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To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.

An Escherichia coli recombinant fusion protein containing the major core protein of bovine immunodeficiency-like virus (BIV) was used to immunize mice for generation of monoclonal antibodies to BIV p26. Eight hybridomas specific for BIV p26 were identified and two antibodies, designated 104 and 142, were further characterized. Both 104 and 142 antibodies were isotyped as IgG1; they reacted specifically with both BIV p26 and the recombinant fusion protein in Western immunoblot analyses. However, the epitope specificity of the antibodies was different. Immunoperoxidase assays were used to determine if antibodies 104 and/or 142 could detect BIV replication in cell culture. Both antibodies were found to react with BIV-induced syncytia and individual BIV-infected cells. The antibodies were also used successfully in a focal immunoassay for quantitation of BIV-infected cells. These antibodies will provide valuable reagents for detection and quantitation of BIV replication in studies of viral pathogenesis and immunity.

An enzyme immunoassay for the detection of total anti-HBc antibodies in undiluted serum samples was developed. This assay utilizes an anti-HBc monoclonal antibody and a recombinant HBc antigen. The results of the clinical validation are now reported. A total of 1,301 sera were tested using both the Recombinant TOTAL HBc Ab EIA and a reference assay. The specificity was evaluated on a panel of 573 normal human sera and human sera from subjects with pathological findings unrelated to a hepatitis B virus infection. The sensitivity was studied on a total of 455 sera from HBV infected patients at different stages of infection. The final results indicate 99.8% sensitivity and 99.8% specificity. In addition, 273 sera with either isolated anti-HBc antibodies or with anti-HCV antibodies were tested. The agreement between the Recombinant and the reference assay for these two populations, 96.6 and 90.1%, respectively, is discussed.

A monoclonal antibody (mAb) to infectious bursal disease virus (IBDV) was bound to polystyrene latex microspheres. The microspheres agglutinated with extracts of bursae and sera from chickens infected with all strains or isolates of IBDV tested. Agglutination appeared within a 10-min reaction time. The assay could detect a 10(3.7) to 10(4.5) mean embryo infective dose (EID50) of the virus in 0.01 ml and the titer of the assay was 10- to 40-times higher than that of the agar gel precipitin test.

We established the hybridoma producing the monoclonal antibody (mAb) 3C3 by immunizing rats with mouse natural killer (NK)-like cells. The 3C3 mAb seemed to react mainly with T cells and T-lineage cell lines. The 3C3 antigen also seemed to be coincidentally expressed on a part of asialo GM1+ cells from nude mice, suggesting its expression on NK cells. Treatment of effector cells with 3C3 mAb markedly inhibited the killer activity against RL male-1 cells, but less so against YAC-1 cells, in vitro. It is suggested that the cell surface molecule defined by 3C3 mAb was closely associated with the killer activity of T cells and NK cells.

Antibodies specific for polycyclic aromatic hydrocarbon (PAH)-DNA adducts have previously been reported in human sera. In this study, we examined the association between mixed PAH exposure and PAH-DNA adduct specific antibodies in BALB/c mice. Mice were treated either by i.p. injection or by intragastric (i.g.) intubation with a mixture of seven different PAHs [benzo(a)pyrene (BP), benz(a)anthracene (BA), fluoranthene (FA), dibenz(a,h)anthracene (DBA), 3-methyl-cholanthrene (MC), chrysene (Ch), benzo(b)fluoranthene (BF)] at three doses (0, 15, 150 micrograms of each PAH) twice a week for 8 weeks. Sera were screened by direct ELISA for antibodies recognizing DNA modified by diolepoxides or epoxides of each PAH injected. In i.p. treated mice, the sera were slightly more reactive to DNAs modified with diolepoxides of BP, BA, or Ch or an epoxide of DBA than to unmodified DNA. In i.g. treated mice, the sera were more reactive to DNAs modified with diolepoxides of BA or BF than to unmodified DNA. For some PAHs, a dose-response effect was observed between sera reactivity to PAH metabolites and the dose of PAH administered. However, there was considerable variation in the immune responses among individual mice within each treatment group. When tested by competitive ELISA, none of the sera could discriminate between modified and unmodified DNA. This animal study suggests that an assessment of previous carcinogen exposure by measuring DNA adduct-specific antibodies requires further validation prior to its application to the human monitoring of carcinogen exposure.

