Disordered nocturnal prolactin regulation in women with breast cancer.

Mean 24-hr prolactin concentrations were determined in 25 female control subjects, 16 women with benign breast masses, and 23 subjects with breast cancer. This evaluation performed before breast surgery revealed significantly decreased (p less than 0.02) nocturnal (12 a.m. to 7 a.m.) prolactin concentrations in 12 postmenopausal breast cancer subjects that contrasted with significantly increased (p less than 0.05) nocturnal prolactin levels in five luteal-phase premenopausal women with breast cancers. Prolactin concentrations in patients with benign breast disease were not significantly different from control subjects. Two of the premenopausal breast cancer patients had marked preoperative elevations in their mean 24-hr prolactin levels, and they were two of the three subjects who have since expired. Nocturnal prolactin secretion was significantly decreased (p less than 0.03) in four premenopausal breast cancer patients when they were studied 1 year after surgery; however, it remained the same in the eight postmenopausal breast cancer patients similarly evaluated. Although disordered prolactin regulation has been found in these women with breast cancer, its role in the etiology and progression of human cancer is still uncertain.

Twenty-four-hour preoperative endocrine profiles in women with benign and malignant breast disease.

Mean 24-hr growth hormone, luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone concentrations determined preoperatively in 16 women with benign breast masses and 17 patients with breast cancer were similar to those levels found in 25 age- and weight-matched control subjects. Mean 24-hr testosterone levels, however, were significantly elevated in women with breast cancer evaluated in the luteal phase of their cycles and were normal in postmenopausal breast cancer women. In addition, serum thyroid-stimulating hormone, thyroxine, cholesterol, and triglyceride levels were normal in these subjects. Plasma cortisols and urinary 17-hydroxysteroid excretion tended to be higher in both the benign and malignant breast disease group and probably reflected preoperative anxiety. Hence, we have found normal concentrations of a variety of endocrine and other biochemical agents that can stimulate breast tissue growth and/or have been previously reported to be disordered in women with breast cancer.

Prevention of mammary tumors with a chimeric HER-2 B-cell epitope peptide vaccine.

Synthetic peptide vaccines targeting B-cell epitopes of the extracellular domain of the HER-2 oncoprotein were evaluated for their capacity to elicit HER-2-specific antibodies with antiproliferative activity. Several HER-2 B-cell epitopes were identified by computer-aided analysis of protein antigenicity, and selected B-cell epitopes were synthesized colinearly with a promiscuous T-helper epitope (208-302) derived from the measles virus fusion protein at either the NH2 or COOH terminus linked via a four-residue turn sequence (GPSL). In addition, one epitope sequence, 628-647, was mutated to optimize disulfide pairing to mimic the native HER-2 receptor. All of the four selected epitopes elicited high-titered antibodies in outbred rabbits with exceptionally high titers for MVF-HER-2(628-647). These antibodies were cross-reactive with the native HER-2 receptor. Antibodies elicited by MVF HER-2(628-647) inhibited proliferation of human HER-2-overexpressing breast cancer cells in vitro and caused their antibody-dependent cell-mediated cytotoxicity. Furthermore, immunization with MVF-HER-2(628-647) prevented the spontaneous development of HER-2/neu-overexpressing mammary tumors in 83% of transgenic mice. The engineered, chimeric peptide B-cell immunogen MVF-HER-2(628-647) may have applications in the prevention of HER-2-overexpressing cancers.

Sexual transmission of simian T-lymphotropic virus type I: a model of human T-lymphotropic virus type I infection.

Simian T-lymphotropic virus type-I (STLV-I) seronegative females placed together with seropositive males for breeding purposes were followed from 1984-1990 to determined seroconversion rates by enzyme immunoassay and western immunoblot analysis. Two of 26 females and 1 of 4 males previously negative for antibodies to STLV-I seroconverted during the study period. Statistical analysis of sexual encounters indicated that the probability of a seronegative female testing positive for STLV-I after a sexual encounter with a seropositive male is less than 4%. These data indicate that even though sexual contact is important in the transmission of STLV-I, it may not be an efficient mode of viral infection. These data also suggest that female-to-male transmission of STLV-I occurs, as recently reported for human T-lymphotropic virus type-I (HTLV-I) infection. These results are important because HTLV-I and STLV-I share many features in common including routes of viral transmission. In addition, the difficulty of clearly quantitating the risk of sexual transmission in humans makes the primate animal model a valuable alternative to study the human infection.

