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In the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitro stimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy

Intravenous immunoglobulins (IVIg) exert a broad range of immunoregulatory functions that provide a basis for the beneficial effects of IVIg in autoimmune and systemic inflammatory disorders. This review focuses on the effects f IVIg on humoral and cellular immunity that may be of relevance for the treatment of inflammatory neurological diseases.

Natural autoantibodies (NAbs), produced by B-1 B-cells, are directed against autoantigens and pathogens. NAbs can capture and present antigen to T helper cells thereby initiating adaptive immunities. It is proposed that screening for NAbs against pathogens will predict the strength of an antigen-induced immune response and could be used as a tool for vaccine development.

Normal immunoglobulin G for therapeutic use (intravenous immunoglobulin [IVIg]) is used in an increasing number of immune-mediated conditions, including acute and chronic/relapsing autoimmune diseases, transplantation, and systemic inflammatory disorders. Several mutually nonexclusive mechanisms of action account for the immunoregulatory effects of IVIg. Although IVIg inhibits T-cell proliferation and T-cell cytokine production, it is unclear whether these effects are directly dependent on the effects of IVIg on T cells or they are dependent through the inhibition of antigen-presenting cell activity. Here, we examined the effects of IVIg on differentiation, maturation, and function of dendritic cells (DCs). We show that IVIg inhibits the differentiation and maturation of DCs in vitro and abrogates the capacity of mature DC to secrete interleukin-12 (IL-12) on activation while enhancing IL-10 production. IVIg-induced down-regulation of costimulatory molecules associated with modulation of cytokine secretion resulted in the inhibition of autoreactive and alloreactive T-cell activation and proliferation. Modulation of DC maturation and function by IVIg is of potential relevance to its immunomodulatory effects in controlling specific immune responses in autoimmune diseases, transplantation, and other immune-mediated conditions.

Hemophilia A is an X-linked, recessive, bleeding disorder caused by defective or deficient factor VIII (FVIII) molecules. Infusion of purified FVIII to patients with severe hemophilia A results in approximately 25% of the cases, in the emergence of anti-FVIII antibodies (inhibitors) that are known to neutralize the pro-coagulant activity of FVIII by steric hindrance. We recently reported on the proteolysis of FVIII by allo-antibodies in the plasma of high responder patients with severe hemophilia A, demonstrating a new mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. Hemophilia is the first model where a direct link between the hydrolysis of the target molecule and the occurrence of the clinical manifestations may be established. It also represents the first example in humans, of the induction of catalytic antibodies following the exogenous administration of an antigen. The characterization of FVIII inhibitors as site-specific proteases may provide new approaches to the treatment of inhibitors.

Despite representing the majority of the world's foot-and-mouth disease (FMD)-susceptible livestock, sheep and goats have generally been neglected with regard to their epidemiological role in the spread of FMD. In the present investigations, FMD virus quadrivalent double emulsion (Montanide ISA 206) vaccines were tested in sheep. The oil adjuvant elicited a better immune response at any time than did aluminum hydroxide gel vaccine, and the response developed quicker. The animals maintained their neutralizing antibody titers at >3 log(10) for the duration of the trial (90 days). Sheep were found to be late responders to serotypes A, C, and Asia-1; a clear upward shift in titer was observed at 60 days postvaccination. However, development of the immune response to serotype O in sheep was superior to that in cattle and goats.

