This subsequent, 26-week, open-label, uncontrolled assessment period evaluated the safety and efficacy of (i) continued exenatide once-weekly treatment and (ii) switching from sitagliptin or pioglitazone to exenatide once-weekly.In the initial 26-week, double-blind, double-dummy assessment period of the DURATION-2 trial in patients with Type 2 diabetes on metformin, the once-weekly glucagon-like peptide 1 (GLP-1) receptor agonist exenatide once-weekly resulted in greater HbA1c 8.5 ± 1.1% (70 mmol/mol), fasting plasma glucose 9.0 ± 2.5 mmol/l, weight 88 ± 20 kg) who continued into the open-label period, 319 patients (88%) completed 52 weeks.Randomised oral medications were discontinued and all patients received exenatide once-weekly. Of the 364 patients [original baseline HbAse) in HbA1c (−1.6 ± 0.1%), fasting plasma glucose (−1.8 ± 0.3 mmol/l) and weight (−1.8 ± 0.5 kg). Evaluable patients who switched from sitagliptin to exenatide once-weekly demonstrated significant incremental improvements in HbA1c (−0.3 ± 0.1%), fasting plasma glucose (−0.7 ± 0.2 mmol/l) and weight (−1.1 ± 0.3 kg). Patients who switched from pioglitazone to exenatide once-weekly maintained HbA1c and fasting plasma glucose improvements , with significant weight reduction (−3.0 ± 0.3 kg). Exenatide once-weekly was generally well tolerated and adverse events were predominantly mild or moderate in intensity. Nausea was the most frequent adverse event in this assessment period . No major hypoglycaemia was observed.Evaluable patients who received only exenatide once-weekly demonstrated significant 52-week improvements (least square mean ± Patients who switched to once-weekly exenatide from daily sitagliptin or pioglitazone had improved or sustained glycaemic control, with weight loss. In an attempt to address treatment concerns of many traditional medications for Type 2 diabetes, the most recent American Diabetes Association/European Association for the Study of Diabetes (ADA/EASD)  and AmerIn the subsequent, open-label phase of the DURATION-2 trial, all patients received exenatide once-weekly. Rather than adding another medication, as is common in clinical practice, patients who originally received daily sitagliptin or pioglitazone discontinued their respective oral treatment to assess incremental changes in glycaemic control and body weight after switching to once-weekly administration of exenatide. Patients originally randomised to exenatide once-weekly continued treatment and discontinued oral placebo. In this open-label extension period, all patients continued metformin background therapy, and patients and investigators remained blinded to original treatment. This trial was designed to evaluate the glycaemic and non-glycaemic  changes in patients: (i) treated with exenatide once-weekly for 52 weeks; (ii) who switched from daily inhibition of DPP-4  and BMI 25–45 kg/m2 . In the second, open-label phase of DURATION-2, randomised oral medications were discontinued and all patients received 2 mg exenatide once-weekly treatment; patients and investigators were not notified of original treatment. A common clinical protocol was approved for each site by the appropriate Ethical Review Board. Patients provided written informed consent prior to participation. This study was conducted in accordance with the Declaration of Helsinki (1946), including the current Seoul revision (2008), and consistent with Good Clinical Practice and applicable laws and regulations.Eligible patients were male and non-pregnant female patients, at least 18 years of age, with Type 2 diabetes mellitus, but otherwise healthy and treated with a stable regimen of metformin for a minimum of 2 months prior to screening. Additional inclusion criteria included HbA45 kg/m2 . Patient1c, patients achieving HbA1c targets of < 7.0% (< 53 mmol/mol) and ≤ 6.5% (≤ 48 mmol/mol), change in fasting plasma glucose (target ≤ 7 mmol/l), body weight, fasting lipids, fasting insulin, systolic and diastolic blood pressure, cardiovascular risk markers , safety and tolerability. Plasma and urine analytes and HbA1c were quantitated by Quintiles Laboratories  using standard methods. All other measures were performed as previously described  at week 52: −1.6% (−1.9 to −1.3%); 1c improvement [week 52: −1.6% (95% CI −1.8 to −1.3%)], while patients who switched from sitagliptin to exenatide once-weekly exhibited a significant incremental reduction in HbA1c , resulting in a 52-week HbA1c reduction of −1.4% (95% CI −1.7 to −1.2%). No significant changes in the proportion of patients who achieved the HbA1c targets of < 7% (< 53 mmol/mol) and ≤ 6.5% (≤ 48 mmol/mol) at week 52 compared with week 26 were observed for patients who originally received exenatide once-weekly or pioglitazone; significantly more patients achieved both HbA1c targets after switching from sitagliptin to exenatide once-weekly . At the end of this assessment period, the proportion of patients who achieved fasting plasma glucose ≤ 7 mmol/l was unchanged with exenatide once-weekly-only (week 26 → week 52: 62% → 62%) and following the switch from pioglitazone to exenatide once-weekly (week 26 → week 52: 59% → 59%). Significantly more patients achieved fasting plasma glucose ≤ 7 mmol/l after switching from sitagliptin to exenatide once-weekly .Exenatide once-weekly-only and pioglitazone → exenatide once-weekly patients also maintained fasting plasma glucose improvements from week 26 to week 52 , while pP < 0.0001; P = 0.0006). Patients who received exenatide once-weekly throughout the trial on average had a + 0.7 kg (P = 0.0325) increase in weight from week 26 to week 52, resulting in a −1.8 ± 0.5 kg (P = 0.0002) reduction from original baseline.Patients who switched from pioglitazone to exenatide once-weekly exhibited a significant weight reduction  exhibited greater systolic blood pressure improvements [−12.2 mmHg (95% CI −16.1 to −8.3)]. Patients who exhibited abnormal systolic blood pressure at week 26 after treatment with sitagliptin (n = 46) or pioglitazone (n = 34) also demonstrated a greater systolic blood pressure reduction . Changes in concomitant anti-hypertensive medications were only allowed if deemed necessary by the investigator. Of the 186 patients who used an agent to treat hypertension at the start of the open-label assessment period, 172 patients  did not change dose and two patients  initiated medication after the start of the open-label period.Systolic blood pressure changes are presented in n = 3 exenatide once-weekly-only; n = 5 sitagliptin → exenatide once-weekly; n = 5 pioglitazone → exenatide once-weekly) started a lipid-lowering medication after the start of the open-label period.Patients who continued exenatide once-weekly for 52 weeks maintained improvement in HDL cholesterol from baseline; all other lipid variables were not significantly changed from original baseline . PatientExenatide once-weekly for 52 weeks was associated with improvements in the urinary albumin/creatinine ratio, B-type natriuretic peptide and high-sensitivity C-reactive protein . Althougn = 2), but did not lead to withdrawal and was deemed unrelated to study medication. Of the 45 patients who withdrew during this study period, the majority (64%) withdrew because of withdrawal of consent, investigator decision, protocol violation, lost to follow-up or for administrative reasons (n = 4) or an adverse event (n = 12) during this treatment period. The only treatment-emergent adverse events leading to withdrawal in more than one patient were abdominal pain and nausea (n = 5 (1%)] and was similar among cohorts .The most frequent treatment-emergent adverse events in this assessment period were nausea and diarrhoea . Nine pa reasons . Sixteend nausea . No case1c reduction, with similar weight reduction, compared with the twice daily exenatide formulation [1c and fasting plasma glucose observed during the first 26 weeks of treatment (52-week improvements: −1.6% and −1.8 mmol/l) and exhibited a significant improvement from original baseline in body weight (−1.8 kg). Similar 52-week improvements in HbA1c, fasting plasma glucose and weight have previously been demonstrated [Exenatide once-weekly elicits continuous exposure to therapeutic concentrations of the GLP-1 receptor agonist, exenatide, which acts to increase glucose-dependent insulin section, decrease inappropriate elevations in postprandial glucagon, increase satiety and slow gastric emptying . The conmulation . In the nstrated .et al. comparing exenatide twice daily with sitagliptin [m; [m; [1c, fasting plasma glucose and body weight observed in the present study after switching from sitagliptin to exenatide once-weekly may be attributable to the continuous exposure to higher circulating concentrations of the GLP-1 receptor agonist exenatide.DPP-4 inhibitors (e.g. sitagliptin) and GLP-1 receptor agonists (e.g. exenatide) elicit effects through the GLP-1 pathway; however, distinct differences exist in the mechanism of action of these compounds. Namely, inhibition of the DPP-4 leads to a two- to threefold elevation of endogenous GLP-1, while administration of GLP-1 receptor agonists results in higher plasma concentrations of agents that bind directly to the GLP-1 receptor. In a short-term trial by DeFronzo agliptin ,9, was aptin [m;  was like [m; [m; ) achieve1c improvement and similar fasting plasma glucose improvement compared with patients treated with exenatide once-weekly in the first phase of this trial [1c and fasting plasma glucose improvements; however, weight significantly decreased to a value similar to that observed prior to pioglitazone treatment.Pioglitazone, which exerts its anti-hyperglycaemic effects primarily through decreasing insulin resistance, resulted in a significantly smaller HbAis trial . In the Consistent with a previous study of exenatide once-weekly , patientIn contrast to a previous assessment of exenatide once-weekly , a signi1c [Overall, treatment with exenatide once-weekly for 52 weeks, or switching to exenatide once-weekly from sitagliptin, was associated with favourable 52-week changes in the urinary albumin/creatinine ratio, B-type natriuretic peptide and high-sensitivity C-reactive protein. Patients who switched from pioglitazone