Effects of a beta-human chorionic gonadotropin subunit immunogen administered in aqueous solution with a novel nonionic block copolymer adjuvant in patients with advanced cancer.

The clinical and immunological effects of a vaccine consisting of CTP37, a synthetic peptide corresponding to the COOH-terminal peptide (CTP) of beta-human chorionic gonadotropin (beta-hCG) conjugated to diphtheria toxoid, combined with CRL 1005, a novel synthetic nonionic block copolymer adjuvant, were examined. Twenty-one patients with metastatic, nontrophoblastic cancers received up to four immunizations by i.m. injection of a fixed dose of CTP37 and escalating doses of CRL 1005. Doses of CRL 1005 adjuvant as high as 75 mg were administered with 1 mg of CTP37 without evidence of significant local or systemic toxicity. Immunizations resulted in the production of IgG antibody to beta-hCG. CRL 1005 doses of 3-25 mg appeared to be optimal for antibody induction. Immunizations also resulted in increases in the cellular response of peripheral blood mononuclear cells (PBMCs) to the unconjugated CTP, hCG, and diphtheria toxoid. Responding PBMCs specifically secreted the TH1-associated cytokines IFN-gamma and interleukin (IL)-2 as well as the TH2-associated IL-5 and IL-10. Increased expression of IFN gamma and IL-5 mRNAs by PBMCs 4 h after immunization was also observed. CRL 1005 administered with CTP37 in aqueous solution is well tolerated. The CTP37-CRL 1005 subunit vaccine has the capacity to stimulate potentially beneficial humoral and cellular immune responses in patients with advanced cancer.

Levels of adrenal and gonadal hormones in rhesus monkeys during chronic hypokinesia.

The purpose of this study was to evaluate the effect of chronic immobilization on the hypophysial-adrenal and hypophysial-gonadal axes of adult male rhesus monkeys and the effect such manipulation has on the ability of these axes to respond to exogenous corticotropin, gonadotropin, and GnRH administration. A comparison was also made of the effects of immobilization on testosterone secretion at periods of low (April) and high (November) gonadal activity in this animal. Adult male rhesus monkeys were immobilized in a horizontal position for periods of up to 20 days during March/April. The function of the hypophysial-adrenal and hypophysial-gonadal axes was studied by monitoring plasma levels of cortisol, 17-hydroxylated precursors, 11 deoxycortisol, and testosterone during the period of restraint. Groups of immobilized and control animals also received iv injections of ACTH, FSH, and LH or LHRH on day 18 of the experiment. An additional group of animals was immobilized for 20 days, but did not receive exogenous hormone treatment. This group was used for comparison of seasonal differences in testosterone secretion with another group of animals subjected to the same treatment in November. During the first 3 h of immobilization, levels of cortisol, 17-hydroxylated precursors, and 11-deoxycortisol increased markedly from initial levels. Cortisol levels remained elevated for 3 days, whereas levels of the other three adrenal hormones declined to near-initial levels within 24 h. Testosterone levels declined steadily during the first 6 h of immobilization in males studied at a time of high testicular activity (November), while an increase during the first hour of restraint followed by a decline during the next 3 days were observed in males studied during a period of low testicular activity (April). Animals injected with ACTH on day 18 of immobilization had cortisol levels similar to those of control animals, but other groups of animals restrained for a similar period exhibited a lower level of plasma testosterone than controls after the injection of FSH and LH or LHRH. These data suggest that adaptation to stress results in a reduced demand for corticosteroid production and that the adrenals of chronically stressed animals are capable of responding to exogenous corticotropin, or alternatively, the immobilization imposed was stressful for only a limited time, and after a few days, animals no longer reacted as in response to stress. Also, secretion of testosterone in male monkeys is markedly influenced by the functional state of the gonads at the time of stress initiation.

