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The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs., (Copyright © 2015. Published by Elsevier B.V.)

Aim: To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs., Methods: Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines., Results: Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894-6051 pg ml⁻¹) compared with muromonab-CD3 (62-262 pg ml⁻¹) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype., Conclusions: The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release., (© 2013 Medicines and Healthcare products Regulatory Agency MHRA. The British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

In 2006, a first-in-man phase-I clinical trial of an immunomodulatory mAb, TGN1412, ended in disaster when six healthy recipients suffered a life-threatening systemic inflammatory response, termed a 'Cytokine Storm'. A subsequent investigation concluded that these serious adverse events, not predicted by pre-clinical safety testing, were unforeseen biological effects in man. However, the adverse events had been exacerbated by administration of a near-maximum immuno-stimulatory dose to volunteers, because the calculation of a safe starting dose in man had been based upon results from pre-clinical safety testing in a non-responsive species. In hindsight, many lessons have been learnt from this experience and these have prompted a revision of the European guidelines for first-in-man phase-I clinical trials of biologics. Perhaps the most important lesson is that greater caution needs to be exercised when evaluating new biologics.

The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.

Inflammatory mediators have been shown to play a major role in the complex series of co-ordinated events that occur in wound healing responses following injury. However, to date most of the studies carried out have addressed the expression, interactions and role of only one or two cytokines that are thought to be involved in wound repair. This study has evaluated, in murine skin samples taken at 0, 3, 12, 18, 24, 48, 72, 120 and 168 h post-wounding, the expression of a wide range of cytokines with potential for a role in wound repair. Various techniques (reverse transcription polymerase chain reaction (RT-PCR), bioassays and ELISA) were used to evaluate cytokine expression in these samples at both the mRNA and protein expressions level. Semi-quantitative analysis using RT-PCR revealed that IL-1beta, IP10, bFGF, and TGFbeta3 up-regulated in wounded samples, compared to non-injured control samples. Expression of mRNA for other cytokines and inflammatory mediators, IL-1alpha, IL-6, TGFbeta1, TNFalpha, MIP-1alpha, MIP-2, JE, KC, PDGFalpha and PDGFbeta, were found to be down-regulated in injured adult murine samples compared to normal control samples. Interestingly we failed to find evidence of mRNA expression for the cytokines IL-2, IL-4, IL-12, GM-CSF, IFNgamma and RANTES, in both non-injured and injured samples. These observations were also generally supported by the results obtained using bioassays for IL-1 and IL-6 and ELISA for IL-1alpha, IL-1beta, IL-6, TNFalpha, and IFNgamma.

Aims/hypothesis: Tolerance to orally administered antigens may be generated through the induction of T helper cell type 2 and 3 (Th2/Th3) regulatory cells. We previously reported that treatment of recent onset type 1 diabetes with oral insulin had no effect on residual beta cell function. The aim of this study was to evaluate whether this treatment produces a deviation in the immune response, with polarisation of the cytokine pattern and the induction of a Th2-like antibody response., Methods: Mononuclear cells were collected from a total of 20 patients with type 1 diabetes before and after 12 months of treatment with oral insulin (n=11) or placebo (n=9). Following stimulation of the cells with insulin or phytohaemagglutinin, levels of Th2 and Th3 cytokines (including TGF-beta, IFN-gamma, IL-4 and IL-5) in the culture supernatants were assessed by ELISA. In addition, levels of total and specific insulin antibody IgG subclasses were measured by radioimmunoassay in serum samples drawn from 33 patients with type 1 diabetes before and after 3, 6 and 12 months of therapy with oral insulin (n=18) or placebo (n=15)., Results: After 12 months of treatment, the release of TGF-beta was significantly higher in patients who received oral insulin compared with those who received placebo (p=0.025 and p=0.006 for lymphocytes challenged with insulin and phytohaemagglutinin respectively). The two groups had similar levels of IL-4 and IL-5 both at baseline and after 12 months of treatment. The release of IFN-gamma was markedly reduced in patients treated with oral insulin compared with those who received placebo at the 12-month follow-up. Circulating levels of IgG1 and IgG3 subclasses directed against insulin were significantly lower in the oral insulin group than in the placebo group after 12 months of treatment (p=0.05 for IgG1 and p=0.014 for IgG3)., Conclusions/interpretation: The increased TGF-beta release observed in patients treated with oral insulin suggests that a regulatory response can be induced in vivo by this treatment. The lower levels of insulin antibody IgG1 and IgG3 subclasses present in patients exposed to oral insulin are consistent with a Th2 deviation of the immune response. The failure of oral insulin treatment to provide any measurable clinical benefit may be due to the timing of treatment initiation.

