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The detection of Clostridioides difficile infections (CDI) relies on testing the stool of patients by toxin antigen detection or PCR methods. Although PCR and antigenic methods have significantly reduced the time to results, delays in stool collection can significantly add to the turnaround time. The use of rectal swabs to detect C. difficile could considerably reduce the time to diagnosis of CDI. We developed a new rapid PCR assay for the detection of C. difficile and evaluated this PCR assay on both stool and rectal swab specimens. We recruited a total of 623 patients suspected of C. difficile infection. Stool samples and rectal swabs were collected from each patient and tested by our PCR assay. Stool samples were also tested by the cell cytotoxicity neutralization assay (CCNA) as a reference. The PCR assay detected C. difficile in 60 stool specimens and 61 rectal swabs for the 64 patients whose stool samples were positive for C. difficile by CCNA. The PCR assay detected an additional 35 and 36 stool and rectal swab specimens positive for C. difficile, respectively, for sensitivity with stools and rectal swabs of 93.8% and 95.3%, specificity of 93.7% and 93.6%, positive predictive values of 63.2% and 62.9%, and negative predictive values of 99.2% and 99.4%. Detection of C. difficile using PCR on stools or rectal swabs yielded reliable and similar results. The use of PCR tests on rectal swabs could reduce turnaround time for CDI detection, thus improving CDI management and control of C. difficile transmission. IMPORTANCE: Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, resulting in high morbidity, mortality, and economic burden. In clinical laboratories, CDI testing is currently performed on stool samples collected from patients with diarrhea. However, the diagnosis of CDI can be delayed by the time required to collect stool samples. Barriers to sample collection could be overcome by using a rectal swab instead of a stool sample. Our study showed that CDI can be identified rapidly and reliably by a new PCR assay developed in our laboratory on both stool and rectal swab specimens. The use of PCR tests on rectal swabs could reduce the time for the detection of CDI and improve the management of this infection. It should also provide a useful alternative for infection-control practitioners to better control the spread of C. difficile.
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Clostridioides difficile , Infecciones por Clostridium , Heces , Reacción en Cadena de la Polimerasa , Recto , Sensibilidad y Especificidad , Humanos , Heces/microbiología , Clostridioides difficile/aislamiento & purificación , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/microbiología , Reacción en Cadena de la Polimerasa/métodos , Recto/microbiología , Femenino , Masculino , Anciano , Persona de Mediana Edad , Manejo de Especímenes/métodos , Adulto , Anciano de 80 o más AñosRESUMEN
Cardiovascular disease (CVD) remains an important comorbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed in the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) > 5%) revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by computed tomography angiography scan (CTAScan) as the total (TPV) and low attenuated plaque volume (LAPV), in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4+ T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1, and triglyceride levels; lower Th17/Treg ratios; and classical monocyte expansion. Among HIV+, TPV+ versus TPV- exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9lowHLADRhigh monocytes, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9lowHLADRhigh monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.
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Aterosclerosis , Enfermedades Cardiovasculares , Enfermedad de la Arteria Coronaria , Infecciones por VIH , VIH-1 , Humanos , Adulto , Monocitos , Estudios de Cohortes , Receptores de Lipopolisacáridos , Células Th17 , Canadá , Enfermedad de la Arteria Coronaria/complicaciones , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológicoRESUMEN
Chronic inflammation is associated with higher risk of cardiovascular disease (CVD) in people living with HIV (PLWH). We have previously shown that interleukin-32 (IL-32), a multi-isoform proinflammatory cytokine, is chronically upregulated in PLWH and is linked with CVD. However, the mechanistic roles of the different IL-32 isoforms in CVD are yet to be identified. In this study, we aimed to investigate the potential impact of IL-32 isoforms on coronary artery endothelial cells (CAEC), whose dysfunction represents a major factor for atherosclerosis. Our results demonstrated that the predominantly expressed IL-32 isoforms (IL-32ß and IL-32γ) have a selective impact on the production of the proinflammatory cytokine IL-6 by CAEC. Furthermore, these two isoforms induced endothelial cell dysfunction by upregulating the expression of the adhesion molecules ICAM-I and VCAM-I and the chemoattractants CCL-2, CXCL-8 and CXCL-1. IL-32-mediated expression of these chemokines was sufficient to drive monocyte transmigration in vitro. Finally, we demonstrate that IL-32 expression in both PLWH and controls correlates with the carotid artery stiffness, measured by the cumulated lateral translation. These results suggest a role for IL-32-mediated endothelial cell dysfunction in dysregulation of the blood vessel wall and that IL-32 may represent a therapeutic target to prevent CVD in PLWH.
