Experimentally Induced Reductions in Alcohol Consumption and Brain, Cognitive, and Clinical Outcomes in Older Persons With and Those Without HIV Infection (30-Day Challenge Study): Protocol for a Nonrandomized Clinical Trial.

BACKGROUND: Both alcohol consumption and HIV infection are associated with worse brain, cognitive, and clinical outcomes in older adults. However, the extent to which brain and cognitive dysfunction is reversible with reduction or cessation of drinking is unknown. OBJECTIVE: The 30-Day Challenge study was designed to determine whether reduction or cessation of drinking would be associated with improvements in cognition, reduction of systemic and brain inflammation, and improvement in HIV-related outcomes in adults with heavy drinking. METHODS: The study design was a mechanistic experimental trial, in which all participants received an alcohol reduction intervention followed by repeated assessments of behavioral and clinical outcomes. Persons were eligible if they were 45 years of age or older, had weekly alcohol consumption of 21 or more drinks (men) or 14 or more drinks (women), and were not at high risk of alcohol withdrawal. After a baseline assessment, participants received an intervention consisting of contingency management (money for nondrinking days) for at least 30 days followed by a brief motivational interview. After this, participants could either resume drinking or not. Study questionnaires, neurocognitive assessments, neuroimaging, and blood, urine, and stool samples were collected at baseline, 30 days, 90 days, and 1 year after enrollment. RESULTS: We enrolled 57 persons with heavy drinking who initiated the contingency management protocol (mean age 56 years, SD 4.6 years; 63%, n=36 male, 77%, n=44 Black, and 58%, n=33 people with HIV) of whom 50 completed 30-day follow-up and 43 the 90-day follow-up. The planned study procedures were interrupted and modified due to the COVID-19 pandemic of 2020-2021. CONCLUSIONS: This was the first study seeking to assess changes in brain (neuroimaging) and cognition after alcohol intervention in nontreatment-seeking people with HIV together with people without HIV as controls. Study design strengths, limitations, and lessons for future study design considerations are discussed. Planned analyses are in progress, after which deidentified study data will be available for sharing. TRIAL REGISTRATION: ClinicalTrials.gov NCT03353701; https://clinicaltrials.gov/study/NCT03353701. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/53684.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Dysregulated Cyclic Nucleotide Metabolism in Alcohol-Associated Steatohepatitis: Implications for Novel Targeted Therapies.

BACKGROUND: Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. METHODS: Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). RESULTS: Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. CONCLUSIONS: These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

Tributyrin Mitigates Ethanol-Induced Lysine Acetylation of Histone-H3 and p65-NFκB Downregulating CCL2 Expression and Consequent Liver Inflammation and Injury.

PURPOSE: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. METHODS: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. RESULTS: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. CONCLUSION: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.

Osteopontin is associated with dementia in the presence of cerebral small vessel disease.

BACKGROUND: Osteopontin (OPN) is a proinflammatory cytokine that has been recently implicated in neuroinflammation and neurodegeneration. We hypothesized that an increase in plasma osteopontin is a deleterious neuroinflammatory marker in people with dementia and cerebral small vessel disease (CSVD). METHODS: A pilot study was conducted on participants in the Northern Manhattan Study (NOMAS). Three groups were selected based on their dementia status and evidence of subclinical CSVD and chosen to be similar in age, sex, and education attainment: No dementia/No CSVD (n=19), Dementia/No CSVD (n=22), and Dementia+CSVD (n=21). Dementia (any type) was diagnosed by consensus adjudication following a series of comprehensive neuropsychological assessments and a review of the medical history. CSVD was indicated by silent brain infarcts, enlarged perivascular spaces, cerebral microbleeds, and white matter hyperintensity volumes (WMHV) on MRI. Multinomial logistic regression was used to examine the difference in OPN levels across groups, adjusting for key determinants of CSVD and neurodegeneration. RESULTS: Plasma osteopontin levels were elevated in the Dementia+CSVD group (mean=70.69±39.00 ng/ml) but not in the Dementia/No CSVD group (mean=45.46±19.11 ng/ml) compared to the No dementia/No CSVD group (mean=36.43±15.72 ng/ml). Osteopontin was associated with Dementia+CSVD (Odds Ratio (OR) per ng/ml=1.06, 95%CI 1.02-1.11) after adjusting for covariates, including brain volume. OPN was strongly correlated with WMHV (Spearman's rank correlation =0.46, p=0.0001), but not with other components of CSVD. CONCLUSION: In this pilot, greater levels of plasma osteopontin were associated with dementia with evidence of CSVD. This link was predominately driven by the contribution of OPN to dementia through the burden of white matter lesions.

