Exploring the Role of Platelets in Virus-Induced Inflammatory Demyelinating Disease and Myocarditis.

Theiler's murine encephalomyelitis virus (TMEV) infection has been used as a mouse model for two virus-induced organ-specific immune-mediated diseases. TMEV-induced demyelinating disease (TMEV-IDD) in the central nervous system (CNS) is a chronic inflammatory disease with viral persistence and an animal model of multiple sclerosis (MS) in humans. TMEV infection can also cause acute myocarditis with viral replication and immune cell infiltration in the heart, leading to cardiac fibrosis. Since platelets have been reported to modulate immune responses, we aimed to determine the role of platelets in TMEV infection. In transcriptome analyses of platelets, distinct sets of immune-related genes, including major histocompatibility complex (MHC) class I, were up- or downregulated in TMEV-infected mice at different time points. We depleted platelets from TMEV-infected mice by injecting them with platelet-specific antibodies. The platelet-depleted mice had significantly fewer viral antigen-positive cells in the CNS. Platelet depletion reduced the severities of TMEV-IDD and myocarditis, although the pathology scores did not reach statistical significance. Immunologically, the platelet-depleted mice had an increase in interferon (IFN)-γ production with a higher anti-TMEV IgG2a/IgG1 ratio. Thus, platelets may play roles in TMEV infection, such as gene expression, viral clearance, and anti-viral antibody isotype responses.

Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.

Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG)35-55, MOG92-106, or myelin proteolipid protein (PLP)139-151, with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP139-151-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.

Effectiveness of exercise interventions in animal models of multiple sclerosis.

Multiple sclerosis (MS) is associated with an impaired immune system that severely affects the spinal cord and brain, and which is marked by progressive inflammatory demyelination. Patients with MS may benefit from exercise training as a suggested course of treatment. The most commonly used animal models of studies on MS are experimental autoimmune/allergic encephalomyelitis (EAE) models. The present review intends to concisely discuss the interventions using EAE models to understand the effectiveness of exercise as treatment for MS patients and thereby provide clear perspective for future research and MS management. For the present literature review, relevant published articles on EAE animal models that reported the impacts of exercise on MS, were extracted from various databases. Existing literature support the concept that an exercise regimen can reduce the severity of some of the clinical manifestations of EAE, including neurological signs, motor function, pain, and cognitive deficits. Further results demonstrate the mechanisms of EAE suppression with information relating to the immune system, demyelination, regeneration, and exercise in EAE. The role for neurotrophic factors has also been investigated. Analyzing the existing reports, this literature review infers that EAE is a suitable animal model that can help researchers develop further understanding and treatments for MS. Besides, findings from previous animal studies supports the contention that exercise assists in ameliorating MS progression.

Adjuvant Injections Altered the Ileal and Fecal Microbiota Differently with Changes in Immunoglobulin Isotypes and Antimycobacterial Antibody Responses.

Alterations in the gut microbiota, "dysbiosis," have been reported in autoimmune diseases, including multiple sclerosis (MS), and their animal models. Although the animal models were induced by injections of autoantigens with adjuvants, including complete Freund's adjuvant (CFA) and pertussis toxin (PT), the effects of adjuvant injections on the microbiota are largely unknown. We aimed to clarify whether adjuvant injections could affect the microbiota in the ileum and feces. Using 16S rRNA sequencing, we found decreased alpha diversities of the gut microbiota in mice injected with CFA and PT, compared with naïve mice. Overall, microbial profiles visualized by principal component analysis demonstrated dysbiosis in feces, but not in the ileum, of adjuvant-injected mice, where the genera Lachnospiraceae NK4A136 group and Alistipes contributed to dysbiosis. When we compared the relative abundances of individual bacteria, we found changes in 16 bacterial genera in feces and seven genera in the ileum of adjuvant-injected mice, in which increased serum levels of antibody against mycobacteria (a component of CFA) and total IgG2c were correlated with the genus Facklamia. On the other hand, increased IgG1 and IgA concentrations were correlated with the genus Atopostipes. Therefore, adjuvant injections alone could alter the overall microbial profiles (i.e., microbiota) and individual bacterial abundances with altered antibody responses; dysbiosis in animal models could be partly due to adjuvant injections.

Multiple Sclerosis in Mongolia; the First Study Exploring Predictors of Disability and Depression in Mongolian MS Patients.

