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Results for “antigen expression and secretion”

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PubMed1989

[Expression and secretion of carcinoembryonic antigen in tumors of the large intestine].

The correlation between the expression of the carcinoembryonic antigen (CEA) on cell membranes and the intensity of its secretion into the circulation is studied in patients with malignant tumours of the rectum and the transverse colon. With the use of the monoclonal antibodies against CEA and the immunoperoxidase technique on the frozen sections, the localization of the primary tumour, microinvasion and micrometastases in the lymph nodes are shown. The dependence of the serum CEA concentration upon the histologic type of the tumour and the stage of the disease is not established. The trend towards the inverse correlation between the CEA level in the tumour tissue extract and the degree of the tumour cell differentiation is noted. The opinion is expressed of the applicability of the monoclonal antibody anti-CEA-1 for the morphologic differential diagnosis of the colon tumours, the evaluation of the thoroughness of the surgical treatment and for the metastases radioimmunolocalization.

Antibodies, Monoclonal↗

PubMed1987

Expression of HLA class II antigens and secretion of interleukin-1 by monocytes and macrophages from adults and neonates.

HLA class II antigen expression and IL-1 production by mononuclear phagocytes are important for antigen-stimulated T-cell activation. We examined these surface antigens and a monocyte marker antigen on fresh cord and adult blood monocytes, macrophages (M phi) derived from monocytes in vitro, human placental (fetal) M phi, from adult women. By FACS analysis, we found less DR on cord blood monocytes (80 +/- 7) than on adult monocytes (96 +/- 1) with greater heterogeneity in density of DR due to a weakly staining subpopulation of cord monocytes. There were markedly fewer DR and DQ positive placental M phi, 62 +/- 11% and 19 +/- 3%, compared to adult peritoneal M phi, 91 +/- 11% and 87 +/- 13%. DQ was more intense on peritoneal M phi than on any other cell type. Fresh cord monocytes secreted equal or greater amounts of interleukin-1 (IL-1) in response to lipopolysaccharide (LPS) or Group B streptococci than adult monocytes, although results with individual preparations varied. By Northern blot analysis, LPS-stimulated cord blood and adult monocytes contained similar amounts of IL-1 alpha, IL-1 beta and DR alpha mRNA. Each placental M phi preparation secreted IL-1 (200 +/- 85 U/ml to LPS). Peritoneal M phi preparations from women in the pre-luteal phase did not release detectable IL-1, whereas those from women in the post-luteal phase released as much as monocytes. Cultured monocytes failed to secrete IL-1 and expressed less DQ than fresh monocytes. Exposure to IFN gamma augmented IL-1 release by adult and cord cells and DQ expression on cord cells. These data indicate that class II antigen expression and IL-1 secretion by mononuclear phagocytes are only in part co-ordinately modulated. The differences between placental (fetal) M phi and adult peritoneal M phi may reflect both tissue-specific differences and generally diminished class II antigen expression on fetal and neonatal mononuclear phagocytes.

Adult↗

PubMed1994

Protective immunity elicited by rBCG vaccines.

The live attenuated Mycobacterium bovis strain Bacille Calmette-Guerin (BCG) is still widely used as a vaccine against tuberculosis and therefore offers potential advantages as a safe, live vaccine vehicle for the expression and delivery of protective recombinant antigens. As an attenuated intracellular bacterium residing in macrophages, BCG would seem to be particularly suited for eliciting cellular responses and not humoral responses. Efforts to improve the potential for recombinant BCG (rBCG) vaccines to elicit protective humoral responses were undertaken by developing vectors systems which export or secrete foreign antigens. Expression of the OspA antigen of Borrelia burgdorferi, as a chimaeric membrane-associated lipoprotein, resulted in high-titred protective humoral responses when compared to cytoplasmic or secreted expression of OspA. Expression of the PspA antigen of Streptococcus pneumoniae as a secreted or membrane-associated lipoprotein by rBCG did not result in higher-titred humoral responses in comparison to PspA expressed cytoplasmically but apparently improved the quality of protective PspA specific antibodies. Based on these pre-clinical studies, rBCG vaccines for Lyme and pneumococcal diseases are being developed for clinical trials in humans. The potential for eliciting mucosal responses to antigens delivered by rBCG was also investigated. Nasal immunization was superior at eliciting substantial lasting mucosal responses at multiple mucosal sites and also resulted in systemic responses comparable to those obtained with parenteral immunization.

Animals↗

PubMed1984

Antigenic expression of human melanoma cells in serum-free medium.

