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Biomedical subjects

K Grünewald

Publications and source records attributed to K Grünewald.

At least 37 records · Page 2Linked to original sources
PubMed1995

Detection of hepatitis C viral sequences in serum by 'nested' polymerase chain reaction (PCR) and a commercial single-round PCR assay.

BACKGROUND: Demonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed. OBJECTIVE: The aim of the present study was to compare the new Amplicor assay with an 'in-house' PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified. STUDY DESIGN: Sera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h. RESULTS: A total of 52 patients were found to be positive for anti-HCV by the third-generation EIA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay. CONCLUSION: Because of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.

Journal Article↗
PubMed1995

Necrotizing vasculitis associated with hepatitis C virus infection: successful treatment of vasculitis with interferon-alpha despite persistence of mixed cryoglobulinemia.

Type II cryoglobulinemia may be associated with chronic hepatitis C virus (HCV) infection and may be characterized by vascular purpura. We report on a case of histologically proven necrotizing vasculitis associated with type II cryoglobulinemia and HCV infection. Within 14 days of interferon-alpha therapy (3 x 3 million IU/ml/week), purpuric skin lesions disappeared as well as fatigue and arthralgia; 9 months after initiation of therapy, liver enzyme values were nearly normal despite persistence of HCV RNA tested by PCR and mixed cryoglobulinemia. Rheumatoid factor activity, however, decreased markedly. To our knowledge, our patient is the first reported case with histologically proven necrotizing vasculitis with a beneficial effect of interferon-alpha. Because of the persistence of cryoglobulins, but reduction of the IgM fraction in the cryoglobulin complex under interferon-alpha treatment, it would seem worthwhile to further elucidate the pathogenic role of qualitative instead of quantitative changes of cryoglobulins and the mechanism of action of interferon-alpha.

Arthralgia↗
PubMed1995

Expression of c-fos correlates with IFN-alpha responsiveness in Philadelphia chromosome positive chronic myelogenous leukemia.

This study evaluates (i) constitutive levels of oncogene and p53 transcripts in chronic phase CML patients and (ii) their modulations subsequent to in vivo therapy with rIFN-alpha 2c. Peripheral blood mononuclear cells (pbmc) and bone marrow cells of 26 patients were examined for c-fos, c-myc, p53 and the hybrid bcr/abl mRNA levels. Results indicated that (i) constitutive c-fos transcript levels are significantly higher in patients subsequently responding to IFN-alpha therapy (p < 0.01) and positively correlated with the proportion of lymphocytes (r = 0.6895, p < 0.01) and negatively with the proportion of immature cells (r = -0.568, p < 0.01) contained in the pbmc preparations tested, (ii) constitutive mRNA levels of the hybrid bcr/abl, c-myc and p53 are positively correlated with each other, but failed to relate to disease parameters, and (iii) acute and chronic in vivo exposure to IFN-alpha is accompanied by upregulation of c-fos and downregulation of c-myc mRNA levels in responder patients.

Bone Marrow Cells↗
PubMed1994

Iron regulates nitric oxide synthase activity by controlling nuclear transcription.

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Amino Acid Oxidoreductases↗
PubMed1994

Syringotropic cutaneous T-cell lymphoma: a variant of mycosis fungoides?

We report two patients with the typical clinical (patches/plaques studded with brownish-red papules) and histopathological (hyperplastic eccrine ducts and glands surrounded and infiltrated by lymphocytes) features of syringotropic cutaneous T-cell lymphoma. Immunohistochemical studies confirmed the T-cell character of the infiltrate, and gene rearrangement studies its monoclonality (in one case). Our patients did not present with visceral involvement.

Adult↗
PubMed1994

Persistence of virus and viral genome in myocardium after coxsackievirus B3-induced murine myocarditis.

Following infection with Coxsackievirus B3 (CVB3), A-strain mice develop ongoing myocarditis that persists after the virus ceases to be cultivatable from heart tissue. We studied the natural history of this virus-induced but apparently autoimmune inflammation by means of in situ hybridization (ISH) and by polymerase chain reaction (PCR). Both ISH and culture allowed detection of virus up to 2 weeks post-infection in virtually all heart tissues. In contrast, PCR revealed the presence of viral genome for a substantially longer period of time, i.e. at least 34 days after CVB3 infection. Similarly, the majority of mice showed myocardial inflammation at this time point. However, the persistence of virus did not correlate with ongoing myocarditis, and vice versa. Most mice with ongoing myocarditis produced heart myosin autoantibodies, most probably as a result of tissue damage. The lack of correlation between presence of ongoing inflammation and persistence of virus supports our previous view that the late phase of CVB3-induced myocarditis is mediated by autoimmunological mechanisms.