exhibited a significant decrease in B-type natriuretic peptide, and increases in high-sensitivity C-reactive protein and plasminogen activator inhibitor-1, such that these markers were not significantly changed from original baseline; the urinary albumin/creatinine ratio significantly improved from original baseline. It should be noted that the low mean baseline values of these markers make clinical interpretations difficult. Although the mechanism responsible for these improvements cannot be elucidated from this study, it has been demonstrated that improvements in microalbuminuria are associated with improvements in systolic blood pressure and HbA1c ; B-type 1c . The dif1c ; Table 1As in previous trials with exenatide, mild to moderate gastrointestinal events were the primary adverse events. Of note, patients who received exenatide once-weekly during the initial 26-week treatment period experienced a lower rate of nausea compared with patients who switched (5 vs. 10–11%), presumably because of prior exenatide exposure. Consistent with the glucose-dependent nature of enhanced insulin secretion with exenatide, there were no episodes of major hypoglycaemia throughout the study. Overall, 16 patients (4%) withdrew during this period as a result of an adverse event or loss of glucose control.1c and body weight compared with titrated insulin glargine, with a lower incidence of hypoglycaemia but with a greater incidence of gastrointestinal adverse events [1c improvement when directly compared with three agents that are commonly used after metformin . The current findings suggest that exenatide once-weekly may also be a suitable treatment alternative in patients with Type 2 diabetes on a background of metformin who are not currently achieving adequate glycaemic control with a DPP-4 inhibitor, or in whom thiazolidinedione-related body weight gain is a concern.The open-label, uncontrolled, study design limits the conclusions that can be drawn from this study period; however, it should be noted that, in this phase of the study, neither the patients nor the investigators were informed of original treatment assignment  during the initial 26-week treatment period. The 52-week improvements in glycaemic control and body weight associated with exenatide once-weekly-only treatment were similar to those previously reported . The impe events . Thus, e


V. vulnificus infection are analyzed and molecular features of isolates are described.Clinical features of 84 patients with  Vibrio vulnificus infection from 1995 to 2000 and describe the molecular epidemiologic features of pathogens isolated from these patients. The spectrum of clinical manifestations and outcomes, options of antimicrobial therapy, and virulence mechanisms were investigated. Results of molecular typing of isolates from humans and marine environment in this country had a high genetic divergence among these isolates. Education and measures are needed to prevent this emerging disease.Residents in Taiwan are often exposed to marine microorganisms through seafood and occupational exposure. The number of reported cases of infection attributable to this organism has increased since the first case was reported in 1985. The increasing number of cases may be caused by greater disease activity or improved recognition by clinicians or laboratory workers. We analyze a clinical-case series of 84 patients with  Vibrio vulnificus as a threat to human health has evolved during the past 30 years , the environment, and the popularity of preparing and eating raw or undercooked seafood  from 1985 to 2000 in Taiwan (6 persons) and the other in 1996 to 2000 (0.606–1.237/106 persons). Most reported cases (>90%) occurred in residents of southern Taiwan. In Taiwan, the temperature of surface seawater is usually >18°C, except for February, when it is 17°C–23°C   avirulent for eels, which were recovered from water and oysters, were reported (V. vulnificus isolates recovered from the northeastern coast of Taiwan (The occurrence of n Taiwan . A monthary 1994 . Among t7 (5.7%) . This fi7 (5.7%) . Strainsreported . Ribotypreported . More threported . In addiV. vulnificus infection treated from 1995 to 2000 was obtained from medical records from five hospitals in Taiwan  had some form of surgical treatment . The overall case-fatality rate was approximately 30% , which wEnterobacteriaceae recommended by the National Committee for Clinical Laboratory Standards : ampicillin (1 µg/mL), carbenicillin (4 µg/mL), cephalothin (4 µg/mL), cefamandole (2 µg/mL), cefotaxime (<0.03–0.06 µg/mL), ceftriaxone (<0.03 µg/mL), cefoperazone (0.12 µg/mL), aztreonam (8 µg/mL), imipenem (<0.03–0.12 µg/mL), gentamicin (4 µg/mL), amikacin (8 µg/mL), tetracycline (0.25 µg/mL), minocycline (0.06–0.25 µg/mL), chloramphenicol (0.5 µg/mL), and fluoroquinolones: ofloxacin (<0.03 µg/mL), lomefloxacin (0.12 µg/mL), ciprofloxacin (<0.03–0.03 µg/mL), levofloxacin (0.03 µg/mL), moxifloxacin (0.06 µg/mL), gatifloxacin (0.06 µg/mL), and sparfloxacin (0.06 µg/mL) (20–23). Few isolates were not susceptible to ceftazidime (MIC 32 µg/mL) and moxalactam (MIC 32 µg/mL) (V. vulnificus isolates was documented by time-kill study (V. vulnificus for >48 hours (MICs were determined and interpreted by using the MIC interpretive criteria for tandards –23. All 2 µg/mL) . All iso6 µg/mL) –23. Few ll study . Time-kiV. vulnificus infection clearly indicated that combination therapy with cefotaxime and minocycline is distinctly superior to therapy with cefotaxime or minocycline alone  and minocycline (100 mg every 12 h intravenously) was recommended for treating adult patients with bacteremia and severe soft-tissue infection caused by lnificus ,22. For V. vulnificus isolates whose biotypes were determined belonged to biotype I, which is well known to be pathogenic for humans   (vvp and empV) encoding the metalloprotease and gene (vllY) encoding a novel hemolysin of V. vulnificus were subsequently cloned and characterized  . Lee et hlegeli) . Genes  compared with those of the wild-type strains  and smcR , were not required for V. vulnificus virulence in mice  mutant of al route . A metal strains . These f in mice ,32.V. vulnificus, which quickly reached a lethal concentration with enhanced cytotoxicity in the iron-overloaded mice  were previously reported  among 13 clinical and environmental (from seawater and eels in southern Taiwan) isolates of reported ,35. BothV. vulnificus in recent clinical and environmental isolates in southern Taiwan indicates the possibility of clonal spread in this area. In this study, 50 isolates of V. vulnificus collected from 1995 to 2000 from southern (46 isolates) and northern (4 isolates) Taiwan were analyzed. These isolates included those from various clinical specimens (blood and wound pus) of 50 patients with V. vulnificus infection. All isolates of V. vulnificus were identified by using conventional methods and the O/129 susceptibility tests. Identification of the isolates was further confirmed by the API 32 GN system . Pulsed-field gel electrophoresis (PFGE) analysis was performed by a method described previously by Tenover et al. . All isolates were not identical in PFGE profiles (50 pulsotypes were found), and only two isolates from southern Taiwan were closely related (within three bands of difference). These findings support the high degree of heterogeneity among isolates of V. vulnificus that cause human infections in Taiwan.The concentration of r et al. ,37. DNA V. vulnificus infections. This bacterium is present in warm coastal waters around Taiwan during the summer months, particularly in the southern region. Exposing open wounds or broken skin to warm salt or brackish water or to raw marine animals harvested from such waters should be avoided. Patients at high risk should wear protective clothing  when handling seafood  and not eat raw or improperly cooked seafood. Because this disease is rapidly progressive and deadly if not recognized promptly and treated aggressively, any illness (such as fever or skin lesions), which develops in patients at risk after contact with marine animals or waters or ingestion of seafood requires immediate medical care.Residents of Taiwan, particularly those with preexisting liver and other chronic, underlying medical conditions , should be educated in measures to prevent acquiring The government in Taiwan  should encourage food companies to put warning labels on seafood containers, menus, and public health brochures. The wording of such labeling should be similar to the label required by the Florida Department of Natural Resources for all wholesale shell food and shucked products: "Consumer Information—There is a risk associated with consuming raw oysters or any raw animal protein. If you have chronic illness of the liver, stomach, or blood or have immune disorders, you are at a greater risk of serious illness from raw oysters and should eat oysters fully cooked. If unsure of your risk, consult a physician" .V. vulnificus isolates from humans and the environment indicates that this virulent bacterium is ubiquitous in nature. When V. vulnificus is suspected as the cause of sepsis, empiric therapy that includes a third-generation cephalosporin and minocycline should be administered. It should be standard practice for physicians to advise patients with underlying medical illness to avoid eating raw or undercooked seafood and to avoid exposing wounds to seawater.Residents of Taiwan have a high prevalence of chronic liver disease and are often exposed to marine microorganisms present in the sea that surrounds the island or rivers, lakes, or ponds inside the island. The presence of high genetic divergence among


Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing , and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture.Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria.Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling.One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture. Chronic wounds impact the health of over 8 million people in the United States each year, and the direct healthcare associated cost of these wounds is over 25 billion of dollars each year. These cPrevious studies have compared culturing to molecular methods,4-10, anProviding more accurate information to the clinician via more sensitive testing is typically the goal of the clinical microbiology laboratory. More sensitive microbial testing, such as culture-free sequencing of bacterial DNA that is used in this study, can identify numerous microbial taxa in a single clinical sample. Unfortunately, the clinician can have difficulty discerning which bacteria could be most clinically relevant. Being able to correlate the results of this newer testing method with a familiar testing method (culturing) would be helpful for the clinician. Culture-free sequencing of bacterial DNA reveals qualitatively which bacteria are present, but it also reports a relative abundance score for each organism. Correlating this relative abundance score with the likelihood that the same bacterium would be detected by culture has not been performed until now. Correlating the results of molecular microbial diagnostic tests with the results from culturing will give clinicians insight into understanding these new tests and their clinical relevance. Improving clinicians’ ability to interpret test results will enable them to better customize their patient care and overcome each individual patient’s barriers to healing.In this study, the chronic wounds from 51 subjects were sampled for study in accordance with Western Institutional Review Board protocol #20062347. All subjects signed informed consent documents. All samples were obtained at the Southwest Regional Wound Care Center  in 2009. Types of wounds included chronic wounds such as venous stasis ulcers, diabetic foot ulcers, pressure ulcers, and non-healing surgical wounds. Each sample of devitalized tissue was obtained using sharp debridement as part of standard of care. Each chronic wound site was sampled in duplicate for parallel testing. One portion was analyzed by culture in an academic, hospital-based microbiology laboratory while the other portion was analyzed by molecular methods at PathoGenius laboratory . Both labs are accredited by the College of American Pathologists.Fifty-one (51) consecutive subjects that presented to the Southwest Regional Wound Care Center with full thickness wounds  were included in the study if they had a chronic wound as defined as a wound that fails to progress through normal wound healing trajectory, if sharp debridement was required as part of their standard of care, and if enough debridement material was obtained in order to perform both culture and molecular testing.Cultures were obtained as per laboratory protocol. Samples from the host-bioburden interface were obtained at the Southwest Regional Wound Care Center and transported to the hospital microbiology laboratory for aerobic culture and identification. Some of the isolated bacteria were then analyzed by the Research & Testing Laboratory  to verify the isolates’ identities using molecular testing. A second, parallel sample was obtained and placed in a 2 mL microcentrifuge tube and tested by Pathogenius .The methods have been thoroughly described previously. BrieflyStaphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis). Each detected taxon comprised a percentage of the total relative abundance in the sample. For some of the analyses, these percentages were divided into three categories based upon the frequency of taxon detection. Taxa with a relative abundance of more than 10% were considered to be “Dominant” contributors to the sampled microbiota. Taxa with relative abundance of 1-10% were considered to be “Major” contributors to the sampled bacterial population, and taxa with relative abundance of less than 1% were considered to be “Minor” components of the bacterial population in the wound. This arbitrary scoring system was employed to the genera within the dataset.The total number of sequence matches for an organism in each sample was considered 100% of the relative abundance for that sample. Most of the analysis was performed at the genus level, but some analysis was also performed at the species level  was used to cross-check the culture isolates using molecular testing. The clinical microbiology laboratory provided cultured isolates to the research laboratory for verification of identification. The research laboratory extracted 16S DNA and sequenced a portion of the gene to determine each isolate’s species. The bacterial species determined by molecular testing in the research laboratory was then compared to the clinical laboratory’s identification of the isolate.The combined microbiome of all samples was illustrated using a rank-abundance plot. This plots each genus’ relative abundance (assessed via molecular methods) as a function of its rank within the entire community, and graphically illustrates the overall diversity of the wound microbiome. The relationship between the likelihood of a bacterium being cultured and its relative abundance was graphically illustrated using a mosaic plot and was statistically evaluated using logistic regression. Specifically, we used a generalized linear model that assumed a binomial error distribution and a logit link function. Significance of the relationship was assessed using a likelihood ratio test, and results are reported as an odds ratio with a 95% confidence interval. The odds ratio is a measure of the increase in the odds of being detected via culture based methods given an increase in relative abundance. Statistical analyses were conducted in R[in situ hybridization), which reported, “the absence of a correlation between the bacterial species Staphylococcus aureus and Pseudomonas aeruginosa detected in wound by culture and what is in fact present”[Like previous studies, this study demonstrates that culture testing and molecular testing of wound bacteria produce differing and often conflicting results because molecular methods are more sensitive than culture methods-10. The present”.Staphylococcus and Enterococcus comprised the majority of the detected genera; they were detected 28 and 21 times, respectively. When considering only the 92 detected genera, 16S DNA sequencing identified the same genus in the sample 78 times. Of those 78 times, the bacterial population was determined to be a dominant population (>10% of the wound microbiota relative abundance) 43 times, a major population (1-10% of the wound microbiota relative abundance) 18 times, and a minor population (<1% of the wound microbiota relative abundance) 17 times. Fourteen (14) times, the bacterial genus identified by culture was not detected by molecular testing. It is possible that some of the 14 bacterial genera that were identified by culture but not identified by 16S testing may have been misidentified by one of the testing methods  were detected , and these 753 positive results were found within 145 unique genera  of results attributed to bacteria that occurred with less than 1% relative abundance in their samples were environmental contaminants within the wounds. If this is the case, it is important to note that 18% (17/92) of the bacteria that were isolated by culture were in this group, so culturing also detects bacteria that may be environmental contaminants. Another 15% (14/92) of the cultured bacteria were not detected at all using molecular methods, which could be because of very low levels of the bacteria within the wound. In total, one-third (31/92) of the genera detected by culture were either not detected using 16S sequencing or detected to be present at less than 1% of the relative abundance within the sample. The clinical usefulness of attempting to manage bacteria present at such low abundance within a sample is questionable. The molecular testing results suggest that culture testing may be insufficiently sensitive in some cases or overly sensitive in other cases. Therefore, careful clinical interpretation of culture results is necessary,12.Staphylococcus in 61% (28/46) of the samples that were positive by molecular testing (average relative abundance of 28% per sample), but Corynebacterium was only identified by culture in 16% (6/38) of the samples that were positive by molecular testing (average relative abundance of 24% per sample). Both the organism and its relative abundance affect the ability to detect the organism by culture.Some bacteria were more commonly detected by culture than others Table . In someMany of the bacteria that were identified by culture contributed to a large portion of the relative abundance of bacteria in the samples, but about half of the bacteria that were determined to comprise a dominant portion of the microbiota were not detected by culture Figure . Some ofIn general, the likelihood of an organism being cultured was positively related to its relative abundance Figure . This trPseudomonas aeruginosa  and Staphylococcus aureus , and approached significance for Enterococcus faecalis .Similar results were identified at the species level: species with a higher relative abundance in the sample as determined by molecular testing were more likely to be cultured Figure . Three sExhaustively culturing all of the bacteria within a clinical sample has proven difficult,10,14. DMolecular methods can overcome some of cultures’ shortcomings. Molecular testing can detect biofilm bacteria, anaerobes, and VBNC bacteria regardless of how well the bacteria may or may not grow in culture,12. MoleP. aeruginosa was identified by culture, but Salmonella enterica was identified by molecular testing. As mentioned previously, identification of bacteria by genotype is considered superior to the identification of bacteria by phenotype, so culture testing reported significant misidentifications of bacterial taxa 7% (3/46) of the time.Forty-six (46) culture isolates were cross-checked for identification using molecular methods, and discrepancies were observed in 20% (9/46) of the isolates  are more likely to be detected by culture, and bacteria with lower relative abundance scores are less likely to be detected by culture. Culture testing is not as sensitive as molecular testing, so culturing often fails to detect bacteria, even bacteria that are present in a high relative abundance. Bacteria that are present in high relative abundance are likely of high clinical relevance, including the bacteria that are not detected by culture. By considering the relative abundance scores when interpreting molecular results, clinicians can gain a clearer picture as to which bacteria may be most clinically relevant.Prospective studies comparing patient and wound outcomes when using culture testing versus molecular microbial testing have not yet been performed, but retrospective studies suggest molecular microbial testing can improve patient outcomes,17.DDR has formerly been employed by WCC and RTL. SBC, EJR, and YS are employed by RTL. RDW is the Medical Director of WCC, and has equity positions in RTL and PGL. PGL & RTL engage in business transactions. PGL has applied for patents regarding molecular microbial testing.EJR helped to acquire and analyze the data and draft the manuscript. DDR & SBC analyzed and interpreted the data and drafted the manuscript. YS performed molecular testing and participated in drafting the manuscript. RDW designed the study and participated in drafting the manuscript. All authors read and approved the final manuscript.DDR is a Medical Laboratory Scientist and a Clinical Pathology resident. SBC is a Ph.D. biologist and statistician. YS is a Ph.D. molecular scientist. EJR is a Ph.D. computer scientist and bioinformatics researcher. RDW is a medical doctor and key opinion leader in the wound care community. He focuses his efforts on improving patient care by developing and using biofilm-based wound care practices.