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Aterosclerosis , Enfermedades Cardiovasculares , Interleucinas , Rigidez Vascular , Humanos , Vasos Coronarios , Citocinas/metabolismo , Células Endoteliales/metabolismo , Interleucinas/metabolismo , Isoformas de ProteínasRESUMEN
We report that people with human immunodeficiency virus (HIV) diagnosed with coronary artery atherosclerotic plaques display higher levels of HIV DNA compared with those without atherosclerotic plaques. In a multivariable prediction model that included 27 traditional and HIV-related risk factors, measures of HIV DNA were among the most important predictors of atherosclerotic plaque formation.
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Enfermedades Cardiovasculares , Infecciones por VIH , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/complicaciones , Placa Aterosclerótica/diagnóstico , VIH , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/complicaciones , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/diagnóstico , Factores de RiesgoRESUMEN
Cardiovascular disease (CVD) remains an important co-morbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed on the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) >5%), revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by Computed tomography angiography scan (CTAScan) as total (TPV) and low attenuated plaque volume (LAPV) in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4 + T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells, were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1 and triglyceride levels, lower Th17/Treg ratios, and classical monocyte expansion. Among HIV + , TPV + versus TPV - exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9 low HLADR high monocyte, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9 low HLADR high monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.
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BACKGROUND: The study objectives were to ascertain the efficacy of vitamin D supplementation in rapidly increasing serum vitamin D and of implementation of a hybrid (virtual and in-person) trial. METHODS: In a randomized triple-blind controlled trial, healthcare workers were allocated to receive an oral bolus of 100,000 IU with 10,000 IU/week of vitamin D3 or placebo. The co-primary outcomes were the change from baseline in serum 25-hydroxyvitamin D [(Δ) 25(OH)D] and proportion with vitamin D sufficiency (25(OH)D ≥ 75 nmol/L), at endpoint. Adherence to supplements and procedures as well as adverse event rates were documented. RESULTS: Thirty-four (19 intervention, 15 control) subjects were randomized, with 28 (41%) virtual visits. After 44.78 ± 11.00 days from baseline, a significant adjusted group difference of 44.2 (34.7, 53.8) nmol/L was observed in the Δ 25(OH)D (95% CI) in favor of supplementation; 77.8% of intervention, and 13.3% of control, patients were vitamin D sufficient (OR:6.11, 95% CI:1.6, 22.9). The adherence to intervention was 94.7% in the intervention and 100% in the control groups. Irrespective of visit type, high adherence was observed in sampling procedures and completion of fortnightly online questionnaire. No adverse events attributable to vitamin D were reported. CONCLUSION: The vitamin D supplementation rapidly and safely raised 25(OH)D levels to sufficient levels for a biological effect. Similarly high adherence to study procedures was observed with virtual and in-person participation. TRIAL REGISTRATION: This trial was registered at https://clinicaltrials.gov on July 23, 2020 (# NCT04483635 ).
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Deficiencia de Vitamina D , Vitamina D , Humanos , Método Doble Ciego , Calcifediol , Colecalciferol/efectos adversos , Vitaminas , Suplementos Dietéticos/efectos adversos , Grupo de Atención al Paciente , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/tratamiento farmacológicoRESUMEN
Activated-to-memory transitioning CD4+ T cells display elevated expression of the HIV-1 co-receptor CCR5 and are more prone to HIV-1 latent infection. Here, we show that p53-regulated miRNA-103 downmodulates CCR5 levels in CD4+ T lymphocytes. We reveal that miRNA-103 mimics, as well as Nutlin-3, an inhibitor of Mdm2-mediated p53 degradation, decrease CCR5-dependent HIV-1 infection. Using a dual-reporter virus, we subsequently validate that in transitioning CD4+ T cells, Nutlin-3 treatment decreases the frequency of both productively and latently infected cells via upregulation of miRNA-103. Importantly, we provide evidence that CD4+ T cells from HIV-1 elite controllers express less CCR5 than those from antiretroviral therapy-naïve progressors, an effect linked to a significant increase in miRNA-103 levels. By contributing to the control of CCR5 expression in CD4+ T cells, miRNA-103 is likely to play a key role in countering the establishment of latent HIV-1 reservoirs in vivo.
