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Introduction: Cannabis is widely used for recreational and medical purposes, but its therapeutic efficacy remains unresolved for many applications as data from retrospective studies show dramatic discrepancy. We hypothesized that false self-reporting of cannabis use and lack of differentiation of heavy users from light or occasional users contribute to the conflicting outcomes. Objective: The goal of this study was to develop an objective biomarker of cannabis use and test how application of such biomarker impacts clinical study outcomes and dose-response measures. Methods and Analysis: Population pharmacokinetic (PK) models of (-)-trans-Δ9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC) were developed based on published studies reporting cannabinoid disposition in individual subjects following intravenous administration or smoking of cannabis. Plasma 11-COOH-THC concentration distributions in different cannabis user groups smoking cannabis were generated via Monte Carlo simulations, and plasma concentration cutoff values of 11-COOH-THC were developed to differentiate light and heavy daily cannabis users in clinical studies. The developed cutoff value was then applied to a retrospective study that assessed the impact of cannabis use on T cell activation in subjects with HIV who self-reported as either nonuser or daily user of cannabis. Results: The developed population PK models established plasma 11-COOH-THC concentration of 73.1 μg/L as a cutoff value to identify heavy daily users, with a positive predictive value of 80% in a mixed population of equal proportions of once daily and three times a day users. The stratification allowed detection of changes in T cell activation in heavy users which was not detected based on self-reporting or detectability of plasma cannabinoids. A proof-of-concept power analysis demonstrated that implementation of such cutoff value greatly increases study power and sensitivity to detect pharmacological effects of cannabis use. Conclusions: This study shows that the use of plasma 11-COOH-THC concentration cutoff value as an objective measure to classify cannabis use in target populations is critical for study sensitivity and specificity and provides much needed clarity for addressing dose-response relationships and therapeutic effects of cannabis.

Gastrointestinal (GI) mucosal dysfunction predicts and likely contributes to non-infectious comorbidities and mortality in HIV infection and persists despite antiretroviral therapy. However, the mechanisms underlying this dysfunction remain incompletely understood. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here we used a flow cytometry approach to investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for gastrointestinal dysbiosis. We report that increased neutrophil survival contributes to neutrophil accumulation in colorectal biopsy tissue, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Additionally, we characterized the intestinal microbiome of colorectal biopsies using 16S rRNA sequencing. We found that a reduced Lactobacillus: Prevotella ratio associated with neutrophil survival, suggesting that intestinal bacteria may contribute to GI neutrophil accumulation in treated HIV infection. Finally, we provide evidence that Lactobacillus species uniquely decrease neutrophil survival and neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions.

BackgroundCannabis is a widely used drug in the United States, and the frequency of cannabis use in the human immunodeficiency virus (HIV)-infected population is disproportionately high. Previous human and macaque studies suggest that cannabis may have an impact on plasma viral load; however, the relationship between cannabis use and HIV-associated systemic inflammation and immune activation has not been well defined.MethodsThe impact of cannabis use on peripheral immune cell frequency, activation, and function was assessed in 198 HIV-infected, antiretroviral-treated individuals by flow cytometry. Individuals were categorized into heavy, medium, or occasional cannabis users or noncannabis users based on the amount of the cannabis metabolite 11-nor-carboxy-tetrahydrocannabinol (THC-COOH) detected in plasma by mass spectrometry.ResultsHeavy cannabis users had decreased frequencies of human leukocyte antigen (HLA)-DR+CD38+CD4+ and CD8+ T-cell frequencies, compared to frequencies of these cells in non-cannabis-using individuals. Heavy cannabis users had decreased frequencies of intermediate and nonclassical monocyte subsets, as well as decreased frequencies of interleukin 23- and tumor necrosis factor-α-producing antigen-presenting cells.ConclusionsWhile the clinical implications are unclear, our findings suggest that cannabis use is associated with a potentially beneficial reduction in systemic inflammation and immune activation in the context of antiretroviral-treated HIV infection.

