Gluten-Free Diet Induces Rapid Changes in Phenotype and Survival Properties of Gluten-Specific T Cells in Celiac Disease.

BACKGROUND & AIMS: The treatment of celiac disease (CeD) with gluten-free diet (GFD) normalizes gut inflammation and disease-specific antibodies. CeD patients have HLA-restricted, gluten-specific T cells persisting in the blood and gut even after decades of GFD, which are reactivated and disease driving upon gluten exposure. Our aim was to examine the transition of activated gluten-specific T cells into a pool of persisting memory T cells concurrent with normalization of clinically relevant biomarkers during the first year of treatment. METHODS: We followed 17 CeD patients during their initial GFD year, leading to disease remission. We assessed activation and frequency of gluten-specific CD4+ blood and gut T cells with HLA-DQ2.5:gluten tetramers and flow cytometry, disease-specific serology, histology, and symptom scores. We assessed gluten-specific blood T cells within the first 3 weeks of GFD in 6 patients and serology in an additional 9 patients. RESULTS: Gluten-specific CD4+ T cells peaked in blood at day 14 while up-regulating Bcl-2 and down-regulating Ki-67 and then decreased in frequency within 10 weeks of GFD. CD38, ICOS, HLA-DR, and Ki-67 decreased in gluten-specific cells within 3 days. PD-1, CD39, and OX40 expression persisted even after 12 months. IgA-transglutaminase 2 decreased significantly within 4 weeks. CONCLUSIONS: GFD induces rapid changes in the phenotype and number of gluten-specific CD4+ blood T cells, including a peak of nonproliferating, nonapoptotic cells at day 14. Subsequent alterations in T-cell phenotype associate with the quiescent but chronic nature of treated CeD. The rapid changes affecting gluten-specific T cells and disease-specific antibodies offer opportunities for clinical trials aiming at developing nondietary treatments for patients with newly diagnosed CeD.

Altered Genome-Wide DNA Methylation in the Duodenum of Common Variable Immunodeficiency Patients.

PURPOSE: A large proportion of Common variable immunodeficiency (CVID) patients has duodenal inflammation with increased intraepithelial lymphocytes (IEL) of unknown aetiology. The histologic similarities to celiac disease, lead to confusion regarding treatment (gluten-free diet) of these patients. We aimed to elucidate the role of epigenetic DNA methylation in the aetiology of duodenal inflammation in CVID and differentiate it from true celiac disease. METHODS: DNA was isolated from snap-frozen pieces of duodenal biopsies and analysed for differences in genome-wide epigenetic DNA methylation between CVID patients with increased IEL (CVID_IEL; n = 5) without IEL (CVID_N; n = 3), celiac disease (n = 3) and healthy controls (n = 3). RESULTS: The DNA methylation data of 5-methylcytosine in CpG sites separated CVID and celiac diseases from healthy controls. Differential methylation in promoters of genes were identified as potential novel mediators in CVID and celiac disease. There was limited overlap of methylation associated genes between CVID_IEL and Celiac disease. High frequency of differentially methylated CpG sites was detected in over 100 genes nearby transcription start site (TSS) in both CVID_IEL and celiac disease, compared to healthy controls. Differential methylation of genes involved in regulation of TNF/cytokine production were enriched in CVID_IEL, compared to healthy controls. CONCLUSION: This is the first study to reveal a role of epigenetic DNA methylation in the etiology of duodenal inflammation of CVID patients, distinguishing CVID_IEL from celiac disease. We identified potential biomarkers and therapeutic targets within gene promotors and in high-frequency differentially methylated CpG regions proximal to TSS in both CVID_IEL and celiac disease.

Duodenal inflammation in common variable immunodeficiency has altered transcriptional response to viruses.

