Role of macrophages in early protective immune responses induced by two vaccines against foot and mouth disease.

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.

Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants.

It is well documented that adjuvants improve the immune response generated by traditional viral vaccines; however, less is known about their effects on the immune response elicited by DNA vaccines. In this study, we have investigated the use of adjuvants, and have analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the BoHV-1 glycoprotein D (secreted version) in pCIneo vector with and without Montanide ISA25 (O/W), ISA206 VG (SEPPIC) and Cliptox™ (natural microparticles of clinoptilolite). The comparison of the immune response induced in mice by pCIgD formulated with or without adjuvants showed that the immunomodulators affect the total specific humoral and cellular response. The isotypes induced by these adjuvants were of the type Th1/Th2. A significant increase in the mac-3+ and F4/80+ populations of the groups receiving pCIneo with ISA25, ISA206; and an increase in CD4+ populations of the group receiving pCIneo ISA25, in comparison with the pCIneo group was observed. On the other hand, mice vaccinated with pCIgD/ISA25, pCIgD/ISA206, or pCIgD/Cliptox developed a significantly higher specific cytotoxic activity against BoHV-1 than the pCIgD and pCIneo groups. In this report we propose the use of ISA25, ISA206 or Cliptox as adjuvants in a DNA vaccine since they are able to induce not only a specific humoral immune response but also a specific cellular immune response.

Adjuvant effect of Cliptox on the protective immune response induced by an inactivated vaccine against foot and mouth disease virus in mice.

Foot and Mouth Disease (FMD) is an acute disease caused by Foot and Mouth Disease Virus (FMDV) which causes important economy losses, this is why it is necessary to obtain a vaccine that stimulates a rapid and long-lasting protective immune response. Cliptox is a mineral microparticle that in earlier studies has shown adjuvant activity against different antigens. In this study we have examined the effects of Cliptox on the magnitude and type of immunity elicited in response to inactivated FMDV (iFMDV) vaccine. It was demonstrated that iFMDV-Cliptox stimulates a specific antibody response detected in mucosal and in sera. The different isotype profiles elicited by inoculation with this vaccine indicate a Th1/Th2 response. Also, an increase in dendritic cells and macrophages in the spleen in comparison with the iFMDV vaccine iFMDV-Cliptox was detected. The Cliptox-iFMDV formulation was non toxic by using egg embryos and yielded increased protection against challenge with FMDV in the murine model. Our results show that the incorporation of Cliptox into FMDVi vaccine induces an increase of the specific protective immune response in mice and clearly indicate that Cliptox TM exert an (important) up-regulation on DC and MPhi. Additionally, Cliptox TM adjuvant can be used in vaccines for induction of mucosal immune response.

Beneficial activity of Enterococcus faecalis CECT7121 in the anti-lymphoma protective response.

AIMS: To study the anti-tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T-cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated. METHODS AND RESULTS: In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose-dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre-treated i.p. with Ent. faecalis CECT7121 (2.5 × 10(8 ) CFU) were challenged i.p. with LBC cells (1.0 × 10(6) cells) the day after. On day 30 post-inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre-treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re-challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL-12 and IL-10 levels were observed in surviving animals. CONCLUSIONS: Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down-regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re-challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice.

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4(+)T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

RANKL expression in a case of follicular lymphoma.

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.

BHV-1 DNA vaccination: effect of the adjuvant RN-205 on the modulation of the immune response in mice.

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.

Characterization of the immunophenotype and the metastatic properties of a murine T-lymphoma cell line. Unexpected expression of cytoplasmatic CD4.

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) yCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2-/dim, CD3-/dim, TCRbeta-/dim, TCRgammadelta, CD154 , CD40-, and CD45R. Coexpress cyTCRbeta, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4- CD8 CD44-/low, CD25+) or DN4 (CD4-low, CD8-/low, CD44-, CD25-) to double-positive (DP: CD4+CD8+) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-relatel clisorders.

Induction of apoptosis in murine lymphoma cells by cyclosporin A.

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.

Splice variant expression of CD44 in patients with breast and ovarian cancer.

CD44 is an adhesion molecule involved in many biological functions and has been described to play a role in tumor progression as well as in promotion of metastasis. It has also been suggested that expression of certain CD44 isoforms could be useful for breast and ovarian cancer screening, detection or staging. However, many other reports document no correlation between CD44 isoform expression and tumor malignancy. In light of such contradictory findings, we evaluated by exon-specific RT-PCR whether the expression of CD44 isoforms in breast and ovarian tumors correlated with any of the diagnostic criteria used to assess these diseases. We found a deregulation in the CD44 expression pattern in malignant tumors of both type of cancer compared with the one in benign tumors or normal tissue. However, we could not find a clear correlation between this deregulation or a given CD44 isoform and any diagnostic criteria evaluated, such as age, clinical data, tumor size, hormone receptor status, histological grade or aggressiveness.

Evaluation of soluble CD44 in patients with breast and colorectal carcinomas and non-Hodgkin's lymphoma.

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.

Alternative exon-specific PCR method for the analysis of human CD44 isoform expression.

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.

Interleukin 2 exerts autocrine stimulation on murine T-cell leukaemia growth.

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.

Enhancement of anti-tumour immunity in syngeneic mice after MHC class II gene transfection.

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.

[Immunobiological characterization of murine LB leukemia and the LBC cell line]. / Caracterización inmunobiológica de la leucemìa murina LB y la línea celular LBC.

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.

Induction of anti-tumour immunity in syngeneic mice by a leukaemic cell line.

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.

Inhibitory activity of soluble IL-2R in sera, ascites and culture supernatants from murine leukaemic cells.

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.