<<62.5 (again assuming cu k radiation) so that both the ferh (001) and (002) peaks, as well as the substrate peak, are captured . Perform a measurement of the temperature dependence of the sample resistivity to determine the transition temperature . Make electrical contacts to the sample such that a standard 4-point measurement can be made to avoid contact resistance problems . If a dc method is being used, make measurements for forward and reverse current directions and the resistances averaged in order to null off any thermal e.m.f . Then place the sample on a temperature controlled hot stage (in this set - up the stage is in a small turbo - pumped high vacuum chamber to be sure of avoiding any oxidation), and measure the resistance as a function of temperature on both heating and cooling sweeps so that any hysteresis in the first order magnetostructural phase transition can be determined . We show typical results obtained using the most common characterization procedures for a selection of representative samples . Results such as these are expected for samples in the thickness range 20 - 50 nm . Other methods we have used to characterize our material in more depth include x - ray magnetic circular dichroism, grazing incidence x - ray scattering, and polarized neutron reflectometry . We have also studied the effects of doping the alloy with au . Further data on the properties that can be expected from this material can be found in those reports, and the references contained therein . The structure of one of our epilayers is shown in detail in the transmission electron micrographs shown in figure 1 . The sample cross - section was prepared by the conventional dimpling and ion polishing technique- a standard specimen preparation method (see, for instance williams and carter)- and observed using a 200 kv electron beam . The overall layer structure can be seen in figure 1(a). In this case, a 30 nm ferh film was epitaxially grown onto an mgo substrate, followed by a ~4 nm cr layer and a ~1 nm thick al layer . (the cr layer was included here for a particular experiment and is not needed in general). The roughness of the ferh / mgo and ferh / cr interfaces are 0.6 nm and 2.8 nm, respectively, as measured from the image . In figure 1(b) a high resolution micrograph of the mgo / ferh interface is shown . The epitaxial relationship the lattice matching across the interface demonstrates the high quality of the epitaxial growth . We do not show the data here but have used energy dispersive x - ray spectroscopy in the tem to check the composition on a selection of samples -ray reflectometry data are shown in figure 2 for a nominally 25 nm thick ferh epilayer capped with a thin, polycrystalline layer of al . The measurement was performed in a standard two - circle diffractometer in the bragg - brentano geometry, using cu k radiation (= 0.1541 nm), with a ni filter to attenuate the k radiation . The pronounced kiessig fringes, which arise from the interference of x - ray beams that reflect from the various interfaces in the layer stack, indicate that those interfaces are smooth and well - correlated . The solid red line shows a fit to the data that has been performed using the genx software . The fact that a portion of the al layer will have oxidized and self - passivated once the sample is exposed to the air is accounted for in the model . -ray diffraction data for the same sample are shown in figure 3, collected on the same instrument . The (002) reflection of the mgo substrate is strong and sharp enough to just resolve the cu k1and k2 lines . There is some broadening due to the finite thickness of the epilayer and strain gradients . The (002) ferh b2 peak is centered at 2 = 61.30.02, yielding an average out - of - plane lattice constant of 3.020.05 . It is possible to determine the chemical order parameter s of the ferh b2 structure from the relative integrated intensities of these two peaks . This quantity is defined as s = rfe+rrh -1, where is the fraction of fe(rh) sites occupied by fe(rh) atoms . A brief inspection of the formula shows that when rfe = rrh = 1and the structure is perfect, s = 1, whereas when rfe = rrh = 0.5, so that all the lattice sites are randomly occupied, s = 0 . The reason is that when s = 0 the site - averaged structure is bcc, for which the structure factor forbids the (001) reflection, whereas when s = 1 the structure is primitive cubic, for which the (001) reflection is allowed . This means that in practical terms,, where and are the experimental and theoretical intensities of the (00i) bragg reflection, respectively . For the calculation of the theoretical intensities the debye - waller factors from exafs measurements on ferh were used . In this case, s = 0.8550.001, typical for a sputtered thin film of this material . The lattice expansion that accompanies the metamagnetic transition may be detected by the shift in the position of the bragg peaks; however, this requires a diffractometer with a heater stage . Perhaps the most obvious method is to detect the appearance of the ferromagnetic moment as the sample is heated through tt . This can be done using any temperature dependent magnetometer with sufficient sensitivity, for instance using the magneto - optical kerr effect or a vibrating sample magnetometer . In figure 4 we show the temperature dependence of the magnetization m, measured using a superconducting quantum interference device (squid) magnetometer . Measurements were made in the temperature range of 275 - 400 k with a temperature sweep rate of 2 k / min . The curve shown displays the anticipated af fm transition (heating) and fm af transition (cooling) with a 15 k thermal hysteresis . This measurement was made at high field (50 koe) and yielded a transition temperature tt 365 k. the transition temperature is field - dependent, as a higher magnetic field reduces the free energy of the fm phase with respect to the af phase . Note that the magnetic moment in the af phase is not quite zero, but is a few tens of emu / cmwhen averaged over the volume of the entire sample . This moment resides in the near - interface regions of the ferh epilayer, which remain ferromagnetic (albeit with a reduced magnetization) when the bulk of the sample transforms into the af phase . A way to detect the transition that uses simpler equipment and the simplest measurement is of the resistivity of the film, since in the fm phase is much less than in the af phase . The temperature dependence of for the same 25 nm ferh epilayer for which x - ray data were shown is plotted in figure 5, measured using a standard four - point probe method: spring - loaded, gold - plated pins were pressed on to the sample surface to make contact to the sample, which was mounted on a heater stage in a small custom vacuum chamber to prevent any sample oxidation when hot . A linear, metallic (t) dependence is seen in both the af and fm phases, but there is a marked drop in resistivity between the two . The hysteresis seen in figure 5 is a clear fingerprint of the magnetostructural phase transition taking place and is a convenient method to measure the transition temperature, which is given by the minimum point in d/dt (shown in the inset of figure 5). Another easily measured transport property, the hall effect, can also be used to confirm the presence of the transition, as there is a large difference in the hall coefficient between the two phases . The ferh is 30 nm thick with a further ~4 nm cr layer and ~1 nm al cap deposited on top . The amorphous region at the top of the image is an epoxy resin used during cross - section sample preparation . (b) a high resolution image of the mgo ferh interface . The epitaxial matching across the interface is seen here, and the associated relationship, as confirmed from selected area diffraction, is ferh(001)\|\|mgo(001). Click here to view larger figure figure 2 . X - ray reflectometry spectrum from a 25 nm thick ferh epilayer capped with polycrystalline al . The solid line is a fit as described in the text, using the parameters given in table 1 . The inset shows the scattering length density profile associated with that set of fitting parameters . X - ray diffraction spectrum from a 25 nm thick ferh epilayer capped with polycrystalline al . The presence of the (001) ferh peak indicates that b2 ordering has taken place . The chemical order parameter is s = 0.8550.001, as determined using the method described in the text . Temperature dependence of magnetization m of a 50 nm thick ferh epilayer capped with polycrystalline al . The transition temperature tt is seen to be ~365 k with a hysteresis width of about 15 k. click here to view larger figure figure 5 . Temperature dependence of the resistivity of a 25 nm thick ferh layer capped with al . Inset is the derivative of with respect to temperature t. the transition temperature tt is seen to be 447 k on warming into the fm phase and 375 k on cooling into the af phase . Fitting parameters for the x - ray reflectivity spectrum shown in figure 2, leading to the scattering length density profile shown in the inset of that figure . Here we have demonstrated that this method can be used to prepare epilayer samples of ferh of good crystallographic quality and a high degree of b2 chemical ordering . The method is suited to the preparation of a wide variety of epitaxial metallic layers, including ordered alloys . Whilst we have used the b2-ordered ferh alloy as an example here, as it shows a dramatic phase transition when the stoichiometry is correct and chemical ordering is present, this method can also be used for other materials . For instance, both fepd and fept have l10 phases, which leads to a very strong uniaxial magnetocrystalline anisotropy . We have successfully grown this material in the past, showing domain wall resistance in fept, and large anomalous hall effects in both fepd and fept . With an appropriate adjustment of growth temperatures and rates and a suitable choice of substrate, this method ought to be useful for preparing a wide variety of different magnetic and non - magnetic metal epilayers displaying chemical order . Nevertheless, a limitation of this approach is the need for a single crystal substrate to achieve epitaxy . This means difficulties will be encountered in performing experiments such as plan - view transmission electron or x - ray microscopy or integration into a technology built on another substrate wafer such as the near - ubiquitous si . A possible means to get around this problem is to grow a thin mgo layer on which the ferh can then be deposited . This can yield out - of - plane texture that nucleates local epitaxial growth on top of each mgo grain . Remarkably, it is possible to grow a thin mgo layer that has both (001) texture and in - plane crystallographic alignment on an amorphous surface using a method with an ion - beam assist gun that is oriented at 45 to the substrate normal . This could permit growth of b2-ordered ferh on e.g. Electron or x - ray transparent si3n4 membranes, which are capable of surviving the high growth temperatures required in our protocol, or on the native oxide layer of a si wafer . Further refinements of the method include the use of b2-ordered underlayers, such as nial, to promote b2-ordering in the ferh epilayer when it is ultrathin, or its use to build heterostructures involving multiple chemically - ordered layers . Since ferh can be doped on the rh site to adjust the transition temperature tt up (for example using ir or pt) or down (e.g. Using au or pd), the creation of doping profiles in ferh layers can lead to designed - in magnetic profiles as the sample is heated and cooled . This opens up a route to generating purely magnetic stratification of an epilayer in a controllable way.
To critically inspect written evidence and cover all available information relating to the presence of tunga spp . In peru, we searched for all documented names ascribed to this parasite over the past 4 centuries (1619). We used 35 local terms (nigua, nihua, niua, pique, pigue, piqui, piki, pico, sico, seccec, chegoe, chego, chigger, puce - chique, puce de sable, chique, chica, bicho de p, bicho do porco, pulga de areia, jatecuba, jigger, chicque, sand flea, tchike, tschike, sike, xique, ckicke, aagrani, atten, tom, t, tungay, and tunga) and 9 scientific terms (pulex minimus cutem penetrans americanus, pulex minutissimun nigricans, acarus fuscus sub cutem nidulans proboscide acutiore, pulex penetrans, rhynchoprion penetrans, sarcophaga penetrans, dermatophilus penetrans, sarcopsylla penetrans, and tunga penetrans). Using on - site electronic catalogs, we screened all available manuscripts, books, doctoral theses, journals, bulletins, monographs, and periodicals in their original english, spanish, or french from 2 major sources: the main campus library of the school of medicine at cayetano heredia peruvian university, in lima, peru, and the william h. welch medical library, institute of history of medicine at johns hopkins university, in baltimore, maryland, usa . These searches were complemented by using the pubmed, lilacs, scielo, and medline electronic databases with no publication date- or language - based restrictions . After screening the written material to identify the locations of ceramics portraying tungiasis, we assessed earthenware representations through visits to selected private collections of pre - incan pottery at the amano museum foundation in miraflores, lima, peru, and the halls of mexico, central and south american peoples at the american museum of natural history in new york, new york, usa . These museums were the only facilities cited at least 1 time as potential depositories of pottery depicting pre - incan tungiasis . All anthropomorphic ceramics that depicted> 1 nodule - like representations on the lower or upper extremities, either localized or clustered, with or without representations of holes in the soles of the feet and irrespective of the presence of a central depression, were deemed possible depictions of tunga spp . Infection . From each museum, 50 pieces were screened; data on the date and location of findings were recorded when they fulfilled the criteria for possible depiction of tungiasis . A complete screening of the entire collection of ceramics representing diseases of ancient peruvians was possible only at the amano museum foundation . We found written evidence of tungiasis in pre - incan or incan times in 17 documents (7 in english, 4 in french, and 6 in spanish) (table 1). The documents were 1 unique 17th - century manuscript written by the indigenous peruvian chronicler guaman poma de ayala (finalized during 16151616), 1 monograph, 1 bulletin, 2 doctoral theses, 5 books, and 7 journals . All sources are cited exactly as they appeared on the date of publication and in original languages . This ancient and unique manuscript by the indigenous peruvian chronicler guaman poma de ayala has been digitalized by the department of manuscripts and rare books, the royal library of denmark, and is available at www.kb.dk/permalink/2006/poma/info/en/frontpage.htm sources in which reproductions of tungiasis - depicting potteries are available . As for the earthenware representations, we identified 4 anthropomorphic figures representing pre - incan tungiasis (table 2). Of these 4 figures, 3 were reproduced in the written materials surveyed (1 from an unknown location and 2 from the american museum of natural history), and 1 was a piece of polychromic ceramic, located in the amano museum foundation, which had not been previously described . * estimated flourishing period of the culture . The 2 vessels described by ashmead, and subsequently reproduced by moodie and dharcourt, and the fragment identified during this study are not publicly exhibited . 1219, is stored with many other vessels that represent diseases of the ancient peruvians . The anthropomorphic pottery shown in figure 2 originated from the chimu culture (c. ad 12001470). It is a single - spout bottle that represents a man holding a pointed object and depicts multiple holes in the sole of his left foot . It was found in the chicama valley, ascope, la libertad (figure 1, panel b), and its current location is unknown . Chimu culture huaco depicting a person extracting parasites with an awl from the sole of the left foot . The 2 pieces of anthropomorphic pottery shown in figure 3 also originated from the chimu culture . They depict 2 men observing the soles of their feet, which show multiple holes of varying sizes . The pieces are located in the american museum of natural history but are not on display . They had been found in the pachacamac valley, a sandy area in modern southern lima (figure 1, panel b). Two globular chimu huacos found in pachacamac, a sandy land area in northern lima . Each person is examining the soles of the feet, on which multiple punch - out lesions can be detected . Panels b and d are close - up views of the feet of the huacos shown in panels a and c, respectively . Catalogs b/8853 and b/8854, courtesy of the department of anthropology, american museum of natural history . The anthropomorphic piece shown in figure 4 originated from the maranga culture (c. ad 150650). It is a fragment that portrays a person whose right arm, upper torso, and head are broken off . The left arm and leg are decorated with black, triangle - shaped tattoos arranged in a linear distribution . The person is using a stick to extract foreign bodies from a cluster of elevated lesions with central holes in the heel of the left foot . There are also 8 holes in the posterior external aspect of the sole, which are clustered and highlighted by a brick - red background . This piece was located in a storage room at the amano museum foundation and, to our knowledge, has not been previously described or reproduced . It was originally found in las pampas, surquillo, lima (figure 1, panel b). A) polychromic maranga culture fragment that portrays a torso and a tattooed left leg of a person holding a stick while extracting foreign bodies . Cluster lesions with elevated nodules and a central black depression suggest tunga spp . Infection . C) details of the sole of the left foot, showing multiple holes over a brick - red surface, suggesting residual tungiasis lesions . No . 1219, courtesy of the amano museum foundation . Tungiasis is an old disease that has been endemic to peru for centuries and has been illustrated by anthropomorphic pottery showing pathognomonic lesions at various stages of progression . Although the incas and their ancestors lacked a written language, they used pottery to depict diseases, customs, ceremonies, rituals, and many other activities, thus creating a visual record of their knowledge of a disease process that existed for centuries; such pieces of pottery now provide vivid documentation of their sufferings . The huaco from chicama valley (figure 2) was described by the harvard - educated peruvian archeologist julio c. tello (18801947). Tello was the first indigenous archeologist of america and is considered the father of peruvian archeology . In 1924, he reproduced this vessel in a collection of 280 pictures of pottery originating from the mochica (moche or muchik) culture titled arte antiguo peruano, volume ii; all pieces depicted in this work are distributed among various museums in lima (20). Multiple holes in the left plantar surface of the depicted person, a distinguishing feature of tungiasis, can be observed in this figure . Furthermore, the depicted person holds an awl - like instrument in its right hand, which was then commonly used for removing the parasite from the skin . As a physician, tello easily recognized these lesions as signs of tungiasis or piquinosis (pique infection) (22). However, this collection of pictures of mochican pottery lacks detailed information about where they were originally found or where they were at the time of its publication . Although it was reproduced as part of a mochica collection, this huaco actually originates from the chimu culture . Indeed, the mochica culture (bc 100ad 700) developed earlier than the chimu culture (ad 13001470); although both cultures flourished in the moche valley (figure 1, panel b), the chimu culture was a continuation of the mochica culture (31). In other words, the chimu culture was the same generation as the mochica culture, but the chimu culture had a somewhat different ceramic style . A reproduction of the same huaco (plate 65 in tello s mochica collection) was published 14 years earlier, in 1910, by albert s. ashmead (18501911) (30). Ashmead was one of the first north american physicians to study peruvian potteries that depicted diseases, predominantly leprosy and syphilis, at the beginning of the 20th century (32). He received a diverse array of pictures of huacos (including that in figure 2) directly from tello, with whom he corresponded regularly . With tello s permission, ashmead subsequently published his reproduction of this huaco (22). Unlike tello, ashmead documented the site at which this huaco was originally found, the chicama valley (this information was probably provided by the archeologist who discovered the piece). Nevertheless, ashmead did not associate these lesions with tungiasis and instead thought they were a product of syphilis . In a letter addressed to ashmead, tello uses the word piquinosis to describe to the tungiasis depicted on huaco 1; unfortunately, ashmead did not recognize this regional term (piquinosis = pique infection) used to designate tungiasis (22). The 2 huacos from the pachacamac valley (figure 3) were first published by ashmead in 1907 (21). As with the huaco from the chicama valley (figure 2), ashmead erroneously concluded that the lesions depicted on the soles of the feet of persons depicted on these 2 jars represented signs of uta, which he believed to be skin tuberculosis (the etiology of uta, or cutaneous leishmaniasis, in peru was later unveiled during the 1913 harvard expedition to the amazon region led by richard p. strong (33). Because he was interested in prehistoric syphilis and peruvian earthenware representing diseases, ashmead maintained correspondence with several renowned physicians from lima, including tello . However, ashmead never associated these 2 huacos with tungiasis, arguing that the holes in the feet were too prominent to represent tungiasis (32). It was paleopathologist roy l. moodie (18801934) and then americanist raoul dharcourt (18791971) who later reevaluated the significance of these vessels, both concurring that the holes on the feet of these 2 huacos represent residual lesions left by nigua infections (12,23,24,26). Pachacamac, the site at which these 2 jars were located, was not part of the chimu culture s territory (figure 1, panel b). Because the old sanctuary of pachacamac was the major place of worship of the pre - hispanic peruvian coast for> 1,500 years (31), its temples were visited by masses of pilgrims from the entire andean world, who carried with them diverse offerings, including huacos, during religious rituals and ceremonies . Thus, archeological pieces from the coastal, highland and amazon regions of peru can be found in pachacamac . During our visit to the amano museum foundation in 2009, we found the fragment of a huaco from las palmas (figure 4) in a private collection storage room . It had originally been excavated by yoshitaro amano (18981982), a prosperous japanese businessman who arrived in peru in 1951 and was captivated by its history . Pedro weiss (18931985), a peruvian pathologist who dedicated part of his life to the study of these potteries, mentioned that there were representations of niguas in this museum in his 1980 article la enfermedad en las creencias de los primitivos americanos; however, he neither photographed nor described any huacos (29). In contrast to the evidence we have for the previously described huacos, we do not have strong evidence proving that this fragment was the one described by weiss in his above - mentioned work . Together with the first 3 vessels described here, which were also cited by hoeppli in 1959 as early documentation of parasites in the western hemisphere (28), to our knowledge, this fragment is the fourth representation of tunga spp . Furthermore, it is the only vessel that depicts different stages of tungiasis, thus representing explicit evidence of its endemicity in ancient peru . Along with these 4 huacos, additional evidence suggests the presence of tungiasis in pre - incan peru . The 2 most common names attributed to the sand flea in peru and other countries of south america nigua and pique come from the arawak and quechua languages, respectively . Indeed, quechua was the official language of the incan empire and is currently the second most commonly spoken language in peru, after spanish . Furthermore, the incas named it seccec from the verb seccen, a quechua word that means itching (16,17). Currently, it is called huchuy piqui (or huchhuy piqui, according to lavoveria) or ushtuchi piki by amerindian communities in the highlands . Another aspect of pre - incan tungiasis is documentation of the therapeutic approaches by historians, anthropologists, and physicians . For example, in his book la mdecine dans lancien prou, dharcourt mentioned that peruvian natives used a stick to remove fleas from their feet (26), similar to what is observed on our fragment . In addition, lastres, in his compendious historia de la medicina peruana, mentioned nigua as being endemic to peru and described the application of sweet potatoes leaves to the feet to treat infections (27). Until now, numerous factors have impeded our understanding of the history of tungiasis in peru . First, the sand flea has been given multiple names by populations living in parasite - endemic areas, making literature searches difficult . Nigua, pique, jigger, chigoe, puce - chique, and tchique are only a few of the many names that have been given to this burrowing flea . Second, it has been taxonomically reclassified multiple times with different names by entomologists over the past 3 centuries (1619,34). Finally, the high rates of endemicity, along with a relatively uncomplicated clinical course, have made it a disease that is underreported and neglected among physicians in peru (8). Our search had some limitations . The dispersed distribution of these peruvian anthropomorphic pieces in art museums throughout the world made it difficult to document the exact number of pottery pieces that depict tungiasis (35). An unknown number of disease - illustrating huacos remain to be located and investigated . At the beginning of the spanish conquest, the conquerors looted religious places in their quest for gold, leaving behind innumerable pieces of pottery made by the incas and their predecessors . Later, at the beginning of the 20th century, theories about the people of the americas were propounded along with the study of pre - hispanic cultures . As a result, sacred places, ceremonial paraphernalia, and other anthropologic pieces in the coast and the andes were unearthed . In fact, ashmead and tello clearly stated that a large number of peruvian archeological pieces were highly prized on the black market in their time (36,37). Even today, substantial illicit traffic of ceramics from ancient peru continues, which has forced the international council of museums to include mochica vessels in the red list of latin american cultural objects at risk (38). Our photograph of the newly identified fragment depicting tungiasis provides additional evidence of tungiasis among ancient peruvians . The knowledge of this disease in pre - incan cultures is a valuable legacy that gives a historical insight into the endemicity of this arthropod in south america . Their identification and analytic evaluation is critical for enhancing our understanding of the history and effects of this flea that continues to affect peruvians today as it did in pre - incan times.
To evaluate pregnancy outcomes, complications and neonatal outcomes in women who had previously undergone uterine arterial embolization . A retrospective study of 187 patients treated with uterine arterial embolization for symptomatic uterine fibroids between 2005 - 2008 was performed . Uterine arterial embolization was performed using polyvinyl alcohol particles (500 - 900 m in diameter). Of these, 12.5% were miscarriages (n = 2), and 87.5% were successful live births (n = 14). The gestation time for the pregnancies with successful live births ranged from 36 to 39.2 weeks . The mean time between embolization and conception was 23.8 months (range, 554). The newborn weights (n = 14) ranged from 2.260 to 3.605 kg (mean, 3.072 kg). One (7.1%) was considered to have a low birth weight (2.260 kg). There were two cases of placenta accreta (12.5%, treated with hysterectomy in one case [6.3%]), one case of premature rupture of the membranes (prm) (6.3%), and one case of preeclampsia (6.3%). There were no other major obstetric risks, suggesting that pregnancy after uterine arterial embolization is possible without significant morbidity or mortality . Uterine artery embolization (uae), which has been described in numerous reports since 1995, is a recognized treatment for symptomatic uterine fibroids.1 the technical goal is to deliver particulate material (typically polyvinyl alcohol [pva] particles or gelatin - coated polymer microspheres) into both uterine arteries to induce ischemic alterations to the myomas without causing permanent damage to the uterus.2 - 4 despite many studies that have assessed uterine myomas and infertility, the mechanism underlying their detrimental reproductive effects remains to be determined . Similarly, studies evaluating the effects that uae may have on subsequent fertility and pregnancy have yielded conflicting and limited data.5,6 although there are valid concerns regarding the effects of uae on women who wish to retain fertility, pregnancy after this procedure is well documented . Pregnancy after uae has been described in case reports, a review article,7 retrospective series,8 - 10 and a retrospective cohort study that compared uae and laparoscopic myomectomy.11 a retrospective analysis by walker and mcdowell found 56 completed pregnancies in a series of 1200 patients who underwent uae . This cohort included 108 women who attempted pregnancy, and 30.5% of these pregnancies were categorized as successful . The authors noted that 14 of the women with successful pregnancies had failed myomectomies and had been offered a hysterectomy as their only option before undergoing uae.12 the main purpose of this study is to present the pregnancy outcomes and complication rates of uae . Between july 2005 and december 2008 all of the patients provided informed consent to participate in this study, and the local ethics committee approved the related protocol . This study was supported by the department of gynecology and the department of image diagnosis of the federal university of so paulo . The indications for uae were as follows: menorrhagia subjectively reported by the patient as increased or prolonged menstrual blood loss that caused dysfunction in daily life, pelvic pain, compressive symptoms in the urinary / gastrointestinal tract, and infertility related to fibroids . The exclusion criteria for uae were as follows: subserosal and submucosal fibroids, pelvic infections, gynecologic malignancy, an undiagnosed pelvic mass outside of the uterus, unexplained abnormal menstrual bleeding, coagulopathy, a history of pelvic irradiation, a fsh level> 40 iu / l, and adenomyosis indicated by magnetic resonance imaging . The patients were treated with prophylactic antibiotics (1 g of oral azithromycin on the day before the procedure and 2 g of intravenous cefalotin about sixty minutes before the procedure). A 4-f or 5-f catheter was introduced into the right femoral artery and advanced over the aortic bifurcation and into the contralateral internal iliac artery to identify the origin of the uterine artery . Uae was performed with pva particles (500 - 900 m in diameter). The patient preparation, anesthesia and analgesia details, procedure information, and discharge information were recorded . All of the patients provided informed consent to participate in this study, and the local ethics committee approved the related protocol . This study was supported by the department of gynecology and the department of image diagnosis of the federal university of so paulo . The indications for uae were as follows: menorrhagia subjectively reported by the patient as increased or prolonged menstrual blood loss that caused dysfunction in daily life, pelvic pain, compressive symptoms in the urinary / gastrointestinal tract, and infertility related to fibroids . The exclusion criteria for uae were as follows: subserosal and submucosal fibroids, pelvic infections, gynecologic malignancy, an undiagnosed pelvic mass outside of the uterus, unexplained abnormal menstrual bleeding, coagulopathy, a history of pelvic irradiation, a fsh level> 40 iu / l, and adenomyosis indicated by magnetic resonance imaging . The patients were treated with prophylactic antibiotics (1 g of oral azithromycin on the day before the procedure and 2 g of intravenous cefalotin about sixty minutes before the procedure). A 4-f or 5-f catheter was introduced into the right femoral artery and advanced over the aortic bifurcation and into the contralateral internal iliac artery to identify the origin of the uterine artery . Uae was performed with pva particles (500 - 900 m in diameter). The patient preparation, anesthesia and analgesia details, procedure information, and discharge information were recorded . A retrospective study of 187 women treated over a period of 3.5 years was performed . The mean patient age was 34.3 years (range, 26 - 40 years). Of the 187 women who underwent uae, 75 had been seeking to become pregnant at some point, and 15 of these 75 women became pregnant at least once . However, their outcomes were variable . The characteristics of the pregnancies are shown in table 1 . In the 187 women who underwent uae, the mean time between embolization and conception was 23.8 months (range, 554 months). One pregnancy occurred in the first year, 8 occurred in the second year, and 6 occurred in the third year following embolization . The pregnancies lasted at least 36 weeks, with only one pre - term delivery . All 13 pregnancies were delivered by cesarean section due to the obstetricians fearing complications from the pregnancies uterine artery embolizations . The indications for a cesarean section were as follows: the decision of the attending obstetrician, risk avoidance in patients who had long and painful histories of trying to conceive, an older primiparous patient with preeclampsia, a history of prior myomectomy, a macrosomic fetus, and a labor induction that had previous failed . The weights of the newborns ranged from 2260 - 3605 g. no cases exhibited chronic or acute fetal distress, and there was no impairment of vitality . Abnormal placental implantation (placenta accreta) occurred in two patients, and one case terminated in a puerperal hysterectomy . There was one case of premature membrane rupture at 29 weeks, which lasted until a cesarean section delivery at 36 weeks . This meta - analysis documented that uae results in a shorter hospitalization time and a reduction in blood loss (85% compared to pretreatment) and uterine size (30 - 49% compared to pretreatment).4 however, maintenance of fertility after uae is still controversial . Until now, the impact of this treatment on fertility has not been established.13,14 theoretically, uae may contribute to difficulties during embryonic implantation or the maintenance of pregnancy by decreasing the vascularity of the uterine myometrium and endometrium . In fact, embolization particles have been identified in structures adjacent to leiomyomas (e.g., myometrium, parametrium, and mesovarium) after uae.15 data are insufficient to conclude that uae is a safe option for women who wish to retain their fertility.16 normal pregnancy and delivery can be achieved after uae for uterine leiomyomata, as has been shown for postpartum hemorrhages . Comparing pregnancy outcomes after uae with leiomyomata is complicated by the size and location of the myomata and the fact that non - subserosal leiomyomas reduce fertility.17 uae pregnancy outcomes are reduced most severely when submucosal myomas cause endometrial distortion . Reports have not documented the number of women who have attempted pregnancy after embolization; therefore, cycle conception and fecundity rates cannot be calculated.18 in this series, we do not associate complication rates with their direct impacts on fertility, such as ovarian insufficiency . The authors reported that 27% of these pregnancies ended in miscarriage, with two terminations and one ectopic pregnancy . Of the 16 deliveries that occurred after 24 weeks, first- and second - trimester bleeding occurred in 40% and 33% of these deliveries, respectively, and 25% had preterm deliveries . Thirteen percent of the women had spontaneous preterm membrane ruptures, and the rate of primary postpartum hemorrhage was 20% . The mean birth weight of the term babies was 3.39 kg, and none required admission into the neonatal intensive care unit . There was one case of fetal growth restriction.19 another retrospective analysis was undertaken to evaluate the incidence and outcomes of pregnancies after uae for symptomatic uterine fibroids . Fifty - six completed pregnancies were identified in approximately 1200 women . Of the 108 patients that were attempting to become pregnant, premature delivery occurred in 18.2% of these cases, and 30.4% of the pregnancies miscarried ., the authors found a significant increase in cesarean section deliveries and increases in preterm deliveries, postpartum hemorrhages, and miscarriage that were associated with lower pregnancy rates.12 instances of placental alterations after uae, such as placenta previa, placenta accreta, and abruptio placentae, have been reported in the literature.10,19 there were two cases of abnormal placental implantation in our patient series . The frequency of spontaneous abortion is known to increase with maternal age, ranging from a rate of 18% in the late 30s to 34% in the early 40s in the general population . The 13.3% spontaneous abortion rate in our series does not appear to be higher than the general population rates . Relatively high rates (41.2%) of early miscarriages after laparoscopic uterine artery coagulation were observed by chen et al.20 only one of the 15 live newborns in our study had a low birth weight, and the gestation time reached 36 weeks for all of the mothers . In contrast, preterm delivery rates ranging from 10% to 42.8% have been recorded in other studies.10,19 a high rate of pregnancy complications was not observed in our study, probably because a limited embolization (of only the tumor vessels) was performed . However, the degree of dominant fibroid ischemia after uae, which is very important, was over 50% in every patient . Although the sample size is small, when using a relatively new procedure such as uae, it is important to identify complications that could make pregnancy more hazardous . When interpreting the findings from this study, it must be remembered that this cohort is not typical of the general obstetric population . The number of deliveries in this study was too small to allow us to make any recommendations on delivery management or draw conclusions . However, the pregnancy rate reported here adds to the growing number of reported cases of pregnancy after uterine artery embolization as a treatment for fibroids and suggests that it may be appropriate to reconsider the contraindication for this procedure in younger women who wish to preserve their fertility . Randomized controlled trials comparing myomectomy and embolization in a younger population desiring pregnancy are necessary . The efficacy of uae has been published in the cochrane database . This meta - analysis documented that uae results in a shorter hospitalization time and a reduction in blood loss (85% compared to pretreatment) and uterine size (30 - 49% compared to pretreatment).4 however, maintenance of fertility after uae is still controversial . Until now, the impact of this treatment on fertility has not been established.13,14 theoretically, uae may contribute to difficulties during embryonic implantation or the maintenance of pregnancy by decreasing the vascularity of the uterine myometrium and endometrium . In fact, embolization particles have been identified in structures adjacent to leiomyomas (e.g., myometrium, parametrium, and mesovarium) after uae.15 data are insufficient to conclude that uae is a safe option for women who wish to retain their fertility.16 normal pregnancy and delivery can be achieved after uae for uterine leiomyomata, as has been shown for postpartum hemorrhages . Comparing pregnancy outcomes after uae with leiomyomata is complicated by the size and location of the myomata and the fact that non - subserosal leiomyomas reduce fertility.17 uae pregnancy outcomes are reduced most severely when submucosal myomas cause endometrial distortion . Reports have not documented the number of women who have attempted pregnancy after embolization; therefore, cycle conception and fecundity rates cannot be calculated.18 in this series, we do not associate complication rates with their direct impacts on fertility, such as ovarian insufficiency . The authors reported that 27% of these pregnancies ended in miscarriage, with two terminations and one ectopic pregnancy . Of the 16 deliveries that occurred after 24 weeks, first- and second - trimester bleeding occurred in 40% and 33% of these deliveries, respectively, and 25% had preterm deliveries . Thirteen percent of the women had spontaneous preterm membrane ruptures, and the rate of primary postpartum hemorrhage was 20% . The mean birth weight of the term babies was 3.39 kg, and none required admission into the neonatal intensive care unit . There was one case of fetal growth restriction.19 another retrospective analysis was undertaken to evaluate the incidence and outcomes of pregnancies after uae for symptomatic uterine fibroids . Of the 108 patients that were attempting to become pregnant, 33 became pregnant, and 58.9% of the pregnancies had successful outcomes . Premature delivery occurred in 18.2% of these cases, and 30.4% of the pregnancies miscarried ., the authors found a significant increase in cesarean section deliveries and increases in preterm deliveries, postpartum hemorrhages, and miscarriage that were associated with lower pregnancy rates.12 instances of placental alterations after uae, such as placenta previa, placenta accreta, and abruptio placentae, have been reported in the literature.10,19 there were two cases of abnormal placental implantation in our patient series . The frequency of spontaneous abortion is known to increase with maternal age, ranging from a rate of 18% in the late 30s to 34% in the early 40s in the general population . The 13.3% spontaneous abortion rate in our series does not appear to be higher than the general population rates . Relatively high rates (41.2%) of early miscarriages after laparoscopic uterine artery coagulation were observed by chen et al.20 only one of the 15 live newborns in our study had a low birth weight, and the gestation time reached 36 weeks for all of the mothers . In contrast, preterm delivery rates ranging from 10% to 42.8% have been recorded in other studies.10,19 a high rate of pregnancy complications was not observed in our study, probably because a limited embolization (of only the tumor vessels) was performed . However, the degree of dominant fibroid ischemia after uae, which is very important, was over 50% in every patient . Although the sample size is small, when using a relatively new procedure such as uae, it is important to identify complications that could make pregnancy more hazardous . When interpreting the findings from this study, it must be remembered that this cohort is not typical of the general obstetric population . The number of deliveries in this study was too small to allow us to make any recommendations on delivery management or draw conclusions . However, the pregnancy rate reported here adds to the growing number of reported cases of pregnancy after uterine artery embolization as a treatment for fibroids and suggests that it may be appropriate to reconsider the contraindication for this procedure in younger women who wish to preserve their fertility . Randomized controlled trials comparing myomectomy and embolization in a younger population desiring pregnancy are necessary.