Mongolia is located at 45° north latitude in the center of the Asian continent, and about 80% of the territory is at 1000 m above sea level. Epidemiologically, multiple sclerosis (MS) has not been investigated in Mongolia, although there have been a few MS case reports. We investigated the characteristics of MS in Mongolia for the first time, focusing on the association between MS-related parameters and depression levels. We initiated cross-sectional analyses, using data from 27 MS patients aged 20 to 60 years in Ulaanbaatar, Mongolia. The patients completed a questionnaire on their lifestyles and clinical information. We classified the MS patients on the basis of disability levels using the expanded disability status scale (EDSS) scores: 11.1% mild disability and 88.9% moderate to severe disability (median EDSS score, 5.5). We also classified the patients on the basis of depression levels using the 9-item patient health questionnaire (PHQ-9) scores: 44.4% mild depression, 40.7% moderate depression, and 14.8% severe depression (mean PHQ-9's score, 9.96 ± 5.05). We used multivariate logistical regression analyses to identify predictors of EDSS or PHQ-9 scores. Disability levels were associated with vision and balance problems. Depression levels were associated with corticosteroid treatment; no patients were treated with disease-modifying drugs (DMDs). The odds ratios for disease onset age and treatment duration were associated with EDSS scores. In conclusion, MS onset age and treatment duration were independent predicting factors influencing the level of disability. Appropriate DMD treatment would lower the disability and depression levels.

Critical evaluation on roles of macrophagic myofasciitis and aluminum adjuvants in HPV vaccine-induced adverse events.

Cervical cancer is caused by human papillomavirus (HPV) infection, which is preventable by HPV vaccines. In Japan, the HPV vaccination rate has remained extremely low due to the concerns for alleged neuropsychological symptoms or "diverse symptoms" following injections of two HPV vaccines, Cervarix and Gardasil, in HPV vaccine lawsuits. In the lawsuits, the attorneys' group has used several manuscripts proposing that aluminum (Al) adjuvant contained in HPV vaccines causes an immune-mediated disease, called macrophagic myofasciitis (MMF), as well as pathology in the central nervous system (CNS). We scientifically evaluated these manuscripts describing the "Al adjuvant-induced pathologies," particularly MMF. Although MMF patients have been reported to develop clinical symptoms/signs in various organs, including the CNS, muscle biopsy of the patients and animal experiments demonstrated that MMF pathology was localized only at the injected muscle. No muscle pathology which characterizes MMF was observed in any other muscles; thus, the systemic and neurological signs of MMF cases were irrelevant to localized MMF pathology. We evaluated that MMF-like pathology was induced as a local inflammatory response following vaccinations; MMF pathology was not the cause of systemic inflammation or "diverse symptoms." Lastly, MMF cases have been reported after vaccinations with Al-hydroxide-containing vaccines exclusively. As Al-hydroxide is a component of Cervarix, but not Gardasil, "diverse symptoms" following two HPV vaccinations in Japan cannot be explained by MMF. Our evaluation would help readers understand the validity of the manuscripts on the role of Al adjuvants or MMF for the alleged "diverse symptoms."

Exploratory factor analysis determines latent factors in Guillain-Barré syndrome.

Exploratory factor analysis (EFA) has been developed as a powerful statistical procedure in psychological research. EFA's purpose is to identify the nature and number of latent constructs (= factors) underlying a set of observed variables. Since the research goal of EFA is to determine what causes the observed responses, EFA is ideal for hypothesis-based studies, such as identifying the number and nature of latent factors (e.g., cause, risk factors, etc.). However, the application of EFA in the biomedical field has been limited. Guillain-Barré syndrome (GBS) is peripheral neuropathy, in which the presence of antibodies to glycolipids has been associated with clinical signs. Although the precise mechanism for the generation of anti-glycolipid antibodies is unclear, we hypothesized that latent factors, such as distinct autoantigens and microbes, could induce different sets of anti-glycolipid antibodies in subsets of GBS patients. Using 55 glycolipid antibody titers from 100 GBS and 30 control sera obtained by glycoarray, we conducted EFA and extracted four factors related to neuroantigens and one potentially suppressive factor, each of which was composed of the distinct set of anti-glycolipid antibodies. The four groups of anti-glycolipid antibodies categorized by unsupervised EFA were consistent with experimental and clinical findings reported previously. Therefore, we proved that unsupervised EFA could be applied to biomedical data to extract latent factors. Applying EFA for other biomedical big data may elucidate latent factors of other diseases with unknown causes or suppressing/exacerbating factors, including COVID-19.

