Biomedical subjects

R M Bohle

Publications and source records attributed to R M Bohle.

At least 19 recordsLinked to original sources

PubMed2006

Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome.

We report the isolation of the 5' flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive.

Acute Disease↗

PubMed2006

[Gastric cancer in pregnancy -- a case report].

Nausea and vomiting are common sufferings of pregnant woman. No gynaecologist would consider carcinoma of the stomach as a probable differential diagnosis according the extremely rare probability of this disease during pregnancy. Consequently, a late diagnosis in pregnancy can result in spreading throughout the whole abdomen. In this advanced stage, it is only possible to recommend palliative care to the patient followed by short survival. Fetal metastasis is a rare entity, therefore caesarean section and chemotherapy should not be performed until fetal maturity. If vomiting and nausea are prolonged after the sixteenth week of pregnancy a malignant disease of the stomach should be excluded. Only in case of short delay between symptoms and diagnosis, the stomach cancer can be resected totally followed by a better overall survival of the patient.

Adult↗

PubMed2005

Cellular localization of estrogen receptor-alpha (ERalpha) and -beta (ERbeta) mRNA in the boar testis.

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes alpha and beta (ERalpha, ERbeta). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERalpha and ERbeta gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERalpha and ERbeta mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERalpha mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V-VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERbeta mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERalpha nor ERbeta mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERbeta and germ cell formation via ERalpha.

Animals↗

PubMed2005

Atherosclerosis: humoral and cellular factors of inflammation.

In the past decade, atherosclerosis has come to be recognized as active and inflammatory rather than simply a passive process of lipid infiltration or a reparative process after endothelial injury. In general, atherosclerosis can be considered as an intramural chronic inflammation resulting from interactions between modified lipoproteins, monocyte-derived macrophages, lymphocytes, and the normal cellular elements of the arterial wall. The process of inflammation occurs in response to functional and structural injury through a variety of known and unknown stimuli and is active over years and decades. Here, we review recent experimental and human studies of inflammatory mechanisms underlying the pathogenesis of atherosclerosis.

Animals↗

PubMed2005

Cardiac angiosarcoma: case report and review of the literature.

Angiosarcoma of the heart, the most common primary malignant cardiac tumor in adults is known to carry a dismal prognosis. The diagnosis is often delayed because of the nonspecific clinical presentation. Symptoms are determined by the size and location of the tumor. Echocardiography has become the primary diagnostic technique because of its high degree of accuracy, noninvasiveness, and cost effectiveness. Complete surgical resection is required for improved survival. Conventional postoperative chemotherapy does not appear to modify the clinical course. We report a case of cardiac angiosarcoma with a large mural mass infiltrating the right atrial and ventricular walls and critically review the pertinent literature.

Female↗

PubMed2005

Quantification of in-stent restenosis parameters in rabbits by Micro-CT.

PURPOSE: The well-defined model of angioplasty and stent implantation in the rabbit aorta is useful in experimental studies of restenosis. This study was performed to evaluate the feasibility of Micro-CT for quantification of in-stent restenosis in a rabbit aortic in-stent-restenosis model. MATERIAL AND METHODS: To induce in-stent restenosis seven female New Zealand rabbits underwent balloon dilation and denudation prior to stenting the abdominal aorta. After six weeks on atherogenic diet, animals were killed and the aorta was perfused with contrast agent. Micro-CT morphometric analysis of balloon and stent injured arteries obtained by 700 transverse sections (voxel size 10 microm) was compared to conventional histological analysis. RESULTS: Intimal hyperplasia was present in stented and balloon injured arteries with a moderate intimal area assessed by Micro-CT and conventional histology (3.91 +/- 0.5 mm(2) vs. 4.18 +/- 0.42; r = 0.9). High significant correlations between Micro-CT image analysis and conventional histomorphometry were obtained for lesion size, size of media, size of lumen and stent area (r = 0.84 to 0.96). CONCLUSION: Micro-CT is feasible for quantitative information about restenosis following balloon angioplasty and stent implantation and has the potential to become a standard technique in many laboratories which will augment serial histology as the reference method for ex-vivo studies of restenosis.

