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Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 91 records · Page 5Linked to original sources
PubMed1995

Association of changes in monocyte antigen presentation and cytokine production in haemophilic boys with treatment and blood-borne virus infection.

Aspects of monocyte function (antigen presentation and cytokine production (e.g. IL-1, IL-6) have been studied in a normal control population and three groups of haemophilic boys: group 1 HIV and HCV seronegative and treated only with a single heat-treated factor VIII (FVIII) concentrate; group 2 HIV seronegative but HCV seropositive; group 3 all HIV and HCV seropositive. Groups 2 and 3 have been previously treated with unheated and heated FVIII concentrate. Group 1 boys (HIV/HCV uninfected) showed no significant reduction in monocyte antigen presentation function nor IL-1 or IL-6 production when compared with a control population. Group 2 boys (HCV infected) showed a reduced monocyte antigen presentation activity (P < 0.05), but no significant reduction in IL-1 or IL-6 production. Group 3 boys (HIV and HCV infected) showed a significantly reduced monocyte antigen presentation activity (P < 0.001) and an impairment of IL-1 and IL-6 production (P < 0.05). A significant reduction of IL-1 and IL-6 production was only seen in the HIV and HCV infected haemophilic boys, implicating HIV as an aetiological agent. In contrast, reduced monocyte antigen presentation activity was seen in haemophilic boys with both HIV and HCV infection or HCV alone. The HIV and HCV seronegative boys had normal antigen presentation. The absence of immune modulation in haemophilic boys that have not acquired HIV and HCV infection suggests that chronic blood-borne virus infections as contributory to immune modulation seen in haemophiliacs with virus infections.

Adolescent↗
PubMed1995

Fragmentation of therapeutic human immunoglobulin preparations.

Some commercial batches of human therapeutic immunoglobulins (Ig) have been found to show evidence of molecular fragmentation when examined by molecular sizing methodologies including sodium dodecyl sulphate polyacrylamide gel electrophoresis [SDS-PAGE] and size exclusion high performance liquid chromatography (SE-HPLC). These batches all demonstrated impaired immunobiological activity (efficacy) as assessed by Fc function measured using a rubella haemolytic assay and as such are likely to be subpotent for therapeutic use. Fragmented Igs were characterized by the presence of at least three protein bands and peaks additional to monomeric IgG. Incubation of Igs with blood enzymes (plasmin and kallikrein) reproduced the fragmentation patterns observed for intrinsically degraded batches, suggesting that fragmentation occurred by contamination with these proteases from the source material (human blood) during manufacture. Intravenous Igs (IVIG) were found to be more susceptible to proteolysis than intramuscular Igs, probably as a consequence of the post-fractionation processing that some IVIGs receive which may induce molecular alterations, allowing enzyme access and fragmentation. Two of the products examined were found to be relatively resistant to proteolysis and both were formulated by processes that limit enzyme activity. These processes were inclusion of an enzyme inhibitor, alpha 2-macroglobulin, and formulation at acidic pH. Enzyme carry-over into the final product is a likely cause of Ig fragmentation, and reduction in levels of such contamination should lead to improvements in product stability and efficacy.

Chemical Phenomena↗
PubMed1995

Factor VIII concentrates and the immune system--laboratory investigations.

Evidence suggests that haemophiliacs treated with factor VIII concentrates show abnormalities in immune functions. The basis of this is not clear, but some factor VIII concentrates down-regulate Fc receptors on monocytes which may explain the impaired function of these cells. Some concentrates inhibit lymphocyte proliferation and interleukin-2 secretion by human T-cell lines and peripheral blood lymphocytes. They can also inhibit activity of other cytokines such as interleukin-4 and interleukin-5 and secretion of cytokines such as interleukin-1 and granulocyte macrophage colony stimulating factor. These effects are product-related and vary from total inhibition to virtually no detectable inhibition. Of particular significance is that the degree of inhibition is not related to the purity or gross protein composition of the products. The inhibitory activity is not due to factor VIII itself as antibody affinity purified factor VIII products are entirely non-inhibitory. The main inhibitory protein components appear to be of approximately 200 kDa and 60 kDa (by gel filtration). Recent evidence suggests that transforming growth factor-beta (TGF-beta), derived from the plasma used for fractionation, is a major contaminant of 'inhibitory' concentrates and is responsible for the effects, observed in vitro, of concentrates on cytokine secretion or activity. The levels of TGF-beta varied between products and correlated with inhibition of interleukin-2 secretion from stimulated T-cells. The presence of TGF-beta in concentrates may therefore explain the immunosuppression observed in recipients of these products. Correlation of the inhibitory effects with clinically important consequences such as increased susceptibility to infections or decreased CD4 counts also remains to be established.

