Johann Lorenzen

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats

BACKGROUND & AIMS Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.

Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C

Abstract: 
Background: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients.

The presence of non-organ-specific autoantibodies is associated with a negative response to combination therapy with interferon and ribavirin for chronic hepatitis C

BackgroundNon-organ-specific autoantibodies are found in a considerable number of anti-HCV positive patients. Previous studies investigated the clinical relevance of these antibodies in patients treated with interferon monotherapy, but not combination therapies.MethodsAnti-nuclear, anti-smooth muscle, anti-mitochondrial, anti-neutrophil-cytoplasmatic and anti-liver/kidney microsomal antibodies were determined in 78 consecutive anti-HCV positive patients by indirect immunofluorescence. The presence of these antibodies was related to demographic variables and to the outcome of antiviral combination therapy with interferon-α and ribavirin in 65 patients.ResultsIn our study, positivity for autoantibodies was associated with higher alanine aminotransferase levels and higher mean values for HCV-RNA (p < 0.01). Furthermore, negativity for non-organ-specific autoantibodies was associated with a favourable treatment outcome of combination therapy with at least one negative RT-PCR for HCV-RNA during treatment (OR 4.65, 95% CI 1.31 to 16.48, p = 0.02). ANA and SMA staining patterns and titers were not correlated to treatment response. With multiple logistic regression analysis, positivity for autoantibodies and HCV genotype were independently associated with outcome of antiviral combination therapy (p = 0.02).ConclusionsThe absence of non-organ-specific autoantibodies might indicate a significantly higher chance for viral clearance in response to combination therapy for chronic hepatitis C infection. Therefore, despite of an overall higher treatment response, the addition of the immunomodulatory drug ribavirin could accentuate immunological differences that affect treatment outcome and might have been less obvious in earlier studies analysing interferon monotherapy.

CC chemokine receptor 5 Δ32 polymorphism in two independent cohorts of hepatitis C virus infected patients without hemophilia

Recently CC chemokine receptor 5 (CCR5) related immune mechanisms and a functional mutation of the CCR5 gene have been implicated in hepatitis C virus (HCV) infection in a cohort of predominantly hemophiliac patients. The present study investigated the frequency and clinical consequences of the CCR5 Δ32 mutation in two genetically homogeneous populations of HCV infected patients with a different risk profile for infection. Genomic DNA samples from 333 German patients with chronic HCV infection were screened by PCR for the presence of the CCR5 Δ32 polymorphism. In-hospital patients admitted for other diseases than viral hepatitis but with a comparable risk for HCV exposure were used as control population (n=125). Allele frequencies of CCR5 Δ32 polymorphism did not differ significantly between the two groups (7.6% and 9.5%, respectively) and control subjects (10.4%), and did not deviate from Hardy-Weinberg equilibrium in any group. Furthermore, there were no major differences between patients with respect to HCV genotypes, viral loads, liver enzymes, or fibrosis scores in relation to the presence or absence of the heterozygous CCR5 Δ32 mutation. Differences in inflammatory scores in liver biopsy samples and response to antiviral therapy in CCR5 Δ32 heterozygotes in one cohort could neither be reproduced in the other group of patients nor when both cohorts were pooled. These results argue against a strong effect of the CCR5 Δ32 deletion regarding these phenotypes. In conclusion, we found no increased frequency of the CCR5 Δ32 polymorphism in two independent cohorts of patients with HCV infection but without hemophilia as the main risk factor for infection. As the major difference to investigations demonstrating an association between CCR5 Δ32 and HCV infection is the selection of cases and controls, our study emphasizes the importance of epidemiological criteria for association studies of HCV infection.

