Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells.

Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell-intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag-specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen-specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients.

Background and Objectives: Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for ß-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Materials and Methods: Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Results: Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. Conclusion: We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay.

Host metabolome predicts the severity and onset of acute toxicities induced by CAR T-cell therapy.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is a highly effective treatment option for patients with relapsed/refractory large B-cell lymphoma. However, widespread use is deterred by the development of clinically significant acute inflammatory toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), that induce significant morbidity and require close monitoring. Identification of host biochemical signatures that predict the severity and time-to-onset of CRS and ICANS may assist patient stratification to enable timely mitigation strategies. Here, we report pretreatment host metabolites that are associated with CRS and ICANS induced by axicabtagene ciloleucel or tisagenlecleucel therapy. Both untargeted metabolomics analysis and validation using targeted assays revealed a significant association between the abundance of specific pretreatment biochemical entities and an increased risk and/or onset of clinically significant CRS (q < .1) and ICANS (q < .25). Higher pretreatment levels of plasma glucose and lower levels of cholesterol and glutamate were associated with a faster onset of CRS. In contrast, low baseline levels of the amino acids proline and glycine and the secondary bile acid isoursodeoxycholate were significantly correlated with clinically significant CRS. Lower concentration of the amino acid hydroxyproline was associated with higher grade and faster onset of ICANS, whereas low glutamine was negatively correlated with faster development of ICANS. Overall, our data indicate that the pretreatment host metabolome has biomarker potential in determining the risk of clinically significant CRS and ICANS, and may be useful in risk stratification of patients before anti-CD19 CAR T-cell therapy.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii with Special Reference to Carbapenemases: A Systematic Review.

Carbapenemases are ß-lactamase enzymes that hydrolyze a variety of ß-lactams including carbapenem and belong to different Ambler classes (A, B, D). These enzymes can be encoded by plasmid or chromosomal-mediated genes. The major issues associated with carbapenemases-producing organisms are compromising the activity and increasing the resistance to carbapenems which are the last resort antibiotics used in treating serious infections. The global increase of pathogen, carbapenem-resistant A. baumannii has significantly threatened public health. Thus, there is a pressing need for a better understanding of this pathogen, to know the various carbapenem resistance encoding genes and dissemination of resistance genes from A. baumannii which help in developing strategies to overcome this problem. The horizontal transfer of resistant determinants through mobile genetic elements increases the incidence of multidrug, extensive drug, and Pan-drug resistant A. baumannii. Therefore, the current review aims to know the various mechanisms of carbapenem resistance, categorize and discuss carbapenemases encoding genes and various mobile genetic elements, and the prevalence of carbapenemase genes in recent years in A. baumannii from various geographical regions.

Ezrin Promotes Antigen Receptor Diversity during B Cell Development by Supporting Ig H Chain Variable Gene Recombination.

Genome-level rearrangements of Ig genes during B cell development are critical for generation of a diverse repertoire of BCRs that bind to a multitude of foreign Ags and some self Ags. Bone marrow B cell development involves a variety of cell-cell interactions, cell migration, and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins toward BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next-generation sequencing to investigate whether Ezrin plays a role in IgH rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDRs (CDR3s) in the BCRs and reduced Shannon entropy. Ezrin-deficient pre-B cells revealed similar utilization of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length and nucleotide additions and deletions, was not altered in Ezrin-deficient pre-B cells. Mechanistically, Ezrin-deficient cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate that Ezrin shapes the BCR repertoire through combinatorial diversification.

Molecular Characterization Identifies Upstream Presence of IS Aba1 to OXA Carbapenemase Genes in Carbapenem-Resistant Acinetobacter baumannii.

