The role of Epstein-Barr virus in autoimmune and autoinflammatory diseases.

Epstein-Barr Virus (EBV), a dsDNA herpesvirus, is believed to play a significant role in exacerbating and potentially triggering autoimmune and autoinflammatory maladies. Around 90% of the world is infected with the virus, which establishes latency within lymphocytes. EBV is also known to cause infectious mononucleosis, a self-limited flu-like illness, in adolescents. EBV is often reactivated and it employs several mechanisms of evading the host immune system. It has also been implicated in inducing host immune dysfunction potentially resulting in exacerbation or triggering of inflammatory processes. EBV has therefore been linked to a number of autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, and Sjögren's syndrome. The review examines the molecular mechanisms through which the virus alters host immune system components thus possibly resulting in autoimmune processes. Understanding the mechanisms underpinning EBV-associated autoimmunity is pivotal; however, the precise causal pathways remain elusive. Research on therapeutic agents and vaccines for EBV has been stagnant for a long number of years until recent advances shed light on potential therapeutic targets. The implications of EBV in autoimmunity underscore the importance of developing targeted therapeutic strategies and, potentially, vaccines to mitigate the autoimmune burden associated with this ubiquitous virus.

Epstein-Barr Virus DNA Exacerbates Arthritis in a Mouse Model via Toll-like Receptor 9.

Epstein-Barr virus (EBV) DNA is known to be shed upon reactivation of latent EBV. Based on our previous findings linking Toll-like receptor-9 (TLR9) to an EBV DNA-driven surge in IL-17A production, we aimed to examine the therapeutic potential of TLR9 inhibition in EBV DNA-exacerbated arthritis in a collagen-induced arthritis (CIA) mouse model. C57BL/6J mice were administered either collagen, EBV DNA + collagen, EBV DNA + collagen + TLR9 inhibitor, or only the TLR9 inhibitor. After 70 days, paw thicknesses, clinical scores, and gripping strength were recorded. Moreover, affected joints, footpads, and colons were histologically scored. Furthermore, the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in joint sections was determined by immunofluorescence assays. Significantly decreased paw thicknesses, clinical scores, and histological scores with a significantly increased gripping strength were observed in the group receiving EBV DNA + collagen + TLR9 inhibitor, compared to those receiving EBV DNA + collagen. Similarly, this group showed decreased IL-17A+ IFN-γ+, IL-17A+ FOXP3+, and IL-17A+ IFN-γ+ FOXP3+ foci counts in joints. We show that inhibiting TLR9 limits the exacerbation of arthritis induced by EBV DNA in a CIA mouse model, suggesting that TLR9 could be a potential therapeutic target for rheumatoid arthritis management in EBV-infected individuals.

Endosomal Toll-Like Receptors Mediate Enhancement of Interleukin-17A Production Triggered by Epstein-Barr Virus DNA in Mice.

We previously demonstrated that Epstein-Barr virus (EBV) DNA increases the production of the proinflammatory cytokine interleukin-17A (IL-17A) in mice. This property may contribute to the established association between EBV and autoimmune diseases. The objective of the present study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To determine whether endosomal Toll-like receptors (TLRs) played a role in this pathway, the expression of TLR3, -7, or -9 was assessed by real-time reverse transcription-PCR in mouse spleens after injection of EBV DNA. Moreover, specific inhibitors were used for these TLRs in mouse peripheral blood mononuclear cells (PBMCs) cultured with EBV DNA and in mice injected with this viral DNA; IL-17A levels were then assessed using an enzyme-linked immunosorbent assay. The expression of the endosomal receptors TLR3, -7, and -9 was increased in mice injected with EBV DNA. When mouse immune cells were cultured with EBV DNA and a TLR3, -7, or -9 inhibitor or when mice were injected with the viral DNA along with either of these inhibitors, a significant decrease in IL-17A levels was detected. Therefore, endosomal TLRs are involved in the EBV DNA-mediated triggering of IL-17A production in mice. Targeting these receptors in EBV-positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.IMPORTANCE Epstein-Barr virus is a pathogen that causes persistent infection with potential consistent viral DNA shedding. The enhancement of production of proinflammatory cytokines by viral DNA itself may contribute to autoimmune disease development or exacerbation. In this project, we identified that endosomal Toll-like receptors are involved in triggering proinflammatory mediators in response to viral DNA. Pathways and receptors involved may serve as future therapeutic targets for autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.