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Background and Objectives: With the implementation of universal white blood cell (WBC) reduction in the UK, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) and apheresis methods are used routinely for the production of WBC-reduced PCs. While these strategies meet the specification for WBC reduction (< 5 x 10(6) WBCs/unit), the products from these processes may differ depending on the process employed and its performance. The aim of this study was therefore to investigate whether PCs prepared using various WBC-reduction processes are sufficiently depleted of WBCs to limit cytokine accumulation during storage and to assess if cytokine levels detected in platelet products can serve as indicators of acceptable platelet activation as a result of the WBC-reduction process., Materials and Methods: We measured the levels of cytokines predominantly derived from WBCs [e.g. interleukin-8 (IL-8)] and platelets [e.g. regulated on activation, normal, T-cell expressed, and secreted (RANTES) and transforming growth factor-beta(1) (TGF-beta(1))] under the present experimental conditions in different WBC-reduced PCs, i.e. PCs prepared from three different WBC-reduction filters and control non-filtered PCs using pooled BCs from the same donors and three apheresis types. Supernatant plasma was collected at the beginning (day 1) and end (day 5) of the shelf life of each PC, and the cytokine content was determined using appropriate enzyme-linked immunosorbent assays (ELISAs). Process efficiency was assessed by platelet yield and residual WBC count., Results: We found that products from the apheresis process involving a filtration step (Haemonetics MCS+) showed a lower cytokine content on both day 1 and day 5 in comparison with the fluidized bed (COBE Spectra) or elutriation (Amicus) processes. WBC reduction of BC-PCs of the same origin using three different filters showed comparable levels of cytokines on day 1 in all units. After storage for 5 days, the levels of IL-8 remained essentially unchanged in filtered BC-PCs but increased by more than threefold in control non-filtered BC-PCs, suggesting IL-8 release by residual WBCs present in the control PCs. The concentration of platelet-derived cytokines such as RANTES and TGF-beta(1), however, increased significantly in all filtered and control non-filtered PCs during the storage period., Conclusion: These results show that markers of cytokine release from both WBCs and platelets are useful indicators of the performance and efficacy of the WBC-reduction process and of platelet quality.

Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 x 10(6) leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-beta1 in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10-24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-beta1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-beta1 are useful quality indicators for validation of leucoreduced red cell preparations.

Meningococcal A and C but not B capsular polysaccharides stimulated a low level primary antibody response, predominantly IgM, and no secondary response in 21-day-old CBA/A mice. However, in 56-day-old mice a higher proportion of IgG antibody and a secondary response were produced. When the polysaccharides were injected in conjunction with rDNA derived human interleukin 2 (IL-2) the IgG antibody responses were increased in both age groups and memory cells were primed in the younger mice. IL-2 increased significantly the IgG antibody response to conjugates of A and C polysaccharides with diphtheria mutant protein but exerted a minimal effect on the IgG response to B polysaccharide complexed with aluminium hydroxide and outer membrane proteins. The stimulatory effect of IL-2 on the antibody responses to the polysaccharide antigens was not mediated by T-cells as similar results were obtained in athymic (nu/nu) and thymocompetent (nu/+) mice. However, the response to the A and C oligosaccharide conjugates was T-cell dependent and occurred only in the heterozygotes. In this case the adjuvant effect of IL-2 was seen only in the response to the C polysaccharide conjugate and was transferable with T-lymphocytes from primed animals.

