High Responder Strains Research Articles

Kappa-deficient mice are non-responders to dextran B512: is this unresponsiveness due to specialization of the kappa and lambda Ig repertoires?

In the dextran B512 high-responder strain C57BL, the response to dextran is restricted to the preferential expression of the V(H)B512 and the V(kappa)OX1 gene combination. The importance of the heavy chain is suggested by the fact that mice with the Ig C(H) allotype, different from C57BL, are low or non-responders to dextran, but the light chain could also play a role. All anti-dextran B512 mAb described to date (>200) use kappa light chains. No anti-dextran antibody using lambda has ever been observed. To ascertain if the restriction of the use of V(kappa) genes in response to dextran B512 was more stochastic or due to other factors, we have studied the response to dextran B512 in C57BL/6 mice where the C(kappa) domain has been disrupted (C57BL.C(kappa)T). These mice are unable to express kappa light chains and their humoral antibodies bear light chains of the lambda type. We found that C(kappa) knockout mice are unable to respond to dextran given in a thymus-independent or -dependent form. The lack of responsiveness is specifically directed to the dextran epitopes since these mice are fully competent to respond to other antigenic structures present in the same immunogenic molecule. These mice are also apparently normal regarding the expression of V(H) genes. Finally, we tested the response to dextran in C57BL.C(kappa)T mice carrying the lpr mutation that was introduced to favor an increase in the life span and make the response to dextran more easily detectable. The introduction of the lpr mutation was not sufficient to change the pattern of unresponsiveness in the C57BL.C(kappa)T mice. We concluded that there are deficiencies in the light chain repertoire because the V lambda light chain could not reconstitute the response to dextran. We discuss the possible mechanisms for this new type of unresponsiveness to dextran B512.

Intragraft overexpression of interleukin-4 is neither sufficient nor essential for tolerance induction to cardiac allografts in a high-responder strain combination.

Recently we have demonstrated that the nondepleting anti-CD4 monoclonal antibody (mAb) RIB5/2 induces long-term acceptance of kidney and heart allografts in all rat strain combinations tested. Cytokine gene expression studies by reverse transcriptase-polymerase chain reaction revealed a reversed intragraft interleukin (IL)-4/interferon-gamma ratio. Whether IL-4 mediated immune deviation contributes to transplantation tolerance is not clear so far. To learn more about the functional relevance of the relative IL-4 up-regulation, IL-4 was overexpressed in rat heart allografts by using ex vivo adenoviral gene transfer. The efficiency of gene transfer was analyzed by reporter gene assays as well by reverse transcriptase-polymerase chain reaction analysis of IL-4 mRNA expression. The intragraft overexpression of IL-4 did not prolong the allograft survival compared with controls. Moreover, neutralization of IL-4 by OX81 mAb did not prevent tolerance induction by RIB5/2 treatment. Anti-CD4 mAb-induced tolerance is associated with an intragraft type1/type2 shift, however, the up-regulation of IL-4 alone is neither sufficient nor essential to induce tolerance to cardiac allografts in a high-responder strain combination.

Low responsiveness to immunization with immunoglobulin E/antigen and immunoglobulin G/antigen complexes in H-2Ab mice.

Immunoglobulin (Ig)E and IgG antibodies specific for 2,4, 6-trinitrophenyl (TNP) are able to enhance the carrier-specific antibody response to TNP-conjugated soluble proteins such as bovine serum albumin (BSA). We have recently reported that mice carrying the MHC class II Ab molecule are low responders to immunization with IgE/antigen complexes and now show that H-2Ab mice are also low responders to IgG/antigen complexes. In addition, we found that spleen cells from naive low- and high-responder mice captured IgE/antigen complexes exclusively on B cells, and that the binding was completely inhibited by monoclonal antibodies (MoAbs) against the low-affinity receptor for IgE (FcepsilonRII or CD23). The IgG/antigen complexes were targeted both to B cells and macrophages. The binding of IgG/antigen to B cells primarily seemed to be dependent on the low-affinity receptor for IgG (FcgammaRII or CD32), although some influence of complement receptor 2 (CR2 or CD21) was seen. Capture of IgG/antigen complexes on macrophages was partially blocked by MoAbs against FcgammaRII/III. There was no difference in expression of FcepsilonRII, FcgammaRII/III, CR1, CR2, and CR3 between low- and high-responder strains, thus excluding low levels of these FcRs and CRs as a reason for low responsiveness in H-2Ab mice.

