Characterization of Mycobacterium orygis, Mycobacterium bovis, and Mycobacterium caprae Infections in Humans in Western Canada.

Epidemiologic research on zoonotic tuberculosis historically used Mycobacterium bovis as a surrogate measure, however, increased reports of human tuberculosis caused by other animal-associated Mycobacterium tuberculosis complex members like Mycobacterium orygis necessitates their inclusion. We performed a retrospective cohort study including persons infected with any animal-lineage M. tuberculosis complex species in Alberta, Canada, from January 1995 to July 2021, identifying 42 patients (20 M. bovis, 21 M. orygis, one M. caprae). Demographic, epidemiologic and clinical characteristics were compared against persons with culture-confirmed M. tuberculosis infection. The proportion of culture-positive infections caused by M. orygis increased continuously from 2016-2020. Significantly more females at a higher median age were impacted by M. orygis, with all patients originating from South Asia. M. bovis caused significantly more extra-pulmonary disease, and disproportionately impacted young females, particularly those pregnant or post-partum. All infections were acquired abroad. These findings can aid in developing targeted public health interventions.

Continuity obstetric care demonstrates greater vaginal birth after caesarean success.

BACKGROUND: Australia's caesarean rate is higher than Organisation for Economic Co-operation and Development (OECD) average, and is rising. Vaginal birth after caesarean (VBAC) is safe for selected women. Midwifery continuity of care (CoC) is associated with higher rates of vaginal birth compared to other models; however, impacts on VBAC attempts and success are unknown. AIMS: The primary aim was to determine if there is a difference in achieving VBAC between CoC and non-CoC (NCoC) models. The secondary aim was to determine if there is a difference in the proportion of women attempting VBAC between these models. MATERIALS AND METHODS: Retrospective review of antenatal records and birthing data of all women who birthed in 2021 with one or more previous caesareans. Women were included if they had two or fewer caesareans. Women were excluded if contraindications to VBAC existed. RESULTS: There were 142/1109 (12.8%) women who had previous caesareans and were eligible to attempt VBAC. There were 47/109 (43.1%) women who attempted vaginal birth after one caesarean with 78.7% success. After one caesarean, women in CoC were more likely to achieve VBAC than NCoC (45.2% vs 26.1%; relative risk (RR) 1.76, 95% CI 1.04-3.00), although when stratified by private and midwifery CoC models, women in midwifery CoC models were more likely to be successful (private RR 0.69, 95% CI 0.23-2.07 vs midwifery RR 2.48, 95% CI 1.50-4.11). Women in CoC were more likely to attempt VBAC (54.7% vs 34.8%; RR 1.57, 95% CI 1.02-2.41), and receive counselling about VBAC (92.5% vs 62%; RR 1.48, 95% CI 1.41-3.11). CONCLUSION: CoC improves the rate of attempted and successful VBAC through several factors, including increased counselling and greater provision of birth choices.

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Whole genome sequencing-based identification of human tuberculosis caused by animal-lineage Mycobacterium orygis.

A recently described member of the Mycobacterium tuberculosis complex (MTBC) is Mycobacterium orygis, which can cause disease primarily in animals but also in humans. Although M. orygis has been reported from different geographic regions around the world, due to a lack of proper identification techniques, the contribution of this emerging pathogen to the global burden of zoonotic tuberculosis is not fully understood. In the present work, we report single nucleotide polymorphism (SNP) analysis using whole genome sequencing (WGS) that can accurately identify M. orygis and differentiate it from other members of the MTBC species. WGS-based SNP analysis was performed for 61 isolates from different provinces in Canada that were identified as M. orygis. A total of 56 M. orygis sequences from the public databases were also included in the analysis. Several unique SNPs in the gyrB, PPE55, Rv2042c, leuS, mmpL6, and mmpS6 genes were used to determine their effectiveness as genetic markers for the identification of M. orygis. To the best of our knowledge, five of these SNPs, viz., gyrB 277 (A→G), gyrB 1478 (T→C), leuS 1064 (A→T), mmpL6 486 (T→C), and mmpS6 334 (C→G), are reported for the first time in this study. Our results also revealed several SNPs specific to other species within MTBC. The phylogenetic analysis shows that the studied genomes were genetically diverse and clustered with M. orygis sequences of human and animal origin reported from different geographic locations. Therefore, the present study provides a new insight into the high-confidence identification of M. orygis from MTBC species based on WGS data, which can be useful for reference and diagnostic laboratories.

Prevalence of Invasive Bacterial Infection in Hypothermic Young Infants: A Multisite Study.