Increased expression of phosphodiesterase 4 in activated hepatic stellate cells promotes cytoskeleton remodeling and cell migration.

Activation and transdifferentiation of hepatic stellate cells (HSC) into migratory myofibroblasts is a key process in liver fibrogenesis. Cell migration requires an active remodeling of the cytoskeleton, which is a tightly regulated process coordinated by Rho-specific guanine nucleotide exchange factors (GEFs) and the Rho family of small GTPases. Rho-associated kinase (ROCK) promotes assembly of focal adhesions and actin stress fibers by regulating cytoskeleton organization. GEF exchange protein directly activated by cAMP 1 (EPAC1) has been implicated in modulating TGFß1 and Rho signaling; however, its role in HSC migration has never been examined. The aim of this study was to evaluate the role of cAMP-degrading phosphodiesterase 4 (PDE4) enzymes in regulating EPAC1 signaling, HSC migration, and fibrogenesis. We show that PDE4 protein expression is increased in activated HSCs expressing alpha smooth muscle actin and active myosin light chain (MLC) in fibrotic tissues of human nonalcoholic steatohepatitis cirrhosis livers and mouse livers exposed to carbon tetrachloride. In human livers, TGFß1 levels were highly correlated with PDE4 expression. TGFß1 treatment of LX2 HSCs decreased levels of cAMP and EPAC1 and increased PDE4D expression. PDE4 specific inhibitor, rolipram, and an EPAC-specific agonist decreased TGFß1-mediated cell migration in vitro. In vivo, targeted delivery of rolipram to the liver prevented fibrogenesis and collagen deposition and decreased the expression of several fibrosis-related genes, and HSC activation. Proteomic analysis of mouse liver tissues identified the regulation of actin cytoskeleton by the kinase effectors of Rho GTPases as a major pathway impacted by rolipram. Western blot analyses confirmed that PDE4 inhibition decreased active MLC and endothelin 1 levels, key proteins involved in cytoskeleton remodeling and contractility. The current study, for the first time, demonstrates that PDE4 enzymes are expressed in hepatic myofibroblasts and promote cytoskeleton remodeling and HSC migration. © 2023 The Pathological Society of Great Britain and Ireland.

SmartPill™ Administration to Assess Gastrointestinal Function after Spinal Cord Injury in a Porcine Model-A Preliminary Study.

Gastrointestinal (GI) complications, including motility disorders, metabolic deficiencies, and changes in gut microbiota following spinal cord injury (SCI), are associated with poor outcomes. After SCI, the autonomic nervous system becomes unbalanced below the level of injury and can lead to severe GI dysfunction. The SmartPill™ is a non-invasive capsule that, when ingested, transmits pH, temperature, and pressure readings that can be used to assess effects in GI function post-injury. Our minipig model allows us to assess these post-injury changes to optimize interventions and ultimately improve GI function. The aim of this study was to compare pre-injury to post-injury transit times, pH, and pressures in sections of GI tract by utilizing the SmartPill™ in three pigs after SCI at 2 and 6 weeks. Tributyrin was administered to two pigs to assess the influences on their gut microenvironment. We observed prolonged GET (Gastric Emptying Time) and CTT (Colon Transit Time), decreases in contraction frequencies (Con freq) in the antrum of the stomach, colon, and decreases in duodenal pressures post-injury. We noted increases in Sum amp generated at 2 weeks post-injury in the colon, with corresponding decreases in Con freq. We found transient changes in pH in the colon and small intestine at 2 weeks post-injury, with minimal effect on stomach pH post-injury. Prolonged GETs and CTTs can influence the absorptive profile in the gut and contribute to pathology development. This is the first pilot study to administer the SmartPill™ in minipigs in the context of SCI. Further investigations will elucidate these trends and characterize post-SCI GI function.