Potentiation of immune response against the RESA peptides of Plasmodium falciparum by incorporating a universal T-cell epitope (CS.T3) and an immunomodulator (polytuftsin), and delivery through liposomes.

Synthetic peptides representing repeat sequences of ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum have shown poor immunogenicity and protection. In this study, the RESA peptides [(EENVEHDA)2 and (DDEHVEEPTVA)2] were chemically linked to a universal T-cell determinant, CS.T3, derived from the CS protein of P. falciparum. Polytuftsin (TKPR)40, a polymer of naturally occurring immunomodulator "tuftsin," was physically mixed with these conjugates. These preparations in alum and liposomes were immunized in four inbred strains of mice with different genetic backgrounds to study the humoral response. In the case of liposome-entrapped preparations, a 10 microg dose of antigen showed the optimum antibody response. Mice immunized with liposome containing RESA peptide(s)-CS.T3 conjugate along with polytuftsin showed the highest antibody levels in all the strains, whereas the RESA peptide(s) alone, adsorbed on alum or entrapped in liposomes, showed either poor or moderate antibody levels. The antibodies raised against liposome-entrapped preparations in both high-responder strain (SJL/J H-2s) and low-responder strain (FVB/J H-2q) showed 2 4-fold lower Kd values as compared to the alum adsorbed preparations, suggestive of high affinity antibodies. All the antigen preparations predominantly induced IgG2a and IgG2b isotype response, suggesting that the T-helper response involved is of the CD4 Thl type. The in vitro merozoite reinvasion inhibition assay showed 50-92% inhibition with sera raised against different antigen formulations. The highest percentage inhibition was observed with the RESA peptide-CS.T3 conjugate containing polytuftsin in liposomes. Thus, the incorporation of peptide antigens inside liposomes not only reduced the antigen dose by 5-fold but also elicited a high titre with high affinity antibodies and the inhibition of merozoites to RBC in vitro. Therefore, we conclude that the incorporation of these synthetic constructs in liposomes could be a useful strategy for the development of a subunit immunogen against malaria.

A COMBINATION OF INTRATHYMIC AND PORTAL VENOUS INJECTION OF DONOR BONE MARROW CELLS PRODUCES LONG-TERM SURVIVAL OF CARDIAC ALLOGRAFTS IN HIGH RESPONDER RAT STRAINS.

256 Purpose: Intrathymic (IT) and portal venous (PV) injection of donor antigens were previously reported to induce significant prolongation in survival of subsequently transplanted donor-type allografts. These effects were not easy to achieve in high responder rat combinations. Our objective was to determine whether we could prolong the survival of cardiac allografts by combining IT and PV injection of donor bone marrow cells (BMC) at the time of transplantation in a high responder rat strain combination. Methods. ACI (RT1Aa) and Wistar-Furth (WF; RT1Au) rats were used as donors and recipients, respectively. WF recipients received one single injection of 1×108 ACI BMC per each route (IT, PV or IT+PV) at the time of cardiac transplantation, and a five-day course of tacrolimus (FK506; 1.5mg/kg/day, IM, days 0-4). WF recipients who received syngeneic BMC (IT+PV) or allogeneic BMC (intravenous, instead of IT or PV) served as controls. Grafts that survived >150 days (d) were examined for the severity of coronary artery vasculopathy (CAV), lymphocytic infiltration, and fibrosis. Results. The mean graft survival time (MST) in controls that received no treatment was 6.5d. Without FK506, no prolongation of graft survival was observed when BMC were injected via IT (MST: 6.5d), PV (MST: 6.0d) or IT+PV (MST: 5.5d). Graft survival was modestly prolonged when FK506 was combined with IT (MST: 38.6d) or PV (MST: 49.3d) donor BMC, as compared with FK506 alone (MST: 29.0d). The graft survival in WF recipients that received syngeneic (WF) IT+PV BMC plus FK506 (MST: 29.6d) was similar with that of animals receiving FK506 alone. By contrast, in animals that received IT+PV donor BMC together with FK506, 70% (7/10) of the transplanted hearts survived >150 days (MST: 124.4d). When PV route was substituted with the IV route, (i.e., IT+IV donor BMC and FK506), only 30% (3/10) of grafts survived >150d (MST: 74.4d). Moreover, long-term (>150d) surviving grafts in animals that received IT+PV donor BMC had a marked reduction in the prevalence of CAV (p<0.0001), lymphocytic infiltration (p<0.0001), and fibrosis (p=0.0495) as compared with grafts in animals receiving IT+IV donor BMC. Conclusions. In a high responder strain combination, combining IT and PV injections of donor BMC prolongs the survival of cardiac allografts in a setting applicable to clinical transplantation. The adjunct use of immunosuppressive agents (FK506) is critical for attaining prolonged graft survival.

