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. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products. An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.

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. Using a statistical model that takes into consideration the unique donation characteristics of remunerated plasma donors, combined with 1998 CBR virus reactive rates, an estimated residual likelihood of an undetected donation entering a plasma pool was determined. These estimates, for each million donations, were 0, 1.64, and 4.68 donations for HIV, HBV, and HCV, respectively, and were far below those previously reported for remunerated or volunteer donations. These estimates were confirmed by subsequent PCR testing, which allowed for the additional removal of positive units before manufacture. The low virus load of this plasma supply, combined with increasingly effective virus removal and inactivation procedures, has resulted in the safest ever supply of plasma derivatives.

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. To create such an environment, international virus standards, as well as guidelines for validation, assessment of robustness, and quality assurance of NAT have been released. This paper is a critical review of currently available standards and national reference preparations, detection limits, and national regulations of NAT in view of the specific nature of NAT.

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. C1 inhibitor concentrate or albumin (placebo) infusions were administered in a blind fashion to HAE patients who came to the hospital for treatment no later than 5 hours after an attack began. RESULTS: Relief was almost twice as fast in persons receiving C1 inhibitor concentrate than in the controls: 7.62 hours (mean; SD 7.08) versus 15.35 hours (mean; SD 8.31), respectively. The difference for time-to-relief was highly significant (p = 0.007, Mann-Whitney U test). The median time-to-relief was 6.17 hours (interquartile range 0.33-15.35) in the treatment group and 15.35 hours (interquartile range 14.00-22.83) in the control group. Resolution of symptoms was one-third faster in the C1 inhibitor concentrate group than in the placebo group: 23.98 hours (mean; SD 14.81) and 34.58 hours (mean; SD 13.56), respectively (p = 0.09, Mann-Whitney U test). Recovery of functional C1 inhibitor was 119.65 percent (mean; SD 50.80), and half-life was 37.87 hours (mean; SD 19.75). Recovery of antigenic C1 inhibitor was 147.75 percent (mean; SD 97.68), and half-life was 24.01 hours (mean; SD 9.70). There were no viral infections or serious adverse effects from the drug after 70 attacks in the treatment group and 96 attacks in the control group. CONCLUSIONS: C1 inhibitor concentrate is a safe, effective treatment for acute attacks of HAE.

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. The other Mo subpopulation (on average, 75% of total Mo) was far less active in phagocytosing L. monocytogenes and restricted intracellular L. monocytogenes growth. Electron microscopy revealed that the Listeria-permissive Mo subset allowed the bacteria to escape to the cytosol, a mechanism by which these bacteria evade the lethal attack of phagocytes. The Listeria-restrictive Mo subset, on the other hand, confined the bacteria to the phagolysosomes, where they were exposed to the killing mechanisms of the Mo. Permissiveness for L. monocytogenes growth was further associated with differences in the capacity of the Mo subsets to synthesize tumor necrosis factor alpha TNF-alpha), an important mediator in the defense against intracellular bacteria. Following challenge with L. monocytogenes, the Listeria-restrictive Mo subset secreted two to six times more TNF-alpha than did the Listeria-permissive Mo subset. Enhanced TNF-alpha secretion was paralleled by increased accumulation of TNF-alpha mRNA as assessed by quantitative PCR. Despite these functional differences, the two Mo subsets were indistinguishable with respect to expression of cell surface markers known to be involved in adherence and phagocytosis of microbes. A speculative physiological role of the two Mo subsets may lie in the dual function of Mo as microbicidal effector cells and accessory cells for antigen-specific immune reactions.

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. However, productive HIV-1 infection of monocytes was not required to induce the observed effects. These studies suggest that HIV-1 may interfere with innate mononuclear phagocyte function. This may be of physiologic importance in the late stages of AIDS, when an impaired T cell immunity can no longer provide proper immune-activating signals, and may help to explain the undue susceptibility to MAC infections in these patients.

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. Fe2+ and Mn2+ chelates did not bind any of the isolated fibronectin domains. Combined metal chelate affinity chromatography opens a possibility to isolate particular fibronectin domains.

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. We found that DAGs stimulated the release of FN in a concentration- and time-dependent manner. The stimulation of the release of FN correlated with the translocation-activation of PKC by DAG. Retinoic acid reversed the action of DAG with respect to stimulation of FN release and inhibited this release even in the absence of DAG. These results suggest that the release of FN is in some way related to translocation-activation of PKC.

