Biomedical subjects

Rainer Haas

Publications and source records attributed to Rainer Haas.

At least 73 records · Page 4Linked to original sources

PubMed2003

Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.

Helicobacter pylori (Hp) vacuolating cytotoxin VacA induces cellular vacuolation in epithelial cells. We found that VacA could efficiently block proliferation of T cells by inducing a G1/S cell cycle arrest. It interfered with the T cell receptor/interleukin-2 (IL-2) signaling pathway at the level of the Ca2+-calmodulin-dependent phosphatase calcineurin. Nuclear translocation of nuclear factor of activated T cells (NFAT), a transcription factor acting as a global regulator of immune response genes, was abrogated, resulting in down-regulation of IL-2 transcription. VacA partially mimicked the activity of the immunosuppressive drug FK506 by possibly inducing a local immune suppression, explaining the extraordinary chronicity of Hp infections.

Apoptosis↗

PubMed2003

VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N-terminal domains exhibit a collection of rigid-body conformations. Nucleotide binding 'locks' the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide-dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Biochemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.

Adenosine Triphosphatases↗

PubMed2003

Identification and characterization of Helicobacter pylori genes essential for gastric colonization.

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

Animals↗

PubMed2003

Molecular biology of hematopoietic stem cells.

Human CD34+ hematopoietic stem and progenitor cells are capable of maintaining a life-long supply of the entire spectrum of blood cells dependent on systemic needs. Recent studies suggest that hematopoietic stem cells are, beyond their hematopoietic potential, able to differentiate into nonhematopoietic cell types, which could open novel avenues in the field of cellular therapy. Here, we concentrate on the molecular biology underlying basic features of hematopoietic stem cells. Immunofluorescence analyses, culture assays, and transplantation models permit an extensive immunological as well as functional characterization of human hematopoietic stem and progenitor cells. New methods such as cDNA array technology have demonstrated that distinct gene expression patterns of transcription factors and cell cycle genes molecularly control self-renewal, differentiation, and proliferation. Furthermore, several adhesion molecules have been shown to play an important role in the regulation of hematopoiesis and stem cell trafficking. Progress has also been made in elucidating molecular mechanisms of stem cell aging that limit replicative potential. Finally, more recent data provide the first molecular basis for a better understanding of transdifferentiation and developmental plasticity of hematopoietic stem cells. These findings could be helpful for non-hematopoietic cell therapeutic approaches.

Antigens, CD34↗

PubMed2003

Refractory anemia with excess of blasts in transformation: analysis of reclassification according to the WHO proposals.

The category of "refractory anemia with excess blasts in transformation" (RAEB-T) has been abandoned in the new WHO-classification of myelodysplastic syndromes (MDS). The majority of patients previously belonging to this category are now classified as acute myeloid leukaemia (AML). In the FAB-classification, patients had been assigned to the RAEB-T category if they had either (1) a medullary blast count between 20 and 30% or (2) a peripheral blast count of at least 5%, or (3) Auer rods detectable, irrespective of the blast count. We analyzed these subtypes of RAEB-T in terms of hematological characteristics, karyotype anomalies, and prognosis. Patients with more than 20% medullary blasts and patients with at least 5% peripheral blasts as the sole defining parameter for RAEB-T had a median survival of 6 months, as compared to 11 months in patients with Auer rods as the sole defining parameter. The presence of Auer rods therefore does not convey a particularly bad prognosis and does not justify placing patients in a high-risk category of MDS or even classifying them as AML. This finding supports the elimination of Auer rods as a parameter for classification in the new WHO system. On the other hand, the reclassification into RAEB II (according to WHO proposals) of previous RAEB-T patients with a peripheral blast count of at least 5% is problematic, because this feature predicts a median survival not different from that of AML patients.

