Localization Of Antigen Research Articles

S-adenosylmethionine upregulates the angiotensin receptor-binding protein ATRAP via the methylation of HuR in NAFLD

Nonalcoholic fatty liver disease (NAFLD) has emerged globally and is associated with inflammatory signaling. The underlying mechanisms remain poorly delineated, although NAFLD has attracted considerable attention and been extensively investigated. Recent publications have determined that angiotensin II (Ang II) plays an important role in stimulating NAFLD progression by causing lipid metabolism disorder and insulin resistance through its main receptor, Ang II type 1 receptor (AT1R). Herein, we explored the effect of supplementary S-adenosylmethionine (SAM), which is the main biological methyl donor in mammalian cells, in regulating AT1R-associated protein (ATRAP), which is the negative regulator of AT1R. We found that SAM was depleted in NAFLD and that SAM supplementation ameliorated steatosis. In addition, in both high-fat diet-fed C57BL/6 rats and L02 cells treated with oleic acid (OA), ATRAP expression was downregulated at lower SAM concentrations. Mechanistically, we found that the subcellular localization of human antigen R (HuR) was determined by the SAM concentration due to protein methylation modification. Moreover, HuR was demonstrated to directly bind ATRAP mRNA and control its nucleocytoplasmic shuttling. Thus, SAM was suggested to upregulate ATRAP protein expression by maintaining the export of its mRNA from the nucleus. Taken together, our findings suggest that SAM can positively regulate ATRAP in NAFLD and may have various potential benefits for the treatment of NAFLD.

Protective Effect of Pyrus ussuriensis Maxim. Extract against Ethanol-Induced Gastritis in Rats.

Pyrus ussuriensis Maxim (Korean pear) has been used for hundreds of years as a traditional herbal medicine for asthma, cough, and atopic dermatitis in Korea and China. Although it was originally shown to possess anti-inflammatory, antioxidant, and antiatopic properties, its gastroprotective effects have not been investigated. In the present study, we evaluated the protective effects of Pyrus ussuriensis Maxim extract (PUE) against ethanol-induced gastritis in rats. The bioactive compound profile of PUE was determined by gas chromatography mass spectroscopy (GC-MS) and high-performance liquid chromatography (HPLC). The gastroprotection of PUE at different doses (250 and 500 mg/kg body weight) prior to ethanol ingestion was evaluated using an in vivo gastritis rat model. Several endpoints were evaluated, including gastric mucosal lesions, cellular degeneration, intracellular damage, and immunohistochemical localization of leucocyte common antigen. The gastric mucosal injury and ulcer score were determined by evaluating the inflamed gastric mucosa and by histological examination. To identify the mechanisms of gastroprotection by PUE, antisecretory action and plasma prostaglandin E2 (PGE2), gastric mucosal cyclic adenosine monophosphate (cAMP), and histamine levels were measured. PUE exhibited significant antioxidant effects with IC50 values of 56.18 and 22.49 µg/mL for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′- azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) inhibition (%), respectively. In addition, GC/MS and HPLC analyses revealed several bioactive compounds of PUE. Pretreatment with PUE significantly (p < 0.05) decreased the ulcer index by preventing gastric mucosal lesions, erosion, and cellular degeneration. An immunohistochemical analysis revealed that PUE markedly attenuated leucocyte infiltration in a dose-dependent manner. The enhancement of PGE2 levels and attenuation of cAMP levels along with the inhibition of histamine release following PUE pretreatment was associated with the cytoprotective and healing effects of PUE. In contrast, the downregulation of the H+/K+ ATPase pathway as well as muscarinic receptor (M3R) and histamine receptor (H2R) inhibition was also involved in the gastroprotective effects of PUE; however, the expression of cholecystokinin-2 receptors (CCK2R) was unchanged. Finally, no signs of toxicity were observed following PUE treatment. Based on our results, we conclude that PUE represents an effective therapeutic option to reduce the risk of gastritis and warrants further study.

Immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidate, BBV152 in rhesus macaques

The COVID-19 pandemic is a global health crisis that poses a great challenge to the public health system of affected countries. Safe and effective vaccines are needed to overcome this crisis. Here, we develop and assess the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine in rhesus macaques. Twenty macaques were divided into four groups of five animals each. One group was administered a placebo, while three groups were immunized with three different vaccine candidates of BBV152 at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which exhibited interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. This vaccine candidate BBV152 has completed Phase I/II (NCT04471519) clinical trials in India and is presently in phase III, data of this study substantiates the immunogenicity and protective efficacy of the vaccine candidates.

Alpha-enolase (ENO1), identified as an antigen to monoclonal antibody 12C7, promotes the self-renewal and malignant phenotype of lung cancer stem cells by AMPK/mTOR pathway

BackgroundTumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs.MethodsImmunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen.ResultsTargeted antigen showed a surface antigen expression pattern, and the 43–55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs.ConclusionsENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

Hepatitis C Virus Detection In Oral Squamous Cell Carcinoma.

Background: Hepatitis C Virus infection is a major health problem causing significant morbidity and mortalityworldwide. The oral cavity is one of the most frequently reported extra-hepatic disease site associated withit. It has been suggested that HCV is a possible triggering factor for precancerous and cancerous oral lesions.The aim of this study was to detect HCV core antigen in oral squamous cell carcinoma in comparison withnormal mucosa tissues (control) using immunohistochemistry, to detect the possible relationship betweenHCV and OSCC.Material and Methods: A total of 50 archival paraffin embedded specimens were collected (25 OSCCspecimens with 25 control specimens) and HCV core antigen was evaluated by immunohistochemistry.The localization of HCV core antigen were evaluated in both groups. All cells displaying nuclear HCV coreantigen immunostaining were counted.Results: Overall frequency of HCV in OSCC cases was higher than in controls, and a highly significantdifference was observed between them (p &lt; 0.01). Correlation of HCV positivity with the characteristicsof OSCC cases were noncontributory. The greatest cell count was recorded in poorly-differentiated OSCC,whereas the lowest value was recorded in control (P&lt;0.001). Conclusion: HCV core antigen was frequentlydetected in OSCC. HCV is suggested to be a potential risk factor for oral squamous cell carcinoma.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Characterization and localization of antigens for serodiagnosis of human paragonimiasis

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76–99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Immunohistochemical Analysis of Expression, Phosphorylation, and Nuclear Translocation of NF-κB Proteins in Human Tissues.

Immunohistochemistry (IHC) is a technique aimed at detecting specific antigens on tissue sections by the use of targeting reagents labeled with reporter molecules. This technique allows a snapshot of the structure of tissue and determines the cellular and subcellular localization of a target antigen. This chapter describes how to identify and localize NF-κB proteins in human tissue using immunohistochemical staining on formalin-fixed paraffin-embedded and frozen tissue.

Neuroprotective effect of anthocyanin oligomers demonstrated by ischemic insults and lipid peroxidation

PurposeThe purpose of the present study was to determine whether the flavonoid, anthocyanin oligomers is effective at blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of various insults to a retinal precursor cells (R28) in culture.MethodsR28 in culture were given one of three different insults, light (1000 lux for 2 days), hydrogen peroxide (200 µM H2O2 for 24 hours) or serum deprivation (48 hours). Cell survival and reactive oxygen species (ROS) formation were assayed. Lipid peroxidation assay was used to compare the antioxidant capacity of anthocyanin oligomers. In animal studies, anthocyanin oligomers were administered intraperitoneally just before and after an ischemic insult, and ischemia was delivered by raising the intraocular pressure above the systolic blood pressure(50 min). Retinas were analysed for the localisation of various antigens, and retinal extracts were also analysed for various mRNAs.ResultsIschemia/reperfusion to the retina affected the localisation of Thy‐1 and choline acetyltransferase (ChAT) and the content of various proteins (optic nerve and retina) and mRNAs (retina). Importantly, anthocyanin Oligomers statistically blunted most of the effects induced by ischemia/reperfusion. Only the increase in caspase‐8 caused by ischemia/reperfusion was unaffected by anthocyanin oligomers treatment. Anthocyanin oligomers also attenuated significantly the negative insult of light, hydrogen peroxide and serum withdrawal to R28 cells. In the lipid peroxidation studies, anthocyanin oligomers was also found to be equally effective as EGCG to act as an antioxidant. Significantly, the negative insult of serum withdrawal on R28 cell survival was blunted by anthocyanin oligomers but not by EGCG revealing the different properties of the two flavonoids.ConclusionsOur results demonstrates the powerful antioxidant characteristics of anthocyanin oligomers. Together with anti‐apoptotic effects of anthocyanin oligomers suggest its potential use for the treatment of various ocular diseases such as glaucoma, where oxidative stress has been suggested to be involved in many aspects of pathogenesis.

Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody.

Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei-infected cell types in naturally infected horses.

Abstract 5706: Identifying regulators of cancer heterogeneity with phenotypic sorting and single cell sequencing

Abstract Intratumoral heterogeneity is a major challenge to the effective treatment of cancer. The genomic, transcriptomic, and epigenetic differences among tumor cells give rise to diverse phenotypes, some of which can persist in dysregulated environmental conditions and survive therapeutic attempts. Single cell sequencing (scSeq) technologies have improved our understanding of the transcriptional variation among tumor cells. However, it remains unclear how cell behavior is related to these alterations. Here, we link genotype to phenotype by employing a method of fluorescently labeling and isolating tumor cell subpopulations based on user-defined phenotypic characteristics from within a biologically relevant three-dimensional (3D) environment for subsequent scSeq and molecular analyses (Pheno-scSeq). We compare this Pheno-scSeq technique to traditional agnostic clustering of scSeq data and find significant differences. We then use Pheno-scSeq to guide our dissection of the mechanistic basis of heterogeneity in the invasive capacity of triple negative breast cancer cells. After seeding cells in 3D collagen and photoconverting based on invasion phenotype, we find that cells undergoing collective invasion are defined by a transcriptional program of migration, anabolism, and proliferation, suggesting a “go and grow” state contrasting with the “go or grow” hypothesis. Cells that do not invade form acini and are defined by a transcriptional program enriched for catabolism, senescence, and immune regulation. Functional assessment of the gene sets validates the relevance of the transcriptional program and reveals differential Human Leukocyte Antigen localization associated with differential migration phenotypes. Treatment with doxorubicin and paclitaxel reveals that acini cells are resistant to drug-induced death, while collectively invasive cells are more susceptible. Modulation of cell-matrix adhesion is explored to determine the potential for reprogramming between the two phenotypes and to identify couplings between migration, immune, and metabolic phenotypes. This study lends deeper insight into the mechanisms underlying phenotypic heterogeneity and may inform the success of precision oncology initiatives. Citation Format: Kevin Chen, Stephanie Irene Fraley. Identifying regulators of cancer heterogeneity with phenotypic sorting and single cell sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5706.

Significance of Autoantibodies in Autoimmune Encephalitis in Relation to Antigen Localization: An Outline of Frequently Reported Autoantibodies with a Non-Systematic Review.

Autoantibodies related to central nervous system (CNS) diseases propel research on paraneoplastic neurological syndrome (PNS). This syndrome develops autoantibodies in combination with certain neurological syndromes and cancers, such as anti-HuD antibodies in encephalomyelitis with small cell lung cancer and anti-Yo antibodies in cerebellar degeneration with gynecological cancer. These autoantibodies have roles in the diagnosis of neurological diseases and early detection of cancers that are usually occult. Most of these autoantibodies have no pathogenic roles in neuronal dysfunction directly. Instead, antigen-specific cytotoxic T lymphocytes are thought to have direct roles in neuronal damage. The recent discoveries of autoantibodies against neuronal synaptic receptors/channels produced in patients with autoimmune encephalomyelitis have highlighted insights into our understanding of the variable neurological symptoms in this disease. It has also improved our understanding of intractable epilepsy, atypical psychosis, and some demyelinating diseases that are ameliorated with immune therapies. The production and motility of these antibodies through the blood-brain barrier into the CNS remains unknown. Most of these recently identified autoantibodies bind to neuronal and glial cell surface synaptic receptors, potentially altering the synaptic signaling process. The clinical features differ among pathologies based on antibody targets. The investigation of these antibodies provides a deeper understanding of the background of neurological symptoms in addition to novel insights into their basic neuroscience.

