Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.

Linking iodine with autoimmune thyroiditis.

A great deal of circumstantial evidence has linked iodine with the rising incidence of autoimmune thyroiditis in the United States. In our investigations, we have shown directly that T cells from humans with chronic lymphocytic thyroiditis proliferate in the presence of iodinated but not in the presence of noniodinated human thyroglobulin. Moreover, the proliferative response is restored when the thyroglobulin is iodinated artificially in vitro. Using a panel of monoclonal antibodies, we found evidence that the presence of iodine induces a number of stereochemical changes in the conformation of the molecule, resulting in the loss of some antigenic determinants and the appearance of others. One prominent determinant was associated with the iodine-containing amino acid thyroxine. Both the number and position of the iodine substituents determine the precise specificity of this epitope. A new model for the study of the role of iodine in inducing thyroid autoimmunity has become available in the form of the nonobese diabetic (NOD)-H2(h4) mouse. This animal develops autoimmune thyroiditis spontaneously but in relatively low prevalence. However, if iodine is added to the drinking water, the prevalence and severity of the thyroid lesions increase markedly. The immune response is specific for thyroglobulin, both in terms of the antibody response and T-cell proliferation. In fact, the appearance of lesions can be predicted by the presence of thyroglobulin-specific IgG2b antibody. The disease, moreover, can be transferred adoptively, using spleen cells from iodine-fed donors treated in vitro with iodinated thyroglobulin. The effects of iodine feeding are greater in conventional animals compared with those maintained under specific pathogen-free conditions. Based on T-cell proliferation, it appears that the NOD-H2(h4) strain of mice has innately a greater response to murine thyroglobulin than do other mouse strains and that the proliferation is increased even more by feeding iodine. We suggest, therefore, that the presence of iodine increases the autoantigenic potency of thyroglobulin, a major pathogenic antigen in the induction of autoimmune thyroiditis. This animal model provides a unique opportunity for investigating in detail the mechanisms by which an environmental agent can trigger a pathogenic autoimmune response in a susceptible host.

Iodine is essential for human T cell recognition of human thyroglobulin.

Here we describe for the first time that recognition by human T cells of human thyroglobulin depends upon its iodine content. We have examined the proliferation of lymphocytes from blood of autoimmune thyroiditis patients and normal individuals to thyroglobulin preparations containing different amounts of iodine. A minimal degree of iodination was required to elicit the proliferative response of both patients and normal individuals since thyroglobulin preparations containing no detectable iodine did not induce proliferation. A non-iodinated thyroglobulin preparation that was iodinated in vitro produced significant proliferation of both patient and normal lymphocytes. Addition of IL-2 to the culture medium enhanced proliferation but did not change the pattern of response.

Down-regulation of surface antigens recognized by systemic lupus erythematosus antibodies on embryonal cells following differentiation and exposure to corticosteroids.

We have previously suggested that anti-DNA antibodies present in systemic lupus erythematosus patients can bind directly to tissues as a result of cross-reactivity with embryonal tissue-based antigens. Here we have analyzed the interaction between polyclonal and monoclonal mouse and human lupus autoantibodies and an embryonal cell line. We report that a murine embryonal stem cell line (ES) expresses a surface antigen which is recognized by mouse and human lupus autoantibodies. This surface antigen is down-regulated following maturation of the cells or incubation with corticosteroids. Adhesion molecules may serve as the target membrane antigen in ES cells since preincubation with these antibodies decreases the ability of ES cells to adhere to the plate.

In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food.

The role of iodine in autoimmune thyroiditis.

Like most cancers, autoimmune diseases generally are due to the interaction of a number of genetic traits with an environmental trigger. Autoimmune thyroiditis, a model of organ-specific autoimmune disease, is associated with iodine as a precipitating environmental factor. T cells from patients with chronic thyroiditis proliferate in response to normal human thyroglobulin, but fail to react with non-iodinated thyroglobulin. Using a selected monoclonal antibody, we were able to identify a binding site on thyroglobulin containing iodinated thyronine. The greatest affinity was for tetraiodothyronine and binding depended upon the number as well as the positions of iodines. We have also studied an inbred strain of mice, NOD-H2h4, that developed thyroiditis spontaneously. The onset of disease was hastened in a dose-dependent manner by adding iodine to the drinking water. The occurrence of disease was greater in conventional than in specific pathogen-free mice and correlated with T-cell proliferation and IgG2b antibody to thyroglobulin.

Iodine-induced autoimmune thyroiditis in NOD-H-2h4 mice.