Animals↗
PubMed1994

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Base Sequence↗
PubMed1994

Early diagnosis of gastric lymphoma: gene rearrangement analysis of endoscopic biopsy samples.

The diagnosis of gastric lymphoma in endoscopic biopsy specimens remains difficult despite the emergence of accepted criteria for the histologic diagnosis of lymphomas originating from mucosa-associated lymphoid tissue (MALT). The sensitivity and validity of immunoglobulin (Ig) gene rearrangement analysis of mucosal biopsies for the diagnosis of malignant B-cell lymphoma were investigated in comparison with conventional histology and immunohistology. Biopsy specimens from 34 different endoscopies of 20 patients with a previous history, or tentative diagnosis of gastric lymphoma, and 12 control samples were analyzed for the presence of clonal Ig gene rearrangements. A clonal B-cell population was detected by Southern blot analysis in all patients with a definitive histologic diagnosis of lymphoma. In addition, in two patients the detection of clonal rearrangements in biopsy specimens preceded by several months the histologic diagnosis of lymphoma, and clonality was confirmed in three further patients where histology remained inconclusive. In some cases of low-grade MALT-lymphoma, discrete spreading of malignant cells within chronically inflamed mucosa was suggested by the presence of identical clonal rearrangements in all simultaneously obtained biopsies, with or without histologically detectable involvement by lymphoma. Our results show that immunoglobulin gene rearrangement studies of endoscopic biopsy samples are an additional powerful tool for the diagnosis of gastric lymphoma, especially for detecting early recurrence, and improve the preoperative assessment of the extent of mucosal involvement.

Biopsy↗
PubMed1993

Detection of enteroviral ribonucleic acid in myocardial biopsies from patients with idiopathic dilated cardiomyopathy by polymerase chain reaction.

Infection by enteroviruses, especially by Coxsackie B viruses, has been incriminated in pathogenesis of dilated cardiomyopathy. We developed polymerase chain reaction tests for the detection of enteroviral and Coxsackie B3 genomes, respectively, in myocardial biopsies obtained from a homogeneous group of 19 patients with idiopathic dilated cardiomyopathy. To determine unambiguously the incidence of enteroviruses and Coxsackie B3 viruses in these patients, we used two primer pairs, one common to all enteroviruses and the other specific for Coxsackie B3 viruses. In six patients of the dilated cardiomyopathy group, enteroviral ribonucleic acid (RNA) could be detected; only one was subspecified as Coxsackie B3 RNA. In contrast, no enteroviral RNA could be detected in a contrast group of 21 patients with other cardiac disorders. These results suggest that enteroviruses other than Coxsackie B3 are causally linked to the pathogenesis of dilated cardiomyopathy.

Biopsy↗
PubMed1993

Long-term follow-up of patients with hairy cell leukaemia treated with pentostatin: lymphocyte subpopulations and residual bone marrow infiltration.

Peripheral blood lymphocyte (PBL) subsets and bone marrow biopsies were analysed in six patients with hairy cell leukaemia (HCL) treated with 2'-deoxycoformycin (pentostatin, DCF) according to a phase II trial of the EORTC Leukemia Cooperative Group. All patients responded to DCF with four complete and two partial remissions according to conventional criteria. Within the PBL subsets, major changes concerned the CD4+ T cells, which during DCF therapy were distinctly suppressed to nadir values of 0.038-0.18 (median 0.126) x 10(9)/l. In five patients these cells returned to normal 3.0-49.5 (median 14.5) months after the last DCF injection. CD8+ cells were decreased to a lesser extent, and NK cell numbers improved during treatment. Bone marrow immunohistology applying the MoAb B-ly7 demonstrated residual hairy cells (HCs) in all of the six patients following DCF treatment with nadir HC numbers of 0.2-3.0% of bone marrow cells. Immunoglobulin gene rearrangement analysis of DNA obtained from these biopsies showed only germline bands, whereas rearranged bands had been present on the pretreatment specimens. Within the observation period of 15.5-54.0 (median 47.0) months after discontinuation of DCF therapy, immunohistology demonstrated a continuous increase in HC numbers in five of the six patients with clonal rearrangement detectable in bone marrow specimens from three of these patients at last follow-up date. Although established on the basis of a small number of patients, these data suggest that DCF treatment as currently employed in HCL is unable to eradicate the malignant B cell clone.

Adult↗
PubMed1993

Immunoglobulin gene rearrangement in plasma cell dyscrasias: detection of small clonal cell populations in peripheral blood and bone marrow.

