Bacterial Antigens Research Articles

Increased IgA-mediated responses to the gut paracellular pathway and blood-brain barrier proteins predict delirium due to hip fracture in older adults.

Delirium is accompanied by immune response system activation, which may, in theory, cause a breakdown of the gut barrier and blood-brain barrier (BBB). Some results suggest that the BBB is compromised in delirium, but there is no data regarding the gut barrier. This study investigates whether delirium is associated with impaired BBB and gut barriers in elderly adults undergoing hip fracture surgery. We recruited 59 older adults and measured peak Delirium Rating Scale (DRS) scores 2-3 days after surgery, and assessed plasma IgG/IgA levels (using ELISA techniques) for zonulin, occludin, claudin-6, β-catenin, actin (indicating damage to the gut paracellular pathway), claudin-5 and S100B (reflecting BBB damage), bacterial cytolethal distending toxin (CDT), LPS-binding protein (LBP), lipopolysaccharides (LPS), Porphyromonas gingivalis, and Helicobacter pylori. Results from univariate analyses showed that delirium is linked to increased IgA responses to all the self-epitopes and antigens listed above, except for LPS. Part of the variance (between 45-48.3%) in the peak DRS score measured 2-3 days post-surgery was explained by independent effects of IgA directed to LPS and LBP (or bacterial CDT), baseline DRS scores, and previous mild stroke. Increased IgA reactivity to the paracellular pathway and BBB proteins and bacterial antigens is significantly associated with the activation of M1 macrophage, T helper-1, and 17 cytokine profiles. Heightened bacterial translocation, disruption of the tight and adherens junctions of the gut and BBB barriers, elevated CDT and LPS load in the bloodstream, and aberrations in cell-cell interactions may be risk factors for delirium.

P193 Fatigued patients with Inflammatory Bowel Disease exhibit distinct systemic antibody epitope repertoires

Abstract Background Patients with inflammatory bowel diseases (IBD) frequently experience fatigue, affecting up to 80% of those with active disease and approximately 50% with quiescent disease. The exact cause of IBD-associated fatigue is often unknown, making clinical management very challenging. In this study we aimed to explore whether patients with quiescent IBD reporting fatigue exhibit specific systemic antibody responses, which could provide insight into immune reactivities underlying fatigue. Methods Systemic antibody epitope repertoires were profiled in 327 patients with IBD (156 Crohn’s disease [CD]; 171 ulcerative colitis [UC]) leveraging phage-display immunoprecipitation sequencing (PhIP-Seq) against 344,000 rationally selected peptide antigens. Fatigue severity was assessed on a 10-point Likert scale, ranging from 1 (no fatigue) to 10 (highest fatigue severity). Quiescent IBD was defined as clinical (Harvey-Bradshaw Index [HBI] <5 or Simple Clinical Colitis Activity Index [SCCAI] <2.5) and biochemical remission (C-reactive protein [CRP] <5 mg/L) at time of sampling. Multivariable logistic regression analyses, allowing adjustment for potential confounding factors e.g. age, sex, and smoking, were performed to identify associations between fatigue and systemic antibody responses. Results A total of 105 different antibody-bound peptides were associated with fatigue (nominal P-value<0.05), albeit none passed adjustment for multiple comparisons. Among these antibodies, 50 (47.6%) were found to be less frequent in highly fatigued patients (fourth quartile, Q4), while 55 (52.4%) were identified as more frequent in highly fatigued patients compared to those with low fatigue scores (first quartile, Q1). Among highly fatigued patients, antibody responses were primarily directed towards viral antigens, notably several antigens from Epstein-Barr virus (EBV), as well as bacterial antigens, including functional proteins from Streptococcus and Staphylococcus species. Fatigued patients with CD exhibited elevated systemic antibody responses against allergens, Staphylococcus, Pseudomonas aeruginosa, and Shigella spp. Fatigued patients with UC showed higher frequencies of antibody responses against herpes simplex virus (HSV), influenza viruses, and few responses against allergens and Streptococcus bacteria. These results remained materially unchanged when repeating analyses in patients with quiescent IBD. Conclusion This study may suggest a potential role of viral antigens, particularly EBV, in the pathophysiology of fatigue in patients with IBD. However, larger confirmatory studies are needed to validate these findings. PhIP-Seq may represent a valuable strategy to approach the investigation of immune responses underlying complex symptoms such as fatigue.