The identification of peptide sequences of human chorionic gonadotropin containing a conformational epitope.

A series of overlapping peptides were synthesized representing the entire amino acid sequence of the beta-subunit of human chorionic gonadotropin (hCG) and these were reacted with a monoclonal antibody shown to be specific for hCG. One linear peptide (residues 40-52 of the sequence) reacted significantly with the monoclonal antibody but a conjugate of this peptide to diphtheria toxoid (DT) failed to elicit significant levels of antibodies reactive to hCG in rabbits. The subsequent preparation of an extended peptide (residues 38-57) in which the two cysteines were oxidized to form a loop peptide yielded a highly immunogenic antigen when conjugated to DT. Antibody levels reactive with hCG from loop peptide immunizations of rabbits exceeded those found after immunization with a 37 residue peptide representing the carboxyl terminus of the beta-hCG subunit. The antisera did not react with pituitary glycoprotein hormones with similar sequences.

Studies of the human chorionic gonadotrophin-like substance of human pituitary glands and its significance.

Radioimmunoassays utilizing antisera specific for the carboxyl-terminal portion of human chorionic gonadotrophin (hCG) beta-subunit were used to measure the concentration in human pituitary extracts of an immunoactive hCG factor (hCG') which was different from human LH (hLH). The content of hCG' from different human pituitary pools collected between 1966 and 1979 was relatively constant at 0.5-1.1 microgram per gland. The hCG' concentrations observed in acetone-dried powder of individual human pituitary glands (0.4-26 ng/mg) were close to those reported for full-term placental powder. After separation and partial purification of human pituitary glycoprotein hormones, pituitary hCG' was found mainly in the crude human FSH (hFSH) fraction. It was separated from hFSH by diethylaminoethyl-cellulose chromatography at pH 7 and by gel filtration on Sephadex G-100. On gel filtration its molecular size was larger than that of hLH or hFSH and it was strongly bound to Concanavalin A-Sepharose. The most active preparations of pituitary hCG' obtained by these procedures were approximately 5 per cent as potent by specific radioimmunoassay as hCG purified from pregnancy urine. Although the hCG' content in individual pituitary glands was more variable than the hLH content, on average pituitary hCG' was estimated to be around 25- to 50-fold less than the content of hLH, hFSH or human TSH in the human pituitary gland.

Purification and properties of the subunits of human pituitary follicle-stimulating hormone.

Highly purified human pituitary FSH was partially dissociated by treatment with 8M-urea, and alpha- and beta-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated alpha-subunit had less than 1%, and beta-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with thepurified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the beta-subunit.

Characterization of solubilized microvillous membrane proteins and glycoproteins from human placental syncytiotrophoblast.

Microvillous membrane fractions from human term placentae were prepared by differential centrifugation. Extration of membranes with PBS-EDTA or KCI removed soluble cytoplasmic components and serum proteins excepting trace amounts of albumin and transferrin. PAGE-SDS revealed 11 components in the Triton solubilized crude fraction after PBS-EDTA extraction. Membrane components solubilized with Triton were not fractionated by gel filtration on Bio-Gel A-50 m but DEAE-cellulose chromatography partially resolved these components. Three fractions were obtained by stepwise elution of absorbed materials using increasing concentrations of NaCl in the equilibrating buffer. These fractions were characterized using SDS-PAGE. The material unabsorbed to the DEAE contained two components of small molecular weight and one of them showed a positive PAS stain. The first eluted protein peak showed nine components, seven of which stained with PAS. The bulk of glycoproteins with molecular weights greater than 130 000 daltons were found in this fraction. The second eluted peak from DEAE was rich in components with molecular weights less than 42 000 daltons. Four components in this fraction were not identified in the other two ion-exchange fractions. Bands representing mobilities of albumin, transferrin and alkaline phosphatase were observed in DEAE-cellulose fractions; however, 12 components of unknown structure were revealed.

