Lymphocytic Choriomeningitis Virus Infection in FVB Mouse Produces Hemorrhagic Disease

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-gamma, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVBxC57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing

Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

Background Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. Findings Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. Conclusions The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin

Alternative RNA splicing: contribution to pain and potential therapeutic strategy

Since the sequencing of metazoan genomes began, it has become clear that the number of expressed proteins far exceeds the number of genes. It is now estimated that greater up to 98% of human genes give rise to multiple proteins through alternative pre-mRNA splicing. This review highlights the known alternative splice variants of many channels, receptors and growth factors important in nociception and pain. Recently, pharmacological control of alternative splicing has been proposed as potential therapy in cancer, wet age-related macular degeneration, retroviral infections and pain. In this review we consider the effects that known splice variants of molecules key to nociception/pain have on nociceptive processing and/or analgesic action, and the potential for control of alternative pre-mRNA splicing as a novel analgesic strategy

Analysis of immune modulators in rainbow trout (Oncorhynchus mykiss)

The immune systems of various teleost fish have been studied in some detail for the past several decades. One aspect of fish immunity, that of endogenously produced modulating factors, has recently received a great deal of attention. Understanding the functions and roles of endogenous factors that regulate fish immunity is paramount to expanding the fields of fish immunology and vaccinology. It is know that several lymphoid cell derived factors are detectable in in vitro cell culture systems and exhibit immune modulating effects similar to well studied proteins in mammals. However in comparison, few genes or gene products involved in the modulation of the trout immune responses have been isolated, cloned and characterized. The studies described herein were designed to isolate specific genes from rainbow trout (0ncorhynchus mykiss) and characterize their involvement in the modulation and regulation of the trout immune system. Two distinct genes were isolated cloned and sequenced. The first, non-specific cytotoxic cell enhancement factor (NCEF) gene is closely related to a human gene termed "natural killer enhancement factor" (NKEF) which is important in the modulation of human natural killer cell activity. The second gene is closely related to a group of recently characterized mammalian genes involved in the signal transduction of cytokines termed "STATs". The role of these genes and their respective protein products will be examined and discussed. The antigenic structures of the fish proteins (NCEF and STAT5) were examined by western blot and immunohistochemistry. Monoclonal antibodies derived against the respective human proteins were found to cross react with the corresponding trout proteins, demonstrating antigenic relatedness. The monoclonal regents were also used to analyze the expression of these proteins in fish cells of lymphoid and non lymphoid origin. In vitro cell culture analysis was used to determine the effects and roles of NCEF and STAT5 gene products in the trout immune system. The cytolytic and apoptotic killing activities of spleen, head kidney and peripheral blood leukocytes were found to be enhanced by NCEF. Mitogenic stimulation of peripheral blood lymphoid cells resulted in the trout STAT5 protein binding to a know sequences contained with in the promoters of genes transcriptionally activated in response to cytokine exposure

Human Herpesvirus 8 Detection in Nasal Secretions and Saliva

The presence of human herpesvirus 8 (HHV-8) was determined by polymerase chain reaction (PCR) in nasal secretions and saliva from 14 HHV-8-seropositive persons, including 8 Kaposi's sarcoma patients: 7 were human immunodeficiency virus type 1-infected, 6 of whom were asymptomatic. HHV-8 was detected in one or both body fluids in 8 (57%) of 14 subjects. Parallel PCR testing revealed the concomitant presence of cytomegalovirus, Epstein-Barr virus, and HHV-6 in various combinations in these body fluids. These data indicate frequent shedding of multiple herpesviruses in nasal secretions and saliva, particularly in Kaposi's sarcoma patients. Both body fluids are therefore potential sources HHV-8 by nonsexual transmission