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/321/prepubBacteria identified by 16S and culture testing according to each subject.Click here for file


Prior to the rollout of potent antiretroviral therapy (ART), Kaposi’s sarcoma (KS) was the most commonly reported malignancy in many resource-limited settings, such as most of sub-Saharan Africa. In resource-replete settings , the advent of ART—and the availability of established research networks—resulted in documentation of a marked decrease in KS incidence. In contrast, in resource-limiting settings like Africa, the heretofore lack of epidemiologic infrastructure has limited our knowledge about the effect of ART on KS incidence.We prospectively followed HIV-infected adults, without prior KS, attending 26 HIV/AIDS clinics at the Mbarara (Uganda) or AMPATH (Kenya) sites of East Africa IeDEA. Time zero was October 2008, when we introduced skin punch biopsy for KS diagnosis, which was available same-day free of charge. Biopsies were adjudicated by board-certified UCSF dermatopathologists. Patients were followed until they developed KS, death, loss-to-follow-up, or administrative closure. Once started on ART, patients were considered on ART irrespective of adherence/interruptions. Incident KS was defined as any new occurrence of KS 30 days after clinic enrollment that was documented either pathologically or by clinical diagnosis in the absence of pathology.3 (IQR: 137-453). Patients were followed for 144,182 person-years (median 1.8 years/patient) for 499 incident KS diagnoses, 43% of which were pathologically confirmed. KS incidence during non-ART use was 1876 cases/100,000-person-years in Uganda and 596 in Kenya; incidence during ART use was 201/100,000-person-years in Uganda and 270 in Kenya. After adjustment for age and gender, ART-users had a substantially reduced rate of KS compared to non-users: 88% reduction in Uganda (p<0.001), and 50% in Kenya (p<0.001). Further adjustment by CD4+ count showed a persistent ART effect, suggesting ART benefits for KS prevention above and beyond CD4 restoration (Table). In ART-users who achieved a CD4+ count of >350 cells/mm3, KS incidence declined to 18/100,000-person-years in Uganda and 93/100,000-person-years in Kenya.We followed 98,024 HIV-infected adults: 31% men, 66% ever on ART, and median values at study enrollment of 35 years old (IQR: 29-43) and 277 CD4+ T-cells/mmIn a prospective study in East Africa, KS incidence was very high in untreated HIV-infected adults but was substantially reduced by ART — similar to that observed in resource-replete settings. Despite ART, absolute rates of KS remained considerable until a CD4+ count of >350 was achieved, suggesting the need for earlier ART initiation. The IeDEA platform provides unique opportunities for prospective African KS research.


To determine human herpesvirus 8 (HHV-8) K1 genotypes in patients with Kaposi sarcoma (KS) from Peru, we characterized HHV-8 in 25 KS biopsy samples. Our findings of 8 A, 1 B, 14 C, and 2 E subtypes showed high HHV-8 diversity in these patients and association between E genotype and KS development. HHV-8 infection was determined by nested PCR to obtain a 220-bp (variable region [VR] 1–inner fragment) and a 240–300-bp (VR2-inner) fragment of the ORF-K1  was characterized by well-delineated sheets and bundles of spindle cells, which coalesced to form nodules. The proportion of spindle cells labeled with the monoclonal antibody LANA-1 varied according to the histopathologic pattern; the bundles and sheets of spindle cells in the third pattern displayed the strongest signal  and in 4 cases for VR2 (VR2-outer) amplification. After nested PCR, a signal was obtained in 25 cases for VR1-inner and in 17 cases for VR2-inner . After cPhylogenetic analyses were performed by using 2 sets of sequences; 25 VR1-inner  and 11 VTwo AIDS-KS mestizo patients  were thuHHV-8 K1 gene characterization of KS tumor biopsy samples demonstrated high molecular genotype diversity, including 4 of the 5 main known molecular subtypes. The most frequent were the A and C subtypes, typically of European origin. Other patients were infected by a sub-Saharan African HHV-8 genotype (1 A5 and 1 B) or by HHV-8 genotype E strains of Amerindian origin.,Such findings were not unexpected because persons in Peru are of many ethnicities. Indeed, the genetic background of the Peruvian population is diverse, reflecting the multiple waves or populations that colonized the country during the last millennium. Schematically, this began with different Amerindians groups in the Lithic period (infected possibly with E subtype), followed by the Spanish colonization of the Americas mainly from southern Europe (infected possibly by A and C subtypes) and later slave trade from Africa (infected by A5 or B subtypes). Such HHV-8 strain diversity has been previously observed in French Guiana (,HHV-8 genotype E previously has been found exclusively in Amerindians from Central and South America, including Brazil (We demonstrated that KS can occur in HHV-8 subtype E–infected persons. Indeed, in 2 AIDS-KS patients, an E genotype was characterized in the tumor lesions. Further studies are ongoing to provide new insights into the distribution and genetic epidemiology of such HHV-8 infection in Amerindian populations.


Spike frequency adaptation (SFA) is defined as the decline in motoneuron (MN) firing rate during constant current injection. In classical study of Granit et al.  this phein vitro and often supported by computer simulations  has no effect on SFA. However, SFA would be enhanced through blocking IK, when other IKs  were blocked. Thus, the contribution of IK to MN firing patterns depends on the activity of other K+ currents.One piece of evidence supporting this theory of redundancy was presented by Goh et al.  through Considerable part of the research on SFA was produced by Brownstone's lab . This task evokes a sudden increase in synaptic inflow to MN pool, resulting in recruitment of a few MNs with initial firing rates of 60–120 imp/s, which quickly decrease thereafter. The time course of this decrease is similar to those observed in animal MNs in response to constant current injection Figure .The reaction of the MN to the rectangular current step was investigated by Ito and Oshima . They shIn contrast, during ramp contractions MN firing rate is gradually increasing until the contraction force reaches the target level  was applied to cat MNs in the study of Baldissera et al. , who invLate SFA develops in MN over several minutes and does not depend on the type of excitation, but on the type of muscle fibers that the MN innervates. MNs supplying F muscle fibers have significantly higher initial firing rates and faster late adaptation (Kernell and Monster, The reason that SFA in MNs is such a robust phenomenon is presumably related to the unique role of MNs as the Sherrington's “final common pathway” leading to muscle contraction. For MNs to play this role, their properties must be properly matched to those of their muscle units. This matching is indeed observed (Kernell et al., As a result of this matching, MN firing rate is adjusted to the requirements of motor task performance. A short initial ISI, for example, is beneficial for smoothness and speed of F contractions (Baldissera and Parmiggiani, Initially, human MN rate slowing during sustained contractions was thought to be mediated by a peripheral reflex from the fatiguing muscle (Bigland-Ritchie et al., Moreover, the match between MN and muscle fiber properties is preserved in normal aging. The firing rate slowing is related to increases in MN AHP duration (Piotrkiewicz et al., SFA has usually been studied in animal experiments in response to long-lasting rectangular current steps. The physiological analogue of this artificial condition may occur in extreme situations when the animal or human being is falling from a dangerous height so that survival depends on the ability to quickly catch possible support and to hold on to it long enough for rescue. In this case, SFA would help economize necessary forces.However, there are certain motor control tasks in which the firing rate decline is not desirable, such as in breathing or locomotion. Rhythmic movements may be continued without fatigue for periods lasting much longer than sustained contractions of constant force. In this case, neuromodulators may reverse SFA through metabotropic pathways. This reversal has been recently observed during fictive locomotion (Brownstone et al., In summary, there is adequate evidence that SFA is not an artefact, but rather a manifestation of “neuromuscular wisdom,” which assures proper functioning of the healthy neuromuscular system under all physiologic circumstances.


Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA. The immunophilin-binding agents cyclosporine A (CsA), FK506 and rapamycin represent potent immunosuppressive agents that have revolutionized bone marrow and solid organ transplantation as well as treatment of autoimmune diseases. Sanglifehrin A (SFA) is a novel immunophilin-binding immunosuppressive drug isolated from the actinomycetes strain Streptomyces A92-308110 exhibiting high affinity binding to Cyclophilin A, but unknown mechanism of action In contrast to CsA, the immunobiology of SFA is not well understood. Previous reports demonstrated that SFA is different from known immunosuppressive agent Different groups have reported that SFA exerts suppressive effects on human and mouse DC. SFA suppresses antigen uptake, IL-12 and IL-18 production of DC in vitro and in vivo but it does not inhibit DC differentiation and surface costimulatory molecule expression DCs are professional antigen presenting cells that play a central role in the initiation and modulation of innate and adaptive immunity In an experimental transplant model, SFA monotherapy did not suppress acute organ allograft rejection supporting the hypothesis that it does not represent a primary T cell inhibitor Current knowledge indicates that SFA represents a novel class of immunophilin-binding metabolite both with respect to chemical structure and functional activity In this report we describe the results of the first systematic analysis of the immunobiological effects of the novel immunophilin-binding agent SFA on human monocyte-derived DC (moDC) using a combination of genome-wide expression profiling with subsequent confirmation on the protein level and functional in vitro and in vivo assays. Results indicate that SFA represents a novel DC chemokine and migration inhibitor.http://www.ncbi.nlm.nih.gov/geo/) with the GSE15956 accession number.To systematically identify specific gene expression changes by SFA, we compared human moDCs cultured in the presence of either vehicle or drug. Human DCs were generated by differentiation of monocytes with GM-CSF and IL-4 for five days. On day 5 human moDCs were either treated with 1 µM SFA or vehicle for 1 hour followed by 12 h LPS stimulation. The RNA of SFA vs. vehicle-treated human moDCs was analysed by whole genome Oligo Microarray. The Microarray data showed 260 significantly regulated genes . 190 genes are significantly down regulated and 70 genes are up-regulated. The complete data are deposited in the Gene Expression Omnibus database (http://vortex.cs.wayne.edu/ontoexpress/) to get information about the biological functions We analyzed the gene expression changes with PathwayExpress from OntoExpress  of human moDCs with 100 nM SFA resulted in significant suppression of CCL5, CCL17, CXCL9 and CXCL10 production . 100 nM Having established that short-term SFA treatment of moDC significantly inhibited chemokine gene and protein expression we next analysed the potency of SFA to suppress CCL5, CCL17 and CCL19 in moDC. Administration of 50 nM SFA for 4 h resulted in >80% CCL5 suppression , and 100Both, SFA and CsA bind with high affinity to cyclophilin in cells To address the question whether SFA's inhibitory activity on chemokine expression is dependent on cyclophilin A binding, we performed competitive experiments with a 100-fold molar excess of CsA (10 µM). CsA has been described to potently inhibit the binding of SFA to cyclophilin A + T cell migration and moDC migration towards supernatant from SFA-exposed maturing moDCs and vehicle-exposed controls . Given the fact that SFA efficiently inhibited moDC migration in a CCR7-independent manner and previous reports demonstrated that SFA can abrogate IL-12 production in human DCs CD38 is an ectoenzyme and signalling receptor and was reported to represent a novel human DC marker +FITC+ skin DC quantified after 24 h by flow cytometry  and the animal experiments were approved by the animal ethics review board of the Regierungspräsidium Gießen (File GI 20/8 Nr 49/2006).SFA and N-Methyl-Val-4-cyclosporin (4-Cs) were provided by Novartis Pharmaceuticals . Stock solutions were prepared in absolute ethanol (vehicle) and the control DCs were treated with drug vehicle. Dexamethasone and Cyclosporin A were provided by Sigma-Aldrich  and dissolved in vehicle. The drugs were used at the indicated concentrations and time points. The stock solutions were diluted on the day of experiment with culture medium.+ monocytes were purified (>95%) using CD14 immunomagnetic microbeads  and 1×106 cells per ml were cultured in six-well flat-bottom plates, in 3 mL of DC medium, comprising RPMI 1640, L-glutamine, penicillin/streptomycin, sodium-pyruvate, hepes, nonessential amino acids, 10% heat-inactivated FCS Gold , 1000 IU/ml recombinant human (rh) GM-CSF , and 1000 IU/ml rhIL-4 . On day 6, CD1a+ DC represented >90% of cultured cells. The in vitro studies of human blood samples and the animal experiments were approved by the Institutional Review Board.Human PBMC were isolated from buffy coats of healthy blood donors by Ficoll-Paque  density gradient centrifugation. CD14+-T cells were positively selected with magnetic beads and subsequently sorted with autoMACS . Reagents were used according to the manufacturer's instructions. Purity of positively selected T cells was >98% determined by flow cytometry. 1×106 cells per ml were cultured in six-well flat-bottom plates, in 3 mL of medium, comprising RPMI 1640, L-glutamine, penicillin/streptomycin, 10% heat-inactivated FCS Gold .CD4Escherichia coli 026:B6; Sigma-Aldrich, Taufkirchen, Germany) was added to the culture. After 12 hours stimulation, the RNA of 1.5×107 cells was isolated by using the Qiagen RNeasy Mini Kit  according to the manufacturer's instructions.On day 5, 1 µM SFA or vehicle (absolute ethanol) was added to the DC medium. After one hour 1 µg/mL Lipopolysaccharid  using Limma The RNA from four donors was pooled and the RNA was stored at −70°C until use. A total of n = 28 donors were analyzed by microarray. The RNA quality was confirmed with RNA 6000 Nano LabChips . 1 µg RNA was amplified with Low RNA Input Fluorescent Linear Amplification Kit  and labeled with Cyanin 3- or Cyanin 5-CTP . cRNA of SFA and vehicle cRNA was mixed and 40 µg cRNA was used for hybridization with whole human genome oligo Microarray G4112A . A total number of 7 arrays were hybridized, each with a pool of RNA from control samples and a pool of RNA from stimulated samples . Hybridization and washing was done following the Agilent protocol. Images were scanned with the Axon 4100A  and processed with GenePix 5.0. Data analysis was done with R software version 2.10.1 . Phenotypic maturation of moDC after LPS stimulation was controlled by flow cytometry analysis of surface CD83, CD86 and MHC-II (HLA-DR) expression  and appropriate isotype controls  according to the manufacturer's instructions.6 moDCs or CD4+ T cells were suspended in supplemented medium. 100 µl were placed in the upper well. moDC migrated towards 100 ng/ml CCL19 or in other experiments human moDCs and T cells migrated towards cell-free supernatant of SFA-exposed moDC or vehicle-treated moDCs in the lower well. For the “SFA carry over control” supernatant of vehicle-treated moDC was used and spiked with 1 µM SFA. We treated human moDCs with 1 µM SFA or vehicle for 4 hours and collected the supernatant after 12 h stimulation with 100 ng/ml LPS. The transwells were incubated for 4 hours at 37°C in a 5% CO2 moist atmosphere. Migrated cells were quantitated with Leukocount-Kit  on a FACSCalibur flow cytometer .Migration was analysed by using a 24-well microchemotaxis chamber technique with polycarbonate transwells  8–10 wk-old female C57BL/6NCrl mice were treated with SFA or CsA (10 mg/kg/for 2 days). Drug stock solution was diluted freshly in 2.5% Polysorbate 80, 51% PEG300 (Sigma-Aldrich) and 46.5% sterile water. On day two, the abdomen was shaved and painted with 200 µl FITC (10 mg/ml), in a 50∶50 (vol/vol) acetone-dibutylphtahalate mixture, as described by Macatonia et al. Statistical analyses were performed using the Mann Whitney U test. All tests were performed two-tailed. A probability of <0.05 was considered significant. Analyses were performed using the SPSS software version 16.0 . Microarray results were analyzed as described above.