Algorithms↗

PubMed2004

[Cardio-pulmonary vascular system. Three-dimensional quantitative evaluation by microcomputed tomography].

In recent years microcomputed tomography (microCT) has become more and more important in basic research. Now commercial microCT scanners are available. Thus, it is very likely that this new, accurate and promising method for three-dimensional and non-destructive quantitative evaluation of intact tissues including vessels will be applied more frequently. The review provides a survey of the basic technology of microCT and its current use for high resolution three-dimensional morphometric and functional analysis within the cardio-pulmonary vascular system.

Aorta, Thoracic↗

PubMed2004

Analysis of the fetal placental vascular tree by X-ray micro-computed tomography.

The current understanding of the placental vascular tree largely derives from time-consuming morphometric analyses performed by conventional histology, electron microscopy of corrosion casts and three-dimensional reconstructions based on physical tissue sections. In the present study, we demonstrate for the first time that micro-computed tomography (micro-CT) emerges as a new, non-destructive and fast tool for imaging and quantifying fetoplacental vasculature. Term placentae (n=5) were perfused with contrast agent consisting of barium-sulfate, gelatine and thymol shortly after Caesarean-section-delivery. Samples (1 cm(3)) from eight different regions of the placenta were subsequently scanned in a micro-CT. Using tomographic reconstruction algorithms, three dimensional images were obtained by micro-CT allowing total stereoscopic visualization and continuous quantitative analysis of the vascular structure of the investigated samples. These samples were compared regarding vascular surface (VS) and vascular density (vascular volume fraction, TCVF). Quantitative assessment showed an average vascular density of 16 per cent (SD+/-0.4) and a vascular surface of 475 mm(2)(SD+/-8) per total tissue volume (including intervillous space) of 125 mm(3). Micro-CT image-analysis showed no significant differences in the fetal vascularization among term placentae. Micro-CT imaging is feasible for imaging and analysis of the villous vascular tree, allows further morphologic studies and immunohistochemistry of the placental specimens and may emerge as an additional tool in the investigation of the physiology and pathophysiology of the placental vasculature.

Barium Sulfate↗

PubMed2004

[Micro-computed tomography of the vasculature in parenchymal organs and lung alveoli].

Micro-CT has become a powerful technique in non-destructive 3D imaging and morphometric analysis. First results were limited to the investigation of osteoporosis in cancellous bone. But the availability of systems with almost microscopic resolution and sufficient soft tissue contrast has opened up entirely new applications for laboratory investigation of blood vessels and soft tissues. This article gives an overview of micro-CT technology and the potential of three-dimensional imaging of the vessel wall and soft-tissue architecture imaging in different organs using different contrast perfusion and staining techniques. Micro-CT provides quantitative information on human plaque morphology equivalent to histomorphometric analysis. Based on differences in grey-scale attenuations, micro-CT also correctly identifies atherosclerotic lesions that are histologically classified as fibrous plaques, calcified lesions, fibroatheroma, and lipid rich lesions. Micro-CT is a promising method to visualize the architecture of the renal vasculature and, importantly, to separate cortex and medulla for the visualization of glomeruli and their afferent and efferent arterioles. Micro-CT can determine the vascular surface in a defined placental volume. Combining of micro-CT data and total placental volume enables an estimation of the approximate surface of the placental vasculature. The diameter of opacified vessels in the investigated samples ranged from 2 mm (chorion plate artery) to 14 micro m (smallest vessel diameter, terminal loop). Recognizing that lung parenchyma can only be visualized if the alveoli are completely expanded and the contrast of the thin alveolar walls is enhanced, we tested two preparation methods: (1) fixation of lung tissue with formalin vapour and staining with silver nitrate, and (2) intravenous injection of a barium sulfate-gelatine-thymol mixture in vivo in the anesthetized animal. We evaluated the ability of this mixture to enter the pulmonary microcirculation and the technical feasibility of micro-CT to assess lung micro-architecture.

