Virulence gene expression of Vibrio parahaemolyticus in the viable but nonculturable state

Vibrio parahaemolyticus is a food-borne pathogen commonly associated with the consumption of raw or undercooked seafood resulting in primary infections of the human gastrointestinal tract. It is estimated to cause about 4500 illnesses each year in the United States. However, infection from this food-borne pathogen can be avoided if this organism is detected in the implicated food, prior to consumption. Current standard methods of detecting this organism are dependent on the culturability of the bacteria. Detection based on an organism’s culturability may be problematic as V. parahaemolyticus has been known to exist in a viable but nonculturable (VBNC) state. Bacteria in the VBNC state are characterized by low levels of metabolic activity and the inability to be cultured by standard laboratory practices. When bacteria enter the VBNC state, their gene expression profile may be different than the culturable counterpart. We were interested in comparing the expression of two virulence-associated genes between VBNC and culturable cells of V. parahaemolyticus. V. parahaemolyticus RIMD2210633 was incubated at 4°C in modified Morita mineral salt solution supplemented with 0.5% NaCl (MMS) or trypticase soy broth supplemented with 2% NaCl (TSBS), which represented nutrient poor and rich conditions, respectively. The number of VBNC and culturable cells were determined by standard plate count and fluorescence microscopy. The expression levels of virulence-associated genes tdh2 and escU, were measured relative to the housekeeping gene, pvsA, by qRT-PCR. Nutrient availability and temperatures exerted variable effects on the virulence gene expression. It is possible that VBNC V. parahaemolyticus cells may retain their pathogenicity potential

Implementing the use of Deliberative Practice on Teaching Endotracheal Intubation To First Year Medical Students

Purpose: Deliberate practice is defined as a learning theory that values a learner’s holistic ability to learn a task while integrating improvements in targeted skills. The theory states that in order to achieve maximal efficiency, a learner needs time for self-reflection and instantaneous feedback to make improvements before engaging in the next task. Here, we created an Intubation Training Module to study the effectiveness of deliberate practice on teaching endotracheal intubation to first year medical students. Methods: A two-part intubation workshop was created to investigate the effectiveness of deliberate practice and to compare intubation competency between the number of intubation cycles assigned. In Part 1, subjects (n=6) were given a lesson on airway intubation by an attending emergency medicine physician. An Airway Checklist was used as a guide. Deliberate practice and feedback was provided to students in real-time. The number of intubation cycles completed by each student varied: 1x, 5x, and 7x. Part 2 of the workshop occurred a week after where students were asked to recall the Airway Checklist steps to assess their retention rate. Performance and retention rates of the 3 groups were compared. Results and Conclusions: A survey was provided after the initial workshop to collect qualitative feedback; the initial endotracheal intubation workshop was well received by medical students and student mean satisfaction was 9.5 (0 being completely unsatisfied, 10 being extremely pleased). An ANOVA test was conducted among the 3 groups to compare retention rate; using the Airway Checklist, the number of checkboxes for each subject was calculated to assess retention rate (p-value = 0.664). Statistical significance could not be determined; future work will include increased sample size and higher intubation cycles for comparison

Priming Leukemia with 5-Azacytidine Enhances CAR T Cell Therapy

Purpose: Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence. Methods and Results: Using patient-derived xenograft (PDX) mouse models of CD19+ B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ+ effector T cells in co-cultures with CD19+ leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is TNFSF4 which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. TNFSF4 is heterogeneously expressed in a panel of pediatric PDXs, and high TNFSF4 expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing. Conclusion: We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered

Clonal Deletion Prunes but Does Not Eliminate Self-Specific αβ CD8+ T Lymphocytes

SummaryIt has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8+ T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus)

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the genera Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus (Ursus americanus circovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanus parvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases by in situ hybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears

Emergence of the rtA181T/sW172* mutant increased the risk of hepatoma occurrence in patients with lamivudine-resistant chronic hepatitis B

<p>Abstract</p> <p>Background</p> <p>Development of the hepatitis B virus (HBV) rtA181T/sW172* mutant could occur during prolonged lamivudine (LAM) therapy, conferring cross resistance to adefovir. Recent studies demonstrated an increased oncogenic potential of this mutant in NIH3T3 cells. In this study, we aimed to investigate the clinical significance of this finding.</p> <p>Methods</p> <p>Serum samples from 123 LAM-resistant chronic hepatitis B patients were submitted for virological assays. A highly sensitive amplification created restriction enzyme site (ACRES) method was devised to detect small amounts of the rtA181T mutant in the serum. Virological factors including HBV-DNA level, genotype, precore G1896A, BCP A1762T/G1764A, rtM204I/V, rtA181T and pre-S internal deletion mutations as well as clinical variables including subsequent use of rescue drugs were submitted for outcome analysis.</p> <p>Results</p> <p>By use of the highly sensitive ACRES method, the rtA181T mutant was detectable in 10 of the 123 LAM-resistant patients. During the mean follow-up period of 26.2 ± 16.4 months (range 2 to 108 months), 3 of the 10 (30.0%) rtA181T-positive patients and 2 of the 113 (1.8%) rtA181T-negative patients developed hepatocellular carcinoma (HCC). Kaplan-Meier analysis indicated that the presence of rtA181T mutation (P < 0.001), age > 50 years (P = 0.001), and liver cirrhosis (P < 0.001) were significantly associated with subsequent occurrence of HCC. All 5 HCC patients belonged to the older age and cirrhosis groups.</p> <p>Conclusions</p> <p>Emergence of the rtA181T/sW172* mutant in LAM-resistant patients increased the risk of HCC development in the subsequent courses of antiviral therapy.</p

Enteropathy-associated T cell lymphoma subtypes are characterized by loss of function of SETD2

Enteropathy-associated T cell lymphoma (EATL) is a lethal, and the most common, neoplastic complication of celiac disease. Here, we defined the genetic landscape of EATL through whole-exome sequencing of 69 EATL tumors. SETD2 was the most frequently silenced gene in EATL (32% of cases). The JAK-STAT pathway was the most frequently mutated pathway, with frequent mutations in STAT5B as well as JAK1 , JAK3 , STAT3 , and SOCS1 . We also identified mutations in KRAS , TP53 , and TERT . Type I EATL and type II EATL (monomorphic epitheliotropic intestinal T cell lymphoma) had highly overlapping genetic alterations indicating shared mechanisms underlying their pathogenesis. We modeled the effects of SETD2 loss in vivo by developing a T cell–specific knockout mouse. These mice manifested an expansion of γδ T cells, indicating novel roles for SETD2 in T cell development and lymphomagenesis. Our data render the most comprehensive genetic portrait yet of this uncommon but lethal disease and may inform future classification schemes

Calcium Flux in Neutrophils Synchronizes β2 Integrin Adhesive and Signaling Events that Guide Inflammatory Recruitment

Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity β2 integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner