RESUMO
SARS-CoV-2 induced neuroinflammation contributing to neurological sequelae is one of the critical outcomes of long-COVID, however underlying regulatory mechanisms involved therein are poorly understood. We deciphered the profile of dysregulated microRNAs, their targets, associated pathways, protein-protein interactions (PPI), transcription factor-hub genes interaction networks, hub genes-microRNA co-regulatory networks in SARS-CoV-2 Spike-1 (S1) stimulated microglial cells along with candidate drug prediction using RNA-sequencing and multiple bioinformatics approaches. We identified 11 dysregulated microRNAs in the S1-stimulated microglial cells (p < 0.05). KEGG analysis revealed involvement of important neuroinflammatory pathways such as MAPK signalling, PI3K-AKT signalling, Ras signalling and axon guidance. PPI analysis further identified 11 hub genes involved in these pathways. Real time PCR validation confirmed a significant upregulation of microRNA-30b-5p and let-7a-5p; proinflammatory cytokines- IL-6, TNF-α, IL-1ß, GM-CSF; and inflammatory genes- PIK3CA and AKT in the S1-stimulated microglial cells, while PTEN and SHIP1 expression was decreased as compared to the non-stimulated cells. Drug prediction analysis further indicated resveratrol, diclofenac and rapamycin as the potential drugs based on their degree of interaction with hub genes. Thus, targeting of these microRNAs and/or their intermediate signalling molecules would be a prospective immunotherapeutic approach in alleviating SARS-CoV-2-S1 mediated neuroinflammation; and needs further investigations.
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HIV infection impairs host immunity, leading to progressive disease. An anti-retroviral treatment efficiently controls viremia but cannot completely restore the immune dysfunction in HIV-infected individuals. Both host and viral factors determine the rate of disease progression. Among the host factors, innate immunity plays a critical role; however, the mechanism(s) associated with dysfunctional innate responses are poorly understood among HIV disease progressors, which was investigated here. The gene expression profiles of TLRs and innate cytokines in HIV-infected (LTNPs and progressors) and HIV-uninfected individuals were examined. Since the progressors showed a dysregulated TLR-mediated innate response, we investigated the role of TLR agonists in restoring the innate functions of the progressors. The stimulation of PBMCs with TLR3 agonist-poly:(I:C), TLR7 agonist-GS-9620 and TLR9 agonist-ODN 2216 resulted in an increased expression of IFN-α, IFN-ß and IL-6. Interestingly, the expression of IFITM3, BST-2, IFITM-3, IFI-16 was also increased upon stimulation with TLR3 and TLR7 agonists, respectively. To further understand the molecular mechanism involved, the role of miR-155 was explored. Increased miR-155 expression was noted among the progressors. MiR-155 inhibition upregulated the expression of TLR3, NF-κB, IRF-3, TNF-α and the APOBEC-3G, IFITM-3, IFI-16 and BST-2 genes in the PBMCs of the progressors. To conclude, miR-155 negatively regulates TLR-mediated cytokines as wel l as the expression of host restriction factors, which play an important role in mounting anti-HIV responses; hence, targeting miR-155 might be helpful in devising strategic approaches towards alleviating HIV disease progression.
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Infecções por HIV , MicroRNAs , Humanos , Infecções por HIV/tratamento farmacológico , Receptor 7 Toll-Like , Receptor 3 Toll-Like/metabolismo , Citocinas/metabolismo , Imunidade Inata , MicroRNAs/genética , MicroRNAs/uso terapêutico , Progressão da Doença , Antivirais/uso terapêutico , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNARESUMO
High Risk Human Papilloma Viruses (HR-HPV) persistently infect women with Human Immunodeficiency Virus-1 (HIV-1). HPV-16 escapes immune surveillance in HIV-1 positive women receiving combined antiretroviral therapy (cART). HIV-1 Tat and HPV E6/E7 proteins exploit Notch signaling. Notch-1, a developmentally conserved protein, influences cell fate from birth to death. Notch-1 and its downstream targets, Hes-1 and Hey-1 contribute to invasive and aggressive cancers. Cervical cancer cells utilize Notch-1 and hyper-express CXCR4, a co-receptor of HIV-1. Accumulating evidence shows that HIV-1 affects cell cycle progression in pre-existing HPV infection. Additionally, Tat binds Notch-1 receptor for activation and influences cell proliferation. Oncogenic viruses may interfere or converge together to favor tumor growth. The molecular dialogue during HIV-1/HPV-16+ co-infections in the context of Notch-1 signaling has not been explored thus far. This in vitro study was designed with cell lines (HPV-ve C33A and HPV-16+ CaSki) which were transfected with plasmids (pLEGFPN1 encoding HIV-1 Tat and pNL4-3 encoding HIV-1 [full HIV-1 genome]). HIV-1 Tat and HIV-1 inhibited Notch-1expression, with differential effects on EGFR. Notch-1 inhibition nullified Cyclin D expression with p21 induction and increased G2-M cell population in CaSki cells. On the contrary, HIV-1 infection shuts down p21 expression through interaction of Notch-1 downstream genes Hes-1-EGFR and Cyclin D for G2-M arrest, DDR response and cancer progression. This work lays foundations for future research and interventions, and therefore is necessary. Our results describe for the first time how HIV-1 Tat cancers have an aggressive nature due to the interplay between Notch-1 and EGFR signaling. Notch-1 inhibitor, DAPT used in organ cancer treatment may help rescue HIV-1 induced cancers. Graphical abstract: The illustration shows how HIV interacts with HPV-16 to induce Notch 1 suppression for cancer progression (Created with BioRender.com). Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00809-y.
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The present study was conducted to compare the performance of patient self-collected oral swab (OS) with healthcare worker (HCW)-collected nasopharyngeal swab (NPS) for SARS-CoV-2 detection by reverse transcription polymerase chain reaction (RT-PCR) in real-world setting. Paired OS and NPS were collected from 485 consecutive individuals presenting with symptoms of coronavirus disease-19 (COVID-19) or asymptomatic contacts of COVID-19 cases. Both specimens were processed for RT-PCR and cycle threshold (Ct) value for each test was obtained. Positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA) and kappa were calculated for OS RT-PCR compared with NPS RT-PCR as reference. A total of 116/485 (23.9%) participants were positive by NPS RT-PCR. OS had PPA of 71.6%, NPA of 98.8%, OPA of 92.4% and kappa of 0.771. Almost all participants (483/485, 99.6%) reported OS as a convenient and comfortable sample for SARS-CoV-2 testing over NPS. All participants with Ct values <25 and majority (90.8%) with Ct values <30 were detected by OS. To conclude, OS self-sampling was preferred in comparison with NPS due the ease and comfort during collection. The performance of OS RT-PCR for SARS-CoV-2 detection, however, was sub-optimal in comparison with NPS RT-PCR.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Bochecha , Pessoal de Saúde , Humanos , Nasofaringe , SARS-CoV-2/genética , Manejo de Espécimes , LínguaRESUMO
An HIV outbreak investigation during 2017-2018 in Unnao District, Uttar Pradesh, India, unearthed high prevalence of hepatitis C virus (HCV) antibodies among the study participants. We investigated these HCV infections by analyzing NS5B and core regions. We observed no correlation between HIV-HCV viral loads and clustering of HCV sequences, regardless of HIV serostatus. All HCV isolates belonged to genotype 3a. Monophyletic clustering of isolates in NS5B phylogeny indicates emergence of the outbreak from a single isolate or its closely related descendants. The nucleotide substitution rate for NS5B was 6 × 10-3 and for core was 2 × 10-3 substitutions/site/year. Estimated time to most recent common ancestor of these isolates was 2012, aligning with the timeline of this outbreak, which might be attributable to unsafe injection practices while seeking healthcare. HIV-HCV co-infection underlines the need for integrated testing, surveillance, strengthening of healthcare systems, community empowerment, and molecular analyses as pragmatic public health tools.
