Hypervariable digital DNA codes for human paternal lineages: MVR-PCR at the Y-specific minisatellite, MSY1 (DYF155S1)

We describe the first haploid minisatellite, the human Y chromosome- specific locus, MSY1. It consists of an array of 48-114 AT-rich 25 bp repeats of at least five different variant types. A minisatellite variant repeat PCR (MVR-PCR) system gives Y-specific DNA codes, with a virtual heterozygosity of 99.9%, making MSY1 by far the single most variable locus on the Y. African populations contain the most diverged MSY1 structures. MSY1 is the only Y-chromosomal system where the characteristics of large numbers of mutations can be studied in detail: it provides a uniquely powerful tool both for the investigation of mutation in a haploid system, and for the dating of paternal lineages.      

A retrospective analysis of therapy for acute graft-versus-host disease: initial treatment

We have reviewed results of therapy in 740 patients with grades II-IV acute graft-versus-host disease (GVHD) after allogeneic marrow transplantation. At the beginning of therapy, 597 patients (81%) had rash, 369 (50%) had liver dysfunction and 396 (54%) had gut dysfunction. Initial treatment was with glucocorticoids (n = 531), cyclosporine (n = 170), antithymocyte globulin (ATG) (n = 156) or monoclonal antibody (n = 3) either singly (n = 633) or in combination (n = 107). Parameters of GVHD severity in each organ were recorded weekly, and evaluation of response was made using values at the initiation of secondary treatment or, for patients without such treatment, using values on day 29 of primary treatment or the last recorded value before death, whichever occurred first. Minimal criteria for improvement or progression were defined for each organ, but no attempt was made to define liver or gut outcome if another complication such as venocclusive disease or infectious enteritis was present. Improvement rates were 43% for skin disease, 35% for evaluable liver disease and 50% for evaluable gut disease. Overall complete or partial responses were seen in 44% of patients. Multivariate analyses were carried out to identify patient, disease or treatment factors associated with likelihood of overall improvement and likelihood of response in at least one organ. A similar analysis was also carried out to identify covariates associated with time to treatment failure (defined as initiation of secondary therapy or death not due to relapse of malignancy). In all three models, GVHD prophylaxis using cyclosporine combined with methotrexate was associated with favorable GVHD treatment outcome compared to prophylaxis with either agent alone, and treatment with glucocorticoids or cyclosporine was more successful than treatment with ATG. Other factors associated with unfavorable outcome in the model of time to treatment failure and also entered in one of the response models were recipient HLA disparity with the donor, presence of a liver complication other than GVHD, and early onset of GVHD. Results of this analysis indicate that glucocorticoids represent the best initial therapy available for treatment of acute GVHD, although much room for improvement remains.     

Platelet adhesion to fibronectin in flow: dependence on surface concentration and shear rate, role of platelet membrane glycoproteins GP IIb/IIIa and VLA-5, and inhibition by heparin

Platelet adhesion to purified surface-immobilized fibronectin under flow conditions was investigated. Fibronectin was found to support attachment and spreading of platelets. The extent of platelet spreading depended on the amount of immobilized fibronectin. An antiglycoprotein (anti-GP) IIb/IIIa antibody and an Arg-Gly-Asp (RGD)-containing peptide inhibited adhesion almost completely, whereas antibodies directed against platelet GP Ic/IIa (very late antigen 5) inhibited by 50%. Similar results with the antibodies and the peptide were found in a static system. A comparison of different anticoagulants showed no difference in adhesion using citrate or hirudin. However, unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) as the only anticoagulant or in combination with citrate maximally inhibited adhesion by 80% and 60%, respectively. Preincubation of the immobilized fibronectin with UFH resulted in a maximal inhibition of 90%, whereas preincubation with LMWH had no effect. When we preincubated the surface with heparins of different size, we observed 40% inhibition of adhesion with heparins with an average MW of up to 18 kD, whereas a heparin with an average MW of 21 kD almost completely blocked adhesion. These results indicate that platelet adhesion to fibronectin in flow involves several receptors, is highly RGD-mediated, does not require physiologic levels of divalent cations, and can be inhibited by direct binding of heparin to the fibronectin surface.     

Clinical implications of and lessons learnt from external assessment of Mers-CoV diagnostics

With the emergence of new viral infections, it is necessary to set up new target-specific assays, based on existing molecular techniques such as real-time PCR, as quickly as possible. Without these diagnostic tools, the geographical spread of new infections, follow-up of the disease outbreak and analysis of the pathogenesis of the disease are not possible. Therefore, the genomic information of the emerging pathogen, diagnostic protocols and standards allowing quality control need to be available in a few days. This can only be implemented with good quality experienced laboratories having suitable infrastructure to establish in-house assays. Even though these molecular tools are available quickly, challenges still remain with what sample types to select for a proper diagnostic value.