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). Addition of recombinant TNF-alpha (rTNF-alpha) to the cultures of human monocytes for 24 h yielded comparable results (minimal dose required for induction of anti-mycobacterial activity, 10 U ml). Addition of anti-TNF-alpha antibody completely abrogated the effect. A similar treatment protocol failed to activate enhanced anti-listerial activity. To trigger anti-listerial activity, sequential treatment of human monocytes with rTNF-alpha and IL-2 was required. Treatment of monocytes with 10 U ml rTNF-alpha for 24 h followed by incubation in the presence of 200 U/ml of IL-2 for an additional 24 h yielded a reduction of listerial growth which was moderate but statistically significant (P less than 0.001). The activation of monocytes observed with rTNF-alpha/IL-2 treatment was (i) dependent on both cytokines; (ii) sequence dependent (i.e. when IL-2 was added prior to rTNF-alpha, no effect was observed); and (iii) absent in cells treated with one cytokine only. Enhancement of anti-listerial activity by sequential use of cytokines was not accompanied by an increase in oxidative burst, which indicated that oxidative mechanisms were not the reason for the observed Listeria monocytogenes growth restriction. Further support for this hypothesis was obtained after interferon-gamma treatment of human monocytes which led to an augmented PMA-inducible release of active oxygen radicals, but was not paralleled by growth restriction of L. monocytogenes. Our results indicate that TNF-alpha plays a crucial role in the activation of monocytes for growth restriction of intracellular microbes. Activation of human monocytes to restrict the growth of the facultative intracellular bacteria Mycobacterium avium intracellulare and L. monocytogenes, however, follows different patterns, the initial trigger in both cases being provided by TNF-alpha-induced signals.

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. With TPA present, accumulation of 32P-labeled FN at the cell layer of HLF was much slower than under control conditions. On the other hand, treatment of the cells with TPA caused a marked increase in the amount of medium-released FN. This increase in released FN, however, was caused by unlabeled FN and was not paralleled by an increase in 32P-labeled FN. We investigated the ability of FN from normal and TPA-treated cells to bind to normal HLF monolayers. We found no difference in re-binding ability, regardless of whether FN was derived from cell extracts of control cultures (showing highly phosphorylated FN) or from the medium of TPA-treated cultures with low phosphorylation of FN.

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. By quantitative enzyme-linked immunosorbent assay (ELISA), fibronectin was found almost exclusively in the subcellular fraction of isolated nuclear matrices. Using immunoblotting techniques fibronectin was detected in nuclear matrices isolated from cells grown in standard and in fibronectin-depleted medium. The data demonstrate that fibronectin in HeLa tumour cells is preferentially associated with the nuclear matrix.

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. Our findings suggest a role for ATP in the binding of retinol-cRBP complex to DNA.

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. We conclude that TPA does not interfere with the fibronectin receptor on substrate-attached fibroblasts, but may influence intracellular fibronectin before it is bound to the extracellular matrix.

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. Hormone treatment also increased cell associated alkaline phosphatase activity up to twofold and altered morphology such that treated cells were more spread out on the culture dish and contained more cytoplasmic processes. Significantly, 1 alpha, 25-dihydroxyvitamin D3 increased cellular and medium concentrations of fibronectin, a glycoprotein known to be involved in cellular adhesiveness. MG-63 cells contain a specific 1 alpha, 25-dihydroxyvitamin D3 receptor which may mediate these responses.

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. We investigated whether pretreatment of these cultures with a brief pulse of TPA would be sufficient to cause this effect, or whether continuous presence of TPA is required. We found that a pretreatment of 5-15 min with TPA caused the increased FN release when followed by a 2-h TPA-free incubation. The increased release did not cease upon removal of TPA. Longer exposure to TPA (1-2 h) also gave higher FN release than control cultures, but the effect was less pronounced. Pretreatment with retinoic (RA) for up to 30 min did not counteract the subsequent TPA effect after the RA was removed, even though we could show with labeled RA that it had entered the cells. Therefore, RA must be present simultaneously with TPA; it presumably acts on the cell surface when antagonizing the effect of TPA on FN release. When all cell-surface FN was removed by trypsinization and the cells were incubated with TPA, an increased release of FN into the medium occurred and a decreased accumulation of cell-associated FN compared to controls. These results suggest that FN is released even if the pericellular matrix is absent and that TPA interferes with the subsequent build-up of the matrix.

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. Mezerein, a non-phorbol diterpene and second-stage tumor promoter was also found to be active in causing enhanced FN release in promotable but not in non-promotable clones. The vitamin A derivative retinoic acid (RA) antagonized the TPA-caused FN-release in promotable clones. RA had, however, no effect on the basic release patterns, when given alone or given to non-promotable clones together with TPA. These results suggest that the increased release of FN may be a required event for promotion to transformation. Our view is derived from the observation that promotable clones of the JB 6 cell line release increased amounts of FN into their medium upon promoter exposure while non-promotable clones are unaffected.

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., age, sex, nutritional state, trauma, shock, inflammation, certain drugs), sometimes giving conflicting responses. Furthermore, unrecognized pitfalls exist in fibronectin determinations with respect to blood sampling methods, plasma preparation, plasma storage, and re-use and assay methods. It is concluded that the plasma fibronectin level, as frequently reported, has little value as a marker for cancer and other disease states.