Antibodies endowed with hydrolytic properties have been described in humans for over a decade in a variety of pathological conditions such as systemic lupus erythematosus (SLE), autoimmune thyroiditis, asthma, and Bence Jones disease. Although the identified target substrate molecules have always been autoantigens (i.e., DNA, thyroglobulin, vasoactive intestinal peptide), a direct role of hydrolysis of the autoantigen in pathology of the disease has not been clearly documented. We have described in multitransfused patients with hemophilia A the presence of anti-factor VIII (FVIII) IgG antibodies that hydrolyze FVIII. The estimated kinetic parameters derived for FVIII cleavage by anti-FVIII antibodies are in line with the previously described catalytic antibodies. The identified cleavage sites are evenly spread throughout the FVIII molecule and are located after an arginine or a lysine in most cases. We have recently shown that the catalytic antibodies are highly prevalent among hemophilia A patients with FVIII inhibitors. Catalytic antibodies to FVIII are the first example where the hydrolysis of the target molecule by hydrolytic antibodies may be directly relevant to the etiology of the disease. The characterization of FVIII inhibitors as site-specific proteases may provide novel strategies in the design of therapy against FVIII inhibitors in patients with hemophilia A.

Background: Viscum album (VA) preparations consist of aqueous extracts of different types of lectins of VA. Mistletoe lectins have both cytotoxic and immunomodulatory properties that support their study for the development for cancer therapy. However, the mechanisms of the anti- tumoral properties in vivo of mistletoe lectins are not fully understood. Because endothelial cells (EC) play a pivotal role in tumor angiogenesis, we tested the hypothesis that VA extracts induce endothelial cell death and apoptosis., Materials and Methods: We investigated the effect of various VA preparations on both human venous endothelial cell (HUVEC) and immortalized human venous endothelial cell line (IVEC) using morphologic assessment of EC, FACScan analysis after propidium iodine and annexin V labeling, and detection of cleavage of poly(A)DP-ribose polymerase (PARP)., Results: All tested VA preparations, except Iscador P, were cytotoxic in IVEC. Apoptosis, assessed by morphologic examination, annexin V labeling, and Western blot analysis for PARP cleavage, was involved in HUVEC cell death induced by VA preparations derived from plants that grow on oak trees (VA Qu FrF)., Conclusions: Results from the present study suggest that VA extract-induced endothelial apoptosis may explain the tumor regression associated with the therapeutic use of VA preparations and support further investigations to develop novel anti-angiogenic compounds based on mistletoe compounds.

The epidemiological role of small ruminants in foot-and-mouth disease (FMD) outbreaks has been generally neglected. Although, the disease in these species is sub-clinical in nature, their role as virus carriers represents a reservoir for further infection and spread of disease. Data on the usefulness of polyvalent FMD vaccine (FMDV) in goats is scant. Thus, the present study was undertaken to evaluate the benefits of a highly potent polyvalent FMDV in goats. In the present investigations, FMDV quadrivalent double oil emulsion (Montanide ISA 206) vaccines were tested in goats at reduced doses of 2 ml per animal (antigen payload 3.5 microg per serotype per dose). The oil adjuvant elicited superior immune response at any given period than aluminium hydroxide gel (AGS) vaccine and the rapidity of development of response was quicker. The duration of immunity also appeared to be maintained for long period. The differences in immune response between two adjuvant groups were statistically significant (P<0.05). The differences were apparent even in kinetics of immune response. Unlike cattle, goats were found to be late responders for oil-adjuvanted vaccine. Our results indicate possible universal usage of double oil emulsion vaccines for disease control programs irrespective of species of animals.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.

The occurrence of factor VIII (FVIII) inhibitors is one of the major complications of the treatment of haemophilia A. We present this review as a description of the major players of the antiFVIII immune response, with particular emphasis on the nature and properties of the different antiFVIII antibodies, their mechanisms of action in inhibiting FVIII activity, their potential neutralization by anti-idiotypic antibodies, and the importance of the T cell in participating in the induction of FVIII inhibitors. We briefly conclude on the avenues that remain to be explored in order to establish efficient therapeutic approaches aimed at eliminating FVIII inhibitors in patients with haemophilia A.

