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Foot and Mouth Disease (FMD) is an acute disease of cloven-hoofed species. We studied the protection and early immune response induced in the murine model by vaccines formulated with inactivated virus and two different adjuvants. The presence of IMS12802PR or ISA206VG adjuvants yielded protection against viral challenge at early times post vaccination and induced FMDV-specific, but non neutralizing, antibody titers. In vivo macrophage depletion in vaccinated mice severely decreased the protection levels after virus challenge, indicating a central role of this cell population in the response elicited by the vaccines. Accordingly, opsonophagocytosis of FITC-labelled virus was augmented in 802-FMDVi and 206-FMDVi vaccinated mice. These results demonstrate the ability of the studied adjuvants to enhance the protective responses of these inactivated vaccines without the increase in seroneutralizing antibodies and the main role of opsonization and phagocytosis in the early protective immune responses against FMD infection in the murine model.

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The TNF-family molecule, RANKL, is a key regulator of bone remodeling and essential for the development and activation of osteoclasis. Bone involvement signals diesease activity in non-Hodgkin's lymphoma and influences the progenesis. The molecular mechanism and soluble factors involved in osteoclastic activation in haematological malignancies remain unclear except for Multiple Myeloma and Adult T-cell Leukemia. The aim of this paper is to report the first case of Follicular Lymphoma with bone involvement displaying an aberrant expression of RANKL in malignant cells. The detection of RANKL in Follicullar Lymphoma may help to prevent bone lesion in patients by determining an appropriate treatment.

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It is well documented that adjuvants improve the immune response generated by traditional viral vaccines, but less is known about the effects of adjuvants on the immune response elicited by DNA vaccines. In this study, we have investigated the use of RN-205 (immunomodulator containing a membrane rich in lipopolysaccharide from gram-negative bacteria) as an adjuvant and analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD). The comparison of the antibody response induced in mice by a mixture of the three different versions of DNA gD (membrane-anchored, secreted and cytosolic) formulated with or without RN-205 showed that the immunomodulator did not affect the total specific humoral response. The cellular immune response induced in mice immunized with vaccines plus RN-205 was higher than that obtained in mice vaccinated without RN-205, not only in the indexes of proliferation tests but in the number of IL-4 and gammaIFN secreting cells. When total spleen cells were marked with specific monoclonal antibodies against surface markers, a significant increase in the macrophage population of all the groups receiving RN-205 was observed. CD8 and CD4 positive cells were also increased but to a lesser extent. Our results indicate that the incorporation of RN-205 into DNA vaccines induces an increase of the cellular specific immune response in mice.

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The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

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CD44 is a transmembrane glycoprotein involved in cell-cell and cell-substrate interactions. As a cell surface molecule, CD44 may be shed or released into the circulation by proteolytic enzymatic mechanisms. Therefore, soluble CD44 can be found in cell culture supernatants as well as in plasma. In this study we evaluated the levels of soluble total CD44 (sCD44) in serum samples of patients with breast and colorectal carcinoma as well as non-Hodgkin's lymphoma in order to correlate prognosis with sCD44 expression. Besides, we evaluated other clinical tumour markers routinely used, Cancer Antigen (CA) 15.3 and CA 19.9. We investigated 132 serological samples from breast cancer patients, 48 sera from colorectal tumours, 48 samples from stage IV non-Hodgkin's lymphoma and sera from 80 individuals without evidence of cancer or autoimmune disease. Breast cancer patients were divided into three groups: a) patients with no clinical evidence of positive nodules and no metastatic disease; b) patients with positive nodules; and c) patients with metastasis. sCD44 mean serum levels in these groups were 198+/-54 ng/ml, 221+/-78 ng/ml and 242+/-119 ng/ml, respectively, while the marker CA 15.3 values were 15.6+/-6.6 U/ml, 14.0+/-5.8 U/ml and 211.5+/-358.9 U/ml, respectively. sCD44 levels for colorectal tumour were 243+/-72 ng/ml, while CA 19.9 serum levels were 230+/-270 U/ml. Stage IV non-Hodgkin's lymphoma sCD44 levels were 398+/-160 ng/ml. sCD44, CA 15.3 and CA 19.9 values for healthy individuals without evidence of any cancer pathology were 223+/-58 ng/ml, 16.4+/-6.2 U/ml and 33+/-14 U/ml, respectively. From these results we conclude that sCD44 might be used as a reliable marker for patients with non-Hodgkin's lymphoma. However, sCD44 levels failed to correlate with prognosis, tumour burden or metastasis in breast and colorectal cancer patients. Neither was any correlation found between high CA 15.3 or CA 19.9 levels and soluble CD44 serum level.