Background: Several studies have suggested that cytokine accumulation during storage of platelet concentrates (PCs) may mediate nonhemolytic febrile transfusion reactions and that a reduction in WBC numbers prevents the generation of cytokines. Despite efforts to minimize WBC contamination in apheresis PCs, high numbers of WBCs and increased cytokine levels may still occur, depending on the quality of the apheresis device employed., Study Design and Methods: This study was undertaken to investigate whether PCs collected with WBC-reduction devices (Spectra LRS, COBE;or MCS+ LDP, Haemonetics) were sufficiently depleted of WBCs to limit cytokine accumulation during storage. The study evaluated 1) the levels of cytokines of WBC and platelet origin in two types of apheresis PCs during storage and 2) the effects of prestorage filtration on cytokine levels in the Spectra LRS PCs., Results: In the Spectra LRS PCs, low levels of IL-6, IL-8, and monotype chemoattractant protein 1 (MCP-1) were detected in Day 1 PCs, and they remained consistent during the shelf life. RANTES, platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and transforming growth factor (TGF)-beta1 were also detected in these PCs, and their levels increased significantly on storage. Prestorage filtration of Spectra LRS PCs did not further reduce the levels of IL-6, IL-8, MCP-1, PF4, beta-TG, and TGF-beta1 in the filtered component. In the MCS+ LDP PCs, IL-6 was detected on Day 1, and its level increased significantly on storage, whereas the levels in the Spectra PCs remained steady. IL-8 levels were lower in MCS+ LDP PCs than in Spectra LRS PCs of the same age. MCP-1 levels were similar in both products on Day 1 and marginally increased in stored MCS+ LDP PCs. Substantial amounts of RANTES, PF4, beta-TG, and TGF-beta1 occurred in Day 1 MCS+ LDP PCs, and, on storage, these levels rose significantly., Conclusion: Despite a significant reduction in levels of WBC-derived cytokines, platelet-derived cytokines were present in different amounts in the two products.

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.

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.

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.

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.

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.

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.

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.

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.

In summary, many factors influence the estimates of cytokine levels provided by immunoassays. If immunoassays are to supply data which are valid and useful to researchers and clinicians, these factors must be fully investigated during assay design and construction.

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.

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.

Viral infection has been suggested to play a triggering role in the pancreatic beta cell destruction which occurs in insulin-dependent diabetes (IDDM). However, the underlying mechanism of this phenomenon is unknown. In this study a human insulinoma cell line has been infected with measles, mumps and rubella viruses since a temporal association is reported between the clinical onset of IDDM and diseases caused by these viruses. The infection with measles and mumps viruses induced the release of interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cell line as assessed by a bioassay and up-regulated the expression of human leucocyte antigen (HLA) class I and class II antigens as evaluated by cytofluorimetric analysis. Stimulation with rubella virus induced the release of IL-6 only and had no effect on HLA antigen expression. These data show for the first time that IL-1 and IL-6 secretion by an insulinoma cell line may occur after viral infection and suggest that cytokine release and increased expression of HLA molecules by beta cells may act to induce the immune response towards beta cells in IDDM.

Cytokines are known to play an important role in autoimmunity and have been suggested to be involved in the pathogenesis of insulin-dependent diabetes (IDDM). In the present study we have measured IL-1, IL-2, IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) (using both immunoassays and bioassays) in sera from 50 patients affected by IDDM at the time of clinical diagnosis and 51 age and sex matched controls. Detectable levels of IL-1, IL-2, IL-6 and IFN-gamma were found in the serum of a small percentage of subjects and were not significantly different between patients and controls. IL-4 was detectable in a higher number of both patients and controls and circulating TNF-alpha (greater than 1 U/ml) was found in a percentage of patients (24%) significantly higher than controls (P less than 0.01). Raised levels of TNF-alpha were detectable using an immunoenzymatic assay whereas TNF bioactivity in these samples was negligible. We conclude that the presence of immunoreactive TNF-alpha in the patient's sera may reflect an increased localized production of this cytokine at pancreatic level. However, the measurement in serum of other cytokines does not add information on the role that they may play in the pathogenesis of IDDM.