Perforin is a cytoplasmic granule protein expressed in cytotoxic lymphocytes, and is capable of lysing target cells. This protein is induced as cytotoxic T cells are activated, and the mRNA expression is modulated by various stimulators. These observations suggest possible changes in the level of perforin transcripts and protein when killer lymphocytes meet specific target cells leading to target cell death. To address this question, we examined three murine T-cell clones and primary human NK cells in perforin expression. When the cytotoxic lymphocytes were exposed to sensitive targets, perforin mRNA disappeared within 5 to 30 min and appeared within an hour thereafter. Among the murine T cell clones, L3 and OE4 showed two phases of mRNA decrease while human NK cells and the third murine T cell clone, AB.1, showed only one phase of mRNA loss during a 240 min period. The data indicate that when cytotoxic lymphocytes receive signals from a sensitive target, the cells rapidly degrade previously accumulated perforin mRNA and synthesize new transcripts. Interestingly, heat shock protein 70 mRNA was induced as the perforin mRNA levels recovered, while P55 Il-2 receptor mRNA was downregulated within 5 min after exposure to targets. The perforin protein level also rapidly decreased immediately after the interaction with the target, followed by a recovery, and then another decrease as seen in primary human NK cells, OE4 and L3 cells. However, in the AB.1 clone, no change in perforin content was detectable, despite the loss of perforin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Seven monoclonal antibodies (MAs) generated against porcine zona pellucida glycoprotein, ZP3 (comprising both ZP3 alpha and ZP3 beta) were characterized for their specificities to ZP3 alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3 alpha and MA 30 for ZP3 beta. All the antibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3 alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3 beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.

The tissue distribution, molecular weight, and biochemical nature of an antigen detected by a mouse monoclonal antibody designated 283D3 and raised against rabbit visceral yolk sac endodermal cells, has been investigated. The antigen is located on the luminal side of apical tubules and large sub-apical vesicles in rabbit yolk sac endoderm and proximal kidney tubule epithelial cells. It is expressed in a similar polarised fashion in epithelial cells lining the epididymis. Western blotting showed the antigen to comprise proteins of molecular weight 330-380 kDa. The antigen has been affinity purified from yolk sac and kidney and is predominantly protein in nature with a small percentage of N-linked carbohydrate. In terms of tissue distribution and molecular weight it has close similarity to Heymann nephritis antigen but differs in not being confined to coated pits. Its function is not known, but the association with endocytic elements implies a possible role in non-specific protein absorption.

Mouse anti-fibrous sheath antisera (MAFA) produced by immunizing mice with purified preparations of human sperm tail fibrous sheath (FS) reacted with the principal piece of less than 10% of freshly isolated spermatozoa which were immotile and probably dead. Following their demembranation by detergents or repeated freezing and thawing, all spermatozoa were stained. This was also demonstrated on spermatozoa dried onto slides, but the undiluted xenoantisera showed additional reactivity with the acrosomal zone (AZ). Using immunogold electron microscopy, the target antigens were ultrastructurally localized to the FS, and a few spermatozoa showed some reaction at the AZ subacrosomal perinuclear theca. Following titration of the antibodies, the anti-AZ-reaction became undetectable at a dilution of 1:20 while their reactivity with the principal piece continued to a 1:400-dilution. These results indicated that the xenoantisera probably contained an additional unrelated antibody component which reacted with the AZ. Western blotting and staining of purified FS with MAFA detected seven major protein bands with MW ranging between 25 kDa and 97.4 kDa. In human testes, the 1:50 diluted MAFA reacted with sperm tails only, indicating the late expression of the antigenic determinants during spermatogenesis. MAFA did not react with oesophagus, stomach, duodenum, ileum, nasal lining tissues, uterus, pericardium, pancreas, thyroid gland, or cultured fibroblasts. The xenoantisera did, however stain the skin epidermis and cultured keratinocytes which exhibited filamentous cytoplasmic staining although their target antigens could not be biochemically identified. These results indicate that the FS proteins express antigenic determinants which are not shared with other cytoskeletal elements within the sperm flagellum or a variety of somatic tissues.

Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80-100, 120, 160-180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

A new monoclonal antibody designated Mab D11 is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. When tested by light and electron microscopic immunoperoxidase methods, Mab D11 specifically reacts with blood monocytes and stains resident macrophages in a wide variety of human tissues; it does not mark the macrophages of other species, i.e., rat, swine and mouse. Antigen-presenting cells, e.g., Langerhans cells, are Mab D11 negative. Mab D11 reveals the antigen on cryostat and paraffin tissue sections. Ultrastructurally the antigen recognized by Mab D11 in all macrophage types studied is located on the plasma membrane and within cytoplasmic structures including lysosomes. On immunoblotting, Mab D11 detects the 125-kDa antigen in human liver and the 135-kDa protein in tumours of histiocytic origin. The similarity of Mab D11 to known "pan-macrophage" monoclonal antibodies is discussed.

Immunization of naive or specifically primed C3H/HEJ with irradiated B10.BR spleen cells via the hepatic portal vein leads to an antigen specific decrease in the proliferative and cytotoxic response to B10.BR antigen assayed in vitro (and to increased graft survival of B10.BR grafts in vivo). This effect seems to be mediated in the main by a decrease in IL-2 production from CD4+ T lymphocytes of mice given antigen by the portal route, which is in turn caused by a decreased precursor frequency of IL-2-producing cells. No clear decrease in IL-4 production was seen. Hepatic APC isolated from mice receiving antigen via the portal vein were unable to induce IL-2 production from a C3H/HEJ anti-B10.BR cell line in vitro, in contrast to splenic APC derived from the same mice. Even when antigen was given by conventional (systemic) intravenous routes (in this case via the lateral tail vein) hepatic APC isolated from those mice were unable to stimulate IL-2 production from this cell line. Furthermore, 24 h exposure of a cell line to antigen pulsed hepatic APC left those cells refractory to a subsequent restimulation with antigen presented by splenic APC. Spleen lymphoid cells from primed mice challenged in vivo with B10.BR liver cells (i.v.) were similarly unable to produce IL-2 on rechallenge in vitro with irradiated B10.BR spleen cells, though no defect was seen if in vivo challenge was with B10.BR spleen cells. These data imply that presentation of multiple minor cell surface antigens by hepatic APC leads to specific anergization of IL-2 producing T cells, in a fashion which seems to be distinct from that previously reported as due to 'veto-like' activity.

Mouse eggs and pre-implantation stage embryos express on their surfaces a carbohydrate epitope, TEC-2, defined by an IgM monoclonal antibody, TEC-02. The TEC-2 epitope involves the oligosaccharide sequence GalNAc beta 1----4Gal beta 1----4 that is expressed on the plasma membrane and zona pellucida of mouse eggs and on a very limited number of other cell types. In this study we addressed the question whether or not the binding of TEC-02 antibody to the mouse eggs would interfere with their fertilization. Our data showed that the TEC-2 epitope is carried by two zona pellucida glycoproteins, ZP2 and ZP3. Binding of TEC-02 antibody to mouse eggs inhibited specifically and in a dose-dependent manner their fertilization in vitro. The inhibitory effect of TEC-02 antibody was dependent on the presence of an intact zona pellucida. Direct radioantibody binding assays indicated that the TEC-02 antibody completely inhibited fertilization at a concentration at which one quarter of all available TEC-2 binding sites was occupied. Binding of TEC-02 antibody to an egg did not interfere with initial attachment of the sperm to the egg but inhibited maintenance of sperm binding to the zona pellucida, the secondary binding. The combined data indicate that TEC-2, which is a well-defined zona pellucida specific carbohydrate epitope, might be a part of the secondary sperm receptor.

Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One antibody, K5A6, was specific for MT-1, the others recognized all isoMTs present in the human fetal liver. Each of these antibodies showed a unique cross-reactivity pattern when tested with MTs from the livers of different mammals. A double-antibody sandwich enzyme-linked-immunosorbent-assay (ELISA) was developed with the antibodies L2E3 and L5G2. This assay allows the detection of 60 pg human fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and Hg-MT.