CYTOKINE GENE TRANSFER IN HEART ALLOGRAFTS DOES NOT INDUCE TOLERANCE IN HIGH RESPONDER STRAIN COMBINATION

243 Background: E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently it has been shown that overexpression of Th2-cytokines may induce prolongation of allograft acceptance in different rat or mouse transplantation models. However we have observed that overexpression of the Th2-cytokine Interleukin-4 (IL-4) in rat hearts different both in MHC class I and MHC class II molecules does not induce graft acceptance despite detection of intragraft IL-4 by RT-PCR. Aim: In this study we have investigated the role of other immuneregulatory cytokines in our high responder transplantation model (DA, RT1av1→ Lewis, RT11). Methods: For this purpose adenovirus vectors encoding for viral IL-10 and rat IL-12p40 - known as endogenous inhibitor of the Th1-cytokine IL-12 - have been constructed. Following adenovirus gene transfer by injection of 5×109 plaque forming units (pfu) heterotopic heart transplantation was performed in abdominal localization. The graft survival was checked by daily palpation. Graft rejection was defined as absence of palpable heart beat. Results/Conclusions: Neither injection of AdvIL-10 nor AdIL-12p40 prolonged survival of allogeneic heart transplants (Table 1). Moreover co-application of AdvIL-10 and AdIL-12p40 or AdvIL-10 and AdrIL-4 did not lead to the prolongation of heart allograft survival. Our experimental results indicate that overexpression of Th2-cytokines or Th1-inhibitors after adenovirus-mediated gene transfer in rat allogeneic hearts is not able to prevent acute rejection of heart transplants in high responder strain combination.Table 1: Survival of rat allogeneic grafts after adenovirus-mediated gene transfer of cytokines in high responder strain combination (DA→Lewis)

Effect of IL-4 deletion on cardiac allograft survival in the BALB/c to 129Sv x C57BL/6 strain combination.

To investigate the effect of IL-4 deletion on cardiac allograft survival, vascularized BALB/c cardiac allografts were placed in C57BL/6, 129Sv x C57BL/6 (IL-4 +/+) or 129Sv x C57BL/6 IL-4 knockout mice (IL-4-/-). Untreated recipients rejected allografts in < 15 days while isografts survived indefinitely (> 100 days). Treatment with anti-CD4 (GK1.5) for 4 days at the time of allografting increased mean survival to > 100 days in C57BL/6, 90+/-16 days in 129Sv x C57BL/6 (IL-4 +/+) and 68 +/- 36 days in 129Sv x C57BL/6 (IL-4-/-) recipients. Although there was a trend towards shorter survival times in the IL-4-/- mAb-treated mice, survival in the three recipient groups was not significantly different (P = 0.07). A 30-day course of anti-CD4 did not further prolong BALB/c heart survival. All long-surviving hearts had histological evidence of parenchymal damage and transplant vascular disease. None of these recipients developed antigen-specific tolerance, since both donor and third party skin graft challenges were rejected when challenged at > 60 days post-graft and all primary grafts failed by 120 days. Thus the effects of IL-4 deletion were subtle and were seen only with low doses of immunosuppression in this high responder strain combination.

