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The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

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The role of sperm specific lactate dehydrogenase-C4 (LDH-C4) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generation in vitro and local graft versus host (LGVH) reaction and allograft enhancement in vivo has been ascertained. LDH was purified from testes (LDH-C4) and kidney (LDH-B4) of C57Bl/Ks mice. MLC and CTL were performed using C57Bl/Ks-anti A/J lymphocytes in presence of 10(-3)-1 micrograms LDH-B4 or LDH-C4 per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C4. Early MLC culture gave significantly low stimulation index at 10(-2)-10(-1) micrograms LDH-C4 as compared to non-treated control cultures. However, the MLC response in presence of LDH-C4 was not different from the LDH-B4 treated one which showed a similar biphasic trend. On the other hand, 51Cr release from YAC-222 target cells was practically abolished by LDH-C4 at 10(-3)-1(-1) micrograms, and this was strikingly different from LDH-B4 or non-treated cultures. LGVH reactivity as performed by using C57Bl/Ks lymphocytes along with LDH-C4 in (C57Bl/Ks x A/J) F1 hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C4 or LDH-B4) LGVH. Allograft enhancement of Sa I (A/J) in C57Bl/Ks mice in presence of LDH-C4, was delayed slightly but significantly during primary or secondary transplantation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

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Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.

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The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.

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Glycoproteins were fractionated from placentae of syngeneic mice (CBA female xCBA male - H-2k) and evaluated in vivo and in vitro for the modulation of allo-immune responses against the H-2 incompatible A/J (H-2a) strain. It became apparent that the placenta contained high (309-800 kDA) and low (less than 13 kDA) molecular weight proteins, which favoured Sa1 allograft enhancement, leading to 75-100% lethal tumors, and inhibited mixed lymphocyte cultures (MLC). Though the low molecular weight fraction did not modify the mast cell degranulating (DAAD) antibody response, high molecular weight proteins caused a low production of DAAD allo-antibodies of the IgG1 + IgE isotypes. Moreover, addition of the placental fraction representing a specific band of 119 kDA resulted in the production of allo-immune sera rich in DAAD antibodies, which, however, were not associated with allograft enhancement. Beside these components, placenta is endowed with other proteins which modify humoral immune responses in different ways, as ascertained by the C-mediated cytotoxic antibody assay; fractions, rich in a 105 and 55 kDA bands, were immunosuppressive, whereas another, rich in a specific 100 kDA band, was active in eliciting enhanced production of alloantibodies of the IgG2 isotype. Moreover, the fractions representing specific bands of 50, 68 and 75 kDA, which were most effective in inhibiting MLC, neither caused lethal tumor enhancement nor modified IgG2 antibody responses. Based on in vivo and in vitro modulation of immune responses by placental products, it is concluded that: 1) allograft enhancement and high production of IgG1 antibodies are not linked to the same glycoprotein, 2) the immunomodulators in relation to the protection of viviparity appear to be located at the exclusion limits of Sephacryl S-200 (i.e. greater than 250 kDA and less than 13 kDA proteins) and 3) in vitro assays which depend on the inhibition of MLC by placental components should not be taken as the sole criterion for defining a given immunomodulator.

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CBA/N (Xid) mice classically present a genetically determined immune deficiency. This is characterized by the absence of certain B lymphocyte subpopulations (Lyb 3, 5, 7) and a hyporeactivity towards class II T independent antigens. These mice were examined in comparison with conventional CBA/Ca (H-2k) histocompatible controls for their immune reactivity, using different systems linked to allogenic transplantation: the local graft-versus-host reaction (LGVHR), allogeneic tumor (Sa 1 A/J) rejection, mixed lymphocyte reaction (MLR) and cell mediated lymphocytotoxicity (CML). It is concluded that a difference in reactivity is also present in some responses to classically T dependent antigens. This difference can result either in an increased reaction in the case of a primary LGVHR or a decrease in the same but late secondary reaction. The rejection of a tumor allograft (Sa 1 A/J - H-2a) is delayed in the CBA/N (H-2k) substrain with respect to conventional CBA mice. Cell mediated lymphocytotoxicity (CML) does not seem to be affected by the Xid defect in immunized cell donors. On the contrary, MLR responses are strikingly increased. It is suggested that the poor seeding of B cells into peripheral lymphoid organs alters the T/B ratio, favoring either T effector cells or T suppressors (Ts) according to the models used. Antibody responses to allogeneic H-2 antigens are not significantly modified.

