Localization of a molecule immunochemically similar to eosinophil major basic protein in human placenta.

We have recently reported that human pregnancy is characterized by a 10- to 20-fold elevation of eosinophil major basic protein (MBP) immunoreactivity in maternal blood. Here we show, by immunofluorescence, that placental tissue specifically binds antibody to MBP in and around the placental X cells and placental-site giant cells and, using thin plastic sections, that placenta has no infiltrating eosinophils. The X cells line the inner aspects of placental septal cysts, and the cyst fluid, obtained by aspiration, contains immunoreactive MBP at concentrations of 100 micrograms/ml, a sixfold greater concentration than the highest levels measured in maternal blood. The soluble MBP immunoreactivities in placental homogenates and in maternal serum chromatograph identically on Sephadex G-50, and both these gestational MBP molecules migrate as though substantially larger than the MBP found in serum from patients with hypereosinophilic syndrome or purified from the eosinophil granule. Our inability to demonstrate eosinophils in maternal blood or placental tissue, coupled with the large quantities of immunoreactive MBP highly localized in placental cysts and the chromatographic behavior of this molecule, suggest that the MBP detected in human gestation is produced by placenta.

Inhibition of lymphocyte cytotoxicity and mitogen-induced transformation of lymphocytes by human skin homogenates.

Limitation of the inflammatory cascade has been demonstrated in man by an inhibitor(s) of the prostaglandin system and by inhibitor(s) for chemotaxis of leukocytes. Other factors may exist in vivo that inhibit other inflammatory sequences. In this report, homogenates from human skin interfered with 2 standard assays of lymphocyte function, mitogen-induced transformation and cytotoxicity while homogenates of human placenta did not. Differential centrifugation studies located inhibitory factor(s) primarily in the nuclear fractions. Therefore, reversible transitory depressions of the cell-mediated immune response that are observed concomitantly with chronic dermatoses may reflect release of inhibitory material dermatitic skin in vivo.

Public determinants of HLA indicated by pregnancy antibodies.

A tentative table of public specificities in the HLA-A and -B loci is provided. It is based on an all-inclusive survey of placenta extracts from 50,000 pregnancies. We postulate that most of the specificities found are directed against public epitopes. In support of this postulate are the facts that certain combinations occur very frequently, monoclonal antibodies have been made to some of the epitopes, and some have already been established by absorption experiments as being a single specificity. The immunogenicity score for each private and public specificity was computed by taking into account the chance of immunization. It was shown that immunogenicity can vary by factors of more than ten between different specificities. Significantly, immunogenicity of the public epitopes was just as high as against the private ones. This indicates that the public epitopes should be considered as independent, separate antigens in transplantation. Establishment of a table of public specificities and the recognition of each by international nomenclature would be the first step in evaluating public epitopes for transplantation matching.

The ability of placental extracts to modulate a direct PFC response to SRBCs in mice.

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.

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). II. Inhibition of priming by placental extracts.

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.

Effects of antisera raised against native and denatured human alpha-glucosidase and beta-hexosaminidases on native enzyme activity.

Antisera were raised in rabbits against native and sodium dodecylsulfate denatured forms of human acid alpha-glucosidase and beta-hexosaminidases A and B. Anti-native enzyme antisera were able to precipitate all or nearly all enzyme activity from cell extracts, and to eliminate all stainable activity on electrophoresis. Antisera prepared against denatured enzymes precipitated only a minor part of enzyme activity. Electrophoretic analysis showed that these antisera were able to bind to the enzyme molecule. The result was a slowing down of the anodic migration but not immobilization. The use of variants with hexosaminidase deficiencies helped to clarify the action of the antisera on the various hexosaminidase isozymes.

TRH-like peptides in prostate gland and other tissues.

