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Anthony Meager

Publications and source records attributed to Anthony Meager.

13 recordsLinked to original sources
PubMed2006

Thermal stability of the WHO international standard of interferon alpha 2b (IFN-alpha 2b): application of new reporter gene assay for IFN-alpha 2b potency determinations.

A World Health Organization requirement for biological standards is that they should exhibit long-term stability at their recommended storage temperature. Thermal stability is usually predicted in accelerated thermal degradation studies, where ampoules of the lyophilized standard are stored at elevated temperatures for relatively short times before testing. To confirm the predicted thermal stability of the 2nd international standard of human interferon alpha 2b (IFN-alpha2b; 95/566), we tested the potency of the ampouled contents of this standard after 9 years storage at the customary storage temperature of -20 degrees C in comparison with ampoules of the IS which had been stored continuously at temperatures ranging from -150 degrees C to 56 degrees C. Since IFN-alpha2b potency estimates derived from the results of antiviral assays (AVA) showed high within-assay variability, we investigated a novel reporter gene assay (RGA) based on induction of secreted alkaline phosphatase (SEAP) for comparability and precision of such estimations. We show that this RGA generated comparable estimates with overall lower variation. Additionally, the SEAP conversion of p-nitrophenyl phosphate to yellow product could be followed kinetically. Absorbance readings were shown to increase with time in proportion with increasing concentration of IFN-alpha2b. When the time-dependent increments of absorbance were plotted graphically, the slopes of lines corresponded to concentration. This approach enabled single dilutions of IFN samples, identical in molecular structure to an IFN-alpha2b standard, to be used for potency estimates by interpolation of slope value against those of the standard at fixed concentrations. It appears attractive for high through-put potency testing of various R&D IFN-alpha2b samples.

Cell Line, Tumor↗
PubMed2006

Autoimmune polyendocrine syndrome type 1 in Norway: phenotypic variation, autoantibodies, and novel mutations in the autoimmune regulator gene.

CONTEXT: The autoimmune polyendocrine syndrome type I (APS I) is a rare disease that previously was difficult to diagnose. Autoantibody screening as well as mutational analysis of the disease gene autoimmune regulator (AIRE) are important diagnostic tools for this life-threatening syndrome. OBJECTIVE: The objective of the study was to identify all patients with APS I in Norway and correlate their clinical features with their autoantibody profiles and mutations in the AIRE gene. PATIENTS: We identified 36 Norwegian patients from 24 families with APS I (20 males, 16 females) during a nationwide survey for patients with Addison's disease and polyendocrine syndromes, seven of them only after their death. RESEARCH DESIGN AND METHODS: Clinical data were collected from questionnaires and patient records. AIRE mutations were determined by DNA sequencing. Most autoantibodies were measured in RIAs against recombinant autoantigens, but anti-type I interferon (IFN) antibodies were titrated in ELISA or antiviral interferon neutralization assays. RESULTS: The prevalence of APS I in Norway was estimated to be about 1:90,000. Several patients exhibited a milder phenotype with few APS I disease components and onset only in late adolescent or adulthood. The others showed about the same distribution of disease components as reported in Finnish patients. Eleven different mutations were identified in the AIRE gene, six of these were novel, i.e. c.22C>T (p.Arg8Cys), c.290T>C (p.Leu97Pro), c.402delC (p.Ser135GlnfsX12), c.879 + 1G>A (p.IVS7 + 1G>A), c.1249dupC (p.Leu417ProfsX7), and c.1336T>G (p.Cys446Gly). The 13-bp deletion in exon 8 (c.967-979del13) was the most prevalent mutation, present in 23 of 48 (48%) of the alleles. The presence of neutralizing autoantibodies against IFN-omega was the most specific marker of APS I, being found in all but one Norwegian patient. Some other common APS I-associated autoantibodies appeared de novo during long-term follow-up of younger patients. CONCLUSIONS: Norwegian patients with APS I clinically resemble those from Finland and other European countries, but some have milder phenotypes. In total, six new mutations were identified in the Norwegian APS I patients. Anti-type I IFN autoantibodies are easily detectable; their APS I specificity and persistently high titers render them reliable markers of APS I, even in prodromal or atypical cases. Both the clinical features and the AIRE mutations are more diverse in the Norwegian population than previously thought.

