Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA.

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation.

Technical considerations for the performance of Nucleic acid Amplification Technology (NAT). The NAT Task Force Group.

The complexity of Nucleic acid Amplification Technology (NAT(1)), comprising sample preparation, amplification and detection methods, requires specific design considerations for both the laboratory and the procedures utilized in such testing. The purpose of this paper is to establish technical considerations for the performance of NAT. These include the collection, handling and assay of specimens and the design of laboratories to routinely and reliably detect low levels of nucleic acid sequences.

A quantitative, internally controlled real-time PCR Assay for the detection of parvovirus B19 DNA.

Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml.

A safer plasma supply from remunerated donors--"The Immuno/Community Bio-Resources experiment".

With the goal of increasing the safety of plasma used in the manufacture of therapeutic products, Immuno and its subsidiary Community Bio-Resources (now a division of Baxter Healthcare Corporation), have developed a comprehensive plasma quality programme. This programme includes four main safety initiatives: a plasma centre location/appearance programme, a Qualified Donor programme, an Inventory Hold, and the PCR testing of plasma pools. Many of these initiatives have been adopted in part by the plasma collection and fractionation industry.

Assessment of validation studies from a fractionator's point of view.

NAT testing has become an integral part in the safety programs of both plasma fractionators and transfusion services. NAT testing for HCV RNA is now mandatory for plasma fractionators in Europe and for transfusion services in Germany and Austria. Before NAT testing of plasma could become mandatory, a defined environment had to be created to allow comparison of different NAT procedures.

A randomized, controlled trial to study the efficacy and safety of C1 inhibitor concentrate in treating hereditary angioedema.

Background: No effective treatment exists in the United States for acute attacks of hereditary angioedema (HAE).

Study Design And Methods: To evaluate the efficacy and safety of C1 inhibitor concentrate in treating HAE, a large primary care and referral center hospital conducted a randomized, placebo-controlled, double-blind trial with intent-to-treat analysis. Of the 36 patients enrolled in the study, 23 received treatment, and 22 completed the trial.

Monocytes of individual human subjects display heterogeneous bacterial uptake and antilisterial activity.

Peripheral blood monocytes (Mo) of normal human donors simultaneously exhibit two subsets differing in their functional activity towards the facultative intracellular bacterium Listeria monocytogenes. One subset (on average, 25% of total Mo) was characteristically able to ingest a large number of L. monocytogenes bacteria and permitted intracellular growth of these bacteria.

Interaction with human immunodeficiency virus type 1 modulates innate effector functions of human monocytes.

The effect of human immunodeficiency virus (HIV) type 1 on human mononuclear phagocyte effector functions in response to infection with bacteria of the Mycobacterium avium-intracellular complex (MAC) was investigated. The results showed that interaction of HIV-1 or its constituents with CD4 expressed in the monocyte membrane led to substantial impairment of monocyte capacity to restrict the intracellular growth of MAC. This was accompanied by substantially decreased production of tumor necrosis factor-alpha by HIV-1-exposed and MAC-infected monocytes.

Affinity binding of distinct functional fibronectin domains to immobilized metal chelates.

Fibronectin has been found to bind metal ions. Using metal chelate chromatography and limited proteolysis to generate the distinct functional domains of fibronectin we set out to address the metal binding sites to well-defined regions of fibronectin. The results show that the affinity binding of fibronectin to Co2+ is mediated exclusively via the collagen binding domain of the molecule, whereas fibronectin will bind to Zn2+, Ni2+, and Cu2+ by metal binding sites located in two, three, and four well-defined regions of fibronectin, respectively.

The effect of diacylglycerols on fibronectin release and its reversal by retinoic acid in cell culture.

Previous work from our laboratory showed that tumor promoters such as phorbol ester (TPA) stimulated the release of fibronectin (FN) from the surface of several cell types in culture, and that this stimulation was counteracted by retinoic acid. Diacylglycerols (DAGs) are the endogenous ligands of the TPA receptor and can activate and translocate protein kinase C (PKC) in a manner similar to TPA. To show that the release of FN is related to activation of PKC, we tested the action of DAGs on FN release from human lung fibroblasts and its counteraction by retinoic acid.

Induction of anti-mycobacterial and anti-listerial activity of human monocytes requires different activation signals.

The requirements for activation of anti-mycobacterial and anti-listerial activity of human monocytes were investigated. Human monocytes could be activated to display enhanced anti-mycobacterial activity by a 24-h treatment with lipopolysaccharide. The mediator induced by this treatment was identified as being tumour necrosis factor-alpha (TNF-alpha).

