Eric Claassen

STRAIN-DEPENDENT INDUCTION OF CYTOKINE PROFILES IN THE GUT BY ORALLY ADMINISTERED LACTOBACILLUS STRAINS

Different Lactobacillus strains are frequently used in consumer food products. In addition, recombinant lactobacilli which contain novel expression vectors can now be used in immunotherapeutic applications such as oral vaccination strategies and in T cell tolerance induction approaches for autoimmune disease. Both for food and clinical applications of lactobacilli, proper selection of wild type strains is crucial. For that purpose, eight different common Lactobacillus strains were analysed with respect to mucosal induction of pro- and anti-inflammatory cytokines, IgA-producing plasma cells in the gut, as well as systemic antibody responses against a parenterally administered antigen. Immunohistochemical analysis of cytokine-producing cells in the gut villi showed no significant induction of the cytokines IL-1alpha, IFN-gamma, IL-4 or IL-10 after oral administration of wild type Lactobacillus strains. In contrast, oral administration of L. reuteri and L. brevis induced expression of the proinflammatory/Th1 cytokines TNF-alpha, IL-2 and/or IL-1beta. Oral administration of these two strains and L. fermentum also significantly enhanced the IgG response against parenterally administered haptenated chicken gamma globulin (TNP-CGG). The five other strains did not show this adjuvanticity. L. reuteri induced relatively high levels of IgG2a compared to L. murines, a nonadjuving Lactobacillus strain. These findings imply that different Lactobacillus strains induce distinct mucosal cytokine profiles and possess differential intrinsic adjuvanticity. This suggests that rational Lactobacillus strain selection provides a strategy to influence cytokine expression and thereby influence immune responses.

An approach to the control of disease transmission in pig-to-human xenotransplantation

Abstract: Although several major immunologic hurdles need to be overcome, the pig is currently considered the most likely source animal of cells, tissues and organs for transplantation into humans. Concerns have been raised with regard to the potential for the transfer of infectious agents with the transplanted organ to the human recipient. This risk is perceived to be increased as it is likely that the patient will be iatrogenically immunocompromised and the organ‐source pig may be genetically engineered in such a way to render its organs particularly susceptible to infection with human viruses. Furthermore, the risk may not be restricted to the recipient, but may have consequences for the health of others in the community. The identification of porcine endogenous retroviruses and of hitherto unknown viruses have given rise to the most concern. We document here the agents we believe should be excluded from the organ‐source pigs. We discuss the likelihood of achieving this aim and outline the potential means by which it may best be achieved.

Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.

The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono-specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono-specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action.

Expression of accessory molecules and cytokines in acute EAE in marmoset monkeys (Callithrix jacchus)

Accessory molecules and cytokines are involved in the immunopathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) in rodent models, and are potential targets for immunotherapy. Evaluation of such experimental therapies requires appropriate animal models. Therefore, we analysed the expression of selected accessory molecules and cytokines in the brain of marmoset monkeys (Callithrix jacchus) with acute EAE, a newly described non-human primate model for MS. All animals experienced active disease clinically and histopathologically with strong resemblance to MS. Perivascular infiltrates of mononuclear cells showed abundant expression of CD40. CD40 was expressed on macrophages, indicating that T cell priming and macrophage effector functions may result from local CD40-CD40L interactions. CD40 ligand (CD40L) and B7-2 (CD86) were also expressed, but to a lower extent, while B7-1 (CD80) expression was limited. Both pro-inflammatory and anti-inflammatory cytokines were produced within individual lesions during active disease (IFN-alpha, IFN-gamma, TNF-alpha, IL-1alpha, IL-1beta, IL-2, IL-4, IL-10 and IL-12). This suggests that relative levels rather than sequential expression of Th1- and Th2-type cytokines determine disease activity. These findings demonstrate the value of EAE in marmoset monkeys as a model to assess the role of accessory molecules and cytokines in multiple sclerosis, and to evaluate targeted intervention.

Orally administered lactobacillus strains differentially affect the direction and efficacy of the immune response

In mice, strain dependent cytokine production profiles are induced after oral administration of Lactobacillus. Such a cytokine profile seems to determine the direction and efficacy of the humoral response. In SJL mice lactobacilli are able to enhance or inhibit the development of disease after induction of experimental autoimmune encephalomyelitis (EAE). Immuno-histochemical analysis of cytokine profiles showed that differential modulation is obtained dependent on the Lactobacillus strain applied. Serum antibody responses to i.p. immunisation with chicken gamma globulin in BALB/c mice are also modulated by oral application of Lactobacillus. Lactobacilli are now being developed as safe live antigen carriers for application in vaccine technology, but also for the excretion of autoantigens in order to induce tolerance. The findings of this study imply that by proper strain selection the direction of the response can be influenced by the induction of a specific cytokine profile.