Haplotype‐tagging RANTES gene variants influence response to antiviral therapy in chronic hepatitis C

The response to antiviral therapy for chronic hepatitis C virus (HCV) is complex and is determined by both environmental and genetic factors. Recently, interacting gene polymorphisms of the chemokine RANTES have been shown to affect HIV disease progression. Our aim was to assess if these RANTES variants are associated with response to anti‐HCV therapy. Three linked RANTES single nucleotide polymorphisms (403 G/A, Int1.1 T/C, and 3′ 222 T/C) were determined in 297 Caucasian patients who were treated for chronic HCV infection and 152 control subjects. Characteristic nucleotide combinations on single chromosomes (haplotypes) were reconstructed and tested for disease association. Four common RANTES haplotypes (prevalence 73%) were identified in patients and controls. There was a strong association of RANTES haplotype distribution with outcome of antiviral combination therapy (P = .007). Specifically, RANTES haplotypes carrying Int1.1 C and 3′ 222 C alleles were more frequent in nonresponders than in patients with a sustained response to antiviral therapy (odds ratio 1.9, P = .01). The influence of these RANTES haplotypes on the outcome of therapy was more pronounced in patients infected with HCV genotypes 1 and 4 (odds ratio 2.3, P = .02). Because RANTES haplotypes carrying Int1.1 C are known to down‐regulate RANTES transcriptional activity in vitro, the haplotype analysis fits the hypothesis of a diminished T helper 1 lymphocyte response in patients with a negative response to antiviral therapy. In conclusion, RANTES haplotypes might contribute to the polygenic interaction between HCV and the host immune system and could help to risk stratify patients prior to antiviral therapy. (HEPATOLOGY 2004;40:327–334.)

Association of echocardiographic atrial size and atrial fibrosis in a sequential model of congestive heart failure and atrial fibrillation

BACKGROUND Cardioversion (CV) success of atrial fibrillation (AF) inversely correlates to the size of the left atrium (LA). Atrial fibrillation and its most important risk factor, congestive heart failure (CHF), both induce atrial structural enlargement and fibrosis. To investigate the effect of AF and CHF on atrial dilatation and fibrosis, and to estimate whether echocardiographically determined atrial size may be used as a marker for atrial fibrosis. METHODS In six dogs, pacemakers were implanted followed by HIS bundle ablation. After 4 weeks of rapid ventricular stimulation (185 bpm) for CHF induction, additional rapid atrial stimulation (500 bpm) was maintained for 7 weeks to induce AF. Serial determinations of echocardiographic atrial size were performed. Seven dogs with sinus rhythm served as histological controls. Postmortem tissue was obtained to determine the degree and composition of atrial fibrosis. RESULTS While the ejection fraction of the AF/CHF dogs decreased significantly from 57+/-5% to 19+/-7% (P<.01), an increased degree of atrial fibrosis was found (right atrium [RA], 4.9+/-2.0% to 19.9+/-5.4%; LA, 4.4+/-1.6% to 22.2+/-3.2%; P<.01), accompanied by a significant increase of atrial volumes (LA: 21+/-4 to 44+/-4 mm3; P<.01; RA: 10+/-3 to 18+/-6 mm3; P<.05) and LA diameters (34+/-4 to 43+/-2 mm, P<.05). Atrial fibrosis and size significantly correlated. CONCLUSIONS Atrial fibrillation/CHF leads to a significant atrial fibrosis and dilation. The increased echocardiographic size correlates to the degree of atrial fibrosis and may be used as clinical marker for atrial fibrosis. The fibrosis accompanying atrial dilatation may also explain why LA size, as determined by echocardiography, is a strong predictor of CV success.

Atrial fibrillation is associated with cardiac hypoxia.

BACKGROUND Atrial fibrillation (AF), the most common human arrhythmia, is responsible for substantial morbidity and mortality and may be promoted by selective atrial ischemia and atrial fibrosis. Consequently, we investigated markers for hypoxia and angiogenesis in AF. METHODS Right atrial appendages (n=158) were grouped according to heart rhythm [sinus rhythm (SR) or AF]. The degree of fibrosis and microvessel density of all patients were determined morphometrically using Sirius-Red- and CD34/CD105-stained sections, respectively. Next, sections (n=77) underwent immunostaining to detect hypoxia- and angiogenesis-related proteins [hypoxia-inducible factor (HIF)1 alpha, HIF2 alpha, vascular endothelial growth factor (VEGF), VEGF receptor 2 (KDR), phosphorylated KDR (pKDR), carboanhydrase IX, platelet-derived growth factor] and the apoptosis-related B-cell lymphoma 2 protein. RESULTS Fibrosis progressed significantly from 14.7+/-0.8% (SR) to 22.3+/-1.4% (AF). While the positive cytoplasmic staining of HIF1 alpha, HIF2 alpha, VEGF, KDR, and pKDR rose significantly from SR to AF, their nuclear fractions fell (only pKDR significantly). The median CD34/CD105-positive microvessel size increased significantly from SR to AF. CONCLUSIONS AF is closely associated with an atrial up-regulation of hypoxic and angiogenic markers. Whether this is cause, effect, or co-phenomenon of fibrosis remains to be investigated. It is conceivable that fibrosis might lead to an increased O(2) diffusion distance and thus induce ischemic signaling, which, in turn, leads to angiogenesis.