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Infecções por HIV , Hepatite C , Surtos de Doenças , Infecções por HIV/epidemiologia , Hepacivirus , Hepatite C/epidemiologia , Humanos , Índia/epidemiologia , FilogeniaRESUMO
TGF-ß has been shown to play a differential role in either restricting or aiding HIV infection in different cell types, however its role in the cervical cells is hitherto undefined. Among females, more than 80% of infections occur through heterosexual contact where cervicovaginal mucosa plays a critical role, however the early events during the establishment of infection at female genital mucosa are poorly understood. We earlier showed that increased TGF-ß level has been associated with cervical viral shedding in the HIV infected women, however a causal relationship could not be examined. Therefore, here we first established an in vitro cell-associated model of HIV infection in the cervical epithelial cells (ME-180) and demonstrated that TGF-ß plays an important role as a negative regulator of HIV release in the infected cervical epithelial cells. Inhibition of miR-155 upregulated TGF-ß signaling and mRNA expression of host restriction factors such as APOBEC-3G, IFI-16 and IFITM-3, while decreased the HIV release in ME-180 cells. To conclude, this is the first study to decipher the complex interplay between TGF-ß, miR-155 and HIV release in the cervical epithelial cells. Collectively, our data suggest the plausible role of TGF-ß in promoting HIV latency in cervical epithelial cells which needs further investigations.
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Colo do Útero/virologia , Células Epiteliais/virologia , HIV-1/fisiologia , MicroRNAs/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Eliminação de Partículas Virais , Fatores de Restrição Antivirais/genética , Linhagem Celular , Colo do Útero/citologia , Colo do Útero/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , MicroRNAs/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
OBJECTIVE: To determine the reference ranges of various lymphocyte subsets in healthy Indian children. DESIGN: Descriptive cross-sectional study. SETTING: Four centers in India representing four geographical regions. PARTICIPANTS: 1104 children from neonatal age to 18 years of age. MEASUREMENT: One time measurement of absolute count and percentages of different lymphocyte subsets i.e. T lymphocytes (CD3+T, CD4+T, CD8+T cells), B lymphocytes (CD19+B cells) and Natural Killer lymphocytes (CD15/16+NK cells) in whole blood using multicolor flow cytometry. RESULTS: The absolute cell counts of various lymphocytes were found to increase from newborn to 10 months of age, followed by gradual decline until 18 years; however, the proportion of immune cells remained largely similar. Gender did not have a significant impact on the reference ranges, whereas counts were found to vary as per the geographical locations. CONCLUSIONS: These reference ranges will be useful to monitor and predict the immune status in pediatric population. The variation in region wise ranges could be confirmed by testing more number of samples in the specific age groups.
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Antígenos CD19 , Subpopulações de Linfócitos , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Contagem de Linfócitos , Valores de ReferênciaRESUMO
BACKGROUND: HIV-specific Antibody Dependent Cell Cytotoxicity (ADCC) has shown to be important in HIV control and resistance. The ADCC is mediated primarily by natural killer cell activated through the binding of FcγRIIIa receptor to the Fc portion of antibody bound to the antigen expressed on the infected cells. However, no data is available on the influence of the polymorphism in FcγRIIIa receptor on HIV-specific ADCC response. METHODS: The Sanger's method of sequencing was used to sequence the exon of FcγRIIIa receptor while the ADCC activity was determined using NK cell activation assay. The polymorphism in FcγRIIIa receptor was assessed in HIV-infected Indian individuals with or without HIV-specific ADCC antibodies and its influence on the magnitude of HIV-specific ADCC responses was analyzed. RESULTS: Two polymorphisms: V176F (rs396991) and Y158H (rs396716) were observed. The Y158H polymorphism is reported for the first time in Indian population. Both, V176F (V/V genotype) (p = 0.004) and Y158H (Y/H genotype) (p = 0.032) were found to be significantly associated with higher magnitude of HIV-specific ADCC response. CONCLUSION: The study underscores the role of polymorphism in the FcγRIIIa receptor on HIV-specific ADCC response and suggests that the screening of the individuals for FcγRIIIa-V176F and Y158H polymorphisms could be useful for prediction of efficient treatment in monoclonal antibody-based therapies aimed at ADCC in HIV infection.