МОНОКЛОНАЛЬНЫЕ АНТИТЕЛА ПРОТИВ Mycobacterium avium В ИССЛЕДОВАНИИ МИКОБАКТЕРИОЗА ЛЕГКИХ

We used hybridoma technology to obtain 15 monoclonal antibodies (mAbs) to antigens of whole-cell suspension of M. avium. The isotypes and specificity of mAbs to mycobacterial and bacterial antigens were characterized. The chemical nature of the antigens/antigenic determinant recognized by the mAbs under treatment with proteinase K and metaperiodate Na and using extracted with ethanol and benzene M. avium suspension was investigated. All mAbs were purified by affinity chromatography and used in heterologous indirect two-site ELISA to determine diagnostic properties in the study of IgG antibodies in sera of patients with pulmonary tuberculosis, pulmonary mycobacteriosis or healthy donors. The presented method makes it possible to individually identify and take into account non-specific “natural” IgG antibodies of each serum under study (patent for invention No. 2794855). mAbs 1B11 showed diagnostic specificity in the detection of mycobacteriosis − 62.47% and sensitivity – 74.78%, 3E12 (anti-54kDa) − 88.89% and 52.63%, and 1H7G6 − 66.81% and 83.80%, respectively.

Application of Nanobiosensors in Detection of Pathogenic Bacteria: An Update

Bacterial infections remain a critical public health concern worldwide, necessitating the development of efficient and sensitive diagnostic tools. Nanobiosensors, comprising nanomaterials, offer a novel approach to bacterial pathogen detection. The present review aimed to explore the current research and applications of nanobiosensors for bacterial pathogen detection. Recent discoveries in nanotechnology have facilitated the development of nanobiosensors with remarkable sensitivity and specificity. These nanoscale sensors are designed to detect specific bacterial pathogens through various mechanisms, including aptamers, antibodies, and molecular recognition elements. Furthermore, miniaturization and integration with microfluidic systems have enabled the rapid and point-of-care detection of bacterial infections. Incorporating nanomaterials such as carbon nanotubes, quantum dots, and graphene into biosensing platforms has significantly enhanced their performance, leading to ultrasensitive detection of bacterial antigens and nucleic acids. Additionally, using nanobiosensors with advanced analytical techniques, such as electrochemical, optical, and piezoelectric methods, has expanded the possibilities for accurate and real-time monitoring of bacterial pathogens. Nanobiosensors represent a promising frontier in the battle against bacterial infections. Their exceptional sensitivity, rapid response times, and potential for multiplexed detection make them invaluable tools for the early diagnosis and monitoring of bacterial pathogens. Developing cost-effective and portable nanobiosensors for resource-limited settings becomes increasingly possible as nanotechnology advances.

Bacterial antigens unleash tumor-targeting immunity

The composition of the gut microbiome has been shown to influence disease outcome in patients with colorectal cancer (CRC). In a recent Nature Biotechnology article, Wang etal. demonstrate that killing CRC-associated bacteria with a liposomal antibiotic elicits CRC-targeting immune responses of therapeutic relevance as a consequence of epitope mimicry.