Factor VIII↗
PubMed1994

Identification of transforming growth factor-beta as a contaminant in factor VIII concentrates: a possible link with immunosuppressive effects in hemophiliacs.

In previous studies, we have shown that some, but not all low-, intermediate-, and high-purity factor VIII concentrates inhibit interleukin-2 (IL-2) secretion from phytohemagglutinin (PHA)-stimulated T lymphocytes. We now present evidence that this inhibitory action of concentrates is, at least in part, due to contamination with transforming growth factor-beta (TGF-beta). Originally identified in platelets, TGF-beta is a 25-kD homodimer that has been shown to be a natural and potent inhibitor of many immunologic responses. Using a specific bioassay, we have measured TGF-beta in various factor VIII concentrates. While some concentrates contained substantial amounts of the cytokine, there was a wide variation in concentrations of TGF-beta in different products. These levels correlated with the degree of inhibition of IL-2 secretion from T cells exhibited by each product (P = .0001). Noninhibitory concentrates contained no detectable TGF-beta. Addition of a specific TGF-beta 1 antibody reversed the inhibitory effect of some concentrates on IL-2 secretion by PHA-stimulated Jurkat T cells and interleukin-5 (IL-5)-induced proliferation of an erythroleukemic cell line. These findings suggest that TGF-beta contamination is a major contributory factor to the inhibitory activity of some factor VIII concentrates on cytokine secretion or activity, and may partially explain the reported immunosuppressive effects in recipients of these blood products.

Antibodies↗
PubMed1994

Transforming growth factor-beta 1 blocks interleukin 4 induced cell proliferation by inhibiting a protein tyrosine phosphatase essential for signal transduction.

Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine which exhibits pleiotropic effects on many cell types and cellular systems. TGF-beta 1 has been shown to play a modulatory role in haematopoiesis and immunoregulation, expressed through its ability to inhibit the activities induced by other cytokines; however, the mechanisms underlying this activity are currently unclear. The potency of this activity varies according to the selected stimulatory cytokine and we have found that the proliferation of leukaemic cell lines induced by interleukin 4 (IL-4) is particularly sensitive to inhibition by TGF-beta 1 and provides a useful model to study the mechanism of action of TGF-beta. We have previously shown that IL-4 mediated mitogenic signal transduction in human systems involves the induction of phosphatase activity leading to the dephosphorylation of an 80-kDa protein (p80). We now show that TGF-beta 1 inhibits IL-4 induced dephosphorylation of p80 in a dose responsive manner closely correlated with its ability to inhibit the biological activity of IL-4. This suggests that TGF-beta 1 is inhibiting the same protein-tyrosine-phosphatase required by IL-4 to transduce its mitogenic signal. The biochemical mechanism underlying the biological activity of TGF-beta 1 in inhibiting IL-4 bioactivity is therefore the blocking of post receptor binding signal transduction processes.

Amino Acids↗
PubMed1994

Cytokines and autoimmunity.

Although the immunopathology of most autoimmune diseases has been well defined, the mechanisms responsible for the breakdown of self-tolerance and which lead to the development of systemic and organ-specific autoaggression are still unclear. Evidence has accumulated which supports a role for a disregulated production of cytokines by leucocytes and possibly other cells in the pathogenesis of some autoimmune diseases. However, due to the complexity and heterogeneity of cytokine effects in the regulation of the immune response, it is difficult to determine whether abnormalities in the patterns of cytokine production are primary or secondary to the pathological process. Confusion is also caused by the fact that the biological activities of cytokines are multiple and often overlapping, and consequently it is difficult to focus on a unique effect of any one cytokine. Characterization of the potential and actual involvement of cytokines is important not only for a better understanding of the pathogenesis of autoimmune conditions, but particularly because of the implications for the development of immunotherapeutic strategies for the prevention and treatment of the diseases.