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C57BL/Ks (H-2d) female mice were transplanted with early (stage 2) embryos of the A/J (H-2a) strain. Spleens from mice exhibiting successful pregnancies were tested at days 16 to 19 of gestation in a local graft versus host (LGVH) assay using (C57BL/Ks X A/J)F1 recipients and proved to be significantly more reactive than virgin controls or mice carrying transplanted syngeneic fetuses. This increased reactivity was specific for the transplanted embryo's strain. Other controls included donors with semi-allogeneic (F1) transplanted fetuses and females naturally pregnant by allogeneic males which did not give reactions significantly different from virgin control spleen cells. Para-aortic lymph node cells (PALN) obtained from the same A/J embryo-transplanted females showed a strong T suppressive activity both on their own spleen cell (SC) reaction as well as on the reaction obtained with virgin SC. This suppressive activity also appeared to be embryo-strain specific. Serological tests revealed the presence of mast cell-degranulating (anaphylactic) antibodies but not of hemagglutinating or complement-fixing cytotoxic activities. The A/J offspring obtained after embryo transfer to C57BL/Ks females presented at the age of two months significantly lower LGVH reactivity against the surrogate mother's strain. The differences in the responsiveness of the mice transplanted with allogeneic embryos compared with those with conventional pregnancies are discussed.

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The effect of lentil lectin (LCA) on various lymphocyte subpopulations in the spleen, lymph nodes and thymus of CBA mice was studied. The most marked effect after five 1-mg LCA doses was observed in the spleen. LCA caused a drop in both the total and relative number of cells carrying all the markers examined, i.e., Thy 1, L3/T4a, Lyt 2, and sIg, so that large numbers of lymphocytes did not express any of them. A dramatic decrease in the Thy 1-positive cell number is mainly due to depletion of the L3/T4a (helper) T cell subpopulation, whereas the Lyt 2+ (suppressor/cytotoxic) cells are less affected. The decrease in the Thy 1+, Lyt 1+ and L3/T4a+ cell numbers in the thymus and lymph nodes is smaller, the percentage of Lyt 2+ cells is even increased after LCA treatment. Our results indicate that two mechanisms play a role in the induction of allotransplantation tolerance by LCA: 'depletion' of helper T cells and a relative increase in the regulatory (suppressor) cell ratio, which may support allograft survival.

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Gestation can induce a priming for a GVHR towards paternal strain antigens, although this priming is significantly lower than the one induced by experimental immunization. A role has been sought for placental substances in decreasing this priming through immunomodulation. BALB/c (H-2d) spleen cells do not usually induce a systemic, lethal GVHR in DBA/2 (H-2d) newborn mice except when the donors are preimmunized with DBA/2 cells. Placental extracts (as well as RPMI medium or liver extracts used as controls) were added to DBA/2 cells injected into BALB/c mice used as cell donors for GVH induction. The latter's spleen cells, harvested on day 6 after immunization, were used for systemic and local GVHR. In the systemic assay (lethal effect on DBA/2 newborn mice injected i.v. with BALB/c spleen cells) a significant protection was observed. In the local assay (popliteal lymph node assay in F1 hybrids injected with BALB/c spleen cells into the foot-pad) a highly significant inhibition of priming was detected in recipients injected with spleen cells from placental extract-treated donors. The stimulation index was even lower than that obtained with unprimed BALB/c spleen cells. The same type of local GVHR in (CBA/Ca X A/J) F1 hybrids injected with CBA cells led to similar results. In both situations (systemic and local GVHR) the observed inhibition was found to be specific to the priming cell strain. These results support the working hypothesis that placental substances are able to modify the systemic response of an organism towards both H-2 and non-H-2 alloantigens.

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The kinetics and quality of the alloimmune reaction were studied in CBA (H-2k) mice treated for passive enhancement of tumor allografts (Sa 1 indigenous of A/J (H-2a or H-2k/d) mice). Serum samples of treated animals were tested for their biological properties relevant to different antibody isotypes in vitro (hemagglutination, complement-dependent cytotoxicity, and anaphylaxis, i.e., mast cell degranulation involving all main Ig isotypes; IgM, IgG2, and IgG1, IgE, respectively) as well as in vivo (allograft enhancement). Spleen cells from these treated animals were examined for their capacity to interfere with the rejection of tumor allografts by adoptive transfers into syngeneic recipients. In vitro, 51Cr release cytolysis assays were performed in order to test their cytolytic and regulatory activities in comparison to rejecting control animals. It has been shown that: grafted mice, pretreated for passive enhancement, kept their grafts longer and synthetized anaphylactic antibodies (mainly IgG1) earlier and at higher titers than normal serum controls, which rejected the same Sa 1 allografts. Mice with enhanced tumors synthetized cytotoxic antibodies (mainly IgG2) later than rejecting controls. Serum samples from treated and control animals, harvested 10 days (early sera) and 30 days (late sera) after grafting, were injected with a "normal dose" (0.2 ml) and a "high" dose (0.4 ml) to new CBA recipients grafted with Sa 1. Early immune sera were only enhancing at high doses when derived from animals previously treated for enhancement (at the low dose both immune sera were enhancing). Late sera, presenting both complement-fixing, cytotoxic (predominantly IgG2), and IgG1 anaphylactic alloantibodies in the two groups, induced enhancement in all cases, but more strongly when derived from the group treated for Sa 1 enhancement. Adoptive transfer of spleen cells from animals treated for passive enhancement were able either to inhibit the accelerated rejection (Day 10) or to promote enhancement of Sa 1 allogeneic cells (Day 30) while similar cells taken (Day 10 and Day 30) from control graft-rejecting mice transferred accelerated rejection. Among the transferred T-cell sub-populations, the suppressive effect was mediated by Lyt 2 T cells. In vitro, these spleen cells showed a weaker cytolytic activity than those of allograft-rejecting mice. Moreover, they were able to regulate the cytolytic activity of cytotoxic effector cells from specifically immunized CBA mice.

