Kinetics of Cytokine mRNA Expression in the Central Nervous System Following Lethal and Nonlethal Coronavirus-Induced Acute Encephalomyelitis

AbstractThe potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mRNA expression following infection with the neurotropic JHM strain of mouse hepatitis virus. Mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. Infection with both viruses induced the accumulation of mRNAs associated with Th1- and Th2-type cytokines, including IFN-γ, IL-4, and IL-10. Peak mRNA accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. TNF-α mRNA was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. Rapid transient increases in the mRNAs encoding IL-12, iNOS, IL-1α, IL-1β, and IL-6 occurred following infection. Nonlethal infections were associated with increased IL-12, IL-1β, and earlier expression of IL-6, while lethal infections were associated with increased iNOS and IL-1α mRNA. These data suggest a rapid but differential response within the central nervous system cells to infection by different JHMV variants. However, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. Accumulation of both Th1 and Th2 cytokines in the central nervous system of JHMV-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis

Mouse hepatitis virus neurovirulence: evidence of a linkage between S glycoprotein expression and immunopathology.

Differences in disease outcome between the highly neurovirulent MHV-JHM and mildly neurovirulent MHV-A59 have been attributed to variations within the spike (S) glycoprotein. Previously, we found that MHV-JHM neurovirulence was marked by diminished expression of interferon-gamma (IFN-gamma) mRNA and a reduced presence of CD8 T cells in the CNS concomitant with heightened macrophage inflammatory protein (MIP)-1 transcript levels and greater macrophage infiltration relative to MHV-A59 infection. Here, the ability of the S and non-spike genes to regulate these immune responses was evaluated using chimeric viruses. Chimeric viruses WTR13 and S4R22 were made on MHV-A59 variant backgrounds and, respectively, contained the S gene of MHV-A59 and MHV-JHM. Unexpectedly, genes other than S appeared to modulate events critical to viral replication and survival. Unlike unresolving MHV-JHM infections, the clearance of WTR13 and S4R22 infections coincided with strong IFN-gamma transcription and an increase in the number of CD8 T cells infiltrating into the CNS. However, despite the absence of detectable viral titers, approximately 40% of S4R22-infected mice succumbed within 3 weeks, indicating that the enhanced mortality following S4R22 infection was not associated with high viral titers. Instead, similar to the MHV-JHM infection, reduced survival following S4R22 infection was observed in the presence of elevated MIP-1alpha and MIP-1beta mRNA accumulation and enhanced macrophage numbers within infected brains. These observations suggest that the S protein of MHV-JHM influences neurovirulence through the induction of MIP-1alpha- and MIP-1beta-driven macrophage immunopathology

Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted

In-Vitro Investigation of Very Long Defibrillation Shocks: Design and Testing of a Capacitor-Free Defibrillator

Abstrac

Functional diversity of chemokines and chemokine receptors in response to viral infection of the central nervous system.

Encounters with neurotropic viruses result in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign infection. One of the principal factors that control the outcome of infection is the localized tissue response and subsequent immune response directed against the invading toxic agent. It is the role of the immune system to contain and control the spread of virus infection in the central nervous system (CNS), and paradoxically, this response may also be pathologic. Chemokines are potent proinflammatory molecules whose expression within virally infected tissues is often associated with protection and/or pathology which correlates with migration and accumulation of immune cells. Indeed, studies with a neurotropic murine coronavirus, mouse hepatitis virus (MHV), have provided important insight into the functional roles of chemokines and chemokine receptors in participating in various aspects of host defense as well as disease development within the CNS. This chapter will highlight recent discoveries that have provided insight into the diverse biologic roles of chemokines and their receptors in coordinating immune responses following viral infection of the CNS

Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia

Karen Day and colleagues show that increased microcytic erythrocyte count may contribute substantially to the protection of α+-thalassaemia-homozygous children against severe malaria anaemia

RNase L Mediated Protection from Virus Induced Demyelination

IFN-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. However, the IFN-α/β dependent mechanisms underlying innate anti-viral functions within the CNS are poorly understood. The role of RNase L in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the IFN-α/β pathway through RNA degradation intermediates. Infection of RNase L deficient (RL−/−) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. However, RNase L deficiency did not affect overall control of infectious virus, or diminish IFN-α/β expression in the CNS. Furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the CNS. The unique phenotype of infected RL−/− mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. Increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. These data demonstrate a novel protective role for RNase L in viral induced CNS encephalomyelitis, which is not reflected in overall viral control or propagation of IFN-α/β mediated signals. Protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material

A Protective Role for ELR+ Chemokines during Acute Viral Encephalomyelitis

The functional role of ELR-positive CXC chemokines in host defense during acute viral-induced encephalomyelitis was determined. Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice resulted in the rapid mobilization of PMNs expressing the chemokine receptor CXCR2 into the blood. Migration of PMNs to the CNS coincided with increased expression of transcripts specific for the CXCR2 ELR-positive chemokine ligands CXCL1, CXCL2, and CXCL5 within the brain. Treatment of JHMV-infected mice with anti-CXCR2 blocking antibody reduced PMN trafficking into the CNS by >95%, dampened MMP-9 activity, and abrogated blood-brain-barrier (BBB) breakdown. Correspondingly, CXCR2 neutralization resulted in diminished infiltration of virus-specific T cells, an inability to control viral replication within the brain, and 100% mortality. Blocking CXCR2 signaling did not impair the generation of virus-specific T cells, indicating that CXCR2 is not required to tailor anti-JHMV T cell responses. Evaluation of mice in which CXCR2 is genetically silenced (CXCR2−/− mice) confirmed that PMNs neither expressed CXCR2 nor migrated in response to ligands CXCL1, CXCL2, or CXCL5 in an in vitro chemotaxis assay. Moreover, JHMV infection of CXCR2−/− mice resulted in an approximate 60% reduction of PMN migration into the CNS, yet these mice survived infection and controlled viral replication within the brain. Treatment of JHMV-infected CXCR2−/− mice with anti-CXCR2 antibody did not modulate PMN migration nor alter viral clearance or mortality, indicating the existence of compensatory mechanisms that facilitate sufficient migration of PMNs into the CNS in the absence of CXCR2. Collectively, these findings highlight a previously unappreciated role for ELR-positive chemokines in enhancing host defense during acute viral infections of the CNS