Novel paradigms for the gut-brain axis during alcohol withdrawal, withdrawal-associated depression, and craving in patients with alcohol use disorder.

Introduction: Patients with alcohol use disorder (AUD) exhibit symptoms such as alcohol withdrawal, depression, and cravings. The gut-immune response may play a significant role in manifesting these specific symptoms associated with AUD. This study examined the role of gut dysfunction, proinflammatory cytokines, and hormones in characterizing AUD symptoms. Methods: Forty-eight AUD patients [men (n = 34) and women (n = 14)] aged 23-63 years were grouped using the Clinical Institute Withdrawal Assessment of Alcohol Scale (CIWA) as clinically significant (CS-CIWA [score > 10] [n = 22]) and a clinically not-significant group (NCS-CIWA [score ≤ 10] [n = 26]). Clinical data (CIWA, 90-day timeline followback [TLFB90], and lifetime drinking history [LTDH]) and blood samples (for testing proinflammatory cytokines, hormones, and markers of intestinal permeability) were analyzed. A subset of 16 AUD patients was assessed upon admission for their craving tendencies related to drug-seeking behavior using the Penn-Alcohol Craving Score (PACS). Results: CS-CIWA group patients exhibited unique and significantly higher levels of adiponectin and interleukin (IL)-6 compared to NCS-CIWA. In the CS group, there were significant and high effects of association for the withdrawal score with gut-immune markers (lipopolysaccharide [LPS], adiponectin, IL-6, and IL-8) and for withdrawal-associated depression with gut-immune markers (scored using MADRS with LPS, soluble cells of differentiation type 14 [sCD14], IL-6, and IL-8). Craving (assessed by PACS, the Penn-Alcohol Craving Scale) was significantly characterized by what could be described as gut dysregulation (LBP [lipopolysaccharide binding protein] and leptin) and candidate proinflammatory (IL-1ß and TNF-α) markers. Such a pathway model describes the heavy drinking phenotype, HDD90 (heavy drinking days past 90 days), with even higher effects (R2 = 0.955, p = 0.006) in the AUD patients, who had higher ratings for cravings (PACS > 5). Discussion: The interaction of gut dysfunction cytokines involved in both inflammation and mediating activity constitutes a novel pathophysiological gut-brain axis for withdrawal symptoms and withdrawal-associated depression and craving symptoms in AUD. AUD patients with reported cravings show a significant characterization of the gut-brain axis response to heavy drinking. Trial registration: ClinicalTrials.gov, identifier: NCT# 00106106.

Intermittent Hiatus Hernia on High-Resolution Manometry Associates With Abnormal Reflux Burden Similar to Persistent Hiatus Hernia.

INTRODUCTION: Hiatus hernia is characterized by axial separation between the lower esophageal sphincter and the crural diaphragm, and higher reflux burden. Impact on reflux is unclear if such separation is intermittent rather than persistent. METHODS: Reflux burden off antisecretory therapy was compared between no hernia (n = 357), intermittent hernia (n = 42), and persistent hernia (n = 155) after review of consecutive high-resolution manometry and reflux monitoring studies. RESULTS: Proportions with pathologic acid exposure was similar between intermittent and persistent hernia (45.2% vs 46.5%, respectively), and both were significantly different from no hernia (28.7%, P ≤ 0.002). DISCUSSION: Intermittent hiatus hernias are clinically relevant in gastroesophageal reflux pathophysiology.

One-month assessment of Th-cell axis related inflammatory cytokines, IL-17 and IL-22 and their role in alcohol-associated liver disease.

Introduction: Changes in the expression of cyto- and chemokines due to alcohol-associated liver disease (ALD) have been reported to be both protective and pathogenic. This study examined plasma levels of two key cytokines, Il-17 and Il-22, which construct the proinflammatory vs. anti-inflammatory axes across the spectrum of alcohol use disorder (AUD) and ALD including alcohol-associated hepatitis (AH) to determine the underlying status of the inflammation. Methods: Forty-two males and females aged 25-63 yrs. were grouped as healthy controls (HV[n=8]), AUD with no liver injury (AUDNLI [n=8]), AUD with liver injury (AUDLI [n=8]), non-severe alcohol-associated hepatitis (NSAH [n=9]), and severe alcohol-associated hepatitis (SAH [n=9]). Demographic, drinking, and clinical data were collected. Blood samples were collected at baseline (BL, all subjects) and during week 4 (W4, only patients) for IL-17 and IL-22; and statistically analyzed. Results: IL-17 was highly elevated in the SAH group both at BL and post-SOC. LTDH and BL IL-22 in non-severe AH patients were associated significantly. LTDH significantly predicted W4 IL-22 levels, positively (increasing) in NSAH and inversely (lowering) in SAH patients. BL and W4 IL-22 levels were significantly higher (4-fold, p≤0.001) in all AH patients compared to all AUD patients (AUROC=0.988, p≤0.001). IL-22 showed significant affinity with AST, AST: ALT ratio, total bilirubin, INR, and PT both at BL and W4. IL-22 was inversely associated with IL-1ß; and positively with TNF-α and IL-8 both at BL, and W4. BL IL-17 showed a positive correlation with MELD (p=0.017) in all AH patients. In SAH, > 2-fold W4 IL-17 level compared to BL showed significant within subjects' effects, p=0.006. In AUD patients without AH, the drop in IL-17 at W4 vs. BL showed a significant within subjects' effect, p=0.031. Discussion: Drinking chronicity predicted opposite effects in IL-22 levels in NSAH (antiinflammatory) and SAH (pro-inflammatory) patients at post-SOC. BL IL-22 levels differentiated AH patients robustly from the AUD patients (with or without liver injury); and showed corresponding increases stepwise with the stages of ALD. IL-22 was closely associated with progression and injury markers of the liver; and response to the cytokines of pro-inflammatory nature. Pro-inflammatory indicator of IL-17 cell axis, IL-17 showed a strong positive association with MELD, a severity indicator of AH.

