Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Evaluation of antibody response to an adjuvanted hapten-protein vaccine as a potential inhibitor of sexual maturation for farmed Atlantic salmon.

An experimental contraceptive vaccine was evaluated in Atlantic salmon (Salmo salar). A peptide derived from the beta subunit of luteinizing hormone (LH) was conjugated to two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and formulated with one of four immunostimulants in a water-in-oil emulsion. Specific antibody responses to the peptide and each carrier protein were evaluated. While the antibody response to KLH was stronger than the response to BSA, both carrier proteins stimulated comparable antibody responses to the LH peptide. The immunostimulant proved to be more important for enhancing the LH peptide antibody response than the carrier protein selection; vaccines containing a combination of Aeromonas salmonicida and Vibrio anguillarum stimulated significantly greater LH peptide antibody production than any of the other three immunostimulants evaluated at 12 weeks post-vaccination. This study provides proof-of-concept for specific antibody production against a hapten-carrier protein antigen in Atlantic salmon and reinforces the importance of vaccine immunostimulant selection.

Characterization of tilapia (Oreochromis niloticus) gonadotropins by modeling and immunoneutralization.

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone-receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHß]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHß significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia.

Autoantibodies and gastrointestinal symptoms in infertile women in relation to in vitro fertilization.

BACKGROUND: Prior reports suggest a link between gonadotropin-releasing hormone (GnRH) and gastrointestinal function. The aim of the study was to prospectively investigate women subjected to in vitro fertilization (IVF) using the GnRH analog buserelin, taking into account gastrointestinal symptoms and antibody development against buserelin, GnRH, luteinizing hormone (LH), and their receptors. METHODS: Gastrointestinal symptoms were registered by the Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS) before and after IVF treatment, and five years later. Health-related quality of life was evaluated by the 36-item Short-Form questionnaire (SF-36). ELISA was used for antibody analyses before and after treatment. Data were compared with women from the general population. RESULTS: In total, 124 patients were investigated before and after IVF, and 62 were re-evaluated after five years. Buserelin treatment led to significant impairment of constipation (p = 0.004), nausea and vomiting (p = 0.035), psychological well-being (p = 0.000), and the intestinal symptoms' influence on daily life (p = 0.027). At 5-year follow-up, abdominal pain was worsened (p = 0.041), but psychological well-being was improved (p = 0.036), compared to prior treatment, and 15% had an observable deterioration in gastrointestinal symptoms. None developed severe dysmotility. Patients had higher prevalence of IgG antibodies against LH (p = 0.001) and its receptor (p = 0.016), and IgM antibodies against the GnRH receptor (p = 0.001) prior treatment compared with controls, but no antibody development was observed after IVF. CONCLUSION: Patients experience gastrointestinal symptoms during buserelin treatment, and abdominal pain is still increased after five years, but buserelin does not increase antibody formation against GnRH, LH or their receptors.

Quantitative multianalyte microarray immunoassay utilizing upconverting phosphor technology.

A quantitative multianalyte immunoassay utilizing luminescent upconverting single-crystal nanoparticles as reporters on an antibody array-in-well platform was demonstrated. Upconverting nanoparticles are inorganic rare earth doped materials that have the unique feature of converting low energy infrared radiation into higher energy visible light. Autofluorescence, commonly limiting the sensitivity of fluorescence-based assays, can be completely eliminated with photon upconversion technology because the phenomenon does not occur in biological materials. Biotinylated antibodies for three analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone) were printed in an array format onto the bottom of streptavidin-coated microtiter wells. Analyte dilutions were added to the wells, and the analytes were detected with antibody-coated upconverting nanoparticles. Binding of the upconverting nanoparticles was imaged with an anti-Stokes photoluminescence microwell imager, and the standard curves for each analyte were quantified from the selected spot areas of the images. Single analyte and reference assays were also carried out to compare with the results of the multianalyte assay. Multiplexing did not have an effect on the assay performance. This study demonstrates the feasibility of upconverting single-crystal nanoparticles for imaging-based detection of quantitative multianalyte assays.