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Citotoxicidade Celular Dependente de Anticorpos/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Polimorfismo de Nucleotídeo Único/genética , Receptores de IgG/genética , Adolescente , Adulto , Anticorpos Monoclonais/uso terapêutico , Feminino , Frequência do Gene/genética , Genótipo , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/terapia , Humanos , Imunoterapia , Índia , Masculino , Pessoa de Meia-Idade , Prognóstico , Resultado do Tratamento , Proteínas do Envelope Viral/farmacologia , Adulto JovemRESUMO
Progressive HIV infection is marked with reduced frequency and impaired function of Th17 cells. Since T-cell functional responses are regulated by various inhibitory receptors; we examined the expression profile of CTLA-4, PD-1, Tim-3 and apoptotic marker: Caspase-3 on virus-specific Th17 cells from HIV-infected Long-Term Non-Progressors (LTNPs), chronic disease progressors and HIV-uninfected healthy controls (HC) using flowcytometry. Real-time PCR was done to analyze the mRNA expression of Th17 and Treg specific genes. LTNPs showed higher frequencies of HIV-specific Th17 cells; higher mRNA expression of IL-17, IL-22 while lower expression of IL-10; along with lower Caspase-3 expression than the progressors. Among inhibitory receptors, expression of CTLA-4 was 27 and 8 fold; PD-1 was 8 and 6 fold while Tim-3 was 7 and 6 fold higher in progressors and LTNPs respectively than HC. Among HIV-infected patients, LTNPs had 3-fold lower expression of CTLA-4 on HIV-specific Th17 cells than progressors (pâ¯=â¯0.06). Caspase-3+ve Th17 cells were associated with HIV-specific Th17 cells expressing CTLA-4 (râ¯=â¯0.66;pâ¯<â¯0.0001), PD-1 (râ¯=â¯0.40;pâ¯=â¯0.02) and Tim-3 (râ¯=â¯0.35;pâ¯=â¯0.04). To conclude, virus-specific Th17 cells from LTNP maintained IL-17 production, expressed low levels of CTLA-4 and reduced apoptosis. The study suggests that such functional competence of Th17 cells could be one of the factors in maintenance of non-progressive HIV infection.
Assuntos
Antígeno CTLA-4/metabolismo , Infecções por HIV/imunologia , HIV-1/fisiologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Apoptose , Antígenos CD4/metabolismo , Antígeno CTLA-4/genética , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Doença Crônica , Estudos Transversais , Progressão da Doença , Seguimentos , Sobreviventes de Longo Prazo ao HIV , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Interleucina-17/genética , Interleucina-17/metabolismoRESUMO
OBJECTIVE: The activation of effector immune cells at the cervicovaginal mucosa (CVM) might influence the cervical HIV load and thus the secondary transmission; however, limited information is available about the innate effector cells at CVM during HIV infection. In this study, we quantified and assessed the activation of the effector immune cells at the CVM of HIV-infected women with different disease outcomes: nonprogressive HIV disease (LTNPs) and chronic HIV-infected (CHI) and their relationship with cervical viral shedding. METHOD: The phenotype and frequency of cytobrush-derived effector immune cells like natural killer cells, T cells, and dendritic cells and their degranulation status (CD107a expression as a surrogate marker of activation) was determined using flow cytometry in age-matched HIV- infected and uninfected women and their association with cervical HIV load was determined. RESULT: The frequencies of dendritic cells, CD56, CD56 natural killer cell subsets were similar in both the study groups and also within the HIV-infected women with and without progressive disease. The frequencies of CD56CD16 natural killer cells (Pâ=â0.04) and degranulating CD56 natural killer cells were significantly higher among HIV-infected women (Pâ<â0.05). Among HIV-infected women, LTNP women showed reduced degranulation of natural killer and CD8 T cells than seen in the CHI women, which was also associated with lower cervical viral load (Pâ<â0.05). CONCLUSION: The present study showed that increased degranulation of natural killer and T cells is associated with higher HIV shedding at the CVM of HIV-infected women. Hence reduction of the local immune activation at CVM could be an effective strategy to reduce the cervical viral load.