This minireview is aimed to recapitulate the occurrence of TRH-like peptides in the prostate gland and other tissues and to discuss their known functions in the organism. The hypothalamic thyrotropin-releasing hormone (TRH) was the first chemically defined hypophyseotropic hormone with the primary structure pGLU-HIS-PRO.NH2. However, the presence of extrahypothalamic TRH-immunoreactive peptides was reported in peripheral tissues including the gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. It was supposed that this TRH immunoreactivity can partially originate from TRH-homologous peptides and that these peptides have significant cross-reactions with the antibody specific against authentic TRH. This assumption was confirmed by the identification of prostatic TRH immunoreactivity as pyroGLU-GLU-PRO.NH2 using fast atom bombardment mass spectrometry and gas phase sequence analysis. TRH-like peptides are characterized by substitution of the basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acids, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological role of TRH-like peptides in peripheral tissues is not precisely known, but they possess a C-terminal amide group which is characteristic for many biologically active peptides. The occurrence of these peptides in the male reproductive system can influence male fertility. They are also closely related to circulating thyroid and steroid hormones. There might be an important connection of TRH-like peptides to the prostatic local autocrine/paracrine network mediated by extrahypothalamic TRH immunoreactivity corresponding to TRH-like peptides and extrapituitary thyrotropin (TSH) immunoreactivity also found in the prostatic tissue. A similar system of intraepithelial lymphocyte hormonal regulation due to the local paracrine network of TRH/TSH has been described in the gastrointestinal tract. The local network of TRH-like peptides/TSH may be involved in possible regulation of prostatic growth.

Detection of a pregnancy-associated protein.

Monospecific antiserum was produced with the use of a placental extract showing high activity of a proteinase which hydrolyses a synthetic substrate, N-benzoyl-D,L-arginine p-nitroanilide, used for measuring tryptic activity. An immunological study showed that antibodies were not generated against the component with this activity but against an antigen which was not related to several well-known pregnancy-associated proteins. Pregnancy sera contained an antigen which immunologically was completely identical to the antigen of placental origin. Single radial immunodiffusion showed an elevated level of reactive antigen in pregnant subjects: 70% of 333 cases were positive. Cross-reacting antigen was also detected in sera as well as ascitic fluid from cases of advanced ovarian cancer. Sephadex G-200 gel filtration indicated that the antigen has an apparent molecular weight of 94000, and immunoelectrophoresis showed the molecule to be of beta-mobility. These properties suggest that the substance may represent an additional pregnancy-associated protein entity. Partial purification and some immunological properties of this antigen are described.

Immune modulation effect of porcine placenta extracts in weaned the pig.

In a previous study, we established a collection of appropriate porcine placental extracts using PBS at 80°C (PE-PBS80) as a food supplement to increase immune activities in a mice model. In this study, piglets were treated with 0.1%, 0.3%, and 0.5% PE-PBS80 for 3 wk after weaning. Experiments were performed at 2 separate farms using 2 different pig varieties. Composition of white blood cells, lymphocyte activation, and cytokine concentrations were analyzed to assess the immune modulation effect. In Exp. 1, the number of white blood cells increased significantly in the PE-PBS80 treatment and T- and B-cell activation increased as well (P < 0.01). Interestingly, piglets in all treatments in Exp. 2 were naturally infected by a rotavirus at the third day of the experiment but recovered after d 10. Increased lymphocyte activation was observed in the PE-PBS80 treatment (P < 0.01) regardless of viral infection. Additionally, unlike in Exp. 1, the percentage of granulocytes and concentrations of interferon-γ, IL-1ß, and IgG increased in the PE-PBS80 treatment (P < 0.01) and were more active in the 0.3% PE-PBS80 treatment compared with the control and the other treatment. In conclusion, 0.3% PE-PBS80 treatment modulated immune activities in antigen-infected piglets. Therefore, the PE-PBS80 pig placental extract, particularly the 0.3% supplement to the normal diet, could be useful as an alternative feed supplement to modulate immune activity during the early piglet period.

Human placental extract offers protection against experimental visceral leishmaniasis: a pilot study for a phase-I clinical trial.

An aqueous extract of human placenta (HPE) was found to offer protection against established experimental visceral leishmaniasis in BALB/c mice and hamsters, whether the Leishmania donovani strain involved was one that was sensitive or resistant to pentavalent antimony. Intraperitoneal administration of the extract, into mice or hamsters that had been infected 2 months previously, led to antileishmanial T-cell proliferation among splenic mononuclear cells, the generation of host-protective cytokines (interferon-gamma, tumour necrosis factor and interleukin-12) and the upregulation of the expression of inducible nitric oxide synthase (and subsequent NO generation) in splenocytes. Furthermore, splenic macrophages from the HPE-treated mice showed increased generation of reactive oxygen species and enhanced surface expression of antigens of major histocompatibility complex class II (MHCII), and the extract restored the otherwise-defective antigen-presenting ability of the macrophages. Thus, in mice and hamsters infected with L. donovani, HPE therapy can stimulate both arms of the host's immune system and favour the complete resolution of the leishmanial infection. Among five human cases of visceral leishmaniasis, 30 daily intramuscular injections of HPE, at doses much lower than those used in the experimental infections, also gave very promising results. Based on the results of this pilot study, a further evaluation of the efficacy of HPE therapy, which may offer a cost-effective way of improving the treatment of antimony-resistant cases of visceral leishmaniasis, is being undertaken.