Ontogeny of immunoreactive Lh and Fsh cells in relation to early ovarian differentiation and development in protogynous hermaphroditic ricefield eel Monopterus albus.

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control many aspects of gonadal development and function in teleosts. In the present paper, the specific antisera against ricefield eel Lhb (Lh beta subunit), Fshb (Fsh beta subunit), and Cga (the common pituitary glycoprotein hormone alpha subunit) were generated, and the cellular localization, initial appearance, and subsequent development of gonadotrophs in relation to early ovarian differentiation and development in the ricefield eel, a protogynous sex-changing teleost, were examined with immunochemistry. Lhb- and Fshb-immunoreactive signals were identified in distinct pituitary cells that occupied primarily the peripheral regions of the adenohypophysis. During ontogeny, Lhb-immunoreactive signals were first detected in the pituitary around 40 days after hatching (dah) when the oogonia transitioned into early primary growth oocytes, and the intensity of immunoreactivity increased concomitantly with the growth of primary oocytes from 60 to 140 dah. During overwintering from 170 to 230 dah, Lhb-immunoreactive signals were significantly decreased when a large proportion of perinucleolus oocytes contained intense Balbiani bodies. In contrast, Fshb-immunoreactive signals were not detectable in the pituitary until around 230 dah (in the spring after hatching) and slightly increased from 285 dah when the late perinucleolus oocytes began to enter the secondary growth phase. Both Lhb- and Fshb-immunoreactive cells were increased when the early cortical alveoli oocytes emerged at 300 dah. The mRNA expression of lhb and fshb coincided with their immunoreactive signals. Taken together, these results suggest that only Lh is involved in primary oocyte growth in ricefield eels, but both Fsh and Lh are important for the secondary ooctye growth.

Epitope-based in silico peptide design yields peptide-directed antibodies that recognize the buffalo luteinizing hormone.

We present a novel peptide sequence identified through in silico epitope design and the later generation of peptide-directed antibodies recognizing the buffalo luteinizing hormone. Peptides and antibodies, specific to reproductive hormones, are valuable tools for developing point-of-care immunodiagnostic tools. The study predicted an epitope peptide in silico from buffalo luteinizing hormone and the generation of polyclonal antibodies against this peptide sequence. In this quest, we identified a novel epitope peptide sequence (luteinizing hormone peptide, LHP) through bioinformatics tools. The peptide was further synthesized and characterized. The polyclonal antibodies (anti-LHP) were raised against the peptide in the rabbit. Thereafter, we explored a strategy for detecting buffalo luteinizing hormone (LH) using the anti-peptide antibodies developed. The affinity of the peptide, bovine lutropin beta, and crude LH (prepared from buffalo pituitary) towards the raised antibodies was established by dot blot and ELISA. Specific recognition of the luteinizing hormone by the raised polyclonal antibodies highlights the ability of the identified peptide (LHP) and developed polyclonal antibodies (anti-LHP) as suitable diagnostic reagents for sensing the buffalo luteinizing hormone. Through this work, we analyzed and translated the "-omics" information in the LH gene sequence for the development of a novel peptide and antibodies as valuable immuno-reagents.

Induction of pituitary lactotrope differentiation by luteinizing hormone alpha subunit.

Addition of gonadotropin releasing hormone to cultures of fetal rat pituitary induced differentiation of lactotropes as revealed by immunocytochemistry. Antiserum to luteinizing hormone (LH) (recognizing native LH), but not antiserum to LH-beta (recognizing both native LH and its beta subunit), inhibited this induction. Further addition of highly purified LH-alpha subunit in culture medium also induced lactotrope differentiation. Thus, the alpha subunit may have a specific biological activity of its own with probable practical use in clinical investigations.

Effect of bacterial lipopolysaccharide on the reproductive axis of prepubertal and peripubertal female rats. Ontogenic changes in the immune-neuroendocrine interactions.

The immune, endocrine and nervous systems are closely interrelated, which allows the organism to respond to different types of stress such as infection. Chronic infectious and inflammatory conditions are often accompanied by an impaired reproductive function. Leptin, a hormone produced by adipose tissue, exerts a regulatory function on the reproductive axis. It has homology with other proinflammatory cytokines and could be modified by lipopolysaccharide (LPS). Therefore, these studies were designed to investigate the effect of LPS administration on the neuroendocrine mechanisms involved in the regulation of the reproductive axis during sexual maturation. Fifteen- and 30-day-old female rats were injected with a single dose of LPS 250 microg/kg (i.p.) and then nitric oxide synthase (NOS) activity, hypothalamic excitatory/inhibitory amino acids and Gn-RH content, serum LH and leptin concentration were studied. In 15-day-old female rats LPS treatment did not modify hypothalamic inducible (iNOS) and constitutive (cNOS) NOS activity, Gn-RH, glutamate (GLU) and GABA content. Also serum LH and leptin levels were not modified. In 30-day-old rats LPS increased iNOS and cNOS activity (p < 0.001) and hypothalamic Gn-RH content (p < 0.001). At this age hypothalamic GABA content was significantly decreased (p < 0.001) without changes in GLU content, and serum LH (p < 0.001) and leptin (p < 0.0001) decreased significantly. In summary, current studies have demonstrated that LPS administration to 15- and 30-day-old female rats results in a different response of the hypothalamus-pituitary-gonadal axis and of the adipose tissue, demonstrating an ontogenic response of the immune-neuroendocrine system to LPS administration.

Production of effective luteinizing hormone antisera by two immunization methods.

This paper addresses the production of effective luteinizing hormone antisera by two different immunization methods; the traditional and modified methods. The main difference between these two methods is in the immunization procedure. In the modified method, an additional injection of emulsion with complete Freund's adjuvant and only one booster are applied at the third and 28th day from the first injection, respectively. The results of the study indicated the possibility of producing the antiseria using the modified method in short time and better quality than those produced by the traditional methods. The details of both methods are presented together with the results obtained from the antibodies detection. Comparison between these two methods is also presented along with discussions.

Immunocytochemical localization and ultrastructural study of gonadotroph cells in the female desert lizard Uromastyx acanthinura.

The pars distalis from the pituitary gland of adult female desert lizards (Uromastyx acanthinura), captured during vitellogenesis (late may) and hivernal period, was studied with immunocytochemical methods using specific antisera against human FSH (hFSH) and LH (hLH). The immunostaining with anti-hLH and anti-hFSH allowed the identification of only FSH-like containing cells. The FSH-like immunoreactive cells were affected differently by a physiological stage and showed some heterogenous cytological characteristics. During vitellogenesis, four aspects of rostral FSH-like immunoreactive cells could be recognized. The expression of FSH-like in mainly immunoreactive cells was parallel to an intense synthetic activity and to the presence of ultrastructural features indicating an intense release of the hormone. This release was considerably altered in winter, the immunoreactive cells stored an important amount of secretion granules which increased in size and undergo a crinophagic process.

Assessment of the immunogenicity of gonadotrophins during controlled ovarian stimulation.

PROBLEM: Gonadotrophin hormones are used for the controlled ovarian stimulation (COS) as part of the in vitro fertilization techniques. Therapeutic proteins have the potential to induce an unwanted immune response. METHOD OF STUDY: The presence of anti-FSH, anti-LH and anti-hCG antibodies were determined in patients from two different clinical trials after the repeated administration of hMG or FSH. RESULTS: In the first study, 27 subjects were screening for the presence of anti-FSH antibodies. From the 27 patients, only one patient showed the presence of low levels of antibodies. In a second study, 25 patients were screened for the presence of anti-FSH, anti-LH and anti-hCG antibodies. At the end of the study, no patients showed the presence of antibodies. CONCLUSION: The results of this study suggest that repeated treatment cycles with FSH or hMG in patients undergoing COS for in vitro fertilization can be safely and effectively applied without concerns for immunogenicity.