Addison Disease↗
PubMed2006

Continuous delivery of human type I interferons (alpha/beta) has significant activity against acute myeloid leukemia cells in vitro and in a xenograft model.

In this study, we focused primarily on the antileukemic activity of interferon-beta (IFN-beta) in a murine xenograft model of acute myeloid leukemia (AML). Bolus administration of recombinant IFN-beta via the subcutaneous or intravenous route failed to show efficacy in mice injected with AML cells despite achieving peak plasma IFN-beta levels of more than 200 IU/mL. In contrast, stable expression of IFN-beta following adeno-associated virus (AAV) vector-mediated gene transfer resulted in significant antileukemic activity against primary AML cells derived from patients with poor prognostic markers. An almost linear relationship was observed with stable plasma levels of IFN-beta and antileukemic activity in mice. Even levels below 10 IU/mL were able to reduce tumor load by 50-fold when compared with control animals. These levels of IFN-beta are likely to be nontoxic in humans. Therefore, approaches capable of maintaining stable plasma levels of IFN-beta merit further clinical evaluation in patients with AML.

Acute Disease↗
PubMed2006

Measurement of cytokines by bioassays: theory and application.

A large number of cytokines have been characterized, of which several have proved successful in the clinic as biotherapeutic agents for malignant, infectious or autoimmune diseases. As biologically active proteins, they cannot be fully characterized by physicochemical methods alone. Thus, biological assays (bioassays) have become increasingly important for their biological characterization and potency determinations. Since cytokines exert various biological activities in vitro, cultured cell line-based bioassay methods have mainly been developed to quantify potency. Such bioassays, like all biological systems, are inherently variable. Thus, measurement of potency of a particular cytokine must be made relative to a common, stable, reference preparation of the same cytokine to permit valid inter-assay and inter-laboratory comparisons. The development and establishment of appropriate primary reference preparations as World Health Organization (WHO) International standards (IS) and reference reagents (RR) is essential for the standardization of bioassays. This review addresses the practical and statistical considerations for the development of valid bioassays, the preparation and use of WHO IS and RR and, in brief, the types of bioassay methods applicable to potency measurements of individual cytokines. More extensive details for the potency determinations of tumor necrosis factor-alpha (TNF-alpha), related cytokines, and biotherapeutic anti-TNF-alpha products are provided.

Animals↗
PubMed2006

Anti-interferon autoantibodies in autoimmune polyendocrinopathy syndrome type 1.

