F. Cormont

Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants.

A technique for purifying rat monoclonal antibodies quickly and efficiently from in vitro culture supernatants is described. It is based on the fact that more than 95% of rat immunoglobulins carry kappa light chains. A mouse monoclonal antibody with suitable binding affinity for rat kappa light chains is immobilized on solid support and used to purify rat immunoglobulins. Milligrams of rat monoclonal antibodies may be rapidly concentrated from culture supernatants with high recovery. Rat monoclonal antibodies expressing lambda light chains (about 5% of the total) may be purified in a similar way with an appropriate anti-rat lambda chain monoclonal antibody.

Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children

Background Allergic diseases are believed to be due to T helper (Th)2‐like immunity to allergens in affected tissues, and immune responses to allergens are characterized by a cross‐regulation between Th1 and Th2 cells. Atopic individuals may develop IgE antibodies to only one or more allergens. However, the mechanisms behind sensitization to a specific allergen, e.g. why an individual develops IgE to cat but not birch, are not known. Our aim was to study birch‐ and cat‐induced Th1 and Th2 cytokine secretion in children who were sensitized to birch but not to cat, and vice versa.

Rat monoclonal antibodies. II. A rapid and efficient method of purification from ascitic fluid or serum

A technique for purifying rat monoclonal antibodies from ascitic fluid or serum is described which is based on 2 facts. First, approximately 95% of rat immunoglobulin light chains are of the kappa type. Second, an allotypy in the rat species is located on the constant part of the kappa light chain. By use of a mouse monoclonal antibody with specific binding affinity for the Ig kappa-1a allotype on the kappa light chains of the LOU inbred rat strain, it is possible with immunoaffinity chromatography to isolate LOU Ig kappa-1a-bearing immunoglobulins from the serum proteins, including the immunoglobulins, of rats of Ig kappa-1b allotype. LOU histocompatible hybridomas synthesizing the Ig kappa-1a allotype can be transplanted into rats congenic with the LOU inbred strain carrying the Ig kappa-1b allotype, since LOU rats with the Ig kappa-1a kappa light chain allotype and congenic LOU Ig kappa-1b rats with the Ig kappa-1b kappa light chain allotype are fully histocompatible. The serum or ascitic fluid of the recipients is applied to an immunoabsorbent column to which mouse monoclonal antibody against the Ig kappa-1a allotype is coupled. The serum proteins, including the host immunoglobulins pass through the column. An appropriate buffer is used to elute the monoclonal antibodies in a second step. The same technique may be employed for other monoclonal antibodies. A reciprocal system using mouse monoclonal antibodies against Ig kappa-1b rat allotype can be used, a plasmacytoma or hybridoma synthesizing Ig kappa-1b kappa light chain being transplanted into an Ig kappa-1a kappa light chain synthesizing rat. The method is rapid, efficient and inexpensive. Its limitation is with respect to lambda-type monoclonal antibodies, which are relatively rare.

Monoclonal antibodies in prophylactic immunosuppression after liver transplantation. A randomized controlled trial comparing OKT3 and anti-IL-2 receptor monoclonal antibody LO-Tact-1.

A prospective trial was conducted to assess the efficacy of induction immunosuppression with antilymphocyte monoclonal antibodies in 129 primary liver transplant patients who were randomly divided into three groups according to immunosuppression during the first 10 days post-OLT: triple drug therapy only (TDIS: cy-closporine, steroids, azathioprine) (group I: n=42); TDIS with a 10-day course of OKT3 (group II: n=44); and LO-Tact-1 (anti-IL-2 receptor mAb) (group III: n=43). Biopsy-proved acute rejection (AR) was treated using the same biopsy-guided protocol in the 3 groups. One-year patient survival rates were 67%, 84%, and 93% in groups I, II, and III, respectively (I vs. II, NS; I vs. III, P=0.001; II vs. III, P=0.044). Incidences of AR were studied in the subgroup of 100 patients who were exposed to the risk of developing rejection, with an

Allergen‐induced interleukin‐9 production in vitro: correlation with atopy in human adults and comparison with interleukin‐5 and interleukin‐13

Background The contribution of IL‐9 to human atopy is supported by genetic studies. However, IL‐9 production in response to allergen in vitro has been reported only in children.

Enhancement of autoantibody pathogenicity by viral infections in mouse models of anemia and thrombocytopenia.

Abstract Viral infections are involved in the pathogenesis of blood autoimmune diseases such as hemolytic anemia and thrombocytopenia. Although antigenic mimicry has been proposed as a major mechanism by which viruses could trigger the development of such diseases, it is not easy to understand how widely different viruses might induce these blood autoimmune diseases by this sole mechanism. In mice infected with lactate dehydrogenase-elevating virus (LDV), or mouse hepatitis virus, and treated with anti-erythrocyte or anti-platelet monoclonal autoantibodies at a dose insufficient to induce clinical disease by themselves, the infection sharply enhances the pathogenicity of autoantibodies, leading to severe anemia or thrombocytopenia. This effect is observed only with antibodies that induce disease through phagocytosis. Moreover, the phagocytic activity of macrophages from infected mice is increased and the enhancing effect of infection on autoantibody-mediated pathogenicity is strongly suppressed by treatment of mice with clodronate-containing liposomes. Finally, the disease induced by LDV after administration of autoantibodies is largely suppressed in animals deficient for gamma-interferon receptor. Together, these observations suggest that viruses may trigger autoantibody-mediated anemia or thrombocytopenia by activating macrophages through gamma-interferon production, a mechanism that may account for the pathogenic similarities of multiple infectious agents.

