Role of atrial natriuretic peptide in the suppression of lysophosphatydic acid-induced rat aortic smooth muscle (RASM) cell growth.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. In the present study we investigated the possible role of atrial natriuretic peptide (ANP), a hormone affecting cardiovascular homeostasis and inducing antimitogenic effects in different cell types, on LPA-induced cell growth and reactive oxygen species (ROS) production in rat aortic smooth muscle (RASM) cells. Both LPA effects on cell growth and levels of ROS were totally abrogated by physiological concentrations of ANP, without modifying the overexpression of LPA-receptors. These effects were also affected by cell pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Moreover, the LPA-induced activation of Akt, a downstream target of PI3K, was completely inhibited by physiological concentrations of ANP, which were also able to inhibit p42/p44 phosphorylation. Taken together, our data suggest that PI3K may represent an important step in the LPA signal transduction pathway responsible for ROS generation and DNA synthesis in RASM cells. At same time, the enzyme could also represent an essential target for the antiproliferative effects of ANP.

Effective anti-tumor immunity induced in mice by a two-step vaccination protocol.

BACKGROUND: TS/A cells (a Balb/c-derived tumor cell line), when injected into syngenic mice, give rise to rapidly growing tumors. In this study, a vaccination protocol was established which was able to elicit an immune response effective in controlling tumor growth. MATERIALS AND METHODS: T19.2.1, a TS/A clone enginereed to stably express the mycobacterial cell wall-associated 19-kDa lipoprotein, was used as cell vaccine to immunize Mycobacterium Bovis-BCG pre-immunized Balb/c mice. RESULTS: Mice receiving the two-step vaccination protocol were able to develop a strong anti-TS/A DTH reaction. Moreover, following a challenge with wild-type TS/A cells, some vaccinated animals rejected the tumor and the remaining animals showed a significantly increased survival in respect to controls. CONCLUSION: The expression on TS/A cells of the mycobacterial 19-kDa antigen, recognised in the context of a pre-existing memory immune response, promotes the immunological recognition of the otherwise non-immunogenic wild-type TS/A cells.

Different expression of CD44, ICAM-1, and HSP60 on primary tumor and metastases of a human pancreatic carcinoma growing in scid mice.

Surface adhesion molecules play an important, but still not completely clarified, role in tumor metastasization. In this research, FACS analysis was employed to analyze surface expression of CD44H, CD44v5, CD44v6, ICAM-1 and HSP60 in human pancreatic adenocarcinoma cells growing in vitro or collected ex vivo from primary tumors and lung metastases of tumor-engrafted SCID mice. It was found that, in metastatic cells, the standard form of CD44 (CD44H) is down,-regulated, while a large fraction of cells express on membrane the splice variants v5/v6 and, in addition, ICAM-1 and HSP60. It was also apparent that two cell populations are present in lung metastases: a CD44neg population, including cells expressing CD44v5/v6, ICAM-1 and HSP60 and a population of CD44pos, CD44v5/v6neg, ICAM-1neg and HSP60neg cells. These results demonstrate that, in pancreatic adenocarcinomas, metastasization is correlated with expression of the CD44 variants v5 and v6. Moreover, this is the first report demonstrating HSP60 surface expression on metastatic cells.

Induction and membrane expression of heat shock proteins in heat-treated HPC-4 cells is correlated with increased resistance to LAK-mediated lysis.

In human pancreatic carcinoma cells (HPC-4), a hyperthermic treatment at 43 degrees C for 30 min resulted in the vigorous induction of Hsp72, along with a less pronounced increase in the rate of synthesis of Hsp90, Hsp60 and Hsp 27. Biotinylation of surface-exposed proteins, followed by isolation of biotin-tagged proteins by affinity chromatography, demonstrated that both Hsp72 and Hsp60 are expressed on plasma membrane. Membrane expression of these two Hsps was confirmed by immunoprecipitation of surface biotinylated proteins with anti-Hsp72 and anti-Hsp60 specific antibodies. Cytotoxic assays showed that untreated HPC-4 cells are intrinsically resistant to NK-mediated lysis, while they were efficiently killed by LAK lymphocytes, as well as by exposure to TNFalpha. Following heat-treatment, cells became much more resistant to LAK-mediated lysis, while their sensitivity to NK-mediated lysis and to TNFalpha cytotoxicity remained unmodified.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Identification of a 66 kD heat shock protein (HSP) induced in M-14 human melanoma cells by severe hyperthermic treatment.