Identification of a butanol-extractable human placenta-specific antigen with alkaline phosphatase activity.

N-Butanol extracts of whole-term placenta from different individuals were prepared, and used as immunogens to raise heterologous hyperimmune sera in rabbits. Upon immunoelectrophoresis the anti-placenta antisera could recognize at least six antigenic components in the placental extract even after they had been completely absorbed with pooled male serum proteins. However, the antisera so absorbed, designated (-PMS) antisera, could still react strongly with several normal adult tissue extracts including kidney. Systematic and quantitative absorptions of the (-PMS) antisera were thus further carried out with individual butanol extracts of normal adult liver, lung, intestine, stomach, kidney, bone, pancreas, spleen, heart, cerebrum, cerebellum, breast, and packed red cells, as well as a composite extract containing equal amounts of each of the 13 adult tissue extracts. Of the six antigenic components in the placental extracts reacting with the (-PMS) antisera the only one which retained its reactivity with the antisera throughout exhaustive absorptions was associated with alkaline phosphatase activity. This immunologic and enzymologic identity was confirmed with homogeneous placental alkaline phosphatase. Extracts from each of three placentae injected into three pairs of rabbits all produced an identical antibody reaction with the unique determinant(s) of placental alkaline phosphatase. The same identity of precipitin reaction was also found with extracts of 14 other placentae against each of these antisera. It thus firmly establishes that placental alkaline phosphatase is a characteristic placenta-specific fetal protein.

Fertility control through active immunization using placenta proteins.

Numerous studies have demonstrated that antibodies to placental proteins in a variety of species are capable of preventing or disrupting gestation. Early work in this area was primarily directed towards the passive immunization of rodents with heterologous antisera to whole placental extracts. Toxicity and renal damage often accompanied fertility inhibition. More recent studies reported less toxicity and a higher specificity of antibodies to reproductive function when anti-placental antibodies were absorbed with serum and extracts of non-reproductive organs. Few studies have been reported in which active immunization with placental proteins was employed. The most detailed studies of active immunization have employed highly purified placental hormones. Immunizations of rats and rabbits with human placental lactogen have resulted in marked reduction in reproductive function. Immunization of human females with chemically altered (hapten-coupled/ HCG resulted in the production of antibodies reacting with unaltered HCG and pituitary LH. These antibodies were capable of reducing the level of endogenous serum LH in pre- and post-menopausal women. The also altered the events of the menstrual cycle in premenopausal women. More specific inhibition of chorionic gonadotrophin has been obtained by immunization of baboons with the beta subunit of HCG. Antifertility effects without alterations in the menstrual cycle of female baboons immunized with the beta subunit of HCG have been reported. The antibodies produced in these animals reacted significantly with human LH in vitro. The possibility of using hormonal and non-hormonal placental proteins as antigens for the specific immunological inhibition of fertility remains.

Soluble HLA antigens in normal human immunoglobulin preparations.

Nine lots of normal human immunoglobulin produced in Bulgaria and India, prepared from venous or placental blood, were assayed for the presence of HLA antigens by the inhibition of cytotoxicity of HLA alloantisera using two different absorption procedures. The results indicate that the normal human immunoglobulin preparations tested here contain soluble HLA antigens as demonstrated by the inhibition of HLA alloantisera. Most of the higher frequency antigens of the HLA-A, B, C and DR locus appear to be present in the Ig-preparations. In samples from India there appears to be a lack of HLA-A3 antigen, which corresponds to the low frequency of A3 in this population. Immunoglobulins produced from different raw materials contain different amounts of soluble HLA antigens.

Immunoactive products of placenta. IV. Impairment by placental cells and their products of CTL function at effector stage.

Trophoblast-enriched cell suspensions prepared by collagenase digestion from midterm murine placentae were found resistant to CTL-mediated lysis. Treatment of such cells by trypsin or neuraminidase rendered these cells susceptible to such lytic effectors. Collagenase-prepared cell suspensions could impair CTL action, whereas neuraminidase- or trypsin-treated cells did not retain this property. This effect was also observed with extracts. These results indicate that soluble factors (which we will characterize in another paper) released by trophoblast cells (in fact, spongiotrophoblast) can interfere in a dose-dependent fashion with the action of lytic effectors. We suggest that such active mechanisms are physiologic components of the placental barrier and might be defective in some cases of immunologic abortions.