BACKGROUND: A substantial proportion of common variable immunodeficiency (CVID) patients has duodenal inflammation of largely unknown etiology. However, because of its histologic similarities with celiac disease, gluten sensitivity has been proposed as a potential mechanism. OBJECTIVE: We aimed to elucidate the role of the duodenal microenvironment in the pathogenesis of duodenal inflammation in CVID by investigating the transcriptional, proteomic, and microbial signatures of duodenal biopsy samples in CVID. METHODS: DNA, total RNA, and protein were isolated from snap-frozen pieces of duodenal biopsy samples from CVID (with and without duodenal inflammation), healthy controls, and patients with celiac disease (untreated). RNA sequencing, mass spectrometry-based proteomics, and 16S ribosomal DNA sequencing (bacteria) were then performed. RESULTS: CVID separated from controls in regulation of transcriptional response to lipopolysaccharide and cellular immune responses. These differences were independent of mucosal inflammation. Instead, CVID patients with duodenal inflammation displayed alterations in transcription of genes involved in response to viral infections. Four proteins were differently regulated between CVID patients and healthy controls-DBNL, TRMT11, GCHFR, and IGHA2-independent of duodenal inflammation. Despite similar histology, there were major differences in CVID with duodenal inflammation and celiac disease both at the RNA and protein level. No significant difference was observed in the bacterial gut microbial signature between CVID, celiac, and healthy controls. CONCLUSION: Our findings suggest the existence of altered functions of the duodenal epithelium, particularly in response to lipopolysaccharide and viruses. The latter finding was related to duodenal inflammation, suggesting that viruses, not gluten sensitivity, could be related to duodenal inflammation in CVID.

Navigation-Assisted Surgery for Locally Advanced Primary and Recurrent Rectal Cancer.

BACKGROUND: In some surgical disciplines, navigation-assisted surgery has become standard of care, but in rectal cancer, indications for navigation and the utility of different technologies remain undetermined. METHODS: The NAVI-LARRC prospective study (NCT04512937; IDEAL Stage 2a) evaluated feasibility of navigation in patients with locally advanced primary (LARC) and recurrent rectal cancer (LRRC). Included patients had advanced tumours with high risk of incomplete (R1/R2) resection, and navigation was considered likely to improve the probability of complete resection (R0). Tumours were classified according to pelvic compartmental involvement, as suggested by the Royal Marsden group. The BrainlabTM navigation platform was used for preoperative segmentation of tumour and pelvic anatomy, and for intraoperative navigation with optical tracking. R0 resection rates, surgeons' experiences, and adherence to the preoperative resection plan were assessed. RESULTS: Seventeen patients with tumours involving the posterior/lateral compartments underwent navigation-assisted procedures. Fifteen patients required abdominosacral resection, and 3 had resection of the sciatic nerve. R0 resection was obtained in 6/8 (75%) LARC and 6/9 (69%) LRRC cases. Preoperative segmentation was time-consuming (median 3.5 h), but intraoperative navigation was accurate. Surgeons reported navigation to be feasible, and adherence to the resection plan was satisfactory. CONCLUSIONS: Navigation-assisted surgery using optical tracking was feasible. The preoperative planning was time-consuming, but intraoperative navigation was accurate and resulted in acceptable R0 resection rates. Selected patients are likely to benefit from navigation-assisted surgery.

Evaluation of diagnostic factors used to refer children with constipation for rectal biopsies.

PURPOSE: Children with constipation and suspected Hirschsprung's disease are referred for rectal biopsy. Since this is an invasive procedure, appropriate indications should be applied to minimize the number of "unnecessary" biopsies. METHODS: We reviewed all constipated children who underwent a rectal biopsy to diagnose a possible Hirschsprung's disease at a tertiary referral hospital over a 6-year period (2013-2018). We registered clinical and demographic factors in these children and conducted correlation and multivariate regression analysis to evaluate the relation between these factors and a diagnosis of Hirschsprung's disease. RESULTS: We identified 225 children, aged 0-17 years. In total, Hirschsprung's disease was diagnosed in only 49/225 (22%). Among the 49 children with Hirschsprung's disease, 29 (59%) were diagnosed in the neonatal period. Among girls, HD was confirmed in only 10/101 (10%) children, and only 1 of these 10 girls was older than 6 months at the time of the biopsy. The following factors correlated significantly with Hirschsprung's disease diagnosis in children older than 1 month: "male sex", "failure to thrive", "gross abdominal distention plus vomiting" and "fulfils the Rome 4 criteria for functional constipation". CONCLUSION: In children referred for rectal biopsy, the factors most indicative of Hirschsprung's disease were "male sex", "failure to thrive", "gross abdominal distention plus vomiting" and "fulfils the Rome 4 criteria for functional constipation". Notably, the prevalence of Hirschsprung's disease decreased with the increasing age of the children. Girls referred for a biopsy rarely had Hirschsprung's disease, especially those older than 1 month.

Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry.

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.

Can we ablate liver lesions close to large portal and hepatic veins with MR-guided HIFU? An experimental study in a porcine model.

OBJECTIVES: Invasive treatment of tumors adjacent to large hepatic vessels is a continuous clinical challenge. The primary aim of this study was to examine the feasibility of ablating liver tissue adjacent to large hepatic and portal veins with magnetic resonance imaging-guided high-intensity focused ultrasound (MRgHIFU). The secondary aim was to compare sonication data for ablations performed adjacent to hepatic veins (HV) versus portal veins (PV). MATERIALS AND METHODS: MRgHIFU ablations were performed in six male land swine under general anesthesia. Ablation cells of either 4 or 8 mm diameter were planned in clusters (two/animal) adjacent either to HV (n = 6) or to PV (n = 6), with diameter ≥ 5 mm. Ablations were made using 200 W and 1.2 MHz. Post-procedure evaluation was made on contrast-enhanced MRI (T1w CE-MRI), histopathology, and ablation data from the HIFU system. RESULTS: A total of 153 ablations in 81 cells and 12 clusters were performed. There were visible lesions with non-perfused volumes in all animals on T1w CE-MRI images. Histopathology showed hemorrhage and necrosis in all 12 clusters, with a median shortest distance to vessel wall of 0.4 mm (range 0-2.7 mm). Edema and endothelial swelling were observed without vessel wall rupture. In 8-mm ablations (n = 125), heat sink was detected more often for HV (43%) than for PV (19%; p = 0.04). CONCLUSIONS: Ablations yielding coagulative necrosis of liver tissue can be performed adjacent to large hepatic vessels while keeping the vessel walls intact. This indicates that perivascular tumor ablation in the liver is feasible using MRgHIFU. KEY POINTS: • High-intensity focused ultrasound ablation is a non-invasive treatment modality that can be used for treatment of liver tumors. • This study shows that ablations of liver tissue can be performed adjacent to large hepatic vessels in an experimental setting. • Liver tumors close to large vessels can potentially be treated using this modality.

HLA-DQ:gluten tetramer test in blood gives better detection of coeliac patients than biopsy after 14-day gluten challenge.

OBJECTIVE: Initiation of a gluten-free diet without proper diagnostic work-up of coeliac disease is a frequent and demanding problem. Recent diagnostic guidelines suggest a gluten challenge of at least 14 days followed by duodenal biopsy in such patients. The rate of false-negative outcome of this approach remains unclear. We studied responses to 14-day gluten challenge in subjects with treated coeliac disease. DESIGN: We challenged 20 subjects with biopsy-verified coeliac disease, all in confirmed mucosal remission, for 14 days with 5.7 grams per oral gluten daily. Duodenal biopsies were collected. Blood was analysed by multiplex assay for cytokine detection, and by flow cytometry using HLA-DQ:gluten tetramers. RESULTS: Nineteen participants completed the challenge. Villous blunting appeared at end of challenge in 5 of 19 subjects. Villous height to crypt depth ratio reduced with at least 0.4 concomitantly with an increase in intraepithelial lymphocyte count of at least 50% in 9 of 19 subjects. Interleukin-8 plasma concentration increased by more than 100% after 4 hours in 7 of 19 subjects. Frequency of blood CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells increased by more than 100% on day 6 in 12 of 15 evaluated participants. CONCLUSION: A 14-day gluten challenge was not enough to establish significant mucosal architectural changes in majority of patients with coeliac disease (sensitivity ≈25%-50%). Increase in CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells in blood 6 days after gluten challenge is a more sensitive and less invasive biomarker that should be validated in a larger study. TRIAL REGISTRATION NUMBER: NCT02464150.