Prolongation of allograft survival by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, is now believed to play a significant role in the immune responses, both in vitro and in vivo, preventing the development of several autoimmune diseases. These studies suggest that 1,25-dihydroxyvitamin D3 may be effective in prolonging allograph survival. To test the hypothesis that 1,25-dihydroxyvitamin D3 would prolong allograft survival, neonatal heart grafts were transplanted to allogeneic recipients receiving either 19-nor-1,25-dihydroxyvitamin D2 (200 ng/day) or 1,25-dihydroxyvitamin D3 (50 ng/mouse/day) orally through the diet. The efficacy of 1,25-dihydroxyvitamin D3 in prolonging graft survival in a vascularized model was determined by heterotopic ACI to Lewis heart transplants. The provision of exogenous 1,25-dihydroxyvitamin D3 or an analog, 19-nor-1,25-dihydroxyvitamin D2, to mice markedly prolonged the survival of neonatal mouse heart allografts. Similar results were obtained with a vascularized heterotopic heart transplant model in rats. Cyclosporine at a maximum 25 mg/kg dose for mice proved less effective than 1,25-dihydroxyvitamin D3. Graft survival in mice differing at class I and class II loci (B10.A(4R) --> C57BL/10) increased from 13.0+/-1.1 days to 51.0+/-5.6 days and was significantly better than cyclosporine monotherapy (33.2+/-3.6). Rat heart survival in a high responder strain combination (ACI --> Lewis) increased from 6.2+/-0.3 to 25.2+/-2.8 days. The increased survival of the transplants brought about with 1,25-dihydroxyvitamin D3 was not accompanied by hypercalcemia in rats. These results suggest that 1,25-dihydroxyvitamin D3 can be used as an effective agent in preventing graft rejection.

Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody.

Experimental systemic lupus erythematosus (SLE) can be induced in naive mice by immunization with a murine monoclonal anti-DNA antibody (mAb), 5G12, that bears a major idiotype designated 16/6 Id. Strain-dependent differences were observed in the proliferative responses of lymph node cells of mice immunized with two peptides based on the sequences of the complementarity determining region (CDR) 1 and 3 of mAb 5G12. The capacity of the peptides to bind to major histocompatibility complex class II molecules correlated with the proliferative responses. Immunization of high responder strains with the CDR-based peptides led to production of autoantibodies and clinical manifestations characteristic to experimental SLE. The CDR-based peptides could prevent autoantibody production in neonatal mice that were immunized later either with the peptide or with the pathogenic autoantibody. Furthermore, the peptides inhibited specific proliferation of lymph node cells of mice immunized with the same peptide, with mAb 5G12 or with the human mAb anti-DNA, 16/6 Id. Thus, the CDR-based peptides are potential candidates for therapy of SLE.

Specific antibody responses to subtilisin Carlsberg (Alcalase) in mice: development of an intranasal exposure model.

An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.

Genetic Control ofin VivoTumor Necrosis Factor Production in Mice

We report on the genetic effect onin vivoproduction of tumor necrosis factor (TNF)-α induced by lipopolysaccharides (LPS) using various congenic mouse strains. B10.A, B10.A(3R), B10.AQR, B10.A(5R), and B10.S(7R) produced significantly high TNF-α compared with B10.BR, B10.S, C57BL/10, B10.A(2R), B10.A(4R), B10.G, B10.DA(80NS), and B10.RIII(71NS). This suggests that LPS-induced TNF-α production is genetically controlled by H-2. Mice with the same alleles on K, A, E, or S loci produced various (high or low) levels of TNF-α, thus indicating that regulatory genes are located outside these loci. All strains with H-2Ddproduced significantly high levels of TNF-α, but strains with other alleles in the H-2D locus produced low levels. Thus, TNF-α production appears to be genetically linked to H-2D itself or H-2D linked genes and the allele d is linked to a high responder gene. This was the case with the A background. C3H/HeN (H-2k), however, showed a high TNF-α production, suggesting the presence of another controlling gene outside H-2. In addition, high TNF-α productivity was transmitted into F1 mice (B10.A × B10.BR) in a dominant fashion. Both LPS-stimulated and unstimulated TNF-α mRNA expression in splenic macrophages were enhanced in high responder strains. Thus, we conclude that TNF-α production is closely related to genes within or linked to the H-2D locus as well as others outside H-2.

