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Biomedical subjects

Hans Kreipe

Publications and source records attributed to Hans Kreipe.

At least 55 records · Page 3Linked to original sources
PubMed2004

Different expression levels of the telomerase catalytic subunit hTERT in myeloproliferative and myelodysplastic diseases.

Telomerase is the enzyme required for the addition of telomeric repeats to the ends of chromosomes and thus for chromosome stability. Most somatic human cells lack telomerase activity because they do not express the telomerase reverse transcriptase (hTERT) gene. In contrast, in almost every neoplasia, cancer cells express hTERT and therefore prevent progressive telomere shortening during each cell division. Consecutively, cancer cells obtain the ability to divide without limits and overcome replicative senescence. Gene expression level of the telomerase catalytic subunit hTERT in normal and neoplastic haematopoiesis has not yet been described. Using a quantitative real-time RT-PCR assay, we analysed the level of hTERT expression in various haematologic stem cell disorders and normal bone marrow. We could demonstrate that hTERT is differentially expressed in various haematologic stem cell disorders with significant higher levels in refractory anemia (RA) and chronic myeloid leukemia (CML) compared to other haematopoietic stem cell disorders and non-neoplastic haematopoiesis which may be used as a prognostic indicator in these entities.

Catalytic Domain↗
PubMed2004

Overexpression of delta-like (Dlk) in a subset of myelodysplastic syndrome bone marrow trephines.

The mRNA expression level of the gene delta-like (Dlk), coding for a signal transducer related to the Delta-Notch family, was measured using real-time PCR methodology in a large series of control biopsies (n = 61) and bone marrow trephines from patients with myelodysplastic syndrome (MDS) (n = 93) and related myeloid disorders (AML, n = 16 and chronic myeloproliferative disease (CMPD), n = 38). It turned out that dlk is strongly overexpressed in a subset of MDS (16%) cases, however with higher frequency in blast rich MDS cases. Therefore, the quantitative detection of dlk mRNA can support the morphological diagnosis of MDS in selected cases.

Bone Marrow Examination↗
PubMed2004

Aberrant expression of insulin-like growth factor-2 (IGF-2) in Philadelphia chromosome negative chronic myeloproliferative disorders.

Philadelphia chromosome negative chronic myeloproliferative disorders (Ph- CMPD) comprise haematopoietic stem cell disorders with currently unknown underlying molecular defect. Insulin-like growth factor 2 (IGF-2) is an imprinted gene that is known to be involved in the regulation of normal cell growth and that is overexpressed by a variety of tumors. The expression of IGF-2 in bone marrow cells is largely unknown. In order to elucidate gene expression level, protein expression pattern, and a potential role of IGF-2 in the pathogenesis of Ph- CMPD, we quantitatively analyzed the expression of the IGF-2 gene in bone marrow cells of 69 cases with Ph- CMPD and 31 control cases by applying real-time RT-PCR. IGF-2 gene expression in idiopathic myelofibrosis (IMF) was significantly increased by up to 11-fold as compared to the control group (P < 0.0001). IMF also expressed higher IGF-2 gene level as compared to essential thrombocythaemia (ET) and polycythaemia vera (PV) (P < 0.0001, P = 0.005, respectively). Paranuclear IGF-2 protein could be demonstrated in IMF, ET, and PV exclusively in megakaryocytes and myeloid progenitor cells in contrast to undetectable IGF-2 protein in control cases. We conclude that overexpression of the IGF-2 gene is a pathogenic feature in IMF. In addition, an abundant translational and post-translational processing could explain the accumulation of IGF-2 protein detectable in all Ph- CMPD entities in contrast to non-neoplastic haematopoiesis. We conclude that IGF-2 represents a new molecular target for evaluation of underlying fundamental pathomechanisms in Ph- CMPD.

Adult↗
PubMed2004

Chimerism of metanephric adenoma but not of carcinoma in kidney transplants.

