The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Brèves.

TITLE: Brèves. ABSTRACT: L'unité d'enseignement « Immunopathologie ¼ qui propose les brèves de ce numéro est suivie par des étudiants des sept parcours recherche du Master Biologie Santé de l'Université de Montpellier. On y étudie les bases physiopathologiques des maladies immunologiques, les cibles thérapeutiques et les mécanismes d'échappement des microorganismes et des tumeurs. Ce Master rassemble des étudiants issus du domaine des sciences et technologies et de celui de la santé. Les articles présentés ont été choisis par les étudiants selon leur domaine de prédilection.

Superinfection and cell regeneration can lead to chronic viral coinfections.

Molecular diagnostic techniques have revealed that approximately 43% of the patients hospitalized with influenza-like illness are infected by more than one viral pathogen, sometimes leading to long-lasting infections. It is not clear how the heterologous viruses interact within the respiratory tract of the infected host to lengthen the duration of what are usually short, self-limiting infections. We develop a mathematical model which allows for single cells to be infected simultaneously with two different respiratory viruses (superinfection) to investigate the possibility of chronic coinfections. We find that a model with superinfection and cell regeneration has a stable chronic coinfection fixed point, while superinfection without cell regeneration produces only acute infections. This analysis suggests that both superinfection and cell regeneration are required to sustain chronic coinfection via this mechanism since coinfection is maintained by superinfected cells that allow slow-growing infections a chance to infect cells and continue replicating. This model provides a possible mechanism for chronic coinfection independent of any viral interactions via the immune response.

Superinfection Exclusion between Two High-Risk Human Papillomavirus Types during a Coinfection.

Superinfection exclusion is a common phenomenon whereby a single cell is unable to be infected by two types of the same pathogen. Superinfection exclusion has been described for various viruses, including vaccinia virus, measles virus, hepatitis C virus, influenza A virus, and human immunodeficiency virus. Additionally, the mechanism of exclusion has been observed at various steps of the viral life cycle, including attachment, entry, viral genomic replication, transcription, and exocytosis. Human papillomavirus (HPV) is the causative agent of cervical cancer. Recent epidemiological studies indicate that up to 50% women who are HPV positive (HPV+) are infected with more than one HPV type. However, no mechanism of superinfection exclusion has ever been identified for HPV. Here, we show that superinfection exclusion exists during a HPV coinfection and that it occurs on the cell surface during the attachment/entry phase of the viral life cycle. Additionally, we are able to show that the minor capsid protein L2 plays a role in this exclusion. This study shows, for the first time, that superinfection exclusion occurs during HPV coinfections and describes a potential molecular mechanism through which it occurs.IMPORTANCE Superinfection exclusion is a phenomenon whereby one cell is unable to be infected by multiple related pathogens. This phenomenon has been described for many viruses and has been shown to occur at various points in the viral life cycle. HPV is the causative agent of cervical cancer and is involved in other anogenital and oropharyngeal cancers. Recent epidemiological research has shown that up to 50% of HPV-positive individuals harbor more than one type of HPV. We investigated the interaction between two high-risk HPV types, HPV16 and HPV18, during a coinfection. We present data showing that HPV16 is able to block or exclude HPV18 on the cell surface during a coinfection. This exclusion is due in part to differences in the HPV minor capsid protein L2. This report provides, for the first time, evidence of superinfection exclusion for HPV and leads to a better understanding of the complex interactions between multiple HPV types during coinfections.

Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

Interleukin-33 modulates the expression of human ß-defensin 2 in human primary keratinocytes and may influence the susceptibility to bacterial superinfection in acute atopic dermatitis.

BACKGROUND: Interleukin (IL)-33 is a member of the IL-1 family and has been implicated in Th2-driven allergic diseases such as atopic dermatitis (AD) and asthma. The principal Th2 cytokine IL-4, found highly expressed in acute allergic eczema, is known to downregulate human ß-defensin 2 (hBD2) expression in human keratinocytes and this is associated with superinfection in patients with AD. OBJECTIVES: To investigate the effect of IL-33 on the expression of hBD2 in human keratinocytes. METHODS: hBD2 production by stimulated keratinocytes was measured by enzyme-linked immunosorbent assay. RESULTS: Our results showed that serum is a very potent inducer of hBD2 and 2·5% human serum was much more potent in inducing hBD2 than 20 ng mL(-1) of tumour necrosis factor-α. Interestingly, serum from patients with AD showed an impaired ability to induce hBD2 in normal keratinocytes. IL-33 significantly downregulated serum-induced hBD2. The downregulatory capacity of IL-33 was found to be 1·5- to 2-fold weaker compared with IL-4. CONCLUSIONS: Our data suggest that IL-33 can significantly contribute to the decreased expression of hBD2 in acute eczematous reaction clinically characterized by spongiosis and oozing - thus indicative for contact of the epidermis with serum components.

Downregulation of CD4 is required for maintenance of viral infectivity of HIV-1.

Downregulation of virus receptors on the cell surface is considered to be important in preventing superinfection. HIV-1 encodes multiple gene products, Env, Vpu, and Nef, involved in downregulation of CD4, a major HIV-1 receptor. We found that simultaneous mutations in both vpu and nef severely impaired virus replication. We examined the involvement of CD4 downregulation mediated by Vpu and Nef in the modification of virus infectivity. The mutation in vpu increased CD4 incorporation into virions without affecting the Env content in it, inhibiting the attachment step of virions to the CD4-positive cell surface. Although a single mutation in nef suppresses virus infectivity via a CD4-independent mechanism, it could augment CD4 incorporation in virions in combination with a vpu mutation. These results indicated that CD4 downregulation was necessary for maintenance of Env function in the virion.

