Localization Of Antigen Research Articles

Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

The most common inherited cause of vascular dementia and stroke, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is caused by mutations in NOTCH3. Post-translationally altered NOTCH3 accumulates in the vascular media of CADASIL arteries in areas of the vessels that exhibit profound cellular degeneration. The identification of molecules that concentrate in the same location as pathological NOTCH3 may shed light on processes that drive cytopathology in CADASIL. We performed a two phase immunohistochemical screen of markers identified in the Human Protein Atlas to identify new proteins that accumulate in the vascular media in a pattern similar to pathological NOTCH3. In phase one, none of 16 smooth muscle cell (SMC) localized antigens exhibited NOTCH3-like patterns of expression; however, several exhibited disease-dependent patterns of expression, with antibodies directed against FAM124A, GZMM, MTFR1, and ST6GAL demonstrating higher expression in controls than CADASIL. In contrast, in phase two of the study that included 56 non-SMC markers, two proteins, CD63 and CTSH, localized to the same regions as pathological NOTCH3, which was verified by VesSeg, a customized algorithm that assigns relative location of antigens within the layers of the vessel. Proximity ligation assays support complex formation between NOTCH3 fragments and CD63 in degenerating CADASIL media. Interestingly, in normal mouse brain, the two novel CADASIL markers, CD63 and CTSH, are expressed in non-SMC vascular cells. The identification of new proteins that concentrate in CADASIL vascular media demonstrates the utility of querying publicly available protein databases in specific neurological diseases and uncovers unexpected, non-SMC origins of pathological antigens in small vessel disease.

Porcine dermatitis and nephropathy syndrome on natural infection of Porcine Circovirus Type-2 in pigs in Kerala, southern India

Porcine circovirus type-2 (PCV-2), is a widely studied pathogen in the swine population during the last decade. Even though, PCV-2 has many disease manifestations, the objective of the current study was to investigate the cutaneous and renal lesions in PCV-2 infected cases. Swine carcasses presented for post-mortem examination with a history of skin lesions, inappetance, dyspnoea and diarrhoea, during September 2021 to April 2022 from different parts of Kerala were utilised in this study. The samples were collected and screened with Polymerase chain reaction (PCR) after which, histological and immunohistochemical examination were performed. Three samples were found positive for PCV-2 out of 25 samples using PCR. Microscopically, skin revealed congestion, haemorrhages and infiltration of mononuclear infiltration cells and neutrophils in dermis and spongiosis in epidermis. Kidney revealed haemorrhages, congestion, tubular necrosis and degenerative alterations, interstitial mononuclear cell infiltration with occasional glomerulitis and vasculitis in the renal pelvis. Localisation of PCV-2 antigen was observed in renal tissue and lymph node with immunohistochemistry. Altogether, these findings were suggestive of porcine dermatitis and nephropathy syndrome (PDNS) in pigs. These results indicate the importance of including PCV-2 in the differential diagnosis of cutaneous lesions caused by bacterial and viral etiologies in pigs.

ИЗУЧЕНИЕ РЕПРОДУКЦИИ ВИРУСА МЕШОТЧАТОГО РАСПЛОДА ПЧЕЛ ИММУНОЦИТОХИМИЧЕСКИМ МЕТОДОМ IN VITRO В КУЛЬТУРАХ КЛЕТОК

The article presents the results of studying the reproduction of the sacbrood virus (SBV) in cells of heterologous origin using the method of immunocytochemical analysis. To identify the localization of the SBV antigen of bees during its reproduction in sensitive cell cultures of heterologous lines, the method of immunoperoxidase staining of infected cells (IPS) was used, as a result of which sites of specific antibody/antigen interaction were observed on the cell membrane and in the form of inclusions in the cytoplasm.

