GM-CSF Promotes Immune Response and Survival in a Mouse Model of COVID-19.

COVID-19 results in increased expression of inflammatory cytokines, but inflammation-targeting clinical trials have yielded poor to mixed results. Our studies of other disorders with an inflammatory component, including Alzheimer's disease, chemobrain, Down syndrome, normal aging, and West Nile Virus infection, showed that treatment with the 'pro-inflammatory' cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in humans or mouse models alleviated clinical, behavioral, and pathological features. We proposed that human recombinant GM-CSF (sargramostim) be repurposed to promote both the innate and adaptive immune responses in COVID-19 to reduce viral load and mortality1. Here, we report the results of a placebo-controlled study of GM-CSF in human ACE2 transgenic mice inoculated intranasally with SARS-CoV2 virus, a model of COVID-19. Infection resulted in high viral titers in lungs and brains and over 85% mortality. GM-CSF treatment beginning one day after infection increased anti-viral antibody titers, lowered mean lung viral titers proportionately (p=0.0020) and increased the odds of long-term survival by up to 5.8-fold (p=0.0358), compared to placebo. These findings suggest that, as an activator of both the innate and adaptive immune systems, GM-CSF/sargramostim may be an effective COVID-19 therapy with the potential to protect from re-infection more effectively than treatment with antiviral drugs or monoclonal antibodies.

Metagenomic Investigation of Idiopathic Meningoencephalomyelitis in Dogs.

BACKGROUND: Meningoencephalomyelitis of unknown origin (MUO) is a common and life-threatening neuroinflammatory disease in dogs. Features of the disease are suggestive of an underlying immune-mediated process, but the association of this disease with a pathogen is still unknown. HYPOTHESIS/OBJECTIVES: To search for candidate etiologic agent associated with cases if MUO using next generation metagenomic sequencing. ANIMALS: Twenty-two dogs diagnosed with either MUO (11/22; 10 CSF and 3 brain), or noninflammatory CNS diseases inconsistent with MUO (11/22; 11 CSF and 2 brain) that served as negative controls. METHODS: A case control study was performed by identifying MUO and non-MUO cases. Samples were blindly processed and then unblinded for comparative analyses. Inclusion criteria for MUO cases included consistent MRI lesions and inflammatory CSF with a negative PCR panel for infectious agents or histopathologic diagnosis. Dogs with glucocorticoid therapy within 2 weeks of sample collection were excluded. Fresh-frozen cerebrospinal fluid (CSF; 21) and brain (5) samples were collected and RNA and DNA were extracted separately for shotgun metagenomic sequencing. Known positive samples were used as controls to validate our sequencing and analysis pipelines and to establish limits of detection. Sequencing results were analyzed at a nucleotide and protein level for broad comparison to known infectious organisms. RESULTS: No candidate etiologic agents were identified in dogs with MUO. CONCLUSIONS AND CLINICAL IMPORTANCE: These results support but do not prove the hypothesis that MUO is not associated with infectious agents and might be an autoimmune disease.

CSF findings in 250 patients with serologically confirmed West Nile virus meningitis and encephalitis.

