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Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 73 records · Page 4Linked to original sources
PubMed1997

The use of bioassays for the characterisation and control of biological therapeutic products produced by biotechnology.

The primary value of bioassays is that they alone directly assess biological activity of bioactive substances and products. Appropriately designed bioassays reflect the fundamental aspects of the biological activity of a bioactive molecule, including ligand-receptor binding, signal transduction processes (often poorly understood) and the final observed biological effects. Biological assays therefore complement physicochemical and biochemical procedures which normally only assess precise molecular structural features of complex molecules produced by biotechnology. Bioassays provide valuable information concerning the potency of biological products. This is essential for evaluating batch-to-batch consistency and stability. Bioassay data are crucial at all stages in the development of biological products, from early research work to final quality control of finished products. However, the type and design of bioassays may differ according to the information required and its intended use. The assays may or may not directly relate to the clinical use of the product. Bioassays can be difficult to perform and be time consuming, although this often reflects bad assay choice and/or design. Correct analysis of the assay results is essential if valid data are to be obtained. Standardisation, using correctly calibrated primary and secondary standards, is essential. In vivo bioassays are normally more unreliable than in vitro procedures, but in some cases the latter are either not available or do not address important biological characteristics of a product. Bioassays must be validated for their intended purpose and for the types of samples to be measured. Appropriate statistical analysis should be used to derive the significance and specifications of results. This needs to address both variability in samples and assay performance. Specifications (limits) for product acceptability need to be derived from real data using several batches of product.

Animals↗
PubMed1996

An anti-cytokine bioactivity assay for interferons-alpha, -beta and -omega.

Interferons-alpha and -beta (IFN-alpha and -beta) are cytokines that are widely known to induce potent anti-viral activity. However, it has become increasingly apparent that IFN-alpha and -beta exert a variety of other biological effects, including anti-tumour and immunomodulatory activities and are increasingly used clinically to treat a range of malignancies, myelodysplasias and autoimmune diseases, e.g., IFN-beta for multiple sclerosis. The most widely used bioassays for the IFNs are based on their anti-viral activity, but these do not predict the biological activity of the IFNs in anti-tumour and immunomodulatory therapies. Thus, we have developed anti-cytokine-based bioassays that may be more reflective of such activity and which have several advantages over existing anti-viral bioassays. The anti-cytokine bioassay is based on the ability of IFN-alpha, -beta and -omega to inhibit granulocyte-macrophage-colony-stimulating factor (GM-CSF) induced proliferation of the erythroleukaemic cell line TF-1. This assay can take only 24 h, is sensitive to 200 fg (0.04 IU) IFN-alpha or -beta and 100 fg (0.02 IU) IFN-omega and is able to detect down to these levels in serum or plasma samples. The usefulness of anti-cytokine bioassays for IFN-alpha, -beta and -omega is not restricted to the GM-CSF/TF-1 cell format and other alternatives are available, such as erythropoietin (EPO)/TF-1 cells and EPO/UT-7-EPO cells. These assays can be made specific for each of the IFNs by including neutralising antibodies in the bioassay.

Humans↗
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

IL-4 and TNF-alpha-mediated proliferation of the human megakaryocytic line M-O7E is regulated by induced autocrine production of GM-CSF.

In this study, the authors examined the effects of recombinant human interleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alpha (rhTNF-alpha) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e. While rhIL-4 or rhTNF-alpha alone induced only a weak proliferative response, a synergistic proliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-alpha is mediated predominantly by the 55-kDa TNF receptor because the agonistic monoclonal antibody htr-9 and the Trp32 Thr86 TNF-alpha mutant protein specific for this receptor type produced similar results to rhTNF-alpha. In contrast, the Asn143 Arg145 TNF-alpha mutant protein specific for the 75-kDa TNF receptor produced only minimal proliferation of M-O7e cells. Using RT-PCR, we found that rhTNF-alpha rapidly and strongly induced granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient inducer of GM-CSF mRNA. However, there was little evidence of the TNF-alpha/IL-4 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with IL-4 or TNF-alpha alone. Thus, the observed synergistic effect of TNF-alpha/IL-4 costimulation of M-O7e cells seems to be mediated via induction of GM-CSF secretion rather than an enhanced production of GM-CSF mRNA. Higher levels of GM-CSF were detectable in supernatants of cells treated with both rhIL-4 and rhTNF-alpha than in cells stimulated with either cytokine alone. Furthermore, addition of a neutralising antibody against GM-CSF abrogated the observed synergistic effect of rhIL-4 and rhTNF-alpha treatment, indicating that the rhIL-4/TNF-alpha combination acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.

Antibodies, Monoclonal↗
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↗
PubMed1996

Testing plasma pools for markers of viral contamination: the UK experience.

The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.

Antibodies, Viral↗
PubMed1996

The biological properties, assay, and standardization of interferon-alpha: a need for a WHO collaborative study.

Interferon-alpha (IFN-alpha) exists as a range of closely related, biologically active proteins and has been the subject of extensive research and clinical investigation. Standardization of IFN-alpha and the uniform reporting of IFN-alpha activity in International Units (IU) is critical to preclinical research and the clinical development of IFN-alpha products as therapeutic agents. Currently, several different IFN-alpha-containing reference preparations, established as World Health Organization (WHO) International Standards (IS) for particular IFN-alpha proteins (mixtures or single molecular species) are available for assay calibration. Nevertheless, the heterogeneous nature of IFN-alpha has raised standardization issues relating to the activity of individual IFN-alpha proteins, hence-forth termed subtypes, in the various biologic assays used for determining IFN-alpha levels. These issues include the question of parallelism of dose-response curves among particular IFN-alpha subtypes and different, naturally produced IFN-alpha subtype mixtures, for example, leukocyte IFN-alpha, and the applicability of IU of IFN-alpha activity defined by antiviral assays to alternative biologic assays, for example, antiproliferative assays. To address such issues, a WHO Consultative Group on Cytokine Standardization requested that the National Institute for Biological Standards and Control (NIBSC) and the Centre for Biologics Evaluation and Research (CBER) organize an international collaborative study to compare the activities and relative potencies of the several available IFN-alpha preparations, including those derived from human cells containing mixtures of IFN-alpha subtypes and those derived by rDNA methods containing single IFN-alpha subtypes, in different assays. To date, 111 participants in 32 countries have been recruited to the study and have agreed to assay a total of 17 different natural and recombinant IFN-alpha preparations or a defined subset thereof in specific in-house assays. The assay results generated will be statistically analyzed and evaluated to address the stated issues and to assess whether any individual IFN-alpha preparation is suitable to serve as an IS for all IFN-alpha preparations or whether more than one IS will be needed for this purpose.

Animals↗
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↗