A study of once-daily amikacin with low peak target concentrations in intensive care unit patients: pharmacokinetics and associated outcomes.

PURPOSE: The aim of this study was to assess the pharmacokinetics of individualized amikacin single-dosage regimens targeting low peak serum concentrations in a population of intensive care unit (ICU) patients, and to describe the outcomes associated with this dosing strategy. MATERIALS AND METHODS: In this prospective, noncomparative, pharmacokinetic study, 36 ICU patients with adequate renal function were treated with amikacin (intravenous infusion), in combination with other antibiotics, for hospital-acquired infections. The initial doses of amikacin were calculated based on individual creatinine clearance values whereas subsequent doses were calculated by using the amikacin pharmacokinetic parameters estimated from the serum concentration-time data of each individual patient (samples were drawn postinfusion, 1 h, 3 h, 6 h, and 10 h after the onset of the infusion and just before the next dose on days 2, 4, and 7 of therapy). RESULTS: Results showed moderate only interpatient and lack of intrapatient (except for the volume of distribution) variability in amikacin pharmacokinetic parameters. There were no significant differences between achieved and target peak levels. Clinical response was noted in 94% and bacteriologic response was noted in 86% of patients. Nephrotoxicity did not occur during or after treatment. CONCLUSIONS: Amikacin dosage individualization with low peak target concentrations was successful for the 36 ICU patients. This dosing strategy was not associated with nephrotoxicity, but conclusions on clinical efficacy cannot be drawn from this limited study.

Human neuron-committed teratocarcinoma NT2 cell line has abnormal ND10 structures and is poorly infected by herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.

Expression of mouse mammary tumor virus envelope protein does not prevent superinfection in vivo or in vitro.

Inbred mice expressing endogenous mouse mammary tumor virus envelope proteins can be infected with exogenous virus, and the mammary tumors that develop in these mice usually have many proviruses integrated in their genomes, indicating that this virus is not subject to receptor interference. We show here that transgenic mice expressing an exogenous mouse mammary tumor virus (C3H) envelope protein can still be infected with this virus. Moreover, cultured mammary gland cells expressing the mouse mammary tumor virus (C3H) envelope protein can be superinfected with pseudotyped viruses bearing that same protein. Thus cellular expression of the mouse mammary tumor virus envelope protein does not block superinfection in vivo or in vitro.

Modulation of chronic enteric infection by distant oral infection.

This investigation determined the effects of a distant oral infection (Porphyromonas gingivalis) on a concurrent local enteric infection (Echerichia coli). A modified murine subcutaneous tissue chamber model was used. Subcutaneously implanted chambers with different microbial makeups were monitored for sloughing and their contents assayed for prostaglandin E2. Bilaterally implanted chamber experiments revealed that a distant "chronic" (immunization with heat-killed organism, followed by live challenge) P. gingivalis infection offered protection against a local chronic E. coli infection, as evidenced by delaying the time for 50% of the chambers to reject from day 19 to day 25 and a statistically significant prostaglandin E2 decrease from 529.4 +/- 176.6 ng/ml to 191.5 +/- 100.9 ng/ml (p < 0.01) (Mann-Whitney test). An acute (live challenge only) distant P. gingivalis infection or immunization with the heat-killed organism alone had no effect on "chronic" E. coli infection in this model. These data suggest that the presence of low-grade chronic oral infection may modify the responses to other infectious challenges.

Syphilis superinfection activates expression of human immunodeficiency virus I in latently infected rabbits.

Superinfection of latently human immunodeficiency virus (HIV)-infected rabbits with either Treponema pallidum or Shope fibroma virus (SFV) activates HIV expression. In addition, HIV-infected rabbits demonstrate prolonged cutaneous lesions (chancres) after intracutaneous challenge with T. pallidum, the causative agent of syphilis. Rabbits were infected by intravenous inoculation of 3 x 10(7) human T-cell lymphotrophic virus type III (HTLV-III)/B10 (HIV-1)-infected H9 (human) cells. Five weeks after initial infection, integrated HIV-1-specific DNA sequences were detected in the DNA of the peripheral blood lymphocytes of only one of eight rabbits using polymerase chain reactions (PCR); human DNA could not be detected at this time. Furthermore HIV infection could not be demonstrated by either seroconversion or PCR during the next 6 months. All HIV-infected rabbits remained clinically healthy and had normal white blood cell counts. Six months after HIV infection, four HIV-infected and two noninfected controls were superinfected with 10(6) T. pallidum in eight skin sites in the shaved skin of the back, and four infected and two control animals were challenged with an intradermal injection with SFV. After infection with either syphilis or SFV, the DNA from the white blood cells of all eight HIV-infected rabbits contained HIV sequences, and HIV sequences were demonstrated in dermal mononuclear cells of the syphilitic lesions by in situ hybridization. The SFV-induced tumors were rejected normally in the HIV-infected rabbits, but four of the four rabbits challenged with T. pallidum had delayed development of cutaneous lesions and three of four demonstrated larger and more prolonged lesions. White blood counts, mitogen responses, and interleukin-2 production remained within normal limits, and seroconversion for HIV was not detected. Three of four rabbits in a second group, challenged with T. pallidum 4 months after HIV-inoculation, also had delayed healing of syphilitic lesions. These results indicate that latent HIV-infection of rabbits may be activated by immunostimulation and that latently HIV-infected rabbits have impaired delayed hypersensitivity reactions. It is hypothesized that true latent HIV-infection in the rabbits is in monocytes and postulated that further immunostimulation may produce infection of lymphocytes and activation of disease.