Compared to young heterosexual men, young sexual and gender minorities (YSGM) have elevated systemic inflammation and unique intestinal microbial profiles, influenced by HIV infection and substance use. However, links between cannabis use and microbial dysbiosis in this population have not been well described. In this pilot study, we aimed to characterize the complex interrelationships between cannabis use and microbial community structure in YSGM in relationship to HIV status. Cannabis use was assessed by self-administered Cannabis Use Disorder Identification Test (CUDIT) questionnaires and rectal microbial community alpha-diversity metrics were assessed via 16S ribosomal ribonucleic acid (rRNA) sequencing in a subset of YSGM (n = 42) in the RADAR cohort (aged 16–29) in Chicago. Multivariable regression models were used to assess the relationship between cannabis use and microbiome alpha-diversity metrics, adjusting for HIV status and other risk characteristics, including inflammation, which was evaluated by plasma levels of C-reactive protein (CRP). Problematic cannabis use, but not general use, was significantly inversely associated with microbial community richness (Adj. Beta = −8.13; 95% confidence interval [CI]: −15.68 to −0.59) and Shannon diversity (Adj. Beta = −0.04; 95% CI: −0.07 to 0.009). No significant association was observed between CUDIT score and community evenness, nor was any significant moderation observed by HIV status. We observed that problematic cannabis use was associated with reduced microbial community richness and Shannon diversity, adjusting for within population differences in inflammation and HIV status. Future research should aim to assess how cannabis use contributes to microbiome-related health factors among YSGM and if decreasing cannabis use can restore gut microbial community structure. [ABSTRACT FROM AUTHOR]

Multisystem inflammatory syndrome in children (MIS-C) occurs during or recently following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and is characterized by persistent fever, inflammation, and severe illness requiring hospitalization. The majority of patients with MIS-C also present with gastrointestinal (GI) symptoms, including abdominal pain, vomiting, and diarrhea. In this issue of the JCI, Yonker, Gilboa, and colleagues identified zonulin as a biomarker of GI permeability in children with MIS-C and present the results of an intriguing proof-of-concept study indicating that zonulin may represent a potential therapeutic target for MIS-C treatment and prevention. Their findings suggest that intestinal mucosal dysfunction and epithelial barrier breakdown may represent a biological mechanism underlying the development of MIS-C in SARS-CoV-2-infected children., Identification of MIS-C in SARS-CoV-2-infected children Since the first reported cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) (1), the WHO [...]

Background Conflicting evidence exists on the impact of cannabis use on antiretroviral therapy (ART) adherence among people with human immunodeficiency virus (PWH). We leveraged data collected among older PWH to characterize longitudinal associations between cannabis use and ART adherence. Methods AIDS Clinical Trials Group (ACTG) A5322 study participants were categorized as <100% (≥1 missed dose in past 7 days) or 100% (no missed doses) ART adherent. Participants self-reported current (past month), intermittent (past year but not past month), and no cannabis (in past year) use at each study visit. Generalized linear models using generalized estimating equations were fit and inverse probability weighting was used to adjust for time-varying confounders and loss to follow-up. Results Among 1011 participants (median age, 51 years), 18% reported current, 6% intermittent, and 76% no cannabis use at baseline; 88% reported 100% ART adherence. Current cannabis users were more likely to be <100% adherent than nonusers (adjusted risk ratio [aRR], 1.53 [95% CI, 1.11–2.10]). There was no association between ART adherence and current versus intermittent (aRR, 1.39 [95% CI,.85–2.28]) or intermittent versus no cannabis use (aRR, 1.04 [95% CI,.62–1.73]). Conclusions Among a cohort of older PWH, current cannabis users had a higher risk of <100% ART adherence compared to nonusers. These findings have important clinical implications as suboptimal ART adherence is associated with ART drug resistance, virologic failure, and elevated risk for mortality. Further research is needed to elucidate the mechanisms by which cannabis use decreases ART adherence in older PWH and to advance the development of more efficacious methods to mitigate nonadherence in this vulnerable population. [ABSTRACT FROM AUTHOR]

In Sub-Saharan Africa, young women 15–24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population. [ABSTRACT FROM AUTHOR]