A human melanoma cell line, M14 , adapted to grow in serum free synthetic media was examined for its expression and secretion of several serologically defined melanoma associated antigens (MAA) previously described in this laboratory. Melanoma associated antigen expression and secretion was identical to that of M14 cells grown in parallel in serum supplemented medium. Spent synthetic media was found to be an enriched serum free source for the initial isolation of 100 kilodalton secreted glycoprotein MAA. M14 melanoma cells grown in synthetic media were also shown to be adaptable to the double agar clonogenic assay facilitating the examination of clonal heterogeneity in functional studies of MAA in melanoma tumor biology. Recent investigations from this laboratory have focused on characterizing human melanoma associated antigens (MAA) found either as secreted or cell surface associated glycoproteins in human melanoma cell lines. In these studies, monoclonal and polyclonal antiserums to melanoma cell components have been developed to specifically identify these MAAs immunochemically and provide a means to study the structural biochemistry of these determinants. At this time we have identified two antigens on which our research efforts are targeted: 1) a 100,000 dalton secreted glycoprotein (100K) common to melanoma, sarcoma and neuroblastoma tumor cell lines, and 2) a 250,000 dalton-high molecular weight component glycoprotein-proteoglycan complex which is thus far restricted to melanoma cells. The ultimate goal of our efforts is two-fold. Initially, we hope to develop schemes to isolate these melanoma associated antigens in sufficient quantities to obtain detailed structural information on these molecules, and secondly, we wish to implicate these glycoproteins in functional aspects of the biology of metastatic human melanoma in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Neoplasm↗

PubMed1999

Enhancement of antibody responses by DNA immunization using expression vectors mediating efficient antigen secretion.

The immune responses elicited in mice, after intradermal (i.d.) immunization with plasmids encoding secreted or intracellular forms of HIV-1 nef, HIV-1 tat or C. pneumoniae omp2 proteins, respectively, were compared. To mediate secretion of these proteins the genes were fused to a heterologous signal sequence from murine heavy chain IgG. The nef- and omp2-specific antibody responses were dramatically increased when mice were inoculated with the plasmid encoding the secreted form of these proteins. In contrast, HIV-1 tat comprising an internal strong nuclear targeting sequence could not be induced to secretion and subsequently no enhanced antibody response was observed. Slight improvement of the HIV-1 nef antibody response was achieved after co-inoculation with a granulocyte-macrophage colony-stimulating factor (GM-CSF) expression vector. Further, nef-specific T-cell responses were induced after nef DNA injections, and were of Th1-like phenotype regardless of whether the nef protein was secreted or not. The system described in this study, using a plasmid vector with a strong heterologous signal sequence that mediate efficient antigen secretion in vivo, may have wide applicability for the induction of high antibody levels to normally non-secreted antigens.

Animals↗

PubMed1990

Expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system.

The expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system was investigated. The human TK- 143 cells were infected with the recombinant vaccinia viruses vTMS-1 or vTLS-1. Cells infected with vTMS-1, which contains the preS2 + S gene, produced preS2 containing middle HBsAg proteins. Similarly, cells produced preS1 containing large HBsAg proteins upon infection with vTLS-1, which carries the preS1 + preS2 + S gene. The expression products could be secreted and form 22 nm particles. They reacted specifically with anti-preS1 and/or anti-preS2 monoclonal antibodies, and exhibited pHSA-receptor (for polymerized human serum albumin) activity. In addition, the major S components of hepatitis B surface antigen were also present in the products expressed by vTMS-1 and vTLS-1.

Cells, Cultured↗

PubMed1986

Expression of a secreted transplantation antigen gene during murine embryogenesis.

We examined the midgestation mouse embryo for transcripts related to the secreted transplantation antigen Q10 and show here that this gene is transcribed in the endoderm of the visceral yolk sac. Its level of expression is highest at day 14 and then declines as development proceeds. Concurrently with the decrease in yolk sac expression, the amount of transcripts accumulating in the fetal liver increases during late embryogenesis.

Animals↗

PubMed2005

Leishmania donovani: effect of therapy on expression of CD2 antigen and secretion of macrophage migration inhibition factor by T-cells in patients with visceral leishmaniasis.

Visceral leishmaniasis (VL) commonly known as Kala-azar in India is one of the several clinically important infections, where Th1 sub-population of CD4+ T-cells, despite a pre-requisite, fails to express macrophage migration inhibition factor (MIF) and interferon-gamma which both activate the macrophage and coordinate the immune response to intra-cellular Leishmania sp. Expression of CD2 receptors before and after antileishmanial therapy on CD4+ T-cells of VL patients and their corresponding effect on MIF were examined. Before treatment the number of T-cells expressing CD2 was low which incorporated insignificant MIF response. The immunological reconstitution was, however, observed after treatment as manifested through upregulation of CD2+ T-cells with pronounced MIF generation response. The study, therefore, identifies a possible role of CD2 antigen in immunity to VL.