Animals↗
PubMed1993

A novel, sensitive bioassay for transforming growth factor beta.

We have developed a simple, sensitive bioassay for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) based on the ability of these cytokines to inhibit the interleukin-5 induced proliferation of the erythroleukaemia cell line, TF-1. This assay is rapid, reproducible and sensitive to less than 500 fg/ml of TGF-beta 1, and 5-10 pg/ml TGF-beta 2. The assay is 100-1000-fold less sensitive to other inhibitory molecules such as interferon-beta, interferon-gamma and TNF-alpha. The assay can be made specific for TGF-beta 1 or TGF-beta 2 by including specific neutralising antibodies for TGF-beta 1 or TGF-beta 2. The assay can recognise all the readily available recombinant molecular species of these molecules as well as the natural proteins produced from human and bovine platelets and detects TGF-beta in serum samples.

Biological Assay↗
PubMed1992

Detection and measurement of cytokines.

Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.

Animals↗
PubMed1992

Restriction of serum antibody reactivity to the V3 neutralizing domain of HIV gp120 with progression to AIDS.

OBJECTIVE: To identify epitopes on HIV-1 gp120 that correlate with disease resistance and/or prognostic indication. DESIGN: The identification of epitopes on HIV-1 gp120 was determined by testing the reactivity by immunoblotting of anti-HIV-positive human sera against partially cleaved Chinese hamster ovary (CHO) cell-derived recombinant gp120. Cleavage of recombinant gp120 occurs in the V3 loop region resulting in 70 and 50K cleavage bands if the protein is subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Antibodies reactive with the 120 Mr band alone on immunoblotting indicate that binding is restricted to this cleavage site. Reactivity to either of the cleavage products is independent of gp120 cleavage and indicates that binding occurs in sites other than the V3 cleavage region. METHODS: A panel of anti-HIV-positive human sera was tested for virus neutralizing activity and reactivity by immunoblotting against CHO cell-derived gp120. RESULTS: All sera reacted with the uncleaved from of gp120 but reacted either weakly or did not react with its cleavage products. There was a statistically significant correlation between serum reactivity to cleavage products and clinical stage of disease [Centers for Disease Control (CDC) criteria]. Sera of asymptomatic individuals (CDC stage II/III) were more likely to recognize either one or both of the cleavage products compared with sera from patients presenting with symptoms of disease (CDC stage IV). Furthermore, sera reacting with either one or both of the cleavage products were more likely to have higher neutralizing antibody titres than those that were unreactive. CONCLUSIONS: There is a restriction of serum antibody reactivity (when tested by immunoblotting) to the V3 loop with progression to disease. Raised neutralizing antibody titres may be dependent on regions outside the V3 cleavage site.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Mechanisms of inhibition of T cell IL-2 secretion by factor VIII concentrates.

We have continued our previous study of the inhibitory effects of factor VIII concentrates on IL-2 secretion by T cells. Experiments with an extended range of products confirm our previous conclusion that some but not all low, intermediate and high purity concentrates possess inhibitory activity on IL-2 secretion. The inhibition occurs almost immediately after addition of factor VIII concentrate and it was not possible to adsorb inhibitory activity with activated or non-activated cells; this suggests that the mechanism of inhibition involves interference with early T cell activation events rather than simple blocking of cell surface components by inhibitory molecules. The inhibitory components were shown to reside in different molecular weight fractions of concentrates. A strongly inhibitory component of approximately 200 kD and a minor species of approximately 60 kD were identified in strongly inhibitory concentrates. Some products contained a dialysable inhibitory substance which is most likely a salt as it was also present in some formulation buffers. The proportions of the inhibitory components varied widely between products. We have found that the pattern of inhibition using in vitro systems reflects that observed using a mouse in vivo antigen challenge method. In addition we have shown that the previously reported concentrate mediated inhibition of lectin induced low affinity IL-2 receptor (CD25) is mainly a consequence of diminished IL-2 secretion rather than a 'direct' effect on CD25 expression. Considering the wide variation between products of the same purity group, caution should be exercised in drawing conclusions concerning the immunosuppressive effects of a particular type of concentrate in haemophilia patients from study with only one product from that group.

Animals↗