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Degranulação Celular , Colo do Útero/imunologia , Colo do Útero/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Carga Viral , Adolescente , Adulto , Estudos Transversais , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Eliminação de Partículas Virais , Adulto JovemRESUMO
HECT domain and RCC1-like domain-containing protein 5 (HERC-5) is one of the novel host restriction factors that is known to inhibit HIV release in vitro. Polymorphisms in other host restriction factors have been associated with HIV infection and disease progression. However, no report is available on the HERC-5 polymorphism in HIV-infected individuals. We studied the HERC-5 gene polymorphism in HIV-infected individuals and explored whether it is associated with different disease outcomes. Genomic DNA was isolated from 41 HIV-1 progressors, 39 long-term nonprogressors, and 74 HIV seronegative healthy donors for amplification of HERC5 Exon-18 and other regulatory regions followed by sequencing. We found no genetic variation in the known single-nucleotide polymorphism (SNP)-rs34457268 (Exon-18) of HERC-5 in HIV-infected individuals. Instead, a synonymous mutation at rs6857425 (T-C) was present in the same region among all study groups (p > .05), irrespective of their HIV status. We further noted two novel SNPs in Intron-18 region. To the best of our knowledge, this is first study to report the HERC5 gene polymorphism among HIV-infected groups.
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Predisposição Genética para Doença , Infecções por HIV/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Polimorfismo Genético , Adolescente , Adulto , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Adulto JovemRESUMO
Persistent immune activation in human immunodeficiency virus (HIV) infection is responsible for alterations in immune system such as activation, apoptosis, and reduced frequencies. Reduced immune activation is known to be associated with virus control. Limited information is available on the influence of pan-immune activation on memory responses. Hence, we compared the T cell activation and memory profile in HIV-infected individuals exhibiting disease control such as long-term nonprogressors (LTNPs) and progressors. The activated CD4+ and CD8+ T cells were significantly lower and the CD4+ and CD8+ central memory T cell phenotypes were significantly higher in the LTNPs compared to the progressors. In addition, we observed significant inverse association between the T cell activation and frequencies of central memory T cells. Our findings indicate that patients with absence of disease progression have preserved central memory T cell population associated with lesser immune activation.
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Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , Memória Imunológica , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-IdadeRESUMO
The purpose of this study was to determine the in vitro and ex vivo susceptibility of human corneal cells to West Nile virus (WNV) infection and evaluate the ability of the virus to disseminate to the corneas of infected mice. Human corneal epithelial cells were challenged with WNV, incubated for 1-6 days, and tested for evidence of WNV infection. Viral RNA and antigen were detected at every time point, and the virus reached a peak titer of 2.5 × 107 plaque-forming units (pfu)/mL at 3 days postinoculation (PI). Corneas procured from donors were incubated in culture dishes containing WNV for 1-5 days and tested for evidence of WNV. Viral RNA and antigen were detected, and the virus reached a mean peak titer of 4.9 × 104 pfu/mL at 5 days PI. Mice were inoculated intraperitoneally with WNV, and their eyes were harvested at 2, 5, and 8 days PI and tested for evidence of WNV. Viral RNA was detected in corneas of four of nine systemically infected mice as early as 2 days PI. We conclude that human corneal cells support WNV replication in vitro and ex vivo, and WNV may disseminate into the corneas of experimentally infected mice. These findings indicate that corneal transmission cannot be ruled out as a novel mode of human-to-human WNV transmission and additional experiments should be conducted to assess this risk further.