Probiotic-derived nanoparticles inhibit ALD through intestinal miR194 suppression and subsequent FXR activation.

BACKGROUND AND AIMS: Intestinal farnesoid X receptor (FXR) plays a critical role in alcohol-associated liver disease (ALD). We aimed to investigate whether alcohol-induced dysbiosis increased intestinal microRNA194 (miR194) that suppressed Fxr transcription and whether Lactobacillus rhamnosus GG-derived exosome-like nanoparticles (LDNPs) protected against ALD through regulation of intestinal miR194-FXR signaling in mice. APPROACH AND RESULTS: Binge-on-chronic alcohol exposure mouse model was utilized. In addition to the decreased ligand-mediated FXR activation, alcohol feeding repressed intestinal Fxr transcription and increased miR194 expression. This transcriptional suppression of Fxr by miR194 was confirmed in intestinal epithelial Caco-2 cells and mouse enteriods. The alcohol feeding-reduced intestinal FXR activation was further demonstrated by the reduced FXR reporter activity in fecal samples and by the decreased fibroblast growth factor 15 (Fgf15) messenger RNA (mRNA) in intestine and protein levels in the serum, which caused an increased hepatic bile acid synthesis and lipogeneses. We further demonstrated that alcohol feeding increased-miR194 expression was mediated by taurine-upregulated gene 1 (Tug1) through gut microbiota regulation of taurine metabolism. Importantly, 3-day oral administration of LDNPs increased bile salt hydrolase (BSH)-harboring bacteria that decreased conjugated bile acids and increased gut taurine concentration, which upregulated Tug1, leading to a suppression of intestinal miR194 expression and recovery of FXR activation. Activated FXR upregulated FGF15 signaling and subsequently reduced hepatic bile acid synthesis and lipogenesis and attenuated ALD. These protective effects of LDNPs were eliminated in intestinal FxrΔIEC and Fgf15-/- mice. We further showed that miR194 was upregulated, whereas BSH activity and taurine levels were decreased in fecal samples of patients with ALD. CONCLUSIONS: Our results demonstrated that gut microbiota-mediated miR194 regulation contributes to ALD pathogenesis and to the protective effects of LDNPs through modulating intestinal FXR signaling.

A conceptual analysis of SBIRT implementation alongside the continuum of PrEP awareness: domains of fit and feasibility.

Screening, Brief Intervention, and Referral to Treatment (SBIRT) is a supplementary intervention that can be incorporated into the Pre-Exposure Prophylaxis (PrEP) Care Continuum, complementing initiatives and endeavors focused on Human Immunodeficiency Virus (HIV) prevention in clinical care and community-based work. Referencing the Transtheoretical Model of Change and the PrEP Awareness Continuum, this conceptual analysis highlights how SBIRT amplifies ongoing HIV prevention initiatives and presents a distinct chance to address identified gaps. SBIRT's mechanisms show promise of fit and feasibility through (a) implementing universal Screening (S), (b) administering a Brief Intervention (BI) grounded in motivational interviewing aimed at assisting individuals in recognizing the significance of PrEP in their lives, (c) providing an affirming and supportive Referral to Treatment (RT) to access clinical PrEP care, and (d) employing client-centered and destigmatized approaches. SBIRT is uniquely positioned to help address the complex challenges facing PrEP awareness and initiation efforts. Adapting the SBIRT model to integrate and amplify HIV prevention efforts merits further examination.

Novel Liposomal Rolipram Formulation for Clinical Application to Reduce Emesis.