Animals↗

PubMed2003

Design and evaluation of chemically synthesized siRNA targeting the NPM-ALK fusion site in anaplastic large cell lymphoma (ALCL).

The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.

Base Sequence↗

PubMed2001

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia.

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5'-MLL/FBP17-3' fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Artificial Gene Fusion↗

PubMed2001

Heart transplantation in children and infants: short-term outcome and long-term follow-up.

Since 1988, 82 heart transplants have been performed in 80 infants and children. Diagnoses pretransplant were: hypoplastic left heart syndrome (HLHS) (n = 43); cardiomyopathy (n = 19); endocardial fibroelastosis (n = 6); and other complex congenital heart diseases (n = 12). Age at transplantation was < 1 yr in 61 patients. Overall survival rate was 79% at 1 yr and 73% at 5 and 10 yr. To date, 20 patients have died after transplantation. Causes of death were: rejection (eight patients); right ventricular failure (four patients); transplant coronary artery disease (TCAD) (two patients); and other causes (six patients). In the majority of patients somatic growth is not impaired, and renal function is reduced (but stable) in all patients. Two patients developed post-transplant lymphoproliferative disease, which was treated successfully. Major long-term morbidity is neurologic deficit - severe in three patients and minor in six. TCAD was present or suspected in six surviving patients. We conclude that heart transplantation in infants and children can be performed with good early and late results. Quality of life is excellent in most patients. TCAD, however, will become an increasing problem in the long term.

Child↗

PubMed2001

Biosynthesis of constitutive nitric oxide synthase-derived nitric oxide attenuates coronary vasoconstriction and myocardial depression in a model of septic heart failure induced by Staphylococcus aureus alpha-toxin.

OBJECTIVE: Myocardial depression, which frequently occurs in the course of septic shock, has been attributed to the cardiodepressant properties of nitric oxide (NO) generated by either the inducible NO synthase (iNOS) or the constitutive isoform (cNOS). We have previously demonstrated that alpha-toxin from Staphylococcus aureus induces thromboxane-mediated vasoconstriction accompanied by severe cardiodepression in isolated rat hearts. In the present study, we investigated the role of NO in the alpha-toxin-induced vascular and contractile abnormalities. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified staphylococcal alpha-toxin for 60 mins. MEASUREMENTS AND MAIN RESULTS: At a concentration of 0.25 and 0.5 microg/mL, alpha-toxin induced a rise in coronary perfusion pressure, depressed myocardial contractility, and caused edema formation. Simultaneously, a time- and dose-dependent rapid release of NO into the perfusate was noted as quantified by a chemiluminescence technique. L-NMMA, a nonselective inhibitor of NOS, but not PBITU, an iNOS-selective inhibitor, blocked NO synthesis, markedly increased the rise in coronary perfusion pressure and the loss in contractility, and enhanced edema formation in response to alpha-toxin. In contrast, zaprinast, a selective inhibitor of phosphodiesterase type V that is used for stabilization of cyclic guanosine monophosphate, attenuated the toxin-induced coronary vasoconstrictor response and the myocardial depression. L-arginine, the substrate of NOS, had similar, yet less potent, effects as zaprinast and slightly increased the release of NO caused by alpha-toxin. Immunohistochemical analysis of the myocardium at the end of the perfusion period demonstrated a positive staining for cNOS but not for iNOS. In addition, no up-regulation of iNOS mRNA was detected in the tissue of toxin-exposed hearts. CONCLUSIONS: Staphylococcal alpha-toxin provokes NO biosynthesis via activation of cNOS in rat hearts. NO partly antagonizes the deleterious effects of this pathogenicity factor on coronary vasoregulation and myocardial performance.