HIV-2 infection is characterized by low viremia and slow disease progression as compared to HIV-1 infection. Circulating CD14++CD16+ monocytes were found to accumulate and CD11c+ conventional dendritic cells (cDC) to be depleted in a Portuguese cohort of people living with HIV-2 (PLWHIV-2), compared to blood bank healthy donors (HD). We studied more precisely classical monocytes; CD16+ inflammatory (intermediate, non-classical and slan+ monocytes, known to accumulate during viremic HIV-1 infection); cDC1, important for cross-presentation, and cDC2, both depleted during HIV-1 infection. We analyzed by flow cytometry these PBMC subsets from Paris area residents: 29 asymptomatic, untreated PLWHIV-2 from the IMMUNOVIR-2 study, part of the ANRS-CO5 HIV-2 cohort: 19 long-term non-progressors (LTNP; infection ≥8 years, undetectable viral load, stable CD4 counts≥500/μL; 17 of West-African origin -WA), and 10 non-LTNP (P; progressive infection; 9 WA); and 30 age-and sex-matched controls: 16 blood bank HD with unknown geographical origin, and 10 HD of WA origin (GeoHD). We measured plasma bacterial translocation markers by ELISA. Non-classical monocyte counts were higher in GeoHD than in HD (54 vs. 32 cells/μL, p = 0.0002). Slan+ monocyte counts were twice as high in GeoHD than in HD (WA: 28 vs. 13 cells/μL, p = 0.0002). Thus cell counts were compared only between participants of WA origin. They were similar in LTNP, P and GeoHD, indicating that there were no HIV-2 related differences. cDC counts did not show major differences between the groups. Interestingly, inflammatory monocyte counts correlated with plasma sCD14 and LBP only in PLWHIV-2, especially LTNP, and not in GeoHD. In conclusion, in LTNP PLWHIV-2, inflammatory monocyte counts correlated with LBP or sCD14 plasma levels, indicating a potential innate immune response to subclinical bacterial translocation. As GeoHD had higher inflammatory monocyte counts than HD, our data also show that specific controls are important to refine innate immunity studies. [ABSTRACT FROM AUTHOR]

Background Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively. Results CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition. Conclusions In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation. [ABSTRACT FROM AUTHOR]

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated Τ cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1Bal. Addition of heatkilled E. coli to HIV-1-exposed LPMCs resulted in increases in HIV-1 replication, CD4 Τ cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 Τ cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1-exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c+ LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/ Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Moreover, there is limited data available on the impact of Plasmodium co-infection on antiretroviral (ART)-treated HIV infection. Here, we used the rhesus macaque (RM) model to conduct a pilot study to establish a model of Plasmodium fragile co-infection during ART-treated simian immunodeficiency virus (SIV) infection, and to begin to characterize the immunopathogenic effect of co-infection in the context of ART. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VLs) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was also associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in the neutrophil functionality during co-infection. Finally, we characterized the levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these pilot data verify the utility of using the RM model to examine ART-treated SIV/ P. fragile co-infection, and indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/ P. fragile co-infection pathogenesis.

Background: The use of stimulants, such as methamphetamine, has been associated with greater immune activation in treated HIV infection. However, relatively little is known about whether concomitant cannabis use is associated with lower immune activation among HIV-positive stimulant users., Setting: HIV-positive, sexual minority men with biologically confirmed, recent methamphetamine use were enrolled in San Francisco, CA., Methods: In total, 78 methamphetamine-using sexual minority men with an undetectable HIV viral load (<40 copies/mL) completed self-report measures of cannabis use and substance use disorder severity. Plasma biomarkers of monocyte activation (ie, sCD14 and sCD163) and intestinal barrier integrity (iFABP) were measured. The associations of hazardous cannabis use with these measurements were examined after adjusting for substance use disorder severity, age, antiretroviral therapy regimen, CD4 T-cell count, and interleukin-6., Results: Hazardous cannabis users had the highest mean sCD14 levels (2181 ng/mL) compared with nonhazardous users (1991 ng/mL) and nonusers (1859 ng/mL; P = 0.05). In adjusted analyses, greater cannabis use severity was associated with higher sCD14 compared with nonusers (unstandardized beta = 133.6 ng/mL, P = 0.03). Cannabis use severity was not significantly associated with sCD163 or iFABP., Conclusions: Hazardous cannabis use is independently associated with elevations in a clinically relevant marker of immune activation in methamphetamine users with treated HIV.

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16(+) monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14(+) monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16(+) monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16(+) monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.