Introduction: The phosphodiesterase 4 (PDE4) inhibitor, rolipram, has beneficial effects on tissue inflammation, injury and fibrosis, including in the liver. Since rolipram elicits significant CNS side-effects in humans (ie, nausea and emesis), our group developed a fusogenic lipid vesicle (FLV) drug delivery system that targets the liver to avoid adverse events. We evaluated whether this novel liposomal rolipram formulation reduces emesis. Methods: C57Bl/6J male mice were used to compare the effect of three doses of free and FLV-delivered (FLVs-Rol) rolipram in a behavioral correlate model of rolipram-induced emesis. Tissue rolipram and rolipram metabolite levels were measured using LC-MS/MS. The effect of FLVs-Rol on brain and liver PDE4 activities was evaluated. Results: Low and moderate doses of free rolipram significantly reduced anesthesia duration, while the same doses of FLVs-Rol had no effect. However, the onset and duration of adverse effects (shortening of anesthesia period) elicited by a high dose of rolipram was not ameliorated by FLVs-Rol. Post-mortem analysis of brain and liver tissues demonstrated that FLVs affected the rate of rolipram uptake by liver and brain. Lastly, administration of a moderate dose of FLVs-Rol attenuated endotoxin induced PDE4 activity in the liver with negligible effect on the brain. Discussion: The findings that the low and moderate doses of FLVs-Rol did not shorten the anesthesia duration time suggest that FLV delivery prevented critical levels of drug from crossing the blood-brain barrier (BBB) to elicit CNS side-effects. However, the inability of high dose FLVs-Rol to prevent CNS side-effects indicates that there was sufficient unencapsulated rolipram to cross the BBB and shorten anesthesia duration. Notably, a moderate dose of FLVs-Rol was able to decrease PDE4 activity in the liver without affecting the brain. Taken together, FLVs-Rol has a strong potential for clinical application for the treatment of liver disease without side effects.

Age-Associated Gut Dysbiosis, Marked by Loss of Butyrogenic Potential, Correlates With Altered Plasma Tryptophan Metabolites in Older People Living With HIV.

BACKGROUND: Imbalance in tryptophan (TRP) metabolism and its neuroactive metabolites, serotonin and kynurenine (KYN), is a known pathogenic mechanism underlying neurocognitive impairment. Gut microbiota plays an important role in TRP metabolism, and the production of these neuroactive molecules affects neurocognitive function. Although both HIV infection and normal aging independently induce gut dysbiosis and influence TRP metabolism, their interactive effects on compositional/functional changes in gut microbiota and consequent alterations in TRP metabolites remain largely undetermined. METHODS: Older people living with HIV infection (PLWH, aged 50-70 years, n = 22) were enrolled in this cross-sectional pilot study. Metagenomic analysis of fecal microbiome using 16S Ribosomal ribonucleic acid gene sequencing and metabolomics analysis of plasma using mass spectrometry with a reverse-phase iquid chromatography tandem mass spectrometry were performed. Statistical analyses included the univariate linear regression and Spearman correlation analyses. RESULTS: Age-associated changes in plasma levels of key neuroactive TRP metabolites, serotonin and KYN, were seen in PLWH. Specifically, we observed age-dependent decreases in serotonin and increases in KYN and KYN-to-TRP ratio, indicative of dysfunctional TRP metabolism. Furthermore, the gut dysbiosis seen in older PLWH is characterized by a reduction of Firmicutes/Bacteroidetes ratio and butyrate-producing microbial families Lachnospiraceae and Lactobacillaceae. Of importance, correspondent with gut dysbiosis, increasing age was significantly associated with decreased plasma butyrate levels, which in turn correlated positively with serotonin and negatively with KYN/TRP ratio. CONCLUSIONS: Age-dependent gut microbial dysbiosis distinguished by a decrease in butyrogenic potential is a key pathogenic feature associated with the shift in TRP metabolism from serotonin to KYN in older PLWH.

Serological assessment of SARS-CoV-2 infection during the first wave of the pandemic in Louisville Kentucky.

Serological assays intended for diagnosis, sero-epidemiologic assessment, and measurement of protective antibody titers upon infection or vaccination are essential for managing the SARS-CoV-2 pandemic. Serological assays measuring the antibody responses against SARS-CoV-2 antigens are readily available. However, some lack appropriate characteristics to accurately measure SARS-CoV-2 antibodies titers and neutralization. We developed an Enzyme-linked Immunosorbent Assay (ELISA) methods for measuring IgG, IgA, and IgM responses to SARS-CoV-2, Spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins. Performance characteristics of sensitivity and specificity have been defined. ELISA results show positive correlation with microneutralization and Plaque Reduction Neutralization assays with infectious SARS-CoV-2. Our ELISA was used to screen healthcare workers in Louisville, KY during the first wave of the local pandemic in the months of May and July 2020. We found a seropositive rate of approximately 1.4% and 2.3%, respectively. Our analyses demonstrate a broad immune response among individuals and suggest some non-RBD specific S IgG and IgA antibodies neutralize SARS-CoV-2.

