Medizin - Open Access LMU - Teil 13/22 Archiv

Background. Tumor necrosis factor-alpha (TNF-alpha) is involved in the pathogenesis of chronic active hepatitis C. Polymorphisms in the promoter region of the TNF-alpha gene can alter the TNF-alpha expression and modify the host immune response. The present study aimed at the correlation of the G308A TNF-alpha polymorphism with the response to antiviral combination therapy in chronic hepatitis C. Patients and Methods: 62 patients with HCV and 119 healthy unrelated controls were genotyped for the G308A TNF-alpha promoter polymorphism. The patients received 3 x 3 million units of interferon alfa-2a and 1,0001,200 mg ribavirin daily according to their body weight. A response was defined as absence of HCV-RNA and normalization of S-ALT after 6 months of combination therapy. Results:With respect to the allele and genotype frequency, a significant difference was not observed between controls and patients with chronic hepatitis C. Furthermore, such a difference was also not observed if responders and non-responders to antiviral therapy were compared. Conclusions: The promoter polymorphism of the TNF-alpha gene investigated herein is equally distributed in healthy individuals and patients with hepatitis C and does not seem to predict the response to therapy with interferon alfa-2a and ribavirin. Copyright (C) 2003 S. Karger AG, Basel.

The recent identification of the CARD15/NOD2 gene as a susceptibility locus for Crohn's disease represents an important step towards the delineation of the immuno-pathogenesis of inflammatory bowel disease. CARD15 functions as an intracellular receptor for bacterial components and thus represents an important link between inflammatory bowel disease and innate immunity. Three major CARD15/NOD2 gene mutations have been associated with Crohn's disease in Caucasians in several independent studies. Together, they explain about 20% of the genetic susceptibility for Crohn's disease. Genotype-phenotype analyses demonstrated an association of these mutations with ileum-specific disease, an increased incidence of the fibrostenotic phenotype and an earlier age of disease onset. Beside these associations, no other relationship between the CARD15/NOD2 genotype and disease behavior or response to treatment has been detailed so far. Thus, the clinical impact of knowing the patient's genotype is limited at this time. Screening for CARD15 mutations in order to identify high-risk individuals or to introduce an individualized disease management is therefore currently not recommended. Copyright (C) 2003 S. Karger AG, Basel.

This retrospective study aims to determine whether there is a difference in the additional consumption of alcohol between addicts treated with methadone or dihydrocodeine (DHC) and untreated addicts injecting heroin. 1,685 patients admitted for opioid withdrawal between 1991 and 1997 were reviewed. Cross-reference tables and multiple logistic regression analyses were carried out. 28% of patients take more than 40 g of alcohol daily (on average 176 g). We found that patients who are treated with methadone or DHC drink alcohol significantly more often daily than the heroin-dependent patients (p

Substance use, especially alcoholism, has been recognized as a significant problem in schizophrenic patients, though only a few studies on the effects of pharmacotherapy in these patients have been conducted so far. The thioxanthene neuroleptic flupenthixol, which can be given intramuscularly (i.m.) for improving compliance, has been studied as a possible anti-craving drug both in animal models of alcoholism and some clinical studies. Pilot studies suggest that comorbid schizophrenics with substance use may benefit from treatment with flupenthixol. Efficacy of flupenthixol (10-60 mg i.m.) in reducing alcohol consumption of dual diagnosis patients was studied in an open 6-month clinical trial in 27 schizophrenics with comorbid alcoholism. Twenty-one patients entered the intention-to-treat analysis. Fourteen subjects were completers, 13 dropped out. Six patients completely abstained from alcohol during treatment. Alcohol consumption was significantly reduced compared to baseline (4 weeks before treatment as measured by timeline follow-back interview). In general, while patients showed a marked improvement concerning alcohol consumption, only a slight improvement in psychopathology was recorded. Overall tolerability was good. These data indicate a probable beneficial effect of flupenthixol in schizophrenic patients with comorbid alcoholism. Although the efficacy of flupenthixol as an anti-craving drug in dual diagnosis patients has to be explored in further studies, the drug may be considered a promising medication for these patients. Copyright (C) 2003 S. Karger AG, Basel.

We examined an obese 58-year-old patient with a bilateral posterior hypothalamic lesion of unknown etiology. A 24-hour polysomnography revealed a markedly increased total sleep time (17.6 h). During daytime, only 3 continuous wake phases occurred. REM periods occurred only between 5 p.m. and 6 a.m. We conclude from our results that, similar to the results from animal experiments, the posterior hypothalamus in humans plays a critical role in the maintenance of wakefulness. Copyright (C) 2003 S. Karger AG, Basel.

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 x 10(6) spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 +/- 1.1 and 8.5 +/- 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 +/- 1.1 to 6.0 +/- 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 +/- 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF. Copyright (C) 2003 S. Karger AG, Basel.

