Immunomodulatory activity of glycodelin: implications in allograft rejection.

Glycodelin is an immunomodulator, indispensable for the maintenance of pregnancy in humans. The glycoprotein induces apoptosis in activated CD4+ T cells, monocytes and natural killer (NK) cells, and suppresses the activity of cytotoxic T cells, macrophages and dendritic cells. This study explores the immunosuppressive property of glycodelin for its possible use in preventing graft rejection. Because glycodelin is found only in certain primates, the hypothesis was investigated in an allograft nude mouse model. It is demonstrated that treatment of alloactivated mononuclear cells with glycodelin thwarts graft rejection. Glycodelin decreases the number of activated CD4+ and CD8+ cells and down-regulates the expression of key proteins known to be involved in graft demise such as granzyme-B, eomesodermin (EOMES), interleukin (IL)-2 and proinflammatory cytokines [tumour necrosis factor (TNF)-α and IL-6], resulting in a weakened cell-mediated immune response. Immunosuppressive drugs for treating allograft rejection are associated with severe side effects. Glycodelin, a natural immunomodulator in humans, would be an ideal alternative candidate.

Epitope mapping of Brugia malayi ALT-2 and the development of a multi-epitope vaccine for lymphatic filariasis.

Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.

An evaluation of antigen capture assays for detecting active filarial antigens.

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n= 110), microfilaraemic (MF, n= 65), chronic pathology (CP, n= 45) and non-endemic normal (NEN, n= 10) individuals. Of the 230 samples tested, VAH capture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters.

The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form.

A common epitope of beta-lactoglobulin and serum retinol-binding proteins: elucidation of its core sequence using synthetic peptides.

One of the monoclonal antibodies raised against bovine beta-lactoglobulin reacted with human serum retinol binding protein. The finding that this monoclonal antibody also reacted with the serum retinol binding proteins isolated from other animals, suggested that this epitopic conformation is conserved among these proteins. Using ELISA and various synthetic peptides of defined sequence, we show in this paper that the epitope defined by this monoclonal antibody comprises of the highly conserved core sequence of DTDY present in beta-lactoglobulin and retinol binding proteins.

A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice.

In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus.

Antigenic determinants of human serum retinol binding protein as probed with monoclonal antibodies.

Monoclonal antibodies raised against human serum retinol-binding protein (hRBP) were used as probes for the study of the antigenic determinants of hRBP and those shared with the same protein from other species. The antibodies could be classified into four distinct groups and react with the homologous proteins from the rat as well as the rabbit sera. Three of these antibodies recognize sequential or continuous epitopes while the remaining antibody is directed against a discontinuous or conformational epitope. By chemical cleavage with cyanogen bromide, the domains recognized by the monoclonal antibodies could be delineated. By solid-phase synthetic approach, the core sequences recognized by two of these monoclonal antibodies were identified to amino acid sequences 45-51 and 128-131 of the primary amino acid sequence of hRBP.

Immunological characterization of riboflavin carrier proteins using monoclonal antibodies.

Monoclonal antibodies to chicken riboflavin carrier protein have been produced by fusing immunized mouse spleen cells with myeloma SP2/O-Ag 14. The three different monoclonal antibodies specifically bound 125I-labelled chicken riboflavin carrier protein and were characterized with respect to their affinities to bind the antigen, subclass and isotype. These three monoclonal antibodies had similar affinities for holo-, apo- and SDS-denatured riboflavin carrier protein but were unable to recognize the reduced and carboxymethylated protein indicating that they were directed to specific conformational epitopes on the native avian protein. Succinylation of the vitamin carrier protein while still retaining flavin binding characteristics totally abolished the cross-reactivity with all the three monoclonal antibodies indicating that lysine residues were involved at the antigenic sites of the protein. This shows that the antigenic loci may be distinct from the flavin binding sites in the protein. All three antibodies were able to recognize riboflavin carrier protein present in the sera of pregnant rats, monkeys and humans indicating that the epitopes to which they are directed are conserved throughout evolution. These antibodies can therefore be effectively used for radioimmunoassays and further studies on the functional aspects of this protein in higher mammals.

A rapid method of epitope analysis using Superose 12 gel filtration. A study with monoclonal antibodies to chicken riboflavin carrier protein.

A novel and rapid method is described for determining the epitope specificities of monoclonal antibodies. The method utilises a highly reproducible, wide range gel filtration medium, Superose 12. nmol of monoclonal antibodies are incubated with pmol quantities of radiolabelled antigen, and the mixture filtered through Superose 12. Fractions are collected and radioactivity monitored. The size of the resultant immune complex indicates whether two monoclonal antibodies (mixed with the antigen) recognise the same or different epitope(s) of the antigen. The applicability of the above analytical method is illustrated with monoclonal antibodies raised against chicken egg riboflavin carrier protein.

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus.

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I-III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.

Establishment of immunological probes to study human gonadotropin-releasing hormone receptors.

Monoclonal antibodies were raised to a synthetic peptide corresponding to amino acids 1-29 of the human gonadotropin-releasing hormone (GnRH) receptor. One of the two antibodies was found to recognise GnRH receptors on human pituitary gonadotrophs as determined by immunohistochemistry and supported by Western blotting. The antibody also bound to T47D human breast carcinoma cell line as determined by flow cytometric analysis.

Differential influence of recombinant non-glycosylated and glycosylated glycodelin on human sperm function: comparative studies with hamster spermatozoa.