Decrease in acetyl-CoA pathway utilizing butyrate-producing bacteria is a key pathogenic feature of alcohol-induced functional gut microbial dysbiosis and development of liver disease in mice.

Emerging research evidence has established the critical role of the gut-liver axis in the development of alcohol-associated liver disease (ALD). The present study employed 16S rRNA gene and whole genome shotgun (WGS) metagenomic analysis in combination with a revised microbial dataset to comprehensively detail the butyrate-producing microbial communities and the associated butyrate metabolic pathways affected by chronic ethanol feeding. Specifically, the data demonstrated that a decrease in several butyrate-producing bacterial genera belonging to distinct families within the Firmicutes phyla was a significant component of ethanol-induced dysbiosis. WGS analysis of total bacterial genomes encompassing butyrate synthesizing pathways provided the functional characteristics of the microbiome associated with butyrate synthesis. The data revealed that in control mice microbiome, the acetyl-coenzyme A (CoA) butyrate synthesizing pathway was the most prevalent and was significantly and maximally decreased by chronic ethanol feeding. Further WGS analysis i) validated the ethanol-induced decrease in the acetyl-CoA pathway by identifying the decrease in two critical genes but - (butyryl-CoA: acetate CoA transferase) and buk - (butyrate kinase) that encode the terminal condensing enzymes required for converting butyryl-CoA to butyrate and ii) detection of specific taxa of butyrate-producing bacteria containing but and buk genes. Notably, the administration of tributyrin (Tb) - a butyrate prodrug - significantly prevented ethanol-induced decrease in butyrate-producing bacteria, hepatic steatosis, inflammation, and injury. Taken together, our findings strongly suggest that the loss of butyrate-producing bacteria using the acetyl-CoA pathway is a significant pathogenic feature of ethanol-induced microbial dysbiosis and ALD and can be targeted for therapy.

Association of Syndemic Unhealthy Alcohol Use, Smoking, and Depressive Symptoms on Incident Cardiovascular Disease among Veterans With and Without HIV-Infection.

Unhealthy alcohol use, smoking, and depressive symptoms are risk factors for cardiovascular disease (CVD). Little is known about their co-occurrence - termed a syndemic, defined as the synergistic effect of two or more conditions-on CVD risk in people with HIV (PWH). We used data from 5621 CVD-free participants (51% PWH) in the Veteran's Aging Cohort Study-8, a prospective, observational study of veterans followed from 2002 to 2014 to assess the association between this syndemic and incident CVD by HIV status. Diagnostic codes identified cases of CVD (acute myocardial infarction, stroke, heart failure, peripheral artery disease, and coronary revascularization). Validated measures of alcohol use, smoking, and depressive symptoms were used. Baseline number of syndemic conditions was categorized (0, 1, ≥ 2 conditions). Multivariable Cox Proportional Hazards regressions estimated risk of the syndemic (≥ 2 conditions) on incident CVD by HIV-status. There were 1149 cases of incident CVD (52% PWH) during the follow-up (median 10.1 years). Of the total sample, 64% met our syndemic definition. The syndemic was associated with greater risk for incident CVD among PWH (Hazard Ratio [HR] 1.87 [1.47-2.38], p < 0.001) and HIV-negative veterans (HR 1.70 [1.35-2.13], p < 0.001), compared to HIV-negative with zero conditions. Among those with the syndemic, CVD risk was not statistically significantly higher among PWH vs. HIV-negative (HR 1.10 [0.89, 1.37], p = .38). Given the high prevalence of this syndemic combined with excess risk of CVD, these findings support linked-screening and treatment efforts.

Exosome-Like Nanoparticles From Lactobacillus rhamnosusGG Protect Against Alcohol-Associated Liver Disease Through Intestinal Aryl Hydrocarbon Receptor in Mice.