The route by which infectivity reaches the follicles is unknown, however, intestinal epithelial cells may participate in intestinal antigenic presentation by delivering exosomes as vehicles of luminal antigens. In a previous study using an intestinal loop model, following inoculation of scrapie brain homogenate, inoculum associated PrPd was detected by light microscopy shortly (15 minutes to 3.5 hours) after inoculation in the villous lacteals and sub-mucosal lymphatics. No PrPd was located within the follicle-associated epithelium (FAE), sub-FAE domes or the PP follicles. To evaluate this gut loop model and the transportation routes in more detail, we used electron microscopy (EM) to study intestinal tissues exposed to scrapie or control homogenates for 15 minutes to 10 days. In addition, immuno-EM was used to investigate whether exosomes produced in the FAE may possess small amounts of PrPd that were not detectable by light microscopy. This study showed that the integrity of the intestinal epithelium was sustained in the intestinal loop model. Despite prominent transcytotic activity and exosome release from the FAE of the ileal PP in sheep, these structures were not associated with transportation of PrPd across the mucosa. The study did not determine how infectivity reaches the follicles of PPs. The possibility that the infectious agent is transported across the FAE remains a possibility if it occurs in a form that is undetectable by the methods used in this study. Infectivity may also be transported via lymph to the blood and further to all other lymphoid tissues including the PP follicles, but the early presence of PrPd in the PP follicles during scrapie infection argues against such a mechanism.In natural or experimental oral scrapie infection of sheep, disease associated prion protein (PrP The detection of PrPd generally indicates the presence of infectivity, but the correlations between PrPd and infectivity are not always precise as infectivity may be detected in the absence of PrPdThe oral route of infection is considered to be important in the natural pathogenesis of transmissible spongiform encephalopathies (TSEs), which are fatal neurodegenerative disorders described in man, domestic and wild ruminants and carnivores d occurs in the gut associated lymphoid tissues prior to clinical disease d has been detected in the Peyer's patch (PP) follicles as early as 21 days after inoculation in experimentally infected sheep d in gut associated lymphoid tissues provides circumstantial evidence for uptake from the gut but does not give any direct insight into the route of intestinal transport of infection.In natural TSE infections such as scrapie in sheep and various experimental animal models, the early accumulation of PrPd is taken up by the follicle associated epithelium (FAE) of PPs and transported across the sub-FAE dome region to enter the PP follicles, but direct evidence for this route of transmission is lacking. It is widely accepted that FAE, and particularly M cells, transport luminal antigens to subjacent antigen presenting cells as part of the maintenance of immune surveillance and oral tolerance, and that this physiological process is exploited as a route of entry for various pathogens in vivo studies of rodents orally challenged with scrapie which show PrPd within the FAE, possibly within M cells in vitro studies which demonstrate transcytosis of prions within M cell cultures d or infectivity from the gut lumen to mesenteric lymph nodes The mechanisms and route by which infectivity reaches the follicles after crossing the intestinal barrier remain unclear. The assumption is that PrPin vivo for various periods of time d positive material was found to be transported rapidly across the absorptive epithelium (AE) to reach the lacteals of the villi within 15 minutes of inoculation. PrPd positive material was not detected along the presumed route of transmission, namely in the FAE, the domes and PP follicles during the initial 24 hours in which the inoculum was detectable. Nevertheless, when de novo generated PrPd was detected after 30 days, it was present in the PP follicles. These observations invited speculation on the relationship between infectivity and immunohistochemically detectable PrPd. Thus, infectivity may have been carried across the FAE separately from PrPd. Alternatively, PrPd and infectivity may have been carried to the PP at levels below the threshold of immunohistochemical detection.The intestinal transmission of scrapie in sheep has been studied in a gut loop model in which scrapie brain homogenates were incubated in vivo observations correlate with the recent description of the release of prions in association with exosomes from sheep scrapie infected cultured cells Active macromolecular transport exists between the FAE and ileal PP follicles in cattle and sheep et al.d across the FAE, we undertook to determine whether immunogold-EM would reveal the presence of PrPd in the PPs, and in particular whether exosomes could be carriers of PrPd.The object of the present study was to use electron microscopy (EM) to evaluate the gut loop model, especially the transcytotic activity in the FAE and transport of luminal material into the follicles, shown to occur under normal conditions in ileal PP of sheep Generally, observations of all the intestinal tissues showed normal cell morphology, with normal nuclei and cellular organelles. The study of the intestine from loops exposed to scrapie infected brain inoculum, normal uninfected brain inoculum or sucrose-inoculum, did not reveal differences that could be attributed to the nature of the inoculum. Likewise, differences were not observed between surgically ligated/clamped loops and intestine not ligated or incised. However, variations between intestinal tissues sampled at the earliest time points (15 minutes to 3.5 hours) and later time points (24 hours to 10 days) were observed .Exosomes were variably present in the sub-FAE dome. At 15 minutes to 3.5 hours post exposure, large numbers of heterogeneous appearing vesicles, including exosomes (30–80 nm), showed a multifocal distribution in intercellular spaces . At timeMaterial from loops taken 15 minutes to 3.5 hours post-exposure to inoculum showed wide spaces between the cells in the central zone of the PP follicles. These spaces contained large amounts of granular material and vesicles, in addition to a sparse electron dense amorphous material surrounding the follicular dendritic cell (FDC) dendrites . The vesd and GFAP . Control animals had a low grade level of gold particles dispersed with no concentration at any specific localisation (not shown).Amorphous material was observed in lacteals of some villi of intestinal loops exposed to both scrapie brain inoculum  and sucrose loops  at early time points (15 minutes to 2 hours). These sections with villi lacteals containing amorphous material were labelled with antibodies against PrPin vivo events occurring in the intestinal wall at specified times after exposure of the gut to a particular agent or substance to be studied et al.d was detectable in the intestinal wall for 24 hours following exposure but contrary to expectation it was present in lacteals of the villi and not in the dome and follicles of the PP. One explanation for the absence of uptake of scrapie inoculum across the FAE was that the experimental procedure had disturbed the function and/or structure of the intestinal epithelium. As surgical trauma may induce loss of the macromolecular barrier in the intestine, the importance of caution in the interpretation of loop experiments was pointed out by Rhodes et al The experimental intestinal loop model enables d across an intact AE, and a concurrent exclusion of PrPd from a functional FAE, as depicted in Ultrastructural evidence of transcytosis was present in the FAE including pitting of the luminal membrane and the presence of many vacuoles, tubulovesicular structures and MVBs in the apical cytoplasm. The evidence of uptake of extracellular material in macrophages also argues for transcytosis by the FAE and transport of luminal material into the follicles as shown in the ileal PP of lambs et al.et al.Another strong indication of a functional FAE in the isolated intestinal loop was the presence of abundant exosomes in the domes and follicles. Landsverk in vitroin vivod. Immunohistochemistry and light microscopy of intestinal wall compartments known to harbour exosomes did not reveal the presence of PrPd in FAE, dome or lymphoid follicles of isolated intestinal loops exposed to scrapie-infected brain material d on exosomes below the detection limit of light microscopy that could be detected by immunogold labelling and EM had not been ruled out. The present study tested this hypothesis but found that the exosomes were PrPd-negative . In naturally or orally infected animals, numerous studies have reported that PrPd accumulation appears initially in lymphoid follicles of PPs followed by accumulation in other lymphoid tissues at later stages It is possible that infectivity could be transported across the villi to the lymph then to regional lymph nodes and back to the PP follicle via the blood. The presence of TSE infectivity in blood is established, as BSE has been shown to be transmitted from orally infected donors to recipients after a single blood transfusion The animal experiments carried out within VLA were supervised by a named Veterinary surgeon as required under UK legislation and were approved by UK government Home office inspectors; approval ID E04 50.ARQ/ARQ genotype were weaned at two months of age, and then starved, anaesthetised and a midline laparotomy performed. The distal ileum with its continuous PP was formed into a large loop by sectioning the intestine proximally and distally. The isolated segment was flushed with saline containing antibiotics and its ends were either closed or clamped, depending on whether the lamb was to be allowed to recover from anaesthesia or was scheduled for loop removal at an early time point (see below). The remaining bowel was re-anastomosed or clamped. The large isolated loop was then sub-divided into two or four smaller loops by ligation. In each lamb, alternate loops were inoculated with 10 ml  of a scrapie brain pool inoculum or with either normal brain homogenate or with sucrose solution .Brains from 10 scrapie-confirmed clinically affected ARQ/ARQ sheep were obtained from an intensely studied Suffolk flock d using the above antibodies by immunogold methods as previously described d and GFAP.A wide range of visceral and CNS tissues were sampled at necropsy and light microscopy results of these tissues have previously been published To ascertain whether there were any morphological features which might be attributed to early scrapie infection, grids from each loop were coded and examined blind. Features present in each section were listed and each feature was subjectively assessed as large/abundant, moderate, narrow/sparse or absent. To compare effects of scrapie challenge with the controls these categories were converted to scores of 3-0 .


An affiliation of the last author was incorrect. Bruno Spire is correctly indicated as affiliated with institutions 1, 2 and 3, but is also affiliated with institution 8, Aides, Pantin, France, not with institution 7. In addition, there was an error in affiliation institution 1 for authors Nicolas Lorente, Marie Preau, Marion Mora, Jerome Blanche, Maria Patrizia Carrieri, Marie Suzan-Monti and Bruno Spire. Institution 1 should be: INSERM UMR912 (SESSTIM), Marseille, France.