Fouling, i.e. The colonization of bacteria, plankton and animals on underwater structures, is a significant problem for shipping and leisure boating as attached organisms increase friction and drag, resulting in both higher fuel consumption and hull maintenance costs (almeida et al ., 2007). The most commonly used method to prevent fouling is to coat the hull with antifouling paints that contain and leach biocides . The efficacy of the paint depends on the prevailing fouling pressure in the areas where the boat or ship operates, what biocide(s) are used in the paint formulation and the biocidal release rate from the painted surface to the ambient water . Hence, knowing the release rate of biocides required to prevent fouling in the area where the ship operates is fundamental in order to develop efficient antifouling paints and to meet environmental standards . The mechanisms controlling the release rate of biocides from antifouling paints are mainly chemical reactions and diffusion phenomena (yebra et al ., 2004) where sea water soluble pigment dissolution, binder reaction and paint polishing are key processes . Most of today's paints contain toxic soluble pigments where cuprous oxide (cu2o) and zinc oxide (zno) dominate the market (ciriminna et al ., 2015). The dissolution rate of these pigments will control the leaching of biocides from the paint surface (yebra et al ., 2006). The dissolution rate of the binder (matrix) system also governs the leaching rate as it directly affects the paints' polishing rate . Antifouling paints are based on either insoluble matrices (e.g. Epoxy, acrylates, chlorinated rubber and vinyls), soluble matrixes (mainly rosins) or, in the case of self - polishing paints (spc), on a matrix consisting of acrylic polymers such as copper acrylate (almeida et al ., 2007). The thickness of the so - called leached layer, which is a result of the relative dissolution rate of the pigments at the paint - water interface and the polishing rate of the binder, has a strong impact on the diffusion rate of e.g. Dissolved copper species from the paint to the water (kiil et al ., 2003). For example, salinity has shown to both increase the dissolution of cu2o particles (yebra et al ., 2004) and to increase the solubility of rosin (rascio et al ., 1988). Thus, the release rate of biocides can be assumed to be lower in brackish and fresh water compared to fully marine conditions, and the effect is presumably higher for rosin - based and self - polishing antifouling systems as compared to paints based on insoluble matrixes . Today, several methods exist to determine the biocidal release rates from anti - fouling paints . Rotating cylinder methods, developed under the american society for testing materials (astm) and international organization for standardization (iso), have frequently been used in the laboratory to determine release rates of both organic biocides (e.g. Tbt) and inorganic biocides (e.g. Cu2o) (iso, 2000a, iso, 2000b). These methods involve application of the coating on test cylinders that are immersed in artificial standardized seawater for a 45 d period and are recognized to be time consuming, costly and to not reflect environmentally realistic conditions (finnie, 2006). A few field methods have been developed to obtain more reliable and realistic release rate measurements . The most commonly used is the dome method which was originally developed by the us navy to measure organotin and cu release rates in situ from a coated ship hull by using a closed re - circulating system (valkirs et al . However, the use of this method has been restricted to the us navy and also entails practical and economical challenges, including the use of divers . Hence, even though the method is recognized to be the most reliable indicator of environmental release rate, it has never been recommended to be adopted as a standardized method to be used in e.g. Environmental risk assessment (finnie, 2006). Other, strictly mathematically based methods are also used to calculate theoretical release rate of biocides, e.g. The mass - balance method developed by the the advantage with the cepe - method is its low cost, but since all the input data to the mass - balance formula, e.g. Paint film thickness and service life time, comes from the paint manufacturer it is not possible to validate if the calculated release is environmentally realistic or not . Thus, it is apparent that current methods for measuring release rates of biocides from antifouling paints have disadvantages of either being too costly (dome method and rotating cylinder method), not represent field conditions (rotating cylinder method) or hold large uncertainties and to not be fully validated (cepe - method). The aim of the current study was to bridge that gap by developing a cheap, fast and reliable release rate method based on x - ray spectrometry . The method was subsequently used to determine cumulative released cu and zn concentrations from five different coatings applied on static panels deployed in six different marinas located in the baltic sea and in kattegat . An additional aim was to determine how salinity affects the release of cu and zn . Five different antifouling paints, holding a cu2o concentration between 6.9% and 34.6% (w / w), were used for the release study . Information about the tested paints and area of use in swedish waters is presented in table 1 . The selected antifouling coatings were applied on one side of sandpaper rugged poly - methyl - meta acrylate (pmma plexi glass) panels (20 20 cm) following the recommendations on to the product data sheet . Hence, one layer of primer was applied with a roller and left overnight to dry, and thereafter two layers of the antifouling paint were applied with sufficient drying time in between (> 24 h). Three points were analyzed on each panel for total area concentrations of cu and zn (in g / cm) with the xrf method, as described in section 2.2 . The paint thickness on the plexiglas panels was estimated by applying two rolled layers of the different antifouling coatings on plastic films (melinex, putz folien) holding a film thickness of 125 m . For every paint, 10 replicates of plastic films were coated and 3 randomized points were measured on each film for paint thickness using a coating thickness gauge probe (elcometer 456). The coated panels (four replicates per paint) were attached, 30 cm apart and in random order, to a plastic rope . The panels were immersed along a pier in five different marinas in the baltic sea and kattegat, i.e. Stockholm (59.298415n 18.653049e), karlskrona (56.166362n 15.825231e), simrishamn (55.560325n 14.354432e), kiel (54.429409n 10.172572e) and gothenburg (57.647232n 11.853132e) (fig . 1). The exposure depth was approximately 1 m and the panels were submerged over the summer of 2014, from mid - may to mid - october, corresponding to 150 days exposure under static conditions . After the exposure period, the panels were transported back to the laboratory and left to dry for> 48 h. the same measurement points analyzed before deployment were then measured again for total area concentrations of cu and zn with the xrf . The cumulative release of the two metals were determined for all measurement points by subtracting the end concentration from the start concentration . Moreover, the panels were checked for fouling under a stereo microscope and all paints tested completely deterred fouling of macroorganisms during the entire experiment . Handheld xrf analyzers are widely used for elemental measurements on e.g. Soils and alloys and therefore most commercial instruments are equipped with calibrations for measurements of these matrices . However, since no commercial method for metal analysis in antifouling paints exists on the market, the first step was to develop an empirical method for metal analysis in antifouling paints . Two different commercial biocide - free antifouling paints were used to develop antifouling paint standards . Increasing amounts of cu (as cu2o, alfa aeser, 99%) and zn (as zno, alfa aeser, 98%) were added to the paints (both separately and in combination) to obtain wet weight (w / w) concentrations of 2, 4, 8, 16 and 32% . All paints were thoroughly mixed using wooden paint stirrers and subsequently applied on a 6.3 m thick mylar film with a quadruplex film applicator (vf2170, tqc) to obtain a wet film thickness of 100 m (corresponding to 2535 m in dry thickness). After a drying period of> 12 h, 25 mm round discs were punched out and weighed, before measurement with the handheld xrf (delta-50, innovx) attached to its benchtop test stand . The standards were positioned directly on top of the xrf window and a circular area (28.3 mm) was analyzed using a 50 kv, 35.4 a, 4 w setting beam for 30 s. the calibration was performed using the empirical mode in the pc software (innov - x delta advanced pc software). The empirical mode allows the user to utilize their own standards to create and store calibration models for custom applications directly on the analyzer ., the calibration was performed on the adjusted intensity of kalpha signals, i.e. The intensity rates were (through the pc software) adjusted for air background and peak overlap, elemental interference from other elements in the sample that have peak energies close to the element of interest . Peak area integration was performed between 7.87 and 8.22 kev for cu and between 8.45 and 8.81 kev for zn . After the xrf analysis, the standards were chemically analyzed for their total concentration of cu and zn . Sample digestion and chemical analysis were performed by a commercial accredited laboratory (als scandinavia). The standards were digested in aqua regia on a hotplate for 1 h. the sample solutions were analyzed by inductively coupled plasma - sector field mass spectrometry (icp - sfms) according to epa method 200.8 rev 5.4 (1994) and ss en iso 17294 - 1 (2006). The area concentration, carea (g / cm), of the standards were calculated with the following equation: carea = cpaintmawhere cpaint is the (chemically analyzed) total concentration of cu and zn (g / g), m is the weight (g) and a is the area of the standards (cm). The calibration curves were established on the instrument, using the relationship between the measured adjusted k intensities of cu and zn, respectively, and the calculated area concentrations carea of the standards . A regression analysis (for each element) was performed in jmp 11.0.0 to evaluate the linearity of the calibration curves . To accurately determine the metal concentration in any matrix at least two processes should be taken into consideration: (1) the penetration of the primary x - ray beam into the sample and (2) the escape of fluorescent secondary x - rays which will be analyzed by the detector (gauglitz and vo - dinh, 2006). The x - rays capacity to both penetrate into and escape the higher the energy, the further the x - ray can travel through the sample . The penetration depth of the x - rays will therefore vary between elements since heavier elements (such as cu and zn) emit higher energy x - rays compared to lighter elements (such as mg and al) (gauglitz and vo - dinh, 2006). Furthermore, the composition of the sample also plays a major role: the higher the concentration of heavy elements which can absorb the x - rays, the shallower the penetration depth will be (potts and webb, 1992). The critical penetration depth or saturation depth, dthick is defined as the depth from which 99% of the signal will originate (smith and cresser, 2003). Beyond this depth, almost all photons are absorbed by the sample and will not be detected by the instrument . For bulk sample analysis, e.g. Soil analysis, it is important to ensure that the sample is infinitely thick (> dthick) so that the xrf signal is not dependent on the sample amount . On the other hand, for thin film analysis, it is important to instead define the thickness beneath which attenuation effects due to absorption can be neglected i.e. The depth beneath which the response range is linear (fig . 2). This thickness is known as the critical thickness, dthin, and is by convention defined as the thickness where 1% attenuation has occurred (gauglitz and vo - dinh, 2006). As thin film analysis was utilized in this study, the thickness of the antifouling paint should be below dthin to avoid absorption effects and consequent underestimation . Therefore, we designed an experiment to determine dthin for cu and zn in two different antifouling paints . A paint with low cu content (biltema baltic sea, 7.5% cu2o) and one with high cu content (mille xtra, 34.6% cu2o) were chosen to investigate potential differences in dthin due to differences in heavy element content . Plastic films (melinex, putz folien) holding a film thickness of 125 m was used as paint substrate . A motorized film applicator (elcometer 4340) equipped with different applicators was used to produce wet paint thicknesses of 30, 50, 60, 90, 100, 120, 150, 200, 300 and 400 m . The coated films were left to dry for one week . On each film, three measurement points were selected where xrf (n = 1) and dry film thickness (n = 5) measurements were performed . Statistical analysis was performed in jmp 11.0.0 to investigate the pairwise correlations (= 0.05) for all three parameters i.e. Cu loss, zn loss and salinity . Multiple regression analysis was also performed to examine any potential combined contribution from zn loss and salinity on cu loss . Salinity and zn loss were set as explanatory variables for cu loss according to the following statistical model: cu loss = 0 + 1 * salinity + 2 * zn loss . The partial slopes 1 and 2 were then tested for significance at = 0.05 . The regression analyses of cu and zn kalpha net intensities and chemically analyzed metal concentrations are shown in fig . 3 . Both metals showed strong linear relationships between kalpha net intensities and chemically analyzed concentrations; r values were 0.998 (cu) and 0.998 (zn). The effect of dry paint thickness on kalpha net intensities is presented in fig . 4a and b. the paints show a linear increase in signal intensity up until 50 m, followed by an attenuation of the signal as the thickness increases further . The data points were fitted to a polynomial second degree curve (r 0.995 for all). Linear regressions between 0 and 50 m yielded with r values of 0.974 (cu) and 0.975 (zn) for mille xtra and of 0.919 (cu) and 0.917 (zn) for biltema baltic sea (fig . Was then calculated as the thickness at which 1% attenuation is observed between the linear regression and the polynomial second degree curve . For mille xtra, dthin was calculated to 40 m (cu) and 41 m (zn). For biltema baltic sea, the determined dthin was slightly higher at 52 m (cu) and 54 m (zn). The coated (rolled) plastic films of mille xtra and biltema baltic sea (see section 2.1) showed an average dry paint thickness of 64 16 m (sd) and 78 15 m, respectively . Provided that such thicknesses were also obtained on the plexiglas panels exposed in the field, an average underestimation of 8% (mille xtra) and 7% (biltema baltic sea) are expected for cu . For zn, the corresponding underestimation is calculated to be 7% (mille xtra) and 6% (biltema baltic sea). For the remaining three paints, the underestimation at day 0 is likely of similar magnitude as their thicknesses were either lower or in the same range as mille xtra and biltema baltic sea: thicknesses of 62 10 m, 61 16 m and 65 16 m, were measured respectively for biltema west coast, mille light cu and cruiser one . The thicknesses of the coatings after the field exposure (at day 150) were not determined . However, due to polishing, the coatings thickness will be thinner after 150 d of exposure and presumably close to or within the dthin . The total amount of cu released during the exposure period (150 d) is presented in fig . 5 . For all the tested paints, the leached amount of cu was lowest in the locality with the lowest salinity, i.e. Stockholm the pairwise correlations between leached amount of cu and salinity were significant for all paints except mille xtra (table 2), i.e. The leaching increased with higher salinity . For zn, no statistical salinity effect was observed . With the exception of cruiser one, no statistically significant correlation between the leached amount of copper and the leached amount of zn the statistical analysis of the multiple regressions showed that the leached amount of cu can be explained by both the loss of zn and the salinity for two of the paints: cruiser one and mille xtra . Hence, for mille xtra, even though neither the loss of zn nor the salinity alone showed any correlation with the loss of cu, these two parameters can together explain the leaching of cu . As emphasized previously, several release rate methods exist on the market that have the drawback of either being too expensive, not reflect field conditions or hold large uncertainties . In the present study, we have developed a new method based on xrf that has the advantage that it can be used to analyze discharges of cu and zn from antifouling paints exposed in the field . The calibration curves for cu and zn, i.e. The relationship between cu and zn kalpha net intensities and cu and zn concentrations, showed that the xrf release rate model can be used for quantitative measurements (r = 0.998 for both elements) (fig . The critical thickness (dthin) of cu and zn for biltema baltic sea was determined to be 52 and 54 m, respectively . Hence, for both coatings, dthin was similar for cu and zn, likely due to the fact that the secondary x - rays for these two elements have similar energy levels at 8.04 kev and 8.64 kev, respectively . Mille xtra, the paint with the highest combined metal concentration (cu and zn), showed the lowest dthin, presumably due to a higher degree of absorption of x - rays by the heavy elements in the paint . As the coatings' thicknesses were likely thicker than dthin, the start concentrations of cu and zn were calculated to be underestimated by 68% . As a consequence, the total released amounts of cu and zn presented here would also be slightly underestimated . In future studies it is thus recommended to use coatings with dry film thicknesses <40 m . The cumulative release of cu and zn during the 150 d exposure time is presented in fig . The variation in leaching between the replicates for the respective paints was in general low within each harbor, shown by the low standard deviation . The data show the leaching of cu to be governed by salinity, at least for the rosin - based paints for which a significant correlation between leached amount of cu and salinity was observed . Salinity has shown to increase both the rate of cu2o dissolution (kiil et al ., 2001, singh and turner, 2009) and the polishing rate, where the latter is expected to be very low or to stop completely in areas with brackish or fresh water, at least for spc paints (kiil et al ., 2002). However, salinity did not show any significant effect on the leached amount of zn . The observed pattern may partly be described by the difference in dissolution rates, where zno dissolution rates have been reported to be markedly lower than cu2o particles (yebra et al ., 2006). The effect of salinity on the dissolution rate of zno is not known (yebra et al ., 2006), but recent studies on spent antifouling paint particles have shown the leaching of zn to decrease with increasing salinity up until 15 after which any further increase in salinity does not appear to have any effect on the leaching (singh and turner, 2009). However, in the study by singh and turner (2009), the form of zn in the paint matrix was unknown and it cannot be ruled out that forms other than zno (e.g. Zn acrylates, zn pyrithione) were also present in the paint matrixes . Thus, the observed difference in leaching behavior of cu and zn may be explained by the different dissolution rates of the two pigments, with salinity having a larger impact on the dissolution rate of cu2o particles then on zno particles . The current data also suggest the release of both cu and zn to be paint specific and that neither zn nor salinity alone can explain the release of cu for all the investigated paints . In light of this, it seems quite problematic to set up a general model such as the cepe model to generate paint specific release rates of biocides . Therefore, it is recommended that the developed xrf method is used to derive site- or area - specific release rates of metallic biocides . Such knowledge is highly valuable for both environmental regulators to set limit values for cu for water quality as well as for the paint industry to design area - specific paints that meet environmental standards . A direct method for environmentally realistic release rate measurement with the potential for standardization has long been requested by regulatory bodies, industry and academia . This new xrf method allows for the first time the direct measurement of the release of metallic biocides in the field, in a cost and time efficient manner . As exemplified in this work, this method makes it possible to identify parameters key to the release of cu and zn . It is therefore not only useful for the measurement of environmentally relevant release rate, but also as a tool for designing more efficient and optimized antifouling coatings . Using this new technique, more realistic release models based on field data furthermore, it could allow the identification of the minimum release of cu necessary to avoid fouling at different sites, enabling the design of antifouling paints that are efficient while at the same time minimizing the negative effects on the environment.
About 60% of stroke patients experience posture and balance problems during gait1 . Generally, they prefer to use the less affected side than the affected side for weight - bearing load during gait . This can lead to an asymmetric body posture, and the development of abnormal weight shifting in gait1 . These conditions can limit the motion of stroke patients, and increase their risk of falls2 . When stroke patients return to their homes, about 56% of them experience falls within 2 months3 . Such falls can lead to the development of secondary impairments and disability in the nervous system and orthopedic problems . According to a previous study, stroke patients may have problems with the quality of their gait cycle (e.g., gait speed, poor endurance, reduced quality, and adaptability in walking patterns, and coordination of the legs)4 . Also, these conditions cause deceleration control to increase, while acceleration control decreases during gait . Again, this can be lead to increased compensatory movements and asymmetric gait patterns4, 5 . Consequently, these gait problems can result in balance impairment, and then falls . Previous studies have demonstrated that stroke patients have increased fear or anxiety during gait, and a high level of anxiety increases the risk of depression . Many studies have demonstrated a relationship between falls and quality of life, even though they did not compare gait parameters with psychological aspects, such as fall anxiety . Thus, in this study, we investigated the gait parameters and fear and anxiety of stroke patients . Twelve patients with stroke (mean age: 50.7511.07 years; five females, seven males) participated in this study . The inclusion criteria were a history of only one documented stroke event, no orthopedic disease, a mini - mental state examination score above 25, and the ability to walk more than 10 m without a cane . The biodex gait trainer 2 is a device which is used to assess and train the walking performance of patients with neurologic gait dysfunction . It consists of a treadmill with an instrumented deck that monitors and records kinematic gait parameters including gait speed, step cycle, step length, and time on each foot (step symmetry). Lachman et al.7 developed the survey of activities and fear of falling in the elderly (safe) measure that assesses fear of falling and also provides an index of activity avoidance due to fear . Responses use a four - point likert scale (1 = not at all sure to 4 = very sure). Gait analysis was performed on the biodex gait trainer 2 for 5 min . Characteristic gait parameters, including differences between the affected and unaffected sides, were assessed . All subjects walked on the gait trainer treadmill at a speed of 3 km / h wearing normal shoes . Before the experiment, they received instructions concerning the test procedure and walked for 3 min as a warm up and to adapt to the step speed and equipment . We told the subjects that they should try to maintain a comfortable gait speed, continuously . 22.0; spss inc ., chicago, il, usa) was used to analyze correlations between gait parameters and fear of falling . Comparisons of correlations between gait parameters and fear of falling were assessed using spearman s rank correlation test . Correlation between gait parameters and fear of falling scores were calculated . A moderate degree of correlation between fear of falling score and the step cycle item of gait parameters was found (p <0.05). No other gait parameter showed a significant degree of correlation with the fear of falling score (p> 0.05) (table 1table 1.correlation of gait parameters and fall anxiety (n=12)gait parametersgait speedstep cyclestep length(affected)step length(unaffected)time on each foot(affected)time on each foot(unaffected)safe0.4320.581*0.1120.0700.2890.289). Many stroke patients feel a sense of loss over problems of gait ability . Indeed, gait disability can limit the independent living of stroke patients8 . Many stroke survivors have a hard time maintaining their balance due to decreased weight shifting ability and stability8 . A previous study found that gait patterns of stroke patients are slow and require excessive effort, and the patients showed uncoordinated movements in both legs1 . It has also been demonstrated that gait speed is related to the ability to maintain balance . Patients with balance impairment show increased feet support time, and reduction in gait speed is a risk for falls, increasing anxiety9 . Our study confirmed the possibility that certain gait parameters are associated with increased fear and anxiety . We found a moderate degree of correlation between the fear of falling score and the step cycle . A previous study demonstrated that the slow gait speed phenomenon was related to a decrease in cadence and a shortened stride length . Moreover, compared with healthy people, stroke patients show decreased cadence and increased gait cycles, which lead to an increased double - stance phase and stance phase of the unaffected side leg4 . Indeed, our study showed that a decreased step cycle was associated with fear of falling scores, indicating that a decreased step cycle leads to increased fear of falling during gait . It can be demonstrated that asymmetrical limb support slows the step cycle10, 11 . We hypothesize that one cause of this is the development of abnormal weight shifting in the step cycle, which makes subjects feel uneasy about falling10, 11 . Generally, the step cycle is considered to be a temporal gait parameter10, 11 . Thus, we suggest that temporal parameters are likely to be more closely related to falling anxiety than spatial gait parameters . Moreover, problems with functional independence skills affect the quality of life of stroke patients . Mence12 indicated that people with stroke could feel joy just by walking in the street . Thus, enhancing gait ability is an important means of improving the lives of stroke patients.
A 45-year - old male patient reported with a gingival growth on the right anterior region of the mandible for one year and also complained of pain in the same region for 6 to 7 months . The exophytic lesion was small initially and increased gradually to the current size displacing the adjacent teeth . Extraorally, a diffuse swelling measuring around 1.51.5 cm in size was seen on the right lower portion of the face . 1a) at the interdental gingiva between canine and the first premolar, which was roughly circular, measuring about 3.53.5 cm in size, extended labially from the attached gingiva to the lingual sulcus of the right mandibular incisors, canine, and premolar . 1b) was pebbled and rough with indentations of the opposing teeth with mesially displaced canine . Color of the lesion was normal as that of the surrounding mucosa and firm in consistency . 2) showed the presence of characteristic multilocular soap bubble appearance in the region of canine and first premolar, and knife edge shaped root resorption pattern in relation to the first premolar . 3) showed radiolucency measuring 44 cm in diameter extending from the distal aspect of lateral incisor to the mesial aspect of second premolar . The follicles were lined by a single layer of tall columnar ameloblast - like cells and little cystic degeneration . Ameloblastoma usually manifests as an intraosseous or central lesion or rarely as a peripheral epulis - like lesion.1 the lesion may arise most commonly from cell rests of enamel organ, epithelium of odontogenic cysts, disturbances of the developing enamel organ, and basal cells of oral epithelium . Clinico - radiographically, ameloblastoma can be divided into 3 distinct patterns: (1) the conventional solid / multicystic (86% of all cases), (2) the unicystic (13% of all cases), and (3) the peripheral (1% of all cases) (extraosseous).2 various histologic forms have been described which include follicular, plexiform, acanthomatous, granular cell, desmoplastic, and basal cell patterns.2 follicular ameloblastoma presents as a painless swelling or slow expansion of the jaws, and it is described as multilocular expansile radiolucency that occurs most frequently in mandibular molar / ramus area.9 desmoplastic variant occurs more frequently in the anterior mandibular region10 and presents a mixed radiopaque and radiolucent appearance . Unicystic ameloblastoma is usually seen in younger age and most commonly associated with pericoronal radiolucency with unerupted 3rd molar (38%).11 in this particular case, the clinical presentation of the follicular ameloblastoma was different from the usual presentation, which included exophytic growth, uncommon location, and absence of expansion of the cortex . Clinically differential diagnosis of this exophytic growth included peripheral ameloblastoma, peripheral odontogenic fibroma, peripheral giant cell granuloma, and other peripheral hyperplastic swellings superficial to the alveolar ridge.9 the final diagnosis was made by the clinical, radiological, and histopathological features after the complete excision of the lesion . Kuru first reported an intraosseous ameloblastoma having penetrated through the alveolar bone, fused with the oral epithelium, and eventually presenting itself clinically as a' peripheral lesion'.6 our case had the similar clinical and radiological features of the previously reported case by kuru but with no expansion of the cortex . Two other similar cases have been reported by tongdee and ganggakavin7 in 1978 and stevenson and austin8 in 1990, respectively . The exophytic growth is also a characteristic finding of the peripheral (extraosseous) ameloblastoma which is very rare . Radiographically, or at surgery, a superficial erosion of the bone or a superficial bony depression -known as cupping or saucerization- may be noticed, a finding that is thought to be due to pressure resorption, in contrast to resorption caused by neoplastic invasion.5 the diagnostic criteria of peripheral ameloblastoma include the origin from the overlying epithelium, presence of odontogenic epithelium islands in the lesion, and lack of potential to bone infiltration.12 also, peripheral odontogenic fibroma and peripheral giant - cell granuloma share the same clinical characteristic of exophytic growth most commonly at mandibular premolar region, despite the histopathological difference . However, radiographically, both of them create depression / resorption in the underlying bone very rarely.13 most oral surgeons and pathologists unquestionably consider peripheral ameloblastoma to be a nonaggressive lesion without actual infiltration into the underlying bone.14 one of the main problems regarding peripheral ameloblastoma is its possible origin . The two main theories are the following: (1) origin from the extra osseous epithelial remnants of dental lamina and its organ derivatives within the underlying connective tissue; (2) origin from the basal cell layer of the oral mucosa, which is believed to have odontogenic potential.15 those lesions that are entirely separated from the overlying surface epithelium probably arise from the odontogenic remnants, however this hypothesis can be questioned if there is continuity between the tumor and the surface epithelium . In conclusion, even though the incidence of intraosseous ameloblastoma manifesting as exophytic lesion is very rare, it may have to be considered in the differential diagnosis of exophytic lesions at the region of mandibular canine premolar region along with peripheral ameloblastoma, peripheral odontogenic fibroma, peripheral giant cell granuloma, and rarely peripheral hyperplastic swellings superficial to the alveolar ridge.
Inner mongolia cashmere goat (capra hircus, imcg) is a diploid (2n = 60) mammal that belongs to the family of bovidae . It plays an important role in the world animal fiber industry because it can produce high quality underhair (cashmere is the commercial name) and is one of the world's largest breeding groups . Cashmere produced by imcg, which is of a small diameter (1418 m) and is soft to touch, is grown from the secondary hair follicle (hf) of the body skin [1, 2]. Fiber diameter and length determine both the quality and the amount of cashmere produced by an animal . The longer the length and the smaller the diameter of the cashmere fibers, the higher the price becomes . Imcgs exhibit seasonal rhythm and annual cycle of cashmere growth that are controlled by daylength . During the period from the summer solstice to midwinter, when the length of day is reduced, cashmere fiber has a high growth speed; in contrast, it becomes low during the period from midwinter to the next summer solstice [3, 4]. This photoperiodic characteristic of cashmere fiber growth is convenient for cashmere harvest and formulating a management strategy of cashmere production . During the past decades, many mammalian genomic and transcript sequences have become available, including homo sapiens, mus musculus, and bos taurus which play important roles in understanding hf formation and hair growth . However, only a total of 561 ests are pertinent to goat skin or hair follicle indicating that few studies focused on understanding the gene expression pattern of goat skin or hair follicle . On the other hand, almost all the deposited goat skin associated ests were sequenced by the traditional approach from randomly picked cdna clones which did not guarantee that the less - abundant transcripts could be efficiently detected . In addition, we also observed that cashmere fibers of imcg are mainly produced by the back (bk) and side of the body (bs) skin of the trunk coat, but few are produced by the belly (bl) skin . This indicated that the gene expression patterns of bk and bs skin are different from bl . Therefore, the gene expression patterns and differential gene expression profiling of three skin sites during active hair growth phase (anagen) are important to understanding the underlying molecular mechanism associated with cashmere fiber growth . The advents of the ultra - high - throughput, cost - effective next - generation sequencing (ngs) technologies make the whole transcriptome sequencing and analysis feasible, even in the absence of genomic data . In the past few years, ngs has been widely used in rna sequencing (rna - seq) which provided researchers with more information about gene expression, regulation, and networks under specific physiological conditions or developmental stages in both model and nonmodel organisms [68]. It offers accurate quantitative and digital gene expression profiles of sequenced transcripts; moreover, it has very low background noise and a large dynamic range of gene expression levels compared with dna microarray . In the present study, we utilized illumina / solexa paired - end mrna - seq approach to sequence and de novo assemble the goat skin transcriptome during anagen phase, and further investigated the gene expression profiles of three different skin types based on the assembled contigs, skin from the bl, bk, and bs of the trunk coat, which have a large discrepancy in cashmere production . Generally, according to the different skin types of trunk coat, skin from the back and body side of imcg produce either wool or cashmere, whereas belly skin produces mainly wool fiber and few cashmere . A breeding, two - year - old female goat was sampled from yi wei white cashmere goat breeding farm at ulan town of erdos in inner mongolia autonomous region, china . During anagen phase (nov, 2010), the hair (wool and cashmere) of belly, back, and body sides of the goat were sheared and further shaved . After sterilization with 70% alcohol, full - thickness skin sections of each body part were excised and immediately frozen in liquid nitrogen for storage and transport until rna isolation . Goat skin tissue collections were carried out in accordance with the guidelines of inner mongolian animal society ethics committee . This study has been checked and approved by inner mongolian animal society which is responsible for animal care and use in inner mongolia autonomous region, china . Skin excision was performed after xylazine hydrochloride anesthesia, and all efforts were made to minimize suffering . Before rna extraction, each sample was washed with 10 ml pbs (ph 7.2) and 0.5 mm edta . Total rna of each sample was isolated by using a trizol plus rna purification kit according to the manufacturer's protocol (invitrogen). Total rna quality and concentration were determined by a 2100 bioanalyzer nanochip (agilent). Enrichment of mrna from total rna was performed with a ribominus rna - seq kit according to the manufacturer's instructions (invitrogen). It has been reported that mrna fragmentation can result in a more even coverage along the entire gene body, whereas cdna fragmentation is more biased towards the 3 end of the transcript . Therefore, each ploy(a)-enriched rna sample was chemically fragmented into small pieces by using divalent cations at 94c for 5 min . The fragmented rna was reverse - transcribed into cdna by using random hexamer primers containing a tagging sequence at the 3 end with the use of a superscript iii double - stranded cdna synthesis kit according to the manufacturer's protocol (invitrogen). The double - stranded cdna was subjected to end - repair and further 3 terminal tagging by the addition of 5 dna adaptors and t4 dna ligase with overnight incubation at 16c for 16 hours . The targeted di - tagged cdna was purified by polyacrylamide gel electrophoresis (page) and gel excision (200 25 bp). The clean di - tagged cdna was enriched by limited - cycle pcr amplification (18 cycles) with primer pairs that annealed to the tagging sequences of the di - tagged cdna . Library purification by page removed any residual nucleotides, pcr primers, and small amplicons . The initial illumina short reads from this study have been submitted to the ncbi sequence read archive (sra, http://www.ncbi.nlm.nih.gov/traces/sra/sra.cgi/) with the accession number sra055764 . Due to the fact that the base quality requirement for de novo assembly is more strict than that of the resequencing project, customized perl scripts were used to remove reads which contained adaptor contamination, low quality bases (> 2% base smaller than q20 per read), and undetermined bases (> 2% ns per read) from each dataset generated from different skin types . Three datasets were concatenated in a left - to - left and right - to - right manner . Next, the clean high - quality reads dataset was de novo assembled with default parameters by using inchworm assembler which is a component of trinity software . All of the sequence reads were initially trimmed to 50 bp (nucleotides 21 to 70 of each read) in length and then used in mapping experiments and statistical analysis . Mapping short reads uniquely back to the contigs was performed by soapaligner 2.20 with two mismatched bases per read permitted . The contigs which contain two or three predicted cdss may attribute to a small proportion of false positive discovery of predicting coding sequences from mature transcript sequences . Ten randomly selected putative full length transcripts which did not assign with known protein functions and another ten transcripts that are associated with hair cycling were subjected to perform reverse transcription polymerase chain reaction (rt - pcr) and sanger sequencing . Primer sequences used for rt - pcr are available upon request . The assembled transcriptome sequences (49,115 contigs greater than 300 bp in length) in this study have been deposited to the ncbi's transcriptome shotgun assembly (tsa) database under the consecutive accession numbers from ka304519 to ka353633 . Mouse and cow refseq protein sequences were downloaded from the ncbi refseq database (ftp://ftp.ncbi.nih.gov/refseq/). Homology searches against the swiss - prot and nr databases were performed by using blastx algorithms with an e - value cutoff of 10 . Mouse and cow refseq rna data were also downloaded as reference sequences for short reads mapping to calculate hit numbers compared with de novo assembled contigs . Blastx was used to perform kog and kegg annotation [13, 14], followed by retrieval of the functional proteins and assignment to each of the classification entries (e - value 10). Gene ontology (go) against the nr database was conducted by blast2go (e - value 10). Wego and go terms classifications counter (http://www.animalgenome.org/tools/catego/) were used for assignment of each go i d to the related ontology terms . According to the ac statistical framework, the p value of differential expression significance of each transcript - derived contigs between two samples was calculated by using the following equation: (1)p(y x)=(n2n1)y(x+y)!x!y! (1+n2/n1)(x+y+1).n1 and n2 represent the total uniquely mapped reads in sample one and sample two, respectively . X is the number of reads mapped to a certain gene in sample one, and y represents the number of reads mapped to the same gene in sample two . After the calculation of p value, multiple hypothesis testing was performed to correct p value by using phyper function of the r tool (http://www.r-project.org/). Rpkm values of each contig were estimated by aligning trimmed reads back to the contigs . Both a q - value of less than 10and a rpkm value with at least 2-fold difference between the two samples were used as criteria to determine significant degs . Kog enrichment analysis was conducted by hypergeometric distribution test by using the phyper function in the r software package . The significantly enriched functional clusters were selected when the corrected p value (q - value) was less than 10 . Quantitative real - time pcr (qrt - pcr) was performed on the same individual of the corresponding body part skin . Total rna was firstly treated with dnase i before reverse transcription by superscript iii double - stranded cdna synthesis kit (invitrogen). Each cdna sample was used as template for qrt - pcr by using the sybr premix ex taq ii kit (takara) on a 7300 real - time pcr system (applied biosystems), and at least three technical repeats were performed for all the genes within each template . Acetyl - coa carboxylase 1 (a7431; 102) was used to normalize gene expression quantities between samples . To obtain comprehensive transcripts of skin tissue that provide an overview of gene expression profile during anagen in the cashmere goat, skin tissues from the belly (bl), back (bk), and the side of the body (bs) during anagen were sampled . The skin sections were made to show primary hair follicles and secondary hair follicles (figure 1). Three rna - seq libraries were constructed and sequenced by using illumina / solexa technology . As a result, a total of approximately 130 million raw reads (65 million paired - end reads, 2 100 bp) that represented roughly 13 gb of sequence data were generated from three independent 200 bp insert libraries . After the removal of reads with low quality and containing ambiguous bases (> 2% n per reads), we employed inchworm assembler to de novo assemble high quality reads which were generated from three different skin types . De novo assembly mrna - seq reads yielded 49,115 contigs over 300 bp comprising 45.4 mb of total sequence length, with an average length of 924 bp and n50 length of 1380 bp . Of the 49,115 contigs, there were 12,892 (26.2%) contigs greater than 1 kb, 13,768 (28.1%) varying from 501 bp to 1 kb, and the remaining 22,455 (45.7%) ranging from 301 bp to 500 bp in length (table 2). To identify the protein encoding regions, we used genscan to perform the ab initio prediction of the coding sequence (cds) of 49,115 contigs . We found that 23,039 putative cdss were identified from 22,734 (46.3%) assembled contigs . Of the 22,734 cds - contained contigs, 22,440 have one putative cds, 283 have two, and 11 contain three cdss . Further analysis indicated 8,184 out of 23,039 contained a putative full - length cdss (i.e., containing start and stop codons). Further, 6,889 cdss contained a start but no stop codon, 3,171 predicted to have a stop but no start codon, and 4,795 have neither . The average length of putative 8,184 full - length cdss reached 1,326 bp, while the partial cdss have an average length of 605 bp . Among 8,184 predicted full - length cdss, 127 of them cannot be annotated by known proteins . To validate sequence assemblies, we randomly selected ten predicted full - length cdss that did not possess blastx hits in the nr database and ten genes which have been demonstrated that are specific to hair cycling and hair growth to perform rt - pcr and sanger sequencing . These genes include the signaling molecules such as wnt, insulin - like growth factor 1 (igf-1), members of fibroblast growth factor (fgf), and their receptors encoding genes such as frizzled and igf-1r . The result showed that 19 pcrs are positive and the sanger sequencing results all showed higher than 97% identities with de novo assembled transcripts indicating the relatively high credibility of sequences assemblies (table s1 and table s2 in supplementary material available online at http://dx.doi.org/10.1155/2013/269191). To further validate the quality and sequencing depth of the assembled contigs, we mapped the total short reads back to the assembled contigs . The sequencing depth ranged from 2- to 164,789-fold, with an average sequencing depth of 249-fold . Specifically, 87.4%, 87.5%, and 85.9% corresponding to the bl, bk, and bs skin of high - quality reads, respectively, the relatively small proportion of unmapped reads may be involved in comprising the contigs which are shorter than 300 bp . This suggests that the majority of short reads in our rna - seq data were efficiently assembled into relatively larger contigs . Examining the codon usage of 23,039 predicted cdss showed that the most abundant amino acids encoded by triplet codons are nonpolar (hydrophobic) amino acids (44.1%), and then the uncharged polar amino acids (29.5%), while the acidic and basic amino acids accounted for 12.3% and 14.1%, respectively (table s3). Among the 23,039 predicted cdss, the average gc content reaches 54.9%, while the maximal gc content is 86.7% and minimum is 31.3% . Scanning the stop codon of 11,355 cdss (8,184 predicted full lengths cdss plus 3,171 stop codon containing cdss) indicated that the stop codon most frequently used in goat is tga which account for 53.8%, whereas tag (23.2%) and taa (23.0%) have the approximately equal utility frequency . We used misa (http://pgrc.ipk-gatersleben.de/misa/misa.html) to search the potential simple sequence repeats (ssrs) existed in our assembled transcripts . In this study, the repeat sequence which consists of dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide tandem repeats with at least 18 bp in size was considered as an ssr . We found a total of 2,011 transcripts - derived ssrs that represent 158 unique repeat motifs scattered in 1,850 contigs of which 141 contigs contain at least 2 ssrs . The frequency of ssr occurrence is 4.09% and the average distance is 22.6 kb in our assembled 49,115 large contigs . 43.6% of the total ssrs are trinucleotide repeats, followed by dinucleotide (26.3%), hexanucleotide repeats (20.4%), and only a small proportion of them are pentanucleotide and tetranucleotide repeats (5.5% and 4.2%), respectively . The ac / gt (24.1%) motif comprises the highest frequency among all the identified ssrs, followed by ccg / cgg (18.4%), agc / ctg (11.5%), and agg / cct (7.9%). The types of ssrs and their occurrence frequencies that we found in the goat are similar to the findings in other mammals but different from the results in plants . For example, the ac / gt repeat type, most abundant in the goat transcriptome sequence, is also very abundant in the human genome and in other vertebrates, whereas this repeat type is rarely observed in rice and sweet potato [20, 21]. The microsatellites identified in this work will become a valuable resource for goat genetic mapping . To functionally annotate the assembled contigs, a sequence similarity search was performed against bos taurus refseq protein sequences (32,242 sequences), the swiss - prot protein database, and the nonredundant (nr) protein database by using blastx algorithm with a constant e - value (10). With this approach, 21,104, 21,193, and 22,146 contigs were annotated by bos taurus refseq protein sequences, swiss - prot protein database, and the nr database, respectively . 20,984 out of 22,146 contigs showed 70% sequence identity with nr top hit at matched region (figure 2). Among the 22,146 blastx - hit possessing contigs, it is worth to note that only 103 (0.47%) contigs corresponding to the nr database top hits match goat itself, which could explain the limited number of goat gene and protein sequences currently available in the public database . Examining the 22,146 (45.1%) contigs with a high similarity to nr, we found that 19,040 contigs also harbored a predicted cds, demonstrating that many putative cdss cannot be annotated by known functions and thus may indicate some new genes existed in our assembled contigs . The remaining 26,969 contigs which had no significant hits in the nr database are more likely 5 and 3-untranslated regions (utrs) or previously uncharacterized ests (or genes specifically expressed in capra hircus). These transcripts are shorter in average length and relatively less abundant in their sequencing depth compared with those contigs which significantly hit to the nr database (data not shown). However, we also noted that many contigs with high sequencing depth showed no hits with the nr database . In the top 1,000 most reads - abundant contigs, 69 contigs with an average length of 893 bp showed no hits with known proteins . In fact, when we aligned sequenced reads to the 36,442 mus musculus and 34,573 bos taurus refseq mrna, only 8,717 (25.2%) and 14,836 (43.0%) sequences were mapped to each, respectively . This suggested that capra hircus is not phylogenetically close to the other two mammals, even though capra hircus belongs to the same family as bos taurus . Since hair (wool and cashmere) mainly consists of highly compressed dead keratinocytes, we elucidated the relative abundance by calculating the values of reads per kilobase per million reads (rpkm) of 49,115 contigs which enabled us examine the expression level of the keratin - encoding genes relative to other genes . Through blast searching, we found a total of 126 keratin or keratin - associated protein (kap) encoding sequences presented in our contig database . As expected, of the total 49,115 contigs, the top ten most abundant exclusively encode keratin or kap, including k5, k14, kap3.1, k33b, and kap1.1 . The remaining 116 keratins or kap associated contigs also showed a greater average abundance than other genes . We also noted that some of the keratin - related contigs exhibited relatively higher amino acid diversity (6 out of 126 with <80% identities and 11 with <90% identities at blast matched region) when compared with the nr database top hits, suggesting a series of novel keratin variants may be synthesized in skin tissue undergoing rapid hair (anagen phase) growth . The expression level of keratin or kap may also give an insight into the promoter efficiency and selection while performing exogenous gene expression in the skin tissue . We used blastx to search against functional proteins from the kog (eukaryotic orthologous groups) database which is a component of the clusters of orthologous groups (cog) database . 16,036 contigs had significant hits (e - value 10), and these were classified into 4 groups and 25 functional clusters . Apart from 4,750 poorly characterized genes, cellular process, and signaling appeared as the largest group, which consisted of three highly abundant clusters including signal transduction mechanisms (3,106 genes), intracellular trafficking, secretion, vesicular transport (906 genes), and cytoskeleton (864 genes). Furthermore, to analyze pathway - based biological activities of genes which were expressed in goat skin, we annotated the 49,115 contigs against the kegg (http://www.genome.jp/kegg/) protein database . From our contig database, 15,020 (30.6%) contigs were assigned to the 291 kegg pathways . Among them, 3,948 genes can be further assigned to 23 signaling pathways, including the mapk, wnt, insulin, hedgehog, tgf - beta, vegf, and notch pathways which have been previously demonstrated to play various important roles in hf development and hair shaft differentiation . In addition, gene ontology (go) (http://www.geneontology.org/) analysis was also performed by using the blast2go program to further classify the transcript - derived contigs . 18,069 contigs were cataloged into three main go domains with a total of 129,669 go ids, and further subdivided into 47 subcategories (figure 3). Of these assigned go terms, biological process was the predominant domain followed by molecular function and cellular component . Under the biological process category, we found that cellular process and metabolic process are prominently represented, as they were in the kog and kegg classification, suggesting complicated metabolic activities occurred in anagen phase goat skin . A high correlation among kog, kegg, and go classifications may reflect that goat hair growth is mediated by the complicated metabolic processes . A popular method of global measurement of differentially expressed genes is to take the quantity of ngs reads as an indicator for calculating transcript abundance [57]. Quantitative measurement of gene expression by ngs technologies has been suggested to be accurate and highly correlated with other methods of detecting gene expression levels, such as qrt - pcr and dna microarray . To identify differentially expressed genes (degs) among the three different skin types reflected in our short reads dataset, we mapped the short reads datasets from three libraries to the 49,115 contigs by soapaligner with the seed length of 50 bp . After mapping skin type - specific short reads to the reference, we calculated rpkm values for all contigs which can be used to quantify the expression of contigs both within and between samples . An ac statistical framework was applied to calculate the p value of each transcript - derived contig expression difference by two - sample comparison . Then, we performed multiple hypothesis testing by controlling the false discovery rate (fdr, q - value) to correct the p - value . In this study, both the fdr was less than 10 and the rpkm values were greater than 2-fold (or less than 1/2-fold) different between two samples, then they were considered as a statistically significant degs (table s4). From bl - bk comparison, we observed that 3,532 transcript - derived contigs were upregulated expression from bk when compared to bl skin, and 9,927 were downregulated (figure s1). Similarly, 3,128 upregulated and 6,811 downregulated contigs were detected in the comparison of bs skin with bl skin . Further analysis revealed that 1,360 transcript - derived contigs were consistently upregulated and 4,973 were downregulated in bk and bs compared to bl skin (figure 4). However, the number of degs was sharply reduced to 5,367, of which 3,338 were upregulated and 2,029 were downregulated from bs to bk skin, indicating that the genes expression patterns between bk and bs are more similar than those two comparisons . The bk and bs skin of the cashmere goat mainly produce cashmere fiber, whereas bl skin mostly grows wool fiber but few cashmere fibers . Therefore, to investigate differences between these two kinds of skin types, we annotated the 6,333 consistent degs and further performed kog enrichment analysis compared with transcriptome background . We found that the clusters involved in the cell cycle control, cell division, and chromosome partitioning in kog classification were overrepresented by these degs (table s5). The evidence is that 71 out of 1442 (4.92%) annotated degs (kog database annotation) which derived from 538 counterparts of total 17,594 (2.97%) annotated transcriptome sequences (p <8.47e 6, q - value <2e 4). Furthermore, ten significant degs that enriched in this cluster were selected to perform qrt - pcr and to investigate gene expression difference among three skin types . Although the exact fold difference for each deg by qrt - pcr were different from the rna - seq method but all the comparison pairs had the similar trends with rna - seq approach suggesting the relative high consistency between rna - seq and qrt - pcr (table s6). To obtain the comprehensive transcripts of goat and its gene expression profiles reflected in different skin types during anagen phase, we sequenced and assembled mrna from bl, bk, and bs skin of body coat . The assembler used in this work is inchworm which is a major component of trinity software . Initially, the assembler generated 265,169 contigs over 100 bp (average contig length 299 bp and n50 length 417 bp) corresponding to ~79.3 mb sequence length, 87,962 contigs of which are longer than 200 bp (average contig length 622 bp and n50 length 1001 bp) representing ~54.8 mb nonredundant sequence length . When we use these smaller contigs for functional annotation, more redundant hit accessions were obtained, and the functional classification of transcripts - derived contigs would be more redundantly represented in each functional cluster, which gave us biased interpretation and overview of the transcripts functions . In this study, we used the contigs over 300 bp for annotation, which yielded 22,146 hits and represented 17,472 nonredundant accessions, indicating that the large proportion of the contigs belonged to unigene clusters . On the other hand, approximately 87% of the total short reads can uniquely be mapped to the 49,115 contigs also suggesting that the majority of reads contributed to comprising those larger contigs . As the different skin - regions of the cashmere goat body coat, cashmere fibers are mainly produced from skin on the bk and bs, with few growing from the bl part . The molecules that are differentially expressed among these skin types may have underlying or potential roles associated with cashmere growth . Through calculating the short reads mapped on each contig from different libraries, we identified 6,333 consistently differentially expressed transcripts from bk and bs responded to bl skin (figure 4). These degs were mainly enriched in the cell cycle control, cell division, and chromosome partitioning in the kog functional clusters, indicating that the gene expression pattern associated with cell cycle and the cell division between two types of skin are significantly different . For instance, a kinetochore - bound protein kinase, named budding uninhibited by benzimidazoles 1 (bub1), was identified 4- and 3-fold downregulation from bk and bs compared with bl skin (contig i d a91887;15). This kinase functions in part by phosphorylating a member of the miotic checkpoint complex and activating the spindle checkpoint [22, 23]. Similarly, we found another important kinetochore protein known as ndc80 (a132791;11) which is responsible for chromosomes segregation during m phase of the cell division that was also identified as a significant downregulated gene from bk and bs compared with bl skin . In addition to the genes involved in the spindle checkpoint during cell division, we also noted that some important regulators which directly participate in the regulation of cell cycle progress are differentially expressed . For example, we found that cyclin - dependent kinase 7 (cdk7, a53471;29) as well as its partner, cyclin - h (a65131;18), which form a complex to directly regulate cycle division were both identified as downregulated degs in bk and bs libraries compared within bl part . Furthermore, profiling of cell cycle associated degs by using qrt - pcr method also showed relativelyhigh consistent result with rna - seq . This may suggest that the hair synthesis rates between cashmere- and wool - producing skins are significantly different . A large body of literature has focused on revealing the molecular mechanisms of hf initiation, patterning, and hair cycling in mammalian model organisms such as mus musculus . Many significant studies mainly focused on the function of upstream molecules of the signal transduction pathway such as wnt/-catenin, tgf-, eda, hedgehog, igf, and their receptors [2642]. Generally, conditional knockout or skin tissue - specific overexpression of ligand, receptor, or adaptor molecules from these pathways during the embryogenesis or postnatal stages usually has diverse effects on hf and hair shaft formation . . Sustained epithelial -catenin activation by a transgenic approach caused excessive induction and fusion of hf with severely impaired fiber shaft formation . The most compelling molecule discovered to promote hair growth is fgf-5, a secreted protein that when ablated from mice leads to abnormally long hair (~1.5-fold longer than wild type) by either the elongation of the anagen phase or retardation of catagen initiation . Igf-1, another important mitogen associated with hf development, has been reported as an elongation factor of mouse whiskers and was recently demonstrated to promote body hair growth by overexpression of igf-1 in mouse skin through transgenic approach . But this growth was accompanied by an absence of two types of body coat hair and disorientation of a small proportion of hf . Nevertheless, we did not identify that these molecules encoding genes were significantly differentially expressed . This may ascribe to the three samples that were all derived from anagen phase of the hair cycle, because many previous studies have demonstrated that these signaling molecules and their receptors function as important regulators during the transition from telogen (resting phase) to anagen, such as wnt, sonic hedgehog, and tgf- family members [4547], suggesting that the expression levels of these signals are different between two periods . In our deg catalogue, only the genes associated with cell division and cell cycle are significantly enriched, which might indicate that the efficiency of hair synthesis is different between two skin types . The function of these enriched degs involved in the hair cycling should be further characterized . Taking into consideration the read abundance, average contig length, n50 length, and total contig size, our assembled contig catalogue provided a relatively complete and comprehensive dataset which could reflect the goat skin transcriptome during the anagen phase . The identification of numerous genes, including those showing differential expression in three skin types, especially those degs which are enriched in the functional cluster, will provide us good launching points and resources for further characterizing gene functions associated with hair growth . Our dataset was generated solely with the use of an illumina hiseq2000 platform, demonstrating that this ultra - high - throughput sequencing technology is a suitable tool for investigation of the large eukaryotic organism transcriptome and global measurement of gene expression profile . Finally, the extremely abundant paired - end reads generated from anagen phase will be very useful for subsequent studies, such as comparisons with gene expression patterns from catagen or telogen phase, which will be very helpful for further identifying genes associated with hair follicle development and fiber growth.