Helicobacter pylori infection in the stomach induces neuroinflammation: the potential roles of bacterial outer membrane vesicles in an animal model of Alzheimer's disease.

Helicobacter pylori (HP) is a Gram-negative bacterium that colonizes the human stomach chronically. Colonization of HP in the gastric mucosa not only causes gastrointestinal diseases, but also is associated with extra-gastric diseases, such as idiopathic thrombocytopenic purpura and neurological diseases. Among neurological diseases, epidemiological studies have shown that HP infection increases the prevalence of Alzheimer's disease (AD) and Parkinson's disease (PD). Since HP does not invade the central nervous system (CNS), it has been considered that systemic immunological changes induced by HP infection may play pathogenic roles in AD and PD. Here, we investigated the effects of HP infection on the CNS in vivo and in vitro. In the CNS, chronically HP-infected mice had microglial activation without HP colonization, although systemic immunological changes were not observed. This led us to explore the possibility that HP-derived outer membrane vesicles (HP-OMVs) could cause neuroinflammation. OMVs are small, spherical bilayer vesicles (20-500 nm) released into the extracellular space from the outer membrane of Gram-negative bacteria; OMVs contain lipopolysaccharide, proteins, peptidoglycan, DNA, and RNA. OMVs have also been shown to activate both innate and acquired immune cells in vitro, and to disrupt the tight junctions of the gastric epithelium ("leaky gut") as well as cross the blood-brain barrier in vivo. Thus, in theory, OMVs can activate immune responses in the remote organs, including the lymphoid organs and CNS, if only OMVs enter the systemic circulation. From the exosome fraction of sera from HP-infected mice, we detected HP-specific DNA, suggesting the presence of HP-OMVs. We also found that microglia incubated with HP-OMVs in vitro increased the cell proliferation, inflammatory cytokine production, and migration. On the other hand, HP-OMVs suppressed the cell proliferation of neuroblastoma in vitro. Lastly, we found that AD model mice infected with HP had amyloid plaques adjacent to activated microglia and astrocytes in vivo. Based on the literature review and our experimental data, we propose our working hypothesis that OMVs produced in chronic HP infection in the gut induce neuroinflammation in the CNS, explaining the higher prevalence of AD in HP-infected people.

Scientific evaluation of alleged findings in HPV vaccines: Molecular mimicry and mouse models of vaccine-induced disease.

Cervical cancer is caused by infections of the human papillomavirus (HPV), which can be prevented by vaccinations. In Japan, although about 3000 people die of cervical cancer annually, the HPV vaccination rate has remained extremely low in the eligible population since many Japanese have been concerned that "diverse symptoms," such as chronic pain, movement disorders, and cognitive impairment, may occur as adverse reactions after HPV vaccination. The concern has been raised by media coverage of the ongoing HPV vaccine lawsuits, in which the plaintiffs complained of their symptoms caused by HPV vaccination. The claims have been based on the alleged pathogenic findings in research articles on HPV vaccines, summarized in the document prepared by the plaintiffs' attorneys. We critically evaluated these articles, in which the authors proposed the following findings/hypothesis: (i) molecular mimicry between HPV L1 and human proteins leads to the production of cross-reactive antibodies; and (ii) HPV vaccine injection in mice causes damage in the brain, a mouse model for HPV vaccine associated neuro-immunopathic syndrome (HANS). We found that these hypotheses were based mainly on the findings from a few research groups and that all the articles had flaws in the method, result, or discussion sections. Our current evaluation should help better understand the validity of the findings, which have been often misunderstood as the truth by the general public. We propose to accumulate high-quality data on potential adverse events following HPV vaccination and to continue critically evaluating them.

Bacterial and fungal isolation from face masks under the COVID-19 pandemic.