OBJECTIVE: To provide a large, comprehensive evaluation of the CSF findings in patients with serologically confirmed West Nile virus (WNV), CNS disease, and their correlation with outcome. METHODS: CSF samples from 334 WNV-infected hospitalized patients were analyzed. Information was available and extracted from the medical records of 250 of these patients, and CSF parameters correlated with clinical and epidemiologic features of disease (e.g., patient age, sex, outcome). RESULTS: Patients with meningitis had a mean of 226 cells/mm3, and those with encephalitis had a mean of 227 cells/mm3. Three percent of meningitis patients and 5% of encephalitis patients had fewer than 5 cells/mm3, and approximately 8% of both groups had more than 500 cells/mm3. Patients with meningitis had a mean of 41% neutrophils, and those with encephalitis had 45%. Forty-five percent of meningitis patients and 37% of encephalitis patients had at least 50% neutrophils in their initial CSF specimen. Neither the mean percent neutrophils nor their distribution differed significantly between groups. Forty-seven percent of encephalitis patients and 16% of meningitis patients had CSF protein of 100 mg/dL or greater (p < 0.01). Although specific CSF parameters, including nucleated cell count and protein concentration, correlated significantly with outcome, multivariate analysis suggested that their total predictive value was modest. Age was an additional predictor of outcome independent of CSF variables in all patients. CONCLUSIONS: Serologically confirmed West Nile virus meningitis and encephalitis produce similar degrees of CSF pleocytosis and are frequently associated with substantial CSF neutrophilia. Patients with encephalitis have higher CSF protein concentrations and are more likely to have adverse outcomes, including admission to long-term care facilities or even death after their acute illness. CSF findings were only a modest predictor of disease outcome, with patient age adding important independent prognostic information.

Inhibition of NF-kappa B activity and cFLIP expression contribute to viral-induced apoptosis.

Virus-induced activation of nuclear factor-kappa B (NF-kappaB) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-kappaB is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-kappaB activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-kappaB regulation, where NF-kappaB activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-kappaB activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-kappaB is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-kappaB also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-kappaB-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-kappaB activation in reovirus-infected cells. Further, inhibition of NF-kappaB activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-kappaB and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.

Mechanisms of reovirus-induced cell death and tissue injury: role of apoptosis and virus-induced perturbation of host-cell signaling and transcription factor activation.

Reoviruses have provided insight into the roles played by specific viral genes and the proteins they encode in virus-induced cell death and tissue injury. Apoptosis is a major mechanism of cell death induced by reoviruses. Reovirus-induced apoptosis involves both death-receptor and mitochondrial cell death pathways. Reovirus infection is associated with selective activation of mitogen activated protein kinase (MAPK) cascades including JNK/SAPK. Infection also perturbs transcription factor signaling resulting in the activation of c-Jun and initial activation followed by strain-specific inhibition of NF-kappaB. Infection results in changes in the expression of genes encoding proteins involved in cell cycle regulation, apoptosis, and DNA damage and repair processes. Apoptosis is a major mechanism of reovirus-induced injury to key target organs including the CNS and heart. Inhibition of apoptosis through the use of caspase or calpain inhibitors, minocycline, or in caspase 3(-/-) mice all reduce virus-associated tissue injury and enhance survival of infected animals. Reoviruses induce apoptotic cell death (oncolysis) in a wide variety of cancer cells and tumors. The capacity of reoviruses to grow efficiently in transformed cells is enhanced by the presence of an activated Ras signaling pathway likely through mechanisms involving inhibition of antiviral PKR signaling and activation of Ras/RalGEF/p38 pathways. The potential of reovirus-induced oncolysis in therapy of human cancers is currently being investigated in phase I/II clinical trials.

Mechanisms of apoptosis during reovirus infection.

Reovirus infection has proven to be an excellent experimental system for studying mechanisms of virus-induced pathogenesis. Reoviruses induce apoptosis in a wide variety of cultured cells in vitro and in target tissues in vivo, including the heart and central nervous system. In vivo, viral infection, tissue injury, and apoptosis colocalize, suggesting that apoptosis is a critical mechanism by which disease is triggered in the host. This review examines the mechanisms of reovirus-induced apoptosis and investigates the possibility that inhibition of apoptosis may provide a novel strategy for limiting virus-induced tissue damage following infection.

Genes Induced by Reovirus Infection Have a Distinct Modular Cis-Regulatory Architecture.