Acute Disease↗

PubMed2003

Quantitative real-time reverse-transcription polymerase chain reaction for diagnosis of BCR-ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) (qPCR) of the BCR-ABL mRNA is a suitable technique to measure the amount of circulating leukemic cells in chronic myelogenous leukemia (CML). In this study, we evaluated a BCR-ABL-specific qPCR method using the LightCycler technology in 95 patients with Philadelphia chromosome positive acute leukemia (n = 7) or CML in different stages (n = 88). Primers and hybridization probes were chosen to detect the most prevalent variants of BCR-ABL (b2a2, b3a2, b2a3, b3a3, e19a2, e1a2) with a sensitivity of 10-5 for b2a2 and b3a2. With median BCR-ABL/G6PDH ratios of 10.7% in the untreated chronic phase, 43.2% in the newly diagnosed accelerated phase, and 131.4% in newly diagnosed blast crisis the BCR-ABL mRNA levels varied significantly between different stages of CML whereas no difference was found between blast crisis and untreated acute leukemias (136.9%). There was a strong relationship between qPCR results and cytogenetics in patients treated with imatinib, interferon-alpha, or following allografting. Thirteen patients with CML were sequentially examined by qPCR following myeloablative or non-myeloablative allogeneic peripheral blood stem cell transplantation. Five patients received donor lymphocytes and became BCR-ABL negative as confirmed by nested RT-PCR. The gradual disappearance of BCR-ABL positive cells could be monitored by qPCR following non-myeloablative transplantation. Comparison of BCR-ABL levels with the degree of donor chimerism showed that 91% of samples with complete donor chimerism were BCR-ABL negative. In 22% of BCR-ABL negative samples chimerism between 71% and 98% was observed, indicating the persistence of normal recipient's hematopoietic cells. In conclusion, the qPCR protocol used in this study is a reliable and fast method for monitoring molecular response in CML.

Antineoplastic Agents↗

PubMed2003

Non-small lung cancer cells are prime targets for p53 gene transfer mediated by a recombinant adeno-associated virus type-2 vector.

In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy.

Adenoviridae↗

PubMed2003

A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system.

Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag-PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin-8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src-family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10-homologous protein. HP0527 is also clustered in the outer membrane. The VirB7-homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS-associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.

Antigens, Bacterial↗

PubMed2003

Topology and membrane interaction of Helicobacter pylori ComB proteins involved in natural transformation competence.

The human gastric pathogen Helicobacter pylori is naturally competent for genetic transformation. The H. pylori comB gene duster encodes the VirB4-homologous ATPase ComB4 and the structural proteins ComB7-ComB10, which share significant sequence identity to the Agrobacterium tumefaciens virB-encoded type IV secretion system. To study the topology of the ComB7-10 proteins, we applied TnMax transposon mutagenesis by generating fusions of ComB proteins with mature beta-lactamase (BlaM) or alkaline phosphatase (PhoA). Our data show that the putative lipoprotein ComB7 is secreted and is found membrane-attached, probably by its lipid anchor. According to our topology mapping ComB8 is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space. ComB9 was verified as a periplasmic protein, tightly attached to the membrane. The N-terminus of ComB10 is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm. Limited protease digestion and protein extraction under different salt and pH conditions confirmed the periplasmic localization and the tight membrane association of the ComB protein complex. A hypothetical model of the ComB DNA transformation pore in H. pylori is presented.

Alkaline Phosphatase↗

PubMed2003

Topological analysis of a putative virB8 homologue essential for the cag type IV secretion system in Helicobacter pylori.

The type IV secretion system encoded by the cag pathogenicity island of Helicobacter pylori is recognized as a major virulence determinant, governing the translocation of the CagA protein to eukaryotic cells. Despite the presence of 18 genes within the cag pathogenicity island which are essential for this process, several homologues encoding basic functions of a type IV secretion apparatus, such as a virB8 homologue, are still missing. We show here the presence of conserved VirB8 motifs in the cag-encoded apparatus protein HP530 and demonstrate that this protein adopts in E. coli a transmembrane topology similar to VirB8 and its homologues. Thus, we propose that HP530 is a type IV secretion component homologous to VirB8.

Amino Acid Sequence↗

PubMed2003

Differential gene expression underlying the functional distinctions of primary human CD34+ hematopoietic stem and progenitor cells from peripheral blood and bone marrow.

The restorative capacity of human CD34(+) hematopoietic cells is clinically used in the autologous and allogeneic transplant setting to support cytotoxic therapy. We examined gene expression patterns of highly enriched bone marrow CD34(+) (BM-CD34(+)) or G-CSF-mobilized peripheral blood CD34(+) (PB-CD34(+)) cells by cDNA array technology, quantitative real-time RT-PCR, and flow cytometry, to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. The greater cell cycle and DNA synthesis activity of BM-CD34(+) compared to PB-CD34(+) cells was reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle, 11 genes regulating DNA synthesis, and the cell cycle-initiating transcription factor E2F-1. The 2- to 3-fold greater expression of 5 pro-apoptotic genes in PB-CD34(+) cells indicated a higher apoptotic activity, which could functionally be corroborated by apoptosis assays. Thrombin receptor (PAR1), known to play a role in trafficking of malignant cells, was 3.6-fold higher expressed in circulating CD34(+) cells than in BM-CD34(+) cells. Guidance via thrombin receptor might molecularly mediate stem cell migration. In summary, our study provides gene expression profiles of primary human CD34(+) hematopoietic cells of blood and marrow. Our data molecularly confirm and explain the finding that CD34(+) cells residing in the bone marrow are cycling more rapidly, whereas circulating CD34(+) cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data give novel molecular insights into stem cell migration and differentiation.

