Toll-like receptor 4 and NOD2/CARD15 mutations in Hungarian patients with Crohn's disease: Phenotype-genotype correlations

AIM: To determine common NOD2/CARD15 mutations and TLR4 D299G polymorphism in Hungarian patients with CD. METHODS: A total of 527 unrelated patients with CD (male/female: 265/262, age: 37.1 (SD 7.6) years) and 200 healthy subjects were included. DNA was screened for possible NOD2/CARD15 mutations by denaturing high-performance liquid chromatography (confirmed by direct sequencing). TLR4 D299G was tested by PCR-RFLP. RESULTS: NOD2/CARD15 mutations were found in 185 patients (35.1%) and in 33 controls (16.5%, P< 0.0001). SNP8/R702W (10.8% vs 6%, P = 0.02), SNP13/3020insC (19.4% vs 5%, P< 0.0001) and exon4 R703C (2.1% vs 0%, P = 0.02) mutations were more frequent in CD, while the frequency of SNP12/G908R was not increased. The frequency of TLR4 D299G was not different (CD: 9.9% vs controls: 12.0%). Variant NOD2/CARD15 allele was associated with an increased risk for CD (OR(het) = 1.71, 95% CI = 1.12-2.6, P = 0.0001, OR(two-risk alleles) = 25.2, 95% CI = 4.37-, P< 0.0001), early disease onset (carrier: 26.4 years vs non-carrier: 29.8 years, P = 0.0006), ileal disease (81.9% vs 69.5%, OR = 1.99, 95% CI = 1.29-3.08, P = 0.02, presence of NOD2/CARD15 and TLR4: 86.7% vs 64.8%), stricturing behavior (OR = 1.69, 95% CI = 1.13-2.55, P = 0.026) and increased need for resection (OR= 1.71, 95% CI: 1.13-2.62, P = 0.01), but not with duration, extraintestinal manifestations, familial disease or smoking. TLR4 exhibited a modifier effect: age of onset in wt/TLR4 D299G carriers: 27.4 years vs NOD2mut/TLR D299G: 23 years (P = 0.06), in NOD2mut/wt: 26.7 years. CONCLUSION: These results confirm that variant NOD2/CARD15 (R702W, R703C and 3020insC) alleles are associated with earlier disease onset, ileal disease, stricturing disease behavior in Hungarian CD patients. In contrast, although the frequency of TLR4 D299G polymorphism was not different from controls, NOD2/TLR4 mutation carriers tended to present at earlier age. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved

Origin of myofibroblasts in liver fibrosis

Most chronic liver diseases of all etiologies result in progressive liver fibrosis. Myofibroblasts produce the extracellular matrix, including type I collagen, which constitutes the fibrous scar in liver fibrosis. Normal liver has little type I collagen and no detectable myofibroblasts, but myofibroblasts appear early in experimental and clinical liver injury. The origin of the myofibroblast in liver fibrosis is still unresolved. The possibilities include activation of endogenous mesenchymal cells including fibroblasts and hepatic stellate cells, recruitment from the bone marrow, and transformation of epithelial or endothelial cells to myofibroblasts. In fact, the origin of myofibroblasts may be different for different types of chronic liver diseases, such as cholestatic liver disease or hepatotoxic liver disease. This review will examine our current understanding of the liver myofibroblast

Repeated determination of moxifloxacin concentrations in interstitial space fluid of muscle and subcutis in septic patients

Background: For an effective antimicrobial treatment, it is crucial that antibiotics reach sufficient concentrations in plasma and tissue. Currently no data exist regarding moxifloxacin plasma concentrations and exposure levels in tissue under septic conditions. Objectives: To determine the pharmacokinetics of moxifloxacin in plasma and interstitial space fluid over a prolonged period. Patients and methods: Ten septic patients were treated with 400 mg of moxifloxacin once a day; on days 1, 3 and 5 of treatment plasma sampling and microdialysis in the subcutis and muscle of the upper thigh were performed to determine concentrations of moxifloxacin in different compartments. This trial was registered in the German Clinical Trials Register (DRKS, register number DRKS00012985). Results: Mean unbound fraction of moxifloxacin in plasma was 85.5 +/- 3.4%. On day 1, C-max in subcutis and muscle was 2.8 +/- 1.8 and 2.5 +/- 1.3 mg/L, respectively, AUC was 24.8 +/- 15.1 and 21.3 +/- 10.5 mgh/L, respectively, and fAUC(0-24)/MIC was 100.9 +/- 62.9 and 86.5 +/- 38.3 h, respectively. C-max for unbound moxifloxacin in plasma was 3.5 +/- 0.9 mg/L, AUC was 23.5 +/- 7.5 mg.h/L and fAUC(0-24)/MIC was 91.6 +/- 24.8 h. Key pharmacokinetic parameters on days 3 and 5 showed no significant differences. Clearance was higher than in healthy adults, but tissue concentrations were comparable, most likely due to a lower protein binding. Conclusions: Surprisingly, the first dose already achieved exposure comparable to steady-state conditions. The approved daily dose of 400 mg was adequate in our patient population. Thus, it seems that in septic patients a loading dose on the first day of treatment with moxifloxacin is not required

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients

Abstract Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19