Illustration of Gut-Thyroid Axis in Alcohol Use Disorder: Interplay of Gut Dysfunction, Pro-Inflammatory Responses, and Thyroid Function.

(1) Background: Heavy and chronic alcohol drinking leads to altered gut dysfunction, coupled with a pro-inflammatory state. Thyroid-associated hormones and proteins may be dysregulated by heavy and chronic alcohol intake; however, the mechanism for altered gut-derived changes in thyroid function has not been studied thus far. This study investigates the role of alcohol-induced gut dysfunction and pro-inflammatory cytokine profile in the thyroid function of patients with alcohol use disorder (AUD). (2) Methods: Male and female AUD patients (n = 44) were divided into Gr.1, patients with normal thyroid-stimulating hormone (TSH) levels (n = 28, 0.8 ≤ TSH ≤ 3 mIU/L); and Gr.2, patients with clinically elevated TSH levels (n = 16, TSH > 3 mIU/L). Demographics, drinking measures, comprehensive metabolic panels, and candidate thyroid markers (TSH, circulating triiodothyronine (T3), and free thyroxine (fT4)) were analyzed. Gut-dysfunction-associated markers (lipopolysaccharide (LPS), LPS-binding protein (LBP), and soluble LPS-induced pathogen-associated protein (sCD14)), and candidate pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, MCP-1, PAI-1) were also evaluated. (3) Results: Patients in both groups presented with a borderline overweight BMI category. Gr.2 reported numerically higher indices of chronic and heavy drinking patterns than Gr.1. The fT4 levels were elevated, while T3 was within normal limits in both groups. The gut dysfunction markers LBP and sCD14 were numerically elevated in Gr.2 vs. Gr.1, suggesting subtle ongoing changes. Candidate pro-inflammatory cytokines were significantly elevated in Gr.2, including IL-1 ß, MCP-1, and PAI-1. Gr.2 showed a strong and statistically significant effect on the gut-immune-thyroid response (r = 0.896, 36 p = 0.002) on TSH levels in a multivariate regression model with LBP, sCD14, and PAI-1 levels as upstream variables in the gut-thyroid pathway. In addition, AUROC analysis demonstrated that many of the cytokines strongly predicted TSH in Gr.2, including IL-6 (area = 0.774, 39 p < 0.001) and TNF-α (area = 0.708, p = 0.017), among others. This was not observed in Gr.1. Gr.2 demonstrated elevated fT4, as well as TSH, which suggests that there was subclinical thyroiditis with underlying CNS dysfunction and a lack of a negative feedback loop. (4) Conclusions: These findings reveal the toxic effects of heavy and chronic drinking that play a pathological role in thyroid gland dysregulation by employing the gut-brain axis. These results also emphasize potential directions to carefully evaluate thyroid dysregulation in the overall medical management of AUD.

Theragnostic Efficacy of K18 Response in Alcohol Use Disorder with Clinically Significant Fibrosis Using Gut-Liver Axis.