Antigens, CD34↗

PubMed2003

Generation of Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates.

Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria to obtain vaccine candidates. We describe here the application of this technology to Helicobacter pylori. The lysis gene cassette was cloned into an Escherichia coli-Helicobacter pylori shuttle vector and introduced into an H. pylori recipient strain by bacterial conjugation. Temperature induction of the lysis gene cassette revealed a quantitative killing of the H. pylori culture without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact H. pylori. Prophylactic oral vaccination experiments using these H. pylori ghosts in the BALB/c mouse model showed a significant reduction of the bacterial load in the ghost group, as measured by a quantitative bacterial reisolation procedure. Ten of 10 and 5 of 10 mice were protected, respectively, without the use of a mucosal adjuvant. Coadministration of ghosts with cholera toxin as mucosal adjuvant resulted in a complete protection of 10 of 10 and 8 of 8 mice against H. pylori challenge, with three animals showing a sterile immunity.

Animals↗

PubMed2003

Colonization of C57BL/6J and BALB/c wild-type and knockout mice with Helicobacter pylori: effect of vaccination and implications for innate and acquired immunity.

The gram-negative bacterial pathogen Helicobacter pylori is a major cause of peptic ulcer disease and a risk factor for gastric cancer in humans. Adapted H. pylori strains, such as strain SS1, are able to infect mice and are a useful model for gastric colonization and vaccination studies. In this study we used a streptomycin-resistant derivative of H. pylori SS1 to analyze the colonization behavior and the success of vaccination in wild-type (wt) and various knockout mice of the BALB/c and C57BL/6J genetic backgrounds. We here report that BALB/c interleukin-4 knockout (IL-4(-/-)) mice are weakly overcolonized compared to the wt strain but that the IL-12(-/-) knockout results in a strong overcolonization (500%). Unexpectedly, in the C57BL/6J background the same knockouts behaved in diametrically opposed manners. The IL-4(-/-) mutation caused a 50% reduction and the IL-12(-/-) knockout caused a 95% reduction compared to the wt colonization rate. For C57BL/6J mice we further analyzed the IL-18(-/-) and Toll-like receptor 2 knockout mutations, which showed reductions to 66 and 57%, respectively, whereas mice with the IL-10(-/-) phenotype were hardly infected at all (5%). In contrast, the tumor necrosis factor receptor knockout (p55(-/-) and p55/75(-/-)) mice showed an overcolonization compared to the C57BL/6J wt strain. With exception of the low-level infected C57BL/6J IL-10(-/-) and IL-12(-/-) knockout mice, all knockout mutants were accessible to a prophylactic vaccination and their vaccination behavior was comparable to that of the wt strains.

Administration, Oral↗

PubMed2003

Two-component systems of Helicobacter pylori contribute to virulence in a mouse infection model.

Helicobacter pylori encodes three histidine kinases and five response regulators belonging to the family of two-component regulatory systems which are involved in transcriptional control. Here we demonstrate that isogenic mutants of H. pylori P76 with deletions of the response regulator open reading frame (ORF) HP1365 and ORFs HP244, HP165, and HP1364 encoding histidine kinases are unable to colonize the stomachs of BALB/c mice, suggesting an essential role of these systems in the regulation of important virulence properties of H. pylori. Furthermore, we demonstrate that the genes under the control of the P(HP1408) and P(HP119) promoters which are regulated by the two-component system HP166-HP165 are not essential for single mutant colonization of mice but are required under competitive colonization conditions.

Animals↗

PubMed2002

Treatment of secondary myelodysplastic syndrome after heart transplantation with chemotherapy and nonmyeloablative stem-cell transplantation.