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Córnea/virologia , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/crescimento & desenvolvimento , Animais , Linhagem Celular , Córnea/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Viral/isolamento & purificaçãoRESUMO
West Nile virus (WNV), a mosquito-borne, single positive-stranded RNA virus, has been the leading cause of arboviral encephalitis in the U.S. and other parts of the world over the past decade. Up to 50 % of WNV convalescent patients were reported to have long-term neurological sequelae or chronic kidney diseases. However, there are neither antiviral drugs nor vaccines available for humans. The underlying mechanism of the long-term sequelae is not clearly understood either. Animal models have been an effective tool to investigate viral pathogenesis and host immunity in humans. Here, we will review several commonly used murine models of WNV infection.
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Encefalopatias/etiologia , Insuficiência Renal Crônica/etiologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Culicidae/virologia , Modelos Animais de Doenças , Humanos , Insetos Vetores/virologia , Camundongos , Febre do Nilo Ocidental/complicações , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/imunologiaRESUMO
Resistance to azole antifungals is a significant problem in Candida albicans. An understanding of resistance at molecular level is essential for the development of strategies to tackle resistance and rationale design of newer antifungals and target-based molecular approaches. This study presents the first evaluation of molecular mechanisms associated with fluconazole resistance in clinical C.albicans isolates from India. Target site (ERG11) alterations were determined by DNA sequencing, whereas real-time PCRs were performed to quantify target and efflux pump genes (CDR1, CDR2, MDR1) in 87 [Fluconazole susceptible (n = 30), susceptible-dose dependent (n = 30) and resistant (n = 27)] C.albicans isolates. Cross-resistance to fluconazole, ketoconazole and itraconazole was observed in 74.1% isolates. Six amino acid substitutions were identified, including 4 (E116D, F145L, E226D, I437V) previously reported ones and 2 (P406L, Q474H) new ones. CDR1 over-expression was seen in 77.7% resistant isolates. CDR2 was exclusively expressed with CDR1 and their concomitant over-expression was associated with azole cross-resistance. MDR1 and ERG11 over-expression did not seem to be associated with resistance. Our results show that drug efflux mediated by Adenosine-5'-triphosphate (ATP)-binding cassette transporters, especially CDR1 is the predominant mechanism of fluconazole resistance and azole cross-resistance in C. albicans and indicate the need for research directed towards developing strategies to tackle efflux mediated resistance to salvage azoles.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Candida albicans/genética , Candida albicans/isolamento & purificação , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Fúngico/isolamento & purificação , Genes Fúngicos , Humanos , Índia , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
γδ T cells express several different toll-like receptor (TLR)s. The role of MyD88- dependent TLR signaling in TCR activation of murine γδ T cells is incompletely defined. Here, we report that Pam3CSK4 (PAM, TLR2 agonist) and CL097 (TLR7 agonist), but not lipopolysaccharide (TLR4 agonist), increased CD69 expression and Th1-type cytokine production upon anti-CD3 stimulation of γδ T cells from young adult mice (6-to 10-week-old). However, these agonists alone did not induce γδ T cell activation. Additionally, we noted that neither PAM nor CL097 synergized with anti-CD3 in inducing CD69 expression on γδ T cells of aged mice (21-to 22-month-old). Compared to young γδ T cells, PAM and CL097 increased Th-1 type cytokine production with a lower magnitude from anti-CD3- stimulated, aged γδ T cells. Vγ1+ and Vγ4+ cells are two subpopulations of splenic γδ T cells. PAM had similar effects in anti-CD3-activated control and Vγ4+ subset- depleted γδ T cells; whereas CL097 induced more IFN-γ production from Vγ4+ subset-depleted γδ T cells than from the control group. Finally, we studied the role of MyD88-dependent TLRs in γδ T cell activation during West Nile virus (WNV) infection. γδ T cell, in particular, Vγ1+ subset expansion was significantly reduced in both MyD88- and TLR7- deficient mice. Treatment with TLR7 agonist induced more Vγ1+ cell expansion in wild-type mice during WNV infection. In summary, these results suggest that MyD88-dependent TLRs provide co-stimulatory signals during TCR activation of γδ T cells and these have differential effects on distinct subsets.
