Activation and pro-inflammatory cytokine production by unswitched memory B cells during SARS-CoV-2 infection.

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells are debated in the literature, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells have been described to be reduced in frequency with severe SARS-CoV2 infection and here we characterize their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those who have had mild and severe SARS-CoV2 infection, confirm a reduction in the frequency of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this reduction is associated with increased levels of systemic TNFα. We further document how severe viral infection is associated with an increased frequency of 'IgD+' only memory B cells that correlate with increased IgG autoantibody levels. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with a concomitant reduction in the expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling-competent BCR that displayed enhanced BCR signaling activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection are poised to secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data indicate that B cells contribute to the inflammatory milieu during viral infection.

Brain Abscesses in the Tropics.

Purpose of Review: This review aims to elucidate the etiologies of brain abscesses in the tropics. Despite the similarities in causes of brain abscesses across global regions, tropical settings manifest distinguishing characteristics, prominently observed on computed tomography or magnetic resonance imaging. Recent Findings: In tropical climates, the leading conditions predisposing individuals to brain abscesses are polymicrobial bacterial infections originating from paranasal sinuses, dental sources, and otitis media. However, the tropics present unique etiologies to be aware of, including Trypanosoma cruzi (Chagas disease), free-living amoebas like Balamuthia mandrillaris, infections from Burkholderia pseudomallei (melioidosis), fungi such as Talaromyces marneffei, and Mycobacterium tuberculosis. Given the differential diagnoses, which include neoplastic, inflammatory, and demyelinating diseases, a stereotactic biopsy coupled with a microbiological assessment remains valuable for accurate diagnosis. Summary: In tropical regions, brain abscesses are a concern when confronted with mass-occupying or other types of brain lesions. Successful clinical management of brain abscesses typically combines surgical intervention and extended anti-microbial treatment. However, specific parasitic invasions like Chagas disease, free-living amoebas, and Entamoeba histolytica necessitate targeted anti-parasitic therapies. Furthermore, international policy efforts should focus on prevention measures in resource limited regions with heightened risks and disease burden.

Autoantibodies elicited with SARS-CoV-2 infection are linked to alterations in double negative B cells.

Double negative (DN) B cells (CD27-IgD-) comprise a heterogenous population of DN1, DN2, and the recently described DN3 and DN4 subsets. In autoimmune disease, DN2 cells are reported to be precursors to autoreactive antibody secreting cells and expansion of DN2 cells is linked to elevated interferon levels. Severe SARS-CoV-2 infection is characterized by elevated systemic levels of pro-inflammatory cytokines and serum autoantibodies and expansion of the DN2 subset in severe SARS-CoV-2 infection has been reported. However, the activation status, functional capacity and contribution to virally-induced autoantibody production by DN subsets is not established. Here, we validate the finding that severe SARS-CoV-2 infection is associated with a reduction in the frequency of DN1 cells coinciding with an increase in the frequency of DN2 and DN3 cells. We further demonstrate that with severe viral infection DN subsets are at a heightened level of activation, display changes in immunoglobulin class isotype frequency and have functional BCR signaling. Increases in overall systemic inflammation (CRP), as well as specific pro-inflammatory cytokines (TNFα, IL-6, IFNγ, IL-1ß), significantly correlate with the skewing of DN1, DN2 and DN3 subsets during severe SARS-CoV-2 infection. Importantly, the reduction in DN1 cell frequency and expansion of the DN3 population during severe infection significantly correlates with increased levels of serum autoantibodies. Thus, systemic inflammation during SARS-CoV-2 infection drives changes in Double Negative subset frequency, likely impacting their contribution to generation of autoreactive antibodies.

Defining diagnostic approaches and outcomes in patients with inflammatory CSF: A retrospective cohort study.