Traditional and Recent Approaches for the Development of Animal Vaccines. A Review

Objective: To show new trends in the field of vaccinology and spread awareness among population regarding vaccination of animals and successfully controlling spread of diseases Study Design: This is a review study for the development of animal vaccines and was conducted from September, 2021 to June, 2022 at IMBB Department, The University of Lahore, Lahore, Pakistan. Review of Literature was collected on traditional and recent approaches for the development of veterinary vaccines and gathered for the awareness among the field of veterinary vaccinology. Methodology: Animals provide food and clothing in addition to other value-added products. Changes in diet and lifestyle have increased the consumption and the use of animal products. Infectious diseases in animals are a major threat to global animal health and its welfare; their effective control is crucial for agronomic health, for safeguarding food security and also alleviating rural poverty. Development of vaccines has led to increased production of healthy poultry, livestock, and fish. Animal production increases have alleviated food insecurity. Before year 2000, most veterinary vaccines were from inactivated organisms that were formulated with an oil-based adjuvant or live attenuated vaccines. Results: The discovery of antigen/gene delivery systems has facilitated the development of novel prophylactic and therapeutic veterinary vaccines. Uses several bioinformatics algorithms to predict antigen localization and it has been successfully applied to immunize against many veterinary diseases. Vectors and pathogens that may lead to emergent diseases in animals. Preventing transmission of emerging infectious diseases at the animal–human interface is critically important for protecting the world population from epizootics and pandemics. Hence, there is a need to develop new vaccines to prevent diseases in animals. An area of veterinary vaccination that needs more research and discussion is vaccine interference. The phrase itself is ambiguous and might mean either a condition in which immunization against one disease may weaken the protective immunity established by immunization against another, or a circumstance in which the presence of maternally derived antibodies prevent immunization in newborn animals. Practical implication: This study will provide awareness among community about veterinary vaccines and will develop a disease-free state for pets.Vetrinary vaccines not only prevent diseases in animals but also stops their spread among humans. Conclusion: This review examines some of the main topics that have emerged in the veterinary vaccine field with the use of modern biotechnology techniques. In addition, development of effective vaccines has led to healthier companion animals. However, challenges remain including climate change that has led to enhancement in vectors and pathogens that may lead to emergent diseases in animals. Preventing transmission of emerging infectious diseases at the animal–human interface is critically important for protecting the world population from epizootics and pandemics. Hence, there is a need to develop new vaccines to prevent diseases in animals. Keywords: Vaccines, Toxin, Immunization, Antibodies, Antigen, Host.

Subcellular localization of the enterobacterial common antigen GT-E-like glycosyltransferase, WecG.

Enterobacterales have developed a specialized outer membrane polysaccharide (enterobacterial common antigen [ECA]). ECA biosynthesis begins on the cytoplasmic side of the inner membrane (IM) where glycosyltransferases sequentially add sugar moieties to form a complete repeat unit which is then translocated across the IM by WzxE before being polymerized into short linear chains by WzyE/WzzE. Research into WecG, the enzyme responsible for generating ECA lipid-II, has not progressed beyond Barr et al. (1988) who described WecG as a membrane protein. Here we revise our understanding of WecG and re-characterize it as a peripherally associated membrane protein. Through the use of Western immunoblotting we show that WecG in Shigella flexneri is maintained to the IM via its three C-terminal helices and further identify key residues in helix II which are critical for this interaction which has allowed us to identify WecG as a GT-E glycosyltransferase. We investigate the possibility of protein complexes and ultimately show that ECA lipid-I maintains WecG to the membrane which is crucial for its function. This research is the first since Barr et al. (1988) to investigate the biochemistry of WecG and reveals possible novel drug targets to inhibit WecG and thus ECA function and cell viability.