The interplay between the microbiota, diet and T regulatory cells in the preservation of the gut barrier in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is becoming more common in the Western world due to changes in diet-related microbial dysbiosis, genetics and lifestyle. Incidences of gut permeability can predate IBD and continued gut barrier disruptions increase the exposure of bacterial antigens to the immune system thereby perpetuating chronic inflammation. Currently, most of the approved IBD therapies target individual pro-inflammatory cytokines and pathways. However, they fail in approximately 50% of patients due to their inability to overcome the redundant pro inflammatory immune responses. There is increasing interest in the therapeutic potential of T regulatory cells (Tregs) in inflammatory conditions due to their widespread capability to dampen inflammation, promote tolerance of intestinal bacteria, facilitate healing of the mucosal barrier and ability to be engineered for more targeted therapy. Intestinal Treg populations are inherently shaped by dietary molecules and gut microbiota-derived metabolites. Thus, understanding how these molecules influence Treg-mediated preservation of the intestinal barrier will provide insights into immune tolerance-mediated mucosal homeostasis. This review comprehensively explores the interplay between diet, gut microbiota, and immune system in influencing the intestinal barrier function to attenuate the progression of colitis.

De novo identification of bacterial antigens of a clinical isolate by combining use of proteosurfaceomics, secretomics, and BacScan technologies.

Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.

KLF2 Regulates NLRP3 Inflammasome Priming and Activation in Murine Myeloid Cells

Introduction: Krüppel-like factor-2 (KLF2) is a critical nodal transcription factor and a tonic repressor of myeloid cell activation. Loss of KLF2 is associated with a pro-inflammatory and pro-thrombotic state. Mice deficient in myeloid-KLF2 (MK2KO) experience significantly higher mortality from lipopolysaccharide (LPS), and have increased serum and tissue levels of pro-inflammatory cytokines such as IL-1β. IL-1β is a product of NLRP3 inflammasome priming and activation, which has been implicated in numerous inflammatory and immune-mediated diseases. Neutrophils are the first responders of the innate immune system and are responsible for bacterial antigen detection. We thus aimed to elucidate the role of KLF2 in the regulation of NLRP3 inflammasome activation and subsequent IL-1β release in the neutrophil compartment. Methods: 8-week old adult MK2KO ( Lyz2Cre/ Klf2fl/fl) and control ( Lyz2Cre) mice were used for obtaining neutrophils from the bone marrow. Neutrophils were plated in RPMI-1640 media and stimulated with 10 ng/mL of LPS for 1 hour (h) and 2 h, followed by nigericin for 30 minutes. LPS acts as a priming agent for NLRP3 inflammasome assembly, and nigericin is a potassium ionophore which enables the activation step and subsequent IL-1β release. Supernatant was used for IL-1β ELISA and cell lysate was used for protein extraction and Western blotting to quantify NLRP3 protein expression. GraphPad Prism was used for statistical analyses. Results: Neutrophils from both MK2KO and Cre mice have significantly increased production of IL-1β when stimulated with LPS followed by nigericin (Figure 1), and loss of KLF2 leads to significantly increased IL-1B release from neutrophils. MK2KO neutrophils also have significantly more NLRP3 protein expression as compared to Cre neutrophils at baseline, and in response to LPS and nigericin (Figure 2). This increase in NLRP3 protein expression was time dependent and dose-dependent (Figure 2). Conclusion: KLF2 negatively regulates the transcriptional expression of NLRP3 and loss of KLF2 leads to increased NLRP3 inflammasome priming, at baseline and in response to LPS. Loss of KLF2 also affects the second step of the NLRP3 inflammasome pathway - the activation step, leading to increased IL-1β release in neutrophils in adult mice. Ongoing studies are examining the mechanisms underlying KLF2 control of NLRP3 transcription and other aspects of inflammasome activation. Inflammasome activation has been implicated in sepsis-related mortality due to the pro-inflammatory cytokine storm, and thus modulating KLF2 might help in developing novel targets to reduce this burden of mortality.