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Résumé En l'absence de traitement antirétroviral, l'infection par le VIH progresse normalement vers le syndrome d'immunodéficience acquise. Une minorité de sujets infectés par le VIH sont toutefois capables de contrôler la réplication virale en l'absence de traitement. Ces patients appelés sujets contrôleurs d'élite (EC pour elite controllers) représentent un exemple de guérison fonctionnelle de l'infection par le VIH. Certains EC sont infectés par des virus défectifs, alors que d'autres ont des provirus intégrés dans des zones non transcrites de la chromatine. Cependant, la plupart des EC se distinguent des sujets non-contrôleurs parce qu'ils développent de fortes réponses T CD4 et CD8 spécifiques au VIH. Les cellules tueuses naturelles (NK pour Natural Killer) sont des cellules du système immunitaire inné qui fonctionnent à l'interface entre l'immunité innée et l'immunité acquise. Les cellules NK sont capables de reconnaître et de répondre à des cellules infectées dès les stades précoces de l'infection. Les cellules NK peuvent être activées de fac¸on indépendante et dépendante des anticorps afin d'exercer des fonctions antivirales et éliminer les cellules infectées. Ce manuscrit discutera du rôle des cellules NK dans le contrôle de l'infection par le VIH.
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Controladores de Élite , Infecciones por VIH , Humanos , Células Asesinas NaturalesRESUMEN
Untreated HIV infection usually leads to disease progression and development of the acquired immunodeficiency syndrome. A rare subset of people living with HIV control HIV without anti-retroviral therapy. These individuals, known as Elite Controllers (ECs), represent examples of a functional HIV cure. ECs differ from non-controllers is many aspects. Some are infected with defective virus, most have potent CD4 and CD8 virus-specific T cell responses and proviruses in these individuals tend to be inserted into regions with characteristics of deep latency. Natural Killer (NK) cells are innate immune cells that function at the intersection of innate and adaptive immunity. They have the capacity to recognize and respond to HIV-infected cells from the earliest stages in infection. NK cells can be activated through antibody independent and antibody dependent mechanisms to elicit functions that control HIV and kill infected cells. This manuscript will review the role of NK cells in HIV control.
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Síndrome de Inmunodeficiencia Adquirida , Infecciones por VIH , Inmunidad Adaptativa , Controladores de Élite , Infecciones por VIH/tratamiento farmacológico , Humanos , Células Asesinas NaturalesRESUMEN
HIV elite controllers (ECs) are characterized by the spontaneous control of viral replication, and by metabolic and autophagic profiles which favor anti-HIV CD4 and CD8 T-cell responses. Extracellular acyl coenzyme A binding protein (ACBP) acts as a feedback inhibitor of autophagy. Herein, we assessed the circulating ACBP levels in ECs, compared to people living with HIV (PLWH) receiving antiretroviral therapy (ART) or not. We found lower ACBP levels in ECs compared to ART-naïve or ART-treated PLWH (p < 0.01 for both comparisons), independently of age and sex. ACBP levels were similar in ECs and HIV-uninfected controls. The expression of the protective HLA alleles HLA-B*27, *57, or *58 did not influence ACBP levels in ECs. ACBP levels were not associated with CD4 or CD8 T-cell counts, CD4 loss over time, inflammatory cytokines, or anti-CMV IgG titers in ECs. In ART-treated PLWH, ACBP levels were correlated with interleukin (IL)-1ß levels, but not with other inflammatory cytokines such as IL-6, IL-8, IL-32, or TNF-α. In conclusion, ECs are characterized by low ACBP plasma levels compared to ART-naïve or ART-treated PLWH. As autophagy is key to anti-HIV CD4 and CD8 T-cell responses, the ACBP pathway constitutes an interesting target in HIV cure strategies.