Following severe hyperthermic treatment M-14 cells synthesize at high rate a new protein of about 66 kD, in addition to the three well known major HSPs (HSP 28, HSP 70 and HSP 90). This 66 kD protein is constitutively expressed at low levels and its rate of synthesis is not enhanced by mild hyperthermic exposures (40 degrees C for 2-4 h; 42 degrees C for 1-3 h), sufficient to induce the three major HSPs. The 66 kD protein is induced whenever the thermal dose administered to cells attains a threshold, roughly corresponding to a 50% reduction in survival. The 66 kDa protein is not induced by a variety of compounds (disulfiram, arsenate, cadmium) able to elicit a stress response in M-14 cells, as indicated by enhanced synthesis of the three major HSPs. Once induced by a treatment at 45 degrees C for 15 min, the rate of synthesis of the 66 kD protein remains above the control level for 16-20 h during recovery from the stress, while the synthesis of HSP 70 is shut off between 8 and 12 h. Immunoblotting and immunoprecipitation studies showed that the 66 kD protein shares immunological determinant(s) with HSP 70. Pulse-chase experiments demonstrated that the 66 kD protein is not a degradation product or a late post-transcriptional modification of HSP 70. It is proposed that the 66 kD protein is a previously unrecognized heat shock protein (HSP 66), characterized by an unusually high threshold for its induction.

Identification of a specific Mycobacterium tuberculosis protein able to activate T lymphocytes.

The recent advances in the field of immunology and molecular biology allow to consider not distant the identification of protective antigens to be used in new prophilactic and diagnostic tools. In our laboratory we have analysed a M. tuberculosis expression library by murine monoclonal antibodies, human sera and human T lymphocytes. We have identified a 35 Kd protein present only on the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). This 35 Kd protein is also detected, by immunohistological analysis, inside the alveolar macrophage of pulmonary tuberculosis patients. Both recombinant beta-galactosidase and MS2 polymerase-fused proteins have been produced and used to perform western blotting and T cell proliferation assay. As the cloned fragment contains an internal EcoRI restriction site, it was possible to identify two fragments of 1.7 and 2.2 kb respectively. Southern blot analysis showed that both the fragments hibridized with the genomic DNA from M. tuberculosis complex but not with the DNA from other mycobacterial isolates from 12 pulmonary tuberculous patients hibridized with the 1.7 kb fragment. The possible use of this insert for the molecular diagnosis of tuberculosis is under investigation.

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

Immunohistologic analysis of mycobacterial antigens by monoclonal antibodies in tuberculosis and mycobacteriosis.

Four monoclonal antibodies (MoAbs), 60.15, 61.3, 105.10, and 2.16, directed to different proteins of Mycobacterium tuberculosis were used by an indirect avidin-biotin complex peroxidase-antiperoxidase method to detect mycobacterial antigens in lung, lymph node, and joint tissue specimens of tuberculous patients. Using MoAb 60.15, which recognizes a broad range of cross-reactive mycobacterial proteins with a molecular mass of 28 kilodaltons (kD), scattered materials (mycobacterial in origin) were observed, many of which were located within phagocyte cytoplasm. With MoAb 61.3, which reacts with a 35 kD protein present in M tuberculosis, Mycobacterium africanum, and Mycobacterium bovis, many clumped particles similar in size and shape to acid-fast bacilli were observed within the phagocyte cytoplasm (lung tissue) and positive macrophages with lysosomes were distributed throughout the cytoplasm (bronchoalveolar lavage). The specificity of this MoAb (61.3) was confirmed by the negative staining of positive lymph node specimens obtained from a patient infected with Mycobacterium kansasii. MoAbs 105.10 and 2.16 bind to the cross-reactive 65 kD heat shock protein that is present in mycobacteria and stain scattered particles and dark clumps of bacilli within the phagocyte cytoplasm. On the basis of this study, immunohistochemical detection of mycobacterial antigens appears to be useful in establishing the mycobacterial etiology of caseating granulomas and in avoiding the false-negative results obtained by traditional staining methods.