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

The effects of murine recombinant IL-1 alpha (muIL-1 alpha) on lymphocyte migration in the mouse have been investigated. Continuous infusion of muIL-1 alpha had marked effects on patterns of lymphocyte migration into a site of chronic inflammation, inflammatory exudate and spleen; the numbers of lymphocytes migrating to the inflamed tissue and spleen were reduced in a dose-dependent manner. The number of lymphocytes in the blood of muIL-1 alpha-treated animals was increased in a dose-related manner. The decrease in numbers of lymphocytes present in the chronically inflamed site may either be due to a direct inhibitory action of muIL-1 alpha or reflect an increased rate of cell migration through the inflamed tissues accompanied by a more rapid return to the circulation. These findings suggest that IL-1 alpha may act not only as an inflammatory cytokine, but also as a modulator with anti-inflammatory activity during chronic inflammation.

Immunocytochemical and immunoblotting technique have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytocin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.

This study was designed to address the question; does expression of paternal histocompatibility antigens by fetal cells make them susceptible to immune attack in vivo during normal pregnancy? The experimental design was based on the rationale that, if alloantigens are presented by trophoblasts or other fetal cells in a manner which allows accessibility, in vitro-generated immune effector cells of combined helper/cytotoxic phenotype should produce fetal rejection of abortion. Similarly generated effector cells are capable of accelerating skin graft rejection and, when combined with IL-2 in vivo, are capable of causing regression of antigenic, but operationally non-immunogenic, tumors. The alloimmune effector cells generated in vitro during the current study were highly cytotoxic against normal adult target cells, whereas placental cells were completely resistant to cytolysis and fetal cells were only slightly susceptible. Adoptive transfer of effector cells to mice at different stages of gestation had no apparent effect on pregnancy outcome. In vivo administration of IL-2 and/or indomethacin, which expand effector cell numbers in vivo and block PGE2-mediated immune suppression, respectively, failed to potentiate the cellular effect. The data provide additional evidence that paternal histocompatibility antigens are not expressed in a format which allows susceptibility to immune attack during pregnancy. The data are discussed with respect to the role of the trophoblast in protecting developing embryos.

Intravenous immunization of BALB/c mice with irradiated Leishmania mexicana slows the growth of a subsequent intradermal inoculation of virulent parasites. Prior subcutaneous immunization with irradiated parasites before i.v. immunization blocks the protective effect of the latter. Parasites harvested from vaccinated mice grow more slowly in naive mice than the initial inoculated clone, and have a diminished capacity to immunize mice against this initial clone when used as (irradiated) i.v. immunogen. However, parasites harvested from vaccinated mice are as effective as the initial clone in blocking protection when used as subcutaneous immunogen. Understanding the nature of this differential response in expression of protecting/suppressor determinants in parasites harvested from vaccinated or naive mice will likely be important to developing a suitable vaccination strategy for human use.

Whole rat spleen cell populations in culture release constitutively a factor which suppresses the NK activation in response to interleukin-2. Culture supernatants derived from adherent (AD) and non-adherent (NAD) cell preparations obtained from rat spleen cells, did not have a suppressor activity comparable to that present in the culture supernatants of whole spleen cells. When reconstituted mixtures of the AD and NAD cells were cultured, high levels of suppressor activity could again be demonstrated in the culture supernatants, indicating that some kind of co-operation between AD and NAD cells was needed for efficient generation of the suppressor. Non-suppressive culture supernatants derived from NAD cells became suppressive when AD cells were cultured in them. On the other hand, relatively little suppressor activity was generated when NAD cells were cultured in non-suppressive culture supernatants from AD cells. Our results seem to indicate that NAD cells may release some agent which is not itself a suppressor but either (a) induces the generation of suppressor activity from AD cells, or (b) is "processed" into a suppressor agent by AD cells.

We have investigated the capacity of the lymphokine gamma-interferon (IFN-gamma) to induce class II major histocompatibility complex (MHC) antigens on astrocytes cultured from BALB/c mice. This is a mouse strain resistant to experimental allergic encephalomyelitis (EAE), and in a recent report Massa et al. (Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 4219-4223) indicated that BALB/c astrocytes in vitro were not susceptible to class II MHC antigen induction by IFN-gamma. We observed, in agreement with this previous report, that when primary cultures of astrocytes from neonatal BALB/c mice were just at confluence (7-10 days in vitro), IFN-gamma did not stimulate expression of class II MHC antigens. However, after 14-16 days in vitro, a population of astrocytes emerged in the cultures on which class II MHC antigens could be induced. These cells expressed the astrocyte marker glial fibrillary acidic protein, and were found in close association to small round superficial cells (multipotential precursor cells), and to microglia. These results indicate that the ability of astrocytes to respond to lymphokine stimulation is not completely correlated with susceptibility to EAE, and further suggest the importance of central mechanisms in the development of inflammatory brain disease.