Prophylactic immunosuppression with anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 versus OKT3 in liver allografting. A two-year follow-up study.

A prospective trial was conducted in 129 recipients of primary liver transplantation, to compare induction immunosuppression using triple drug therapy (cyclosporine, steroids, and azathioprine; group 1, n = 42), versus triple drug therapy with a 10-day course of OKT3 (group 2, n = 44) or of the anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 (group 3, n = 43). Two-year actual patient survival rates were 64%, 79%, and 93% in groups 1, 2, and 3, respectively (1 vs. 2, NS; I vs. III, P = 0.003; 2 vs. 3, NS). Up to 2 years after transplantation, 18%, 44%, and 53% of the grafts in groups 1, 2, and 3, respectively, had not experienced steroid-resistant acute rejection (1 vs. 2, P = 0.002; 1 vs. 3, P = 0.007; 2 vs. 3, NS). The overall incidence of chronic rejection was 4%. OKT3 therapy, but not LO-Tact-1, significantly increased the incidence of cytomegalovirus infections (P = 0.019). In conclusion, immunoprophylaxis with LO-Tact-1 seemed to provide a liver graft acceptance rate at least as satisfactory as that with OKT3, without an increase in the incidence of infections.

Dexamethasone inhibits IL-9 production by human T cells.

BackgroundInterleukin 9 (IL-9) is produced by activated CD4+ T cells. Its effects include stimulation of mucus production, enhanced mast cell proliferation, enhanced eosinophil function, and IgE production. These effects are consistent with a role in allergic diseases. Glucocorticoids have potent anti-inflammatory effects, including suppression of cytokine synthesis, and are widely used in the treatment of allergic conditions.MethodsWe examined the effect of the glucocorticoid dexamethasone (Dex) on IL-9 mRNA expression and protein secretion with real-time RT-PCR and ELISA. Peripheral blood mononuclear cells (PBMC) were prepared from human volunteers and activated with OKT3. CD4+ T cells were purified from PBMC and activated with OKT3 plus PMA.ResultsIL-9 mRNA abundance and protein secretion were both markedly reduced following treatment of activated PBMC with Dex. mRNA levels were reduced to 0.7% of control values and protein secretion was reduced to 2.8% of controls. In CD4+ T cells, Dex reduced protein secretion to a similar extent. The IC50 value of Dex on mRNA expression was 4 nM.ConclusionThese results indicate that IL-9 production is very markedly inhibited by Dex. The findings raise the possibility that the beneficial effects of glucocorticoids in the treatment of allergic diseases are in part mediated by inhibition of IL-9 production.

Alloreaction increases or restores CD40, CD54, and/or HLA molecule expression in acute myelogenous leukemia blasts, through secretion of inflammatory cytokines: dominant role for TNFβ, in concert with IFNγ

We have previously reported that alloreaction can lead to activation of dendritic cells through secretion of inflammatory cytokines. Here, we addressed whether alloreaction-derived cytokines may also lead to acute myelogenous leukemia (AML) blast differentiation. With this aim, supernatant (sn) harvested from major or minor histocompatibility antigen-mismatched mixed lymphocyte reaction (MLR) were used to culture French American Bristish (FAB) type M4 or M5 AML blasts. Our results showed that the secreted factors induced upregulation of CD40, CD54, and/or HLA molecules in AML blasts. Protein fractionation, blockade experiments and exogenous cytokine reconstitution demonstrated the involvement of TNF in the upregulation of CD54, CD40 and HLA-class II molecules, and of IFNγ in the increase of HLA-class I and class II molecule expression. But, in line of its much higher levels of secretion, TNFβ, rather than TNFα, was likely to play a preponderant role in AML blast differentiation. Moreover TNFβ and IFNγ were also likely to be involved in the AML blast differentiation-mediated by HLA-identical donor T-cell alloresponse against recipient AML blasts. In conclusion, we show herein that upon allogeneic reaction, TNFβ secretion contributes, in concert with IFNγ, to increase or restore surface molecules involved in AML blast interaction with T cells.

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells.

BACKGROUND The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.Background. The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-d and anti-m injections was analyzed in adult mice. Sequential treatment with anti-d and then anti-m induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. Methods. Adult mice were injected with anti-m, anti-d, anti-d then anti-m, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. Results. Anti-m injections induced a depletion of IgM high , immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-d injections induced mature conventional IgD high B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-d then anti-m induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. Conclusions. These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-m mAb depleted IgM high B cells (MZ and B1) and anti-d, IgD high B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgGXNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-d then anti-m mAb depleted all B-cell populations and suppressed the whole XNA production.