OBJECTIVE: Define the etiologies, clinical features, time to diagnosis, and outcomes of patients that present with cerebral spinal fluid (CSF) pleocytosis. PATIENTS AND METHODS: This is retrospective cohort study of patients with CSF pleocytosis, defined as WBC count >5 cells/mm3 in the CSF, from July 2015 to June 2016 at a large tertiary care hospital. The proportion of patients within specific diagnostic categories were analyzed for differences in diagnostic testing and outcomes. RESULTS: 53% of patients had CSF pleocytosis due to an unknown etiology. The leading known cause of neuroinflammation was CNS infection (n = 43/244, 18%), followed by malignancy (n = 28/244, 11%). Mean time to diagnosis was 125 days in patients with autoimmune neuroinflammation and was 16 days in patients with an infection or malignancy. CSF parameters and peripheral white blood cell counts did not distinguish between categories of disease. The presence of CSF oligoclonal bands or a positive biopsy result most commonly supported a diagnosis of an autoimmune disease or malignancy, respectively. Neuroimaging changes were present in only 44% of infections but were found in 80-90% of other categories of neuroinflammation. Patients presenting with a severe neurologic deficit had 22.29 higher odds of a severe deficit at the last neurologic assessment, and mortality was highest (29%) in patients with malignancy-associated neuroinflammation. CONCLUSIONS: This study to defines general diagnostic categories of neuroinflammatory disease in patients and provides new insight on the value of specific diagnostic testing, time to diagnosis, and outcomes in these patient populations.

Cryptococcosis and cryptococcal meningitis: New predictors and clinical outcomes at a United States academic medical centre.

As the diagnosis of cryptococcosis is challenging in low-prevalence settings, uncovering predictive factors can improve early diagnosis and timely treatment. The aim of the study was to relate clinical outcomes to predictive variables for the presence of cryptococcosis. A retrospective case-control study matched by collection date, age and gender at a 1:2 ratio (55 cases and 112 controls) was performed in case patients diagnosed with Cryptococcus infection at the University of Colorado Hospital between 2000 and 2017 (n = 167). A bivariate and a forward, stepwise multivariable logistic regression model were performed to identify predictors of cryptococcosis infection. In an adjusted multivariable model, cryptococcal infection was significantly associated with the presence of respiratory symptoms, hyponatremia, lung disease or corticosteroids. Additionally, cryptococcal meningitis was associated with headaches, corticosteroids or increased CSF protein. Conversely, a reduced risk of cryptococcosis was associated with hypertension or peripheral monocytosis. Cryptococcal meningitis leads to subsequent hearing impairment (16% vs 4% (control), P = .013), muscle weakness (40% vs 20%, P = .021), cognitive deficits (33% vs 6%, P = .0001) or any adverse outcome (84% vs 29%, P = .0001). We uncovered novel clinical predictors for the presence of cryptococcal infection or cryptococcal meningitis. This study in patients at a low-prevalence US medical centre underscores the importance of early diagnosis in this population.

Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease.

The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure that is responsible for ZIKV sfRNA production forms a complex fold that is likely found in many pathogenic flaviviruses. Mutations that disrupt the structure affect exonuclease resistance in vitro and sfRNA formation during infection. The complete ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies features that may modulate function in diverse flaviviruses.

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5' cap with 2'-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

Risk Factors for Cryptococcal Meningitis: A Single United States Center Experience.

Cryptococcal meningitis carries a high mortality. Further understanding of immune suppression factors associated with neuroinvasive infection will improve risk stratification and enhance early diagnosis and treatment with antifungal therapy. The aim of the study was to corroborate established or find novel clinical predictors for cryptococcal meningitis. We performed a matched case-control study of Cryptococcus infection in immunocompromised patients with or without cryptococcal meningitis. Data of all patients with a diagnosis of cryptococcal disease were collected at University of Colorado Hospital between 2000 and 2015 (n = 51). Thirty patients were diagnosed with cryptococcal meningitis. We built a logistic regression model for risk factors associated with cryptococcal meningitis. The single-predictor univariate model found that a positive blood culture, positive serum cryptococcal antigen, current malignancy, and headaches were significantly associated with cryptococcal meningitis (p = 0.02). In the adjusted multivariate model, central nervous system disease was significantly associated with a diagnosis of HIV infection (OR 24.45, 95 % CI 1.62-350.37; p = 0.022) and a positive serum cryptococcal antigen test (OR 42.92, 95 % CI 3.26-555.55; p = 0.0055). In patients with HIV infection or a positive serum cryptococcal antigen, the pretest probability of neuroinvasive Cryptococcus infection is increased and an aggressive diagnostic evaluation should be conducted to exclude infection and consider empiric therapy.

