Christine Mazingue

[3H]serotonin release: An improved method to measure mast cell degranulation

A method based on the release of tritium-labelled serotonin by activated mast cells in rodents is described. Mast cells incorporate labelled serotonin selectively and release the label after activation by non-specific stimulators (compound 48/80, polymyxin B sulphate, ATP, bovine chymotrypsin and L-alpha-lysophosphatidylcholine) or anaphylactic antibody and the corresponding antigen. These two types of activation were investigated in comparison with the toluidine blue microscopic rat mast cell degranulation test, and a methodological study of the release of [3H]serotonin is described. The measurement of labelled serotonin release provides a simple and quick assay of mast cell degranulation compared to the time required for the classical rat mast cell degranulation technique and achieves a greater sensitivity.

Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide.

The aim of this study was to compare the antigenicity and the immunogenicity of five constructs of a peptide, including the peptide in single copy, a tandem repeat containing three copies, a copolymer with glutaraldehyde and two constructs based on the MAP (Multiple Antigenic Peptide) model, one containing two copies (MAP-2) and the other, eight copies of the peptide (MAP-8). The peptide used in this test was the 115-131 sequence derived from the rSm28-GST antigen of Schistosoma mansoni. All constructs were recognized by rSm28-GST specific antibodies in solid phase immunoassays. However, the binding was higher when the MAP-8 was used as antigen at least partly because of its better coating on the microtiter plates. In vitro lymphoproliferative assays showed that polymer was mitogenic, repeat and MAP-2 did not stimulate rSm28-GST specific T cells while MAP-8 induced a slight response. The injection of MAP-8 to rats led to important antibody and T cell responses higher than those obtained with the other constructs. The IgG2a (cytotoxic antibody in schistosomiasis)/IgG2c (blocking antibody) ratio was independent of the immunogen. Taken together these results demonstrate that both the antigenicity and the immunogenicity of a peptide containing T and B cell epitope(s) are strongly related to the molecular form whereby it is presented and that the MAP-8 construct can be useful in serodiagnosis or in vaccination trials using synthetic peptides.

Effect of a lipopeptidic formulation on macrophage activation and peptide presentation to T cells

We studied a 45-69 lipopeptide obtained by N-terminal modification with a N epsilon-palmitoyl lysine residue of the 45-69 peptide derived from the nef protein of HIV. T cells from animals immunized intraperitoneally with 45-69 lipopeptide proliferated in vitro in the presence of 45-69 peptide while no response was obtained after intraperitoneal immunization with 45-69 peptide. The efficiency of the 45-69 lipopeptide is supported by the covalent association to the N epsilon-palmitoyl lysine moiety. The immunogenicity of the 45-69 lipopeptide or of the unmodified peptide is dependent on the route of immunization but is not related to a mitogenic effect on cells or to an increase of the peptide antigenicity. Moreover, only 45-69 lipopeptide induces the secretion of cytokines such as IL-1, IL-6 and TNF-alpha by peritoneal macrophages. Finally, the use of 45-69 lipopeptide permits the activation of highly purified T cells without the addition of antigen-presenting cells. These results have implications for the formulation of synthetic vaccines.

In vitro and in vivo inhibition of mast cell degranulation by a factor from Schistosoma mansoni.

Schistosome incubation product (SIP) obtained from Schistosoma mansoni was tested on mast cell degranulation in vitro or in vivo elicited by chemical compounds or anaphylactic reactions. Normal mast cells from Wistar rats were labeled with 3H-serotonin and incubated with the SIP. Serotonin release normally induced by the chemical compounds 48/80, polymyxin B or an anaphylactic system (ovalbumin-antiovalbumin) was inhibited when mast cells were preincubated with the SIP. Cutaneous reactions elicited by the compound 48/80 or polymyxin B and passive or active cutaneous anaphylactic reactions, were inhibited by the intradermal injection of the SIP before the challenge. The anaphylactic shock by ovalbumin was also inhibited in guinea pigs by a previous intraperitoneal injection of the SIP. This inhibitor appeared dialysable, heat resistant and soluble in trichloroacetic acid (TCA). The inhibitory activity was found in the fraction eluted from an antischistosome immunosorbent. However, no inhibition of cutaneous reactions was observed with the circulating (TCA-soluble, thermostable) M antigen previously described and purified from schistosomes. No effect of the SIP was observed on the growth of HeLa cells or the J 111 human monocytic cell line. The increase of intracellular cAMP levels in mast cells incubated with the SIP suggests that modulation of cAMP is involved in the mechanism of inhibition. The SIP inhibited also the mast cell-dependent eosinophil cytotoxicity for schistosomula sensitized with IgG2a antibody. The SIP appeared to act selectively on mast cell degranulation without effect on eosinophils or the mast cell mediators. This inhibitor, denominated schistosome-derived inhibitory factor, released by the parasite, which acts on mast cells could partly explain the low incidence of clinical allergic manifestations observed in parasitic diseases and might represent an escape mechanism of the parasite to the antibody-dependent eosinophil cytotoxicity mechanism.

