Rift Valley fever virus detection in susceptible hosts with special emphasis in insects

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes

Arachidonic acid pathway alterations in cerebrospinal fluid of dogs with naturally occurring spinal cord injury

BACKGROUND: Canine intervertebral disc πherniation causes a naturally-occurring spinal cord injury (SCI) that bears critical similarities to human SCI with respect to both injury pathomechanisms and treatment. As such, it has tremendous potential to enhance our understanding of injury biology and the preclinical evaluation of novel therapies. Currently, there is limited understanding of the role of arachidonic acid metabolites in canine SCI. RESULTS: The CSF concentrations of PLA2 and PGE2 were higher in SCI dogs compared to control dogs (p = 0.0370 and 0.0273, respectively), but CSF LCT4 concentration in SCI dogs was significantly lower than that in control dogs (p < 0.0001). Prostaglandin E2 concentration in the CSF was significantly and positively associated with increased severity of SCI at the time of sampling (p = 0.041) and recovery 42 days post-injury (p = 0.006), as measured by ordinal behavioral scores. CONCLUSION: Arachidonic acid metabolism is altered in dogs with SCI, and these data suggest that these AA metabolites reflect injury severity and recovery, paralleling data from other model systems

Th17-skewed immune response and cluster of differentiation 40 ligand expression in canine steroid-responsive meningitis-arteritis, a large animal model for neutrophilic meningitis

Background: Steroid-responsive meningitis-arteritis (SRMA) is an immune-mediated disorder characterized by neutrophilic pleocytosis and an arteritis particularly in the cervical leptomeninges. Previous studies of the disease have shown increased levels of IL-6 and TGF-beta(1) in cerebrospinal fluid (CSF). In the presence of these cytokines, naive CD4+ cells differentiate into Th17 lymphocytes which synthesize interleukin 17 (IL-17). It has been shown that IL-17 plays an active role in autoimmune diseases, it induces and mediates inflammatory responses and has an important role in recruitment of neutrophils. The hypothesis of a Th17-skewed immune response in SRMA should be supported by evaluating IL-17 and CD40L, inducing the vasculitis. Methods: An enzyme-linked immunosorbent assay (ELISA) was performed to measure IL-17 and CD40L in serum and CSF from a total of 79 dogs. Measurements of patients suffering from SRMA in the acute state (SRMA A) were compared with levels of patients under treatment with steroids (SRMA T), recurrence of the disease (SRMA R), other neurological disorders, and healthy dogs, using the two-part test. Additionally, secretion of IL-17 and interferon gamma (IFN-gamma) from the peripheral blood mononuclear cells (PBMCs) was confirmed by an enzyme-linked immunospot (ELISpot) assay. Results: Significant higher levels of IL-17 were found in CSF of dogs with SRMA A compared with SRMA T, other neurological disorders and healthy dogs (p < 0.0001). In addition, levels of CD40L in CSF in dogs with SRMA A and SRMA R were significantly higher than in those with SRMA T (p = 0.0004) and healthy controls (p = 0.014). Furthermore, CSF concentrations of IL-17 and CD40L showed a strong positive correlation among each other (rSpear = 0.6601;p < 0.0001) and with the degree of pleocytosis (rSpear = 0.8842;p < 0.0001 and rSpear = 0.6649;p < 0.0001, respectively). IL-17 synthesis from PBMCs in SRMA patients was confirmed;however, IL-17 is mainly intrathecally produced. Conclusions: These results imply that Th17 cells are inducing the autoimmune response in SRMA and are involved in the severe neutrophilic pleocytosis and disruption of the blood-brain barrier (BBB). CD-40L intrathecal synthesis might be involved in the striking vasculitis. The investigation of the role of IL-17 in SRMA might elucidate important pathomechanism and open new therapeutic strategies

Canine central nervous system neoplasm phenotyping using tissue microarray technique

Tissue microarrays (TMAs) represent a useful technique for the simultaneous phenotyping of large sample numbers and are particularly suitable for histopathologic tumor research. In this study, TMAs were used to evaluate semiquantitatively the expression of multiple antigens in various canine central nervous system (CNS) neoplasms and to identify markers with potential discriminative diagnostic relevance. Ninety-seven canine CNS neoplasms, previously diagnosed on hematoxylin and eosin sections according to the World Health Organization classification, were investigated on TMAs, with each tumor consisting of 2 cylindrical samples from the center and the periphery of the neoplasm. Tumor cells were phenotyped using a panel of 28 monoclonal and polyclonal antibodies, and hierarchical clustering analysis was applied to group neoplasms according to similarities in their expression profiles. Hierarchical clustering generally grouped cases with similar histologic diagnoses; however, gliomas especially exhibited a considerable heterogeneity in their positivity scores. Multiple tumor groups, such as astrocytomas and oligodendrogliomas, significantly differed in the proportion of positive immunoreaction for certain markers such as p75NTR, AQP4, GFAP, and S100 protein. The study highlights AQP4 and p75NTR as novel markers, helping to discriminate between canine astrocytoma and oligodendroglioma. Furthermore, the results suggest that p75NTR and proteolipid protein may represent useful markers, whose expression inversely correlates with malignant transformation in canine astrocytomas and oligodendrogliomas, respectively. Tissue microarray was demonstrated to be a useful and time-saving tool for the simultaneous immunohistochemical characterization of multiple canine CNS neoplasms. The present study provides a detailed overview of the expression patterns of different types of canine CNS neoplasms

Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods

Canine Central Nervous System Neoplasm Phenotyping Using Tissue Microarray Technique

Tissue microarrays (TMAs) represent a useful technique for the simultaneous phenotyping of large sample numbers and are particularly suitable for histopathologic tumor research. In this study, TMAs were used to evaluate semiquantitatively the expression of multiple antigens in various canine central nervous system (CNS) neoplasms and to identify markers with potential discriminative diagnostic relevance. Ninety-seven canine CNS neoplasms, previously diagnosed on hematoxylin and eosin sections according to the World Health Organization classification, were investigated on TMAs, with each tumor consisting of 2 cylindrical samples from the center and the periphery of the neoplasm. Tumor cells were phenotyped using a panel of 28 monoclonal and polyclonal antibodies, and hierarchical clustering analysis was applied to group neoplasms according to similarities in their expression profiles. Hierarchical clustering generally grouped cases with similar histologic diagnoses; however, gliomas especially exhibited a considerable heterogeneity in their positivity scores. Multiple tumor groups, such as astrocytomas and oligodendrogliomas, significantly differed in the proportion of positive immunoreaction for certain markers such as p75NTR, AQP4, GFAP, and S100 protein. The study highlights AQP4 and p75NTR as novel markers, helping to discriminate between canine astrocytoma and oligodendroglioma. Furthermore, the results suggest that p75NTR and proteolipid protein may represent useful markers, whose expression inversely correlates with malignant transformation in canine astrocytomas and oligodendrogliomas, respectively. Tissue microarray was demonstrated to be a useful and time-saving tool for the simultaneous immunohistochemical characterization of multiple canine CNS neoplasms. The present study provides a detailed overview of the expression patterns of different types of canine CNS neoplasms

Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods