Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.

In vitro immunomodulatory effects of placental substances on preparatory MLR and resulting modifications of lymphocyte reactivities.

The immunomodulatory effects of murine placental extracts (PE) were studied in vitro using mixed lymphocyte culture (MLC) and resulting cell-mediated lympholysis (CML). The results showed that preparative cultures in the presence of PE syngeneic to the responding cells led to a low secondary MLR response with a concomitant generation of suppressor cells. At the efferent phase, cells from the same preparative culture showed a weaker cytotoxic activity than controls cultured in the absence of extract. Furthermore, the induction of regulatory cells able to inhibit CTL in vitro activity was also observed. The active substances can be found in the 30% ammonium sulphate precipitate as well as in some gel filtration fractions showing several main bands from 115 to 43 kDa in SDS-PAGE.

[Isolation and characterization of monoclonal antibody directed against antigenic peptide presented by class I histocompatibility molecule]. / Isolement et caractérisation d'un anticorps monoclonal dirigé contre un peptide antigénique présenté par une molécule d'histocompatilité de classe I.

We describe the isolation and several characteristics of a monoclonal antibody (X5.3.7) which recognizes a peptide derived from influenza virus nucleoprotein and presented by the murine class I major histocompatibility molecule Kd. X5.3.7 is thus an example of an antibody capable of recognizing an epitope normally recognized by T cells.

Monoclonal antibodies directed against T cell epitopes presented by class I MHC antigens.

Monoclonal antibodies which recognize a synthetic peptide derived from influenza virus nucleoprotein in association with a murine class I MHC molecule (Kd) have been isolated. One such antibody has been characterized and shown to react neither with the peptide nor with the Kd molecule, but only with the Kd-peptide complex. Evidence is given that it recognizes the naturally processed peptide located in the peptide binding groove, i.e. the antigenic moiety presented to T cells.

Electron microscopic study of thymus and bone marrow derived mouse lymphocytes: morphological differences and evolution of surface markers before and after in vitro mitogenic stimulation.

T and B lymphocytes from spleen, lymph nodes, thymus and bone marrow of unstimulated CBA mice have distinct ultrastructural features: respectively the Th type (dense dark appearance, smooth margin, high nucleocytoplasmic ratio, rare cytoplasmic organelles) and the Bm type (low electronic density, villous margin, low nucleocytoplasmic ratio, numerous cytoplasmic organelles). Correlations between Th or Bm morphology and presence of specific T or B surface markers (theta antigen or surface Ig) have been established. The Th/T and Bm/B equivalence do not however hold in all circumstances: first, there are morphologically intermediate types termed In (less than 13%) that may be theta- or Ig-positive; second, some Bm lymphocytes are theta-positive in CBA thoracic duct and some Th lymphocytes are Ig-positive in Nude mice spleen. Purified T-or B lymphocyte populations stimulated by selective mitogens (ConA or LPS respectively) undergo ultrastructural modifications before their surface markers (theta or Ig respectively) disappear. A time, some theta-positive T lymphocytes show a Bm-like morphology. The results suggest that the basis for the usual T-B ultrastructural differences in unstimulated mice resides in the normally different functional state of metabolic activity of these two types of cells: the cell metabolism would be higher in Bm (the usual form of unstimulated B lymphocytes) than in Th (the usual form of unstimulated T lymphocytes). This view may explain the paradoxical results observed in thoracic duct and Nude mice spleen as well as conflicting data reported by several authors.

An ultrastructural study of two different responses of mouse mast cells to transplantation antibodies directed against the same transplantation antigens.

The present paper describes an ultrastructural study of two different kinds of behavior of mouse mast cells during two immunological reactions induced by transplantation antibodies: the direct allogeneic anaphylactic degranulation and the serocytotoxicity. In both situations, the same alloantigens, born by the mast cells themselves, are the targets of the reaction, but the first one is mediated by anaphylactic alloantibodies whereas the second one is mediated by cytotoxic alloantibodies in the presence of complement. The comparison of the ultrastructural aspects of the cells in these two systems demonstrated that mast cells can behave in two different ways depending on the nature of the immunological agents utilized. First, a physiological degranulation process with active granule expulsion was observed. This process was shown to be identical to the one induced in classical in vitro anaphylaxis or by histamine releasers such as compound 48/80. A second type of behavior was a lethal, complement-dependent, cell lysis without active granule expulsion.

[Autoantigen T in guinea pig germinal cells: fine localization and ultrastructural lesions induced in vitro by specific anti-T autoantibody and complement]. / L'autoantigène T des cellules germinales de cobaye: localisation par immunoperoxydase et lésions ultrastructurales induites in vitro par l'autoanticorps qui lui est spécifique en présence du complément

Four autoantigens (S, P, T, Z), are known to be present in guinea pig spermatozoa. The only anti-T antibody is able to fix complement and is spermotoxic. Using immunoenzymatic technics, autoantigen T has been localized on the plasma membrane of spermatozoa and spermatids. A quantitative ultrastructural study has shown the anti-T induced irreversible, specific lesions on the germinal cells in presence of complement. A few minutes after addition of complement, almost all the cells are injured. Control sere (normal serum, anti-ova or anti-S or anti-P sera are inefficient. These findings are related to the mechanisms of autoimmune aspermatogenetic orchitis.