Recipient-derived cells integrate into renal allografts inducing organ-specific microchimerism. Circulating pluripotent progenitor cells with high plasticity for differentiation were suggested as a potential source of allograft chimerism. Whether or not these cells also contribute to tumor formation in renal transplants is unknown. We analyzed six histologically different tumors in renal allografts for the presence of recipient-derived cells. To circumvent dependency on gender mismatch, a polymerase chain reaction assay for highly polymorphic short tandem repeat marker (DNA fingerprinting) in combination with laser microdissection was applied. Pure tumor cell populations were harvested by laser microdissection after immunohistochemical (CD45/CD68) marking of contaminating leukocytes. In cases of gender mismatch (n = 2), results were confirmed by sex chromosome in situ hybridization. Two metanephric adenomas demonstrated microchimerism comprising both donor- and recipient-derived tumor cells. Two clear cell carcinomas, one transitional cell carcinoma, and one renal cortical adenoma were all of donor origin without chimerism. We conclude that except for metanephric adenomas, tumors arising in renal transplants originate completely from graft cells. The mixed derivation of metanephric adenomas indicates an incorporation of recipient-derived progenitor cells. This finding suggests that adult stem cells can assume neoplastic phenotypes.

Adenocarcinoma, Clear Cell↗
PubMed2004

Constitutive expression of the FK506 binding protein 51 (FKBP51) in bone marrow cells and megakaryocytes derived from idiopathic myelofibrosis and non-neoplastic haematopoiesis.

OBJECTIVES: Overexpression of FK506 binding protein 51 (FKBP51) in megakaryocytic progenitor cells generated from purified CD34+ cells in patients with idiopathic myelofibrosis (IMF) has been demonstrated. It has been suggested that FKBP51 is involved in the dysregulation of the apoptotic programme with consecutive prolongation of cell survival. The knowledge of FKBP51 and its expression in bone marrow cells and mature megakaryocytes in non-neoplastic haematopoiesis and IMF is sparse. METHODS: To evaluate a potential overexpression of FKBP51 in patients with IMF (n = 37) compared with non-neoplastic haematopoiesis (n = 31), total bone marrow cells as well as single megakaryocytes, isolated by laser microdissection, were quantitatively analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). By applying immunohistochemistry, FKBP51 gene expression was correlated with staining pattern and cellular localisation of the corresponding FKBP51 protein. RESULTS: We demonstrated that FKBP51 is constitutively expressed in non-neoplastic haematopoiesis. FKBP51 gene expression by total bone marrow cells as well as megakaryocytes was not significantly different in IMF. FKBP51 protein expression could be localised to myeloid progenitor cells as well as megakaryocytes. In particular, megakaryocytes were stained almost exclusively nuclear for FKBP51. No differences in expression patterns between both IMF and control cases could be demonstrated. CONCLUSIONS: For the first time, FKBP51 expression, in particular gene expression and subcellular localization was described in bone marrow cells of non-neoplastic and neoplastic haematopoiesis grown in vivo. We conclude that FKBP51 could be temporarily overexpressed in megakaryocytic progenitors rather than contribute to the accumulation of mature megakaryocytes in IMF.

Adult↗
PubMed2004

Tubular chimerism occurs regularly in renal allografts and is not correlated to outcome.

Recent studies have demonstrated an integration of recipient-derived progenitor cells into solid allografts with differentiation into parenchymal cells. Whether or to what extent this phenomenon influences allograft outcome has still to be elucidated. To detect epithelial chimerism tubular cells were harvested from sequential renal allograft biopsy samples by laser microdissection in 36 patients. Recipient-derived cells were detected by short-tandem repeat-based genotyping. In cases with gender-mismatched transplantation, chimerism was semiquantitatively evaluated by in situ hybridization for the Y-chromosome. Findings were correlated to different pathomechanisms of epithelial injury as well as to morphologic and clinical outcome. Epithelial chimerism was detectable as early as 8 d after transplantation and lasted for 8 yr. A total of 88% of the patients showed an epithelial chimerism; 72% had a stable chimerism in sequential biopsy samples. Evaluation of Y-chromosome by in situ hybridization revealed low percentages of chimerical tubular epithelial cells (2.4% to 6.6%). No correlation to morphology was found. Chimerism was detectable in inconspicuous protocol biopsy samples, cases of drug toxicity, and rejected allografts with and without chronic changes. No correlation was found to allograft function. Epithelial microchimerism is an early and persistent phenomenon after renal transplantation. There is no correlation to morphologic or functional outcome. Probably recipient-derived stem cells contribute in a minor fashion to tissue homeostasis, and cell turnover in renal allografts is predominantly enabled by donor cell renewal.

Biopsy↗
PubMed2004

Aberrant expression of beta-catenin discriminates acute myeloid leukaemia from acute lymphoblastic leukaemia.