The role of viral infections in the development of autoimmune diseases.

The exact aetiology of most autoimmune diseases remains unknown, nonetheless, several factors contributing to the induction or exacerbation of autoimmune reactions have been suggested. These include the genetic profile and lifestyle of the affected individual in addition to environmental triggers such as bacterial, parasitic, fungal and viral infections. Infections caused by viruses usually trigger a potent immune response that is necessary for the containment of the infection; however, in some cases, a failure in the regulation of this immune response may lead to harmful immune reactions directed against the host's antigens. The autoimmune attack can be carried out by different arms and components of the immune system and through different possible mechanisms including molecular mimicry, bystander activation, and epitope spreading among others. In this review, we examine the data available for the involvement of viral infections in triggering or exacerbating autoimmune diseases in addition to discussing the mechanisms by which these viral infections and the immune pathways they trigger possibly contribute to the development of autoimmunity.

Effect of rifampicin and gentamicin on Shiga toxin 2 expression level and the SOS response in Escherichia coli O104:H4.

BACKGROUND: A novel pathotype, Shiga toxin-producing Escherichia coli O104:H4, was the cause of a severe outbreak that affected European countries, mainly Germany, in 2011. The effect of different regimens of rifampicin and gentamicin were evaluated to determine possible treatment modes for the novel strain, and to evaluate the SOS response and its effect on toxin release. MATERIALS AND METHODS: Pulsed-field gel electrophoresis (PFGE) was performed on the novel E. coli O104:H4 pathotype and two pre-outbreak E. coli O104:H4 CDC strains. Transcript levels of the stx2 and recA gene (SOS response inducer) were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in the novel E. coli O104:H4 samples subjected to different regimens of rifampicin and gentamicin. Consequently, reverse passive latex agglutination (RPLA) was used to determine the Stx2 titers in these samples. Western blot was performed to determine the LexA levels (SOS response repressor) in E. coli O104:H4. The efficacy of treatment with antimicrobial agents was assessed in BALB/c mice. RESULTS: The outbreak and pre-outbreak strains are closely related as shown by PFGE, which demonstrated slight genomic differences between the three strains. The transcription level of the stx2 gene in the new pathotype was 1.41- and 1.75-fold that of the 2009 EL-2050 and 2009 EL-2071 pre-outbreak strains, respectively. Moreover, the transcription level of the stx2 gene in the new pathotype was substantially decreased as a result of treatment with the different concentrations of the antimicrobial agents, but was enhanced when the antibiotics were administered at two subinhibitory levels. RPLA data were in accordance with the qRT-PCR results. E. coli O104:H4 exposed to gentamicin at both sub-minimum inhibitory concentration (MIC) levels led to high transcription levels of the recA gene and lack of expression of the LexA protein, implying that the SOS response was activated. Rifampicin at both sub-MIC levels resulted in low transcript levels of the recA gene, indicating that the SOS response was not induced. In vivo, the highest survival rate in BALB/c mice was observed in the group that was treated with the minimum bactericidal concentration (MBC) of gentamicin. CONCLUSION: The use of antimicrobial agents in E. coli O104:H4 infection seems to be effective at the MIC and MBC levels. This provides a promising ground for treatment of E. coli O104:H4.

An IL-17A-centric response to Epstein-Barr virus DNA mediated by dendritic Cell-T cell interactions.