Localization and immunohistochemical detection of swine influenza A virus subtype H1N1 antigen in formalin-fixed, paraffin-embedded lung tissues from naturally infected pigs

BackgroundSwine influenza A viruses (SIV) infection is among the leading causes of respiratory diseases in a number of animal species and human, and has been reported to cause substantial losses to pig industry. Previous reports of serological, molecular, and surveillance studies in commercial piggeries in Nigeria indicated the presence of SIV subtypes H1N1 and H3N2 in infected pigs; hitherto, there exists lack of studies on the pulmonary pathology and pathogenicity of SIV in Nigeria. This study investigates the presence of SIV subtype H1N1 antigen in the formalin-fixed paraffin-embedded lung sections obtained from apparently healthy pigs slaughtered at abattoirs located in Lagos, Ogun, and Oyo States, Southwest Nigeria using a streptavidin-biotin (ABC) immunoperoxidase (IP) staining. Two hundred four lungs consisting of 144 grossly pneumonic lungs and 60 apparently normal lungs were randomly collected, fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and processed for histopathological examination and immunohistochemistry.ResultsThe main gross lesions were marked pulmonary edema and mild bilateral consolidation of cranial lobes. Histopathology revealed suppurative bronchitis, and bronchiolitis with or without concurrent widespread degeneration and necrosis of epithelial cells (52.08%) and thickening of alveolar septa due to cellular infiltration consisting predominantly of neutrophils and mononuclear cells (macrophages and plasma cells) (39.58%). The lumina of most airways contained exudate consisting of neutrophils, desquamated epithelia cells, and necrotic debris. SIV antigen was immunohistochemically detected in 7/204 (3.43%) samples using SIV-specific (H1N1) monoclonal antibody. Positive cells exhibited a typical dark-brown reaction in the infected cells. A strong positive immunohistochemical staining was detected mainly in the alveolar macrophages and bronchial submucosal glandular epithelial cells while less intense staining was observed in the bronchiolar epithelial cells.ConclusionsThe present study describes the distribution and localization of SIV subtype H1N1 antigens in the lung tissues of the infected pigs and provides public awareness on the presence of the virus in pig population in Nigeria and the risk factors associated with the infection. Therefore, people working in pig farms should maintain high level of biosafety and personal hygiene. This is the first report of immunohistochemical detection of SIV subtype H1N1 antigen in naturally infected pigs in Nigeria and may indicate rapid dissemination of the virus in susceptible pigs in the study area. A further molecular epidemiological study to investigate other SIV subtypes circulating in Nigerian pig population is warranted.

Localisation of antigens in the gut post-challenge with Streptococcus iniae in vaccinated and non-vaccinated red hybrid tilapia (Oreochromis sp.)

This study was conducted to determine the localisation of antigen in the gut of vaccinated and non-vaccinated red hybrid tilapia post-challenge via immunohistochemistry and scanning electron microscopy. All groups (1, 2, 3, and 4) were vaccinated orally with feed-based killed whole-cells of Streptococcus iniae (FKV). Group 1 was fed with the FKV for 3 days consecutively only on day 0; group 2 was fed with the FKV for 3 days consecutively on day 0 and booster on day 14; group 3 was fed with the FKV for 3 days consecutively on day 0, followed by a booster on day 14 and 21; and group 4 was fed with the FKV for 5 days consecutively on day 0, followed by a booster on day 14 and 21. The remaining groups, group 5 (negative control group) and group 6 (positive control group), only fed with a commercial feed. Five fish from each group were sacrificed on a weekly basis for the entire 6 weeks for a collection of fresh small intestinal samples including post-challenge starting from 4 to 72 h. Immunohistochemical staining was conducted using the avidin-biotin-peroxidase complex method. Scanning electron microscopy was also used to view the hindgut samples from each group. Positive control group recorded significantly the highest (p < 0.05) antigen positivity, followed by groups 1 and 2, meanwhile, lowest positivity in groups 3 and 4 at 12 h, 24 h, and 72 h, respectively. Immunohistochemistry and scanning electron microscopy showed the presence of antigens in red hybrid tilapia hindgut, protruding in goblet cells, gut-associated lymphoid tissues, and villi dome epithelium in different hours of post-challenge. Significantly, boosted group 4 with a low number of antigens in 72 h showed the stimulation and irregular projection of M-cells with variable morphology and antigen distribution pattern.

Localization of Pasteurella multocida antigens in the brains of pigs naturally infected with Pasteurellosis revealing a newer aspect of pathogenesis

Pasteurella multocida is an economically important respiratory pathogen of pigs confronting swine industry worldwide. Despite extensive research over the decades, its pathogenesis is still poorly understood. Recent reports have demonstrated the nervous system affection as a newer aspect of pathogenesis by Pasteurella multocida type B:2 in Haemorrhagic Septicemia, but there are no reports of the involvement of nervous system by P. multocida in pigs. Therefore, the study was aimed to explore the neurovirulence of Pasteurella multocida in naturally infected pigs. A total of 15 brains were collected from the natural cases of pig mortality suggestive of Pasteurellosis. Grossly, the leptomeninges were markedly congested and brains were oedematously swollen. Histologically, there was moderate to severe fibrinohaemorrhagic and mononuclear cells exudates present in the leptomeningeal tissue and cerebrospinal spaces. Similar vascular inflammatory lesions (perivascular and perineuronal) along with gliosis, neuronal degeneration and necrosis were noted in various subanatomical sites of the brain (cerebrum, cerebellum, brainstem and spinal cord). The culture and biochemical tests showed the presence of P. multocida within the brain tissue. P. multocida type specific antibody staining in the brain tissues revealed intense distribution of antigens in the inflammatory exudates of meningeal vessels, neurons, glial cells and endothelial cells of the blood vessels contributing its association with neuropathological lesions. Pasteurella multocida specific PCR amplification of capsular polysaccharide gene yielded 460 bp and multiplex PCR showed the involvement of capsular serogroups A &D. All the isolates showed the presence of 10 genes for virulence factors. The disease confirmation of both serotypes was proven by Koch's postulates using Swiss albino mice. Further, histopathological brain lesions along with the immunohistochemical detection of bacterial antigens were corroborated with natural cases of P. multocida as described above. To the best of our knowledge, we first time report the neuroinvasion of P. multocida in naturally infected pigs.

Immunohistochemical, histological and ultrastructural evaluation of protection provided by cholera vaccine against V. cholerae O139

In our previous study, complete protection was observed in rabbit immunized with 1 × 1010 CFU of live attenuated VCUSM21P vaccine against challenge with 1 × 109 CFU Vibrio cholerae O139. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological, immunohistochemical and ultrastructural techniques. Severe pathology is evident in wild type injected ileum in non-immunized, showing extensive villous destruction, edema, necrosis and inflammation with infiltration of large numbers of inflammatory cells, extensive damage to the villi and microvilli with pore formation. Histology of ileum injected with wild type in immunized rabbit shows no significant pathological changes except for a few inflammatory cells in lamina propria with mild edema in mucosa and submucosa. immunohistochemical staining revealed O139 antigens of wild type are seen in the lamina propria of edematous villi, muscularis mucosa and submucosa with weak presence in the muscle coat in non-immunized rabbit after challenged with wild type in non-immunized rabbits, but in immunized rabbit localisation of the O139 LPS antigen is seen at the tips of the intact villi, within lamina propria and muscularis mucosa only. These observations suggest that the vaccine can effectively protect animals from any pathologic changes and eliminate V. cholerae O139 from the immunized animals.

Serum Levels of Autoantibodies Against Extracellular Antigens and Neutrophil Granule Proteins Increase in Patients with COPD Compared to Non-COPD Smokers.

