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Hepatocyte growth factor (HGF) is a multifunctional growth factor implicated in a variety of tissue restructuring processes. Since HGF acts as a highly potent mitogen, HGF expression is suggested to be under a well-defined transcriptional control. The 5′ sequence of the HGF gene clusters a set of several binding sites for transcription factors in a so-called multiconsensus region (MCR) located between −230 and 260. Our studies demonstrate that a NF1-like element and the E1-box of the MCR form the main complexes with nuclear proteins and that both are involved in transcriptional silencing of the HGF gene in non-HGF expressing cell types. The E1-box of two tandemly arranged E-boxes was shown to be a binding site of high affinity interacting with the upstream stimulatory factor (USF). While recombinant expression of a wild-type USF did not affect gene expression, a USF variant lacking the DNA binding domain restored the MCR mediated transcriptional repression. In conclusion, our data provide evidence that USF is a central factor of cell-type specific HGF regulation, acting in cooperation with additional regulatory proteins as a bivalent mediator of transcriptional activation or repression. [Copyright &y& Elsevier]

Objective: Alpha-smooth muscle actin (SMA), is a main member of the cytoskeleton of differentiated vascular and visceral smooth muscle cells (SMC). In addition, SMA is expressed transiently during embryonic differentiation of cardiac and skeletal muscle cells and during myofibroblastic differentiation of organ-specific precursor cells when wound healing or scarring processes occur in chronically diseased tissues. In this study we characterized the potential of the SMA promoter to direct hepatic or renal myofibroblast-specific gene expression. Methods: Hepatic stellate cells (HSC) were isolated from rats and maintained in primary culture. From quiescent (freshly isolated) and myofibroblastic-activated HSC (culture day 3, 5, 7) nuclear proteins were extracted. The 5′-promoter sequence of the SMA gene was analysed by reporter assays and mobility shift assays. Differentiation-dependent promoter activities were investigated during embryonic body development of stably transfected embryonic stem cells carrying the promoter-GFP reporter fusion plasmid. For in vivo studies the SMA promoter combined with the first intron of the β-actin gene was used to control GFP reporter expression in a transgenic reporter mouse model. Myofibroblastic differentiation was induced by ureter obstruction (UUO) in the SMA-GFP reporter mice. Kidneys were taken after 3, 7 and 14 days of UUO and cell type specific reporter expression and fibrosis were analysed. Results: The SMA promoter contains several conserved protein interaction sites which we show to be bound by transcription factors in a cell-type specific and differentiation dependent manner. The characterised regulatory sequences of the SMA promoter were able to drive transient gene transcription in embryonic skeletal precursors and in hepatic stellate cells (HSC) after in vitro induced differentiation into contractile myofibroblastic cells. In addition to these in vitro studies the SMA promoter combined with the first intron of the β-actin gene directs cell-type specific reporter gene transcription in cardiomyocytes and SMC during development of embryonic bodies, thereby mimicking the native embryonic expression pattern of SMA in muscle cell types. In order to test the identified promoter fragment for myofibroblastic-specific control we established a transgenic reporter mouse model. In adult mice, the promoter regulates gene expression in visceral SMC. Furthermore, the differentiation and cell-type specific control of the SMA promoter already shown in vitro was confirmed in vivo during embryogenesis and myofibroblastic transition in renal tissue after ureter ligation as model of progressive renal insufficiency. At day 7 after UUO, GFP-reporter expression was observed in agreement to the occurrence of myofibroblastic differentiation shown by SMA immunohistology. Corresponding to the degree of interstitial fibrosis and myofibroblastic transition SMA promoter-directed reporter expression has been increased at day 14 after UUO. Conclusions: In conclusion, the SMA promoter is a useful tool to target myofibroblastic cell types and to direct transient gene expression in myofibroblasts in different organ systems such as liver and kidney. [ABSTRACT FROM AUTHOR]