Differentiation and secretory activities of cultured human placental cytotrophoblast.

Ultrastructural, autoradiographic, immunofluorescent and biochemical techniques were used to characterize primary cultures of term placental cytotrophoblast in order to gain insight into the differentiation and secretory capacities of the cellular component of human trophoblast. Trypsin treatment of placental villi allowed isolation of a predominantly cytotrophoblast cell population that maintained viability up to 13 weeks in monolayer culture. Autoradiographic studies of tritiated thymidine incorporation identified a smaller diameter mononucleated cell population that was mitotically active and developed into larger diameter mononucleated cells and into multinucleated cells during culture. Ultrastructurally, cultured cells formed desmosomes, had an extensive network of cytoplasmic microfilaments and contained the organelles for hormone synthesis and secretion. These cells secreted steroid hormones, secreted Schwangerschafts protein I, actively incorporated tritiated glycoprotein precursors and expressed surface immunoreactivity for the beta-subunit of human chorionic gonadotrophin (hCG). However, medium concentrations of hCG and human placental lactogen dropped rapidly to undetectable levels after 14 days in primary culture. Cells grown beyond confluence differentiated into 1 to 2 mm structures with a villus-like histology. Our studies indicate that cytotrophoblast can secrete steroids, cytotrophoblast differentiation occurs in vitro in the absence of maternal tissues, hCG synthesis occurs in cultured cytotrophoblast and medium concentrations of placental protein hormones are not the best indicators of cell viability for cultures of cytotrophoblast.

Antibody reactivity against trophoblast and trophoblast products.

Sensitive immunoassays have been applied to WHO reference bank sera from fertile and infertile women in order to assess any naturally occurring antibody reactive with isolated human placental trophoblast membranes or two separate trophoblast protein products (hCG and SP1). A very low incidence of antibody reactive with solubilised trophoblast membrane was detected, and no significant antibody to either hCG or SP1 could be detected. Infertile states represented within this serum bank appear unlikely to involve adverse immune reactions to trophoblast.

Immunization of female baboons with hapten-coupled gonadotropins.

PIP: Female baboons (papio anubis) were immunized with gonadotropin preparations that had been structurally altered by hapten-coupling and the effects of such immunizations upon the events of the menstrual cycle were observed. Antibodies were detected against the immunizing antigen within 3-5 weeks. Cross-reaction of antibodies to sulphanil-azo-human luteinizing hormone (LH), human chorionic gonadotropin and ovine LH with unaltered baboon LH was marked only during the first 8 weeks, the period of immunoglobulin M (IgM) abundance, then dropped sharply as the antisera shifted to nearly all IgG. This drop did not occur with monkey and baboon immunizations. Ovulation was disrupted for 2-3 months with human and ovine gonadotropin immunizations but sulphanil-azo-monkey or baboon LH caused anovulation for 4-8 months. A reduction of hormone secretion correlated with titers of cross-reactivity. This evidence suggested that hapten-coupling produced changes in gonadotropins with enhance their antigenicity and the cross-reactivity of antibodies raised to them.^ieng

Birth control vaccines and immunological approaches to the therapy of noninfectious diseases.

Vaccination usually means the immunization of persons or animals against foreign infectious organisms for disease prevention. However, it has now been demonstrated that immunization against certain self substances to which tolerance normally exists can elicit beneficial effects to humans and other animals without inducing autoimmune disease. Clinical trials in women have been conducted with vaccines against reproductive antigens for the prevention of pregnancy and research is under way to develop more advanced formulations. Other self antigens have been described that might be used for developing methods of immunological therapy for such diseases as cancer, ulcers, and complications of diabetes. Emphasis is placed on the need for careful studies in appropriate animal models before any clinical application of such procedures is suggested.