The COVID-19 pandemic has led people to wear face masks daily in public. Although the effectiveness of face masks against viral transmission has been extensively studied, there have been few reports on potential hygiene issues due to bacteria and fungi attached to the face masks. We aimed to (1) quantify and identify the bacteria and fungi attaching to the masks, and (2) investigate whether the mask-attached microbes could be associated with the types and usage of the masks and individual lifestyles. We surveyed 109 volunteers on their mask usage and lifestyles, and cultured bacteria and fungi from either the face-side or outer-side of their masks. The bacterial colony numbers were greater on the face-side than the outer-side; the fungal colony numbers were fewer on the face-side than the outer-side. A longer mask usage significantly increased the fungal colony numbers but not the bacterial colony numbers. Although most identified microbes were non-pathogenic in humans; Staphylococcus epidermidis, Staphylococcus aureus, and Cladosporium, we found several pathogenic microbes; Bacillus cereus, Staphylococcus saprophyticus, Aspergillus, and Microsporum. We also found no associations of mask-attached microbes with the transportation methods or gargling. We propose that immunocompromised people should avoid repeated use of masks to prevent microbial infection.

Corrigendum: Curcumin ß-D-Glucuronide Modulates an Autoimmune Model of Multiple Sclerosis with Altered Gut Microbiota in the Ileum and Feces.

[This corrects the article DOI: 10.3389/fcimb.2021.772962.].

Curdlan, a Microbial ß-Glucan, Has Contrasting Effects on Autoimmune and Viral Models of Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease characterized by inflammatory demyelination and axonal degeneration in the central nervous system (CNS). Bacterial and fungal infections have been associated with the development of MS; microbial components that are present in several microbes could contribute to MS pathogenesis. Among such components, curdlan is a microbial 1,3-ß-glucan that can stimulate dendritic cells, and enhances T helper (Th) 17 responses. We determined whether curdlan administration could affect two animal models for MS: an autoimmune model, experimental autoimmune encephalomyelitis (EAE), and a viral model, Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD). We induced relapsing-remitting EAE by sensitizing SJL/J mice with the myelin proteolipid protein (PLP)139-151 peptide and found that curdlan treatment prior to PLP sensitization converted the clinical course of EAE into hyperacute EAE, in which the mice developed a progressive motor paralysis and died within 2 weeks. Curdlan-treated EAE mice had massive infiltration of T cells and neutrophils in the CNS with higher levels of Th17 and Th1 responses, compared with the control EAE mice. On the other hand, in TMEV-IDD, we found that curdlan treatment reduced the clinical scores and axonal degeneration without changes in inflammation or viral persistence in the CNS. In summary, although curdlan administration exacerbated the autoimmune MS model by enhancing inflammatory demyelination, it suppressed the viral MS model with reduced axonal degeneration. Therefore, microbial infections may play contrasting roles in MS depending on its etiology: autoimmunity versus viral infection.

Curcumin ß-D-Glucuronide Modulates an Autoimmune Model of Multiple Sclerosis with Altered Gut Microbiota in the Ileum and Feces.

We developed a prodrug type of curcumin, curcumin monoglucuronide (CMG), whose intravenous/intraperitoneal injection achieves a high serum concentration of free-form curcumin. Although curcumin has been reported to alter the gut microbiota and immune responses, it is unclear whether the altered microbiota could be associated with inflammation in immune-mediated diseases, such as multiple sclerosis (MS). We aimed to determine whether CMG administration could affect the gut microbiota at three anatomical sites (feces, ileal contents, and the ileal mucosa), leading to suppression of inflammation in the central nervous system (CNS) in an autoimmune model for MS, experimental autoimmune encephalomyelitis (EAE). We injected EAE mice with CMG, harvested the brains and spinal cords for histological analyses, and conducted microbiome analyses using 16S rRNA sequencing. CMG administration modulated EAE clinically and histologically, and altered overall microbiota compositions in feces and ileal contents, but not the ileal mucosa. Principal component analysis (PCA) of the microbiome showed that principal component (PC) 1 values in ileal contents, but not in feces, correlated with the clinical and histological EAE scores. On the other hand, when we analyzed the individual bacteria of the microbiota, the EAE scores correlated with significant increases in the relative abundance of two bacterial species at each anatomical site: Ruminococcus bromii and Blautia (Ruminococcus) gnavus in feces, Turicibacter sp. and Alistipes finegoldii in ileal contents, and Burkholderia spp. and Azoarcus spp. in the ileal mucosa. Therefore, CMG administration could alter the gut microbiota at the three different sites differentially in not only the overall gut microbiome compositions but also the abundance of individual bacteria, each of which was associated with modulation of neuroinflammation.

Galectin-3 as a Therapeutic Target for NSAID-Induced Intestinal Ulcers.