Microsatellite analysis is an important tool in clinical research and molecular diagnostics because microsatellite instability (MSI) occurs frequently in various types of cancer. Approximately 10-15% of colorectal, gastric and endometrial carcinomas are associated with MSI, and this has an impact on clinical prognosis. The microsatellite loci Bat25, Bat26, D2S123, D5S346 and D17S250, recommended by the Bethesda guidelines, were analysed by microfluidic-based on-chip electrophoresis in 40 cases of colon carcinoma with known MSI status. In all cases, microfluidic separation of the PCR amplicons resulted in highly resolved, distinct patterns of each of the five microsatellite loci. Detection of MSI could be demonstrated by microsatellite-loci-associated, well-defined deviations in the electropherogram profiles of tumour and non-tumour material, and confirmed the classification of MSI cases performed by conventional technology. In conclusion, microfluidic chip technology is a simple and reliable approach for MSI detection that allows label-free and very fast analysis of microsatellite amplicons. [ABSTRACT FROM AUTHOR]

Objective: Glomerulosclerosis describes the destruction of the glomerular tuft by a transient mesangial cell proliferation followed by obliteration of the capillary loops through expansive mesangial matrix deposition. In this complex process, the mesangial production of alpha-smooth muscle actin (SMA) has been shown to be a major feature, highlighting the so-called myofibroblastic differentiation. To study this process, we developed a convenient system in living cells allowing readily detection of early mesangial activation using different mediators useful for in vitro and in vivo investigation. Methods: Commercially available human mesangial cells in subculture 6-9 were subjected to different commercially available transfection reagents (Lipofectin, Fugene, Lipofectamine, CaCl2, DEAE-dextran) or electroporation with the nucleofector (Amaxa) using a CMV-β Gal reporter plasmid for evaluation of transfection efficiency. In addition, adenoviral-mediated infection was performed and recombinant reporter expression driven by the CMV or the SMA promoter has been studied. Infection efficiency was monitored by GFP reporter fluorescence of CMV reporter infected mesangial cells. Recombinant reporter expression directed by the SMA promoter was quantitatively analysed by the Bioanalyzer (Agilent) during myofibroblastic transition upon glucose (4 vs. 30 mM) and mechanical strain (60 cycles/min) treatment of mesangial cells for 24 hrs. To test the inducibility of this construct in vivo, a unilateral renal perfusion after 1.5 nephrectomy leading to glomerulosclerosis is used. Results: Adenovirus-mediated transgene transmission and recombinant expression in mesangial cells was successful in 100% of the cells after 24 hrs of infection with 10 MOI (Multiplicity of Infection) of an adenoviral CMV promoter driven reporter construct. Lower MOI resulted in retarded initiation of transgenic reporter expression but recombinant reporter synthesis was shown to extend up to seven days. From the commercially available transfection mediators, however, only DEAE-dextran yielded positive cells (about 5%). Electroporation failed. In contrast to constitutive expression of CMV driven GFP, fluorescence of the SMA promoter GFP construct could be induced after 24 by mechanical strain and by glucose. Maximal GFP intensity was achieved after glucose stimulation for 7d, lasting for at least 3 weeks after switching to the normal feeding medium. The inducibility of the adenoviral mediated transgenic expression in the in vivo model is currently under investigation. Conclusions: In conclusion, an adenovirus-mediated infection seems the only efficient and reliable method of mesangial cell genetic manipulation. Using GFP, the inducibility of any promoter construct can be easily monitored and potentially quantitated with in vitro and in vivo applicability. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC. [ABSTRACT FROM AUTHOR]

Objectives: Lymph node ratio (LNR) is an established predictor in different entities of carcinoma, including head and neck malignancies. In oropharyngeal squamous cell carcinoma (OPSCC), lymph node involvement differs between human papilloma virus (HPV)‐positive and HPV‐negative tumours. Herein, we evaluate the impact of HPV association on the concept of LNR. Methods: 88 surgically treated patients were included in this retrospective chart review. HPV‐positive and HPV‐negative OPSCC were evaluated for prediction of outcome by LNR separately. The endpoints were 5‐year overall survival (OS) and recurrence‐free survival (RFS). Results: The OS of all patients was 60.1%. In univariate analysis, LNR was a significant predictor of overall survival rate (P=.008) in OPSCC independently of the HPV status, as well as extracapsular spread (ECS). T‐classification was only a significant predictor in the univariate analysis in HPV‐positive OPSCC carcinoma. However, in the multivariate analysis LNR remained predictor of prognosis in all OPSCC and in HPV‐negative OPSCC. In patients with HPV‐positive OPSCC, only T‐classification reached significance to predict OS. Conclusion: Prognosis of primarily operated HPV‐positive patients might be more dependent on the extent of primary tumour site, whereas prognosis of HPV‐negative patients is based more on cervical metastatic spread, represented by LNR. [ABSTRACT FROM AUTHOR]