The present study shows that interleukin 4 (IL4) can exert either stimulatory or inhibitory effects on an antigen-specific IgE response in vitro. Spleen cells from BALB/c mice that had been primed with trinitrophenyl keyhole limpet hemocyanin (TNP-KLH) were cultured with the same antigen for 2 days, washed, transferred to an antigen-free culture medium, and then cultured for 4 days. Anti-TNP IgE antibodies secreted into the medium were determined by an enzyme immunoassay developed in our laboratory. Anti-TNP IgE response was elicited by TNP-KLH in cultures of the spleen cells from mice that had been primed twice with the antigen at an interval of 3 weeks, but not in cells from animals that had received only a single injection of the antigen. When the latter cells were cultured with the antigen in the presence of mouse recombinant IL4, a considerable level of the antigen-specific IgE response was induced. In contrast, the IgE response elicited in the cells of mice that had been primed twice with the antigen was markedly down-regulated by added IL4. These observations suggest a novel function of IL4 in the regulation of IgE antibody responses.

In the H-2-incompatible donor-recipient combination (BALB/c----CBA/H) local administration of cyclosporin allows a 4-fold reduction in a single dose prolonging survival of heart tissue allografts. These results suggest that local administration of cyclosporin may be useful for certain grafts.

This report describes a suitable model for analysing heterogeneous T cell responses to complex foreign antigens using coated polystyrene beads. The advantage of this technique is that it allows the simple removal of detergents from bound antigen so that biochemically separated antigens or crude antigen mixtures can be used. Furthermore, due to the enhanced uptake of latex-bound antigens by phagocytic antigen-presenting cells (APC), very small amounts of antigen will suffice for activation of T cells in vitro. The potential use of this technique to analyse relevant T cell responses to antigens which are difficult to obtain purified in bulk quantities, is discussed.

We report here that the high rate of spontaneous resorption observed in CBA/J female mice mated with DBA/2 J males can be dramatically reduced by vaccination with Balb/c male spleen cells, but not by CBA/J or DBA/2 J male spleen cells. This effect correlates with the differential ability of Balb/c spleen cells to induce MLR suppressor activity in CBA/J female mice, and should lead to a better understanding of the immunology of the materno-fetal relationship.

Human SP-1, a glycoprotein synthesized by the placenta during pregnancy, was shown to exist as polymers in maternal serum by a rapid passive transfer immunoblotting technique following conventional agarose electrophoresis. Moreover, the SP-1 polymers in serum were shown to dissociate into one main component upon treatment with 8 M urea prior to electrophoresis. However, an unexpected observation was the existence of an SP-1-like immunoreactive species in male serum with the sensitive immunoblotting technique. This SP-1-like protein in male serum had similar properties to its placentally derived counterpart in pregnancy serum, namely a propensity for complex formation and a reduced electrophoretic mobility following neuraminidase treatment. The relationship between the two SP-1 proteins was demonstrated by isolating them from their respective sera using an immobilized monoclonal antibody raised to purified SP-1 from pregnancy serum. Immunoblotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two major SP-1 species in pregnancy serum. These two SP-1 species had apparent molecular weights of 68,000 and 64,000. In addition, there were two minor bands at 35,000 and 32,000. These smaller SP-1 species did not appear to be subunits of the larger entities since they were detectable in the absence of reducing conditions. SDS-PAGE and immunoblotting showed that the immunoaffinity-purified SP-1 species from male and pregnancy sera had similar, but not identical, molecular weights.

The immunization with the flagellar (F) fraction from epimastigotes of Trypanosoma cruzi has been shown to protect mice against a challenge of bloodstream trypomastigotes of the parasite, both in terms of mortality and decrease in parasitemia. We have compared the immunoprotective properties of the fresh F fraction with those of a lyophilized F (LF) fraction, alone or together with Bordetella pertussis (Bp) as adjuvant. The best results were obtained with LF + Bp: after challenge with 1 X 10(3) metacyclic trypomastigotes, 100% of the mice immunized with LF + Bp survived, and 60% of them showed no signs of parasitemia. Only the animals in which patent parasitemia was demonstrated presented heart and muscle infiltrates.