BACKGROUND: Treatment of secondary cancer after solid-organ transplantation is difficult because of coexisting medical conditions, reduced organ function, or advanced age in most patients. METHODS: A 60-year-old man developed treatment-related myelodysplastic syndrome (refractory anemia with excess blasts in transformation) 10 years after orthotopic heart transplantation. After remission induction with chemotherapy, the patient received peripheral blood stem cells from his HLA-identical ABO-mismatched (A-->O) sister after conditioning with fludarabine and low-dose total-body irradiation (200 cGy). Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine A. RESULTS: The patient experienced little toxicity from the conditioning regimen and achieved complete donor chimerism within 65 days. Five months after transplant, the patient developed pure red cell aplasia that resolved after treatment. At present, he is alive in complete remission with 100% donor hematopoiesis 940 days after stem-cell transplantation. CONCLUSIONS: Allogeneic blood stem-cell transplantation using minimal conditioning is a new, promising treatment option for patients with hematologic malignancies after solid-organ transplantation.

Antibiotics, Antineoplastic↗

PubMed2002

P53 is the strongest predictor of survival in high-risk primary breast cancer patients undergoing high-dose chemotherapy with autologous blood stem cell support.

Our purpose was to determine the predictive value of tumor biologic parameters in patients with HRPBC who received HDCT with ASCT as first-line treatment. From September 1992 to May 2000, 149 stage II or III HRPBC patients were enrolled in a single-arm trial using a tandem HDCT regimen followed by ASCT. Her2/neu, p53, Ki67 and bcl-2 protein expression was studied using immunohistochemic staining on formalin-fixed, paraffin-embedded primary tumor sections. DNA content of tumor cells (DNA index) and tumor cell proliferation (SPF) were measured by DNA flow cytometry. The relationship between these tumor biologic parameters, on the one hand, and DFS, DDFS and OS, on the other, was analyzed. With a median follow-up of 43 months (range 7-106), p53 protein accumulation (p = 0.000004), negative combined hormone receptor status (p = 0.003) and Her2/neu overexpression (p = 0.02) were significant negative predictors of OS in univariate analysis. A poorer DFS was associated with p53 positivity (p = 0.04) and nodal ratio > or = 0.8 (p = 0.008). Poorer DDFS was associated with p53 positivity (p = 0.03). In multivariate analysis, Her2/neu overexpression (RR = 3.86, 95% CI 1.48-10.1, p = 0.006) and p53 overexpression (RR = 6.06, 95% CI 2.22-16.52, p < 0.001) proved to be independent predictors of adverse OS. p53 overexpression was the only independent predictor of DFS (RR = 2.21, 95% CI 1.07-4.57, p = 0.03). p53 overexpression and Her2/neu overexpression are independent negative predictors of survival in HRPBC treated with HDCT. The adverse impact of these biologic features was probably not altered by HDCT. For HRPBC patients with tumors not overexpressing Her2/neu or p53, HDCT may be an appropriate approach to achieve long-term survival and tumor control.

Adult↗

PubMed2002

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells.

CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+ (BM-CD34+) or granulocyte-colony-stimulating factor-mobilized peripheral blood CD34+ (PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle-initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+ cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

Antigens, CD34↗

PubMed2002

Primary human cells differ in their susceptibility to rAAV-2-mediated gene transfer and duration of reporter gene expression.

The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell sorting analysis. The most effective target cells for rAAV-2-mediated gene transfer were bronchial epithelial, artery endothelial as well as smooth and skeletal muscle cells with mean transduction rates ranging from 34.3 to 81.6%. Lower transduction rates between 4.3 and 19.5% were found in chondrocytes, dermal papilla follicle epithelial cells and fibroblasts. No transduction was observed in melanocytes, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) cells or malignant CD19(+) cells from patients with chronic lymphocytic leukemia. A proportion of EGFP-expressing skeletal muscle and smooth muscle cells was maintained over a period of 6 weeks after transduction (42.7+/-5.4 and 67.1+/-0.9%, respectively). Interestingly, among hair follicle epithelial cells the proportion of transduced cells increased from 8+/-0.5 to 36+/-7.7% in the course of 6 weeks. In contrast, for endothelial cells, bronchial epithelial cells and fibroblasts, a rapid decline in the number of EGFP expressing cells were noted. An inverse relationship between the proportion of cells in G2/M phase of cell cycle and long-term gene expression was observed. All rAAV-2 susceptible primary cells expressed FGFR-1 and the alphaV integrin consistent with their role as co-receptors for AAV-2. In conclusion, AAV-2 is a suitable vector system for transduction and evaluation of functional effects of long-term gene expression in primary human muscle and hair follicle cells.

Cell Line↗