The cases were identified at nepal red cross society (nrcs), central blood transfusion service (cbts), kathmandu, nepal . Information of the subjects such as age and gender was accessed from the standard blood donor questionnaire form of cbts recorded by health professionals from the blood donors . Samples were tested with confidentiality and identified by the sample code number given during the sample collection . Ethical approval to conduct the study was taken from cbts . Who strategy 2 for surveillance diagnosis of hiv was used for the test of the samples (9). Blood samples were tested for hcv using elisa test kits eiagen hcv ab kit (adaltis, italy) confirmed with sd bioline hcv (standard diagnostics, inc . Hcv seropositive cases were tested for hiv, hbv and syphilis with elisa test kits enzygnost anti - hiv plus (dade behring, germany), enzygnost hbsag 5.0 (dade behring, germany), sd syphilis elisa 3.0 (standard diagnostics, inc ., korea) respectively . The positive test results for hiv, hbv and syphilis were confirmed with rapid immunochromatographic test kits sd bioline hiv- 3.0 (standard diagnostics, inc ., korea) and virucheckhbsag (orchid biomedical systems, india) respectively while syphilis was confirmed with the same elisa test kit . Test results with seropositivity for hiv, hbv and syphilis among hcv positive samples were considered to determine the co - infection rate . Among the 139 hcv seropositive cases, eight of them were determined to have co - infections with total co - infection rate of 5.75% (95% ci=2.52 - 11.03). Co - infection rate of hiv among hcv was 3.59%, hbv among hcv was 0.71% and syphilis among hcv was 1.43% (table 1). All the co - infection cases were male though 11 hcv seropositive females were included in the study . Hiv / hcv co - infection was seen in the age group between 2150 years . Hbv / hcv co - infection in the age group 3140 years and syphilis / hcv in the age group 2130 years . Prevalence of hiv, hbv and syphilis co - infection among hcv cases indicates that the infections could be transmitted simultaneously due to their common modes of transmission . Hcv is more commonly transmitted among idus and the co - infection of hiv, hbv and syphilis with hcv can be due to transmission modes such as sharing of needles or unsafe sexual transmission among such risk groups . Hiv / hcv was the most common co - infection followed by syphilis / hcv and hbv / hcv . Prevalence of higher hiv / hcv co - infection in this study can be due to the practice of injecting drug use as many idus who become infected with hiv are already infected with hcv . Hiv / hcv co - infection determined is lower than the reported co - infection rate of 10.8% with hcv and hiv (4). Co - infection rate of hiv in hcv estimated is similar to the reported rate of hcv co - infection in hiv patients in north india (2.43%) and 2.2% reported in south india (10, 11). Hbv / hcv co - infection is lower than the hcv / hbv co - infection rate of 1.67% reported in nepalese blood donors (5). Lower co - infections rate in this study is due to the low - risk group of population (healthy looking blood donors) considered; decreasing prevalence of transfusion transmissible infections (3). Higher hiv / hcv co - infection indicates hiv risks associated with the injecting drugs users who are usually infected with hcv . Prevalence of co - infections observed in the study in only males indicates indulgence of such gender groups in unsafe practices that could have transmitted the multiple infections . Monitoring of co - infections is necessary to commence immediate treatment of the cases besides prevention of transfusion associated risks with such blood donors . Co - infection of hiv, hbv and syphilis with hcv is prevalent in the blood donors of kathmandu, nepal . Further investigations can be carried out with larger sample size and in high risk groups with other infections sharing common modes of transmission to estimate the co - infections in the general population of the country . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
Learning and mastering procedural skills are major challenges in anesthesia practice and are essential in the process of achieving clinical competence . Anesthesiologists carry out many complex clinical tasks in their routine work which the trainee is expected to learn and master during training . An increased public awareness of healthcare related issues has led to greater accountability of healthcare professionals . This has very rightly led to an increasing focus on patient safety in clinical practice . The supervisors have to undertake the important responsibility of deciding when a trainee can be allowed to perform the various procedures without direct supervision while ensuring patient safety . Supervisors and trainers must accept that not all trainees can be equally quick in learning and equally competent in performing practical procedures and reliable, and objective assessment is, therefore, mandatory . Airway management is an inherent part of the routine day - to - day work of anesthesiologists . They are required to perform this procedure not only in the operation theater, but, also in the intensive care unit, the wards and the emergency department . Failure to perform the technique promptly and correctly can lead to serious consequences including death . It is important to ensure that an anesthesia trainee is capable of performing tracheal intubation independently before he or she could be included in a cardiac arrest team, where direct supervision by a senior colleague is not always possible . This requires robust and reliable assessment techniques such as direct observation by senior anesthesiologists using procedure - specific tools while the trainee is performing the procedure on actual patients . When constructing an assessment tool, it is important to explore the literature to see whether there is an already existing instrument that is appropriate and has established reliability and validity . We were successful in retrieving tools for assessment of procedures performed by anesthesiologists, including rapid sequence induction of anesthesia and management of difficult airways . However, we could not identify a structured tool for assessment of routine airway management with established reliability and validity . The objectives of this study were to evaluate the inter - rater and test - retest reliability and construct validity of a tool designed to assess competence in bag - mask ventilation and tracheal intubation . Reliability of a tool is its ability to assess skills consistently by different assessors at different times while construct validity is the ability of the tool to differentiate among varying levels of expertise . Approval was granted by the university ethics review committee (1398-ane - erc-09) and written informed consent was obtained from all participants . Junior trainees were described as those having had more than two and <4 months of anesthesia training, while senior residents recruited were those in the fourth year of training and already performing airway management independently . The study protocol was presented in the departmental faculty meeting so as to share it with all faculty members . The purpose of the study was explained to the participating residents at the time of informed consent . The participants' bag - mask ventilation and tracheal intubation skills were assessed by the use of a structured procedure - specific assessment tool . All three authors participated in the construction of the tool and advice was taken from two other senior anesthesia consultants . The tool comprised of five major categories with further sub - categories in each, in order to evaluate the performance of the trainee in all the essential steps involved in the procedure [table 1]. A simple 3-point scale was used to assess each step, where: steps of bag - mask ventilation and tracheal intubation assessed by direct observation in anesthesia trainees 1 (one) meant step not performed2 (two) meant performance below expectations3 (three) meant performance meets expectationsa column was added for steps not applicable during the performance . 1 (one) meant step not performed 2 (two) meant performance below expectations 3 (three) meant performance meets expectations a column was added for steps not applicable during the performance . Performance below expectation was defined in the tool as unsuccessful attempt or incorrectly performed step, while meets expectation was defined as step performed adequately and successfully . The procedural steps used for assessment of bag - mask ventilation and tracheal intubation skills are provided in table 1 . Before finalizing the tool for the study, we conducted a pilot study to identify any missing steps and to assess the practicality of using the tool in the operation theater . The pilot study provided a chance for a final check on the content validity and served as a means of training the investigators in rating trainees' performance by direct observation . The authors also attended a half - day workshop on direct observation of procedural skills . The residents were assessed while working in their assigned operation theater under the supervision of the assigned consultant anesthesiologist . Trainee's assessment was not done if the patient being anesthetized was pregnant or had oral, faciomaxillary or neck pathology or anatomic anomaly, obesity (body mass index> 30), rheumatoid arthritis, ankylosing spondylitis, a history of difficult airway in the past or was found to have limited mouth opening, buck teeth, short thick neck with limited mobility, and mallampati grade iii or iv . The assessment was done simultaneously by two of the investigators who are senior consultant anesthesiologists and registered supervisors for anesthesia training . The trainee was observed while managing the airway with bag - mask ventilation and intubating the trachea with a tracheal tube . The assessment time began once the patient was transferred to the operating table for induction of anesthesia and monitors were attached and ended when the endotracheal tube position was confirmed, and the tube was fixed . Any decision to take over the procedure, in case the trainee was unable to intubate the patient's trachea, was left to the discretion of the supervising consultant . It was planned to allow two attempts at laryngoscopy and intubation, and if the trainee was unsuccessful after two attempts, it was to be considered a failed attempt . Each resident was observed performing the same procedure again after 3 - 4 weeks by the same assessors to evaluate the test - retest reliability of the tool . Sample size was calculated using pass version 11 (ncss llc, kaysville, utah). In a test for agreement between raters using the kappa statistic, a sample size of 20 subjects achieves 80% power to detect a true kappa value of 0.90 in a test of h0: kappa = 0.50 versus h1: kappa 0.50 using a two - tailed level of significance of 0.05 . Statistical analysis was performed using statistical packages for social sciences version 19 (spss inc ., inter - rater and test - retest reliability were computed by percent agreement and kappa statistic . Kappa statistic was used to evaluate the level of agreement between assessors' ratings and between the same assessor's ratings at two points in time for each item of the structured assessment form . Kappa is positive when the agreement exceeds what is expected by chance; kappa is negative when the observed agreement is less than the chance agreement . For the interpretation of kappa values the rating indicators are: 0.0 - 0.2 slight agreement, 0.21 - 0.40 fair agreement, 0.41 - 0.60 moderate agreement, 0.61 - 0.80 substantial agreement, and 0.81 - 1.0 almost perfect or perfect agreement . Average agreement and the average kappa value was also calculated . For construct validity, the score of sub - categories of the main criteria were added for each rater in order to perform the analysis by using independent sample t - test and mann - whitney u - test (as per rule of normality of the data) to compare the scores between junior and senior residents . Statistical analysis was performed using statistical packages for social sciences version 19 (spss inc ., inter - rater and test - retest reliability were computed by percent agreement and kappa statistic . Kappa statistic was used to evaluate the level of agreement between assessors' ratings and between the same assessor's ratings at two points in time for each item of the structured assessment form . Kappa is positive when the agreement exceeds what is expected by chance; kappa is negative when the observed agreement is less than the chance agreement . For the interpretation of kappa values the rating indicators are: 0.0 - 0.2 slight agreement, 0.21 - 0.40 fair agreement, 0.41 - 0.60 moderate agreement, 0.61 - 0.80 substantial agreement, and 0.81 - 1.0 almost perfect or perfect agreement . Average agreement and the average kappa value was also calculated . For construct validity, the score of sub - categories of the main criteria were added for each rater in order to perform the analysis by using independent sample t - test and mann - whitney u - test (as per rule of normality of the data) to compare the scores between junior and senior residents . The inter - rater agreement between scores at the two assessments is presented in table 2 . Percent agreement and kappa values were found to be high for patient positioning, bag - mask ventilation, chin lift / jaw thrust, and leak around the facemask among the two assessors, and the options of absence of co2 trace, and difficulty in bag - mask ventilation exhibited 100% agreement . The average kappa value for inter - rater reliability for the first assessment session was 0.91 and for the second assessment 0.99, with an average agreement of 95% [table 2]. Inter - rater reliability of the tool for assessment of bag - mask ventilation and tracheal intubation (percentage agreement and kappa values) kappa values and percent agreement for test - retest reliability are presented in table 3 . The average agreement for test - retest reliability was 82% with a kappa value of 0.39 . Determination of construct validity [table 4] showed that senior trainees obtained higher scores compared to the junior trainees in all areas of assessment . This difference was statistically significant for the sums of scores for patient positioning, preoxygenation, and laryngoscopy technique . Test - retest reliability of the tool for assessment of bag - mask ventilation and tracheal intubation (percentage agreement and kappa values) construct validity of the assessment tool for bag - mask ventilation and tracheal intubation assessment of competence in cognitive knowledge, judgment, communication, including history taking, physical examination, etc ., is routinely done by written, oral, and objective structured clinical examinations . However, procedural skills have historically been assessed with subjective evaluations done by senior colleagues and supervisors without well - defined criteria or through procedure logs maintained by trainees . Work has been done on defining a minimum number of procedures required to attain competency in anesthetic procedures . However the relationship between experience, as judged by number of procedures performed, and competence is difficult to define and differs markedly in trainees . End - of - rotation global rating forms are often filled out by supervising faculty members who have not directly observed trainees performing the procedure on patients . This form of assessment cannot reliably assess procedural skills in their entirety and cannot be justified for use in decisions about allowing trainees to perform procedures without direct supervision . Direct observation of the trainee, while performing a procedure on an actual patient, is recommended for a more reliable assessment of competence in procedural skills to enhance the quality of clinical training and ensure patient safety . The construction of procedure - specific assessment tools is therefore required for all complex procedural skills . It is essential to ensure that the trainee masters the principal components of airway management before he / she is allowed to perform this procedure without direct supervision . The tool employed in this study was designed specifically for novices in anesthesia and hence the technique was broken down into each of its basic steps forming a checklist with a simple rating scale of 1 - 3 so that the procedure could be assessed in its entirety as recommended for assessment of procedural skills . The inter - rater reliability for the tool was high . During their training, the anesthesia trainees work at multiple sites with multiple consultants who are responsible for their assessment and provision of feedback . Good inter - rater reliability is, therefore, a basic requirement for this assessment tool . This would allow the tool to be used by different assessors in different locations depending upon the initial rotations of the trainee . Many other researchers studying the inter - rater reliability of procedure - specific assessment tools for medical trainees have obtained good to excellent results for inter - rater reliability . The test - retest reliability for the assessment tool does not show as high agreement or kappa values as for inter - rater reliability . The most probable reason for this seems to be the learning effect involved due to the 3 - 4 weeks interval between the two assessment sessions . The anesthesia trainees get frequent opportunities to perform bag - mask ventilation and tracheal intubation on a daily basis and thus get the adequate practice to learn and master the skills in the early months of their training . Therefore, their performance might have improved in the 3 - 4 weeks between the two assessments in this study . We found that the senior trainees obtained higher scores for all steps of bag - mask ventilation and intubation, the difference being significant in many of the steps [table 4]. This indicates that this procedure - specific structured assessment tool has the ability to discriminate between junior and senior trainees, thus depicting good construct validity . Obtained similar results when testing validity and reliability of an assessment tool for brachial plexus regional anesthesia performance and have recommended their tool for routine use during anesthesia training . The main use of the tool employed in the current study will be for assessment of junior anesthesia trainees in their first 6 months of training . Bag - mask ventilation and tracheal intubation are among the first few procedural skills that anesthesia trainees learn at the beginning of training and then use it for the rest of their professional career . The authors hope to use the instrument for formative assessment in novices and for judgment of competence to perform the procedure without direct supervision . The average assessment score obtained by the group of senior trainees could be used to ascertain the score that the junior trainees must reach before they are trained and assessed for more advanced airway management skills required during difficult intubations and rapid sequence induction . Both percent agreement and kappa statistics were used to analyze the reliability of the tool to increase the strength of the analysis . The percent agreement does not take account of the possibility that raters may guess on some scores due to uncertainty . It is therefore advised to calculate both percent agreement and kappa for analysis of inter - rater reliability . A limitation of our study is that the assessments were done in real time, and, therefore, the assessors were not blinded to the trainees being assessed . Similar studies on assessment tools have been performed by assessing videotaped performance of procedural skills after masking the identity of the trainees or by employing assessors not known to the trainees and vice versa . Efforts were made to reduce this bias by the inclusion of residents who were not rotating with either of the two assessors at the time of assessment . Another limitation of this study is that a relatively long interval was allowed between the two assessment sessions . This could have affected the value of test - retest reliability due to learning effect, which is the main shortcoming of test - retest reliability studies . We recommend that the second assessment should be done after shorter intervals to ascertain the test - retest reliability of tools used for assessment of frequently performed procedure such as endotracheal intubation . The absence of criteria for passing or failing the assessment may be considered as a limitation of the tool . This has been overcome by adding a sentence: demonstrates ability to perform all aspects of the procedure independently with a yes / no option at the end of the procedural steps . This section must be carefully filled by the assessors as it identifies whether or not the candidate was able to perform the entire procedure successfully and thus indicates that he / she has passed or not passed in performing the skill . Simulation - based skill assessment is now being described for assessment of residents' ability to perform anesthetic skills . However, financial constraints are a limiting factor in developing countries, where reliable and valid assessment tools like ours would be feasible and practical for routine assessment of trainees . As stated by cuschieri et al . Development of objective procedure - specific assessment tools for evaluation of procedural skills and their integration into training programs are the needs of the day . We believe that objective assessment with direct observation using well - defined criteria and rating scales has the potential to greatly improve assessment of procedural skills . Future research should focus on assessing improvement in procedural skills and quality of patient care with implementation of procedure - specific tools for assessment of skills in anesthesia training programs . Our results show that the tool designed by us to assess bag - mask ventilation and tracheal intubation skills in anesthesia trainees demonstrates good construct validity, excellent inter - rater reliability, and fair test - retest reliability.
Metabolic syndrome (mets) comprises a group of conditions, including central obesity, dyslipidemia elevated blood pressure (bp), and abnormal glucose metabolism . It is associated with increased risk of type 2 diabetes (t2 dm) and cardiovascular disease (cvd). The prevalence of mets has been increasing dramatically in china during the past decade, accompanied by the rapid economic growth and adoption of a sedentary lifestyle [13]. Pathophysiologically, mets is characterized by chronic low - grade inflammatory responses which are associated with abnormal levels of cytokines and other inflammatory signaling markers [46]. Fractalkine (cx3cl1), the only known member of the cx3c class of chemokines, is known to convey its signals through a single g - protein - coupled receptor, cx3cr1, thereby promoting leukocyte activation and survival . Fractalkine expression has been detected in activated or stressed endothelial, smooth muscle cells, skeletal muscle, macrophages, neurons, hepatocytes [812], and adipocytes . It is characterized as a structurally unique chemokine, with both membrane - bound and soluble forms that act, respectively, to promote cell - to - cell adhesion of circulating leukocyte or as a classical chemoattractant of monocytes and lymphocytes [9, 1416]. The soluble fractalkine is generated by cleavage of the membrane - bound form by two peptidases, adam10 and adam17 [17, 18]. Patients with unstable angina pectoris and plaque rupture or cvd show strongly enhanced activation of the fractalkine / cx3cr1 axis, and this signal has been implicated in the development of these pathogenic processes . A recent study reported that inflammation upregulates fractalkine, particularly in the adipose tissue of obese individuals and t2 dm patients . A putative explanation for the association between fractalkine and mets was published recently, but the evidence of such a relationship remains scarce . Therefore, this study was designed to investigate the relationship between baseline serum fractalkine and the development of mets using a group of middle - aged chinese adults . In addition, the fractalkine - mets association was evaluated to determine any potential dependence upon well - established risk factors of mets, such as central obesity, c - reactive protein (crp), insulin resistance, and dyslipidemia . This population - based cross - sectional survey was conducted from march to may 2010 in the caihe community of hangzhou, zhejiang province, china . A total of 887 eligible han chinese participants, aged 4065 years, were recruited in the baseline study . None of the participants had a previous diagnosis of diabetes, moderate to severe hypertension (resting bp> 170/100 mmhg), other cvd, chronic renal disease, acute infectious disease or chronic inflammatory disease, endocrine disease, cancer, or treatment with lipid - lowering drugs . During the 2-year follow - up period, 428 participants dropped out because of death (n = 8), loss of contact (n = 143), or withdrawal from the study (n = 277). At the end of the study, the study protocol was approved by the ethics committee of sir run run shaw hospital and conducted in accordance with the declaration of helsinki . Written informed consent was obtained from all participants . Face - to - face interviews were conducted by trained medical staff using a standardized questionnaire to collect participant demographic data and to obtain baseline lifestyle and health status information . Participants visited local community health care centers between 7 and 8 am following an overnight fast . Venous blood samples were collected at 0 and 2 hours following a 75-g oral glucose tolerance test (ogtt). Blood samples obtained for laboratory testing were immediately centrifuged, and the serum was stored at 80c . Serum glucose concentrations, triglyceride (tg), total cholesterol (tc), low density lipoprotein - cholesterol (ldl - c), high density lipoprotein - cholesterol (hdl - c), and crp were assayed with an autoanalyzer (aeroset, chicago, il, usa). Glycosylated hemoglobin a1c (hba1c) was measured by ion - exchange high - performance liquid chromatography (hemoglobin testing system; bio - rad, hercules, ca, usa). Serum insulin levels were measured by a radioimmunoassay using an insulin detection kit (beijing north institute of biological technology, china). Homeostatic model assessment of insulin resistance (homa - ir) was calculated using the following formula: [fasting serum insulin (fins; mu / l) fasting serum glucose (fpg; mmol / l)/22.5]. Fractalkine concentration was determined with a commercially available enzyme - linked immunosorbent assay (r&d systems, minneapolis, mn, usa). Intra- and interassay coefficient of variation were 1.7% to 4.3% and 3.5% to 7.9%, respectively . Body mass index (bmi) was calculated by dividing body weight by height squared (kg / m). Waist circumference (wc) was measured at the midpoint between the lower border of the rib cage and the iliac crest . Hip circumference was measured at the widest point of the hips, and the waist - to - hip ratio (whr) was calculated and recorded for each patient . Body fat percentage (fat%) was measured by bioelectrical impedance analysis (tbf-300, tanita co., tokyo, japan). Systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured in triplicate using a mercury sphygmomanometer, and the average of the three measurements was recorded . Abdominal adipose tissue was measured using a whole - body imaging system (smt-100, shimadzu co., kyoto, japan) with tr-500 and te-200 of se . Magnetic resonance imaging (mri) was performed at the umbilical level with the participant in a supine position; abdominal visceral adipose tissue area (vfa) and abdominal subcutaneous adipose tissue area (sfa) were calculated with the accompanying software . Mets was defined according to criteria established by the joint committee for developing chinese guidelines on prevention and treatment of dyslipidemia in adults (jcdcg). Individuals with three or more of the following abnormalities were considered as having mets: central obesity (wc> 90 cm for men and> 85 cm for women); hypertriglyceridemia (1.70 mmol / l); elevated bp (130/85 mmhg or current treatment for hypertension); and hyperglycemia (fpg 6.1 mmol / l or 2 h postprandial glucose (2 h pg) 7.8 normally distributed variables were expressed as mean standard deviation (sd); variables with a skewed distribution, including fractalkine, insulin, glucose, homa - ir, tc, tg, crp, sfa, and vfa, underwent a lg(x) transformation to achieve a normal distribution and were reported as median value (interquartile range) [m(iqr)]. Fractalkine levels of the 459 participants with 2-year follow - up data were grouped into quartiles to simplify the interpretation of the results of subsequent analyses . The chi - squared test was used to compare categorical variables between groups . For continuous variables, t - test was used to compare between 2 groups, and anova test was used for comparison of multiple groups . Bivariate correlation analyses between fractalkine and the metabolic parameters were performed using pearson's correlation analysis . The adjusted odds ratios (ors) for the development of mets at year 2 according to the baseline fractalkine quartiles were calculated in multivariate logistic regression models . Potential confounders, including age, sex, and lifestyle factors, were carefully controlled . Potential interactions between vfa, crp, homa - ir, tg, hdl - c, and fractalkine were also examined . All statistical analyses were performed with spss 20.0 (ibm, armonk, ny, usa) and considered statistically significant when the 2-sided p value was <0.05 . The mean (sd) age was 56.90 (7.28) years, and 39.5% of the participants were male . Among the participants, the median (range) for serum fractalkine was 0.44 (0.280.65) ng / ml for males and 0.40 (0.250.62) ng / ml for females (p = 0.379). As expected, participants with mets at baseline had a greater number of adverse risk factors than participants without mets, including higher bmi, whr, fat%, insulin, homa - ir, hba1c, tc, crp, sfa, vfa, and mets defining parameters (table 1, p <0.05 for all parameters). In addition, the fractalkine concentration was significantly higher in participants with the mets (table 1, p <0.001). Serum fractalkine concentration was positively correlated with bmi, wc, whr, fat%, bp, blood glucose, insulin, homa - ir, tc, tg, sfa, and vfa but negatively correlated with serum hdl - c after adjustment for age, sex, education, smoking, and drinking at baseline (table 2). Of all the metabolic parameters, fractalkine showed the strongest correlation with vfa (r = 0.28, p <0.001). There was no significant difference in baseline characteristics between these subjects and the general participants (see table 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/715148). Among them, 399 participants did not have mets at baseline . Participants in the higher fractalkine quartiles at baseline exhibited higher levels for bmi, wc, whr, fat%, blood glucose, insulin, homa - ir, tc, and tg (all p <0.05) than participants in the lower quartile after 2 years . In addition, participants with higher fractalkine levels had lower hdl - c levels (p = 0.035). The prevalence of mets and each component at year 2 increased along with the elevation of baseline fractalkine concentration (table 3 and as presented in table 4, the baseline age-, sex-, education-, smoking-, and drinking - adjusted fractalkine was found to have significant positive correlations with multiple adverse metabolic parameters at year 2, including high bmi, wc, whr, fat%, blood glucose, insulin, homa - ir, hba1c, and tg . On the other hand, a significant negative correlation was found between baseline fractalkine and hdl - c level at year 2 . In addition, the number of mets components, indicated as 1, 2, 3, and 4, increased gradually across the baseline fractalkine concentration at the 2-year follow - up (figure 2). Among 399 participants who did not have mets at baseline, nine of 399 participants were on statins at year 2 but did not have pretreatment lipid profiles for accurate classification of the mets status and hence were excluded from year 2 analysis . The baseline fractalkine concentrations were significantly higher in participants who had progressed to mets by year 2 than in participants without mets [0.51 (0.360.68) versus 0.40 (0.240.58), p <0.001]. In the multiple stepwise logistic regression analysis, participants in the higher quartiles for fractalkine had higher or for the development of mets and its components by year 2.table 5 (model 2) showed that, compared with the lowest quartile of fractalkine concentration, the ors in the highest quartile were 7.18 (95% ci: 2.2818.59) for mets, 4.83 (95% ci: 2.0911.19) for central obesity, 1.03 (95% ci: 0.551.93) for elevated bp, 3.61 (95% ci: 1.638.02) for hyperglycemia, 2.63 (95% ci: 1.305.34) for hypertriglyceridemia, and 1.59 (95% ci: 0.604.24) for low hdl - c . Further adjustment for vfa (model 3), vfa and crp (model 4), homa - ir (model 5), or tg and hdl - c (model 6) only slightly reduced the magnitude of the association of baseline fractalkine with the development of mets (or = 5.31, 95% ci: 1.6514.09 for model 3, or = 5.17, 95% ci: 1.6013.74 for model 4, or = 5.73, 95% ci: 1.7914.34 for model 5, and or = 5.94, 95% ci: 1.8515.09 for model 6). These results suggest that the association between fractalkine and the development of mets is independent of central obesity, crp, insulin resistance, and dyslipidemia . In addition, circulating fractalkine concentration was significantly associated with the development of each mets component (table 5). The associations of fractalkine with hyperglycemia were particularly strong and independent of vfa; the association between fractalkine and the other mets components was largely explained by central obesity . Altered circulating cytokine levels can be used as early abnormal markers and may contribute to mets development . This study addressed the relationship between fractalkine and the development of mets in a 2-year prospective study . We found that elevated serum fractalkine concentrations were significantly correlated with the development of mets . And the mets severity at the 2-year follow - up defined as the number of mets components increased along with the elevation of baseline fractalkine concentration . Central obesity, insulin resistance, inflammatory marker (crp), and dyslipidemia are well - established risk factors of mets [2325]. However, in this study, adjustments for vfa, crp, homa - ir, or tg and hdl - c and other potential confounders yielded only minor reductions in the risk of mets development across fractalkine quartiles . Thus, the observed association between fractalkine concentrations and development of mets cannot be attributed mainly to central obesity, crp, insulin resistance, or dyslipidemia . In this study, body composition was assessed not only by bmi, wc, whr, and fat%, but also by sfa and vfa . Body fat distribution, especially visceral fat accumulation, is more strongly correlated with obesity - related metabolic disorders than the overall amount of body fat [26, 27]. Compared with subcutaneous fat, visceral adipose tissue is known to have more extensive inflammatory leukocyte infiltration and adipocytokines content . Shah et al . Reported that fractalkine levels in subcutaneous adipose were significantly higher in obese individuals compared to their lean counterparts and that fractalkine concentrations were more strongly correlated with visceral than subcutaneous adiposity . However, they did not report an observation of increased serum fractalkine concentrations in the obese participants . Recent studies in 3306 middle - aged uk women and in a group of obese mexican - american children both showed higher fractalkine levels in obese participants with mets than in nonobese participants . Our analysis showed a positive correlation between serum fractalkine levels and bmi, wc, whr, fat%, sfa, and vfa . The discrepancies between those studies and our results might be explained by differences in study design and in the methods of selecting the study participants . There are also differences in the available study data describing the association between fractalkine levels and hyperglycemia . Shah et al . Reported that serum fractalkine concentrations were significantly higher in 281 patients with t2 dm than in 274 nondiabetic participants . Another study using a cohort of middle - aged uk women showed that higher fractalkine levels were correlated with elevated insulin levels . Our data from both cross - sectional and prospective studies also suggest that serum fractalkine is positively associated with glucose and insulin . However, in another study of cvd patients with and without t2 dm or with and without mets, no differences in circulating fractalkine concentration or expression of cx3cr1 were observed . The lack of correlation between fractalkine levels and diabetes has also been reported by others [8, 33, 34]. Accumulating evidence, mainly from cell culture and animal studies, suggests that high glucose concentrations, similar to those seen in type 2 diabetes, promote the expression of fractalkine by smooth muscle cells and endothelial cells in vitro, which may then enhance monocyte adhesion and potentially promote atherogenesis [35, 36]. Relationships between circulating fractalkine concentrations and the lipoprotein - lipid profile have been observed in some studies . Franco et al . Reported that increased fractalkine levels correlated with elevated levels of apo - b and ldl - c . Statin therapy can significantly reduce the expression of fractalkine and cx3cr1 . In the present study, we found significant correlations between circulating fractalkine and tg and hdl - c at baseline and at the 2-year follow - up . And the fractalkine concentrations were associated with the development of hypertriglyceridemia, however, which was largely mediated by vfa or homa - ir . Therefore, it may also be possible that relationships reported in other cross - sectional studies between the lipid profile and fractalkine levels were not causal but largely explained by the concomitant variation in central obesity or insulin resistance . Recent studies have shown that inflammatory cytokines, such as tnf-, ifn-, and il-1, may upregulate membrane - bound fractalkine expression and the release of functional, soluble fractalkine from the bound form [9, 37, 38]. Notably, in our study, we did not observe a significant correlation between fractalkine and crp . Together, this data suggested that fractalkine might provide incremental value in mets prediction beyond current approaches . Further studies are required to determine whether an increase in circulating fractalkine is merely a reflection of obesity - related inflammation or the result of specific regulation by common mediators in adipocytes . First, because of the relatively short follow - up time of 2 years, only a small number of participants developed mets . Whether serum fractalkine levels can be useful in predicting mets has to be confirmed in studies involving larger populations with different genetic and environmental backgrounds . Secondly, although participants with a higher baseline serum fractalkine level present a higher risk of developing mets, we did not have sufficient data on cardiovascular end points to investigate whether this would translate into a greater risk of cardiovascular mortality or morbidity . Thirdly, the dietary intake and work - related physical activity were not assessed in our study . Thus, the data are subject to potential under- or overestimation . In conclusion, in this population - based middle - aged chinese cohort, we have shown that serum fractalkine levels could predict the development of the mets . Our findings suggest that fractalkine plays a potential role in the pathogenesis of mets that is independent of its relationship with central obesity as reflected by vfa, insulin resistance as reflected by homa - ir, systemic inflammation as reflected by crp, and dyslipidemia as reflected by tg and hdl - c . Further studies are required to investigate the efficacy of fractalkine as a biomarker or intervention target for mets.
Constipation is a major debilitating problem in children, which can be caused by various factors . It is a common complaint comprising 3% of referrals to pediatricians and 25% of referrals to pediatric gastroenterologists . In other words, it can be caused by any anatomical or organic reasons; it is caused by taking medications . Fecal incontinency is the most obvious complication of constipation, which is seen in 34% of affected patients . Chronic abdominal pain, as well as rectal and anal pain, is observed in nearly half of the children suffering from constipation . Other complications of constipation are urinary signs such as enuresis or urinary incontinency . According to issenmen's report from north america, 16% of parents of 22-month - old infants complained from their children's constipation . In england, yong and bettie reported that 34% of 4 - 11 year - old healthy children had short term constipation with few complications, while chronic constipation was often the result of a poorly treated acute episode affecting 5% of such british children . Evaluating the blood level of lead is the gold standard for determining the effect of lead on health status . The center for disease control, the american academy of pediatrics, and many national and international organizations consider a blood lead level 10 microgram / dl as lead poisoning . It has been estimated that 99% of lead poisoning cases can be identified through screening . Previous studies have shown a prevalence of 7 - 41% for lead poisoning in different parts of the world . Lead most often enters children's bodies through swallowing surface dust or ingesting peeled pieces of paint, and less frequently by drinking water from lead taps and eating food that is in contact with lead - stained ceramic surfaces . Other sources of lead poisoning include paint, fishing rods, pots soldered with lead, vinyl miniblind, old walls, electric cables, car exhaust fumes . Gastrointestinal signs of lead poisoning include frequent and recurrent loss of appetite, abdominal pain, vomiting, and constipation, which last for some weeks . Children with a serum lead level> 20 microgram / dl complain about gastroenterological problems two times more than those with serum level <20 microgram / dl . Having considered the prevalence of constipation in children and its complications, and as one of the less discussed causes of constipation is lead poisoning, we aimed to assess the serum lead levels of 2 - 13 year - old children complaining from constipation who referred to our center in guilan province, northern iran . To the best of our knowledge no studies have been done so far on the prevalence of lead poisoning in children in our region . In this cross - sectional study, we evaluated 90 2 - 13 year - old children complaining from constipation who referred to 17shahrivar hospital, rasht, guilan province, northern iran, over a one year period from march 2009 to april 2010 . We took 2 - 13 year - old children with a history of constipation lasting for longer than one month as the case group . The control group consisted of 90 2 - 13 year - old guilani children admitted to pediatric surgery ward for non - emergency surgeries without any complaint of constipation . The patients who did not agree to participate in the study or those who did not consent to blood withdrawal were excluded from the study . The demographic data of both groups including their full name, age, sex, place of residence, and parents occupation were recorded . To determine the serum lead level, 2cc of whole blood the blood was kept in a plastic tube containing edta anticoagulant, and transferred to the laboratory while maintaining the cold chain . The serum lead level was measured using the atomic absorption method by graphite furnace method with standard lead (merek device, germany). With respect to their age, the children were divided into two groups; preschool (below 7 years) and over 7 years . Occupations such as painting and battery manufacturing, working in lead mines, and any occupation linked to lead were classified as high risk jobs . The place of residence was assessed considering three aspects: rural or urban areas, high traffic or low traffic, and old building (more than 15 years old) or new building (less than 15 years old). The amount of traffic was measured based on the residential address stated by the parents . The mean (sd) age of the participants in the case and control group was 4.1282.268 and 4.8783.027 years, respectively . The mean serum lead level of the children in the case and control group was 11.643 g / dl and 4.924 g / dl, respectively . Demographic data of children with constipation and the controls on the basis of low and high serum lead levels is shown in table 1 . Comparison of the demographic data in children with constipation and controls on the basis of low and high serum lead levels in the case group, 34 (37.8%) patients had a serum lead level of 10 g / dl compared with 8 (8.9%) patients in the control group (p<0.05). The odds ratio of having constipation in patients with a serum lead level of 10 g / dl was 6.22 times more than those with a serum lead level of <10 g / dl (ci: 2.682 - 14.441). Comparison of the confidence interval and odds ratio in children with constipation and their controls on the basis of lead level and demographic data according to mantel haenszel test is shown in table 2 . Comparison of the confidence interval and odds ratio in children with constipation and their controls on the basis of lead level and demographic data (mantel haenszel test) we found that lead poisoning was significantly more frequent in the children who were less than 7 years old and had constipation compared with those who did not (p=0.001). However, this difference was not significant for the children over 7 years old (p=0.48). Mantel haenszel test showed that age was not a confounding factor in our study since no significant difference was found between both groups (p>0.05) (table 2). With respect to sex, lead poisoning was significantly more frequent in the boys and girls of the case group compared with the control group (p=0.0002 and 0.005 respectively). Statistical analysis showed that sex was not a confounding factor in our study because no significant difference was found between both sexes (p>0.05) (table 2). In the case group, children living in both old and new houses had significantly higher serum lead levels compared with the control group . We found a significant difference between lead level of children residing in old houses and those living in table 2: comparison of the confidence interval and odds ratio in children with constipation and their controls on the basis of lead level and demographic data (mantel haenszel test) new ones which shows higher levels in old houses (or: 5.55 - 6.22). Therefore, living in old or new houses was a confounding factor (table 2). Although the participants in the case group who lived in high and low traffic areas both had higher serum lead levels than the control groups, we found no significant difference between the case and control group in those living in high traffic areas . We found a significant difference between children residing in low traffic areas and those living in high traffic areas (or: 4.87 - 6.22, mantel haenzel test); therefore, the amount of traffic was a confounding factor which is confirmed by statistical methods (table 2). In the case group, more children had significantly higher serum lead levels than the control group regardless of the low - risk or high - risk occupation of their parents (p=0.006 and 0.001 respectively). We found no significant difference between the lead level of children with respect to their parents occupation between the two groups, and therefore, the parents occupation was not a confounding factor (p>0.05) (table 2). In the children living in urban areas although the same figure existed in comparison of cases and controls in the rural areas, and there was no significant difference between rural and urban areas residents (p = 0.3). Therefore, the place of residence was not considered as a confounding factor (table 2). Evidence exists on the negative effects of high concentrations of serum lead on children's health and its negative behavioral, social, and intellectual consequences . The need for control, supervision, and management to decrease the risks of lead exposure is deeply felt . In our study, more boys suffered from lead poisoning in the case group (40.9%) compared with the control group (9.3%). More girls in the case group (34.8%) suffered from lead poisoning compared with the control group (8.3%). In a study by verbel and coworkers on 189 5 - 9 year - old children of 10 different schools in columbia, 7.4% of the children had serum lead levels of 10g / dl . Consistent with our study, they found no significant difference in serum lead levels with respect to sex . However, a review article that assessed the serum lead levels of chinese children from 1994 - 2004 showed inconsistent results . G / l) was significantly higher (89.4 g / l) than that of the girls . We found that in the case group, the odds ratios of those suffering from lead poisoning in the boys and girls were 6.785 and 5.867, respectively, compared with the control group . We also found that boys were at a higher risk of lead poisoning which shows that boys are possibly more exposed to lead . Considering the significant difference between the children living in old and new houses in our study, the age of the building was a confounding factor . In a randomized controlled trial, the effects of decreasing lead pollution on children's serum lead levels were evaluated after a one - year follow - up . 152 children aged less than 4 years with a serum lead level of 7 - 24 g / dl were enrolled . The researchers found that lead - contaminated soil led to lead poisoning in children living in urban areas . Consistent with this study, we found that living in old houses was linked to lead poisoning because of the existing dust and used paint . We found no significant difference between the children residing in urban or rural areas with respect to their serum lead levels . Therefore, the place of residence was not a confounding factor . In a study performed by kumar and colleagues on 150, 5 - 15 year - old indian children, the serum lead levels of the children living in rural areas were evidently lower than the children residing in urban areas . Consistently wang and coworkers found that the mean serum lead level of the children living in industrial or urban areas was significantly higher than those living in suburban or rural areas . We also found similar results which is quite likely considering the higher load of traffic and air pollution in urban areas . Considering the significant difference between the children living in high and low traffic areas, the amount of traffic was considered as a confounding factor . In a study done in belize on 164, 2 - 8 year - old children in four different areas, the researchers found that 7% of the studied children had serum lead levels of 10 g / dl . Moreover, the study showed that children residing in more crowded areas had a higher concentration of serum lead . Researchers then evaluated the mean serum lead levels and the prevalence of high serum lead levels in primary school children in jakarta and assessed the risk factors of exposure to lead before using unleaded diesel . In children who lived near highways or a large intersection, the serum lead levels were significantly higher compared with children who lived in low traffic areas . As shown, consistent with our study, all studies indicate the role of traffic in lead poisoning . In our study, in the children whose parents had low - risk jobs, the odds ratio of having lead poisoning in the case group compared with the control group was 4.071 . The corresponding figure for the children whose parents had high - risk jobs was 17.188 . In khan and colleagues study in pakistan, the frequency of lead poisoning in children whose parents had high - risk jobs (31%) was significantly higher . In a descriptive study, nuwayhid and coworkers measured the serum lead level of 281 healthy 1 - 3 year - old children referring to a medical center in beirut . Logistic regression analysis showed that an increased serum lead level was related to the fathers job . Furthermore, olewe and colleagues studied the potential factors related to increased serum lead levels on 387 under 5 year - old children in kibera slums in nairobi . They found that high levels of serum lead was related to having unhealthy and unsuitable housing, eating and playing in contaminated soil . Queirolo and coworkers conducted a study in montevideo, uruguay on 222 pre - school children to identify the predicting factors for high serum lead levels . They concluded that the lead - related job of the fathers was related to higher serum lead levels in the children . Considering the previously mentioned studies, the high - risk job of fathers is related to lead poisoning in the children, which is consistent with our study . In this study no significant difference was found between the two groups with respect to their sex, age, father's job, and living in urban or rural areas . We suggest screening high risk children such as those living in dusty and smoggy areas . Also, serum lead levels should be measured in children presenting with similar symptoms to lead poisoning such as constipation and loss of appetite . Performing extensive studies on children's serum lead levels and recording their demographic data and other factors could be beneficial.