Non-steroidal anti-inflammatory drugs (NSAIDs) induce ulcers in the gastrointestinal tract, including the stomach and small intestine. NSAID-induced gastric ulcers can be prevented by taking acid-neutralizing/inhibitory drugs and cytoprotective agents. In contrast, there are no medicines to control NSAID-induced small intestinal ulcers, which are accompanied by a mucosal invasion of bacteria and subsequent activation of immune cells. Galectin-3 (Gal3), an endogenous lectin, has anti-microbial and pro-inflammatory functions. In the small intestine, since Gal3 is highly expressed in epithelial cells constitutively and macrophages inducibly, the Gal3 level can affect microbiota composition and macrophage activation. We hypothesized that the modulation of Gal3 expression could be beneficial in NSAID-induced intestinal ulcers. Using Gal3 knockout (Gal3KO) mice, we determined whether Gal3 could be a therapeutic target in NSAID-induced intestinal ulcers. Following the administration of indomethacin, an NSAID, we found that small intestinal ulcers were less severe in Gal3KO mice than in wild-type (WT) mice. We also found that the composition of intestinal microbiota was different between WT and Gal3KO mice and that bactericidal antibiotic polymyxin B treatment significantly suppressed NSAID-induced ulcers. Furthermore, clodronate, a macrophage modulator, attenuated NSAID-induced ulcers. Therefore, Gal3 could be an exacerbating factor in NSAID-induced intestinal ulcers by affecting the intestinal microbiota population and macrophage activity. Inhibition of Gal3 may be a therapeutic strategy in NSAID-induced intestinal ulcers. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03832946.

Bioinformatics Analysis of Gut Microbiota and CNS Transcriptome in Virus-Induced Acute Myelitis and Chronic Inflammatory Demyelination; Potential Association of Distinct Bacteria With CNS IgA Upregulation.

Virus infections have been associated with acute and chronic inflammatory central nervous system (CNS) diseases, e.g., acute flaccid myelitis (AFM) and multiple sclerosis (MS), where animal models support the pathogenic roles of viruses. In the spinal cord, Theiler's murine encephalomyelitis virus (TMEV) induces an AFM-like disease with gray matter inflammation during the acute phase, 1 week post infection (p.i.), and an MS-like disease with white matter inflammation during the chronic phase, 1 month p.i. Although gut microbiota has been proposed to affect immune responses contributing to pathological conditions in remote organs, including the brain pathophysiology, its precise role in neuroinflammatory diseases is unclear. We infected SJL/J mice with TMEV; harvested feces and spinal cords on days 4 (before onset), 7 (acute phase), and 35 (chronic phase) p.i.; and examined fecal microbiota by 16S rRNA sequencing and CNS transcriptome by RNA sequencing. Although TMEV infection neither decreased microbial diversity nor changed overall microbiome patterns, it increased abundance of individual bacterial genera Marvinbryantia on days 7 and 35 p.i. and Coprococcus on day 35 p.i., whose pattern-matching with CNS transcriptome showed strong correlations: Marvinbryantia with eight T-cell receptor (TCR) genes on day 7 and with seven immunoglobulin (Ig) genes on day 35 p.i.; and Coprococcus with gene expressions of not only TCRs and IgG/IgA, but also major histocompatibility complex (MHC) and complements. The high gene expression of IgA, a component of mucosal immunity, in the CNS was unexpected. However, we observed substantial IgA positive cells and deposition in the CNS, as well as a strong correlation between CNS IgA gene expression and serum anti-TMEV IgA titers. Here, changes in a small number of distinct gut bacteria, but not overall gut microbiota, could affect acute and chronic immune responses, causing AFM- and MS-like lesions in the CNS. Alternatively, activated immune responses would alter the composition of gut microbiota.

Evaluation of Five International HBV Treatment Guidelines: Recommendation for Resource-Limited Developing Countries Based on the National Study in Nepal.