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

Strain-dependent effects of probiotic lactobacilli on EAE autoimmunity

In this study we present new data showing strain-specific differences on the effect of commercially available probiotic drinks in an EAE rat autoimmune model for multiple sclerosis. In this particular model, we conclude that these drinks do not enhance but rather suppress the disease. We suggest that conclusions on probiotics are limited to specific strains and models and not generalised. We further suggest that physiological use (normal route, normal dose, normal growth phase, specific strain or substrain/species) is studied in all cases, so as not to overwhelm (high dose) or circumvent natural immune processing.

The involvement of specific anti myelin basic protein antibody-forming cells in multiple sclerosis immunopathology.

Irrespective of the large body of literature on the putative role of antibodies in the development of multiple sclerosis (MS), the detection of specific antibody-forming B cells (AFCs) in the central nervous system (CNS) tissues has not been described. In this study we show that autoantigen-specific AFCs can be found in CSN tissue sections of MS patients. Applying a newly developed myelin basic protein (MBP)-enzyme conjugate technique, we have detected MBP-specific AFCs in autopsy periventricular white matter and cerebellum tissue sections of MS patients. We demonstrated the presence of MBP-specific AFCs in CNS tissue sections in five out of 12 MS patients. No MBP-specific AFCs were detected in CNS tissue sections of 11 patients with other neurological diseases, such as Parkinsons and Alzheimers disease, or in brain tissue sections of eight deceased persons without neurological diseases. In MS patients, anti-MBP AFCs were present in brain tissue sections both with and without plaques. The proportion of MBP-specific AFCs in some of the MS patient brain tissues reached over 50% of all AFCs. The high relative frequency of the anti-MBP AFCs and their localization in periventricular white matter and cerebellum of MS patients only, suggests that anti-MBP AFCs represent a cell population, which could play an important role in MS immunopathology.

Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits.

In three experiments we evaluated several types of adjuvants as an alternative to Freunds adjuvant (FA). In the first experiment three adjuvant preparations (a water-in-oil emulsion (Specol), a combination preparation of monophosphoryl lipid A + trehalose dimycolate + cell wall skeleton and a non-ionic block polymer surfactant (TiterMax)) were evaluated. The adjuvants were combined with three different types of weak immunogenic antigens (synthetic peptide, glycolipid and particulate antigen) and administered following the intramuscular and subcutaneous route. The evaluation was based on clinical, pathological and immunological parameters. The animals did not appear to be severely or chronically impaired by the experiment. After injection of the RIBI adjuvant, side effects of the same severity as with FA were induced, while low antibody titers were produced. TiterMax caused few side effects, while antibody responses were very low. In comparing Specol and FA, Specol had far fewer adverse effects than FA. However, Specol had immunostimulating properties of the same level as FA. In the second experiment, the effect of injected volume of FA on side effects and antibody titer was studied. Immunization of rabbits with a total of 0.5 ml FA at different sites does not seem to increase the immune response when compared with the immune response seen after injection of 0.5 ml FA at one site. However side effects were seen in all the animals. In the third experiment, the side effects following intradermal (i.d.) injection of the adjuvants were studied. After i.d. injection of FA or RIBI, undesirable effects were found. No side effects occurred after i.d. injection of Specol or TiterMax. From the studies it is concluded that Specol is an alternative to FA for hyperactivation of the immune response in rabbits.

A novel carbodiimide coupling method for synthetic peptides: Enhanced anti-peptide antibody responses

Coupling of peptides to immunogenic protein carriers is required for the generation of anti-peptide antibody responses. Carbodiimides are hetero bi-functional coupling reagents that are utilized for coupling reactions through carboxyl and amino groups. The procedures generally used for carbodiimide coupling of peptides and proteins result in conjugates which generate immunodominant antibody responses directed against the neodeterminants on the carrier protein. These determinants are induced by the reaction of carrier and/or peptide with the coupling agent. We have investigated the potential inhibiting effect of an imidazole intermediary on the formation of unwanted neodeterminants during carbodiimide coupling. The serum antibody responses elicited with the peptide-protein conjugates produced were evaluated in ELISA. We have modified and improved the coupling with a watersoluble carbodiimide (EDC) in such a way that a high response to the coupled peptide is obtained in association with negligible levels of anti-neodeterminant antibodies.