The bone marrow (BM) and peripheral blood (PB) samples of 71 patients with plasma cell dyscrasias were analysed by the Southern blot technique for the presence of clonal immunoglobulin (Ig) gene rearrangements. 53% of BM samples examined were archival material such as air dried BM slides or frozen trephine biopsies. The results were related to bone marrow plasmacytosis as determined by cytology and flow cytometry, and other clinical parameters. Clonal Ig gene rearrangements were found in BM samples of 45 (83%) of 54 MM patients and in 3 of 6 patients with monoclonal gammopathy of unknown significance (MGUS). Clonal cell populations in the PB were detected in 11 (30%) of 37 examined MM patients, but in none of the patients with MGUS or solitary plasmacytoma of bone. PB involvement was associated with progressive disease. Circulating monoclonal cells were significantly associated with higher M-protein levels (p < 0.05). Thus, circulating clonal precursor cells are encountered more frequently in active MM.

Adult↗
PubMed1993

[Proliferative activity and p53 expression in transitional cell carcinoma of the urinary bladder].

Mutations of the p53 gene are important mechanisms in malignant transformation and are associated with dysregulation of normal cell growth. In the present study the expression of mutated p53-protein and proliferating cell nuclear antigen (PCNA) was investigated in a series of 31 human transitional cell carcinomas (TCC) by immunohistochemistry (IHC). The number of PCNA-positive cells and the pattern of expression was distinct in normal urothelium being confined to the basal cell layer. In dysplastic urothelium and in Carcinoma in situ (CIS) PCNA-immunoreactive nuclei were irregularly distributed throughout all layers. In tumor cell complexes the pattern of PCNA-immunoreactivity was different in papillary and primary infiltrating TCCs. Densitometric quantification of the intensity of the PCNA-reactivity using image analysis revealed an increase from normal to dysplastic urothelium and from dysplastic urothelium to invasive tumors. 21/31 (68%) of the tumors and tumor-associated CIS showed overexpression of p53 varying in percentage, pattern and reaction intensity. The percentage of PCNA-positive cells was higher in tumors overexpressing p53. Double IHC showed colocalization of both molecules in a significant proportion of tumor cells suggesting a link of p53 overexpression and the abnormal proliferative activity. The present results show that p53 over-expression is found in a significant percentage of TCCs and indicate a close association with a defective growth regulation resulting in increased PCNA-levels and enhanced cellular proliferation.

Analysis of Variance↗
PubMed1992

A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.

High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.

Animals↗
PubMed1992

[Opportunistic malignant lymphomas in SIV infected primates--a model for Epstein-Barr virus associated lymphomas in AIDS].

In a series of 33 cynomolgus monkeys (Macaca fascicularis) experimentally infected with Simian Immunodeficiency virus (SIV), strain smm3, 13 animals developed malignant Non-Hodgkin lymphomas. These lymphomas presented with unusual primary manifestations like in the orbita, testes, and brain. The morphological features and immunophenotyping identified the tumors as high malignant B-cell lymphomas. In all tumors as well as in tumor-derived cell lines a cynomolgus B-lymphotropic herpes virus (CBLV) with structural homogeneity to the Epstein-Barr virus (EBV) could be demonstrated by Southern blotting with EBV-specific probes. The lymphoma cells also expressed CBLV-associated nuclear antigens involved in B-cell transformation crossreacting with EBNA-specific human sera and monoclonal antibodies. Ig-gene rearrangement studies revealed clonal populations, however, no translocations of the c-myc oncogene could be detected. The lymphomas developing with high frequency in SIV-induced immunodeficiency resemble a major subtype of human EBV-associated AIDS lymphomas. This animal model can therefore be used to further elucidate interactions of HIV and EBV in AIDS-related lymphomagenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1992

P53 mutations in myelodysplastic syndromes.

Point mutations in the p53 tumor-suppressor gene are the most frequently identified genetic alterations in human malignancies. In order to evaluate the role of p53 mutations in the multistep process of leukemogenesis we studied 61 patients with myelodysplastic syndromes using single-strand conformation polymorphism analysis of polymerase chain reaction products as well as direct sequencing. Mutant alleles were observed in 1/14 refractory anemia with excess of blasts (RAEB) and 2/5 RAEB in transformation. The three mutations represented G:C to A:T transitions at codon 141 (exon 5) and codons 245 and 248 (exon 7), respectively. These data suggest that p53 mutations may contribute, albeit rarely, to the development of preleukemic disorders of the myeloid cell lineage.

Anemia, Refractory↗