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Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 19 recordsLinked to original sources
PubMed1996

Implications for the assay and biological properties of interleukin-3. Results of a WHO international collaborative study.

Five preparations of interleukin-3 (IL-3) have been evaluated by 28 laboratories in 12 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for IL-3 and interleukin-4 (IL-4). The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the biological assays contributed to this study that different recombinant preparations of IL-3 can have very different biological specific activities, including those from the same source (i.e., E. coli). Biological assays of IL-3 were significantly more consistent in their estimates of levels of IL-3 than the immunoassays, suggesting an unusual pattern of epitope recognition amongst the antibodies included in the immunoassays. This study also illustrates the point that the level of cytokine measured by immunoassay does not necessarily reflect the biological potency of the cytokine. On the basis of results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-3 (91/510) was established as the international standard for interleukin-3 with an assigned unitage of 1700 IU/ampoule.

Biological Assay↗
PubMed1996

Implications for the assay and biological activity of interleukin-4. Results of a WHO international collaborative study.

Five ampouled preparations of interleukin-4 (IL-4) have been evaluated by 36 laboratories in 14 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for interleukin-3 (IL-3) and IL-4. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-4 can have very different biological specific activities, including those from the same source (i.e., E. coli). In addition, immunoassay estimates of IL-4 levels did not correlate with those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. It is of interest that the estimates provided by the different bioassays were less variable than those produced by the immunoassays, suggesting that bioassays can be as accurate, if not more so, than immunoassays. The large reduction in the variability of estimates with the inclusion of a single reference preparation clearly illustrates the need for a single standard to assay IL-4. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-4 (88/656) was established as the international standard for interleukin-4 with an assigned unitage of 1000 IU/ampoule.

Biological Assay↗
PubMed1996

An antiproliferative bioassay for interleukin-4.

Interleukin-4 (IL-4) is currently being used for therapeutic intervention in a wide range of malignant diseases as an antitumour agent. Although bioassays have been developed that measure the proliferative capacity of IL-4, none measure the antiproliferative activity of this molecule. We have developed a simple, sensitive bioassay for human IL-4 based on the ability of this cytokine to inhibit the proliferation of the human lung carcinoma line, CCL-185, an easy to maintain, cytokine independent, cell line. It is rapid, reproducible and sensitive, able to detect 2 pg/ml IL-4. The assay is completely unresponsive to all other interleukins from IL-2 to IL-12, to the colony stimulating factors and transforming growth factor beta and is 100-fold less sensitive to interferon-alpha, tumour necrosis factor-alpha, IL-1 beta and IL-13. The assay can be made completely specific for IL-4 by including specific neutralizing antibodies for IL-4 and is suitable for the estimation of IL-4 in both plasma and serum samples.

Biological Assay↗
PubMed1996

A sensitive human cell line based bioassay for megakaryocyte growth and development factor or thrombopoietin.

We have developed a simple, rapid, sensitive bioassay for megakaryocyte growth and development factor (MGDF) or thrombopoietin (TPO) based on its ability to stimulate the proliferation of the human megakaryoblastic cell line MO7e. The bioassay takes only 24 h, is reproducible and sensitive to less than 10 pg/ml of MGDF. It can be made specific for MGDF by including neutralizing antibodies specific for MGDF, and is suitable for detection of MGDF in both plasma and serum samples.

Antibodies↗
PubMed1996

Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays.

Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte-derived inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) may contribute to a larger number of unexplained non-antibody-mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. we have monitored the plasma of PCs prepared by the platelet-rich plasma method (PRP), the buffy-coat method or by apheresis for IL-6, IL-1, transforming growth factor-beta (TGF-beta), TNF and interferon gamma (IFN gamma). Biologically active IL-6 increased in stored PRP-PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL-8, as detected by immunoassay, were evident in PRP-PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL-1 with only minimal biological activity were present in some PRP-PCs by day 5/6. No significant increase in the levels of IL-8, IL-6 or IL-1 were seen in buffy-coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL-8 content, but not in IL-6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGF beta were present, but there was no evidence of any biologically active or immunoreactive TNF alpha. Pre-storage filtration of PRP-PCs for depletion of leucocytes prevented the increase in IL-8 and IL-6 levels of these PCs. Our results show that leucocyte reduction by buffy-coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines.

Biological Assay↗
PubMed1996

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.

In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

Adolescent↗
PubMed1995

Quantitative cell line based bioassays for human cytokines.

Research in cytokine biology is ever increasing and it is clear that cytokines are involved in a wide range of pathological and physiological processes. The validity of such research relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Amongst the currently available methods for measuring cytokine levels, it is only the biological assay of samples that can directly provide estimates of biologically active cytokines present in test samples. Of the several bioassay systems available for detecting cytokines, cell line based bioassays are the easiest to perform and provide the most precise and accurate data. The suitability of any cell line for bioassaying a particular cytokine depends on several criteria such as sensitivity, ease of growth maintenance, and cytokine specificity. The design and analysis of cell line bioassays is also important in providing valid estimates of cytokine levels. We review the most useful cell lines currently available for bioassaying cytokines and discuss the design advantages and limitations of cytokine bioassays.

Animals↗
PubMed1995

Evidence for a signaling role for the alpha chains of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors: divergent signaling pathways between GM-CSF/IL-3 and IL-5.

In the present study, we have used a human erythroleukemia cell line, TF-1, that proliferates in response to granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) to investigate the role of receptors for these cytokines in signal transduction mechanisms involved in proliferative responses. The receptors for GM-CSF, IL-3, and IL-5 each possess a cytokine specific alpha subunit, but all three share a common beta chain. Using an immunoblotting system designed to detect phosphotyrosine containing proteins and a permeabilized cell system to detect rapid changes in phosphate turnover on proteins, we show that while GM-CSF and IL-3 use tyrosine phosphorylation to mediate mitogenic signal transduction, IL-5 uses tyrosine dephosphorylation in its signaling pathway. The use of different signaling pathways by these cytokines can be confirmed in a biologic system whereby the proliferation induced in culture by GM-CSF and IL-3 is inhibited by tyrosine kinase inhibitors, but that induced by IL-5 is enhanced. Conversely, GM-CSF- and IL-3-induced proliferation is stimulated by a tyrosine phosphatase inhibitor, yet IL-5-induced proliferation is inhibited. Inhibitors of protein kinase C inhibit IL-3- and GM-CSF-, but not IL-5-induced proliferation. We suggest that, because all these cytokines share the identical beta chain of their receptors, the cytokine specific alpha chain mediates the linkage of each receptor to the individual biochemical signal transduction pathways responsible for the different biologic activities of these cytokines.

Alkaloids↗
PubMed1995

The international standard for macrophage colony stimulating factor (M-CSF). Evaluation in an international collaborative study.

Three ampouled preparations of macrophage colony stimulating factor (M-CSF) were evaluated by 23 laboratories in nine countries for their suitability to serve as international standards for this material. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), the preparation in ampoules coded 89/512 was established as the international standard for M-CSF.

Animals↗
PubMed1995

The international standard for granulocyte colony stimulating factor (G-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Three ampouled preparations of granulocyte colony stimulating factor (G-CSF) have been evaluated by 29 laboratories in 11 countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/502) was established as the international standard for G-CSF.

Biological Assay↗
PubMed1995

The international standard for granulocyte-macrophage colony stimulating factor (GM-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Two ampouled preparations of granulocyte-macrophage colony stimulating factor (GM-CSF) have been evaluated by twenty nine laboratories in eleven countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/646) was established as the international standard for GM-CSF.

Biological Assay↗
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↗