PIP: By deliberately upsetting the body's recognition of self, and inducing an immune response against specific self antigens, birth control vaccines and disease therapies may be possible. The vaccine must be highly specific, the antigen must be present in small amounts or at limited times, and the benefit-to-risk ratio must be thoroughly evaluated. Target antigens being considered for birth control vaccines include sperm antigens or enzymes, zona pellucida of the ova and the pituitary hormone hCG. Anti-sperm immunity has been observed naturally occurring, and induced experimentally. Anti-ovum vaccines have the drawback of possibly reacting with ovarian tissue. The gonadotropin hormone receiving the most attention is hCG, or specifically its carboxy-terminal subunit. The rest of the peptide is too similar to hLH. Anti-hCG could act by preventing the luteotrophic effect of the hormone, or by attacking the hCG-producing cells of the blastocyst. Vaccines based on hCG with a tetanus toxoid carrier and a synthetic hCG peptide vaccine have been tested successfully in Phase 1 clinical trials. Other therapies based on immunization against self antigens include anticancer therapy, excess growth hormone and the Zollinger- Ellison syndrome of gastric ulcer disease. All of these approaches need to be monitored carefully to ensure that no damage is done to the host from circulating immune complexes.

Immunocontraceptives.

The advent of immunocontraceptives represents the first truly novel approach to the development of family planning methods in over 30 years. Such products would have many advantages over existing contraceptives in that they would not elicit metabolic disturbances, would provide long-acting (i.e. 6 to 12 months) protection from pregnancy, be easy to administer, be economical to manufacture and distribute, and could, depending on their composition, be used by either men or women. Several lines of research and development currently in progress are aimed at the development of safe and effective immunocontraceptives based on reproductive hormones, components of the gametes (sperm and ova) and products of the early pre-implantation conceptus. The only prototype immunocontraceptives to have reached the stage of clinical trials in women are those based on the hormone human chorionic gonadotropin, and in men that based on follicle-stimulating hormone. However, extensive research is also underway on immunocontraceptives based on sperm and ovum components for use by women, and on immunocontraceptives based on sperm components and gonadotropin-releasing hormone for use by men. Before such preparations can be made available for wide-scale use, further research is needed on ways to overcome genetically determined variations in individual immune responses so that protective responses of a predetermined duration can be elicited in all recipients. It is anticipated that these technical problems can be solved and the clinical testing of lead products will be completed in the next decade. Almost all of the financial support for the research and development of immunocontraceptives has been provided by academic institutions and public sector agencies. In general, the pharmaceutical industry has not been willing to engage in new contraceptive development, largely because of concerns about product liability claims, anticipated low profitability and/or the risk of negative publicity. Therefore, the further development, manufacture and distribution of immunocontraceptives will probably require the collaboration of public sector agencies, governments and industry in order to overcome the current paucity of effort being put into the development and provision of new, safe, effective and acceptable methods of family planning. The purpose of this review is to provide information on the current status of research and development of potential immunocontraceptives and to attempt to stimulate pharmaceutical companies to reassess their positions with regard to the development, manufacture and distribution of these products.

Suppression of luteal and placental function in pregnant baboons with agonist analogs of luteinizing hormone-releasing hormones.

Implantation of pelleted agonistic analogs of luteinizing hormone-releasing hormone (LH-RH) into six pregnant baboons, ranging between 14 and 21 days after mating in individual animals, resulted in two abortions: one at 39 and the other at 67 days after mating. Plasma progesterone levels were low in treated animals through the 23rd day after mating, in spite of rising levels of plasma chorionic gonadotropin. Plasma levels of progesterone then rose rapidly to normal in five of six animals. Plasma estradiol levels were low from treatment on in the late-aborting animal but were normal until 5 days before delivery in the early abortant. The profile of circulating chorionic gonadotropin was truncated in treated animals, which may be the first indication of a paradoxical effect of LH-RH agonist on the trophoblast.

Reduction of fertility in female baboons immunized with lactate dehydrogenase C4.