Hepatitis B virus (HBV) infects the liver, causing cirrhosis and cancer. In developed countries, five international guidelines have been used to make a decision for the management of patients with chronic HBV infection. In this review, since the guidelines were established by clinical and epidemiological data of developed countries, we aimed to evaluate whether (1) HBV patient profiles of developing countries are similar to developed countries, and (2) which guideline can be applicable to resource-limited developing countries. First, as an example of the most recent data of HBV infections among developing countries, we evaluated the national HBV viral load study in Nepal, which were compared with the data from other developing countries. In Nepal, the highest number of patients had viral loads of 20-2000 IU/mL (36.7%) and belonged to the age group of 21-30 years; HBV epidemiology in Nepal, based on the viral loads, gender, and age groups was similar to those of not only other developing countries but also developed countries. Next, we reviewed five international HBV treatment guidelines of the World Health Organization (WHO), American Association for the Study of Liver Diseases (AASLD), National Institute for Health and Care Excellence (NICE), European Association for the Study of the Liver (EASL), and Asian Pacific Association for the Study of the Liver (APASL). All guidelines require the viral load and alanine aminotransferase (ALT) levels for decision making. Although four guidelines recommend elastography to assess liver cirrhosis, the WHO guideline alternatively recommends using the aspartate aminotransferase (AST)-to-platelet ratio index (APRI), which is inexpensive and conducted routinely in most hospitals. Therefore, in resource-limited developing countries like Nepal, we recommend the WHO guideline for HBV treatment based on the viral load, ALT, and APRI information.

Bioinformatics Analyses Determined the Distinct CNS and Peripheral Surrogate Biomarker Candidates Between Two Mouse Models for Progressive Multiple Sclerosis.

Previously, we have established two distinct progressive multiple sclerosis (MS) models by induction of experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) in two mouse strains. A.SW mice develop ataxia with antibody deposition, but no T cell infiltration, in the central nervous system (CNS), while SJL/J mice develop paralysis with CNS T cell infiltration. In this study, we determined biomarkers contributing to the homogeneity and heterogeneity of two models. Using the CNS and spleen microarray transcriptome and cytokine data, we conducted computational analyses. We identified up-regulation of immune-related genes, including immunoglobulins, in the CNS of both models. Pro-inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-17, were associated with the disease progression in SJL/J mice, while the expression of both cytokines was detected only at the EAE onset in A.SW mice. Principal component analysis (PCA) of CNS transcriptome data demonstrated that down-regulation of prolactin may reflect disease progression. Pattern matching analysis of spleen transcriptome with CNS PCA identified 333 splenic surrogate markers, including Stfa2l1, which reflected the changes in the CNS. Among them, we found that two genes (PER1/MIR6883 and FKBP5) and one gene (SLC16A1/MCT1) were also significantly up-regulated and down-regulated, respectively, in human MS peripheral blood, using data mining.

Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130.

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated ß-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

IL-1ß reduces cardiac lymphatic muscle contraction via COX-2 and PGE2 induction: Potential role in myocarditis.

The role of lymphatic vessels in myocarditis is largely unknown, while it has been shown to play a key role in other inflammatory diseases. We aimed to investigate the role of lymphatic vessels in myocarditis using in vivo model induced with Theiler's murine encephalomyelitis virus (TMEV) and in vitro model with rat cardiac lymphatic muscle cells (RCLMC). In the TMEV model, we found that upregulation of a set of inflammatory mediator genes, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-αand COX-2 were associated with disease activity. Thus, using in vitro collagen gel contraction assays, we decided to clarify the role(s) of these mediators by testing contractility of RCLMC in response to IL-1ß and TNF-α individually and in combination, in the presence or absence of: IL-1 receptor antagonist (Anakinra); cyclooxygenase (COX) inhibitors inhibitors (TFAP, diclofenac and DuP-697). IL-1ß impaired RCLMC contractility dose-dependently, while co-incubation with both IL-1ß and TNF-α exhibited synergistic effects in decreasing RCLMC contractility with increased COX-2 expression. Anakinra maintained RCLMC contractility; Anakinra blocked the mobilization of COX-2 induced by IL-1ß with or without TNF-α. COX-2 inhibition blocked the IL-1ß-mediated decrease in RCLMC contractility. Mechanistically, we found that IL-1ß increased prostaglandin (PG) E2 release dose-dependently, while Anakinra blocked IL-1ß mediated PGE2 release. Using prostaglandin E receptor 4 (EP4) receptor antagonist, we demonstrated that EP4 receptor blockade maintained RCLMC contractility following IL-1ß exposure. Our results indicate that IL-1ß reduces RCLMC contractility via COX-2/PGE2 signaling with synergistic cooperation by TNF-α. These pathways may help provoke inflammatory mediator accumulation within the heart, driving progression from acute myocarditis into dilated cardiomyopathy.