OBJECTIVE: To determine the prevalence of bacteremia and meningitis (invasive bacterial infection [IBI]) in hypothermic young infants, and also to determine the prevalence of serious bacterial infections (SBI) and neonatal herpes simplex virus and to identify characteristics associated with IBI. STUDY DESIGN: We conducted a retrospective cohort study of infants ≤90 days of age who presented to 1 of 9 hospitals with historical or documented hypothermia (temperature ≤36.0°C) from September 1, 2017, to May 5, 2021. Infants were identified by billing codes or electronic medical record search of hypothermic temperatures. All charts were manually reviewed. Infants with hypothermia during birth hospitalization, and febrile infants were excluded. IBI was defined as positive blood culture and/or cerebrospinal fluid culture treated as a pathogenic organism, whereas SBI also included urinary tract infection. We used multivariable mixed-effects logistic regression to identify associations between exposure variables and IBI. RESULTS: Overall, 1098 young infants met the inclusion criteria. IBI prevalence was 2.1% (95% CI, 1.3-2.9) (bacteremia 1.8%; bacterial meningitis 0.5%). SBI prevalence was 4.4% (95% CI, 3.2-5.6), and neonatal herpes simplex virus prevalence was 1.3% (95% CI, 0.6-1.9). Significant associations were found between IBI and repeated temperature instability (OR, 4.9; 95% CI, 1.3-18.1), white blood cell count abnormalities (OR, 4.8; 95% CI, 1.8-13.1), and thrombocytopenia (OR, 5.0; 95% CI, 1.4-17.0). CONCLUSIONS: IBI prevalence in hypothermic young infants is 2.1%. Further understanding of characteristics associated with IBI can guide the development decision tools for management of hypothermic young infants.

Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal. There is a dire need for better therapeutic targets. Cancer cells have increased demand for sugars, amino acids, and lipids and therefore up-regulate various nutrient transporters to meet this demand. In PDAC, SLC6A14 (an amino acid transporter (AAT)) is up-regulated, affecting overall patient survival. Previously we have shown using in vitro cell culture models and in vivo xenograft mouse models that pharmacological inhibition of SLC6A14 with α-methyl-l-tryptophan (α-MLT) attenuates PDAC growth. Mechanistically, blockade of SLC6A14-mediated amino acid transport with α-MLT leads to amino acid deprivation, eventually inhibiting mTORC1 signaling pathway, in tumor cells. Here, we report on the effect of Slc6a14 deletion on various parameters of PDAC in KPC mice, a model for spontaneous PDAC. Pancreatic tumors in KPC mice show evidence of Slc6a14 up-regulation. Deletion of Slc6a14 in this mouse attenuates PDAC growth, decreases the metastatic spread of the tumor, reduces ascites fluid accumulation, and improves overall survival. At the molecular level, we show lower proliferation index and reduced desmoplastic reaction following Slc6a14 deletion. Furthermore, we find that deletion of Slc6a14 does not lead to compensatory up-regulation in any of the other amino transporters. In fact, some of the AATs are actually down-regulated in response to Slc6a14 deletion, most likely related to altered mTORC1 signaling. Taken together, these results underscore the positive role SLC6A14 plays in PDAC growth and metastasis. Therefore, SLC6A14 is a viable drug target for the treatment of PDAC and also for any other cancer that overexpresses this transporter.

Deletion of autophagy related, ATG1 and F-box motif encoding YDR131C, together, lead to synthetic growth defects and flocculation behavior in Saccharomyces cerevisiae.

Ubiquitin proteasome system (UPS) and autophagy both pathways are involved in clearing the nonessential cellular components and also crosstalk during cellular response to normal and stress conditions. The F-box motif proteins constitute the SCF-E3 ligase complex of the UPS pathway in Saccharomyces cerevisiae and are involved in the substrate recruitment for ubiquitination. The ATG1 encoded Atg1p, a conserved serine-threonine kinase is crucial for the autophagy process. Here in this study, we report that loss of F-box motif encoding YDR131C and ATG1 together results in growth defects, floc formation, sensitivity to hydroxyurea, methyl methanesulfonate, and hydrogen peroxide. Both the genes also interact with the flocculation-related genes (FLO) and associate with gene ontology terms "ubiquitin-protein transferase activity" and "cellular catabolic process." Based on in silico analysis and experimental evidence we conclude that YDR131C and ATG1 function in parallel pathways to regulate the growth, flocculation, and stress response.

Compilation of 10 Years of MIRU-VNTR Data: Canadian National Tuberculosis Laboratory's Experience.