The tumor-promoter-induced release of phosphorylated fibronectin from human lung fibroblasts.

The role of fibronectin (FN) phosphorylation was examined for its involvement in tumor-promoter-induced FN release from normal fibroblasts. We investigated phosphorylated FN in spent media and in cell layers of human lung fibroblasts (HLF) cultured in the presence and absence of the tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data, obtained by metabolic labeling of HLF with [32P]orthophosphate, revealed that 32P-labeled FN accumulated rapidly in the cell layer in the absence of TPA.

Fibronectin observed in the nuclear matrix of HeLa tumour cells.

We have investigated the intracellular distribution of insoluble fibronectin in HeLa tumour cells. By indirect immunofluorescence microscopy fibronectin was detected in the nuclear region, but not in the region of the cell surface. Isolated nuclei and isolated nuclear matrices also stained for fibronectin.

ATP stimulates binding of retinol-cellular retinol binding protein complex to DNA-cellulose.

In calf uterus cytosol a cellular retinol-binding protein (cRBP) was detected which was found to bind to DNA-cellulose. The binding to DNA-cellulose could be enhanced by ATP in a dose-dependent manner. ATP treatment did not change the physico-chemical properties of the retinol-cRBP complex.

Influence of tumor promoter on binding and release of fibronectin added to human lung fibroblasts.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) did not alter the binding and release kinetics of externally added 125I-labeled plasma fibronectin to human lung fibroblasts in culture. Cell layer-bound plasma fibronectin was found to be chased into the medium at the same rate in tumor-promoter-treated as in non-treated cells. Unlabeled fibronectin accumulated to a much higher degree in the medium when tumor promoter was present.

1 alpha, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line.

The ability of the hormonally active vitamin D metabolite, 1 alpha, 25-dihydroxyvitamin D3, to affect cell growth, morphology and fibronectin production has been examined using the MG-63 human osteosarcoma cell line. Hormone treatment reduced cell growth rate, saturation density and [3H]thymidine incorporation. Inhibition was specific for 1 alpha, 25-dihydroxyvitamin D3 relative to other vitamin D metabolites (1 alpha, 25-dihydroxyvitamin D3 greater than 25-dihydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3 greater than D3), antagonized by high concentrations of serum and readily reversed by removal of 1 alpha, 25-dihydroxyvitamin D3 from the culture medium.

Studies on the tumour promoter-induced release of fibronectin from human lung fibroblasts, and its counteraction by retinoic acid.

The purpose of the experiments reported here is to improve our understanding of the mechanism whereby tumour promoters (e.g., 12-O-tetradecanoylphorbol-13-acetate, TPA) stimulate increased release of fibronectin (FN) from human lung fibroblasts (HLF) in culture.

Release of fibronectin is linked to tumor promotion: response of promotable and non-promotable clones of a mouse epidermal cell line.

By use of indirect immunofluorescence technique and enzyme-linked immunosorbent assay we show that JB 6 mouse epidermal cells have cell surface fibronectin (FN) and release FN into the culture medium. The addition of 10(-8) M 12-O-tetradecanoylphorbol-13-acetate (TPA) to promotable clones caused a 2-fold enhancement of the FN release over solvent control. On the other hand, in non-promotable clones, TPA in concentrations of 10(-8) M or 10(-7) M did not cause increased FN release.

Plasma fibronectin as a marker for cancer and other diseases.

The blood concentration of the high-molecular-weight glycoprotein fibronectin showed great promise as a marker for cancer and certain other disease states. However, it now appears that plasma fibronectin levels are influenced by many factors (e.g.

Kinetics of fibronectin release from fibroblasts in response to 12-O-tetradecanoylphorbol-13-acetate and retinoic acid.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its in vivo and in vitro antagonist retinoic acid (RA) on the synthesis and release of a major extracellular glycoprotein, fibronectin (FN), in human lung fibroblasts (HLF). The studies reported here investigate the question of whether the increased amounts of FN released by TPA treatment are cell-surface derived or require de novo synthesis of FN. Untransformed HLF continuously released FN into the medium.

Vitamin A deficiency and serum or plasma fibronectin in the rat and in human subjects.

Fibronectin, a high-molecular-weight glycoprotein occurring in plasma and on the surface of many cells, is involved in cell adhesion and other cell-surface phenomena. Vitamin A deficiency in rats resulted in a threefold increase in the concentration of fibronectin in serum, as measured immunochemically. In vitamin A-depleted human subjects, on the other hand, no correlation could be found between plasma retinol and fibronectin levels.