Analysis of Mycobacterium tuberculosis genoma and production of a recombinant protein containing specific B and T cell antigenic determinants--new approaches to second generation antituberculosis vaccines.

Tuberculosis is still a major health problem in almost all over the world. Thus, new directions in basic and applied research of tuberculosis are under investigation in several laboratories. In this paper, we provide recent data obtained in our laboratory with the recombinant DNA technology which allow a systematic survey of the microbial genome. Screening of the M. tuberculosis genomic DNA library in the phage lambda gt11 expression vector, using E. coli as surrogate host, has evidenced the possibility of producing recombinant M. tuberculosis proteins recognized by sera from tuberculosis patients and by specific monoclonal antibodies. Using this technology, we have isolated a recombinant protein (molecular weight 130 kilodaltons) which is identified by a murine monoclonal antibody recognizing a mycobacterial cell wall antigenic determinant present on a mycobacterial protein. This protein is only present on the mycobacterial species related to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) and does not crossreact with nonpathogenic Mycobacteria. Since the immune response to mycobacterial infections is cell-mediated, the question arises about the use of M. tuberculosis-specific T lymphocytes to screen this gene bank. Thus, the recombinant mycobacterial protein isolated by antibodies has been then used to stimulate the proliferation of T lymphocytes from patients with tubercular pleuritis. This experiment indicates that the recombinant protein contains antigenic determinants recognized by T cells. Moreover, such protein is able to elicit delayed type hypersensitivity skin reaction in mice immunized or infected with M. tuberculosis and M. bovis. Finally, gene mapping and hybridization studies with native M. tuberculosis DNA confirme the mycobacterial nature of the recombinant DNA insert. Thus a good candidate for the prophylaxis and the immunodiagnosis of tuberculosis has been identified. The identification and selection of genes encoding antigenic determinants recognized by mycobacteria-specific T cells with protective functions will allow in the near future the construction by genetic engineering of recombinant vaccines effective in the control of this disease. This paper will briefly discuss the present strategy used in our laboratory to reach this goal.

Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis.

Three monoclonal antibodies (H60.15, H61.3, and H105.10) directed to protein antigens of Mycobacterium tuberculosis were obtained and characterized. H60.15 recognizes a protein with a molecular mass of 28 kilodaltons (kDa) with broad cross-reactivity on a panel of 12 species and strains of mycobacteria. H61.3 reacts with a 35-kDa protein present in M. tuberculosis, Mycobacterium bovis BCG, and M. africanum. On the basis of the antigen molecular masses and competition experiments with other monoclonal antibodies, H60.15 and H61.3 seem to be the first described monoclonal antibodies to these M. tuberculosis proteins. H105.10 binds to the cross-reactive 65-kDa protein present in mycobacteria. Epitope mapping of H105.10 was performed by using the M. leprae DNA sublibrary available in bacteriophage lambda gt11 for this antigen and revealed that its epitope resides in the region from amino acids 20 to 54. The 28-, 35-, and 65-kDa antigens isolated by immunoblotting and presented on nitrocellulose to pleural effusion T cells from tuberculosis patients induced a proliferative response, indicating the presence of T-cell epitopes. These observations indicate that two protein antigens should be added to the list of antigens detectable in M. tuberculosis by monoclonal antibodies. The common feature of such proteins, the elicitation of an immune response of limited or broad cross-reactivity for mycobacteria, encourages the search for their role in the pathogenesis of mycobacterioses.