Establishment of a surgical bile duct injection technique giving direct access to the bile ducts for studies of the murine biliary tree.

Cholangiopathies are progressive disorders with largely unknown pathoetiology and limited treatment options. We aimed to develop a novel surgical technique with direct access to the bile ducts that would complement existing mouse models of cholestasis, biliary inflammation, and fibrosis and present a new route of administration for testing of potential treatment strategies. We developed a surgical technique to access the murine biliary tree by injection of different solvents through catheterization of the gall bladder with simultaneous clamping of the common bile duct. To demonstrate the applicability of the technique, we injected either phosphate-buffered saline or dimethyl sulfoxide in concentrations of 50 or 65% and compared these groups with sham-operated mice. The surgery was optimized to achieve a mortality rate close to 0. There were no significant changes in pain, activity level, or mortality from the day of the surgery until euthanization for any groups. Injection of phosphate-buffered saline or 50% dimethyl sulfoxide was generally well-tolerated, whereas 65% dimethyl sulfoxide led to higher weight loss, an increase of serum alanine transaminase, and histological portal inflammation. There were no signs of inflammation in the gut. We have developed a bile duct injection technique that is well-tolerated, easily reproducible, and that may complement existing models of cholangiopathies. Direct access to the bile ducts without causing harm to the hepatobiliary or intestinal tissue may be valuable in future studies of normal biliary physiology and different pathophysiological mechanisms of disease and to test novel therapeutic strategies. NEW & NOTEWORTHY To evaluate tolerability of the bile duct to injection of both polar and nonpolar compounds, we established a novel biliary injection technique. This technique is well-tolerated, easily reproducible, and with direct access to the bile ducts for studies of the murine biliary tree. The bile duct injection technique may complement existing animal models and be a valuable tool in future studies of normal biliary physiology or pathophysiology and to test novel therapeutic strategies.

The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation.

BACKGROUND & AIMS: A strong association between human inflammatory biliary diseases and gut inflammation has led to the hypothesis that gut microbes and lymphocytes activated in the intestine play a role in biliary inflammation. The NOD.c3c4 mouse model develops spontaneous biliary inflammation in extra- and intrahepatic bile ducts. We aimed to clarify the role of the gut microbiota in the biliary disease of NOD.c3c4 mice. METHODS: We sampled cecal content and mucosa from conventionally raised (CONV-R) NOD.c3c4 and NOD control mice, extracted DNA and performed 16S rRNA sequencing. NOD.c3c4 mice were rederived into a germ free (GF) facility and compared with CONV-R NOD.c3c4 mice. NOD.c3c4 mice were also co-housed with NOD mice and received antibiotics from weaning. RESULTS: The gut microbial profiles of mice with and without biliary disease were different both before and after rederivation (unweighted UniFrac-distance). GF NOD.c3c4 mice had less distended extra-hepatic bile ducts than CONV-R NOD.c3c4 mice, while antibiotic treated mice showed reduction of biliary infarcts. GF animals also showed a reduction in liver weight compared with CONV-R NOD.c3c4 mice, and this was also observed in antibiotic treated NOD.c3c4 mice. Co-housing of NOD and NOD.c3c4 mice indicated that the biliary phenotype was neither transmissible nor treatable by co-housing with healthy mice. CONCLUSIONS: NOD.c3c4 and NOD control mice show marked differences in the gut microbiota. GF NOD.c3c4 mice develop a milder biliary affection compared with conventionally raised NOD.c3c4 mice. Our findings suggest that the intestinal microbiota contributes to disease in this murine model of biliary inflammation. LAY SUMMARY: Mice with liver disease have a gut microflora (microbiota) that differs substantially from normal mice. In a normal environment, these mice spontaneously develop disease in their bile ducts. However, when these mice, are raised in an environment devoid of bacteria, the disease in the bile ducts diminishes. Overall this clearly indicates that the bacteria in the gut (the gut microbiota) influences the liver disease in these mice.

A Cross-Sectional Study of the Prevalence of Gastrointestinal Symptoms and Pathology in Patients With Common Variable Immunodeficiency.

