T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from and infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Prevention of bone marrow graft failure by an anti LFA-1 monoclonal antibody

Graft rejection is the major cause of failure of HLA mismatched bone marrow transplantation because of residual host immunity. we have proposed to use a monoclonal murine antibody specific for the LFA-1 molecule (25-3) to prevent graft failure in HLA mismatched bone marrow transplantation (BMT). The rationale for this approach is three fold: LFA-1 deficient patients (3/3) do not reject HLA mismatched BMT; anti LFA-1 blocka in vitro the induction of T cell responses and T/ non T cytotoxic functions; LFA-1 is not expressed by other cells than leucocytes. We have accordingly treated twenty two patients with inherited diseases and 8 with leikemia. The bone marrow was T cells depled by E rosetting of Campath antibody. The antibody was given at days -3, -1, +1, +3, +5 at dose of .1 mg/kg/d for the first 9 and then .2mg/kg/d from day -3 to +6. Engraftment occured in 23/30 patients as shown by at least HLA typing. Hematological recovery was rapid, GVH was limited. Side effects of antibody infusion included fever and possibly an increased incidence of early bacteral infection (sepsis, 1 death). Immunological reconstitution occured slowly leading in six cases to EBV-induced B cell poliferation (1 death and in two others to transient auto immune hemolytic anemia. There has been only one secondary graft rejection. Sisteen patients are alive 3 to 26 months post transplant with functional grafts. Although the number of patients treated is still low the absence of late rejection so far, gives hope for long term maintenance of the graft using anti LFA-1. Since the antibody is an IgG 1 unable to bind human complement, and since it is known to inhibit phagocytosis, there is a good suggestion that 25-3 act through functional blocking of host T and non T luymphocytes at both induction and effector levels.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

Role of a mouse monoclonal IgE antibody in passive transfer of immunity to Schistosoma japonicum infection

We have been able to produce a mouse monoclonal IgE antibody specific to an adult worm antigen extracted from Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1:256,000 but did not cross-react with S. mansoni antigen. The antibody recognized a 97-kDa molecule expressed on the surface of mechanically transformed schistosoma of S. japonicum. Passive transfer of the antibody into mice in the early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. Induction of eosinophilia by an oral administration of embryonated eggs of Toxocara canis prior to challenge infection enhanced the reduction.

Levels of lethal antibody during the course of infection with Schistosoma mansoni in rats and mice

Schistosoma mansoni infected hosts produce an IgG that mediates the complement-dependent killing of schistosomula in vitro. In this study, we followed the levels of serum lethal antibody during infection of rats and mice. Rats presented detectable lethal activity early in the course of infection with a peak in the 6-8th week of infection. This activity declined to non-detectable levels within 2 weeks, remaining low up to the 20-26th week. In mice, lethal antibody was not detected before 7-12 weeks of infection, but raised to higher levels, as compared to non-infected animals, up to 20-24 weeks after infection. We correlate lethal antibody and protective immunity suggesting that the antibody-mediated complement-dependent cytotoxicity to schistosomula play a role in the immunity to reinfection.

Demyelination in brain cell aggregate cultures, induced by a monoclonal antibody against the myelin/oligodendrocyte glycoprotein (MOG).

Previous work has shown that aggregate cultures prepared from fetal rat telencephalon and grown in a chemically defined medium offer a useful model to study developmental processes such as myelin synthesis. Since compact myelin is formed in these cultures, we investigated the possibility to use this culture system to study demyelinating mechanisms. In particular, we examined the effect of a monoclonal antibody (8-18C5) directed against the myelin/oligodendrocyte glycoprotein (MOG). We found that addition of anti-MOG antibodies and complement to aggregate cultures led to a highly significant decrease in myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) specific activity. These results indicate that, in our culture system, anti-MOG antibodies have a strong demyelinating effect.

Evaluation of antibody responses in american visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (asorbance [greater than or equals to] 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frenquently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease of cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated prarasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.