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Inhibidor de la Unión a Diazepam , Infecciones por VIH , Linfocitos T CD4-Positivos , Citocinas/metabolismo , Inhibidor de la Unión a Diazepam/metabolismo , Controladores de Élite , HumanosRESUMEN
Persistent immune activation and inflammation in people living with HIV (PLWH) are associated with immunosenescence, premature aging and increased risk of non-AIDS comorbidities, with the underlying mechanisms not fully understood. In this study, we show that downregulation of the T-cell immunoglobulin receptor CD96 on CD8+ T cells from PLWH is associated with decreased expression of the co-stimulatory receptors CD27 and CD28, higher expression of the senescence marker CD57 and accumulation of a terminally differentiated T-cell memory phenotype. In addition, we show that CD96-low CD8+ T-cells display lower proliferative potential compared to their CD96-high counterparts and that loss of CD96 expression by HIV-specific CD8+ T-cells is associated with a suboptimal response to HIV antigens. In conclusion, our results suggest that CD96 marks CD8+ T-cells with competent responses to HIV and the loss of its expression might be used as a biomarker for CD8+ T-cell senescence and dysfunction in PLWH.
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Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Adulto , Antígenos CD/biosíntesis , Diferenciación Celular/inmunología , Femenino , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Persistent inflammation in HIV infection is associated with elevated cardiovascular disease (CVD) risk, even with viral suppression. Identification of novel surrogate biomarkers can enhance CVD risk stratification and suggest novel therapies. We investigated the potential of interleukin 32 (IL-32), a proinflammatory multi-isoform cytokine, as a biomarker for subclinical carotid artery atherosclerosis in virologically suppressed women living with HIV (WLWH). METHODS AND RESULTS: Nested within the Women's Interagency HIV Study, we conducted a cross-sectional comparison of IL-32 between 399 WLWH and 100 women without HIV, followed by a case-control study of 72 WLWH (36 carotid artery plaque cases vs. 36 age-matched controls without plaque). Plasma IL-32 protein was measured by ELISA, and mRNA of IL-32 isoforms (IL-32α, ß, γ, D, ε, and θ) was quantified by reverse transcription polymerase chain reaction from peripheral blood mononuclear cells. Plasma IL-32 protein levels were higher in WLWH compared with women without HIV (P = 0.02). Among WLWH, although plasma IL-32 levels did not differ significantly between plaque cases and controls, expression of IL-32 isoforms α, ß, and ε mRNA was significantly higher in peripheral blood mononuclear cells from cases (P = 0.01, P = 0.005, and P = 0.018, respectively). Upregulation of IL-32ß and IL-32ε among WLWH with carotid artery plaque persisted after adjustment for age, race/ethnicity, smoking, systolic blood pressure, body mass index, and history of hepatitis C virus (P = 0.04 and P = 0.045); the adjusted association for IL-32α was marginally significant (P = 0.07). CONCLUSIONS: IL-32 isoforms should be studied further as potential CVD biomarkers. This is of particular interest in WLWH by virtue of altered IL-32 levels in this population.
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Aterosclerosis/complicaciones , Enfermedades de las Arterias Carótidas/complicaciones , Infecciones por VIH/complicaciones , Interleucinas/metabolismo , Placa Aterosclerótica , Aterosclerosis/metabolismo , Biomarcadores , Enfermedades de las Arterias Carótidas/epidemiología , Estudios de Casos y Controles , Estudios Transversales , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , Humanos , Interleucinas/genética , Leucocitos Mononucleares , Persona de Mediana Edad , Isoformas de Proteínas , ARN MensajeroRESUMEN
OBJECTIVES: Untreated HIV infection was previously associated with IL-32 overexpression in gut/intestinal epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people with HIV (PWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN: Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PWH (HIVâ+âART; nâ=â17; mean age: 56âyears; CD4+ cell counts: 679âcells/µl; time on ART: 72âmonths) and age-matched HIV-uninfected controls (HIVneg; nâ=â5). The IEC line HT-29 was used for mechanistic studies. METHODS: Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A and HIV. IL-32α/ß/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS: IL-32ß/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32ß mRNA levels being predominantly expressed in both compartments and at significantly higher levels in HIVâ+âART compared to HIVneg. IL-17A transcripts were only detectable in SCB, with increased IL-17A levels in HIVneg compared with HIVâ+âART and negatively correlated with IL-32ß mRNA levels. IL-32ß/γ/ε isoform mRNA were detected in HT-29 cells upon exposure to TNF-α, Poly I:C (TLR3 agonist), Flagellin (TLR-5 agonist) and HIV. IL-17A significantly decreased both IL-32 ß/γ/ε mRNA and cell-associated IL-32 protein levels induced upon TNF-α and Poly I:C triggering. CONCLUSION: We document IL-32 isoforms abundant in the colon of ART-treated PWH and reveal the capacity of the Th17 hallmark cytokine IL-17A to attenuate IL-32 overexpression in a model of inflamed IEC.
