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R Thorpe

Publications and source records attributed to R Thorpe.

At least 55 records · Page 3Linked to original sources
PubMed1999

Immunogenicity of granulocyte-macrophage colony-stimulating factor (GM-CSF) products in patients undergoing combination therapy with GM-CSF.

In this study, we have assessed the development of neutralizing and nonneutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in two groups of patients with metastatic colorectal carcinoma receiving two different GM-CSF products. Three clinical trials were carried out, and a combination of GM-CSF and a colon carcinoma-reactive antibody was used in the absence of any concomitant chemotherapy. Two different GM-CSF products, both rDNA-derived and produced in Escherichia coli, were used. Patients in Trial 1 received product X, and those in Trials 2 and 3 received product Y. Patients in Trial 2 also received interleukin 2 in an attempt to potentiate immune responses. After the first cycle of treatment, no GM-CSF antibodies were detected, but on subsequent therapy, 28 of the 38 patients tested receiving product Y (Trials 2 and 3) developed antibodies that bound to the GM-CSF product used for therapy. However, none of the patients developed antibodies that neutralized the biological activity of GM-CSF, as assessed using an in vitro bioassay. Furthermore, there was no in vivo impairment in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in the patients. In contrast, 19 of the 20 patients given product X (Trial 1) developed GM-CSF binding antibodies, and 9 of these patients were shown to develop antibodies that neutralized the biological activity of GM-CSF. The presence of the latter was associated with a significant reduction in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in patients. Therefore, product X appears to be more immunogenic than product Y. Immunochemical characterization confirmed that the specificity of the antibody responses varied depending on the product used for therapy. Whereas sera from Trial 1 patients treated with product X showed the presence of antibodies with strong recognition of GM-CSF proteins, sera from patients treated with product Y showed varied recognition of GM-CSF ranging from fairly strong to very weak but bound predominantly to two E. coli-derived, non-GM-CSF-related proteins of Mr approximately 20,000 and Mr approximately 30,000. Therefore, in sera from patients receiving product Y, the antibody specificity appeared to be directed not only against GM-CSF but also against non-product-related host cell contaminants. This study shows that GM-CSF products used for therapy are potentially immunogenic and generate antibodies to GM-CSF and/or other non-product-related contaminants. However, only antibodies that neutralize the biological activity of GM-CSF compromise therapeutic efficacy of the cytokine.

Adult↗
PubMed1999

Bioassays for the characterisation and control of therapeutic cytokines; determination of potency.

The primary value of bioassays is that they alone directly assess the biological activity of bioactive substances and products like cytokines. Appropriately designed bioassays reflect the fundamental aspects of the biological activity of a cytokine 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 cytokines. Bioassays provide valuable information concerning the potency of cytokine products. This is essential for evaluating batch-to-batch consistency, appropriate formulations and stability. Bioassay data are crucial at all stages in the development of cytokine products, from early research to final quality control of finished product. 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 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 in vitro systems 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 cytokine product.

Biological Assay↗
PubMed1998

Cytokine immunoassays: recommendations for standardisation, calibration and validation.

Accurate and sensitive methods for measurement and detection of cytokines are important for the understanding of cytokine biology and biochemistry and the assessment of cytokine involvement in pathology and physiological processes. Various types of assays are available for estimation of cytokine levels in biological fluids. Often, immunoassays are used for this purpose but in many cases inappropriate assay choice, design and standardisation have led to confusing or erroneous data and conclusions. Only if well characterized, validated and standardized methods are used and results carefully analysed can useful unambiguous information be obtained. This article contributes significantly towards the standardisation, calibration and validation aspects of immunoassays for cytokines.

Antibody Specificity↗
PubMed1998

Developments in immunological standardization.

In the century since Paul Erlich's innovative immunological standardization work with diphtheria anti-toxin, the field of immunological standardization has expanded dramatically. Biological standards for a diverse range of immunological substances have been produced e.g. immunoglobulins, complement components, autoantibodies and blood group reagents. The concept of calibration of such materials in biological units of potency is now widely accepted for many such substances. Most recently much effort has been devoted to producing biological standards for cytokines which can be used to calibrate and validate biological assays for these analytes. Immunoassays have been found to be particularly problematical from the standardization view point although provision of a single international standard for distribution world-wide is clearly advantageous and helps reduce assay variability. It is hoped that the considerable progress with immunological standardization achieved during the past century will continue and expand to ensure the validity of existing and new immunological assays which will be required in the future.