In most cases geriatrics does not address the problems of death, which is nothing else but the limit of old age, there is also a " physiological death ", unknown as such, as a closing of the vital cycle . Death issues are generally left to other disciplines such as tanatology, tanatopsychiatry, pathological anatomy and forensic . If this is normal, the aforementioned disciplines with their role physicians concerns and practice . The purpose of our study is to pass in review the available data in order to determine the physician s attitude from the bioethics perspective in front of the end of the existence . We intended to approach within this study three aspects: the physician s attitude towards death, death within the medical practice and death assistance . The physician's attitude towards death, a phenomenon which he frequently encounters in his work practice, is most of the times ambiguous, uncertain, lacking a philosophical significance coherent enough . If he sometimes manifests some interest, scientific curiosity for the cause of death, this happens regarding the corpse or specific organs viewed as anatomopathological pieces deprived of identity, inherently depersonalized . But the period that precedes it, which corresponds to the transition from life to death, period of time which includes preagony and agony, when the human being who is about to relinquish life for good suffers, understands, needs assistance, imposes to the doctor most of the time a sort of detachment, of distance . When it seems that " there is nothing else to be done " for the purposes of effective treatments intended to prevent the end, there is still a lot to be in the study of death, it is not normal that the end of old age to be ignored and separate from old age itself, because it harms the very understanding of the ageing process as a whole stage of human existence . It can be seen that the problem of assisting the dying person has rarely been until a few decades ago, and for many physicians, even nowadays, a topic of interest in the medical literature in the medical education in general within the physicians concerns and practice . The purpose of our study is to pass in review the available data in order to determine the physician s attitude from the bioethics perspective in front of the end of the existence . We intended to approach within this study three aspects: the physician s attitude towards death, death within the medical practice and death assistance . It is considered that the phrase " there is nothing else to be done ", more sententious, is right in the case of the elderly person, because old age also associates reality to the natural closure of the vital cycle and it has to represent the phase completion of the assistance granted by the physician and the beginning of another one, the last one, because one can talk of assisting death, assisting the dying person, which represents a problem with real content, not to be neglected . As a matter of fact, the preexitus period is considered to be a distinct period where takes place the transition life - death, the sequences well defined from the bioethical, clinical, therapeutical point of view being as follows: sickness (interventions may be effective in the sense of recovering for health or survival), preterminal phase, (the interventions become palliative: pain relief, moral and spiritual preparation life ending) and terminal phase (the exitus). One can easily notice that the problem of assisting the dying person in general and the dying elderly person in particular is only rarely a topic of interest in the physicians concerns . The content mostly psychological, sociological, moral pushes it to the side in the concerns of the physician beneficiary of a biological and medical training, who avoids to give too much importance to a situation which constitutes rather a failure of his work or of the medicine and this may happen as well out of a reflex of defense, according to psychologists (the death of another human being makes the physician think, more or less, of his own death). At this time assistance, healthcare depend on the ability of the person offering assistance - care to put up with the fear of death which influences him as well . The reasons of physicians reserves towards the issue of death, apart from the anatomopathologic interest mentioned before, are varied . To those deriving from his training as a physician, are added those specific to man as a mortal human being . A general reflex of self - protection that tries to chase away the serious or sad things makes him limit his attachment, the deepening of this domain in addition the nowadays society interested in putting forward the health, vigor and youth rejects, by contrast, old age, decrepitude, death . Furthermore, the society invests physicians with the duty to heal, and education generally trains the physician according to a pharmacotherapist status able to heal everything " . As a result, physicians are themselves invested only with the power to heal . Death is a biological phenomenon, implacable, compulsory, of an inexorable fatality, but the attitudes towards death represent a cultural manifestation . The physician gets his action force from his professional culture, and equally from the culture thesaurus . The attitude towards death has to represent the fusion of the concepts offered by biology, medicine, psychology, philosophy, culture, religion . The life research polarizes a greater interest than death research, although it could serve a better understanding, in general, of the ageing process, knowing that death is obligatorily associated with ageing that it puts an end to . The benefit principle (charity, the care to do well) and non - injury (the care not to do any harm) applies both in the case of palliative care and medicine . In the light of these two basic principles of medical ethics, only death in a climate of collective participation, medical care, human permanence gives the person in question the feeling of accomplishment, of human detachment from the life and the world . Death, and especially the preceding period, cannot be excluded from health care and, therefore, the physician may not avoid obligations (especially moral ones) that devolved on him . Yet, unfortunately, sometimes things are totally different . The dying person, maybe often consciously, spends his last moments in a dramatic isolation . Most of the times the dying elderly person spends his last sufferings in hospital, where he feels the isolation in a more painful way . He has the feeling that he is removed from life earlier, all the more so as he sometimes still hopes that death will go away . Whether he is in hospital or close to his family, the physician will always be that person who has to provide him assistance until the end . Even if he is at home, although there is no hope, the family provides the physician's presence as a psychological support to solace the sufferings, to calm his pain . This demonstrates that the assistance of death represents a reality that must be included in the training of the medical practitioner who is going to embrace in the assembly of his knowledge and attitudes the elements necessary to ensure this assistance to deal with the attitude in the face of death . Taking into account that any elderly patient will reach at a certain point the end of life, the attending physician may question himself about the following three attitudes: to keep a tight hold on the fight against the disease using all therapeutic means, to hasten the inevitable end in order to put an end to sufferings, we are not talking here about an active acceleration but rather about a passivity attitude or to accompany the patient staying close to him without abandoning him, giving him care in order to relieve his sufferings . It is obvious that the most natural behaviour is the last one . Giving effective and qualitative palliative care represents the moral answer to the immortality of euthanasia . There are authors who do not approve to the practice of therapeutic methods and laborious investigations in the case of agonizing patients, as other authors claim that the treatment of the dying person will not be suspended until the last moment . If this issue can be further discussed being different from one case to another, what there cannot be discussed is the moral therapy that has to be always applied by the physician, well trained for this purpose, convinced of its necessity when all the other means become ineffective . The elements of such therapies are represented by ensuring the human presence, communication, then patience, understanding, moderate optimism, warmth, kindness . The doctor has the responsibility to also prepare the environment around the dying person, care staff, relatives, and caregivers . In order to comply with this duty it depends on each person to draw from these great intensity moments the lesson of life and understand that the presence alongside the dying person is an integral part of the medical profession, a profession which never ceases to exist alongside with the sentence " there is nothing to be done ", or when the subject comes of a certain age . This is an attitude in accordance with the principle of respect for life, which animates our profession, life coming to an end only with the last breath and the last heart beating . The physician patient relationships evolved from the paternalistic aspect, where the physician is the one who makes the choice for the well - being " of the patient, to a negotiation between two partners . The problem of palliative and terminal care highlights the need for information and effective communication of prognosis and therapy goals . The convention on human rights and biomedicine (convention from oviedo), besides the provisions with general character, it refers to the situation of patients in terminal phase that do not have the legal capacity to decide on the medical action, showing that: the wishes regarding a medical intervention, that was previously expressed by a patient who, at the moment of the intervention is no longer capable to express his wishes, should be taken into consideration . It has to meet his fundamental needs (of comfort, hygiene, feeding, breathing, rest), his specific needs: (to eliminate or calm the pain), his personal needs (human presence, communication, ensuring his respect as a person, self - esteem). It is said that assistance shall not be provided in order to prevent death, but in order to prevent the suffering . Hereinafter we will present some simple objectives that can become the support and guide of an individualized human relationship with the elderly patient in imminent terminal phase, both at home as in hospital . A first objective concerns the avoidance of a change in the physician s behaviour that might detach himself from the patient the moment the latter is dying . This change of attitude consists most often either in abandoning the patient, shorter, more sporadic and more formal visits, or in discussions in his presence with the caregivers, with the staff, discussions having as subject the aforementioned sentence, even if these discussions are whispered or are made in a foreign language . Another objective of the palliative and terminal medical care is to maintain the normal atmosphere around the elderly person and avoid his isolation, which is so easily practiced from psychological reasons . Being surrounded by an adequate environment allows him to keep hold of the space and faces that are familiar to him in his last moments . In this way he feels safe and another imperative of the terminal phases assistance is ensuring the human beings presence, of an accompanying person, important element which gives the dying person a feeling of security and peace . On the one hand, the treatment and healthcare persons will continue to be close to the dying person even more than before, thus avoiding the short and rare visits, the short lasting and formal contact . It will also be ensured the permanent presence of relatives, of those persons close to the patient . Not only that the forbiddances are not justified, being opposed to the medicine humanism, but they appear as repressive measures that do not belong in the hospital environment . Creating the adequate conditions for the close contact with relatives shall be a rule not only in the assistance of the dying elderly person, but throughout the entire geriatrics assistance . We refer in this sense to the " therapy with the help of the relatives " which is a reality . The good influence on the elderly person s psyche, the feeling that he is not abandoned and isolated from the loved ones has a genuine therapeutic effect often superior to tranquilizers . If all the things mentioned before represent measures intended to ease the mental sufferings, another important measure, component of this assistance phase, is the stopping of physical suffering . Within this framework, another component in palliative and terminal medical care is the proper nutrition and hydration of the patient . A particular imperative in the case of the elderly person s assistance is taking care of the oral cavity, organ that changes during this phase (" non - functional mouth "), and may be a mirror of the sufferings evolution (dehydration, azotemia, infections). Starting with the basic needs, the habitual healthcare represents a very important component in terminal phases assistance . It is about ensuring the general comfort of the subject to which the entire entourage must participate and which will start from the fact that the dying person often finds himself in a situation of external dependence in relation to his entourage . One aspect depending mostly on moral assistance, which has a significant importance in, palliative and terminal medical care is represented by the concern we have to express towards the personal needs and desires of the dying person, which should not be understood as a concession, but as a moral duty of the first order . It is about recognizing the unique identity of the personality of each dying elderly person . His (last) desires should be listened to, the subject should be encouraged to express them and his entourage must try to satisfy them . Throughout the assitance period, maintaining the communication remains an essential objective . At home, as well as at the hospital, efforts must be made to ensure a " permanent human presence, sympathetic, receptive, able to face up to the needs and wishes of the dying person until the last moment . The peace and relief felt by the dying person in the presence of the priest make his presence necessary . The exception is represented by the situations when he does not want this presence or, when having the hope of healing, associates this presence to his end . All the gestures and material concerns mentioned before represent the base of maintaining communication and in fact it means the support and guide of the individualized human relationship with the dying elderly person . It is imperative to know to listen, to understand, to ask, to guess his questions and answers, by examining his look, his facial expression . This non - verbal communication becomes very important and has a benefic effect on him, of tranquility, of peace, of satisfaction . Simple gestures of touching the dying person, shaking his hand, touching his forehead becomes an essential way of communication, an effective moral therapy . The dying person assistance can be a life lesson that has the power to make us better . It is an integral part of the medical profession and completes in a necessary way the technical assistance, which changes from one epoch to another, while moral therapy is only once acquired . Terminally ill patient is regarded as a living person, until the end, and death if viewed as a natural process within the human evolution . The physician beneficiary of medical training of healing avoids giving too much importance to the dying person, a situation which represents more of a failure of his work or of medicine, it can be from a defense reflex, because as psychologists say, the death of another always makes the physician reflects more or less on his own death . The natural consequence of such beliefs induced and accepted by most physicians determines the disinterest towards the people in terminal phase illness, underestimates the palliative care it considers unnecessary, but so essential and vital for the patient), attaches most of the importance to the healing and neglects the quality of life . A physician trained in this spirit feels helpless, the lack of healing is perceived by him as a defeat, as a failure.
Peripheral artery disease (pad) is a prevalent chronic condition that increases with age, affecting 20% of persons over the age of 75 years, and is associated with exceptionally high risks for cardiac and cerebrovascular events [1, 2]. Intermittent claudication, defined as the onset of pain in the leg or gluteal muscles with exertion which resolves with rest, is a sentinel symptom of pad and, in most cases, indicative of disease severity . The effects of claudication on walking performance, that is the ability to walk without pain, vary within patients who have similar clinical profiles [36], suggesting that there are other factors that influence walking performance . The primary goal of conservative clinical management of pad is to minimize disease progression and optimize performance; thus, the ability to easily evaluate the effects of treatment, including lifestyle modification, on walking performance is clinically important . Walking performance has been measured via patient - report questionnaire tools [712] or standardized treadmill tests [13, 14]. However, many clinicians may not use these measures and rely solely on patient's subjective responses to their questioning . Thus it is difficult to quantify, monitor and accurately assess actual or changes in performance across the continuum of this chronic condition . Walking performance in patients with pad has been assessed with continuous and graded treadmill tests; the graded test is a more reliable measure of performance in patients with pad . Patients participate in a standardized protocol and walk on the treadmill at increasing elevations until they experience pain . A number of measures are obtained, including the absolute claudication distance (acd) defined as the maximal distance the patient can reach before they can no longer walk on the treadmill because of claudication pain or peak walking time (pwt) defined as the greatest time of exercise achieved . While these tests provide accurate assessment of the impact of claudication on walking performance and are considered the gold standard of assessment, they are not feasible to conduct in most clinical settings . The walking impairment questionnaire is a common patient - reported measure, first developed by regenstiener et al ., designed to assess walking ability in patients with pad [710]. The wiq has been used to describe walking performance and to assess the efficacy of clinical interventions . The tool has been translated and employed in different countries [1719] and in different conditions . The tool can be either interview administered or completed by the patient, with no significant impact on responses . Concerns have been raised about the complexity of the wording of some of the items on the tool (i.e., patients rate a lower difficulty for higher intensity tasks) and the need to correct responses . While these studies have provided important normative data and descriptions and have suggested that this tool could be used to clinically monitor walking performance, the wiq remains predominantly a research tool . Therefore, the primary aim of this study was to further validate the wiq as a clinical tool for patients with peripheral artery disease . Specifically, we hoped to identify valid cut - off points for identifying patients with low and high walking ability, as indicated by the wiq . Categorization of high and low performers, in combination with knowledge of a patient's clinical condition could allow clinicians to more effectively prescribe treatment strategies for patients' symptoms, monitor progress and make changes to patient management as needed . The research protocol was reviewed and approved by the queen's university health sciences research ethics board . All consecutive pad patients seen in the vascular clinic at kingston general hospital between may 2010 and may 2011 who met the inclusion criteria were identified by two attending vascular surgeons . The identified patients were telephoned, consented and invited to return to the hospital for a study visit . Patients were included if they had a resting ankle - brachial index (abi) of 0.90 . Participants were excluded if they had (a) severe ischemia requiring intervention, (b) comorbid conditions that limited walking (angina, congestive heart failure, chronic obstructive pulmonary disease or severe arthritis), (c) wheel chair, cane or walker requirement, (d) non - compressible arteries, and/or (e) severe cognitive impairment . The exclusion criteria were selected to ensure that participants were able to walk safely on a treadmill, and to ensure that claudication due to pad was the limiting factor for walking performance . The treadmill test was similar to protocols followed in previous pad studies [23, 24] and consisted of a progressive, graded treadmill protocol (constant speed at 3.2 km / hr after initial increase, 0% grade initially with 2% increases in grade every two minutes after the initial speed increase to a maximum of 10%) conducted until maximal claudication pain was reached or to a maximum duration of 30 minutes (about 1.6 km). The peak walking time (pwt) and distance of absolute claudication (acd) were identified as the time and distance to maximal pain, classified as 8/10 on the borg perceived exertion scale . Participants familiarized themselves with the treadmill by starting at an initial speed of 1.7 km / hr . This was increased by 1.6 km every 10 seconds for the first 90 seconds until the maximum speed of 3.2 km / hr was reached . The test start time was the beginning of the treadmill testing session (i.e., included familiarization and graded protocol). Participants were excluded from the analysis if they failed to complete the treadmill test for reasons other than claudication, such as shortness of breath . The walking impairment questionnaire utilized in this study contained 14 items that contributed to 3 subscales (distance, speed and stair) and an overall score . A copy of the questionnaire is included in the appendix . It should be noted that this version of the wiq differs from the original version first validated by regensteiner et al . ; the distance scale includes one more level; the speed questions are the same and there is an added stair subscale . Participants answer each item on a likert scale from 0 for unable to do to 4 for no difficulty . Each response is weighted based on the difficulty of the task (e.g., the weight for walk slowly is 1.5 whereas for the weight for run or jog is 5). Subscale scores are determined by dividing the weighted answers by the maximum possible weighted score and multiplying by 100 . The overall score is the average of all 3 subscores and combined scores are the average of 2 distinct subscales (e.g., distance and speed, distance and stairs, speed and stairs). Items coded as didn't do for other reasons or missing are removed from the denominator of the weighted score to calculate a percent score based on the items that remained (i.e., limitation, if any, was due only to intermittent claudication). In example, if a participant responded with much difficulty for walking 1500 feet (5 blocks), some difficulty for walking 900 feet (3 blocks) and no difficulty for the remaining distance questions the distance score would be: [4(20) + 4(50) + 4(150) + 4(300) + 4(600) + 3(900) + 1(1500)]/14080100 = (8680/14080)100 = 61.65 . If more than half of the items in a subscale are coded as such the subscore is coded as missing . In this study, participants completed the questionnaire after the treadmill test session, after a rest period . The administrator provided no additional guidance to the participant . When reviewing the questionnaire responses we noted that some participants seemed to misunderstand the around the home question and based their answer on the presence of stairs in the house rather than the ability to walk on level ground . Category if the participant's answer for around the home was lower . The abi was obtained from previous testing completed at the vascular testing centre, within 6 months of testing . Weight and height were measured using a medical scale to determine the participant's body - mass index (weight over height squared). Diabetic status (yes or no), smoking status (current, former or never) were self reported . Age was determined based on the participant's self - reported birth date and year of testing date . Number of pack years was determined based on the number of cigarettes smoked daily divided by 20 (standard pack size) multiplied by the estimated duration of smoking in years . Comparison of the abi between those who participated and those contacted and who did not participate was determined using two - sample independent t - tests . Scores for each subscale, combined subscales and overall scale of the walking impairment questionnaire were determined . Participants' walking performance was classified as low, medium and high based on the distribution of the acd scores by tertiles . Receiver operating characteristic (roc) curves were generated for identifying high and low walking ability for each subscale score, combined and overall scale scores . These curves were obtained by plotting the sensitivity against 1-specificity using 0.5 score increments of the scale scores . The cut - off values for the questionnaire were identified for varying levels of sensitivity and specificity (at least 0.80 and at least 0.90). Positive and negative predictive values for the cut - offs of the score with the highest area under the roc curve for 0.90 sensitivity for low performers and 0.90 specificity for high performers were calculated . 174 of the 381 pad patients screened were deemed ineligible based on the exclusion criteria . Of the 207 eligible patients 132 (63.8%) patients consented and participated in testing . 8 participants stopped the test prior to the onset of claudication (e.g., due to shortness of breath) and one additional patient stopped prior to maximum claudication . 123 patients were, therefore, included in the analysis (figure 1). The characteristics of the pad patients who participated in the study (n = 123) are described in table 1 . There was no significant difference in abi between those who participated and those contacted who did not participate (mean abi, 0.58 and 0.60 resp . ). In this study, 11 participants self - reported no claudication; 9 of these 11 experienced claudication with treadmill testing . The subscale and overall wiq scores, categorized according to the acd tertiles are shown in table 2 . The scores increased consistently across the three performance groups, as classified by the acd obtained via the graded treadmill test . Despite a large standard deviation in scores within each performance group, all comparisons achieved a high level of statistical significance . In a bivariate analysis (data not shown) men reported higher wiq speed scores in comparison to women (p = 0.02); there were no other sex difference in other subscale scores, acd and pwt . As well, there were no significant age differences in the wiq scores when age was dichotomized at 60 and above . The associations between the acd, pwt and wiq scores, when controlling for age, sex, and abi were all moderate to strong (partial correlation coefficient, r> 0.5, p <.001). (see table 3) figure 2 plots the linear trend model between the wiq distance scores and pwt (log transformed) within each sex category . The area under the curve of the receiver operating characteristics (roc) curve provides information about the ability of a test to identify true positives and true negatives . The closer the area under the curve is to 1, the better the test is at distinguishing between patient groups . In all analyses we used the unadjusted acd score to identify non - performers and performers as our focus was on actual walking distance; as well the acd and pwt scores (transformed and non - transformed) were highly correlated (r 0.92). The area under the curve values for the roc ranged from 0.80 to 0.89 with the value for the overall wiq score providing the highest value (table 4). Based on this analysis, a wiq overall score of less than or equal to 39.0 permitted identification of a low performer with a sensitivity of at least 0.80 while maximizing specificity (0.75). A wiq overall score of 42.5 increased the sensitivity to at least 0.90 but decreased the specificity to 0.73 . Similar cut - off values are shown for 0.8 specificity and 0.9 specificity in table 4 . The area under the curve values for the roc ranged between 0.73 and 0.81, with the value for the combined distance and stair climbing ability being the highest (table 4). A combined distance and stair climbing ability score of 58.0 permitted identification of a high performance with a specificity of at least 0.80 . Choosing a cut - off of 75.5 increased the specificity to at least 0.90 but decreased the sensitivity to 0.41 . Similar cut - off values are shown for 0.8 sensitivity and 0.9 sensitivity in table 5 . The area under the curve values for identifying high walking performance were lower than those for identifying low walking performance . Negative predictive values were higher for low performers (0.94) in comparison to high performers (0.75). In both cases the positive predictive value was lower (0.62 for identifying low performers, 0.70 for identifying high performers). The ability to objectively measure walking performance in patients with pad with a range of claudication symptoms is relevant to conservative management . While recent guidelines acknowledge the ability of treadmill tests to objectively quantify performance, it is not recommended as a routine measure for practice; thus the need to consider less invasive yet reliable tools . The walking impairment questionnaire (wiq) is the most commonly reported self - report tool that has been used to evaluate patient's walking ability . Although it is typically used as a research tool, it is also has potential to be applied to routine clinical management of patients with pad . We, therefore, built upon previous research and determined cut - off values for the wiq for the potential classification of low and high walking performance in a diverse pad patient population; information that could easily be used by clinicians to make more informed decisions concerning a patient's treatment plan . The wiq scores reported in this study (39.5 for distance, 47.6 for speed and 58.0 for stair climbing) are similar to those of previous studies which ranged from 38 to 55 for distance, 37 to 52 for speed and 48 to 68 for stair climbing [810, 29]. In our sample, the mean acd was 418 meters, similar to other studies with similar populations [23, 24]. Thus both of our standard measures of walking performance are consistent with the most current reports . Previous studies have reported moderate to strong correlations between scores on the wiq and walking performance as measured by treadmill tests in patients with intermittent claudication [9, 30, 31]. Regensteiner et al . Reported that the wiq distance and speed scores correlated moderately and significantly with the peak treadmill walking time (pwt) (r = 0.68, p <0.05, n = 26); myers et al . Reported significant and moderate correlations between the wiq distance and speed scores and the acd (spearman's rank correlations 0.41 and 0.39, resp ., p <0.05, n = 48); and verspaget et al ., using a dutch version of the questionnaire, reported similar correlations: distance, speed and stair climbing scores as well as the overall score were moderately correlated with the acd (0.45, 0.43, 0.37, 0.52, resp ., all p <0.01, n = 130). Our findings support these previous associations and the linear association between patient - reported scores and pwt and acd (data not shown). While the correlation values suggest that moderate to strong associations exist between self - report assessment and actual walking ability, these do not provide clinicians with cut points or indicators of performance . Based on the area under the curve of the roc analysis, we were able to determine that the overall wiq score was the most appropriate score for identifying low performers while the combined distance and stair score was the most appropriate for identifying high performers . However, the 95% confidence intervals of the area under the curve of the roc for all scores or combination of scores overlap . Thus there may not be a significant difference between the accuracy of a particular score or combination of scores in classifying performance . Since no score appears to be significantly more accurate than another, consideration could be given to consistently using the overall wiq score . The accurate identification of low performers is important as these patients' symptoms are impacting walking performance more . This translates into a test for identifying low performers with high sensitivity . In our study, to obtain a sensitivity of at least 0.80 or 0.90 the cut - off values for the overall score were 39.0 (specificity = 0.77) and 42.5 (specificity = 0.73), respectively . Thus, with a cut off score of 42.5 or less we could identify 90% of low performers (i.e., participants who were only able to walk, on average,.96.6 meteres). With this score we would also identify 30% of the participants who were actually performing well but scored lower on the wiq . From a clinical perspective, this would be reassuring as we would accurately assess patients as low performers most of the time and plan care accordingly . High performers may not require further invasive or different interventions as their current conservative management and lifestyle (i.e., exercise) is adequate . Therefore, it is important to have a test with high specificity for identifying high performers with low false positives to ensure that low performers are identified and receive the intervention that they need . To obtain a specificity of at least 0.80 or 0.90, the cut - off values for the combined distance and stair score were 58.0 (sensitivity = 0.62) and 75.5 (sensitivity = 0.41), respectively . Less than 10% of low performers would have a combined distance and stair scores of 75.5 or more; however, 59% of high performers would be identified as being low performers with that same cut - off . Again, from a clinical perspective these cut - offs would be reassuring . The cut - off value for identifying low performers had both high sensitivity and specificity (0.90 and 0.73). It also had a very high negative predictive value (0.94) but a lower positive predictive value (0.62) indicating that this score was very good at identifying low performers in this population but may result in the overtreatment of patients who are misclassified as low . The cut - off value for identifying high performers had high specificity (0.90) but low sensitivity (0.41). In this population it had high positive predictive value and high negative predictive value (0.70 and 0.75, resp .) Indicating that, despite a low specificity, the cut - off may be effective at differentiating between high performers and non - high performers . The population used in this study appears to be a clinically diverse patient sample reflective of the typical pad population; therefore, the positive and negative predictive values identified may be generalizable to the greater pad population . The specific strengths of this study were as follows: validation testing in a large, clinically diverse patient sample reflective of the typical pad population; comparison of the wiq with a graded treadmill test; analysis of varied score combinations for the wiq and detailed roc curve analysis to determine clinically useful cut - off values . Approximately twenty participants perceived they had a higher level of difficulty walking around their home than walking 50 feet . Participants commented that in - home walking ability included stair climbing and this was more difficult than walking on level ground as the question states . Adjusting for this was, therefore, done as described in the methods . This problem, highlighted in previous research needs to addressed through item or question modification . Further testing is warranted . The timing of questionnaire administration after the treadmill test may have influenced the participant's responses . Participants who were pleased with their treadmill test results may have overinflated their ability; participants who were not pleased may have scored lower . Regardless, self reports of patient activity are reliable estimates of activity [32, 33]. Repeat administration of the wiq, perhaps during clinical follow - up, would allow for comparison of reported scores . The population studied, while a diverse population of pad patients (from severe impairment to no claudication), was limited to individuals able to safely participate in a treadmill test and whose walking was limited by claudication and not other factors . This was also a strength of the study as the findings are generalizable to a group who could potentially participate in exercise interventions designed to alleviate symptoms and promote performance . A large number of patients either refused or were unable to participate; however, they were not clinically different than those who did participate . The high number of patients unwilling to participate in a treadmill test or unable to attend a test date does highlight the importance of a having a non - invasive tool such as the wiq with established cut - off points for use in future studies and/or practice . Our findings further support that the wiq could be used to classify the walking performance of patients with pad in a clinical setting, with an acceptable level of sensitivity and specificity . In a diverse population of patients able to safely participate in a treadmill test, an overall wiq score of 70 or higher or a combined distance and stair score of 76 or higher similarly identified high performers . Individuals in the middle range of these scores could also be classified as moderate performers, with an opportunity to improve . This ability to classify walking performance, a sentinel indicator of disease impact, when combined with other patient characteristics could inform clinical decisions and guide patient management . Given the documented evidence that daily activity and exercise enhances walking performance [24, 3436] clinicians need tools to assess and monitor progress and/or decline . Reasons behind the poor uptake of validated self - report measurement tools in the clinical setting are complex, but likely related to factors such as the: (a) validity and usefulness of the results, (b), ease of application and (c) integration of assessment tool into current clinical flow . Integration of validated self - report measures, through a variety of means (i.e., electronic kiosks) are becoming an important component of clinical symptom management and practice in other conditions and settings [37, 38]. The poor uptake of a valid measure of an important symptom for patients with pad is concerning . Further research may need to explore the development and validation of revised and shorter versions of the wiq in similar patient populations . However, a standardized version of the tool for adoption into clinical practice guidelines would be helpful for clinicians, and likely facilitate uptake . As well, a cohort or natural history study of patients with the recommended cut - offs should be conducted to assess the prognostic potential and clinical utility of the suggested wiq cut - offscores.
Rosacea is a common skin disease characterized by transient or persistent central facial erythema, visible blood vessels, and frequently, papules and pustules . Since the 1950's, tetracycline and erythromycin are the most commonly used oral antibiotics (1 - 3). However, long - term treatment with antibiotics is not well tolerated because of side - effects including gastrointestinal intolerance, photosensitivity and candidiasis (3). Moreover, given the chronic nature of the disease, there is a possibility of developing bacterial resistance (1). Azithromycin is a newly developed macrolide which offers some advantages compared to previously used antibiotics (4). Fernandez - obregon (5) reported that all of ten patients who were not tolerated or controlled by conventional treatment demonstrated a significant improvement with the oral use of azithromycin . After then, azithromycin has been found to be effective in the intractable rosacea in several clinical reports (1, 6, 7). We report a case of a 52-yr - old rosacea patient who was refractory and complicated by reactions to conventional topical and oral medications . After 10 weeks of oral azithromycin, her lesions had mostly disappeared and the patient suffered no specific side - effects related to the medication . Our case showed that oral azithromycin could be a viable new treatment option for intractable rosacea cases . A 52-yr - old postmenopausal woman complained of a 10-month history of multiple erythematous papules and some pustules on both cheeks on february 11, 2008 (fig . Physical examination revealed multiple pinhead to rice - sized erythematous papules and pustules on both cheeks . Laboratory studies including complete blood cell count (cbc), blood chemistry, and anti - nuclear antibody (ana) were within normal limits . More than ten demodex mites were observed on the demodex study . On the standard patch test, only nickel sulfate showed a positive response . On the basis of medical history and clinical findings her skin lesions were not controlled by topical application of benzoyl peroxide, metronidazole twice a day or crotamiton once a day, or oral administration of doxycycline 200 mg for a month, metronidazole 500 mg for two weeks or isotretinoin 10 mg for a month . Rather her rosacea was aggravated or complicated by these medications . After taking conventional treatment of rosacea, she complained of diffuse facial erythema, swelling and aggravated papules (fig . We then prescribed oral azithromycin, which has both anti - inflammatory effects and leads to a reduction in reactive oxygen species . Two weeks after oral administration of 500 mg per day of azithromycin, facial erythema and swelling was reduced . Since then, papular lesions which are presented on the cheek were begun to decrease . After 10 weeks of oral azithromycin, the lesions had mostly disappeared, and no specific side - effects related to the medication were noted (fig . 3). Also until 6 months after treatment, the patient had not experienced any recurrence or aggravation . Rosacea is a common cutaneous disorder which occurs most frequently in light - skinned middle aged women . There are variable cutaneous signs of rosacea such as flushing, erythema, telangiectasia, edema, papules and pustules (2). While the pathogenesis of rosacea remains unknown, several factors recent reports suggest that the effects of reactive oxygen species can contribute to the development of rosacea (8). Conventional treatment of rosacea is based on a combination of systemic and topical antibiotics . Since the 1950's, tetracycline and erythromycin are the most commonly used oral antibiotics (1 - 3). The therapeutic activity of commonly used antimicrobials including tetracycline, doxycycline has been mainly attributed to their anti - inflammatory activities (9, 10). These results indicate that antibiotics used in the treatment of rosacea affect various inflammatory processes such as the migration of neutrophils, production of pro - inflammatory cytokines including interleukin-1, 6, 8, 10, tumor necrosis factor-, leukotriene b4, and oxidative burst in phagocytes (11 - 13). Topical metronidazole and oral tetracyclines effectively treat about 80%-90% of rosacea patients (6). However, long - term treatment with antibiotics is not well tolerated due to requiring frequent administration, poor compliances and side - effects including gastrointestinal intolerance, photosensitivity and candidiasis (5, 7). In addition, some patients do not respond to variable combination therapies and rather aggravated by their complications . (1) reported a rosacea patient who were developed tense bullae and hypopigmented scarring after taking oral doxycycline 100 mg . Oral azithromycin is a newly developed macrolide which offers some advantages over previously used antibiotics . It has unique pharmacokinetics that allows it to penetrate into intracellular compartments rapidly and maintain prolonged tissue levels . This allows for less frequent application and shorter duration of treatment which may increase compliance . With its high affinity for inflammatory tissues, azithromycin can achieve steady state tissue levels in approximately 72 - 96 hr (14). Moreover, after administration of oral azithromycin, tissue reactive oxygen species, an etiologic factor in the development of rosacea, were greatly decreased in some cases (7). Fernandez - obregon (5) reported that all of ten patients who were not tolerated or controlled by conventional treatment of rosacea demonstrated a significant improvement with the oral use of azithromycin . (1) treated a 67-yr old man who had photosensitivity to the doxycycline and hyperpigmented dyschromia to the minocycline with an oral use of azithromycin in a dose of 250 mg 3 times weekly . (4) reported that treatment with oral azithromycin led to 75% decreases in the total number of lesions and an 89% decrease in inflammatory lesions compared with basal status . Another open - label study showed that azithromycin is as effective as standard dose of doxycycline and has a positive impact on the quality of life of patients compared with conventional treatment regimens (15). Azithromycin may also be an acceptable agent for those using other medications, because it has no known major drug interactions . Additionally, azithromycin shows lower incidences of gastrointestinal discomfort compared with erythromycin (16). According to one clinical study, only two of 32 patients complained of mild gastric discomfort and no one withdrew from the study due to the side - effects of aztithromycin (17). In our case, we report a case of a 52-yr - old woman who presented with intractable multiple, pinhead to rice - sized erythematous papules on her face, which were controlled by oral azithromycin . Our case shows that azithromycin could be a new treatment option for the treatment of intractable rosacea.
In an attempt to develop a uniform classification of the most common demyelinating disorder of childhood, the international pediatric multiple sclerosis study group proposes the definition of acute disseminated encephalomyelitis (adem) as the first clinical event with a polysymptomatic encephalopathy, with acute or subacute onset, showing focal or multifocal hyperintense lesions predominantly affecting the cns white matter . Beyond that, evidences of previous destructive white matter changes or clinical setting of a demyelinating event must not be present in the patient's history . The risk of developing multiple sclerosis after adem has been focused by many studies in the literature. [13] the clinical features of adem are well known among pediatric neurologists and the outcome usually shows complete recovery in up to 50%, even in those patients who are not treated . Nevertheless, some forms of presentation have peculiarities and they might be a challenge . Acute hemorrhagic encephalomyelitis (ahem) is considered a rare form of adem's presentation due to acute brain vasculitis . Immediate and aggressive treatment is required because this clinical scenario shows high mortality . Herein, the authors report a case of ahem with remarkable abnormalities of brain magnetic resonance imaging (mri) who had an unfavorable outcome . Besides, a review of similar pediatric cases previously reported in the literature has been given . A 2-year - old, previously healthy girl was admitted to the hospital with a 1 week history of extreme irritability . Associated with irritability she did not have any antecedent history of infection or vaccination preceding the present symptoms . The neurological examination on the first evaluation showed impairment of consciousness, ranging from irritability to numbness . Brisk deep tendon reflexes, bilateral babinski sign and ankle clonus were present . Computed tomography scan was normal and the cerebrospinal fluid showed the following: white cell count 15 cells / mm(92% lymphocytes, 3% monocytes, 3% neutrophils), red blood cells 15/mm, protein levels 70 mg / dl, gamma globulin levels 18.3% on protein electrophoresis, and glucose levels 47 mg / dl . Acid - fast bacilli staining was negative as was polymerase chain reaction for herpes simplex and cytomegalovirus . The first mri showed hyperintense flair / t2 lesions in cerebellar white matter [figure 1a], and also in central, periventricular and juxtacortical white matter [figure 1b and c]. Axial flair images demonstrating hyperintense extensive and confluent lesions in cerebellar white matter (a), affecting the corpus callosum (b) and compromising the central and juxtacortical white matter (c) she had significant improvement after iv administration of high - dose intravenous methylprednisolone (30 mg / kg / day) for 5 days, followed by oral prednisolone (2 mg / kg / day) taper for 6 weeks . After 2 months of the initial symptoms, she had recurrence of her symptoms, associated with rapidly progressive refractory status epilepticus . An electroencephalogram obtained showed delta wave activity that was consistent with diffuse, severe encephalopathy . It was necessary to administer iv midazolam (23 g / kg / min) followed by thiopental (50 mg / kg / hour) to control the seizures . The second mri, in addition to the impairment of cerebral and cerebellar white matter [figure 2a and b], showed hemorrhagic lesions in the corpus callosum and right centrum semiovale [figure 2c and d]. She was submitted to a new high - dose iv steroid therapy and iv immunoglobulin, but no improvement was observed and she died after a nosocomial pneumonia following 2 months of intubation . Axial flair image (a) demonstrating hyperintense extensive and confluent lesions in central and juxtacortical white matter (dense arrow). Sagittal reformation (b) shows involvement of pericallosal and cerebellar white matter, sparing the u - fibers (dense arrows). Axial t2 gradient echo - weighted images (c and d) showing areas of very low signal, corresponding to breakdown products of hemoglobin (thin arrows), in the corpus callosum (c) and in the right centrum semiovale (d) whereas adem is more commonly diagnosed in children, ahem is seen most frequently among adults . Ahem is usually fatal, whereas full recovery is the rule for patients with adem . In 1997, rosman et al . Reported a pediatric case of ahem with a good outcome, and they did a review of the cases published before, since the first description by hurst in 1941 . At that time, there were nine pediatric cases published with pathological or radiologic confirmation . After 2000, the largest adem series in childhood have demonstrated only three cases of ahem . In tenembaum and coworkers series, dale et al . Have published 35 cases of adem, and only one scan had evidence of secondary hemorrhage . Three additional cases were published as case reports by leake, takeda, and mader in 2004 . Overall, 16 pediatric cases have been reported, including the one on this report [table 1]. The mortality of these 16 cases was 50% (8/16). Among the eight survivors, clinical information was available in seven: four patients recovered with sequelae and three patients made a full recovery . Despite the severe presentation of most adem cases, the outcome of nonhemorrhagic forms usually is favorable in childhood, with full recovery to normal neurological state in more than 5060% of patients, as previously demonstrated by the main series . Pediatric cases of acute hemorrhagic encephalomyelitis because of the epidemiological data, and especially the outcome between adem and ahem, some authors have tried to separate these conditions . However, distinction between adem and ahem is not well established and they may be a continuation of disease spectrum . The case presented herein is in agreement with the concept that both disorders are considered as autoimmune - mediated entities, with pathological features of prominent multifocal perivascular demyelination . The patient had a typical presentation of adem with an initial good response to steroids, and in an unexpected way, she relapsed with recurrence of symptoms associated with refractory status epilepticus, and a new mri disclosing hemorrhagic features . As the relapse took place within the first 3 months from the initial event, it was considered temporally related to the same acute monophasic condition, but with subsequent vessel occlusion leading to a secondary hemorrhage . Even though some authors have reported favorable neurologic outcome in adult patients, the high rate of ahem mortality mandates a quick and aggressive treatment using combinations of corticosteroids, immunoglobulin, cyclophosphamide, and plasma exchange. [1451719] the case reported here emphasizes that adem may present a severe outcome, making this well - known condition a challenge . Ahem must be properly investigated with mri whenever a patient presents with unexpected neurological worsening.
Basal cell carcinoma (bcc) is a common skin cancer arising from the basal layer of the epidermis and its appendages . It is particularly common in caucasian people, increases with age and it is a malignant locally invasive epidermal tumour with a good prognosis due to a slow growth - rate and low metastatic potential . Local invasion and tissue destruction, however, cause patient morbidity . The most common types are nodular and superficial bcc, which occur for the most part on the face . The choice of appropriate therapy is dependent upon the characteristics of the lesion and patient - specific factors . Treatment modalities include electrodesiccation and curettage, cryotherapy, surgical excision including mohs surgery, topical 5-fluouracil or imiquimod, photodynamic therapy, and radiotherapy [25]. Determining tumour extension, and defining accurate lateral and deep safety margins are very important aspects in the treatment approach for bcc . It is not possible to determine lesion depth based on clinical observations alone, because there might be an overestimation of the extension, which may lead to unnecessary tissue excision or radiation . In addition, the rate of incomplete excision of bccs has been reported to be 5 - 25% [714]. Although surgery is the first - line treatment for nonmelanoma skin cancers, radiotherapy can be indicated in selected cases . When radiotherapy is the treatment of choice, brachytherapy (bt) may be a good option for shallow, widespread lesions, or lesions on anatomic sites (e.g., hand, full scalp) that lie immediately above structures, which are vulnerable to irradiation . High - dose - rate (hdr) bt approaches offer significant advantages in this setting due to adaptability, patient protection, and variable dose fractionation schedules, and achieve excellent cure rates and cosmetic results . Several innovative applicators have been introduced to the bt community, and the use of skin bt has significantly increased over the years . The valencia applicator [1720] (nucletron, an elekta company, elekta ab, stockholm, sweden) is a new superficial device that improves the dose distribution compared with that of the leipzig applicator [2124] (elekta, stockholm, sweden and varian medical systems, palo alto, ca, usa). Recently, electronic brachytherapy using specific applicators has also become available, as xoft [15, 25] (xoft inc ., san jose, ca, usa) and esteya (nucletron, an elekta company, elekta ab, stockholm, sweden). Brachytherapy provides minimal dose delivery to surrounding healthy tissue, thus enabling good functional and cosmetic results . Brachytherapy appears to be most effective for small, primary, and/or superficial squamous cell carcinomas and basal cell carcinomas, where it is associated with excellent cosmetic results . The primary benefit of bt compared to external beam radiation therapy is the ability of bt to deliver radiation to the target tissue, with less injury to surrounding normal - appearing skin . Lateral and deep tumour delimitations are the main challenges when treating basal cell carcinomas with bt . Lateral delimitation may be aided by dermoscopy, and deep demarcation can be estimated by biopsy and/or imaging techniques . In superficial bt, the dose is prescribed to the deepest point of the target, which results in a higher dose between the source and this prescription point [20, 28]. A punch biopsy provides confirmation of the tumour's histopathology as well as determining its depth . However, it is an invasive technique, which only measures the depth in a portion of the tumour which cannot be representative . Ultrasonography, on the other hand, is a non - invasive, painless, non - ionizing, low risk, and non - expensive method, which is of academic interest in diagnosing bcc . In normal skin, basal cell carcinomas will appear more hypoechogenic than adjacent, normal dermis due to a medium change . The use of high frequency ultrasonography (hfus) between 10 mhz and 50 mhz has made it possible to visualize deep layers of skin, and to define very small hypo - echoic masses . Using the refraction of ultrasonography waves at the interface between the perilesional hyper - echoic area and the hypoechoic area of the tumour itself, it is possible to precisely define the lesion . High frequency ultrasonography has been shown to be potentially quite useful in bcc for both tumour measurement (for planning surgical resection) and as a diagnostic technique [33, 34]. Most of the published research in this field deals with the study of tumour size, delineation of surgical margins, and comparison of ultrasound findings with histologic results obtained following subsequent excisional biopsy of the lesion [3539]. Concordance rates between hfus findings and histology results for tumour size are between 73 - 98% . Published rates of tumour - free margins assessed by hfus are as high as 95%, but this has never been studied prior to radiotherapy treatment . The purpose of this study is to compare both hfus and punch biopsy methods in determining the depth of basal cell carcinomas prior to brachytherapy . We also present the strategy adopted at our department as a result of this present study . This study included 10 men and 10 women, all of them caucasian, with 10 superficial and 10 nodular bccs . All tumours were primary, maximum 20 mm in diameter and were located in a regular or flat area that was not adjacent to or over a burn, scar or inflammatory process . Only clinically apparent exclusion criteria included other bcc varieties, recurrent bccs, and bccs that were in locations difficult to image or treat with isotope or electronic brachytherapy applicators . All lesions were studied by histopathology and hfus to determine tumour depth (breslow thickness) prior to bt treatment . The mean time between the two techniques was 53 days (range 30 - 92 days). This technique allowed us to confirm the diagnosis of bcc and to measure the microscopic depth of the tumour that represents the clinical target volume (ctv) depth . Subsequently, hfus imaging was done to measure the macroscopic depth of the tumour, which represents the gross tumour volume (gtv) depth . In every case, hfus was done at least one month after the biopsy in order to avoid peritumoral inflammation due to the biopsy scar . To investigate whether hfus is sufficient to determine a correct prescription depth dose, this study was conducted under helsinki ii ethical principles after approval by the medical ethics committee at our hospital . A 3 mm diameter punch biopsy, including the whole dermis, was performed in all lesions . The deepest site estimated clinically was the site chosen for the biopsy; this is the usual method practised by dermatologists . An intralesional injection of mepivacaine was administered prior to the biopsy and a silk suture was used to close the wound . Histopathologic assessment of depth was done with the leica dmd108 digital microimaging network (leica microsystems slu, barcelona, spain). Tumour thickness was measured from the granular layer to the deepest portion of the tumour, as shown in figure 1 . Example of depth histopathologic assessment (breslow rate) using a leica dmd108 digital microimaging network after the biopsy, a radiologist who was an expert in skin lesions, estimated the depth of the lesions . All bccs were scanned in vivo using a high resolution b - scan with an 18 mhz hand - held transducer (siemens acuson s2000, munich, germany). A 2 cm 9 cm gel pad (aquaflex, pallej, barcelona, spain) was applied over the skin to enhance the air - skin interface (fig . High frequencies have better resolution, but lower frequencies are often used in hospital, and it has been reported in the literature that there is a good correlation between ultrasonic and histologic measurements (with complete lesion excision), even with probes emitting frequencies of 15 mhz or lower . In each lesion, the depth (from the epidermal surface to the deepest hypo - echoic point of the tumour) was measured . Because the epidermis thickness is approximately 0.1 mm, when hfus did not show any value, 0.1 mm was assigned . Illustration of the probe plus gel pad use during the acquisition high frequency ultrasonography examples of depth measurement . This study included 10 men and 10 women, all of them caucasian, with 10 superficial and 10 nodular bccs . All tumours were primary, maximum 20 mm in diameter and were located in a regular or flat area that was not adjacent to or over a burn, scar or inflammatory process . Only clinically apparent exclusion criteria included other bcc varieties, recurrent bccs, and bccs that were in locations difficult to image or treat with isotope or electronic brachytherapy applicators . All lesions were studied by histopathology and hfus to determine tumour depth (breslow thickness) prior to bt treatment . The mean time between the two techniques was 53 days (range 30 - 92 days). This technique allowed us to confirm the diagnosis of bcc and to measure the microscopic depth of the tumour that represents the clinical target volume (ctv) depth . Subsequently, hfus imaging was done to measure the macroscopic depth of the tumour, which represents the gross tumour volume (gtv) depth . In every case, hfus was done at least one month after the biopsy in order to avoid peritumoral inflammation due to the biopsy scar . To investigate whether hfus is sufficient to determine a correct prescription depth dose, this study was conducted under helsinki ii ethical principles after approval by the medical ethics committee at our hospital . A 3 mm diameter punch biopsy, including the whole dermis, was performed in all lesions . The deepest site estimated clinically was the site chosen for the biopsy; this is the usual method practised by dermatologists . An intralesional injection of mepivacaine was administered prior to the biopsy and a silk suture was used to close the wound . Histopathologic assessment of depth was done with the leica dmd108 digital microimaging network (leica microsystems slu, barcelona, spain). Tumour thickness was measured from the granular layer to the deepest portion of the tumour, as shown in figure 1 . Example of depth histopathologic assessment (breslow rate) using a leica dmd108 digital microimaging network after the biopsy, a radiologist who was an expert in skin lesions, estimated the depth of the lesions . All bccs were scanned in vivo using a high resolution b - scan with an 18 mhz hand - held transducer (siemens acuson s2000, munich, germany). A 2 cm 9 cm gel pad (aquaflex, pallej, barcelona, spain) was applied over the skin to enhance the air - skin interface (fig . High frequencies have better resolution, but lower frequencies are often used in hospital, and it has been reported in the literature that there is a good correlation between ultrasonic and histologic measurements (with complete lesion excision), even with probes emitting frequencies of 15 mhz or lower . In each lesion, the depth (from the epidermal surface to the deepest hypo - echoic point of the tumour) was measured . Because the epidermis thickness is approximately 0.1 mm, when hfus did not show any value, 0.1 mm was assigned . Illustration of the probe plus gel pad use during the acquisition high frequency ultrasonography examples of depth measurement . The clinical and histological characteristics of the lesions of the 20 patients studied are presented in table 1 . There were 10 men and 10 women, with 10 superficial and 10 nodular bccs . The mean age of the patients was 67 years (range 51 - 89 years). Resulting lesion depths with hfus and breslow are presented in figure 4 for both superficial and nodular lesions, respectively . In the superficial lesions, the breslow rate was similar or higher than hfus in most cases (8/10). In the nodular lesions, a) histopathology (breslow rate) vs. high frequency ultrasonography (hfus) depth determination for the 10 patients evaluated with superficial basal cell carcinoma (bcc). B) the same but with nodular bcc clinical and histological characteristics hfus high frequency ultrasonography statistical analyses were performed calculating covariance and correlation matrices for the hfus and breslow depths . So, although it could be considered that some correlation exists for the scatter plot in figure 4, it does not show any clear dependence between both variables . Conceptually, hfus should determine the gtv while histopathology is able to detect the ctv . Histopathologic measurements are done just on a small lesion section, which is not necessarily the deepest one . The large ultrasound probe makes access difficult in certain tumour locations, although brachytherapy is also not typically used in these locations anyway . Small tumour aggregates are not detected by hfus and it is also not possible to differentiate between the tumour and adjacent inflammation ., hfus has an important advantage over punch - biopsy as it allows a three - dimensional analysis of the tumour, whereas clinical measurements only permit a two - dimensional view . In this study, all us acquisitions were done by the same radiologist . In order to explore the intraobserver variability, the images were reviewed by the radiologist 3 times with a sufficient time interval in between . The resulting differences were negligible . According to the results, hfus was less accurate at very shallow depths . The nodular cases presented with larger depth differences than the superficial ones . In most cases of superficial bccs, the hfus depth measurement was lower than the histopathologic one, which was in contrast to the nodular cases . Neither clear tendency nor significance was observed from this depth comparison after applying standard statistical tests to search for depth measurement correlations between the two techniques . It has been reported in the literature that there is a good correlations between ultrasonic and histologic measurements, even with probes emitting frequencies of 15 mhz or lower . In our study, punch biopsy was done prior to hfus to confirm the diagnosis before measuring the tumour depth . This can lead to two problems: the biopsy could potentially remove the deepest part of the tumour, and both the scar and the inflammation after biopsy could distort / change the echographic image . Trying to avoid the latter problem, however, the number is sufficient to demonstrate that there is no clear correlation between these two methods . It has been reported in the literature that there is a good correlations between ultrasonic and histologic measurements, even with probes emitting frequencies of 15 mhz or lower . In our study, punch biopsy was done prior to hfus to confirm the diagnosis before measuring the tumour depth . This can lead to two problems: the biopsy could potentially remove the deepest part of the tumour, and both the scar and the inflammation after biopsy could distort / change the echographic image . Trying to avoid the latter problem, however, the number is sufficient to demonstrate that there is no clear correlation between these two methods . High frequency ultrasonography vs. histopathologic depth determination have been compared for 10 superficial and 10 nodular basal cell carcinomas . Neither a clear trend nor a significant difference in histopathology compared to hfus depth determination was observed . As a result of: 1) the comparison results of the present study, 2) the depth dose gradient, 3) the maximum skin dose using radionuclide applicators or electronic bt, and 4) the cosmesis experienced in clinical practice, we have decided in our protocol to prescribe to 3 mm depth when hfus measurements give lesion depths smaller than this threshold depth . This study was supported within a collaborative project with elekta brachytherapy (elekta company, veenendaal, the netherlands). This study was also partially supported by generalitat valenciana (project prometeoii/2013/010) and by spanish government under project no.