OBJECTIVES: The objective of this study was to study the prevalence of gastrointestinal (GI) symptoms and histopathology in patients with common variable immunodeficiency (CVID) as well as linking the findings to GI infections and markers of systemic immune activation. METHODS: In this cross-sectional study, we addressed GI symptoms in 103 patients and GI histopathological findings in 53 patients who underwent upper and lower endoscopic examination. The most frequent histopathological findings were linked to GI symptoms, B-cell phenotype, and markers of systemic immune activation (soluble (s)CD14, sCD25, and sCD163). Microarray analysis compared "celiac-like disease" in CVID to celiac disease. Screening for selected bacterial and viral infections in fecal samples and gut mucosal biopsies was performed. RESULTS: The main findings of this study were as follows: most common GI symptoms were bloating (34%), pain (30%), and diarrhea (26%). The most frequent histopathological findings were increased intraepithelial lymphocytes in the descending part of the duodenum, i.e., "celiac-like disease" (46% of patients), decreased numbers of plasma cells in GI tract mucosa (62%), and lymphoid hyperplasia (38%), none of which were associated with GI symptoms. Reduced plasma cells in GI mucosa were associated with B-cell phenotypic characteristics of CVID, and increased serum levels of sCD14 (P=0.025), sCD25 (P=0.01), and sCD163 (P=0.04). Microarray analyses distinguished between CVID patients with "celiac-like disease" and celiac disease. Positive tests for bacterial and viral infections were scarce both in fecal samples and gut mucosal biopsies, including PCR test for norovirus in biopsy specimens (0 positive tests). CONCLUSIONS: In conclusion, GI pathology is common in CVID, but does not necessarily cause symptoms. However, reduced plasma cells in GI mucosa were linked to systemic immune activation, "celiac-like disease" in CVID and true celiac disease appear to be different disease entities, as assessed by gene expression, and infections (including norovirus) are rarely a cause of the CVID enteropathy.

High screening blood pressure at young age predicts future masked hypertension: A 17 year follow-up study.

OBJECTIVE: Approximately 10-20% of the general population have masked hypertension. However, how best to identify affected individuals is uncertain, and what predicts future masked hypertension is largely unknown. This study aimed to identify longitudinal predictors of masked hypertension. METHODS: A long-term follow-up study of 100 healthy young men who had normal (n = 28) or high (n = 72) screening blood pressure (BP) at the compulsory military draft was carried out. They were examined in a detailed and highly standardized way for cardiovascular risk markers at baseline and at follow-up after a mean of 17.4 years. RESULTS: At follow-up, 40% had masked hypertension. Participants with high screening BP had a 4.8 times higher likelihood of having masked hypertension at follow-up compared to men with low screening BP (odds ratio 4.8, 95% confidence interval 1.7-13.5, p = 0.003). Furthermore, only 25% of the men with masked hypertension had high normal office BP at follow-up, and the remaining 75% would, according to guidelines, not be recommended ambulatory BP measurements, and thus go undiagnosed. CONCLUSION: Our data suggest that high screening BP at a young age is an important predictor of future masked hypertension in young men, and that BP measurement according to guidelines is insufficient to uncover masked hypertension.

Multiregional transcriptomics identifies congruent consensus subtypes with prognostic value beyond tumor heterogeneity of colorectal cancer.

Intra-tumor heterogeneity compromises the clinical value of transcriptomic classifications of colorectal cancer. We investigated the prognostic effect of transcriptomic heterogeneity and the potential for classifications less vulnerable to heterogeneity in a single-hospital series of 1093 tumor samples from 692 patients, including multiregional samples from 98 primary tumors and 35 primary-metastasis sets. We show that intra-tumor heterogeneity of the consensus molecular subtypes (CMS) is frequent and has poor-prognostic associations independently of tumor microenvironment markers. Multiregional transcriptomics uncover cancer cell-intrinsic and low-heterogeneity signals that recapitulate the intrinsic CMSs proposed by single-cell sequencing. Further subclassification identifies congruent CMSs that explain a larger proportion of variation in patient survival than intra-tumor heterogeneity. Plasticity is indicated by discordant intrinsic phenotypes of matched primary and metastatic tumors. We conclude that multiregional sampling reconciles the prognostic power of tumor classifications from single-cell and bulk transcriptomics in the context of intra-tumor heterogeneity, and phenotypic plasticity challenges the reconciliation of primary and metastatic subtypes.