The control of membrane and secreted heavy chain biosynthesis varies in different immunoglobulin isotypes produced by a monoclonal B cell lymphoma.

The control of production of the membrane (m) vs secreted (s) forms of immunoglobulin heavy chains was investigated in a panel of cell lines expressing different heavy chain classes but identical light chains (lambda) and variable regions. These cell lines could be induced towards Ig secretion by mitogen treatment. During this process a shift from m to s heavy chain production takes place. Here we show that, similarly to IgA- and IgE-producing B cells, in IgG2a-producing I.29 cells the gamma m-gamma s shift was accompanied by a shift in the corresponding mRNAs, with a decrease of gamma m mRNA and an increase of the gamma s mRNA in LPS-stimulated cells. By contrast, the micron mRNA was increased in LPS-stimulated IgM-producing cells, albeit these cells synthesized reduced amounts of micron polypeptides. The utilization of the translational level in the early steps of B lymphocyte maturation thus distinguishes the mode of regulation of mu chains from those of the other isotypes. In addition, in B cells a post-translational event blocks the secretion of IgM but not of IgG or IgE.

Epstein-Barr virus-transformed B cells process and present Mycobacterium tuberculosis particulate antigens to T-cell clones.

We have analyzed the presentation of mycobacterial antigens by Epstein-Barr virus-transformed human B (EBV-B) cells to mycobacteria-specific T-cell clones and lines, and to purified resting T cells. EBV-B cells were able to process and present not only soluble forms of antigen, such as PPD and the expressate preparation of M. tuberculosis strain H37Rv, but also particulate forms of antigen, such as whole mycobacterial H37Rv or M. bovis organisms. Electron microscopy studies demonstrated the capacity of EBV-B cells to phagocytose mycobacterial cells in 18 hr and pulsing experiments confirmed that an 18-hr of incubation is required for an efficient processing and presentation of mycobacterial determinants to T cells. The processing of whole-H37Rv particulate antigen by EBV-B cells was inhibited by the lysosomotrophic compound chloroquine and by high doses of irradiation. Finally, the analysis of the presentation of soluble and particulate mycobacterial antigens by PPD-positive and PPD-negative EBV-B cell clones has shown a preferential presentation of both forms of antigen by PPD-positive EBV-B clones.

Differentiation in the murine B cell lymphoma I.29: individual mu + clones may be induced by lipopolysaccharide to both IgM secretion and isotype switching.

Cells from the monoclonal B cell lymphoma I.29 expressing surface IgM (mu +) are capable of differentiating in vitro to IgM secretion and of switching to IgA or IgE production in response to lipopolysaccharide (LPS) stimulation. To determine whether a single mu + B cell is capable of undertaking both differentiative pathways (isotype switch and plasma cell differentiation) I.29 mu + cells were cloned by limiting dilution and a panel of clones were analyzed by immunofluorescence, endogenous labeling and Northern blotting. While 100% of the clones could differentiate toward IgM secretion, only a proportion of them (greater than 70%) also switched to IgA and/or IgE production. Certain clones switched preferentially to a specific isotype. Taken together with the observation that C gamma genes were never the target of switching in our experiments, these data suggest that individual mu + clones from the I.29 lymphoma are "precommitted" as for their switching potentials. The subclones that showed a high frequency of switching to IgA transcribed the germ line C alpha gene(s), suggesting a role for chromatin structure in determining the isotype switch specificity. Switch variant clones expressing either IgA or IgE on the cell surface were isolated and found capable of further differentiating toward Ig secretion in response to LPS. On the contrary, we could not induce switch to IgA in IgE-producing cells. Unlike mu + and alpha + cells, all the switch variant clones expressing IgE tested by endogenous labeling constitutively secreted large amounts of IgE in the supernatants even in the absence of LPS stimulation.

Immunology of tuberculosis: new directions in research.