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Infecciones por VIH , VIH-1 , Infecciones por VIH/tratamiento farmacológico , Humanos , Interleucina-17 , Persona de Mediana Edad , Isoformas de Proteínas , Células Th17RESUMEN
Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) are still at higher risk for cardiovascular diseases (CVDs) that are mediated by chronic inflammation. Identification of novel inflammatory mediators with the inherent potential to be used as CVD biomarkers and also as therapeutic targets is critically needed for better risk stratification and disease management in PLWH. Here, we investigated the expression and potential role of the multi-isoform proinflammatory cytokine IL-32 in subclinical atherosclerosis in PLWH (n=49 with subclinical atherosclerosis and n=30 without) and HIV- controls (n=25 with subclinical atherosclerosis and n=24 without). While expression of all tested IL-32 isoforms (α, ß, γ, D, ϵ, and θ) was significantly higher in peripheral blood from PLWH compared to HIV- controls, IL-32D and IL-32θ isoforms were further upregulated in HIV+ individuals with coronary artery atherosclerosis compared to their counterparts without. Upregulation of these two isoforms was associated with increased plasma levels of IL-18 and IL-1ß and downregulation of the atheroprotective protein TRAIL, which together composed a unique atherosclerotic inflammatory signature specific for PLWH compared to HIV- controls. Logistic regression analysis demonstrated that modulation of these inflammatory variables was independent of age, smoking, and statin treatment. Furthermore, our in vitro functional data linked IL-32 to macrophage activation and production of IL-18 and downregulation of TRAIL, a mechanism previously shown to be associated with impaired cholesterol metabolism and atherosclerosis. Finally, increased expression of IL-32 isoforms in PLWH with subclinical atherosclerosis was associated with altered gut microbiome (increased pathogenic bacteria; Rothia and Eggerthella species) and lower abundance of the gut metabolite short-chain fatty acid (SCFA) caproic acid, measured in fecal samples from the study participants. Importantly, caproic acid diminished the production of IL-32, IL-18, and IL-1ß in human PBMCs in response to bacterial LPS stimulation. In conclusion, our studies identified an HIV-specific atherosclerotic inflammatory signature including specific IL-32 isoforms, which is regulated by the SCFA caproic acid and that may lead to new potential therapies to prevent CVD in ART-treated PLWH.
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Aterosclerosis/complicaciones , Caproatos/metabolismo , Ácidos Grasos Volátiles/metabolismo , Tracto Gastrointestinal/metabolismo , Regulación de la Expresión Génica , Infecciones por VIH/complicaciones , Interleucinas/genética , Aterosclerosis/diagnóstico , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Biomarcadores , Electrocardiografía , Femenino , Microbioma Gastrointestinal , Infecciones por VIH/diagnóstico , Humanos , Interleucinas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Metagenoma , Metagenómica/métodos , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: HIV-1 transmitted/founder viruses (TF) are selected during the acute phase of infection from a multitude of virions present during transmission. They possess the capacity to establish infection and viral dissemination in a new host. Deciphering the discrete genetic determinant of infectivity in their envelope may provide clues for vaccine design. METHODS: One hundred twenty-six clade B HIV-1 consensus envelope sequences from untreated acute and early infected individuals were compared to 105 sequences obtained from chronically infected individuals using next generation sequencing and molecular analyses. RESULTS: We identified an envelope amino acid signature associated with TF viruses. They are more likely to have an isoleucine (I) in position 841 instead of an arginine (R). This mutation of R to I (R841I) in the gp41 cytoplasmic tail (gp41CT), specifically in lentivirus lytic peptides segment 1 (LLP-1), is significantly enriched compared to chronic viruses (OR = 0.2, 95% CI (0.09, 0.44), p = 0.00001). Conversely, a mutation of lysine (K) to isoleucine (I) located in position six (K6I) of the envelope signal peptide was selected by chronic viruses and compared to TF (OR = 3.26, 95% CI (1.76-6.02), p = 0.0001). CONCLUSIONS: The highly conserved gp41 CT_ LLP-1 domain plays a major role in virus replication in mediating intracellular traffic and Env incorporation into virions in interacting with encoded matrix protein. The presence of an isoleucine in gp41 in the TF viruses' envelope may sustain its role in the successful establishment of infection during the acute stage.