Allogeneic bone marrow transplantation for patients with treatment-refractory Crohn's Disease.

Background & aims: Durable remissions of Crohn's Disease (CD) have followed myeloablative conditioning therapy and allogeneic marrow transplantation. For patients with treatment-refractory disease, we used reduced-intensity conditioning to minimize toxicity, marrow from donors with low Polygenic Risk Scores for CD as cell sources, and protracted immune suppression to lower the risk of graft-versus-host disease (GVHD). Our aim was to achieve durable CD remissions while minimizing transplant-related complications. Methods: DNA from patients and their HLA-matched unrelated donors was genotyped and Polygenic Risk Scores calculated. Donor marrow was infused following non-myeloablative conditioning. Patient symptoms and endoscopic findings were documented at intervals after transplant. Results: We screened 807 patients, 143 of whom met eligibility criteria; 2 patients received allografts. Patient 1 had multiple complications and died at day 332 from respiratory failure. Patient 2 had resolution of CD symptoms until day 178 when CD recurred, associated with persistent host chimerism in both peripheral blood and intestinal mucosa. Withdrawal of immune suppression was followed by dominant donor immune chimerism in peripheral blood and resolution of CD findings. Over time, mucosal T-cells became donor-dominant. At 5 years after allografting, Patient 2 remained off all medications but had mild symptoms related to a jejunal stricture that required stricturoplasty at 6 years. At 8 years, she remains stable off medications. Conclusions: The kinetics of immunologic chimerism after allogeneic marrow transplantation for CD patients depends on the intensity of the conditioning regimen and the magnitude of immune suppression. One patient achieved durable improvement of her previously refractory CD only after establishing donor immunologic chimerism in intestinal mucosa. Her course provides proof-of-principal for allografting as a potential treatment for refractory CD, but an immunoablative conditioning regimen should be considered for future studies.(ClinicalTrials.gov, NCT01570348).

Impact of alcohol habits and smoking on the risk of new-onset atrial fibrillation in hypertensive patients with ECG left ventricular hypertrophy: the LIFE study.

BACKGROUND: The incidence of new-onset atrial fibrillation (AF) is increased by uncontrolled hypertension, and antihypertensive treatment reduces new-onset AF. However, it is unclear whether alcohol intake and smoking influence the risk of new-onset AF during antihypertensive treatment. METHODS: In the Losartan Intervention For Endpoint reduction in Hypertension (LIFE) study, a double-blinded, randomized, parallel-group study, 9193 hypertensive patients with electrocardiogram (ECG)-documented left ventricular hypertrophy (LVH), randomized to once-daily losartan- or atenolol-based antihypertensive therapy were followed for a mean of 4.8 years. At baseline, 8831 patients (54% women, mean age 67 years, mean blood pressure 174/98 mmHg after placebo run-in) had neither a history of AF nor AF on ECG, and they were thus at risk of developing this condition during the study. RESULTS: New-onset AF occurred in 353 (4%) patients. Univariate Cox analyses showed that intake of alcohol > 10 units/week compared with less or no alcohol intake predicted new-onset AF (Hazard ratio, HR = 1.60 [95% CI 1.02-2.51], p = 0.043). Multivariate Cox regression analysis showed that intake of alcohol > 10 units/week predicted new-onset AF (p = 0.010) independently of most other univariate predictors, except when also baseline serum cholesterol, serum potassium and urinary albumin/creatinine ratio were included in the model (HR = 1.60 [95% CI 0.94-2.72], p = 0.081). Impact of smoking was not significant in Cox univariate or multivariate analyses, and there were no significant interactions between high alcohol intake and either smoking or gender on the risk of getting AF. CONCLUSIONS: Up to 10 drinks of alcohol per week appears to be safe with respect to the risk for AF in hypertensive patients with LVH. Our data suggest that alcohol intake above this level may be marginally deleterious, while no effect of smoking on risk of AF was detected in hypertensive patients with LVH.