Tuberculosis is still one of the major health problems in almost all over the world. Thus, new directions in basic and applied research on tuberculosis are under investigation. In this review we have provided recent data obtained in our laboratories on three main aspects of the immunology of tuberculosis, namely: i. the role of B lymphocytes in the processing and presentation of Mycobacterium tuberculosis antigens to T cells; ii. the activation and characterization of mycobacterial-specific T cell clones; iii. the T cell regulation of the immune response to M. tuberculosis. The analysis of the antigenic determinants of M. tuberculosis relevant in the antimycobacterial immunity is the major goal of the WHO programme on the immunology of tuberculosis. In fact, the attempt to develop a second generation vaccine against this microorganism is now possible by analyzing recombinant genomic DNA libraries of M. tuberculosis with monoclonal antibodies and T cell clones. In the near future, the identification of epitopes recognized by mycobacterial-specific T cells with helper, cytotoxic and suppressor functions will allow the preparation of recombinant and synthetic vaccines effective in the control of this disease.

Regulation of immune interferon production during the response to soluble microbial antigens.

Human peripheral blood mononuclear cells (PBMC) stimulated in vitro with purified protein derivative (PPD) or with a Candida albicans polysaccharide extract (MPPS) released immune interferon (IFN) and interleukin 2 (IL-2). Kinetic studies showed a biphasic production of IFN with maximum levels at days 3-4 and days 5-6 of culture. In contrast, the IL-2 production is only observed at days 2-3 of culture. The relationship between IFN and IL-2, analysed both in responder and nonresponder PBMC cultures, showed that the early peak of IFN production appears to be IL-2 independent whereas the second peak seems strictly related to the presence of IL-2 culture. Furthermore, monoclonal antibodies against class I and class II products of the major histocompatibility complex (MHC) inhibited IFN production when added at the beginning of culture, whereas only anti-class I antibodies interfered with the release of IFN when added to antigen-primed lymphocytes.

Mechanism of action of an antigen nonspecific inhibitory factor produced by human T cells stimulated by MPPS and PPD.

Human T lymphocytes cultured in vitro for 5 days with C. albicans purified polysaccharide (MPPS) and with purified protein derivative (PPD) from M. tuberculosis produce an antigen nonspecific inhibitory factor(s) (nsINH). nsINH blocks antigen-driven cell proliferation and the development of natural killer cells (NK) when added at the beginning of peripheral blood mononuclear cell culture. Analysis of the mechanism of action shows that nsINH inhibits the production of interleukin 2 (IL-2), the expression of IL-2 receptor (Tac antigen), and the synthesis of immune interferon (IFN). The biochemical characterization of nsINH shows that the suppressive activity is acid (pH 2.5) and temperature (56 degrees C) resistant. Gel filtration analysis indicates a molecular weight of 30-35K and 60-65K. These results suggest a role for nsINH in the down regulation of the lymphokine cascade.

Effects of dexamethasone on human natural killer cell cytotoxicity, interferon production, and interleukin-2 receptor expression induced by microbial antigens.

Dexamethasone inhibits the expression of the interleukin-2 receptor, the synthesis of immune interferon, and the development of natural killer cells when added to peripheral blood mononuclear cells cultured with soluble microbial antigens (purified protein derivative and a polysaccharide extract from Candida albicans [MPPS]) or human recombinant interleukin-2.

Monocyte subsets in the production of inhibitory factor by Candida albicans-activated human T cells.

Macrophages are essential for the proliferative response of human T lymphocytes to a purified polysaccharide extract from Candida albicans (MPPS). The role of macrophages as antigen-presenting cells in the production of an antigen non-specific inhibitory factor (nsINH) by MPPS-activated T cells was also investigated. Fc receptor positive (FcR+) or negative (FcR-) plastic-adherent mononuclear cells were used as MPPS-presenting cells, and the results show that the FcR- subset is mainly responsible for the release of nsINH from activated T cells.