Glomus tumor was first described by wood as early as 1821, but the characteristic histological description was given by masson . They are mostly located in the subungual region but occur less frequently in other nail unit region and extradigital sites . Characteristic triad of symptoms of temperature sensitivity, severe pain and localized tenderness can be noted in 63 - 100% of the patients . A 36-year - old female was referred from surgery department, with history of pain in the left thumb since 10 years . She noticed splitting of nail 3 years back, which gradually progressed to involve the whole length of nail ., there was a small swelling with indistinct margins just behind the proximal nail fold . There was also longitudinal split in the nail which was extending from the free end of the nail plate to the proximal nail fold [figures 1 and 2]. No color changes either in the nail plate or in the proximal nail fold were noted . Intraoperatively, a semitranslucent mass of 2 mm 3 mm size was found . It was excised and sent for histopathological examination . These tumor cells had sharply punched out round to oval nucleus and well - defined borders . There were some dilated vessels with clusters of tumor cells in their walls [figure 3]. Fullness in posterior nail fold region glomus tumor during exploration tumor cells invading vessel wall (magnification 10) subungual location is the preferred site for digital glomus tumor, but can occur in other areas also . In our patient, the site involved was in posterior nail fold region, which is a rare occurrence . Reported the same in only one of his 28 glomus tumor patients evaluated by magnetic resonance (mr) imaging . The most common site in his study was the reticular dermis of nail bed in rest of the patients . Our patient had the classical triad of symptoms - temperature sensitivity, severe pain and localized tenderness . Bhaskaranand and navadgi reviewed different clinical tests to diagnose glomus tumors and opined that cold sensitivity test is the most accurate test to diagnose glomus tumors . Positive cold sensitivity is also reported by vasisht et al . In 84% of their patients . Erythronychia and distal onycholysis were seen in all the glomus tumor patients studied by dominguez - charit et al . Noted the same in 76% of patients and only 24% of his patients had blue blush of nail in their study . Radiographic examination was inconclusive in our patient and the same was reported by bhaskaranand and navadgi in their study . Use of high resolution magnetic resonance imaging (hr - mri) and high variable frequency ultrasound (hvfus) in preoperative assessment will help in the outcome of surgical treatment . Bhaskaranand and navadgi employed the use of double tourniquet, one at the mid arm and the other at the base of digit, for better visualization of the tumor during exploration . Patient was followed up and it was noticed that she had recurrence of pain after 6 months small size and difficulty in locating the tumor by traditional methods were the contributory factors . But due to their high cost, glomus tumors continue to cause diagnostic difficulty to the treating physician because of their small size and lack of simple cost - effective investigation.
It is a developmental epilepsy with a complex genetic inheritance that has yet to be elucidated . Re is also known as benign rolandic epilepsy of childhood (brec) or benign epilepsy with centro - temporal spikes (bects). Rolandic means that the seizures begin in the part of the brain called the rolandic area . The seizures are classified as a partial seizure because only this one part of the brain is involved [46]. Benign because it has a good outcome: nearly all children with re will outgrow it during puberty . It affects almost one in five of all children who have epilepsy . Which makes it is one of the most common types of epilepsy in children . Re usually begins between the ages of 3 and 10 years, and often stops around puberty (age 1418). Some children who have this type of epilepsy are usually well otherwise and do not have learning difficulties, although some may have specific difficulties with reading and language or with drawing and visuo - spatial skills, and some have associated neuropsychiatric deficits resembling the symptoms of attention deficit - hyperactivity disorder (adhd), the most common neurobehavioral disorder of childhood . It was gradually realized that there was a close relationship between benign partial epilepsy with rolandic spikes (bpers) and acquired epileptic aphasia (landau - kleffner syndrome), which was the first example of a mainly cognitive epilepsy in children . Prolonged reversible oral - motor deficits were subsequently recognized during the active epilepsy phase in some children with an otherwise typical syndrome and good final prognosis . Several neuropsychological studies confirmed the clinical experience that children with bpers had normal intelligence but that a certain percentage of them showed variable attention or selective deficits (linguistics, visuospatial etc . ), as compared to normal controls . Recent studies on the frequent temporary cognitive - behavioral disorders encountered in bpers have placed emphasis on their probable direct epileptic origin . This was thought to possibly explain some of the learning and school problems that many of these children experienced in the active phase of the syndrome . Rare longitudinal correlative eeg - neuropsychological studies have recently shown that acquired temporary cognitive - behavioral problems correlate with epileptic activity (eeg) in some children . It is now an open question whether this epilepsy can cause a specific developmental learning disability, or more general cognitive disability, if the onset is severe, early, and affects brain areas other than the strictly rolandic . To this end, eeg spectra and erps in a patient with rolandic interictal spikes were compared with the normative data (hbidatabase) in order to estimate the main neurophysiological deficits found in this patient . The deviations from the normality are discussed in term of well - known pathophysiological patterns in the adhd population, such as increased in the theta - beta ratio, decrease of the p3b component [68] and decrease of the p3 nogo potential . A quantitative analysis technique to analyze the erp data, without any a priori the frequency of rolandic spikes in children with adhd is significantly higher than that expected from epidemiologic studies . Also, erps are of significantly higher amplitude in the epilepsy group compared to the control group over frontal and central regions within the time window between 250 and 425 ms post - stimulus, which coincides with the time window of target - nontarget stimulus discrimination . The question arises how adhd symptoms are related to rolandic spikes in this adhd subgroup and how these symptoms can be treated by neurotherapy . The aim of this study was to find out: whether this boy with rolandic epilepsy shows different cortical activation patterns compared to non - epileptic children during the performance of a working memory task; does relative beta training, which activates the frontal cortex by enhancing beta activity recorded over the frontal electrodes, helps: to decrease the number of spikes? To reduce the neuropsychiatric symptoms, executive dysfunction, and behavioral changes, rendering him able to function independently in school and in many situations of everyday life . Age 9.3 . Suffered from neuropsychiatric symptoms, cognitive dysfunction, especially attention deficits1, and behavioral changes, rendering him unable to function effectively in school and in many situations of everyday life . In the initial descriptions of the syndrome, his mother and teachers observed behavioral and learning disabilities which caused serious school problems . At the age of 7.5 . It was diagnosed as acquired isolated graphomotor deficit, as an example of selective epileptic developmental deficit (figure 1). At the age of 7.6 . The first seizure was observed, and later active epilepsy (mainly in sleep), which was slowing the normal process of his development . At the age of 9.9 . The seizures were simple partial motor and sensory seizures involving the lower face and the pharyngeal region, the so - called sylvian seizures, and tended to occur during sleep (after falling asleep or before arousal in the morning) sometime with extension to the hemibody or with generalization . There was a feeling of tingling (like pins and needles) on one side of the mouth involving the tongue, lips, gum and inner side of the cheek . Sometimes the seizure also involved the throat which may have caused speech to be unclear and therefore difficult to understand . The child made strange throaty or gurgling noises from time to time, and it was often this which alerted the parents to the fact that something may be wrong . He often knew what he wanted to say but he was not able to speak properly . Although he was treated for epilepsy (carbamazepine), his condition was worsening, and a dramatic regression of acquired skills occurred (figure 2). However, all this could be explained by the psychological consequences of the disease [1114]. In brain mri, frfset2 sequence, coronal plane dysgenesis of the fornix was found, along with asymmetry of the lateral ventricles, more prominent on the left . In the axial plane, after contrast, in the axial plane there was local atrophy of the brain parenchyma in the right frontal area parasagittal . In frfset2 sequence, coronal plane, local atrophy of the brain parenchyma was found in the right frontal area parasagittal (figure 3a d). The patient took part in the neurotherapy program which included 20 sessions of relative beta training; the goal of the training was to activate the frontal cortex by enhancing the beta activity recorded over the frontal electrodes . In more detail the procedure was as follows: electrodes were placed at fz and cz bipolar recording . The procedure was to increase the ratio of beta eeg power / eg power in the theta and alpha frequency bands . We used neuropsychological testing as well as erps before the experiment, as well as after the completion of the program . The experiment was reviewed and approved by the respective medical ethics committees, and the parents gave written informed consent for the anonymous publication of his case history . The patient underwent standardized neuropsychological testing: electroencephalogram recording, wechsler intelligence scale for children - revised, wechsler memory scale - iii polish version (wms - iii, polish version), peabody picture vocabulary test - iii (ppvt - iii) and boston naming test, polish version (bnt - vpl), at baseline during active disease (exam 1) and at follow - up after the completion of the neurotherapy program, during which recovery from epilepsy occurred (exam 2). Neuropsychological testing at baseline (exam 1) showed multiple deficits (see table 1). At follow up, after conclusion of the neurotherapy program (exam 2), patient e.z . His verbal and non - verbal iq, and most of his cognitive functions increased significantly, including immediate and delayed logical and visual recall on the wms - iii (cf . His results for maintaining attention on the wms - iii also improved (34/40 points). In other cognitive functions e.zs results also improved in the 2 examination . On the auditory learning task, he had forgotten all the words after a 15-minute filled delay in the 1 examination, and achieved 5 words in recognition; however, in the 2 examination he remembered 2 words after the delay, and achieved all the words in recognition . Neuropsychological testing at baseline (exam 1) showed also executive dysfunction in drawing of semantic figure (figure 4). Patient e.z . Was not able to copy the semantic figure (figure 4a), as he worked very fast and then abandoned the task . However, at follow up, after completion of the neurotherapy program (exam 2), the child showed major improvement in executive functions (figure 4c), even though these were the most disturbed of all his neuropsychological functions . Event related potentials (erps) were used to assess the functional changes in the patient induced by rehabilitation programs . First, erps have a superior temporal resolution (on the order of milliseconds) as compared to other imaging methods, such as fmri and pet (which have time resolution of 6 seconds and more), secondly, erps have been proven to be a powerful tool for detecting changes induced by neurofeedback training in adhd children . And finally, in contrast to spontaneous eeg oscillations, erps reflect the stages of information flow within the brain . The diagnostic power of erps has been enhanced by the recent emergence of new methods of analysis, such as independent component analysis (ica) and low resolution electromagnetic tomography (loreta). A modification of the visual two - stimulus go / no go paradigm was used (figure 5). Three categories of visual stimuli were selected: 20 different images of animals, referred to later as a; 20 different images of plants, referred to as p; 20 different images of people of different professions, presented along with an artificial novel the randomly varying novel sounds consisted of five 20-ms fragments filled with tones of different frequencies (500, 1000, 1500, 2000, and 2500 hz). Each time a new combination of tones was used, while the novel sounds appeared unexpectedly (the probability of appearance was 12.5%). The trials consisted of presentations of paired stimuli with inter - stimulus intervals of 1 s. the duration of stimuli was 100 ms . Four categories of trials were used (see figure 2): a - a, a - p, p - p, and p-(h+sound). The trials were grouped into four blocks with one hundred trials each . In each block a unique set of five a, five p, and five h stimuli were selected . The task was to press a button with the right hand in response to all a - a pairs as fast as possible, and to withhold button pressing in response to other pairs: a - p, p - p, p-(h+sound) (figure 5). According to the task design, two preparatory sets were distinguished: a continue set, in which a is presented as the first stimulus and the subject is presumed to prepare to respond; and a discontinue set, in which p is presented as the first stimulus, and the subject does not need to prepare to respond . In the continue set a - a pairs will be referred to as go trials, a - p pairs as no go trials . Omission errors (failure to respond in go trials) and commission errors (failure to suppress a response to no go trials) were also computed . The eeg was recorded referentially to linked ears, allowing computational re - referencing of the data . The analysis consists of the following steps: 1) eye movement artifact correction and elimination: a) using a spatial filtration technique based on zeroing the activation curves of individual independent component analysis (ica) components corresponding to horizontal and vertical eye movements, as well as b) excluding epochs with an excessive amplitude of eeg and excessive faster and slower frequency activity; 2) fast - fourier transformation (fft) of the corrected eeg for extracting eeg power and coherence for all 0.25 hz bins in the frequency band from 0.5 to 30 hz; 3) computation of event related potentials by averaging eeg over trials for each category of trial and each channel with a time resolution of 4 ms; 4) decomposition of an individual erps into independent components by applying spatial filters extracted by means of the ica from the collection of erps computed for the corresponding group of healthy subjects; 5) comparison of each extracted electrophysiological and behavioral variable against the corresponding variable computed for a carefully constructed and statistically controlled age - regressed, normative database in which the variables have been transformed and confirmed for their gaussian distribution . Visual inspection of raw eeg was made in order to search for paroxysmal patterns that pop out of the background eeg . Besides the visual inspection, an automated spike detection was performed . The method of automated spike detection is based on the temporal parameters of spikes, and the spatial location of the corresponding spike dipole2 . The amplitude - temporal parameters have been defined on the basis of comparison spike detection by the program and by experienced experts from a data base of more than 300 eeg recordings in epileptic patients; paroxysmal character, high degree of sharpness, and short duration . The automatic spike detection was performed on eeg in the common average montage for both eyes open and eyes closed conditions . The results of automatic spike detection in the fragment of eeg recorded during 22 min of the go / nogo task are presented in figure 7 . The number of spikes dramatically decreased after treatment, especially at the left temporal and central electrodes . Moreover, in eeg after treatment we were not able to detect any spikes at the cz electrode . The results of a comparison of the patient s behavioral parameters during the go / nogo task are presented in table 2 . The number of omission errors decreased by more than 50% after treatment, so that no deviation in behavior from norms was observed after treatment . Deviations from normality in the eeg spectra computed for 20 minutes of the go / nogo task before treatment are presented in figure 8 left . As one can see the eeg pattern is characterized by excessive slow (around 6 hz) activity over frontal - temporal areas . One can see dramatic changes of the nogo erps with increase of the nogo potential over the cz electrode and decrease over the left temporal areas . The results of a comparison of the patient s behavioral parameters during the go / nogo task are presented in table 2 . The number of omission errors decreased by more than 50% after treatment, so that no deviation in behavior from norms was observed after treatment . Deviations from normality in the eeg spectra computed for 20 minutes of the go / nogo task before treatment are presented in figure 8 left . As one can see the eeg pattern is characterized by excessive slow (around 6 hz) activity over frontal - temporal areas . One can see dramatic changes of the nogo erps with increase of the nogo potential over the cz electrode and decrease over the left temporal areas . Deviations from normality in the eeg spectra computed for 20 minutes of the go / nogo task before treatment are presented in figure 8 left . As one can see the eeg pattern is characterized by excessive slow (around 6 hz) activity over frontal - temporal areas . One can see dramatic changes of the nogo erps with increase of the nogo potential over the cz electrode and decrease over the left temporal areas . Rolandic epilepsy, in which interictal epileptiform discharges appear, is associated with neuropsychological disorders such as cognitive impairment and behavioral problems, even in the absence of clinical epilepsy . Uncontrolled reports and three preliminary randomised controlled trials of the antiepileptic treatment of interictal epileptiform discharges have suggested that suppression of discharges is associated with a significant improvement in psychosocial function . However, a greater number of controlled studies needs to be carried out in order to confirm this hypothesis [1012]. The etiology is multifactorial, being affected by the type of epileptic syndrome, the cause of epilepsy, the high frequency of epileptic seizures, a previous history of status epilepticus, the age at the onset of epilepsy, the antiepileptic treatment selected, and the role of interictal epileptiform discharges . Several studies have sought to analyze to what extent cognitive impairment can be attributed to interictal epileptiform discharges among the other epilepsy factors . The disruptive effect of interictal epileptiform discharges on cognition is supported by a wide range of factors, such as the concept of transient cognitive impairment, the definition of epileptic encephalopathy, the natural course of epileptic syndromes with continuous spike and wave activity during slow sleep, the concept of autistic regression related to epileptiform activity, the cognitive profile of benign rolandic epilepsy, and the cognitive impact of non convulsive status epilepticus . According to this information it has been suggested that the treatment of interictal epileptiform discharges with antiepileptic drugs could improve cognition and behaviour in these children . Convergent clinical data and new evidence drawn from electrophysiological studies and functional imaging suggest that the cognitive and behavioral dysfunction is directly related to the particular role that the affected cortical area plays when the epileptic process becomes active . The continuous spike - waves during slow wave sleep [csws] phenomenon is probably due to a disturbance of the corticothalamic oscillatory mechanisms at work during slow sleep, which seem to play a role in the consolidation of material acquired during waking [1921]. One especially dramatic example of how partial epilepsy can lead to a progressive dementia and/or a massive behavioral regression in children is the acquired epileptic frontal syndrome . Cases of partial epilepsy with csws are increasingly described in a variety of developmental and acquired focal cortical pathologies in children (also in association with thalamic lesions) [2226]. However, as in our case, acquired focal cortical pathologies may be not in association with thalamic lesions, however, the child may have an increased frequency of rolandic spikes as in other adhd children, as was confirmed by kropotov, and have associated neuropsychiatric deficits resembling the symptoms of attention deficit - hyperactivity disorder (adhd), the most common neurobehavioral disorder of childhood . Graphomotor deficit is not indicated to be criterial for adhd, though it occurs almost commonly . However, it is still not clear whether there lie at its basis motor activity problems or rather the planning and organisation of behaviour . Point rather to a connection of the disturbances with all these factors cumulatively . Not without significance is the fact that the lack of allocation of brain resources in the case of performing the most difficult tasks is linked with the occurrence of epileptic brain activity . Traditional therapies for the recovery of a child with benign partial epilepsy with rolandic spikes (bpers), associated with neuropsychiatric deficits resembling symptoms of attention deficit - hyperactivity disorder (adhd), are still not satisfactory . To date the best approach seems to be cognitive therapy and behavioral training, however, the results are limited . Adjunct interventions that can augment the response of the brain to the behavioral and cognitive training might be useful to enhance therapy - induced recovery in patients with rolandic epilepsy . In this context, neurofeedback self - regulation appears to be an additional intervention to standard neuropsychological therapies . Bpers is a model for the study of the cognitive manifestations of focal epileptic discharges in a developing brain, although the prolonged fluctuating and cognitive manifestations and their dynamics of onset and recovery cannot be explained in terms of simple ictal - postictal symptoms which suggest that several different mechanisms are probably involved . This theory differs from other theories of brain function in that it emphasizes: process and change, rather than data processors connected to each other by neural cables, as though the brain were a computer; the creative nature of perception, which is not just a passive collection of stimuli, but a process of creating an image of reality; understanding the symptom as a segment of normal behavior that is revealed prematurely by pathology, and is not just a deficit, that is, the absence of the correct behavior; the development of mental processes that evolve on different scales of time, assuming that the laws of behavior are the laws of evolution expressed on another temporal plane; processing of information from whole to part, and not, as in standard theory, from bits to stacks of information . In particular, a fuller understanding of the essence of adhd brings us closer to grasping the process of symptom formation . In figure 10, the afferent pathways bring impulses received from the sense organs to the brainstem, which constitutes the oldest and most primitive part of the brain . The brainstem reacts with a general activation of the organism, which is transmitted upwards (a), to the limbic system and cerebellum . From these somewhat younger structures the activation signal becomes more complex (b), so that in the cortex it spreads to highly specialized areas . Signals from the cortex then travel by pyramidal pathways back down to the brainstem, and from there by efferent pathways to effectors in the musculoskeletal system . Understanding this somewhat simplified diagram of activation makes it easier to interpret the mosaic of diverse disturbances that occur in children with ad / hd, associated with disturbances that are both structural (involving different areas and different levels of the brain) and functional (resulting from changes on the level of neurotransmission), as well as the resolution of these disturbances in the course of rehabilitation . The process of symptom formation responsible for the heterogeneity and changeability of behavioral disturbances in adhd children is explained by figure 11, which illustrates the bidirectional transition from emotion to mentation and action . The state of arousal in the mind, which in a healthy brain can be reinforced or inhibited by the executive functions, cannot be controlled in the brains of adhd children . Thus the behavior which these children exhibit can be diverse, variable, and capricious, depending on a whole range of factors both structural and functional in nature . Bpers is a model for the study of the cognitive manifestations of focal epileptic discharges in a developing brain, although the prolonged fluctuating and cognitive manifestations and their dynamics of onset and recovery cannot be explained in terms of simple ictal - postictal symptoms which suggest that several different mechanisms are probably involved . This theory differs from other theories of brain function in that it emphasizes: process and change, rather than data processors connected to each other by neural cables, as though the brain were a computer; the creative nature of perception, which is not just a passive collection of stimuli, but a process of creating an image of reality; understanding the symptom as a segment of normal behavior that is revealed prematurely by pathology, and is not just a deficit, that is, the absence of the correct behavior; the development of mental processes that evolve on different scales of time, assuming that the laws of behavior are the laws of evolution expressed on another temporal plane; processing of information from whole to part, and not, as in standard theory, from bits to stacks of information . In particular, a fuller understanding of the essence of adhd brings us closer to grasping the process of symptom formation . In figure 10, the afferent pathways bring impulses received from the sense organs to the brainstem, which constitutes the oldest and most primitive part of the brain . The brainstem reacts with a general activation of the organism, which is transmitted upwards (a), to the limbic system and cerebellum . From these somewhat younger structures the activation signal becomes more complex (b), so that in the cortex it spreads to highly specialized areas . Signals from the cortex then travel by pyramidal pathways back down to the brainstem, and from there by efferent pathways to effectors in the musculoskeletal system . Understanding this somewhat simplified diagram of activation makes it easier to interpret the mosaic of diverse disturbances that occur in children with ad / hd, associated with disturbances that are both structural (involving different areas and different levels of the brain) and functional (resulting from changes on the level of neurotransmission), as well as the resolution of these disturbances in the course of rehabilitation . The process of symptom formation responsible for the heterogeneity and changeability of behavioral disturbances in adhd children is explained by figure 11, which illustrates the bidirectional transition from emotion to mentation and action . The state of arousal in the mind, which in a healthy brain can be reinforced or inhibited by the executive functions, cannot be controlled in the brains of adhd children . Thus the behavior which these children exhibit can be diverse, variable, and capricious, depending on a whole range of factors both structural and functional in nature . Deficits of cognitive functions characteristic for adhd are detected in a child with benign partial epilepsy with rolandic spikes (bpers). . The parameters of attention were improved after the neurotherapy program intended to activate the frontal lobes . The improvement in attention induced by the neurotherapy program was accompanied by a decrease in the number of spikes and an increase of the p3nogo component of event related potentials in the go / nogo task . Event related potentials can be used to assess functional brain changes induced by neurotherapeutical programs.
It is a relatively rare condition, typically benign and resolves with conservative therapies (1). This has been occasionally described in the surgical published work, usually in the context of thoracic surgery or as a complication of spinal instrumentation (2). It initially described by gordon and hardman in 1977 as intraspinal air (3). Since then, especially with the advent of computed tomography (ct), there has been an increasing number of case reports describing the presence of air within the spinal canal (4). In 1987, pneumorrhachis may be an indication of substantial vertebral column injury, especially when paraspinal hematoma, rib fractures, transverse process fractures, clavicle fractures, subcutaneous air, intramuscular air, pneumothorax, or hemothorax are also present (2, 6 - 8). Pneumorrhachis has also been reported with isolated head trauma and in the absence of an identifiable fracture within the skull or spine (9, 10). Nontraumatic causes of pneumorrhachis include infection (11). Despite known conditions that can result in pneumorrhachis the clinical significance and neurologic outcome for patients with pneumorrhachis, we report a case of pneumorrhachis with fracture in lumbar spine and history of ankylosing spondylitis . The axial thoracic spinal ct scan of patient a 39-year - old man was admitted because of low back pain and dyspnea after locating between motor vehicle and wall 3 days before admission . He suffered from ankylosing spondylitis and was under corticosteroid therapy . On arrival, his glasgow coma scale was 15/15 with normal neurological examination . He was able to move his lower extremity, but it was limited by pain . Vital signs were stable and in radiographic examination, there was rib fracture in three ribs, hemothorax, subcutaneous emphysema, and lumbar vertebra fracture in l2-l4 . Ct scan showed bilateral pleural effusion, fracture of ribs number 8, 9 and 10 in lower left side of thorax, fracture of vertebra in l2-l4, and air bubbles in upper thoracic spinal canal (figure 1). The patient underwent lumbar surgery (laminectomy and cord decompression) and fusion of l2-l4 by screw . In according to neurosurgery consult no action performed on air bubbles . Pneumorrhachis, as the presence of free intra - spinal air, is also called aerorachia or epidural emphysema . It usually occurs in the epidural space but may be spread within the subarachnoid space with the distraction of dura meter (1). A significant increase in intra alveolar pressure and intra alveolar pressure was increased in several conditions such as acute asthma, recurrent vomiting and closed thoracic trauma . This led to alveolar rupture and air movement along the bronchovascular axis up to the mediastinum . The collected air then disassociates the pleura from the aorta and the parietal pleura from the spine, subsequently inflowing the extradural space via the intervertebral foramina (12). In addition, subarachnoid air may easily move cranially and caudally and may cause back and/or local pain and headache and/or nervous tissue compression (brain and spinal cord) by a valve mechanism . However, in the present case, there are not strong evidence of air diffusion between the pleura and the epidural space, it is the only pathophysiological mechanism stated in the literature (1). Magnetic resonance imaging is the most accurate investigation for the assessment of the extent of the condition . The best explanation for the radiologic findings in our patient is that air, under pressure in the pleural space, entered the spinal canal and then the subarachnoid space directly through tears of the parietal pleura and the spinal meninges . Tension pneumothorax and thoracic spinal fracture should be considered in the differential diagnosis of both pneumorrhachis and pneumocephalus . Intraspinal air is usually asymptomatic, self - limiting and resolves with conservative therapies, but in a rare number of cases, pneumorrhachis can cause cord compression and may even require decompressive surgery . Appropriate antibiotic prophylaxis must be considered, however, due to increased risk of infection of the underlying breach in the dura in traumatic etiologies (2). In comparison with others, sinha and mantle has been described a case of pneumorrhachis with rapid deterioration to death (14)., they presented a case of tension pneumocephalus and pneumorrhachis secondary to a subarachnoid pleural fistula after thoracic spinal surgery (16). Valente et al reported a 21-year - old male with severe pneumocephalus and pneumorrhachis who was made a full recovery without any neurological complication (17). It is usually asymptomatic and self - limiting, but its presence should alert the attending trauma physician to carry out diagnostic workup for associated injury and treat the underlying cause (18). When seen in a trauma patient all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors.
A 73-year - old female patient (height: 160 cm, weight: 58 kg) was diagnosed with degenerative osteoarthritis and admitted for bilateral total knee arthroplasty replacement . The patient was previously diagnosed and medicated for hypertension for 5 years, and diagnosed with suspected angina 5 months prior to the surgery receiving aspirin but was discontinued 7 days prior to the surgery . The chest radiography examination showed no special findings except cardiomegaly, bronchiectasis or nonspecific fibrosis in right lower lung zone . Upon electrocardiogram examination and echocardiography, a normal sinus rhythm of 66 beats per minute (bpm) along with a nonspecific t wave abnormality and about 57% ejection fraction without abnormalities of regional wall motion, thrombi, and vegetation and normal left atrium and ventricle size, normal valve function were revealed . To perform the surgery under combined spinal epidural anesthesia (cse), 8 mg of 0.5% heavy marcaine (marcaine, astrazeneca, sweden) was injected into the subarachnoid space using a needle through the needle technique, and 3 ml of a test dose using 0.375% of levobupivacaine containing epinephrine (1: 200,000) was given following insertion of an epidural catheter . Surgery began after sensory blocked level reached the t10 dermatome and maintained with intermittent injections of 0.5% levobupivacaine . The patient was monitored using routine methods [ekg with st depression, pulse oximeter (spo2), noninvasive blood pressure, arterial blood pressure, urine output (uo), and bis]. 5 l / min of 100% oxygen was administered via facemask and monitored spo2 was 98 to 100%, and intermittent midazolam was given for sedation with a bis level of 65 to 80 . The patient remained hemodynamically stable [blood pressure (bp); 130 - 150/70 - 85 mmhg, heart rate (hr); 65 - 85 bpm, respiration rate (rr); 15 - 18/min, spo2; 100%] through the intraoperative period immediately before tourniquet release of the second knee arthroplasty, and uo was 900 ml during the 5 hours procedure . The estimated blood loss was 400 ml, and intraoperative fluid replacement consisted of 1,600 ml of crystalloid and 500 ml of hetastarch solution . Immediately after tourniquet release of the second knee arthroplasty, twenty - five minutes after tourniquet release and initiation of prbc transfusion, the patient's blood pressure and spo2 decreased to 87/46 mmhg and 90 - 95%, respectively, with tachypnea and cyanosis so normal saline and a second packed rbc transfusion were rapidly replaced along with a 5 mg injection of ephedrine . Her arterial blood gas analysis (abga) showed a ph of 7.32, a paco2 of 43 mmhg, a pao2 of 73 mmhg, and oxygen saturation (sao2) at 93% . At the end of the operation (about 30 minutes after transfusion), sudden paroxysmal atrial fibrillation developed with systolic bp dropping below 70 mm hg, and a spo2 of 80% and her mental state changed to drowsy with intact light, eyerish, and gag reflex upon her neurological examination . Respiration and reviving the patient to a conscious state was encouraged, but consciousness continually diminished . Since the oxygen saturation was reduced to 80% with rigidity of temporomandible joint, etomidate and midazolam were injected, and positive pressure ventilation was performed using a ventilator after endotracheal intubation . The patient's spo2 increased to 95 - 98%, but pulmonary edema was suspected because a rale was heard by auscultation from both lungs in addition to the secretion of a frothy sputum . Suddenly, ventricular tachycardia occurred and the hr dropped to less than 30 bpm; therefore, cardiopulmonary resuscitation was done . After epinephrine was injected three times and cardioversions with 200 j biphasic waveform shocks, the patient's rhythm returned to a normal state, and thus, a joint diagnosis was done by the cardiology and neurology departments . There was no specific abnormal findings neurologically, and her transthoracic echocardiography in the or did not show any fine emboli or embolic mass or pulmonary hypertension or ventricular dysfunction, but did show a decrease in volume and a hyperdynamic status of the heart . Upon portable chest x - ray examination, it showed bilateral pulmonary infiltrates (fig . The patient was given 20 mg of furosemide and 500 mg of methylprednisolone iv with inotrophic support (dopamine: 5 - 15 g / kg / min, dobutamine: 5 - 15 g / kg / min) and manually assisted ventilation for pulmonary edema and 60 semifowel's position, and yet, her condition of respiratory insufficiency with frothy secretions did not improve over time . She was transferred to icu for further management by department of cardiogy 1 hour after an advent event . The femoral vein was catheterized, and we continued to focus on treatment for her pulmonary edema, which consisted of medical treatment with furosemide, dopamine: (10 - 20 g / kg / min), dobutamine (10 - 20 g / kg / min), norepinephrine (> 20 g / kg / min), and volume maintenance using packed rbcs and least crystalloid fluid therapy, but copious pinkish frothy secretions began to flow out from her tracheal tube, and her vital signs and spo2 were aggravated with anuria and dic . At this time, her abga showed a ph of 7.25, a paco2 of 43.8mmhg, a pao2 of 59.7 mmhg, and a sao2 of 87.7% . Despite her mechanical ventilatory support (fio2 of 1.0 with peep 10 - 15 cmh2o) and inotropic treatment, her spo2 was still low (70 - 90%), and her blood pressure (60 - 90/40 - 50 mmhg) was unstable and decreased gradually . Abga, cardiac enzymes including n - terminal probrain natriuretic peptide bnp (nt - probnp) and troponin t, and other blood chemistry tests were done (table 1). Ekg and tte also were done reevaluate and determine the origin of the pulmonary edema . Her cardiac enzymes including troponin t were slightly increased though her nt - probnp level was within normal limits, her transthoracic echocardiography in the icu did not show any signs of heart failure but did show a hyperdynamic and more hypovolemic heart . Although she was again given a total of 7 units of packed rbc for volume maintenance and 2 units of ffp for correction of a coagulation abnormality 3 hours after the initial adverse event during the icu stay, her hr began to decrease to <40 bpm . Since there is no consensus with respect to the risk factors and clinical diagnostic criteria of trali, recognition of it is confused and not easy in most cases, and thus, only a small percentage of trali cases are published . Hence, the incidence of trali is estimated to be 0.08 - 8% among patients who undergo transfusions . The american european consensus conference (aecc) modified the definition of trali and published it, and recently, canadian consensus conference further modified the working group criteria for trali . The trali was classified into " suspected trali " and " possible trali " and stated that it should be differentially diagnosed from transfusion - associated circulatory overload (taco). " Suspected trali " is defined as a case in which new acute pulmonary damage occurs as bilateral diffuse infiltration on a chest radiograph during or within six hours after transfusion with a pao2/fio2 ratio less than 300 mmhg, hypoxemia of spo2 less than 90% in room air, or clinical evidence of hypoxemia in a patient who has not showed any evidence of hypertension related to pulmonary edema or to the left atrium . A case with clinical circulatory overload (e.g. Hypertension related to the left atrium) is differentially diagnosed as " taco " . " Possible trali " is defined by the same diagnostic criteria with those of the suspected trali, but it refers to the case in which the patient has other acute pulmonary risk factors such as sepsis, aspiration, neardrowning, disseminated intravascular coagulation, trauma, pneumonia, drug overdose, fracture, burns, and cardiopulmonary bypass in addition to a transfusion . It is reported that trali occurs within six hours after transfusion, but nonspecific symptoms and signs such as tachypnea, frothy pulmonary secretions, hypotension, hyperthermia, tachycardia, and cyanosis are found within 30 - 120 minutes after transfusion in most of the cases, and a diffuse rale is heard from both sides by auscultation . One important factor in trali diagnosis is that there should be no evidence of circulatory overload such as s3 gallop or jugular distension . It is known that most of the trali patients have euvolemic state or may be hypovolemia because of lung parenchymal excessive fluid leakage . Since the use of diuretics and rapid volume restriction can be the treatment of choice in the case of taco trali's cvp, pulmonary capillary wedge pressure, and b - natriuretic peptide (bnp) values are within the normal ranges . However, taco and trali can be differentiated because taco shows a bnp measurement of 100 pg / ml or higher . The patient in our case had a nt - probnp value of 227 after the occurrence of trali, which was in the normal range, but hypovolemia was exacerbated by the use of diuretics and fluid volume restriction because of the misdiagnosis of not trali but taco by physicians in charge . Consequentially, her vital sign showed still unstable despite the treatment with inotropics and vasoconstrictors, and copious pinkish frothy secretions were persisted . Her prognosis became worse as the unnecessary transfusion was in addition done to correct for unexplained hypovolemia that was later found in the echocardiogram without ever knowing causes . It is generally known that trali occurs usually when ffp is administered, but it can be caused by all the other blood components . The accurate pathogenesis of trali is not known, but it has been reported that trali is related with anti - hla i, ii, anti - neutrophil antibody, and biologically active lipids of the donor plasma . Although it is known that trali pathogenesis is triggered by a trace amount of plasma, the relationship between the administered dose and the severity is not certain . Almost nothing is known about the effects of antibody titer, avidity, or leukocyte - antigen density on the severity or frequency of trali reactions, but antibodies were not detected in 17% of the cases . Hence, considering the amount of time it takes and the cost to obtain results, serological tests are not necessary to diagnose a patient; instead, clinical symptoms are used as the diagnostic criteria . The fda recently recommended that clinicians should be alert for trali as it publishes its characteristics and morbidity . Kopko and holland insisted that " possible trali " should be kept in mind primarily when respiratory symptoms are accompanied by fever or hypotension within two hours following transfusion . Suggested an approach for the management of respiratory patients after blood transfusion based on the aecc definition . The approach may be helpful to differentiate the diagnosis of trali and hydrostatic and permeability pulmonary edema, although both can happen simultaneously in some cases . Generally, for the treatment of trali, transfusions should be stopped; supplemental oxygen should be administered, and auxiliary treatment should be done . Different from acute pulmonary damage due to other causes, recovery is rapid with recovery from diffuse pulmonary infiltration known to be within 96 h following transfusion in 80% of the cases . The reason why our case was diagnosed as " possible trali " was that the possibility of ali by fat embolism syndrome (fes) could hardly be excluded . It is known that fes occurs in relationship to many traumatic or nontraumatic conditions, but it usually occurs within 24 to 48 hours after lower extremities long bone fracture operations because of the high intramedullary pressure occurring during prosthesis implantation . Particularly, fes in total knee replacement arthroplasty (tkra) usually takes place in cemented tkra that employs a long intramedullary stem, and there is a high probability of pulmonary fat microembolism, especially in the case of bilateral tkra, in which a tourniquet is installed for a long period . Since the laboratory and radiographic findings are nonspecific, gurd's diagnosis criteria based on the clinical symptoms are often used . With these criteria, fes can be diagnosed if the symptoms correspond to one major criterion and four minor criteria . In our case, we observed two major criteria including symptoms in the respiratory system and central nervous system, and other minor criteria such as tachycardia, oliguria, thrombocytopenia, and anemia . Various changes in brain functions are found in 86% of the patients from drowsy to coma . Additionally, it is known that the cutaneous symptom, petechial rash, is found at the conjunctiva, around the neck, in the peri - axillary area or upper body within 24 - 36 hours in 20 - 50% of the patients, but it spontaneously disappears within one week . Additional tests were not done to confirm the diagnosis in our case because the patient rapidly exacerbated after the symptoms and eventually died within 7 hours . In addition, for the diagnosis of trali, we should distinguish the changes in hemodynamic status that can develop after deflation of a tourniquet or changes that can develop from movement of thromboemboli or fat particles from the lower part of the body to the right atrium, right ventricle, and pulmonary artery . Especially in surgery performed on the lower part of the body under combined epidural spinal anesthesiasedation with preservation of spontaneous respiration as in our case, respiration and metabolic variables from the release of the tourniquet were promptly recovered within 3 - 5 minutes from the compensation effect of spontaneous breath . However, the recovery of hypotension was slow due to the loss of sympathetic tone . The decrease in blood pressure that develops after the deflation of the tourniquet was significantly low 3 minutes after release due to a loss of blood, reflexive vascular relaxation of the ischemic area, and systemic inflow of metabolites produced from anaerobic metabolism such as adenosine and lactic acid . However, it is known that any tendency of hypotension can be simply managed through vasopressor agents . In our patient, the ph, paco2, potassium, and lactic acid measured after the release of the second tkra tourniquet were within normal range, which rules out the possibility of hypotension from the expansion of the blood vessels and myocardial depression . In addition, since the last injection of the epidural local anesthetic was 55 minutes before the deflation of the tourniquet, which was 80 minutes before the event occurred, the possibility of sympathetic block influence is thought to be negligible . Park and kim reported that in cases of bilateral tkra, the decrease in cardiac index and mean arterial pressure is larger in the second tkra compared to the first; thus, properly maintaining preload is very important . When injection of crystalloid is increased to the speed of 20 ml / kg / h after deflation of the tourniquet and 3 units of prbc is transfused to increase the full load, there were no differences in arterial pressure after the deflation of the tourniquet . In our case, hypotension and arrhythmia occurred 25 minutes after the deflation of the tourniquet; therefore, the volume state of the patient was euvolemia, and according to the duration of time, we suspected that recovery from tourniquet syndrome was possible . In addition, it is necessary to distinguish whether it was hydrostatic pulmonary edema from circulatory overload or permeability pulmonary edema from ali to determine the cause of hypoxia and pulmonary edema that developed in our patient; thus, the possibility of circulatory and respiratory suppression from the tourniquet seems unlikely . The patient in our case had been taking aspirin since angina was suspected by a local clinic because of dyspnea that had occurred five months before . The joint diagnosis with the department of cardiology did not show any distinctive cardiovascular symptoms, and the electrocardiography and echocardiography did not show any specific findings . The cardiac enzyme levels were within the normal limits indicating that there would be no specific problems during the operation, and the chest pulmonary radiograph did not show any findings by which acute pulmonary damage could be suspected . However, before the end of the operation, just over five hours, that is, 25 minutes after the transfusion, symptoms began to appear such as hypotension, tachycardia, and cyanosis immediately followed by diminished consciousness, tachypnea, frothy pulmonary secretions, and arrhythmia . The possibility of " possible trali " was un - recognized and misdiagnosed . Since the auxiliary treatment was originally for cardiogenic pulmonary edema, we did mechanical ventilator therapy, loop diuretics, inotropics, and crystalloids volume restriction . Additionally, two or three units of blood were transfused to maintain the circulatory volume, and even two units of ffp were administered to correct the dic one hour before respiratory arrest occurred . These treatments might have exacerbated the symptoms rather than providing conservative treatments for the patient . Conclusively trali has been the major cause of transfusion - related morbidity and mortality, recently . Differential diagnosis of trali from taco and other diseases is difficult since it is suspected and diagnosed by the clinical symptoms and findings; use of consensus definitions that were recently introduced can help to improve the prognosis of trali patients by doing the diagnosis and treatment early . Moreover, blood should be transfused to patients based on strict strategies so that transfusion - related complications including trali can be reduced.