Tuberculosis is a significant cause of morbidity worldwide and is a priority at the provincial and federal levels in Canada. It is known that tuberculosis transmission networks are complex and span many years as well as different jurisdictions and countries. MIRU-VNTR is a universal tuberculosis genotyping method that utilizes a 24-loci pattern and it has shown promise in identifying inter and intrajurisdictional clusters within Canada. MIRU-VNTR data collected over 10 years from the National Reference Centre for Mycobacteriology (NRCM) were analyzed in this study. Some clusters were unique to a single province/territory, while others spanned multiple provinces and/or territories in Canada. The use of a universal laboratory test can enhance contact tracing, provide geographical information on circulating genotypes, and hence, aid in tuberculosis investigation by public health. The housing of all data on one platform, technical ease of the method, easy exchange of data between jurisdictions, and strong collaboration with laboratories and surveillance units at the provincial and federal levels have the potential to identify possible outbreaks in real time.

Molecular Epidemiology of Mycobacterium tuberculosis To Describe the Transmission Dynamics Among Inuit Residing in Iqaluit Nunavut Using Whole-Genome Sequencing.

BACKGROUND: In the last decade, tuberculosis (TB) incidence among Inuit in the Canadian Arctic has been rising. Our aim was to better understand the transmission dynamics of TB in this remote region of Canada using whole-genome sequencing. METHODS: Isolates from patients who had culture-positive pulmonary TB in Iqaluit, Nunavut, between 2009 and 2015 underwent whole-genome sequencing (WGS). The number of transmission events between cases within clusters was calculated using a threshold of a ≤3 single nucleotide polymorphism (SNP) difference between isolates and then combined with detailed epidemiological data using a reproducible novel algorithm. Social network analysis of epidemiological data was used to support the WGS data analysis. RESULTS: During the study period, 140 Mycobacterium tuberculosis isolates from 135 cases were sequenced. Four clusters were identified, all from Euro-American lineage. One cluster represented 62% of all cases that were sequenced over the entire study period. In this cluster, 2 large chains of transmission were associated with 3 superspreading events in a homeless shelter. One of the superspreading events was linked to a nonsanctioned gambling house that resulted in further transmission. Shelter to nonshelter transmission was also confirmed. An algorithm developed for the determination of transmission events demonstrated very good reproducibility (κ score .98, 95% confidence interval, .97-1.0). CONCLUSIONS: Our study suggests that socioeconomic factors, namely residing in a homeless shelter and spending time in a gambling house, combined with the superspreading event effect may have been significant factors explaining the rise in cases in this predominantly Inuit Arctic community.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Identification and Characterization of a Novel FosA7 Member from Fosfomycin-Resistant Escherichia coli Clinical Isolates from Canadian Hospitals.

Here, we characterize the fosA genes from three Escherichia coli clinical isolates recovered from Canadian patients. Each fosA sequence was individually overexpressed in E. coli BW25113, and antimicrobial susceptibility testing was performed to assess their role in fosfomycin resistance. The findings from this study identify and functionally characterize FosA3, FosA8, and novel FosA7 members and highlight the importance of phenotypic characterization of fosA genes.

A real-time PCR assay for rapid identification of inducible and acquired clarithromycin resistance in Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. METHODS: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. RESULTS: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. CONCLUSION: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

Peptide Vaccine Formulation Controls the Duration of Antigen Presentation and Magnitude of Tumor-Specific CD8+ T Cell Response.

Despite remarkable progresses in vaccinology, therapeutic cancer vaccines have not achieved their full potential. We previously showed that an excessively long duration of Ag presentation critically reduced the quantity and quality of vaccination-induced T cell responses and subsequent antitumor efficacy. In this study, using a murine model and tumor cell lines, we studied l-tyrosine amino acid-based microparticles as a peptide vaccine adjuvant with a short-term Ag depot function for the induction of tumor-specific T cells. l-Tyrosine microparticles did not induce dendritic cell maturation, and their adjuvant activity was not mediated by inflammasome activation. Instead, prolonged Ag presentation in vivo translated into increased numbers and antitumor activity of vaccination-induced CD8+ T cells. Indeed, prolonging Ag presentation by repeated injection of peptide in saline resulted in an increase in T cell numbers similar to that observed after vaccination with peptide/l-tyrosine microparticles. Our results show that the duration of Ag presentation is critical for optimal induction of antitumor T cells, and can be manipulated through vaccine formulation.

Generation of Catalytic Antibodies Is an Intrinsic Property of an Individual's Immune System: A Study on a Large Cohort of Renal Transplant Patients.