Impact of Investigational Microbiome Therapeutic SER-155 on Pathogen Domination: Initial Results from a Phase 1b Study in Adults Undergoing Allogeneic Hematopoietic Cell Transplantation (HCT)

Introduction: During allogeneic hematopoietic cell transplantation (allo-HCT), conditioning regimens disrupt the epithelial barrier of the gastrointestinal (GI) tract and broad-spectrum antibiotics cause microbiome disruption characterized by low microbial diversity and high abundance of pathogens that can dominate gut communities. Microbiome disruption is associated with increased translocation of pathogenic bacteria leading to risk of bloodstream infections (BSIs) and acute graft-vs-host disease (aGvHD). Nonclinical studies have demonstrated that domination with pathogens leads to immune activation and subsequent GvHD following exposure to bacterial antigens in the gut-associated lymphoid tissue [ Jenq 2015 Biol Blood Marrow Transplant, Stein-Thoeringer 2019 Science, Peled 2020 NEJM]. SER-155, an oral investigational cultivated microbiome therapeutic comprised of 16 bacterial Firmicutes strains, was designed to engraft in the GI tract, improve GI barrier integrity, and restructure the GI microbiome by introducing bacteria with immunomodulatory and anti-inflammatory properties and driving a reduction in abundance of proinflammatory bacteria [ Halvorsen 2021 OFID, 2022 Bone Marrow Transplant]. Herein, we present preliminary clinical data through Day 100 from an open-label cohort of a Phase 1b study in patients undergoing allo-HCT (NCT04995653) and compare the incidence of GI pathogen domination in this cohort to a historical cohort. Methods: Study patients received two treatment courses of SER-155 (before conditioning and after neutrophil engraftment), each comprised of microbiome conditioning with 4 days oral vancomycin (to facilitate engraftment of SER-155 strains into the GI microbiome) followed by 10 days of oral SER-155. The primary endpoint was safety. Stool samples collected pre- and post-HCT were used to evaluate secondary endpoints, including engraftment of SER-155 strains (following each course of SER-155) and GI pathogen domination, defined as a relative abundance of a specific taxa at ≥30% ( Peled, 2020). Engraftment of SER-155 strains was assessed via strain-specific molecular probes (NanoString). Relative abundance of Enterobacteriaceae, Enterococcaceae, Streptococcaceae and Staphylococcaceae was assessed with whole metagenomic shotgun sequencing (WMS). A post-hoc comparison of GI domination was made with a historical cohort of 459 HCT patients with similar patient characteristics from one of the study sites (MSKCC) using stool samples profiled by 16S-amplicon sequencing. Results: Fifteen adults (median age 67 years; 54% male) were enrolled and 13 received study drug. SER-155 was generally well-tolerated. Following each course of SER-155, the majority of the 16 strains in SER-155 were detected in evaluable patients (median = 11 strains in 8 patients before conditioning; median = 12 strains in 7 patients after neutrophil engraftment).Between the day of transplant (Day 0) and Day 30, GI domination with potentially pathogenic bacteria occurred in only 1 of 9 (11%) evaluable patients (Figure 1). In contrast, in the historical comparator cohort, 64% of patients (294 of 459) had pathogen domination during the same timeframe, with domination by Enterococcus being most common. Conclusions: The observation of SER-155 strain engraftment before transplant and low incidence of GI pathogen domination after transplant through Day 30 are encouraging, although these preliminary data are limited by the small size of this open-label cohort of HCT patients. These observations are consistent with the design and preclinical evaluation of SER-155, supporting further development of this investigational cultivated microbiome therapeutic. Enrollment in the second double-blind, randomized, placebo-controlled cohort (approximately 60 participants) has initiated following a preplanned review by the Data Safety Monitoring Committee.

Neonatal Sepsis: Insight into Incidence, Classification, Risk Factors, Causative Organisms, Pathophysiology, Prognosis, Clinical Manifestations, Complications, Systemic Examination, and Treatment