Inhibition of Cytotoxic T Lymphocytes by a Schistosome-Derived Inhibitory Factor Is Independent of an Inhibition of the Production of Interleukin 2

Schistosome-derived inhibitory factor (SDIF) was shown to inhibit lymphocyte proliferation. While SDIF is not toxic to lymphoid cells, the generation of cytotoxic effector cells was inhibited by SDIF in a mixed lymphocyte culture. This inhibitory effect was not attributable to the induction of suppressor cells, as SDIF also inhibited the development of nonspecific suppressor cell activity in 7-day cultures of unstimulated spleen cells. The interleukine 2-dependent proliferation of cytotoxic T lymphocytes of or of blast cells was markedly reduced by the addition of SDIF. In contrast, the production of interleukine 2 itself was not impaired by SDIF. These results support the hypothesis of an inhibition by SDIF of a particular step of the mitotic cycle, probably posterior to the G0-G1 transition. An inhibitory activity of SDIF on the expression of the cytolytic activity of cytotoxic T lymphocytes was also observed.

T-cell responsiveness towards various synthetic peptides of the P28 antigen in rat and mouse models during Schistosoma mansoni infection.

It has recently been demonstrated that the Schistosoma mansoni P28 antigen can induce a strong protective immunity after direct immunization in various experimental models. T lymphocytes from Fischer rats immunized with the recombinant P28 antigen were cultured in vitro in the presence of seven synthetic peptides derived from the amino acid sequence of the P28. The most significant and reproducible proliferation was obtained with the 24-43 and 115-131 synthetic peptides. In order to analyze whether these located determinants were also exposed to the hosts immune system during the natural S. mansoni infection or after immunization with crude antigenic extracts of various development stages of the parasite, the T-cell responsiveness of infected or immunized Fischer rats and BALB/c mice was tested towards these synthetic peptides. The results showed that, in both permissive (mouse) and non-permissive (rat) hosts, 24-43 and 115-131 synthetic peptides are recognized during the course of infection and that there is a dynamic variation of this recognition. These peptides are also recognized by T cells educated against crude antigenic extracts of different developmental stages of the parasite which contained the native form of the P28 molecule. Taken together, the results indicated that these synthetic peptides derived from the recombinant P28 antigen can activate T lymphocytes educated against the native P28 molecule during the development and maturation of the parasite in their hosts. Therefore, they might be useful for the construction of synthetic vaccines against schistosomiasis.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitophorus vacuole antigen of Plasmodium falciparum has been shown to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and the antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in terms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced antibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

Effect of Schistosome-Derived Inhibitory Factor on the Cell Cycle of T Lymphocytes

A schistosome-derived inhibitory factor (SDIF) with immunosuppressive properties has been investigated for its effect on human T cell proliferation. We show here that SDIF has no effect on the process of lymphocyte activation because peripheral blood leukocytes (PBL) stimulated with lectin in the presence of SDIF increased normally their RNA content and showed normal acquisition of interleukin 2 (IL-2) and transferrin receptors. IL-2 production was not altered by SDIF but utilization of IL-2 was decreased, suggesting that SDIF blocked cells before or in the early s phase. Jurkat T cell line cells physically enriched for G1 cells were also more susceptible to SDIF inhibition. On the contrary, normal PBL or Jurkat cells which were already in the s phase were no more inhibited by SDIF. While SDIF has no effect on T lymphocyte activation and on production of regulatory lymphokines it selectively blocks T cell proliferation at G1 transition of the cell cycle.

Immunoregulation by Schistosoma mansoni

Schistosoma mansoni is known to release an inhibitory factor of lymphocyte proliferation elicited in vitro. The effect of this dialyzable schistosome incubation product (DSIP) was tested in vivo on different aspects of the cell-mediated immune response. First, the DSIP injected into C57B1/6 mice markedly inhibited the delayed type hypersensitivity to sheep red blood cells (SRBC). Furthermore, the DSIP injected into S. mansoni infected Fisher rats at the beginning of the infection induced an inhibition of the specific lymphocyte response to S. mansoni antigen and of the spleen cell response to concanavalin A (Con A). The DSIP injected into uninfected rats also inhibited the spleen cell response to Con A. In uninfected as in infected rats injected with the DSIP, the lymphocyte response to Con A was restored after purification of the spleen cells on a nylon wool column. Moreover spleen cells from rats injected wtih the DSIP reduced the proliferative response of normal syngeneic spleen cells induced by Con A. This inhibition was not observed when cells from DSIP-injected rats were previously passed through a nylon wool column. In contrast, nylon wool depletion of spleen cells from infected rats injected with the DSIP did not restore the lymphocyte response to S. mansoni antigen. It seems tht DSIP could partly explain the modulation of the cellular immune responses observed during S. mansoni infection and could represent one of the mechanisms of this parasites survival in the immunized host.

H-2b restriction of the immune response to the pl26 Plasmodium falciparum antigen

Inbred BALB/c (H‐2d), CBA (H‐2k) and C57Bl/6 (H‐2%) mice immunized with Plasmodium falciparum schizonls or culture supernatcs develop antibodies of different anligcnic specificities. It has been observed that C57B1/6 mice were unable to produce deteelable antibodies against the pl26 antigen (native molecule and p73 or p50 processed fragments) compared with other inbred miee. Similar results were obtained using BALBeongenlc mice with a lack of p126 antibody response in H‐2b mice, while H‐2d and H‐2k mice produced antibodies against the pl26. Lymphoeyte proliferation assays performed by incubation of spleen cells with immunopurified pl26 were positive for immunized BALB/c (H‐2d) and congenic H‐2d or H‐2k mice. On the other hand, no lymphocyte stimulation was observed with either C57B1/6 (H‐2b) or congenic H‐2b miee. These results suggest an MHC restriction ofthe immune response against the entire pi 26 (found in schizonts) and its p73 and p50 naturally processed fragments (found in culture supernates).