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Infecciones por VIH/virología , VIH-1/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Enfermedad Aguda , Aminoácidos , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/química , VIH-1/clasificación , Humanos , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Señales de Clasificación de Proteína/genética , Virión/metabolismo , Replicación Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
BACKGROUND: Human IL-32 is a polyfunctional cytokine that was initially reported to inhibit HIV-1 infection. However, recent data suggest that IL-32 may enhance HIV-1 replication by activating the HIV-1 primary targets, CD4 T-cells. Indeed, IL-32 is expressed in multiple isoforms, some of which are proinflammatory, whereas others are anti-inflammatory. SETTING AND METHODS: Here, we aimed to determine the relative expression of IL-32 isoforms and to test their inflammatory nature and potential to induce HIV-1 production in latently infected cells from virologically suppressed HIV-infected individuals. IL-32 and other cytokines were quantified from plasma and supernatant of CD4 T-cells by ELISA. Transcripts of IL-32 isoforms were quantified by qRT-PCR in peripheral blood mononuclear cells. The impact of recombinant human IL-32 isoforms on HIV-1 transcription was assessed in CD4 T-cells from HIV-1cART individuals by qRT-PCR. RESULTS: All IL-32 isoforms were significantly upregulated in HIV-1cART compared to HIV individuals with IL-32ß representing the dominantly expressed isoform, mainly in T-cells and NK-cells. At the functional level, although IL-32γ induced typical proinflammatory cytokines (IL-6 and IFN-γ) in TCR-activated CD4 T-cells, IL-32α showed an anti-inflammatory profile by inducing IL-10 but not IL-6 or IFN-γ. However, IL-32ß showed a dual phenotype by inducing both pro- and anti-inflammatory cytokines. Interestingly, consistent with its highly pro-inflammatory nature, IL-32γ, but not IL-32α or IL-32ß, induced HIV-1 production in latently infected CD4 T-cells isolated from combined antiretroviral therapy-treated individuals. CONCLUSIONS: Our data report on the differential expression of IL-32 isoforms and highlight the potential role of IL-32, particularly the γ isoform, in fueling persistent inflammation and transcription of viral reservoir in HIV-1 infection.
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Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/sangre , VIH-1/genética , Interleucinas/sangre , Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/virología , Estudios de Casos y Controles , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/farmacología , Leucocitos Mononucleares , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba , Carga ViralRESUMEN
The identification of transmission clusters (TCs) of HIV-1 using phylogenetic analyses can provide insights into viral transmission network and help improve prevention strategies. We compared the use of partial HIV-1 envelope fragment of 1,070 bp with its loop 3 (108 bp) to determine its utility in inferring HIV-1 transmission clustering. Serum samples of recently (n = 106) and chronically (n = 156) HIV-1-infected patients with status confirmed were sequenced. HIV-1 envelope nucleotide-based phylogenetic analyses were used to infer HIV-1 TCs. Those were constructed using ClusterPickerGUI_1.2.3 considering a pairwise genetic distance of ≤10% threshold. Logistic regression analyses were used to examine the relationship between the demographic factors that were likely associated with HIV-1 clustering. Ninety-eight distinct consensus envelope sequences were subjected to phylogenetic analyses. Using a partial envelope fragment sequence, 42 sequences were grouped into 15 distinct small TCs while the V3 loop reproduces 10 clusters. The agreement between the partial envelope and the V3 loop fragments was significantly moderate with a Cohen's kappa (κ) coefficient of 0.59, p < .00001. The mean age (<38.8 years) and HIV-1 B subtype are two factors identified that were significantly associated with HIV-1 transmission clustering in the cohort, odds ratio (OR) = 0.25, 95% confidence interval (CI, 0.04-0.66), p = .002 and OR: 0.17, 95% CI (0.10-0.61), p = .011, respectively. The present study confirms that a partial fragment of the HIV-1 envelope sequence is a better predictor of transmission clustering. However, the loop 3 segment may be useful in screening purposes and may be more amenable to integration in surveillance programs.