Differential modulation of the in vitro lymphocyte activation pathways by soluble and solubilized placental substances.

Soluble and detergent-solubilized placental extracts were studied for their modulatory effects upon the proliferation of lymphocytes stimulated by various activating agents. It was shown that soluble placental extract (SPE) exerted an inhibitory effect on the lymphoproliferation triggered by alloantigen or LPS but not by Con A or the combined action of PMA + calcium ionophore A 23187. This effect was also observed with SPE precipitated by 30% of ammonium sulfate (SPE30). On the other hand, a solubilized placental extract (SzPE) that was obtained by using octyl-beta-D-glucopyranoside inhibited the stimulation triggered by alloantigen, LPS, and Con A but did not affect the protein kinase C pathway. The modulatory effects were observed not only when SPE (or SPE30) and SzPE were added at the time of culture initiation but also at 24 h before or after the activating agents. Preincubation with SPE30 or SzPE immobilized on plastic surface, however, transduced an enhanced lymphoproliferative response to alloantigen and mitogen Con A but not to LPS. The above results suggest that placental substances exerted their modulatory effects by interfering mainly with the antigen or mitogen lymphoproliferation pathways.

Comparative evaluation of in vitro and in vivo immunosuppressive potential of cyclosporin G with cyclosporin A and FK-506.

Cyclosporin G (CsG), a promising cyclosporin A (CsA) analogue, was examined and compared with two reference immunosuppressive drugs: CsA and FK-506, regarding their inhibitory effects on different lymphocyte activation pathways as well as on graft-versus-host reaction (GvHR) across differences at major or minor histocompatibility loci. The results showed that, at different concentrations, CsG efficiently inhibited proliferation induced by alloantigens (mixed lymphocyte culture), mitogens (concanavalin A, pokeweed mitogen) and the combination of phorbol myristate acetate + ionomycin, to the same extent as observed with CsA and FK-506. It was also shown that CsG exhibited the same strong inhibitory effects as the two other immunosuppressants upon stimulation triggered by viral (MLs-1a) or bacterial (staphylococcal enterotoxin B) superantigen. Determination of IL-2 activity in the supernatant of MLC also confirmed similar strong inhibitory effects, exerted by CsG compared to CsA and FK-506. In systemic and local GvHR across major or minor histocompatibility barriers, CsG as well as CsA and FK-506 presented an equivalent immunosuppressive potential. In conclusion, from various experiments involving different modes of activation, it was shown that CsG was as strongly immunosuppressive as CsA and FK-506.

In vitro and in vivo comparative studies on immunosuppressive properties of cyclosporines A, C, D and metabolites M1, M17 and M21.

Cyclosporine A (CsA) and its major metabolites: M1, M17 and M21 and two analogues: cyclosporines C (CsC) and D (CsD), were studied for their capacity to interfere with different in vitro activation pathways. Their inhibition potentials against the reaction of Graft-versus-Host (GvH) were also studied. The results showed: CsA, CsC and metabolite M17 were the most active compounds upon the inhibition of lymphocyte proliferation induced by different mitogens (ConA, PHA, PWM) and also on the proliferation of mixed lymphocyte cultures (MLC). The same results were observed concerning the direct activation by protein kinase C using a combined action of phorbol ester + calcium ionophore. In vivo using local GvH reaction, CsA and CsC proved more active than M17 in the two different combinations: H-2d --> (H-2b x H-2d)F1 and H-2k --> (H-2b x H-2k)F1 CsD and two metabolites M1 and M21 showed no or weak immunosuppressive effects. Overall, the immunosuppressive potency of six compounds could be schematized as: CsA > or = CsC > M17 > M1 > or = CsD > M21.

In vitro and in vivo immunosuppressive potential of thalidomide and its derivative, N-hydroxythalidomide, alone and in combination with cyclosporin A.

Thalidomide (Thd) has been shown to have interesting immunosuppressive properties and strong action against TNF-alpha. It is used for treating a variety of immune-mediated pathology and inflammatory diseases. The purpose of this work was to evaluate the in vitro and in vivo immunosuppressive effects of Thd and its derivative, N-Hydroxythalidomide (H-Thd), alone and in combination with cyclosporin A (CsA), upon different in vitro lymphocyte activation pathways and in vivo local graft-versus-host-reaction (GvHR). At different concentrations, both Thd and H-Thd alone inhibited the lymphocyte proliferation induced by alloantigen (MLR), mitogens (Con A, PWM) and superantigen (SEB) with an activity of 50-75% that of CsA, however, in some tests, immunosuppressive potency of H-Thd was shown to be higher than that of Thd. In vivo using GvHR, Thd and H-Thd alone proved as active as CsA. The association in vitro and in vivo of each compound with CsA at different low concentrations, produced an additive effect as strong as CsA used alone at high therapeutic concentrations. In summarizing, this study revealed that: (1) despite its weaker potency in vitro than that of CsA, H-Thd presents interesting immunosuppressive properties similar to, and in some cases, better than Thd, and (2) the combination of H-Thd or Thd with CsA at suboptimal concentrations leads to high activity.