Medizin - Open Access LMU - Teil 17/22

Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.

Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn's disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10⁻⁸). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10⁻⁵). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10⁻²) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10⁻²) but none of these associations remained significant after Bonferroni correction.

Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.

Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy.

Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling.

The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait "anxiety". We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4).Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

Inclusion in systematic reviews is one important component in judging the potential impact of clinical studies upon practice and hence the 'value for money' of spending for clinical research. This study aims to quantify the distribution of countries of origin of clinical studies used in Cochrane Reviews (CRs), and to link these data to the size of a country and to its spending on research.

Random sample of publications used for CRs published in Issue 1 2008 and of publications used in CRs in the field of complementary and alternative medicine (CAM). Publications without original data were excluded. Likely countries of origin determined based on abstracts/full texts. CIA World Factbook (population data) and OECD database (economic data) were used.

1,000 random entries out of 140,005 references available in all specialities. In 876 (91.4%) of 959 eligible studies, country of origin was determined. The USA was the leading contributor (36.0% of the studies), followed by UK (13.4%), Canada (5.3%), Australia and Sweden (3.7%). In the CAM sample, country of origin was determined in 458 (93.5%) of 497 assessed studies. Again, the USA was the leading contributor (24.9%), with China also emerging as a significant contributor (24.7%) in this field. For both samples, the contribution of smaller countries (especially Scandinavian countries, Greece, and Ireland) became more noteworthy when considered in relation to population size and research spending.

Our results support the leading roles of both the USA and the UK in publishing clinical papers. The emerging role of China can be seen, particularly related to CAM studies. Taking into account size of population and economic power, countries like France, Germany, Italy, and Spain provide small contributions. In contrast, smaller countries like Australia, Denmark, Finland, Ireland, New Zealand, and Sweden also play major roles.

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.

BACKGROUND/AIM OF THE STUDY: The study aimed to determine the cost impacts of antiretroviral drugs by analysing a long-term follow-up of direct costs for combined antiretroviral therapy, cART, -regimens in the nationwide long-term observational multi-centre German HIV ClinSurv Cohort. The second aim was to develop potential cost saving strategies by modelling different treatment scenarios.

Antiretroviral regimens (ART) from 10,190 HIV-infected patients from 11 participating ClinSurv study centres have been investigated since 1996. Biannual data cART-initiation, cART-changes, surrogate markers, clinical events and the Centre of Disease Control- (CDC)-stage of HIV disease are reported. Treatment duration was calculated on a daily basis via the documented dates for the beginning and end of each antiretroviral drug treatment. Prices were calculated for each individual regimen based on actual office sales prices of the branded pharmaceuticals distributed by the license holder including German taxes.

During the 13-year follow-up period, 21,387,427 treatment days were covered. Cumulative direct costs for antiretroviral drugs of €812,877,356 were determined according to an average of €42.08 per day (€7.52 to € 217.70). Since cART is widely used in Germany, the costs for an entire regimen increased by 13.5%. Regimens are more expensive in the advanced stages of HIV disease. The potential for cost savings was calculated using non-nucleotide-reverse-transcriptase-inhibitor, NNRTI, more frequently instead of ritonavir-boosted protease inhibitor, PI/r, in first line therapy. This calculation revealed cumulative savings of 10.9% to 19.8% of daily treatment costs (50% and 90% substitution of PI/r, respectively). Substituting certain branded drugs by generic drugs showed potential cost savings of between 1.6% and 31.8%.

Analysis of the data of this nationwide study reflects disease-specific health services research and will give insights into the cost impacts of antiretroviral therapy, and might allow a more rational allocation of resources within the German health care system.

Main contributors to adverse outcomes in severely burned pediatric patients are profound and complex metabolic changes in response to the initial injury. It is currently unknown how long these conditions persist beyond the acute phase post-injury. The aim of the present study was to examine the persistence of abnormalities of various clinical parameters commonly utilized to assess the degree hypermetabolic and inflammatory alterations in severely burned children for up to three years post-burn to identify patient specific therapeutic needs and interventions.

Nine-hundred seventy-seven severely burned pediatric patients with burns over 30% of the total body surface admitted to our institution between 1998 and 2008 were enrolled in this study and compared to a cohort non-burned, non-injured children. Demographics and clinical outcomes, hypermetabolism, body composition, organ function, inflammatory and acute phase responses were determined at admission and subsequent regular intervals for up to 36 months post-burn. Statistical analysis was performed using One-way ANOVA, Student's t-test with Bonferroni correction where appropriate with significance accepted at p<0.05. Resting energy expenditure, body composition, metabolic markers, cardiac and organ function clearly demonstrated that burn caused profound alterations for up to three years post-burn demonstrating marked and prolonged hypermetabolism, p<0.05. Along with increased hypermetabolism, significant elevation of cortisol, catecholamines, cytokines, and acute phase proteins indicate that burn patients are in a hyperinflammatory state for up to three years post-burn p<0.05.

Severe burn injury leads to a much more profound and prolonged hypermetabolic and hyperinflammatory response than previously shown. Given the tremendous adverse events associated with the hypermetabolic and hyperinflamamtory responses, we now identified treatment needs for severely burned patients for a much more prolonged time.

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/-) mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G(0)/G(1) phase. Furthermore, low expression of K(v1.5), a voltage-gated potassium channel known to alter myoblast proliferation during the G(1) phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K(v1.5) and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K(v1.5) within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K(v1.5) with FHL1C in Xenopus laevis oocytes markedly reduced K(+) currents when compared to oocytes expressing K(v1.5) only. We here present the first evidence on a biological relevance of FHL1C.