Background Evaluating the expression pattern of oxacillinases (OXA) carbapenemases is essential to understand the prevalence and spread of carbapenem resistance Acinetobacter baumannii . Objectives The aim of the study is to evaluate the presence of OXA carbapenemase genes and IS Aba1 upstream to these genes in carbapenem-resistant A. baumannii clinical isolates. Materials and Methods A. baumannii isolated from clinical samples were phenotypically identified and antibiotics sensitivity was performed. Multiplex polymerase chain reaction (PCR) was used to detect OXA51-like gene, OXA carbapenemases genes (OXA-23-like, OXA-24-like, and OXA-58-like), and IS Aba1 in carbapenem-resistant isolates. Results Out of 55 Acinetobacter isolates, 54 were confirmed as A. baumannii by PCR. Bla OXA-23 -like gene was observed in 51 isolates of A. baumannii and none of the isolates showed the presence of bla OXA-24 -like and bla OXA-58 -like genes. Presence of IS Aba1 upstream to OXA-23-like gene, OXA-51-like gene, and both OXA-51-like/OXA-23-like genes was observed in 51, 7, and 4 A. baumannii isolates, respectively. Conclusion The genetic pattern of carbapenem-resistant A. baumannii isolated in this study was unique, which should be factored for clinical protocols to manage infections caused by emerging resistant strains of A. baumannii .

Tyrosine phosphorylation of NLRP3 by the Src family kinase Lyn suppresses the activity of the NLRP3 inflammasome.

In response to microbes and other danger signals, the NLRP3 inflammasome in immune cells triggers the activation of the protease caspase-1, which mediates the maturation of the inflammatory cytokine IL-1ß. Here, we investigated how the NLRP3 inflammasome is regulated. We found that its activation in primary mouse macrophages induced the Src family kinase Lyn to phosphorylate NLRP3 at Tyr918, which correlated with a subsequent increase in its ubiquitination that facilitated its proteasome-mediated degradation. NLRP3 tyrosine phosphorylation and ubiquitination was abrogated in Lyn-deficient macrophages, which produced increased amounts of IL-1ß. Furthermore, mice lacking Lyn were more susceptible to LPS-induced septic shock in an NLRP3-dependent manner. Our data demonstrate that Lyn-mediated tyrosine phosphorylation is a prerequisite for the ubiquitination that dampens NLRP3 inflammasome activity.

Cutting Edge: Myosin 18A Is a Novel Checkpoint Regulator in B Cell Differentiation and Antibody-Mediated Immunity.

We investigated the function of the newly discovered myosin family protein myosin 18A (Myo18A) in Ab-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Myo18A deficiency led to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia and increased splenic IgM-secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing Abs against the major Ag hemagglutinin, associated with persistent accumulation of Ag-specific germinal center B cells and more Ag-specific bone marrow plasma cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into Ab-secreting cells, associated with higher AID and Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, and humoral immunity.

Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

Cutting Edge: Deletion of Ezrin in B Cells of Lyn-Deficient Mice Downregulates Lupus Pathology.

Genetic deletion of the Src family tyrosine kinase Lyn in mice recapitulates human systemic lupus erythematosus, characterized by hyperactive BCR signaling, splenomegaly, autoantibody generation, and glomerulonephritis. However, the molecular regulators of autoimmunity in Lyn-deficient mice and in human lupus remain poorly characterized. In this study, we report that conditional deletion of the membrane-cytoskeleton linker protein ezrin in B cells of Lyn-deficient mice (double knockout [DKO] mice) ameliorates B cell activation and lupus pathogenesis. B cells from DKO mice respond poorly to BCR stimulation, with severe downregulation of major signaling pathways. DKO mice exhibit reduced splenomegaly as well as significantly lower levels of autoantibodies against a variety of autoantigens, including dsDNA, histone, and chromatin. Leukocyte infiltration and deposition of IgG and complement component C3 in the kidney glomeruli of DKO mice are markedly reduced. Our data demonstrate that ezrin is a novel molecular regulator of B cell-associated lupus pathology.

Knowledge of hepatitis B virus infection and its control practices among dental students in an Indian city.

Background Hepatitis B virus infection is a general cause of chronic hepatitis, liver cirrhosis and hepato-cellular carcinoma worldwide. It is highly contagious. It is an important reason for morbidity and mortality in the Indian population. Oral health professionals are at the highest risk. Vaccination for hepatitis B can prevent this deadly disease. Methods The present study was designed to evaluate the degree of awareness, knowledge of hepatitis B infection and status of hepatitis B vaccination among dental students. A cross-sectional study was conducted among 240 students of 3rd year, 4th year and interns of a professional dental course. A pre-tested questionnaire was given to the students of each year. All the data management and analysis were carried out using SPSS software version 16. Results Eighty-six percent of the students had knowledge about hepatitis B infection. The majority of the students had correct knowledge regarding mode of transmission, however, 21% failed to recognize saliva as the mode of hepatitis B transmission. Forty-five percent of the students were vaccinated for hepatitis B. Conclusion The present study concludes that there is reasonable awareness of hepatitis B infection hazards, its transmission and vaccination, among the dental students who will be entering into the profession. However, half of the students were not vaccinated for hepatitis B in our study group, which keeps them at risk to the disease. The Indian Health Ministry should make hepatitis B vaccination mandatory for all health care professionals. A strategy should be executed for health education and compulsory vaccination of all students joining the health care professional colleges. Antibody titers should be routinely checked among those who are vaccinated.