BACKGROUND: The autoimmune regulator (AIRE) gene influences thymic self-tolerance induction. In autoimmune polyendocrinopathy syndrome type 1 (APS1; OMIM 240300), recessive AIRE mutations lead to autoimmunity targetting endocrine and other epithelial tissues, although chronic candidiasis usually appears first. Autoimmunity and chronic candidiasis can associate with thymomas as well. Patients with these tumours frequently also have high titre immunoglobulin G autoantibodies neutralising type I interferon (IFN)-alpha and IFN-omega, which are secreted signalling proteins of the cytokine superfamily involved in both innate and adaptive immunity. METHODS AND FINDINGS: We tested for serum autoantibodies to type I IFNs and other immunoregulatory cytokines using specific binding and neutralisation assays. Unexpectedly, in 60/60 Finnish and 16/16 Norwegian APS1 patients with both AIRE alleles mutated, we found high titre neutralising immunoglobulin G autoantibodies to most IFN-alpha subtypes and especially IFN-omega (60% homologous to IFN-alpha)-mostly in the earliest samples. We found lower titres against IFN-beta (30% homologous to IFN-alpha) in 23% of patients; two-thirds of these (from Finland only) also had low titres against the distantly related "type III IFN" (IFN-lambda1; alias interleukin-29). However, autoantibodies to the unrelated type II IFN, IFN-gamma, and other immunoregulatory cytokines, such as interleukin-10 and interleukin-12, were much rarer and did not neutralise. Neutralising titres against type I IFNs averaged even higher in patients with APS1 than in patients with thymomas. Anti-type I IFN autoantibodies preceded overt candidiasis (and several of the autoimmune disorders) in the informative patients, and persisted for decades thereafter. They were undetectable in unaffected heterozygous relatives of APS1 probands (except for low titres against IFN-lambda1), in APS2 patients, and in isolated cases of the endocrine diseases most typical of APS1, so they appear to be APS1-specific. Looking for potentially autoimmunising cell types, we found numerous IFN-alpha(+) antigen-presenting cells-plus strong evidence of local IFN secretion-in the normal thymic medulla (where AIRE expression is strongest), and also in normal germinal centres, where it could perpetuate these autoantibody responses once initiated. IFN-alpha2 and IFN-alpha8 transcripts were also more abundant in antigen-presenting cells cultured from an APS1 patient's blood than from age-matched healthy controls. CONCLUSIONS: These apparently spontaneous autoantibody responses to IFNs, particularly IFN-alpha and IFN-omega, segregate like a recessive trait; their high "penetrance" is especially remarkable for such a variable condition. Their apparent restriction to APS1 patients implies practical value in the clinic, e.g., in diagnosing unusual or prodromal AIRE-mutant patients with only single components of APS1, and possibly in prognosis if they prove to predict its onset. These autoantibody responses also raise numerous questions, e.g., about the rarity of other infections in APS1. Moreover, there must also be clues to autoimmunising mechanisms/cell types in the hierarchy of preferences for IFN-omega, IFN-alpha8, IFN-alpha2, and IFN-beta and IFN-lambda1.

Adolescent↗
PubMed2005

Biological standardization of human interferon beta: establishment of a replacement world health organization international biological standard for human glycosylated interferon beta.