Antibody-mediated rejection (AMR) is gaining increasing recognition as a major complication after heart transplantation, posing a significant risk for allograft failure, cardiac allograft vasculopathy, and poor survival. AMR results from activation of the humoral immune arm and the production of donor-specific antibodies (DSA) that bind to the cardiac allograft causing myocardial injury predominantly through complement activation. The diagnosis of AMR has evolved from a clinical diagnosis involving allograft dysfunction and the presence of DSA to a primarily pathologic diagnosis based on histopathology and immunopathology. Treatment for AMR is multifaceted, targeting inhibition of the humoral immune system at different levels with emerging agents including proteasome and complement inhibitors showing particular promise. While there have been significant advances in our current understanding of the pathogenesis, diagnosis, and treatment of AMR, further research is required to determine optimal diagnostic tools, therapeutic agents, and timing of treatment. Antibody-mediated rejection (AMR) is a diagnostic and therapeutic challenge in human heart transplantation. Although the true incidence of AMR is unknown, it has been reported in 10–20% of patients after heart transplant, typically occurring within a few months after transplant .AMR was first described as a clinical entity in 1987 by Herskowitz et al. who identified a subset of heart transplant patients with arteriolar vasculitis and poor outcomes . HammondAMR occurs due to a humoral immune response with antibodies binding to endothelium on the transplanted heart . The antDSA binding to the allograft causes myocardial injury and allograft dysfunction predominantly through immune complex activation of the classical pathway of the complement cascade . AntigenAnti-HLA antibody binding may also lead to endothelial cell activation by complement independent mechanisms. Direct cross-linking of HLA molecules on the cell surface can activate endothelial cells and lead to the production of growth factors such as fibroblast growth factor, platelet-derived growth factor, monocyte chemotactic protein as well as cytokines and adhesion molecules , 14. ImmThe role of non-HLA antibodies in AMR remains an area of contention. Recently, Nath et al. showed that non-HLA antibodies directed against cardiac myosin and vimentin were elevated in heart transplant recipients who subsequently developed AMR and CAV . The appThe diagnostic criteria for AMR have undergone significant revision. A key difference between the 2005 and 2011 ISHLT guidelines is the proposed shift from a clinical to pathologic diagnosis. The 2005 guidelines recommended diagnosis of AMR based on interpretation of pathologic changes in conjunction with cardiac allograft dysfunction and/or hemodynamic compromise and the presence of DSA . AMR hasScreening for DSA is an important component of immune surveillance following heart transplantation. Screening is typically performed with solid phase assays using recombinant HLA-protein-coated beads . DetectiHistologic features of AMR as defined by the 2005 ISHLT guidelines include “myocardial injury with endothelial cell swelling and intravascular macrophage accumulation.” Additional features include “interstitial edema and hemorrhage” as well as “intravascular thrombi and myocyte necrosis” . ImportaDetection of AMR may be improved by immunopathologic examination of endomyocardial biopsy specimens. The 2005 ISHLT guidelines recommended immunofluorescence or immunohistochemical staining on frozen and paraffin sections, respectively. Immunofluorescence stains detect immunoglobulin and complement  deposition within capillaries, while immunohistochemistry stains detect capillary CD68 positive macrophages and C4d deposition .C4d deposition has been evaluated in multiple studies as a potential marker for AMR. Gupta et al. evaluated the routine use of C4d immunofluorescence on endomyocardial biopsies after transplant and found that positive C4d staining correlated with a positive retrospective crossmatch and the presence of DSA . RodriguA potentially useful marker of AMR is CD68, a cell surface antigen present on macrophages. Rodriguez et al. showed that patients with graft dysfunction likely related to AMR had positive staining for intravascular macrophages while patients with preserved graft function did not display CD68 staining . SeveralThe ISHLT recently proposed a new AMR grading scheme that is focussed on a shift towards a pathologic diagnosis . The proA proposed timeline for AMR screening is also proposed in the new ISHLT guidelines . The guiThere has been growing interest in the use of microarray technology to monitor gene expression and diagnose AMR. Microarrays were used to study endothelial-associated gene transcripts (ENDATs) in kidney allograft biopsies , 33. In The development of evidence-based guidelines for the management of AMR in heart transplantation is greatly needed. Persisting controversy over the diagnostic criteria for AMR, continued development of diagnostic techniques and a paucity of robust data to substantiate traditional therapies represent important challenges. At present, the management of AMR is nonstandardized, and optimal surveillance, treatment, and timing of intervention are undefined. AMR is difficult to treat, and therapy remains empiric with supporting evidence limited to small, nonrandomized studies with short followup. Furthermore, although it is widely accepted that AMR associated with allograft dysfunction should be aggressively treated, the management of asymptomatic AMR is less clear. While close surveillance is advocated for this group of patients, the ideal frequency and mode of assessment including evaluation of allograft function, performance of endomyocardial biopsies, and HLA antibody screening are unknown. Additionally, although studies suggest that asymptomatic AMR is associated with reduced patient survival, and an increased risk for development of CAV, implementation of surveillance and treatment has not been proven to improve outcomes , 21.A proposed management algorithm for AMR is summarized in A potential DSA monitoring algorithm is shown in Altered humoral immunity with plasma cell production of antibodies directed against donor antigens on the allograft is primarily responsible for myocardial injury in AMR. Plasma cells are generated by T-lymphocyte-dependent activation and generation of memory B-lymphocytes. Treatment strategies for AMR are directed at inhibiting the humoral response at various levels by targeting (1) removal and blockade of circulating antibodies, (2) depletion of B-lymphocytes, (3) depletion of plasma cells, (4) suppression of T-lymphocyte dependent antibody responses, and (5) inhibition of the complement cascade. While several agents have been used successfully and others show promise, AMR remains difficult to treat and is often refractory to therapy.B-lymphocyte responses, including B-lymphocyte activation, the generation of memory B-lymphocytes, and high-affinity antibodies are dependent on T-lymphocytes. Suppression of T-lymphocytes, therefore, indirectly inhibits B-lymphocytes. Cytolytic antibodies , corticosteroids, calcineurin inhibitors , mycophenolate mofetil, and proliferation signal inhibitors  inhibit T-cell proliferation and/or activation through a variety of mechanisms and may be used to treat AMR. Many of these drugs also exhibit direct effects on B-lymphocytes. For example, thymoglobulin is a polyclonal antibody containing multiple anti-T-lymphocyte antibodies that target classes I and II, HLA molecules, associated ligands , costimulatory molecules, cell adhesion molecules as well as surface molecules expressed on B-lymphocytes (CD20) and plasma cells (CD38 and CD138) . CalcinePlasmapheresis removes circulating plasma antibodies by extracorporeal separation of plasma from cellular blood components using either centrifugation or membrane filtration. As antibody production is unaffected, antibody levels may increase after discontinuation of plasmapheresis. Potential adverse effects include complications related to vascular access, hypotension due intravascular volume contraction, bleeding from depletion of clotting factors as well as allergic reactions and blood-borne infection from plasma repletion with replacement fluids such as albumin and fresh frozen plasma.n < 15) followed. Although these small studies have consistently demonstrated that plasmapheresis is an effective treatment for AMR, variation in protocols with differing duration and frequency of treatment and adjunctive treatment confounds interpretation. Similarly, an ideal plasmapheresis treatment regimen has not been established with the number of treatments in studies varying from days to weeks.In 2001, Grauhan et al. published the first clinical case series of plasmapheresis for the treatment of AMR after heart transplantation . BetweenIntravenous immunoglobulin (IVIG) is comprised of 90% IgG, extracted from pooled plasma from between 50,000 and 100,000 blood donors . A numbeExperience with IVIG in transplantation is largely derived from renal transplantation. In this patient population, IVIG has mostly been evaluated for desensitization and shown to reduce PRA and increase conversion to a negative complement dependent cytotoxic crossmatch . RecentlRituximab is a chimeric monoclonal IgG antibody, comprised of human constant regions and mouse variable regions directed against the CD20 pan-B lymphocyte surface molecule. Rituximab depletes B-lymphocytes by antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and induction of apoptosis through CD20 cellular crosslinking and caspase activation . Rituximn = 8) [2/week for up to 4 weeks. Pharmacodynamic studies in patients with lymphoma have shown maximal B-lymphocyte depleting effects at three to four months with low or undetectable numbers in the peripheral blood for up to six months and full recovery within 12 months [The efficacy of Rituximab for AMR treatment after heart transplant has only been examined in case reports and one small case series (n = 8) . The maj2 months . Interes2 months . Further2 months , 51.The safety and tolerability of rituximab is well established in the context of hematologic malignancies. Mild-to-moderate first dose infusion-related reaction with fever and hives account for the majority of adverse effects. Concerns have, however, been raised over an increased risk of serious infections with rituximab therapy after transplantation. In a study of 22 renal transplant recipients, rituximab was shown to be effective in treating AMR but associated with a high incidence of infection . Eighty-Proteasomes are responsible for the processing and degradation of ubiquitin-labeled proteins. Bortezomib is a selective 26S proteasome inhibitor depleting plasma cells and antibody production by inducing apoptosis via cell cycle arrest as well as inducing the unfolded protein response due to increased endoplasmic reticulum stress from accumulation of misfolded proteins , 54. Addn = 81), but also included five heart transplant recipients. Bortezomib was administered in four doses, each preceded by plasmapheresis, and all patients also received a single dose of rituximab. Over an average 9 month followup, 58% of patients showed histologic improvement, with 81% and 96% graft and patient survival, respectively. Additionally, there was a >50% reduction in DSA in 40% of patients at 14 days after treatment. An 11.2% incidence of opportunistic infection was reported. Studies in transplantation and multiple myeloma series have reported high tolerability of Bortezomib with common adverse effects including fatigue, gastrointestinal toxicity, thrombocytopenia, neutropenia, and peripheral neuropathy.Bortezomib is currently approved for the treatment of multiple myeloma. Small studies in renal transplant patients have shown that bortezomib effectively decreases HLA antibodies in patients with stable renal function and is effective in treating refractory AMR –59. WoodEculizumab is a humanized monoclonal antibody directed against C5, thereby preventing complement activation by inhibiting cleavage of C5 to C5a and C5b, and formation of the membrane attack complex. Eculizumab is currently approved in the United States for the treatment of paroxysmal nocturnal hemoglobinuria. Experimental studies in sensitized mice have shown that eculizumab as part of a multi-immunosuppressive regimen prevents allograft rejection and improves allograft survival . At presThe diagnosis and management of AMR in heart transplantation has evolved significantly over the last decade. Current international multidisciplinary consensus recommend a pathologic-based diagnosis, combining specific histologic and immunopathologic features on endomyocardial biopsy and utilizing accompanying allograft dysfunction and/or circulating DSA as additional supporting evidence. AMR as a disease entity will continue to evolve and become better defined with advances in diagnostic techniques and improved understanding of key pathogenic molecular and immunologic mechanisms. Current available therapeutic options modify the humoral immune response by targeting the removal and blockade of antibodies, and the depletion of T- and B-lymphocytes. Emerging therapies showing promise include agents that directly deplete plasma cells and antibody production, such as bortezomib, as well as complement inhibiting agents such as eculizumab. The scarcity of heart transplantation as a clinical resource and current poor patient and allograft outcomes associated with AMR provide significant impetus for the development of effective treatment strategies. It is anticipated that the 2011 ISHLT consensus guidelines on AMR diagnosis and management will further facilitate the goal of developing a standardized evidence based management approach to AMR in heart transplantation.