Animals↗

PubMed1991

Multilineage antigen expression and possible autocrine secretion of an interleukin 1 by Ph1-positive leukemic cell line KOPM-28.

A newly established Ph1-positive cell line, KOPM-28 was analyzed for immunological phenotype and the release of biological substance. The surface marker analysis revealed myelomonocytoid antigens, erythroid antigens and megakaryocyte-platelet antigens. Also, the cells could be induced for hemoglobin synthesis by the addition of hemin. The conditioned medium of this cell line showed the presence of interleukin 1 (IL-1) activity but not interleukin 2. Exogenous IL-1 and the conditioned medium stimulated the DNA synthesis of the cells themselves. Consequently, the yield substance works on the growth of original cells as an autocrine fashion, and might act on other normal cells as a paracrine factor.

Antigens, Neoplasm↗

PubMed1992

Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner.

The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.

Animals↗

PubMed2005

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

Amino Acid Sequence↗

PubMed2004

Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA.

Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

Animals↗

PubMed2003

Expression of SA5K, a secretion antigen of Mycobacterium tuberculosis, inside human macrophages and in sputum from tuberculosis patients.

An 8.3 kDa protein (SA5K), secreted by Mycobacterium tuberculosis/Mycobacterium bovis bacillus Calmette-Guérin (BCG) in culture filtrate, has been previously described in our laboratory. In the present study, analysis of the distribution of SA5K gene (Rv1174c) among M. tuberculosis strains, isolated from a wide variety of clinical specimens, revealed that the gene is present in all clinical isolates analyzed (29/29). SA5K expression inside human macrophages infected with BCG was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) on RNA extracted from bacterial cells following 24 and 48 h of infection. In addition, in order to evaluate whether SA5K gene was also expressed at the site of infection in the lung, a nested RT-PCR assay was developed to detect specific mRNA in sputum samples collected from smear positive tuberculosis patients. SA5K mRNA was detected in all the samples containing high numbers of tubercle bacilli demonstrating that the corresponding gene is expressed during the course of clinical infection.

Acyltransferases↗

PubMed1990

Effects of gamma-interferon on DR antigen expression, growth, 3,5,3'-triiodothyronine secretion, iodide uptake, and cyclic adenosine 3',5'-monophosphate accumulation in cultured human thyroid cells.

We have examined, using the same system of human thyroid cells in culture, the effects of the cytokine human gamma-interferon (gamma IFN) on the expression of DR antigen, cell pro-liferation, cAMP accumulation, and the differentiated functions, iodide uptake and T3 secretion. gamma IFN elicited a dose- and time-dependent increase in DR expression, with a maximum effect on day 5 of culture. The cytokine, at the same concentrations and experimental conditions as those found to be effective in inducing DR antigen expression, caused on day 5 of culture a dose-dependent inhibition of [3H]thymidine incorporation, DNA content, cell count, as well as TSH-stimulated (but not basal) iodide uptake and T3 secretion. The gamma IFN suppressive influence on the differentiated functions was not merely due to a reduction in cell number, but was also apparent when results were expressed per micrograms DNA. Since the cytokine did not inhibit TSH- or forskolin-stimulated cAMP accumulation and showed a suppressive influence toward 8-bromo-cAMP- and forskolin-stimulated T3 secretion, its inhibitory effect seems to be exerted at a site located distal to cAMP formation. Although gamma IFN alone was devoid of any effect on cAMP accumulation, it enhanced forskolin-stimulated (as well as TSH-activated) cAMP in the presence of 3-isobutyl-1-methylxanthine, an inhibitor of cAMP degradation. Thus, it would seem that gamma IFN also exerts an influence on cAMP formation (rather than degradation) at a step subsequent to TSH binding to its receptor. The effects we observed seem specific to gamma IFN, since alpha IFN, although capable of inhibiting human thyrocyte multiplication, lacked any influence on DR antigen expression, cAMP accumulation, or T3 secretion by human thyroid cells. In what way, if any, is gamma IFN-induced DR antigen expression on human thyrocytes, an event believed to be critical in the pathophysiology of autoimmune thyroid disease, related to decreased thyroid function and growth is presently unknown.

Cell Count↗

PubMed1989

Analysis of surface antigen expression of human immunoglobulin-secreting cells: phenotypic heterogeneity in normal counterparts of myeloma cells.