The availability of complete genomes and global gene expression profiling has greatly facilitated analysis of complex genetic regulatory systems. We describe the use of a bioinformatics strategy for analyzing the cis-regulatory design of genes diferentially regulated during viral infection of a target cell. The large-scale transcriptional activity of human embryonic kidney (HEK293) cells to reovirus (serotype 3 Abney) infection was measured using the Affymetrix HU-95Av2 gene array. Comparing the 2000 base pairs of 5' upstream sequence for the most differentially expressed genes revealed highly preserved sequence regions, which we call "modules". Higher-order patterns of modules, called "super-modules", were significantly over-represented in the 5' upstream regions of transcriptionally responsive genes. These supermodules contain binding sites for multiple transcription factors and tend to define the role of genes in processes associated with reovirus infection. The supermodular design encodes a cis-regulatory logic for transducing upstream signaling for the control of expression of genes involved in similar biological processes. In the case of reovirus infection, these processes recapitulate the integrated response of cells including signal transduction, transcriptional regulation, cell cycle control, and apoptosis. The computational strategies described for analyzing gene expression data to discover cis-regulatory features and associating them with pathological processes represents a novel approach to studying the interaction of a pathogen with its target cells.

Reovirus-induced apoptosis: A minireview.

Reoviruses infect a variety of mammalian hosts and serve as an important experimental system for studying the mechanisms of virus-induced injury. Reovirus infection induces apoptosis in cultured cells in vitro and in target tissues in vivo, including the heart and central nervous system (CNS). In epithelial cells, reovirus-induced apoptosis involves the release of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) from infected cells and the activation of TRAIL-associated death receptors (DRs) DR4 and DR5. DR activation is followed by activation of caspase 8, cleavage of Bid, and the subsequent release of pro-apoptotic mitochondrial factors. By contrast, in neurons, reovirus-induced apoptosis involves a wider array of DRs, including TNFR and Fas, and the mitochondria appear to play a less critical role. These results show that reoviruses induce apoptotic pathways in a cell and tissue specific manner. In vivo there is an excellent correlation between the location of viral infection, the presence of tissue injury and apoptosis, indicating that apoptosis is a critical mechanism by which disease is triggered in the host. These studies suggest that inhibition of apoptosis may provide a novel strategy for limiting virus-induced tissue damage following infection.

Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death.

Apoptosis plays an important role in the pathogenesis of many viral infections. Despite this fact, the apoptotic pathways triggered during viral infections are incompletely understood. We now provide the first detailed characterization of the pattern of caspase activation following infection with a cytoplasmically replicating RNA virus. Reovirus infection of HEK293 cells results in the activation of caspase-8 followed by cleavage of the pro-apoptotic protein Bid. This initiates the activation of the mitochondrial apoptotic pathway leading to release of cytochrome c and activation of caspase-9. Combined activation of death receptor and mitochondrial pathways results in downstream activation of effector caspases including caspase-3 and caspase-7 and cleavage of cellular substrates including PARP. Apoptosis is initiated by death receptor pathways but requires mitochondrial amplification producing a biphasic pattern of caspase-8, Bid, and caspase-3 activation.

Reovirus infection activates JNK and the JNK-dependent transcription factor c-Jun.

Viral infection often perturbs host cell signaling pathways including those involving mitogen-activated protein kinases (MAPKs). We now show that reovirus infection results in the selective activation of c-Jun N-terminal kinase (JNK). Reovirus-induced JNK activation is associated with an increase in the phosphorylation of the JNK-dependent transcription factor c-Jun. Reovirus serotype 3 prototype strains Abney (T3A) and Dearing (T3D) induce significantly more JNK activation and c-Jun phosphorylation than does the serotype 1 prototypic strain Lang (T1L). T3D and T3A also induce more apoptosis in infected cells than T1L, and there was a significant correlation between the ability of these viruses to phosphorylate c-Jun and induce apoptosis. However, reovirus-induced apoptosis, but not reovirus-induced c-Jun phosphorylation, is inhibited by blocking TRAIL/receptor binding, suggesting that apoptosis and c-Jun phosphorylation involve parallel rather than identical pathways. Strain-specific differences in JNK activation are determined by the reovirus S1 and M2 gene segments, which encode viral outer capsid proteins (sigma1 and mu1c) involved in receptor binding and host cell membrane penetration. These same gene segments also determine differences in the capacity of reovirus strains to induce apoptosis, and again a significant correlation between the capacity of T1L x T3D reassortant reoviruses to both activate JNK and phosphorylate c-Jun and to induce apoptosis was shown. The extracellular signal-related kinase (ERK) is also activated in a strain-specific manner following reovirus infection. Unlike JNK activation, ERK activation could not be mapped to specific reovirus gene segments, suggesting that ERK activation and JNK activation are triggered by different events during virus-host cell interaction.

Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in susceptible cells by binding to death receptors 4 (DR4) and 5 (DR5). TRAIL preferentially induces apoptosis in transformed cells and the identification of mechanisms by which TRAIL-induced apoptosis can be enhanced may lead to novel cancer chemotherapeutic strategies. Here we show that reovirus infection induces apoptosis in cancer cell lines derived from human breast, lung and cervical cancers. Reovirus-induced apoptosis is mediated by TRAIL and is associated with the release of TRAIL from infected cells. Reovirus infection synergistically and specifically sensitizes cancer cell lines to killing by exogenous TRAIL. This sensitization both enhances the susceptibility of previously resistant cell lines to TRAIL-induced apoptosis and reduces the amount of TRAIL needed to kill already sensitive lines. Sensitization is not associated with a detectable change in the expression of TRAIL receptors in reovirus-infected cells. Sensitization is associated with an increase in the activity of the death receptor-associated initiator caspase, caspase 8, and is inhibited by the peptide IETD-fmk, suggesting that reovirus sensitizes cancer cells to TRAIL-induced apoptosis in a caspase 8-dependent manner. Reovirus-induced sensitization of cells to TRAIL is also associated with increased cleavage of PARP, a substrate of the effector caspases 3 and 7.

Polymerase chain reaction as a diagnostic adjunct in herpesvirus infections of the nervous system.

Polymerase chain reaction (PCR) is a powerful technique that allows detection of minute quantities of DNA or RNA in cerebrospinal fluid (CSF), vesicle and endoneurial fluids, blood, fresh-frozen, and even formalin-fixed tissues. Various infectious agents can be detected with high specificity and sensitivity, including bacteria, parasites, rickettsia and viruses. PCR analysis of CSF has revolutionized the diagnosis of nervous system viral infections, particularly those caused by human herpesviruses (HHV), and has now replaced brain biopsy as the gold standard for diagnosis of herpes simplex virus (HSV) encephalitis. PCR analysis of both CSF and nervous system tissues has also broadened our understanding of the spectrum of disease caused by HSV-1 and -2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and HHV-6. Nonetheless, positive tissue PCR results must be interpreted cautiously, especially in cases that lack corroborating clinical and neuropathologic evidence of infection. Moreover, positive PCR results from tissues do not distinguish latent from productive (lytic) viral infections. In several neurological diseases, negative PCR results have provided strong evidence against a role for herpesviruses as the causative agents. This review focuses on the use of PCR tests to diagnose HSV and VZV infections of the nervous system.

Reovirus-induced sigma1s-dependent G(2)/M phase cell cycle arrest is associated with inhibition of p34(cdc2).

Serotype 3 reoviruses inhibit cellular proliferation by inducing a G(2)/M phase cell cycle arrest. Reovirus-induced G(2)/M phase arrest requires the viral S1 gene-encoded sigma1s nonstructural protein. The G(2)-to-M transition represents a cell cycle checkpoint that is regulated by the kinase p34(cdc2). We now report that infection with serotype 3 reovirus strain Abney, but not serotype 1 reovirus strain Lang, is associated with inhibition and hyperphosphorylation of p34(cdc2). The sigma1s protein is necessary and sufficient for inhibitory phosphorylation of p34(cdc2), since a viral mutant lacking sigma1s fails to hyperphosphorylate p34(cdc2) and inducible expression of sigma1s is sufficient for p34(cdc2) hyperphosphorylation. These studies establish a mechanism by which reovirus can perturb cell cycle regulation.