Objective Lymph node ratio ( LNR) has been shown to be an independent predictor of recurrence risk and survival in different entities of carcinoma. Methods In this retrospective chart review, 128 patients with parotid gland cancer (PGC) subsequently treated by primary surgery were included. About 64% ( n = 82) of these patients were additionally treated with adjuvant radiotherapy. Five-year overall survival rates were determined by subgroups based on LNR value. Results Lymph node ratio was found to be significantly associated with overall survival rate ( P < 0.001). Using univariate analyses, pathological tumour-node-metastasis ( TNM)-stage, UICC-stage grouping and extracapsular spread were found to be significant predictors of overall survival ( P < 0.001). However, with a multivariate analyses, LNR remained the only independent predictor of overall survival ( P = 0.043). Conclusions After surgery for PGC, evaluation of the neck using LNR was found to reliably stratify the overall survival rate. [ABSTRACT FROM AUTHOR]

Objectives: We tested the hypothesis whether selective blunting of platelet-derived growth factor (PDGF)-dependent vascular smooth muscle cell (VSMC) proliferation and migration is sufficient to prevent neointima formation after vascular injury.Background: To prevent neointima formation and stent thrombosis after coronary interventions, it is essential to inhibit VSMC proliferation and migration without harming endothelial cell function. The role of PDGF-a potent mitogen and chemoattractant for VSMC that does not affect endothelial cells-for neointima formation remains controversial.Methods: To decipher the signaling pathways that control PDGF beta receptor (βPDGFR)-driven VSMC proliferation and migration, we characterized 2 panels of chimeric CSF1R/βPDGFR mutants in which the binding sites for βPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase [PI3K], GTPase activating protein of ras, SHP-2, phospholipase Cγ 1 [PLCγ]) were individually mutated. Based on in vitro results, the importance of PDGF-initiated signals for neointima formation was investigated in genetically modified mice.Results: Our results indicate that the chemotactic response to PDGF requires the activation of Src, PI3K, and PLCγ, whereas PDGF-dependent cell cycle progression is exclusively mediated by PI3K and PLCγ. These 2 signaling molecules contribute to signal relay of the βPDGFR by differentially regulating cyclin D1 and p27(kip1). Blunting of βPDGFR-induced PI3K and PLCγ signaling by a combination mutant (F3) completely abolished the mitogenic and chemotactic response to PDGF. Disruption of PDGF-dependent PI3K and PLCγ signaling in mice expressing the F3 receptor led to a profound reduction of neointima formation after balloon injury.Conclusions: Signaling by the activated βPDGFR, particularly through PI3K and PLCγ, is crucial for neointima formation after vascular injury. Disruption of these specific signaling pathways is sufficient to attenuate pathogenic processes such as vascular remodeling in vivo. [ABSTRACT FROM AUTHOR]