Human myeloma cells are malignant counterparts of plasma cells which represent the most differentiated B cells. Myeloma cells are, however, heterogeneous in their surface antigen expression (Katagiri et al, 1984, 1985), which may reflect that normal plasma cells have a spectrum of differentiation. To test this hypothesis, immunoglobulin-secreting cells (ISC) of non-neoplastic nature were studied with regard to their surface antigen expression by using a combination of reverse haemolytic plaque assay and complement-dependent cytolysis. Non-neoplastic ISC were found to have a broad spectrum of differentiation stages from the immature type of CD20+, HLA-DR+, CD38+ in the peripheral blood to the mature type of CD20-, HLA-DR-, CD38+ in the bone marrow. In patients with polyclonal B cell activation (PBA), ISC showed a more immature antigen expression in comparison with ISC in normal controls or patients without PBA. The surface antigen development of ISC was clearly demonstrated throughout the stages in the analysis of mitogen-induced ISC in vitro. No significant difference in the surface phenotype of ISC was found among heavy chain classes. Thus, non-neoplastic ISC show a spectrum of differentiation similar to that of myeloma cells, depending on the site where ISC are located, and on the degree of PBA in vivo.

Antibody-Producing Cells↗

PubMed2000

Humoral immune responses to DNA vaccines expressing secreted, membrane bound and non-secreted forms of the Tania ovis 45W antigen.

The antibody response to DNA vaccines expressing secreted, membrane bound and non-secreted forms of the same antigen was investigated. The antigen gene selected for these studies was the full length 45W antigen gene from Taenia ovis. This gene encodes a host protective membrane bound antigen with a native secretion signal at the amino terminus and a hydrophobic anchor domain at the carboxyl terminus. Full length and rationally truncated forms of the 45W antigen gene were generated and used to construct DNA vaccines encoding membrane bound, secreted and non-secreted forms of the 45W antigen. The cellular localisation of these antigen forms was confirmed by Western blot studies. BALB/c mice were immunised intramuscularly with plasmid DNA and serum antibody responses measured by enzyme linked immunosorbant assay (ELISA). The cellular localisation of DNA vaccine antigen had a significant effect on the magnitude but not the subclass of antibody responses. Immunisation with DNA expressing secreted 45W generated three-fold higher antibody titres than immunisation with DNA expressing membrane bound 45W, and 18-fold higher antibody titres than DNA expressing non-secreted 45W. All mice generated a predominantly IgG1 antibody response indicative of a TH-2 type immune response. These results indicate that the optimal induction of humoral immune responses to intramuscular genetic immunisation with the 45W antigen, requires the active secretion of antigen. This observation may be of value during the design of DNA vaccines in the future.

Animals↗

PubMed1992

Identification and characterization of epitopes shared between the mycobacterial 65-kilodalton heat shock protein and the actively secreted antigen 85 complex: their in situ expression on the cell wall surface of Mycobacterium leprae.

Both mycobacterial hsp65 and the actively secreted antigen 85 complex of 30-kDa region proteins are considered to be major immune targets in mycobacterial diseases. In this study, by using a novel series of monoclonal antibodies (MAbs) directed to these antigens, we identified and partially characterized three unique epitopes (Rb2, Pe12, and A2h11) that are shared between mycobacterial hsp65 and the individual components of the antigen 85 complex. Dot blot assays with native purified proteins revealed that all three MAbs are strongly bound to hsp65 and antigens 85A (MPT44) and 85B (MPT59), while a weak reaction or no reaction was found with antigen 85C (MPT45). Immunoblotting showed that MAb Rb2 reacted strongly with both hsp65 and the antigen 85 complex proteins, whereas MAbs Pe12 and A2h11 reacted strongly with the former but weakly with the latter. Moreover, these MAbs did not react with other closely related MPT51 and MPT64 secreted proteins. Further characterization of these epitopes was performed by using recombinant fusion and truncated proteins of Mycobacterium bovis BCG hsp65 (MbaA) and the M. leprae 30- and 31-kDa antigen 85 complex fusion proteins. In hsp65, Rb2-Pe12- and A2h11-reactive epitopes were found to reside in the C-terminal region of amino acid residues 479 to 540 and 303 to 424, respectively. In the M. leprae 30- and 31-kDa antigen 85 complex, all three epitopes were located in an N-terminal region of amino acid residues 55 to 266, one of the known fibronectin-binding sites of the M. leprae antigen 85 complex. Comparison of these MAb-reactive amino acid sequence regions between mycobacterial hsp65 and the components of the antigen 85 complex revealed that these regions show certain amino acid sequence identities. Furthermore, by immunoperoxidase and immunogold ultracytochemistry, we demonstrated that Rb2-, Pe12-, and A2h11-reactive epitopes are expressed both on the cell wall surface and in the cytosol of M. leprae bacilli within the lesions of lepromatous leprosy patients and in M. leprae-infected armadillo liver tissue.

Amino Acid Sequence↗

PubMed1990

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Animals↗