Background: Neonatal sepsis is a clinical syndrome of bacteremia with systemic signs and is the most important cause of neonatal death in developing country. Mortality increase with delay in diagnosis and initiation of antibiotics. So, improvements in diagnosis and management of sepsis can significantly decrease the sepsis complications and improve its outcome. Objectives: The current review aims to high light on the incidence of neonatal sepsis, classification, risk factors, causative organisms, pathophysiology, prognosis, clinical manifestations, complications, systemic examination, and treatment. The incidence of neonatal sepsis varies among the different geographic areas, the highest being registered in Africa and Asia and the lowest in the U.S.A and Australia. The sepsis has been classified as early-onset sepsis (EOS) and the late-onset sepsis. Late-onset infections onset after 1 month of life, which occurs particularly in VLBW preterm infants or term infants requiring prolonged neonatal intensive care for other chronic problems. Neonatal sepsis includes maternal risk factors and neonatal risk factors. Septic or traumatic risk factors delivery, maternal poverty, pre-eclampsia, cardiac disease and diabetes mellitus are significant risk factors. Prolonged rupture of fetal membranes before the onset of labour, chorio-amnionitis, and maternal genitourinary tract infections have been significantly associated with a wide range of adverse perinatal and maternal outcomes, including miscarriage, stillbirth, preterm birth, fetal growth restriction, neonatal and puerperal sepsis, neonatal encephalopathy and neonatal and maternal mortality. Improvement in the socio-economic status of the population and availability of affordable antenatal care would reduce the incidence of neonatal septicemia. Amniocentesis, cervical cerclage, transcervical chorionic villus sampling, or percutaneous blood sampling, can permit entry of skin or vaginal organisms, causing amnionitis and secondary fetal infection. Infection of skin abrasions after use of obstetric forceps increases the risk of infection in neonates. Infant birth weight is inversely related to risk of early- onset sepsis. Prematurity and low birth weights are the most important neonatal factors predisposing to infection. Endotracheal intubation, insertion of an umbilical vessel catheter, or both, is associated with an increased risk of bacterial infection. Prolonged intravascular access and mechanical ventilation, the use of intravenous intralipids, and the duration of total parenteral nutrition are among the proposed risk factors associated with NICU-acquired coagulase- negative staphylococcus infections. Males have an approximately two-fold higher incidence of sepsis than females. Systemic antibiotics potentiate the overgrowth of certain organisms as Staphylococcus epidermidis. Corticosteroid treatment increases the risk of invasive candidiasis among premature infants. Hospital Acquired Infections are one of the leading causes of mortality and morbidity in (NICU). Group B Streptococcus (GBS) and E. coli as the dominant early onset sepsis (EOS) pathogens and coagulase- negative staphylococci (CONS) as the dominant late onset sepsis (LOS) pathogen followed by GBS and Staph aureus. In developing countries, Gram negative organisms are more common and are mainly represented by Klebsiella, E.coli and Pseudomonas of the Gram positive organisms, Staph aureus, CONS, Streptococcus pneumoniae, and Streptococcus pyogenes are most commonly isolated. Agents that commonly cause nosocomial infection are coagulase-negative staphylococci, gram-negative bacilli, Enterococci, and S. aureus. Causative organisms are bacterial, fungal, viral, and protozoal organism's infections. Mode of infection is prenatal, natal, and postnatal infections. Soon after birth, the newborns are exposed to the "hostile world" of bacteria, viruses, fungi and parasites, so they must immediately defend themselves. Necotizing enterocolitis, Neonatal thrombocytopenia, endophthalmitis, abdominal aortic-iliac thrombosis, fatal complete atrioventricular block, and wide range of signs and symptoms that may be observed in neonates with septicemia frequently suggest a number of alternative diagnoses. Identification of infection may be made by isolating the etiologic agent from a sterile body fluid (blood, CSF, and urine), or by demonstrating endotoxin or bacterial antigen in a body fluid. Investigations are including isolation of the microorganism, blood culture, urine culture, tracheal aspirate culture, isolation of organisms from CSF, complete blood cell count, cerebrospinal fluid analysis, C-Reactive protein, and tissue release of procalcitonin. Purpose of supportive care is to normalize the temperature, stabilize the cardiopulmonary status, correct hypoglycemia, and prevent bleeding tendency. Treatment of sepsis includes antibiotics, antiviral, antifungal, intravenous immunoglobulin, granulocyte transfusion, .......