Alcohol-associated liver disease (ALD) is a major cause of mortality. Gut barrier dysfunction-induced bacterial translocation and endotoxin release contribute to the pathogenesis of ALD. Probiotic Lactobacillus rhamnosus GG (LGG) is known to be beneficial on experimental ALD by reinforcing the intestinal barrier function. In this study, we aim to investigate whether the protective effects of LGG on intestinal barrier function is mediated by exosome-like nanoparticles (ELNPs) released by LGG. Intestinal epithelial cells and macrophages were treated with LGG-derived ELNPs (LDNPs) isolated from LGG culture. LDNPs increased tight junction protein expression in epithelial cells and protected from the lipopolysaccharide-induced inflammatory response in macrophages. Three-day oral application of LDNPs protected the intestine from alcohol-induced barrier dysfunction and the liver from steatosis and injury in an animal model of ALD. Co-administration of an aryl hydrocarbon receptor (AhR) inhibitor abolished the protective effects of LDNPs, indicating that the effects are mediated, at least in part, by intestinal AhR signaling. We further demonstrated that LDNP administration increased intestinal interleukin-22-Reg3 and nuclear factor erythroid 2-related factor 2 (Nrf2)-tight junction signaling pathways, leading to the inhibition of bacterial translocation and endotoxin release in ALD mice. This protective effect was associated with LDNP enrichment of bacterial tryptophan metabolites that are AhR agonists. Conclusions: Our results suggest that the beneficial effects of LGG and their supernatant in ALD are likely mediated by bacterial AhR ligand-enriched LDNPs that increase Reg3 and Nrf2 expression, leading to the improved barrier function. These findings provide a strategy for the treatment of ALD and other gut barrier dysfunction-associated diseases.

A specific low-density neutrophil population correlates with hypercoagulation and disease severity in hospitalized COVID-19 patients.

SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19-associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.

Epigenetic Mechanisms Underlying HIV-Infection Induced Susceptibility of CD4+ T Cells to Enhanced Activation-Induced FasL Expression and Cell Death.

BACKGROUND: Chronic immune activation and CD4 T cell depletion are significant pathogenic features of HIV infection. Expression of Fas ligand (FasL), a key mediator of activation-induced cell death in T cells, is elevated in people living with HIV-1 infection (PLWH). However, the epigenetic mechanisms underlying the enhanced induction of FasL expression in CD4 T lymphocytes in PLWH are not completely elucidated. Hence, the current work examined the effect of HIV infection on FasL promoter-associated histone modifications and transcriptional regulation in CD4 T lymphocytes in PLWH. METHOD: Flow cytometric analysis was performed to examine the Fas-FasL expression on total CD4 T cells and naïve/memory CD4 T cell subsets. Epigenetic FasL promoter histone modifications were investigated by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis using freshly isolated total CD4 T lymphocytes from HIV-1 infected and noninfected individuals. RESULTS: All naïve/memory CD4 T cell subsets from PLWH showed markedly greater frequency of FasL expression. Notably, examination of functional outcome of FasL/Fas co-expression demonstrated the preferential susceptibility of Tcm and Tem subsets to activation-induced apoptosis. Importantly, these CD4 T cells collectively demonstrated a distinct FasL promoter histone profile involving a coordinated cross-talk between histone H3 modifications leading to enhanced FasL gene expression. Specifically, levels of transcriptionally permissive histone H3K4-trimethylation (H3K4Me3) and histone H3K9-acetylation (H3K9Ac) were increased, with a concomitant decrease in the repressive H3K9-trimethylation (H3K9Me3). CONCLUSION: The present work demonstrates that epigenetic mechanisms involving promoter-histone modifications regulate transcriptional competence and FasL expression in CD4 T cells from PLWH and render them susceptible to activation-induced cell death.

Cathelicidin-related antimicrobial peptide alleviates alcoholic liver disease through inhibiting inflammasome activation.