Idiotypic vaccination has proven successful in several animal models and human trials. Here we suggest that the expression of cross-reactive idiotypes on factor VIII (FVIII) inhibitors of patients with haemophilia A, patients with anti-FVIII autoimmune disease and natural anti-FVIII antibodies of healthy individuals, together with the ability of anti-idiotypic reagents to neutralize anti-FVIII antibodies, provides a rationale for designing a vaccine strategy aimed at preventing the occurrence of or suppressing inhibitors, based on the induction of protective anti-idiotypes. Here we discuss the rationale supporting the concept of using idiotypic vaccination to prevent the occurrence of FVIII inhibitors in patients with haemophilia A.

Background: Factor VIII inhibitors are IgG alloantibodies that arise during replacement therapy in 25 to 50 percent of patients with severe hemophilia A. The hydrolysis of factor VIII by anti--factor VIII antibodies has been proposed as a mechanism of inactivation of factor VIII., Methods: We purified IgG from patients with severe hemophilia A. The proteolytic activity of the antibodies was assessed by incubating the IgG with biotinylated human factor VIII and analyzing patterns of factor VIII cleavage by sodium dodecyl sulfate--polyacrylamide-gel electrophoresis and immunoblotting. The controls were normal human IgG and IgG purified from plasma of patients with hemophilia who did not have inhibitory antibodies., Results: Significant proteolytic activity was detected in IgG from 13 of 24 inhibitor-positive patients. No hydrolytic activity was detected in control antibodies of IgG from patients without inhibitors. The rate of hydrolysis of factor VIII by purified IgG correlated positively with the factor VIII--neutralizing activity of IgG in plasma (r2=0.67, P=0.029). Principal-component analysis of migration profiles of digestion fragments demonstrated the heterogeneity of the catalytic potential of factor VIII inhibitors among patients., Conclusions: Proteolysis is a mechanism by which IgG antibodies against factor VIII can inactivate factor VIII.

Anti-Factor VIII (FVIII) antibodies represent a unique model to study the relationship between natural autoreactivity (natural antibodies to FVIII of healthy individuals) and disease-associated autoimmunity ('spontaneous' FVIII inhibitors of patients with anti-FVIII autoimmune disease) to a single human protein antigen. Although natural and disease-associated anti-FVIII antibodies are not readily distinguished based on the comparison of their isotypic distribution and epitope mapping, available studies of cross-reacting idiotypes suggest that FVIII inhibitors in patient's plasma encompass two populations of anti-FVIII antibodies - some antibodies result from the clonal expansion of B lymphocytes that exist previous to the treatment with FVIII and secrete anti-FVIII antibodies with properties similar to those of natural anti-FVIII antibodies present in healthy individuals, other inhibitors are produced by B cell clones that have undergone affinity-maturation and hypermutation of the V-regions of the antibodies they produce. The implications for the treatment of autoimmune patients with anti-FVIII inhibitors are discussed.

Vaccine against foot-and-mouth disease (FMD) is prepared after inactivating the virus produced in cell culture. Inactivation of the FMD virus (FMDV) was earlier done by formaline. However, several vaccine outbreaks, which occurred in Europe revealed that the formaline treatment is not highly effective for virus inactivation. Subsequently, binary ethyleneimine (BEI) was identified as an effective inactivation reagent for FMDV. However, these chemical reagents are likely to have effect on whole virus particle whose integrity is essential for vaccine potency. Therefore, a need is felt to develop non-chemical methods. We have studied induction of endonucleolytic activity as an alternative method for virus inactivation. This method of inactivation was compared with the chemical methods, and found to be highly effective for virus inactivation. The effects of endonucleolytic activity on the integrity of virus capsid was studied using antibodies raised against recombinant proteins, which elicited antibodies against major epitopes present on the surface of the virus. Further, the effect of the agents on the integrity of the virus capsid was studied by using antigen capture PCR (Ag-RT/PCR) which detects the whole virus. The studies showed that inactivation of the virus by induction of endonucleolytic activity is more effective besides maintaining virus integrity. The effect of various inactivating agents on four serotypes of FMDV has also been studied and found to have varying effects, depending on serotype.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.