Biomimetic Metal–Organic Framework Combats Biofilm‐Associated Infections via Hyperthermia‐Enhanced Bacterial Metabolic Interference and Autophagy‐Promoted Adaptive Immunity

AbstractRobust bacterial metabolism and the immunosuppression on peripheral immune cells cause biofilm‐associated infections (BAIs) extremely refractory to be eradicated via antibiotics alone. Herein, hierarchical mesoporous UiO‐66 metal–organic framework is decorated with selenite, polypyrrole, and macrophage membrane (MM) to develop a biomimetic nanosphere (USPM). Following the recruitment of USPM to the biofilm microenvironment (BME) via the pathogen‐targeting ability derived from MM. The BME‐responsive USPM can precisely release selenite to penetrate the loosened biofilm in synergy with near‐infrared‐induced mild photothermal therapy (mPTT). Selenite can quickly react with reducing substances to generate hydrogen selenide (H2Se) inside the biofilm. H2Se can competitively inhibit bacterial metabolic processes and disrupt biofilm metabolic homeostasis by cascade amplification effects. Furthermore, H2Se inside the biofilm further sensitizes photothermia to exert a precise local photothermal effect. Outside the biofilm, USPM can simultaneously promote the phagocytosis and autophagy of macrophages to kill and decompose the phagocytosed bacteria. Finally, the well‐decomposed bacterial antigens in macrophages can be presented to antigen‐presenting cells to arouse adaptive immune responses and enhance anti‐biofilm effectiveness further. Such powerful mPTT‐enhanced bacterial metabolic disruption and macrophagic autophagy‐promoted adaptive immune activation suggest an alternative therapeutic strategy to cure refractory BAIs.

Intestinal damage is required for the pro-inflammatory differentiation of commensal CBir1-specific T cells

Commensal-specific CD4+ T cells are expanded in inflammatory bowel disease (IBD) patients compared to healthy individuals. How and where commensal-specific CD4+ T cells get activated is yet to be fully understood. We used CBir1 TCR-transgenic CD4+ T cells, specific to a commensal bacterial antigen, and different mouse models of IBD to characterize the dynamics of commensal-specific CD4+ T cells activation. We found that CBir1 T cells proliferate following intestinal damage and cognate antigen presentation is mediated by CD11c+ cells in the colon-draining mesenteric lymph nodes (cMLNs). Using assay for transposase-accessible chromatin sequencing (ATAC-seq) and flow cytometry, we showed that activated CBir1 T cells preferentially acquire an effector rather than regulatory phenotype, which is plastic over time. Moreover, CBir1 T cells, while insufficient to initiate intestinal inflammation, contributed to worse disease outcome in the presence of other CD4+ T cells. Our results suggest that the commensal-specific T cell responses observed in IBD exacerbate rather than initiate disease.

PSXIV-28 Post-Confluence Cell Age Affects Lipopolysaccharide-Induced Interleukin-8 Secretion in Ipec-J2 Cells

Abstract Porcine intestinal epithelial cell line IPEC-J2 is routinely used to investigate epithelial cell interactions with common enteric porcine antigens and pathogens. Lipopolysaccharides (LPS) are bacterial antigens metabolized from Gram negative bacteria that can induce a cellular inflammatory cascade, resulting in changes to cell structure and integrity, and are often used to challenge IPEC-J2 cells. Although numerous investigators employ the IPEC-J2 LPS model, consistent methodology regarding cell culture conditions and treatment times is not well defined. The objective of the current study was to determine the optimal post-confluence cell age for LPS induction of Interleukin-8 (IL-8) secretion and related cell integrity changes. For this study, cells were seeded at 2x104/cm2 in 24 well plates on day zero and incubated at 37°C, 5% CO2 for 3 days (confluence) prior to treatment in complete growth media (CGM) containing 5% fetal bovine serum. One day prior to treatment, CGM was removed and replaced with serum free (SF), phenol red-free media. On days 0, 5, 7, 12, 14 and 18 post confluence, cells were treated with LPS (0 or 10μg/mL) in SF and phenol red-free media for 24 hours. There were 5 replicates per treatment per timepoint. Lactate dehydrogenase (LDH) release, trans-epithelial electrical resistance (TEER), and IL-8 secretion were measured at each experimental timepoint. Data were statistically analyzed for the effect of time, treatment, and the subsequent interactive effect. There was no effect of treatment, time or their interaction on LDH release (P > 0.05), indicating cellular health was maintained over time and 10μg/ml LPS did not influence cellular toxicity. There was an effect of treatment, time and the respective interaction that showed IL-8 secretory responsiveness was increased with LPS challenge (P < 0.01), was increased on d 7 regardless of LPS challenge (P < 0.01) and peaked on d 5 post-confluence (P < 0.01). Furthermore, TEER was maximized at d 5 post-confluence and remained consistently greater relative to the d 0 baseline throughout the experiment (P < 0.01). These data indicate that LPS-induced IL-8 secretion and related cellular integrity are optimized at d 5 post-confluence in IPEC-J2 cells. In conclusion, in-vitro research utilizing the IPEC-J2 LPS model should allow cells to age 5 days post-confluence to maximize responses.