Introduction: The Epstein-Barr virus has been associated with a considerable number of autoimmune diseases. We have previously demonstrated that EBV DNA enhances the production of IL-17A, a pro-inflammatory cytokine, via endosomal Toll-like receptor signalling. Methods: We used RNA-seq to analyze the transcriptional profile of mouse immune cells treated with EBV DNA. Results: We observed that EBV DNA upregulates an IL-17A-centric network of mediators. Ensemble Gene Set Enrichment Analysis (EGSEA) showed enriched expression of sets involved in inflammatory responses including IFNγ and TNF-α-associated pathways as well as proinflammatory diseases. On the other hand, while macrophages and B cells were somewhat able to induce an IL-17A response from T cells to EBV DNA, they were less potent than dendritic cells. EBV virions were also capable of eliciting the production of inflammatory mediators from dendritic cell-T cell cultures largely mirroring responses to the viral DNA. Conclusions: Given the wide prevalence of EBV in the population, our analyses reveal a network of mediators and cell types that may serve as therapeutic targets in a large proportion of people affected by autoimmune diseases.

The Effects of Endosomal Toll-like Receptor Inhibitors in an EBV DNA-Exacerbated Inflammatory Bowel Disease Mouse Model.

Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.

Effects of subinhibitory concentrations of antimicrobial agents on Escherichia coli O157:H7 Shiga toxin release and role of the SOS response.

Treatment of Escherichia coli O157:H7 by certain antimicrobial agents often exacerbates the patient's condition by increasing either the release of preformed Shiga toxins (Stx) upon cell lysis or their production through the SOS response-triggered induction of Stx-producing prophages. Recommended subinhibitory concentrations (sub-MICs) of azithromycin (AZI), gentamicin (GEN), imipenem (IMI), and rifampicin (RIF) were evaluated in comparison to norfloxacin (NOR), an SOS-inducer, to assess the role of the SOS response in Stx release. Relative expression of recA (SOS-inducer), Q (late antitermination gene of Stx-producing prophage), stx1, and stx2 genes was assessed at two sub-MICs of the antimicrobials for two different strains of E. coli O157:H7 using reverse transcription-real-time polymerase chain reaction. Both strains at the two sub-MICs were also subjected to Western blotting for LexA protein expression and to reverse passive latex agglutination for Stx detection. For both strains at both sub-MICs, NOR and AZI caused SOS-induced Stx production (high recA, Q, and stx2 gene expression and high Stx2 production), so they should be avoided in E. coli O157:H7 treatment; however, sub-MICs of RIF and IMI induced Stx2 production in an SOS-independent manner except for one strain at the first twofold dilution below MIC of RIF where Stx2 production decreased. Moreover, GEN caused somewhat increased Stx2 production due to its mode of action rather than any effect on gene expression. The choice of antimicrobial therapy should rely on the antimicrobial mode of action, its concentration, and on the nature of the strain.

The interplay between Epstein-Barr virus DNA and gut microbiota in the development of arthritis in a mouse model.

Epstein-Barr virus (EBV) DNA may influence the development of autoimmune diseases by increasing the production of proinflammatory cytokines. Such cytokines have been associated with inducing the dysbiosis of colonic microbiota, which, in turn, is a risk factor for autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we investigated the role that EBV DNA may play in modulating the intestinal microbiota and consequent exacerbation of arthritis in a mouse model. Mice were treated with collagen (arthritis-inducing agent), EBV DNA and collagen, EBV DNA, or water. Fecal samples were collected from arthritic and control mice, and 16S rRNA sequencing was performed to determine the effect of EBV DNA on the composition of colonic microbiota. EBV DNA causes a change in the alpha diversity of the microbiota resulting in an increased Chao1 microbial richness and decreased Shannon diversity index in the RA mouse model. In addition, the abundance of particular genera/genus clusters was significantly altered among the various groups, with the EBV DNA-exacerbated arthritic group having the highest number of altered genera/genus cluster abundances. This group also had the highest number of cells co-expressing IL-17A, FOXP3, and IFNγ in the colons. Antimicrobial-cleared mice transplanted with fecal samples from EBV DNA-exacerbated arthritic mice showed a higher incidence and enhanced severity of RA compared to those transplanted with fecal samples from water or collagen-treated mice. IMPORTANCE Epstein-Barr virus (EBV) DNA alters the composition and diversity of the gut microbiota in a rheumatoid arthritis (RA) mouse model. These induced changes are associated with enhanced severity of symptoms. This better understanding of the various factors involved in the development of RA will possibly help in creating individualized treatments for RA patients including target mediators triggered by viral DNA. Given that a large swathe of the population harbors EBV, a significant proportion of subjects with arthritis may benefit from possible approaches that target EBV or mediators triggered by this virus.