Excess iodine ingestion has been implicated in induction and exacerbation of autoimmune thyroiditis in human populations and animal models. We studied the time course and sex-related differences in iodine-induced autoimmune thyroiditis in NOD-H-2h4 mice. This strain, derived from a cross of NOD with B10.A(4R), spontaneously develops autoimmune thyroiditis but not diabetes. NOD-H-2h4 mice were given either plain water or water with 0.05% iodine for 8 weeks. Approximately 54% of female and 70% of male iodine-treated mice developed thyroid lesions, whereas only 1 of 20 control animals had thyroiditis at this time. Levels of serum thyroxin (T4) were similar in the treatment and control groups. Thyroglobulin-specific antibodies were present in the iodine-treated group after 8 weeks of treatment but antibodies to thyroid peroxidase were not apparent in the serum of any of the animals. Levels of thyroglobulin antibodies increased throughout the 8-week iodine ingestion period; however, no correlation was seen between the levels of total thyroglobulin antibodies and the degree of thyroid infiltration at the time of autopsy. The thyroglobulin antibodies consisted primarily of IgG2a, IgG2b, and IgM antibodies with no detectable IgA, IgG1, or IgG3 thyroglobulin-specific antibodies. The presence of IgG2b thyroglobulin-specific antibodies correlated well with the presence of thyroid lesions.

The urine of SLE patients contains antibodies that bind to the laminin component of the extracellular matrix.

Direct immunofluorescence of tissues derived from patients affected with SLE demonstrates antibodies bound to the extracellular matrix (ECM). In the present work we have tested whether such antibodies are found in the serum and urine of lupus patients and mice. We found that the urine of patients with active SLE and of MRL/lpr/lpr mice contains antibodies that bind ECM and that a major target for these antibodies is the 200 kDa light chain of laminin which is one of the matrix components. The level of the anti-ECM, anti-laminin antibodies correlates with disease activity.

Polyclonal autoreactive IgA increase and mesangial deposition during vomitoxin-induced IgA nephropathy in the BALB/c mouse.

To establish the relationship between autoreactive antibodies and vomitoxin-induced immunoglobulin A (IgA) nephropathy, the effects of dietary vomitoxin exposure on the antigen specificity of serum IgA, IgA-producing cells and accumulated mesangial IgA in BALB/c mice were assessed. Exposure to dietary vomitoxin for 8 wk caused a significant increase in total serum IgA. There was a concurrent significant increase in serum IgA specific for trinitrophenol (TNP), phosphorylcholine, cardiolipin and sphingomyelin compared with controls, suggesting an elevation of autoreactive IgA. Casein, a protein found in the AIN-76A diet, could inhibit binding of serum IgA to sphingomyelin and cardiolipin, indicating that these antibodies may be polyspecific. When enzyme-linked immunospot assay was used to monitor autoreactive IgA production, trends were observed towards increased IgA-secreting cells specific for TNP, cardiolipin and sphingomyelin in Peyer's patches from vomitoxin-fed mice compared with control mice. IgA-producing cells reactive with TNP were increased in the spleen of vomitoxin-fed mice whereas effects on IgA-secreting cells for the other antigens were marginal. Marked deposition of mesangial IgA was also observed in vomitoxin-fed mice compared with controls. When IgA was eluted from the kidney sections of treated mice and tested by enzyme-linked immunosorbent assay, it exhibited a strong binding to the above antigen panel as well as inulin, DNA and casein. These data suggest that dietary vomitoxin induced the polyclonal activation of IgA-producing cells and that resultant autoreactive IgA was subsequently deposited in the kidney mesangium.

Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer's patches of vomitoxin-fed mice: characterization and possible pathogenic role in IgA nephropathy.

A total of 122 immunoglobulin (Ig)A-producing hybridoma clones were isolated from the Peyer's patches of vomitoxin-fed BALB/c mice and the resultant antibodies were characterized for their antigenic specificity and pathogenic potential. When reactivity was tested against a panel consisting of DNA, sphingomyelin, thyroglobulin, collagen, casein, cardiolipin and bovine serum albumin conjugates of phosphorylcholine, inulin and trinitrophenol that were representative of self and non-self antigens, approximately 95% of the monoclonal IgAs bound to at least one of the panel antigens and 80% bound to more than one antigen. The polyspecificity of some of the monoclonal IgAs was further suggested by demonstrating the capacity of one antigen to inhibit binding of monoclonal IgA to another antigen. Protein staining and Western blotting of gradient native polyacrylamide gels, indicated that trimeric IgA predominated in the isolated monoclonal IgAs. Repeated injections of mice with representative monoclonal IgAs induced microhaematuria in three of four of the clones tested but not IgA deposition in the kidney glomerulus. In addition, three of the four monoclonal IgAs caused IgG and C3 deposition in the kidney mesangium. These and previous results suggest that dietary vomitoxin promotes the polyclonal activation and expansion of IgA-secreting B cells at the Peyer's patch level and that resultant polyspecific, autoreactive IgA may contribute to kidney pathogenesis.

Vomitoxin-induced dysregulation of serum IgA, IgM and IgG reactive with gut bacterial and self antigens.