A monoclonal antibody has been obtained which binds to the cell surface of cultured chick myotubes and retinal and tectal neurons but not fibroblasts, myoblasts and embryonic liver cells. Indirect immunocytochemistry reveals that antigen is present in all layers of the chick neural retina. The antibody therefore recognizes an antigen common to most, if not all, chick neurons. The antigen has been identified by staining SDS gels with [125I]monoclonal antibody and appears to be a polydisperse collection of polypeptides with molecular weights centered about 250 kdalton.

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and the antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBA/2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas others antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.

This paper investigates the requirement for genetic restriction in the action of T suppressor afferent cells (Ts-aff) which regulate the induction phase of contact sensitivity to picryl chloride. The results here reported show that Ts-aff which inhibit both the induction of contact sensitivity and DNA synthesis in the draining lymph nodes of sensitized mice, act across both H-2 and Igh genetic barrier and constitute new evidence which discriminates between two T suppressor cell populations in contact sensitivity.

Significant differences in susceptibility to the induction of experimental allergic orchitis (EAO) were found to exist among the various substrains of BALB/c mice. Substrains which were susceptible to disease induction include BALB/cKa, BALB/cByJ, BALB/cWtJ, BALB/cCmcCr, BALB/cCrgl, BALB/cHeA, BALB/cAnNCr, BALB/cPt, BALB/cWehiUnm, BALB/cOrnl and BALB/cArg whereas BALB/cJ and BALB/cSki BoyPt were resistant to EAO. Aspermatogenesis and autoimmune vasitis, two additional lesions characteristic of murine EAO, were found to correlate with susceptibility to autoimmune orchitis. Susceptibility and/or disease resistance did not segregate within any of the evolutionary branches of the BALB/c genealogic tree nor did it correlate with any of the previously described allelic differences distinguishing BALB/c substrains. Therefore, differential disease susceptibility may represent the manifestation of mutational and/or retroviral induced genotypic differences in the regulatory genes controlling testicular autoimmunity.

We have previously demonstrated that high frequency (1/20) of potent cytotoxic cells reactive with the nonimmunogenic lymphoma PIR-2 of C57BL/6 (B6, H-2b) origin, can be obtained by allosensitization of syngeneic B6 splenocytes against BALB/c (H-2d) splenocytes in limiting dilution cultures (LDC). Since a high concentration (250 U/ml) of exogenous interleukin 2 (IL-2), sufficient for the elicitation of lymphokine-activated killer (LAK) cells, was used in the LDC, and because the LDC-derived cytotoxic cells were active against a wide spectrum of target cells, we investigated whether the anti PIR-2 effector cells are LAK cells or cytotoxic T lymphocytes (CTL). We found that depletion from the B6 responder cell population of Lyt2+ (CTL precursors), but not of asialo GM1+ (LAK cell precursors), prior to LDC, results in the ablation of anti PIR-2 activity. When B6 splenocytes were plated in LDC with IL-2, in the absence of allogeneic stimulating cells, the resulting anti PIR-2 activity was greater than 10- to 500-fold lower than that obtained in LDC in the presence of allogeneic stimulating cells and IL-2. These and other observations suggest that the cytotoxic response against syngeneic tumors elicited by alloantigens in LDC is mediated by CTL rather than LAK cells, and that allogeneic sensitization in LDC can provide a means for the generation of CTL against syngeneic, nonimmunogenic tumors.

Five xenogeneic anti-idiotypic antisera (anti-id) were produced against individual BALB/c-derived monoclonal antibodies (mAb) which bound to different peptidic determinants on the human thyroglobulin molecule (Tg). Idiotypic analysis performed using sensitive radioimmunoassays revealed that: (1) the anti-id highly precipitated their homologous ligands; (2) two anti-id displayed minor cross-reactivities with one or two heterologous mAb; (3) each unlabelled homologous mAb was able to inhibit the idiotype binding of the corresponding anti-id; (4) no significant inhibition of homologous idiotype binding was observed with large excess of heterologous mAb; (5) efficient inhibition of mAb binding to Tg was observed only when homologous anti-id served as inhibitor. The data support the conclusion that xenogeneic anti-id may detect on their corresponding ligands individual idiotypic specificities that can be located at the mAb-combining site. Such reagents may constitute appropriate probes for further studies on anti-Tg autoimmunity.