Expression of interleukin-5, interleukin-8, and interleukin-10 mRNA in the osteomeatal complex in nasal polyposis.

Background Several cytokines are expressed in chronic sinusitis with and without underlying allergy. Their local production and regulation in the osteomeatal complex, the key area of paranasal sinuses, still is not fully understood. This study was performed to investigate differences of cytokine messenger RNA (mRNA) expression between the medial and the lateral part of the middle turbinate and anterior ethmoid mucosa of allergic and nonallergic patients. Methods Using the LightCycler system for real-time reverse-transcription polymerase chain reaction, we investigated the content of interleukin (IL)-5, IL-8, and IL-10 mRNA in tissue samples from middle turbinates and anterior ethmoids of 18 patients with chronic sinusitis and nasal polyps. Inferior turbinate mucosa of six control subjects without sinusitis and allergy served as control. Results IL-5 mRNA was detectable in 32 (60%) of 54 samples (two of six controls) in significant different amounts between the various locations (p ≤ 0.001). Anterior ethmoid mucosa (0.96 ± 0.99) expressed the highest amount of IL-5 mRNA followed by the lateral (0.37 ± 0.54) and the medial portion of the middle turbinate (0.12 ± 0.29) with no difference between allergic and nonallergic subgroups. IL-8 was detected in significant higher amounts in all three origins with no significant difference in concentrations between the examined locations as compared with controls. Patients expressed either IL-5 or IL-8 or both cytokine mRNA. IL-10 was expressed in all three specimens from five of eight allergic patients. All five individuals with clinical symptoms of allergy at the time of operation expressed IL-10 in at least one specimen. Conclusions IL-5 cytokine expression in the osteomeatal complex is linked to the presence of nasal polyps, whereas IL-8 is up-regulated without distinct correlation to nasal polyps. IL-10 expression was detectable in five of eight allergic patients.

Morphological effects of nanosecond- and femtosecond-pulsed laser ablation on human middle ear ossicles

We evaluate the feasibility of nanosecond-pulsed and femtosecond-pulsed lasers for otologic surgery. The outcome parameters are cutting precision (in micrometers), ablation rate (in micrometers per second), scanning speed (in millimeters per second), and morphological effects on human middle ear ossicles. We examine single-spot ablations by a nanosecond-pulsed, frequency-tripled Nd:YAG laser (355 nm, beam diameter 10 microm, pulse rate 2 kHz, power 250 mW) on isolated human mallei. A similar system (355 nm, beam diameter 20 microm, pulse rate 10 kHz, power 160-1500 mW) and a femtosecond-pulsed CrLi:SAF-Laser (850 nm, pulse duration 100 fs, pulse energy 40 microJ, beam diameter 36 microm, pulse rate 1 kHz) are coupled to a scanner to perform bone surface ablation over a defined area. In our setups 1 and 2, marginal carbonization is visible in all single-spot ablations of 1-s exposures and longer: With an exposure time of 0.5 s, precise cutting margins without carbonization are observed. Cooling with saline solution result is in no carbonization at 1500 mW and a scan speed of 500 mms. Our third setup shows no carbonization but greater cutting precision, although the ablation volume is lower. Nanosecond- and femtosecond-pulsed laser systems bear the potential to increase cutting precision in otologic surgery.