Diabetic nephropathy is a significant complication in diabetic patients, and it is becoming the most common cause of end stage renal disease (1). Childhood diabetic patients live long enough for nephropathy to develop because they develop diabetes early, usually before reaching adolescence . Therefore, preventing diabetic nephropathy or delaying the disease progression by way of early detection is very important (2, 3). The determination of microalbuminuria has been suggested as an early predictor of diabetic nephropathy . However, the standard clearance technique necessitates timed urine collection, which is not only time - consuming but also subject to error . Particularly, children with diabetes tend to collect 24-hr urine inaccurately because they are young and immature . Furthermore, 24-hr urinary albumin excretion rates can be altered by various conditions, such as intra - individual variability and day - to - day variation during the evolution of albuminuria (4). Recently, the albumin to creatinine ratio (acr), measured from a random urine sample, was suggested to be an effective surrogate to 24-hr urine collection for detecting microalbuminuria (5 - 7). Acr is convenient to perform, and is less affected by variation in urine concentration because it is a ratio between two measured substances . Also, serum creatinine has been widely used as a marker of gfr, but it is not sensitive enough to detect decreased renal function . Therefore, various plasma low molecular weight proteins have been suggested as valuable markers of decreased renal function in place of serum creatinine (8). Among these markers, previous studies demonstrated that serum cystatin c (cysc) might be a superior marker for the evaluation of renal function than serum creatinine (9). However, the effectiveness of cysc for estimating gfr has not been sufficiently demonstrated in children with diabetes . Therefore, the aim of this study was to investigate whether spot urine acr and serum cysc are accurate and effective in assessing renal function instead of 24-hr urine microalbumin in children and adolescents with diabetes . A total of 113 children and adolescents (age 12 - 19 yr) with type 1 or 2 diabetes at childhood diabetes clinic of severance children's hospital were included in this study from january 2008 to august 2010 . Ninety - eight (41 males and 57 females) patients were type 1 and 15 (6 males and 9 females) were type 2 . Age, duration of disease, mean glycated hemoglobin (hba1c), 24-hr urine microalbumin, acr in spot urine, serum cr, serum cysc, high sensitivity c - reactive protein (crp), which rises in response to inflammation, were checked in all patients . Estimated gfr (egfr) was calculated with the schwartz formula for patients under the age of 18 yr: egfr (ml / min/1.73 m) = k * height (cm)/serum cr (mg / dl) (k = 0.55 in children to 13 yr of age, k = 0.70 in adolescent males [the constant remains 0.55 for females]) (10) and modification of diet in renal disease (mdrd) formula for those of ages of 18 to 19: egfr = 186 * (serum cr) - 1.154 * (age) - 0.203 * (0.742 if female) (11). Serum creatinine was determined by an enzymatic method (kodak ektachem 700 xr - c system, eastman kodak, rochester, ny, usa). Serum cysc was measured by automated particle - enhanced immunonephelometry using a bn100 nephelometer (dade behring, marburg, germany). Data were analyzed by the sas program (version 9.1; sas institute, cary, nc, usa). We used the generalized linear regression model and chi - squared test to verify the demographic trends of continuous and categorical variables over the ordinal variable of time, respectively . The sensitivity and specificity of serum cysc, serum creatinine, and creatinine clearance for the detection of reduced gfr were assessed by the roc curve . This study was approved by the institutional review board of yonsei university severance hospital (irb number 4 - 2012 - 0001). This study was approved by the institutional review board of yonsei university severance hospital (irb number 4 - 2012 - 0001). Written informed consent there were no significant differences between the type 1 and 2 diabetes groups regarding mean hba1c, serum creatinine, cysc and creatinine clearance . Ninety six patients (85%) had normoalbuminuria, 17 (15%) had microalbuminuria and no patient had macroalbuminuria . Age, duration of disease, and mean hba1c levels were significantly higher in the microalbuminuria group than in the normoalbuminuria group, suggesting that the incidence of diabetic nephropathy increases according to the progress of the disease and poor glycemic control . Serum creatinine level was not significantly different between the two groups, but serum cysc was significantly higher in the microalbuminuria group (p = 0.040). Spot urine acr was positively correlated with 24-hr urine albumin excretion (r = 0.828 and p = 0.001) (fig . 1) and negatively correlated with creatinine clearance (r = 0.249 and p = 0.017). We analyzed serum creatinine, cysc and spot urine acr between the patients with creatine clearance <60 ml / min/1.73 m (group i) and those with creatine clearance> 60 ml / min/1.73 m (group ii) (table 3). Serum creatinine did not differ between the two groups, but spot urine acr and serum cysc were significantly greater in group i. the sensitivity and specificity of serum cysc and creatinine compared with creatinine clearance were estimated via roc curves (fig . The area under the curves (auc) with the cut - off value of 60 ml / min/1.73 m was 0.732 for serum cysc and 0.615 for serum creatinine . The auc was significantly higher for serum cysc (p = 0.028), but not for serum creatinine (p = 0.069). The sensitivity and specificity of cysc were 87.3% and 66.2%, respectively, with the upper reference limit as the cut - off . The roc curve analysis of serum cysc demonstrated higher diagnostic accuracy than that of serum creatinine . The gold standard for the assessment of diabetic nephropathy in children is still the quantitative analysis of microalbumin through 24-hr urine collection . However, many factors such as exercise, hypertension, hyperglycemia can influence daily urinary albumin excretion (12). Day - to - day variation according to the volume of collected urine can hinder the diagnosis of diabetic nephropathy (4). Discovery of microalbuminuria through 24-hr urine collection can also be erroneous because of improper collection, which commonly happens with children and adolescents . Therefore, a test using timed urine collection has been suggested to be repeated three times to confirm microalbuminuria, but this is very hard to apply to children . Recent studies have demonstrated good correlation between spot urine acr and 24-hr urinary albumin excretion (6, 13). Spot urine acr was thought to be more accurate because it eliminates the possibility of improper collection . The method using spot urine samples may ensure better compliance than timed urine test, especially in childhood patients . Furthermore, the spot urine acr has the advantage of being less affected by urine volume because it is a ratio of two measured substances (14). Nevertheless, few studies have reported the efficacy and effectiveness of spot urine acr compared with creatinine clearance through 24-hr urine collection in childhood patients with diabetes . The present study showed that spot urine acr was closely correlated with 24-hr urine albumin excretion and creatinine clearance in children and adolescents with diabetes . However, we cannot definitively support that spot urine acr should be used in place of 24-hr urine collected miacroalbumin due to the small study population . We do, however, feel that spot urine acr would be useful as a screening tool when timed urine collection is diffcult to obtain in out - patient settings . The use of endogenous markers to evaluate renal function is also important in the evaluation of diabetic nephropathy . Various low molecular proteins, such as cysc, 2-microglobulin, and collagen type iv, instead of serum creatinine have been suggested as useful endogenous markers for evaluating renal function (8). Among these, previous studies have demonstrated that cysc might be a more sensitive indicator of gfr than serum creatinine, but there have been few studies on cysc as an endogenous marker reflecting gfr in children with diabetic nephropathy (15 - 17). In the present study, the level of serum cysc, but not serum creatinine, was significantly different between the normoalbuminuria group and the microalbuminuria group . Estimated gfr according to the schwartz and mdrd ii formulas did not demonstrate any difference between the two groups, although creatinine clearance was significantly lower in the microalbuminuria group . It is thought that serum creatinine is unable to reflect the early signs of decreased renal function in childhood diabetic patients, while serum cysc can . However, we cannot say that serum cysc might be surrogate marker of diabetic nephropathy, because diabetic patients have the increased possibility of decreased renal function due to causes other than diabetic nephropathy . Nevertheless, cysc based gfr is suggested to be a useful method for estimating renal function (18). Recently, schwartz et al . Proposed an updated schwartz formula using cysc (19). We can estimate that cysc based gfr may be a better method than creatinine based gfr on the findings in this study . Our studies have some limitations in that we did not perform the measurement of exogenous substances such as insulin, -edta, -diethylenetriaminepentaacetic acid, iohexol (20) or dtpa renogram, which has been suggested as the most accurate method to evaluate renal function . However, dtpa renogram or other accurate measurements based on the measurement of exogenous substances are hard to perform, especially in childhood diabetic patients . More studies with a large sample population are needed to confirm that spot urine acr can replace 24-hr urine microalbumin in childhood diabetes . There was a discrepancy between egfr and creatinine clearance through 24-hr urine collection in this study . Second, the mdrd formula within the normal range of creatinine might not reflect accurate renal function . Nevertheless, to the best of our knowledge, the present study is the first to simultaneously check both serum cysc and spot urine acr in children and adolescents with diabetes, and our study demonstrated that spot urine acr might be a more accurate, convenient, and effective indicator for the detection of microalbuminuria instead of 24-hr urine microalbumin in diabetic children . Cysc based gfr may be a more accurate method than creatinine based gfr to evaluate renal function.
A 25-year - old male (height 167.7 cm, weight 52.2 kg) was admitted for surgical resection of a 5.0 1.8 cm right - cheek mass, suspected to be a lymphangioma . The patient had undergone a tonsillectomy under general anesthesia 8 years previously without any perioperative complication . Therefore, anesthesia was applied according to the american society of anesthesiologists physical status classification 1 . The blood pressure upon arrival in the operating theater was 158/76 mmhg, with a heart rate of 80 beats / min (bpm), and with a peripheral oxygen saturation of 99% of room air . Anesthesia was induced using an injection of 50 g fentanyl, 150 mg propofol and 50 mg rocuronium . One hour after commencing surgery, the surgeon requested a reversal of the neuromuscular blockade to monitor patency of the facial nerve . Anesthesia was consequently maintained using desflurane alone . At this time, the blood pressure was 105/74 mmhg and the heart rate 92 bpm . After approximately 5 min, the blood pressure was decreased to 75/42 mmhg and the heart rate was 104 bpm . After 5 min, the blood pressure was 67/36 mmhg and the heart rate was 94 bpm . To manage persistent hypotension, 200 g phenylephrine were administered, followed by a rapid infusion of lactated ringer's solution . However, the non - invasive blood pressure decreased further to 57/36 mmhg and the heart rate rose to 138 bpm . As we asked the surgeon invasive blood pressure monitoring was applied to the left dorsalis pedis artery and additional venous access was established . Consequently, the blood pressure was restored to 99/51 mmhg and surgery was resumed . Because the patient was covered with a drape, clinical manifestation of anaphylaxis, including skin rash, could not be observed . Approximately 1 h after the hypotension event, a skin rash was detected over the entire body . A 10 g bolus of epinephrine was given and a continuous epinephrine infusion was initiated at 0.03 g / kg / min . The patient responded to the epinephrine and soon maintained hemodynamic stability; with a blood pressure of 110/52 mmhg and a heart rate of 78 bpm . Norepinephrine infusion was discontinued and epinephrine infusion was tapered to maintain the restored blood pressure . The patient was treated using 4 mg chlorpheniramine together with 5 mg dexamethasone to prevent delayed reactions and halt any further histamine release . After 1.5 h, the blood pressure was 110/46 mmhg without any inotropic or vasopressor support . The skin lesion was improved and no respiratory symptoms were detected throughout the completion of the surgery . The total infused crystalloid, colloid, estimated blood loss and urine output were 1,300 ml, 500 ml, 50 ml, and 450 ml, respectively (fig . The patient was transferred to the intensive care unit for close monitoring and was placed on ventilatory support . An hour after arrival, blood tests were obtained for the tryptase level, which was elevated to 37.9 g / l (normal range: 0 - 11 g / l) and the total serum immunoglobulin e (ige) level, which was increased to 675 ku / l (normal range: negative). The patient was transferred to a general ward 1 day later and was discharged after 1 week without any complications . Skin - prick and intradermal tests were performed after 1 month to determine the cause of the anaphylaxis . The skinprick tests were negative for all agents, whereas the intradermal tests were positive for atropine (wheal of 4 4 mm for a stock concentration of 0.5 mg / ml; wheal of 4 4 mm with 1: 5 dilution) (table 1). The intradermal skin tests also demonstrated a positive result for rocuronium (wheal of 5 5 mm for a stock concentration of 10 mg / ml; wheal of 4 4 mm with 1: 10 dilution). The patient was advised to completely avoid atropine and is currently receiving regular check - ups at the outpatient department of plastic surgery . This case indicates the difficulties anesthesiologists encounter in diagnosing anaphylactic reactions after administration of an atypical antigen . Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mastcell- and basophil - derived mediators into the circulation . The first criterion includes the acute onset of illness with the involvement of the skin or mucosa, including hives, pruritus and angioedema, and either respiratory compromise or a systolic blood pressure <90 mmhg (or symptomatic hypotension). A second criterion consists of two or more signs or symptoms occurring rapidly following exposure to a likely allergen . The third criterion is defined as a systolic blood pressure <90 mmhg or symptomatic hypotension following exposure to a known allergen . In the present case, tachycardia is a classic cardiovascular sign of anaphylaxis, although bradycardia occasionally develops later in the reaction should the patient becomes hypoxemic or develop a heart block . Bronchospasm may present as a sudden increase in the ventilatory pressure required to inflate the lungs, an increase in the end - tidal carbon dioxide or a decrease in the arterial oxygen saturation . A rapidly developing laryngeal edema may present as a difficulty with intubation or stridor postoperatively . In the current case, anaphylaxis during anesthesia may be particularly difficult to recognize for several reasons: early or mild symptoms, including itching and shortness of breath, may go unrecognized because the patient cannot communicate . Secondly, cutaneous signs, including flushing, urticaria and angioedema, may be missed because the patient is draped for surgery . As a result of these factors, the reaction may be detected only upon dramatic respiratory and hemodynamic changes . In the present case, prompt initial treatment is essential in an anaphylactic episode, because even a few minutes' delay may lead to fatal complications, including hypoxic - ischemic encephalopathy and death . Although epinephrine is the first - line therapy for the emergency management of anaphylaxis, it is not always given promptly, even in hospitalized patients . As in the present case, anaphylaxis can be difficult to diagnose promptly during anesthesia; consequently, treatment using epinephrine may be delayed . In a retrospective study, 45% of the patients with anaphylaxis during anesthesia developed shock, circulatory instability or cardiac arrest, yet it is important for the attending physician to consider any agent which may only rarely induce anaphylaxis, including atropine, as in the present case . H1-antihistamines are not the drugs of choice in initial anaphylaxis treatment, because they do not relieve life - threatening respiratory symptoms or shock, although they do decrease urticaria and itching . . However, they remain in use for anaphylaxis because they potentially prevent biphasic anaphylaxis . The results of laboratory tests performed during anaphylaxis may be useful in some patients for subsequently confirming the diagnosis . Serum tryptase is one of the mediators released by activated mast cells in immune - mediated anaphylactic reactions . The half - life of serum tryptase is 90 min, and an elevation in tryptase levels 1 - 2 h after anaphylaxis displays a positive correlation with anaphylaxis severity . Serum or plasma to determine the tryptase level should be obtained ideally within the first 3 h after anaphylaxis . Elevated levels of active or total tryptase in the serum may be useful in distinguishing anaphylaxis from other conditions in the differential diagnosis, including vasovagal reactions, septic shock, seizures, myocardial shock, benign flushing, and carcinoid syndrome . In the present case, the patient displayed anaphylaxis following intravenous administration of neuromuscular reversal agents . Taking into account the clinical features displayed during surgery and the time to reaction onset, we assumed that this case was a typical anaphylaxis . Elevated total ige was further strong etiological evidence for anaphylaxis, which is mediated by ige . We presumed that atropine or neostigmine would be the most likely candidate, because anaphylaxis usually occurs within a few minutes of administration . As far as we are aware, this is the first report of atropine - induced anaphylaxis in korea . Skin testing is an important element in the diagnosis of an ige - mediated allergy . There are two commonly used methods of skin testing for ige - mediated disorders: prick / puncture and intradermal tests . Intradermal tests are more reproducible than prick / puncture skin tests and are approximately 100- to 1000-fold more sensitive . However, false - positive reactions are more common, and this type of testing has a higher risk of inducing a systemic allergic reaction . After an anaphylactic episode, it is standard to delay skin tests for a minimum of 3 - 4 weeks . An anaphylactic episode within the previous month is a contraindication for skin testing because it may yield false - negative results . When the intradermal skin test was conducted in the present case, both rocuronium and atropine displayed a positive reaction . Despite the possibility of the occurrence of delayed anaphylaxis, the positive finding of the intradermal test (1: 10) for rocuronium is not a strong indicator of the reason for anaphylaxis, because of a high false - positive ratio . In the present case, it is less likely that delayed anaphylaxis resulted from rocuronium, because without a specific external stimuli to maintain a constant blood pressure, it suddenly fell, with a systolic blood pressure of 60 mmhg . Delayed anaphylaxis differs from anaphylaxis in that a series of symptoms occur gradually and to a lesser degree . The fact that the vital signs of the patient in the present case were kept steady before atropine injection, in response to which the systolic blood pressure fell abruptly to 60 mmhg while displaying no further symptoms of anaphylaxis, excludes the possibility of delayed anaphylaxis . Because anaphylaxis occurred immediately after intravenous injection of atropine and neostigmine, we could rule out induction agents such as rocuronium, rather suspecting atropine to be the allergen . However, in the present case we cannot with certainty rule out ephedrine or phenylephrine as the causative agent of anaphylaxis . The abrupt decrease in the systolic blood pressure from 105 to 75 mmhg in the absence of any other external stimulus is rather difficult to explain should ephedrine or phenylephrine be the cause of anaphylaxis . Atropine is a muscarinic acetylcholine receptor antagonist frequently used to treat symptomatic sinus bradycardia, av block and the reversal of non - depolarizing neuromuscular blockade . As neuromuscular monitoring becomes more common, atropine can be used as an adjuvant for reversal agents during surgery . Anaphylaxis during surgery is a rare but life - threatening event, and anaphylaxis in response to the administration of rare, unusual agents, including atropine, as in the present case, makes the diagnosis even more difficult . Therefore, we recommend that anesthesiologists should be aware of the potential allergenicity of atropine . In conclusion, anaphylactic reactions can occur in response to even to the most commonly used drugs during general anesthesia, including atropine . Anesthesiologists should always be aware of the possibility of anaphylactic reactions to all drugs used during anesthetic management . The initiation of aggressive therapeutic management is essential when anaphylaxis is strongly suspected, and tests to determine the specific drug(s) responsible for anaphylaxis should be conducted immediately after the operation to prevent further adverse events.
Breast cancer is the most common cancer among women worldwide and the second leading cause of cancer - related mortality in developed countries . Most breast cancer - related deaths are not the result of primary tumor growth but are rather caused by the metastasis of cancer to other organs (1). Metastasis is the last stage in the progression of cancer during which cancer cells detach from the primary tumor, migrate into blood vessels, disseminate throughout the body, and ultimately seeding in distant organs and give rise to new tumors . Several studies have shown that chemokines and chemokine receptors play a key role in cancer metastasis . Chemokines are expressed in specific organs and their interactions with the correspondent receptors which are expressed on tumor cells induce directed cell migration (2). The extent of this response triggered by chemokines depends on the amount of chemokine receptors expressed on the plasma membrane (3). Chemokine receptors may potentially facilitate tumor dissemination via activation of key survival pathways such as mek1/2 and pi3/akt (4). Chemokine receptors belong to a subfamily of g protein - coupled receptors and they are named based on the chemokine ligands to which they bind . So, they are classified into four groups of (i) cxc receptors (cxcr1, 2, 3, 4 and 5), (ii) cc receptors (ccr1, 2, 3, 4, 5, 6, 7, 8, and 9); (iii) cx3c and (iv) the receptor of xc . It has been suggested that interactions of some specific chemokine receptors with their ligands play important roles in many of the critical steps of the metastatic process (5). Among different classes of chemokine receptors, cxcr4 was found to be the most common chemokine receptor overexpressed in different human malignancies including breast cancer . It was revealed that cxcr4 and its ligand (cxcl12) have a key role in initiating and regulating tumor cell migration and metastasis (6). Moreover, it was revealed that the chemokine receptors ccr1, ccr6, ccr9, and cxcr1 are often overexpressed in cancer cells and are involved in cancer metastasis (7 - 10). Regarding the important role of chemokine receptors in cancer metastasis, scientists focus on developing new drugs to target them . Although pharmacological effects of berberine have been previously reported, recently, special attention was paid to its bioactivities including antioxidant, anti - inflammatory anti - diabetic, neuroprotective effects and its ability as a natural drug for breast cancer treatment (11). Moreover, it was shown that berberine is effective in inhibition of cell proliferation and promotion of apoptosis in different cancerous cells (12). Unlike anti - cancer growth effects of berberine, very little is known about the effect of berberine on cancer cell migration and metastasis . Therefore, in this study, we examined the effect of berberine on migration and invasion of breast cancer cells . Moreover, to clarify the underlying mechanism of berberine effect on migration of breast cancer cells, the expression levels of some selected chemokine genes were analyzed by qpcr . Specific chemokine receptors namely, ccr1, ccr6, ccr9, cxcr1, and cxcr4 were selected since they were previously reported to be associated with cancer metastasis (8). The present study aimed to demonstrate berberine effects against the metastasis of breast cancer cells . Mcf-7 human breast cancer cell line was cultured in dulbecco s modified eagle medium (dmem) supplemented with 10% fetal calf serum and antibiotics (1% penicillin- streptomycin 10000 units / ml) at 37 c in a humidified atmosphere of 95% air and 5% co2 . Mtt assay was performed to evaluate the cytotoxic effect of berberine on mcf-7 breast cancer cells (7). Briefly, the cells were seeded at a density of 1.210 cells / ml and incubated with berberine (sigma) at 0, 10, 20, 40, 80 and 160 m for 24 hr . Thereafter, the medium was changed and cells were incubated with mtt (sigma) solution (0.5 mg / ml) for 4 hr . The number of viable cells is directly proportional to the production of a purple dye, after solubilization with isopropanol which can be measured spectrophotometrically (= 545 nm). The percentage of the viable cells was calculated using the following equation: (mean od of treated cells / mean od of control cells)100 . Control groups were treated with media containing 0.1% dmso in all experiments . For the wound - healing migration assay, cells were seeded in 6-well plates (8). When cells were confluent, scratch was made with a 200 l sterile pipette tip, generating a cell - free area of approximately 1 mm in width . Cellular debris removal was done by gentle washing with culture medium and the photos of the wounds (0 thereafter, medium was replaced by culture medium with different concentrations of berberine (0, 10, 20, 40, and 80 m) and the cells were allowed to migrate for 24 hr . At the end of migration experiment, the gap size was analyzed using image - j 1.45 software . In order to assess migration ability of cultured cells, cell - free areas of the scratches at 24 hr post - wounding were subtracted from the area of the scratches (0 hr)and calculated as a percentage of untreated (0 hr) cultures . Total rnas were isolated from cells using tripure isolation reagent (roche, germany) based on the manufacturer s instructions . Extracted rna one microgram of rna from each sample was used for cdna synthesis by reverse transcriptase . Rna samples were incubated at 65c for 5 min with 1 l oligodt (0.5 g/l) at a final volume of 12 l, and then, chilled on ice and mixed with 4 l 5x buffer, 2 l dntps (10 mm), 0.5 l ribolock, and 1 l m - mlv - rtase . After incubation at 42c for 60 min, and at 70c for 10 min, the mixture was transcribed into cdna . Synthesized cdnas were diluted at 1:4 ratio and 2 l cdna of each sample was used for real - time pcr in a 20 l reaction mixture with 10 l of 2x sybr green pcr mastermix (parstous, iran) and 1 l of specific primer pair . Description of the designed primers each plate was run at 95c for 10 min, then 40 cycles of 95c for 15 sec, 60c for 30 sec, and 72c for 30 sec . All real - time rt - pcrs were performed in duplicate, and relative mrna of each target gene was determined by using the formula 2 (ct, cycle threshold) where ct = ct (target gene) ct (-actin). The comparative expression level of each target gene between different samples was calculated by 2 . The significance of difference among the experimental groups and controls was assessed by one - way anova and post hoc tukey test . Mcf-7 human breast cancer cell line was cultured in dulbecco s modified eagle medium (dmem) supplemented with 10% fetal calf serum and antibiotics (1% penicillin- streptomycin 10000 units / ml) at 37 c in a humidified atmosphere of 95% air and 5% co2 . Mtt assay was performed to evaluate the cytotoxic effect of berberine on mcf-7 breast cancer cells (7). Briefly, the cells were seeded at a density of 1.210 cells / ml and incubated with berberine (sigma) at 0, 10, 20, 40, 80 and 160 m for 24 hr . Thereafter, the medium was changed and cells were incubated with mtt (sigma) solution (0.5 mg / ml) for 4 hr . The number of viable cells is directly proportional to the production of a purple dye, after solubilization with isopropanol which can be measured spectrophotometrically (= 545 nm). The percentage of the viable cells was calculated using the following equation: (mean od of treated cells / mean od of control cells)100 . For the wound - healing migration assay, cells were seeded in 6-well plates (8). When cells were confluent, scratch was made with a 200 l sterile pipette tip, generating a cell - free area of approximately 1 mm in width . Cellular debris removal was done by gentle washing with culture medium and the photos of the wounds (0 thereafter, medium was replaced by culture medium with different concentrations of berberine (0, 10, 20, 40, and 80 m) and the cells were allowed to migrate for 24 hr . At the end of migration experiment, the gap size was analyzed using image - j 1.45 software . In order to assess migration ability of cultured cells, cell - free areas of the scratches at 24 hr post - wounding were subtracted from the area of the scratches (0 hr)and calculated as a percentage of untreated (0 hr) cultures . Total rnas were isolated from cells using tripure isolation reagent (roche, germany) based on the manufacturer s instructions . Extracted rna was treated with dnase i enzyme . One microgram of rna from each sample was used for cdna synthesis by reverse transcriptase . Rna samples were incubated at 65c for 5 min with 1 l oligodt (0.5 g/l) at a final volume of 12 l, and then, chilled on ice and mixed with 4 l 5x buffer, 2 l dntps (10 mm), 0.5 l ribolock, and 1 l m - mlv - rtase . After incubation at 42c for 60 min, and at 70c for 10 min, the mixture was transcribed into cdna . Synthesized cdnas were diluted at 1:4 ratio and 2 l cdna of each sample was used for real - time pcr in a 20 l reaction mixture with 10 l of 2x sybr green pcr mastermix (parstous, iran) and 1 l of specific primer pair . Description of the designed primers each plate was run at 95c for 10 min, then 40 cycles of 95c for 15 sec, 60c for 30 sec, and 72c for 30 sec . All real - time rt - pcrs were performed in duplicate, and relative mrna of each target gene was determined by using the formula 2 (ct, cycle threshold) where ct = ct (target gene) ct (-actin). The comparative expression level of each target gene between different samples was calculated by 2 . The significance of difference among the experimental groups and controls was assessed by one - way anova and post hoc tukey test . In this study, using mtt assay, we determined the cytotoxicity of berberine by treating mcf-7 cells with various concentrations berberine for 24 hr . The results from mtt assay revealed that growth retardation was correlated with increased concentrations of berberine in a concentration - dependent manner (figure 1). Cell viability was 99%, 89%, 81%, 79%, and 64% for 10, 20, 40, 80, and 160 m berberine, respectively . Results are represented as mean sd; n=3, (* p <0.05 compared to control) here, we checked whether berberine inhibits cell migration in breast cancer cells . For this purpose, mcf-7 breast cancer cells were subjected to the cell migration assay after 24 hr - treatment with various concentrations of berberine (0, 10, 20, 40 and 80 m), which were relatively non - toxic for the cells (figure 2a). Treatment of the cells with berberine resulted in a concentration - dependent reduction in the cell migration capacity of mcf-7 cells as compared to control (figure 2b). Effect of different concentrations of berberine on cell migration as examined by wound - healing assay . (a) mcf-7 cells grew to 90% confluent and were scratched by a sterile 200 l pipette tip . The ability of cells to migrate into the scratch area was monitored by photographing the same spot with an inverted microscope equipped with a digital camera, 0 and 24 hr after scratch . Wound - healing images show the extent of healing and black lines indicate the wound edge . The graph represents the mean sd for at least three independent experiments . * and * * donate p <0.05 and p <0.01, respectively, as compared to the control after observing the decrease in cell migration, we were interested to investigate whether alternations of chemokine receptors expression at mrna level occurred in mcf-7 cells following berberine treatment . Therefore, qpcr was performed to determine the expression of selected chemokine receptor genes . As shown in figure 3, 24 hr - incubation with berberine caused no significant effect on the expression of ccr1(a); however, ccr6 was significantly down - regulated following treatment with berberine 40 m (b). A similar effect was also observed for ccr9 gene expression following treatment with all concentrations of berberine except 10 m (figure 3c). Moreover, cxcr1 and cxcr4 were significantly down - regulated following treatment with all concentrations of berberine (figures 3d and e). These data suggested that the inhibition of chemokine receptors expression in the presence of berberine may resulted in inhibition of mcf-7 breast cancer cell migration . Mcf7 cells were treated with berberine at concentrations of 0, 10, 20, 40 and 80 m for 24 hr, then, subjected to real - time pcr analysis for determination of alterations in gene expression of selected chemokine receptors including ccr1 (a), ccr6 (b), ccr9 (c), cxcr1 (d), and cxcr4 (e). In this study, using mtt assay, we determined the cytotoxicity of berberine by treating mcf-7 cells with various concentrations berberine for 24 hr . The results from mtt assay revealed that growth retardation was correlated with increased concentrations of berberine in a concentration - dependent manner (figure 1). Cell viability was 99%, 89%, 81%, 79%, and 64% for 10, 20, 40, 80, and 160 m berberine, respectively . Results are represented as mean sd; n=3, (* p <0.05 compared to control) here, we checked whether berberine inhibits cell migration in breast cancer cells . For this purpose, mcf-7 breast cancer cells were subjected to the cell migration assay after 24 hr - treatment with various concentrations of berberine (0, 10, 20, 40 and 80 m), which were relatively non - toxic for the cells (figure 2a). Treatment of the cells with berberine resulted in a concentration - dependent reduction in the cell migration capacity of mcf-7 cells as compared to control (figure 2b). Effect of different concentrations of berberine on cell migration as examined by wound - healing assay . (a) mcf-7 cells grew to 90% confluent and were scratched by a sterile 200 l pipette tip . The ability of cells to migrate into the scratch area was monitored by photographing the same spot with an inverted microscope equipped with a digital camera, 0 and 24 hr after scratch . Wound - healing images show the extent of healing and black lines indicate the wound edge . The graph represents the mean sd for at least three independent experiments . * and * * donate p <0.05 and p <0.01, respectively, as compared to the control after observing the decrease in cell migration, we were interested to investigate whether alternations of chemokine receptors expression at mrna level occurred in mcf-7 cells following berberine treatment . Therefore, qpcr was performed to determine the expression of selected chemokine receptor genes . As shown in figure 3, 24 hr - incubation with berberine caused no significant effect on the expression of ccr1(a); however, ccr6 was significantly down - regulated following treatment with berberine 40 m (b). A similar effect was also observed for ccr9 gene expression following treatment with all concentrations of berberine except 10 m (figure 3c). Moreover, cxcr1 and cxcr4 were significantly down - regulated following treatment with all concentrations of berberine (figures 3d and e). These data suggested that the inhibition of chemokine receptors expression in the presence of berberine may resulted in inhibition of mcf-7 breast cancer cell migration . Mcf7 cells were treated with berberine at concentrations of 0, 10, 20, 40 and 80 m for 24 hr, then, subjected to real - time pcr analysis for determination of alterations in gene expression of selected chemokine receptors including ccr1 (a), ccr6 (b), ccr9 (c), cxcr1 (d), and cxcr4 (e). The anti - cancer effects of berberine against many different types of human cancers have been well documented (13). In this study, our results revealed that berberine decreased the percentage of viable mcf-7 breast cancer cells in a concentration - dependent manner which is in agreement with previous studies (14). However, there was no available information regarding the effect of berberine on migration and invasion of mcf-7 breast cancer cells and associated signaling pathways . In this study, the results of wound - healing assay indicated that berberine decreased the migration and invasion of viable mcf-7 breast cancer cells and these effects were concentration - dependent . Chemokine receptors play an important role in the induction of directed cell migration (3). Therefore, to discover underlying mechanisms involved in decreased cell migration in response to berberine, we also examined the level of chemokine receptors gene expression after berberine treatment in breast cancer cells . Cc chemokine receptor 1 (ccr1) plays a critical role in the recruitment of leukocytes to the site of inflammation (16). Knockdown of ccr1 resulted in a significantly reduced invasive ability of hepatocellular carcinoma cells and human non - small cell lung cancer cell (7). The potential role of ccr1 in breast cancer cells metastasis has not been studied yet . The result of our study indicated that berberine did not change the expression level of ccr1 in breast cancer cells . Ccr6, another chemokine receptor is closely related to disease stages when up - regulated and its down - regulation has been proposed as one of the strategies to stop metastasis (8). According to the results of our study, ccr6 showed a statistically significant down - regulation when breast cancer cells were treated with berberine 40 m . Ccr9, another chemokine receptor associated with invasiveness and metastasis of tumors (9) impaired the migration and invasive potential of prostate cancer cells when its expression was blocked, indicating that these responses were dependent on chemokine and chemokine receptor interactions (17). To our knowledge, for the first time, we demonstrated decreased expression of ccr9 in breast cancer cells treated with berberine . Decreased expression of ccr9 may be the answer to why migration potential of breast cancer cells decreased after berberine treatment . Among the genes over - expressed in the breast cancer stem cell (cscs) population, cxcr1, a receptor that binds to the pro - inflammatory chemokines of il-8/cxcl8 and cxcl6, appeared to have an important role in growth and metastasis of breast cscs (18). It was shown that cxcr1 blockade resulted in apoptotic induction, retarded tumor growth and reduced systemic metastasis of breast cancer in nod / scid mice (10). Human colon cancer liver metastases can be inhibited by small molecule antagonists of cxcr1 (19). Using wound - healing assay, we demonstrated that treatment with berberine resulted in decreased cell migration in breast cancer cells . Consistent with these findings, we suggested that berberine may affect migration of breast cancer cells through down - regulation of cxcr1 . Cxcr4 is the most common chemokine receptor expressed in tumor cells, and plays a vital role in the migration, invasion, and prognosis of breast cancer (20). Furthermore, it was demonstrated that targeted down - regulation of cxcr4 expression in breast cancer cell line via different strategies like rna interference (rnai) or cxcr4 antagonists, significantly decreased metastasis of breast cancer cells (21). It has also been reported that cxcl12-cxcr4 axis stimulates the natural selection of breast cancer cell metastasis (22). Cxcr4 receptors ligands stimulate small gtpases and induce reorganization of the actin cytoskeleton, which indicates that these receptors might be involved in the migration and, perhaps, metastasis of tumor cells (23). In the present study, decreased migration of breast cancer cell line following berberine treatment was observed with decreased expression of cxcr4 . These observations may offer an understanding of the molecular mechanisms that lead to decreased breast cancer migration following berberine treatment . It has also been shown that tumor angiogenesis, growth and metastasis are facilitated by the transcription factor of nf-b which modulates chemokine genes (23). Treatment of melanoma cells with caffeic acid phenethyl ester, an inhibitor of nf-b, resulted in an inhibitory effect on cell migration (24). Furthermore, previous experiments on mcf-7 breast cancer cells showed that berberine triggers apoptotic processes through formation of p53 and ros, over - expression of p21 and inhibition of nf-b and bcl-2 expression (25). These observations support the hypothesis that down - regulation of nf-b target genes as a consequence of decreased chemokine receptors expression, may contribute to berberine - induced inhibition of cell migration . Moreover, it is interesting to know that berberine can inhibit ras / mapk and pi3k pathways, which play a major role in promoting gene expression and might be excessively activated in cancer cells (13). Pi3k signaling can also be activated by the g - protein - coupled chemokine receptor cxcr4, which seems to be the principal chemokine receptor expressed on cancer cells (26). Therefore, chemokine - induced activation of pi3k may allow for enhanced survival and metastasis of cancer cells . Moreover, it was revealed that dysregulation of the cxcr4-cxcl12 axis in melanoma, activates cell cycle progression and migration via stimulation of mapk pathway (27). A better understanding of the gene networks and signaling pathways regulated by berberine will definitely enable us to have a better understanding of breast cancer pathogenesis and therapy . Most cancer - related deaths are not the result of primary tumor growth but are rather caused by the spread of cancer to other organs; hence, therapies designed to minimize metastasis are greatly needed . For this purpose, neutralizing antibodies, small molecule antagonists, peptide - derived inhibitors and sirna nanoparticles have been used (28). Despite extensive pre - clinical literature, there has been limited knowledge on cancer clinical trials because safety and efficacy of each of these strategies in the treatment of patients is currently unknown . Identification of a natural molecule that can specifically target cancer cells with minimal or no toxicity to normal cells would be of great benefit and berberine can be one of promising natural compounds to inhibit the proliferation of cancer cells and to alter key markers involved in development and progress of cancer, directly or indirectly . The significant finding of the current study is that treatment of mcf-7 breast cancer cells with berberine decreased cell migration and invasion . Moreover, down - regulation of key chemokine receptors in mcf-7 breast cancer cells may probably represent one of the mechanisms of anti - metastatic effect of berberine . These data may be helpful for future use of berberine in clinical trials for cancer chemotherapy.