Cardiac Allograft Unresponsiveness Using a Posttransplant Strategy: Characterization of the Graft Infiltrate

We evaluated a combined posttransplant strategy using antilymphocyte serum (ALS) at time of engraftment followed by low dose total lymphoid irradiation (TLI) and donor bone marrow cell (BMC) inoculation administered either intrathymically (IT) or intravenously (IV) in the vigorously rejecting strain combination DA into Lew recipients. Allograft survival was significantly prolonged with administration of ALS in combination with TLI and IT (105 ± 28.6 days) or IV (106.8 ± 28.6 days) BMC compared to administration of ALS combined with either TLI (17.8 ± 0.4 days) or BMC (9.0 ± 0.0 days), or TLI combined with BMC (11.5 ± 0.5 days) (P< 0.0001, experimental vs control animals). There was no difference in survival between those animals who underwent IT or IV BMC inoculation. Third-party (WF) BMC inoculation did not significantly prolong allograft survival (10.0 ± 1.0 days). A mild to moderate cellular infiltrate was present in allograft tissue after 100 days. To further characterize these cells, cytokine mRNA expression in allograft tissue (>100 days posttransplant) was evaluated using nonisotopic in situ hybridization. A similar cytokine profile was demonstrated in allograft tissue compared to naive and isograft tissue, except for a slight increase in IL-2 (P< 0.02, control vs IV BMC;P= NS, other groups). In summary, unresponsiveness was induced in a high-responder strain combination using a combined posttransplant strategy of ALS, TLI, and donor antigen either IT or IV. The cytokine profile of the graft infiltrating cells was similar to that of normal tissue. Unresponsiveness may be the result of functional inactivation of these cells.

Specific Antibody Responses to Subtilisin Carlsberg (Alcalase) in Mice: Development of an Intranasal Exposure Model

An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 × DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-μl aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.

抗原刺激に伴うリンパ洞及び洞周囲領域の病態

In this experiment, we examined the structure and the cellular reaction in lymphatic sinuses and the surrounding regions caused by T-independent (LPS) and T-dependent (SRBC) antigens administration in rats and SMA mice (LPS high responder strain).Lymphatic sinuses are incompletely lined by smooth lymphatic endothelial cells, and a distinctive basement membrane is lacking. Reticulum cells form a loose meshwork not only the regions around the lymphatic sinuses but also in the lumen of the sinuses. On days 1 and 3 after intraperitoneal injections of either LPS or SRBC, the lymphatic sinuses were dilated, and many macrophages, which bear LPS antigens immunostained by IC6H or EO9, were found in and out of the marginal and trabecular sinuses. After the carbon solution was injected into footpad subcutis, carbon laden-macrophages were also found there in a few minutes. That is to say, antigens reached LN via lymphatics, and spreaded around lymphatic sinuses easily in short time. The structure of lymphatic sinuses allows easy passage of antigens in and out of the sinuses, and the meshwork of reticulum cells causes the stasis of lymphatic flow.We also found in lymph node (LN) that B-lymphocytes (B-lys), which have the same phenotypes as marginal zone B-lys (MZB), were induced by LPS injection. These B-lys infiltlated around marginal and trabecular lymphatic sinuses on days 1 and 3 after LPS injection. Moreover, plasmacytic B-lys and plasma cells were found in the same regions as the B-lys infiltlated preceding the formation of activated germinal centers (GC) in SMA mice. These findings suggested that the B-lys mature into plasma cells around the lymphatic sinuses independent of B-lys differentiation in GC. Although marginal zone (MZ) do not exist in LN anatomically, it is also suggested that the regions around marginal and trabecular lymphatic sinuses are functionally identical to MZ.