Animals↗

PubMed2000

Endotoxin-induced myocardial tumor necrosis factor-alpha synthesis depresses contractility of isolated rat hearts: evidence for a role of sphingosine and cyclooxygenase-2-derived thromboxane production.

BACKGROUND: Although endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. METHODS AND RESULTS: Perfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-alpha into the perfusate and preceded by myocardial TNF-alpha mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-alpha antiserum or the ceramidase inhibitor N:-oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A(2) and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-alpha and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. CONCLUSIONS: LPS depresses contractility of isolated rat hearts by inducing TNF-alpha synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2-derived TxA(2) appears to facilitate TNF-alpha synthesis in response to LPS.

Animals↗

PubMed2000

Na(+)/H(+) exchange inhibitor cariporide attenuates cell injury predominantly during ischemia and not at onset of reperfusion in porcine hearts with low residual blood flow.

BACKGROUND: This study investigated whether myocardial protection by inhibition of Na(+)/H(+) exchange (NHE) occurs during ischemia and/or during reperfusion. METHODS AND RESULTS: The left anterior descending coronary artery was occluded in 32 pigs for 60 minutes and then reperfused for 24 hours. Infarct sizes (nitroblue tetrazolium [NBT] stain, histology) were determined at the end of the experiments. An extracorporeal bypass was used to achieve a constant residual blood flow of 3 mL/min in the myocardium at risk during ischemia. The NHE-1 inhibitor cariporide or distilled water was infused into the extracorporeal bypass system. In group 1, active treatment was administered from the onset of ischemia until 10 minutes of reperfusion (n=8). In group 2, active treatment was infused during the first 30 minutes of ischemia only (n=8). The group 3 animals (n=8) received intracoronary cariporide after 45 minutes of ischemia until 10 minutes of reperfusion. The control animals (group 4, n=7) were treated similarly to group 1 animals, with the cariporide solution being replaced by distilled water. Infarct sizes of group 1 (NBT stain, 41.5+/-20%; histology, 44. 6+/-12%) and group 2 (NBT stain, 33.5+/-14%; histology 34.9+/-15%) differed significantly (at least P:=0.012) from infarct sizes of group 3 (NBT stain, 71.6+/-15%; histology, 69.2+/-12%) and the control group (NBT stain, 76+/-9%; histology 72.4+/-12%). Cariporide treatment in group 1 and group 2 significantly improved functional recovery after 24 hours of reperfusion. CONCLUSIONS: Myocardial protection by cariporide is predominantly achieved by NHE inhibition during ischemia and not during early reperfusion.

Animals↗

PubMed2000

[Therapy-resistant ventricular tachycardia in a woman with primary cardiac non-Hodgkin lymphoma].

HISTORY: A 65-year-old woman had suffered from relapsing ventricular tachycardias (VT) since 1996. FINDINGS: Physical examination was normal. An arrhythmogenic substrate was found in the right ventricular outflow tract by electrophysiological examination. Nuclear magnetic resonance imaging (MRI) showed an infiltration of the right heart. Myocardial biopsy revealed a high-grade centroblastic non Hodgkin lymphoma. The patient was now transferred to our hospital for further treatment. Lactate dehydrogenase was elevated (2,030 U/l). Echocardiography showed a thickened and more reflecting right ventricular myocardium. Bone marrow aspiration and MRI/computed tomography of abdomen and thorax excluded a generalized stage. Ventricular tachycardias were caused by a primary cardiac lymphoma. TREATMENT AND COURSE: Combined radio-chemotherapy succeeded in complete remission. High-frequency ablation and amiodarone failed. Although MRI showed no more vital lymphoma after the combined radio-chemotherapy the patient suffered from spontaneous and symptomatic relapses of VT. Therefore this patient with primary cardiac lymphoma was the first in literature to get a defibrillator (ICD). The incidence of VT decreased and up to now the patient showed no relapse of the non Hodgkin lymphoma (follow-up 23 months).

Aged↗

PubMed2000

The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).

Artificial Gene Fusion↗

PubMed2000

Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts.

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.

Animals↗