Expression of apoptotic genes in testicular germ cells and their modulation by luteinizing hormone.

Gonadotropins regulate spermatogenesis by promoting survival and differentiation of germ cells. The molecular markers that are modulated by these hormones to ensure survival however have not been described in great detail. Immunoneutralization of LH in particular leads to apoptotic cell death of the spermatocytes and the round spermatids. In the present study, the expression pattern and regulation of apoptotic markers after specific immunoneutralization of LH in germ cells purified from rats has been investigated at the RNA and protein level. Of the several markers tested, Bax, caspases 1 and 2 and Fadd exhibit differential expression, with the round spermatids expressing higher levels of caspases 1 and 2, and the spermatocytes expressing higher levels of Bax and Fadd. The two cell types therefore exhibit differential expression of apoptotic markers. The cell types also differ with respect to their response to LH antiserum treatment. Fas and Bax both are up-regulated in the round spermatids after 24h of antiserum treatment. In the spermatocytes, Fas was up-regulated as early as 12h after antiserum treatment while Bax was up-regulated after 2 days. These results demonstrate that LH regulates survival of germ cells by modulating the levels of pro and anti-apoptotic proteins.

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.

[Progress in the study of immunocontraceptive antigens].

Fertility management is a global issue of medical, economic, and social consequence. Although many methods have been devised to inhibit reproduction, more acceptable alternatives are still needed. Regulation by immune intervention is a promising technology as applied to human beings. The objective of this review is to indicate several immunocontraceptive antigens.

Dual Positive Regulation of Embryo Implantation by Endocrine and Immune Systems--Step-by-Step Maternal Recognition of the Developing Embryo.

In humans, HCG secreted from the implanting embryo stimulates progesterone production of the corpus luteum to maintain embryo implantation. Along with this endocrine system, current evidence suggests that the maternal immune system positively contributes to the embryo implantation. In mice, immune cells that have been sensitized with seminal fluid and then the developing embryo induce endometrial differentiation and promote embryo implantation. After hatching, HCG activates regulatory T and B cells through LH/HCG receptors and then stimulates uterine NK cells and monocytes through sugar chain receptors, to promote and maintain pregnancy. In accordance with the above, the intrauterine administration of HCG-treated PBMC was demonstrated to improve implantation rates in women with repeated implantation failures. These findings suggest that the maternal immune system undergoes functional changes by recognizing the developing embryos in a stepwise manner even from a pre-fertilization stage and facilitates embryo implantation in cooperation with the endocrine system.

Human pituitary luteinizing hormone. Isolation and characterization of four glycoproteins with luteinizing activity.

Two major and two minor components of human luteinizing hormone (lutropin) were isolated from whole frozen pituitaries by a procedure involving extraction of homogenized pituitaries, (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, Sephadex G-100, and SE-Sephadex C-50 and electrophoresis in polyacrylamide gel. The isolation procedure was monitored by both bioassays and radioimmunoassays. Contamination of the final products by other pituitary hormone activities was very low. The four lutropin components were all homogeneous by polyacrylamide gel electrophoresis (a sieving medium) and by free zone electrophoresis (a non-sieving medium). No heterogeneity was observed when the components were studied in the ultracentrifuge by sedimentation-equilibrium technique. The molecular weights of the components were in the range of 34 000-40 000. Sedimentation velocity experiments with the two major components revealed in each case one boundary with S20,W values of 3.2 S and 3.5 S. Further evidence for the homogeneity of the components was the observation of only one precipitin line for each component upon immunodiffusion against a rabbit anti-human lutropin serum. Amino acid and carbohydrate analyses indicated close similarity among the four components. From the analysis data the molecular weights of the components were calculated to be 31 000-33 000.

Identification of a heterodimer-specific epitope present in human chorionic gonadotrophin (hCG) using a monoclonal antibody that can distinguish between hCG and human LH.

Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the beta subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the beta subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its beta subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCG beta that is absent in hLH beta. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.

Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction.

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased 'steady-state' GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44(MAPK) and P38 (MAPK). These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.