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the current study, we have followed the evolution of the levels of catalytic IgG in a large cohort of renal transplant patients over a 2-y period. Our results demonstrate that, prior to transplant, patients with renal failure present with heterogeneous levels of IgG hydrolyzing the generic proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA) substrate. PFR-MCA hydrolysis was greater for patients' IgG than for a therapeutic preparation of pooled IgG from healthy donors. Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 mo followed by a steady increase during the next 21 mo. Patients who displayed high levels of catalytic IgG pretransplant recovered high levels of catalytic Abs 2 y posttransplant. Interestingly, IgG-mediated hydrolysis of a model protein substrate, procoagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, whereas it did 12 mo posttransplant. Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual's immune system and that recovery of pretransplant levels of catalytic IgG is accompanied by changes in the repertoire of target Ags.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.

Effect of high-protein meal replacement on weight and cardiometabolic profile in overweight/obese Asian Indians in North India.

The aim of the present study was to evaluate the impact of a high-protein meal replacement (HPMR) on weight and metabolic, lipid and inflammatory parameters in overweight/obese Asian Indians. In this 12-week open-label, parallel-arm randomised controlled trial, 122 overweight/obese men and women were administered either a HPMR or a control diet after 2 weeks of diet and exercise run-in. Body weight, waist circumference (WC), percentage body fat (%BF), fasting blood glucose, post-oral glucose tolerance test (post-OGTT) blood glucose, fasting and post-OGTT serum insulin, lipid profile, high-sensitivity C-reactive protein (hs-CRP), kidney function and hepatic aminotransferases were assessed before and after the intervention. Additional improvement in mean values for the following parameters in the HPMR group compared with the control group was observed: body weight, 4·9 % (95 % CI 3·8, 6·1; P<0·001); WC, 3·8 % (95 % CI 2·5, 5·1; P<0·001); %BF, 6·3 % (95 % CI 4·3, 8·2; P<0·001); systolic blood pressure, 2·8 % (95 % CI 0·4, 5·1; P=0·002); diastolic blood pressure, 3·5 % (95 % CI 0·7, 6·3; P= 0·01); post-OGTT blood glucose, 7·3 % (95 % CI 1·4, 13·1; P=0·02); total cholesterol, 2·5 % (95 % CI 1·6, 3·5; P<0·001); LDL-cholesterol, 7·3 % (95 % CI 1·7, 12·9; P<0·01); alanine aminotransferase, 22·0 % (95 % CI 2·1, 42; P=0·03) and aspartate aminotransferase, 15·2 % (95 % CI 0·9, 29·5; P=0·04). The absolute reduction in BMI was 0·9 units in the intervention arm compared with the control arm (-0·9 %, 95 % CI -1·4, -0·5; P<0·001) and in serum TAG was 11·9 mg/dl (-11·9 mg/dl, 95 % CI -21·1, -2·7; P<0·01). The reduction in fasting serum insulin in the intervention v. the control arm was 3·8 v. 0 % (P=0·002); post-OGTT serum insulin was 50·3 v. 77·3 mU/l (P=0·005); and hs-CRP, 16·7 % v. 0 % (P=0·002). These findings show that intervention with HPMR may lead to significant weight loss and improvement in obesity measures, metabolic, lipid and inflammatory parameters and hepatic transaminases in overweight/obese Asian Indians.

Early allograft dysfunction after liver transplantation: an intermediate outcome measure for targeted improvements.

BACKGROUND: The term early allograft dysfunction (EAD) identifies liver transplant (LT) allografts with initial poor function and portends poor allograft and patient survival. Aims of this study are to use EAD as an intermediate outcome measure in a large single center cohort and identify donor, recipient and peri-operative risk factors. MATERIAL AND METHODS: In 1950 consecutive primary LT, donor, recipient and peri-operative data were collected. EAD was defined by the presence of one or more of the following: total bilirubin ≥ 10 mg/dL (171 µmol/L) or, INR ≥ 1.6 on day 7, and ALT/AST > 2,000 IU/L within the first 7 days. RESULTS: The incidence of EAD was 26.5%. 1-, 3-, and 5-year allograft and patient survival for patients who developed EAD were significantly inferior to those who did not (P < 0.01 at all time points). Multivariate analysis demonstrated associations in the development of EAD with recipient pre-operative ventilator status, donation after cardiac death allografts, donor age, allograft size, degree of steatosis, operative time and intra-operative transfusion requirements (all P < 0.01). Patients with EAD had a significantly longer hospitalization at 20.9 ± 38.9 days (median: 9; range: 4-446) compared with 10.7 ± 13.5 days (median: 7; range: 3-231) in patients with no EAD (P < 0.01). CONCLUSIONS: This is the largest single center experience demonstrating incidence of EAD and identifying factors associated with development of EAD. EAD is a useful intermediate outcome measure for allograft and patient survival. Balancing recipient pretransplant conditions, donor risk factors and intra-operative conditions are necessary for avoiding EAD.

Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance. Intravenous immunoglobulin (IVIg), a therapeutic preparation of normal pooled human IgG, expands Tregs in various experimental models and in patients. However, the cellular and molecular mechanisms by which IVIg expands Tregs are relatively unknown. As Treg expansion in the periphery requires signaling by antigen-presenting cells such as dendritic cells (DCs) and IVIg has been demonstrated to modulate DC functions, we hypothesized that IVIg induces distinct signaling events in DCs that subsequently mediate Treg expansion. We demonstrate that IVIg expands Tregs via induction of cyclooxygenase (COX)-2-dependent prostaglandin E2 (PGE2) in human DCs. However, costimulatory molecules of DCs such as programmed death ligands, OX40 ligand, and inducible T-cell costimulator ligands were not implicated. Inhibition of PGE2 synthesis by COX-2 inhibitors prevented IVIg-mediated Treg expansion in vitro and significantly diminished IVIg-mediated Treg expansion in vivo and protection from disease in experimental autoimmune encephalomyelitis model. IVIg-mediated COX-2 expression, PGE2 production, and Treg expansion were mediated in part via interaction of IVIg and F(ab')2 fragments of IVIg with DC-specific intercellular adhesion molecule-3-grabbing nonintegrin. Our results thus uncover novel cellular and molecular mechanism by which IVIg expands Tregs.

Evaluation of host genetics on outcome of tuberculosis infection due to differences in killer immunoglobulin-like receptor gene frequencies and haplotypes.

BACKGROUND: Outcome of Mycobacterium tuberculosis (Mtb) infection is affected by virulence of the infecting strain of Mtb, host environment, co-morbidities, and the genetic composition of the host, specifically the presence or absence of genes involved in immune responses/regulation. It is hypothesized that specific killer immunoglobulin-like receptor (KIR) genes may be associated with Mtb infection and clinical outcome. This cross-sectional study examined the KIR gene frequencies, profiles, and haplotypes of individuals with active tuberculosis, latent tuberculosis infection, compared to TB and HIV negative healthy controls. RESULTS: Analysis of KIR gene frequencies revealed differences among disease status groups, suggesting that enrichment or depletion of specific KIR genes may direct the disease outcome. Mtb infected individuals were more likely to have a centromeric-AA haplotype compared to controls. CONCLUSION: The differences in KIR gene frequencies and haplotypes may result in differential cytokine expression, contributing to different disease outcomes, and suggest a genetic influence on Mtb susceptibility and pathogenesis.

Intravenous gammaglobulin inhibits encephalitogenic potential of pathogenic T cells and interferes with their trafficking to the central nervous system, implicating sphingosine-1 phosphate receptor 1-mammalian target of rapamycin axis.

Despite an increasing use of high-dose therapy of i.v. gammaglobulin (IVIg) in the treatment of various T cell- and Ab-mediated inflammatory and autoimmune diseases, comprehension of the mechanisms underlying its therapeutic benefit has remained a major challenge. Particularly, the effect of IVIg in T cell-mediated autoimmune conditions remains unexplored. Using an actively induced experimental autoimmune encephalomyelitis model, a T cell-mediated autoimmune condition, we demonstrate that IVIg inhibits the differentiation of naive CD4 T cells into encephalitogenic subsets (Th1 and Th17 cells) and concomitantly induces an expansion of Foxp3(+) regulatory T cells. Further, IVIg renders effector T cells less pathogenic by decreasing the expression of encephalitogenic molecular players like GM-CSF and podoplanin. Intriguingly and contrary to the current arguments, the inhibitory FcγRIIB is dispensable for IVIg-mediated reciprocal modulation of effector and regulatory CD4 subsets. Additionally, F(ab')2 fragments also retained this function of IVIg. IVIg or F(ab')2 fragments decrease the sphingosine-1 phosphate receptor on CD4 cells, thus sequestering these cells in the draining lymph nodes and decreasing their infiltration into the CNS. Our study reveals a novel role of Igs in the modulation of polarization and trafficking of T lymphocytes, accounting for the observed beneficial effect in IVIg therapy.