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Modifications of the alloimmune response at both the humoral and the cellular levels by placental extracts (PE) syngeneic to the recipient were studied in the mouse using two different H-2 strain combinations. CBA (H-2k) or C57BL/Ks (H-2d), immunized with A/J (H-2a) spleen cells. The tests included in vivo tumor allograft evolution (accelerated rejection or enhancement reactions), and in vitro analysis of the involved immune agents, both cellular and humoral, using mixed lymphocyte reactions (MLR) and biological activity studies of serum samples. Animals from the recipient strains exhibited a delayed rejection of A/J tumor Sa 1 allografts if preimmunization was carried out with 10(6) A/J spleen cells combined with PE syngeneic to the recipients, as compared to controls immunized with A/J cells only or supplemented with isogeneic liver extracts (LE). The serological analysis revealed that PE treatment did not modify the overall hemagglutinating antibody production but resulted simultaneously in both a decreased production of cytotoxic complement fixing antibodies and an increase of specific anaphylactic mast cell degranulating antibodies, as compared to controls. The sera from PE-treated donors also demonstrated enhancing activity following passive transfer to isogeneic recipients. MLR regulatory activity was exhibited by spleen cells from PE- and immunogen-treated mice although the same or stronger activity was obtained from mice immunized without the addition of PE. However, in vivo transfer of these cells to syngeneic recipients showed that PE treatment erased the accelerated rejection caused by allogeneic immunization in the absence of PE and could even cause some degree of allografted tumor enhancement. The cells responsible for this inhibitory effect were mainly IJ+ lymphocytes, since their elimination with a relevant anti-IJ serum and complement restored a secondary type rejection pattern. These results show that PE present during the onset of immunization can promote the activation of regulatory agents such as enhancing antibodies and suppressor cells favoring allograft survival.

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Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG1 production. This phenomenon was also obtained with the 40 KDa fraction. IgG2 responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG2 and IgM responses and a constant suppression of the IgG1 response. This shows a clear dissociation between IgG1 and C'-fixing (IgM, IgG2) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).

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Placental extracts obtained from CBA (H-2k) mice during syngeneic (CBA female X CBA male) pregnancy were evaluated for their capacity to deviate the immune reaction of CBA mice toward A/J(H-2a) immunizing spleen cells, as tested by alloantibody subclass formation and Sa 1 allograft accelerated rejection-or enhancement. The immunomodulatory activity appeared to be located in the soluble and in the insoluble extracts of placenta. The sodium deoxycholate (SDO) solubilized proteinic material, fractionated on Concanavalin A [Con A] and injected to CBA mice simultaneously with A/J spleen cells caused a suppression of the hemagglutinating titer and C-mediated cytotoxicity (IgG2). However, it also favoured antibody-mediated mast cell degranulation (IgG1) and Sa 1 allograft enhancement. The presence and role of IgG1 antibodies in both tumor enhancement and gestation has been described. It is concluded that placenta contains Con A-binding glycoproteins which are located in the membranes and released in soluble form. They exert their immunomodulatory effect in a way which might help the successful outcome of pregnancy as it helps to enhance the development of allografted sarcoma.

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The immune reactivity of lymphoid cells from pregnant mice was studied during the course of pregnancy in primiparous and multiparous animals either " isopregnant " (male and female of same strain) or " allopregnant " (male and female differing at H-2), using a local GVH assay (CBA lymphoid cells injected into (CBA X A/J)F1 recipients). The findings were as follows: The lymphoid cell number in the para-aortic lymph nodes ( PALN ) was increased at all stages of gestation. The peak occurred in the 2nd week in primiparity and as early as 60 h after fertilization in multiparity. PALN cell alloreactivity was weak at the beginning and higher than normal in the third week of pregnancy. Spleen cell alloreactivity was increased in the second week and decreased in the third week in primiparous compared with multiparous animals. Anti-paternal alloreactivity exhibited by spleen cells of allogestation was decreased (as compared to cells of isogestation ) especially in primiparous mice, particularly in the third week. At this time, the anti-paternal alloreactivity of PALN cells was increased. The influence of the recipient's sex on GVHR intensity was reversed when the cells were obtained from a pregnant donor, becoming stronger in male compared with female hybrids.