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CD44 is a widely expressed cell-surface transmembrane glycoprotein involved in diverse adhesive processes. Its isoforms have been implicated in tumor progression and are considered a promising marker for evaluation of the metastatic potential of various tumors. Several methods have been described for the analysis of CD44 isoforms in tumor cells, including immuno-histochemistry, RT-PCR followed by hybridization and nested RT-PCR. We describe an alternative nested PCR for the analysis of CD44 isoform expression in various malignancies. Total RNA was isolated from various shock-frozen tissues from human tumors, reverse-transcribed and PCR-amplified using CD44-specific primers. Reverse-transcription was performed by two different methods, either using Tth-polymerase or MMLV-RT. Exon-specific amplification was then carried out using specific primers for each variable exon. Amplification products were assayed by agarose gel electrophoresis. Comparison of the patterns obtained from the first amplification and from the exon-specific amplification allowed to identify exons expressed by tumor tissues, as well as the genomic organization of CD44 isoforms. The method developed proved to be sensitive, reliable and inexpensive in comparison with other methods. It can be performed even in solid tumors and for numerous samples, and is suitable for laboratories with limited resources.

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As it has been suggested that an autocrine mechanism may control tumour cell growth, in this work cells from a spontaneous murine T lymphocyte leukaemia (LB) expressing the interleukin-2 receptor (IL-2R) (CD25) were evaluated in vitro for IL-2-mediated autocrine growth. Cells grew readily in culture and proliferation was enhanced by the addition of recombinant IL-2 but inhibited by monoclonal antibodies against either IL-2 or IL-2 receptor, in the absence of exogenous IL-2. Cyclosporin A also inhibited LB cell growth. However, when exogenous IL-2 was added together with cyclosporin A, cell proliferation proved similar to controls. Using reverse transcription polymerase chain reaction (PCR), mRNA for IL-2 was found to be present in tumour cells. Our findings support the hypothesis that LB tumour cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture.

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The relationship between tumorigenicity and expression of MHC class II molecules in a class II-negative murine leukaemia cell line (LBC) was studied. Analysis of structural DNA sequences encoding MHC class II proteins was performed by Southern blot with DNA isolated from both the original LB tumour and LBC cell line, digested with EcoRI, BamHI and HindIII and hybridised with specific probes for I-A alpha d and I-A beta d chains. Similar patterns were obtained for LB, LBC and normal BALB/c lymphocytes. In vitro treatment with IFN-gamma (20 - 1000 IU ml-1) failed to induce the expression of MHC class II antigens in LBC cell line. LBC cells were tri-transfected by a liposome-mediated protocol with I-A alpha d, I-A beta d genes and pSV2neo. Cells were selected for growth in medium containing Geneticin (G418). Surviving transfectants were cloned and three I-A+ clones were obtained after 20 days (LBCT cells). Syngeneic mice inoculated with 1.0 x 10(3) LBCT (I-A+) cells failed to develop a tumour, whereas the DT50 of mice injected with 1.0 x 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). Furthermore, specific CTL response against tumour cells was significantly enhanced upon priming with irradiated LBC-transfected cells (27 +/- 2%) compared with irradiated LBC cells (15 +/- 1.5%) in a 4 h 51Cr-release assay. It is suggested that neoexpression of MHC class II molecules enhances anti-tumour response by transforming tumour cells into professional antigen-presenting cells (APCs), which may be used to improve tumour-specific immunity in the autologous host.