Glycodelin, also known as placental protein 14, has been implicated in endometriosis-related infertility. To determine the role of glycodelin and its glycosylated state, the influence of recombinant nonglycosylated-glycodelin (nongly-glycodelin) and glycosylated-glycodelin (gly-glycodelin) on human sperm function was evaluated. Whereas there was a significant (P<0.001) increase in the capacitation of nongly-glycodelin-treated spermatozoa compared with untreated controls (28.8 +/- 1.0% v. 21 +/- 1.5% respectively), treatment of spermatozoa with gly-glycodelin markedly (P<0.001) inhibited capacitation (10.7 +/- 0.3%); acrosome reaction (AR) remained unaltered in all treatments. In a zona-free hamster egg penetration assay, the egg penetration index was higher (P<0.001) with nongly-glycodelin-treated spermatozoa (3.4 +/- 0.3) than with gly-glycodelin-treated spermatozoa (0.4 +/- 0.1) and untreated spermatozoa (1.6 +/- 0.2). A similar influence of glycodelin on capacitation was observed with hamster spermatozoa. However, the AR rate was higher (P<0.01) in nongly-glycodelin-treated spermatozoa (39.4 +/- 1.6%) than in either gly-glycodelin-treated spermatozoa (19.3 +/- 2.0%) or untreated controls (30.0 +/- 1.2%). Moreover, the in vitro fertilization rate was significantly (P<0.01) higher with nongly-glycodelin treated-spermatozoa compared with untreated spermatozoa (77.5 +/- 2.3% v. 52.9 +/- 4.3%) and gly-glycodelin-treated spermatozoa (38.3 +/- 6.5%; P<0.05). These results indicate that whereas nongly-glycodelin improves, gly-glycodelin inhibits, capacitation and fertilization potential of human and hamster spermatozoa, and that the glycosylation status of glycodelin determines its influence on sperm function.

Subpopulations of human T lymphocytes in patients with Hodgkin's disease before and after treatment.

Circulating T gamma and T mu cells enumerated in the peripheral blood of 51 untreated Hodgkin's disease (HD) patients and 66 treated HD patients tested after few months to more than 3 years of remission, brought about by radiation and/or chemotherapy. 53 age matched normal healthy individuals were studied as controls. Untreated HD patients showed significant increase in T gamma cells (p less than 0.001) and decrease in T mu cells (p less than 0.001) when compared to normal donors. The abnormal percentages of T cell subsets did not correlate with the severity of the disease. A progressive partial restoration in the proportion of these two subsets of T lymphocytes was seen in the disease free condition. However, even after 3 years of remission the recovery was not equivalent to controls. There was no correlation between the recovery of T gamma and T mu cells with the modality of treatment.

In vitro induction of chronic myeloid leukemia associated immune reactivity in normal human lymphocytes by xenogeneic immune RNA.

Peripheral blood lymphocytes from normal human donors were treated with immune RNA (IRNA) prepared from lymphoid tissues of guinea pigs immunized with chronic myeloid leukemia (CML) cells, normal leukocytes (WBCs) and normal bone marrow (BM) cells, in order to study whether IRNA can confer specific immunoreactivity on normal lymphocytes. The IRNA treated lymphocytes were further exposed to solubilized membrane antigens from CML cells, normal WBCs and BM cells in a criss-cross fashion. The migration inhibition factor produced by these lymphocytes was tested in an in vitro leukocyte migration inhibition assay using normal leukocytes. The results indicated that IRNA prepared from guinea pigs immunized with all the three cell types could transfer the immune reactivity to normal cells in response to the respective antigens. The WBC IRNA incubated lymphocytes did not react with BM cell and CML cell antigens. A potent transfer of specific reactivity was shown by CML IRNA. IRNA produced against BM cells and CML cells demonstrated considerable cross reactivity perhaps due to shared immature cell antigens.

Immunohistochemical localization of riboflavin carrier protein in testicular cells of mammals.

Using specific polyclonal antibodies against chicken riboflavin carrier protein (cRCP), immunocytochemical localization of riboflavin carrier protein was carried out in testicular sections and isolated cells of mammals. A positive reaction was observed in the developing germ cells of rat testis, especially in meiotic and post-meiotic germ cells such as pachytene spermatocytes, round spermatids and spermatozoa. In addition both the somatic cells of the testis, viz. Leydig and Sertoli cells with vital function in germ cell proliferation and differentiation, displayed a moderate to strong staining reaction. This was further confirmed using in utero X-irradiated rat testis devoid of germ cells. Different types of cells isolated from testis when subjected to immunostaining showed similar patterns of reaction as in the intact tissue. Mature spermatozoa from different mammals (rat, bull and monkey) exhibited strong staining reaction in their head regions localized mainly in acrosomal caps. It is suggested that the testicular riboflavin carrier protein has a role in cell to cell communication and may be crucial during development of germ cells especially at the meiotic and post-meiotic stages.

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.

Establishment of the functional importance of thiamin carrier protein in pregnant rats by using monoclonal antibodies.

Monoclonal antibodies (mAbs) to chicken thiamin carrier protein (TCP) have been produced by hybridoma technology to identify the crucial epitopes involved in bioneutralization of the vitamin carrier. The monoclonality of these mAbs (A4C4, F3H6, H8H3, C8C1 and G7H10) was sought to be confirmed by sub-class isotyping; they all belong to IgG1, k type. The epitopes recognized by all the five mAbs are conserved in TCP from the chicken to the rat as assessed by liquid phase RIA and immunoprecipitation of 125I-labelled proteins from pregnant rat serum. Among these mAbs. passive immunization of pregnant rats with the mAb C8C1 only on three consecutive days (day 10, 11 and 12) resulted in embryonic resorption. These results demonstrate the importance of epitopic structure specified by the mAb C8C1 on TCP during pregnancy in rats.

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus.

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone.

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of (a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadotropin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.