West nile virus-induced activation of mammalian target of rapamycin complex 1 supports viral growth and viral protein expression.

UNLABELLED: Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth. IMPORTANCE: Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states in the United States and is now the leading cause of epidemic encephalitis in North America. Currently, the mechanism by which flaviviruses such as WNV translate their genomes in host cells is incompletely understood. Elucidation of the host mechanisms required to support WNV genome translation will provide broad understanding for the basic mechanisms required to translate capped viral RNAs. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection. Following inducible genetic knockout of the major mTOR complex cofactors raptor (TORC1) and rictor (TORC2), we now show that TORC1 supports WNV growth and protein synthesis. This study demonstrates the requirement for TORC1 function in support of WNV RNA translation and provides insight into the mechanisms underlying flaviviral RNA translation in mammalian cells.

Fas-mediated apoptotic signaling in the mouse brain following reovirus infection.

Type 3 (T3) reovirus strains induce apoptotic neuronal cell death and lethal encephalitis in infected mice. T3 strain Dearing (T3D)-induced apoptosis in primary neuronal cultures occurs by a Fas-mediated mechanism and requires the activation of caspase 8. We now show that Fas mRNA is upregulated in the brains of mice infected with encephalitic reovirus T3D and T3 strain Abney (T3A) but not following infection with nonencephalitic reovirus type 1 strain Lang. Fas is upregulated in regions of the brain that are injured during infection with T3 reovirus strains and colocalizes with virus antigen in individual neurons. In contrast, levels of FasL mRNA induced by encephalitic and nonencephalitic reovirus strains do not differ significantly. Caspase 8, the initiator caspase associated with Fas-mediated apoptosis, is activated in the cortex and hippocampal regions of both T3D- and T3A-infected mice. Furthermore, Bid cleavage and the activation of caspase 9 in the brains of T3D-infected mice suggest that the caspase 8-dependent activation of mitochondrial apoptotic signaling contributes to virus-induced apoptosis. We have previously shown that the inhibition of c-Jun N-terminal kinase (JNK) signaling blocks T3D-induced apoptosis and improves the outcome of virus-induced encephalitis. We now show that the reovirus-induced upregulation of Fas requires JNK signaling, thereby providing a link between reovirus-induced death receptor signaling and mitogen-activated protein kinase pathways and a potential mechanism for the therapeutic action of JNK inhibition.

Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase.

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.

Respiratory viral infections in patients with chronic, obstructive pulmonary disease.

OBJECTIVES: The purpose of the present study was to apply reverse transcription-PCR (RT-PCR) assays to clinical specimens collected from patients with acute respiratory illness and chronic obstructive pulmonary disease (COPD). METHODS: One hundred and ninety-four samples from two different study cohorts were analysed using RT-PCR assays for picornaviruses, coronaviruses 229E and OC43, influenza A and B viruses, respiratory syncytial virus, parainfluenza types 1-3 viruses, and human metapneumovirus and a PCR assay for adenoviruses. The results were added to results obtained previously using cell culture and serologic methods. RESULTS: RT-PCR assays identified an additional 35 respiratory virus-associated illnesses not identified previously by cell culture or serology (n=46). Picornaviruses and coronaviruses were the most common viral infections identified only by RT-PCR. Overall, 41.8% of the acute respiratory illnesses evaluated were associated with a respiratory virus infection, with picornaviruses, coronaviruses and influenza viruses being the most common infections recognized. No human metapneumovirus infections were identified by RT-PCR assay. CONCLUSIONS: Respiratory viral infections are commonly associated with acute respiratory illness in COPD patients, and the use of RT-PCR assays significantly increases the ability to diagnose these infections.