Abstract 1376: Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions

Abstract T cells have the potential to carry out potent anti-tumor responses. Most studies examining T cell-cancer interactions have been directed toward late-stage tumors, but much less is known about how T cells respond to premalignant lesions. Using a clinically relevant liver cancer mouse model (Albumin-Stop-Large T Antigen; AST) in which the SV40 Large T Antigen (TAG) serves as oncogene and tumor neoantigen, we previously demonstrated that TAG-specific CD8 T cells (TCR-TAG) rapidly become dysfunctional (unable to produce cytokines and cytolytic molecules) after entering premalignant liver lesions, although they persist long-term. We hypothesized that peripheral tolerance mechanisms, which prevent autoimmunity against self-antigens, drive T cell dysfunction in premalignant lesions. To test this, we compared CD8 T cell (TCR-GAG) responses to a liver self-antigen (GAG) to TCR-TAG responses to the TAG neoantigen in premalignant hepatocytes. We found that liver-reactive TCR-GAG were unable to produce cytokines, similar to neoantigen-specific TCR-TAG; however, in contrast, they failed to persist. To test whether oncogenic transformation of hepatocytes promoted T cell persistence, we crossed Alb-GAG and AST mice to generate a model in which GAG and TAG was expressed in premalignant hepatocytes (ASTxAlb-GAG). We found that both TCR-GAG and TCR-TAG transferred into ASTxAlb-GAG mice bearing premalignant lesions driven by the TAG oncogene rapidly lost the ability to produce cytokines; however, only TCR-TAG persisted while TCR-GAG quickly disappeared. To understand why the TCR-GAG failed to persist, we assessed proliferation and cell cycling after T cells encountered their cognate antigen. While both TCR-GAG and TCR-TAG were rapidly activated and began proliferating, TCR-GAG exited cell cycle and began decreasing in number after four cell divisions, while TCR-TAG continued to divide and increase in number. Antigen amount and localization may regulate T cell proliferation and persistence. We found much higher levels of TAG-encoding versus GAG-encoding mRNA in the liver, suggesting that higher antigen expression may be required for T cell persistence. T cell responses can be directed against both shared tumor self-antigens and neoantigens. By dissecting the determinants of CD8 T cell function and persistence in response to self and non-self-antigens, these studies could inform strategies to boost anti-tumor CD8 T cell responses in cancer patients. Citation Format: Jessica J. Roetman, Megan M. Erwin, Minna K. Apostolova, Mary Philip. Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1376.

Comparison of the Immunogenic Properties of Lactiplantibacillus plantarum Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations.

The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that Mycobacterium tuberculosis first enters the body through the mucosal membranes. We have previously demonstrated the immunogenicity of a recombinant Lactiplantibacillus plantarum delivery vector with TB hybrid antigen Ag85B-ESAT-6 anchored to the cell membrane. The goal of the present study was to analyze the impact of antigen localization in the immune response. Thus, we assessed two novel vaccine candidates, with the TB antigen either non-covalently anchored to the cell wall (LysMAgE6) or located intracellularly (CytAgE6). In addition, we compared two expression systems, using an inducible (LipoAgE6) or a constitutive promoter (cLipoAgE6) for expression of covalently anchored antigen to the cell membrane. Following administration to mice, antigen-specific CD4+ T-cell proliferation and IFN-γ and IL-17A secretion were analyzed for lung cell and splenocyte populations. Generally, the immune response in lung cells was stronger compared to splenocytes. The analyses showed that the type of expression system did not significantly affect the immunogenicity, while various antigen localizations resulted in markedly different responses. The immune response was considerably stronger for the surface-displaying candidate strains compared to the candidate with an intracellular antigen. These findings emphasize the significance of antigen exposure and further support the potential of L. plantarum as a mucosal vaccine delivery vehicle in the fight against TB.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Proliferating Cell Nuclear Antigen (PCNA) Localization in the Ovaries of Buffalo, Dog and Pig by Immunohistochemistry

Background: It was expected from the previous studies that the immune histochemical staining for PCNA within follicular cells altered depending on the stage of follicular growth. Therefore, we aimed at determining the expression of proliferating cell nuclear antigen protein (PCNA) in the buffalo, dog and pig ovaries. Methods: The localization of PCNA was demonstrated in formalin fixed, paraffin embedded tissue sections of buffalo, dog and pig ovaries using primary mouse monoclonal anti-PCNA antibody. Result: Staining for PCNA was not observed in pregranulosa cells but observed in oocytes of primordial follicles. In primary to early secondary follicles, in oocytes and in few granulosa cells the positive staining was detected. The late secondary preantral follicles and actively growing small to large antral follicles showed strong PCNA labelling in the layers of granulosa and theca cells. PCNA staining was expressed in nuclei of oocytes in preantral and small antral follicles. The follicles demonstrating advanced atresia showed mild to no PCNA labelled granulosa and theca cells. The results of our study demonstrated that follicular growth and development in ovaries may be effectively monitored by determining the rate of granulosa cell proliferation of PCNA.