Immunization of female rabbits and mice with the sperm-specific isozyme of lactate dehydrogenase, LDH-C4, significantly reduced their fertility. Similar studies have been extended to nonhuman primates. Two female baboons, immunized with human LDH-C4, produced low antibody titers. These titers were markedly enhanced by booster injections of murine LDH-C4. An additional seven female baboons responded with relatively high antibody titers after receiving murine LDH-C4 as both priming and booster dosages. All nine females received injections of murine LDH-C4 at varying times determined by serum titer levels during fertility studies. These antisera reacted with human, mouse, and baboon LDH-C4. In a series of breeding experiments, 22 of 30 matings, or 73%, were infertile as compared with 28% in control matings. This contraceptive effect of the vaccine containing LDH-C4 was related to antibody titer and was reversible. Normal pregnancies ensued in animals in which the titer declined after termination of booster injections of vaccine.

Antifertility effects of immunization of female baboons with C-terminal peptides of the beta-subunit of human chorionic gonadotropin.

Fifteen adult female baboons were immunized with a conjugate of tetanus toxoid coupled with a synthetic carboxy-terminal peptide of the beta-subunit of human chorionic gonadotropin and another 15 were immunized with tetanus toxoid. Freund's complete adjuvant was used in all immunizations. All females were placed with males of proven fertility during the third menstrual cycle after the primary immunization and those not sustaining pregnancy were mated in two additional consecutive cycles. Four pregnancies occurred following 42 matings in the conjugate-immunized group and 15 pregnancies resulted from 21 matings in the tetanus toxoid-immunized animals. Low levels of antibodies reactive to baboon chorionic gonadotropin were found in the pregnant animals in the conjugate-immunized group.

Use of a synthetic peptide adjuvant for the immunization of baboons with denatured and deglycosylated pig zona pellucida glycoproteins.

Baboons were immunized using a synthetic peptide adjuvant with two purified pig zona pellucida glycoproteins. The major zona pellucida glycoprotein (ZPI) was purified by preparative isoelectric focusing, and the 80 K deglycosylated zona pellucida protein (ZPIII) was purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. The immunogenicity as well as the antigenicity of these proteins were evaluated by characterizing antibodies using the enzyme-linked immunoassay and by immunoblotting of zona pellucida proteins separated by high-resolution two-dimensional polyacrylamide gel electrophoresis. Both groups of animals developed antibodies that recognize the major zona pellucida glycoprotein, (ZPI) and immunoblotting procedures provide evidence that two of the major porcine zona pellucida glycoprotein families (ZPI and ZPIII) contain shared antigenic determinants. The animals immunized with ZPI showed decreased levels of estrogen throughout their menstrual cycles, and two of the animals ceased ovulation. All animals in the group immunized with ZPIII had a significant reduction in the numbers of antral follicles as compared with control animals. Although ovarian cyclicity was not altered significantly within a few months after immunization, two of the five animals in this group became amenorrheic by 8 months. Histologic analysis of ovarian tissue shows that follicles were absent or frequently abnormal in animals of both groups following long-term immunization. These studies demonstrate that the synthetic adjuvant is effective in inducing antibodies (to purified zona pellucida glycoproteins) that recognize antigenic determinants to either denatured or deglycosylated zona pellucida glycoproteins, and that some of these antibodies may interfere with normal ovarian function.

The effect of immunizing female rats with modified rat pituitary extract.

Immune reactions to a number of hormones have been induced, but information is lacking on the feasibility of inducing immune reactions to homologous gonadotropins. Female rats immunized with diazotized and tyrosylated rat pituitary extract containing gonadotropic activity and emulsified in complete Freund's adjuvant expressed various reproductive disturbances such as increased cycle length (7.0 days), increased number of sterile matings, reduced implantation in animals becoming pregnant, and reduced ability to carry fetuses to term. These effects were not exhibited by rats immunized similarly with diazotized or tyrosylated rat pituitary extract containing no detectable gonadotropic activity. Rats not becoming pregnant had a significantly higher antibody level to rat luteinizing hormone than did rats becoming pregnant. No significant elevation of rat follicle-stimulating hormone antibodies was noted. It is concluded that auto-immune reactions to gonadotropins can be induced and that very low levels of antibodies have a significant effect on reproduction.