A natural IgM monoclonal antibody, 1.67, was generated from apparently healthy unstimulated BALB/c mice. This antibody reacted with L5178Y murine T cell lymphoma, with human Raji cells, and with several normal cells. Further analysis of its ligand binding capacity disclosed strong binding to single-stranded DNA (ssDNA). However, this naturally-occurring monoclonal antibody binds to different epitopes on cell membranes and on DNA than another anti-DNA monoclonal antibody (18/103/1) from human origin. This conclusion was based on competition assays. Furthermore, NOA 1.67 lacks the 16/6 idiotype expressed on the 18/103/1 antibody. The 16/6 idiotype is shared by human and mouse lupus monoclonal autoantibodies that bind simultaneously to lymphoid cells and DNA. This is a first report on a natural autoantibody that binds to malignant and to normal cell membrane(s) as well as to ssDNA. It may have regulatory functions controlling malignancy and or autoimmunity.

The adherence of granulocytes induces secretion of specific granule contents. The secreted proteins were termed granulocyte factors (GF). The experiments in vivo provide evidence that GF play an essential role in the stimulation of PFC in BALB/c mice immunized with SRBC when applied before challenge three times (5 micrograms per mouse), but 50 micrograms per mouse given in the same way diminishes the response. To elucidate this discrepancy, the effect of GF on the generation of suppressor cells (SC) and helper cells (HC) in vitro has been investigated. Antigen specific nonadherent SC or HC were induced in vitro using CBA mice spleen cells incubated with 100 micrograms/ml or 0.1 mg/ml of TNP-KLH, respectively, for 4 days. GF in concentrations of 0.1 to 1 microgram/ml abolish antigen specific SC generation. SC and HC activity was tested in cooperative cultures. Antigen specific SC in delayed hypersensitivity (DTH) to BCG were induced in an in vitro system as above using normal BALB/c spleen cells and 100 micrograms/ml PPD. Nonadherent suppressor cells were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer. DTH was measured by foot-pad reaction. This reaction was positive to PPD in CY treated mice immunized to BCG, while it was suppressed by the transfer of in vitro induced SC. When the SC were induced in the presence of 1 microgram/ml GF, the suppression was abrogated. The higher GF concentrations stimulated SC activities when they were measured in response to a nonrelated antigen and in specific anti-PPD response, but the HC inhibition could not be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of long-term regular and moderate exercise on antibody production has been studied in mice. Healthy mice were trained to run for 10 min twice a day, and antibody levels following immunization were compared with those of healthy sedentary control mice. The antibody levels of the running mice were significantly higher than those of the sedentary animals at all times tested, from 1 to 13 wk post immunization. Overall, the antibody titres of the runners were 2.76 times that of the controls. The significance of these findings is discussed.

We report here that vaccination of CBA/J female mice with DBA/2 X BALB/c recombinant line that decreases the spontaneous abortion rate increases local active decidua-associated suppressor cell activity. In contrast, vaccination with a recombinant line that increases the abortion rate decreases suppressor cell activity. No correlation was seen between the effect on the abortion rate and the ability of cells from the fetoplacental unit to inhibit cytolysis by NK cells. Successful vaccination against spontaneous abortion may act primarily by augmenting suppressor cell activity in the decidua at the implantation site.

We have produced two monoclonal antibodies against polypeptide-associated antigens of developing rat cerebellum. One antibody recognizes an antigen associated with synaptic vesicles and another binds to a polypeptide which is restricted to the cytoplasm of a subset of cerebellar Purkinje cells. Both antibodies reveal the biochemical differentiation of the rodent cerebellar cortex into antigenically distinct sagittal zones.

Female CBA/J (H-2k) mice mated with male DBA/2J (H-2d) mice show a high level of fetal resorption, which can be reduced by immunization with BALB/c (H-2d) spleen cells. The morphologically defined fetal resorption rate upon which evaluation of the outcome of pregnancy has previously been based in this strain combination is not equivalent to the rate of production of viable neonates.

The development of specific markers for retinal ganglion cells is an area of great interest in retinal research. In this study we report on a monoclonal antibody (AB5) which specifically labels ganglion cells in rabbit, cat and monkey, as well as a variety of other mammalian species. Labelling of ganglion cells was also observed in isolated cell preparations of rabbit retina.