CC chemokine 1 protein from Cromileptes altivelis (CaCC1) promotes antimicrobial immune defense

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%–90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

In situ analysis of hepatitis B virus (HBV) antigen and DNA in HBV-induced hepatocellular carcinoma

AimsHepatitis B Virus (HBV) infection is the major risk factor for hepatocellular carcinoma (HCC) in East Asia. Here we aimed to further investigate the abundance of viral antigen and DNA within HBV-related HCC and surrounding tissues at histological level.MethodIn addition to routine histopathology, in situ hybridization (ISH) of HBV DNA and immunohistochemistry (IHC) of HBsAg were performed in tissues from 131 HBsAg-positive HCC patients undergoing liver resection. Serum α-fetoprotein together with basic biochemical and immunological parameter was also measured.ResultsOverall, the ISH of HBV DNA and IHC of HBsAg showed 31.3% and 92.9% positive rate respectively (p < 0.0001). The level of correlation between these two markers was much more significant in tumor (p < 0.0001) than in tumor-surrounding tissue (p = 0.01). HBsAg exhibited a much higher positive rate in tumor-adjacent tissue than in tumor tissue (86.6% versus 29.9%, p < 0.0001) with significantly different staining pattern. By contrast, the positive rate of HBV DNA ISH was comparable in tumor and surrounding tissue (17.6% versus 22.9%, p = 0.36). Yet the HBV DNA signal in tumor tissue showed predominant nuclear localization (87.0%) whereas staining pattern in adjacent tissue was mixed (43.3% nuclear localization, p = 0.0015). Finally, no significant association between intra-tumor HBV DNA/HBsAg positivity and major histological markers (microvascular invasion, tumor differentiation, etc.) or recurrence after surgery was observed.ConclusionsThese data confirmed the largely integrated state of HBV DNA, weaker expression and altered localization of surface antigen in tumor compared with surrounding tissue. The strikingly different prevalence and localization of HBsAg and HBV DNA reflected the complex and heterogeneous mechanisms leading to HBV-induced tumorigenesis.

Licensed liposomal vaccines and adjuvants in the antigen delivery system.

Liposomes (LSs) are promising nanoparticles with unique properties such as controlled nanosize, large surface area, increased reactivity, and ability to undergo modification. Worldwide, licensed liposomal forms of antibiotics, hormones, antioxidants, cytostatics, ophthalmic drugs, etc., are available on the pharmaceutical market. This review focuses on the adjuvant properties of LSs in the production of vaccines (VACs). LS-VACs have the following advantages: antigens with low immunogenicity can become highly immunogenic; LSs can include both hydrophilic and hydrophobic antigens; LSs allow to achieve a prolonged specific action of antibodies; and LSs reduce the toxicity and pyrogenicity of encapsulated antigens and adjuvants. The immune response is influenced by the composition of the liposomal membrane, physicochemical characteristics of lipids, antigen localization in LSs, interaction of LSs with complement, and a number of proteins, which leads to opsonization. The major requirements for adjuvants are their ability to enhance the immune response, biodegradability, and elimination from the organism, and LSs fully meet these requirements. The effectiveness and safety of LSs as carriers in the antigen delivery system have been proven by the long-term clinical use of licensed vaccines against hepatitis A, influenza, herpes zoster, malaria, and COVID-19.

Astrocyte Cell Surface Antigen 2 and Other Potential Cell Surface Markers of Enteric glia in the Mouse Colon.

Enteric glia regulate gut functions in health and disease through diverse interactions with neurons and immune cells. Intracellular localization of traditional markers of enteric glia such as GFAP, s100b, and Sox10 makes them incompatible for studies that require antigen localization at the cell surface. Thus, new tools are needed for probing the heterogeneous roles of enteric glia at the protein, cell, and functional levels. Here we selected several cell surface antigens including Astrocyte Cell Surface Marker 2 (ACSA2), Cluster of differentiation 9 (CD9), lysophosphatidic acid receptor 1 (LPAR1), and Proteolipid protein 1 (PLP1) as potential markers of enteric glia. We tested their specificity for enteric glia using published single-cell/-nuclei and glia-specific translating mRNA enriched transcriptome datasets, immunolabeling, and flow cytometry. The data show that ACSA2 is a specific marker of mucosal and myenteric glia while other markers are suitable for identifying all subpopulations of enteric glia (LPAR1), glia and immune cells (CD9), or are not suitable for cell-surface labeling (PLP1). These new tools will be useful for future work focused on understanding specific glial functions in health and disease. Summary Statement This study identifies astrocyte cell surface antigen 2 as a novel marker of myenteric glia in the intestine. This, in combination with other markers identified in this study, could be used for selective targeting of enteric glia.