Oxidative damage varies in response to bacterial, fungal and viral antigen challenges in bats.

The immune system plays an important role in defending against pathogens and regulating physiological homeostasis, but the strength of the immune responses depends on the type of pathogen. The immune system of bats shows a high variability in responsiveness towards various pathogens; they can safely harbor certain pathogens that are highly lethal to other mammals. Oxidative stress may act as a pathophysiological cellular mechanism mediating the immunological function of bats because of its potentially detrimental effects on physiological homeostasis, fertility, and longevity. By experimentally exposing greater mouse-eared bats (Myotis myotis) to three antigens, it was previously shown that animals reacted immunologically most strongly to bacterial and viral antigens, but not to fungal one. As a follow up, in this study we observed that both bacterial and fungal antigens induced a significant increase of plasma oxidative damage, whereas viral antigens did not cause any increase of plasma oxidative damage at all albeit the mild immune response. Thus, experimental bats were able to avoid oxidative stress only in the face of a viral antigen, possibly by dampening inflammatory signaling. Bats may be able to handle viral infections and live well beyond expectations by reducing the detrimental effects of molecular oxidation.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Phenotypic Characteristics of Monocytes and Macrophages in Lupus Nephritis with Bacterial Antigen Induction

Phenotypic Characteristics of Monocytes and Macrophages in Lupus Nephritis with Bacterial Antigen Induction

An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1β, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

Glycosylation of bacterial antigens changes epitope patterns.

Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Helicobacter pylori extract induces purified neutrophils to produce reactive oxygen species only in the presence of plasma.

H. pylori is a bacterial pathogen infecting over half of the world's population and induces several gastric and extra-gastric diseases through its various virulence factors, especially cagA. These factors may be released from the bacteria during interactions with host immune cells. Neutrophils play key roles in innate immunity, and their activity is regulated by plasma factors, which can alter how these cells may interact with pathogens. The aim of the present study was to determine whether purified neutrophils could produce reactive oxygen species (ROS), one of the key functions of their anti-microbial functions, in response to extracts of cagA+ and cagA- H. pylori. Extracts from either cagA+ or cagA- H. pylori were co-cultured with human neutrophils in the presence or absence of plasma, and the neutrophil ROS production was measured. In the absence of plasma, extracts from cagA+ and cagA- H. pylori did not induce neutrophil ROS production, whereas in the presence of plasma, extracts from both cagA+ and cagA- H. pylori-induced ROS production. Furthermore, when peripheral blood mononuclear cells (PBMCs) were added to the purified neutrophils in the absence of plasma, there was no neutrophil ROS production after challenging with extracts from either cagA+ or cagA- H. pylori. Thus, it is suggested that plasma contains immunological components that change the responsiveness of neutrophils, such that when neutrophils encounter the bacterial antigens in H. pylori extracts, they become activated and produce ROS. This study also revealed a potential novel immunopathogenic pathway by which cagA activation of neutrophils contributed to inflammatory damage.

Pathology, virulence associated gene profiling, antimicrobial susceptibility and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by PM-PCR assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in IHC confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.