Calpain inhibition protects against virus-induced apoptotic myocardial injury.

Viral myocarditis is an important cause of human morbidity and mortality for which reliable and effective therapy is lacking. Using reovirus strain 8B infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. Proteases play a critical role as effectors of apoptosis. The activity of the cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the dipeptide alpha-ketoamide calpain inhibitor Z-Leu-aminobutyric acid-CONH(CH(2))3-morpholine (CX295). Treatment of reovirus-infected neonatal mice with CX295 protects them against reovirus myocarditis as documented by (i) a dramatic reduction in histopathologic evidence of myocardial injury, (ii) complete inhibition of apoptotic myocardial cell death as identified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, (iii) a reduction in serum creatine phosphokinase, and (iv) improved weight gain. These findings are the first evidence for the importance of a calpain-associated pathway of apoptotic cell death in viral disease. Inhibition of apoptotic signaling pathways may be an effective strategy for the treatment of viral disease in general and viral myocarditis in particular.

Reoviruses and the host cell.

Reovirus infection of target cells can perturb cell cycle regulation and induce apoptosis. Differences in the capacity of reovirus strains to induce cell cycle arrest at G1 and G2/M have been mapped to the viral S1 genome segment, which also determines differences in the ability of reovirus strains to induce apoptosis and to activate specific mitogen-activated protein kinase (MAPK) cascades selectively. Reovirus-induced apoptosis involves members of the tumor necrosis factor (TNF) superfamily of death receptors and is associated with activation of both death receptor- and mitochondrial-associated caspases. Reovirus infection is also associated with the activation of a variety of transcription factors, including nuclear factor (NF)-kappaB. Junctional adhesion molecule (JAM) has recently been identified as a novel reovirus receptor. Reovirus binding to JAM appears to be required for induction of apoptosis and activation of NF-kappaB, although the precise cellular pathways involved have not yet been identified.

The Goltz-Ferrier debates and the triumph of cerebral localizationalist theory.

OBJECTIVE: To analyze the significance of the Goltz-Ferrier debates held at the International Medical Congress of 1881 for the development of ideas on cerebral localization. BACKGROUND: Cerebral localization was the subject of vigorous debate throughout the 19th century. At the Congress of 1881, David Ferrier, a leading proponent of cerebral localization, and Friedrich Leopold Goltz, an equally prominent anti-localizationist, had the opportunity to present their experimental research before 3,000 of the world's leading medical figures. METHODS: The authors reviewed and translated the presentations by Goltz and Ferrier at the Congress and supporting publications in contemporary medical journals. RESULTS: In his presentation to the Physiology Section, Goltz criticized localizationists for their widely divergent conclusions about the exact anatomic sites of cortical centers and for their failure to adequately explain functional restitution after cortical ablations. He noted that localizationist theories could, like an apple, "look very tempting and still have a worm inside." He described his own studies on massive decerebrations in dogs and noted that despite complete destruction of the cortices of both hemispheres these animals failed to exhibit motor weakness or deficits in primary sensation. Ferrier noted that Goltz's results were irreconcilable with his own experiments in monkeys, in which circumscribed lesions produced clear and reproducible functional deficits. Both investigators exhibited animals with cortical ablations. Ferrier's presentation of a hemiplegic monkey prompted Charcot's famous utterance, "C'est un malade!" ["It's a patient!"]. A distinguished committee examined the brains of the animals, and confirmed that Ferrier had indeed succeeded in producing a circumscribed lesion in the frontoparietal cortex, whereas the cortical ablations in Goltz's dogs were much less widespread than anticipated. CONCLUSIONS: Ferrier's dramatic demonstration of the effects produced by localized lesions in macaques triumphed over Goltz's unitary view of brain function, providing a major impetus for the subsequent successful development of neurologic surgery.