CYTOPATHOGENICITY OF INFLUENZA VIRUS (H4 N3)

Monolayer tissue cultures of chicken embryo fibroblast ( CEF ) cells infected with avian influenza virus isolate were examined by the hematoxylie and eosin (H&amp;E) staining and indirect immunoperoxidase test for studying the cytopathogenic effect of the virus. Cytopathological changes which occurred in the uncleus of infected cells included nuclear and nucleolar hypertropy, chromatin margination and intranuclear inclusions. The most striking cytoplasmic change were the presence of perinuclear. eosinophilic inclusions at 22-36 hours post inoculation ( p. i.). Vacuolization, and granulation were also observed. Indirect immunoperoxidase ( IP ) test demonstrated the localization of influenza virus antigens in infected cells. A positive peroxidase reaction observed in the nucleus and cytoplasm were similar to those shown hematoxyline and eosin staining.

Pathological changes and antigen localization in the small intestine of rabbits infected with Eimeria magna

&lt;p&gt;Coccidiosis is a major disease caused by various &lt;em&gt;Eimeria&lt;/em&gt; species in rabbits. The aim of the present study was to investigate the haematological and pathological changes in rabbits infected with &lt;em&gt;E. magna&lt;/em&gt;. Moreover, the localisation of coccidial antigens was examined in the intestines of rabbits with two kinds of serum as primary antibodies. In the present study, forty-five 28-day-old weaned rabbits were randomly divided into three groups and reared in three separate places. Group A was infected with 20×10&lt;sup&gt;3&lt;/sup&gt; sporulated oocysts of &lt;em&gt;E. magna&lt;/em&gt;, group B was only used to produce anti-&lt;em&gt;E. intestinalis&lt;/em&gt; serum by infecting them with 3×10&lt;sup&gt;3&lt;/sup&gt; sporulated oocysts of &lt;em&gt;E. intestinalis&lt;/em&gt;, and group C was designated as the control group. According to histopathological evaluation of group A, the epithelial cells of the jejunum and ileum were parasitised with a large number of oocysts and other stages of &lt;em&gt;E. magna&lt;/em&gt;. The haematological results showed that red blood cell counts, haemoglobin counts, haematocrit levels and the percentage of lymphocytes were significantly decreased in group A compared with group C (&lt;em&gt;P&lt;/em&gt;&amp;lt;0.01), but white blood cell counts and the percentage of neutrophils were significantly increased (&lt;em&gt;P&lt;/em&gt;&amp;lt;0.01). The weight of group A began to decrease on the 5&lt;sup&gt;th&lt;/sup&gt; day after infection, and this decrease continued until the 9th day. Immunohistochemistry staining revealed that two kinds of coccidial antigens were basically located at the same sites of the intestine when anti-&lt;em&gt;E. intestinalis&lt;/em&gt; serum and anti-&lt;em&gt;E. magna&lt;/em&gt; serum were used as primary antibodies. Most likely, &lt;em&gt;E. magna&lt;/em&gt; and &lt;em&gt;E. intestinalis&lt;/em&gt; antigens have some similar antigenic determinants; this finding provides a theoretical basis for screening for common antigens of these two coccidian species.&lt;/p&gt;

Molecular and Pathological Investigations of Selected Viral Neuropathogens in Rabies-Negative Brains of Cats and Dogs Revealed Neurotropism of Carnivore Protoparvovirus-1.

Throughout the year, the Thai Red Cross Society (TRCS), Bangkok, Thailand, received more than 100 animals that died of suspected rabies due to neurological clinical signs. Concerning the role of viral infection in the brain in the outcome of neurological diseases in cats and dogs, a comprehensive study was conducted of 107 brain samples of cats and dogs submitted to the TRCS from August 2019 to August 2020. Selective molecular screening using conventional polymerase chain reaction (PCR) and reverse transcription PCR targeting nine viral pathogens was employed in addition to histopathological investigations. The results showed that carnivore protoparvovirus-1 (CPPV-1) was detected in 18.69% of the cats and dogs sampled (20/107). These results were found in young and old animals; the brain tissue did not show any pathological changes suggesting encephalitis or cerebellar hypoplasia. In addition, feline calicivirus, feline alphaherpesvirus-1, feline coronavirus, and canine distemper virus were also detected, providing a broader range of potential viral infections to consider in the clinical manifestation of neurological disorders in companion animals. The detection of all pathogens was confirmed by the localization of each viral antigen in various resident brain cells using immunohistochemistry. A unique L582S amino acid substitution of the non-structural protein 1 gene coding sequence, speculated to be associated with the neurotropism of CPPV-1 in cats and dogs, was not evident. In conclusion, this study revealed a noteworthy neurotropism of CPPV-1 in both cats and dogs without neurological lesions.