Prevalence of Human Papillomavirus (HPV)-16 in Different Dental Infections in the Lebanese Population.

BACKGROUND: Dental infections, which are the main cause of tooth loss, are known to be caused by bacteria. However, recent research suggests that other organisms, such as viruses, may also play a role. In this study, we aim to detect the presence of human papillomavirus (HPV)-16 and assess its prevalence in tissues infected with various dental infections, including aggressive and chronic periodontitis, pericoronitis, and periapical infection, as well as healthy gingival tissues, saliva, and gingival crevicular fluid, for comparison. METHODS: A cross-sectional study including 124 adult healthy patients presenting with dental infections requiring dental extractions were conducted to assess the prevalence of HPV-16 in saliva, infected, and healthy tissues using quantitative polymerase chain reaction (PCR) tests. Samples were collected and a categorical scale was used for the prevalence. Statistical analyses were performed using Chi-square for the prevalence of HPV-16. RESULTS: Among the HPV-16-positive PCR cases, the prevalence of HPV-16 was highest in periapical infection tissues as compared to chronic periodontitis, aggressive periodontitis, pericoronitis, and control tissues. CONCLUSION: The prevalence of HPV-16 in periapical infection samples was the highest among the studied dental infection samples. Thus, a primary conclusion can be drawn about the presence of an association between HPV-16 and the occurrence of periapical infection.

The impact of prophylactic antiviral agents and statin administration on graft longevity in kidney allograft recipients.

CONTEXT: In an earlier study, we compared the duration of kidney graft survival between two groups of recipients; one on triple (cyclosporine, prednisone and mycophenolate mofetil) and the other on quadruple (cyclosporine, prednisone, mycophenolate mofetil, and sirolimus) immunosuppressive therapy. OBJECTIVE: The aim of this study was to examine the impact of antiviral and statin therapy on graft longevity. MATERIALS AND METHODS: One hundred five kidney allograft recipients were preoperatively assessed for serological markers of infection with various viral agents. All patients were on a prophylactic antiviral regimen of acyclovir and gancyclovir. Seventeen patients were on a statin. Patients were monitored for viral infections and graft rejection or loss for period of 3 years posttransplantation. RESULTS: We detected a high preoperative prevalence rate of IgG immunoglobulins versus the latency-establishing Herpesviridae viruses. Two patients who were preoperatively IgG positive for CMV had cytomegalovirus disease after transplantation. One patient who was preoperatively IgG positive for VZV had shingles after the surgery. No other confirmed viral infections were reported. Thirteen of 88 patients (14.77%) whose treatment regimen did not include a statin suffered a rejection episode or lost the graft whereas 1 of 17 patients (5.88%) on a statin had a rejection episode. CONCLUSIONS: The low rate of viral infections observed in our study population supports the utility of prophylactic administration of antiviral agents to transplant recipients. However, statins seem to have a protective effect on graft longevity (odds ratio [OR] = 0.361, 95% confidence interval [CI] = 0.044-2.957).

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line.

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world's adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles. Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 °C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

Decrease in Shiga toxin expression using a minimal inhibitory concentration of rifampicin followed by bactericidal gentamicin treatment enhances survival of Escherichia coli O157:H7-infected BALB/c mice.