The prevalence of chronic alcoholism is estimated to range between 50 60% in trauma patients, and alcohol withdrawal syndrome (aws) occurs in 31% of these patients . Aws usually develops within 6 h to 72 h after the cessation of drinking alcohol . Delirium tremens (dt) typically appears later in the withdrawal process, occurring on average 48 72 h after the last drink . Although dt occurs in approximately 5 20% of patients treated for aws, the mortality rate of patients with dt has been previously reported to be as high as 15% . Due to early detection and effective treatments, the mortality rate of dt has decreased to 1 5% in recent years . While pain and opioid use have both been reported as risk factors for post - operative delirium, clinicians sometimes hesitate to administer opioids for acute pain management due to their potential adverse effects . Meperidine and fentanyl had been known to increase the likelihood of post - operative delirium, while intravenous pain control with morphine appears less likely to cause delirious symptoms . A recent study provided further evidence that improved pain control with intravenous patient - controlled analgesia (iv - pca) is associated with a lower rate of post - operative delirium . Patients with chronic alcoholism are more sensitive to painful stimuli due to lowered pain thresholds; therefore, the adoption of a multimodal approach and administration of iv - pca to effectively manage acute pain has been recommended for these patients . However, to the best of our knowledge, there have been no studies reported in the literature investigating the effects of iv - pca on the development of aws . Using a framework provided by the care statement, we present a case report documenting the occurrence of dt after discontinuation of a 4-day treatment with an iv - pca . In this case, dt manifested more than 5 days after the patient's last drink of alcohol, far exceeding the typical 48 h 72 h time interval . The delayed onset of dt led to delayed diagnosis and incorrect treatment recommendations made by the consulting psychiatrist during the patient's initial evaluation . A 41-year - old married man, who was a taxi driver by profession at the time of admission, was admitted to the plastic surgery unit because of a right ankle abscess . On admission, he reported drinking between 200 ml and 300 ml of whisky daily, beginning each evening only with dinner after work, for the preceding 20 years . He was treated 2 months previously for an alcohol withdrawal seizure at the neurology unit . At that time, valproate 500 mg every 12 h effectively suppressed the signs and symptoms of the seizure . The abscess was debrided three times and then a split - thickness skin graft reconstruction with a gracilis muscle flap was performed . He received an iv - pca for 4 days after the reconstruction surgery for acute pain control set to self - administered 2 ml boluses without a continuous background infusion (morphine 1 mg / ml and ketorolac 1.2 mg / ml). The lockout interval was 10 min with a 4-h morphine limitation of 20 mg . The total amount administered to the patient was 142 ml and he used 12 ml in the last 24 h before it was discontinued . The patient received continuous valproate 500 mg every 12 h and no aws or delirium was observed throughout the 37-day hospitalization . He was discharged on valproate 1000 mg daily and was able to continue working as a taxi driver . Two years later, the patient presented to the emergency department (ed) with a 2-day history of intermittent epigastric pain . Abdominal computed tomography (ct) revealed a linear foreign body in the second portion of the duodenum penetrating the right mesentery with surrounding inflammation . The patient's mother recalled that he accidentally swallowed a toothpick after being drunk two days ago . A preliminary diagnosis of intra - abdominal foreign body with peritonitis was made and an emergent laparotomy was performed under general anesthesia . He was induced with fentanyl 2.5 mcg / kg, thiamylal sodium 5 mg / kg, and cisatracurium 1.5 mg / kg intravenously, and general anesthesia was maintained using desflurane - cisatracurium . After the 3-hour operation, the patient had a smooth recovery with minimal blood loss and stable vital signs . In the post - anesthesia care unit, after a 3 mg intravenous bolus of morphine, an iv - pca was initiated and set at self - administered 2 mg boluses without a continuous background infusion (morphine 1 mg / ml and ketorolac 1.2 mg / ml). The lockout interval was 10 min with a 4-h morphine limitation of 20 mg . The patient was transferred to the general surgical unit with a modified aldrete recovery score of 10 . Since the patient could nt take anything by mouth following duodenal repair, valproate 500 mg every 12 h was discontinued . His condition remained stable for the next 4 days and the iv - pca was administered for 96 h (the total amount used was 146 ml, and 12 ml were used in the last 24 h). Twelve hours after discontinuing the iv - pca, the patient became tachycardic (his heart rate increased from 66 beats / min in the morning to> 100 beats / min in the afternoon) and diaphoretic (his mother changed his hospital clothes four times that afternoon) with tremulous extremities . Central nervous system (cns) symptoms, such as disorganized speech with intermittent curse words, disorientation to time, place, and people, visual hallucination (e.g., he saw some transformers fighting near the ceiling), and disruptive behaviors (e.g., pulling out his nasogastric and iv tubes) were observed 2 h later . Since it had been more than 5 days since his arrival to the ed, he had not had any alcohol for at least 120 h. an injection of 2 mg of lorazepam was immediately administered by slow - push and an additional 2 mg was prescribed 2 h later . Intramuscular haloperidol 5 mg was injected and was repeated 2 h later; however, the patient's agitation continued throughout the entire night . The patient received iv cefmetazole 1 g every 8 h following the operation, and the follow - up laboratory tests revealed leukocytosis (19,900/ul) and mildly elevated c - reactive protein (5.4 mg / dl). Renal and liver functions as well as serum electrolyte, ammonia, hemoglobin, blood glucose, and albumin levels were all within normal limits . Psychiatric consultation was initiated the next day because, despite the administration of iv lorazepam every 8 h, the patient had become increasingly delirious . Since more than 5 days had passed since the patient's last alcoholic drink at the time delirium developed, dt was not considered by the consulting psychiatrist who diagnosed delirium due to multiple causes (chronic alcoholism, recent major surgery, and residual focal infection). Ongoing monitoring for underlying medical and surgical conditions was recommended along with the administration of oral quetiapine 25 mg bid and 50 mg at bedtime . The eeg showed a moderate degree of nonspecific diffuse cortical dysfunction; however, no seizure waves were found . The brain ct did not reveal edema, acute ischemia, intracranial hemorrhage, mass lesion, or mass effect . Two days later, because the patient remained tachycardic and diaphoretic with tremulous extremities, visual hallucination, and disorientation during the second evaluation, a diagnosis of dt was considered by the psychiatrist . The dose of lorazepam was increased to 2 mg every 4 h, quetiapine was reduced to 50 mg at bedtime, and valproate 500 mg every 12 h was reinitiated . Within 8 h, the patient's agitation, disorientation, hallucinations, delusions, and tachycardia began to diminish . No further signs or symptoms of delirium a 41-year - old married man, who was a taxi driver by profession at the time of admission, was admitted to the plastic surgery unit because of a right ankle abscess . On admission, he reported drinking between 200 ml and 300 ml of whisky daily, beginning each evening only with dinner after work, for the preceding 20 years . He was treated 2 months previously for an alcohol withdrawal seizure at the neurology unit . At that time, valproate 500 mg every 12 h effectively suppressed the signs and symptoms of the seizure . The abscess was debrided three times and then a split - thickness skin graft reconstruction with a gracilis muscle flap was performed . He received an iv - pca for 4 days after the reconstruction surgery for acute pain control set to self - administered 2 ml boluses without a continuous background infusion (morphine 1 mg / ml and ketorolac 1.2 mg / ml). The lockout interval was 10 min with a 4-h morphine limitation of 20 mg . The total amount administered to the patient was 142 ml and he used 12 ml in the last 24 h before it was discontinued . The patient received continuous valproate 500 mg every 12 h and no aws or delirium was observed throughout the 37-day hospitalization . He was discharged on valproate 1000 mg daily and was able to continue working as a taxi driver . Two years later, the patient presented to the emergency department (ed) with a 2-day history of intermittent epigastric pain . Abdominal computed tomography (ct) revealed a linear foreign body in the second portion of the duodenum penetrating the right mesentery with surrounding inflammation . The patient's mother recalled that he accidentally swallowed a toothpick after being drunk two days ago . A preliminary diagnosis of intra - abdominal foreign body with peritonitis was made and an emergent laparotomy was performed under general anesthesia . He was induced with fentanyl 2.5 mcg / kg, thiamylal sodium 5 mg / kg, and cisatracurium 1.5 mg / kg intravenously, and general anesthesia was maintained using desflurane - cisatracurium . After the 3-hour operation, the patient had a smooth recovery with minimal blood loss and stable vital signs . In the post - anesthesia care unit, after a 3 mg intravenous bolus of morphine, an iv - pca was initiated and set at self - administered 2 mg boluses without a continuous background infusion (morphine 1 mg / ml and ketorolac 1.2 mg / ml). The lockout interval was 10 min with a 4-h morphine limitation of 20 mg . The patient was transferred to the general surgical unit with a modified aldrete recovery score of 10 . Since the patient could nt take anything by mouth following duodenal repair, valproate 500 mg every 12 h was discontinued . His condition remained stable for the next 4 days and the iv - pca was administered for 96 h (the total amount used was 146 ml, and 12 ml were used in the last 24 h). Twelve hours after discontinuing the iv - pca, the patient became tachycardic (his heart rate increased from 66 beats / min in the morning to> 100 beats / min in the afternoon) and diaphoretic (his mother changed his hospital clothes four times that afternoon) with tremulous extremities . Central nervous system (cns) symptoms, such as disorganized speech with intermittent curse words, disorientation to time, place, and people, visual hallucination (e.g., he saw some transformers fighting near the ceiling), and disruptive behaviors (e.g., pulling out his nasogastric and iv tubes) were observed 2 h later . Since it had been more than 5 days since his arrival to the ed, he had not had any alcohol for at least 120 h. an injection of 2 mg of lorazepam was immediately administered by slow - push and an additional 2 mg was prescribed 2 h later . Intramuscular haloperidol 5 mg was injected and was repeated 2 h later; however, the patient's agitation continued throughout the entire night . The patient received iv cefmetazole 1 g every 8 h following the operation, and the follow - up laboratory tests revealed leukocytosis (19,900/ul) and mildly elevated c - reactive protein (5.4 mg / dl). Renal and liver functions as well as serum electrolyte, ammonia, hemoglobin, blood glucose, and albumin levels were all within normal limits . Psychiatric consultation was initiated the next day because, despite the administration of iv lorazepam every 8 h, the patient had become increasingly delirious . Since more than 5 days had passed since the patient's last alcoholic drink at the time delirium developed, dt was not considered by the consulting psychiatrist who diagnosed delirium due to multiple causes (chronic alcoholism, recent major surgery, and residual focal infection). Ongoing monitoring for underlying medical and surgical conditions was recommended along with the administration of oral quetiapine 25 mg bid and 50 mg at bedtime . The eeg showed a moderate degree of nonspecific diffuse cortical dysfunction; however, no seizure waves were found . The brain ct did not reveal edema, acute ischemia, intracranial hemorrhage, mass lesion, or mass effect . Two days later, because the patient remained tachycardic and diaphoretic with tremulous extremities, visual hallucination, and disorientation during the second evaluation, a diagnosis of dt was considered by the psychiatrist . The dose of lorazepam was increased to 2 mg every 4 h, quetiapine was reduced to 50 mg at bedtime, and valproate 500 mg every 12 h was reinitiated . Within 8 h, the patient's agitation, disorientation, hallucinations, delusions, and tachycardia began to diminish . To date, there has been only a single case report addressing the issue of delayed - onset dt . The authors reported the case of a young man with onset of dt activity on day 15 of his hospitalization for alcohol abstinence and, theoretically, the dt were attributed to the delayed effects of indian liquor that contains a high percentage of alcohol . In the present case, while receiving continuous treatment with valproate, dt did not appear during the patient's first hospitalization for skin grafting and reconstruction, including during the administration of an iv - pca and after its discontinuation . During the second hospitalization, valproate was discontinued due to the patient's inability to take anything by mouth post - surgery and the dt appeared 14 h after discontinuation of an iv - pca that had been administered for 96 h. although a few studies have found anticonvulsants, with the potential exception of carbamazepine, have a limited effect on dt, a recent retrospective cohort study revealed that valproate may have better efficacy and tolerability as an adjunct treatment for aws . Therefore, if valproate had been continued by iv, as the patient could not take anything by mouth during the second hospitalization, the occurrence of dt could potentially have been prevented . There were no generalized tonic - clonic movements or epileptic waves on the eeg during the patient's delirium; therefore, his agitation could not be explained by seizures, interictal, or postictal confusion . Laboratory findings, including blood ammonia level, were not suggestive of a clinically significant metabolic derangement . The patient received only a limited amount of morphine as an analgesic for a brief period; therefore, neither the limited exposure to morphine nor the patient's signs and symptoms supported a diagnosis of opioid withdrawal . Furthermore, the elimination half - life of valproate is 14h and it had been approximately 5 days (120 h) since the last valproate dose . Therefore, as more than eight half - lives had passed by the time the dt began, the delirium could not be related to the discontinuation of valproate . A total of 146 mg of morphine was administrated over the 96 h iv - pca was administered . Since the elimination half - life of iv morphine is approximately 2 h in healthy volunteers, the appearance of the dt correlated with the clearance of the morphine . First, the patient received general anesthesia with the cns suppressive agents fentanyl, thiamylal sodium, and desflurane . There was no post - anesthetic emergence delirium following the 3 h of general anesthesia . A recent study showed that post - anesthetic emergence delirium usually develops in the immediate postoperative period, with the primary manifestation comprising hypoactivity . However, delirium in this patient was noticed 5 days after the general anesthesia and manifested itself as extreme agitation . Second, the patient's delirium indicated signs of alcohol withdrawal including autonomic hyperactivity (tachycardia and sweating) and limb tremors . The presence of these autonomic hyper - arousal signs occurred 12 h after the discontinuation of 96 h of iv - pca and dt appeared 2 h later . The signs and symptoms of alcohol withdrawal and agitated delirium persisted concurrently throughout the 2-day course of the delirium, matching the diagnostic criteria of alcohol withdrawal.autonomic hyperactivity is the core feature of dt, but not of post - operative delirium . Third, the autonomic hyperactivity and agitated delirium did not subside despite the continued administration of an atypical antipsychotic agent and low dose lorazepam; however, it did remit 8 h after the lorazepam dose was titrated up, the antipsychotics were titrated down, and valproate was added . In post - operative delirium, the administration of benzodiazepines could be a precipitating or maintaining factor; only patients withdrawing from alcohol or sedatives - hypnotic - anxiolytic benefit from the timely and adequate use of benzodiazepines . The signs and symptoms of dt in this patient subsided within 23 days, consistent with previous studies . Foy et al noted that the median time of onset of dt was 46 h after arriving at the hospital and the median time for resolution was 23h . Monte et al reported that the minimum time of onset of dt was 4 h, the first quartile was 22 h, the median time was 24 h, the third quartile was 48 h, and the maximum time of onset was 87 h; the median time to resolution was found to be 72 h. however, the time of onset of dt in present case was more than 120h after the patient's last drink, a time interval far exceeding the documented range . It is worth noting that dt appeared sequentially after cessation of the 4-day iv - pca morphine administration, occurring approximately 14 h after discontinuation of the iv - pca . We therefore hypothesize that administration of iv - pca morphine could delay the onset of dt . Alcohol is concurrently a gamma - aminobutyric acid type a (gabaa) receptor agonist and a glutamate n - methyl - d - aspartate (nmda) receptor antagonist . The chronic consumption of alcohol can down - regulate the gabaa receptors and up - regulate the nmda receptors . When a chronic alcoholic suddenly stops or significantly reduces alcohol intake, the excessive downhill gaba effect and uphill glutamate effect induce aws . Aws initially manifests as a hyperactive autonomic nervous system and may progress to seizures and even dt in patients with multiple risk factors . Carbamazepine and propofol are effective treatments via their dual actions as gabaa agonists and nmda antagonists . Other drugs, including clomethiazole, valproate, baclofen, and barbiturates may also be effective due to their ability to boost the gaba system . Opioids are known to evoke an analgesic effect via mu receptors; however, only a few studies have discussed their effect on aws . Blum et al reported a suppressive effect of morphine on alcohol withdrawal seizures in mice; the authors proposed that the increase of dopamine in the cns after the administration of morphine played a significant role . Tsueda et al reported the use of opioids to suppress dt in three patients and postulated that opioid deficiency in the cns may be related to aws . Other potential mechanisms explaining the suppressive effect of opioids on aws and dt include an inhibitory effect on the locus coeruleus . Increased firing rates of the norepinephrine neurons in the locus coeruleus can be found in alcohol withdrawal or opioid withdrawal and account for most of the withdrawal signs and symptoms, such as anxiety, tremors, tachycardia, hypertension, sweating, nausea, and insomnia . Furthermore, alpha-2 receptors, gaba receptors, and opioid receptors can be found on the neurites of the locus coeruleus neurons and comprise the sites that exert inhibitory effects . Therefore, alpha-2 agonists help to relieve not only aws but also opioid withdrawal; however, benzodiazepinesare also known to relieve the symptoms of opioid withdrawal . In conclusion, the case described here illustrates that, while iv - pca is one of the most common and effective methods of providing postoperative analgesia in patients with chronic alcoholism, its administration may delay the onset of dt . This highlights the importance of taking into consideration trauma patients psychiatric history including answers to questions on alcoholism, so that when an iv - pca is administered and then discontinued, adequate interventions to prevent further morbidity associated with aws and dt can be initiated in sufficient time . The authors disclose no proprietary or commercial interest in any product mentioned or concept discussed in this article.
Alzheimer's disease (ad), the most common cause of dementia in the elderly, is an irreversible progressive neurodegenerative disorder clinically characterized by memory loss and cognitive decline . Ad is characterized pathologically by synaptic loss and by the accumulation of extracellular beta - amyloid (a), neuritic plaques, and hyperphosphorylated tau in intracellular neurofibrillary tangles (nft) [24]. Of these, synaptic loss most closely correlates with cognitive decline, whereas beta - amyloid accumulation, the presence of neuritic plaques, and nft are the pathological markers required to make a definitive diagnosis of ad . Failure of synaptic plasticity has been proposed as the mechanism underlying memory impairment in ad [7, 8]. The chromatin - remodeling enzyme poly(adp - ribose) polymerase-1 (parp-1) plays important roles in synaptic plasticity and memory consolidation in both aplysia and rodents [911]. This enzyme engages in poly(adp)-ribosylation (par), using nicotinamide adenine dinucleotide (nad+) to form branched adp - ribose polymers on nuclear acceptor proteins, such as dna polymerases, ligases, and histones . This epigenetic modification results in the loosening of chromatin structure allowing repair proteins and transcription factors to access the dna [12, 13]. Parp-1 activation leads to the expression of genes required for memory consolidation such as immediate early genes and ribosomal rna genes (rdnas) in the nucleolus . In addition, parp-1 has also been shown to regulate multiple areas of nucleolar function, including the inheritance of rdna chromatin structure, editing of precursor rrna, and biogenesis of ribosomes in the nucleolus [16, 17]. Since synaptic plasticity has been shown to be impaired in ad, we hypothesized that this impairment may be due to a loss of parp-1 and a disruption of parp's role in the nucleolus in maintaining nucleolar integrity . To begin addressing this hypothesis, we compared parp-1 expression in postmortem hippocampal brain tissue derived from patients with neuropathologically confirmed ad to control hippocampal brain tissue from patients without significant neuropathology . We show that parp-1 positive staining of nucleoli in ca1 and ca4 hippocampal pyramidal cell neurons in ad is significantly reduced compared to controls . We suggest that memory impairment in ad may be due, in part, to this novel finding . This loss of nucleolar parp-1 in ad appears due in part to a mislocalization of the protein from the nucleolus . Here, we present a model in which the loss of nucleolar parp-1 precedes changes in nucleolar function and integrity seen in early stages of ad . Paraffin - embedded tissue blocks from the hippocampus were collected from deidentified archived material from the alzheimer's disease research center (adrc) at emory university school of medicine, sun health research institute brain and body donation program of sun city, arizona [18, 19], kings county hospital center, and state university of new york downstate medical center . Postmortem brain tissue was acquired from two groups of individuals (table 1): (1) the ad group consisted of tissue from male and female patients with neuropathologically confirmed ad that meet the criteria for the diagnosis of definite alzheimer's disease according to the consortium to establish a registry for alzheimer's disease and a high likelihood that dementia was due to ad by nia reagan criteria and (2) the control group consisted of individuals, both male and female, of similar age to the ad group with no known history of dementia or neurologic disorder and without significant neuropathology . The ad cases had braak scores of v - vi and the controls had braak scores of 0, i, or ii (table 1). The samples were deparaffinized, hydrated, and submerged in 10 mm citrate buffer (ph 6.0) and microwave irradiated (15 min) for antigen retrieval ., slides were rinsed for 5 min with 0.1% triton x-100 in phosphate - buffered saline (pbs - triton), treated with 3% h2o2 for 20 min, rinsed with pbs - triton for 5 min, blocked in 2% normal horse serum in pbs - triton for 30 min, and incubated with primary antibody (anti - par polyclonal, 1: 200; cat #4336-bcp-100, trevigen; and parp-1 monoclonal antibody, 1: 200; cat #1522 g, abd serotec) overnight in a humidity chamber . The sections were then rinsed in pbs - triton and incubated for 1 h in biotinylated secondary antibody horse anti - mouse (1: 200) diluted in blocker (vectastain abc systems, vector laboratories), rinsed again, and developed using the abc system (vector laboratories, burlingame, ca), using standard histologic procedures . For controls, sections were treated as mentioned above with omission of primary antisera (1: 200). For single immunofluorescent visualization, the samples were blocked for 1 h with 2% normal goat serum (ngs) in pbs - triton and then incubated overnight with parp-1 monoclonal antibody (1: 200) diluted in blocker . After rinsing 3 times for 10 min each in pbs - triton, the samples were incubated 4 h with goat anti - mouse - biotin f(ab) fragment (1: 200) in blocker buffer, rinsed 3 times for 10 min each in pbs - triton, and incubated for two hours with strep alexa 647 (1: 200) and dapi (1: 500) in blocker buffer . The sections were then rinsed in pbs - triton and in distilled water, immersed for 5 minutes in 70% ethanol containing 0.3% sudan black, rinsed in distilled water, and mounted on glass slides with prolong gold (molecular probes, eugene, or). For controls, sections were treated as mentioned above with omission of primary antisera . The double immunohistochemistry was similar to the single immunohistochemistry except for (a) a second primary antibody (rabbit anti - fibrillarin antibody, 1: 100; cat #ab5821, abcam) which was used during the incubation overnight and (b) a second secondary antibody (fluorescein goat anti - rabbit; 1: 200 invitrogen, thermo fisher scientific, glen island, ny) which was used during the incubation with secondary antibodies . Qualitative assessment of the immunohistochemistry using light and confocal microscopy was performed and staining was determined to be either strong (for light microscopy) or high intensity (for confocal microscopy), weak or absent . Images were taken of each slide at a magnification of 400x and all the cells in three randomly chosen fields within the designated region were counted for presence or absence of nucleolar staining . For confocal microscopy, all images were taken at the same parameters preset on sections stained with no primary antibodies . We found no significant differences in the nuclear staining of par in neurons in hippocampal regions ca14, entorhinal and temporal cortices, or subiculum (data not shown). In contrast, parp-1 immunohistochemistry showed positive staining in the nucleus with strong staining of the nucleolus in controls and weak nuclear staining with little to no staining in the nucleoli within neurons in ad (figure 1 compare (a) and (b)). Interestingly, the only exception was dentate gyrus where no differences between ad and controls were observed . In controls, the percentage of pyramidal neurons with parp-1 positive nucleoli was 63.9% in ca1 and 51.1% in ca4 . In contrast, the percentage of parp-1 positive nucleoli in pyramidal neurons in ad was 28.7% in ca1 and 30.4% in ca4 (figures 1(c) and 1(d)). We used confocal microscopy to confirm our results showing loss of parp-1 nucleolar staining in ad . Consistent with the light microscopy data, we found that 66.1% and 62.2% of ca1 and ca4 hippocampal pyramidal cell nucleoli stained positive for parp-1 in controls, whereas, in ad, nucleolar parp-1 staining was present in only 29.3% and 32.0% of ca1 and ca4 pyramidal cells, respectively (figure 2). To test whether other nucleolar proteins are affected in ad, we performed double immunohistochemistry with parp-1 and fibrillarin, a nucleolar protein involved in pre - rrna processing . If the loss of parp-1 nucleolar staining was due to general damage and structural loss of nucleoli from cells, then we would also expect to see a comparable loss of fibrillarin and other nucleolar proteins . However, a loss of parp-1 with preserved fibrillarin staining in ad would indicate that loss of parp-1 is selective . Control cases exhibited high intensity nucleolar staining and a higher percentage of parp-1 and fibrillarin colocalization (figures 3(a)3(d)) compared to ad (figures 3(e)3(h)). There is a significant loss (p = 0.017) of parp-1 nucleolar staining in ca1 pyramidal cells in ad compared to controls . In contrast, fibrillarin staining in ca1 is not significantly different between ad and controls (tables 2 and 3). The loss of parp-1 from the nucleolus, therefore, appears to be a selective departure and may reflect a departure from the nucleolus due to mislocalization of the protein (tables 2 and 3). In this study, we demonstrated that there is a loss of parp-1 from hippocampal pyramidal cell nucleoli in ad, suggesting that parp-1 nucleolar function may be compromised in ad . Recently, our group demonstrated that the maintenance of late - phase long - term potentiation (l - ltp), a model for long - term memory, requires nucleolar integrity and the expression of new rrnas the latter being regulated by parp-1 . Therefore, we hypothesize that parp-1 and nucleolar integrity are required for long - term memory . Recently, in a study complementary to ours, it was demonstrated that chronic deficits in nucleolar function alter synaptic plasticity and learning and memory . In addition, parp-1 has also been shown to regulate multiple areas of nucleolar function, including the inheritance of rdna chromatin structure, editing of precursor rrna, and biogenesis of ribosomes in the nucleolus [16, 17]. There is a previous immunohistochemical study of parp-1 and par staining in ad, which found an increase in nuclear parp-1 and par in frontal and temporal lobe tissues . It is possible that the parp-1 nucleolar loss has a differential degree of sensitivity in different areas of the brain in ad and may be a finding specific to the hippocampus . We found that the ca1 and ca4 subregions of the hippocampus exhibit vulnerability to the nucleolar parp-1 loss in ad, which mirrors the vulnerability to ad neuropathological change and to ischemic damage . Interestingly, chronic deficits in nucleolar function have been shown to lead to neurodegeneration with differential cellular vulnerability in the hippocampus . Parp-1 has shown to be activated secondary to oxidative stress and dna damage [24, 2628] and, in mild to moderate stress, is thought to be part of the repair mechanism but may lead to cell death via consumption of nad+ when overactivated . We hypothesize that the loss of parp-1 from nucleoli of hippocampal pyramidal cells may be an early and persistent finding in ad . This loss of nucleolar parp-1 from hippocampal pyramidal neurons may lead to deficits in synaptic plasticity and, thus, to cognitive impairment . In contrast, late in ad, it is possible that parp-1 is overactivated and contributes to cell death in frontal and temporal cortices as shown by love et al . . Furthermore, we speculate that the loss of parp-1 from hippocampal pyramidal cells in ad may help to explain some of the selective vulnerability of the ca1 and ca4 regions of the hippocampus . That is, there is a loss of the physiologic parp-1 activation required for long - term synaptic plasticity and memory consolidation [911, 14, 15] and also a region specific loss of the reparative activation of parp-1 associated with mild to moderate stress . The nucleolus has emerged as an important structure to study in relation to ad neuropathology . In a study of postmortem brains from the nun study of aging and alzheimer's disease, a longitudinal study examining the onset of ad, it was found that asymptomatic ad cases, in which autopsied brain samples revealed common ad lesions in spite of the subjects having had normal cognition, exhibited significant hypertrophy of nucleoli (+ 80.2%) in ca1 neurons compared to mci or controls . There was also hypertrophy of cell bodies and nuclei but it was the nucleoli which had the largest change . This suggests a compensatory mechanism preventing the impairment of cognition despite the presence of typical ad pathology . Based on these findings, we hypothesize that it was the maintenance of nucleolar function (and, therefore, rrna synthesis) which prevented the cognitive deficits in these individuals with ad neuropathology . Aberrations in the epigenetic code of acetylation, methylation, and parylation are a common denominator of neurodegenerative diseases [3032]. Nucleolar impairment may also be a common denominator in several neurodegenerative disorders such as huntington's, parkinson's, and alzheimer's' disease . Epigenetic silencing of rdna by dna methylation has been found to be a common feature of mild cognitive impairment (mci) and ad and may represent a new marker of the disease . The rdna silencing occurs in the nucleolus, perturbing nucleolar functions such as global chromatin regulation and biogenesis of ribosomes . This gene silencing is consistent with previous reports of a decrease in ribosomes in the inferior parietal lobe of mci and ad patients . Impairing the expression of rrnas (essential components of ribosomes) or any of the steps of ribosome biogenesis can produce nucleolar stress, leading to changes in gene expression and a reduction in ribosomes and protein synthesis resulting in cellular dysfunction . To date, the factors leading to increased rdna methylation in mci and ad are unknown . Since parp-1 has been shown to regulate genomic methylation patterns by inhibiting the activity of dna methyl - transferase, we propose that parp-1 displacement from the nucleolus in ad leads to hypermethylation of rdna . There is then downregulation of rrna expression and of ribosomal biogenesis (see figures 4(a) and 4(b)). Without new ribosomes,
Microsatellites are repetitive nucleotide elements that have a core repeat structure of 2 to 6 nucleotides (chambers and macavoy, 2000). Microsatellite allele mutations predominantly occur via strand slippage during chromosome replication at a rate of 10 to 10 (eisen, 1999), and is influenced by repeat unit length, number of repeat units in the array, microsatellite flanking sequence, and recombination (chambers and macavoy, 2000). Ostrinia nubilalis (hbner), the european corn borer, is an invasive agricultural pest in north america introduced from europe around 1917 . The herbivorous o. nubilalis larvae are adapted to feeding on cultivated zea mays (l.) and annually cause major economic losses (mason et al . Phenotypic diversity in the north american population is present in two sex pheromone races, and three voltinism types (showers 1993). Females of the two sex pheromone races are differentiated by their emission of either e- or z-11-tetradecenyl acetate as the dominant pheromone component . Pheromone races show little allozyme marker differentiation (harrison and vawter 1977; card et al . They interbreed in the laboratory and under natural conditions continual gene flow between the races apparently occurs (roelofs et al . 1985; durant et al ., 1995). In contrast, diapause ecotypes differ in the number of degree days required prior to adult emergence (showers 1993), and the resultant asynchrony of adult mating cycles between voltinism types was suggested to minimize genetic exchange (roelofs et al . 1985). Reduced gene flow between voltinism ecotypes was supported by rapd - pcr marker data (pornkulwat et al . 2000) or mitochondrial rflp data (marcon et al ., 1999). To date, locus specific genetic markers used for o. nubilalis population analysis have inherent limitations that restrict the levels of variation detected, which might contribute to the conflicting evidence provided between o. nubilalis ecotypes . Intraspecific diversity of allozyme markers is reduced by negative selection on nonsynonymous changes and environmental influence on post translational modification pathways (hartel 1988). Mitochondrial dna is also prone to fixation by genetic drift due to a reduced effective population size of the molecule compared to chromosomes (avise et al ., 1988). Given the limitations of previously used markers, unresolved questions regarding gene flow among north american ecotypes, and our desire to estimate both male- and female - based population differentiation (movement), we developed sex - specific o. nubilalis nuclear microsatellite markers . In the following allele variation of the north american population is characterized at two such markers, onw1 (ostrinia nubilalis w - chromosome 1) and onz1 (ostrinia nubilalis z - chromosome 1). Genotypic and haplotypic data were used to evaluate intraspecies differences, infer the presence of any population subdivision, and estimate the levels of genetic exchange (migration) among o. nubilalis voltinism ecotypes and geographically distinct north american subpopulations . Genomic dna from o. nubilalis was enriched for ca microsatellites by methods described by lyall et al . Resultant pcr products were ligated into pgem - t easy cloning vector (promega, www.promega.com) according to manufacturer instructions . Electroporation competent e. coli sure cells (stratagene, www.stratagene.com) were transformed on a micropulsar apparatus (biorad, www.bio-rad.com), and clone selection and blue white screening was performed . Positive clones were propagated overnight at 37 c in 25 ml terrific broth that contained ampicillin . Plasmid dna was isolated with a qiaprep spin miniprep kit (qiagen, www.qiagen.com) according to manufacturer's directions . Template was sequenced at the dna sequencing and synthesis facility at iowa state university, ames, ia, usa . Ostrinia nubilalis samples were collected from three light traps in iowa, and samples obtained from collaborators (table 1). Dna pellets were diluted to 50ng/l with tle (10 mm tris, 0.1 mm edta, ph 7.5) and stored at 20 c prior to use . Primer pairs onw1-f (5-tggaagttgatcggaataagaagtc 3) with onw1-r (5-tggaagagcggtaacctcct 3), and onz - m1f (5-ggtgggacctccatgcgc-3) with onz - m1r (5-gctggggcgtcttcgaggt-3) were designed from respective dna sequence data using primer3 (rozen and skaletsky 1998). Primers were synthesized at integrated dna technologies (www.idtdna.com), and 5 pmol of each used along with 0.425 u taq polymerase (promega), 1.25 l 10x thermal polymerase buffer (promega), 2.5 mm mgcl2, and 150 m dntps to pcr amplify 150 ng of individual o. nubilalis dna sample in a 12.5 l reaction volume . Thermocycler reactions used 94 c for 3 min ., followed by 40 cycles of 94 c for 20 sec . Onw1 locus amplification from eighteen other species used modified amplification conditions (table 2). Pcr products were separated at 150v for 10.5 hr . On a 0.1 20 cm 8% polyacrylamide (19:1 acrylamide: bisacrylamide) 1x tbe gel with a 25 base pair ladder (promega) for size comparison . Bands were visualized after ethidium bromide staining, and image capture took place on a pc - foto / eclipse electronic documentation system (fotodyne, www.fotodyne.com). Basis of variation was determined by cloning 2 loci of each observed fragment size for onw1 and onz1 . Onw1 allele and onz1 genotypic frequency data was used to evaluate o. nubilalis population structure, and estimate male and female migration (m) between subpopulations . Population structure assumed genetic separation of univoltine z - pheromone subpopulations (uz; i d 1 to 4), from bivoltine and multivoltine z - pheromone (table 1; i d 5 to 11). Calculations of geographic - based population subdivision assumed three groups: group 1, subpopulations with i d 1 to 4; group 2, i d 5 to 9; group 3, i d 10 and 11 . A second analysis assumed north - south population subdivision assumed two subpopulation groups; north: subpopulations with i d 1 through 4, and south i d 5 to 11 (table 1). Statistical evaluations included analysis of molecular variance (amova), and f - statistic or haploid allele data modified f - statistic (; excoffier et al . The hardy - weinberg equilibrium of subpopulation onz1 was tested using 10,000 markov chain steps, and significance p values set at 0.05 . All calculations were performed with the arlequin software package (schneider et al ., 1997). Genomic dna from o. nubilalis was enriched for ca microsatellites by methods described by lyall et al . Resultant pcr products were ligated into pgem - t easy cloning vector (promega, www.promega.com) according to manufacturer instructions . Electroporation competent e. coli sure cells (stratagene, www.stratagene.com) were transformed on a micropulsar apparatus (biorad, www.bio-rad.com), and clone selection and blue white screening was performed . Positive clones were propagated overnight at 37 c in 25 ml terrific broth that contained ampicillin . Plasmid dna was isolated with a qiaprep spin miniprep kit (qiagen, www.qiagen.com) according to manufacturer's directions . Template was sequenced at the dna sequencing and synthesis facility at iowa state university, ames, ia, usa . Ostrinia nubilalis samples were collected from three light traps in iowa, and samples obtained from collaborators (table 1). Dna pellets were diluted to 50ng/l with tle (10 mm tris, 0.1 mm edta, ph 7.5) and stored at 20 c prior to use . Primer pairs onw1-f (5-tggaagttgatcggaataagaagtc 3) with onw1-r (5-tggaagagcggtaacctcct 3), and onz - m1f (5-ggtgggacctccatgcgc-3) with onz - m1r (5-gctggggcgtcttcgaggt-3) were designed from respective dna sequence data using primer3 (rozen and skaletsky 1998). Primers were synthesized at integrated dna technologies (www.idtdna.com), and 5 pmol of each used along with 0.425 u taq polymerase (promega), 1.25 l 10x thermal polymerase buffer (promega), 2.5 mm mgcl2, and 150 m dntps to pcr amplify 150 ng of individual o. nubilalis dna sample in a 12.5 l reaction volume . Thermocycler reactions used 94 c for 3 min ., followed by 40 cycles of 94 c for 20 sec . Onw1 locus amplification from eighteen other species used modified amplification conditions (table 2). Pcr products were separated at 150v for 10.5 hr . On a 0.1 20 cm 8% polyacrylamide (19:1 acrylamide: bisacrylamide) 1x tbe gel with a 25 base pair ladder (promega) for size comparison . Bands were visualized after ethidium bromide staining, and image capture took place on a pc - foto / eclipse electronic documentation system (fotodyne, www.fotodyne.com). Basis of variation was determined by cloning 2 loci of each observed fragment size for onw1 and onz1 . Onw1 allele and onz1 genotypic frequency data was used to evaluate o. nubilalis population structure, and estimate male and female migration (m) between subpopulations . Population structure assumed genetic separation of univoltine z - pheromone subpopulations (uz; i d 1 to 4), from bivoltine and multivoltine z - pheromone (table 1; i d 5 to 11). Calculations of geographic - based population subdivision assumed three groups: group 1, subpopulations with i d 1 to 4; group 2, i d 5 to 9; group 3, i d 10 and 11 . A second analysis assumed north - south population subdivision assumed two subpopulation groups; north: subpopulations with i d 1 through 4, and south i d 5 to 11 (table 1). Statistical evaluations included analysis of molecular variance (amova), and f - statistic or haploid allele data modified f - statistic (; excoffier et al ., 1992; the hardy - weinberg equilibrium of subpopulation onz1 was tested using 10,000 markov chain steps, and significance p values set at 0.05 . All calculations were performed with the arlequin software package (schneider et al ., 1997). Two polymorphic tandem repetitive loci, o. nubilalis z - chromosome 1 (onz1) and w - chromosome 1 (onw1), were sequenced from separate clones in a microsatellite enriched partial genomic library . The onw1-containing clone, pgem - onca09, had two repetitive regions, ten tandem gt / ca repeat units and four consecutive gaaaat repeats (fig . 1a; genbank af442958), and population analysis revealed three alleles of 141 (3 repeats; allele wa3), 147 (4 repeats; allele wa4), and 153 base pairs (5 repeats; allele wa5). Two samples from each of three observed onw1 allelic types (electromorphs) were dna sequenced, and full gaaaat repeat units were identified as the basis of size variation without flanking region mutation (data not shown). Successful pcr results were obtained from the onw1 hexanucleotide repeat only from female dna samples (data not shown). The onz1-containing clone pgem - onca01 had a fifteen nucleotide - long imperfect repeat element, caycarcgtcactaa, (underlined nucleotides optional; fig 1b; genbank ay102618 to ay102620). Onz1 was pcr amplified from male derived dna and all attempts to amplify female dna failed (data not shown). Two onz1 allele size range variants 95 to 97 (za1) and 106 to 107 base pairs (za2) were characterized with one insertion / deletion and two substitutions within both the repeat and the flanking region (fig 1b). Since single base changes were not resolved by polyacrylamide gel electrophoresis methods, two alleles (za1 and za2) were scored and used for genotype data . Three pedigrees with 46 f2 progeny confirmed the male specificity of onz1 and female specificity of onw1 . More specifically, onw1 and onz1 were hemizygous in females (single locus with no corresponding homologous pair), and males were diploid at onz1 and lacked the onw1 locus . Onz1 and onw1 were pcr amplified from 448 female, and onz1 from 493 male, dna samples from 11 north american subpopulations of o. nubilalis . Male onz1 genotypic and onz1 and female onw1 haplotype frequencies were calculated (table 1). Male genotypic and female haplotype data were separately evaluated by amova and fixation indices, and independently showed stronger evidence for geographic - compared to voltinism - based population structuring (table 2). Low allele size diversity was present at o. nubilalis w chromosome (onw1) and z - chromosome (onz1) microsatellite loci . The onw1 allele with four repeat units [(gaaaat)4; wa4), was present among 76% of north american o. nubilalis females, and described from 87.1% of 41 female o. furnicalis (gune) and a single o. pentitalis (grote) sample, indicating that the allele likely is ancestral to the genus . The molecular basis of low onw1 allele size variation might lie in the repetition of a hexanucleotide repeat that is less prone to strand slippage during recombination, the short array of 3 to 5 gaaaat repeats, and the potential location of onw1 in the nonrecombining region of the w chromosome that eliminates unequal crossover as a mechanism generating allele diversity . Onw1 wa3 alleles outnumbered wa5 alleles, 19.3% to 4.7% (table 1), indicating a preponderance of microsatellite repeat unit loss, as opposed to gain that occurs typically at microsatellite loci (rubinsztein et al ., 1995, weber and wong, 1993). Rose and falush (1998) determined that a minimum of eight repeats was required for evolution of hypervariable microsatellites, and array lengths such as onw1 that are below this threshold suggest insensitivity toward repeat number expansion bias . The onz1 locus is biallelic, with the dominant za1 allele frequency at 95% (937 of 986) in males and 99% (445 of 448) in females . Near fixation of za1 in female o. nubilalis and not in males might result from differences in effective population sizes (ne) of the w chromosome compared to z chromosomes . Assuming a 1:1 sex - ratio, lepidopteran w chromosomes have a three- and fourfold reduction in effective population size compared to z and autosomal chromosomes respectively (charlesworth et al ., 1987; hartel, 1988), whereby associated loci are be more prone to fixation and interpopulation divergence (charlesworth et al ., 1987). Despite being less prone to molecular fixation, onz1 locus allele diversity was lower than onw1 that might result from its longer imperfect repeat, or be a result of population effects (see below). No genetic divergence was detected between female onw1 and onz haplotypes based on voltinism ecotype, as indicated by similarly at the onz1 locus, grouping samples by voltinism showed an fst = 0.028, and 0.83% of total population variance among ecotypes (remaining data not shown). Results indicated no evidence of o. nubilalis voltinism - based ecotype structure and are in agreement with mitochondrial pcr - rflp (marcon et al . 1999) and allozyme data (bourget et al . 2000), but in conflict with voltinism ecotype variation detected by rapd - pcr (pornkulwat et al . Additionally, a single e - pheromone subpopulation from newark, delaware did not show significant onz1 allele or genotype variance with z - pheromone race subpopulations . A triose phosphate isomerase (tpi) marker was previously mapped to the z chromosome and linked to inter - pheromone race behavioral or response differences in males (glover et al . 1991), suggesting a z chromosome location of onw1 distant from tpi and male pheromone response genes . Lack of both voltinism ecotype - based population differentiation or a correlation of onz1 with pheromone race might be an artifact of recent o. nubilalis range expansion across north america (showers 1993), suggesting slowed onw1 and onz1 lineage sorting (avise et al . 1984) and allele extinction by genetic drift (takahata 1983) between ecotypes . Low but significant differentiation was previously shown between o. nubilalis ecotypes (harrison and vawter 1977; card et al . 1978), but not between geographically distant samples (bourget et al . 2000). Both sex - linked genetic markers, onw1 and onz1, indicated greater contribution of geography to total genetic variance compared to voltinism . Amova analysis indicated that 3.78% of total population variance was between two groups; a northern group consisting of minnesota and south dakota subpopulations (i d 1 to 4), and all other subpopulations classified as onz1 and onw1 female haplotype data indicated that 4.02% of total population variance was accounted for among northern and southern groups . Low - level geographic divergence of four subpopulations from northern latitudes might reside in their omission of the za1wa5 female haplotype (table 1), and significant deviation of onz1 data from hardy - weinberg expectation at three of the four sites . In the northern geographic region, chi - square () tests detected significant departure of onz1 genotypes from hardy - weinberg equilibrium within lamberton, minnesota bivoltine z - pheromone (p <0.001), lamberton, minnesota univoltine z - pheromone (p = 0.007), and south shore, south dakota univoltine z - pheromone subpopulations (p = 0.016). Deviation for hwe was not exhibited from any other subpopulation . Except for the brookings, south dakota sample, the basis for hardy - weinberg deviations among northern subpopulations likely reside in their decreased the average heterozygosity (hs= 2 81 0.025) compared to the average among the remaining subpopulations (ht = 19 317 0.060). Reduced heterozygosity might be attributed to the presence of null alleles, or a stronger effect of the 1/4 reduction in z chromosome effective population size in smaller fringe populations . Alternatively, recent or recurrent population size effects might disproportionately disrupt o. nubilalis population hardy - weinberg equilibrium in northern regions . Increased severity of cyclical genetic bottlenecks due to larval over - wintering mortality in northern regions (hudson and leroux 1986) might disturb mutation - drift equilibrium since ne likely does not remain stable for 2ne to 4ne generations (nei and li 1976). Additionally, entry of univoltine o. nubilalis moths into minnesota in the early 1940s (chiang, 1961), south dakota in 1948, and north dakota in 1950 (chiang 1972), followed by the northern migration of bivoltine subpopulations starting in the early 1950s (chiang 1965), suggest recent range expansion might have caused a deviation from migration - drift equilibrium (takahata 1983). The level of migration was inferred from f - statistics derived from male genotype and female haplotype data (table 2). Female haplotype analysis indicated substantial effects of inbreeding and genetic drift (st = 0.227; is = 0.274), whereas male onz1 genotypic analysis suggested little evidence of either (fst = 0.028; fis = 0.020; table 2). Inbreeding prevalence among female o. nubilalis might be used to infer lower migration rates as compared to males . When considered in conjunction with increased levels of genetic drift among females (st = 0.227) in relation to males (fis = 0.020), the reduced effective population size of the w chromosome and lack of homologous recombination during female gametogenesis might result in a strong influence of nucleotide fixation in female lineages . Thus differences between loci used in this study might reflect a background of chromosome position effects, and not truly be representative of population - based influences . Corroborating biological estimations of male and female flight distances are required to verify these results . Low onz1 and onw1 allele diversity was attributed to large repeat unit sizes, low repeat number, reduced effective population (ne) size of sex chromosomes, the result of a genetic bottleneck that occurred when o. nubilalis was introduced, or genetic drift caused by continued population expansion . Analysis of male onz1 genotypes and female onw1 and onz1 haplotypes showed no evidence for voltinism - based population structure . The similarity of west - central minnesota and eastern south dakota samples, compared to all other subpopulations, for both onw1 and onz1 provided evidence of geographic population structure . Recent o. nubilalis population expansion into northern regions of the central united states and recurrent seasonal bottlenecks likely explain the maintenance of hw disequilibrium, and form the basis of geographic population subdivision instead of inbreeding . A) 272 bp insert dna sequence from clone pgem - onca09 showing three tandem gaaaat repeats (underlined) and primer binding sites (underscored by arrows indicating direction) used to pcr amplify the locus ostrinia nubilalis w - chromosome marker number 1 (onw1; genbank accession: af442958). B) multiple dna sequence alignment of the pcr amplified o. nubilalis z - chromosome linked marker number 1 (onz1) alleles . Consensus sequence of imperfect alleles, caycarcgtcactaa (underlined nucleotides optional), and primer binding sites (arrows indicating direction) are underscored, and representing an invariable nucleotide identical to clone onca01, and * is a deletion . North american o. nubilalis male onz1 genotypic, and heterogametic female onw1 and onz1 haplotype frequencies in twelve subpopulations . Wa3, wa4, and wa5 represent onw1 allele with three, four, and five gaaaat repeats, respectively . Za1 = onz1 allele with one caycarcgtcactaa consensus repeat, and za3 = onz1 allele with three caycarcgtcactaa consensus repeats . Male amova and fst, and female amova and st values from test of o. nubilalis geographic variation assuming four south dakota and minnesota (north latitude) subpopulations are subdivided from the remainder of the north american o. nubilalis population.