Human interferon beta (IFN-beta) has been developed as a major biotherapeutic agent for the treatment of multiple sclerosis. Since World Health Organization (WHO) international standards (IS) for IFN-beta were established several years prior to the development of clinical grade IFN-beta products, a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-beta products have emerged. In order to address these issues, an international collaborative study to evaluate WHO IS and candidate international standards (CIS) of IFN-beta was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2000 and was carried out in the succeeding year. Sixteen expert laboratories from 8 countries worldwide participated in the study. They performed titrations on 8 different IFN-beta preparations, including IS and new CIS, in a variety of mainly antiviral- but also including some antiproliferative- and reporter gene-assays, and contributed raw data from these assays to NIBSC for statistical analysis and calculation of potencies. While both intra- and inter-laboratory variation of potency estimates was evident, overall validity of the study as a whole was clearly shown by comparison of two pairs of internal coded duplicates, which gave the expected relative potency of 1 and the lowest inter-laboratory variability of potency estimates in all assay types. The CIS containing Chinese hamster ovary (CHO) cell- or human fibroblast-derived, glycosylated, IFN-beta gave similar low inter-laboratory variation in potency estimates one to another as the coded duplicates, which was significantly less than to the 2nd WHO IS of IFN-beta, human fibroblast-derived, Gb23-902-531. One of these CIS, designated 00/572, containing CHO cell-derived IFN-beta and formulated with both bovine casein and human serum albumin, could be assigned a potency, consistent for all assay types, of 40,000 international units (IU) per ampoule relative to the IU of the 2nd IS of IFN-beta, Gb23-902-531. Other CIS containing glycosylated IFN-beta, either CHO cell- or human-fibroblast-derived, could also be assigned potency values that were continuous with the IU of Gb23-902-531 and 00/572. However, greater inter-laboratory variations in estimates were evident from comparisons of Gb23-902-531 or 00/572 with either the 1st IS for E. coli-derived, non-glycosylated, IFN-beta with serine substitution at position 17 (IFN-beta Ser 17 mutein), Gxb02-901-535, or with a CIS (00/574) containing IFN-beta Ser 17 mutein. Indeed, variations in potency estimates for preparations containing IFN-beta Ser 17 mutein were sufficiently large to indicate that assays could distinguish preparations of IFN-beta Ser 17 mutein from preparations of glycosylated IFN-beta. Thus, neither the 2nd IS of IFN-beta, Gb23-902-531, containing fibroblast-derived IFN-beta, nor CIS, 00/572, containing CHO cell-derived IFN-beta, was appropriate for standardisation of preparations of IFN-beta Ser 17 mutein. Conversely, neither the IS of IFN-beta Ser 17 mutein, Gxb02-901-535, or a CIS of IFN-beta Ser 17 mutein, 00/574, was appropriate for the standardisation of preparations of glycosylated IFN-beta. CIS 00/572, containing CHO cell-derived, glycosylated IFN-beta, was clearly shown to be suitable to serve as a primary standard for glycosylated forms of IFN-beta, especially clinical grade IFN-beta-1a products. It was further shown to exhibit high thermal and long-term stability. Since the CHO cell-derived IFN-beta used for preparation of 00/572 was of a greater purity than the IFN-beta used for the 2nd IS of IFN-beta, Gb23-902-531, it was recommended by the WHO Informal Consultation on the Standardisation of Cytokines, Growth Factors and Other Endocrinological Substances, which met in October 2003, that 00/572 should replace Gb23-902-531 as the IS for glycosylated IFN-beta. This recommendation was accepted by the WHO Expert Committee on Biological Standardization (ECBS) at its annual meeting in November 2003 and 00/572 was established as the 3rd IS for human glycosylated IFN-beta with an assigned potency of 40,000 IU. As this study identified no advantage to replacing the existing 1st IS for IFN-beta Ser 17 mutein, Gxb02-901-535, WHO ECBS accepted that this should continue to serve as the IS for this material.

Animals↗
PubMed2005

Biological activity of interleukins-28 and -29: comparison with type I interferons.

Despite binding to receptors distinct from those of type I interferons (IFNs), human interleukins-28A, -28B and -29 (IL-28A, IL-28B and IL-29; alternatively named IFN lambda-2 {IFN-lambda2}, IFN-lambda3 and IFN-lambda1, respectively, or collectively, type III IFNs), a small family of three structurally-related cytokines, are, like IFNs, known to induce antiviral activity. To further biologically characterize IL-28A and IL-29, we compared their activities with those of IFNs in a range of human cell lines. We found that they induced antiviral activity in fewer cell lines and more weakly than IFNs; also IL-28A was less active than IL-29. Additionally, we showed IL-28A and IL-29 induced reporter genes--protein MxA promoter linked to luciferase, or interferon stimulated response element (ISRE) linked to secreted alkaline phosphatase (SEAP)--more weakly than IFN. Antiproliferative activity was induced by IFNs in most cell lines, but only in one human glioblastoma cell line, LN319, was dose-dependent IL-29-growth inhibition demonstrable. Polymerase chain reaction (PCR) quantification of messenger (m) RNA of IL-28/29 receptor subunits, IL-28Ralpha and IL-10Rbeta, indicated variable expression levels; although their expression was highest in the responsive LN319 cell line, lower but significant expression of both mRNAs was found in relatively unresponsive cell lines. In conclusion, we found IL-28A and IL-29 act similarly to IFNs, but are less effective generally and have activity in a more limited range of cell lines.

Cell Division↗
PubMed2003

Spontaneous production of anti-IFN-alpha and anti-IL-12 autoantibodies by thymoma cells from myasthenia gravis patients suggests autoimmunization in the tumor.