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Análisis por Conglomerados , Genes env , Infecciones por VIH/transmisión , VIH-1/clasificación , Filogenia , Enfermedad Aguda , Adolescente , Adulto , Secuencia de Aminoácidos , Enfermedad Crónica , Secuencia de Consenso , Femenino , Variación Genética , Proteína p24 del Núcleo del VIH/sangre , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/genética , Vigilancia de la Población , Valor Predictivo de las Pruebas , Quebec/epidemiología , Factores de Riesgo , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
Classical CD16- vs intermediate/nonclassical CD16+ monocytes differ in their homing potential and biological functions, but whether they differentiate into dendritic cells (DCs) with distinct contributions to immunity against bacterial/viral pathogens remains poorly investigated. Here, we employed a systems biology approach to identify clinically relevant differences between CD16+ and CD16- monocyte-derived DCs (MDDCs). Although both CD16+ and CD16- MDDCs acquire classical immature/mature DC markers in vitro, genome-wide transcriptional profiling revealed unique molecular signatures for CD16+ MDDCs, including adhesion molecules (ITGAE/CD103), transcription factors (TCF7L2/TCF4), and enzymes (ALDH1A2/RALDH2), whereas CD16- MDDCs exhibit a CDH1/E-cadherin+ phenotype. Of note, lipopolysaccharides (LPS) upregulated distinct transcripts in CD16+ (eg, CCL8, SIGLEC1, MIR4439, SCIN, interleukin [IL]-7R, PLTP, tumor necrosis factor [TNF]) and CD16- MDDCs (eg, MMP10, MMP1, TGM2, IL-1A, TNFRSF11A, lysosomal-associated membrane protein 1, MMP8). Also, unique sets of HIV-modulated genes were identified in the 2 subsets. Further gene set enrichment analysis identified canonical pathways that pointed to "inflammation" as the major feature of CD16+ MDDCs at immature stage and on LPS/HIV exposure. Finally, functional validations and meta-analysis comparing the transcriptome of monocyte and MDDC subsets revealed that CD16+ vs CD16- monocytes preserved their superior ability to produce TNF-α and CCL22, as well as other sets of transcripts (eg, TCF4), during differentiation into DC. These results provide evidence that monocyte subsets are transcriptionally imprinted/programmed with specific differentiation fates, with intermediate/nonclassical CD16+ monocytes being precursors for pro-inflammatory CD103+RALDH2+TCF4+ DCs that may play key roles in mucosal immunity homeostasis/pathogenesis. Thus, alterations in the CD16+ /CD16- monocyte ratios during pathological conditions may dramatically influence the quality of MDDC-mediated immunity.
Asunto(s)
Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Cadenas alfa de Integrinas/metabolismo , Monocitos/metabolismo , Receptores de IgG/metabolismo , Retinal-Deshidrogenasa/metabolismo , Factor de Transcripción 4/metabolismo , Transcripción Genética , Familia de Aldehído Deshidrogenasa 1 , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/citología , Lipopolisacáridos/farmacología , MicroARNs/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Transcripción Genética/efectos de los fármacosRESUMEN
Myeloid cells such as monocytes, dendritic cells (DC) and macrophages (MΦ) are key components of the innate immune system contributing to the maintenance of tissue homeostasis and the development/resolution of immune responses to pathogens. Monocytes and DC, circulating in the blood or infiltrating various lymphoid and non-lymphoid tissues, are derived from distinct bone marrow precursors and are typically short lived. Conversely, recent studies revealed that subsets of tissue resident MΦ are long-lived as they originate from embryonic/fetal precursors that have the ability to self-renew during the life of an individual. Pathogens such as the human immunodeficiency virus type 1 (HIV-1) highjack the functions of myeloid cells for viral replication (e.g., MΦ) or distal dissemination and cell-to-cell transmission (e.g., DC). Although the long-term persistence of HIV reservoirs in CD4+ T-cells during viral suppressive antiretroviral therapy (ART) is well documented, the ability of myeloid cells to harbor replication competent viral reservoirs is still a matter of debate. This review summarizes the current knowledge on the biology of monocytes and DC during homeostasis and in the context of HIV-1 infection and highlights the importance of future studies on long-lived resident MΦ to HIV persistence in ART-treated patients.