The role of beta-catenin in epithelial neoplasms has been widely studied whereas current knowledge regarding beta-catenin gene and protein expression in bone marrow cells derived from normal haematopoiesis and clonal haematological disorders is lacking. beta-Catenin gene expression was quantitatively investigated in bone marrow cells derived from clonal haematological disorders [acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), Philadelphia chromosome-positive chronic myeloid leukaemia (Ph+ CML], Ph- myeloproliferative disorders, n = 96) compared with non-neoplastic haematopoiesis (n = 33) by real-time reverse transcription polymerase chain reaction. Cellular localization of beta-catenin protein was detected by immunocytochemistry. beta-Catenin gene expression was significantly increased in AML compared with ALL cases (P < 0.0001), Ph+ CML (P < 0.0001) and non-neoplastic haematopoiesis (P = 0.019). Immunocytochemistry revealed that, in non-neoplastic haematopoiesis, the granulopoietic lineage as well as megakaryocytes showed membranous and cytoplasmic staining to various degrees along with unlabelled nuclei. Besides haematopoiesis, beta-catenin prominently marked bone marrow vascularity and diverse stroma cells. beta-Catenin gene was inversely expressed in AML and ALL with a lack of protein expression in neoplastic cells in ALL. In contrast, the other haematological disorders under study, except for Ph+ CML, did not show significant alterations of overall beta-catenin gene expression compared with normal bone marrow. These data suggest different regulatory mechanisms in the expression and function of beta-catenin in haematopoietic cells.

Acute Disease↗
PubMed2004

Real-time PCR-based assay for quantitative determination of methylation status.

The best studied epigenetic modification in mammals is the methylation of cytosine. During the development and progression of malignant neoplasia, a global hypomethylation is often accompanied by a locus-specific increase in methylation. Also, during normal development specific alterations in DNA methylation patterns take place. In recent years it has become clear that in many situations only quantitative changes in methylation levels occur and that the pure qualitative detection of cytosine methylation misses important biological and pathophysiological information. Therefore, several protocols were developed for the quantitative detection of cytosine methylation. Here, we describe a real-time polymerase chain reaction-based assay for the sensitive and precise quantification of methylated and unmethylated alleles after bisulfite treatment of genomic DNA. In addition to providing quantitative methylation data, this methodology is suitable for high-throughput analysis.

Animals↗
PubMed2003

Role of epigenetic changes in hematological malignancies.

Inactivation of tumor suppressor genes is an important event contributing to the development of neoplasia. In addition to the classic genetic mechanisms of deletion or inactivating point mutations, growth regulatory genes can be functionally inactivated without alterations of the primary sequence by methylation of cytosine residues in the promoter regions of the genes. After introducing epigenetic phenomena in general and the molecular basis of DNA methylation in more detail, this review will present the broad spectrum of alterations in DNA methylation patterns found in hematopoietic malignancies. In addition, the implications for therapy and prognosis will be discussed.

Animals↗
PubMed2003

IGF-1R, IGF-1 and IGF-2 expression as potential prognostic and predictive markers in colorectal-cancer.

Insulin-like growth factors (IGF-1 and IGF-2) and the IGF-1 membrane receptor (IGF-1R) have been found to play a critical role in the carcinogenesis of several tumors, among them colorectal cancer (CRC). To study the prognostic impact of these molecules, a total number of 713 cases of CRC were examined for the expression of IGF-1, IGF-2, and IGF-1R. The results were correlated with other clinicopathological data and clinical follow-up. IGF-1 expression was noted in 53 (7.5%), IGF-2 in 88 (12.6%), and IGF-1R in 698 (99.6%) of the cases. There were significant associations between the two growth factors ( P<0.00001), between IGF-1 and Ki-67 proliferation activity ( P<0.05), and between IGF-2 and tumor stage ( P<0.005). IGF-2 positivity was significantly correlated to a worse clinical outcome ( P<0.005) only in univariate, but not in multivariate, survival analysis. A similar trend was obtained for patients with IGF-1-positive CRC, but reached statistical significance only in limited tumor stages (pT1/pT2; P<0.01). Although the synchronous expression of IGF-1, IGF-2, and IGF-1R in a subset of CRC is consistent with an auto-/paracrine loop of cancer cell autostimulation, the prognostic effect of IGF-1 and IGF-2 expression seems to be of limited value. However, the identification of IGF-positive CRC might be beneficial for predictive reasons, as new molecular therapeutic approaches are aimed at the IGF system and related pathways.