Background: The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC.Methods: Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification.Results: Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05).Conclusion: Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs have recently taken centre stage as short non-coding RNAs that regulate mRNA expression. Aim/Methods: To assess the feasibility of using microRNA techniques on routinely processed tissues, the accessibility of two representative microRNAs was examined by real-time quantitative PCR in 86 human formalin-fixed paraffin-embedded (FFPE) samples from liver, breast, bone marrow, lymphatic tissues and colon. Murine liver was used to analyse the influence of fixation time and different fixatives. Results: High-quality microRNA was successfully extracted from routinely processed formalin-fixed tissues, resembling PCR amplification results from snap-frozen material analysed in parallel. While fixation time did not affect microRNA accessibility, non-buffered formalin or fixative supplements such as glutaraldehyde influenced PCR results. Storage of human tissues for up to 7 years did not cause a significant deterioration of microRNA. However, microRNA quality in human archival material following routine processing 10-20 years ago was decreased. Oxidation by ambient air during storage and fixation in non-buffered formalin is a possible reason for loss of microRNA quality. Conclusion: The assessment of microRNAs in readily obtained formalin-fixed paraffin-embedded samples is a highly promising tool in molecular pathology when similarly treated samples are analysed. Therefore, microRNA analyses will gain wider acceptance as an adjunct to morphological tissue assessment in routine pathology and retrospective studies. [ABSTRACT FROM AUTHOR]

Objective: Tubulointerstitial injury (TI) is a common pathway of chronic renal disease and determines the loss of renal function. Chronic proteinuria causes TI, but it is still controversial by what mechanism. Purpose of the present study was to find out whether ET-1/ETB-receptor is involved in the TI caused by proteinuria. We made use of the COL4A3-/- animal model mimicking Alport syndrome (AS), which is characterized by hematuria and proteinuria leading to progressive renal failure. Untreated COL4A3-/- mice die from renal failure after about 70 days. However, treatment with ramipril delays onset and reduces extent of proteinuria thereby increasing the lifespan by more than 100%. -- The model of the ramipril treated COL4A3-/- mice was used to study the role of the endothelin (ET) -- system in the development of the proteinuria caused tubulointerstitial fibrosis. Methods: COL4A3 wild type and knockout mice were treated with 10 mg/kg/day ramipril in the drinking water starting at 4 wks of age and compared to controls (placebo). Animals were sacrificed following 7.5 and 9.5 wks. Kidneys were rapidly harvested and either processed for immunohistochemistry or used for extraction of total RNA. TI was characterized by incubation with a-smooth muscle actin (aSMA) antibody identifying myofibroblasts. Expression of ET-1 and ETB receptor was quantified by real time RT-PCR and results were normalized for b-actin expression. Results: Wildtype controls showed normal kidneys and no upregulation of ET-1 or ETB receptor expression. However, untreated COL4A3-/- mice showed increasing amounts of aSMA positive cells and a with time increasing expression of ET-1 (7.5wks: 2.05; 9.5 wks: 4.73) but not of ETB receptor (7.5wks: 0.29; 9.5 wks: 0.13). Ramipril treatment did not affect renal histology in wildtype control animals, nor did it alter expression of ET-1 or ETB receptor. However, ramipril treated COL4A3-/- mice developed less TI; ET-1 expression was increased (7.5wks: 2.47; 9.5 wks: 2.98) but did not reach the level of placebo treated COL4A3-/- animals. ETB receptor expression was only slightly different from placebo treated animals (7.5 wks: 0.30; 9.5 wks: 0.59). Conclusions: We conclude that (1) renal ET-1 expression is increased and ETB receptor expression is decreased in chronic proteinuria and that (2) the protective effect of ramipril treatment against renal TI is linked to a reduced increase of ET-1 expression. We hypothesize that ET-1/ETB receptor interaction plays an important role in mediation of the TI caused by proteinuria. This will be investigated in further studies. [ABSTRACT FROM AUTHOR]

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.Gene Therapy (2008) 15, 443–451; doi:10.1038/sj.gt.3303077; published online 15 November 2007 [ABSTRACT FROM AUTHOR]