Feline morbillivirus-1 in dogs with respiratory diseases.

Feline morbillivirus-1 (FeMV-1) is a viral pathogen associated with kidney disease in domestic cats and wild felids. We initially identified the FeMV-1 from the lung of a necropsied dog with severe pulmonary disease by the reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we investigated FeMV-1 in nasal and oral swab samples from 73 healthy and 113 dogs with respiratory illnesses. We found polymerase chain reaction (PCR)-positive FeMV-1 from only 14/113 (12.39%) dogs with respiratory disease (p=.001). Of these 14 dogs, six were co-infected with other canine respiratory viruses (6/14; 42.86%). Two independent immunohistochemistry procedures, using antibodies against matrix and phosphoprotein of FeMV-1, confirmed the presence of FeMV-1 in lung tissues of two necropsied dogs (out of a total of 22 dogs, 9.09%) that died from respiratory disease. This finding corresponded to transmission electron microscopy findings that paramyxoviral particles exist in lung epithelia. FeMV-1 antigen localization was also evident in the kidney, lymphoid and brain tissues of two deceased dogs. FeMV-1 was successfully isolated from a necropsied dog and from two living dogs, all with respiratory illnesses, which supports FeMV infection in dogs. The detection of FeMV-1 in dog tissues expands the known tropism of this virus to a non-felid host. Our findings indicate that FeMV-1, alone or in co-infection with other viral pathogens, might contribute to respiratory illness and death in dogs.

Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against Both Diseases.

GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg−) Salmonella Typhimurium GMMA. Only the OAg− GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg− GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg− GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

Simple SummaryThe glycocalyx (GCX) is a hydrated, gel-like layer of biological macromolecules attached to the cell membrane. The GCX acts as a barrier and regulates the entry of external substances into the cell. The function of the GCX is highly dependent on its structure and composition. Pathogenic factors can affect the protective structure of the GCX. We know very little about the three-dimensional organization of the GXC. The tiny and delicate structures of the GCX are difficult to study by microscopic techniques. In this study, we evaluated a method to preserve and label sensitive GCX components with antibodies for high-resolution microscopy analysis. High-resolution microscopy is a powerful tool because it allows visualization of ultra-small components and biological interactions. Our method can be used as a tool to better understand the role of the GCX during the development and progression of diseases, such as viral infections, tumor invasion, and the development of atherosclerosis.The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.

Expression and Localisation of CD44 Antigen as a Prognostic Factor of Oral Leukoplakia

Abstract It is essential to identify markers that could indicate the presence of early molecular changes in premalignant tissues like oral leukoplakia (OL). CD44 adhesion molecule is not only a stem cell marker, but also determines cell proliferation and migration in malignant processes. The aim of our study was to assess the amount and pattern of CD44 antigen expression by epithelial and mononuclear cells in the lamina propria under OL and their role in premalignant lesions. The current study included 102 cases of OL and ten biopsies from healthy oral mucosa. Immunohistochemical CD44 antigen expression was determined in 34 cases by a standard EnVision imaging system in three points of OL: both edges and centre. Statistical analysis was done using GraphPad Prism software version 8.4.0. In OL, statistically significant overexpression of membranous CD44 was demonstrated compared to healthy mucosa (p &lt; 0.0001). The intra-cytoplasmatic CD44 expression of epithelium together with characteristic nuclear changes may be used as a predictive factor for potential malignant transformation of non-homogenous leukoplakia. CD44 expression in mononuclear cells under the basal membrane in OL (p &lt; 0.05) possibly influences the process of premalignant lesion transformation into intraepithelial cancer. Further study of CD44 antigen expression in intra-cytoplasmatic structures is required for better explanation of the role of this glycoprotein.