History, 20th Century↗
PubMed1998

Laboratory protocols for the quantitation of cytokines by bioassay using cytokine responsive cell lines.

Cytokines have been shown to be involved in many physiological processes, including the maintenance and control of a competent haematopoietic/immune system. Abnormal cytokine secretion or synthesis can cause a wide range of pathological disorders. Cytokines have also been shown to have therapeutic potential in many diseases. In order for the role of cytokines to be evaluated in normal and pathogenic processes, it is vital that appropriate assay systems are used to measure their levels in different biological fluids. Whilst immunoassays maybe a more convenient method for quantitating cytokines, they only measure immunologically reactive material. They may or may not detect biologically inactive material, such as cytokines bound to soluble receptors or degraded cytokine molecules. Bioassays, however, detect biologically active cytokines and can be as accurate and precise as immunoassays. The purpose of these protocols is to provide practical stepwise methods for the bioassay of cytokines using cytokine responsive cell lines. They include tables of the most useful currently available cell lines for the detection of cytokines and how to maintain them. In addition, these protocols provide all the materials and methods in a logical step-by-step procedure to carry out bioassays. Information is provided on possible pitfalls and general problems associated with bioassays and how to overcome them. These protocols should be valuable to scientists new to the cytokine field as well as experienced scientists who require a consensus methodology and access to information on cell lines useful for cytokine bioassays.

Animals↗
PubMed1998

Interferon alpha primes early proliferative response of bone marrow cells in vivo.

Using colony-forming assays, a number of previous studies established that interferon alpha (IFN-alpha) could cause bone marrow cell (BMC) suppression. In this study, the suppressive effect of IFN-alpha is however shown to be time-dependent, occurring only 7-8 days after transfer of BMC obtained from IFN-alpha-treated mice to growth factor-containing culture medium. In contrast, in the interval before suppression is observed, BMC obtained from IFN-alpha-treated mice initially proliferated more rapidly than BMC from placebo-treated mice. These findings suggest that IFN-alpha acts in vivo to prime the proliferative responses of BMC, a hitherto unexpected action which may have clinical relevance.

Animals↗
PubMed1998

IgG purification.

Explore the source record for details and available documents.

Ammonium Sulfate↗
PubMed1998

Human monoclonal antibodies encoded by the V4-34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy-chain variable region.

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.

Adult↗
PubMed1997

Implications for the assay and biological activity of interleukin-8: results of a WHO international collaborative study.

Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study. 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-8 can have different biological specific activities, even though all were produced using E. coli. It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays. In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8. 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-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.

Biological Assay↗
PubMed1997

Cytokines in platelet concentrates: a comparison of apheresis platelet (haemonetics) and filtered and unfiltered pooled buffy-coat derived platelet concentrates.

Variable degrees of platelet activation, shape changes, microvesiculation and fragmentation may occur during collection, processing and storage of platelet concentrates (PCs), contributing to different rate of platelet storage lesion. Leukocytes contribute to both the frequency of transfusion reactions and the acceleration of the rate of platelet storage lesion hence leukocyte removal of platelet concentrates has been introduced to overcome these problems. However transfusion reaction can still occur with the use of leuko-reduced products and it is not fully elucidated that the rate of storage lesion is equivalent for filtered and unfiltered counter parts. This issue has been addressed in this manuscript comparing the generation of cytokines during storage in PCs derived from pooled buffy coat with the standard apheresis products, with a similar level of leukocyte contamination. The EDTA-induced shape change in platelet was used as an index of platelet functional integrity. In addition IL-8 and TGF beta were used as indicators of filtration process-inducing stimulation of cytokines. Our results clearly indicate that a rapid disc/spheric conversion occurs during storage of buffy-coat derived PC, and while prestorage filtration reduces both IL-8 content immediately after filtration and at the end of platelet shelf life but such a process may lead to slight enhancement of the rate of TGF beta generation indicating that any additional process may have some bearing in stimulation of TGF beta release.

Blood Platelets↗
PubMed1997

Are cytokines in platelet concentrates responsible for febrile transfusion reactions?