Adenocarcinoma↗
PubMed2003

Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury.

Chimerism on the parenchymal level has been shown for several human allografts, including liver, heart, and kidney, with the integrated recipient-derived cells most likely originating from multipotent bone marrow precursors. We investigated whether chimerism also occurs within epithelial structures of the lung. For this purpose archival tissue biopsies from seven explanted human lung allografts were obtained. We performed laser microdissection of the target structures with subsequent short tandem repeat analysis to detect chimerism within the isolated cells. We found integration of recipient-derived cells in the bronchial epithelium, in type II pneumocytes and in seromucous glands lying adjacent to larger bronchi in all lung allografts studied. Quantitative analysis revealed that the epithelial structures displaying signs of chronic injury, such as squamous metaplasia, showed a markedly higher degree of chimerism (24% versus 9.5%). We therefore conclude that in human lungs, epithelial chimerism occurs at least within bronchi, type II pneumocytes, and seromucous peribronchial glands. Although a bone marrow origin of immigrating host-derived stem cells has been suggested by previous studies in rodents, analysis of lung biopsies from bone marrow-transplanted patients (n = 3) could not prove such delineation in this study. The observation of an enhanced integration of recipient cells into chronically damaged epithelial structures suggests that extrapulmonary precursor cells are able to contribute to pulmonary regeneration.

Adolescent↗
PubMed2003

Quantitative intra-individual monitoring of BCR-ABL transcript levels in archival bone marrow trephines of patients with chronic myeloid leukemia.

We established a quantitative real-time RT-PCR assay for the detection of chimeric BCR-ABL transcripts in archival formalin-fixed bone marrow trephines, both acrylate-embedded and paraffin-embedded. This new methodology enables determination of transcript levels in direct comparison to histopathological findings and therapeutic interventions during the time course of the disease in a retrospective and a prospective manner. We found an excellent correlation between the quantitative molecular data and the morphological evaluation as well as the clinical outcome for a cohort of chronic myeloid leukemia patients (n = 10). To the best of our knowledge, this is the first study demonstrating the feasibility of large-scale quantitative expression analysis in archival bone marrow trephines for monitoring molecular markers over several years or even decades.

Base Sequence↗
PubMed2003

Expression profiling of breast cancer cells by differential peptide display.

Expression profiling of RNAs or proteins has become a promising means to investigate the heterogeneity of histopathologically defined classes of cancer. Peptides, representing degradation as well as processing products of proteins offer an even closer insight into cell physiology. Peptides are related to the turnover of cellular proteins and are capable to reflect disease-related changes in homoeostasis of the human body. Furthermore, peptides derived from tumor cells are potentially useful markers in the early detection of cancer. In this study, we introduced a method called differential peptide display (DPD) for separating, detecting, and identifying native peptides derived from whole cell extracts. This method is a highly standardized procedure, combining the power of reversed-phase chromatography with mass spectrometry. This technology is suitable to analyze cell lines, various tissue types and human body fluids. Peptide-based profiling of normal human mammary epithelial cells (HMEC) and the breast cancer cell line MCF-7 revealed complex peptide patterns comprising of up to 2300 peptides. Most of these peptides were common to both cell lines whereas about 8% differed in their abundance. Several of the differentially expressed peptides were identified as fragments of known proteins such as intermediate filament proteins, thymosins or Cathepsin D. Comparing cell lines with native tumors, overlapping peptide patterns were found between HMEC and a phylloides tumor (CP) on the one hand and MCF-7 cells and tissue from a invasive ductal carcinoma (DC) on the other hand.

Biomarkers, Tumor↗
PubMed2003

The polycythaemia rubra vera-1 gene is constitutively expressed by bone marrow cells and does not discriminate polycythaemia vera from reactive and other chronic myeloproliferative disorders.

Little is known about the expression of the polycythaemia rubra vera-1 (PRV-1) gene in bone marrow cells. To determine the expression level of PRV-1 in the bone marrow, we analysed PRV-1 quantitatively in Polycythaemia vera, other chronic myeloproliferative disorders, and reactive states. We demonstrated that PRV-1 was constitutively expressed in both myeloproliferative and reactive states. We concluded that, rather than an upregulation of the PRV-1 gene in the clonal haematopoiesis of polycythaemia vera, a failure to downregulate PRV-1 in granulocytes emigrating from the bone marrow might be responsible for the increase of transcripts in peripheral cells.