Alcoholic liver disease (ALD) is associated with gut dysbiosis and hepatic inflammasome activation. While it is known that antimicrobial peptides (AMPs) play a critical role in the regulation of bacterial homeostasis in ALD, the functional role of AMPs in the alcohol-induced inflammasome activation is unclear. The aim of this study was to determine the effects of cathelicidin-related antimicrobial peptide (CRAMP) on inflammasome activation in ALD. CRAMP knockout (Camp-/-) and wild-type (WT) mice were subjected to binge-on-chronic alcohol feeding and synthetic CRAMP peptide was administered. Serum/plasma and hepatic tissue samples from human subjects with alcohol use disorder and/or alcoholic hepatitis were analyzed. CRAMP deficiency exacerbated ALD with enhanced inflammasome activation as shown by elevated serum interleukin (IL)-1ß levels. Although Camp-/- mice had comparable serum endotoxin levels compared to WT mice after alcohol feeding, hepatic lipopolysaccharide (LPS) binding protein (LBP) and cluster of differentiation (CD) 14 were increased. Serum levels of uric acid (UA), a Signal 2 molecule in inflammasome activation, were positively correlated with serum levels of IL-1ß in alcohol use disorder patients with ALD and were increased in Camp-/- mice fed alcohol. In vitro studies showed that CRAMP peptide inhibited LPS binding to macrophages and inflammasome activation stimulated by a combination of LPS and UA. Synthetic CRAMP peptide administration decreased serum UA and IL-1ß concentrations and rescued the liver from alcohol-induced damage in both WT and Camp-/- mice. In summary, CRAMP exhibited a protective role against binge-on-chronic alcohol-induced liver damage via regulation of inflammasome activation by decreasing LPS binding and UA production. CRAMP administration may represent a novel strategy for treating ALD. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

cAMP Signaling in Pathobiology of Alcohol Associated Liver Disease.

The importance of cyclic adenosine monophosphate (cAMP) in cellular responses to extracellular signals is well established. Many years after discovery, our understanding of the intricacy of cAMP signaling has improved dramatically. Multiple layers of regulation exist to ensure the specificity of cellular cAMP signaling. Hence, disturbances in cAMP homeostasis could arise at multiple levels, from changes in G protein coupled receptors and production of cAMP to the rate of degradation by phosphodiesterases. cAMP signaling plays critical roles in metabolism, inflammation and development of fibrosis in several tissues. Alcohol-associated liver disease (ALD) is a multifactorial condition ranging from a simple steatosis to steatohepatitis and fibrosis and ultimately cirrhosis, which might lead to hepatocellular cancer. To date, there is no FDA-approved therapy for ALD. Hence, identifying the targets for the treatment of ALD is an important undertaking. Several human studies have reported the changes in cAMP homeostasis in relation to alcohol use disorders. cAMP signaling has also been extensively studied in in vitro and in vivo models of ALD. This review focuses on the role of cAMP in the pathobiology of ALD with emphasis on the therapeutic potential of targeting cAMP signaling for the treatment of various stages of ALD.

Acetylator Genotype-Dependent Dyslipidemia in Rats Congenic for N-Acetyltransferase 2.

Recent reports suggest that arylamine N-acetyltransferases (NAT1 and/or NAT2) serve important roles in regulation of energy utility and insulin sensitivity. We investigated the interaction between diet (control vs. high-fat diet) and acetylator phenotype (rapid vs. slow) using previously established congenic rat lines (in F344 background) that exhibit rapid or slow Nat2 (orthologous to human NAT1) acetylator genotypes. Male and female rats of each genotype were fed control or high-fat (Western-style) diet for 26 weeks. We then examined diet- and acetylator genotype-dependent changes in body and liver weights, systemic glucose tolerance, insulin sensitivity, and plasma lipid profile. Male and female rats on the high fat diet weighed approximately 10% more than rats on the control diet and the percentage liver to body weight was consistently higher in rapid than slow acetylator rats. Rapid acetylator rats were more prone to develop dyslipidemia overall (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats. Total cholesterol (TC)-to-HDL ratios were significantly higher and HDL-to-LDL ratios were significantly lower in rapid acetylator rats. Our data suggest that rats with rapid systemic Nat2 (NAT1 in humans) genotype exhibited higher dyslipidemia conferring risk for metabolic syndrome and cardiovascular dysfunction.