BACKGROUND: Treatment of Escherichia coli O157:H7 infections with antimicrobial agents is controversial due to an association with potentially fatal sequelae. The production of Shiga toxins is believed to be central to the pathogenesis of this organism. Therefore, decreasing the expression of these toxins prior to bacterial eradication may provide a safer course of therapy. METHODS: The utility of decreasing Shiga toxin gene expression in E. coli O157:H7 with rifampicin prior to bacterial eradication with gentamicin was evaluated in vitro using real-time reverse-transcription polymerase chain reaction. Toxin release from treated bacterial cells was assayed for with reverse passive latex agglutination. The effect of this treatment on the survival of E. coli O157:H7-infected BALB/c mice was also monitored. RESULTS: Transcription of Shiga toxin-encoding genes was considerably decreased as an effect of treating E. coli O157:H7 in vitro with the minimum inhibitory concentration (MIC) of rifampicin followed by the minimum bactericidal concentration (MBC) of gentamicin (> 99% decrease) compared to treatment with gentamicin alone (50-75% decrease). The release of Shiga toxins from E. coli O157:H7 incubated with the MIC of rifampicin followed by addition of the MBC of gentamicin was decreased as well. On the other hand, the highest survival rate in BALB/c mice infected with E. coli O157:H7 was observed in those treated with the in vivo MIC equivalent dose of rifampicin followed by the in vivo MBC equivalent dose of gentamicin compared to mice treated with gentamicin or rifampicin alone. CONCLUSIONS: The use of non-lethal expression-inhibitory doses of antimicrobial agents prior to bactericidal ones in treating E. coli O157:H7 infection is effective and may be potentially useful in human infections with this agent in addition to other Shiga toxin producing E. coli strains.

IL-17A in COVID-19 Cases: a meta-analysis.

INTRODUCTION: Numerous reviews, commentaries and opinion pieces have suggested targeting IL-17A as part of managing Coronavirus disease 2019 (COVID-19), the notorious pandemic caused by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IL-17A is a proinflammatory cytokine attributed with homeostatic roles but that is also involved in autoimmune disease pathogenesis. While some studies have reported an increase in IL-17A in COVID-19 cases, no significant associations were found by others. Hence, we undertook this meta-analysis to study serum IL-17A levels in COVID-19 patients in relation to disease severity. METHODOLOGY: Multiple databases were systematically reviewed for literature published on the topic from January 1, 2019 to April 30, 2021. A random effects model was used to calculate weighted mean differences (WMDs) and 95% confidence interval (CIs) as well as the t2 and I2 statistics for heterogeneity analysis. RESULTS: We report that IL-17A increases in COVID-19 subjects irrespective of disease severity compared to controls [WMD = 2.51 pg/ml (95% CI 1.73-3.28), p < 0.00001]. It is also higher in patients with moderate disease compared to controls [WMD = 2.41 pg/ml (95% CI:1.40-3.43), p < 0.00001] as well as higher in patients with severe COVID-19 [WMD = 4.13 pg/ml (95% CI:1.65-6.60), p = 0.001]. While the increase in serum levels in subjects with severe disease over those with moderate disease was statistically significant, the association was not as robust as the other comparisons [WMD = 2.07 pg/ml (95% CI:0.20-3.95), p = 0.03]. Variable heterogeneity was observed in the various analyses with no significant publication bias detected. CONCLUSIONS: Hence, IL-17A may be of relevance when considering management approaches to COVID-19.

Epstein-Barr Virus DNA Exacerbates Colitis Symptoms in a Mouse Model of Inflammatory Bowel Disease.

Infection with EBV has been associated with various inflammatory disorders including inflammatory bowel diseases (IBD). Contribution of this virus to intestinal disease processes has not been assessed. We previously detected that EBV DNA triggers proinflammatory responses via the activation of endosomal Toll-like receptor (TLR) signaling. Hence, to examine the colitogenic potential of EBV DNA, we used the dextran sodium sulfate (DSS) mouse colitis model. C57BL/6J mice received either DSS-containing or regular drinking water. Mice were then administered EBV DNA by rectal gavage. Administration of EBV DNA to the DSS-fed mice aggravated colonic disease activity as well as increased the damage to the colon histologic architecture. Moreover, we observed enhanced expression of IL-17A, IFNγ and TNFα in colon tissues from the colitis mice (DSS-treated) given the EBV DNA compared to the other groups. This group also had a marked decrease in expression of the CTLA4 immunoregulatory marker. On the other hand, we observed enhanced expression of endosomal TLRs in colon tissues from the EBV DNA-treated colitis mice. These findings indicate that EBV DNA exacerbates proinflammatory responses in colitis. The ubiquity of EBV in the population indicates that possible similar responses may be of pertinence in a relevant proportion of IBD patients.