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LB leukemia is a nonimmunogenic T cell tumor which spontaneously arose in a BALB/c mouse; efforts to induce immunological rejection of the leukemic cells have always failed. The leukemic cells grow rapidly and progressively in the syngeneic host invading spleen, lymph nodes and liver. A cell line (LBC) was developed from the original tumor. Both the original tumor and the cell line have been characterized as expressing the Thy 1+, CD3-, CD25+, MHC class I+, class II-, CD4- (original tumor), CD4+ (cell line), CD8+, gp70-, J11d.2+ phenotypes. Immunization of syngeneic mice with irradiated LBC cells induced cytotoxic T lymphocytes as well as anti-LBC antibodies which reacted with components of 14, 16 and 27 kDa present on LB tumor cells, LBC cell line and normal thymocytes but not on normal lymph node cells. Immunization of syngeneic mice with LBC cells partially protected them against subsequent challenge with the original tumor cells. The effect of sera from tumor-bearing mice and the super-natants from short term cultures were studied on cell proliferation. An inhibitory activity was demonstrated in these fluids, which was abrogated by addition of exogenous IL-2. ELISA showed the presence of soluble IL-2R alpha chain both in the conditioned medium as well as in the serum, which was demonstrated to be responsible for the inhibitory activity. The soluble IL-2R was produced by LB leukemic cells and exerted the inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2. Using reverse-transcription PCR, mRNA for IL-2 was found to be present in tumor cells. Our findings indicate that LB cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. The relationship between tumorigenicity and expression of MHC class II was also investigated. In vitro treatment with IFN-gamma failed to induce the expression of class II antigens in LBC cell line. Therefore these cells were tri-transfected by a liposome-mediated protocol with 1-A alpha d, I-A beta d genes and pSV2neo. Cells were selected to grow in medium containing Genetecin (G418) and surviving transfectants were cloned. Three I-A+ clones were obtained (LBCT) and were used to induce a specific CTL response against tumor cells. Syngeneic mice inoculated with 10(3) LBCT cells failed to develop a tumor while the DT50 of mice injected with 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). It is suggested that neoexpression of MHC class II molecules enhances anti-tumor response by transforming tumor cells into professional antigen-presenting cells, which may be used to improve tumor-specific immunity in the autologous host.

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Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

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The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24-48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non-specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 10(5)-1.3 x 10(5) M(r) serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsin or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing-thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL-2 abrogated inhibitory activity. ELISA showed the presence of soluble IL-2R both in LB conditioned medium and in above serum fraction. It is demonstrated that the inhibitory factor, soluble IL-2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL-2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2.

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A methodology for the preparation of a polymerized antigen from house dust mites Dermatophagoides Pteronyssinus is described. Polymerization was carried out by glutaraldehyde treatment in PBS and Sephacryl S300 column fractionation. A polymer of 135 Kd was obtained. Rabbits immunized with the soluble polymer (HDMP) elaborated specific antibodies as measured by a Dot-immunobinding assay. The reaction was inhibited by previous incubation with the HDMP antigen. It is suggested that the polymerized antigen may be employed in the treatment of patients with house dust mite anaphylaxis.

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This paper presents a model of spontaneous BALB/c lymphoma (LB) useful for the study of the relationship between the tumor and the immune system. Characterization of this tumor was done by FACS and it was determined that the tumor cells are T lymphocytes which express both CD4 and CD8 membrane markers but not the viral protein gp 70. In addition, tumor cells express IL-2 receptors and class I, but not class II, H-2 antigens. Antibodies were not detected in sera or ascitic fluid of syngeneic mice bearing a tumor transplant while both sera and ascites contained a suppressor factor that inhibited in vitro the proliferation of tumor cells and blastogenesis of normal BALB/c splenocytes after Concanavalin A stimulation. The results indicate that this tumor may be used as an experimental model for adult T cell leukemia as well as for the isolation and characterization of cytokines which may be responsible for the immunological unresponsiveness found in certain cancer patients.

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This paper presents a model of spontaneous BALB/c lymphoma (LB) useful for the study of the relationship between the tumor and the immune system. Characterization of this tumor was done by FACS and it was determined that the tumor cells are T lymphocytes which express both CD4 and CD8 membrane markers but not the viral protein gp 70. In addition, tumor cells express IL-2 receptors and class I, but not class II, H-2 antigens. Antibodies were not detected in sera or ascitic fluid of syngeneic mice bearing a tumor transplant while both sera and ascites contained a suppressor factor that inhibited in vitro the proliferation of tumor cells and blastogenesis of normal BALB/c splenocytes after Concanavalin A stimulation. The results indicate that this tumor may be used as an experimental model for adult T cell leukemia as well as for the isolation and characterization of cytokines which may be responsible for the immunological unresponsiveness found in certain cancer patients.