CD6 Receptor Regulates Intestinal Ischemia/Reperfusion-induced Injury by Modulating Natural IgM-producing B1a Cell Self-renewal.

Intestinal ischemia/reperfusion (I/R) injury is a relatively common pathological condition that can lead to multi-organ failure and mortality. Regulatory mechanism for this disease is poorly understood, although it is established that circulating pathogenic natural IgM, which is primarily produced by B1a cells outside of the peritoneal cavity, are integrally involved. CD6 was originally identified as a marker for T cells and was later found to be present on some subsets of B cells in humans; however, whether CD6 plays any role in intestinal I/R-induced injury and, if so, the underlying mechanisms, remain unknown. Here we report that CD6-/- mice were significantly protected from intestinal inflammation and mucosal damage compared with WT mice in a model of intestinal I/R-induced injury. Mechanistically, we found that CD6 was selectively expressed on B1 cells outside of the bone marrow and peritoneal cavity and that pathogenic natural IgM titers were reduced in the CD6-/- mice in association with significantly decreased B1a cell population. Our results reveal an unexpected role of CD6 in the pathogenesis of intestinal IR-induced injury by regulating the self-renewal of B1a cells.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

A Review of Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) and Enamel Remineralization.

White-spot lesions are the earliest macroscopic evidence of enamel caries. In such a situation, the enamel surface layer stays intact during subsurface demineralization, but without treatment the subsurface loss will continue, and eventually the surface layer will collapse and lead to a cavity formation. By introducing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) at this stage the lesion can be reversed. CPP-ACP is a unique, naturally derived protein-based remineralizing technology that is now used globally in chewing gums and topical creams. The aim of this review is to expound on the potential for reversal of demineralization through the use of products such as CPP-ACP, and to provide guidance to clinicians considering remineralization as a viable treatment option.

Cutting Edge: Ezrin Regulates Inflammation by Limiting B Cell IL-10 Production.

IL-10 produced by B cells is important for controlling inflammation, thus underscoring the need to identify mechanisms regulating its production. In this study, we demonstrate that conditional deletion of ezrin in B cells increases IL-10 production induced by TLR4 ligation. The MyD88-independent Toll/IL-1R domain-containing adapter inducing IFN-ß-IFN regulatory factor 3 pathway is required for Ezrin-deficient B cells to produce higher IL-10 upon LPS stimulation. Treatment of B cells with a novel small-molecule inhibitor of ezrin induces its dephosphorylation and increases LPS-induced NF-κB and IFN regulatory factor 3 activation and IL-10 secretion, indicating a role for threonine 567 phosphorylation of ezrin in limiting IL-10. Loss of ezrin in B cells results in dampened proinflammatory response to a sublethal dose of LPS in vivo, which is dependent on increased IL-10 production. Taken together, our data yield new insights into molecular and membrane-cytoskeletal regulation of B cell IL-10 production and reveal ezrin as a potential therapeutic target in inflammatory diseases.

Isolation and identification of Acinetobacter species with special reference to antibiotic resistance.