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The purpose of the present work was to examine the persistence of regulatory cells implicated in the inhibition of allotransplantation reactions in recipient mice in the absence of antigenic material. Two experimental systems were used: (1) When neonatally induced tolerance (obtained as a result of cell-free spleen extract administration in the B10.D2 versus M504 strain combination) was adoptively transferred, the persistence of suppressor cells was detected in secondary recipients receiving a second homograft as long as 30 days after removal of a first homograft to the tolerance inducing haplotype. The removal of the skin homograft presenting a source of antigen for 60 days, resulted in tolerance interruption when a second test graft was applied. (2) When tolerance was induced in CBA mice with spleen-cell antigenic extracts and CFA and spleen cells from such animals were transferred to secondary recipients the following results were observed: Secondary recipients grafted with SaI tumour cells derived from the tolerance inducing A strain 20 days after tolerance induction presented an enhanced tumour growth. The adoptive transfer of this effect was at least partly due to IJ+ lymphocytes, since their removal reduced adoptive enhancement. However, if the cell transfer was delayed for 56 days after tolerance induction, the depletion of IJ+ or Thy 1.2+ cells did not affect the capacity of spleen cells to cause an enhanced growth of the homografted SaI cells. A delay of 100 days following donor enhancement induction did not result any adoptive transfer of SaI enhancement.

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Placental extracts were injected in conjunction with sheep or pigeon RBC to CBA female mice. Spleen cells from these animals were then transferred to isogeneic recipients. The immune response of these recipients, actively immunized towards the immunogen used for priming of cell donors, was studied. (1) It was shown that placental extracts were most efficient in inducing suppressor cell activity in a direct (IgM) plaque-forming assay when injected simultaneously with the immunogen used for cell donor priming. (2) Suppressor cells were generated both in anti-SRBC and anti-PRBC reactions and suppressed the corresponding IgM response after transfer to isogeneic recipients. (3) Combined experiments consisting of the transfer of cells, primed by either SRBC or PRBC, into recipients immunized with a mixture of both immunogens, resulted in a selective suppression of the response directed against the type of RBCs used for the induction of suppressor cells. Results are discussed in the light of previous experiments from this and other laboratories, which showed suppressor effects of placental extracts operating in transplantation systems.

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Immune reactivity of the female recipients, made artificially pregnant, of fully allogeneic embryos was investigated. Allogeneic blastocysts were transferred into hormonally synchronized foster mothers in two strain combinations A (H-2a)--CBA (H-2k) and C57BL/6 (H-2b)--DBA/2 (H-2d). Immunogenicity was tested by growth of tumours Sa 1 (syngeneic with A strain recipients) and EL 4 (syngeneic with C57BL/6 strain recipients). Allogeneic blastocysts implanted in the host's uterus are able to elicit a stronger immune response than in semi-syngeneic pregnancies. The immune reaction was manifested by enhanced growth of tumour Sa 1 and accelerated rejection of EL 4.

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Two parameters of enhancing major histocompatibility complex (MHC) antibodies, previously separately studied, namely, Ig class and antigen specificity, have been treated simultaneously. In the experimental model used, Sa 1 tumor cells, indigenous of A/J (H-2a) were grafted on CBA (H-2k) or C57BL/Ks (H-2d) mice. Immune sera specific for the H-2 K/D- or H-2 I coded antigens of the A/J haplotype (anti-Kk, or IAk, IBk, IJk, IEk, or ICd, Sd, Gd, or Dd) and their immunoglobulin fractions (separated on protein A-Sepharose columns) were injected either i.v. or locally as mixture with the challenging Sa 1 cells. Within the limits of the studied system, the following results were obtained: (1) Sa 1 cells do possess Iak antigens at their surface detected by C-dependent cytotoxicity; no ICd, Sd, or Gd products were detected. (2) The bulk of enhancing activity is concentrated in IgG1 anti-K/D antibodies (anti-Dd when Sa 1 was grafted on CBA mice and anti-Kk, on C57BL/Ks). (3) Anti-Iak antibodies have some activity on Sa 1 cells grafted on C57BL/Ks mice. This activity is significant for IgG1 anti-Iak and suggestive for IgG2 of the same specificity. (4) No enhancing activity was detected in the other antibodies: IgG2 anti-Dd, IgG2 anti-Kk, IgG1, or IgG2 anti-ICd, Sd, Gd as well as in fractions containing IgM and IgA antibodies directed against any studied portion of the MHC products. This results are discussed in terms of the mechanisms involved in enhancement.

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