Myasthenia gravis (MG) is mediated by autoantibodies to the acetylcholine receptor (AChR), expressed in muscle and rare thymic myoid cells. Most early-onset cases show thymic lymph node-type infiltrates, including pre-activated plasma cells spontaneously producing anti-AChR antibodies. Since these are not evident in the associated thymomas found in another 10% of MG patients, AChR-specific B cells must be autosensitized elsewhere. Unexpectedly, at diagnosis, >70% of MG/thymoma patients also have high-titer neutralizing autoantibodies to IFN-alpha, and >50% to IL-12; moreover, titers increase strikingly if the thymomas recur, indicating a closer tumor relationship than for anti-AChR. To investigate this, we have measured autoantibody production by cells cultured from thymomas, any available thymic remnants and blood, with or without the B cell stimulant pokeweed mitogen (PWM). To check autoantibody specificity and clonal origins, we isolated Fabs from two combinatorial libraries from producer thymus/thymoma cells. Surprisingly, thymoma cells spontaneously produced antibodies to IFN-alpha and/or IL-12 in >40% of seropositive cases, showing typical plasma cell behavior, whereas they produced anti-AChR only after PWM stimulation. We isolated 15 combinatorial Fabs to IFN-alpha (versus only one to AChR). Their strong binding in radio-immunoprecipitation and Western blots implies high affinities. The four Fabs tested neutralized anti-viral actions of IFN-alpha. The diverse V genes clearly showed ongoing antigen-driven selection. These results imply pre-activation in situ by native IFN-alpha/IL-12 expressed within a 'dangerous' tumor microenvironment. With these molecules, it should be easier to identify provoking cell type(s) that may give novel additional clues to autoimmunization against T-cell epitopes from the more complex AChR.

Adult↗
PubMed2002

Biological assays for interferons.

Interferons (IFN) are potent biologically active proteins synthesised and secreted by somatic cells of all mammalian species. They have been well characterised, especially those of human origin, with respect to structure, biological activities, and clinical therapeutic effects. While structural differences are known to exist among the IFN species that constitute the "IFN family" and despite the existence of different receptors for type I and type II IFN, all species have been shown to exert a similar spectrum of in vitro biological activities in responsive cells. Principal among the biological activities induced by IFN is antiviral activity, the activity used to originally define IFN. Antiviral activity of IFN is mediated via cell receptors and is dependent on the activation of signalling pathways, the expression of specific gene products, and the development of antiviral mechanisms. Sensitivity of cells to IFN-mediated antiviral activity is variable, and depends on a number of factors including cell type, expression of IFN receptors and downstream effector response elements, effectiveness of antiviral mechanisms, and the type of virus used to infect cells. Nevertheless, by the judicious use of sensitive cell lines in combination with appropriate cytopathic viruses, effective assays to measure the antiviral activity have been developed. Historically, "antiviral assays" (AVA) were the first type of biological assays that were developed to measure the relative activity or potency of IFN preparations. However, the subsequent discoveries of several other biological activities of IFN has opened the way to the development of assays based on one or other of these activities. The latter include inhibition of cell proliferation, regulation of functional cellular activities, regulation of cellular differentiation and immunomodulation. More recently, the cloning of IFN responsive genes has led to the development of "reporter gene assays". In this case, the promoter region of IFN responsive genes is linked with a heterologous reporter gene, for example, firefly luciferase or alkaline phosphatase, and transfected into an IFN-sensitive cell line. Stably transfected cell lines exposed to IFN increase expression of the reporter gene product in direct relation to the dose of IFN, the readout being a measure of this product's enzymic action. The current review aims to give a critical overview of the development, specificity, standardisation and present use of the various biological assay methods now available for the quantification of IFN activity.

Animals↗
PubMedDate not supplied

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region.

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.

Amino Acid Sequence↗