Human molecular cytogenetics integrates the knowledge on chromosome and genome organization at the molecular and cellular levels in health and disease. Molecular cytogenetic diagnosis is an integral part of current genomic medicine and is the standard of care in medical genetics and cytogenetics, reproductive medicine, pediatrics, neuropsychiatry and oncology. Regardless numerous advances in this field made throughout the last two decades, researchers and practitioners who apply molecular cytogenetic techniques may encounter several problems that are extremely difficult to solve. One of them is undoubtedly the occurrence of somatic genome and chromosome variations, leading to genomic and chromosomal mosaicism, which are related but not limited to technological and evaluative limitations as well as multiplicity of interpretations. More dramatically, current biomedical literature almost lacks descriptions, guidelines or solutions of these problems. The present article overviews all these problems and gathers those exclusive data acquired from studies of genome and chromosome instability that is relevant to identification and interpretations of this fairly common cause of somatic genomic variations and chromosomal mosaicism. Although the way to define pathogenic value of all the intercellular variations of the human genome is far from being completely understood, it is possible to propose recommendations on molecular cytogenetic diagnosis and management of somatic genome variations in clinical population. It is hard to find a cytogeneticist who has not encountered the problem of interpreting mosaicism. It seems to be relatively simple when the majority of cells are abnormal, but is extremely difficult in low-level mosaics or in cases of detecting cryptic, complex or dynamic mosaic cell populations. With the introduction of molecular cytogenetic techniques into basic research and diagnostic practice, this has become not only a problem of practicing cytogeneticists, but a major focus of biomedicine encompassing genomics, medical genetics, neuropsychiatry, aging research and reproductive medicine . Since pThere are two main problems surrounding SGV detection during molecular cytogenetic diagnosis. The first one is technological. The majority of the researchers’ audience is unfamiliar with techniques providing for the highest resolution of SGV evaluation, even though such technologies do exist . AdditioThis article overviews both problems in the light of new developments in molecular cytogenetics. Further, we have attempted to define the way it can be managed in molecular cytogenetic diagnosis of chromosome imbalances in normal and clinical population on the basis of rare data on SGV. Additionally, associations between this phenomenon and disease are described in order to simplify interpretations of mosaicism. Finally, brief troubleshooting and perspectives of SGV research in this extent are given to assist researchers in molecular diagnosis of mosaicism.To get a view of SGV impact on molecular cytogenetic diagnosis, one has to refer to the diseases that are associated with. These are malignant , 6, braiin situ hybridization) and CGH (comparative genomic hybridization) or, more precisely, array CGH. However, the most applicable methods for detection of SGV are based on FISH . Table via molecular genetic techniques . Therefore, a molecular cytogenetic technique applied in this instance has to provide simultaneously for high resolution and for single-cell analysis [As one can see, morbid conditions, which are in need of molecular cytogenetics, are either those caused by subtle genomic rearrangements (detected at molecular level) or those associated with mosaicism in a proportion of cases. These are more commonly manifested as cases of chromosomal mosaicism . Syndromanalysis . The nexRecently, a series of reviews has addressed the level of excellence in molecular cytogenetics with respect to identification of SGV , 21, 38.Regardless developments in molecular cytogenetics that allow to analyze either interphase nuclei or DNA isolated from any cell type, the major part of cases requiring cytogenetic evaluation are firstly assessed through metaphase-analysis-based techniques , 21, 48.via transcriptional changes [As previously noted, the confirmation of mosaicism  in an individual leads to the problem of the interpretation. In other words, pathogenicity of mosaicism or SGV is always a matter of conjecture. It seems to be easier when abnormal cell lines are prevalent, but the borderline between pathogenic condition and benign variation is undetermined . This is changes , 53. Stu changes -42, 54.Since the data on SGV were rarely confirmed to be pathogenic, it is better to consider such genomic variations rather a neutral or a susceptibility factor than a causative genetic abnormality. Unless repeatedly described in the literature, low-level mosaics or SGV cannot be defined as disease-causing mutations. It does not mean that some disease with strong genetic background do not require molecular cytogenetic diagnosis respecting the presence of SGV, but suggests more critical assessment as to constitutional genomic rearrangements. In summary, even though several lines of evidence suggest an association between low-level mosaics, SGV and an abnormal phenotype, their clinical interpretation and diagnostic value seem to require additional case-control studies.in vitro  [2 summarizes experimentally tested recommendations for detection of mosaicism (SGV).Molecular cytogenetic diagnosis of SGV means: (i) scoring of rare events; (ii) possible lack of reproducibility; (iii) different approaches may not yield similar results; (iv) detection of SGV does not imply guaranteed diagnostic success. Together, it makes an impression that the solution of all the difficulties surrounding this task is hardly possible. Notwithstanding, either separate or sequential application of numerous molecular cytogenetic techniques gives extremely high resolution for scoring single-cell rare events , 36-42. nalyses) , 40-47. nalyses) , 40-47. 2 are unlikely to be combined for producing unified guidelines. The latter is mainly caused by different techniques applied for the development and area of application. Therefore, to solve the problem, a large-scale study of several cell populations acquired from different somatic human tissues appears to be strongly required. Nonetheless, before such data will be available, one has to follow these recommendations or to develop new ones on basis of original molecular cytogenetic investigation.The recommendations presented in Table Despite of difficulties in interpreting results, there are positive associations between SGV and several morbid conditions -47, 54. SGV manifesting as chromosome-specific mosaicism or instability are common among spontaneous abortions in the first trimester ; chromos1) depicts the proposed scheme of molecular cytogenetic diagnosis taking into account all the points discussed herein. It is noteworthy that previous schemes of molecular diagnosis in the available biomedical literature have never addressed the detection of SGV manifested at chromosomal or subchromosomal level . Together, one can get an idea of the huge amount of “diagnostic work” that has to be performed for high-resolution diagnosis of the mosaicism. Additionally, “starting techniques” have to be those providing for whole genome screen  CGH or array CGH). However, it is to keep in mind that CGH-based techniques are poorly applicable for low-level mosaicism and SGV detection in large cell populations [1 and [Taking into account all the developments in the field of molecular cytogenetics as well as main characteristics of more popular FISH-, CGH- and array-CGH-based techniques , 63, 64,s [1 and , 38). We1). Thus, to provide a highly technological medical care , these have to be introduced. It is hard not to recognize that current biomedical diagnostic research and practice are more focused on constitutional genomic rearrangements that are easier to interpret and to use for disease-association studies , 64. As The problems that surround diagnosis of SGV and mosaicism remain to be thoroughly addressed by large-scale forthcoming studies targeted at definition of causative amount of abnormal cells per analysis, local effect on cellular/tissular physiology with respect to potential effects on the whole organism, correlations between metaphase and interphase analyses in cases of molecular cytogenetic diagnosis of chromosome abnormalities. To this end, we would like to point out that current molecular cytogenetics possess tools for high-resolution detection of SGV, which have to be used for uncovering the biomedical significance of this common and, probably, most mysterious type of genomic variations.Hopefully, further research will be able to solve this complex and widely relevant problem.