Bone Marrow Cells↗
PubMed2003

Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases.

The contribution of the acute phase inducer interleukin 6 (IL-6) in the pathogenesis of liver diseases is yet unclear. Our analysis showed enhanced expression of IL-6 in livers derived from patients with acute and chronic liver diseases. Additionally, IL-6 plasma levels were significantly increased in patients with chronic liver diseases and showed an inverse correlation with biochemical markers of liver function and a positive correlation with inflammatory markers, signs of portal hypertension, and the degree of liver fibrosis. To prove the relevance of these clinical findings, we applied the tetrachlorcarbonide (CCl(4)) model to conditional knockout animals (Cre/loxP system) for gp130, the common signal transducer of IL-6 family cytokines. Cre recombinases were expressed through a hepatocyte (AlfpCre) and a ubiquitous (MxCre) control element. Gp130 deleted mice had a totally abolished STAT3 activation and acute phase response induction, but gp130 deletion had no effect on the degree of acute liver injury and subsequent hepatocyte proliferation. In contrast, during chronic liver injury induced by biweekly application of CCl(4), deletion of the gp130 receptor in nonparenchymal liver cells and not hepatocytes resulted in fibrosis progression. In conclusion, our experiments indicate an involvement of IL-6 in the pathogenesis of liver diseases and suggest a protective role of IL-6/gp130-dependent pathways in nonparenchymal liver cells during fibrosis progression in chronic liver diseases. (Hepatology 2003;38:218-229).

Acute Disease↗
PubMed2003

Incidence of polyomavirus-nephropathy in renal allografts: influence of modern immunosuppressive drugs.

BACKGROUND: In recent years an increasing number of cases with polyomavirus (PV)-nephropathy after renal transplantation were reported from several transplant centres. New, highly potent immunosuppressive drugs like tacrolimus or mycophenolate mofetil were accused as risk factors for this increase. However, data about the incidence of PV-nephropathy in correlation to different immunosuppressive therapy concepts are lacking. METHODS: All renal transplant biopsies performed at Hannover Medical School between 1999 and 2001 (n=1276) were immunohistochemically screened for the presence of PV-specific proteins. The results were correlated to the different immunosuppressive therapy protocols and patients with PV-nephropathy were compared with a matched control group. RESULTS: PV-nephropathy was found in <1% of all investigated allograft biopsies (11/1276) and in approximately 1% of all patients (7/638), respectively. All patients being immunohistochemically positive for PV-specific proteins also showed the typical morphological changes of PV-nephropathy. Four out of seven patients with PV-nephropathy were under triple immunosuppression comprising tacrolimus and mycophenolate mofetil. Under this immunosuppressive therapy protocol an eight times higher incidence and a 13 times higher risk (multivariate odds ratio 12.7) of PV-nephropathy was observed in our patients compared with the control group. CONCLUSIONS: PV-nephropathy is a rare but serious complication after renal transplantation. A small group of patients under intensive immunosuppression comprising tacrolimus in combination with mycophenolate mofetil has a significantly increased risk of acquiring this deleterious complication.

Adolescent↗
PubMed2003

Multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts in paraffin-embedded tissues from patients with chronic myeloid leukemia and acute lymphoblastic leukemia.

Diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia requires the investigation of the Philadelphia chromosome translocation t(9;22) or the molecular detection of BCR-ABL fusion transcripts. Determination of the type of fusion transcript is crucial for quantitative molecular monitoring the course of the disease during treatment. Histopathologists, who usually use formalin-fixed tissues, may be confronted with the need to investigate the BCR-ABL rearrangement when evaluating tumor forming infiltrates and bone marrow trephines from patients presenting with chronic myeloproliferative disorders. Therefore, we have established a one-tube multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts (b2a2, b3a2, e1a2) in routinely processed tissues and bone marrow trephines with respect to the inevitable fragmentation of ribonucleic acids in these specimens. RT-PCR products allow distinct and unequivocal differentiation of the underlying fusion in either the Major- or minor-breakpoint cluster region. Detection of BCR-ABL fusion transcripts by multiplex RT-PCR in routinely processed and fixed tissues is a time- and cost-sparing tool for definite diagnosis of typical chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.

Bone Marrow Cells↗