(1) Background: Fibrosis in early-stage alcohol-associated liver disease (ALD) is commonly under-diagnosed in routine clinical practice. This study characterized the liver-injury and cell death response in alcohol use disorder (AUD) patients with ALD who also exhibited fibrosis and assessed the efficacy of standard of care (SOC) treatment in the improvement in liver injury. (2) Methods: Forty-eight heavy-drinking AUD patients aged 21−65 yrs. without clinical manifestations of liver injury were grouped by Fibrosis-4 (FIB-4) score, as negative (Gr.1 < 1.45, n = 21) or positive (Gr.2 ≥ 1.45, n = 27). Patients received 2-weeks (2 w) inpatient SOC. Data on demographics, drinking patterns, liver-injury, immune markers, and liver cell death (K18s) markers were analyzed at baseline (BL) and after 2 w SOC. (3) Results: Lifetime drinking (LTDH, yrs.) and acute heavy drinking (Heavy Drinking Days Past 90 Days [HDD90]) markers were significantly higher in Gr.2 vs. Gr.1. BL ALT, AST, AST:ALT and K18M65 were considerably higher in Gr.2. Dysregulated gut dysfunction and elevated immune activity were evident in Gr.2 characterized by TNF-α, IL-8 and LPS levels. After SOC, Gr.2 showed improvement in AST, ALT, AST/ALT ratio; and in the K18M65, K18M30 and K18M65/M30 ratio vs. Gr.1. The true positivity of BL IL-8 response to predict the improvement in K18M65 to normal levels among Gr.2 patients against those who did not have improvement after 2 w SOC was very high (AUROC = 0.830, p = 0.042). (4) Conclusions: Gut dysfunction, elevated cytokine response and necrotic liver cell death were elevated in AUD patients with early-stage ALD. K18 showed promise as a predictive theragnostic factor to differentiate among the AUD patients with early-stage ALD and baseline fibrosis who had improvement in liver injury against those who did not, by the levels of baseline IL-8.

Anti-inflammatory effects of interleukin-4 on intervertebral disc cells.

BACKGROUND CONTEXT: Inflammation has been associated with a number of pathological conditions including intervertebral disc (IVD) degeneration, increased risks of low back pain and other spinal diseases. Downregulating disc inflammation may be a strategy to reduce degeneration and more importantly back pain. Interleukin (IL)-4 was first discovered as a T-cell secreted factor that enhanced the proliferation of anti-IgM stimulated B cells and is now known as a cytokine that can stimulate cell proliferation and differentiation, tissue regeneration and neurological functions. IL-4 has been shown to be effective in inhibiting inflammatory pathways in chondrocytes. Immunohistochemical studies have shown that disc tissues are immunopositive for IL-4 receptor α (IL-4Rα) and IL-4. Yet, the roles of IL-4 and IL-4R in disc biology remain unknown. PURPOSE: The purpose of this study is to understand the roles of IL-4 and IL-4Rα in IVDs and to determine if IL-4 can function to inhibit inflammation in IVD cells. STUDY DESIGN/SETTING: In vitro experiment. METHODS: Deidentified patient IVD tissues were collected after surgery under the Orthopedic Information, Tissue and Implant Repository (ORA L00011021). IVD cells were isolated and cultured in monolayer. IL-4R protein expression was analyzed using immunocytochemistry. To test if the IL-4R was responsive to its ligand, signal transducer and activator of transcription 6 (STAT6) phosphorylation was analyzed on cell lysates of IVD cells treated with recombinant human IL-4 for 30 minutes using enzyme linked immunosorbent assay kit. Gene expression analysis of IL-4 up- and downregulated genes were analyzed using real-time RT-PCR. Anti-inflammatory effects of IL-4 were determined by cotreating disc cells with lipopolysaccharide (LPS) and IL-4 and measuring gene expression and protein release of inflammatory markers, IL-6 and IL-8. The significance of differences among means of data on gene expression and protein analyses were analyzed by one-way analysis of variance or student t test. Differences were considered significant when the p value was below 0.05. RESULTS: Immunocytochemistry staining for IL-4Rα in primary IVD cells (n=8) showed the majority of immunopositive staining was intracellular. After IVD cells (n=3-7) were treated with different concentrations of recombinant human IL-4 (0.1-100 ng/mL) for 30 minutes, phospho-STAT6 levels significantly increased by two- to four-fold at all concentrations tested compared with untreated cells. Gene expression of IL-4Rα and IL-6 increased significantly in cells undergoing IL-4 treatment for 24 hours compared with control treated IVD cells (n=5-10). LPS stimulated inflammatory gene expression of interferon (IFN)ß, IL-12, IL-6, and IL-8 were downregulated significantly in the presence of IL-4 (n=7). Lastly, protein release of IL-6 and IL-8 were reduced significantly in cells treated with IL-4 and LPS compared with those treated with LPS alone (n=7). CONCLUSIONS: This study was the first to explore the function of IL-4 and IL-4R in IVD cells. Immunocytochemistry studies confirmed that the majority of cells isolated from patient IVDs expressed IL-4Rα at the protein level. Also, IVD cells can respond to IL-4 by up-regulating IL-4Rα and IL-6 genes and inhibiting inflammatory genes and proteins induced by LPS. Further studies to test the anti-inflammatory effects of IL-4 in the IVD would be needed in animal models. CLINICAL RELEVANCE: Biological therapies which include intradiscal delivery of cells, anti-inflammatories or growth factors are being investigated to treat disc degeneration and back pain in animal models and in the clinic. Based on our findings that IL-4 has anti-inflammatory effects on IVD cells, the results of this study suggest including recombinant IL-4 delivery into the intervertebral disc may be a beneficial therapeutic strategy to treat patients with back pain by reducing disc inflammation.