The failure of regulatory science to keep pace with and support the development of new biological medicines was very publically highlighted in March 2006 when the first-in-man Phase I clinical trial of the immunomodulatory CD28-specific monoclonal antibody (mAb) TGN1412 ended in disaster when all six volunteers suffered a life-threatening adverse reaction termed a 'Cytokine Storm'. The poor predictive value of standard pre-clinical safety tests and animal models applied to TGN1412 demonstrated the need for a new generation of immunotoxicity assays and animal models that are both sensitive and predictive of clinical outcome in man. The non-predictive result obtained from pre-clinical safety testing in cynomolgus macaques has now been attributed to a lack of CD28 expression on CD4+ effector memory T-cells that therefore cannot be stimulated by TGN1412. In contrast, high levels of CD28 are expressed on human CD4+ effector memory T-cells, the source of most TGN1412-stimulated pro-inflammatory cytokines. Standard in vitro safety tests with human cells were also non-predictive as they did not replicate in vivo presentation of TGN1412. It was subsequently shown that, if an immobilized therapeutic mAb-based assay or endothelial cell co-culture assay was used to evaluate TGN1412, then these would have predicted a pro-inflammatory response in man. New in vitro assays based on these approaches are now being applied to emerging therapeutics to hopefully prevent a repeat of the TGN1412 incident. It has emerged that the mechanism of pro-inflammatory cytokine release stimulated by TGN1412 is different to that of other therapeutic mAbs, such that standard pro-inflammatory markers such as TNF α and IL-8 are not discriminatory. Rather, IL-2 release and lymphoproliferation are optimal readouts of a TGN1412-like pro-inflammatory response. [ABSTRACT FROM AUTHOR]

Background and Purpose: In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412. To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established. Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology.Experimental Approach: Polychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing.Key Results: In vitro IL-2 and IFN-γ release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response. This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells. In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412.Conclusions and Implications: It is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm. Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a 'cytokine storm' in humans. [ABSTRACT FROM AUTHOR]

Background and Objectives The accumulation of platelet-derived cytokines in platelet concentrates (PC) during storage may contribute towards non-haemolytic transfusion reactions (NHTR). We investigated the effect of platelet storage medium on platelet activation, complement activation and cytokine levels in leucocyte-reduced PC. Materials and Methods Hyperconcentrated platelets (3000–6000 × 109 /l) were collected by apheresis and diluted in 100% plasma, 70% PASIII, or 70% or 80% PASIII supplemented with magnesium and potassium (PAS IIIM). Results Levels of transforming growth factor-β (TGF-β) and regulated on activation, normal, T-cell expressed, and secreted (RANTES) increased during storage, as did the expression of P-selectin (CD62P), and were highest in PC stored in PASIII. In PC stored in PASIIIM, however, levels of TGF-β and RANTES were not significantly different from PC stored in plasma. Levels of CD62P expression, however, remained higher in PASIIIM PC than in those stored in plasma by day 5, but were lower than PC stored in PASIII. C3a des arg levels increased during storage in all media with the exception of PASIII and, on day 7, were higher in PC stored in plasma compared to PC stored in the other media. Conclusions Our results indicate that replacing plasma in PC with unmodified PASIII for storage results in higher levels of platelet-derived cytokines in PC. Furthermore, it appears that the nature of the medium used for storage of PC has a significant impact on platelet activation and cytokine levels of the PC. These implications should be taken into account when considering replacement of plasma with PAS. [ABSTRACT FROM AUTHOR]

Presents a commentary on a study by Hodge on the immunological effects of factor VIII concentrates. Study findings on the effects of coagulation factor concentrates on cytokin secretion confirmed by the Hodge's study; Correlations between concentrates.

Biological therapeutics, particularly those produced by the biotechnology industry, have been perceived as highly complex, novel and having ‘unknown’ adverse potential. This has led to the rapid growth of biological regulatory control of such products, with a plethora of regulations (often originally based on classical chemical drugs) in addition to case-by-case treatment by control authorities. There is a need for a relationship between industry and regulators using a scientifically based dialogue in order to establish appropriate scientifically based guidelines. These guidelines need to take into account increasing experience in manufacturing, sophisticated analytical techniques and the clinical use of biotherapeutics. Such dialogue must include a rationale of risk/benefit that is not unique to these products and therefore redresses the concept that complexity equates to potential hazard. Current regulatory issues are focused on the use of scientific experience and understanding to relate regulations produced during the early development of the bioindustry to the present use of these materials in the clinic and the realistic potential for difficulties when compared to chemical drugs. This covers such issues as toxicity, heterogeneity and analysis in relation to quality. When considering the significance of any issue for the regulation of a biological product it must be in relation to the type of product (e.g. homogeneous or heterogeneous) its clinical use (e.g. single or multiple dose form, life-threatening disease or not) and efficacy (e.g. complete cure, palliation or treatment adjunct) as each of these impinges on the scientific appropriateness of any control decision. [ABSTRACT FROM AUTHOR]