BackgroundChronic obstructive pulmonary disease (COPD) is a highly prevalent disease leading to irreversible airflow limitation and is characterized by chronic pulmonary inflammation, obstructive bronchiolitis and emphysema. Etiologically, COPD is mediated by toxic gases and particles, eg, cigarette smoke, while the pathogenesis of the disease is largely unknown. Several lines of evidence indicate a link between COPD and autoimmunity but comprehensive studies are lacking.MethodsBy using a protein microarray assaying more than 19,000 human proteins we determined in this study the autoantibody profiles of COPD and non-COPD smokers. The discovery cohort included 5 COPD patients under acute exacerbation (AECOPD) and 5 age- and gender-matched non-COPD smokers. One putative candidate autoantibody, anti-lactoferrin IgG, was further investigated by using immunoblotting with a large validation cohort containing 124 healthy controls, 92 patients with AECOPD and 52 patients with stable COPD.ResultsWe show that i) autoantigens targeted by autoantibodies with higher titers in COPD patients were enriched in extracellular regions, while those with lower titers in COPD patients were enriched in intracellular compartments. ii) levels of IgG autoantibodies against many neutrophil granule proteins were significantly higher in COPD patients than in non-COPD smokers. Furthermore, increased levels of anti-lactoferrin antibodies in COPD patients were confirmed in a cohort with a large number of samples.ConclusionThe comprehensive autoantibody profiles from COPD patients established in this study demonstrated for the first time a shift in the cellular localization of antigens targeted by autoantibodies in COPD.

Morphological analysis of Apolipoprotein E binding to A\u03b2 Amyloid using a combination of Surface Plasmon Resonance, Immunogold Labeling and Scanning Electron Microscopy

BackgroundImmunogold labeling in combination with transmission electron microscopy analysis is a technique frequently used to correlate high-resolution morphology studies with detailed information regarding localization of specific antigens. Although powerful, the methodology has limitations and it is frequently difficult to acquire a stringent system where unspecific low-affinity interactions are removed prior to analysis.ResultsWe here describe a combinatorial strategy where surface plasmon resonance and immunogold labeling are used followed by a direct analysis of the sensor-chip surface by scanning electron microscopy. Using this approach, we have probed the interaction between amyloid-β fibrils, associated to Alzheimer’s disease, and apolipoprotein E, a well-known ligand frequently found co-deposited to the fibrillar form of Aβ in vivo. The results display a lateral binding of ApoE along the amyloid fibrils and illustrates how the gold-beads represent a good reporter of the binding.ConclusionsThis approach exposes a technique with generic features which enables both a quantitative and a morphological evaluation of a ligand-receptor based system. The methodology mediates an advantage compared to traditional immunogold labeling since all washing steps can be monitored and where a high stringency can be maintained throughout the experiment.

Glucose Transporter 2 and 4 Are Involved in Glucose Supply during Pulpal Wound Healing after Pulpotomy with Mineral Trioxide Aggregate in Rat Molars

IntroductionPulp capping materials allow healing of injured pulp with a layer of reparative dentin. Glucose is needed to cure the injured area. Glucose is transported by glucose transporter (Glut) 2 and Glut4, which are transmembrane proteins that act as gatekeepers. We hypothesized that the transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing. Therefore, we explored Glut2 and Glut4 expression during reparative dentinogenesis after mineral trioxide aggregate capping. MethodsThe upper left first molar of 8-week-old Wistar rats underwent pulpotomy with mineral trioxide aggregate. At 1, 3, 5, 7, and 14 days after treatment, localization and colocalization of Glut2, Glut4, nestin (odontoblast marker), and antiendothelial cell antigen 1 (RECA-1; endothelial cell marker) were analyzed with immunohistochemical staining. Messenger RNA expression levels of Slc2a2 (encoding Glut2), Slc2a4 (encoding Glut4), Igf-1r (encoding insulinlike growth factor 1 receptor), and nestin were analyzed in the extracted teeth using real-time polymerase chain reaction. ResultsGlut2 and Glut4 were localized within odontoblasts and endothelial cells in normal control teeth. Three days after pulpotomy, Glut2- and Glut4-positive cells were detected; 7 days after pulpotomy, immunoreactivity for Glut2 and Glut4 was confined to newly differentiated odontoblastlike cells arranged beneath reparative dentin. Messenger RNA expression levels of Slc2a2 and Slc2a4 were significantly up-regulated after pulpotomy. ConclusionsGlut2 and Glut4 regulate glucose transport during wound healing beneath the injured area. This may contribute to the development of new vital pulp therapy for patients with deep caries.

Rhoptry and Dense Granule Secreted Effectors Regulate CD8+ T Cell Recognition of Toxoplasma gondii Infected Host Cells.

Toxoplasma gondii secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8+ T cells. Here, we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8+ T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8+ T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8+ T cell recognition and clearance of Toxoplasma gondii infected host cells.