BACKGROUND: Acinetobacter is clinically important pathogen with widespread resistance to various antibiotics. We assessed the incidence of Acinetobacter infection at a tertiary care hospital, analyze their resistance pattern and identify the production of extended spectrum ß-lactamases (ESBLs) and metallo ß-lactamases (MBLs). MATERIALS AND METHODS: The study was conducted in tertiary care hospital, India over a period of 2 years. Acinetobacter species were isolated from various clinical samples received in Department of Microbiology. After identification, Acinetobacter isolates were speciated and antibiotic susceptibility was determined by the standard disc diffusion method. ESBL and MBL production was detected by the double disc synergy test and combined disc diffusion test respectively. RESULTS: Of 3298 infected samples, 111 (3.36%) were found to be Acinetobacter. The most predominant species was Acinetobacter calcoaceticus-A. baumannii (Acb) complex (72%). High incidence of resistance was recorded for piperacillin (55%), followed by ceftriaxone (46%) and ceftazidime (46%). Isolation rate and antibiotic resistance was higher in the Intensive Care Units (ICUs) of the hospital. ESBL and MBL production was detected in 31.5% and 14.4% of the isolates respectively. DISCUSSION AND CONCLUSION: A high level of antibiotic resistance was observed in our study and maximum isolation rate of Acinetobacter was in the ICUs. Acb complex was the most predominant and most resistant species. The analysis of susceptibility pattern will be useful in understanding the epidemiology of this organism in our hospital setup, which will help in treating individual cases and controlling the spread of resistant isolates to other individuals.

The ezrin-radixin-moesin family of proteins in the regulation of B-cell immune response.

Dynamic reorganization of the cortical cytoskeleton is essential for numerous cellular processes, including B- and T-cell activation and migration. The ezrin-radixin-moesin (ERM) family of proteins plays structural and regulatory roles in the rearrangement of plasma membrane flexibility and protrusions through its members' reversible interaction with cortical actin filaments and the plasma membrane. Recent studies demonstrated that ERM proteins not only are involved in cytoskeletal organization but also offer a platform for the transmission of signals in response to a variety of extracellular stimuli through their ability to cross-link transmembrane receptors with downstream signaling components. In this review, we summarize present knowledge relating to ERMs and recent progress made toward elucidating a novel role for them in the regulation of B-cell function in health and disease.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins.

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

Primary splenic tubercular abscess in an immunocompromised patient-rapid diagnosis by line probe assay.

Diagnosing extra-pulmonary tuberculosis is a challenge that can confound even the most practiced clinicians as clinical manifestations are vague, non-specific and typical chest radiograph findings may not be evident till late in the disease. Conventional methods for mycobacteriological culture and drug susceptibility testing are slow and cumbersome. Novel technologies for rapid detection of Mycobacterium tuberculosis and its anti-TB drug resistance have therefore become a priority hence with the development of molecular line probe assays are most advanced. Herewith we are reporting a case of splenic tuberculosis in an immunocompromised patient for its rarity and to emphasis the fact that such patients can be diagnosed early for better treatment outcome to enhance the longevity if a health setup possesses all the modern diagnostic services.

Intraoperative Crede maneuver for tape adjustment during transobturator sling placement: does it improve continence?

OBJECTIVE: This study evaluated the efficacy of intraoperative extrinsic manual compression on the bladder, or Crede maneuver (CM) for tape adjustment during transobturator tape (TOT) sling procedure versus the traditional method where tension-free tape is adjusted the same for all patients. METHODS: All patients undergoing TOT sling procedure for stress urinary incontinence (SUI) between May 2008 and June 2011 by the first author were assessed. Tape adjustment was either performed in a traditional manner, leaving a tonsil clamp-size space between the sling and posterior urethra, or by using CM after filling the bladder to 300 ml capacity. Patients were considered cured at postoperative visits if they had no SUI symptoms and negative Cough Stress Test (CST) result, improved if they had some SUI symptoms and negative CST result, and failed if symptomatic and had positive CST result. The Fisher exact test and the Wilcoxon rank sum test were used to evaluate the baseline differences between the 2 groups, along with multiple logistic regression to evaluate independent predictors of cure. RESULTS: The continence rate was 77.67% in the traditional group (87/112) and 79.65% (137/172) in the CM group (P = 0.76). Older patients and smokers were less likely to be continent (odds ratio, 0.95; P = 0.015; and odds ratio, 0.22; P = 0.003, respectively). Five (4.5%) of the 112 patients in the traditional group and 12 (6.9%) of the 172 patients in the CM group had adverse outcomes including transient urinary retention, mesh erosion, or dysuria (P = 0.45). CONCLUSION: Using CM for intraoperative tape adjustment does not improve continence rates compared to the traditional method of TOT sling placement.