TOLERANCE INDUCED BY DIRECT INOCULATION OF DONOR ANTIGEN INTO THE THYMUS IN LOW AND HIGH RESPONDER RODENTS

A novel approach to tolerance induction in rats was recently described where donor antigen is inoculated directly into the thymus along with a brief period of immunosuppression in a pretransplant strategy. To develop a strategy that has more clinical appeal, we evaluated the timing of donor antigen inoculation in relation to allografting, the use of frozen bone marrow as the antigen, and the dose response of purified T cells as the antigen in a low responder heterotopic heart allograft combination. Additionally, the success of this pretransplant strategy in different low and high responder strain combinations was defined. In tolerant low responder animals we evaluated in vitro and in vivo cellular immunity. Tolerant host strain naive CD8+ T cell responses to donor and third party stimulators were compared to determine if tolerance is related to the strength of the response of the T cell subsets to donor antigen. Frozen bone marrow can induce tolerance in a low responder combination. Additionally, the dose of purified T cells necessary for tolerance induction was 5 x 10(5) cells. The pretransplant strategy was successful in two low responder strain combinations, Lewis into Wistar Furth and Lewis into DA, but unsuccessful in all high responder strain combinations evaluated. Low responder animals unresponsive to donor heart allografts demonstrate intact cell-mediated immunity and donor-specific tolerance in vivo by rejecting third party but not second donor strain hearts. The in vitro responses of tolerant animals demonstrated donor-specific suppression of the MHC class II response but an intact (normal) response to third party stimulators by proliferation assays and IFN-gamma production, suggesting suppression at the CD4+ T cell subset level.

Immunological parameters associated with antigenic competition in a multivalent footrot vaccine

A murine model for antigenic competition with multivalent D. nodosus pili vaccine has been established that parallels the phenomenon observed in sheep where levels of antibody, specific for any particular serogroup of pili, are significantly lower following vaccination in the presence of multiple serogroups of pili than with that serogroup alone. This competition was observed in both high and low responder strains of mice and was not dependent on the multiplicity of the antigens in the multivalent vaccine but could be observed with a large excess of a single heterologous serogroup. Competition was manifest by a reduction in the number of serogroup-specific antibody secreting cells elicited in response to vaccination. The antibody response to a single serogroup of pili reached a plateau at high doses and it was at these doses that antigenic competition was most pronounced, under conditions where both B- and T-cell responses were limiting. The limit in T-cell responsiveness was not imposed at the level of presentation of antigen. Pili-specific T cells were largely cross-reactive for different serogroups, and under conditions of limiting T-cell stimulation within a lymph node the available T cells would have to be shared between B cells specific for each serogroup of pili, which may in turn result in the decrease of serogroup-specific antibody induced following inoculation with the multivalent vaccine.

Transient immunosuppressive treatment leads to long-term retention of allogeneic myoblasts in hybrid myofibers.

Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in muscular dystrophies. A limitation is the immune response of the host to the transplanted cells. Allogeneic myoblasts are rapidly rejected unless immunosuppressants are administered. However, continuous immunosuppression is associated with significant toxic side effects. Here we test whether immunosuppressive treatment, administered only transiently after allogeneic myoblast transplantation, allows the long-term survival of the transplanted cells in mice. Two immunosuppressive treatments with different modes of action were used: (a) cyclosporine A (CSA); and (b) monoclonal antibodies to intracellular adhesion molecule-1 and leukocyte function-associated molecule-1. The use of myoblasts genetically engineered to express beta-galactosidase allowed quantitation of the survival of allogeneic myoblasts at different times after cessation of the immunosuppressive treatments. Without host immunosuppression, allogeneic myoblasts were rejected from all host strains tested, although the relative time course differed as expected for low and high responder strains. The allogeneic myoblasts initially fused with host myofibers, but these hybrid cells were later destroyed by the massive immunological response of the host. However, transient immunosuppressive treatment prevented the hybrid myofiber destruction and led to their long-term retention. Even four months after the cessation of treatment, the hybrid myofibers persisted and no inflammatory infiltrate was present in the tissue. Such long-term survival indicates that transient immunosuppression may greatly increase the utility of myoblast transplantation as a therapeutic approach to the treatment of muscle and nonmuscle disease.

Role of the bowel flora for development of immunity to hsp 65 and arthritis in three experimental models.

An infectious aetiology in rheumatoid arthritis (RA) has for long been suggested, although no conclusive evidence for this is at present available. Lately a large interest has been devoted to the involvement of heat shock proteins (hsps) in autoimmune disorders due to their conserved structure and immunogenic properties. Immunity to hsps has been observed both in human autoimmune conditions and in experimental models of autoimmune disease. We have studied the role of the bacterial flora and hsp immunity in the arthritic response in three experimental models of arthritis; type II collagen arthritis (CIA), adjuvant arthritis (AA) and oil induced arthritis (OIA); by using germ free and conventional DA rats. A high incidence of severe arthritis developed in all the models evaluated irrespectively of whether the animals were in the conventional or germ free state. This confirms earlier results which show a minor effect of the bacterial flora in CIA and AA in high responder strains. These results also show that a severe OIA can develop in germ free animals. Despite the severe arthritic response induced, no serum antibody levels to hsp 65 could be detected in the germ free animals, which was in contrast to the conventional animals where a positive anti-hsp 65 serum response was seen in 35-80% of the animals with CIA, AA or OIA. These results show that development of a humoral response to hsp 65 in these models of arthritis is dependent on the presence of a bacterial flora. Further, the lack of humoral immunity in germ free animals despite a severe arthritic response indicates that humoral immunity to hsp 65 is not involved in development of disease in these three models of experimental arthritis.

Induction of tolerance to heart allografts in rats using posttransplant total lymphoid irradiation and anti-T cell antibodies.

This study examined whether posttransplant anti-T cell monoclonal or polyclonal antibody therapy could provide a window of treatment to allow posttransplant total lymphoid irradiation (TLI) to induce tolerance. These experiments were conducted in a high responder strain combination of an ACI cardiac allograft into a Lewis rat. In this situation, treatment with antibody or posttransplant TLI alone is insufficient to induce tolerance, while similar treatments alone have been shown to induce tolerance in low responder strains. The affects of three anti-T cell therapies were compared: anti-CD4 mAb therapy, anti-CD3 mAb, and rabbit antithymocyte globulin (RATG). None of these antibody therapies alone prolonged graft survival indefinitely. Combining anti-CD4 therapy with posttransplant TLI markedly delayed rejection but failed to induce long-term graft survival. Tolerance could be induced by a combination of anti-pan T cell antibody (anti-CD3) and TLI, and, all grafts survived beyond 100 days. RATG failed to prevent graft rejection when used alone or in combination with TLI. However, posttransplant therapy with a combination of RATG, TLI, and single-donor blood transfusion resulted in graft survival beyond 100 days. Recipients bearing long-term donor grafts rejected third-party (PVG) grafts within 2 weeks. Low density donor bone marrow cells used instead of a blood transfusion did not facilitate tolerance. The results indicate that monoclonal or polyclonal anti-pan T cell antibodies, TLI, and a donor blood cell infusion function synergistically in facilitating tolerance to allografts in the posttransplant period.

Inhibition of T-cell reactivity to myasthenogenic epitopes of the human acetylcholine receptor by synthetic analogs.

The synthetic peptides p195-212 and p259-271, representing amino acids 195-212 and 259-271 of the alpha subunit of the human acetylcholine receptor, preferentially stimulate T cells of patients with myasthenia gravis and are immunodominant T-cell epitopes in SJL and BALB/c mice, respectively. We designed and synthesized analogs of these peptides that contain single amino acid substitutions. An analog of peptide p195-212, no. 455 (Met-207-->Ala), was capable of inhibiting up to 100% of the proliferative responses of a p195-212-specific T-cell line originating from the high-responder strain SJL. Similarly, an analog of p259-271, no. 306 (Glu-262-->Lys), was capable of inhibiting up to 93% of the proliferative responses of the p259-271-specific T-cell line originating from high-responder BALB/c mice. Analog 306 also inhibited up to 43% of the proliferative responses of p259-271-primed lymph node cells in an in vitro proliferation assay. To test the in vivo inhibitory activity of the analogs, mice were primed with the myasthenogenic peptides in complete Freund's adjuvant concomitant with administration of the analogs in aqueous solution. Administration of analogs 455 and 306 led to decreased proliferative responses of up to 70% by peptide p199-212-primed lymph node cells and up to 85% by peptide p259-271-primed lymph node cells. Similar results were obtained whether the analogs were administered i.v. or i.p. Thus, these analogs are good candidates for specific immunomodulatory therapy for patients with myasthenia gravis.