Effect of Epstein-Barr Virus DNA on the Incidence and Severity of Arthritis in a Rheumatoid Arthritis Mouse Model.

Objective: We recently demonstrated that EBV DNA is correlated with proinflammatory responses in mice and in rheumatoid arthritis (RA) patients; hence, we utilized an RA mouse model to examine whether EBV DNA enhances the risk and severity of arthritis and to assess its immunomodulatory effects. Methods: C57BL/6J mice were treated with collagen (arthritis-inducing agent), EBV DNA 6 days before collagen, EBV DNA 15 days after collagen, Staphylococcus epidermidis DNA 6 days before collagen, EBV DNA alone, or water. Mice were then monitored for clinical signs and affected joints/footpads were histologically analysed. The relative concentration of IgG anti- chicken collagen antibodies and serum cytokine levels of IL-17A and IFNϒ were determined by ELISA. The number of cells co-expressing IL-17A and IFNϒ in joint histological sections was determined by immunofluorescence. Results: The incidence of arthritis was significantly higher in mice that received EBV DNA prior to collagen compared to mice that only received collagen. Similarly, increased clinical scores, histological scores and paw thicknesses with a decreased gripping strength were observed in groups treated with EBV DNA and collagen. The relative concentration of IgG anti-chicken collagen antibodies was significantly increased in the group that received EBV DNA 6 days prior to collagen in comparison to the collagen receiving group. On the other hand, the highest number of cells co-expressing IFNϒ and IL-17A was observed in joints from mice that received both collagen and EBV DNA. Conclusion: EBV DNA increases the incidence and severity of arthritis in a RA mouse model. Targeting mediators triggered by viral DNA may hence be a potential therapeutic avenue.

Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases.

The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since we have previously reported immune-stimulating capabilities of EBV DNA in various models, in this study we investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. We have generated inflamed gastrointestinal tracts in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing our understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. Our findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators.

Triple Immunotherapy Overcomes Immune Evasion by Tumor in a Melanoma Mouse Model.

Background: Melanoma is a malignancy with increasing incidence that underlies most skin cancer-related deaths. Advanced melanoma patients still have poor prognosis despite recently developed immunotherapies. This study devises a triple immunotherapy to treat melanoma in a mouse model. The combination includes anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, Monophosphoryl-lipid-A (MPLA), and an Indolamine-Dioxygenase-1 (IDO1) inhibitor. The aim of the study is, first, to rule out any major toxic effects related to this therapy and, second, to assess its antitumor effects. Methods: Cancer-free C57BL/6 mice were randomized into control groups and groups receiving single, dual, or triple therapies of the defined treatments. Clinical signs, weight gain, and histological sections from their main organs were assessed. Then, melanoma-bearing mice were segregated into similar groups, monitored for survival, and their tumor size was measured repeatedly. Finally, flow cytometry was used to analyze immune cell populations in the tumor masses including CD4+, CD8+, and regulatory T cells in addition to natural killer cells. Results: No adverse effects were detected in any of the treated groups. Survival analysis indicated that the groups receiving dual or triple therapies had prolonged survival compared to the controls. However, the group receiving triple therapy was the only group to show statistically significant increase in survival compared to the controls. Tumor size progression paralleled the survival outcome. The group receiving the triple therapy showed statistically significant smaller tumor sizes compared to all the other groups throughout the whole monitoring period. Flow cytometry used to analyze immune cell populations in the tumor mass indicated that the triple immune therapy was capable of significantly enhancing the natural killer cell counts as well as the CD3+CD4+/Treg and CD3+CD8+/Treg ratios possibly enhancing the anti-tumorigenic environment. Conclusions: Generated data rule out any major adverse events pertaining to the triple immunotherapy and reveal its enhanced effectiveness in thwarting melanoma progression over all other tested treatments.

ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.