Objective: Infants with gastroschisis (GS) still face severe morbidity. Prenatal closure may prevent gastrointestinal organ damage, but intrauterine GS repair (GSR) has not been established yet. Methods: In New Zealand White rabbits we developed and compared GS versus GSR: creation of GS was achieved by hysterotomy, right-sided laparotomy of the fetus and pressure on the abdominal wall to provoke evisceration. GSR was accomplished by careful reposition of eviscerated organs and a running suture of the fetal abdominal wall. For study purposes, 18 animals were divided equally into 3 groups: GS, GS with GSR after 2 h, and unmanipulated controls (C). Vitality was assessed by echocardiography. After 5 h all animals were sacrificed. Results: GSR inflicted no increased mortality, because all fetuses survived GS or GS with GSR. All fetuses with GS demonstrated significant evisceration of abdominal organs. In contrast, the abdominal wall of the fetuses from GSR was intact. Conclusion:The present animal model demonstrated the technical feasibility and success of an intrauterine repair of GS for the first time. However, further long-term studies (leaving GS and GSR in utero for several days) will be necessary to compare survival rates and intestinal injury, motility or absorption. The clinical application of GSR in utero remains a vision so far. Copyright (C) 2003 S. Karger AG, Basel.

The linkage of vascular genes to specific functions will lead to a better understanding of cardiovascular pathophysiology. We developed an experimental model that enables the introduction of one or multiple gene(s) into vascular smooth muscle cells (VSMCs) of isolated resistance arteries. Exposure of the arteries to a green fluorescent protein (GFP)-encoding plasmid in combination with the transfectant Effectene(R) for 20 h resulted in the expression of GFP in virtually all VSMCs in the arterial wall at fully preserved vascular function. For functional validation of the model, plasmids encoding the specific RhoA inhibitors C3 transferase or N19RhoA were transfected. In subsequent functional tests, inhibition of RhoA-dependent constriction induced by sphingosine-1-phosphate was similar to that in arteries treated with exogenous C3 transferase protein or the Rho kinase inhibitor Y27632. Responses to norepinephrine remained unaffected. This novel transfection technique enables gene function to be assessed in direct conjunction with signalling pathways in vascular tissue and provides, therefore, a new tool for microvascular proteomics. Copyright (C) 2003 S. Karger AG, Basel.

Inhibition of angiogenesis might be a therapeutic approach to prevent joint destruction caused by the overgrowing synovial tissue during chronic joint inflammation. The aim of this study was to investigate angiogenesis in the knee joint of mice with antigen-induced arthritis (AIA) by means of intravital microscopy. In 14 mice (C57BL6/129Sv) intravital microscopic assessment was performed on day 8 after AIA induction in two groups (controls, AIA). Synovial tissue was investigated by intravital fluorescence microscopy using FITC-dextran (150 kD). Quantitative assessment of vessel density was performed according to the following categories: functional capillary density (FCD, vessels 10 mum) and FVD of vessels with angiogenic criteria (convoluted vessels, abrupt changes of diameter, vessels which are generated by sprouting and progressively pruned and remodelled). Microvessel count was performed using immunohistochemistry. There was no significant difference in FCD between the control group (337 +/- 9 cm/cm(2); mean +/-SEM) and the AIA group (359 +/- 13 cm/cm(2)). The density of vessels larger than 10 gm diameter was significantly increased in animals with AIA (135 +/- 10 vs. 61 +/- 5 cm/cm(2) in control). The density of blood vessels with angiogenic criteria was enhanced in arthritic animals (79 +/- 17 vs. 12 +/- 2 cm/cm(2) in control). There was a significant increase in the microvessel count in arthritic animals (297 +/- 25 vs. 133 +/- 16 mm(-2) in control). These findings demonstrate that angiogenesis in murine AIA can be assessed quantitatively using intravital microscopy. Further studies will address antiangiogenic strategies in AIA.

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration.

Modification of cellular functions by overexpression of genes is increasingly practised for research of signalling pathways, but restricted by limitations of low efficiency. We investigated whether the novel technique of magnetofection (MF) could enhance gene transfer to cultured primary endothelial cells. MF of human umbilical vein endothelial cells (HUVEC) increased transfection efficiency of a luciferase reporter gene up to 360-fold compared to various conventional transfection systems. In contrast, there was only an up to 1.6-fold increase in toxicity caused by MF suggesting that the advantages of MF outbalanced the increase in toxicity. MF efficiently increased transfection efficiency using several commercially available cationic lipid transfection reagents and polyethyleneimine (PEI). Using PEI, even confluent HUVEC could be efficiently transfected to express luciferase activity. Using a green fluorescent protein vector maximum percentages of transfected cells amounted up to 38.7% while PEI without MF resulted in only 1.3% transfected cells. Likewise, in porcine aortic endothelial cells MF increased expression of a luciferase or beta-galactosidase reporter, reaching an efficiency of 37.5% of cells. MF is an effective tool for pDNA transfection of endothelial cells allowing high efficiencies. It may be of great use for investigating protein function in cell culture experiments.