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Fator 88 de Diferenciação Mieloide/fisiologia , Subpopulações de Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Subpopulações de Linfócitos T/metabolismoRESUMO
In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.
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Neoplasias Encefálicas/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Modelos Animais de Doenças , Terapia Genética , Glioma/mortalidade , Glioma/terapia , Humanos , Lentivirus/genética , Mutação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Análise de SobrevidaRESUMO
Prior work shows that an attenuated West Nile virus (WNV), the nonstructural (NS)4B-P38G mutant infection in mice induced strong immune responses and protected host from subsequent lethal wild-type WNV infection. Here, we investigated NS4B-P38G mutant infection in myeloid differentiation factor 88-deficient (MyD88(-/-)) and Toll-like receptor 7-deficient (TLR7(-/-)) mice and found they had enhanced susceptibility compared to wild-type mice. Both groups had lower WNV-specific IgM response and reduced effector T cell functions. Dendritic cells (DCs) also exhibited a reduced maturation and impaired antigen-presenting functions compared to wild-type DCs. Moreover, infection with NS4B-P38G mutant in TLR7(-/-) and MyD88(-/-) mice provided full and partial protection respectively from subsequent challenge with lethal wild-type WNV. There were reduced T cell responses in MyD88(-/-) and interleukin-1 receptor deficient (IL-1R(-/-)) mice during secondary challenge with wild-type WNV. In contrast, TLR7(-/-) mice displayed normal T cell functions. Collectively, these results suggest that TLR7-dependent MyD88 signaling is required for T cell priming during NS4B-P38G mutant infection, whereas the TLR7-independent MyD88 signaling pathways are involved in memory T cell development, which may contribute to host protection during secondary challenge with wild-type WNV.
Assuntos
Imunidade Adaptativa , Glicoproteínas de Membrana/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 7 Toll-Like/metabolismo , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologia , Sequência de Aminoácidos , Animais , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Interações Hospedeiro-Patógeno , Imunidade Humoral , Memória Imunológica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Vírus do Nilo Ocidental/patogenicidadeRESUMO
BACKGROUND: West Nile virus (WNV) can persist long term in the brain and kidney tissues of humans, non-human primates, and hamsters. In this study, mice were infected with WNV strain H8912, previously cultured from the urine of a persistently infected hamster, to determine its pathogenesis in a murine host. METHODOLOGY/PRINCIPAL FINDINGS: We found that WNV H8912 was highly attenuated for neuroinvasiveness in mice. Following a systemic infection, viral RNA could be detected quickly in blood and spleen and much later in kidneys. WNV H8912 induced constitutive IL-10 production, upregulation of IFN-ß and IL-1ß expression, and a specific IgM response on day 10 post-infection. WNV H8912 persisted preferentially in kidneys with mild renal inflammation, and less frequently in spleen for up to 2.5 months post infection. This was concurrent with detectable serum WNV-specific IgM and IgG production. There were also significantly fewer WNV- specific T cells and lower inflammatory responses in kidneys than in spleen. Previous studies have shown that systemic wild-type WNV NY99 infection induced virus persistence preferentially in spleen than in mouse kidneys. Here, we noted that splenocytes of WNV H8912-infected mice produced significantly less IL-10 than those of WNV NY99-infected mice. Finally, WNV H8912 was also attenuated in neurovirulence. Following intracranial inoculation, WNV persisted in the brain at a low frequency, concurrent with neither inflammatory responses nor neuronal damage in the brain. CONCLUSIONS: WNV H8912 is highly attenuated in both neuroinvasiveness and neurovirulence in mice. It induces a low and delayed anti-viral response in mice and preferentially persists in the kidneys.