Entmethylierung von Methylpervitin zu Pervitin

Journal of Molecular Medicine -     

Detection of human immunodeficiency virus type 1 (HIV-1) in heel prick blood on filter paper from children born to HIV-1-seropositive mothers.

The human immunodeficiency virus type 1 (HIV-1) DNA PCR results of 94 dried blood spot (DBS) samples on filter paper and corresponding venous blood in EDTA obtained from infants born to HIV-1-seropositive mothers were compared. In addition, the results of HIV-1 DNA PCR on DBS and the HIV-1 RNA PCR from plasma of 70 paired samples were compared. A 100% specificity and a 95% sensitivity for HIV-1 DNA PCR on DBS compared with results for venous blood were observed for the 94 paired samples. The results of the DBS HIV-1 DNA PCR and HIV-1 RNA PCR of 70 corresponding plasma samples correlated perfectly (100%). The DBS HIV-1 DNA PCR method proved reliable for HIV-1 detection.     

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.     

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients.

PURPOSE: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA. METHODS: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples. RESULTS: Call rates were 94% on average and heteroplasmy detection levels varied from 5-50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected. CONCLUSIONS: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.     

Heterogeneous malignant non Hodgkin's lymphomas as a causative disorder in lethal midline granuloma

Summary The present report describes the results of a combined morphological, enzyme- and immunohistochemical analysis of nine cases of malignant non Hodgkin's lymphomas (NHL) clinically presenting as lethal midline granuloma. In a previous report written before antibodies directed against B and T lymphocytes were available, a histiocytic origin of such neoplasms had been suggested. A panel of antibodies reactive with most B cells (L26, MB1, KiB3) and a majority of T cells (MT1, UCHL1) was applied on paraffin sections of formalin fixed tissues as well as antibodies directed against leukocyte common antigen (LCA), myeloid/histiocyte antigen (MAC 387), lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, S-100 protein, prekeratin and immunoglobulin light chains. Enzyme histochemistry included tests for non-specific acid esterase, acid phosphatase, betaglucuronidase and chloroacetate esterase. As a result, five T, two B and two unclassified (malignant histiocytosis probable) NHL were identified, indicating distinct heterogeneity of NHL as causative disorders in lethal midline granuloma.     

Expression of calcium-binding proteins MRP8 and MRP14 in inflammatory muscle diseases

The pathophysiological role of infiltrating macrophages and their subtypes in idiopathic inflammatory myopathies such as dermatomyositis, polymyositis, and inclusion body myositis is not fully clear. Monocytes exhibit various phenotypes with different functional properties such as release of pro- or anti-inflammatory mediators. Expression of myeloid-related proteins MRP8 and MRP14, two calcium-binding S100-proteins, characterizes a proinflammatory subtype of macrophages. We immunohistochemically investigated expression of MRP8 and MRP14 in muscle biopsies of 33 patients with dermatomyositis, polymyositis, and inclusion body myositis. We found a clear association of expression of MRP8 and MRP14 by infiltrating macrophages with degeneration of myofibers. Because MRP8 and MRP14 are secreted by activated macrophages we investigated if these proteins would have direct extracellular effects on myocytes. We found that the purified MRP8/MRP14 complex inhibited proliferation and differentiation of C2C12 myoblasts and that it induced apoptosis via activation of caspase-3 in a time- and dose-dependent manner. These results indicate that in the course of inflammatory myopathies, activated macrophages can promote destruction and impair regeneration of myocytes via secretion of MRP8/MRP14.     

A novel molecule expressing HLA-DR antigenic determinants

Our previous studies suggested that the polymorphism of HLA-DR antigens (the human equivalent of murine I-E antigens) was a result of structural variation in the small (beta) subunit. In order to more accurately define this polymorphism we have expanded these studies to include HLA-DR antigens isolated with monoclonal cells derived from genotypically HLA-homozygous DRw2, DR2w5, and DRw7 lymphoblastoid cells derived from offspring of consanguineous relationships. Our results indicate the large (alpha) subunits of DRw2 and DRw7 antigens are nearly identical, while their beta subunits show many differences. In contrast, both the alpha and beta subunits of the DRw5 antigen differ strikingly from the respective subunits of the DRw2 and DRw7 antigens. The significance of the variability of the DRw5 alpha subunit is in question at this point. One intriguing possibility is that DRw5 actually represents the human counterpart of the mouse I-A subregion antigen and that the monoclonal antibody is reacting with a determinant which is shared by the human equivalents of murine I-A and I-E antigens.