The effect of dietary vomitoxin exposure on immunoglobulins that react with naturally occurring gut bacterial and self antigens was assessed in the B6C3F1 mouse. Ingestion of 25 ppm vomitoxin for 4 and 8 wk resulted in significantly elevated total IgA but depressed total IgG and IgM in serum when compared with control mice fed semi-purified diet only. IgA specific for phosphorylcholine (PC) and inulin (haptens associated with intestinal bacteria) increased significantly in mice fed vomitoxin whereas IgM with the identical specificity decreased. When sera were assessed for autoantibodies recognizing DNA and bromelated mouse red blood cells (MRBC), vomitoxin-exposed mice exhibited elevated specific IgA as compared with controls. This occurred together with decreases in DNA-specific IgG and IgM, and decreases in MRBC-specific IgM. Additionally, vomitoxin exposure did not enhance the specific serum IgA response to orally administered trinitrophenylated sheep red blood cells (TNP-SRBC), but significantly depressed TNP-specific serum IgG. The results suggest that hyperelevation of total and specific serum IgA for oral and self antigens occurs during vomitoxin feeding and that may be coupled with down-regulation of total and specific IgM or IgG. These effects could be contributory to the capacity of vomitoxin to induce IgA immune complex glomerulonephritis.

Effect of dietary administration of the trichothecene vomitoxin (deoxynivalenol) on IgA and IgG secretion by Peyer's patch and splenic lymphocytes.

Prolonged dietary exposure of mice to the trichothecene vomitoxin induces abnormally high levels of serum IgA and kidney mesangial IgA accumulation in a manner that is highly analogous to the human glomerulonephritis IgA nephropathy. In this study, the capacity of Peyer's patch and splenic lymphocytes to produce IgA and IgG were compared in B6C3F1 mice that were fed diets with and without 25 ppm vomitoxin for up to 12 wk. Serum IgA increased 2-, 4- and 8-fold after 4, 8 and 12 wk, respectively, of vomitoxin exposure and it became the primary serum isotype, whereas serum IgG was unaffected. On termination of the experiment there were increased numbers of IgA-secreting cells in Peyer's patches after 8 wk of toxin exposure and in the spleen after 4, 8 and 12 wk of toxin exposure. There were also increased numbers of IgG-secreting cells in Peyer's patches on termination of the experiment at 4, 8 and 12 wk but no effects was observed in the spleen. Supernatant IgA and IgA-secreting cell numbers were also markedly elevated in lymphocyte cultures obtained from Peyer's patches and, to a lesser extent, from spleens of treated mice compared with controls. Based on output of treated mice relative to corresponding controls, IgA secretion was greatest in concanavalin-A-stimulated and unstimulated Peyer's patch cultures. Enhanced IgG secretion and IgG-secreting cells were also observed in mitogen-stimulated and unstimulated Peyer's patch lymphocyte cultures of treated relative to control mice, but differences in splenocyte cultures were negligible. Based on total Ig output, IgA production was 8- to 20-fold greater than IgG production in both control and treatment Peyer's patch cultures. In contrast, vomitoxin treatment caused a shift from primarily IgG production in lipopolysaccharide-stimulated spleen cultures to equivalent IgA production. These data provide in vitro evidence that ingestion of vomitoxin promotes terminal differentiation of IgA-secreting progenitors in the Peyer's patch and, to a lesser extent, in the spleen. These functional changes are consistent with the shift from IgG to IgA as the primary serum isotype.

Elevated membrane IgA+ and CD4+ (T helper) populations in murine Peyer's patch and splenic lymphocytes during dietary administration of the trichothecene vomitoxin (deoxynivalenol).

Recent investigations indicate that dietary exposure to the trichothecene vomitoxin increases total and antigen-specific serum immunoglobulin A (IgA) and glomerular IgA accumulation in mice. In this study, the effects of 25 ppm dietary vomitoxin on the histological and lymphocytic profile of component immune organs in the mucosal lymphocyte migratory pathway were evaluated in the B6C3F1 mouse. Vomitoxin administration resulted in marked stimulation of the size and frequency of germinal centres in Peyer's patches, mesenteric lymph nodes and the spleen. A slight increase in the percentage of B cells in the Peyer's patch was observed, although vomitoxin treatment had no effect on the percentage of B cells in the spleen. The percentage of IgA+ cells in Peyer's patches and spleen were approximately twice that of controls at 4, 8 and 12 wk of vomitoxin exposure whereas the percentage of IgG+ cells decreased in these two organs. Exposure to vomitoxin increased the percentage of T cells in Peyer's patches and the spleen. The percentage of CD4+ cells (T helper subset) increased slightly in Peyer's patches and more markedly (30-50%) in the spleen following vomitoxin treatment. Contrastingly, there was only a slight increase in the percentage of CD8+ cells (T cytotoxic/suppressor subset) in the spleens of vomitoxin-treated mice in comparison with controls, and no effect in Peyer's patches. The relative effects of vomitoxin on these two T cells populations was also reflected in increased CD4+: CD8+ ratios in Peyer's patches and spleen. These results are consistent with the hypothesis that dietary vomitoxin modulates normal regulation of the IgA response at the Peyer's patch level and that this is manifested in an altered lymphocyte distribution pattern in both the mucosal and systemic compartment. Notably increased levels of IgA+ and CD4+ cells are indicative of IgA-producing progenitors and T helper subsets, respectively, that in tandem could favour IgA hyperproduction and elevated IgA in serum.