Murine cytomegalovirus (MCMV) has provided useful models for acute, chronic and latent CMV infection because of its similarities in structure and biology with human CMV. We report the induction of acute MCMV hepatitis with different bacterial artificial chromosome (BAC)-cloned virus constructs [MCMV-SEAP which includes the gene for secreted alkaline phosphatase (SEAP) under Rous sarcoma virus (RSV)-promoter control, MCMV-GFP which includes the gene for enhanced green fluorescent protein (eGFP) under HCMV-ie promoter control, MCMV-HBs includes the gene for hepatitis B surface antigen (HBsAg) under simian virus (SV)40-promoter control and the DeltaMC95.21 virus in which the m152 gene was deleted and substituted by the reporter gene lacZ] in order to elucidate the histopathological changes together with different reporter-gene products in the liver tissue and the effect of the deletion of a certain gene. All the virus constructs induced a similar mild acute hepatitis which had its climax from days 3 to 5 post-infection in immunocompetent mice. In situ, the reporter-gene products beta-galactosidase and secreted alkaline phosphatase could be visualized in relation to the inflammatory changes. The composition of the invading cell populations did not change even in the absence of the m152 gene. Additionally discrete inflammatory changes were seen in kidney and serosa while the other organs were not involved. This model helps us understand the immunological and histopathological mechanisms of the CMV-induced hepatitis, which plays an important role especially in the immunocompromised patient. The morphological changes can be analysed while the respective reporter gene product is expressed by the virus construct. [ABSTRACT FROM AUTHOR]

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future. [Copyright &y& Elsevier]

Background: Gastric infection with Helicobacter pylori is the major cause of chronic active gastritis and is associated with the pathogenesis of peptic ulcer and gastric carcinoma. Gastric mucosal damage involves both host and H pylori dependent factors, such as the presence of the cag pathogenicity island and allelic variations of the vacA and iceA genes. Aims: To evaluate the association of these virulence factors with the development of gastric malignancies, a retrospective study was performed on archived tissue routinely obtained for diagnostic histopathology. Methods: DNA was extracted from formalin fixed, paraffin wax embedded gastric tissue sections of 93 patients with chronic active gastritis (n = 39), adenocarcinoma (n = 28), or mucosa associated lymphoid tissue (MALT) lymphoma (n = 24). The extracted DNA was used to perform a polymerase chain reaction based, simultaneous analysis of the following: (1) cagA status, (2) allelic variation of the iceA genes (iceA1, iceA2), allelic variation of the signal peptide (sla, slb, s2) and the midregion (ml, m 1 a, m2) of the vacA gene. Results: The iceA1 gene showed a 3.6 fold and the vacA sla variant a 4.2 fold higher prevalence in gastric adenocarcinoma than in gastritis. The combined presence of both the vacA sla and iceA1 genes had a 5.6 fold higher frequency in adenocarcinoma. The vacA m2 allele was the predominant subtype in MALT lymphoma and the combination of the vacA m2 subtypes with the vacA sl and the iceA1 variants occurred in MALT lymphoma nearly five times more often than in chronic active gastritis. Conclusions: Certain H pylori subtype combinations possess a differentiating and predictive value for the development of gastric adenocarcinoma and MALT lymphoma. [ABSTRACT FROM AUTHOR]

Objective(s): To compare the outcome of patients with stage D1 (TxN+M0) prostate cancer undergoing radical prostatectomy or androgen deprivation alone.Patients and Methods: Eighty-two patients treated for lymph node positive prostate cancer were retrospectively analyzed for time to progression, tumor-specific and overall survival. Furthermore, subsequent tumor and treatment related morbidity requiring intervention including frequency and duration of associated hospital stays was recorded.Results: The extent of lymph node metastasis was significantly lower in 50 patients undergoing radical prostatectomy (± early androgen deprivation) compared to 32 receiving androgen deprivation only. The treatment groups, however, did not differ with regard to other characteristics including age, comorbidity, stage, grade and preoperative PSA. Mean actuarial progression-free, and tumor-specific survival was significantly longer for the radical prostatectomy patients (36% and 47%, respectively at 10 years) compared to androgen deprivation (15% and 32%, respectively). The latter group required more secondary interventions resulting in more frequent and overall longer hospital stays.Conclusions: Patients undergoing radical prostatectomy for stage D1 prostate cancer possibly benefit with regard to the necessity for secondary interventions and, at least for limited (solitary) nodal disease, in terms of progression-free and tumor-specific survival. However, the latter observation may be biased by a larger extent of lymph node metastasis in the androgen deprivation group. [ABSTRACT FROM AUTHOR]