Vertebral erosion determined by an abdominal aortic aneurysm is rare but already described [15]. Very few cases of vertebral lesion caused by false aneurysm secondary to prosthetic stent have been reported [59], and this is the first case of vertebral erosion due to a false aneurysm in a patient who underwent endovascular aneurysm surgery in absence of disco - vertebral infection . An 80-year - old man, with multiple co - morbidities was admitted to the author s hospital for severe low - back pain, lower limbs motor impairment and bilateral thigh pain . Nine months before admission to our department, he underwent endovascular aneurysm repair (evar) for an abdominal aortic aneurysm (fig . 1). A computed tomography (ct) scan performed 1 month after the endovascular abdominal aortic aneurysm repair did not show any signs of lumbar vertebral or disc erosion (fig . 2). Three months after the endovascular procedure, he developed a progressive lower back pain and bilateral thigh pain that did not respond to conservative treatments . At this time, in another hospital, mri of the lumbar spine showed severe bone loss in the anterior half of the third lumbar vertebral body and l3 vertebroplasty was performed without any significant relief of the symptoms (fig . 9 months after evar, clinical evaluation showed severe low - back pain, bilateral thigh pain, motor deficit of the lower limbs and the patient was not able to walk (frankel c). Laboratory findings were within normal limits: haemoglobin, 12.5 g / dl (normal 1216 g / dl), white blood cell count, 6.3 10/l (normal 4.310.8 10/l) with a normal differential, erythrocyte sedimentation rate 25 mm / h (normal 230 mm / h), c - reactive protein 2.4 mg / l (normal 0.003.00 mg a new mri of the lumbar spine, performed at admission, showed the false aneurysm and its relation to l2l3 bodies causing vertebral and disc erosion (fig . Fluoroscopy guided biopsy was performed, but it was negative as regards microbiological and histopathological examination for tumours or infections . A more accurate evaluation of mri of the lumbar spine uncovered an extensive abdominal aortic false aneurysm, corresponding with the prosthetic stents that had eroded the vertebral bodies of l2l3 5) had excluded infection, and for this reason we avoided draining the fluid at the l2l3 disc space, but we opted for a posterior decompression of the central canal stenosis, between l2 and l3, to improve the neurologic condition of the patient . In addition a long and extensive instrumentation of t12l5 was performed to avoid the risk of implant failure due to the presence of severe vertebral osteopenia (fig . Moreover, an autologous iliac bone graft was utilized to obtain better postero - lateral fusion . In the disc space another biopsy of the l2l3 disc performed during surgery confirmed the absence of tumours or infections . Postoperatively, laboratory findings were: haemoglobin 9.2 g / dl, white blood cell count 8.5 10/l, erythrocyte sedimentation rate 35 mm / h, c - reactive protein 3.00 mg / l . After surgery the patient obtained excellent relief of low - back and thigh pain with a satisfactory regain of walking (frankel d). Unfortunately, 1 month after surgery the patient died because of respiratory complications . We excluded sepsis as a possible cause of death because the patient in the postoperative period had no fever and laboratory findings were within normal limits.fig . No signs of rupture are evidentfig . 2computed tomography scan 1 month after endovascular abdominal aortic aneurysm repair . 3lumbar spine x - ray showing vertebroplasty procedure performed 3 months after aneurysm repairfig . 4 a mri 9 months after endovascular abdominal aortic aneurysm repair showed the false aneurysm and its relation to l2l3 vertebrae . B l2l3 central stenosisfig . 5pet - ct section 9 months after endovascular abdominal aortic aneurysm repair was not significative of infectionfig . 6postoperative x - ray of lumbar spine intraoperative angiography before (a) and after (b) the endoprosthesis deployment . No signs of rupture are evident computed tomography scan 1 month after endovascular abdominal aortic aneurysm repair . No signs of vertebral erosion are present lumbar spine x - ray showing vertebroplasty procedure performed 3 months after aneurysm repair a mri 9 months after endovascular abdominal aortic aneurysm repair showed the false aneurysm and its relation to l2l3 vertebrae . B l2l3 central stenosis pet - ct section 9 months after endovascular abdominal aortic aneurysm repair was not significative of infection postoperative x - ray of lumbar spine some reports in the literature report that an abdominal aortic aneurysm can cause erosion of the lumbar vertebral body, due to a progressive aneurysmatic sac expansion [15]. Few authors [6, 7] have reported lumbar vertebral erosion resulting from abdominal aortic contained rupture aneurysm in patients surgically treated for an abdominal aortic aneurysm by a conventional open surgical repair . Other authors have reported vertebral lesions resulting from endovascular abdominal aneurysm repair complicated by an infection [8, 9]. To the best of our knowledge, this is the first report of a case in which an endovascular aneurysm repair (evar) for an abdominal aortic aneurysm was complicated by an abdominal aortic false aneurysm which caused severe erosion of two lumbar vertebral bodies and disc through an inflammatory mechanism, without signs of infective pathogenesis . It is possible to assume that, despite the endovascular procedure, the pseudo - aneurysmatic sac can cause an inflammatory stimulus that is erosive for the adjacent vertebrae and discs . Pre - existing osteopenia, frequently observed in old patients, can contribute to the development of the vertebral erosion . If the patient is in good general condition, an anterior approach with the removal of the prosthesis and l2l3 decompression and fusion should be considered . We assume that when a lytic lesion of a lumbar vertebral body or disc is discovered in a patient treated for an abdominal aortic aneurysm by endovascular repair, an abdominal aortic false aneurysm can be the cause of the vertebral or disc erosion even in cases without infective complication.
Pathogenic plaque micro - flora, host immune responses, and environmental factors play a major etiologic role and cause both direct as well as host - mediated tissue injury . Elimination or modification of these factors is the basic aspect of treatment, which arrests or controls the disease process . In addition, the aim is to regenerate the tissues and restore function with methods, which are predictable and achieve long - term benefits . Complete mechanical debridement being the gold standard of periodontal treatment, still does not eliminate the micro - organisms in the soft tissue wall of the pocket, neither is complete resection of the diseased tissues possible . Additional soft tissue curettage procedures using ultrasonics and other chemicals as well as several adjunctive locally delivered agents such as antimicrobials, antiseptic agents, antiinflammatory agents, and host -modulating agents have been evaluated for enhancing the treatment outcome of chronic periodontitis with varying degrees of success . However, the predictability of these treatments is not certain as well as antimicrobial drugs may lead to the development of resistant microbial strains . Other methods for enhancing regeneration such as bio - mimetics, grafts and barrier membranes have shown to benefit the treatment outcome, but may increase the treatment costs, and additional invasive procedures may not be acceptable to the patient . Laser technology, specifically the diode laser is gaining popularity in general dental practice with potential benefits in a wide range of applications . Lasers have provided us with a potential alternative to simultaneously remove the diseased soft tissues, target the micro - organisms as well as stimulate wound healing . Several lasers such as the carbon dioxide (co2), ho: yag, nd: yag, diode, er: yag have been experimentally utilized for soft tissue periodontal procedures . Laser use produces less postoperative swelling, reduces inflammation and is also relatively painless . Some studies have even reported tissue regeneration on histologic evaluation following laser mediated periodontal therapy utilizing the laser assisted new attachment procedure . Furthermore, several authors have reported enhanced outcomes using lasers to de - epithelize the inner lining of the flap based on the principle of guided tissue regeneration . The diode laser is the most popular choice of laser technology for the general dental practitioner since it is economical, portable and convenient to use . In addition, since it has good tissue penetration, and is well absorbed in pigmented tissues, it can specifically target the pigmented bacteria and granulation tissue . In vitro evaluation has shown the diode laser to achieve a more complete elimination of the epithelial lining of the periodontal pocket . Soft tissue surgical procedures using lasers were found to have good hemostasis, sterilization and minimal postoperative pain when compared to conventional surgical procedures . However, the evidence available so far is conflicting . Systematic reviews have to date not shown the lasers in general to provide any additional benefit over conventional mechanical debridement modalities in nonsurgical therapy, and very few trials have been conducted on the use of diode lasers as an adjunct to periodontal surgery and the parameters for safe as well as effective laser use . Hence, the aim of this study was to evaluate the adjunctive benefit of a diode laser to conventional mechanical debridement in the surgical treatment of chronic periodontitis . A total of 30 patients in the age group of 25 - 60 years having moderate to severe chronic periodontitis, undergoing treatment at the department of periodontology were selected for the study . The treatment plan was explained to the patients and their written informed consent was obtained . Patients who had at least two quadrants with three teeth each having a pocket probing depth (pd) of 5 mm post -phase i therapy were included . Patients having systemic diseases e.g. Diabetes, heart disease, immuno - compromised patients and patients on medications, which could affect the periodontium were excluded . In addition, smokers (current or smoking within the last 5 years), pregnant women, patients having recent history of antibiotic use (within the previous 3 months) and patients allergic to medications to be prescribed were excluded from the study . A split - mouth study design was used by selecting two quadrants in each of the 30 patients . The two assessment groups were -the control was open flap debridement (ofd) and test group prior to the surgery, the selected quadrants were randomly allocated (by the toss of a coin) into control and test group where the control sites were treated with ofd and the test sites with ofd + diode laser [figure 1]. Flowchart of study design all the patients were subjected to clinical periodontal examination by a single examiner . The clinical probing measurements that are pd, clinical attachment level (cal) and gingival recession (gr) were measured using a university of north carolina-15 periodontal probe . 3 teeth / quadrant were selected and the deepest site was recorded of each tooth . Custom made acrylic stents were prepared on study models and an indelible marker was used to record the location as well as the direction of probing to standardize the probe angulation in two dimensions [figure 2]. The recordings were repeated at 3 months and 6 months . The reference point for measuring recording of clinical parameters using a customized occlusal stent plaque index (pi) and gingival index (gi) were calculated at baseline, followed by posttreatment at 3 months and 6 months recall visits . The average of the mesio - buccal, facial, disto - buccal, and lingual values was considered as the index value per tooth . In addition, tooth mobility (tm) was assessed at baseline, 3 months and 6 months using the miller's index . Radiographs were taken at baseline and 6 months postoperatively and assessed for changes in the bone to rule out any detrimental effect of the laser used in close proximity to the bone . Procedural pain experience of the patient was recorded objectively using a visual analog scale (vas), a 10 cm scale, which had markings from 0 to 10 depicting the pain intensity from minimal to maximal . In addition, the development of other complications such as necrosis, swelling, bleeding, delayed healing, infection, and scar formation was checked for and noted 1-week postoperatively . Two weeks later the test side surgery was performed similarly with diode laser irradiation of the inner lining of the flap . Meticulous defect debridement and root planning were carried out using curettes . In the test group, a 940 nm diode laser unit (having a maximum power output of 7 w) was used with a 7 mm long, 300 micron diameter, and disposable fiber - optic tip for energy delivery to the site [figure 3]. 940 nm portable diode laser unit with fibre - optic delivery system the laser at power of 1.5 w in contact mode was used by placing the fiber - optic tip at a 45 angle to the inner aspect of the flap, avoiding directing it toward the bone and teeth [figure 4]. Horizontal overlapping strokes were used on the inner lining of both facial and palatal flaps for about 10 s in relation to each tooth . Following this, the site was irrigated with normal saline . High volume suction apparatus was additionally used to clear the field and the tip debris was removed with a moistened cotton swab . The prelasing and postlasing appearance of the tissue can be appreciated in figures 5 and 6, respectively showing the light brownish appearance of the tissue and a bloodless field postlasing . Intraoperative photograph - application of the diode laser on the inner lining of the flap prelasing appearance of raised muco - periosteal flap postlasing appearance of the flap no osseous re - contouring was done . The surgical area was covered with a noneugenol periodontal dressing - coe - pak (gc america inc . Routine postoperative instructions were given along with antibiotic and antiinflammatory medication -amoxicillin, 500 mg thrice daily and diclofenac sodium 100 mg twice daily for 4 days respectively . The sutures and the periodontal dressing were removed 1-week postoperatively, and the sites were evaluated for any signs of delayed healing, flap necrosis, infection, and scar formation . In addition, the patients were questioned regarding the experience of postoperative swelling and bleeding . Reinforcement of the patients oral hygiene regime was done at all the recall appointments and the patient was advised to use a soft bristled tooth - brush . The data were analyzed using the statistical package for social sciences (spss version 11.5 software, spss inc ., the intra - group variations were analyzed using wilcoxon's signed - rank test, while inter - group comparisons were drawn using the mann the values of the three sites were averaged to obtain the value for the quadrant . A split - mouth study design was used by selecting two quadrants in each of the 30 patients . The two assessment groups were -the control was open flap debridement (ofd) and test group ofd + diode laser . Prior to the surgery, the selected quadrants were randomly allocated (by the toss of a coin) into control and test group where the control sites were treated with ofd and the test sites with ofd + diode laser [figure 1]. The clinical probing measurements that are pd, clinical attachment level (cal) and gingival recession (gr) were measured using a university of north carolina-15 periodontal probe . 3 teeth / quadrant were selected and the deepest site was recorded of each tooth . Custom made acrylic stents were prepared on study models and an indelible marker was used to record the location as well as the direction of probing to standardize the probe angulation in two dimensions [figure 2]. The recordings were repeated at 3 months and 6 months . The reference point for measuring recording of clinical parameters using a customized occlusal stent plaque index (pi) and gingival index (gi) were calculated at baseline, followed by posttreatment at 3 months and 6 months recall visits . The average of the mesio - buccal, facial, disto - buccal, and lingual values was considered as the index value per tooth . In addition, tooth mobility (tm) was assessed at baseline, 3 months and 6 months using the miller's index . Radiographs were taken at baseline and 6 months postoperatively and assessed for changes in the bone to rule out any detrimental effect of the laser used in close proximity to the bone . Procedural pain experience of the patient was recorded objectively using a visual analog scale (vas), a 10 cm scale, which had markings from 0 to 10 depicting the pain intensity from minimal to maximal . In addition, the development of other complications such as necrosis, swelling, bleeding, delayed healing, infection, and scar formation was checked for and noted 1-week postoperatively . Two weeks later the test side surgery was performed similarly with diode laser irradiation of the inner lining of the flap . Meticulous defect debridement and root planning were carried out using curettes . In the test group, a 940 nm diode laser unit (having a maximum power output of 7 w) was used with a 7 mm long, 300 micron diameter, and disposable fiber - optic tip for energy delivery to the site [figure 3]. 940 nm portable diode laser unit with fibre - optic delivery system the laser at power of 1.5 w in contact mode was used by placing the fiber - optic tip at a 45 angle to the inner aspect of the flap, avoiding directing it toward the bone and teeth [figure 4]. Horizontal overlapping strokes were used on the inner lining of both facial and palatal flaps for about 10 s in relation to each tooth . Following this, high volume suction apparatus was additionally used to clear the field and the tip debris was removed with a moistened cotton swab . The prelasing and postlasing appearance of the tissue can be appreciated in figures 5 and 6, respectively showing the light brownish appearance of the tissue and a bloodless field postlasing . Intraoperative photograph - application of the diode laser on the inner lining of the flap prelasing appearance of raised muco - periosteal flap postlasing appearance of the flap no osseous re - contouring was done . The surgical area was covered with a noneugenol periodontal dressing - coe - pak (gc america inc . Statistical package for social sciences - spss inc ., alsip, illinois, usa). Routine postoperative instructions were given along with antibiotic and antiinflammatory medication -amoxicillin, 500 mg thrice daily and diclofenac sodium 100 mg twice daily for 4 days respectively . The sutures and the periodontal dressing were removed 1-week postoperatively, and the sites were evaluated for any signs of delayed healing, flap necrosis, infection, and scar formation . In addition, the patients were questioned regarding the experience of postoperative swelling and bleeding . Reinforcement of the patients oral hygiene regime was done at all the recall appointments and the patient was advised to use a soft bristled tooth - brush . The data were analyzed using the statistical package for social sciences (spss version 11.5 software, spss inc ., chicago, illinois, usa) using nonparametric tests . The intra - group variations were analyzed using wilcoxon's signed - rank test, while inter - group comparisons were drawn using the mann the values of the three sites were averaged to obtain the value for the quadrant . The 30 patients sample comprised of 20 females and 10 males in the age range of 26 - 47 years . 60 quadrants were treated totally-30 as test group and 30 as control in a split - mouth design . The average value of 3 teeth in each quadrant (i.e. Test or control group) for each patient was considered while assessing the clinical parameters . The mean values with standard deviation at baseline, 3 months and 6 months are presented in table 1 and the percentage change in the parameters in table 2 . Mean and sd of all the clinical parameters at baseline, 3 months and 6 months postoperatively inter - group comparison of percentage change in clinical parameters at the 3 months and 6 months follow - up evaluation the mean probing pocket depth at baseline in the ofd group was 6.08 0.91 mm which was reduced to 2.81 0.59 mm at 3 months and 2.56 0.49 mm at 6 months . In the ofd + laser group the baseline value was 6.02 0.81 mm, which reduced to 2.53 0.53 mm at 3 months and 2.35 0.50 mm at 6 months . The ofd and ofd + laser groups showed a percentage reduction in pd of 53.2 and 57.4 at 3 months and 58.2 and 60.2 at 6 months, which was statistically highly significant (p <0.05). However, there was an insignificant difference statistically and clinically between the two groups with p> 0.05 at both 3 and 6 months intervals [graph 1a]. Inter group comparison of baseline and follow - up of clinical parameters (a) probing depth; (b) clinical attachment level; (c) gingival recession the cal showed a significant reduction from baseline in both groups indicating a gain in the attachment level . The mean cal at baseline in the ofd group was 6.64 0.84 mm and ofd + laser was 6.83 1.09 mm, which was reduced to 4.87 0.63 mm and 5.23 0.79 mm at 3 months, showing a percentage reduction of 26.1 and 22.9, which was statistically highly significant (p <0.05). Similarly, at 6 months, cal reduced to 4.75 0.47 mm in ofd group and 5.18 0.78 mm in the ofd + laser group showing a percentage change in cal of 28.8 and 23.6 which was also statistically highly significant (p <0.05). The difference in cal change in the two groups at 3 months and 6 months however, was statistically insignificant with p> 0.05 [graph 1b]. Gingival recession [graph 1c] increased significantly in each group from baseline over the 6-month period . The intra - group comparisons - showed that both groups the control (ofd) group and the test (ofd + laser) group had significant reduction in pi, gi and tm index at all intervals from the baseline as shown in table 1 and graph 2a c . (a) plaque index; (b) gingival index; (c) tooth mobility miller's index however, the inter - group comparison between the test (ofd + laser) and the control (ofd) group showed that the differences for all these parameters were found to be statistically insignificant at all the recorded recall intervals as shown in table 2 except for the gi scores . It was found that the gi scores at all points of time were significantly less in the laser treated group (p = 0.01) than in the control (ofd) group implying a greater reduction in the gingival inflammation sustained in the laser treated test group . The procedural pain evaluated on the vas revealed mean scores of 1.90 1.18 and 2.07 1.17 for the control group and test group, respectively . Complications were seen in a few cases (2/group) postsurgical treatment . These included mild postoperative intraoral swelling which subsided within a week and in others slightly delayed healing where the inflammation persisted over 1-week taking an additional week to subside . Radiographic changes were minimal in both control and laser treated sites with a slight gain 6 months postoperatively . The mean probing pocket depth at baseline in the ofd group was 6.08 0.91 mm which was reduced to 2.81 0.59 mm at 3 months and 2.56 0.49 mm at 6 months . In the ofd + laser group the baseline value was 6.02 0.81 mm, which reduced to 2.53 0.53 mm at 3 months and 2.35 0.50 mm at 6 months . The ofd and ofd + laser groups showed a percentage reduction in pd of 53.2 and 57.4 at 3 months and 58.2 and 60.2 at 6 months, which was statistically highly significant (p <0.05). However, there was an insignificant difference statistically and clinically between the two groups with p> 0.05 at both 3 and 6 months intervals [graph 1a]. Inter group comparison of baseline and follow - up of clinical parameters (a) probing depth; (b) clinical attachment level; (c) gingival recession the cal showed a significant reduction from baseline in both groups indicating a gain in the attachment level . The mean cal at baseline in the ofd group was 6.64 0.84 mm and ofd + laser was 6.83 1.09 mm, which was reduced to 4.87 0.63 mm and 5.23 0.79 mm at 3 months, showing a percentage reduction of 26.1 and 22.9, which was statistically highly significant (p <0.05). Similarly, at 6 months, cal reduced to 4.75 0.47 mm in ofd group and 5.18 0.78 mm in the ofd + laser group showing a percentage change in cal of 28.8 and 23.6 which was also statistically highly significant (p <0.05). The difference in cal change in the two groups at 3 months and 6 months however, was statistically insignificant with p> 0.05 [graph 1b]. Gingival recession [graph 1c] increased significantly in each group from baseline over the 6-month period . The intra - group comparisons - showed that both groups the control (ofd) group and the test (ofd + laser) group had significant reduction in pi, gi and tm index at all intervals from the baseline as shown in table 1 and graph 2a c . (a) plaque index; (b) gingival index; (c) tooth mobility miller's index however, the inter - group comparison between the test (ofd + laser) and the control (ofd) group showed that the differences for all these parameters were found to be statistically insignificant at all the recorded recall intervals as shown in table 2 except for the gi scores . It was found that the gi scores at all points of time were significantly less in the laser treated group (p = 0.01) than in the control (ofd) group implying a greater reduction in the gingival inflammation sustained in the laser treated test group . The procedural pain evaluated on the vas revealed mean scores of 1.90 1.18 and 2.07 1.17 for the control group and test group, respectively . Complications were seen in a few cases (2/group) postsurgical treatment . These included mild postoperative intraoral swelling which subsided within a week and in others slightly delayed healing where the inflammation persisted over 1-week taking an additional week to subside . Radiographic changes were minimal in both control and laser treated sites with a slight gain 6 months postoperatively . The diode laser is an effective soft tissue laser for incision, hemostasis and coagulation . At a wavelength in the range of 800 - 980 nm it is well - absorbed by pigmented tissues and so can selectively target the darkened, inflamed tissues and pigmented bacteria . In fact some trials have confirmed the bactericidal effect of the diode laser . However being deeply penetrating it is important to adjust the parameters within a safe and efficacious range so as to adequately remove the epithelial lining . Effect by retarding the epithelial migration and may achieve a more complete epithelial removal than conventional mechanical methods as shown in vitro . Low - level lasers have shown to reduce inflammatory mediators, facilitate collagen synthesis, angiogenesis and stimulate the release of growth factors . Low - level diode laser therapy in vitro has shown to increase the proliferation of human fibroblasts . Furthermore, reduction in the production of inflammatory mediators has been observed . There are however conflicting reports on the use of lasers as an adjunct to the nonsurgical treatment of chronic periodontitis, with several systematic reviews showing no additional advantage of laser use in general . This had also been stated by the american academy of periodontology . However, since published reports on the use of diode lasers in periodontal flap surgery are relatively few until date it was decided to evaluate the same . The results of the present study indicate that the diode laser can be safely used as an adjunct to conventional therapy . However, no significant additional benefits except for its ability to cause a significant decrease in the gingival inflammation were observed . Clinical attachment level being the gold standard for evaluating the success of periodontal therapy and pd of the pocket being an important factor affecting the long - term stability of the results, these were the primary outcomes measured . No significant differences were found in these parameters using a diode laser as an adjunct . Pi was recorded to monitor the oral hygiene status of the patients and showed a significant decrease posttreatment . However, a slightly increasing trend with time implies the need to re - motivate the patient at each recall interval, which was done . Our study showed a significant reduction in the gingival inflammation in the laser treated group compared to the control group when used as an adjunct to flap surgery . Although, the precise mechanism was not established it could be possibly be attributed to the bacterial reduction achieved by the laser as well as decreased inflammation and stimulation of tissue healing . As it is important for the patient to be comfortable with the treatment, it was decided to evaluate procedural pain on a vas . Interestingly, in the cases where flap surgery was performed along with laser use there was a slightly though insignificantly higher pain experience . This could be attributed to the pressure during contact of the sharp fiber - optic tip of the laser on the inner flap lining . Radiographic evaluation showed that the diode laser used in proximity to the bone did not have detrimental effects . Moreover, so the diode laser can be safely used in the power range of 1 - 1.5 w without any complications . Furthermore, it was observed that adequate hemostasis was achieved using the diode laser on the inner surface of the flap leaving a light brownish tissue, which proceeded to heal normally . In many cases the tissue adaptation postlaser use appeared to be more favorable than in case of the control sites . In addition, certain drawbacks such as the inability to remove hard deposits, risk of thermal damage to the hard tissues, the infrastructure required and high investment cost for the laser have to be weighed along with the benefits . Along with this, the time taken for the laser procedure is more and the laser equipment has to be used cautiously to prevent damage to vision and accidents . From this study, it can be concluded that the use of the diode laser for this application did not significantly benefit the treatment outcome on the whole . Short term benefit of reduction in gingival inflammation could potentially benefit the outcomes in diseased soft tissues with a larger inflammatory component . Also, certain limitations regarding the study such as lack of a blinded design as well as the lack of standardization of the amount of laser energy delivered could have influenced the observations . The results of the present study indicate that diode laser used as an adjunct to in ofd did not significantly enhance the treatment outcome . However, since there was a significant clinical improvement in case of gingival inflammation, it can be safely and effectively used to achieve the same and can aid in tissue healing . Further research is required to provide evidence for the benefit of diode laser use in flap surgery.
There are multiple case reports in the otolaryngology literature of projectiles that have become lodged in the paranasal sinuses . Retained packing gauze from endoscopic sinus surgery has also been reported in the paranasal sinuses . In the endodontic literature, many reports and reviews have described various materials that can cause disease of the maxillary sinuses . In this case report, we present an interesting case of a patient who presented with recalcitrant maxillary sinusitis that was ultimately found to be related to retained endodontic material in her maxillary sinus . A 26-year - old caucasian woman presented with a chief complaint of left - sided maxillary pain with intermittent, discoloured nasal drainage . Seven years prior to current presentation, the patient had reported a history of headaches, nasal congestion and bilateral discolored drainage refractory to prednisone, antibiotics, and endoscopic sinus surgery at an outside facility . Two years prior to current presentation, her left maxillary pain recurred, and a computed tomography (ct) scan revealed a left wisdom tooth projecting into her maxillary sinus . Following wisdom teeth extraction, she had marked improvement in pain and nasal drainage; however 8 weeks later, left maxillary pain returned and was associated with left - sided yellow nasal discharge . Her oral surgeon discovered an infection in the molar adjacent to the extracted wisdom tooth and performed a root canal . Two weeks following the root canal, she presented with continued left maxillary tooth pain and left - sided discolored nasal discharge . Extraction of the molar failed to resolve her pain, and she was subsequently referred to the facial pain / headache clinic by her dentist where she was prescribed gabapentin 300 mg tid, which was ineffective . Her dentist also prescribed her multiple courses of clindamycin 150 mg tid and guaifenesin 600 gm qid, which would improve her pain and discoloured drainage . However, her symptoms would return after completing the antibiotics . On examination with rigid endoscopy, she had widely patent maxillary, ethmoid, and frontal sinus ostia with no purulence or polyposis . On flexible endoscopic examination of the floor of her left maxillary sinus, white to slightly yellow mucus maxillary sinus cultures revealed few polymorphonuclear leukocytes, few mononuclear cells, and no microorganisms . Her most recent ct scan from two years prior to presentation at our facility was remarkable for minimal mucosal thickening of the floor of the left maxillary sinus was otherwise normal (figure 1). An occult dental infection was considered high in the differential diagnosis; however, because no actual dental infection could be demonstrated, a medial maxillectomy was considered in order to facilitate topical washing of the left maxillary sinus . Follow - up appointments in the facial pain / headache clinic found some features of migraine, but it is unclear whether headaches represent primary or secondary headaches with migraine features . Repeat evaluations by her dentist found no evidence of a dental infection . Because of past improvement on an 8-week course of clindamycin 300 mg tid, she was prescribed a 12-week course of clindamycin 300 mg tdi before surgery was considered, and she was referred to an infectious disease specialist . Only mild mucosal thickening of the floor of the left maxillary sinus and post - surgical changes related to endoscopic sinus surgery were noted . A bone scan was positive in the region of the left maxilla; however, a repeat indium scan was negative . Her ct scan was repeated and she was referred to an oral surgeon for evaluation (figure 2 figure 4). The ct scan was evaluated by an oral and maxillofacial surgeon who noted that there were two small remnants of the prior left maxillary root canal that had been performed two and half years ago (figure 2 figure 4). A combined endoscopic and caldwell - luc approach under computer - assisted navigation to drill out retained gutta - percha in the maxillary sinus resolved the patient s pain and drainage immediately, without recurrence at her three month follow - up . Gutta - percha, a product of tropical rubber plants, has been used since the mid-1800s as an endodontic filling material following root canal procedures . The chemical structure of gutta - percha is a trans - isomer of poly - isoprene, or natural rubber; however it is more crystalline than natural rubber and often is formulated with medications such as zinc oxide, iodoform, chlorhexidine and calcium hydroxide, which contribute to both the antibacterial and antifungal activity 4 7 . Animal studies have shown that gutta - percha becomes encapsulated by fibrous connective tissue with little inflammatory reaction . Maxillary sinus complications from gutta - percha from root canals are rare . According to a previous case report, gutta - percha from a maxillary tooth root canal can migrate to and obstruct the maxillary ostium . Gutta - percha has also been reported to migrate into the ethmoid sinus . In the current patient, retained gutta - percha in the maxillary sinus resulted in chronic inflammation and a persistent sinusitis - type picture with nasal congestion, pain and drainage . Her symptoms preceding the sinus surgery may or may not have been related to dental infection, but she clearly improved following wisdom teeth extraction, and she relapsed due to the infection of the adjacent molar . The retained gutta - percha prevented the resolution of infection and symptoms, even following extraction of the affected tooth . Partial improvement with antibiotics directed to usual dental pathogens provided some evidence that the etiology of her symptoms was a dental infection . In patients with persistent sinusitis and a history of endodontic procedures, an evaluation for dental materials retained in or near the sinuses may be warranted to rule out an additional source of infection . Removal of these retained dental materials may require an external approach with drilling, which can be facilitated by endoscopic visualization through the caldwell - luc procedure . A review of the otolaryngology literature did not provide any additional case reports on retained gutta - percha . In fact, only two case reports were found in the oral and maxillofacial surgery literature that described retained gutta - percha in the paranasal sinuses . In the oral surgery and endodontic literature, there are multiple reports of aspergillosis occurring in the maxillary sinus as a result of overextension of root canals of maxillary teeth, especially using materials containing zinc oxide or formaldehyde 3, 11 . One case series of aspergillosis of the maxillary sinus was found in the otolaryngology literature . In this series, 85 cases of aspergillosis of the maxillary sinus in non - immunosuppressed patients were reviewed . Of these, 94% presented evidence of a radio - opaque foreign body in the maxillary sinus, with 85% of the cases related to endodontic dental paste . Our case report highlights the importance of investigating alternative sources of infection in cases of recalcitrant sinusitis . Dental sources of infection as well as retained or overextended endodontic materials should be investigated in patients with unexplained, chronic sinusitis . Written informed consent for publication of clinical details and clinical images was obtained from the patient.
Kyphoscoliosis occurs due to disruption of balance between structural and dynamic components or neuromuscular elements of the spine . The respiratory system is compromised, and its severity depends on the severity of the kyphoscoliosis and concomitant respiratory disease . A 27-year - old, 45 kg female, g1p0l0a0, was admitted at 29 weeks of gestation with complaints of moderate grade fever associated with chills and rigors since 3 weeks, followed by development of cough and difficulty in breathing . Her past history revealed gradually progressing thoracic kyphoscoliosis following poliomyelitis diagnosed at the age of 4 months . On admission, the patient was diagnosed to have signs and symptoms of ards with bilateral lung infiltrates in chest x - ray . Hemogram, renal and liver functions revealed no abnormality except for a total protein of 4.5 gm%, positive peripheral smear for malaria parasite (plasmodium falciparum) and total leukocyte count of 14,700/mm . Pulmonary function test revealed a restrictive lung disease (forced vital capacity [fvc], forced expiratory volume [fev1] and fev1/fvc were 67%, 58% and 110%, respectively). Other causes of fever such as typhoid, hepatitis, urinary tract infection and cholangitis were ruled out . She was admitted to intensive care unit (icu) and ards was managed with intravenous antibiotics cefotaxim 1 g 8 hourly, levofloxacin 500 mg once in 24 h, amikacin 500 mg 12 hourly and metronidazole 500 mg 8 hourly . She was administered oxygen by face mask (5 - 6 l / min), steam inhalation, nebulization and chest physiotherapy . There was gradual worsening of her respiratory condition, subsequently leading to desaturation (spo2 77%) with a respiratory rate (rr) of 50 - 60 breaths / min, and crepts on auscultation of her right chest . Her arterial blood gas (abg) analysis revealed po2 - 64 mmhg, pco2 - 26 mmhg, spo2 of 88% for which noninvasive continuous positive airway pressure (cpap) of 5 cmh2o was applied with a fio2 of 40% . This led to an improvement of her respiratory condition; spo2 rose to 99% and the rr settled to 20 - 22 breaths / min . Repeat abg revealed po2 - 96 mmhg, pco2 - 34 mmhg and spo2 99% . She continued to remain on noninvasive cpap for first 4 days, maintaining hemodynamics and was managed with intravenous antibiotic, steroids and nebulization with bronchodilators . In the icu she developed hyponatremia (na 128 meq / l) and hypoprotenemia (total proteins 4.5 g / dl and albumin 1.8 g / dl). Hypoproteinemia was managed with albumin (20% 100 ml daily for 5 days) and hyponatremia with 0.9% normal saline administration . During her icu she was administered intramuscular betamethasone (12 mg, 2 doses 24 h apart) for fetal lung maturity . Tocolytics were also prescribed for occasional uterine contractions (nifedipine 5 - 10 mg when required). On day 5 of her icu stay, she had respiratory distress (rr-26 - 28 breaths / min) and started having labor pains . On examination, this was followed by deterioration of her respiratory condition and desaturation (spo2 of 73%) even on the support of noninvasive cpap of 5 cmh2o . Her abg revealed po2 - 52 mmhg, pco2 - 45 mmhg and spo2 of 82% . Fetal doppler revealed fetal heart rate of 110 beats / min . In view of such critical condition, rapid sequence induction using thiopentone (200 mg) and succinylcholine (75 mg) was performed, followed by tracheal intubation . Patient was then shifted to the operating room in left lateral position with an endotracheal tube in situ on bain's circuit and with manual assisted ventilation . During the surgery, patient remained hemodynamically stable with systolic blood pressure ranging 110 - 140 mmhg, diastolic blood pressure ranging 60 - 80 mmhg and heart rate ranging from 60 to 80 beats / min . She sustained blood loss of 600 ml that was replaced with balanced salt solution and 1 unit of packed red blood cells . A low birth weight (1181 g) female baby was delivered with an apgar score of 7 and 8 at 1 and 5 min respectively . Baby was shifted to neonatal icu for further management . In view of her poor lung condition, the lung condition further deteriorated, and abg revealed - ph 7.321, pao2 59 mmhg, paco2 46 mmhg, hco3 20 her bronchial alveolar lavage cultures grew acinetobacter and urine culture revealed escherichia coli that were managed with appropriate intravenous antibiotics . During this period in icu, she required sedation with midazolam and morphine as well as intermittent neuromuscular blockade and inotropic support (noradrenalin 5 - 8 mg / kg). Over next 4 days, her lung condition and abg improved with the gradual increase in pao2 of 65 mmhg to 202 mmhg and pao2/fio2 ratio of 81.25 - 505 . This led to gradual weaning of the patient off the ventilator and finally tracheal extubation 10 days later . Anesthetic management of the parturient with thoracic kyphoscoliosis, malaria and ards for emergency lscs is challenging . Pregnant patients with pulmonary compromise (kyphoscoliosis and ards in this case) may not tolerate increased metabolic demand generated by the fetus, placenta, and gravid uterus due to limited respiratory reserves . Labor pains result in marked hyperventilation causing a fall in paco2 and respiratory alkalosis causing cerebral and uteroplacental vasoconstriction . This reduces the release of oxygen from hemoglobin that not only compromises maternal tissue oxygenation, but also has a deleterious effect on fetal oxygen transfer . The presence of kyphoscolisos in parturient may further leads to ventilation / perfusion mismatch and marked dyspnea . It can also interfere with provision of labor analgesia or regional anesthesia for cesarean section . The risk of development of ards in a parturient is higher than in the nonpregnant population . Golden hour we conclude that parturient with associated respiratory comorbidities needs timely management for a better outcome not only of the mother, but also of the fetus.
Reticulohistiocytosis is a spectrum of disorders ranging from solitary and diffuse cutaneous forms without systemic involvement to multicentric reticulohistiocytosis (mr). The primary skin lesion, reticulohistiocytoma, usually presents as a firm, skin colored, yellowish or reddish papule or nodule . In multiple cutaneous reticulohistiocytoma (mcr), there is onset of multiple lesions without underlying systemic illness, whereas in mr there are extensive skin lesions in association with a severe, often destructive arthropathy, and systemic features . Mcr is more common in young adult males and mr in middle aged women associated with systemic symptoms . Both the variants are characterized in histo - pathology by the presence of large mononucleated or multinucleated histiocytes with an abundance of eosinophilic, homogenous to finely granular cytoplasm having a ground glass appearance . We report here a case of mcr in a middle aged female for its rarity and the importance of ruling out mr in such a patient . A 35-year - old female presented with gradual onset of multiple skin colored, asymptomatic pea sized swellings, which started with face and gradually involved back, arms, and legs . There was no history of preceding local trauma, discharge, pain in these lesions . There were no constitutional symptoms like fever, joint pain, and complaints referable to other systems . The patient's main concern was regarding the cosmetic appearance as lesions were present over the face . Her family history was negative and she had no history of any other chronic illness . Mucocutaneous examination revealed multiple (20 in number), discrete, skin colored nodules, 12 cm in size scattered over her face, back, abdomen, thighs, and lower legs [figure 1]. Her complete blood count along with general blood picture, liver, and renal function tests, fasting lipid profile and urinalysis were normal . Rheumatoid factor and antinuclear antibodies were found to be within normal limits . On the basis of history and clinical examination, differential diagnosis of xanthoma, reticulohistiocytoma, sarcoidosis, and histoid leprosy (a) multiple discrete skin colored nodular lesions over back (arrow) (b) discrete skin colored nodular lesions over face, (c) discrete skin colored nodular lesions over back, (d) discrete skin colored nodular lesions over posterior aspect of left thigh and leg excisional biopsy of a nodule over the trunk was done and histopathological examination revealed circumscribed large focus of diffuse dense infiltrate of large histiocytes and histiocytic giant cells with scattering of lymphocytes and a few neutrophils [figure 2]. Several histiocytic giant cells resembling the touton giant cells were also seen [figure 3]. Overlying epidermis showed flattening and thinning and was covered by parakeratosis containing collection of neutrophils . Based on clinical and histopathological findings a diagnosis of multiple cutaneous reticulohistocytoma the nature of the disease was explained to the patient and was referred to the plastic surgeon for the cosmetic correction of the facial lesion . Skin biopsy showing lymphocytic infiltrate with histiocytes and giant cells [h and e, 100] skin biopsy showing multinucleated giant cells (arrow) with an amorphous eosinophilic cytoplasm [h and e, 400] non - langerhans cells histiocytosis represents a broad group of different disorders characterized commonly by proliferation of histiocytes other than langerhans cells . Mcr represents a unique pattern in the spectrum of the reticulohistiocytoses, characterized by histiocytic proliferations of the skin and soft tissues . It may represent an abnormality histiocytic reaction to different stimuli . Local trauma may play a role in the pathogenesis of solitary cutaneous histiocytoma, whereas in diffuse forms, the association with internal malignancies and autoimmune diseases suggest an immunologic basis . Reticulohistiocytomas are usually solitary lesions of less than 1 cm in diameter . However, multiple lesions and large reticulocytomas have been rarely reported . Histopathology of reticulohistiocytoma often shows mid - dermal infiltration of mononuclear histiocytes and multinucleated histiocytes with a ground - glass appearance and a variable number of vacuolated, spindle shaped, and xanthomatized mononuclear histiocytes . Vimentin is universally positive but all other markers like s-100, desmin, and smooth muscle - specific actin remain negative . Pulsed dye laser, oral corticosteroids, and methotrexate has been tried in the treatment of extensive lesions . The only concern in our patient was regarding the physical appearance of the lesions as they were also present over the face . However, patient of mcr needs to be followed up regularly for long time to look for the development of mr which has various life threatening systemic complications . This case thus lays emphasis on considering mcr in the differential diagnosis of asymptomatic skin colored papulo - nodular lesions and adequate systemic evaluation and follow up of such patients.
Dental trauma remains one of the most important oral health problems in childhood causing pain and distress . Children usually encounter many minor accidents during their day to day activities like cycling, skating, running, etc . All these activities result in complete avulsion of teeth.1 the most commonly avulsed teeth are the central incisors . The enigmatic mechanism for tooth avulsion is thought to be the incomplete formation of the roots and the lack of resiliency of the periodontal ligament seen at those ages.2 epidemiological studies indicate that dental trauma is a significant problem in young people and that in the near future the incidence of trauma will exceed that of dental caries and periodontal disease in the young population.3 andreasen and andreasen documented that oral injuries are the fourth most common injuries among the 7 - 30 years age group.4,5 traumatic injuries can thus, have a significant effect on a child s quality of life . 6 walker and brenchley (2000) observed that 16% of dental injuries led to tooth loss.7 most of the children with avulsed tooth present late for treatment due to lack of awareness and knowledge among parents resulting in unfavorable long - term prognosis.1 the prognosis of the avulsed tooth depends solely on the appropriate treatment that in turn relies on the knowledge of the parents regarding the management of the avulsed tooth.2 hence, the aim of the present study was to assess the knowledge and attitude of parents / caretakers in the emergency management of avulsed tooth in the maharashtrian population . An aphoristic questionnaire was formulated to assess the knowledge and attitude of parents and caretakers of 5 - 14 years old children regarding the management of avulsed tooth . The questionnaire consisted of 6 closed - ended questions for the assessment of parents knowledge and attitude toward their child s avulsed tooth and its emergency management . The questionnaire was treatised in both english and vernacular language and was checked by 3 professors of the same department so as to ratify the study . Statistical analysis was performed using statistical package for social sciences version 17.0 (ibm company). Chi - square analysis was used, and the level of significance was found to be p <0.04 . The answer for each question was documented as follows: 90% of parents said their child experienced dental trauma10% did not experience dental trauma . 90% of parents said their child experienced dental trauma 10% did not experience dental trauma . 65% said they put it back into child s mouth25% removed it from the child s mouth10% discarded the tooth . 65% said they put it back into child s mouth 25% removed it from the child s mouth 10% discarded the tooth . 10% in water5% in cloth5% in milknearly 80% did not use any medication to save the tooth . Based on these, the results were graphically represented as shown in graphs 1a - f . Questionnaire formulated and distributed to assess the knowledge and attitude of parents or caretakers of 5 - 14 years old children regarding the avulsed tooth . The questionnaire used in the present survey was aphoristic, direct, and closed - ended questions . Similar studies reported in the literature surveyed the knowledge of parents and teachers concerning the management of avulsed teeth.8 - 11 the prognosis of the avulsed tooth depends on the immediate management of the tooth after its displaced out of the socket . But reports indicate that parents / caretakers present at the site lack knowledge about the immediate management of this tooth . One of the most important requirements is reimplantation of the tooth into its socket after it is cleaned with saline solution so as to preserve the cells of the periodontal ligament viable for healing and revascularization . In the present study, 90% of the parents / caretakers said that their child experienced dental trauma at the age of 5 - 14 years . Furthermore, 65% of them usually placed the avulsed tooth back into the child s mouth, similar to previous studies conducted . 95% of the parent s visited the dentist which was similar to study conducted by oliveira et al . (93%).2 in the present study, only 2.5% of parents would leave the avulsed tooth inside the mouth and it was much lesser than reimplantation of an avulsed tooth, similar to a study reported by al - jundi,11 whereas, the reports by oliveira et al.,2 (39%), raphael and gregory12 (66.6%), and hegde et al.,13 (66.5%) showed that some of the parents would reimplant the avulsed tooth, which clearly indicates the insufficiency in the knowledge about the immediate management of avulsed tooth . Hence the present study found that about 20% of the mothers would discard an avulsed tooth, which was similar to the study conducted by oliveira et al.2 (10%). This may be due to the fact that they consider avulsed tooth as an infected material which needs to be discarded . These results were not similar to the studies conducted by hegde et al.13 (68%). It is a well - documented fact that the best storage media for avulsed tooth is the patient s own socket . But as the tooth is usually contaminated by dirt, it is necessary to clean the tooth and then place it back into its own socket . Before reimplantation of the avulsed tooth, milk and saliva are easy to obtain storage media and normal saline is easily available in drug stores . In the present study, only 2% of the parents / caretakers preserved the tooth in an appropriate media like saline or saliva . This indicated poor knowledge among the population with regards to preserve the tooth which led to the failure of its reimplantation . To summarize, lack of knowledge among the parents / caretakers concerning the avulsed tooth demands the need of more effective communication between the dental professionals and parents . Furthermore, preventive programs and educational campaigns should be conducted to ensure good knowledge about the preservation and management of the avulsed teeth . This study shows the need to provide the general population with information regarding the emergency management of avulsed tooth . This study shows the need of effective communication between dentists and caretakers for better management of avulsed teeth.

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