In recent years, several studies have identified the leukocyte content and the age of the blood components as dominant factors in febrile transfusion reactions (FTRs). At present, extensive efforts are being made to reduce adverse effects by implementation and/or introduction of new methods for leuko-depletion of blood components and by studying the mechanisms responsible for these phenomena. A recent approach has been the evaluation of cytokines in platelet concentrates and this issue has been addressed with some detail in this review. Comparative data currently available on levels of cytokines in the different platelet concentrates is provided along with the functional role of the detected cytokines in including adverse reactions.

Cytokines↗
PubMed1997

Treatment of metastatic renal cell carcinoma with subcutaneous interleukin 2: evidence for non-renal clearance of cytokines.

The circulating cytokine concentrations following administration of subcutaneous recombinant interleukin 2 (IL-2) in combination with interferon alpha and 5-fluorouracil used to treat advanced renal cancer were studied. One patient was anephric and on dialysis, and seven had normal biochemical renal function, although five had undergone single nephrectomy. The pharmacokinetics of IL-2 and changes in IL-6 and tumour necrosis factor (TNF)-alpha were essentially similar in all patients including the anephric patient, irrespective of the periods of dialysis, although at some time points, IL-2 concentrations were slightly higher in the anephric patient than in the others. These results show that for subcutaneous administration of low-dose IL-2, renal clearance of IL-2 is not important. This contrasts with high-dose, intravenous IL-2 where blood concentrations are higher and renal clearance seems to occur, perhaps because of saturation of the non-renal mechanisms of clearance. The subcutaneous route is certainly preferred if IL-2 is used in anephric patients and in those with impaired renal function, and it may be generally preferred for most purposes.

Adult↗
PubMed1997

Fighting, fleeing and having fun: the immunology of physical activity.

Exercise places a spectrum of demands on the body, dependent on the form, intensity, and duration, which are super-imposed on a background of physiological and psychological factors peculiar to the host. Thus the net effect of these factors is both heterogeneous and complex. Studying the effects of exercise is dependent on an understanding of an elaborate network of interactions between the central nervous, endocrine, and immune systems that is yet to be understood. While investigators agree that immune suppression results from exhaustive exercise, opinions vary about its mechanism. Some of this is due to inter- and even intra-subject variation (perceptions, previous experience, gender, age, biological rhythms, other temporally related events, attributions, etc.), yet other is a result of differences in study design, parameters measured, methods and materials used, and a host of other variables. To achieve accord and to define the mechanisms leading to changes in health status, beneficial or harmful, that result from physical activity, we must strive to understand the complex network that exists in the psychoneuroendocrine immune system, design rigorous research models, standardize our methods, and offer sound hypotheses for future study. Lastly, investigations into exercise-induced immune alterations need to be conducted by multidisciplinary teams of individuals expert in each of the fields encompassed by this complex field of study. After offering some examples of the complex interactions between components of the psychoneuroendocrine immune axis, we discuss study design, caveats of laboratory methods, data reduction and interpretation, and a means of perhaps achieving our common goals in studying exercise immunology.

Animals↗
PubMed1997

Production and characterization of monoclonal antibodies to human interleukin-12.

Interleukin-12 is a cytokine that plays a central role in mediating cell mediated immunity via enhancement of a TH1 cell response. IL-12, unusually for a cytokine, has a heterodimeric structure made up of 35 kDa and 40 kDa subunits. The aim of this study was to produce and characterize monoclonal antibodies to recombinant human IL-12. Twenty-two monoclonal antibodies to IL-12 were successfully produced and subunit specificity determined using recombinant human IL-12 and chimeric murine/human IL-12. All antibodies were shown to react with the p40 subunit by ELISA and immunoblotting with three of the MAbs being found to cross-react with murine IL-12. Using two individual bioassays for IL-12, seven of the MAbs were shown to neutralize biological activity of IL-12. Ten of the antibodies were found to be of use in immunocytochemistry, reacting with LPS-stimulated peripheral blood monocytes. The approaches and difficulties encountered in characterizing antibodies to a heterodimeric cytokine are discussed together with possible reasons for the failure to generate antibodies to the p35 subunit.

Animals↗
PubMed1997

Suction dressings: a new surgical dressing technique.

A new technique in surgical dressing is described which is a combination of a semi-permeable dressing and suction drainage. This dressing has been used successfully in 20 orthopaedic patients without any wound complication and with satisfactory comfort to the patient. This form of post-operative wound management appears to retain the nursing and hygiene advantages of suction drainage, whilst avoiding the patient discomfort and the possibilities of wound infection, associated with deep internal drainage.

Adult↗